The Interactive Fly
Genes involved in tissue and organ development
The Drosophila heart is a linear organ formed by the movement of bilaterally specified progenitor cells to the midline and adherence of contralateral heart cells. This movement occurs through the attachment of heart cells to the overlying ectoderm which is undergoing dorsal closure. Therefore heart cells are thought to move to the midline passively. Through live imaging experiments and analysis of mutants that affect the speed of dorsal closure this study shows that heart cells in Drosophila are autonomously migratory and part of their movement to the midline is independent of the ectoderm. This means that heart formation in flies is more similar to that in vertebrates than previously thought. It was also shown that defects in dorsal closure can result in failure of the amnioserosa to properly degenerate, which can physically hinder joining of contralateral heart cells leading to a broken heart phenotype (Haack, 2014).
The movement of the heart progenitor cells to the midline has long been established to be dependent on dorsal closure. The cardioblasts were noted as being several cell diameters away from the dorsal edge of the epidermal primordium and moving relatively little compared to the ectoderm. Heart cell movement to the midline in Drosophila occurs through additional autonomous heart cell migration. Several lines of evidence support this conclusion. Firstly, live imaging of heart cells and ectodermal cells during dorsal closure shows uncoupling of the movement of heart and ectodermal cells. Secondly, cardioblasts make extensive protrusions during dorsal closure. It is speculated that these protrusions are required for motility, but they could be used for attachment to contralateral cardioblasts. In support of the former hypothesis, the protrusions occur from the onset of dorsal closure several hours before heart cells meet and are not suppressed through genetic mechanisms that are sufficient to suppress protrusions of ectodermal leading edge cells. The latter are generally longer (up to 10μm) than cardioblast protrusions (below 2μm) and are required for attachment to contralateral leading edges cells. Thirdly the strength of adhesion of heart cells to the ectoderm, as judged by the ablation experiments, is reduced as the heart cells approach the midline. It is speculated this is because the heart cells are moving partly autonomously at this time. Finally, when dorsal closure is delayed, as occurs in wun wun2 mutants, the heart cells migrate up to the amnioserosa before dorsal closure has completed. This phenotype appears to be a general feature when dorsal closure is delayed as it is reported to occur in other mutants (Haack, 2014).
Several molecular players are implicated in linking heart cells to the ectoderm: Spot adherens junctions (AJ) have been reported between cardioblasts and ectodermal cells. The AJ component DE-Cadherin (encoded by shotgun, shg) is highly expressed in heart cells and shg mutants display defects in cardioblasts reaching the midline as well as lumen formation. The extracellular collagen-like protein Pericardin (Prc) is expressed by pericardial cells and surrounds both them and cardioblasts Reduction in Prc levels causes interruptions in the cardioblasts lines, which appears to result from a loss of interaction with the ectoderm. Finally, disruption of integrin complexes, which are receptors for extracellular matrix proteins, using scab or mys mutants (encoding integrin α and β subunits respectively) causes mislocalisation of pericardial cells. If and how and these complexes and proteins are regulated to allow the heart cells to dynamically attach to the ectoderm remains an open question (Haack, 2014).
How might Wun and Wun2 be working mechanistically to promote heart formation and dorsal closure? The forces for dorsal closure arise from three sources. Firstly, actin rich filopodia from leading edge cells make contact with contralateral partners at the anterior and posterior most ends (canthi) and act in a zippering fashion. These filopodia are also important for correct alignment of the ectoderm with respect to parasegmental boundaries. Secondly, an actin-myosin-rich cable at the leading edge acts as a supracellular purse-string. Finally contractility and coordinated internalization of amnioserosa cells pulls the leading edges towards the midline (Haack, 2014).
This study found in wun wun2 mutants that the actin cable and leading edge filopodia are present, and internalization of amnioserosa cells is seen. Amnioserosa cells have highly wavy edges, normally only observed in much earlier embryos, during germ band retraction. Therefore the idea is favored that there are defects in tension in the ectoderm in wun wun2 mutants. wun and wun2 are expressed in the ectoderm and ectodermal wun2 expression is needed (along with heart cell expression) to rescue the heart defects of wun wun2 mutants. This loss of tension would also explain why the pericardial cells often lie away from the cardioblasts in wun wun2 mutants. In wild type the pericardial cells are strongly associated with the cardioblasts during dorsal closure. However, by the time the embryo is ready to hatch these two cell types are not tightly attached as can be seen during a heartbeat when the pericardial cells are thrust laterally and normally immediately rebound. It is speculated that if tension is lost then this rebound is weak leading to displacement of the pericardial cells (Haack, 2014).
The Hand gene family encodes highly conserved basic helix-loop-helix (bHLH)
transcription factors that play crucial roles in cardiac and vascular
development in vertebrates. In Drosophila, a single Hand
gene is expressed in the three major cell types that comprise the circulatory
system: cardioblasts, pericardial nephrocytes and lymph gland hematopoietic
progenitors. Drosophila Hand functions as a potent transcriptional activator, and converting it into a repressor blocks heart and lymph gland formation. Disruption of Hand function by homologous recombination also results in profound cardiac defects that include hypoplastic myocardium and a
deficiency of pericardial and lymph gland hematopoietic cells, accompanied by
cardiac apoptosis. Targeted expression of Hand in the heart
completely rescues the lethality of Hand mutants, and cardiac
expression of a human HAND gene, or the caspase inhibitor P35, partially rescues the cardiac and lymph gland phenotypes. These findings
demonstrate evolutionarily conserved functions of HAND transcription factors
in Drosophila and mammalian cardiogenesis, and reveal a previously
unrecognized requirement of Hand genes in hematopoiesis (Han, 2006).
The initial steps in heart formation are remarkably conserved from fruit
flies to mammals. In both types of organism, mesodermal progenitors become
committed to a cardiac fate in response to signals from adjacent tissues and
converge along the embryonic midline to form a linear cardiac tube with
rhythmic contractility. In Drosophila, the myocardial cell layer of the
contractile heart tube, composed of cardioblasts, is surrounded by pericardial
nephrocytes, which function as secretory cells, and by
lymph gland hematopoietic cells that give rise to all the major blood cells in
the adult fly. Cardioblasts, pericardial nephrocytes and lymph gland
hematopoietic progenitors -- the three major embryonic cell types that
comprise the Drosophila circulatory system -- arise from the
same cardiac mesoderm, specified by signaling pathways involving
Decapentaplegic (DPP), Wingless (WG) and FGF. In the
more complex mammalian cardiovascular system, cardiac and hematopoietic
progenitors are also derived from the same mesodermal region -- the
lateral mesoderm -- and are specified by conserved signaling pathways
involving bone morphogenetic protein (BMP), WNT and FGF,
exemplifying the conservation of developmental programs for cardiogenesis and
hematopoiesis between Drosophila and mammals (Han, 2006).
NK-type homeodomain proteins and the GATA family of zinc-finger
transcription factors are required for cardiac and hematopoietic development
in Drosophila and mammals. The Drosophila NK family transcription factor, Tinman, and its mammalian ortholog Nkx2.5, are expressed specifically in the developing heart and are both regulated by the DPP/BMP pathway. Both
Tinman and Nkx2.5 play central roles in activation of myocardial genes
required for heart development. The GATA factors, Drosophila Pannier (Pnr) and its mammalian homologues GATA4, GATA5 and GATA6, are also expressed in the
cardiogenic mesoderm and play crucial roles in heart development. Pannier
and GATA4 function as partners of Tinman and Nkx2.5, respectively, to activate
the cardiac gene program in Drosophila and mammals. Another
group of GATA factors, Drosophila Serpent (Srp), and its mammalian
homologues GATA1, GATA2 and GATA3 are required for hematopoiesis in
Drosophila and mammals, respectively. It is
likely that the functions of Tinman, Pannier and Serpent in cardiogenesis and
hematopoiesis reflect the highly conserved but simplified developmental
processes in Drosophila compared with mammals (Han, 2006).
The basic helix-loop-helix (bHLH) transcription factor HAND is the only
transcription factor known to be specific to the three major embryonic cell
types that comprise the Drosophila circulatory system. Cardiac and hematopoietic
expression of Hand is controlled by a 513 bp enhancer that integrates
the activity of Tinman, Pannier and Serpent, the three central transcription
factors that control cardiogenesis and hematopoiesis. Hand expression is activated by Tinman and Pannier in cardioblasts and pericardial nephrocytes in the heart and by Serpent in hematopoietic progenitors in the lymph gland, through evolutionarily conserved DNA-binding sites in this enhancer. These
findings place Hand at a nexus of the transcriptional networks that
govern cardiogenesis and hematopoiesis, but the potential functions of
Hand in these developmental processes have not been explored (Han, 2006).
By contrast, the functions of the two vertebrate Hand genes, Hand1
and Hand2, have been intensively studied. Hand1 and
Hand2 are initially expressed throughout the cardiogenic region but
later display complementary expression patterns in the left and right
ventricular chambers. Mice lacking Hand1 die from placental and
extra-embryonic abnormalities, whereas mice lacking Hand2 die from right ventricular hypoplasia and vascular defects.
Deletion of the Hand1 and Hand2 genes in the heart revealed
their dose-sensitive requirement and functional redundancy for myocardial
growth, and mutation of the single hand gene in zebrafish
results in a dramatic reduction in the number of cardiac cells. In
addition to its cardiac expression, Hand1 is highly expressed in the
lateral plate mesoderm from which the intra-embryonic aorta-gonad-mesonephros (AGM), a major source of hematopoiesis, is derived. The potential functions of Hand genes in hematopoiesis have not been investigated (Han, 2006).
Although HAND factors are essential in vertebrate cardiogenesis, little is
known about their mechanism of action. The requisite role of HAND factors in
growth of the cardiac chambers during vertebrate heart development also raises
interesting questions about the function of the highly conserved
Drosophila Hand gene, since the Drosophila heart is thought to
be a simple linear tube that does not undergo complex morphogenic changes (Han, 2006).
Drosophila Hand is shown to function as a highly potent
transcriptional activator, and converting it into a transcriptional repressor
blocks heart and lymph gland formation. To explore the possible roles of
Hand in cardiogenesis and hematopoiesis, a null mutant was generated
in the gene through homologous recombination. Hand mutant embryos and
larvae display profound cardiac defects, including hypoplastic myocardium, a
deficiency of pericardial nephrocytes, and abnormal cardiac morphology,
suggesting an essential role of Hand during Drosophila
cardiac morphogenesis. Lymph gland hematopoietic progenitors are also
dramatically reduced in most Hand mutant larvae, as well as in a
subset of Hand mutant embryos, indicating an important role of
Hand in Drosophila hematopoiesis. These abnormalities were
prevented by cardiac expression of Drosophila or human Hand genes, as
well as the caspase inhibitor P35. These findings demonstrate evolutionarily
conserved roles of Hand genes in Drosophila and mammalian
cardiogenesis, and suggest a possible requirement of Hand genes in mammalian
hematopoiesis (Han, 2006).
HAND1 and HAND2 have been shown to play essential roles the processes of
cardiac remodeling and chamber specification during mammalian cardiogenesis.
As the Drosophila heart has generally been considered to function as
a linear tube, without a defined chamber, the function of the single highly
conserved HAND factor in Drosophila has been a source of curiosity.
The results show that a substantial fraction of Hand mutant larvae
display cardiac morphological defects, including a thin hypoplastic heart tube
and dramatically reduced pericardial nephrocytes, as well as disruption of the
chamber-like structure. Hand mutant larvae also display abnormal
cardiac function, reflected by their sluggish heart rate and more frequent
discontinuities between continuous periods of heart beating, which could be
the cause of lethality after hatching. These findings suggest
that Hand plays an essential role in Drosophila heart
development (Han, 2006).
HAND transcription factors are expressed during heart development in human,
mouse, chick, frog, zebrafish, ciona and Drosophila embryos. Mouse Hand2 and Drosophila Hand are both regulated by GATA factors during heart development.
Functional studies have suggested that Hand genes are essential for
cardiogenesis in mouse, chick, zebrafish and Drosophila. The finding that cardiac expression of human HAND2 can rescue the early larval cardiac and hematopoietic phenotype of the Drosophila
Hand mutant provides strong evidence that Hand genes play evolutionarily
conserved roles in cardiogenesis (Han, 2006).
Mouse embryos lacking HAND2 exhibit hypoplasia of the right ventricle and
pharyngeal arches and associated apoptosis. Loss of the apoptosis protease-activating factor 1 (Apaf1), a downstream mediator of mitochondrial-induced apoptosis, partially rescues the ectopic apoptosis in Hand2-null embryos and delays embryonic
lethality,
suggesting that HAND2 acts, at least in part, to inhibit apoptosis (Han, 2006).
Ectopic apoptosis is observed in Hand mutant
Drosophila embryos, accompanied by a dramatic reduction in
pericardial nephrocytes and gaps in the cardiac tube (indicative of missing
cardioblasts). Interestingly, both the ectopic apoptosis and the early cardiac
and hematopoietic defects can be rescued by targeted expression the
apoptosis inhibitor P35 in Hand-expressing cells, indicating that one of the
important roles of Hand is to inhibit apoptosis (Han, 2006).
To determine if Hand can generally inhibit apoptosis, tests were performed to see whether overexpression of Hand in transfected Drosophila S2 cells could block apoptosis induced by genes that induce apoptosis, such as Reaper
and HID, or with drugs that induce apoptosis, such as Etoposide and Taxol. However, Hand failed to inhibit apoptosis in response to these stimuli, suggesting that it does not function as a general inhibitor of
apoptosis. The fact that targeted overexpression of P35 could not completely
rescue the cardiac morphological defects in Hand mutant larvae also
suggests that Hand performs functions in addition to inhibiting apoptosis. It
is possible that Hand could control differentiation of the cardiac and lymph
gland cells and the absence of Hand would lead to apoptosis indirectly as a
result of its role in some differentiation event (Han, 2006).
Although Hand family genes have been identified for a long time, their
mechanism of action has not been fully elucidated. The results of this study
demonstrate Drosophila Hand to be a potent transcriptional activator
in vitro and during heart and lymph gland development in vivo. Converting Hand
into a transcription repressor evokes more severe cardiac and hematopoietic
defects than simply removing it, suggesting that its function depends on the
activation of its downstream target genes. Based on the phenotypes resulting
from Hand mutants and from overexpression of Hand-EnR, it is predicted
that these target genes participate in cell growth and survival and in
maintaining cardiac and hematopoietic cell fates. Given the functional
redundancy among Hand genes in mammals, Drosophila offers a powerful
system with which to uncover conserved functions and mechanisms of action of
this gene family in both cardiogenesis and hematopoiesis (Han, 2006).
In Drosophila, adult blood cells originate from the lymph gland
hematopoietic progenitors, which are derived from cardiac mesoderm. The lymph
gland dissociates at the pupal stages to release all the adult blood cells.
Hand is the only transcription factor identified to date that is expressed in
all hematopoietic progenitors and the entire heart. The dramatic reduction of
lymph gland hematopoietic progenitors in Hand mutants suggests that
Hand is essential for Drosophila hematopoiesis (Han, 2006).
In mammals, the adult hematopoietic system originates from the yolk sac and
the intra-embryonic aorta-gonad-mesonephros (AGM) region. Previous studies have suggested a close relationship between the Drosophila cardiac mesoderm and the mammalian cardiogenic and AGM region. In both Drosophila and mammals, the specification of these regions requires the input of BMP, WNT and FGF signaling from the neighboring germ layer and function of NK and GATA factors in the mesoderm. Although the possible role of HAND factors in mammalian hematopoiesis has not been explored, mouse Hand1 is expressed at high levels in the lateral plate mesoderm, from which the cardiogenic region and the AGM region arise. This study provides the first evidence for the requirement of Hand in Drosophila hematopoiesis, suggesting similar functions for its mammalian orthologs (Han, 2006).
Fluid- and gas-transporting tubular organs are critical to metazoan development and homeostasis. Tubulogenesis involves cell polarization and morphogenesis to specify the luminal, adhesive, and basal cell domains and to establish an open lumen. This study explores a requirement for Talin, a cytoplasmic integrin adaptor, during Drosophila embryonic heart tube development. Talin marked the presumptive luminal domain and was required to orient and develop an open luminal space within the heart. Genetic analysis demonstrated that loss of zygotic or maternal-and-zygotic Talin disrupted heart cell migratory dynamics, morphogenesis, and polarity. Talin is essential for subsequent polarization of luminal determinants Slit, Robo, and Dystroglycan as well as stabilization of extracellular and intracellular integrin adhesion factors. In the absence of Talin function, mini-lumens enriched in luminal factors form in ectopic locations. Rescue experiments performed with mutant Talin transgenes suggested actin-binding was required for normal lumen formation, but not for initial heart cell polarization. The study proposes that Talin provides instructive cues to position the luminal domain and coordinate the actin cytoskeleton during Drosophila heart lumen development (Vanderploeg, 2015).
These experiments establish an essential function for the integrin adapter Talin in the assembly of the Drosophila embryonic heart. During the cardioblast (CB) migratory phase preceding tubulogenesis, Talin localizes along the CB apical surface, immediately ventral to the leading edge which extends towards the dorsal midline. As this Talin rich domain persists throughout embryonic heart assembly, eventually surrounding the lumen of the open cardiac tube, this surface is termed the pre-luminal domain. Talin is essential for the dynamic cell morphology and the leading edge features that characterise collective cardial cell migration. Furthermore, following migration, Talin is required to enclose a continuous lumen between the bilateral CB rows (Vanderploeg, 2015).
Analysis of late stage hearts in rhea zygotic mutants reveals that Talin is essential to correctly orient the CB polarity such that a continuous lumen is enclosed along the midline. In wildtype, many membrane receptors including Robo, Dg, Unc5, and Syndecan accumulate along the luminal domain. E-cadherin, Dlg, and other cell-cell adhesion factors are restricted to cell contact points immediately dorsal and ventral to the lumen and to the lateral cell domains between ipsilateral CBs. As evidenced by Robo and Dg immunolabeling experiments, the midline luminal domain is absent or, at best, is discontinuous along the midline in rhea mutant embryos. However, the Robo and Dg enriched luminal domains are not completely absent in null rhea homozygotes, but are found ectopically along lateral membranes between ipsilateral CBs. Robo's ligand, Slit, is also detected within these ectopic lumina. Similar ipsilateral Slit and Robo accumulations were observed in embryos mutant for the integrin subunit genes scab (αPS3) or mys (βPS1). Thus, the expanded Dlg-rich adhesive contact observed in rhea null embryonic hearts is consistent with a model in which integrins and Talin instruct the localization of Slit and Robo. These cues are essential to orient the lumen and to restrict the adhesive regions. In the absence of Talin, other components of the luminal structure, including Dg and the Slit-Robo complex, can self-assemble and create non-adherent luminal domains. However, proper midline positioning of the lumen requires Talin function (Vanderploeg, 2015).
Using an array of Talin transgenes previously shown to modify integrin adhesion strength and actin recruitment, this study assessed and compared the importance of these Talin-dependent processes. Binding of Talin's integrin binding site 1 (IBS1) to a membrane proximal NPxY motif on the β-integrin tail induces conformational changes within the integrin dimer, activating it and increasing the affinity for ECM ligands. Integrin activation is likely required prior to Talin IBS2 binding, an interaction which promotes a strong and stable integrin-cytoplasmic adhesome linkage. The current data indicates that either of Talin's two integrin binding sites are sufficient to promote CB morphogenesis and heart tube assembly. The ability of the heart to form in the presence of only IBS1 or IBS2 suggests that strong, long-lasting integrin-mediated adhesions are unnecessary. This idea is reinforced by the late accumulation of CAP, a protein recruited to more mature muscle adhesions. It is likely that transient adhesions are sufficient for lumenogenesis. It remains possible that an essential role for either IBS1 or IBS2 is masked by the perdurant maternal Talin in zygotic mutants. However, the functional redundancy of these domains is consistent with in vitro and in vivo studies suggesting that a subset of Talin functions can be fulfilled by either IBS domain (Vanderploeg, 2015).
Talin links integrins to the actin cytoskeleton both directly through an actin binding domain, or indirectly through recruitment of actin regulators such as Vinculin. Bond force studies of the C-terminal ABD suggest that although the ABD-actin linkage is direct, it is a weak bond which likely relies on additional direct or indirect Talin-actin linkages to form a strong and stable connection. Supporting this, TalinABD is essential for morphogenetic processes which rely on transient and dynamic integrin-actin linkages, but it is at least partially dispensable for longer-lasting adhesions which are likely stabilized by indirect Talin-actin interactions through Vinculin. The current studies demonstrate that Drosophila heart development is sensitive to disruptions in Talin's C-terminal ABD, which implicates cytoskeletal reorganization as a key process downstream of integrins during tubulogenesis. Supporting this, expression of constitutively active Diaphanous or dDAAM, formin proteins which promote actin polymerization, induced ectopic lumina similar to those that have been characterized in rhea mutants. These data are consistent with Talin promoting CB morphogenesis and lumen formation through direct, but dynamic actin linkages and suggest that formins may act downstream of Talin in apicalizing lumen formation (Vanderploeg, 2015).
To date, most studies on the Drosophila embryonic heart have focused on cell surface factors including receptors and their respective ligands; few studies have moved into the cell to establish the downstream signaling pathways involved. Insights into in vitro models suggest that polarity pathways and vesicle trafficking will be informative areas of study. For example, in the MDCK cyst model, the small GTPases Rab8a and Rab11a coordinate with the exocyst complex to deliver luminal factors to the pre-luminal initiation site. It remains to be determined whether similar exocytosis or secretion mechanisms are required for Drosophila heart lumen initiation or expansion. Furthermore, although it is unclear which classical apical polarity proteins are conserved in the Drosophila heart, epithelial and endothelial models suggest that the Cdc42-Par6-aPKC complex is a conserved master regulator of tube formation in both vertebrates and flies. Indeed, Drosophila heart tubulogenesis fails in embryos with heart specific inhibition of Cdc42 and expression of activated Cdc42 results in lateral lumina reminiscent of those characterized in rhea homozygotes. A mechanism is envisioned of heart tubulogenesis in which Talin provides instructive cues to the vesicle trafficking and polarity networks that target luminal factors and inhibit the assembly of cell-cell adhesion structures within the pre-luminal domain (Vanderploeg, 2015).
Mechanotransduction of tension can govern the remodeling of
cardiomyocytes during growth or cardiomyopathy. Tension is
signaled through the integrin adhesion complexes found at muscle
insertions and costameres but the relative importance of
signalling during cardiomyocyte growth versus remodelling has not
been assessed. Employing the Drosophila cardiomyocyte as
a genetically amenable model, this study depleted the levels of
Talin, a central component of the integrin adhesion complex, at
different stages of heart growth and remodeling. A continuous
requirement for Talin was demonstrated during heart growth to
maintain the one-to-one apposition of myofibril ends between
cardiomyocytes. Retracted myofibrils cannot regenerate appositions
to adjacent cells after restoration of normal Talin expression,
and the resulting deficit reduces heart contraction and lifespan.
Reduction of Talin during heart remodeling after hatching or
during metamorphosis results in pervasive degeneration of cell
contacts, myofibril length and number, for which restored Talin
expression is insufficient for regeneration. Resultant dilated
cardiomyopathy results in a fibrillating heart with poor
rhythmicity. Cardiomyocytes have poor capacity to regenerate
deficits in myofibril orientation and insertion, despite an
ongoing capacity to remodel integrin based adhesions (Bogatan,
2015). In late embryogenesis, the Drosophila heart is a 4
micron diameter tube enclosed by 2 cardiomyocytes, attached at the
dorsal and ventral midline with cadherin based cell junctions, and
an integrin rich lumen. The larval cardiomyocytes are dominated by
myofibrils that terminate in integrin rich insertions at the
dorsal and ventral midline, without a cadherin rich domain.
Therefore early heart development is marked by dramatic
reorganisation of cell adhesion and polarity. For the remainder of
a fly’s life, cardiomyocyte differentiation is remarkable
for increase in cell size but not cell number, and for the pupal
remodelling of posterior aorta myocytes into heart myocytes
(Bogatan, 2015). This study examined the role of Talin production in the
differentiation, growth and remodelling of cardiomyocytes. The
requirement of integrin function for cardiomyocyte adhesion was
verified, and it was noted that, like body wall muscle, the
insertions are integrin-rich, and that the muscle costameres
coincide with myocyte surface integrin adhesions. Normally,
myofibrils of each cell are aligned end-to-end with myofibrils of
the contralateral cardiomyocyte, suggesting that extracellular
matrix (ECM) linkages at the end of myofibrils are different from
the rest of the cell surface, reminiscent of the mammalian
intercalated disc. If levels of Talin production are reduced,
cardiomyocyte insertion, particularly at points of myofibril
termination are vulnerable to degeneration (Bogatan, 2015). During first instar heart differentiation and pupal remodelling,
cardiomyocytes are most susceptible to depletion of Talin,
resulting in significant cell shrinkage. At less susceptible
stages of heart growth, less cell shrinkage, but loss of myofibril
apposition between cells results. The resulting degeneration of
heart structure is likely due to the loss of adhesion caused by
the depletion of Talin. This reflects the ongoing turnover of
Talin and Integrin at adhesions, shown to be modulated by tension
in Drosophila muscle. Remarkably, restoration of normal
Talin expression does not enable regeneration of myofibril length,
inter-cardiomyocyte cell junctions or apposition of myofibril ends
between myocytes at any larval or adult stage. Instead, the
cardiomyocyte perimeter is marked by a broader band of integrin,
suggestive of expanded adhesion to the heart ECM, and hence less
direct transmission of tension between cardiomyocytes.
Nevertheless, affected cardiomyocytes continue to grow as the
larva grows, without restoring cell to cell apposition and
alignment of myofibrils (Bogatan, 2015). Heart contraction is reduced subsequent to Talin reduction at
each larval stage, including during the second instar, when
myocyte degeneration is minimal, but midline apposition of
myofibrils is disrupted. Nevertheless, this disruption does not
reduce the rhythmicity of second instar treated hearts. Heart
dilation, rhythmicity and contraction are most affected by
transient depletion of Talin during cardiomyocyte remodelling in
the first instar, suggesting that synchronicity of cardiomyocyte
contraction requires cell to cell contact, possibly along the
ipsilateral domains of cardiomyocytes. This cell surface domain
contains the costameres, where components of the IAC are
implicated in tension signalling (Bogatan, 2015). Myofibril stability may depend upon linkage to integrin adhesion
at insertions, or at the costameres, as Talin depleted
cardiomyocytes have fewer myofibrils. However muscle insertion
structure is far more sensitive to the level of Talin than the
structure of the costamere. Weakened costameres, observed in Drosophila
mutants of muscle Trim32,
are depleted of Integrin Adhesion complex (IAC) proteins,
including Talin, resulting in unbundling of myofibrils and muscle
“wasting”. Similarly, increased or decreased Integrin
function in vertebrate heart muscle alters intercalated disc
structure and cardiomyocyte contractility. In Drosophila
and vertebrates, integrin adhesion signalling is required for
homeostasis of the contractile apparatus (Bogatan, 2015). ECM is visible on the luminal and abluminal surfaces of
cardiomyocytes. As heart diameter grows normally, new matrix must
be deposited on both surfaces. Similarly, when cardiomyocytes
retract, the remaining ECM likely stretches and expands as the
heart vessel becomes dilated. In the Drosophila model,
this dilation results in the deposition of a more elaborate
network of Pericardin containing ECM fibrils. This process is
analogous to mammalian Dilated Cardiomyopathy (DCM). DCM can be
triggered by mutations in proteins that link the sarcomere to the
ECM, such as IAC proteins vinculin and tintin. Expression of IAC
proteins is elevated in cardiac hypertrophy. Analysis of IAC gene
function in genetic models such as Drosophila reveals
the temporal dimension of the stability and remodeling of
myofibrils. This study indicates myofibril stability requires
ongoing Talin renewal, and that regeneration after perturbation is
very limited. Further study of IAC function subsequent to changes
in cardiac load in Drosophila cardiomyocytes should be
instructive in revealing the signalling pathways activated in DCM
(Bogatan, 2015)
The formation of the cardiac tube is a remarkable example of complex morphogenetic processes conserved from invertebrates to humans. It involves coordinated collective migration of contralateral rows of cardiac cells. The molecular processes underlying the specification of cardioblasts (CBs) prior to migration are well established and significant advances have been made in understanding the process of lumen formation. However, the mechanisms of collective cardiac cells migration remain elusive. This study identified CAP and MSP300 as novel actors involved during CBs migration. They both exhibit highly similar temporal and spatial expression patterns in migrating cardiac cells and are necessary for the correct number and alignment of CBs, a prerequisite for the coordination of their collective migration. These data suggest that CAP and MSP300 are part of a protein complex linking focal adhesion sites to nuclei via the actin cytoskeleton that maintains post-mitotic state and correct alignment of CBs (Dondi, 2021).
