RBPMS2 Is a Myocardial-Enriched Splicing Regulator Required for Cardiac Function.

BACKGROUND: RBPs (RNA-binding proteins) perform indispensable functions in the post-transcriptional regulation of gene expression. Numerous RBPs have been implicated in cardiac development or physiology based on gene knockout studies and the identification of pathogenic RBP gene mutations in monogenic heart disorders. The discovery and characterization of additional RBPs performing indispensable functions in the heart will advance basic and translational cardiovascular research. METHODS: We performed a differential expression screen in zebrafish embryos to identify genes enriched in nkx2.5-positive cardiomyocytes or cardiopharyngeal progenitors compared to nkx2.5-negative cells from the same embryos. We investigated the myocardial-enriched gene RNA-binding protein with multiple splicing (variants) 2 [RBPMS2)] by generating and characterizing rbpms2 knockout zebrafish and human cardiomyocytes derived from RBPMS2-deficient induced pluripotent stem cells. RESULTS: We identified 1848 genes enriched in the nkx2.5-positive population. Among the most highly enriched genes, most with well-established functions in the heart, we discovered the ohnologs rbpms2a and rbpms2b, which encode an evolutionarily conserved RBP. Rbpms2 localizes selectively to cardiomyocytes during zebrafish heart development and strong cardiomyocyte expression persists into adulthood. Rbpms2-deficient embryos suffer from early cardiac dysfunction characterized by reduced ejection fraction. The functional deficit is accompanied by myofibril disarray, altered calcium handling, and differential alternative splicing events in mutant cardiomyocytes. These phenotypes are also observed in RBPMS2-deficient human cardiomyocytes, indicative of conserved molecular and cellular function. RNA-sequencing and comparative analysis of genes mis-spliced in RBPMS2-deficient zebrafish and human cardiomyocytes uncovered a conserved network of 29 ortholog pairs that require RBPMS2 for alternative splicing regulation, including RBFOX2, SLC8A1, and MYBPC3. CONCLUSIONS: Our study identifies RBPMS2 as a conserved regulator of alternative splicing, myofibrillar organization, and calcium handling in zebrafish and human cardiomyocytes.

H3K27me3-mediated silencing of structural genes is required for zebrafish heart regeneration.

Deciphering the genetic and epigenetic regulation of cardiomyocyte proliferation in organisms that are capable of robust cardiac renewal, such as zebrafish, represents an attractive inroad towards regenerating the human heart. Using integrated high-throughput transcriptional and chromatin analyses, we have identified a strong association between H3K27me3 deposition and reduced sarcomere and cytoskeletal gene expression in proliferative cardiomyocytes following cardiac injury in zebrafish. To move beyond an association, we generated an inducible transgenic strain expressing a mutant version of histone 3, H3.3K27M, that inhibits H3K27me3 catalysis in cardiomyocytes during the regenerative window. Hearts comprising H3.3K27M-expressing cardiomyocytes fail to regenerate, with wound edge cells showing heightened expression of structural genes and prominent sarcomeres. Although cell cycle re-entry was unperturbed, cytokinesis and wound invasion were significantly compromised. Collectively, our study identifies H3K27me3-mediated silencing of structural genes as requisite for zebrafish heart regeneration and suggests that repression of similar structural components in the border zone of an infarcted human heart might improve its regenerative capacity.

Coordinating cardiomyocyte interactions to direct ventricular chamber morphogenesis.

Many organs are composed of complex tissue walls that are structurally organized to optimize organ function. In particular, the ventricular myocardial wall of the heart comprises an outer compact layer that concentrically encircles the ridge-like inner trabecular layer. Although disruption in the morphogenesis of this myocardial wall can lead to various forms of congenital heart disease and non-compaction cardiomyopathies, it remains unclear how embryonic cardiomyocytes assemble to form ventricular wall layers of appropriate spatial dimensions and myocardial mass. Here we use advanced genetic and imaging tools in zebrafish to reveal an interplay between myocardial Notch and Erbb2 signalling that directs the spatial allocation of myocardial cells to their proper morphological positions in the ventricular wall. Although previous studies have shown that endocardial Notch signalling non-cell-autonomously promotes myocardial trabeculation through Erbb2 and bone morphogenetic protein (BMP) signalling, we discover that distinct ventricular cardiomyocyte clusters exhibit myocardial Notch activity that cell-autonomously inhibits Erbb2 signalling and prevents cardiomyocyte sprouting and trabeculation. Myocardial-specific Notch inactivation leads to ventricles of reduced size and increased wall thickness because of excessive trabeculae, whereas widespread myocardial Notch activity results in ventricles of increased size with a single-cell-thick wall but no trabeculae. Notably, this myocardial Notch signalling is activated non-cell-autonomously by neighbouring Erbb2-activated cardiomyocytes that sprout and form nascent trabeculae. Thus, these findings support an interactive cellular feedback process that guides the assembly of cardiomyocytes to morphologically create the ventricular myocardial wall and more broadly provide insight into the cellular dynamics of how diverse cell lineages organize to create form.

