The size, shape, and function of the heart depend upon the production of an appropriate number of properly differentiated cardiomyocytes. This process initiates with the specification of a pool of myocardial progenitor cells and continues with their efficient progression through multiple steps of myocardial differentiation. Prior studies have shown that the bHLH transcription factor Hand2 facilitates cardiomyocyte production, yet the precise functions of Hand2 in this context are not well understood. Our recent studies suggest that hand2 plays multiple distinct roles during myocardial specification and myocardial differentiation in zebrafish. During cardiac specification in wild-type embryos, most of the nkx2.5-expressing progenitor pool is assigned a myocardial fate, while a minority of the nkx2.5-expressing cells instead contribute to other mesodermal derivatives such as pharyngeal vasculature and cranial muscle; these pharyngeal mesodermal progenitor cells express both nkx2.5 and tcf21. In hand2 mutants, a normal number of nkx2.5+ progenitor cells form, but an abnormally large proportion of these cells also exhibit tcf21 expression. Conversely, overexpression of hand2 leads to a substantial reduction in the number of nkx2.5+;tcf21+ cells. These data suggest that hand2 promotes the specification of myocardial progenitor cells at the expense of pharyngeal mesodermal progenitors. Furthermore, the small number of cells that do go on to form cardiomyocytes in hand2 mutants also display striking defects in the kinetics of their differentiation. Through transcriptomic and in situ analyses of differential gene expression, we find that hand2 mutants exhibit delayed onset of myocardial gene expression, altered dynamics of the myocardial differentiation gene expression program, and a generally reduced pace of cardiomyocyte accumulation. Together, our data suggest that hand2 controls cardiomyocyte production through at least two distinct roles: first by balancing myocardial and pharyngeal mesodermal fate assignments, and later by insuring timely and effective cardiomyocyte differentiation.

In mammals, cardiomyocytes (CM) and cardiac fibroblasts (CF) undergo coordinated maturation after birth. Recent studies show that FB-CM interactions are essential for heart maturation after birth. Here, we investigate the role of CF-expressed GDF10 in promoting postnatal CM maturation. By in situ hybridization in neonatal wildtype hearts, Gdf10 is expressed specifically in CF, with its highest expression at postnatal day (P)7, coincident with onset of CM cell cycle arrest and transition to hypertrophic growth. In neonatal rat ventricular myocyte (NRVM) cultures, GDF10 treatment promotes CM maturation indicated by increased binucleation and cell size. Additionally, GDF10 treatment leads to an increase in expression of mature sarcomeric protein isoforms Myh6 and TnnI3, while expression of fetal isoforms Myh7 and ssTNI is decreased. Interestingly, GDF10 does not affect CF proliferation in vitro, supporting a paracrine function for Gdf10. RNAseq studies of GDF10-treated NRVM show an increase in gene expression related to myocardial maturation. Interestingly, RNA seq studies also showed altered expression of potassium channel genes, Kcnj2 and Kcnh2, and sodium channel genes, Scn3b and Scn4b, in response to GDF10 treatment. Together these studies demonstrate that Gdf10 promotes CM maturation and suggests a role for Gdf10 in the electrical maturation of the heart. To assess the in vivo functions of Gdf10 in postnatal heart maturation, Gdf10-null mice were obtained. Gdf10-null mice are viable to adulthood with minimal gross anatomical differences to their littermate controls. However, more careful examination revealed Gdf10-null hearts experience a delay in myocardial maturation indicated by a decrease in CM binucleation and increase in mitotic activity by P7. Further, gene expression studies show a delay in mature sarcomeric protein isoform Myh6 and TnnI3 gene expression at P7 and consistent changes in Scn3b, Scn4b, Kcnj2 and Kcnh2 expression observed in NRVM cultures. These indicators of CM maturation are dependent on Gdf10 in the postnatal heart up to P10 but are normalized relative to controls at P30. Together, these results implicate Gdf10 as a novel crosstalk mediator between neonatal cardiomyocytes and fibroblasts, required for cardiomyocyte maturation steps including binucleation, hypertrophy, mature sarcomeric isoform switch in the postnatal period.

