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 (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.

PKCα-specific phosphorylation of the troponin complex in human myocardium: a functional and proteomics analysis.

AIMS: Protein kinase Cα (PKCα) is one of the predominant PKC isoforms that phosphorylate cardiac troponin. PKCα is implicated in heart failure and serves as a potential therapeutic target, however, the exact consequences for contractile function in human myocardium are unclear. This study aimed to investigate the effects of PKCα phosphorylation of cardiac troponin (cTn) on myofilament function in human failing cardiomyocytes and to resolve the potential targets involved. METHODS AND RESULTS: Endogenous cTn from permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was exchanged (∼69%) with PKCα-treated recombinant human cTn (cTn (DD+PKCα)). This complex has Ser23/24 on cTnI mutated into aspartic acids (D) to rule out in vitro cross-phosphorylation of the PKA sites by PKCα. Isometric force was measured at various [Ca(2+)] after exchange. The maximal force (Fmax) in the cTn (DD+PKCα) group (17.1±1.9 kN/m(2)) was significantly reduced compared to the cTn (DD) group (26.1±1.9 kN/m(2)). Exchange of endogenous cTn with cTn (DD+PKCα) increased Ca(2+)-sensitivity of force (pCa50 = 5.59±0.02) compared to cTn (DD) (pCa50 = 5.51±0.02). In contrast, subsequent PKCα treatment of the cells exchanged with cTn (DD+PKCα) reduced pCa50 to 5.45±0.02. Two PKCα-phosphorylated residues were identified with mass spectrometry: Ser198 on cTnI and Ser179 on cTnT, although phosphorylation of Ser198 is very low. Using mass spectrometry based-multiple reaction monitoring, the extent of phosphorylation of the cTnI sites was quantified before and after treatment with PKCα and showed the highest phosphorylation increase on Thr143. CONCLUSION: PKCα-mediated phosphorylation of the cTn complex decreases Fmax and increases myofilament Ca(2+)-sensitivity, while subsequent treatment with PKCα in situ decreased myofilament Ca(2+)-sensitivity. The known PKC sites as well as two sites which have not been previously linked to PKCα are phosphorylated in human cTn complex treated with PKCα with a high degree of specificity for Thr143.

GSK3ß phosphorylates newly identified site in the proline-alanine-rich region of cardiac myosin-binding protein C and alters cross-bridge cycling kinetics in human: short communication.

RATIONALE: Cardiac myosin-binding protein C (cMyBP-C) regulates cross-bridge cycling kinetics and, thereby, fine-tunes the rate of cardiac muscle contraction and relaxation. Its effects on cardiac kinetics are modified by phosphorylation. Three phosphorylation sites (Ser275, Ser284, and Ser304) have been identified in vivo, all located in the cardiac-specific M-domain of cMyBP-C. However, recent work has shown that up to 4 phosphate groups are present in human cMyBP-C. OBJECTIVE: To identify and characterize additional phosphorylation sites in human cMyBP-C. METHODS AND RESULTS: Cardiac MyBP-C was semipurified from human heart tissue. Tandem mass spectrometry analysis identified a novel phosphorylation site on serine 133 in the proline-alanine-rich linker sequence between the C0 and C1 domains of cMyBP-C. Unlike the known sites, Ser133 was not a target of protein kinase A. In silico kinase prediction revealed glycogen synthase kinase 3ß (GSK3ß) as the most likely kinase to phosphorylate Ser133. In vitro incubation of the C0C2 fragment of cMyBP-C with GSK3ß showed phosphorylation on Ser133. In addition, GSK3ß phosphorylated Ser304, although the degree of phosphorylation was less compared with protein kinase A-induced phosphorylation at Ser304. GSK3ß treatment of single membrane-permeabilized human cardiomyocytes significantly enhanced the maximal rate of tension redevelopment. CONCLUSIONS: GSK3ß phosphorylates cMyBP-C on a novel site, which is positioned in the proline-alanine-rich region and increases kinetics of force development, suggesting a noncanonical role for GSK3ß at the sarcomere level. Phosphorylation of Ser133 in the linker domain of cMyBP-C may be a novel mechanism to regulate sarcomere kinetics.

