Wang Hao, Yang Junbo, Cai Yihong, Zhao Yang
State Key Laboratory of Natural and Biomimetic Drugs, Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, College of Future Technology, Peking University, Beijing 100871, China.
Peking-Tsinghua Center for Life Science, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China.
Protein Cell. 2024 Dec 2;15(12):906-929. doi: 10.1093/procel/pwae013.
Direct conversion of cardiac fibroblasts (CFs) to cardiomyocytes (CMs) in vivo to regenerate heart tissue is an attractive approach. After myocardial infarction (MI), heart repair proceeds with an inflammation stage initiated by monocytes infiltration of the infarct zone establishing an immune microenvironment. However, whether and how the MI microenvironment influences the reprogramming of CFs remains unclear. Here, we found that in comparison with cardiac fibroblasts (CFs) cultured in vitro, CFs that transplanted into infarct region of MI mouse models resisted to cardiac reprogramming. RNA-seq analysis revealed upregulation of interferon (IFN) response genes in transplanted CFs, and subsequent inhibition of the IFN receptors increased reprogramming efficiency in vivo. Macrophage-secreted IFN-β was identified as the dominant upstream signaling factor after MI. CFs treated with macrophage-conditioned medium containing IFN-β displayed reduced reprogramming efficiency, while macrophage depletion or blocking the IFN signaling pathway after MI increased reprogramming efficiency in vivo. Co-IP, BiFC and Cut-tag assays showed that phosphorylated STAT1 downstream of IFN signaling in CFs could interact with the reprogramming factor GATA4 and inhibit the GATA4 chromatin occupancy in cardiac genes. Furthermore, upregulation of IFN-IFNAR-p-STAT1 signaling could stimulate CFs secretion of CCL2/7/12 chemokines, subsequently recruiting IFN-β-secreting macrophages. Together, these immune cells further activate STAT1 phosphorylation, enhancing CCL2/7/12 secretion and immune cell recruitment, ultimately forming a self-reinforcing positive feedback loop between CFs and macrophages via IFN-IFNAR-p-STAT1 that inhibits cardiac reprogramming in vivo. Cumulatively, our findings uncover an intercellular self-stimulating inflammatory circuit as a microenvironmental molecular barrier of in situ cardiac reprogramming that needs to be overcome for regenerative medicine applications.
在体内将心脏成纤维细胞（CFs）直接转化为心肌细胞（CMs）以再生心脏组织是一种有吸引力的方法。心肌梗死后（MI），心脏修复通过炎症阶段开始，该阶段由单核细胞浸润梗死区域引发，从而建立免疫微环境。然而，MI微环境是否以及如何影响CFs的重编程仍不清楚。在这里，我们发现与体外培养的心脏成纤维细胞（CFs）相比，移植到MI小鼠模型梗死区域的CFs对心脏重编程具有抗性。RNA测序分析显示移植的CFs中干扰素（IFN）反应基因上调，随后抑制IFN受体可提高体内重编程效率。巨噬细胞分泌的IFN-β被确定为MI后主要的上游信号因子。用含有IFN-β的巨噬细胞条件培养基处理的CFs重编程效率降低，而MI后巨噬细胞耗竭或阻断IFN信号通路可提高体内重编程效率。免疫共沉淀、双分子荧光互补和Cut-tag分析表明，CFs中IFN信号下游的磷酸化STAT1可与重编程因子GATA4相互作用，并抑制心脏基因中GATA4的染色质占据。此外，IFN-IFNAR-p-STAT1信号的上调可刺激CFs分泌CCL2/7/12趋化因子，随后招募分泌IFN-β的巨噬细胞。这些免疫细胞一起进一步激活STAT1磷酸化，增强CCL2/7/12分泌和免疫细胞招募，最终通过IFN-IFNAR-p-STAT1在CFs和巨噬细胞之间形成一个自我强化的正反馈环，从而在体内抑制心脏重编程。累积而言，我们的研究结果揭示了一种细胞间自我刺激的炎症回路，作为原位心脏重编程的微环境分子屏障，在再生医学应用中需要克服这一障碍。
