Disruption of cTnT-Mediated Sarcomere–Mitochondrial Communication Results in Dilated Cardiomyopathy

BACKGROUND: Dilated cardiomyopathy (DCM) is substantially influenced by genetic factors. Sarcomere function is intricately associated with other organelles, particularly the reciprocal regulation between sarcomeres and mitochondria. Mitochondrial stress dysregulation is linked to DCM progression, yet mechanisms remain unclear. In this study, we investigated the effects of cTnT (cardiac troponin T) dysregulation on sarcomere-mitochondrial communication in DCM. METHODS: (c.A553G) sequence variation in iPSCs. A knock-in mouse model harboring the (p.K192E) sequence variation, equivalent to the human cTnT (p.K185E) sequence variation, was subsequently established. The pathological phenotypes were analyzed in iPSC-derived cardiomyocytes, iPSC-derived cardiac organoids, and mice. RNA sequencing, metabolite profiling, and coimmunoprecipitation mass spectrometry were used to elucidate the molecular mechanisms. RESULTS: Through whole exome sequencing, we identified a novel pathogenic variant in cTnT (p.K185E) as the causal sequence variation in a familial DCM cohort. In iPSC-derived cardiomyocytes from patients with DCM, we observed sarcomere disarray and mitochondrial fragmentation accompanied by severe mitochondrial dysfunction. The diminished interaction between cTnT (p.K185E) and 14-3-3 proteins resulted in the dissociation of 14-3-3 proteins from sarcomeric structures. The free 14-3-3 proteins aberrantly engaged in the RAS/RAF1 signaling axis, driving aberrant p44/42 kinase activation that culminated in the phosphorylation of mitochondrial fission regulators DRP1 (dynamin-related protein 1) and MFF (mitochondrial fission factor). These observations were replicated in iPSC-derived cardiac organoids. The knock-in mice bearing the orthologous cTnT sequence variation faithfully recapitulated the hallmark features of human DCM, including cardiac dysfunction, ventricular dilatation, sarcomeric disarray, and mitochondrial fragmentation. Mdivi-1, a mitochondrial fission inhibitor, alleviated DCM phenotypes in vivo. CONCLUSIONS: Our findings delineate a novel pathogenic mechanism underlying DCM, demonstrating that cTnT (p.K185E) sequence variation disrupts sarcomere-mitochondrial communication by weakening the interaction between cTnT and 14-3-3 proteins, thereby accelerating mitochondrial fragmentation through excessive activation of the 14-3-3 protein-mediated RAS/RAF1-p44/42-DRP1/MFF signaling axis. Therefore, therapeutic targeting of 14-3-3 proteins and p44/42 kinase activity may represent a promising strategy for DCM and other cardiac diseases associated with aberrant mitochondrial dynamics.