Pathogenic c-MYBPC3 variants recapitulated HCM phenotypes in zebrafish embryos, altering C0-C2 domain structure and reducing actin binding affinity, with the V5 double mutant showing the greatest instability.
Do c-MYBPC3 missense variants alter protein structure and actin binding, and induce hypertrophic cardiomyopathy phenotypes in zebrafish embryos?
Pathogenic c-MYBPC3 variants alter the functional and structural characteristics of C0-C2 domains, impairing actin interaction and contractility, providing mechanistic insight into hypertrophic cardiomyopathy pathogenesis.
The most common inherited cardiac disorder, hypertrophic cardiomyopathy (HCM), is characterized by thickening of heart muscle, for which genetic mutations in cardiac myosin-binding protein C3 (c-MYBPC3) gene, is the leading cause. Notably, patients with HCM display a heterogeneous clinical presentation, onset and prognosis. Thus, delineating the molecular mechanisms that explain how disparate c-MYBPC3 variants lead to HCM is essential for correlating the impact of specific genotypes on clinical severity. Herein, five c-MYBPC3 missense variants clinically associated with HCM were investigated; namely V1 (R177H), V2 (A216T), V3 (E258K), V4 (E441K) and double mutation V5 (V3 + V4), all located within the C1 and C2 domains of MyBP-C, a region known to interact with sarcomeric protein, actin. Injection of the variant complementary RNAs in zebrafish embryos was observed to recapitulate phenotypic aspects of HCM in patients. Interestingly, V3- and V5-cRNA injection produced the most severe zebrafish cardiac phenotype, exhibiting increased diastolic/systolic myocardial thickness and significantly reduced heart rate compared with control zebrafish. Molecular analysis of recombinant C0–C2 protein fragments revealed that c-MYBPC3 variants alter the C0–C2 domain secondary structure, thermodynamic stability and importantly, result in a reduced binding affinity to cardiac actin. V5 (double mutant), displayed the greatest protein instability with concomitant loss of actin-binding function. Our study provides specific mechanistic insight into how c-MYBPC3 pathogenic variants alter both functional and structural characteristics of C0–C2 domains leading to impaired actin interaction and reduced contractility, which may provide a basis for elucidating the disease mechanism in HCM patients with c-MYBPC3 mutations.
Da’as et al. (Fri,) conducted a other in Hypertrophic cardiomyopathy. c-MYBPC3 missense variants (V1, V2, V3, V4, V5) complementary RNAs vs. Control zebrafish was evaluated on Cardiac phenotype (myocardial thickness, heart rate) and molecular properties (secondary structure, stability, actin binding). Pathogenic c-MYBPC3 variants recapitulated HCM phenotypes in zebrafish embryos, altering C0-C2 domain structure and reducing actin binding affinity, with the V5 double mutant showing the greatest instability.