MFN2 alleviates mitochondrial calcium overload in sepsis-induced myopathy by modulating GRP75–VDAC1 interaction

Abstract

Sepsis-induced myopathy (SIM) is characterized by skeletal muscle dysfunction associated with mitochondrial calcium overload. The mitochondria-associated endoplasmic reticulum membrane (MAM) plays a crucial role in mediating mitochondrial calcium uptake. Although MFN2 and the IP3R–GRP75–VDAC1 complex have each been implicated in mitochondrial calcium regulation, their respective contributions and potential interplay in the pathogenesis of SIM remain poorly understood. A murine sepsis model was established by cecal ligation and puncture (CLP). Skeletal muscle function was assessed by compound muscle action potential (CMAP) recording and forelimb grip strength testing. Gastrocnemius muscle histology was evaluated by H&E and WGA staining. Tibialis anterior muscles were analyzed by immunofluorescence and transmission electron microscopy (TEM) to characterize MAM ultrastructure. Protein expression was quantified by western blotting, and MFN2 mRNA levels were measured by qPCR. Co-immunoprecipitation was performed to assess GRP75–VDAC1 protein interaction. Mitochondrial and cytoplasmic calcium levels were monitored using Rhod-2 AM and Fluo-4 AM, respectively. Sepsis induced skeletal muscle atrophy and dysfunction both in vivo and in vitro. IP3R-dependent calcium transfer at MAM was associated with pathological mitochondrial calcium overload, and pharmacological IP3R inhibition with 2-APB attenuated mitochondrial calcium uptake and ameliorated muscle atrophy. Concurrently, sepsis-induced MFN2 downregulation was associated with reduced ER-mitochondria distance, further promoting mitochondrial calcium overload. AAV-mediated MFN2 overexpression restored ER-mitochondria distance and was associated with reduced VDAC1 protein expression, attenuated GRP75–VDAC1 interaction, collectively contributing to mitigation of mitochondrial calcium overload and amelioration of skeletal muscle atrophy. MFN2 attenuates sepsis-induced mitochondrial calcium overload through two complementary mechanisms: increasing ER-mitochondria distance and modulating GRP75–VDAC1 interaction and IP3R–GRP75–VDAC1 complex assembly. These findings identify MFN2 as a potential regulator of MAM-mediated mitochondrial calcium homeostasis in SIM and suggest it may represent a therapeutic target for sepsis-induced skeletal muscle dysfunction.