This study used discriminative training methods to uncover the chromatin, transcription factor (TF) binding and sequence features of enhancers underlying gene expression in individual cardiac cells. Machine learning with TF motifs and ChIP data for a core set of cardiogenic TFs and histone modifications were used to classify Drosophila cell-type-specific cardiac enhancer activity. The classifier models can be used to predict cardiac cell subtype cis-regulatory activities. Associating the predicted enhancers with an expression atlas of cardiac genes further uncovered clusters of genes with transcription and function limited to individual cardiac cell subtypes. Further, the cell-specific enhancer models revealed chromatin, TF binding and sequence features that distinguish enhancer activities in distinct subsets of heart cells. Collectively, these results show that computational modeling combined with empirical testing provides a powerful platform to uncover the enhancers, TF motifs and gene expression profiles which characterize individual cardiac cell fates (Busser, 2015).
A previous study designed and applied a meta-analysis of gene expression profiles derived from purified mesodermal cells obtained from wild-type (WT) and informative mutants to characterize and predict gene activity in the Drosophila heart. In addition, recent studies have chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) of numerous cardiac TFs to uncover the cis regulatory elements and genes which characterize the cardiac lineage. In order to compile a more comprehensive list of genes with confirmed expression in the Drosophila heart, this analysis consisted of a large-scale validation of these predictions using whole embryo in situ hybridization. Out of 103 tested genes, an additional 50 genes were uncovered with previously uncharacterized expression in the cardiac mesoderm (CM) and/or mature heart. Combining these newly-identified cardiac genes with a complete curation of the literature reveals a total of 284 genes with verified expression in the heart (Busser, 2015).
GO analysis followed by the generation of a condensed summary of the list that was initially obtained was assembled by removing redundant GO terms. The purpose of this analysis was to uncover the functions of this large battery of cardiac genes. Indeed, the non-redundant GO terms revealed a diversity of functions for these genes, identifying both upstream (signaling, transcription, etc.) and downstream (adhesion, chemotaxis, metabolic processes, etc.) components of the heart gene regulatory network. In fact, a more detailed categorization revealed that 165 of these 284 genes are upstream components, with 82 of these being sequence-specific TFs. As there are presently only eight described cardiac cell subtypes (five PC and three CC; referring to paracardial cells and cardial cells), this shows that there are at least 10x the number of TFs than previously characterized cell states, suggesting that there is more extensive diversity in the combinations of TFs utilized to achieve specificity of cardiac gene expression than had been appreciated in prior studies. The diversity of TFs required to achieve cellular specificity of gene expression seems to be mirrored in the enhancers they regulate, since similar diversity was found in the combinations of motifs regulating and TFs binding myogenic enhancers. In total, this work uncovers a large battery of cardiac genes, and both the diversity of their inferred functions and the large number of TFs identified suggest that these genes are under complex combinatorial transcriptional regulation (Busser, 2015).
The molecular mechanisms underlying the coordinate regulation of these heart genes ≈ previous study characterized the motifs, enhancers and TFs that discriminate the two broad populations of the Drosophila heart, PCs and CCs. This study sought to model enhancers with cardiac activity of individual cardiac cell states to gain insights into both the similarities and differences in sequence and chromatin features amongst the eight individual cardiac cell subtypes that are known to exist. To do so, compiled a list of enhancers with previously reported activity in the Drosophila heart was compiled, including those from a preceding study, and transgenic reporter assays were performed to confirm and refine prior findings at the level of single cells of defined identities. To avoid the confounding effects of reporter variability due to insertion site, these reporters were inserted at a specific genomic locus that permits robust and reproducible activity in the mesoderm. In vivo transgenic reporter assays were performed with the 95 curated cardiac enhancer sequences and it was confirmed that 73 are active in the CM and/or heart, with the majority of the enhancer sequences with non-cardiac activity showing activity in the neighboring amnioserosa cells (Busser, 2015).
The activity of these 73 cardiac reporters was monitored in the differentiated heart to compile training sets of enhancers with activity in the different cardiac cell subtypes. As the cells of the heart can be subdivided into individual identities based on morphological differences and the expression pattern of distinct TFs, the expression of Tin, which marks a subset of CCs and PCs, and Zinc Finger Homeobox 1 (Zfh1) which labels all PCs, with anatomical and morphological differences of the cells was used to identify every distinct cardiac cell type. Using these markers and monitoring reporter activity in the differentiated heart, a set of enhancers was uncovered with activity in all the PCs (22 total sequences, hereafter referred to as 'pan-PC') and/or all the CCs (33 total sequences, hereafter referred to as 'pan-CC'). Of these 73 cardiac reporters, 6 to 7 enhancers were identified with activity restricted to the subsets of the CCs (hereafter referred to as 'Tin-CC', 'Tin-Lb-CC' or 'Svp-CC') which is an insufficient quantity to serve as a training set for a machine learning analysis without over-fitting the data. Many enhancer sequences with activity in the different PC subtypes of the heart were identified, including the Svp-PCs, Odd-PCs and Eve-PCs. However, it was not possible to individualize the activity of enhancer sequences in the Tin-alone or Tin-Lb-PCs, with only one enhancer sequence (that associated with the Lb genes) with activity restricted to the Tin-Lb PCs but not the Tin-alone PCs. As enhancer sequences are active in both of these cell types, this class is referred as the 'Tin-PC' enhancers. In total, these results identified sets of enhancers with activities in different subsets of cardiac cells, including pan-PC, pan-CC, Eve-PC, Tin-PC, Odd-PC and Svp-PC (Busser, 2015).
A machine learning approach was used to uncover associated regulatory elements and the discriminating characteristics (sequence motifs and epigenetic features) that differentiate these individual heart cells. Previous work has shown that the distribution of epigenetic modifications of the histone proteins and in vivo binding profiles of relevant TFs can be used to predict cis regulatory elements and gene activity. A recent study has described the distribution of a series of histone modifications in sorted mesodermal nuclei from Drosophila embryos at a developmental stage in which the cardiac precursor cells are being specified. In addition, another study examined the in vivo binding sites of a series of conserved cardiogenic TFs at different developmental time points. These include the T-box TFs (Doc), the GATA4 ortholog Pnr, the Nkx2.5 ortholog Tin and the TFs downstream of the signaling pathways for Wnt (dTCF in Drosophila) and Bmp (phosphorylated Mad (pMad) in Drosophila). In addition to the aforementioned TFs and histone marks, this study also included over 1000 binding motifs from available databases to identify sequence features critical for categorizing enhancer activities. The binding motifs and in vivo binding profiles for cardiogenic TFs and relevant histone modifications were mapped onto the training set and control sequences and a support vector machine (SVM) was used to discriminate the training set from controls. To model cell-type-specific cardiac enhancer activity, separate SVM models were built for pan-PC, pan-CC, Eve-PC, Tin-PC, Svp-PC and Odd-PC sequences (Busser, 2015).
Attempts were made to classify the different cell subtypes against each other. However, this approach failed to discriminate the training set sequences from controls as the area under the receiver operator characteristic (AUC) curve values ranged from 0.46 to 0.67. This result is due to the overlap in the training set sequences, with most sequences showing activity in more than one cell type, which reflects a requirement for the gene products regulated by these enhancers in more than one cell type. To circumvent this issue, separate SVM models were built for training set sequences from GC and length-matched background sequence. Here reliable classification of cardiac cell subtype enhancers were observed as the AUC curve varied for the separate classifiers from 0.96 to 0.99. In addition, enhancers predicted by these models are significantly associated with known heart genes. Finally, it was shown that the enhancer predictions of cardiac cell classifications are cell-type-specific. In total, these results confirm the generation of cardiac cell subtype-specific cardiac classifiers that can reliably discriminate the training set from controls (Busser, 2015).
It was next asked if the enhancer predictions from the individual cell-specific classifications could be used to predict expression patterns of known cardiac genes, and to use these annotated gene expression patterns to uncover the functions of individual heart cells. To do so, the top-scoring cardiac cell subtype enhancer prediction were isolated from each classification for each gene with known heart expression. By focusing this analysis on genes with validated cardiac expression, it was possible to confidently associate a predicted enhancer with bona fide transcriptional targets, findings that are not always available or included in such studies, often due to the lack of known expression patterns for candidate target genes. Underscoring the utility of this approach, 278 out of 284 heart genes (97.9%) were associated with a top-scoring predicted cell-specific cardiac enhancer. Out of these 278 heart genes associated with a predicted enhancer, 196 of these predictions were found within the introns of the heart gene (70.5%), increasing the confidence in its association with this transcriptional target. Hierarchical clustering of the prediction scores was used to group related expression patterns, which uncovered distinct clusters of cell-specific cardiac gene expression. This analysis revealed gene expression clusters specific for the individual cardiac cell subtypes and also for the pan-PC, pan-CC and all cardiac cell expression patterns (Busser, 2015).
With these expression clusters, it was asked if functions associated with these individual cardiac cell subtypes could be inferred. GO analysis for the genes within these expression clusters, followed by the removal of redundant terms, revealed functions for these gene expression clusters. Genes associated with enhancers predicted to be active in all heart cells (pan-PC/pan-CC) were associated with developmental, signaling and transcriptional functions. This result is consistent with these genes playing a role in the upstream regulatory network that specifies the cardiac lineage. Furthermore, genes with predicted expression in all CCs (pan-CC) were enriched for myogenic functions including cell adhesion and the actin cytoskeleton which are expected functions for contractile cells. Interestingly, genes associated with pan-PC enhancers were associated with renal system development, which further supports their proposed role as insect nephrocytes (Busser, 2015).
This analysis also uncovered specialized functions for individual cardiac cell subtypes. For example, the Odd-PCs were enriched for chemotaxis and locomotion functions, suggesting these cells are responsive to migratory cues. Alternatively, in the anterior segments of the embryo, Odd is expressed in the PCs of the neighboring lymph gland which forms the adult blood cells, and it is this population of cells which are responsive to migratory cues. Interestingly, the genes associated with enhancers with predicted activity in Tin-PCs are associated with development of endocrine functions (the ring gland in Drosophila is an endocrine organ). Since the physiological processes of filtration, secretion and reabsorption must be coordinated, this specialized endocrine role for Tin-PCs suggests these cells may act as a cellular relay mechanism between these components of the insect excretory system. Lastly, genes associated with enhancers with predicted activity in Eve-PCs and Svp-PCs specialize in the production of extracellular matrix components, which is an essential aspect of proper filtration of the haemolymph (Drosophila blood). In total, these results confirm that modeling cell-type-specific enhancer activities can be used to both confirm and identify previously uncharacterized functions of individual cardiac cells (Busser, 2015).
To test the in vivo transcriptional activities of the predicted enhancers, transgenic reporter assays inserted at specific genomic loci were used to test 47 enhancer predictions of varying scores in the cell-specific classifications. These results revealed that 46 of these 47 candidate enhancers were active reporters in the Drosophila embryo, with 19 of these 46 active reporters (41.3%) showing activity in the differentiated heart. Analyses of cell-type-specific reporter activity uncovered a concordance between predicted and confirmed activity. For example, a predicted enhancer located within the first intron of CG5522 scores well in the pan-PC and pan-CC classifications and poorly in the classifications of individual cardiac cell subtypes. Transgenic reporter assays confirm this result as this genomic region activates reporter expression in all PCs and CCs of the differentiated heart. The distribution of prediction scores was used to reveal enhancers that are active in individual cardiac cells. For example, another enhancer prediction located within the first intron of the Dscam gene scores very well in the Eve-PC and Odd-PC classifications. In agreement with these cell-specific predictions, this enhancer prediction ws shown to be active in these two cell types with additional activity in the Svp-PCs, thereby confirming the significant but slightly less robust Svp-PC prediction score. Some successful enhancer predictions scored well in a cellular subtype classification as well as in the pan-PC and pan-CC classifications. It is possible that such regulatory elements may be composed of overlapping enhancer signatures, with one DNA segment regulating pan-PC and pan-CC activity while another DNA segment enhances transcription in a different cellular subtype. The transgenic reporter assays used to assay enhancer activity would be insensitive to detecting such minor differences in reporter activity due to in vivo perdurance of the reporter RNA and/or protein. In agreement with this possibility, previous studies uncovered multiple signatures in the enhancers regulating muscle founder cell gene expression. Taken together, these results show that the distribution of prediction scores for individual cardiac cell classifications can be used to predict enhancer activity in individual cardiac cell subtypes (Busser, 2015).
To gain an understanding of the regulatory network required for specifying individual cardiac cell fates, the sequence, TF binding and chromatin features critical for the classification of each subtype of heart cell included in this analyses was assessed. As features in the training set receive positive weights, those in the control set receive negative weights, and irrelevant features receive zero weight in linear SVMs, the classification weights associated with the histone marks, TF binding and sequence features relevant to the previously delineated cell-specific regulatory models were examined (Busser, 2015).
The in vivo binding of cardiogenic TFs was next examined as a feature at two developmental time points: (1) 4-6 h after egg laying, a time point in which the dorsal mesodermal derivatives-which includes the precursors of the CM-are specified; and (2) 6-8 h after egg laying, a time point during which the more differentiated CM is specified. Tin, the Nkx2.5 ortholog in Drosophila, is first expressed in and required to specify the dorsal mesodermal derivatives, its expression and function then become restricted to the CM and later there is a confinement of Tin to subsets of cells comprising the mature heart. Pnr (the Gata4 ortholog in Drosophila) and Doc (Tbx4 ortholog in Drosophila) expression intersect with Tin in the CM, and both of these TFs are required for the differentiation of most cardiac cells. Finally, the overlap of signaling by Wnt (whose downstream effector in Drosophila is dTCF) and Bmp (whose downstream effector in Drosophila is phosphorylated Mad, pMad) is critical for specification of the CM (Busser, 2015).
Among the TFs examined, the greatest enrichment was seen with Tin at 6-8 h, which is consistent with the central role played by Tin in the cardiac transcriptional network in Drosophila. However, this interpretation should be considered with caution as the majority of heart enhancers in the training sets were identified based on the presence of Tin binding sites or in vivo binding. The larger positive classification weight at 6-8 h than at 4-6 h for Tin supports a more critical role for Tin binding to cardiac enhancers when the CM is specified (Busser, 2015).
Surprisingly, since Pnr has previously been shown to be a key regulator of cardiogenesis, the SVM weights reveal a minor role for the GATA TF Pnr binding in regulating cardiac enhancer activity. However, this finding is consistent with a recent report which failed to identify cardiac enhancers due to Pnr binding and suggests either a non-enhancer role for such binding or an inability to accurately assess such enhancers with the transgenic reporter assays used in these studies. For example, as minimal promoters are used in transgenic reporter assays, this result could reflect a requirement for a certain promoter in vivo for enhancer activity driven by Pnr-dependent enhancers (Busser, 2015).
Positive classification weights associated with pMad, Tcf and Doc was noticed among the different cell types. Interestingly, it was found that differential SVM weights are associated with these TFs in the various cardiac subtype classifications. For example, Doc shows the greatest positive weight for the Eve-PC classification, and every newly-identified enhancer with Eve-PC activity is bound by Doc. Furthermore, pMad demonstrates a greater SVM weight amongst the classifications of individual cardiac cell subtypes than amongst the pan-PC or pan-CC classifications. This outcome suggests that differential utilization of this signaling pathway may play a role in specifying individual cardiac cell fates. As 7 out of 11 pan-PC enhancers (63.6%) and 6 out of 8 individual cardiac cell subtype enhancers (75%) of newly-identified cardiac enhancers are bound by pMad, validation of this hypothesis requires further testing. In conclusion, these data show that differential SVM weights of in vivo TF binding can be used to model cell-specific enhancer activities (Busser, 2015).
As numerous studies have shown that the epigenetic modifications of the histone proteins can be used as predictors of cis regulatory element activity, the SVM weights were examined for multiple histone mark modifications for each cardiac cell subtype classification identified in this analyses. These histone modifications were examined at the 6-8 h developmental time point (a time at which the cardiac precursors are specified) from sorted mesodermal nuclei. Surprisingly, the strongest enrichment of any modification is tri-methylation of lysine 27 on histone 3 (H3K27me3) for all cardiac cell subtypes. An enrichment of H3K27me3 on active mesodermal enhancers was shown previously; this was in disagreement with another study that revealed a depletion of H3K27me3 on active mesodermal enhancers. As the polycomb complex. which is associated with silent chromatin. primarily trimethylates lysine 27 on histone 3, the most likely explanation for these data is that they reflect the overall enhancer activity in a heterogenous rather than pure population of cells. Since the cells of the Drosophila heart only correspond to a tiny population of the entire mesoderm, and whole mesoderm was previously studied, the apparently inconsistent observation noted in this study suggests that the enhancer is repressed in the majority of the cells (non-heart mesodermal cells) and is active in the minority of cells examined (the fraction of the mesoderm which comprises the heart and its precursors). In agreement with this interpretation, the SVM weights for H3K27me3 are greater for the cardiac subpopulations than those with activity in all PCs or CCs in which a larger population of total cells would show signs of repression. Furthermore, the enrichment for acetylation of lysine 27 on histone 3 (H3K27ac) on these same enhancers suggests that they are active in a subset of cells. These results argue that an accurate interrogation of the epigenetic signatures of individual genomic loci requires isolating homogenous subpopulations of cells. This point is especially relevant when describing bivalent chromatin signatures which may reflect the presence of either a bivalent locus in a single cell or different epigenetic modifications in some but not all members of a more diverse cell population (Busser, 2015).
Monomethylation of lysine 4 on histone 3 (H3K4me1) is positively weighted amongst all classifications, consistent with its description as an enhancer mark. In contrast, trimethylation of lysine 4 on histone 3 (H3K4me3) and trimethylation of lysine 36 on histone 3 (H3K36me3) received either no weight or negative weights for all classifications, consistent with their description as marks of promoters and gene bodies, respectively. Surprisingly, the SVM weight for the active enhancer mark H3K27ac received no weight among Tin-PC enhancers, which may be due to the fact that H3K27ac was seen to only mark two out of nine training set sequences. This suggests that H3K27ac may not always associate with active enhancers in certain cell types. However, this interpretation should be regarded with caution as the training set was small for these cell types and two out of two newly-identified Tin-PC enhancers were marked by H3K27ac. Trimethylation of lysine 79 on histone 3 (H3K79me3) was positively associated with each cardiac cell subtype classification, a result that is in agreement with a recent study which observed H3K79me3 on a subset of developmental enhancers. Interestingly, H3K79me3 showed greater SVM weights associated with Svp-PC and Odd-PC classifications than with the other models, suggesting that these modifications may be differentially utilized amongst cardiac cell subtypes. A large-scale validation of enhancer activities will be required to test this hypothesis, although six out of seven (85.7%) newly-discovered enhancers with activity in Svp-PCs and/or Odd-PCs are marked by H3K79me3 while 7 out of 11 (63.6%) with pan-PC activity are marked by H3K79me3. In any event, such differential utilization of histone marks amongst cell types and regulatory elements may explain the incomplete association between a particular mark and a class of regulatory element. Furthermore, such a cell- or tissue-specific role for histone modifications likely explains the tissue-specific effects of loss-of-function mutations in histone-modifying enzymes. In total, these results uncover chromatin features that are enriched and that potentially discriminate among cardiac cell subtypes (Busser, 2015).
In order to identify DNA sequence similarities and differences amongst the cardiac cell subtype classifications, this study examined the top 500 scoring sequence motifs amongst all classifications and used hierarchical clustering of their SVM weights to reveal clusters of similarly-acting regulatory motifs. Similar to the clustering of enhancer activities, this analysis revealed motif clusters enriched amongst each cardiac cell subtype classification and depleted or irrelevant to the classification of the other cardiac cells. In addition, this analysis revealed motifs relevant for activity in all cardiac cells. The identification of cell-type-specific clusters suggests a role for these motifs in mediating particular patterns of gene expression that are specific for different subsets cardiac cells (Busser, 2015).
The preceding section identified sequence features that potentially discriminate enhancer activity in individual cardiac cells. In order to test this hypothesis, sequence features were identified that were positively weighted within a cell subtype classification(s) and that were depleted or irrelevant for the other cardiac subtype models. cis mutagenesis of a selected fraction of these sequence motifs was then used in transgenic reporter assays to monitor the effects of their targeted removal from otherwise WT enhancers. For this purpose, the activity of five separate motifs, each of which is predicted to discriminate regulatory element activity within subtypes of cardiac cells was analyzed: V$ZF5_01, V$ETS_Q4, V$TEF_01, V$EVI1_06 and V$MTF_01 (Busser, 2015).
The WT mib1 enhancer (mib1WT) is active in the Odd-PCs and contains two V$ZF5_01 motifs. This motif has a high positive weight within the Odd-PC classification, suggesting that it plays a critical role in Odd-PC enhancer activity In agreement with this hypothesis, mutagenesis of the V$ZF5_01 motifs in the mib1 enhancer (mib1ZF5) leads to a loss of reporter expression in Odd-PCs (Busser, 2015).
Previous studies have documented an essential role for Ets binding sites in enhancers with activity in Eve-PCs. This observation is now extended by showing that V$ETS_Q4 motifs are heavily weighted in the Eve-PC classification, and that the two V$ETS_Q4 motifs in the Doc1 enhancer are critical for activity in Eve-PCs. Interestingly, the V$ETS_Q4 motif is derived from binding sites for the ETS1 TF, whose ortholog in Drosophila is Pointed (Pnt). In prior studies it was also shown that Pnt was critical in trans for enhancer activity in Eve-PCs, a finding which further establishes that motif enrichment in enhancers can be used to reveal cell-type-specific TFs (Busser, 2015).
The V$TEF_01 motif is positively weighted amongst the Eve-PC and Odd-PC classification, suggesting that it contributes a critical function to Eve-PC and Odd-PC enhancer activities. This study now shows that mutagenesis of the two V$TEF_01 motifs in the CG13822 enhancer (CG13822TEF) leads to a loss of reporter expression in Odd-PCs and de-repression into Eve-PCs. The V$TEF_01 motif is recognized by thyrotroph embryonic factor, which is a member of the proline and acidic amino acid-rich (PAR) subfamily of basic region/leucine zipper TFs, whose closest Drosophila ortholog is Par domain protein 1 (Pdp1). The functional role of V$TEF_01 motifs in the CG13822 enhancer suggests a role for Pdp1 in cardiogenesis. In support of this hypothesis, a previous functional genomic screen uncovered a role for Pdp1 in patterning the fly heart. Thus, both cis and trans tests of Pdp1 function are consistent with each other in establishing a key role for this TF in Drosophila cardiogenesis (Busser, 2015).
Finally, the SVM weights enriched amongst pan-PC and pan-CC classifications were used to uncover features that are essential for activity in all heart cells. The SVM weights for V$MTF1_01 and V$EVI1_06 motifs are positive amongst classifications of pan-PC and pan-CC enhancers. The WT sty enhancer (styWT) is active in all PCs and CCs. Mutagenesis of the one V$EVI1_06 motif (styEVI) or the one V$MTF1_01 motif (styMTF) in the sty enhancer abrogates enhancer activity in the majority of PCs and CCs, suggesting a critical role for these motifs in regulating enhancer activity in all heart cells. V$MTF1_01 is recognized by Metal regulatory factor 1 (MTF1) in vertebrates and V$EVI1_06 is recognized by EVI-1 (also known as MECOM and PRDM3) whose Drosophila orthologs correspond to MTF1 and hamlet (ham), respectively. The present identification and characterization of these TFs makes them excellent candidates for regulating cardiogenesis in Drosophila. In support of this model, targeted depletion of ham in the dorsal mesoderm using RNAi causes abnormalities in cardiogenesis (Busser, 2015).
The distribution of histone marks, in vivo TF binding, and the presence of TF binding motifs have all been exploited to reveal the enhancers that govern gene expression. This study has combined all three of these approaches using discriminative machine learning methods on a training set of enhancers with activity in distinct subtypes of cardiac cells to model cell-type-specific enhancer activity in the Drosophila heart. Using this approach, sequence, chromatin and TF binding features were uncovered that appear to underlie enhancer activity in individual cardiac cells. From these findings, it is hypothesized that such features potentially discriminate the unique enhancer specificities of single cardiac cells, which was empirically confirmed for a series of sequence motifs in regulating appropriate patterns of cardiac enhancer activity. Finally, by associating a cardiac gene expression atlas with the predicted enhancers from each cell subtype classification, this study uncovered previously unknown functions of individual cells of the Drosophila heart. Collectively, these results document the utility of computational modeling of enhancers to uncover the sequence motifs, chromatin and TF binding patterns as well as the gene expression profiles and functions of individual cells within the overall cardiac lineage (Busser, 2015).
Methyltransferases regulate transcriptome dynamics during development and aging, as well as in disease. Various methyltransferases have been linked to heart disease, through disrupted expression and activity, and genetic variants associated with congenital heart disease. However, in vivo functional data for many of the methyltransferases in the context of the heart are limited. This study used the Drosophila model system to investigate different histone 3 lysine 36 (H3K36) methyltransferases for their role in heart development. The data show that Drosophila Ash1 is the functional homolog of human ASH1L in the heart. Both Ash1 and Set2 H3K36 methyltransferases are required for heart structure and function during development. Furthermore, Ash1-mediated H3K36 methylation (H3K36me2) is essential for healthy heart function, which depends on both Ash1-complex components, Caf1-55 and MRG15, together. These findings provide in vivo functional data for Ash1 and its complex, and Set2, in the context of H3K36 methylation in the heart, and support a role for their mammalian homologs, ASH1L with RBBP4 and MORF4L1, and SETD2, during heart development and disease (Zhu, 2023).
