RIPK3-driven phosphorylation of MFN2 orchestrates endoplasmic reticulum-mitochondria interaction and cardiomyocyte stress responses.

BACKGROUND: Recent studies have demonstrated that necroptosis is one of the main forms of cardiomyocyte death in heart diseases. However, the crosstalk between the death-receptor necroptosis pathway and the mitochondrial necroptosis pathway remains largely unknown. It has been reported that Mitofusin 2 (MFN2) can promote myocardial injury by inducing Endoplasmic Reticulum (ER)-mitochondria interaction. The purpose of this study was to investigate whether MFN2 promotes cardiac necroptosis and myocardial ischemia/reperfusion (I/R) injury by regulating ER-mitochondrial interactions, and whether this function of MFN2 can be regulated by the death-receptor necroptosis pathway. METHODS: Myocardial necroptosis was induced by H(2)O(2) in H9c2 cardiomyocytes in vitro and through left anterior descending (LAD) ligation and subsequent reperfusion in C57/BL6 mice in vivo. ER-mitochondria interaction was detected by immunofluorescence. Calcium levels were analyzed by Rhod-AM staining. The interaction between MFN2 and Receptor-interacting protein kinase 3 (RIPK3) was explored by co-immunoprecipitation and immunofluorescence. The phosphorylation site of MFN2 was examined and measured via mass spectrometry analysis. Additionally, a customized MFN2 phosphorylation-specific antibody was used to detect the role of the Threonine 130 site of MFN2 in myocardial necroptosis. In vivo, MFN2 cardiac-specific knockout mice were constructed to further explore the effect of MFN2 on myocardial I/R injury and necroptosis. RESULTS: Our results showed that MFN2 participated in H(2)O(2)-induced cardiomyocyte necroptosis by promoting the formation of ER-mitochondrial interactions and ER-mitochondrial Ca(2+) transfer, which could be regulated by RIPK3 via phosphorylating MFN2 at the Threonine 130 site. Moreover, mitochondrial Ca(2+) overload induced mPTP opening and subsequent activation of Calpain1, resulting in the inhibition of mitophagy initiation. Both of these pathways could promote cardiac necroptosis. Furthermore, our results revealed that cardiac-specific knockout of MFN2 could attenuate myocardial I/R injury. CONCLUSION: Our findings reveal that RIPK3 can mediate MFN2 phosphorylation to promote ER-mitochondria interaction and mitochondrial Ca(2+) overload, leading to the induction of cardiac necroptosis.