Unique developmental trajectories and genetic regulation of ventricular and outflow tract progenitors in the zebrafish second heart field.

During mammalian embryogenesis, cardiac progenitor cells constituting the second heart field (SHF) give rise to the right ventricle and primitive outflow tract (OFT). In zebrafish, previous lineage-tracing and mutant analyses suggested that SHF ventricular and OFT progenitors co-migrate to the arterial pole of the zebrafish heart tube soon after their specification in the nkx2.5+ field of anterior lateral plate mesoderm (ALPM). Using additional prospective lineage tracing, we demonstrate that while SHF ventricular progenitors migrate directly to the arterial pole, OFT progenitors become temporarily sequestered in the mesodermal cores of pharyngeal arch 2 (PA2), where they downregulate nkx2.5 expression. While there, they intermingle with precursors for PA2-derived head muscles (HMs) and hypobranchial artery endothelium, which we demonstrate are co-specified with SHF progenitors in the nkx2.5+ ALPM. Soon after their sequestration in PA2, OFT progenitors migrate to the arterial pole of the heart and differentiate into OFT lineages. Lastly, we demonstrate that SHF ventricular and OFT progenitors exhibit unique sensitivities to a mutation in fgf8a Our data highlight novel aspects of SHF, OFT and HM development in zebrafish that will inform mechanistic interpretations of cardiopharyngeal phenotypes in zebrafish models of human congenital disorders.

Complement Receptor C5aR1 Plays an Evolutionarily Conserved Role in Successful Cardiac Regeneration.

BACKGROUND: Defining conserved molecular pathways in animal models of successful cardiac regeneration could yield insight into why adult mammals have inadequate cardiac regeneration after injury. Insight into the transcriptomic landscape of early cardiac regeneration from model organisms will shed light on evolutionarily conserved pathways in successful cardiac regeneration. METHODS: Here we describe a cross-species transcriptomic screen in 3 model organisms for cardiac regeneration: axolotl, neonatal mice, and zebrafish. Apical resection to remove ≈10% to 20% of ventricular mass was carried out in these model organisms. RNA-sequencing analysis was performed on the hearts harvested at 3 time points: 12, 24, and 48 hours after resection. Sham surgery was used as internal control. RESULTS: Genes associated with inflammatory processes were found to be upregulated in a conserved manner. Complement receptors (activated by complement components, part of the innate immune system) were found to be highly upregulated in all 3 species. This approach revealed induction of gene expression for complement 5a receptor 1 in the regenerating hearts of zebrafish, axolotls, and mice. Inhibition of complement 5a receptor 1 significantly attenuated the cardiomyocyte proliferative response to heart injury in all 3 species. Furthermore, after left ventricular apical resection, the cardiomyocyte proliferative response was diminished in mice with genetic deletion of complement 5a receptor 1. CONCLUSIONS: These data reveal that activation of complement 5a receptor 1 mediates an evolutionarily conserved response that promotes cardiomyocyte proliferation after cardiac injury and identify complement pathway activation as a common pathway of successful heart regeneration.

The AP-1 transcription factor component Fosl2 potentiates the rate of myocardial differentiation from the zebrafish second heart field.

