Abstract Body (Do not enter title and authors here): Background: Cardiac muscle contraction and relaxation are regulated by the interaction between myosin neck region (S2) and cardiac myosin binding protein-C (cMyBP-C). Clinically, several mutations in the myosin S2 region, affecting its interaction with cMyBP-C, have been linked to hypertrophic cardiomyopathy (HCM). However, the molecular mechanism by which mutations in myosin S2 affect function is unknown. The interaction of the myosin S2 region with cMyBP-C and whether facilitating this interaction using a small peptide of myosin S2 can efficiently improve cardiac contractility remains unclear.
Objectives: To determine the interactive region of myosin S2 with cMyBP-C on contractility in vitro, and define the impact of an E932del mutation in α-myosin (Myh6E932del) in vivo, which is located in the myosin S2 region affecting its interaction with cMyBP-C.
Methods and Results: To target the cMyBP-C-myosin interaction, we used the proximal region of S2 (126aa) and nine overlapping and shortened variations of the S2 region in solid-phase binding and skinned papillary muscle assays. Based on these experiments, we determined that Amg 27119, a 22-amino acid myopeptide, was highly efficacious in improving contractile function in cardiac muscle. Next, we generated a CRISPR-Cas9 mediated knock-in mouse model expressing the Myh6E932del mutation. Echocardiography and real-time PCR data from heterozygous mice at 9 months of age revealed cardiac hypertrophy with preserved cardiac function and increased expression of cardiac hypertrophic genes compared to wild-type controls, respectively. In vivo catheterization analyses indicated impaired relaxation, with reductions in dP/dt_min and tau in mutant mice versus controls. pCa-force measurements showed significantly increased force production in mutant skinned myocytes (29.289 nM/mm^2, p=0.0039) compared to controls (20.311 nM/mm^2), without affecting myofilament calcium sensitivity and the Hill coefficient.
Conclusion: The myopeptide was determined to target and relieve the cMyBP-C inhibition of myosin, thereby improving contractility in vitro. Disrupting the interaction between myosin S2 and cMyBP-C with HCM mutations results in diastolic dysfunction and hypercontraction.