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, nϭ7; c.2864_2865delCT, nϭ4) and nonfailing donors (nϭ13). 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 33Ϯ5%. 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 84Ϯ5%, indicating divergent phosphorylation of the 2 main contractile target proteins of the ␤-adrenergic pathway. Force measurements in mechanically isolated Triton-permeabilized cardiomyocytes demonstrated a decrease in maximal force per crosssectional area of the myocytes in MYBPC3 mut (20.2Ϯ2.7 kN/m 2 ) compared with donor (34.5Ϯ1.1 kN/m 2 ). Moreover, Ca 2ϩ sensitivity was higher in MYBPC3 mut (pCa 50 ϭ5.62Ϯ0.04) than in donor (pCa 50 ϭ5.54Ϯ0.02), consistent with reduced cardiac troponin I phosphorylation. Treatment with exogenous protein kinase A, to mimic ␤-adrenergic stimulation, did not correct reduced maximal force but abolished the initial difference in Ca 2ϩ sensitivity between MYBPC3 mut (pCa 50 ϭ5.46Ϯ0.03) and donor (pCa 50 ϭ5.48Ϯ0.02). Conclusions-Frameshift MYBPC3 mutations cause haploinsufficiency, deranged phosphorylation of contractile proteins, and reduced maximal force-generating capacity of cardiomyocytes. The enhanced Ca 2ϩ sensitivity in MYBPC3 mut is due to hypophosphorylation of troponin I secondary to mutation-induced dysfunction.

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 state (SRX), 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.

the four important S4 arginine residues on the voltage-sensing paddle is still unknown, with some models placing them in an aqueous crevice, and others a lipid environment. To learn more about the intricate role of lipid in the structure and function of potassium channels we have studied deuterium and phosphate ESEEM on spin-labeled, liposome reconstituted KcsA. By scanning the trans-membrane helices of KcsA, we show that deuterium coupling can be used to determine residue depth within a lipid bilayer. In addition, residues that interact with the phosphate head-groups of the lipid can be determined by phosphate coupling, and their precise location modeled.

Background-Hypertrophic cardiomyopathy (HCM), typically characterized by asymmetrical left ventricular hypertrophy, frequently is caused by mutations in sarcomeric proteins. We studied if changes in sarcomeric properties in HCM depend on the underlying protein mutation. Methods and Results-Comparisons were made between cardiac samples from patients carrying a MYBPC3 mutation (MYBPC3 mut ; nϭ17), mutation negative HCM patients without an identified sarcomere mutation (HCM mn ; nϭ11), and nonfailing donors (nϭ12). All patients had normal systolic function, but impaired diastolic function. Protein expression of myosin binding protein C (cMyBP-C) was significantly lower in MYBPC3 mut by 33Ϯ5%, and similar in HCM mn compared with donor. cMyBP-C phosphorylation in MYBPC3 mut was similar to donor, whereas it was significantly lower in HCM mn . Troponin I phosphorylation was lower in both patient groups compared with donor. Force measurements in single permeabilized cardiomyocytes demonstrated comparable sarcomeric dysfunction in both patient groups characterized by lower maximal force generating capacity in MYBPC3 mut and HCM mn, compared with donor (26.4Ϯ2.9, 28.0Ϯ3.7, and 37.2Ϯ2.3 kN/m 2 , respectively), and higher myofilament Ca 2ϩ -sensitivity (EC 50 ϭ2.5Ϯ0.2, 2.4Ϯ0.2, and 3.0Ϯ0.2 mol/L, respectively). The sarcomere length-dependent increase in Ca Key Words: cardiomyopathy Ⅲ myofilament proteins Ⅲ mutation Ⅲ myocardial contraction H ypertrophic cardiomyopathy (HCM), most often caused by mutations in genes encoding sarcomeric proteins, is a major cause of morbidity and mortality affecting Ϸ1:500 people worldwide at a relatively young age. 1,2 It often is characterized by asymmetrical left ventricular (LV) hypertrophy, predominantly involving the interventricular septum, occurring in the absence of other cardiac or systemic disease (such as hypertension or aortic stenosis). Clinical presentation is very heterogeneous in HCM as some patients reach old age with virtually no complaints, while others progress to end-stage heart failure or die at a young age from sudden cardiac arrest. To develop a targeted treatment to prevent or delay HCM, it is highly relevant to understand the pathophysiology of this disease. Clinical Perspective on p 46During the last 2 decades, many disease causing mutations have been identified, mainly in genes encoding sarcomeric proteins. 3,4 Despite improved genetic testing the causal gene mutation remains unidentified in over 40% of HCM patients. 5 Furthermore, the pathophysiological mechanism leading from a Recently we have provided evidence for sarcomeric dysfunction in manifest HCM patients with truncating MYBPC3 founder mutations (c.2373dupG and c.2864_2865delCT). 12 The sarcomeric dysfunction included a reduction in maximal force generating capacity and a higher myofilament Ca 2ϩ -sensitivity compared with nonfailing human myocardium, which may be the result of altered sarcomeric protein composition as we observed haploinsufficiency (ie, reduced cardiac myosin binding protein C [cMyBP...

Low cardiomyocyte Fmax in HCM patients is largely explained by hypertrophy and reduced myofibril density. MYH7 mutations reduce force generating capacity of sarcomeres at maximal and submaximal [Ca²⁺]. These hypocontractile sarcomeres may represent the primary abnormality in patients with MYH7 mutations.