The vertebrate heart forms through successive phases of cardiomyocyte differentiation. Initially, cardiomyocytes derived from first heart field (FHF) progenitors assemble the linear heart tube. Thereafter, second heart field (SHF) progenitors differentiate into cardiomyocytes that are accreted to the poles of the heart tube over a well-defined developmental window. Although heart tube elongation deficiencies lead to life-threatening congenital heart defects, the variables controlling the initiation, rate and duration of myocardial accretion remain obscure. Here, we demonstrate that the AP-1 transcription factor, Fos-like antigen 2 (Fosl2), potentiates the rate of myocardial accretion from the zebrafish SHF. fosl2 mutants initiate accretion appropriately, but cardiomyocyte production is sluggish, resulting in a ventricular deficit coupled with an accumulation of SHF progenitors. Surprisingly, mutant embryos eventually correct the myocardial deficit by extending the accretion window. Overexpression of Fosl2 also compromises production of SHF-derived ventricular cardiomyocytes, a phenotype that is consistent with precocious depletion of the progenitor pool. Our data implicate Fosl2 in promoting the progenitor to cardiomyocyte transition and uncover the existence of regulatory mechanisms to ensure appropriate SHF-mediated cardiomyocyte contribution irrespective of embryonic stage.

Exploring the Activities of RBPMS Proteins in Myocardial Biology.

Numerous RNA-binding proteins (RBPs) are expressed in the heart, and mutations in several RBPs have been implicated in cardiovascular disease through genetic associations, animal modeling, and mechanistic studies. However, the functions of many more cardiac RBPs, and their relevance to disease states, remain to be elucidated. Recently, we have initiated studies to characterize the functions of the RBPs RBPMS and RBPMS2 in regulating myocardial biology in zebrafish and higher vertebrate species. These studies began when we learned, using an unbiased gene discovery approach, that rbpms2a and rbpms2b in zebrafish are robust markers of embryonic myocardium. This observation, which is consistent with published data, suggests that the encoded proteins are likely to be performing critical functions in regulating one or more aspects of cardiomyocyte differentiation, proliferation, survival, and/or contractility. This notion is supported by recent reports demonstrating that zebrafish embryos with disrupted Rbpms2 function exhibit gross signs of cardiac distress. Interestingly, a 20-year-old study determined that myocardial tissue from the frog, chick, and mouse also express high levels of Rbpms and/or Rbpms2, which is suggestive of evolutionary conservation of function. In this review, we will provide a historical account of how RBPMS and RBPMS2 genes were discovered, attempt to clarify some potentially confusing nomenclature, and summarize published observations that inform our ongoing studies.

Latent TGF-ß binding protein 3 identifies a second heart field in zebrafish.

The four-chambered mammalian heart develops from two fields of cardiac progenitor cells distinguished by their spatiotemporal patterns of differentiation and contributions to the definitive heart. The first heart field differentiates earlier in lateral plate mesoderm, generates the linear heart tube and ultimately gives rise to the left ventricle. The second heart field (SHF) differentiates later in pharyngeal mesoderm, elongates the heart tube, and gives rise to the outflow tract and much of the right ventricle. Because hearts in lower vertebrates contain a rudimentary outflow tract but not a right ventricle, the existence and function of SHF-like cells in these species has remained a topic of speculation. Here we provide direct evidence from Cre/Lox-mediated lineage tracing and loss-of-function studies in zebrafish, a lower vertebrate with a single ventricle, that latent TGF-ß binding protein 3 (ltbp3) transcripts mark a field of cardiac progenitor cells with defining characteristics of the anterior SHF in mammals. Specifically, ltbp3(+) cells differentiate in pharyngeal mesoderm after formation of the heart tube, elongate the heart tube at the outflow pole, and give rise to three cardiovascular lineages in the outflow tract and myocardium in the distal ventricle. In addition to expressing Ltbp3, a protein that regulates the bioavailability of TGF-ß ligands, zebrafish SHF cells co-express nkx2.5, an evolutionarily conserved marker of cardiac progenitor cells in both fields. Embryos devoid of ltbp3 lack the same cardiac structures derived from ltbp3(+) cells due to compromised progenitor proliferation. Furthermore, small-molecule inhibition of TGF-ß signalling phenocopies the ltbp3-morphant phenotype whereas expression of a constitutively active TGF-ß type I receptor rescues it. Taken together, our findings uncover a requirement for ltbp3-TGF-ß signalling during zebrafish SHF development, a process that serves to enlarge the single ventricular chamber in this species.

Notch signaling regulates cardiomyocyte proliferation during zebrafish heart regeneration.

