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Mitochondria as important cellular organelle play essential roles in maintaining cell structure and function. Apart from its pro-inflammatory and pro-apoptosis effects, emerging evidence indicated that TNFα in certain circumstances can promote mitochondrial integrity and function, which could be ascribed to the existence of TNFα receptor 2 (TNFR2). The present study aimed to investigate whether and how TNFR2 activation mediated the effects of TNFα on mitochondria.
Freshly isolated neonatal mouse cardiomyocytes (NMCMs) treated with shRNA targeting TNFR1 were used to study the effects of TNFR2 activation. Mitochondrial network in cells were obtained by transmission electron microscope (TEM). Quantitative real time PCR (RT-PCR) and western blotting were conducted to examine mRNA and protein expression levels associated with mitochondrial dynamic and biogenesis. Mitochondrial membrane potential (ΔΨm) and mitochondrial mass was assessed by flow cytometry using TMRM and MitoTracker Deep Red staining respectively. ATP content in cells was determined with a luciferin/luciferase-based assay. Oxygen consumption rate (OCR) of cardiomyocytes was indicated by OROBOROS instrument. Chromatin immunoprecipitation (CHIP) was used to analyze OPA1 gene transcription. Interactions between corresponding proteins were conducted through co-immunoprecipitation (Co-IP) assay. Heart failure mice model was generated by transverse aortic constriction (TAC) surgery.
TNFR2 activated NMCMs exhibited an increased mitochondrial fusion process, which were associated with increases in mitochondrial membrane potential, intracellular ATP levels and oxygen consumption capacity. Importantly, increased OPA1 protein level induced by TNFR2 activation was responsible for this enhanced mitochondrial function, as these changes can be attenuated by using siRNA targeting OPA1. In addition, we showed that both Stat3 and RelA bind to the promoter region of OPA1, and their interactions synergistically upregulated OPA1 expression at transcriptional levels. Moreover, the acetylation of Stat3, at lysine 370 or lysine 383, plays key roles in its forming a supercomplex with RelA. We also provided evidence that p300 modulated the Stat3 acetylation, which has been tested in HEK293T cells. And this p300 mediated Stat3/RelA interaction was indispensable for upregulated OPA1 expression. Finally, the beneficial effect of TNFR2 activation on enhanced OPA1 expression was validated in an in vivo TAC model where TNFR1 knock out mice showed much improved outcome compared with TNFR1 and TNFR2 double knock out mice.
TNFR2 activation confers protection for cardiomyocytes against stress via upregulating OPA1 expression for which p300 mediated acetylation of Stat3 facilitated its interaction with RelA to activate the transcriptional activity of OPA1, leading to improved mitochondrial morphology and function.