Congenital heart diseases are the major cause of death in newborns, but the genetic etiology of this developmental disorder is not fully known. The conventional approach to identify the disease-causing genes focuses on screening genes that display heart-specific expression during development. However, this approach would have discounted genes that are expressed widely in other tissues but may play critical roles in heart development. This study reports an efficient pipeline of genome-wide gene discovery based on the identification of a cardiac-specific cis-regulatory element signature that points to candidate genes involved in heart development and congenital heart disease. With this pipeline, 76% of the known cardiac developmental genes were retrived and 35 novel genes were predicted that previously had no known connectivity to heart development. Functional validation of these novel cardiac genes by RNAi-mediated knockdown of the conserved orthologs in Drosophila cardiac tissue reveals that disrupting the activity of 71% of these genes leads to adult mortality. Among these genes, RpL14, RpS24, and Rpn8 are associated with heart phenotypes. This pipeline has enabled the discovery of novel genes with roles in heart development. This workflow, which relies on screening for non-coding cis-regulatory signatures, is amenable for identifying developmental and disease genes for an organ without constraining to genes that are expressed exclusively in the organ of interest (Nim 2021).
Developmental patterning and tissue formation are regulated through complex gene regulatory networks (GRNs) driven through the action of transcription factors (TFs) converging on enhancer elements. As a point of entry to dissect the poorly defined GRN underlying cardiomyocyte differentiation, an integrated approach was appled to identify active enhancers and TFs involved in Drosophila heart development. The Drosophila heart consists of 104 cardiomyocytes, representing less than 0.5% of all cells in the embryo. By modifying BiTS-ChIP for rare cells, H3K4me3 and H3K27ac chromatin landscapes were examined to identify active promoters and enhancers specifically in cardiomyocytes. These in vivo data were complemented by a machine learning approach and extensive in vivo validation in transgenic embryos, which identified many new heart enhancers and their associated TF motifs. These results implicate many new TFs in late stages of heart development, including Bagpipe, an Nkx3.2 ortholog, which is shown to be essential for differentiated heart function (Seyres, 2016).
Transcription is initiated at the core promoter, which confers specific functions depending on the unique combination of core promoter elements. The downstream core promoter element (DPE) is found in many genes related to heart and mesodermal development. However, the function of these core promoter elements has thus far been studied primarily in isolated, in vitro or reporter gene settings. tinman (tin) encodes a key transcription factor that regulates the formation of the dorsal musculature and heart. Pioneering a novel approach utilizing both CRISPR and nascent transcriptomics, this study showed that a substitution mutation of the functional tin DPE motif within the natural context of the core promoter results in a massive perturbation of Tinman's regulatory network orchestrating dorsal musculature and heart formation. Mutation of endogenous tin DPE reduced the expression of tin and distinct target genes, resulting in significantly reduced viability and an overall decrease in adult heart function. This study has demonstrated the feasibility and importance of characterizing DNA sequence elements in vivo in their natural context, and accentuate the critical impact a single DPE motif has during Drosophila embryogenesis and functional heart formation (Sloutskin, 2023).
The adult abdominal heart of Drosophila receives extensive innervation from glutamatergic neurons at specific cardiac regions during metamorphosis. The neurons form presynaptic specializations, as indicated by the localization of synaptotagmin and active zone markers, adjacent to postsynaptic sites that have aggregates of glutamate IIA receptors. To determine the role of this innervation in cardiac function, an optical technique was developed, based on the movement of green fluorescent protein-labeled nerve terminals, to monitor heart beat in intact and semi-intact preparations. Simultaneous monitoring of adjacent cardiac chambers revealed the direction of contractions and allowed correlation with volume changes. The cardiac cycle is composed of an anterograde beat in alternation with a retrograde beat, which correlate respectively with systole and diastole of this multichambered heart. The periodic change in hemolymph direction is referred to as cardiac reversal. Intracellular recordings from muscles of the first abdominal cardiac chamber (the conical chamber) revealed pacemaker action potentials and the excitatory effect of local glutamate application, which initiated retrograde contractions in semi-intact preparations. Unilateral electrical stimulation of the transverse nerve containing the glutamatergic neuron that serves the conical chamber causes a chronotropic effect and initiation of retrograde contractions. This effect is distinct from that of peripheral crustacean cardioactive peptide (CCAP) neurons, which potentiate the anterograde beat. Cardiac reversal was evoked pharmacologically by sequentially applying CCAP and glutamate to the heart (Dulcis, 2005).
Normal cardiac performance depends both on intrinsic excitability of cardiac pacemaker cells and on extrinsic neuronal activation or modulation of this specialized class of cardiomyocites. The fine balance between cardiac pacemaker activity, conduction of electrical impulses to the working myocardium, and its regulation by classical neurotransmitters, neuropeptides and amines is, in many cases, still poorly understood. This study investigated the role of glutamatergic innervation in the regular cardiac function of adult Drosophila. Octopamine and neuropeptides are expressed in cardiac neurons of a variety of insects, but the glutamatergic cardiac innervation in adult flies represents a novel finding. Axons grow onto the cardiac muscle in the first abdominal segment and fasciculate during metamorphosis to form a characteristic glutamate-immunoreactive (IR) synaptic structure, the transverse bridge (TB) (Dulcis, 2005).
Glutamate is the major excitatory transmitter of the mammalian CNS, where it mediates not only normal synaptic transmission but also participates in functional plasticity during development and throughout life. The Drosophila neuromuscular junction (NMJ) is glutamatergic and with the availability of powerful genetic tools has served as a valuable model system for investigating synaptic function and plasticity. The relatively large size of the novel cardiac synapses, however, may prove advantageous for many studies. Thus, the goals of this study were to investigate whether presynaptic and postsynaptic specializations accompany the glutamate-IR cardiac innervation and to determine the role of these synapses in cardiac function (Dulcis, 2005).
Adult holometabolous insects display a cardiac cycle composed of two alternating pacemaker phases, the anterograde and the retrograde beats, which correlate with a reversal of hemolymph flow. In other species, cardiac reversal develops during metamorphosis and requires new neuronal input. Drosophila may follow a similar pattern, but this awaits confirmation. During the larval stage in Drosophila, the heart does not receive innervation. The larval cardiac contractions are completely myogenic, originate in the caudal chamber, and produce an anterograde heartbeat. Profound anatomical changes occur during metamorphosis, including the formation of a new conical chamber, which is added posterior to the aorta, and an extensively innervated new muscular ventral layer. Because the conical chamber has an independent development from the rest of the abdominal heart, it has been hypothesized that this region might represent the location of the retrograde pacemaker whose neuronal activation could produce cardiac reversal in adult flies (Dulcis, 2005).
This study investigated whether formation of the glutamatergic innervation correlates with changes in the cardiac function of adult Drosophila. A novel optical technique, based on the movement of green fluorescent protein (GFP)-labeled nerve terminals, used to monitor heartbeat in intact and semi-intact preparations, revealed that cardiac reversal is indeed a feature of adult heart function. The excitatory effect of glutamatergic synapses on the myocardium provides the mechanism for originating the retrograde beat and hence cardiac reversal (Dulcis, 2005).
The adult heart is innervated extensively by glutamate-IR neurons. A large glutamate-IR synaptic structure is formed during metamorphosis in the first cardiac chamber (the conical chamber), which has been suggested as the location of the retrograde pacemaker. Presynaptic and postsynaptic specializations, including extensive synaptotagmin immunoreactivity and clusters of DGluRIIA immunoreactivity, are present along the glutamatergic terminals. In addition, abundant NC82 immunoreactivity, which is a marker that colocalizes with DPAK at the level of active zones, revealed a number of putative release sites both in the transverse bridge and bouton-like terminals (Dulcis, 2005).
Local glutamate application in the conical chamber evokes a long-lasting depolarization of the membrane potential, which initiates pacemaker action potentials in normal saline. Both ionotropic (GluRs) and metabotropic (mGluRs) glutamate receptors have been described in the Drosophila CNS and at the NMJ. Although ionotropic glutamate receptors were localized at the cardiac synapses, the glutamate-evoked depolarization observed in myocardial cells might also be attributable in part to activation of mGluRs, which may cause an increase of postsynaptic excitability by, for example, blocking resting K+ currents or reducing voltage-gated and Ca2+-activated K+ currents. Ultrastructural, immunocytochemical, and additional electrophysiological analyses of these cardiac synapses must be undertaken to understand the mechanism of cardiac pacemaker cell activation in adult Drosophila (Dulcis, 2005).
To determine the influence of cardiac innervation on heart function, the first necessary step has been to produce a detailed description of the regular cardiac activity. The cardiac cycle of resting adult flies is composed of two alternating phases, the anterograde and retrograde beats, displaying different contraction rates. This phenomenon, known as cardiac reversal in other open circulatory systems, is associated with a change in the direction of blood circulation. Because cardiac contraction originates periodically at the two ends of the heart, two putative pacemakers must be alternately active in adult Drosophila. The terminal chamber, where the anterograde contractions originate, has been suggested as the location of the anterograde pacemaker. In contrast, the retrograde pacemaker may reside in the conical chamber (Dulcis, 2005).
In addition to a constant beat, consisting of high-frequency cardiac contractions (mini-systole-mini-diastole cycles), the conical chamber also displays a superimposed lower frequency systole-diastole cycle, which is characterized by a slow change in its diameter and with anterograde and retrograde beats, respectively. Unlike closed circulatory systems in which each cardiac ventricular contraction-relaxation cycle corresponds to a systole-diastole cycle, in open circulatory systems, many anterograde mini-systole-mini-diastole cycles must occur to complete a systolic phase. Similarly, it takes several retrograde mini-systole-mini-diastole cycles before diastole is complete. This ensures that in multichambered hearts, blood moves backward during diastole and forward during systole to achieve complete filling (or emptying) of all four cardiac chambers (Dulcis, 2005).
Larval cardiac activity is characterized by a constant anterograde beat that originates in a pacemaker putatively located in the caudal chamber. During metamorphosis, the adult conical chamber forms between the existing abdominal heart and the thoracic aorta of the larva. Extensive glutamatergic innervation develops, and cyclic cardiac reversal begins. The formation of a new retrograde cardiac pacemaker in the conical chamber, however, is not by itself sufficient to explain cyclic alternation of the two adult cardiac pacemakers and other features of the heart beat in intact animals. It is hypothesized that both intrinsic excitable properties of the myocardium and neuronal inputs participate in producing selective activation-inhibition of the two pacemakers (Dulcis, 2005).
Both bath application of exogenous glutamate and transverse nerves (TN) stimulation have a chronotropic effect in semi-intact preparations, involving an increase of the mini-systole-mini-diastole cycle rate of conical chamber activity. The glutamate-evoked cardiac contractions originate in the conical chamber and travels in the retrograde direction. They are correlated with the glutamate-evoked pacemaker potentials recorded intracellularly from myocardial cells. Thus, cardiac reversal to the retrograde beat can be evoked in hearts that are spontaneously beating in the anterograde direction. Similarly, retrograde contractions are initiated in the conical chamber by glutamate application to hearts that have been preincubated with CCAP, which by itself potentiates the anterograde beat (Dulcis, 2005).
One mechanism that is consistent with these results is that the muscle cells of the conical chamber may have faster intrinsic excitability and/or contractile properties than the more posterior myocardial cells. The mini-systole-mini-diastole cycle is always shorter in the conical chamber with respect to more posterior chambers. This feature would allow the putative retrograde pacemaker in the conical chamber to impose its faster pace on the anterograde pacemaker of the caudal chamber. Although GluRIIA immunoreactivity and glutamatergic innervation are present at every cardiac chamber, a higher sensitivity of the glutamate receptors and/or faster properties of the putative pacemaker localized in the conical chamber may explain why retrograde contractions originate in the conical chamber when glutamate is applied to the entire abdominal heart (Dulcis, 2005).
There are, however, important differences between the results observed in semi-intact preparations and the heartbeat of the intact organism, suggesting that this mechanism alone is not sufficient to explain normal cardiac reversal. Whereas bath application of glutamate or TN stimulation evokes a retrograde beat that is always faster than the ongoing anterograde beat in semi-intact preparations, the retrograde beat that was recorded from intact animals always displays a slower rate. This is analogous to what has been described in other holometabolous insects that show reversal. Perhaps in intact animals, in which neuronal activity and physiological conditions are preserved, the reciprocal alternation of pacemaker dominance is maintained by simultaneous inactivation of the anterograde pacemaker before or during activation of the retrograde pacemaker. In Manduca sexta, for example, the motoneuron that serves the caudal chamber receives inhibitory synaptic input that stops activation of the anterograde pacemaker and allows the slower retrograde beat to begin. Innervation of the caudal chamber also develops during metamorphosis in Drosophila. The activity of these CCAP-IR neurons potentiates the anterograde beat. As in Manduca, the larval myogenic heart of Drosophila does not need innervation to produce the anterograde beat, but once the reversal is established and a new retrograde pacemaker develops, the alternation of the two adult pacemakers may require innervation to stop and/or reactivate the anterograde beat (Dulcis, 2005).
Another factor is that the adult heart is composed of two separate muscle layers -- a circular layer that is present in the larval stage and a ventral longitudinal layer that develops in the adult. The ventral longitudinal muscle layer is well developed in the conical chamber but is absent in the caudal chamber, where the anterograde beat originates. Glutamatergic innervation and glutamate receptors were found only in the ventral longitudinal muscle layer. The anterograde and the retrograde beats may travel along the two cardiac muscle layers independently if the two layers are not electrically coupled. It is not clear whether the relative activation of the two layers is altered in semi-intact preparations (Dulcis, 2005).
Finally, whereas the conical chamber is in diastole during the retrograde phase of cardiac activity in intact adults, bath-applied glutamate causes sustained contraction of the conical chamber while initiating the retrograde beat. This probably reflects differences between sustained bath application and the patterned glutamate release and more restricted access to targets that would occur during normal TN activity. In addition, although glutamate alone is sufficient for initiation of the retrograde beat, TN activity may cause the release of other neurotransmitters that have independent functions. The role of the glutamatergic and peptidergic (CCAP) innervation serving the second and third cardiac chamber is not known. One could hypothesize that each chamber requires innervation to potentiate and coordinate cardiac contractions occurring at different levels of the abdominal heart. To this aim, the pattern of activity of central (glutamatergic) and peripheral (peptidergic) segmental neurons, that is probably sculpted by sensory feedback loops, may be designed to sequentially activate adjacent cardiac chambers to produce a coordinated anterograde and retrograde wave of contraction (Dulcis, 2005).
Cardiac function in adult Drosophila needs to accommodate a variety of physiological conditions (for example, postfeeding vs dehydrated states) and behaviors, such as flight, locomotion, and ovoposition, which require specific variations of hemolymph circulation. Cardiac synapses may, therefore, undergo short-term and long-term synaptic plasticity that ultimately affects the activation of retrograde pacemaker cells. This system provides a unique model in which the effects of genetic manipulation on glutamatergic synaptic transmission can be analyzed not only at the molecular and cellular level, as with the skeletal muscle synapse, but also at the systems level (Dulcis, 2005).
Aging causes cardiac dysfunction, often leading to heart failure and death. This study performed a cardiac-specific gene expression study on aging Drosophila and carried out a comparative meta-analysis with published rodent data. Pathway level transcriptome comparisons suggest that age-related, extra-cellular matrix remodeling and alterations in mitochondrial metabolism, protein handling, and contractile functions are conserved between Drosophila and rodent hearts. However, expression of only a few individual genes similarly changed over time between and even within species. Gene expression was examined in single fly hearts, and significant variability was found as has been reported in rodents. It is proposed that individuals may arrive at similar cardiac aging phenotypes via dissimilar transcriptional changes, including those in transcription factors and micro-RNAs. Finally, the data suggest the transcription factor Odd-skipped, which is essential for normal heart development, is also a crucial regulator of cardiac aging (Cannon, 2017).
Transcriptomic, proteomic, and methylation aging clocks demonstrate that aging has a predictable preset program, while Transcriptome Trajectory Turning Points indicate that the 20 to 40 age range in humans is the likely stage at which the progressive loss of homeostatic control, and in turn aging, begins to have detrimental effects. Turning points in this age range overlapping with human aging clock genes revealed five candidates that were hypothesized could play a role in aging or age-related physiological decline. To examine these gene's effects on lifespan and health-span, this study utilized whole body and heart specific gene knockdown of human orthologs in Drosophila melanogaster. Whole body Loxl2, fz3, and Glo1 RNAi positively affected lifespan as did heart-specific Loxl2 knockdown. Loxl2 inhibition concurrently reduced age-related cardiac arrythmia and collagen (Pericardin) fiber width. Loxl2 binds several transcription factors in humans and RT-qPCR confirmed that a conserved transcriptional target CDH1 (Drosophila CadN2), has expression levels which correlate with Loxl2 reduction in Drosophila. These results point to conserved pathways and multiple mechanisms by which inhibition of Loxl2 can be beneficial to heart health and organismal aging (Bouska, 2021).
The heart is a muscle with high energy demands. Hence, most
patients with mitochondrial disease produced by defects in the
oxidative phosphorylation (OXPHOS) system are susceptible to
cardiac involvement. The presentation of mitochondrial
cardiomyopathy includes hypertrophic, dilated and left ventricular
noncompaction, but the molecular mechanisms involved in cardiac
impairment are unknown. One of the most frequent OXPHOS defects in
humans frequently associated with cardiomyopathy is cytochrome c
oxidase (COX) deficiency caused by mutations in COX assembly
factors such as Sco1 and Sco2. To investigate the molecular
mechanisms that underlie the cardiomyopathy associated with Sco
deficiency, this study interfered with scox (the single
Drosophila Sco orthologue) expression in the heart.
Cardiac-specific knockdown of scox reduces fly lifespan, and it
severely compromises heart function and structure, producing
dilated cardiomyopathy. Cardiomyocytes with low levels of scox
have a significant reduction in COX activity and they undergo a
metabolic switch from OXPHOS to glycolysis, mimicking the clinical
features found in patients harbouring Sco mutations. The major
cardiac defects observed are produced by a significant increase in
apoptosis, which is dp53-dependent.
Genetic and molecular evidence strongly suggest that dp53 is
directly involved in the development of the cardiomyopathy induced
by scox deficiency. Remarkably, apoptosis is enhanced in the
muscle and liver of Sco2 knock-out mice, clearly suggesting that
cell death is a key feature of the COX deficiencies produced by
mutations in Sco genes in humans (Martínez-Morentin, 2015).
Cardiomyopathies are a collection of myocardial disorders in which
the heart muscle is structurally and functionally abnormal. In the
past decade, it has become clear that an important proportion of
cases of hypertrophic and dilated cardiomyopathies are caused by
mutations in genes encoding sarcomeric or desmosomal proteins. In
addition, cardiomyopathies (both hypertrophic and dilated) are
frequently associated to syndromic and non-syndromic mitochondrial
diseases. The importance of oxidative metabolism for cardiac
function is supported by the fact that 25–35% of the
myocardial volume is taken by mitochondria. The current view of
mitochondrial involvement in cardiomyopathy assumes that ETC
malfunction results in an increased ROS production, triggering a
“ROS-induced ROS release” vicious circle which in turn
perpetuates ETC dysfunction via damage in mtDNA and proteins
involved in electron transport. Under this view, accumulated
mitochondrial damage would eventually trigger apoptosis through
mitochondrial permeability transition pore (mPTP) opening other
mechanisms. Under normal circumstances, damaged mitochondria would
be eliminated through mitophagy. Excessive oxidative damage is
supposed to overcome the mitophagic pathway resulting in
apoptosis. Nevertheless, although several potential mechanisms
have been suggested, including apoptosis deregulation, oxidative
stress, disturbed calcium homeostasis or impaired iron metabolism,
the molecular basis of the pathogenesis of mitochondrial
cardiomyopathy is virtually unknown (Martínez-Morentin,
2015). Pathogenic mutations in human SCO1
and SCO2
have been reported to cause hypertrophic cardiomyopathy, among
other clinical symptoms. However, the molecular mechanisms
underlying this cardiac dysfunction have yet to be elucidated.
This study reports the first cardiac-specific animal model to
study human SCO1/2-mediated cardiomyopathy. Cardiac-specific scox
KD in Drosophila provokes a severe dilated
cardiomyopathy, as reflected by a significant increase in the
conical chamber size, due to mitochondrial dysfunction. It
presents a concomitant metabolic switch from glucose oxidation to
glycolysis and an increase in ROS levels, leading to p53-dependent
cell death. Interestingly, previous studies on patients and rat
models have shown that mitochondrial dysfunction is associated
with abnormalities in cardiac function and changes in energy
metabolism, resulting in glycolysis optimization and lactic
acidosis. Furthermore, in the Sco2KI/KO mouse model,
where no evidence of cardiomyopathy has been described, partial
loss of Sco2 function induces apoptosis in liver and skeletal
muscle. In flies scox KD causes a significant reduction
in FS and in the DI, as well as cardiac myofibril disorganization.
This degenerative process is most likely due to mitochondrial
dysfunction rather than to a developmental defect and moreover,
the dilated cardiomyopathy developed by flies resembles that
caused by mitochondrial fusion defects in flies
(Martínez-Morentin, 2015). The ETC is the major site of ROS production in cells, and aging
and many neurodegenerative diseases have been linked to
mitochondrial dysfunction that results in excessive oxidative
stress. Interestingly, there is an increase in ROS formation
associated with oxidative DNA damage in human Sco2−/−
cells. Accordingly, it was found that cardiac-specific knockdown
of scox increases oxidative stress, although it could
not be distinguished whether this increase in free radical
accumulation arises from the mitochondria or whether it comes from
non-mitochondrial sources due to a loss of cellular homeostasis,
as reported in yeast and in a neuro-specific COX-deficient
Alzheimer disease mouse model (Martínez-Morentin, 2015). Sco2 expression is known to be modulated by p53, a transcription
factor that participates in many different processes, including
cancer development, apoptosis and necrosis. p53 regulates
homeostatic cell metabolism by modulating Sco2 expression and
contributes to cardiovascular disorders. In addition, p53
activation in response to stress signals, such as increased
oxidative stress or high lactic acid production, is well
documented. Data from this study, showing that p53 is upregulated
in response to scox KD, but not in response to KD of
another Complex IV assembly factor, Surf1,
suggest a specific genetic interaction between dp53 and
scox. This is corroborated by the dramatic effects
observed in the heart structure and function when dp53 is
overexpressed in scox KD hearts. Furthermore, the
functional and structural defects seen in scox KD hearts
can be rescued in dp53-DN OE or dp53 null
backgrounds, indicating that the scox-induced defects are mediated
by increased p53 expression. Interestingly, opposed to scox
KD, the heart structure defects induced by dp53 OE can
be fully rescued by heart-specific Surf1 KD, further
confirming the specificity of the genetic interaction between dp53
and scox (Martínez-Morentin, 2015). It has recently been shown that SCO2 OE induces
p53-mediated apoptosis in tumour xenografts and cancer cells.
Furthermore, SCO2 KD sensitizes glioma cells to
hypoxia-induced apoptosis in a p53-dependent manner and induces
necrosis in tumours expressing WT p53, further linking the
SCO2/p53 axis to cell death. In Drosophila, there is a
dp53-mediated upregulation of Reaper,
Hid and Grim
in response to scox KD. This, coupled with the
observation that Reaper overexpression in the adult heart enhances
the structural defects caused by cardiac-specific scox
KD, suggests that scox normally prevents the triggering of
dp53-mediated cell death in cardiomyocytes in stress response.
Indeed, it was found that there is massive cell death in the
skeletal muscle and liver of Sco2KI/KO mice, supporting
the hypothesis that Sco proteins might play this role also in
mammals (Martínez-Morentin, 2015). The study provides evidence that scox KD hearts exhibit
partial loss of COX activity, with cardiomyocytes undergoing
apoptosis. There is evidence from vertebrate and invertebrate
models that partial inhibition of mitochondrial respiration
promotes longevity and metabolic health due to hormesis. In fact,
it has recently been shown that mild interference of the OXPHOS
system in Drosophila IFMs preserves mitochondrial
function, improves muscle performance and increases lifespan
through the activation of the mitochondrial unfolded pathway
response and IGF/like
signalling pathways. This study speculates that cell death, rather
than mitochondrial dysfunction itself, is likely to be the main
reason for the profound heart degeneration observed in TinCΔ4-Gal4>scoxi
flies. Expression of dominant negative dp53 in scox
KD hearts rescues dysfunction and cardiac degeneration, and, most
importantly, scox KD in dp53−/−
animals causes no apparent heart defects, which could attribute
the rescue observed to blockade of the p53 pathway. Indeed,
inhibiting apoptosis by p35 or Diap1
OE almost completely rescues the morphological scox KD
phenotype. As scox KD in the absence of dp53 causes no
symptoms of heart disease, coupled with the inability of p35 and
Diap1 to completely rescue the morphological phenotype, suggests
that, in addition to inducing apoptosis, dp53 plays a
key role in the development of cardiomyopathy
(Martínez-Morentin, 2015). The fact that heart-specific Surf1 KD neither
upregulates p53 nor induces apoptosis supports the idea that the
partial loss of scox function itself triggers dp53
upregulation and apoptosis, rather than it being a side effect of
COX dysfunction and the loss of cellular homeostasis. In this
context, it is noteworthy that SCO2 interference in
mammalian cells induces p53 re-localization from mitochondria to
the nucleus. It is therefore tempting to hypothesize that scox
might play another role independent of its function as a COX
assembly factor, perhaps in redox regulation as suggested
previously and that it may act in conjunction with dp53 to fulfil
this role. Another issue deserves further attention, the
possibility of this interaction being a tissue-specific response.
It may be possible that the threshold of COX deficiency tolerated
by the heart might be lower than in other tissues, thus the scox/dp53
genetic interaction may be a tissue-dependent phenomenon or the
consequence of a tissue-specific role of scox. In fact,
mitochondrial dysfunction in mice is sensed independently from
respiratory chain deficiency, leading to tissue-specific
activation of cellular stress responses. Thus, more work is
necessary to test these hypotheses and try to understand how the
partial lack of scox induces cell death through dp53
(Martínez-Morentin, 2015). Although the role of mitochondria in Drosophila
apoptosis remains unclear, there is strong evidence that, as in
mammals, mitochondria play an important role in cell death in
flies. The localization of Rpr, Hid and Grim in the mitochondria
is essential to promote cell death, and fly mitochondria undergo
Rpr-, Hid- and Drp1-dependent
morphological changes and disruption following apoptotic stimulus.
