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 Ca 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 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) 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 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, 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 cardiomyocytes. JP2 hearts are more resistant to pressure-overload stress, having significantly improved Ca homeostasis and transverse tubule organization with significantly attenuated cardiac dysfunction, hypertrophy, lung edema, fibrosis, and gene expression changes relative to wild-type mice. JP2 preserves the integrity of calpain-sensitive E-C coupling-related proteins, including ryanodine receptor 2, Ca1.2, and sarcoplasmic reticulum calcium ATPase 2a, by attenuating transverse aortic constriction-induced increases in calpain activity. Furthermore, JP2 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.