The human heart's failure to replace ischemia-damaged myocardium with regenerated muscle contributes significantly to the worldwide morbidity and mortality associated with coronary artery disease. Remarkably, certain vertebrate species, including the zebrafish, achieve complete regeneration of amputated or injured myocardium through the proliferation of spared cardiomyocytes. Nonetheless, the genetic and cellular determinants of natural cardiac regeneration remain incompletely characterized. Here, we report that cardiac regeneration in zebrafish relies on Notch signaling. Following amputation of the zebrafish ventricular apex, Notch receptor expression becomes activated specifically in the endocardium and epicardium, but not the myocardium. Using a dominant negative approach, we discovered that suppression of Notch signaling profoundly impairs cardiac regeneration and induces scar formation at the amputation site. We ruled out defects in endocardial activation, epicardial activation, and dedifferentiation of compact myocardial cells as causative for the regenerative failure. Furthermore, coronary endothelial tubes, which we lineage traced from preexisting endothelium in wild-type hearts, formed in the wound despite the myocardial regenerative failure. Quantification of myocardial proliferation in Notch-suppressed hearts revealed a significant decrease in cycling cardiomyocytes, an observation consistent with a noncell autonomous requirement for Notch signaling in cardiomyocyte proliferation. Unexpectedly, hyperactivation of Notch signaling also suppressed cardiomyocyte proliferation and heart regeneration. Taken together, our data uncover the exquisite sensitivity of regenerative cardiomyocyte proliferation to perturbations in Notch signaling.

Zebrafish second heart field development relies on progenitor specification in anterior lateral plate mesoderm and nkx2.5 function.

Second heart field (SHF) progenitors perform essential functions during mammalian cardiogenesis. We recently identified a population of cardiac progenitor cells (CPCs) in zebrafish expressing latent TGFß-binding protein 3 (ltbp3) that exhibits several defining characteristics of the anterior SHF in mammals. However, ltbp3 transcripts are conspicuously absent in anterior lateral plate mesoderm (ALPM), where SHF progenitors are specified in higher vertebrates. Instead, ltbp3 expression initiates at the arterial pole of the developing heart tube. Because the mechanisms of cardiac development are conserved evolutionarily, we hypothesized that zebrafish SHF specification also occurs in the ALPM. To test this hypothesis, we Cre/loxP lineage traced gata4(+) and nkx2.5(+) ALPM populations predicted to contain SHF progenitors, based on evolutionary conservation of ALPM patterning. Traced cells were identified in SHF-derived distal ventricular myocardium and in three lineages in the outflow tract (OFT). We confirmed the extent of contributions made by ALPM nkx2.5(+) cells using Kaede photoconversion. Taken together, these data demonstrate that, as in higher vertebrates, zebrafish SHF progenitors are specified within the ALPM and express nkx2.5. Furthermore, we tested the hypothesis that Nkx2.5 plays a conserved and essential role during zebrafish SHF development. Embryos injected with an nkx2.5 morpholino exhibited SHF phenotypes caused by compromised progenitor cell proliferation. Co-injecting low doses of nkx2.5 and ltbp3 morpholinos revealed a genetic interaction between these factors. Taken together, our data highlight two conserved features of zebrafish SHF development, reveal a novel genetic relationship between nkx2.5 and ltbp3, and underscore the utility of this model organism for deciphering SHF biology.

Tbx1 is required for second heart field proliferation in zebrafish.

BACKGROUND: The mammalian outflow tract (OFT) and primitive right ventricle arise by accretion of newly differentiated cells to the arterial pole of the heart tube from multi-potent progenitor cells of the second heart field (SHF). While mounting evidence suggests that the genetic pathways regulating SHF development are highly conserved in zebrafish, this topic remains an active area of investigation. RESULTS: Here, we extend previous observations demonstrating that zebrafish tbx1 (van gogh, vgo) mutants show ventricular and OFT defects consistent with a conserved role in SHF-mediated cardiogenesis. Surprisingly, we reveal through double in situ analyses that tbx1 transcripts are excluded from cardiac progenitor cells and differentiated cardiomyocytes, suggesting a non-autonomous role in SHF development. Further, we find that the diminutive ventricle in vgo animals results from a 25% decrease in cardiomyocyte number that occurs subsequent to heart tube stages. Lastly, we report that although SHF progenitors are specified in the absence of Tbx1, they fail to be maintained due to compromised SHF progenitor cell proliferation. CONCLUSIONS: These studies highlight conservation of Tbx1 function in zebrafish SHF biology.