Moreover, the participation of the mitochondrial fission protein
Drp1 in cell death is conserved in worms and mammals. It has been
proposed that p53 plays a role in the opening of the mPTP that
induces necrotic cell death. According to this model, p53
translocates to the mitochondrial matrix upon ROS stimulation,
where it binds cyclophilin D (CypD) to induce mPTP opening
independent of proapoptotic Bcl-2 family members Bax and Bak, and
in contrast to traditional concepts, independent of Ca2+
(Martínez-Morentin, 2015). Apoptotic and necrotic pathways have a number of common steps and
regulatory factors, including mPTP opening that is thought to
provoke mitochondrial swelling and posterior delivery of necrotic
factors, although Drosophila mPTP activation is not
accompanied by mitochondrial swelling. Interestingly, although the
p53 protein triggers mitochondrial outer membrane permeabilization
(MOMP) in response to cellular stress in mammals, releasing
mitochondrial death factors, MOMP in Drosophila is more
likely a consequence rather than cause of caspase activation and
the release of mitochondrial factors does not appear to play a
role in apoptosis. Thus, in cardiac-specific scox KD
flies, dp53 might induce mPTP opening to trigger cell death, which
in the absence of mitochondrial swelling would result in apoptosis
instead of necrosis, as occurs in mammals. Drosophila
mPTP has been shown to be cyclosporine A (CsA)-insensitive in
vitro, although CsA administration ameliorates the mitochondrial
dysfunction with a severely attenuated ATP and enhanced ROS
production displayed by collagen XV/XVIII mutants. Interestingly,
mice lacking collagen VI display altered mitochondrial structure
and spontaneous apoptosis, defects that are caused by mPTP opening
and that are normalized in vivo by CsA treatment
(Martínez-Morentin, 2015). Cancer and heart diseases are the two leading causes of mortality and morbidity worldwide. Many cancer patients undergo heart-related complications resulting in high incidences of mortality. It is generally hypothesized that cardiac dysfunction in cancer patients occurs due to cardiotoxicity induced by therapeutic agents, used to treat cancers and/or cancer-induced cachexia. However, it is not known if localized tumors or unregulated cell growth systemically affect heart function before treatment, and/or prior to the onset of cachexia, hence, making the heart vulnerable to structural or functional abnormalities in later stages of the disease. This study incorporated complementary mouse and Drosophila models to establish if tumor induction indeed causes cardiac defects even before intervention with chemotherapy or onset of cachexia. Focus was placed on one of the key pathways involved in irregular cell growth, the Hippo-Yorkie (Yki) pathway. The transcriptional co-activator of the Yki signaling pathway was overexpressed to induce cellular overgrowth; Yki overexpression in the eye tissue of Drosophila results in compromised cardiac function. These cardiac phenotypes were rescued using antioxidant treatment, with which it is concluded that the Yki induced tumorigenesis causes a systemic increase in ROS affecting cardiac function. These results show that systemic cardiac dysfunction occurs due to abnormal cellular overgrowth or cancer elsewhere in the body; identification of specific cardiac defects associated with oncogenic pathways can facilitate the possible early diagnosis of cardiac dysfunction (Karekar, 2021).
Cardiomyopathy (CM) is a group of diseases distinguished by morphological and functional abnormalities in the myocardium. It is etiologically heterogeneous and may develop via cell autonomous and/or non-autonomous mechanisms. One of the most severe forms of CM has been linked to the deficiency of the ubiquitously expressed RNase Z endoribonuclease. RNase Z cleaves off the 3'-trailer of both nuclear and mitochondrial primary tRNA (pre-tRNA) transcripts. Cells mutant for RNase Z accumulate unprocessed pre-tRNA molecules. Patients carrying RNase Z variants with reduced enzymatic activity display a plethora of symptoms including muscular hypotonia, microcephaly and severe heart hypertrophy; still, they die primarily due to acute heart decompensation. Determining whether the underlying mechanism of heart malfunction is cell autonomous or not will provide an opportunity to develop novel strategies of more efficient treatments for these patients. This study used CRISPR-TRiM technology to create Drosophila models that carry cardiomyopathy-linked alleles of RNase Z only in the cardiomyocytes. This modification is sufficient for flies to develop heart hypertrophy and systolic dysfunction. These observations support the idea that the RNase Z linked CM is driven by cell autonomous mechanisms (Migunova, 2023).
Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) with a likely oligogenic etiology, but understanding of the genetic complexities and pathogenic mechanisms leading to HLHS is limited. This study performed whole genome sequencing (WGS) on 183 HLHS patient-parent trios to identify candidate genes, which were functionally tested in the Drosophila heart model. Bioinformatic analysis of WGS data from an index family of a HLHS proband born to consanguineous parents prioritized 9 candidate genes with rare, predicted damaging homozygous variants. Of them, cardiac-specific knockdown (KD) of mitochondrial MICOS complex subunit dCHCHD3/6 resulted in drastically compromised heart contractility, diminished levels of sarcomeric actin and myosin, reduced cardiac ATP levels, and mitochondrial fission-fusion defects. These defects were similar to those inflicted by cardiac KD of ATP synthase subunits of the electron transport chain (ETC), consistent with the MICOS complex's role in maintaining cristae morphology and ETC assembly. Five additional HLHS probands harbored rare, predicted damaging variants in CHCHD3 or CHCHD6. Hypothesizing an oligogenic basis for HLHS, 60 additional prioritized candidate genes from these patients were tested for genetic interactions with CHCHD3/6 in sensitized fly hearts. Moderate KD of CHCHD3/6 in combination with Cdk12 (activator of RNA polymerase II), RNF149 (goliath, E3 ubiquitin ligase), or SPTBN1 (β-Spectrin, scaffolding protein) caused synergistic heart defects, suggesting the likely involvement of diverse pathways in HLHS. Further elucidation of novel candidate genes and genetic interactions of potentially disease-contributing pathways is expected to lead to a better understanding of HLHS and other CHDs (Briker, 2023).
Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults. It is caused by the excessive expansion of noncoding CTG repeats, which when transcribed affects the functions of RNA-binding factors with adverse effects on alternative splicing, processing, and stability of a large set of muscular and cardiac transcripts. Among these effects, inefficient processing and down-regulation of muscle- and heart-specific miRNA, miR-1, have been reported in DM1 patients, but the impact of reduced miR-1 on DM1 pathogenesis has been unknown. This study used Drosophila DM1 models to explore the role of miR-1 in cardiac dysfunction in DM1. miR-1 down-regulation in the heart was found to lead to dilated cardiomyopathy (DCM), a DM1-associated phenotype. In silico screening for miR-1 targets was combined with transcriptional profiling of DM1 cardiac cells to identify miR-1 target genes with potential roles in DCM. Multiplexin (Mp) as a new cardiac miR-1 target involved in DM1. Mp encodes a collagen protein involved in cardiac tube formation in Drosophila. Mp and its human ortholog Col15A1 are both highly enriched in cardiac cells of DCM-developing DM1 flies and in heart samples from DM1 patients with DCM, respectively. When overexpressed in the heart, Mp induces DCM, whereas its attenuation rescues the DCM phenotype of aged DM1 flies. Reduced levels of miR-1 and consecutive up-regulation of its target Mp/Col15A1 might be critical in DM1-associated DCM (Souidi, 2023).
In addition to the heart proper, insects possess wing hearts in the thorax to ensure regular hemolymph flow through the narrow wings. In Drosophila, the wing hearts consist of two bilateral muscular pumps of unknown origin. This paper presents the first developmental study on these organs and reports that the wing hearts originate from eight embryonic progenitor cells arising in two pairs in parasegments 4 and 5. These progenitors represent a so far undescribed subset of the Even-skipped positive pericardial cells (EPC) and are characterized by the early loss of tinman expression in contrast to the continuously Tinman positive classical EPCs. Ectopic expression of Tinman in the wing heart progenitors omits organ formation, indicating a crucial role for Tinman during progenitor specification. The subsequent postembryonic development is a highly dynamic process, which includes proliferation and two relocation events. Adults lacking wing hearts display a severe wing phenotype and are unable to fly. The phenotype is caused by omitted clearance of the epidermal cells from the wings during maturation, which inhibits the formation of a flexible wing blade. This indicates that wing hearts are required for proper wing morphogenesis and functionality (Tögel, 2008).
Unlike in vertebrates, where an elaborate closed blood vessel system extends throughout the whole body, insects possess only one vessel, the tubular heart, in their otherwise open circulatory system. Once the hemolymph has left the heart, it moves freely between the internal organs and can not be directed into narrow body appendages such as antennae, legs or wings. To ensure sufficient hemolymph supply of these appendages additional circulatory organs evolved (Pass, 2000; Pass, 2006). In Drosophila, circulation in the wings is maintained by the so-called wing hearts (Krenn, 1995), a pair of autonomous muscular pumps located bilaterally in the scutellum, the dorsal elevation of the second thoracic segment. Due to this location, they are also referred to as scutellar pulsatile organs. Although known for many years, no developmental studies on the origin or morphogenesis of these organs have been performed. Probably, this was due to the lack of available methods to track their differentiation. However, studies on the origin of the thoracic somatic muscles in Drosophila and comparative anatomical investigations in insects suggested that the wing hearts originate from the cardiac mesoderm or from the heart itself (Tögel, 2008).
A previous study identified an enhancer region of the Drosophila hand gene that is able to drive reporter gene activity in the wing hearts (Sellin, 2006). In the present work, this reporter was used to identify the embryonic anlagen of the wing hearts and to elucidate the dynamics of their postembryonic development with in vivo time lapse imaging. It was found that the anlagen of the Drosophila wing hearts indeed derive from the cardiac mesoderm but, astonishingly, not from the muscular cardioblast lineage. Instead, they represent a so far undescribed subpopulation of the well-known Even-skipped (Eve) positive pericardial cells (EPCs) (Tögel, 2008).
In addition to their unknown origin, little is known about the contribution of wing hearts to wing morphogenesis and functionality. After eclosion, wings are unfolded by a sudden influx of hemolymph and subsequently undergo maturation. During this process, the epidermal cells that until then bonded the dorsal and ventral wing surfaces enter programmed cell death, delaminate from the cuticle, and disappear into the thorax (Kimura, 2004). Subsequently, the cuticles of the intervein regions become tightly bonded to form a flexible wing blade, while the cuticles of the vein regions form tubes, lined by living cells, through which hemolymph circulates in mature adult insects. Measurements of hemolymph flow in adult butterflies showed that wing hearts function as suction pumps that draw hemolymph out of the wings starting shortly after wing unfolding. Whether wing hearts might play a role in wing maturation was tested by generating flies lacking wing hearts. The findings demonstrate that the delaminated epidermal cells are removed from the wings by the hemolymph flow generated by the wing hearts. Loss of wing heart function leads to remains of epidermal cells resting between the unbonded dorsal and ventral wing surfaces which results in malformation of the wing blade and flightlessness. It is concluded that wing hearts are essential for wing maturation and, thus, for acquiring flight ability in Drosophila (Tögel, 2008).
A hand-C-GFP reporter was generated (Sellin, 2006) that reflects the described hand expression pattern and was found to be active in wing hearts. To confirm that the hand-C-GFP reporter is expressed in all cells of mature wing hearts, their morphology was examined based on the signal from the reporter in conjunction with histological sections. In the adult fly, wing hearts are located at the lateral angles of the scutellum, which are joined to the posterior wing veins by cuticular tubes. Each organ is curved in anterior–posterior direction as well as dorso-ventrally. It consists of about 7-8 horizontally arranged rows of prominent muscle cells, which are attached at their proximal side to a thin layer of cells that has a greater dorsal extension than the muscle cells. Both cell types are labeled by the reporter. The fine acellular strands that hold the wing hearts to the adjacent epidermal cells were not observed to be marked by the reporter. PubMed ID: Movies are provided to demonstrate the location and the beating of wing hearts (Tögel, 2008).
The hand-C-GFP reporter was tested for expression in earlier stages of wing heart development and it was found to be active throughout the entire organogenesis. This enabled identification of the embryonic anlagen of the wing hearts, which consist of eight progenitor cells located dorsally and anterior to the heart, in two pairs in the second and third thoracic segment from stage 16/17 onward. The progenitors exhibit a flattened triangular shape and are interconnected by thin cytoplasmic extensions. In addition, the second and the fourth pair of the progenitors are closely associated with the dorsal tracheal branches at their interconnection in the second and third thoracic segments. The characteristic pairwise arrangement and the connection to the tracheae are retained during the subsequent three larval stages. Proliferation starts at about the transition from the second to the third larval instar, leading to eight clusters of cells that remain arranged in four pairs in the anterior region until 1h after puparium formation (APF). Between 1 and 10h APF, the cell number increases significantly and the anterior three pairs of cell clusters are retracted to join the last pair of clusters, eventually forming one large median cluster. Between 13 and 50h APF, the single large cluster splits along its anterior-posterior axis into two groups of cells that migrate laterally in the forming scutellum, thereby adopting the characteristic arched appearance of the adult wing hearts. During this process some of the cells on either side form the underlying thin layer while the remaining cells arrange in horizontal rows along that layer. First contractions of the mature organs were observed at about 45-50h APF (Tögel, 2008).
The expression of the bHLH transcription factor Hand in the wing heart progenitors, which serves as a general marker for all classes of heart cells in Drosophila, prompted a to screen for the expression of genes known to be active in cardiac lineages. Analysis of Even-skipped (Eve) expression revealed that the embryonic wing heart progenitors arise through the same lineage as the well described Eve expressing pericardial cells (EPCs). At stage 10 in embryogenesis, 12 Eve clusters are present on either side of the embryo, located in parasegments (PS) 2 to 12. Each cluster gives rise to a pair of EPCs, except for the most posterior cluster in PS 14, which generates only one EPC. During subsequent development, the first and the second pair of EPCs, located in parasegment 2 and 3, turn toward the midline of the embryo to accompany the tip of the heart, which later bends ventrally into the embryo. The third and the fourth pair of EPCs in PS 4 and 5 are shifted anteriorly in relation to the heart. This step is not based on migration but on the remodeling of the embryo during head involution, since the cells remain in their PS close to the likewise Eve positive anlagen of the DA1 muscle. The EPCs in PS 4 and 5 subsequently differentiate into the later wing heart progenitors, while all others become the classical EPCs and accompany the heart in a loosely associated fashion. At least from PS 4 to 12, all pairs of Eve positive cells (wing heart progenitors and classical EPCs) are interconnected by cytoplasmic extensions forming a rope ladder-like strand above the heart after dorsal closure at stage 16/17. This mode of contact between the cells persists in the wing heart progenitors in postembryonic stages and might be essential for proper relocation in the prepupae (Tögel, 2008).
Although the Drosophila wing hearts have been known for many years, their origin and development have remained unknown. This study provides the first developmental approach on these organs using in vivo time lapse imaging as well as genetic and immunohistochemical methods. It was found that the wing hearts develop from embryonic anlagen that consist of eight progenitor cells located anterior to the heart. Analysis of gene expression in these progenitors confirmed the hypothesis that the wing hearts originate from the cardiac mesoderm, but not from the contractile cardioblast lineage, as has been suggested based on anatomical data. Surprisingly, the embryonic anlagen derive from a particular subset of the well-known EPCs. EPCs arise in pairs in PS 2 to 12 from the dorsal progenitor P2, which divides asymmetrically into the founder of the dorsal oblique muscle 2 and the founder of the EPCs in a numb-dependent lineage decision. Additionally, a single EPC arises in PS 14. The subsequent differentiation of the founders into EPCs requires the activity of the transcription factors Zfh1 and Eve. This study shows that the EPCs located in PS 4 and 5 are relocated in relation to the heart during head involution at stage 14/15 of embryogenesis and subsequently differentiate into the wing heart progenitors. Until this step, no difference to the EPCs in the anterior and posterior PS could be detected. Like the classical EPCs, which remain close to the heart, the EPCs that give rise to the wing heart progenitors depend on factors involved in asymmetric cell division, e.g. Insc or Numb, and fail to differentiate in embryos mutant for zfh1 as well as in animals lacking mesodermal Eve. Loss of tinman expression is the only event that could be identified that discriminates between a classical EPC fate and the specification of wing heart progenitors. Consistently, ectopic expression of Tinman in the wing heart progenitors effectively represses their specification, probably by committing them to a classical EPC fate, indicating that Tinman plays a crucial role in the involved regulatory pathway (Tögel, 2008).
So far, the biological role of pericardial cells (PCs), and EPCs in particular, is not well understood. In the embryo, three populations of PCs arise in each segment, which are characterized by the expression of different combinations of genes (Odd positive PCs, Eve positive PCs, and Tinman positive PCs). During postembryonic stages, the number of PCs decreases, raising the question which population contributes to the final set of PCs in the adult and whether all PCs have the same function throughout development. Recent studies have shown that postembryonic PCs express Odd and Eve, a combination which is not observed in the embryo, and are dispensable for cardiac function. Genetic ablation of all larval PCs had no effect on heart rate, but increased sensitivity to toxic stress. In contrast, the specification of the correct number of embryonic PCs is crucial for normal heart function. Loss of mesodermal Eve during embryogenesis results in fewer larval pericardial cells, which causes a reduction in heart rate and lifespan. Conversely, hyperplasia of embryonic PCs has no effect on heart rate but causes decreased cardiac output. This was explained by an excess of Pericardin secreted by the PCs into the extracellular matrix enveloping the heart (Johnson, 2007). Taken together, embryonic PCs seem to influence cardiac development by e.g., secreting substances whereas postembryonic PCs function as nephrocytes. However, in this study, functional data is provided on a subset of embryonic EPCs, which differentiate into adult progenitors giving rise to a myogenic lineage. This represents a completely new function of PCs, raising the question whether EPCs might in general have myogenic potential and rather represent a population of adult progenitors, than PCs in a functional sense (Tögel, 2008).
The organogenesis of the wing hearts is a highly dynamic process, which includes distinct cellular interactions. At first, adjacent EPCs (including the wing heart progenitors) on either side of the embryo establish contact via cytoplasmic extensions. After dorsal closure of the embryo, interconnections are also formed between opposing EPCs resulting in a rope ladder-like strand above the heart. These interconnections are assumed to be needed to retain contact between the wing heart progenitors during the subsequent development. During larval stages, some of the wing heart progenitors establish a second contact to specific tracheal branches and proliferation starts. In the prepupa, a relocation event joins all wing heart progenitors in one large cluster. During this step, the progenitors are probably passively relocated in conjunction with the tracheal branches to which they are connected. Finally, the wing heart progenitors initiate active migration and form the mature wing hearts in the pupa. Considering the complexity of their development, it is proposed that wing hearts provide an ideal model for studying organogenesis on several different levels such as signaling, cell polarity, or path finding (Tögel, 2008).
Elimination of the embryonic progenitors by ectopic expression of tinman or by laser ablation causes the loss of wing hearts, which results in a specific wing phenotype in conjunction with flightlessness. In the identified phenotype, the delaminated epidermal cells are not cleared from the wings during wing maturation and bonding of the dorsal and ventral wing surfaces is omitted. Recently, it was reported that the epidermal cells transform into mobile fibroblasts and actively migrate out of the wings. However, in in vivo time-lapse studies migration of epidermal cells could not be observed during wing clearance. Conversely, their movements correlated with the periods of wing heart beating, indicating that they are passively transported by the hemolymph flow. One-sided ablation of mature wing hearts in pupae, confirms that wing hearts play a crucial physiological role in wing maturation, since the wing phenotype occurs only on the treated side, but in the same genetic background. In contrast, mutations in genes coding for proteins involved in cell adhesion, e.g. integrins, or in adhesion to the extra cellular matrix, cause a blistered wing phenotype. In the latter phenotype, the epidermal cells of the immature wings are not attached to their opposing cells or to the cuticle and the wing surfaces are separated during unfolding by the sudden influx of hemolymph. In contrast, in animals lacking wing hearts the wings resemble those of the wild-type shortly after unfolding. The epidermal cells also delaminate later from the cuticle, as indicated by their disarrayed pattern, but are not removed from the wings due to the missing hemolymph circulation and probably impede spatially the bonding of the dorsal and the ventral cuticle. Thus, the wings remain in their immature state and do not acquire aerodynamic properties, which accounts for the flightlessness. It is concluded that wing hearts are crucial for establishing proper wing morphology and functionality in Drosophila (Tögel, 2008).
Wing hearts occur in all winged insects, but differ considerably in their morphology. However, their function is highly conserved, since they all function as suction pumps that draw hemolymph from the wings. In the basal condition, the heart itself is directly connected to the scutellum and constitutes the pump. This connection was lost several times during evolution and other muscles, e.g. the separate wing hearts in Drosophila, were recruited to retain the function indicating a high selection pressure on wing circulation. It is suggested that this is due to the crucial role of wing hearts during wing maturation. Since proper wing morphogenesis is essential for flight ability, insect flight might not have been possible before the evolution of wing hearts (Tögel, 2008).
miR-1 is a small noncoding RNA molecule that modulates gene expression in heart and skeletal muscle. Loss of Drosophila miR-1 produces defects in somatic muscle and embryonic heart development, which have been partly attributed to miR-1 directly targeting Delta to decrease Notch signaling. This study shows that overexpression of miR-1 in the fly wing can paradoxically increase Notch activity independently of its effects on Delta. Analyses of potential miR-1 targets revealed that miR-1 directly regulates the 3'UTR of the E3 ubiquitin ligase Nedd4 . Analysis of embryonic and adult fly heart revealed that the Nedd4 protein regulates heart development in Drosophila. Larval fly hearts overexpressing miR-1 have profound defects in actin filament organization that are partially rescued by concurrent overexpression of Nedd4. These results indicate that miR-1 and Nedd4 act together in the formation and actin-dependent patterning of the fly heart. Importantly, it was found that the biochemical and genetic relationship between miR-1 and the mammalian ortholog Nedd4-like (Nedd4l) is evolutionarily conserved in the mammalian heart, potentially indicating a role for Nedd4L in mammalian postnatal maturation. Thus, miR-1-mediated regulation of Nedd4/Nedd4L expression may serve to broadly modulate the trafficking or degradation of Nedd4/Nedd4L substrates in the heart (Zhu, 2017).
Unexpectedly, overexpression of miR-1 in the anterior-posterior (AP) organizer of the wing disc results in a dose-dependent loss of L3 vein structures, consistent with de-repression of Notch or weakening of a regulatory mechanism that dampens the Notch signal. Using genetic techniques, it was determined that the loss of the distal aspect of L3 could be phenocopied by reducing the gene dose of Notch co-repressors or Nedd4; in the case of Nedd4, the regulation by miR-1 was direct. An expanded model is proposed in which miR-1 expression in the AP organizer has complex effects on Notch signaling owing to its regulation of ligand availability and receptor trafficking. As lower levels of miR-1 expression (18°C) caused wing-vein thickening and tortuosity, and higher levels (22°C) caused vein loss, Delta and Nedd4 may be differentially sensitive to miR-1 regulation, although these studies were not designed to address this issue. It is also possible that indirect effects, such as reductions in Nedd4-mediated ubiquitylation of positive effectors of the Notch receptor (e.g. Deltex) or perturbations in Delta-mediated cis-inhibition, contributed to the de-repression of Notch in the wing-based assay system (Zhu, 2017).
The findings in the mammalian heart indicate that the genetic and biochemical interaction between miR-1 and Nedd4l is physiologically relevant and may provide developmental or tissue-specific regulation of Nedd4l in the myocardium. It is speculated that the additional bands observed on western blots of heart lysates using an anti-Nedd4L antibody might result from post-translational modifications, because Nedd4L can autoregulate its stability through ubiquitylation of its HECT domain. Alternatively, they might represent heart-specific splice variants, because tissue-specific isoforms of Nedd4L have been found in the heart and the liver (Zhu, 2017).
Importantly, although miR-1-mediated reductions in Nedd4 activity caused wing-vein phenotypes induced by Notch, miR-1-mediated dysregulation of Nedd4L in the heart likely affects proteins outside the Notch pathway. Indeed, protein microarrays comparing human Nedd4 with human Nedd4L, suggest that Nedd4L (also known as Nedd4-2) preferentially targets ion channels, whereas Nedd4 targets are enriched for signaling pathways. Thus, in the heart, where miR-1 and murine Nedd4L are both expressed, their genetic and biochemical interaction might influence the excitability and connectivity of cardiomyocytes. Indeed, susceptibility to cardiac arrhythmias and sudden death in humans is associated with six genes that encode ion channels (SCN5A, KCNQ1, KCNH2, KCNE1, KCNE2 and RYR2). Murine Nedd4L regulates the cell-surface densities of the sodium channel, the voltage-gated type V alpha subunit (Scn5a), the potassium voltage-gated channel, KQT-like subfamily member 1 (Kcnq1) and the human Ether-a-go-go-related (KCNH2, previously hERG) channel. Furthermore, miR-1 directly regulates human KCNJ2, a channel that maintains cardiac resting potential. These findings suggest that the regulation of murine Nedd4l by miR-1 contributes to some of the electrophysiological abnormalities seen in miR-1 null mice. It would be interesting to determine whether Nedd4L is dysregulated in the heart after an infarction or under ischemic conditions, when miR-1 is upregulated and fatal cardiac dysrhythmias are common (Zhu, 2017).
Drosophila melanogaster is a good model species for the study of heart function. However, most previous work on D. melanogaster heart function has focused on the effects of large-effect genetic variants. This study compared heart function among 18 D. melanogaster populations that have been selected for altered development time, aging, or stress resistance. Populations with faster development and faster aging were found to have increased heart dysfunction, measured as percentage heart failure after electrical pacing. Experimental evolution of different triglyceride levels, by contrast, has little effect on heart function. Evolved differences in heart function correlate with allele frequency changes at many loci of small effect. Genomic analysis of these populations produces a list of candidate loci that might affect cardiac function at the intersection of development, aging, and metabolic control mechanisms (Shahrestani, 2017).
Drosophila harbors a simple tubular heart that ensures hemolymph circulation within the body. The heart is built by a few different cell types, including cardiomyocytes that define the luminal heart channel and ostia cells that constitute openings in the heart wall allowing hemolymph to enter the heart chamber. Regulation of flow directionality within a tube, such as blood flow in arteries or insect hemolymph within the heart lumen, requires a dedicated gate, valve, or flap-like structure that prevents backflow of fluids. In the Drosophila heart, intracardiac valves provide this directionality of hemolymph streaming, with one valve being present in larvae and three valves in the adult fly. Each valve is built by two specialized cardiomyocytes that exhibit a unique histology. The capacity to open and close the heart lumen was found to rely on a unique myofibrillar setting as well as on the presence of large membranous vesicles. These vesicles are of endocytic origin and probably represent unique organelles of valve cells. Moreover, the working mode of the cells was characterised in real time. Valve cells exhibit a highly flexible shape and during each heartbeat, oscillating shape changes result in closing and opening of the heart channel. Finally, a set of novel valve cell markers useful for future in-depth analyses of cell differentiation in wildtype and mutant animals were identified (Lammers, 2017).
Drosophila, like all insects, has an open circulatory system for the distribution of haemolymph and its components. The circulation of the haemolymph is essentially driven by the pumping activity of the linear heart. The heart is constructed as a tube into which the haemolymph is sucked and pumped forward by rhythmic contractions running from the posterior to the anterior, where it leaves the heart tube. The heart harbours cardiac valves to regulate flow directionality, with a single heart valve differentiating during larval development to separate the heart tube into two chambers. During metamorphosis, the heart is partially restructured, with the linear heart tube with one terminal wide-lumen heart chamber being converted into a linear four-chambered heart tube with three valves. As in all metazoan circulatory systems, the cardiac valves play an essential role in regulating the direction of blood flow. This study provides evidence that the valves in adult flies arise via transdifferentiation, converting lumen-forming contractile cardiomyocytes into differently structured valve cells. Interestingly, adult cardiac valves exhibit a similar morphology to their larval counterparts, but act differently upon heart beating. Applying calcium imaging in living specimens to analyse activity in valve cells, adult cardiac valves were shown to operate owing to muscle contraction. However, valve cell shape dynamics are altered compared with larval valves, which led the proposal of the current model of the opening and closing mechanisms in the fly heart (Meyer, 2023).
Alary muscles (AMs) have been described as a component of the cardiac system in various arthropods. Lineage-related thoracic muscles (TARMs), linking the exoskeleton to specific gut regions, have recently been discovered in Drosophila Asymmetrical attachments of AMs and TARMs, to the exoskeleton on one side, and internal organs on the other, suggested an architectural function in moving larvae. This study analysed AMs and TARMs striated organisation, and imaged their atypical deformability in crawling larvae. Then AMs and TARMs were selectively eliminated by targeted apoptosis. Elimination of AMs revealed that AMs are required for suspending the heart in proper intra-hemocelic position and opening of the heart lumen, and constrain the curvature of the trachea, the respiratory system, during crawling; TARMs are required for proper positioning of visceral organs and efficient food transit. AM/TARM cardiac versus visceral attachment depends on Hox control, with visceral attachment being the ground state. TARMs and AMs are the first example of multinucleate striated muscles connecting the skeleton to the cardiac and visceral systems in bilaterians, with multiple physiological functions (Bataille, 2020).
