Abstract
BACKGROUND: Excitation-contraction (E-C) coupling processes become disrupted in heart failure (HF), resulting in abnormal Ca(2+) homeostasis, maladaptive structural and transcriptional remodeling, and cardiac dysfunction. Junctophilin-2 (JP2) is an essential component of the E-C coupling apparatus but becomes site-specifically cleaved by calpain, leading to disruption of E-C coupling, plasmalemmal transverse tubule degeneration, abnormal Ca(2+) homeostasis, and HF. However, it is not clear whether preventing site-specific calpain cleavage of JP2 is sufficient to protect the heart against stress-induced pathological cardiac remodeling in vivo. METHODS: Calpain-resistant JP2 knock-in mice (JP2(CR)) were generated by deleting the primary JP2 calpain cleavage site. Stress-dependent JP2 cleavage was assessed through in vitro cleavage assays and in isolated cardiomyocytes treated with 1 μmol/L isoproterenol by immunofluorescence. Cardiac outcomes were assessed in wild-type and JP2(CR) mice 5 weeks after transverse aortic constriction compared with sham surgery using echocardiography, histology, and RNA-sequencing methods. E-C coupling efficiency was measured by in situ confocal microscopy. E-C coupling proteins were evaluated by calpain assays and Western blotting. The effectiveness of adeno-associated virus gene therapy with JP2(CR), JP2, or green fluorescent protein to slow HF progression was evaluated in mice with established cardiac dysfunction. RESULTS: JP2 proteolysis by calpain and in response to transverse aortic constriction and isoproterenol was blocked in JP2(CR) cardiomyocytes. JP2(CR) hearts are more resistant to pressure-overload stress, having significantly improved Ca(2+) homeostasis and transverse tubule organization with significantly attenuated cardiac dysfunction, hypertrophy, lung edema, fibrosis, and gene expression changes relative to wild-type mice. JP2(CR) preserves the integrity of calpain-sensitive E-C coupling-related proteins, including ryanodine receptor 2, Ca(V)1.2, and sarcoplasmic reticulum calcium ATPase 2a, by attenuating transverse aortic constriction-induced increases in calpain activity. Furthermore, JP2(CR) gene therapy after the onset of cardiac dysfunction was found to be effective at slowing the progression of HF and superior to wild-type JP2. CONCLUSIONS: The data presented here demonstrate that preserving JP2-dependent E-C coupling by prohibiting the site-specific calpain cleavage of JP2 offers multifaceted beneficial effects, conferring cardiac protection against stress-induced proteolysis, hypertrophy, and HF. Our data also indicate that specifically targeting the primary calpain cleavage site of JP2 by gene therapy approaches holds great therapeutic potential as a novel precision medicine for treating HF.