Myosin-binding protein C slows cardiac myofibril relaxation kinetics

Abstract: Mutations in cardiac myosin binding protein C (cMyBP-C) are a leading cause of hypertrophic cardiomyopathy (HCM). Patients with HCM often have reduced cMyBP-C expression, reduced protein phosphorylation, and diastolic dysfunction. Relaxation of a single myofibril in response to a sudden drop in activator calcium is biphasic, consisting of a slow isometric phase (k_REL,_slow) followed by a fast exponential phase (k_REL,_fast), considered to reflect cross-bridge-dependent and -independent processes, respectively. Here, we determined the effects of cMyBP-C on myofibril activation and relaxation kinetics by deleting the C0-C7 fragment of cMyBP-C and replacing it using our ‘cut-and-paste’ method. Results show that acute loss of C0-C7 desensitized myofilaments to Ca²⁺ and sped both phases of relaxation. Ligation of recombinant wild-type C0-C7 returned relaxation rates back to baseline, whereas ligation of phosphorylated cMyBP-C left the fast relaxation phase accelerated and increased the rate of activation in response to Ca²⁺ (k_ACT). Mavacamten (Mava), an inhibitor of myosin, accelerated both phases of relaxation independently of the presence or absence of cMyBP-C. Finally, we found that a point mutation in the M-domain of cMyBP-C (L348P) slowed both phases of relaxation. Taken together, we report that cMyBP-C slows both phases of relaxation, suggesting that it affects relaxation via cross-bridge-dependent and -independent mechanisms. (Figure presented.). Key points: Mutations in MYBPC3, the gene encoding cardiac myosin-binding protein C, (cMyBP-C) occur in ∼20%–25% of patients with hypertrophic cardiomyopathy. The majority of these mutations lead to reduced cMyBP-C protein expression in sarcomeres (haploinsufficiency). Here we investigated effects of acute loss of cMyBP-C on relaxation kinetics in mouse cardiac myofibrils using our ‘cut and paste’ approach. Results showed that cMyBP-C slows both phases of myofibril relaxation. Phosphorylation of cMyBP-C accelerated the fast phase of relaxation, whereas a point mutation (L348P) that increases the affinity of cMyBP-C for actin, significantly slowed both phases of relaxation. Mavacamten, a myosin inhibitor, accelerated both phases of relaxation independently of cMyBP-C. Overall, we interpret our results in terms of dual cross-bridge-dependent and cross-bridge-independent mechanisms of action of cMyBP-C on cardiac relaxation.