Actomyosin networks provide the major contractile machinery for regulating cell and tissue morphogenesis during development. These networks undergo dynamic rearrangements, enabling cells to have a broad range of mechanical actions. How cells integrate different mechanical stimuli to accomplish complicated tasks in vivo remains unclear. This study explored this problem in the context of cell matching, where individual cells form precise inter-cellular connections between partner cells. To study the dynamic roles of actomyosin networks in regulating precise cell matching, focus was placed on the process of heart formation during Drosophila embryogenesis, where selective filopodia-binding adhesions ensure precise cell alignment. Non-muscle Myosin II clusters periodically oscillate within cardioblasts with ~4-min intervals. Filopodia dynamics-including protrusions, retraction, binding stabilization, and binding separation-are correlated with the periodic localization of Myosin II clusters at the cell leading edge. Perturbing the Myosin II activity and oscillatory pattern alters the filopodia properties and binding dynamics and results in mismatched cardioblasts. By simultaneously changing the activity of Myosin II and filopodia adhesion levels, it was further demonstrated that levels of Myosin II and adhesion are balanced to ensure precise connectivity between cardioblasts. Combined, a mechanical proofreading machinery of robust cell matching is proposed, whereby oscillations of Myosin II within cardioblasts periodically probe filopodia adhesion strength and ensure correct cell-cell connection formation (Zhang, 2020).
The Drosophila dorsal vessel (DV) is comprised of two opposing rows of cardioblasts (CBs) that migrate toward the dorsal midline during development. While approaching the midline, CBs change shape, enabling dorsal and ventral attachments with their contralateral partners to create a linear tube with a central lumen. Previous studies demonstrated DV closure occurs via a "buttoning" mechanism where specific CBs advance ahead of their lateral neighbors, and attach creating transient holes, which eventually seal. This study investigate the role of the actin-regulatory protein Enabled (Ena) in DV closure. Loss of Ena results in DV cell shape and alignment defects. Live analysis of DV formation in ena mutants shows a reduction in CB leading edge protrusion length and gaps in the DV between contralateral CB pairs. These gaps occur primarily between a specific genetic subtype of CBs, which express the transcription factor Seven-up (Svp) and form the ostia inflow tracts of the heart. In WT embryos these gaps between Svp(+) CBs are observed transiently during the final stages of DV closure. These data suggest that Ena modulates the actin cytoskeleton in order to facilitate the complete sealing of the DV during the final stages of cardiac tube formation (King, 2021).
The Drosophila Forkhead gene jumeau (jumu) controls three categories of cardiac progenitor cell division-asymmetric, symmetric, and cell division at an earlier stage-by regulating Polo kinase activity, and mediates the latter two categories in concert with the TF Myb. Those observations raised the question of whether other jumu-regulated genes also mediate all three categories of cardiac progenitor cell division or a subset thereof. By comparing microarray-based expression profiles of wild-type and jumu loss-of-function mesodermal cells, nebbish (neb), a kinesin-encoding gene activated by jumu, was identified. Phenotypic analysis shows that neb is required for only two categories of jumu-regulated cardiac progenitor cell division: symmetric and cell division at an earlier stage. Synergistic genetic interactions between neb, jumu, Myb, and polo and the rescue of jumu mutations by ectopic cardiac mesoderm-specific expression of neb demonstrate that neb is an integral component of a jumu-regulated subnetwork mediating cardiac progenitor cell divisions. These results emphasize the central role of Fox TFs in cardiogenesis and illustrate how a single TF can utilize different combinations of other regulators and downstream effectors to control distinct developmental processes (Kump 2021).
Development of the Drosophila embryonic mesoderm is controlled through both internal and external inputs to the mesoderm. One such factor is Heartless (Htl), a Fibroblast Growth Factor Receptor (FGFR) expressed in the mesoderm. Although Htl has been extensively studied, the dynamics of its action are poorly understood after the initial phases of mesoderm formation and spreading. To begin to address this challenge, an optogenetic version of the FGFR Heartless was developed in Drosophila (Opto-htl). Opto-htl enables activation of the FGFR pathway in selective spatial (~35 μ section from one of the lateral sides of the embryo) and in temporal domains (ranging from 40 min to 14 h) during embryogenesis. Importantly, the effects can be tuned by the intensity of light-activation, making this approach significantly more flexible than other genetic approaches. Controlled perturbations to the FGFR pathway were performed to define the contribution of Htl signalling to the formation of the developing embryonic heart and somatic muscles. A direct correlation was found between Htl signalling dosage and number of Tinman-positive heart cells specified. Opto-htl activation favours the specification of Tinman positive cardioblasts and eliminates Eve-positive DA1 muscles. This effect is seen to increase progressively with increasing light intensity. Therefore, fine tuning of phenotypic responses to varied Htl signalling dosage can be achieved more conveniently than with other genetic approaches. Overall, Opto-htl is a powerful new tool for dissecting the role of FGFR signalling during development (Yadav, 2021).
The Notch pathway is an ancient intercellular signaling system with crucial roles in numerous cell-fate decision processes across species. While the canonical pathway is activated by ligand-induced cleavage and nuclear localization of membrane-bound Notch, Notch can also exert its activity in a ligand/transcription-independent fashion, which is conserved in Drosophila, Xenopus, and mammals. However, the noncanonical role remains poorly understood in in vivo processes. This study shows that increased levels of the Notch intracellular domain (NICD) in the early mesoderm inhibit heart development, potentially through impaired induction of the second heart field (SHF), independently of the transcriptional effector RBP-J. Similarly, inhibiting Notch cleavage, shown to increase noncanonical Notch activity, suppressed SHF induction in embryonic stem cell (ESC)-derived mesodermal cells. In contrast, NICD overexpression in late cardiac progenitor cells lacking RBP-J resulted in an increase in heart size. This study suggests that noncanonical Notch signaling has stage-specific roles during cardiac development (Miyamoto, 2021).
Mammalian KMT2C, KMT2D, and HCFC1 are expressed during heart development and have been associated with congenital heart disease, but their roles in heart development remain elusive. This study found that the Drosophila Lpt and trr genes encode the N-terminal and C-terminal homologs, respectively, of mammalian KMT2C or KMT2D. Lpt and trr mutant embryos showed reduced cardiac progenitor cells. Silencing of Lpt, trr, or both simultaneously in the heart led to similar abnormal cardiac morphology, tissue fibrosis, and cardiac functional defects. Like KMT2D, Lpt and trr were found to modulate histone H3K4 mono- and dimethylation, but not trimethylation. Investigation of downstream genes regulated by mouse KMT2D in the heart showed that their fly homologs are similarly regulated by Lpt or trr in the fly heart, suggesting that Lpt and trr regulate an evolutionarily conserved transcriptional network for heart development. Moreover, this study showed that cardiac silencing of Hcf, the fly homolog of mammalian HCFC1, leads to heart defects similar to those observed in Lpt and trr silencing, as well as reduced H3K4 monomethylation. These findings suggest that Lpt and trr function together to execute the conserved function of mammalian KMT2C and KMT2D in histone H3 lysine K4 mono- and dimethylation required for heart development. Possibly aided by Hcf, which plays a related role in H3K4 methylation during fly heart development (Huang, 2022).
Establishing a catalog of Congenital Heart Disease (CHD) genes and identifying functional networks would improve understanding of its oligogenic underpinnings. The current studies identified protein biogenesis cofactors Nascent polypeptide-Associated Complex (NAC) and Signal-Recognition-Particle (SRP) as disease candidates and novel regulators of cardiac differentiation and morphogenesis. Knockdown (KD) of the α-subunit (Nacα) or beta-subunit (bicaudal, bic) of NAC in the developing Drosophila heart disrupted cardiac developmental remodeling resulting in a fly with no heart. Heart loss was rescued by combined KD of Nacα with the posterior patterning Hox gene Abd-B. Consistent with a central role for this interaction in cardiogenesis, KD of Nacα in cardiac progenitors derived from human iPSCs impaired cardiac differentiation while co-KD with human HOXC12 and HOXD12 rescued this phenotype. These data suggest that Nacα KD preprograms cardioblasts in the embryo for abortive remodeling later during metamorphosis, as Nacα KD during translation-intensive larval growth or pupal remodeling only causes moderate heart defects. KD of SRP subunits in the developing fly heart produced phenotypes that targeted specific segments and cell types, again suggesting cardiac-specific and spatially regulated activities. Together, this study demonstrated directed function for NAC and SRP in heart development, and that regulation of NAC function depends on Hox genes (Schroeder, 2022).
In Drosophila larvae, the direction of blood flow within the heart tube, as well as the diastolic filling of the posterior heart chamber, is regulated by a single cardiac valve. This valve is sufficient to close the heart tube at the junction of the ventricle and the aorta and is formed by only two cells; both are integral parts of the heart tube. The valve cells regulate hemolymph flow by oscillating between a spherical and a flattened cell shape during heartbeats. At the spherical stage, the opposing valve cells close the heart lumen. The dynamic cell shape changes of valve cells are supported by a dense, criss-cross orientation of myofibrils and the presence of the valvosomal compartment, a large intracellular cavity. Both structures are essential for the valve cells' function. In a screen for factors specifically expressed in cardiac valve cells, the transcription factor Tailup was identified. Knockdown of tailup causes abnormal orientation and differentiation of cardiac muscle fibers in the larval aorta and inhibits the formation of the ventral longitudinal muscle layer located underneath the heart tube in the adult fly and affects myofibrillar orientation of valve cells. Furthermore, this study has identified regulatory sequences of tup that control the expression of tailup in the larval and adult valve cells (Meyer, 2022).
Within a cell, vesicles play a crucial role in the transport of membrane material and proteins to a given target membrane, and thus regulate a variety of cellular functions. Vesicular transport occurs by means of, among others, endocytosis, where cargoes are taken up by the cell and are processed further upon vesicular trafficking, i.e. transported back to the plasma membrane via recycling endosomes or the degraded by fusion of the vesicles with lysosomes. During evolution, a variety of vesicles with individual functions arose, with some of them building up highly specialised subcellular compartments. This study analysed the biosynthesis of a new vesicular compartment present in the valve cells of Drosophila melanogaster. The compartment is formed by invaginations of the plasma membrane and grows via re-routing of the recycling endosomal pathway. This is achieved by inactivation of other membrane-consuming pathways and a plasma membrane-like molecular signature of the compartment in these highly specialised heart cells (Meyer, 2022).
Developmentally programmed polyploidy (whole-genome-duplication) of cardiomyocytes is common across evolution. Functions of such polyploidy are essentially unknown. This study reveals roles for precise polyploidy levels in cardiac tissue. A conserved asymmetry is found in polyploidy level between cardiac chambers in Drosophila larvae and humans. In Drosophila , differential Insulin Receptor (InR) sensitivity leads the heart chamber to reach a higher ploidy/cell size relative to the aorta chamber. Cardiac ploidy-reduced animals exhibit reduced heart chamber size, stroke volume, cardiac output, and acceleration of circulating hemocytes. These Drosophila phenotypes mimic systemic human heart failure. Using human donor hearts, this study revealed asymmetry in nuclear volume (ploidy) and insulin signaling between the left ventricle and atrium. Tese results identify productive and likely conserved roles for polyploidy in cardiac chambers and suggest precise ploidy levels sculpt many developing tissues. These findings of productive cardiomyocyte polyploidy impact efforts to block developmental polyploidy to improve heart injury recovery (Chakraborty, 2023).
Heart development begins with the formation of a tube as cardiac progenitors migrate from opposite sides of the embryo. Abnormal cardiac progenitor movements cause congenital heart defects. However, the mechanisms of cell migration during early heart development remain poorly understood. Using quantitative microscopy, this study found that in Drosophila embryos, cardiac progenitors (cardioblasts) migrated through a sequence of forward and backward steps. Cardioblast steps were associated with oscillatory non-muscle myosin II waves that induced periodic shape changes and were necessary for timely heart tube formation. Mathematical modeling predicted that forward cardioblast migration required a stiff boundary at the trailing edge. Consistent with this, a supracellular actin cable was found at the trailing edge of the cardioblasts that limited the amplitude of the backward steps, thus biasing the direction of cell movement. These results indicate that periodic shape changes coupled with a polarized actin cable produce asymmetrical forces that promote cardioblast migration (Balaghi, 2023).
RNA binding proteins play a pivotal role in post-transcriptional gene expression regulation, however little is understood about their role in cardiac function. Alterations in the levels of Fragile X Related 1 protein, FXR1, the predominant FraX member expressed in vertebrate striated muscle, have been linked to structural and functional defects in mice and zebrafish models. FraX proteins are established regulators of translation and are known to regulate specific targets in different tissues. To decipher the direct role of FraX proteins in the heart in vivo, Drosophila, which harbors a sole, functionally conserved and ubiquitously expressed FraX protein, dFmr1, was investigated. Using classical loss of function alleles as well as muscle specific RNAi knockdown, dFmr1 was shown to be required for proper heart rate during development. Functional analyses in the context of cardiac-specific dFmr1 knockdown by RNAi demonstrate that dFmr1 is required cell autonomously in cardiac cells for regulating heart rate. Interestingly, these functional defects are not accompanied by any obvious structural abnormalities, suggesting that dFmr1 may regulate a different repertoire of targets in Drosophila than in vertebrates. Taken together, these findings support the hypothesis that dFmr1 protein is essential for proper cardiac function and establish the fly as a new model for studying the role(s) of FraX proteins in the heart (Novak, 2015).
The heart has emerged as an important organ in the regulation of systemic lipid homeostasis; however, the underlying mechanism remains poorly understood. Drosophila cardiomyocytes regulate systemic lipid metabolism by producing apolipoprotein B-containing lipoproteins (apoB-lipoproteins), essential lipid carriers that are so far known to be generated only in the fat body. In a genetic screen, this study discovered that when haplo-insufficient, microsomal triglyceride transfer protein (mtp), required for the biosynthesis of apoB-lipoproteins, suppressed the development of diet-induced obesity. Tissue-specific inhibition of Mtp revealed that whereas knockdown of mtp only in the fat body decreases systemic triglyceride (TG) content on normal food diet (NFD) as expected, knockdown of mtp only in the cardiomyocytes also equally decreases systemic TG content on NFD, suggesting that the cardiomyocyte- and fat body-derived apoB-lipoproteins serve similarly important roles in regulating whole-body lipid metabolism. Unexpectedly, on high fat diet (HFD), knockdown of mtp in the cardiomyocytes, but not in fat body, protects against the gain in systemic TG levels. It was further shown that inhibition of the Drosophila apoB homologue, apolipophorin or apoLpp, another gene essential for apoB-lipoprotein biosynthesis, affects systemic TG levels similarly to that of Mtp inhibition in the cardiomyocytes on NFD or HFD. Finally, it was determined that HFD differentially alters Mtp and apoLpp expression in the cardiomyocytes versus the fat body, culminating in higher Mtp and apoLpp levels in the cardiomyocytes than in fat body and possibly underlying the predominant role of cardiomyocyte-derived apoB-lipoproteins in lipid metabolic regulation. These findings reveal a novel and significant function of heart-mediated apoB-lipoproteins in controlling lipid homeostasis (Lee, 2017).
Insects and vertebrates have multiple major physiological systems, each species having a circulatory system, a metabolic system, and a respiratory system that enable locomotion and survival in stressful environments, among other functions. Broadening understanding of the physiology of Drosophila melanogaster requires the parsing of interrelationships among such major component physiological systems. By combining electrical pacing and flight exhaustion assays with manipulative conditioning, this study started to unpack the interrelationships between cardiac function, locomotor performance, and other functional characters such as starvation and desiccation resistance. Manipulative sequences incorporating these four physiological characters were applied to five D. melanogaster lab populations that share a common origin from the wild and a common history of experimental evolution. While exposure to starvation or desiccation significantly reduced flight duration, exhaustion due to flight only affected subsequent desiccation resistance. A strong association was found between flight duration and desiccation resistance, providing additional support for the hypothesis that these traits depend on glycogen and water content. However, there was negligible impact on rate of cardiac arrests from exhaustion by flight or exposure to desiccant. Brief periods of starvation significantly lowered the rate of cardiac arrest. These results provide suggestive support for the adverse impact of lipids on Drosophila heart robustness, a parallel result to those of many comparable studies in human cardiology. Overall, this study underscores clear distinctions among the connections between specific physiological responses to stress and specific types of physiological performance (Kezos, 2017).
The minuteness of Drosophila poses a challenge to quantify performance of its tubular heart and computer-aided analysis of its beating heart has evolved as a resilient compromise between instrumental costs and data robustness. This paper introduces an optical flow algorithm (OFA) that continuously registers coherent movement within videos of the beating Drosophila heart and uses this information to subscribe the time course of observation with characteristic phases of cardiac contraction or relaxation. The OFA combines high discriminatory power with robustness to characterize the performance of the Drosophila tubular heart using indicators from human cardiology. Proof of this concept is provided using the test bed of established cardiac conditions that include the effects of ageing, knockdown of the slow repolarizing potassium channel subunit KCNQ and ras-mediated hypertrophy of the heart tube. Together, this establishes the analysis of coherent movement as a suitable indicator of qualitative changes of the heart's beating characteristics, which improves the usefulness of Drosophila as a model of cardiac diseases (Monck, 2017).
The Drosophila melanogaster heart has become a principal model in which to study cardiac physiology and development. While the morphology of the heart in Drosophila and mammals is different, many of the molecular mechanisms that underlie heart development and function are similar and function can be assessed by similar physiological measurements, such as cardiac output, rate, and time in systole or diastole. This study utilized an intact, optogenetic approach to assess the neural influence on heart rate in the third instar larvae. To simulate the release of modulators from the nervous system in response to environmental influences, expression of channel-rhodopsin variants were directed to targeted neuronal populations to assess the role of these neural ensembles in directing release of modulators that may affect heart rate in vivo. The observations show that the activation of targeted neurons, including cholinergic, dopaminergic, and serotonergic neurons, stimulate the release of cardioactive substances that increase heart rate after the initial activation at both room temperature and in a cold environment. This parallels previous studies suggesting these modulators play a crucial role in altering heart rate when applied to exposed hearts and adds to understanding of chemical modulation of heart rate in intact Drosophila larvae (Malloy, 2017).
One of the primary targets for therapeutic intervention into human heart disease has been the human ether a go-go (hERG; see Drosophila Eag) K+ channel that, together with the KCNQ channel, controls the rate and efficiency of repolarization in human myocardial cells. Neither of these channels plays a major role in adult mouse heart function; however, this study shows that the hERG homolog seizure (sei), along with KCNQ, both contribute significantly to adult heart function in Drosophila as they do in humans. In Drosophila, mutations in or cardiac knockdown of sei channels cause arrhythmias that become progressively more severe with age. Intracellular recordings of semi-intact heart preparations revealed that these perturbations also cause electrical remodeling that is reminiscent of the early afterdepolarizations seen in human myocardial cells defective in these channels. In contrast to KCNQ, however, mutations in sei also cause extensive structural remodeling of the myofibrillar organization, which suggests that hERG channel function has a novel link to sarcomeric and myofibrillar integrity. It is concluded that deficiency of ion channels with similar electrical functions in cardiomyocytes can lead to different types or extents of electrical and/or structural remodeling impacting cardiac output (Ocorr, 2017).
Ocorr, K., Zambon, A., Nudell, Y., Pineda, S., Diop, S., Tang, M., Akasaka, T. and Taylor, E. (2017). Age-dependent electrical and morphological remodeling of the Drosophila heart caused by hERG/seizure mutations. PLoS Genet 13(5): e1006786. PubMed ID: 28542428
Understanding the cellular-molecular substrates of heart disease is key to the development of cardiac specific therapies and to the prevention of off-target effects by non-cardiac targeted drugs. One of the primary targets for therapeutic intervention has been the human ether a go-go (hERG) K+ channel that, together with the KCNQ channel, controls the rate and efficiency of repolarization in human myocardial cells. Neither of these channels plays a major role in adult mouse heart function; however, this study shows that the hERG homolog seizure (sei), along with KCNQ, both contribute significantly to adult heart function as they do in humans. In Drosophila, mutations in or cardiac knockdown of sei channels cause arrhythmias that become progressively more severe with age. Intracellular recordings of semi-intact heart preparations revealed that these perturbations also cause electrical remodeling that is reminiscent of the early afterdepolarizations seen in human myocardial cells defective in these channels. In contrast to KCNQ, however, mutations in sei also cause extensive structural remodeling of the myofibrillar organization, which suggests that hERG channel function has a novel link to sarcomeric and myofibrillar integrity. It is concluded that deficiency of ion channels with similar electrical functions in cardiomyocytes can lead to different types or extents of electrical and/or structural remodeling impacting cardiac output (Ocorr, 2017).
Channel dysfunction, or channelopathies, underlie a number of cardiac disorders such as long QT syndrome (LQTS) and are thought to contribute to sudden cardiac arrest, infant sudden death syndrome and increased risk of cardiac arrhythmias. The human ether-a-go-go related K+ channel (hERG) along with the KCNQ K+ channel (IKr, and IKs respectively) are the major contributors to cardiac repolarization in humans. Defects in KCNQ and hERG channels have been shown to cause LQT1 and LQT2, respectively and cardiac arrhythmias in humans. The hERG channel in particular has been a major target for the development of anti-arrhythmia drugs and can be inhibited by a variety of drugs that do not specifically target the heart. Previous work has shown that the KCNQ K+ channel is functional in the adult fly heart and that mutations in this channel contribute to cardiac arrhythmias. In the fly, seizure and KCNQ mutations do not significantly affect cardiac development, although in the mouse some hERG mutations cause looping defects and embryonic lethality. Importantly for heart function studies, survivorship of adult flies is not acutely affected by badly functioning hearts that would quickly result in death in vertebrates, likely because the fly does not rely on the heart for oxygen distribution, which is carried out by a separate system of tracheoles (Ocorr, 2017).
The current data demonstrate that the sei gene, the fly homolog of hERG, along with a number of other genes encoding K+ channels, are also expressed in fly myocardial cells and likely contribute to the repolarization capacity of the heart. This set of K+ channels is reminiscent of what is observed in vertebrate hearts. The primary effect of sei dysfunction in mutants was bradycardia and this phenotype could be replicated by cardiac-specific and adult, cardiac-specific sei KD as well as acute application of selective hERG antagonists. However, the increases in SI seen in both KD experiments are different from the mutant phenotype and possibly reflect some compensatory genetic alterations in the systemic sei mutants. The current data also show that, as in humans, mutations in sei cause arrhythmias and electrical remodeling in the form of AP bursts that are likely triggered by EADs. In addition this study shows that systemic mutants or cardiac-specific KD of sei, but not of KCNQ, reduced heart contractility, and were associated with structural remodeling (Ocorr, 2017).
These effects appear to be specific to alterations in sei as most of the sei cardiac function phenotypes can be rescued by over-expression of the wt channel. In addition, the morphological/functional effects of channel dysfunction appear not to be due to developmental defects but are adult stage- and cardiac-specific (Ocorr, 2017).
The intracellular electrical activity in adult fly cardiomyocytes appears more nodal or atrial-like and is similar to what has previously been observed in larva although more robust in terms of the resting potential and AP amplitude. Frequent AP bursts were observed in both sei and KCNQ mutants and in hearts from old flies compared to young Wt fly hearts reminiscent of the increase in electrical remodeling with disease and age in human myocardial cells. However, there appears to be a significant difference between mutations in KCNQ and sei in terms of their effects on electrical and morphological remodeling, with mutations in KCNQ resulting in significantly more electrical arrhythmia at younger ages compared to sei mutations. In addition, although 100% of APs recorded from both old KCNQ and sei mutants are arrhythmogenic, the severity of the events appears to be worse in KCNQ mutants. Notably, the ability to simultaneously record both intracellular APs while optically monitoring intact heart function demonstrate that the arrhythmogenic APs that were observed correlate directly with unsustained fibrillatory contractions of the heart wall revealed in M-modes (Ocorr, 2017).
Although both sei and KCNQ channel mutations cause electrical remodeling and arrhythmia in the fly heart model they appear to have differing effects on muscle structure. Mutations in sei appear to be linked to an increase in myofibrillar disorganization and this effect on myocardial structure is manifest by reductions in fractional shortening. In addition, the observed reductions in shortening velocities under loaded conditions suggests that the ability to generate tension is significantly lower in hearts from sei mutants than for controls or KCNQ mutants and are consistent with the reduced ability to sustain an isometric contraction in the sei mutant hearts. Together these results suggest differential roles for these channels on structural and electrical integrity of the adult heart (Ocorr, 2017).
The microarray results suggest that the different effects of the two K+ channel mutants are the result of underlying differences in gene expression. The observation that many pathways involved in metabolism are significantly upregulated in sei mutants is consistent with previous observations in rabbits, although in that study both LQT1 and LQT2 models exhibited similar effects. In particular, in the adult fly heart the Wnt signaling pathway appears to be selectively perturbed in the sei mutant hearts, as evidenced by a downregulation of many of its pathway components. This is consistent with previous results demonstrating that mutation or cardiac KD of the TCF co-factor encoded by pygo cause bradycardia and morphological remodeling similar to that observed for sei mutants. Importantly, this study now demonstrates an interaction between sei channel mutations and mutations in pygo. Wnt signaling has previously been shown to play significant roles in cardiac development in flies and vertebrates and in cardiac disease. This study has shown in the fly that pygo likely plays a role in the maintenance of cardiac function and structure in the adult and the current data suggest that Pygo and Sei channel dysfunction are likely linked genetically. Many components of both the canonical and non-canonical pathways exhibit reduced expression, which would suggest an overall reduction in Wnt signaling. However, downregulation was also observed of potential negative effectors of Wnt signaling, such as APC, the core of the destruction complex, and CtBP, which has been shown to mediate both activation and repression of transcription. Both effects could be expected to result in increased stabilization of β-catenin. Thus, the exact role of different Wnt signaling components in maintaining cardiac structure and function remains to be determined (Ocorr, 2017).
Electrical and morphological remodeling have previously been shown to be linked; for example, knockout of the heart development transcription factor Ptx1 (see Drosophila Ptx1) was shown to affect both electrical and morphological remodeling in mouse and humans. A number of studies have documented pro-fibrotic and apoptotic effects of atrial fibrillation as well as tachypacing in cardiomyocytes and in response to sustained atrial fibrillation in patients. Further, a genetic variant situated close to the long QT syndrome (LQTS) type 2 gene KCNH2 has been shown to be associated with early onset AF. Thus, cellular/molecular links between channel function, electrical activity and morphological remodeling and ultimately heart failure have been suggested but have yet to be clearly elucidated. An understanding of how hERG channels interact with cellular pathways involved in electrical and structural remodeling and with other repolarizing currents such as IKs, mediated by KCNQ channels, will be important to the development of novel anti-arrhythmia therapies. These data using the fly heart model now provides the first clear genetic and physiological evidence that some channelopathies may be contributing to cardiac remodeling during disease progression via Wnt signaling (Ocorr, 2017).
