Background— Mutations in the MYBPC3 gene, encoding cardiac myosin-binding protein C (cMyBP-C), are a frequent cause of familial hypertrophic cardiomyopathy. In the present study, we investigated whether protein composition and function of the sarcomere are altered in a homogeneous familial hypertrophic cardiomyopathy patient group with frameshift mutations in MYBPC3 (MYBPC3 _mut ). Methods and Results— Comparisons were made between cardiac samples from MYBPC3 mutant carriers (c.2373dupG, n=7; c.2864_2865delCT, n=4) and nonfailing donors (n=13). Western blots with the use of antibodies directed against cMyBP-C did not reveal truncated cMyBP-C in MYBPC3 _mut . Protein expression of cMyBP-C was significantly reduced in MYBPC3 _mut by 33±5%. Cardiac MyBP-C phosphorylation in MYBPC3 _mut samples was similar to the values in donor samples, whereas the phosphorylation status of cardiac troponin I was reduced by 84±5%, indicating divergent phosphorylation of the 2 main contractile target proteins of the β-adrenergic pathway. Force measurements in mechanically isolated Triton-permeabilized cardiomyocytes demonstrated a decrease in maximal force per cross-sectional area of the myocytes in MYBPC3 _mut (20.2±2.7 kN/m ² ) compared with donor (34.5±1.1 kN/m ² ). Moreover, Ca ²⁺ sensitivity was higher in MYBPC3 _mut (pCa ₅₀ =5.62±0.04) than in donor (pCa ₅₀ =5.54±0.02), consistent with reduced cardiac troponin I phosphorylation. Treatment with exogenous protein kinase A, to mimic β-adrenergic stimulation, did not correct reduced maximal force but abolished the initial difference in Ca ²⁺ sensitivity between MYBPC3 _mut (pCa ₅₀ =5.46±0.03) and donor (pCa ₅₀ =5.48±0.02). Conclusions— Frameshift MYBPC3 mutations cause haploinsufficiency, deranged phosphorylation of contractile proteins, and reduced maximal force-generating capacity of cardiomyocytes. The enhanced Ca ²⁺ sensitivity in MYBPC3 _mut is due to hypophosphorylation of troponin I secondary to mutation-induced dysfunction.