Altered C10 domain in cardiac myosin binding protein-C results in hypertrophic cardiomyopathy

Abstract

Aims A 25-base pair deletion in the cardiac myosin binding protein-C (cMyBP-C) gene (MYBPC3), proposed to skip exon 33, modifies the C10 domain (cMyBP-CΔC10mut) and is associated with hypertrophic cardiomyopathy (HCM) and heart failure, affecting approximately 100 million South Asians. However, the molecular mechanisms underlying the pathogenicity of cMyBP-CΔC10mutin vivo are unknown. We hypothesized that expression of cMyBP-CΔC10mut exerts a poison polypeptide effect leading to improper assembly of cardiac sarcomeres and the development of HCM. Methods and results To determine whether expression of cMyBP-CΔC10mut is sufficient to cause HCM and contractile dysfunction in vivo, we generated transgenic (TG) mice having cardiac-specific protein expression of cMyBP-CΔC10mut at approximately half the level of endogenous cMyBP-C. At 12 weeks of age, significant hypertrophy was observed in TG mice expressing cMyBP-CΔC10mut (heart weight/body weight ratio: 4.43 ± 0.11 mg/g non-transgenic (NTG) vs. 5.34 ± 0.25 mg/g cMyBP-CΔC10mut, P < 0.05). Furthermore, haematoxylin and eosin, Masson’s trichrome staining, as well as second-harmonic generation imaging revealed the presence of significant fibrosis and a greater relative nuclear area in cMyBP-CΔC10mut hearts compared with NTG controls. M-mode echocardiography analysis revealed hypercontractile hearts (EF: 53.4%±2.9% NTG vs. 66.4% ± 4.7% cMyBP-CΔC10mut; P < 0.05) and early diastolic dysfunction (E/E′: 28.7 ± 3.7 NTG vs. 46.3 ± 8.4 cMyBP-CΔC10mut; P < 0.05), indicating the presence of an HCM phenotype. To assess whether these changes manifested at the myofilament level, contractile function of single skinned cardiomyocytes was measured. Preserved maximum force generation and increased Ca2+-sensitivity of force generation were observed in cardiomyocytes from cMyBP-CΔC10mut mice compared with NTG controls (EC50: 3.6 ± 0.02 µM NTG vs. 2.90 ± 0.01 µM cMyBP-CΔC10mut; P < 0.0001). Conclusion Expression of cMyBP-C protein with a modified C10 domain is sufficient to cause contractile dysfunction and HCM in vivo.

Amino terminus of cardiac myosin binding protein-C regulates cardiac contractility

“…Myofilament-enriched protein fractions from frozen mouse hearts were isolated and subjected to SDS-PAGE and Western blot analysis for total and phosphorylated cMyBP-C and cardiac troponin I as described previously [10,17]. Total RNA isolation, quantitative expression of MYBPC3 and hypertrophic gene markers, and RNA-seq were performed as previously described [10,17,23].…”

Assessment of the Contribution of a Thermodynamic and Mechanical Destabilization of Myosin–Binding Protein C Domain C2 to the Pathomechanism of Hypertrophic Cardiomyopathy–causing Double Mutation MYBPC3Δ25bp/D389V

“…These studies showed that transcripts originating from the affected allele are subject to alternative splicing mechanisms that lead to skipping of exon 33. The resulting protein has a C10 domain with 62 residues removed and 55 added, mislocalizes to the Z-disc and its overexpression is sufficient to cause HCM in transgenic mice [46,47]. However, the probability of the alternative splicing event in human and the resulting amount of mutant protein remains unclear.…”

“…While many previous studies utilized C0-C2 or C1-C2 constructs [13][14][15][16][17][18][19][20], little is known about the biochemical properties of the C2 domain. Since MYBPC3 Δ25bp alone is not a penetrant factor for development of HCM and has been characterized before [45][46][47][48], this study focuses on the impact of the D389V mutation in the C2 domain of MyBPC. Residue D389 lies in a region of the C2 domain that is highly conserved across mammals (Figure 1B).…”

Variation p.R1045H in MYH7 correlated with hypertrophic cardiomyopathy in a Chinese pedigree

“…Inherited hypertrophic cardiomyopathy (HCM) is the common type of ICM 1, 2 . The main pathological manifestation of inherited HCM is left ventricle and ventricular septal asymmetry hypertrophy, which is also the main cardiogenic cause of sudden death in this disease 2,3 . The latest epidemiological investigation shows that most hereditary modes of HCM are autosomal dominant with an incidence rate of about 200/100,000, and there is no difference in genders 4 .…”