Neuropeptides and peptide hormones serve as critical regulators of numerous biological processes, including development, growth, reproduction, physiology, and behaviour. In mammals, peptidergic regulatory systems are complex and often involve multiple peptides that act at different levels and relay to different receptors. To improve the mechanistic understanding of such complex systems, invertebrate models in which evolutionarily conserved peptides and receptors regulate similar biological processes but in a less complex manner have emerged as highly valuable. Drosophila melanogaster represents a favoured model for the characterisation of novel peptidergic signalling events and for evaluating the relevance of those events in vivo. The present study analysed a set of neuropeptides and peptide hormones for their ability to modulate cardiac function in semi-intact larval Drosophila melanogaster. Numerous peptides were identifed that significantly affected heart parameters such as heart rate, systolic and diastolic interval, rhythmicity, and contractility. Thus, peptidergic regulation of the Drosophila heart is not restricted to chronotropic adaptation but also includes inotropic modulation. By specifically interfering with the expression of corresponding peptides in transgenic animals, the in vivo relevance of the respective peptidergic regulation was assessed. Based on the functional conservation of certain peptides throughout the animal kingdom, the identified cardiomodulatory activities may be relevant not only to proper heart function in Drosophila, but also to corresponding processes in vertebrates, including humans (Schiemann, 2018).
Approaches are sought after to regulate ionotropic and chronotropic properties of the mammalian heart. Electrodes are commonly used for rapidly exciting cardiac tissue and resetting abnormal pacing. With the advent of optogenetics and the use of tissue-specific expression of light-activated channels, cardiac cells cannot only be excited but also inhibited with ion-selective conductance. As a proof of concept for the ability to slow down cardiac pacing, anion-conducting channelrhodopsins (GtACR1/2) and the anion pump halorhodopsin (eNpHR) were expressed in hearts of larval Drosophila and activated by light. Unlike body wall muscles in most animals, the equilibrium potential for Cl(-) is more positive as compared to the resting membrane potential in larval Drosophila. As a consequence, upon activating the two forms of GtACR1 and 2 with low light intensity the heart rate increased, likely due to depolarization and opening of voltage-gated Ca(2+) channels. However, with very intense light activation the heart rate ceases, which may be due to Cl(-) shunting to the reversal potential for chloride. Activating eNpHR hyperpolarizes body wall and cardiac muscle in larval Drosophila and rapidly decreases heart rate. The decrease in heart rate is related to light intensity. Intense light activation of eNpHR stops the heart from beating, whereas lower intensities slowed the rate. Even with upregulation of the heart rate with serotonin, the pacing of the heart was slowed with light. Thus, regulation of the heart rate in Drosophila can be accomplished by activating anion-conducting channelrhodopsins using light. These approaches are demonstrated in a genetically amenable insect model (Stanley, 2019).
Understanding the effects of microgravity on human organs is crucial to exploration of low-earth orbit, the moon, and beyond. Drosophila can be sent to space in large numbers to examine the effects of microgravity on heart structure and function, which is fundamentally conserved from flies to humans. Flies reared in microgravity exhibit cardiac constriction with myofibrillar remodeling and diminished output. RNA sequencing (RNA-seq) in isolated hearts revealed reduced expression of sarcomeric/extracellular matrix (ECM) genes and dramatically increased proteasomal gene expression, consistent with the observed compromised, smaller hearts and suggesting abnormal proteostasis. This was examined further on a second flight in which dramatically elevated proteasome aggregates were found co-localizing with increased amyloid and polyQ deposits. Remarkably, in long-QT causing sei/hERG mutants, proteasomal gene expression at 1g, although less than the wild-type expression, was nevertheless increased in microgravity. Therefore, cardiac remodeling and proteostatic stress may be a fundamental response of heart muscle to microgravity (Walls, 2020).
Studies about the relationship between substances consumed by humans and their impact on health, in animal models have been a challenge due to differences between species in the animal kingdom. However, the homology of certain genes has allowed extrapolating certain knowledge obtained in animals. Drosophila melanogaster, studied for decades, has been widely used as model for human diseases as well as to study responses associated with the consumption of several substances. This work explores the impact of tobacco consumption on a model of "smoking flies". These experiments were designed to provide information about the effects of tobacco consumption on cardiac physiology. Intracellular calcium handling, a phenomenon underlying cardiac contraction and relaxation, was assessed. Flies chronically exposed to tobacco smoke exhibited an increased heart rate and alterations in the dynamics of the transient increase of intracellular calcium in myocardial cells. These effects were also evident under acute exposure to nicotine of the heart, in a semi-intact preparation. Moreover, the alpha 1 and alpha 7 subunits of the nicotinic receptors are involved in the heart response to tobacco and nicotine under chronic (in the intact fly) as well as acute exposure (in the semi-intact preparation). The present data would help to understand the implication of the intracellular cardiac pathways affected by nicotine on the heart tissue. Based on the probed genetic and physiological similarity between the fly and human heart, cardiac effects exerted by tobacco smoke in Drosophila would help to know the impact of it in the human heart. Additionally, it may also provide information on how nicotine-like substances, e.g. neonicotinoids used as insecticides, affect cardiac function (Santalla, 2021).
The cross talk between adipose tissue and the heart has an increasing importance for cardiac function under physiological and pathological conditions. This study characterizes the role of fat body lipolysis for cardiac function in Drosophila melanogaster. Perturbation of the function of the key lipolytic enzyme, brummer (bmm), an ortholog of the mammalian ATGL (adipose triglyceride lipase) exclusively in the fly's fat body, protected the heart against starvation-induced dysfunction. Evidence is provided that this protection is caused by the preservation of glycerolipid stores, resulting in a starvation-resistant maintenance of energy supply and adequate cardiac ATP synthesis. Finally, it is suggested that alterations of lipolysis are tightly coupled to lipogenic processes, participating in the preservation of lipid energy substrates during starvation. Thus, this study identified the inhibition of adipose tissue lipolysis and subsequent energy preservation as a protective mechanism against cardiac dysfunction during catabolic stress (Blumrich, 2021).
Mutations in the human LMNA gene cause a collection of diseases known as laminopathies. These include myocardial diseases that exhibit age-dependent penetrance of dysrhythmias and heart failure. The LMNA gene encodes A-type lamins, intermediate filaments that support nuclear structure and organize the genome. Mechanisms by which mutant lamins cause age-dependent heart defects are not well understood. This study modeled human disease-causing mutations in the Drosophila Lamin C gene and expressed mutant Lamin C exclusively in the heart. This resulted in progressive cardiac dysfunction, loss of adipose tissue homeostasis, and a shortened adult lifespan. Within cardiac cells, mutant Lamin C aggregated in the cytoplasm, the CncC(Nrf2)/Keap1 Mutations in the human LMNA gene are associated with a collection of diseases called laminopathies in which the most common manifestation is progressive cardiac disease. This study has generated Drosophila melanogaster models of age-dependent cardiac dysfunction. In these models, mutations synonymous with those causing disease in humans were introduced into Drosophila LamC. Cardiac-specific expression of mutant LamC resulted in (1) cardiac contractility, conduction, and physiological defects, (2) abnormal nuclear envelope morphology, (3) cytoplasmic LamC aggregation, (4) nuclear enrichment of the redox transcriptional regulator CncC (mammalian Nrf2), (5) and upregulation of autophagy cargo receptor Ref(2)P (mammalian p62). These cardiac defects were enhanced with age and accompanied by increased adipose tissue in the adult fat bodies and a shortened lifespan (Bhide, 2018).
To understand the mechanistic basis of cardiolaminopathy and identify genetic suppressors, advantage was taken of powerful genetic tools available in Drosophila. The presence of cytoplasmic LamC aggregates prompted a determination of whether increasing autophagy would suppress the cardiac defects. Cardiac-specific upregulation of autophagy (Atg1 OE) suppressed G489V-induced cardiac defects. Consistent with this, decreased autophagy due to expression of Atg1 DN resulted in enhanced deterioration of G489V-induced cardiac dysfunction. Interestingly, cardiac-specific Atg5 OE and Atg8a OE, two factors that also promote autophagy, showed little to no suppression of G489V-induced heart dysfunction, suggesting that Atg1 might be rate limiting in this context. These findings are consistent with studies in mouse laminopathy models in which rapamycin and temsirolimus had beneficial effects on heart and skeletal muscle through inhibition of AKT/mTOR signaling. These findings are depicted in a model (see Model for the interactions between the autophagy and CncC/Keap1 signaling pathway in mutant lamin-induced cardiac disease) in which cytoplasmic aggregation of mutant LamC results in upregulation of p62, which in turn inhibits autophagy via activation of TOR and inactivation of AMPK. AMPK inactivation leads to the activation of PI3K/Akt/mTOR pathway and inhibition of autophagy Atg1 OE promoted clearance of the LamC aggregates and restored proteostasis in these Drosophila models. Thus, the data suggest that mutant LamC reduces autophagy, resulting in impairment of cellular proteostasis that leads to cardiac dysfunction (Bhide, 2018).
Cardiac-specific expression of mutant LamC altered CncC subcellular localization. Previously, Drosophila larval body wall muscles expressing G489V were shown to experience reductive stress, an atypical redox state characterized by high levels of reduced glutathione and NADPH, and upregulation CncC target genes (Dialynas, 2015). Cardiac-specific CncC RNAi in the wild-type LamC background did not produce major cardiac defects. Consistent with this, Nrf2 deficiency in mice does not compromise cardiac and skeletal muscle performance. Cardiac-specific CncC RNAi suppressed G489V-induced cardiac dysfunction and reduced cytoplasmic LamC aggregation, but not R205W-induced defects. However, cardiac-specific RNAi against CncC did not affect G489V-induced adipose tissue accumulation and lifespan shortening. Similar to the nuclear enrichment of CncC in hearts expressing G489V, human muscle biopsy tissue from an individual with a point mutation in the LMNA gene that results in G449V (analogous to Drosophila G489V) showed nuclear enrichment of Nrf2 (Dialynas, 2015). Disruption of Nrf2/Keap1 signaling has also been reported for Hutchinson-Gilford progeria, an early-onset aging disease caused by mutations in LMNA. In this case, however, the thickened nuclear lamina traps Nrf2 at the nuclear envelope that results in a failure to activate Nrf2 target genes, leading to oxidative stress. In these studies, CncC nuclear enrichment was observed; however, a redox imbalance was not readily observed at the three-time points investigated. This might indicate that there is a window of time in disease progression in which redox imbalance occurs and that mechanisms are in place to re-establish homeostasis (Bhide, 2018).
It has been postulated that there is cross-talk between autophagy and Nrf2/Keap1 signaling. This was tested by manipulating autophagy and CncC (Nrf2) alone and in combination. CncC RNAi suppressed the cardiac defects caused by G489V, but not the lipid accumulation and lifespan shortening, suggesting the latter two phenotypes are not specifically due to loss of cardiac function. In contrast, Atg1 OE suppressed the cardiac and adipose tissue defects and lengthened the lifespan. The double treatment (simultaneous Atg1 OE and RNAi knockdown of CncC) gave the most robust suppression of the mutant phenotypes and completely restored the lifespan. Interestingly, Atg1 DN and RNAi knockdown of CncC simultaneously did not further deteriorate or improve the mutant phenotypes. Taken together, these data suggest that autophagy plays a key role in suppression of the G498V-induced phenotypes and that knockdown on CncC enhances this suppression (Bhide, 2018).
These findings support a model whereby autophagy and Nrf2 signaling are central to cardiac health. It is proposed that cytoplasmic aggregation of LamC increases levels of Ref(2)P (p62), which competitively binds to Keap1, resulting in CncC (Nrf2) translocation to the nucleus. Inside the nucleus, Nrf2 regulates genes involved in detoxification. Continued expression of antioxidant genes results in the disruption of redox homeostasis, defective mitochondria, and dysregulation of energy homeostasis/energy sensor such as AMPK and its downstream targets. Simultaneously, upregulation of Ref(2)P (p62) causes inhibition of autophagy via activation of TOR, which leads to the inactivation of AMPK. AMPK inactivation in combination with activation of the TOR pathway causes cellular and metabolic stress that leads to cardiomyopathy. In support of this model, transcriptomics data from muscle tissue of an individual with muscular dystrophy expressing Lamin A/C G449V (analogous to Drosophila G489V) showed (1) upregulation of transcripts from Nrf2 target genes, (2) upregulation of genes encoding subunits of the mTOR complex, and (3) downregulation of AMPK, further demonstrating relevance of the Drosophila model for providing insights on human pathology (Bhide, 2018).
The structural conservation and activity of the myosuppressin cardioinhibitory peptide across species suggests it plays an important role in physiology, yet much remains unknown regarding its signaling. Drosophila melanogaster myosuppressin (dromyosuppressin, DMS; TDVDHVFLRF-NH(2)) decreases cardiac contractility through a G protein-coupled receptor, DMS-R2. This study showed the DMS N-terminus amino acids influence its structure-activity relationship (SAR), yet how they act is not established. It was predicted that myosuppressin N-terminal amino acids played a role in activity and signaling. This hypothesis was tested in the beetle, Zophobas atratus, using a semi-isolated heart bioassay to explore SAR in a different Order and focus on cardiac signaling. A series of myosuppressin truncated analogs was generated by removing the N-terminal residue and measuring the activity of each structure on cardiac contractility. While DVDHVFLRF-NH(2) decreased cardiac contractility, it was found VDHVFLRF-NH(2), DHVFLRF-NH(2), and HVFLRF-NH(2) increased activity. In contrast, VFLRF- NH(2) decreased activity and FLRF-NH(2) was inactive. Next, molecular docking data was analyzed, and it was found the active truncated analogs interacted with the 3-6 lock in DMS-R2, the myosuppressin cardiac receptor, disrupting the salt bridge between H114 and E369, and K289 and Q372. Further, the docking results showed the inhibitory effect on contractility may be associated with contact to Y78, while the analogs that increased contractility lacked this interaction. The data from this study demonstrated N-terminal amino acids played a role in myosuppressin activity and signaling suggesting the cardiac receptor can be targeted by biased agonists. These myosuppressin cardiac contractility data and predicted receptor interactions describe the presence of functional selectivity in a ligand-directed signaling pathway in heart (Nichols, 2021).
Thermotolerance is a complex trait that can greatly differ between heat-susceptible (HS) and heat-adapted populations of small insects including Drosophila, with short-term effects after a sub-lethal level of heat stress on many physiological functions. Cardiac performance could accordingly be more robust in heat-resistant (HR) than in HS individuals under heat stress. This study tested heart performance under heat-stress effects in two recombinant inbred lines (RIL) of Drosophila melanogaster that dramatically differ in heat knockdown resistance. Heart rate did not strongly differ between heat-susceptible and heat-tolerant flies after a sub-lethal heat stress. Instead, heat-susceptible flies showed a much higher arrhythmia incidence, a longer duration of each heartbeat, and a larger amount of bradycardia than heat-tolerant flies. The highly conserved cardiac proteins SERCA, RyR and NCX that participate in the excitation/contraction coupling, did not differ in activity level between HR and HS flies. Available information for both RIL suggests that heart performance under heat stress may be linked, at least partially, to candidate genes of previously identified quantitative trait loci (QTL) for thermotolerance. This study indicates that HR flies can be genetically more robust in their heart performance than HS flies under even sub-lethal levels of heat stress (Rodriguez, 2021).
Deciphering the genetic architecture of human cardiac disorders is of fundamental importance but their underlying complexity is a major hurdle. This study investigated the natural variation of cardiac performance in the sequenced inbred lines of the Drosophila Genetic Reference Panel (DGRP). Genome-wide associations studies (GWAS) identified genetic networks associated with natural variation of cardiac traits which were used to gain insights as to the molecular and cellular processes affected. Non-coding variants that this study identified were used to map potential regulatory non-coding regions, which in turn were employed to predict transcription factors (TFs) binding sites. Cognate TFs, many of which themselves bear polymorphisms associated with variations of cardiac performance, were also validated by heart-specific knockdown. Additionally, this study showed that the natural variations associated with variability in cardiac performance affect a set of genes overlapping those associated with average traits but through different variants in the same genes. Furthermore, it was shown that phenotypic variability was also associated with natural variation of gene regulatory networks. More importantly, correlations were documented between genes associated with cardiac phenotypes in both flies and humans, which supports a conserved genetic architecture regulating adult cardiac function from arthropods to mammals. Specifically, roles for PAX9 (Drosophila Poxm) and EGR2 (Drosophila Stripe) in the regulation of the cardiac rhythm were established in both models, illustrating that the characteristics of natural variations in cardiac function identified in Drosophila can accelerate discovery in humans (Saha, 2022).
Appropriate cardiac performance depends on a tightly controlled handling of Ca2+ in a broad range of species, from invertebrates to mammals. The role of the Ca2+ ATPase, SERCA, in Ca2+ handling is pivotal, and its activity is regulated, inter alia, by interacting with distinct proteins. This study gives evidence that 4E binding protein (4E-BP) is a novel regulator of SERCA activity in Drosophila melanogaster during cardiac function. Flies over-expressing 4E-BP showed improved cardiac performance in young individuals associated with incremented SERCA activity. Moreover, it was demonstrated that SERCA interacts with translation initiation factors eIF4E-1, eIF4E-2 and eIF4E-4 in a yeast two-hybrid assay. The specific identification of eIF4E-4 in cardiac tissue leads to a proposal that the interaction of elF4E-4 with SERCA may be the basis of the cardiac effects observed in 4E-BP over-expressing flies associated with incremented SERCA activity (Santalla, 2022).
Apolipoprotein B plays an essential role in systemic lipid metabolism, and it is closely related to cardiovascular diseases. Exercise-training can regulate systemic lipid metabolism, improve heart function, and improve exercise capacity, but the molecular mechanisms involved are poorly understood. This study used a Drosophila model to demonstrate that exercise-training regulates the expression of apoLpp (a homolog of apolipoprotein B) in cardiomyocytes, thereby resisting heart insufficiency and low exercise capacity caused by obesity. The apoLpp is an essential lipid carrier produced in the heart and fat body of Drosophila. In a Drosophilla genetic screen, low expression of apoLpp reduced obesity and cardiac dysfunction induced by a high-fat diet (HFD). Cardiac-specific inhibition indicated that reducing apoLpp in the heart during HFD reduced the triglyceride content of the whole-body and reduced heart function damage caused by HFD. In exercise-trained flies, the result was similar to the knockdown effect of apoLpp. Therefore, the inhibition of apoLpp plays an important role in HFD-induced cardiac function impairment and low exercise capacity. Although the apoLpp knockdown of cardiomyocytes alleviated damage to heart function, it did not reduce the arrhythmia and low exercise capacity caused by HFD. Exercise-training can improve this condition more effectively, and the possible reason for this difference is that exercise-training regulates climbing ability in ways to promote metabolism. Exercise-training during HFD feeding can down-regulate the expression of apoLpp, reduce the whole-body TG levels, improve cardiac recovery, and improve exercise capacity. Exercise-training can downregulate the expression of apoLpp in cardiomyocytes to resist cardiac function damage and low exercise capacity caused by HFD. The results revealed the relationship between exercise-training and apoLpp and their essential roles in regulating heart function and climbing ability (Ding, 2021).
Atrial fibrillation (AF) is a common and genetically inheritable form of cardiac arrhythmia; however, it is currently not known how these genetic predispositions contribute to the initiation and/or maintenance of AF-associated phenotypes. One major barrier to progress is the lack of experimental systems to investigate the effects of gene function on rhythm parameters in models with human atrial and whole-organ relevance. This study assembled a multi-model platform enabling high-throughput characterization of the effects of gene function on action potential duration and rhythm parameters using human induced pluripotent stem cell-derived atrial-like cardiomyocytes and a Drosophila heart model. The findings were validated using computational models of human adult atrial myocytes and tissue. As proof of concept, 20 AF-associated genes were screened and identified phospholamban loss of function as a top conserved hit that shortens action potential duration and increases the incidence of arrhythmia phenotypes upon stress. Mechanistically, this study reveals that phospholamban regulates rhythm homeostasis by functionally interacting with L-type Ca2+ channels and NCX. In summary, this study illustrates how a multi-model system approach paves the way for the discovery and molecular delineation of gene regulatory networks controlling atrial rhythm with application to AF (Kervadec, 2023).
Estimation of dynamic change of crossbridge formation in living cardiomyocytes is expected to provide crucial information for elucidating cardiomyopathy mechanisms, efficacy of an intervention, and others. This study established an assay system to dynamically measure second harmonic generation (SHG) anisotropy derived from myosin filaments depended on their crossbridge status in pulsating cardiomyocytes. Experiments utilizing an inheritable mutation that induces excessive myosin-actin interactions revealed that the correlation between sarcomere length and SHG anisotropy represents crossbridge formation ratio during pulsation. Furthermore, the present method found that ultraviolet irradiation induced an increased population of attached crossbridges that lost the force-generating ability upon myocardial differentiation. Taking an advantage of infrared two-photon excitation in SHG microscopy, myocardial dysfunction could be intravitally evaluated in a Drosophila disease model. Thus, this study has successfully demonstrated the applicability and effectiveness of the present method to evaluate the actomyosin activity of a drug or genetic defect on cardiomyocytes. Because genomic inspection alone may not catch the risk of cardiomyopathy in some cases, this study would be of help in the risk assessment of future heart failure (Fujita, 2023).
The Drosophila model has proven tremendously powerful for understanding pathophysiological bases of several human disorders including aging and cardiovascular disease. Relevant high-speed imaging and high-throughput lab assays generate large volumes of high-resolution videos, necessitating next-generation methods for rapid analysis. This study presents a platform for deep learning-assisted segmentation applied to optical microscopy of Drosophila hearts and the first to quantify cardiac physiological parameters during aging. An experimental test dataset is used to validate a Drosophila aging model. Then two novel methods were used to predict fly aging: deep-learning video classification and machine-learning classification via cardiac parameters. Both models suggest excellent performance, with an accuracy of 83.3% (AUC 0.90) and 77.1% (AUC 0.85), respectively. Furthermore, beat-level dynamics are reported for predicting the prevalence of cardiac arrhythmia. The presented approaches can expedite future cardiac assays for modeling human diseases in Drosophila and can be extended to numerous animal/human cardiac assays under multiple conditions. Significance Current analysis of Drosophila cardiac recordings is capable of limited cardiac physiological parameters and are error-prone and time-consuming. This study presents the first deep-learning pipeline for high-fidelity automatic modeling of Drosophila contractile dynamics. Methods are presented for automatically calculating all relevant parameters for diagnosing cardiac performance in aging model. Using the machine and deep learning age-classification approach, aging hearts can be predicted with an accuracy of 83.3% (AUC 0.90) and 77.1% (AUC 0.85), respectively (Pant, 2023).
The structures and activities of invertebrate sulfakinins that influence gut motility and heart rate are like the vertebrate cholecystokinin (CCK) peptides. Typical of sulfakinin precursors Drosophila melanogaster encodes non-sulfated drosulfakinin I (nsDSK I; FDDYGHMRF-NH2) and nsDSK II (GGDDQFDDYGHMRF-NH2) that bind DSK-R1 and DSK-R2. To explore the role of the nsDSK II N-terminal extension (GGDDQ) in gut its structure-activity relationship (SAR) were identified, and novel agonists were identified. Then the nsDSK II extension SAR was predicted to be tissue specific, consistent with cardiac CCK structure activity and signaling being different from gut. To evaluate this hypothesis, single-substituted alanine and asparagine analogs in heart were tested. Alanyl-substituted analogs were less active in heart than nsDSK II; in gut they include a super agonist and a protean agonist. Additionally, it was discovered that ns[N4]DSK II was more active than nsDSK II in pupal heart, while ns[N3]DSK II was inactive. In contrast, ns[N3]DSK II and ns[N4]DSK II were super agonists in adult heart, yet inactive in larva. Although this study reported nsDSK II acts through DSK-R2 in gut, its identity in heart was unknown. This study reviewed ligand-receptor interactions in conjunction with SAR data to suggest nsDSK II acts through DSK-R1 in heart consistent with sulfakinin tissue-specific signaling (Nichols, 2022).
Adjusting to a dynamic environment involves fast changes in the body's internal state, characterized by coordinated alterations in brain activity and physiological and motor responses. Threat-induced defensive states are a classic case of coordinated adjustment of bodily responses, cardiac regulation being one of the best characterized examples in vertebrates. A great deal is known regarding the neural basis of invertebrate defensive behaviors, mainly in Drosophila melanogaster. However, whether physiological changes accompany these remains unknown. This study sets out to describe the internal bodily state of fruit flies upon an inescapable threat and found cardiac acceleration during running and deceleration during freezing. In addition, this study found that freezing leads to increased cardiac pumping from the abdomen toward the head-thorax, suggesting mobilization of energy resources. Concordantly, threat-triggered freezing reduces sugar levels in the hemolymph and renders flies less resistant to starvation. The cardiac responses observed during freezing were absent during spontaneous immobility, underscoring the active nature of freezing response. Finally, this study shows that baseline cardiac activity predicts the amount of freezing upon threat. This work reveals a remarkable similarity with the cardiac responses of vertebrates, suggesting an evolutionarily convergent defensive state in flies. These findings are at odds with the widespread view that cardiac deceleration while freezing has first evolved in vertebrates and that it is energy sparing. Investigating the physiological changes coupled to defensive behaviors in the fruit fly has revealed that freezing is costly yet accompanied by cardiac deceleration and points to heart activity as a key modulator of defensive behaviors (Barrios, 2021).
Despite their highly reactive nature, reactive oxygen species (ROS) at the physiological level serve as signaling molecules regulating diverse biological processes. While ROS usually act autonomously, they also function as local paracrine signals by diffusing out of the cells producing them. Using in vivo molecular genetic analyses in Drosophila, this study provides evidence for ROS-dependent paracrine signaling that does not entail ROS release. Elevated levels of physiological ROS within the pericardial cells activate a signaling cascade transduced by Ask1, c-Jun N-terminal kinase, and p38 to regulate the expression of the cytokine Unpaired 3 (Upd3). Upd3 released by the pericardial cells controls fat body-specific expression of the extracellular matrix (ECM) protein Pericardin, essential for cardiac function and healthy life span. Therefore, this work reveals an unexpected inter-organ communication circuitry wherein high physiological levels of ROS regulate cytokine-dependent modulation of cardiac ECM with implications in normal and pathophysiological conditions (Gera, 2022).
Abnormal heart rhythm is a common cardiac dysfunction in obese patients, and its pathogenesis is related to systemic lipid accumulation. The cardiomyocyte-derived apoLpp (homologous gene in Drosophila of the human apolipoprotein B) plays an important role in whole-body lipid metabolism of Drosophila under a high-fat diet (HFD). Knockdown of apoLpp derived from cardiomyocytes can reduce HFD-induced weight gain and abdominal lipid accumulation. In addition, exercise can reduce the total amount of apoLpp in circulation. However, the relationship between regular exercise, cardiomyocyte-derived apoLpp and abnormal heart rhythm is unclear. This study found that an HFD increased the level of triglyceride (TG) in the whole-body, lipid accumulation and obesity in Drosophila. Moreover, the expression of apoLpp in the heart increased sharply, the heart rate and arrhythmia index increased and fibrillation occurred. Conversely, regular exercise or cardiomyocyte-derived apoLpp knockdown reduced the TG level in the whole-body of Drosophila. This significantly reduced the arrhythmia induced by obesity, including the reduction of heart rate, arrhythmia index, and fibrillation. Under HFD conditions, flies with apoLpp knockdown in the heart could resist the abnormal cardiac rhythm caused by obesity after receiving regular exercise. HFD-induced obesity and abnormal cardiac rhythm may be related to the acute increase of cardiomyocyte-derived apoLpp. Regular exercise and inhibition of cardiomyocyte-derived apoLpp can reduce the HFD-induced abnormal cardiac rhythm (Ding, 2022).
