The Role of Cardiac Myosin Binding Protein C3 Variants in Hypertrophic Cardiomyopathy: Genotype - Phenotype Association in the Zebrafish Model

Hypertrophic cardiomyopathy (HCM) is a complex disorder characterized by high levels of clinical and genetic heterogeneity. Cardiac manifestations include left ventricular myocardial hypertrophy and cardiac deterioration leading to progressive heart failure or sudden cardiac death. Genetically, the most common HCM-linked variants are found in cardiac myosin binding protein C3 (c-MYBPC3); specifically, at N-terminal domains, critical to regulating sarcomere structure and contractility. To validate different N-terminal domains variants predicted to have different pathogenicity classifications, we use the zebrafish model to examine genotype-to-phenotype associations. We test two splice-site variants (SV1 and SV2) in zebrafish and observe cardiac phenotypes recapitulating typical characteristics of human HCM, including thickened ventricular myocardial walls and impaired cardiac function. These phenotypes were partially restored by human mRNA co-injections, suggesting an exon-specific impact of SV1 on cardiomyocyte contractility in contrast to SV2's impact on sarcomere structure. We further assess the effects of four unique missense variants in the N-terminal domains on cardiac phenotypes in zebrafish. Molecular analysis of recombinant C0-C2 protein fragments expressing the missense variants revealed altered protein secondary structure properties and altered interaction/binding-affinity to cardiac actin. Finally, to further elucidate its role in cardiac development, we generate mypbc3 knockout zebrafish that display remodeling and hyperplasia of the myocardium, likely a response to chronic blood volume overload due to an observed increase in ejection fraction. In conclusion, we have utilized the zebrafish model to evaluate allele-specific impact of c-MYBPC3 variants on cardiac morphology and function and their associations with HCM clinical presentation, thus revealing novel mechanistic associations between these genetic variants and HCM disease.