TMBIM5 couples mitochondrial Ca2+ homeostasis to FUNDC1-dependent mitophagy and Drp1-driven fission during MASLD pathogenesis

Mitochondrial dysfunction plays a pivotal role in the progression of metabolic dysfunction-associated steatotic liver disease (MASLD), but the upstream regulators responsible for maintaining mitochondrial integrity remain poorly defined. This study investigated the involvement of Transmembrane Bax Inhibitor Motif-containing 5 (TMBIM5), an inner mitochondrial Ca 2+ /H + exchanger, in MASLD pathogenesis. Bioinformatics analysis identified TMBIM5 as a mitochondrial regulator in MASLD. Its functional role was explored in both mouse and cellular models. In vitro, hepatocytes with TMBIM5 overexpression and knockdown were assessed for cell viability, inflammation, and apoptosis. In vivo, hepatocyte-specific TMBIM5 knockout (TMBIM5-LKO) mice were evaluated by histological staining and molecular assays. To explore the underlying mechanisms, we performed proteomic profiling and mitochondrial functional assays. Molecular alterations were quantified by quantitative PCR (qPCR), western blot, and immunofluorescence. Mitochondrial function was assessed by measuring ROS production, ATP levels, JC-1 staining, mPTP opening, and oxygen consumption rate (OCR). Ionomycin and oleuropein were also used to further explore Ca 2+ regulation mechanisms. A high-fat diet (HFD) significantly reduced TMBIM5 expression in the mouse liver and AML12 cells. TMBIM5 deficiency exacerbated HFD-induced steatosis and liver damage, whereas in vitro its overexpression mitigated these effects. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis revealed that proteins modulated by TMBIM5 overexpression were enriched in mitochondrial pathways, suggesting its critical role in mitochondrial function and regulation. Mechanistically, TMBIM5 overexpression reduced palmitic acid (PA)-induced mitochondrial oxidative stress, preserved mitochondrial integrity, and enhanced mitochondrial function. Furthermore, TMBIM5 facilitated mitochondrial homeostasis by extruding Ca 2+ from the mitochondrial matrix, alleviating inner-membrane stress, and activating FUNDC1-mediated mitophagy, while concurrently inhibiting Drp1-dependent fission. TMBIM5 acts as a key regulator that links Ca 2+ homeostasis to mitochondrial quality control (MQC), providing novel insights into MASLD pathogenesis.