Judiciously tuning heart rates is critical for regular cardiovascular function. The fractal pattern of heartbeats - a multiscale regulation in instantaneous fluctuations - is well known for vertebrates. The most primitive heart system of the Drosophila provides a useful model to understand the evolutional origin of such a fractal pattern as well as the alterations of fractal pattern during diseased statuses. A non-invasive visible optical heart rate recording system especially suitable for long-term recording was developed by using principal component analysis (PCA) instead of fluorescence recording system to avoid the confounding effect from intense light irradiation. To deplete intracellular Ca(2+) levels, the expression of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) was tissue-specifically knocked down. The SERCA group shows longer heart beat intervals as compared to the control group. The multiscale correlation of SERCA group, on the other hand, is weaker than that of the control Drosophila. It is concluded that fractal correlations were presented in control group but were disrupted by the heart specific SERCA depletion (Lin, 2016).
The Bowditch effect or staircase phenomenon is the increment or reduction of contractile force when heart rate increases, defined as either a positive or negative staircase. The healthy and failing human heart both show positive or negative staircase, respectively, but the causes of these distinct cardiac responses are unclear. Different experimental approaches indicate that while the level of Ca(2+) in the sarcoplasmic reticulum is critical, the molecular mechanisms are unclear. This study demonstrates that Drosophila melanogaster shows a negative staircase which is associated to a slight but significant frequency-dependent acceleration of relaxation (FDAR) at the highest stimulation frequencies tested. It was further shown that the type of staircase is oppositely modified by two distinct SERCA mutations. The dominant conditional mutation SERCA(A617T) induced positive staircase and arrhythmia, while SERCA(E442K) accentuated the negative staircase of wild type. At the stimulation frequencies tested, no significant FDAR could be appreciated in mutant flies. The present results provide evidence that two individual mutations directly modify the type of staircase occurring within the heart and suggest an important role of SERCA in regulating the Bowditch effect (Balcazar, 2018).
G-protein signaling is known to be required for cell-cell contacts during the development of the Drosophila dorsal vessel. However, the identity of the G protein-coupled receptor (GPCR) that regulates this signaling pathway activity is unknown. This study describes the identification of a novel cardiac specific GPCR, called Gia, for "GPCR in aorta". Gia is the only heart-specific GPCR identified in Drosophila to date and it is specifically expressed in cardioblasts that fuse at the dorsal midline to become the aorta. Gia is the only Drosophila gene so far identified for which expression is entirely restricted to cells of the aorta. Deletion of Gia leads to a broken-hearted phenotype, characterized by pericardial cells dissociated from cardioblasts and abnormal distribution of cell junction proteins. Both phenotypes are similar to those observed in mutants of the heterotrimeric cardiac G proteins. Lack of Gia also led to defects in the alignment and fusion of cardioblasts in the aorta. Gia forms a protein complex with G-αo47A, the alpha subunit of the heterotrimeric cardiac G proteins and interacts genetically with G-αo47A during cardiac morphogenesis. Gia acts as an essential aorta-specific GPCR that functions upstream of cardiac heterotrimeric G proteins and is required for morphological integrity of the aorta during heart tube formation. These studies lead to a redefinition of the bro phenotype, to encompass morphological integrity of the heart tube as well as cardioblast-pericardial cell spatial interactions (Patel, 2016).
The Drosophila heart, composed of discrete subsets of cardioblasts and pericardial cells, undergoes Hox-triggered anterior-posterior morphogenesis, leading to a functional subdivision into heart proper and aorta, with its most anterior part forming a funnel-shaped cardiac outflow. Cardioblasts differentiate into Tin-positive 'working myocytes' and Svp-expressing ostial cells. However, developmental fates and functions of heart-associated pericardial cells remain elusive. This study shows that the pericardial cells that express the transcription factor Even Skipped adopt distinct fates along the anterior-posterior axis. Among them, the most anterior Antp-Ubx-AbdA-negative cells form a novel cardiac outflow component that is called the outflow hanging structure, whereas the Antp-expressing cells differentiate into wing heart precursors. Interestingly, Hox gene expression in the Even Skipped-positive cells not only underlies their antero-posterior diversification, but also influences heart morphogenesis in a non-cell-autonomous way. In brief, this study has identified a new cardiac outflow component derived from a subset of Even Skipped-expressing cells that stabilises the anterior heart tip, and demonstrate non-cell-autonomous effects of Hox gene expression in the Even Skipped-positive cells on heart morphogenesis (Zmojdzian, 2018).
The Drosophila heart (dorsal vessel) is a relatively simple tubular organ that serves as a model for several aspects of cardiogenesis. Cardiac morphogenesis, proper heart function and stability require structural components whose identity and ways of assembly are only partially understood. Structural components are also needed to connect the myocardial tube with neighboring cells such as pericardial cells and specialized muscle fibers, the so-called alary muscles. Using an EMS mutagenesis screen for cardiac and muscular abnormalities in Drosophila embryos, multiple mutants were obtained for two genetically interacting complementation groups that showed similar alary muscle and pericardial cell detachment phenotypes. The molecular lesions underlying these defects were identified as domain-specific point mutations in LamininB1 and Cg25C, encoding the extracellular matrix (ECM) components laminin beta and collagen IV alpha1, respectively. Of particular interest within the LamininB1 group are certain hypomorphic mutants that feature prominent defects in cardiac morphogenesis and cardiac ECM layer formation, but in contrast to amorphic mutants, only mild defects in other tissues. All of these alleles carry clustered missense mutations in the laminin LN domain. The identified Cg25C mutants display weaker and largely temperature-sensitive phenotypes that result from glycine substitutions in different Gly-X-Y repeats of the triple helix-forming domain. While initial basement membrane assembly is not abolished in Cg25C mutants, incorporation of perlecan is impaired and intracellular accumulation of perlecan as well as the collagen IV alpha2 chain is detected during late embryogenesis. It is concluded that assembly of the cardiac ECM depends primarily on laminin, whereas collagen IV is needed for stabilization. The data underscore the importance of a correctly assembled ECM particularly for the development of cardiac tissues and their lateral connections. The mutational analysis suggests that the beta6/beta3/beta8 interface of the laminin beta LN domain is highly critical for formation of contiguous cardiac ECM layers. Certain mutations in the collagen IV triple helix-forming domain may exert a semi-dominant effect leading to an overall weakening of ECM structures as well as intracellular accumulation of collagen and other molecules, thus paralleling observations made in other organisms and in connection with collagen-related diseases (Hollfelder, 2014).
The correct balance between stiffness and elasticity is essential to the function of numerous tissues, and depends on ECM constituents (the matrisome). However, despite its physiological relevance, the matrisome composition and organization remain poorly understood. Previously, it was reported that the ADAMTS-like protein Lonely heart (Loh) is critical for recruiting the type IV collagen-like protein Pericardin to the cardiac ECM. Thus study utilized Drosophila as a simple and genetically amenable invertebrate model for studying Loh-mediated recruitment of tissue-specific ECM components such as Pericardin to the ECM. Focus was placed on the functional relevance of distinct Loh domains to protein localization and Pericardin recruitment. Analysis of Loh deletion constructs revealed that one thrombospondin type 1 repeat (TSR1-1), which has an embedded WXXW motif, is critical for anchoring Loh to the ECM. Two other thrombospondin repeats, TSR1-2 and TSR1-4, the latter containing a CXXTCXXG motif, appeared to be dispensable for tethering Loh to the ECM, but were crucial for proper interaction with and recruitment of Pericardin. Moreover, the results also suggested that Pericardin in the cardiac ECM primarily ensures the structural integrity of the heart, rather than increasing tissue flexibility. In conclusion, this work provides new insight into the roles of thrombospondin type 1 repeats and advances understanding of cardiac ECM assembly and function (Rotstein, 2018).
Extracellular matrices (ECMs), which support and protect cells and provide mechanical linkage between tissues like muscles and epidermis, are generally assembled in a similar manner. After incorporation of transmembrane receptors, such as integrins and dystroglycans, meshwork-forming components like laminin and collagen IV are able to anchor. By interacting with each other, they form a complex network with distinct biomechanical properties, which is furthermore stabilized by nidogen and allows other proteins (e.g., perlecan) to bind to the matrix as well (Rotstein, 2018).
Whereas these steps can be found ubiquitously, the Drosophila cardiac ECM is different from the matrices of other tissues or organs in several ways. It forms a 3D meshwork that connects the contractile heart tube to the alary muscles and, thereby, to the epidermis. Within this meshwork, embedded pericardial cells differentiate into a distinct population of cell types, such as nephrocytes or wing hearts (Rotstein, 2018).
In flies, the cardiac ECM combines two important biomechanical features: elasticity that accounts for a flexible connection between heart and alary muscle cells and a high tensile strength that withstands forces produced by lifelong heart contractions. One major difference between cardiac ECMs and matrices of other tissues is the presence of the ADAMTS-like adapter protein Lonely heart (Loh), which can be found exclusively at the surface of the heart and chordotonal organs. Lonely heart is essential to proper recruitment of the type IV collagen-like protein Pericardin. Pericardin (Prc) is secreted into the hemolymph by pericardial nephrocytes and adipocytes, and, as soon as it becomes recruited to the cardiac matrix by Lonely heart, it starts to form a stable network. By this mechanism the heart is provided with an exceptional ECM that allows it to withstand the strong mechanical forces of a heartbeat. Lack of Pericardin or its anchor Lonely heart leads to a total collapse of the dorsal vessel and dissociation of the pericardial cells and alary muscles from the heart tube. Concomitants are severely impaired heartbeat and absence of heart-mediated hemolymph transport. Accordingly, corresponding mutant animals exhibit decreased fitness and shortened lifespan (Rotstein, 2018).
The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) superfamily consists of two classes of proteins: ADAMTS and ADAMTS-like proteins. Their main difference is that ADAMTS-like proteins, such as Loh, lack the proteolytically active motif within the ADAM spacer. Both classes share several domains, with most of them being poorly defined. In addition to a spacer region, a changing number of thrombospondin type 1 repeats (TSR1) can be found next to a protease and lacunin (PLAC) domain and a signal peptide. These ancillary domains apparently ensure proper substrate specificity as well as cell-surface or ECM tethering (Rotstein, 2018).
TSR1 motifs were initially discovered in thrombospondins (TSPs), which belong to the family of calcium-binding glycoproteins that are secreted into the extracellular matrix of all complex organisms. TSPs have been shown to bind to fibronectin, laminin, collagen, and other matricellular proteins to form complex networks on the cell surface. TSP superfamily members are involved in regulation of spinal cord outgrowth (e.g., F-spondin) or act as specific anti-angiogenic factors in brain development (e.g., BAI-1). In addition, they can be critical to directed ECM proteolysis. TSPs are modular proteins containing several types of repetitive sequence motifs. One of the most characteristic motifs is the evolutionarily conserved thrombospondin type 1 repeat (TSR1), which is ~60 amino acids in length and supposed to form an antiparallel three-stranded structure that interacts with glycoproteins of the extracellular matrix. The human genome harbors ~90 genes encoding TSR1-containing proteins, whereas ~14 corresponding proteins are present in D. melanogaster. Among these, some have been shown to contribute to heart development during embryogenesis. These proteins are the transmembrane receptor Uncoordinated 5 (Unc5) and the ADAMTS-like protein Lonely heart (Rotstein, 2018).
To understand the molecular mechanism by which Lonely heart ensures proper cardiac ECM formation in more detail, this study analyzed a large set of individually mutated Loh proteins for their capability to incorporate into the ECM and recruit Pericardin. To allow quantitative measurements of Pericardin recruitment efficiency, an in vivo recruitment assay was applied. In addition to the ECM of somatic muscles, this study investigated other types of matrices present in Drosophila for their capability to recruit Pericardin in a Loh-dependent manner. Furthermore, this study analyzed whether Pericardin, once recruited to a target matrix, has an intrinsic capacity to self-assemble into a meshwork independent of Loh. To perform the analysis, imaginal discs were used to express full-length Loh in distinct compartments of the disc and potential spreading was evaluated of the Pericardin meshwork over neighboring zones that lacked Loh (Rotstein, 2018).
Finally, to achieve an initial understanding of the biomechanical relevance of Pericardin, physiological consequences were sought of ectopic Pericardin deposition. In this respect, body wall muscles represent an effective readout system to investigate, for example, altered animal locomotion or lifespan. This study found that incorporation of Pericardin into the matrix of somatic muscles has no influence on lifespan but impairs contraction, thereby affecting the general locomotion performance (Rotstein, 2018).
In contrast to matrices present at the surface of most other tissues, the cardiac ECM is exposed to permanent mechanical stress generated by the regular and repetitive contraction cycles of the heart. These unique biomechanical conditions require ECM adaptation, which is achieved predominantly by incorporating specific structural components into the respective matrices. In Drosophila, one of these components is the type IV collagen-like protein Pericardin, which is recruited specifically to cardiac tissue by its adaptor protein Lonely heart. However, until now, neither the recruitment process itself nor the relevance of Pericardin to the biomechanical properties of the cardiac ECM have been studied in detail. By conducting a recruitment assay based on systematically generated domain-specific Loh mutants, this study found that presence of the first TSR1 domain is critical to localizing Loh to the ECM. Interestingly, mutating only the speculative GAG-binding site embedded within the first TSR1 domain is sufficient to abrogate Loh anchoring, indicating a high functional relevance of this distinct sequence motif. This result was confirmed by expressing the same constructs in Sf21 cells. Also in this system, deletion of the first TSR1 domain or mutation of the embedded putative GAG site resulted in considerably reduced surface localization of the respective Loh constructs. Significantly, Western blot analysis detected the proteins in the culture medium, which indicates that production and secretion still occurred, whereas incorporation into the ECM was impaired. Thus, the data suggest that TSR1-1, with its embedded putative GAG-binding site, is crucial for anchoring Loh to the ECM, which represents a prerequisite for the subsequent recruitment of Pericardin. On the other hand, the second speculative GAG binding site, embedded within the TSR1-4 domain, appears to be dispensable for localizing Loh but is required for efficient Pericardin recruitment. Of note, previous work identified the respective CXXTCXXG motif as a consensus site for O-fucosylation and showed that mutating this motif results in impaired protein secretion. Because the substitution in UAS-LohGAG2* covers this motif, slightly impaired secretion of this construct appears possible. However, its complete inability to recruit Pericardin cannot be attributed to minor deficiencies in secretion. Thus, the findings indicate that both speculative GAG-binding sites are of high functional relevance, with the first site being essential to proper anchoring of Loh, whereas the second one appears to be required for Pericardin recruitment. Interestingly, also lack of the second TSR1 domain results in failure to recruit Pericardin, whereas localization of Loh is not affected. Thus, the TSR1-2 and TSR1-4 domains as well as the putative GAG2-binding site seem to be dispensable for localizing Loh but crucial to proper Pericardin interaction and recruitment. In this context, the distinct position of the respective domains within Loh is probably decisive. According to structural modeling, TSR1-2 and TSR1-4, the latter containing the predicted GAG2 site, exhibit close spatial proximity. The fact that lack of either domain completely abolishes the capacity of Loh to recruit Pericardin indicates that these two domains constitute the interaction site between Loh and Prc, with the embedded speculative GAG binding site being of critical relevance. Subsequent to the initial binding, the nearby TSR1-3 and TSR1-5 domains may support interaction; however, their functional relevance is minor compared with TSR1-2 and TSR1-4. Taking these data into account, it appears likely that the N-terminal part of Loh, including the first TSR1 repeat and the embedded predicted GAG binding site, is facing the plasma membrane and anchors the protein to the cell surface, probably via glycosaminoglycan binding. The C-terminal part of Loh would then be available for interaction with Pericardin, and possibly also with other ECM components, via the second and fourth TSR1 repeats. Of note, a function of the PLAC domain, which is present in several enzymes and ECM proteins, such as ADAMTS-2, -3, -10, and others, was not uncovered by the approach of this study. Deleting the C-terminal PLAC domain in Loh has no distinct consequences, either for Loh secretion or for Pericardin recruitment efficiency, as far as can be stated in view of the sensitivity limitations of the test system (Rotstein, 2018).
Regarding the question of whether ECMs are generally capable of recruiting and incorporating Pericardin, this was found not to be the case. Whereas Loh-dependent recruitment was observed for fat body cells, somatic muscles, glial cells of the central nervous system, and wing discs, salivary gland cells did not incorporate Pericardin into the ECM, although Loh was present at the surface. This result indicates that other, yet unknown ECM components are required, in addition to Loh, for proper recruitment of Pericardin and that at least one of these factors is not present in salivary gland cells. Identification of the respective constituents represents an important objective of future studies because it would complement the current understanding of the interconnections that form the cardiac extracellular matrix. The alternative explanation, the presence of an inhibitory protein that prevents Pericardin incorporation into the ECM of salivary glands, appears unlikely, although this possibility cannot be ruled out for sure (Rotstein, 2018).
In Drosophila, formation of the cardiac extracellular matrix (ECM) starts during embryogenesis. Assembly and incorporation of structural proteins such as Collagen IV, Pericardin, and Laminin A, B1, and B2 into the cardiac ECM is critical to the maintenance of heart integrity and functionality. The cardiac ECM connects the heart tube with the alary muscles; thus, the ECM contributes to a flexible positioning of the heart within the animal's body. Moreover, the cardiac ECM holds the larval pericardial nephrocytes in close proximity to the heart tube and the inflow tract, which is assumed to be critical to efficient haemolymph clearance. Mutations in either structural ECM constituents or ECM receptors cause breakdown of the ECM network upon ageing, with disconnection of the heart tube from alary muscles becoming apparent at larval stages. Finally, the heart becomes non-functional. This study characterised existing and new pericardin mutants and investigated biosynthesis, secretion, and assembly of Pericardin in matrices. Two new pericardin alleles, which turned out to be a null (pericardin3-548) and a hypomorphic allele (pericardin3-21), were identifed. Both mutants could be rescued with a genomic duplication of a fosmid coding for the pericardin locus. Biochemical analysis revealed that Pericardin is highly glycosylated and forms redox-dependent multimers. Multimer formation is remarkably reduced in animals deficient for the prolyl-4 hydroxylase cluster at 75D3-4 (Wilmes, 2018).
By underlying or encasing a multitude of cells or tissues, extracellular matrices (ECMs) are essential to several physiological processes including tissue protection, tissue scaffolding, and cell signalling. Biochemical analysis, which is generally impeded by the insoluble and frequently cross-linked nature of the ECM, has shown that the complexity of matrices is much higher than previously expected. It has been reported that the 'matrisome', which collectively encompasses the proteins that constitute the ECM, comprises more than 300 proteins in mammals, including collagens, proteoglycans, growth factors, and receptors. The complexity of matrices is not only reflected by the number of proteins that constitute the matrix, but also by the different ratio with which the various components contribute and by the appearance of unique components in matrices of specific tissues. For example, while a high amount of Collagen I is characteristic of tendons, basement membranes (BMs) contain large amounts of Collagen IV, Laminins, Perlecan, and other proteins. Due to its diverse physiological function, the ECM is more than a homogeneous mass of proteins and carbohydrates. Within the meshwork of its structural components, the ECM is spatially patterned and thereby provides locally restricted reaction environments and structural micro-compartments (Wilmes, 2018).
The Drosophila heart is considered as a model for a specialised ECM composition that ensures proper tissue integrity, functionality, and organ performance. In Drosophila, at present, only four collagens or collagen-like proteins have been identified. One of these proteins is Pericardin (Prc); the others are Collagen IV alpha2 (Viking, Vkg), Cg25c (Dcg1), and Multiplexin. The Pericardin precursor protein consists of 1713 amino acids and harbours an N-terminal signal peptide as well as a long repeat region separated into a collagen-like domain and a non-collagen-like domain, with the former containing 26 atypical and several typical (Gly-X-Y)n repeats. In addition, a single potential Integrin-binding site (RGD) is present at the C-terminus. In contrast to the ubiquitously distributed Collagen IV, Pericardin assembles specifically within distinct matrices: these include the matrix of the heart tube, the surface of pericardial cells and oenocytes, and the cap cells of chordotonal organs. Lack of Pericardin, or its ECM adapter protein Lonely heart (Loh), causes heart failure upon ageing (Drechsler, 2013; Rotstein, 2018). During development, Pericardin is synthesised and secreted by different tissues: first, during embryogenesis, the pericardial cells secrete Pericardin; later, in first and second instar larvae, the main source of Pericardin secretion is the adipocytes. After biosynthesis, secretion, and release into the haemolymph, Pericardin specifically assembles at the outer surface of the cardiac tube and incorporates into the meshwork formed by typical structural components of basement membranes such as Collagen IV, Perlecan, and Nidogen. Adipocyte-specific knock-down of Sar1 expression inhibits Pericardin secretion and thereby affects the formation of a proper heart ECM in Drosophila (Drechsler, 2013). When Pericardin is not expressed, not secreted, or mislocalised, heart integrity is lost, which ultimately results in heart failure and heart collapse. These findings demonstrate that the assembly of a single structural protein, such as Pericardin, in the larval heart is essential for organ integrity and that adipocytes are the major source of distinct ECM components delivered to the heart tube (Wilmes, 2018).
Aiming to extend the current knowledge on how the specific meshwork of structural ECM constituents that characterise the heart matrix is established, this study investigated aspects of the biosynthesis, secretion and deposition of Pericardin in the cardiac matrix in more detail. The Pericardin protein displays collagen-like features that led to the assumption that Pericardin forms, like Collagen IV, trimeric helices that incorporate into matrices (Drechsler, 2013; Wilmes, 2018 and references therein).
Thus, this study focused particularly on components that are known to play an important role in Collagen IV processing, asking whether these enzymes also process Pericardin. Hydroxylation of proline and lysine residues of collagen proteins, taking place within the ER of the collagen-synthesising cells, leads to dimer- and trimerisation by converting proline or lysine into hydroxyproline or hydroxylysine. This reaction is catalysed by various proteins such as Prolyl 4-hydroxlases (PH4), which map to different loci within the genome. Lysine hydroxylation is performed by Lysyl-hydroxylases of which only one, dPlod, is present in the fly genome. Prolyl 4-hydroxylases are comprised of an α2β2 tetramer; the β-subunit is encoded, in Drosophila, by the pdi gene (Pdi, Protein-disulfide isomerase) (Wilmes, 2018).
This study found that Pericardin processing, i.e. multimerisation, is not blocked in mutants for pdi and dplod, and - to some extent - is inhibited in deficiencies that delete a cluster of PH4-encoding genes, which is in contrast to Collagen IV processing phenotypes seen in mutants for pdi, dplod, or PH4 genes. Possibly, redundant or residual activity of the enzymes is sufficient for Pericardin (but not for Collagen IV) maturation and cardiac assembly. Furthermore, recent results show that Pericardin deposition at the embryonic cardiac matrix is, unlike deposition of Collagen IV), not necessary for the recruitment and incorporation of additional structural ECM proteins such as Laminin, Nidogen, or Perlecan. Western blot analyses provide initial evidence that Pericardin forms intermediate dimers as well as multimeres. Like many other secreted matrix proteins, Pericardin is extensively glycosylated, indicating cross-linking of Pericardin with other ECM proteins via carbohydrate chains. Finally, previous analyses of pericardin mutant phenotypes were extended by characterising two new EMS-induced pericardin alleles, which were identified in a genetic screen for mutants displaying post-embryonic heart malformations. One of the new pericardin alleles turned out to be a protein null allele, whereas the other one represents most likely a hypomorphic allele with Pericardin being expressed but misassembled (Wilmes, 2018).
Pericardin plays a fundamental role in supporting the structural integrity of the cardiac matrix in the developing Drosophila embryo and larvae. Lack of Pericardin or inhibition of Pericardin recruitment to the cardiac matrix results in destabilisation of the larval cardiac ECM meshwork and loss of the alary muscles-pericardial nephrocytes-heart tube connection upon initiation of heart beat activity. Finally, upon ageing, this leads to luminal heart collapse and renders the heart nonfunctional. This study introduced two new EMS-induced pericardin mutants that both display the characteristic cardiac phenotypes; one of the alleles turned out to be a null allele characterised by complete absence of the protein. All pericardin alleles and transheterozygous combinations of pericardin alleles not only show ECM disintegration upon ageing but also heart collapse associated with disorientation of the cardiomyocyte sarcomeres. In wild-type animals the sarcomeres are highly organised and show a helical orientation. In pericardin mutants this orientation is lost, presumably due to the fact that the costameres lose their link -- via integrins -- to the extracellular matrix upon ECM disintegration (Wilmes, 2018).
Pericardin co-localises with type IV Collagen (Viking). However, in contrast to Viking, which assembles into the basal lamina of virtually all tissues within the animal, Pericardin is highly restricted to the cardiac ECM. ECM fibres harbouring Viking and Pericardin connect the heart tube to the alary muscles. A Pericardin::GFP fusion protein expressed from an engineered fosmid carrying an approximately 40 kb genomic region including the pericardin locus with the pericardin gene tagged with GFP, is synthesised, secreted, distributed by haemolymph flow and assembles at the cardiac matrix. Co-staining for Prc::GFP and endogenous Pericardin shows a complete overlap. However, the Prc::GFP fusion protein fails to rescue the cardiac phenotype of pericardin mutants. By contrast, Pericardin, expressed from an identical fosmid but lacking the C-terminal GFP tag, harbours rescue capability. This demonstrates the importance of the C-terminus of Pericardin for full functionality. While future studies are needed to analyse why Prc::GFP fails to rescue, it appears likely that the C-terminal tag affects accessibility of the RGD-site, which is located close to the C-terminus and which might play a role in anchoring Pericardin to the cell surface via Integrin interaction (Wilmes, 2018).
Based on distinct sequence similarities, including a central Collagen-like repeat domain with typical (Gly-X-Y)n repeats, Pericardin was classified as a type IV Collagen-like protein. In addition, it has been speculated that, analogous to collagens, Pericardin has the ability to form triple helices. However, experimental evidence for dimer-, trimer- or multimerisation of Pericardin has not been provided yet. By analysing protein samples under defined redox conditions, this study found that non-reducing conditions result in formation of high molecular weight Prc multimers. Based on the apparent molecular mass (>500 kDa), the largest multimers most likely correspond to trimeric or even higher order multimeric Prc. Thus, similar to collagens, Prc appears to form redox-dependent multimers, probably disulfide-bonded. Considering the fact that Prc is embedded into the cardiac extracellular matrix, which resides in an oxidising environment, multimeric Prc likely represent the mature, functional form, while the monomeric species presumably constitute biosynthesis intermediates. In addition to confirming Prc multimerisation, this study also found that the protein is extensively glycosylated. Application of both, N- as well as O-glycosidic bond-specific enzymes resulted in distinct mass shifts. While the apparent shift of about 2-3 kDa resulting from PNGase F incubation suggests presence of 1-2 N-linked glycans, the huge mass shift that is obvious upon O-glycosidase incubation (~65 kDa) indicates substantial O-glycosylation of the protein in the Golgi. This indication is supported by sequence analysis, which predicts only two N-glycosylation (MotifScan) but 231 O-glycosylation sites (NetOGlyc 4.0). Glycosylation represents a highly prevalent post-translational modification of ECM proteins and accounts for cell-cell and cell-matrix attachment by promoting the formation of ramified networks between the glycosylated proteins present in the matrix. Considering this, as well as the severe effects of Prc knock out, glycosylated Pericardin appears to be a core component of the ECM network present at the heart (Wilmes, 2018).
