Preventing Site-Specific Calpain Proteolysis of Junctophilin-2 Protects Against Stress-Induced Excitation-Contraction Uncoupling and Heart Failure Development

Background: Excitation-contraction (E-C) coupling processes become disrupted in Heart Failure (HF), resulting in abnormal CA²⁺ 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 CA²⁺ 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 CA²⁺ 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_V1.2, 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.