Perturb-seq uncovers pathological obstacles to direct cardiac reprogramming in vivo.

Direct induction of cardiomyocytes from fibroblasts represents a promising strategy for cardiac regeneration. However, the transdifferentiation efficiency in vivo remains low. Leveraging a Perturb-seq platform tailored to complex pathological environments, we systematically compared and ranked 140 potential barriers of in vivo cardiac reprogramming. Based on their shRNA distribution and enrichment along the single-cell RNA-seq trajectory, calreticulin (Calr) emerged as a top inhibitor. Calr knockdown greatly enhanced iCM induction efficiency in vitro, enabling synchronized calcium oscillations in iCMs, and accelerated in situ reprogramming after myocardial infarction, improving cardiac function and reducing fibrosis. Mechanistically, Calr knockdown activates calcium signaling, boosting MEF2C activity to drive reprogramming and even substitute for exogenous MEF2C. Collectively, our study reveals critical regulators hindering in situ cardiomyocyte induction in a pathological microenvironment, providing effective reprogramming factors and a strategic framework for cardiac repair and regeneration after myocardial infarction.