Principle Investigator Peking University Peking University, China (People's Republic)
Abstract: Direct induction of cardiomyocytes (iCMs) from cardiac fibroblasts after myocardial infarction is a promising strategy for heart repair and regeneration. However, compared to in vitro settings, in vivo transdifferentiation remains significantly less efficient, with key barriers—particularly microenvironmental cues—remaining incompletely understood. Here, we present an in vivo Perturb-seq system to systematically identify potential barriers to cardiomyocyte induction. Using 143 hub-centered genes from the in vivo cardiomyocyte induction gene regulatory network as candidates, we investigated their roles in cardiac reprogramming. Cardiac myofibroblasts were transduced with shRNAs targeting these candidate genes, along with reprogramming factors, and transplanted into the infarcted heart to evaluate their effects on in vivo reprogramming. Our analysis revealed seven transcriptionally distinct fibroblast subpopulations, including Thbs4+ fibroblasts predisposed to fibrosis and Tnnt2+ iCMs indicative of successful transdifferentiation. Interestingly, we observed a population of Sca1+ quiescent fibroblasts emerging during late-stage reprogramming, which suggests that many fibroblasts evade excessive activation but fail to fully transition into cardiomyocytes. The fidelity of our Perturb-seq approach was confirmed by identifying known reprogramming barriers, such as IFNAR2. Among newly identified candidates, Calreticulin (CALR) emerged as a critical barrier. CALR knockdown significantly enhanced fibroblast-to-cardiomyocyte transdifferentiation both in vivo and in vitro. Mechanistically, CALR knockdown elevated intracellular free calcium levels, which activated MEF2C, a key transcription factor for cardiomyocyte development. Moreover, treatment with the L-type Ca2+ channel agonist FPL64176 further activated MEF2C and promoted iCM generation, highlighting the role of calcium signaling in overcoming reprogramming barriers. Collectively, our study reveals the pivotal role of CALR and calcium signaling in in vivo cardiac reprogramming and provides a systematic framework for identifying barriers to heart repair and regeneration via direct reprogramming approaches.
