Myosin Binding Protein C Mutations and Hypertrophic Cardiomyopathy: Haploinsufficiency, Deranged Phosphorylation and Cardiomyocyte Dysfunction

Background Mutations in the MYBPC3 gene, encoding for cardiac myosin binding protein C (cMyBP-C), are a frequent cause of familial hypertrophic cardiomyopathy (FHCM). In the present study we investigated if protein composition and function of the sarcomere are altered in a homogenous FHCM patient group with truncating mutations in MYBPC3 (MYBPC3mut).Methods and Results Comparisons were made between cardiac samples from MYBPC3 mutant carriers (c.2373dupG, n=7; c.2864_2865delCT, n=4) and non-failing donors (n=8). Western Immunoblotting using antibodies directed against different parts of cMyBP-C did not reveal truncated cMyBP-C in MYBPC3mut. Protein expression of cMyBP-C was significantly reduced in MYBPC3mut by 33±5%. Cardiac MyBP-C phosphorylation in MYBPC3mut samples was similar to the values in donor samples, whereas the phosphorylation status of troponin I (cTnI) was reduced by 84±5%, indicating divergent phosphorylation of the two main contractile target proteins of the beta-adrenergic pathway. Force measurements in mechanically isolated Triton-permeabilized cardiomyocytes demonstrated a decrease in maximal force per cross-sectional area of the myocytes in MYBPC3mut (21.4±3.9 kN/m2) compared to donor (34.5±1.7 kN/m2). Moreover, Ca2+-sensititivy was higher in MYBPC3mut (pCa50=5.60±0.04) than in donor (pCa50=5.52±0.03), consistent with reduced cTnI phosphorylation. Treatment with exogenous protein kinase A, to mimic beta-adrenergic stimulation, did not correct reduced maximal force, but abolished the initial difference in Ca2+-sensititivy between MYBPC3mut (pCa50=5.46±0.03) and donor (pCa50=5.48±0.02).Conclusions Truncating MYBPC3 mutations cause haploinsufficiency, deranged phosphorylation of contractile proteins and reduced maximal force generating capacity of cardiomyocytes. The enhanced Ca2+-sensititivy in MYBPC3mut is due to hypophosphorylation of troponin I secondary to mutation-induced dysfunction.