Cardiac myosin binding protein C phosphorylation in cardiac disease

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

Protein kinase C α and ε phosphorylation of troponin and myosin binding protein C reduce Ca2+ sensitivity in human myocardium

Previous studies indicated that the increase in protein kinase C (PKC)-mediated myofilament protein phosphorylation observed in failing myocardium might be detrimental for contractile function. This study was designed to reveal and compare the effects of PKCα- and PKCε-mediated phosphorylation on myofilament function in human myocardium. Isometric force was measured at different [Ca2+] in single permeabilized cardiomyocytes from failing human left ventricular tissue. Activated PKCα and PKCε equally reduced Ca2+ sensitivity in failing cardiomyocytes (ΔpCa50 = 0.08 ± 0.01). Both PKC isoforms increased phosphorylation of troponin I- (cTnI) and myosin binding protein C (cMyBP-C) in failing cardiomyocytes. Subsequent incubation of failing cardiomyocytes with the catalytic subunit of protein kinase A (PKA) resulted in a further reduction in Ca2+ sensitivity, indicating that the effects of both PKC isoforms were not caused by cross-phosphorylation of PKA sites. Both isozymes showed no effects on maximal force and only PKCα resulted in a modest significant reduction in passive force. Effects of PKCα were only minor in donor cardiomyocytes, presumably because of already saturated cTnI and cMyBP-C phosphorylation levels. Donor tissue could therefore be used as a tool to reveal the functional effects of troponin T (cTnT) phosphorylation by PKCα. Massive dephosphorylation of cTnT with alkaline phosphatase increased Ca2+ sensitivity. Subsequently, PKCα treatment of donor cardiomyocytes reduced Ca2+ sensitivity (ΔpCa50 = 0.08 ± 0.02) and solely increased phosphorylation of cTnT, but did not affect maximal and passive force. PKCα- and PKCε-mediated phosphorylation of cMyBP-C and cTnI as well as cTnT decrease myofilament Ca2+ sensitivity and may thereby reduce contractility and enhance relaxation of human myocardium

Detrimental effect of combined exercise training and eNOS overexpression on cardiac function after myocardial infarction

de Waard MC, van der Velden J, Boontje NM, Dekkers DH, van Haperen R, Kuster DW, Lamers JM, de Crom R, Duncker DJ. Detrimental effect of combined exercise training and eNOS overexpression on cardiac function after myocardial infarction. Am J Physiol Heart Circ Physiol 296: H1513-H1523, 2009; doi:10.1152/ajpheart.00485.2008.-It has been reported that exercise after myocardial infarction (MI) attenuates left ventricular (LV) pump dysfunction by normalization of myofilament function. This benefit could be due to an exercise-induced upregulation of endothelial nitric oxide synthase (eNOS) expression and activity. Consequently, we first tested the hypothesis that the effects of exercise after MI can be mimicked by elevated eNOS expression using transgenic mice with overexpression of human eNOS (eNOSTg). Both exercise and eNOSTg attenuated LV remodeling and dysfunction after MI in mice and improved cardiomyocyte maximal force development (F-max). However, only exercise training restored myofilament Ca2+-sensitivity and sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA)2a protein levels and improved the first derivative of LV pressure at 30 mmHg. Conversely, only eNOSTg improved survival. In view of these partly complementary actions, we subsequently tested the hypothesis that combining exercise and eNOSTg would provide additional protection against LV remodeling and dysfunction after MI. Unexpectedly, the combination of exercise and eNOSTg abolished the beneficial effects on LV remodeling and dysfunction of either treatment alone. The latter was likely due to perturbations in Ca2+ homeostasis, as myofilament Fmax actually increased despite marked reductions in the phosphorylation status of several myofilament proteins, whereas the exercise-induced increases in SERCA2a protein levels were lost in eNOSTg mice. Antioxidant treatment with N-acetylcysteine or supplementation of tetrahydrobiopterin and L-arginine prevented these detrimental effects on LV function while partly restoring the phosphorylation status of myofilament proteins and further enhancing myofilament Fmax. In conclusion, the combination of exercise and elevated eNOS expression abolished the cardioprotective effects of either treatment alone after MI, which appeared to be, at least in part, the result of increased oxidative stress secondary to eNOS "uncoupling.

