Abstract 97: Ablation of Calpain-Targeted Site in Cardiac Myosin Binding Protein C Is Cardioprotective

Abstract

Rationale: Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is essential for normal heart function. We recently demonstrated a direct correlation between cMyBP-C dephosphorylation and its degradation during ischemia-reperfusion (I-R) injury. Strikingly, cMyBP-C phosphorylation protects the heart from I-R injury. However, the mechanism of cMyBP-C-mediated cardioprotection is unknown. Objective: To determine if preventing cMyBP-C proteolysis and cleavage confers cardioprotection during I-R injury. Methods and Results: cMyBP-C is a substrate for calpains and dephosphorylation makes cMyBP-C more susceptible to proteolysis. In patients with heart failure, such proteolysis leads to the cleavage and release of the predominant N'-fragment (40 kDa) in association with increased calpain activity. MS/MS analysis identified a cardiac-specific phosphorylation motif responsible for the release of the 40 kDa fragment, 272-TSLAGAGRR-280, which we term the calpain-targeted site (CTS). Next, we utilized cardiac-specific transgenic mice expressing cMyBP-C[[Unable to Display Character: △]]CTS in which CTS motif was ablated and bred into cMyBP-C null (t/t) mice. We hypothesized that ablation of the CTS motif would confer resistance to calpain-mediated proteolysis and thus protect the heart from I-R injury. An animal that transgenically expressed the normal cardiac isoform, cMyBP-CWT, served as a control. Histological, electron microscopic and immunofluorescence analyses confirm that cMyBP-C[[Unable to Display Character: ∆]]CTS incorporates normally into the cardiac sarcomere and results in complete rescue of the cMyBP-Ct/t phenotype, compared control groups. Moreover, echocardiography and in vivo hemodynamic studies revealed that cMyBP-C[[Unable to Display Character: ∆]]CTS:t/t mice have normal cardiac function, similar to control mice. Altogether, these data suggest that cMyBP-C[[Unable to Display Character: ∆]]CTS is benign. Compared to cMyBP-CWT, cMyBP-C[[Unable to Display Character: ∆]]CTS protein is protected from calpain-mediated degradation. Finally, we determined that ablation of the CTS reduced infarct size and preserved cMyBP-C stability and cardiac function during I-R injury. Conclusion: Our data suggest that preserving cMyBP-C stability by thwarting calpain-mediated proteolysis protects cardiac structure and function from I-R injury, as a supportive therapy.