Familial hypertrophic cardiomyopathy: functional effects of myosin mutation R723G in cardiomyocytes.

Familial Hypertrophic Cardiomyopathy (FHC) is frequently caused by mutations in the ß-cardiac myosin heavy chain (ß-MyHC). To identify changes in sarcomeric function triggered by such mutations, distinguishing mutation effects from other functional alterations of the myocardium is essential. We previously identified a direct effect of mutation R723G (MyHC723) on myosin function in slow Musculus soleus fibers. Here we investigate contractile features of left ventricular cardiomyocytes of FHC-patients with the same MyHC723-mutation and compare these to the soleus data. In mechanically isolated, triton-permeabilized MyHC723-cardiomyocytes, maximum force was significantly lower but calcium-sensitivity was unchanged compared to donor. Conversely, MyHC723-soleus fibers showed significantly higher maximum force and reduced calcium-sensitivity compared to controls. Protein phosphorylation, a potential myocardium specific modifying mechanism, might account for differences compared to soleus fibers. Analysis revealed reduced phosphorylation of troponin I and T, myosin-binding-protein C, and myosin-light-chain 2 in MyHC723-myocardium compared to donor. Saturation of protein-kinaseA phospho-sites led to comparable, i.e., reduced MyHC723-calcium-sensitivity in cardiomyocytes as in M. soleus fibers, while maximum force remained reduced. Myofibrillar disarray and lower density of myofibrils, however, largely account for reduced maximum force in MyHC723-cardiomyocytes. The changes seen when phosphorylation of sarcomeric proteins in myocardium of affected patients is matched to control tissue suggest that the R723G mutation causes reduced Ca(++)-sensitivity in both cardiomyocytes and M. soleus fibers. In MyHC723-myocardium, however, hypophosphorylation can compensate for the reduced calcium-sensitivity, while maximum force generation, lowered by myofibrillar deficiency and disarray, remains impaired, and may only be compensated by hypertrophy.

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 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. 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.

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.

Transmural heterogeneity of myofilament function and sarcomeric protein phosphorylation in remodeled myocardium of pigs with a recent myocardial infarction.

AIM: Transmural differences in sarcomeric protein composition and function across the left ventricular (LV) wall have been reported. We studied in pigs sarcomeric function and protein phosphorylation in subepicardial (EPI) and subendocardial (ENDO) layers of remote LV myocardium after myocardial infarction (MI), induced by left circumflex coronary artery ligation. METHODS: EPI and ENDO samples were taken 3 weeks after sham surgery (n = 12) or induction of MI (n = 12) at baseline (BL) and during ß-adrenergic receptor (ßAR) stimulation with dobutamine. Isometric force was measured in single cardiomyocytes at various [Ca(2+)] and 2.2 µm sarcomere length. RESULTS: In sham hearts, no significant transmural differences were observed in myofilament function or protein phosphorylation. Myofilament Ca(2+)-sensitivity was significantly higher in both EPI and ENDO of MI compared to sham hearts. Maximal force was significantly reduced in MI compared to sham, but solely in ENDO cells. A higher passive force was observed in MI hearts, but only in EPI cells. The proportion of stiff N2B isoform was higher in EPI than in ENDO in both sham and MI hearts, and a trend toward increased N2B-proportion appeared in MI EPI, but not MI Endo. Analysis of myofilament protein phosphorylation did not reveal significant transmural differences in phosphorylation of myosin binding protein C, desmin, troponin T, troponin I (cTnI), and myosin light chain 2 (MLC-2) both at BL and during ßAR stimulation with dobutamine infusion. A significant increase in MLC-2 phosphorylation was observed during dobutamine only in sham. In addition, the increase in cTnI phosphorylation upon dobutamine was twofold lower in MI than in sham. CONCLUSION: Myofilament dysfunction is present in both EPI and ENDO in post-MI remodeled myocardium, but shows a high degree of qualitative heterogeneity across the LV wall. These heterogeneous transmural changes in sarcomeric properties likely contribute differently to systolic vs. diastolic global LV dysfunction after MI.