Preserved cross-bridge kinetics in human hypertrophic cardiomyopathy patients with MYBPC3 mutations

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 (MYBPC3(mut)). Isometric force and the rate of force redevelopment (k (tr)) at different activating Ca2+ concentrations were measured in mechanically isolated Triton-permeabilized cardiomyocytes from MYBPC3(mut) (n = 18) and donor (n = 7) tissue. Furthermore, the stretch activation response of cardiomyocytes was measured in tissue from eight MYBPC3(mut) 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 MYBPC3(mut) (24.5 +/- 2.3 kN/m(2)) compared to donor (34.9 +/- 1.6 kN/m(2)). The rates of force redevelopment in MYBPC3(mut) and donor over a range of Ca2+ 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 MYBPC3(mut) 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 MYBPC3(mut) 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

Myocardial contraction is 5-fold more economical in ventricular than in atrial human tissue

Cardiac energetics and performance depend on the expression level of the fast (α-) and slow (β-) myosin heavy chain (MHC) isoform. In ventricular tissue, the β-MHC isoform predominates, whereas in atrial tissue a variable mixture of α- and β-MHC is found. In several cardiac diseases, the slow isoform is upregulated; however, the functional implications of this transition in human myocardium are largely unknown. The aim of this study was to determine the relation between contractile properties and MHC isoform composition in healthy human myocardium using the diversity in atrial tissue. Isometric force production and ATP consumption were measured in chemically skinned atrial trabeculae and ventricular muscle strips, and rate of force redevelopment was studied using single cardiomyocytes. MHC isoform composition was determined by one-dimensional SDS-gel electrophoresis. Force development in ventricular tissue was about 5-fold more economical, but nine times slower, than in atrial tissue. Significant linear correlations were found between MHC isoform composition, ATP consumption and rate of force redevelopment. These results clearly indicate that even a minor shift in MHC isoform expression has considerable impact on cardiac performance in human tissue

Prevention of Myofilament Dysfunction by beta-Blocker Therapy in Postinfarct Remodeling

Background-Myofilament contractility of individual cardiomyocytes is depressed in remote noninfarcted myocardium and contributes to global left ventricular pump dysfunction after myocardial infarction (MI). Here, we investigated whether beta-blocker therapy could restore myofilament contractility. Methods and Results-In pigs with a MI induced by ligation of the left circumflex coronary artery, beta-blocker therapy (bisoprolol, MI+beta) was initiated on the first day after MI. Remote left ventricular subendocardial biopsies were taken 3 weeks after sham or MI surgery. Isometric force was measured in single permeabilized cardiomyocytes. Maximal force (F-max) was lower, whereas Ca2+ sensitivity was higher in untreated MI compared with sham (both P<0.05). The difference in Ca2+ sensitivity was abolished by treatment of cells with the beta-adrenergic kinase, protein kinase A. beta-blocker therapy partially reversed F-max and Ca2+ sensitivity to sham values and significantly reduced passive force. Despite the lower myofilament Ca2+ sensitivity in MI+beta compared with untreated myocardium, the protein kinase A induced reduction in Ca2+ sensitivity was largest in cardiomyocytes from myocardium treated with beta-blockers. Phosphorylation of beta-adrenergic target proteins (myosin binding protein C and troponin I) did not differ among groups, whereas myosin light chain 2 phosphorylation was reduced in MI, which coincided with increased expression of protein phosphatase 1. beta-blockade fully restored the latter alterations and significantly reduced expression of protein phosphatase 2a. Conclusions-beta-blockade reversed myofilament dysfunction and enhanced myofilament responsiveness to protein kinase A in remote myocardium after MI. These effects likely contribute to the beneficial effects of beta-blockade on global left ventricular function after MI. (Circ Heart Fail. 2009;2:233-242.

Contractile Dysfunction Irrespective of the Mutant Protein in Human Hypertrophic Cardiomyopathy With Normal Systolic Function

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); 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 phosphory Conclusions-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. (Circ Heart Fail. 2012; 5: 36-46.