TRPC6 inhibition therapy to rescue cardiac muscle dysfunction in muscular dystrophy

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Project Summary This proposal tests the efficacy and mechanisms by which a recently developed, bioavailable, selective small- molecule inhibitor of Transient Receptor Potential Canonical 6 (TRPC6) ameliorates cardiac and skeletal myopathy in Duchenne muscular dystrophy (DMD). This research fulfills the candidate’s long-term goals of advancing novel therapies for dystrophic cardiomyopathy and applying mechanosensitive signaling assays in mice and engineered heart tissues (EHT) to study disease. DMD results from a loss of dystrophin, inducing profound progressive muscle weakness, spinal deformities, fibrosis, heart failure, and early mortality. TRPC6 is a mechanosensitive, non-voltage gated cation channel expressed in muscle cells that is hyper-activated in DMD, mediating excessive mechanical stress-induced force/Ca2+ responses, arrhythmias, cardiac dysfunction, and muscle fibrosis. During the candidate’s postdoctoral training, he led projects assessing the impact of blocking TRPC6 genetically and pharmacologically in models of cardiac fibrosis. Preliminary data in DMD show genetic or pharmacological TRPC6 inhibition prolongs lifespan in severe DMD models by 2-3 fold, ameliorating fibrosis and associated pathology, and improving heart and skeletal muscle function. The candidate first reported on the TRPC6 drug inhibitor (BI 749327) in 2019, and its clinical derivatives are now in human trials for lung and renal disease. In this proposal, the candidate addresses key questions whose answers will importantly inform future DMD translational efforts, and is organized into three aims. Aim 1 tests the efficacy of chronic TRPC6 inhibition by BI 749327 to prevent and reverse DMD skeletal and cardiac muscle dysfunction, histopathology, and TRPC6- NFAT, pro-fibrotic, and inflammatory signaling. Cell-type expression is analyzed by single-cell RNAseq to identify how subpopulations of cardiac cells that express TRPC6 (fibroblasts, vascular, and myocytes) are impacted by the treatment. Aim 2 tests the capacity of chronic TRPC6 suppression to restore mechanical activation-induced defects in force and calcium in isolated DMD mouse cardiomyocytes, and to obviate effects of membrane sealants and other mechanosensitive-activated pathways. I will further test the role of TRPC6 pathobiology in a novel human DMD EHT model of mechanosensitive activation using the same mechanical stimuli as in mouse cardiomyocytes. Aim 3 tests the efficacy of micro-dystrophin (μDys) gene therapy to treat TRPC6 pathobiology in the recently-developed D2.mdx DMD mouse model or the combination of μDys with BI 749327 provides additive benefits. The proposal uniquely combines the candidate’s prior training with expertise from his mentors. The candidate has the unique skills needed to conduct these studies, combining biomedical engineering, muscle physiology, and molecular signaling experience. He will expand into single-cell transcriptomics, stem cell derived EHT and their mechanical analysis, and DMD gene therapy. The environment at Johns Hopkins and expert mentoring team provides every opportunity for success for the candidate and project.