RNA-sequencing analysis of ZsYellow-negative cells and ZsYellow-positive cardiopharyngeal progenitors and cardiomyocytes purified from the same 14-16 somites stage Tg(nkx2.5:ZsYellow) zebrafish embryos

The identification of novel cardiomyocyte-intrinsic factors that support ventricular function will expand the number of candidate genes and therapeutic options for heart failure, a leading cause of death worldwide. Here, we demonstrate that a conserved RNA-binding protein RBPMS2 is required for ventricular function in zebrafish and for myofibril organization and the regulation of intracellular calcium dynamics in zebrafish and human cardiomyocytes. A differential expression screen uncovered co-expression of rbpms2a and rbpms2b in zebrafish cardiomyocytes. Double knockout embryos suffer from compromised ventricular filling during the relaxation phase of the cardiac cycle, which significantly reduces cardiac output. Evaluating rbpms2-null embryos with splicing-sensitive differential expression analysis, quantitative PCR, and in situ hybridization revealed differential alternative splicing of cardiomyopathy genes including myosin binding protein C3 (mybpc3) and phospholamban (pln). Cardiomyocytes in double mutant ventricles and those derived from RBPMS2-null human induced pluripotent stem cells exhibit myofibril disarray and calcium handling abnormalities. Taken together, our data suggest that RBPMS2 performs a conserved role in regulating alternative splicing in cardiomyocytes, which is required for sarcomere organization, optimal calcium handling, and cardiac function. Tg(nkx2.5:ZsYellow) zebrafish embryos at the 14-16 somites stage were dissociated to single cells and their ZsYellow-positive cardiopharyngeal progenitors and cardiomyocytes were purified by fluorescence activated cell sorting. ZsYellow-negative cells from the same embryos were collected in parallel. RNA-sequencing analysis was performed on both populations (two replicates each) and differentially expressed genes were identified.