👤 Kristina Bezold Kooiker

Approximately 40% of hypertrophic cardiomyopathy (HCM) mutations are linked to the sarcomere protein cardiac myosin binding protein-C (cMyBP-C). These mutations are either classified as missense mutations or truncation mutations. One mutation whose nature has been inconsistently reported in the literature is the MYBPC3-c.772G > A mutation. Using patient-derived human induced pluripotent stem cells differentiated to cardiomyocytes (hiPSC-CMs), we have performed a mechanistic study of the structure-function relationship for this MYBPC3-c.772G > A mutation versus a mutation corrected, isogenic cell line. Our results confirm that this mutation leads to exon skipping and mRNA truncation that ultimately suggests ∼20% less cMyBP-C protein (i.e., haploinsufficiency). This, in turn, results in increased myosin recruitment and accelerated myofibril cycling kinetics. Our mechanistic studies suggest that faster ADP release from myosin is a primary cause of accelerated myofibril cross-bridge cycling due to this mutation. Additionally, the reduction in force generating heads expected from faster ADP release during isometric contractions is outweighed by a cMyBP-C phosphorylation mediated increase in myosin recruitment that leads to a net increase of myofibril force, primarily at submaximal calcium activations. These results match well with our previous report on contractile properties from myectomy samples of the patients from whom the hiPSC-CMs were generated, demonstrating that these cell lines are a good model to study this pathological mutation and extends our understanding of the mechanisms of altered contractile properties of this HCM MYBPC3-c.772G > A mutation. Show less

We sought to establish a large animal model of inherited hypertrophic cardiomyopathy (HCM) with sufficient disease severity and early penetrance for identification of novel therapeutic strategies. HCM is the most common inherited cardiac disorder affecting 1 in 250-500 people, yet few therapies for its treatment or prevention are available. A research colony of purpose-bred cats carrying the A31P mutation in MYBPC3 was founded using sperm from a single heterozygous male cat. Cardiac function in four generations was assessed by periodic echocardiography and measurement of blood biomarkers. Results showed that HCM penetrance was age-dependent, and that penetrance occurred earlier and was more severe in successive generations, especially in homozygotes. Homozygosity was also associated with progression from preclinical to clinical disease. A31P homozygous cats represent a heritable model of HCM with early disease penetrance and a severe phenotype necessary for interventional studies aimed at altering disease progression. The occurrence of a more severe phenotype in later generations of cats, and the occasional occurrence of HCM in wildtype cats suggests the presence of at least one gene modifier or a second causal variant in this research colony that exacerbates the HCM phenotype when inherited in combination with the A31P mutation. Show less

Cardiac myosin binding protein-C (cMyBP-C) is a structural and regulatory component of cardiac thick filaments. It is observed in electron micrographs as seven to nine transverse stripes in the central portion of each half of the A band. Its C-terminus binds tightly to the myosin rod and contributes to thick filament structure, while the N-terminus can bind both myosin S2 and actin, influencing their structure and function. Mutations in the MYBPC3 gene (encoding cMyBP-C) are commonly associated with hypertrophic cardiomyopathy (HCM). In cardiac cells there exists a population of myosin heads in the super-relaxed (SRX) state, which are bound to the thick filament core with a highly inhibited ATPase activity. This report examines the role cMyBP-C plays in regulating the population of the SRX state of cardiac myosin by using an assay that measures single ATP turnover of myosin. We report a significant decrease in the proportion of myosin heads in the SRX state in homozygous cMyBP-C knockout mice, however heterozygous cMyBP-C knockout mice do not significantly differ from the wild type. A smaller, non-significant decrease is observed when thoracic aortic constriction is used to induce cardiac hypertrophy in mutation negative mice. These results support the proposal that cMyBP-C stabilises the thick filament and that the loss of cMyBP-C results in an untethering of myosin heads. This results in an increased myosin ATP turnover, further consolidating the relationship between thick filament structure and the myosin ATPase. Show less

Mutations in MYBPC3, the gene encoding cardiac myosin binding protein C (cMyBP-C), are a major cause of hypertrophic cardiomyopathy (HCM). While most mutations encode premature stop codons, missense mutations causing single amino acid substitutions are also common. Here we investigated effects of a single proline for alanine substitution at amino acid 31 (A31P) in the C0 domain of cMyBP-C, which was identified as a natural cause of HCM in cats. Results using recombinant proteins showed that the mutation disrupted C0 structure, altered sensitivity to trypsin digestion, and reduced recognition by an antibody that preferentially recognizes N-terminal domains of cMyBP-C. Western blots detecting A31P cMyBP-C in myocardium of cats heterozygous for the mutation showed a reduced amount of A31P mutant protein relative to wild-type cMyBP-C, but the total amount of cMyBP-C was not different in myocardium from cats with or without the A31P mutation indicating altered rates of synthesis/degradation of A31P cMyBP-C. Also, the mutant A31P cMyBP-C was properly localized in cardiac sarcomeres. These results indicate that reduced protein expression (haploinsufficiency) cannot account for effects of the A31P cMyBP-C mutation and instead suggest that the A31P mutation causes HCM through a poison polypeptide mechanism that disrupts cMyBP-C or myocyte function. Show less