Abstract 19028: Ablation of the 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 calpain and dephosphorylation makes cMyBP-C more susceptible to proteolysis. In patients with congestive heart failure, increased calpain activity was associated with cMyBP-C degradation. During ischemia proteolysis leads to the cleavage and release of the predominant N-terminal fragment (40 kDa) in association with increased calpain activity. MS/MS analysis identified a motif in the cMyBP-C M-domain responsible for the release of the 40 kDa fragment, 272-TSLAGAGRR-280, which we term the calpain-targeted site (CTS). Next, we used cardiac-specific transgenic mice expressing an ablated CTS (cMyBP-C[[Unable to Display Character: ∆]]CTS) which were bred into cMyBP-C null (t/t) mice. We hypothesized that ablation of the CTS would confer resistance to calpain-mediated proteolysis and 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 rescue of the cMyBP-Ct/t phenotype compared to control. Echocardiography and in vivo hemodynamic studies revealed that cMyBP-C[[Unable to Display Character: ∆]]CTS 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 preserved cMyBP-C stability and cardiac function during I-R injury. Conclusions: Our data suggest that preserving cMyBP-C stability by thwarting calpain-mediated proteolysis protects the heart from I-R injury.