The Hippo-Yap pathway is a highly conserved signaling cascade across species, and Lats1/2 is a core kinase of this pathway. Activated Lats1/2 phosphorylates transcriptional regulators Yap and Wwtr1 thereby precluding them from the nucleus and inhibiting cell cycle activity. While the regulatory role of Lats1/2 in cardiomyocytes (CMs) is well-studied, Lats1/2 comprehensive function(s) during CM proliferation remains relatively understudied. Furthermore, a major knowledge gap is in understanding the mechanisms that modulate the cytoarchitecture remodeling of a rigid cell like CM to facilitate cytokinesis. Prior literature reported that Lats1/2 can directly phosphorylate Shroom3 (SHRM3), a cytoskeletal actin-binding protein that is highly expressed in CMs. Here, we investigate how Lats1/2 kinase activity affects SHRM3 binding dynamics and the role of this protein in mammalian CM proliferation. We overexpressed the SHRM3 protein in commonly used HEK293 cells and assessed its interactions with actin using immunoprecipitation. Our in-vitro mechanistic study showed that inhibition of Lats1/2 phosphorylation (activated proliferation) alters the binding affinity of SHRM3 to actin. Accordingly, we developed a novel genetic mouse model to delete SHRM3 specifically in CMs (SHRM3fl/fl;Myh6MCM, SHRM3cKO) to investigate the role of this protein in CM proliferation. We knocked out SHRM3 in CMs at birth in neonatal mice using tamoxifen injections and tracked DNA synthesis by injecting EdU, a thymidine analog, to investigate CM cell cycle activity changes. Interestingly, SHRM3cKO mice showed significantly elevated EdU incorporation in CM nuclei compared to the controls, suggesting that SHRM3 deletion in CMs promotes cell cycle activity. Furthermore, our echocardiography results showed a significant reduction in ejection fraction and fractional shortening and elevated left ventricular inner diameter both in systole and diastole in SHRM3cKO mice compared to controls. Therefore, our findings suggest that SHRM3 deletion in CMs promotes their cell cycle activity parallel with reduced cardiac contractility. Therefore, SHRM3 may provide the cytoarchitectural alterations required for CM cell cycle activity and proliferation.

Sex disparities exist in cardiac anatomy and physiology and the prevalence of types of heart disease. These differences are often attributed to sex hormones. We demonstrated that in addition to sex hormones, genes on the X-chromosome are both necessary and sufficient to regulate cardiac sex-chromosome-derived protein differences (CSCDPs). Consecutive genetic screens in the Four Core Genotype, Turners syndrome, and Klinefelter syndrome mouse models identified the DEAD-Box Helicase 3 X-Linked (Ddx3x) as a candidate for regulating CSCDPs. Consistently, mutations in DDX3X have been shown to cause congenital heart disease in females predominantly. We have generated a novel Ddx3x cardiomyocyte conditional allele in mice. Preliminary data demonstrate that Ddx3x is essential in the cardiomyocyte lineage for embryonic cardiac viability, with females but not males dying before E11.5. Multiple functions have been proposed for DDX3X in RNA biology. As it has been shown that DDX3X ancillary proteins provide DDX3X substrate specificity, we have developed a novel Ddx3x-3XHA allele and determined the composition of the cardiomyocyte DDX3X interactome in males and females. Our results suggest that DDX3X is required for cardiomyocyte mRNA-dependent translation. To identify the DDX3X cardiac target mRNAs and region of binding, we performed Enhanced Crosslinking and Immunoprecipitation (eCLIP). As our results have demonstrated that DDX3X functions in mRNA translation, in parallel, we have used quantitative proteomic analyses and determined those proteins misregulated in DDX3X null female cardiomyocytes. By aligning our eCLIP and proteomic data sets, we have identified the potential cardiac targets of DDX3X. We have gone on to validate a subset of these in tissue culture. Collectively, our results imply that DDX3X regulates translation initiation within the female but not the male mammalian heart at E10.5. Our future studies will focus on determining the translationally dependent targets of DDX3X in the heart, thus providing insight into the basis for cardiac sex differences and potential clinical relevance for CHD patients.

