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.

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.

Histone demethylases Kdm6ba and Kdm6bb redundantly promote cardiomyocyte proliferation during zebrafish heart ventricle maturation.

Trimethylation of lysine 27 on histone 3 (H3K27me3) by the Polycomb repressive complex 2 (PRC2) contributes to localized and inherited transcriptional repression. Kdm6b (Jmjd3) is a H3K27me3 demethylase that can relieve repression-associated H3K27me3 marks, thereby supporting activation of previously silenced genes. Kdm6b is proposed to contribute to early developmental cell fate specification, cardiovascular differentiation, and/or later steps of organogenesis, including endochondral bone formation and lung development. We pursued loss-of-function studies in zebrafish to define the conserved developmental roles of Kdm6b. kdm6ba and kdm6bb homozygous deficient zebrafish are each viable and fertile. However, loss of both kdm6ba and kdm6bb shows Kdm6b proteins share redundant and pleiotropic roles in organogenesis without impacting initial cell fate specification. In the developing heart, co-expressed Kdm6b proteins promote cardiomyocyte proliferation coupled with the initial stages of cardiac trabeculation. While newly formed trabecular cardiomyocytes display a striking transient decrease in bulk cellular H3K27me3 levels, this demethylation is independent of collective Kdm6b. Our results indicate a restricted and likely locus-specific role for Kdm6b demethylases during heart ventricle maturation rather than initial cardiogenesis.

Comparative analysis of the cardiac structure and transcriptome of scallop and snail, perspectives on heart chamber evolution.

The evolution of a two-chambered heart, with an atrium and a ventricle, has improved heart function in both deuterostomes (vertebrates) and some protostomes (invertebrates). Although studies have examined the unique structure and function of these two chambers, molecular comparisons are few and limited to vertebrates. Here, we focus on the two-chambered protostome heart of the mollusks, offering data that may provide a better understanding of heart evolution. Specifically, we asked if the atrium and ventricle differ at the molecular level in the mollusk heart. To do so, we examined two very different species, the giant African land snail (Lissachatina fulica) and the relatively small, aquatic yesso scallop (Mizuhopecten yessoensis), with the assumption that if they exhibited commonality these similarities would likely reflect those across the phylum. We found that, although the hearts of these two species differed histologically, their cardiac gene function enrichments were similar, as revealed by transcriptomic analysis. Furthermore, the atrium and ventricle in each species had distinct gene function clusters, suggesting an evolutionary differentiation of cardiac chambers in mollusks. Finally, to explore the relationship between vertebrate and invertebrate two-chambered hearts, we compared our transcriptomic data with published data from the zebrafish, a well-studied vertebrate model with a two-chambered heart. Our analysis indicated a functional similarity of ventricular genes between the mollusks and the zebrafish, suggesting that the ventricle was differentiated to achieve the same functions in invertebrates and vertebrates. As the first such study on protostomes, our findings offered initial insights into how the two-chambered heart arose, including a possible understanding of its occurrence in both protostomes and deuterostomes. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00202-0.

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.

Deep learning enables automated volumetric assessments of cardiac function in zebrafish.

Although the zebrafish embryo is a powerful animal model of human heart failure, the methods routinely employed to monitor cardiac function produce rough approximations that are susceptible to bias and inaccuracies. We developed and validated a deep learning-based image-analysis platform for automated extraction of volumetric parameters of cardiac function from dynamic light-sheet fluorescence microscopy (LSFM) images of embryonic zebrafish hearts. This platform, the Cardiac Functional Imaging Network (CFIN), automatically delivers rapid and accurate assessments of cardiac performance with greater sensitivity than current approaches.This article has an associated First Person interview with the first author of the paper.

Spatial and temporal control of transgene expression in zebrafish.

Transgenic zebrafish research has provided valuable insights into gene functions and cell behaviors directing vertebrate development, physiology, and disease models. Most approaches use constitutive transgene expression and therefore do not provide control over the timing or levels of transgene induction. We describe an inducible gene expression system that uses new tissue-specific zebrafish transgenic lines that express the Gal4 transcription factor fused to the estrogen-binding domain of the human estrogen receptor. We show these Gal4-ERT driver lines confer rapid, tissue-specific induction of UAS-controlled transgenes following tamoxifen exposure in both embryos and adult fish. We demonstrate how this technology can be used to define developmental windows of gene function by spatiotemporal-controlled expression of constitutively active Notch1 in embryos. Given the array of existing UAS lines, the modular nature of this system will enable many previously intractable zebrafish experiments.