Translational landscape of direct cardiac reprogramming reveals a role of Ybx1 in repressing cardiac fate acquisition.

Direct reprogramming of fibroblasts into induced cardiomyocytes holds great promise for heart regeneration. Although considerable progress has been made in understanding the transcriptional and epigenetic mechanisms of iCM reprogramming, its translational regulation remains largely unexplored. Here, we characterized the translational landscape of iCM reprogramming through integrative ribosome and transcriptomic profiling, and found extensive translatome repatterning during this process. Loss of function screening for translational regulators uncovered Ybx1 as a critical barrier to iCM induction. In a mouse model of myocardial infarction, removing Ybx1 enhanced in vivo reprogramming, resulting in improved heart function and reduced scar size. Mechanistically, Ybx1 depletion de-repressed the translation of its direct targets SRF and Baf60c, both of which mediated the effect of Ybx1 depletion on iCM generation. Furthermore, removal of Ybx1 allowed single factor Tbx5-mediated iCM conversion. In summary, this study revealed a new layer of regulatory mechanism that controls cardiac reprogramming at the translational level.

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.

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.

Isoform Specific Effects of Mef2C during Direct Cardiac Reprogramming.

Direct conversion of cardiac fibroblasts into induced cardiomyocytes (iCMs) by forced expression of defined factors holds great potential for regenerative medicine by offering an alternative strategy for treatment of heart disease. Successful iCM conversion can be achieved by minimally using three transcription factors, Mef2c (M), Gata4(G), and Tbx5 (T). Despite increasing interest in iCM mechanistic studies using MGT(polycistronic construct with optimal expression of M,G and T), the reprogramming efficiency varies among different laboratories. Two main Mef2c isoforms (isoform2, Mi2 and isoform4, Mi4) are present in heart and are used separately by different labs, for iCM reprogramming. It is currently unknown if differently spliced isoform of Mef2c contributes to varied reprogramming efficiency. Here, we used Mi2 and Mi4 together with Gata4 and Tbx5 in separate vectors or polycistronic vector, to convert fibroblasts to iCMs. We found that Mi2 can induce higher reprogramming efficiency than Mi4 in MEFs. Addition of Hand2 to MGT retroviral cocktail or polycistronic Mi2-GT retroviruses further enhanced the iCM conversion. Overall, this study demonstrated the isoform specific effects of Mef2c, during iCM reprogramming, clarified some discrepancy about varied efficiency among labs and might lead to future research into the role of alternative splicing and the consequent variants in cell fate determination.