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.

中文翻译：

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巨噬细胞通过干扰素介导的细胞间自刺激回路抑制体内成纤维细胞的心脏重编程。


在体内将心脏成纤维细胞（CF）直接转化为心肌细胞（CM）以再生心脏组织是一种有吸引力的方法。心肌梗塞 (MI) 后，心脏修复会进入炎症阶段，该阶段由单核细胞浸润梗塞区引发，建立免疫微环境。然而，MI 微环境是否以及如何影响 CF 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（CF）相比，移植到 MI 小鼠模型梗塞区域的 CF 能够抵抗心脏重编程。 RNA-seq 分析揭示了移植 CF 中干扰素 (IFN) 反应基因的上调，随后对 IFN 受体的抑制增加了体内重编程效率。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 CF 表现出重编程效率降低，而 MI 后巨噬细胞耗竭或阻断 IFN 信号通路则增加了体内重编程效率。 Co-IP、BiFC 和 Cut-tag 检测表明，CF 中 IFN 信号下游的磷酸化 STAT1 可以与重编程因子 GATA4 相互作用，并抑制心脏基因中 GATA4 染色质的占据。此外，IFN-IFNAR-p-STAT1信号传导的上调可以刺激CF分泌CCL2/7/12趋化因子，随后招募分泌IFN-β的巨噬细胞。这些免疫细胞共同进一步激活 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 CF 和巨噬细胞之间形成自我强化的正反馈回路，抑制体内心脏重编程。 总的来说，我们的研究结果揭示了细胞间自刺激炎症回路作为原位心脏重编程的微环境分子屏障，再生医学应用需要克服这一屏障。