In vivo Perturb-seq uncovers pathological obstacles to heart repair and regeneration by direct reprogramming

Direct induction of cardiomyocytes (iCMs) from fibroblasts represents a promising strategy for cardiac regeneration. However, the transdifferentiation efficiency in vivo remains low, and the barriers impeding in situ cardiomyocyte induction are incompletely understood. Leveraging an in vivo Perturb-seq platform tailored to complex pathological environments, we evaluated ~140 potential barriers of cardiomyocytes transdifferentiation. CALR was identified as a predominant barrier. Calr knockdown in vitro greatly enhanced iCMs induction efficiency and functionality, and dramatically accelerated iCMs induction in vivo, while concurrently improving cardiac function and mitigating fibrosis. Calr knockdown activates calcium signaling, promoting the activity of MEF2C to facilitate cardiac reprogramming or even substitute for exogenous MEF2C expression during iCMs induction. Overall, by defining the pathological obstacles to cardiomyocyte regeneration with in vivo Perturb-seq, our study offers new insights for achieving effective heart repair. This repository contains the single-cell Perturb-seq data for eGFP-positive cells isolated at two critical time points: seven days and fourteen days post-myocardial infarction. Specifically, the data comprises the quality-controlled and normalized single-cell RNA sequencing expression matrix and the associated metadata, which includes annotations for cell cluster assignment and the specific perturbation identity for each cell.