👤 Nicky Boontje

Mutations in the MYBPC3 gene, encoding cardiac myosin binding protein C (cMyBP-C) are frequent causes of hypertrophic cardiomyopathy (HCM). Previously, we have presented evidence for reduced cMyBP-C expression (haploinsufficiency), in patients with a truncation mutation in MYBPC3. In mice, lacking cMyBP-C cross-bridge kinetics was accelerated. In this study, we investigated whether cross-bridge kinetics was altered in myectomy samples from HCM patients harboring heterozygous MYBPC3 mutations (MYBPC3mut). Isometric force and the rate of force redevelopment (k tr) at different activating Ca(2+) concentrations were measured in mechanically isolated Triton-permeabilized cardiomyocytes from MYBPC3mut (n = 18) and donor (n = 7) tissue. Furthermore, the stretch activation response of cardiomyocytes was measured in tissue from eight MYBPC3mut patients and five donors to assess the rate of initial force relaxation (k 1) and the rate and magnitude of the transient increase in force (k 2 and P 3, respectively) after a rapid stretch. Maximal force development of the cardiomyocytes was reduced in MYBPC3mut (24.5 ± 2.3 kN/m(2)) compared to donor (34.9 ± 1.6 kN/m(2)). The rates of force redevelopment in MYBPC3mut and donor over a range of Ca(2+) concentrations were similar (k tr at maximal activation: 0.63 ± 0.03 and 0.75 ± 0.09 s(-1), respectively). Moreover, the stretch activation parameters did not differ significantly between MYBPC3mut and donor (k 1: 8.5±0.5 and 8.8 ± 0.4 s(-1); k 2: 0.77 ± 0.06 and 0.74 ± 0.09 s(-1); P 3: 0.08 ± 0.01 and 0.09 ± 0.01, respectively). Incubation with protein kinase A accelerated k 1 in MYBPC3mut and donor to a similar extent. Our experiments indicate that, at the cMyBP-C expression levels in this patient group (63 ± 6 % relative to donors), cross-bridge kinetics are preserved and that the depressed maximal force development is not explained by perturbation of cross-bridge kinetics. Show less

High-myofilament Ca(2+) sensitivity has been proposed as a trigger of disease pathogenesis in familial hypertrophic cardiomyopathy (HCM) on the basis of in vitro and transgenic mice studies. However, myofilament Ca(2+) sensitivity depends on protein phosphorylation and muscle length, and at present, data in humans are scarce. To investigate whether high myofilament Ca(2+) sensitivity and perturbed length-dependent activation are characteristics for human HCM with mutations in thick and thin filament proteins. Cardiac samples from patients with HCM harboring mutations in genes encoding thick (MYH7, MYBPC3) and thin (TNNT2, TNNI3, TPM1) filament proteins were compared with sarcomere mutation-negative HCM and nonfailing donors. Cardiomyocyte force measurements showed higher myofilament Ca(2+) sensitivity in all HCM samples and low phosphorylation of protein kinase A (PKA) targets compared with donors. After exogenous PKA treatment, myofilament Ca(2+) sensitivity was similar (MYBPC3mut, TPM1mut, sarcomere mutation-negative HCM), higher (MYH7mut, TNNT2mut), or even significantly lower (TNNI3mut) compared with donors. Length-dependent activation was significantly smaller in all HCM than in donor samples. PKA treatment increased phosphorylation of PKA-targets in HCM myocardium and normalized length-dependent activation to donor values in sarcomere mutation-negative HCM and HCM with truncating MYBPC3 mutations but not in HCM with missense mutations. Replacement of mutant by wild-type troponin in TNNT2mut and TNNI3mut corrected length-dependent activation to donor values. High-myofilament Ca(2+) sensitivity is a common characteristic of human HCM and partly reflects hypophosphorylation of PKA targets compared with donors. Length-dependent sarcomere activation is perturbed by missense mutations, possibly via posttranslational modifications other than PKA hypophosphorylation or altered protein-protein interactions, and represents a common pathomechanism in HCM. Show less

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. Comparisons were made between cardiac samples from patients carrying a MYBPC3 mutation (MYBPC3(mut); n=17), mutation negative HCM patients without an identified sarcomere mutation (HCM(mn); n=11), and nonfailing donors (n=12). 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 33±5%, 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 (26.4±2.9, 28.0±3.7, and 37.2±2.3 kN/m(2), respectively), and higher myofilament Ca(2+)-sensitivity (EC(50)=2.5±0.2, 2.4±0.2, and 3.0±0.2 μmol/L, respectively). The sarcomere length-dependent increase in Ca(2+)-sensitivity was significantly smaller in both patient groups compared with donor (ΔEC(50): 0.46±0.04, 0.37±0.05, and 0.75±0.07 μmol/L, respectively). Protein kinase A treatment restored myofilament Ca(2+)-sensitivity and length-dependent activation in both patient groups to donor values. Changes in sarcomere function reflect the clinical HCM phenotype rather than the specific MYBPC3 mutation. Hypocontractile sarcomeres are a common deficit in human HCM with normal systolic left ventricular function and may contribute to HCM disease progression. Show less

Perturbations in sarcomeric function may in part underlie systolic and diastolic dysfunction of the failing heart. Sarcomeric dysfunction has been ascribed to changes in phosphorylation status of sarcomeric proteins caused by an altered balance between intracellular kinases and phosphatases during the development of cardiac disease. In the present review we discuss changes in phosphorylation of the thick filament protein myosin binding protein C (cMyBP-C) reported in failing myocardium, with emphasis on phosphorylation changes observed in familial hypertrophic cardiomyopathy caused by mutations in MYBPC3. Moreover, we will discuss assays which allow to distinguish between functional consequences of mutant sarcomeric proteins and (mal)adaptive changes in sarcomeric protein phosphorylation. Show less