The cardiovascular system comprises diverse cell lineages that emerge from progenitor cells in the anterior lateral plate mesoderm (ALPM). In zebrafish, nkx2.5 expression initiates during early somitogenesis in the bilateral ALPM that becomes segregated into two main populations: a cardiac fraction of first (FHF) and second heart field (SHF) progenitors that migrates to the midline and gives rise to ventricular myocardium, and a pharyngeal fraction that remains lateral and becomes sequestered in the cores of the pharyngeal arches (PAs). Progenitors in PA2 give rise to three lineages in the outflow tract and craniofacial muscles, while those in PAs3-6 become pharyngeal arch artery (PAA) endothelium. The mechanisms underlying these cell fate decisions remain unclear. By integrating single cell RNA sequencing (scRNAseq) with multiplexed error-robust fluorescence in situ hybridization (MERFISH), we demonstrate that nkx2.5+ progenitors in the ALPM are transcriptionally heterogeneous and anatomically regionalized prior to their physical separation and differentiation into distinct cardiovascular structures. Specifically, scRNAseq of 14 somite-stage nkx2.5:ZsYellow+ progenitors revealed six transcriptionally unique populations including a cardiomyocyte cluster (FHF-derived) and a lateral plate mesoderm (LPM) population that subclustered into three groups; LPM1-3. MERFISH spatial transcriptomics of ~150 marker genes from our scRNAseq dataset and the literature detected cell populations that closely mirrored but futher subclustered those identified by scRNAseq. Along the anterior-posterior axis, the tip regions were organized into cell groups while the middle was split into monolayers with tbx1+ pharyngeal progenitors occupying the dorsal tier and myl7+ FHF cardiomyocytes residing in the ventral tier. SHF progenitors localized medial to the FHF domain. LPM-1 corresponded to the anterior region and the middle pharyngeal tier, LPM-3 to the posterior-most region, and LPM-2 to larger medial- and lateral-most cells. Targeted photoconversion of nkx2.5:Kaede revealed that LPM-1 and LPM-3 seed the heart/PA2 and PAs3-6 respectively, while LPM-2 gives rise to pericardium. Overall, our data provide a high-resolution fate map of the ALPM and demonstrate that progenitor partitioning precedes lineage differentiation.

Cardiomyocyte maturation is a complex process involving major functional and structural reorganization during the progression from fetal to postnatal to adult cell states. In striated muscle cells, the sarco(endo)plasmic reticulum (SR/ER) is a highly specialized organelle critical for excitation-contraction coupling. During development, the SR transitions from an irregular reticulated network into a highly ordered structure, positioned in close proximity to the t-tubules, mitochondria, and sarcomere. However, how this transition occurs remains poorly understood. Here, we investigated the role of ER membrane-shaping proteins in the formation and maturation of the cardiomyocyte SR. First, we interrogated multi-omic datasets (bulk/single cell transcriptome & global proteome) of the embryonic and postnatal mouse heart to establish an atlas of SR/ER signatures across development. Using integrative analyses, we characterized the developmental trajectory of this subcellular compartment and identified the membrane-shaping protein, receptor expression-enhancing protein 5 (REEP5), as a putative maturation signal. Using high-resolution confocal and dSTORM imaging, we demonstrated that REEP5 and other ER-shaping proteins (atlastins, reticulons) show a striated or junctional SR (jSR) organization by postnatal day (P) 8 in the mouse heart (Pearson’s coefficient 0.28±0.05 P6 vs. 0.56±0.06 P8, P<0.05). In vitro experiments in neonatal mouse cardiomyocytes demonstrated that REEP5 organization at the jSR precedes RYR2 (63.0% [REEP5] vs. 21.6% [RYR2]; cells with jSR staining at day 4 in culture). Mechanistically, localization of REEP5 at the jSR required an intact actin cytoskeleton scaffold, but was independent of the microtubule network. Finally, we assessed human pluripotent stem cell-derived cardiomyocytes up to 40 days post-differentiation. We showed increased colocalization of REEP5 and α-actinin with extended time-in-culture, with ongoing work aiming to further elucidate the role of REEP5 in cardiomyocyte maturation, using lentiviral-mediated shRNA-knockdown and overexpression approaches. Collectively, these findings reveal new developmental mechanisms in cardiomyocyte organelle maturation which may also have applications for cardiac regenerative medicine therapies.