TMEM65 ameliorates post-myocardial infarction heart failure by maintaining mitochondrial calcium homeostasis via inhibiting NCLX ubiquitination

Abstract

Disruption of mitochondrial calcium homeostasis drives post-myocardial infarction (MI) heart failure by impairing excitation-contraction coupling . However, the critical upstream molecular mechanisms remain unclear, necessitating novel interventional targets. To elucidate the core mechanisms driving cardiomyocyte mitochondrial calcium imbalance in ischemic cardiomyopathy (ICM) and evaluate targeted therapeutic strategies for post-MI remodeling. Human ICM single-nucleus transcriptome data combined with high-dimensional weighted gene co-expression network analysis (hdWGCNA) were utilized to identify core genes regulating cardiomyocyte calcium homeostasis. Using an oxygen-glucose deprivation (OGD) model in rat cardiomyocytes (H9c2) and a murine MI model, myocardium-specific gene knockdown and overexpression were achieved via AAV9 vectors. The mechanisms governing mitochondrial function by the identified core molecule were elucidated through co-immunoprecipitation (Co-IP), tandem ubiquitin-binding entity (TUBE) pull-down assays, mitochondrial functional assessments, and the IonOptix single-cell contractility system. Furthermore, CellChat was applied to map pathological cell-cell communication networks within the myocardial microenvironment. Bioinformatics analysis identified the transmembrane protein 65 (TMEM65) as the core factor regulating myocardial calcium homeostasis, which was significantly downregulated in ICM and during the ultra-early phase of ischemia. In vitro and in vivo mechanistic studies demonstrated that TMEM65 physically interacts with the mitochondrial Na⁺/Ca²⁺ exchanger (NCLX) , thereby inhibiting its polyubiquitination-mediated degradation. Ischemia-induced loss of TMEM65 triggered NCLX degradation and hindered mitochondrial calcium efflux, culminating in the loss of mitochondrial membrane potential, reactive oxygen species (ROS) bursts, diminished oxygen consumption rate (OCR), and the failure of excitation-contraction coupling. Myocardium-specific knockdown of TMEM65 exacerbated mitochondrial calcium overload in MI mice, significantly worsening cardiac function and ventricular remodeling. Conversely, targeted overexpression of TMEM65 effectively preserved NCLX stability and mitochondrial calcium efflux, dramatically reducing infarct size and fibrotic area, thereby rescuing heart failure. The early and abrupt ischemia-induced downregulation of TMEM65 is a pivotal upstream event that triggers mitochondrial calcium overload and ventricular failure in cardiomyocytes. TMEM65 sustains mitochondrial calcium homeostasis and energy metabolism by preventing the ubiquitin-dependent degradation of NCLX. Targeted restoration of TMEM65 expression in cardiomyocytes can effectively reverse mitochondrial dysfunction and halt pathological remodeling, offering a highly promising novel therapeutic strategy for ischemic heart disease and heart failure.MI