Embryonic alcohol exposure in zebrafish predisposes adults to cardiomyopathy and diastolic dysfunction.

AIMS: Fetal alcohol spectrum disorders (FASDs) impact up to 0.8% of the global population. However, cardiovascular health outcomes in adult patients, along with predictive biomarkers for cardiac risk stratification, remain unknown. Our aim was to utilize a longitudinal cohort study in an animal model to evaluate the impact of embryonic alcohol exposure (EAE) on cardiac structure, function, and transcriptional profile across the lifespan. METHODS AND RESULTS: Using zebrafish, we characterized the aftereffects of embryonic alcohol exposure (EAE) in adults binned by congenital heart defect (CHD) severity. Chamber sizes were quantified on dissected adult hearts to identify structural changes indicative of cardiomyopathy. Using echocardiography, we quantified systolic function based on ejection fraction and longitudinal strain, and diastolic function based on ventricular filling dynamics, ventricular wall movement, and estimated atrial pressures. Finally, we performed RNA sequencing on EAE ventricles and assessed how differentially expressed genes (DEGs) correlated with cardiac function. Here, we demonstrate that embryonic alcohol exposure (EAE) causes cardiomyopathy and diastolic dysfunction through persistent alterations to ventricular wall structure and gene expression. Following abnormal ventricular morphogenesis, >30% of all EAE adults developed increased atrial-to-ventricular size ratios, abnormal ventricular filling dynamics, and reduced myocardial wall relaxation during early diastole despite preserved systolic function. RNA sequencing of the EAE ventricle revealed novel and heart failure-associated genes (slc25a33, ankrd9, dusp2, dusp4, spry4, eya4, and edn1) whose expression levels were altered across the animal's lifespan or correlated with the degree of diastolic dysfunction detected in adulthood. CONCLUSIONS: Our study identifies EAE as a risk factor for adult-onset cardiomyopathy and diastolic dysfunction, regardless of CHD status, and suggests novel molecular indicators of adult EAE-induced heart disease.

The miR-143-adducin3 pathway is essential for cardiac chamber morphogenesis.

Discovering the genetic and cellular mechanisms that drive cardiac morphogenesis remains a fundamental goal, as three-dimensional architecture greatly impacts functional capacity. During development, accurately contoured chambers balloon from a primitive tube in a process characterized by regional changes in myocardial cell size and shape. How these localized changes are achieved remains elusive. Here, we show in zebrafish that microRNA-143 (miR-143) is required for chamber morphogenesis through direct repression of adducin3 (add3), which encodes an F-actin capping protein. Knockdown of miR-143 or disruption of the miR-143-add3 interaction inhibits ventricular cardiomyocyte F-actin remodeling, which blocks their normal growth and elongation and leads to ventricular collapse and decreased contractility. Using mosaic analyses, we find that miR-143 and add3 act cell-autonomously to control F-actin dynamics and cell morphology. As proper chamber emergence relies on precise control of cytoskeletal polymerization, Add3 represents an attractive target to be fine-tuned by both uniform signals, such as miR-143, and undiscovered localized signals. Together, our data uncover the miR-143-add3 genetic pathway as essential for cardiac chamber formation and function through active adjustment of myocardial cell morphology.

Generation and application of endogenously floxed alleles for cell-specific knockout in zebrafish.

The zebrafish is amenable to a variety of genetic approaches. However, lack of conditional deletion alleles limits stage- or cell-specific gene knockout. Here, we applied an existing protocol to establish a floxed allele for gata2a but failed to do so due to off-target integration and incomplete knockin. To address these problems, we applied simultaneous co-targeting with Cas12a to insert loxP sites in cis, together with transgenic counterscreening and comprehensive molecular analysis, to identify off-target insertions and confirm targeted knockins. We subsequently used our approach to establish endogenously floxed alleles of foxc1a, rasa1a, and ruvbl1, each in a single generation. We demonstrate the utility of these alleles by verifying Cre-dependent deletion, which yielded expected phenotypes in each case. Finally, we used the floxed gata2a allele to demonstrate an endothelial autonomous requirement in lymphatic valve development. Together, our results provide a framework for routine generation and application of endogenously floxed alleles in zebrafish.

