The Interactive Fly

Genes involved in tissue and organ development

Heart

  • Drosophila heart cell movement to the midline occurs through both cell autonomous migration and dorsal closure
  • Hand, an evolutionarily conserved bHLH transcription factor required for Drosophila cardiogenesis and hematopoiesis
  • Talin is required to position and expand the luminal domain of the Drosophila heart tube
  • Talin is required continuously for cardiomyocyte remodeling during heart growth in Drosophila
  • Enhancer modeling uncovers transcriptional signatures of individual cardiac cell states in Drosophila
  • Identification and in silico modeling of enhancers reveals new features of the cardiac differentiation network
  • Glutamatergic innervation of the heart initiates retrograde contractions in adult Drosophila melanogaster
  • Vinculin network-mediated cytoskeletal remodeling regulates contractile function in the aging heart
  • Expression patterns of cardiac aging in Drosophila
  • Cardiac deficiency of single cytochrome oxidase assembly factor scox induces p53-dependent apoptosis in a Drosophila cardiomyopathy model
  • The E3 ubiquitin ligase Nedd4/Nedd4L is directly regulated by microRNA 1 in embryonic heart development
  • Experimental evolution and heart function in Drosophila
  • Formation and function of intracardiac valve cells in the Drosophila heart

    Heart physiology
  • Probing the fractal pattern of heartbeats in Drosophila pupae by visible optical recording system
  • Regulation of heart rate in Drosophila via Fragile X mental retardation protein
  • Cardiomyocyte regulation of systemic lipid metabolism by the Apolipoprotein B-containing lipoproteins in Drosophila
  • Starvation but not locomotion enhances heart robustness in Drosophila
  • Using optogenetics to assess neuroendocrine modulation of heart rate in Drosophila melanogaster larvae
  • A new method to characterize function of the Drosophila heart by means of optical flow
  • Age-dependent electrical and morphological remodeling of the Drosophila heart caused by hERG/seizure mutations

    Pericardial cells
  • The Drosophila wing hearts originate from pericardial cells and are essential for wing maturation
  • Gia/Mthl5 is an aorta specific GPCR required for Drosophila heart tube morphology and normal pericardial cell positioning
  • Distinct subsets of Eve-positive pericardial cells stabilise cardiac outflow and contribute to Hox gene-triggered heart morphogenesis in Drosophila
  • Distinct functions of the laminin beta LN domain and collagen IV during cardiac extracellular matrix formation and stabilization of alary muscle attachments revealed by EMS mutagenesis in Drosophila
    Genes expressed in heart morphogenesis

    Drosophila heart cell movement to the midline occurs through both cell autonomous migration and dorsal closure

    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).

    Hand, an evolutionarily conserved bHLH transcription factor required for Drosophila cardiogenesis and hematopoiesis

    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).

    Talin is required to position and expand the luminal domain of the Drosophila heart tube

    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).

    Talin is required continuously for cardiomyocyte remodeling during heart growth in Drosophila

    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)

    Enhancer modeling uncovers transcriptional signatures of individual cardiac cell states in Drosophila

    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).

    Identification and in silico modeling of enhancers reveals new features of the cardiac differentiation network

    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).

    Glutamatergic innervation of the heart initiates retrograde contractions in adult Drosophila melanogaster

    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).

    Vinculin network-mediated cytoskeletal remodeling regulates contractile function in the aging heart
    The human heart is capable of functioning for decades despite minimal cell turnover or regeneration, suggesting that molecular alterations help sustain heart function with age. However, identification of compensatory remodeling events in the aging heart remains elusive. This study presents the cardiac proteomes of young and old rhesus monkeys and rats, from which it was shown that certain age-associated remodeling events within the cardiomyocyte cytoskeleton are highly conserved and beneficial rather than deleterious. Targeted transcriptomic analysis in Drosophila confirmed conservation and implicated vinculin as a unique molecular regulator of cardiac function during aging. Cardiac-restricted vinculin overexpression reinforced the cortical cytoskeleton and enhanced myofilament organization, leading to improved contractility and hemodynamic stress tolerance in healthy and myosin-deficient fly hearts. Moreover, cardiac-specific vinculin overexpression increased median life span by more than 150% in flies. A broad array of potential therapeutic targets and regulators of age-associated modifications, specifically for vinculin, are presented. These findings suggest that the heart has molecular mechanisms to sustain performance and promote longevity, which may be assisted by therapeutic intervention to ameliorate the decline of function in aging patient hearts (Kaushik, 2015).

    Expression patterns of cardiac aging in Drosophila

    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).

    Cardiac deficiency of single cytochrome oxidase assembly factor scox induces p53-dependent apoptosis in a Drosophila cardiomyopathy model

    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).

    The Drosophila wing hearts originate from pericardial cells and are essential for wing maturation

    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).

    The E3 ubiquitin ligase Nedd4/Nedd4L is directly regulated by microRNA 1 in embryonic heart development

    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).

    Experimental evolution and heart function in Drosophila

    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).

    Formation and function of intracardiac valve cells in the Drosophila heart

    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).

    Regulation of heart rate in Drosophila via Fragile X mental retardation protein

    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).

    Cardiomyocyte regulation of systemic lipid metabolism by the Apolipoprotein B-containing lipoproteins in Drosophila

    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).

    Starvation but not locomotion enhances heart robustness in Drosophila

    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).

    A new method to characterize function of the Drosophila heart by means of optical flow

    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).

    Using optogenetics to assess neuroendocrine modulation of heart rate in Drosophila melanogaster larvae

    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).

    Age-dependent electrical and morphological remodeling of the Drosophila heart caused by hERG/seizure mutations

    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).

    Probing the fractal pattern of heartbeats in Drosophila pupae by visible optical recording system

    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).

    Gia/Mthl5 is an aorta specific GPCR required for Drosophila heart tube morphology and normal pericardial cell positioning

    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).

    Distinct subsets of Eve-positive pericardial cells stabilise cardiac outflow and contribute to Hox gene-triggered heart morphogenesis in Drosophila

    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).

    Distinct functions of the laminin beta LN domain and collagen IV during cardiac extracellular matrix formation and stabilization of alary muscle attachments revealed by EMS mutagenesis in Drosophila

    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).


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