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 initiate...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-βwas identified as the dominant upstream signaling factor after MI.CFs treated with macrophage-conditioned medium containing IFN-βdisplayed 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.展开更多
基金supported by the National Key Research and Development Program of China(2018YFA0800504)the National Natural Science Foundation of China(31922020)funding provided by Plastech Pharmaceutical Technology Co.,Ltd.
文摘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-βwas identified as the dominant upstream signaling factor after MI.CFs treated with macrophage-conditioned medium containing IFN-βdisplayed 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.
