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 IFN 介导的细胞间自刺激回路在体内抑制成纤维细胞的心脏重编程。


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 （MI） 后，心脏修复进行，由单核细胞浸润梗死区建立免疫微环境引发的炎症阶段。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 对心脏重编程有抵抗力。RNA-seq 分析显示移植 CF 中干扰素 （IFN） 反应基因的上调，随后对 IFN 受体的抑制提高了体内重编程效率。巨噬细胞分泌的 IFN-β 被确定为 MI 后的主要上游信号因子。用含有 IFN-β 的巨噬细胞条件培养基处理的 CFs 显示重编程效率降低，而 MI 后巨噬细胞耗竭或阻断 IFN 信号通路提高了体内重编程效率。Co-IP、BiFC 和 Cut-tag 检测显示，CFs 中 IFN 信号转导下游的磷酸化 STAT1 可与重编程因子 GATA4 相互作用，并抑制 GATA4 染色质在心脏基因中的占有率。此外，IFN-IFNAR-p-STAT1 信号的上调可刺激 CFs 分泌 CCL2/7/12 趋化因子，随后募集分泌 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 分泌和免疫细胞募集，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。 累积起来，我们的研究结果揭示了一个细胞间自刺激的炎症回路，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。