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Published on: April 24, 2021
RIPK3-driven phosphorylation of MFN2 orchestrates endoplasmic reticulum-mitochondria interaction and cardiomyocyte
1Department of Comprehensive Internal Medicine, The Affiliated Hospital of Qingdao University, Qingdao, 266021, China; Institute for Translational Medicine, The Affiliated Hospital of Qingdao University, Qingdao, 266021, China; School of Basic Medicine, Qingdao University, Qingdao, 266071, China.
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 H2O2 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 H2O2-induced cardiomyocyte necroptosis by promoting the formation of ER-mitochondrial interactions and ER-mitochondrial Ca2+ transfer, which could be regulated by RIPK3 via phosphorylating MFN2 at the Threonine 130 site. Moreover, mitochondrial Ca2+ 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 Ca2+ overload, leading to the induction of cardiac necroptosis.
Insights
Receptor-interacting protein kinase 3 (RIPK3) phosphorylates Mitofusin 2 (MFN2), promoting ER-mitochondria interactions and calcium overload, which induces cardiac necroptosis and myocardial ischemia/reperfusion injury.
Area of Science:
- Cardiovascular Biology
- Cell Death Mechanisms
- Mitochondrial Dynamics
Background:
- Necroptosis is a key cell death pathway in heart disease.
- The interplay between death-receptor and mitochondrial necroptosis pathways is poorly understood.
- Mitofusin 2 (MFN2) exacerbates myocardial injury via endoplasmic reticulum (ER)-mitochondria interactions.
Purpose of the Study:
- To investigate MFN2's role in cardiac necroptosis and ischemia/reperfusion (I/R) injury.
- To determine if MFN2 regulates ER-mitochondria interactions in cardiac necroptosis.
- To explore if the death-receptor necroptosis pathway modulates MFN2's function.
Main Methods:
- In vitro (H9c2 cells) and in vivo (mice) models of myocardial necroptosis and I/R injury.
- Immunofluorescence for ER-mitochondria interaction and RIPK3-MFN2 co-localization.
- Calcium imaging, co-immunoprecipitation, and mass spectrometry for MFN2 phosphorylation.
- Cardiac-specific MFN2 knockout mice were generated.
Main Results:
- MFN2 promotes cardiomyocyte necroptosis by enhancing ER-mitochondria interactions and calcium transfer.
- Receptor-interacting protein kinase 3 (RIPK3) phosphorylates MFN2 at Threonine 130, mediating these effects.
- Mitochondrial calcium overload activates Calpain1, inhibiting mitophagy and promoting necroptosis.
- MFN2 knockout attenuated myocardial I/R injury in vivo.
Conclusions:
- RIPK3-mediated MFN2 phosphorylation is a critical step in cardiac necroptosis.
- MFN2 promotes ER-mitochondria interaction and calcium overload, inducing cell death.
- Targeting the RIPK3-MFN2 axis may offer therapeutic strategies for heart disease.
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