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 Ca 2+ 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 Ca 2+ ( 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. image 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.
Dvornikov et al. (Thu,) studied this question.