Epigenetic Regulation of Cardiomyocyte Maturation by Arginine Methyltransferase CARM1.

BACKGROUND: During the neonatal stage, the cardiomyocyte undergoes a constellation of molecular, cytoarchitectural, and functional changes known collectively as cardiomyocyte maturation to increase myocardial contractility and cardiac output. Despite the importance of cardiomyocyte maturation, the molecular mechanisms governing this critical process remain largely unexplored. METHODS: We leveraged an in vivo mosaic knockout system to characterize the role of Carm1, the founding member of protein arginine methyltransferase, in cardiomyocyte maturation. Using a battery of assays, including immunohistochemistry, immuno-electron microscopy imaging, and action potential recording, we assessed the effect of loss of Carm1 function on cardiomyocyte cell growth, myofibril expansion, T-tubule formation, and electrophysiological maturation. Genome-wide transcriptome profiling, H3R17me2a chromatin immunoprecipitation followed by sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing were used to investigate the mechanisms by which CARM1 (coactivator-associated arginine methyltransferase 1) regulates cardiomyocyte maturation. Finally, we interrogated the human syntenic region to the H3R17me2a chromatin immunoprecipitation followed by sequencing peaks for single-nucleotide polymorphisms associated with human heart diseases. RESULTS: We report that mosaic ablation of Carm1 disrupts multiple aspects of cardiomyocyte maturation cell autonomously, leading to reduced cardiomyocyte size and sarcomere thickness, severe loss and disorganization of T tubules, and compromised electrophysiological maturation. Genomics study demonstrates that CARM1 directly activates genes that underlie cardiomyocyte cytoarchitectural and electrophysiological maturation. Moreover, our study reveals significant enrichment of human heart disease-associated single-nucleotide polymorphisms in the human genomic region syntenic to the H3R17me2a chromatin immunoprecipitation followed by sequencing peaks. CONCLUSIONS: This study establishes a critical and multifaceted role for CARM1 in regulating cardiomyocyte maturation and demonstrates that deregulation of CARM1-dependent cardiomyocyte maturation gene expression may contribute to human heart diseases.

Single-cell dual-omics reveals the transcriptomic and epigenomic diversity of cardiac non-myocytes.

AIMS: The precise cellular identity and molecular features of non-myocytes (non-CMs) in a mammalian heart at a single-cell level remain elusive. Depiction of epigenetic landscape with transcriptomic signatures using the latest single-cell multi-omics has the potential to unravel the molecular programs underlying the cellular diversity of cardiac non-myocytes. Here, we characterized the molecular and cellular features of cardiac non-CM populations in the adult murine heart at the single-cell level. METHODS AND RESULTS: Through single-cell dual omics analysis, we mapped the epigenetic landscapes, characterized the transcriptomic profiles and delineated the molecular signatures of cardiac non-CMs in the adult murine heart. Distinct cis-regulatory elements and trans-acting factors for the individual major non-CM cell types (endothelial cells, fibroblast, pericytes, and immune cells) were identified. In particular, unbiased sub-clustering and functional annotation of cardiac fibroblasts (FBs) revealed extensive FB heterogeneity and identified FB sub-types with functional states related to the cellular response to stimuli, cytoskeleton organization, and immune regulation, respectively. We further explored the function of marker genes Hsd11b1 and Gfpt2 that label major FB subpopulations and determined the distribution of Hsd11b1+ and Gfp2+ FBs in murine healthy and diseased hearts. CONCLUSIONS: In summary, we characterized the non-CM cellular identity at the transcriptome and epigenome levels using single-cell omics approaches and discovered previously unrecognized cardiac fibroblast subpopulations with unique functional states.

Functional coordination of non-myocytes plays a key role in adult zebrafish heart regeneration.

Cardiac regeneration occurs primarily through proliferation of existing cardiomyocytes, but also involves complex interactions between distinct cardiac cell types including non-cardiomyocytes (non-CMs). However, the subpopulations, distinguishing molecular features, cellular functions, and intercellular interactions of non-CMs in heart regeneration remain largely unexplored. Using the LIGER algorithm, we assemble an atlas of cell states from 61,977 individual non-CM scRNA-seq profiles isolated at multiple time points during regeneration. This analysis reveals extensive non-CM cell diversity, including multiple macrophage (MC), fibroblast (FB), and endothelial cell (EC) subpopulations with unique spatiotemporal distributions, and suggests an important role for MC in inducing the activated FB and EC subpopulations. Indeed, pharmacological perturbation of MC function compromises the induction of the unique FB and EC subpopulations. Furthermore, we developed computational algorithm Topologizer to map the topological relationships and dynamic transitions between functional states. We uncover dynamic transitions between MC functional states and identify factors involved in mRNA processing and transcriptional regulation associated with the transition. Together, our single-cell transcriptomic analysis of non-CMs during cardiac regeneration provides a blueprint for interrogating the molecular and cellular basis of this process.

Derivation of stable embryonic stem cell-like, but transcriptionally heterogenous, induced pluripotent stem cells from non-permissive mouse strains.