Voltage-gated sodium channels are required for heart development in zebrafish.

RATIONALE: Voltage-gated sodium channels initiate action potentials in excitable tissues. Mice in which Scn5A (the predominant sodium channel gene in heart) has been knocked out die early in development with cardiac malformations by mechanisms which have yet to be determined. OBJECTIVE: Here we addressed this question by investigating the role of cardiac sodium channels in zebrafish heart development. METHODS AND RESULTS: Transcripts of the functionally-conserved Scn5a homologs scn5Laa and scn5Lab were detected in the gastrulating zebrafish embryo and subsequently in the embryonic myocardium. Antisense knockdown of either channel resulted in marked cardiac chamber dysmorphogenesis and perturbed looping. These abnormalities were associated with decreased expression of the myocardial precursor genes nkx2.5, gata4, and hand2 in anterior lateral mesoderm and significant deficits in the production of cardiomyocyte progenitors. These early defects did not appear to result from altered membrane electrophysiology, as prolonged pharmacological blockade of sodium current failed to phenocopy channel knockdown. Moreover, embryos grown in calcium channel blocker-containing medium had hearts that did not beat but developed normally. CONCLUSIONS: These findings identify a novel and possibly nonelectrogenic role for cardiac sodium channels in heart development.

Latent TGFß-binding proteins 1 and 3 protect the larval zebrafish outflow tract from aneurysmal dilatation.

Aortic root aneurysm is a common cause of morbidity and mortality in Loeys-Dietz and Marfan syndromes, where perturbations in transforming growth factor beta (TGFß) signaling play a causal or contributory role, respectively. Despite the advantages of cross-species disease modeling, animal models of aortic root aneurysm are largely restricted to genetically engineered mice. Here, we report that zebrafish devoid of the genes encoding latent-transforming growth factor beta-binding protein 1 and 3 (ltbp1 and ltbp3, respectively) develop rapid and severe aneurysm of the outflow tract (OFT), the aortic root equivalent. Similar to syndromic aneurysm tissue, the distended OFTs display evidence for paradoxical hyperactivated TGFß signaling. RNA-sequencing revealed significant overlap between the molecular signatures of disease tissue from mutant zebrafish and a mouse model of Marfan syndrome. Moreover, chemical inhibition of TGFß signaling in wild-type animals phenocopied mutants but chemical activation did not, demonstrating that TGFß signaling is protective against aneurysm. Human relevance is supported by recent studies implicating genetic lesions in LTBP3 and, potentially, LTBP1 as heritable causes of aortic root aneurysm. Ultimately, our data demonstrate that zebrafish can now be leveraged to interrogate thoracic aneurysmal disease and identify novel lead compounds through small-molecule suppressor screens. This article has an associated First Person interview with the first author of the paper.

Ruvbl2 Suppresses Cardiomyocyte Proliferation During Zebrafish Heart Development and Regeneration.

Cardiomyocyte proliferation is an important source of new myocardium during heart development and regeneration. Consequently, mutations in drivers of cardiomyocyte proliferation cause congenital heart disease, and infarcted human hearts scar because cardiomyocytes exit the cell cycle postnatally. To boost cardiomyocyte proliferation in either setting, critical regulators must be identified. Through an ENU screen in zebrafish, the liebeskummer (lik) mutant was isolated and described as having elevated cardiomyocyte numbers during embryogenesis. The lik mutation results in a three amino acid insertion into Ruvbl2, a highly conserved ATPase. Because both gain- and loss-of-function properties have been described for ruvbl2 lik , it remains unclear whether Ruvbl2 positively or negatively regulates cardiomyocyte proliferation. Here, we demonstrate that Ruvbl2 is a suppressor of cardiomyocyte proliferation during zebrafish heart development and regeneration. First, we confirmed speculation that augmented cardiomyocyte numbers in ruvbl2 lik/lik hearts arise by hyperproliferation. To characterize bona fide ruvbl2 null animals, we created a ruvbl2 locus deletion allele (ruvbl2 Δ ). Like ruvbl2 lik/lik mutants, ruvbl2 Δ/Δ and compound heterozygote ruvbl2 lik/Δ animals display ventricular hyperplasia, demonstrating that lik is a loss of function allele and that ruvbl2 represses cardiomyocyte proliferation. This activity is autonomous because constitutive myocardial overexpression of Ruvbl2 is sufficient to suppress cardiomyocyte proliferation in control hearts and rescue the hyperproliferation observed in ruvbl2 Δ/Δ mutant hearts. Lastly, heat-shock inducible overexpression of Ruvbl2 suppresses cardiomyocyte proliferation during heart regeneration and leads to scarring. Together, our data demonstrate that Ruvbl2 functions autonomously as a suppressor of cardiomyocyte proliferation during both zebrafish heart development and adult heart regeneration.

