Abstract Body: Background: Cardiac muscle contraction and relaxation are regulated by the interaction between the myosin neck region (S2) and cardiac myosin-binding protein-C (cMyBP-C). Mutations in the myosin S2 region, which alter its interaction with cMyBP-C, have been clinically associated with hypertrophic cardiomyopathy (HCM). However, the precise molecular mechanisms underlying these effects remain unclear.
Objective: To investigate the impact of the pathogenic mutation E930del mutation in β-myosin (hMyh7E930del), located within the S2 region, on its interaction with cMyBP-C and in vitro and utilize a homologous mouse model (mMyh6E932del) to determine in vivo effects.
Methods and Results: To probe the cMyBP-C-myosin interaction, we synthesized the proximal S2 region of hMyh7 (126 amino acids) alongside nine overlapping and truncated S2 variants. Using solid-phase binding and skinned papillary muscle assays, we identified Amg 27119, a 22-amino-acid myopeptide, as highly effective in enhancing cardiac contractile function. Next, A CRISPR-Cas9-mediated knock-in mouse model harboring a homologous mutation (mMyh6E932del) was developed to study in vivo effects of this mutation. Homozygous mice were embryonically lethal, whereas heterozygous mice exhibited cardiac hypertrophy, preserved systolic function, and upregulated hypertrophic gene expression at 6 to 9 months old, as demonstrated by echocardiography, RNA-seq, and real-tme PCR. In vivo catheterization revealed diastolic dysfunction, evidenced by reductions in dP/dtmin and tau. Force-calcium (pCa-force) measurements demonstrated a significant increase in maximal peak force production in skinned myocfibers from mutant mice (29.29 nM/mm^2, p=0.0039) relative to controls (20.311 nM/mm^2), without altering myofilament calcium sensitivity or the Hill coefficient.
Conclusion: The identified myopeptide effectively targeted and alleviated cMyBP-C-mediated inhibition of myosin, improving contractility in vitro. The HCM mutation, E932del, disrupts the myosin S2-cMyBP-C interaction leads to diastolic dysfunction and hypercontractility, highlighting the therapeutic potential of targeting the myosin S2 and cMyBP-C interaction.