Trimerisation of type IV Collagen has been shown to depend on the enzymatic activity of Prolyl (PH4)- and Lysyl (LH)-Hydroxylases. Prolyl-Hydroxylases form a tetrameric complex, with Protein disulfide-Isomerase (Pdi) being present in the complex. Hydroxylation occurs in the Collagen producing cells in the lumen of the ER prior to secretion of Collagen IV molecules. Interestingly, the primary sequence of Pericardin contains a high number of prolines (158, 9.2% of total) as well as numerous type IV Collagen-like repeats, which indicates that Pericardin may undergo a similar biosynthesis pathway as collagens. Therefore, this study analysed whether formation of high molecular mass forms of Pericardin (dimers and trimers) is affected in Pdi, PH4 or LH mutants. Only in PH4 mutants was observed the absence of these multimers observed in Western blot analysis. The PH4 mutants that were used harbour deletions that remove six annotated Prolyl-Hydroxylases at once, the so-called 75D cluster. Single mutant lines for each of these PH4s are not available; therefore whether RNAi-mediated down-regulation of the individual PH4 genes in the cluster results in an inhibition of the formation of high molecular mass forms of Pericardin, which was not the case. This is either caused by either inefficient down-regulation of the target gene or by a redundant function of more than one of the PH4s in the 75D cluster. Pdi and LH seem to play no major role in Pericardin biosynthesis (Wilmes, 2018).
In H5 cells, which are widely used to express high amounts of recombinant protein for further biochemical characterisation, Pericardin is not expressed endogenously at detectable levels. Using a full-length FLAG-tagged version of Pericardin it was found that, after the cells were transfected with the construct, only the monomeric form of Pericardin is produced. It is concluded that H5 cells derived from the cabbage looper Trichoplusia ni lack activity of certain enzymes critical to the biosynthesis of multimeric forms of Pericardin. Interestingly, it has been noticed earlier that the expression of human Collagens is highly efficient in H5 cells, but multimerisation fails due to absence of the appropriate PH4 enzymes in the cultured cells. The current result that a yet unidentified PH, or a combination of several PHs from the 75D cluster, appears to be essential to proper formation of Pericardin multimers indicates that, like in Collagens, Pericardin multimer formation fails due to absence of the required PH in the H5 cells. However, the cell culture system was successfully used to determine the epitope recognised by the widely used EC11 antibody. EC11 recognises Pericardin in the native and the denatured state and the current experiments indicate that the epitope bound by this antibody locates to the N-terminus of Pericardin (Wilmes, 2018).
Septate junction (SJ) complex proteins act in unison to provide a paracellular barrier and maintain structural integrity. This study has identified a non-barrier role of two individual SJ proteins, Coracle (Cora) and Kune-kune (Kune). Reactive oxygen species (ROS)-p38 MAPK signaling in non-myocytic pericardial cells (PCs) is important for maintaining normal cardiac physiology in Drosophila. However, the underlying mechanisms remain unknown. This study has found that in PCs, Cora and Kune are altered in abundance in response to manipulations of ROS-p38 signaling. Genetic analyses establish Cora and Kune as key effectors of ROS-p38 signaling in PCs on proper heart function. It was further determined that Cora regulates normal Kune levels in PCs, which in turn modulates normal Kune levels in the cardiomyocytes essential for proper heart function. These results thereby reveal select SJ proteins Cora and Kune as signaling mediators of the PC-derived ROS regulation of cardiac physiology (Lim, 2019).
Cell-cell interaction is typically maintained and regulated by various multi-protein complexes such as tight junctions, adherens junctions (AJs), and gap junctions. Invertebrate septate junctions (SJs), which have functional and molecular similarity to vertebrate tight junctions (TJs), are specialized, multi-protein junctional complexes that reside between the apposed plasma membranes of adjacent epithelial cells. In Drosophila, more than 20 molecular constituents of the SJ have been identified, and characterization of these proteins reveals their canonical role in in sealing neighboring cells and restricting the free diffusion of solutes between adjacent cells, thereby providing a paracellular permeability barrier. The SJ protein complex is also involved in the coordinated changes in cell shape and rearrangement during tissue morphogenesis at a stage when the SJ structure has not yet formed or matured to become optically visible. For instance, mutations in all tested SJ genes cause defects in head involution, dorsal closure, and salivary gland elongation during early embryonic development before a clear SJ structure has been formed. Mutations in all tested SJ genes also cause cell-cell dissociation in the Drosophila embryonic heart, a tissue that seemingly lacks discernable SJs. Although most studies on the SJ proteins are focused on their canonical barrier function, it has been known that subsets of SJ proteins may have a different, non-barrier role. For instance, the SJ proteins Neurexin-IV (Nrx-IV), the Na+K+ATPase β subunit Nervana 2 (Nrv2), Coracle (Cora), and Yurt form a group with a distinct role in promoting epithelial apical-basal polarity. SJ components have also recently been found to play a role in regulating Hippo signaling to control intestinal stem cell activity and hematopoiesis. Together, these findings support the emerging notion that SJ proteins could serve important roles beyond their canonical barrier function. However, the non-barrier functions of the SJ proteins and the individual SJ proteins that could be involved remain poorly understood (Lim, 2019).
The heart is a heterogeneous organ comprising the contractile cardiomyocytes (CMs) and non-myocytes, such as the epicardial cells and endocardial cells. The non-myocytes have important signaling roles that contribute to CM development, growth, and function. The Drosophila heart is a linear tube comprising two inner rows of contractile CMs closely flanked by two outer rows of non-myocytic pericardial cells (PCs). PCs have been characterized as nephrocytes that are analogous to the mammalian podocytes that function to filter toxins and proteins from the hemolymph, the equivalent of mammalian blood. The PC nephrocytes are characterized by an intricate cell shape that includes elongated infoldings of the plasma membrane to form foot processes and labyrinthine channels. The labyrinthine channels are sealed by the slit diaphragm, which is a highly organized structure composed of similar proteins as the slit diaphragm in mammals. The slit diaphragm serves as a filtration barrier to control the inflow of certain substances into the labyrinthine channels from the hemolymph. In addition, vesicular invaginations of the plasma membrane occur along the labyrinthine channels that are indicative of endocytosis of the sequestered materials from the hemolymph. Materials endocytosed into the nephrocytes, presumably toxic molecules from the hemolymph, are targeted for either degradation in the lysosome or recycling back to the hemolymph. Moreover, the CMs and PCs are separated by a basement membrane composed of extracellular matrix (ECM), which could serve as a filtration system for hemolymph content (Lim, 2019).
On the other hand, accumulating evidence is indicating an important secretory function of PC nephrocytes. An early observation of an increased synthesis of the bactericidal enzyme lysozyme in PCs following the experimental infection of the insect Calliphora erythrocephala with bacteria provided the first indication that PCs could manufacture proteins for release into the hemolymph. More recently, Drosophila PCs have been reported to secrete factors, such as the ECM components and hemolymph proteins that could directly control neighboring CM function. In addition, PCs have been reported to produce reactive oxygen species (ROS) under normal, non-stressed conditions. ROS belong to a group of reactive chemical species produced by the incomplete reduction of molecular oxygen and are now recognized to serve an important role in the regulation of various cardiac physiological processes. Physiological ROS produced in the PCs of the Drosophila heart control the production of downstream signals such as D-p38 MAPK in PCs that then act in a paracrine manner to regulate CM function and morphology. The phenomenon is apparently conserved, as a study on the zebrafish heart reported that injury-induced H2O2 in the epicardial cells promotes the regeneration of the neighboring myocardium through the activation of ERK1/2 MAPK signaling and likely the generation of soluble factors from the epicardial cells. Together, these findings support the notion that a conserved ROS-MAPK signaling axis operates in the epi- or pericardium to influence myocardial function. However, the molecular mechanisms underlying ROS-MAPK-mediated paracrine interactions are currently unknown (Lim, 2019).
This study found that among the SJ proteins tested in adult PCs, only Cora and Kune-kune (Kune) are altered in abundance by ROS-D-p38 signaling in PCs. The results further showed that pericardial ROS-D-p38 signaling regulates CM function and structure through Cora and Kune. It was also found that Cora controls Kune amount in PCs and that pericardial Kune in turn modulates myocardial Kune expression that is essential for normal cardiac physiology. This study thereby unravels an unexpected function of the select SJ proteins Cora and Kune as physiological signaling mediators in PCs, a role that is distinct from their common primary barrier function (Lim, 2019).
On the basis of the results of this study, a model is proposed for the ROS-mediated paracrine regulation of cardiac physiology. In PCs, physiological ROS-p38 level governs Cora amount, which in turn regulates the level of Kune in the cellular surface. Peripheral Kune then directs the abundance of Kune in the CMs, which is essential for proper myocardial function and morphology. As a result, lowering of ROS-p38 signaling to sub-physiological level in PCs reduces pericardial Cora level and heightens pericardial Kune level, thereby raising Kune in CMs to a level that is detrimental to normal cardiac function. Conversely, elevating ROS-p38 signaling to supra-physiological level in PCs increases pericardial Cora quantity and diminishes pericardial Kune content, thereby suppressing Kune in the CMs to a level that perturbs normal heart function (Lim, 2019).
The findings suggest that Cora and/or Kune serve dual roles as structural elements of the SJ complex and as downstream effectors of ROS signaling. Such a dual function of Cora or Kune is unexpected but perhaps not unprecedented. The signaling role of Cora and Kune as core SJ components appears analogous to that of Arm as a core AJ component. Within the AJ, Arm mediates cell-cell adhesion and anchoring of the actin cytoskeleton. However, upon activation by Wingless, the Drosophila homolog of Wnts, Arm accumulates in the cell and serves as a key effector of Wingless signal transduction. In the case of Cora and Kune in the PCs, in response to the ROS signal, p38 is activated which then regulates the abundance and/or activity of these two individual SJ proteins. It is therefore proposed that Cora and/or Kune in the SJ have parallel functions as Arm in the AJ in that they serve as a structural component of the junctional complex and as downstream effector of signaling pathways (Lim, 2019).
The results indicate that Kune level in the PC affects Kune level in the CM; however, the underlying mechanism is unclear. One possibility is that pericardial Kune and cardiomyocyte Kune homotypically interact. In this scenario, one would predict that Kune is likely localized at the cell-cell interface. This was not observed; however, it does not necessarily rule out the homotypic interaction hypothesis. It is possible that in addition to engaging in homotypic interaction to mediate ROS signaling, other obligations of Kune may cause Kune to become more evenly distributed across the cells. For instance, Kune might be involved in the nephrocytic activity of PCs, and hence localization of Kune all over the cell surface is essential to promote the uptake of materials from the hemolymph into PCs. In the myocardium, Kune might be involved in the synchronous contraction of the CMs, a process that could be facilitated by the uniform localization of Kune across the entire CM surface. Alternatively, Kune interaction between the pericardial and cardiac cells might not involve their direct homotypic interaction but rather be mediated by the basement membrane that resides between PCs and CMs, at least in certain regions of the fly heart. In addition, an aberrant change in the pericardial nephrocyte morphology caused by loss of pericardial Kune might also alter Kune level in the CM. Last but not least, paracrine factors could be released from the PC in a Kune-controlled manner, which then influence Kune level in the CM. Regardless of whether intercellular Kune interaction occurs via direct cell-cell contact or indirect mechanisms, the results have demonstrated an interesting phenomenon by which the maintenance of normal Kune abundance in CMs by its pericardial counterpart is essential for proper adult cardiac morphology and physiology. This further raises the question as to how Kune acts in CMs to control proper cardiac performance and morphology. One possibility is that Kune regulates ion channel level and/or activity in the CM plasma membrane, such as the transient receptor potential (TRP) family of Ca2+ channels. As such, alterations in the CM Kune level could perturb intracellular Ca2+ homeostasis, thereby disrupting proper cardiac contractility and structure. These possibilities remain to be investigated in future studies (Lim, 2019).
In summary, these findings reveal that select SJ proteins can act as signaling effectors and suggest that the SJ, like the AJ, could serve to organize signaling centers. This work also provides important insights into the essential mechanisms of ROS-mediated non-myocyte-myocyte signaling interactions, a process that appears to be conserved between invertebrates and vertebrates (Lim, 2019).
The excretory system produces urine by ultrafiltration via a filtration epithelium. Podocytes are widely found as filtration epithelial cells in eucoelomates. In some animal taxa, including insects and crustaceans, nephrocytes serve to separate toxic substances from the body fluid, in addition to podocytes. Drosophila nephrocytes have been recently utilized as a model system to study podocyte function and disease. Pericardial nephrocytes, for example, develop from the cardiogenic mesoderm by the late embryo/early larval stage; they arrange into two rows of 20–25 flanking each side of the heart from the first to the sixth segment. However, functionality and cellular architecture are strikingly different between Drosophila nephrocytes and eucoelomate podocytes, and the phylogenetic relationship between these cells remains enigmatic. Using focused-ion beam-scanning electron microscopy (FIB-SEM) tomography, this study revealed three-dimensional architecture of decapod nephrocytes with unprecedented accuracy-they filled an enormous gap, which can be called "missing link," in the evolutionary diversity of podocytes and nephrocytes. Thus, it is concluded that nephrocytes are part of the spectrum of filtration epithelial diversity in animal phylogeny (Miyaki, 2020).
Peptidergic signaling regulates cardiac contractility; thus, identifying molecular switches, ligand-receptor contacts, and antagonists aids in exploring the underlying mechanisms to influence health. Myosuppressin (MS), a decapeptide, diminishes cardiac contractility and gut motility. Myosuppressin binds to G protein-coupled receptor (GPCR) proteins. Two Drosophila melanogaster myosuppressin receptors (DrmMS-Rs) exist; however, no mechanism underlying MS-R activation is reported. It was predicted that DrmMS-Rs contained molecular switches that resembled those of Rhodopsin. Additionally, it is believed DrmMS-DrmMS-R1 and DrmMS-DrmMS-R2 interactions would reflect structure-activity relationship (SAR) data. It was hypothesized agonist- and antagonist-receptor contacts would differ from one another depending on activity. Lastly, it was expected that this study would apply to other species; this hypothesis was tested in Rhodnius prolixus, the Chagas disease vector. Searching DrmMS-Rs for molecular switches led to the discovery of a unique ionic lock and a novel 3-6 lock, as well as transmission and tyrosine toggle switches. The DrmMS-DrmMS-R1 and DrmMS-DrmMS-R2 contacts suggested tissue-specific signaling existed, which was in line with SAR data. R. prolixus (Rhp)MS-R was identified and it, too, was found to contained the unique myosuppressin ionic lock and novel 3-6 lock found in DrmMS-Rs as well as transmission and tyrosine toggle switches. Further, these motifs were present in red flour beetle, common water flea, honey bee, domestic silkworm, and termite MS-Rs. RhpMS and DrmMS decreased R. prolixus cardiac contractility dose dependently with EC50 values of 140 nM and 50 nM. Based on ligand-receptor contacts, RhpMS analogs were designed that were believed to be an active core and antagonist; testing on heart confirmed these predictions. The active core docking mimicked RhpMS, however, the antagonist did not. Together, these data were consistent with the unique ionic lock, novel 3-6 lock, transmission switch, and tyrosine toggle switch being involved in mechanisms underlying TM movement and MS-R activation, and the ability of MS agonists and antagonists to influence physiology (Leander, 2015).
Peptidergic signaling plays numerous critical roles in transmitting and regulating physiological processes. Therefore, delineating the mechanisms that underlie these events is a powerful approach to identifying target molecules to influence health. An important first step in signaling is when a ligand binds to a G protein-coupled receptor protein (GPCR). Ligand-receptor binding disrupts molecular switches which cause TM movement and ultimately results in receptor activation (Leander, 2015).
Myosuppressin dramatically decreases cardiac contractility and gut motility. First isolated as a cockroach brain peptide that affects spontaneous contractions of the gut, myosuppressin was subsequently found to be distributed throughout the invertebrates. The conservation of its structure and activities, and its widespread distribution are consistent with myosuppressin playing an important role in physiology (Leander, 2015).
Myosuppressins are members of a family of peptides with an identical C-terminal RF-NH2, however, the N-terminal extension is unique and differs in length and sequence. The identical C terminus and variant N terminus are both important in binding and activating signaling pathways. FMRF-NH2, the first RF-NH2-containing peptide isolated, was identified from a neural extract applied to a clam heart preparation. This superfamily of peptides is grouped based on XRF-NH2, where myosuppressins often contain X = L. Myosuppressins are typically represented by X1DVX2HX3FLRF-NH2, where X1 = pQ, P, T, A; X2 = D, G, V; X3 = V, S (Leander, 2015).
Drosophila melanogaster myosuppressin (DrmMS; TDVDHVFLRF-NH2) is representative of its peptide family. DrmMS is pleotropic decreasing the frequency of both cardiac contractility and gut motility. The DrmMS structure-activity relationship (SAR) for its effect on cardiac contractility and gut motility is reported; the data are consistent with DrmMS having distinct signaling pathways in heart and gut. In addition, DrmMS binds to two putative GPCR proteins, DrmMS-R1 and DrmMS-R2. Apart from these facts, little is known about MS signaling. No mechanism which underlies MS receptor activation, a crucial step in signal transduction, is described in literature. And, the design and characterization of MS antagonists in a disease vector are molecularly and physiologically limited in scope in the literature (Leander, 2015).
The kissing bug, Rhodnius prolixus, is a vector of the Chagas disease, an important health problem. RhpMS, pQDIDHVFMRF-NH2, contains two substitutions compared to the MS consensus structure; V3 -> I3 and L8 -> M8. The physicochemical characteristics of myosuppressins are conserved with the residue replacements; RhpMS affects R. prolixus heart rate. The unique MS provides an opportunity to further explore a pathway that affects a crucial physiological function in a disease vector. Previously, little was known about RhpMS signaling; its receptor sequence and structure was unidentified and its SAR uncharacterized (Leander, 2015).
This study tested the prediction that MS signaling would mimic mechanisms involved in Rhodopsin activation. Molecular switch motifs were sought across DrmMS-Rs and the unique ionic lock and novel 3-6 lock, in addition to the transmission and tyrosine toggle switches, were discovered. The belief that DrmMS-DrmMS-R1 and DrmMS-DmrMS-R2 interactions would reflect the SAR data was tested, which it did. When DrmMS and its N-terminal truncation and alanyl-substituted analogs were docked to the DrmMS-Rs, the ligand contacts were distinct between receptors consistent with the SAR data. DrmMS interactions with DrmMS-R2 mirrored the cardiac contractility SAR data and DrmMS-DrmMS-R1 interactions reflected the gut motility data. Additionally, it was hypothesized that agonist- and antagonist-receptor contacts would differ from one another reflecting activity and inactivity. The docking data confirmed this prediction; agonists mirrored DrmMS interactions, yet an inactive analog failed to mimic parent peptide contacts (Leander, 2015).
Lastly, it was expected that this study would apply to other species; this hypothesis was tested in R. prolixus. RhpMS-R structure, binding pockets, ligand contacts, and SAR were tested. The R. prolixus receptor was identified, and it was found to share substantial sequence identity to DrmMS-R1 and DrmMS-R2, 56% and 51%, respectively. The predicted protein was modeled to find the receptor contained typical GPCR features. RhpMS-R contained the unique myosuppressin ionic lock and novel 3-6 lock, and the transmission and tyrosine toggle switches, which, upon ligand binding, promoted TM movement and receptor activation. Further support was obtained for the role of the unique and novel locks by identifying and modeling the red flour beetle, common water flea, honey bee, domestic hornworm, and termite MS-Rs; their structures, too, contained the myosuppressin motifs, which were likely involved in TM movement and receptor activation (Leander, 2015).
Due to the conservation of physicochemistry of the amino acids, which differed between the peptides and the high receptor sequence identity, it was predicted that RhpMS and DrmMS would be alike in activity and ligand contacts. RhpMS and DrmMS decreased R. prolixus cardiac contractility dose dependently with EC50 values of 140 nM and 50 nM, respectively. Based on ligand-receptor contacts, analogs were predicted to be an RhpMS agonist or inactive and act as an antagonist. RhpMS mimicked the full-length peptide; RhpMS applied to R. prolixus heart was inactive and blocked the effect of the parent peptide. Together, data from these studies confirmed tissue specificity in MS signaling, and supported the roles of a unique and a novel lock in MS-R activation (Leander, 2015).
This paper has described molecular switches involved in TM movement that underlies MS-R activation; no prior publication reports these motifs and mechanisms. A unique ionic lock and novel 3-6 lock held MS-Rs in an inactive state that, upon ligand binding, may lead to TM6 movement, a crucial step for receptor activation. A conserved TEFP present in MS-Rs mimicked the Rhodopsin transmission switch, CWLP. Lastly, NF(M/I)(I/L)Y in MS-Rs resembled the Rhodopsin tyrosine toggle switch motif, NPVIY (Leander, 2015).
The motifs were present and made contacts consistent with MS-R activation. Even so, the motifs of the MS-Rs were unique compared to Rhodopsin, in line with the MS peptide, receptor structures, and ligand contacts, and consistent with the DrmMS and RhpMS SAR data. The loss of contacts and networks, and weakened interactions observed in MS-Rs compared to Rhodopsin may indicate their transition from the inactive to active state occurs on a different time scale or energy level (Leander, 2015).
Myosuppressins are likely to play crucial roles in physiology; however, ligand-receptor contact data remained unpublished. DrmMS-R1 and DrmMS-R2 share high sequence identity and bind the same ligand, yet, their binding pockets differed physicochemically. The unique DrmMS-DrmMS-R1 and DrmMS-DrmMS-R2 contact data that this study reports and previous SAR data were consistent with tissue-specific signaling in heart and gut. Next, MS ligand binding and receptor activation were explored in R. prolixus. Although RhpMS-R shared high sequence identity with the DrmMS-Rs, its binding pocket was different in shape and size, and the residues which projected into it. These data were in line with the unique RhpMS SAR compared to DrmMS data, in particular, the differences in the RhpMS active core and antagonist structures for R. prolixus cardiac contractility (Leander, 2015).
Together, the data from this study describe molecular switches involved in receptor activation and ligand contacts which provide insight into how the motifs are involved in MS signaling. Additionally, a bioassay, and binding pockets, size and physicochemistry of the residues available to make contacts supported published data demonstrating tissue-specificity of myosuppressin signaling and yielded information on MS SAR and its receptor in a disease vector (Leander, 2015).
Peptides may act through G protein-coupled receptors to influence cardiovascular performance; thus, delineating mechanisms involved in signaling is a molecular-based strategy to influence health. Molecular switches, often represented by conserved motifs, maintain a receptor in an inactive state. However, once the switch is broken, the transmembrane regions move and activation occurs. The molecular switches of Drosophila melanogaster myosuppressin (MS) receptors were previously identified to include a unique ionic lock and novel 3-6 lock, as well as transmission and tyrosine toggle switches. In addition to MS, cardioactive ligands structurally related by a C-terminal RF-NH2 include sulfakinin, neuropeptide F (NPF), short NPF, and FMRF-NH2-containing peptide subfamilies. It was hypothesized receptor molecular switch motifs were conserved within a RF-NH2 subfamily and across species. Thus, RF-NH2 receptor (RFa-R) molecular switches were investigated in D. melanogaster, Tribolium castaneum, Anopheles gambiae, Rhodnius prolixus, and Bombyx mori. Adipokinetic hormone (AKH), which does not contain a RF-NH2, was also examined. The tyrosine toggle switch and ionic lock showed a higher degree of conservation within a RF-NH2 subfamily than the transmission switch and 3-7 lock. AKH receptor motifs were not representative of a RF-NH2 subfamily. The motifs and interactions of switches in the RFa-Rs were consistent with receptor activation and ligand-specific binding (Rasmussen, 2015).
Heart failure (HF) and the development of chronic kidney disease (CKD) have a direct association. Both can be cause and consequence of the other. Many factors are known, such as diabetes or hypertension, which can lead to the appearance and/or development of these two conditions. However, it is suspected that other factors, namely genetic ones, may explain the differences in the manifestation and progression of HF and CKD among patients. One candidate factor is Rph, a gene expressed in the nervous and excretory system in mammals and Drosophila, encoding a Rab small GTPase family effector protein implicated in vesicular trafficking. Rph is expressed in the Drosophila heart, and the silencing of Rph gene expression in this organ had a strong impact in the organization of fibers and functional cardiac parameters. Specifically, a significant increase was observed in diastolic and systolic diameters of the heart tube, which is a phenotype that resembles dilated cardiomyopathy in humans. Importantly, this study also showed that silencing of Rabphilin (Rph) expression exclusively in the pericardial nephrocytes, which are part of the flies' excretory system, brings about a non-cell-autonomous effect on the Drosophila cardiac system. In summary, this work demonstrates the importance of Rph in the fly cardiac system and how silencing Rph expression in nephrocytes affects the Drosophila cardiac system (Selma-Soriano, 2021).
Previous toxicity assessments of heavy metals on Drosophila are limited to investigating the survival, development rate, and climbing behaviour by oral administration while cardiac toxicity of these elements have not been investigated. This study utilized a microfluidic device to inject known dosages of zinc (Zn) or cadmium (Cd) into the larvae's hemolymph to expose their heart directly and study their heart rate and arrhythmicity. The effect of heart-specific overexpression of metal responsive transcription factor (MTF-1) on different heartbeat parameters and survival of Drosophila larvae was investigated. The heart rate of wild-type larvae decreased by 24.8% or increased by 11.9%, 15 min after injection of 40 nL of 100 mM Zn or 10 mM Cd solution, respectively. The arrhythmicity index of wild-type larvae increased by 58.2% or 76.8%, after injection of Zn or Cd, respectively. MTF-1 heart overexpression ameliorated these effects completely. Moreover, it increased larvae's survival to pupal and adulthood stages and prolonged the longevity of flies injected with Zn and Cd. The microfluidic-based cardiac toxicity assay illustrated that heart is an acute target of heavy metals toxicity, and MTF-1 overexpression in this tissue can ameliorate cardiac toxicity of Zn and Cd. The method can be used for cardiotoxicity assays with other pollutants in the future (Zabihihesari, 2022).
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Ding, M., Li, Q. F., Yin, G., Liu, J. L., Jan, X. Y., Huang, T., Li, A. C. and Zheng, L. (2022). Effects of Drosophila melanogaster regular exercise and apolipoprotein B knockdown on abnormal heart rhythm induced by a high-fat diet. PLoS One 17(6): e0262471. PubMed ID: 35657779
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Kump, A. J., Panta, M., Schwab, K. R., Inlow, M. H. and Ahmad, S. M. (2021). The Drosophila Forkhead/Fox transcription factor Jumeau mediates specific cardiac progenitor cell divisions by regulating expression of the kinesin Nebbish. Sci Rep 11(1): 3221. PubMed ID: 33547352
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Lin, C., Chang, Y. C., Cheng, Y. C., Lai, P. J., Yeh, C. H., Hsieh, W. H., Hu, K., Wu, J. T., Lee, H. H., Lo, M. T. and Ho, Y. L. (2016). Probing the fractal pattern of heartbeats in Drosophila pupae by visible optical recording system. Sci Rep 6: 31950. PubMed ID: 27535299
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