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.直接从成纤维细胞诱导心肌细胞（iCMs）是心脏再生领域极具前景的策略。然而，体内转分化效率仍处于较低水平，阻碍原位心肌细胞诱导的分子屏障尚未完全阐明。我们借助适配复杂病理环境的体内扰动测序（Perturb-seq）平台，对心肌细胞转分化的约140种潜在障碍开展了评估，最终鉴定出CALR为核心阻碍因素。体外敲低Calr可显著提升iCMs的诱导效率与功能，并在体内大幅加速iCMs生成，同时改善心脏功能、减轻心肌纤维化。进一步研究表明，Calr敲低可激活钙信号通路，增强肌细胞增强因子2C（MEF2C）的活性以促进心脏重编程，甚至可在iCMs诱导过程中替代外源性MEF2C的表达。综上，本研究通过体内扰动测序（Perturb-seq）明确了心肌细胞再生的病理障碍，为实现高效心脏修复提供了全新见解。 本数据集仓库包含心肌梗死后7天与14天两个关键时间点分离的增强型绿色荧光蛋白（eGFP）阳性细胞的单细胞扰动测序（Perturb-seq）数据。具体而言，该数据集包含经过质量控制与标准化处理的单细胞RNA测序表达矩阵，以及配套的元数据；元数据涵盖细胞簇分配注释信息与每个细胞的特异性扰动标识。