Intrinsic myocardial defects underlie an Rbfox-deficient zebrafish model of hypoplastic left heart syndrome.

Hypoplastic left heart syndrome (HLHS) is characterized by underdevelopment of left sided structures including the ventricle, valves, and aorta. Prevailing paradigm suggests that HLHS is a multigenic disease of co-occurring phenotypes. Here, we report that zebrafish lacking two orthologs of the RNA binding protein RBFOX2, a gene linked to HLHS in humans, display cardiovascular defects overlapping those in HLHS patients including ventricular, valve, and aortic deficiencies. In contrast to current models, we demonstrate that these structural deficits arise secondary to impaired pump function as these phenotypes are rescued when Rbfox is specifically expressed in the myocardium. Mechanistically, we find diminished expression and alternative splicing of sarcomere and mitochondrial components that compromise sarcomere assembly and mitochondrial respiration, respectively. Injection of human RBFOX2 mRNA restores cardiovascular development in rbfox mutant zebrafish, while HLHS-linked RBFOX2 variants fail to rescue. This work supports an emerging paradigm for HLHS pathogenesis that centers on myocardial intrinsic defects.

Innate Mechanisms of Heart Regeneration.

Heart regeneration is a remarkable process whereby regrowth of damaged cardiac tissue rehabilitates organ anatomy and function. Unfortunately, the human heart is highly resistant to regeneration, which creates a shortage of cardiomyocytes in the wake of ischemic injury, and explains, in part, why coronary artery disease remains a leading cause of death worldwide. Luckily, a detailed blueprint for achieving therapeutic heart regeneration already exists in nature because several lower vertebrate species successfully regenerate amputated or damaged heart muscle through robust cardiomyocyte proliferation. A growing number of species are being interrogated for cardiac regenerative potential, and several commonalities have emerged between those animals showing high or low innate capabilities. In this review, we provide a historical perspective on the field, discuss how regenerative potential is influenced by cardiomyocyte properties, mitogenic signals, and chromatin accessibility, and highlight unanswered questions under active investigation. Ultimately, delineating why heart regeneration occurs preferentially in some organisms, but not in others, will uncover novel therapeutic inroads for achieving cardiac restoration in humans.

Chondroitin sulfate expression is required for cardiac atrioventricular canal formation.

Defects in cardiac valvulogenesis are a common cause of congenital heart disease, and the study of this process promises to provide mechanistic insights and lead to novel therapeutics. Normal valve development involves multiple signaling pathways, and recently roles have been identified for extracellular matrix components, including glycosaminoglycans. We, therefore, explored the role of the glycosaminoglycan chondroitin sulfate during zebrafish cardiac development. Beginning at 33 hr, there is a distinct zone of chondroitin sulfate expression in the atrioventricular (AV) boundary, in the cardiac jelly between the endocardium and myocardium. This expression is both spatially and temporally restricted, and is undetectable after 48 hr. Chemical as well as genetic inhibition of chondroitin synthesis results in AV canal (AVC) defects, including loss of the atrioventricular constriction, blood regurgitation, and failure of circulation. Lack of chondroitin disrupts a marker of cell migration, results in a loss of myocardial and endothelial markers of valvulogenesis, and misregulates bone morphogenetic protein expression, supporting an early role in AVC development. In summary, we have defined a requirement for chondroitin sulfate expression in the normal patterning of the AV boundary, suggesting that this component of the cardiac jelly provides a necessary signal in this critical transition in vertebrate cardiogenesis.