Genetic background is known to play a role in the ability to derive pluripotent, embryonic stem cells (ESC), a trait referred to as permissiveness. Previously we demonstrated that induced pluripotent stem cells (iPSC) can be readily derived from non-permissive mouse strains by addition of serum-based media supplemented with GSK3B and MEK inhibitors, termed 2iS media, 3 days into reprogramming. Here, we describe the derivation of second type of iPSC colony from non-permissive mouse strains that can be stably maintained independently of 2iS media. The resulting cells display transcriptional heterogeneity similar to that observed in ESC from permissive genetic backgrounds derived in conventional serum containing media supplemented with leukemia inhibitor factor. However, unlike previous studies that report exclusive subpopulations, we observe both exclusive and simultaneous expression of naive and primed cell surface markers. Herein, we explore shifts in pluripotency in the presence of 2iS and characterize heterogenous subpopulations to determine their pluripotent state and role in heterogenous iPSCs derived from the non-permissive NOD/ShiLtJ strain. We conclude that heterogeneity is a naturally occurring, necessary quality of stem cells that allows for the maintenance of pluripotency. This study further demonstrates the efficacy of the 2iS reprogramming technique. It is also the first study to derive stable ESC-like stem cells from the non-permissive NOD/ShiLtJ and WSB/EiJ strains, enabling easier and broader research possibilities into pluripotency for these and similar non-permissive mouse strains and species.

Down-regulation of Beclin1 promotes direct cardiac reprogramming.

Direct reprogramming of fibroblasts to alternative cell fates by forced expression of transcription factors offers a platform to explore fundamental molecular events governing cell fate identity. The discovery and study of induced cardiomyocytes (iCMs) not only provides alternative therapeutic strategies for heart disease but also sheds lights on basic biology underlying CM fate determination. The iCM field has primarily focused on early transcriptome and epigenome repatterning, whereas little is known about how reprogramming iCMs remodel, erase, and exit the initial fibroblast lineage to acquire final cell identity. Here, we show that autophagy-related 5 (Atg5)-dependent autophagy, an evolutionarily conserved self-digestion process, was induced and required for iCM reprogramming. Unexpectedly, the autophagic factor Beclin1 (Becn1) was found to suppress iCM induction in an autophagy-independent manner. Depletion of Becn1 resulted in improved iCM induction from both murine and human fibroblasts. In a mouse genetic model, Becn1 haploinsufficiency further enhanced reprogramming factor-mediated heart function recovery and scar size reduction after myocardial infarction. Mechanistically, loss of Becn1 up-regulated Lef1 and down-regulated Wnt inhibitors, leading to activation of the canonical Wnt/ß-catenin signaling pathway. In addition, Becn1 physically interacts with other classical class III phosphatidylinositol 3-kinase (PI3K III) complex components, the knockdown of which phenocopied Becn1 depletion in cardiac reprogramming. Collectively, our study revealed an inductive role of Atg5-dependent autophagy as well as a previously unrecognized autophagy-independent inhibitory function of Becn1 in iCM reprogramming.

An Optimized Protocol for Human Direct Cardiac Reprogramming.

Direct cardiac reprogramming, the conversion of fibroblasts into cardiomyocyte-like cells (iCMs), is an attractive approach to heal the injured heart. Here we present a new approach to human cardiac reprogramming that utilizes a polycistronic three-factor reprogramming cocktail and one microRNA. Our protocol produces cardiac Troponin T positive human iCMs (hiCMs) at an efficiency of 40%-60%, approximately double that of previous protocols, within just 2 weeks. The resulting hiCMs display cardiomyocyte-like sarcomere structure, gene expression, and calcium oscillation. For complete details on the use and execution of this protocol, please refer to Zhou et al. (2019).

Single-Cell Transcriptomic Analyses of Cell Fate Transitions during Human Cardiac Reprogramming.

Direct cellular reprogramming provides a powerful platform to study cell plasticity and dissect mechanisms underlying cell fate determination. Here, we report a single-cell transcriptomic study of human cardiac (hiCM) reprogramming that utilizes an analysis pipeline incorporating current data normalization methods, multiple trajectory prediction algorithms, and a cell fate index calculation we developed to measure reprogramming progression. These analyses revealed hiCM reprogramming-specific features and a decision point at which cells either embark on reprogramming or regress toward their original fibroblast state. In combination with functional screening, we found that immune-response-associated DNA methylation is required for hiCM induction and validated several downstream targets of reprogramming factors as necessary for productive hiCM reprograming. Collectively, this single-cell transcriptomics study provides detailed datasets that reveal molecular features underlying hiCM determination and rigorous analytical pipelines for predicting cell fate conversion.

Permissiveness to form pluripotent stem cells may be an evolutionarily derived characteristic in Mus musculus.

Mus musculus is the only known species from which embryonic stem cells (ESC) can be isolated under conditions requiring only leukemia inhibitory factor (LIF). Other species are non-permissive in LIF media, and form developmentally primed epiblast stem cells (EpiSC) similar to cells derived from post-implantation, egg cylinders. To evaluate whether non-permissiveness extends to induced pluripotent stem cells (iPSC), we derived iPSC from the eight founder strains of the mouse Collaborative Cross. Two strains, NOD/ShiLtJ and the WSB/EiJ, were non-permissive, consistent with the previous classification of NOD/ShiLtJ as non-permissive to ESC derivation. We determined non-permissiveness is recessive, and that non-permissive genomes do not compliment. We overcame iPSC non-permissiveness by using GSK3B and MEK inhibitors with serum, a technique we termed 2iS reprogramming. Although used for ESC derivation, GSK3B and MEK inhibitors have not been used during iPSC reprogramming because they inhibit survival of progenitor differentiated cells. iPSC derived in 2iS are more transcriptionally similar to ESC than EpiSC, indicating that 2iS reprogramming acts to overcome genetic background constraints. Finally, of species tested for ESC or iPSC derivation, only some M. musculus strains are permissive under LIF culture conditions suggesting that this is an evolutionarily derived characteristic in the M. musculus lineage.