Exercise training does not improve cardiac function in compensated or decompensated left ventricular hypertrophy induced by aortic stenosis.

There is ample evidence that regular exercise exerts beneficial effects on left ventricular (LV) hypertrophy, remodeling and dysfunction produced by ischemic heart disease or systemic hypertension. In contrast, the effects of exercise on pathological LV hypertrophy and dysfunction produced by LV outflow obstruction have not been studied to date. Consequently, we evaluated the effects of 8 weeks of voluntary wheel running in mice (which mitigates post-infarct LV dysfunction) on LV hypertrophy and dysfunction produced by mild (mTAC) and severe (sTAC) transverse aortic constriction. mTAC produced ~40% LV hypertrophy and increased myocardial expression of hypertrophy marker genes but did not affect LV function, SERCA2a protein levels, apoptosis or capillary density. Exercise had no effect on global LV hypertrophy and function in mTAC but increased interstitial collagen, and ANP expression. sTAC produced ~80% LV hypertrophy and further increased ANP expression and interstitial fibrosis and, in contrast with mTAC, also produced LV dilation, systolic as well as diastolic dysfunction, pulmonary congestion, apoptosis and capillary rarefaction and decreased SERCA2a and ryanodine receptor (RyR) protein levels. LV diastolic dysfunction was likely aggravated by elevated passive isometric force and Ca(2+)-sensitivity of myofilaments. Exercise training failed to mitigate the sTAC-induced LV hypertrophy and capillary rarefaction or the decreases in SERCA2a and RyR. Exercise attenuated the sTAC-induced increase in passive isometric force but did not affect myofilament Ca(2+)-sensitivity and tended to aggravate interstitial fibrosis. In conclusion, exercise had no effect on LV function in compensated and decompensated cardiac hypertrophy produced by LV outflow obstruction, suggesting that the effect of exercise on pathologic LV hypertrophy and dysfunction depends critically on the underlying cause.

Enhanced myofilament responsiveness upon ß-adrenergic stimulation in post-infarct remodeled myocardium.

Previously we showed that left ventricular (LV) responsiveness to exercise-induced increases in noradrenaline was blunted in pigs with a recent myocardial infarction (MI) [van der Velden et al. Circ Res. 2004], consistent with perturbed ß-adrenergic receptor (ß-AR) signaling. Here we tested the hypothesis that abnormalities at the myofilament level underlie impaired LV responsiveness to catecholamines in MI. Myofilament function and protein composition were studied in remote LV biopsies taken at baseline and during dobutamine stimulation 3 weeks after MI or sham. Single permeabilized cardiomyocytes demonstrated reduced maximal force (F(max)) and higher Ca(2+)-sensitivity in MI compared to sham. F(max) did not change during dobutamine infusion in sham, but markedly increased in MI. Moreover, the dobutamine-induced decrease in Ca(2+)-sensitivity was significantly larger in MI than sham. Baseline phosphorylation assessed by phosphostaining of ß-AR target proteins myosin binding protein C (cMyBP-C) and troponin I (cTnI) in MI and sham was the same. However, the dobutamine-induced increase in overall cTnI phosphorylation and cTnI phosphorylation at protein kinase A (PKA)-sites (Ser23/24) was less in MI compared to sham. In contrast, the dobutamine-induced phosphorylation of cMyBP-C at Ser282 was preserved in MI, and coincided with increased autophosphorylation (at Thr282) of the cytosolic Ca(2+)-dependent calmodulin kinase II (CaMKII-δC). In conclusion, in post-infarct remodeled myocardium myofilament responsiveness to dobutamine is significantly enhanced despite the lower increase in PKA-mediated phosphorylation of cTnI. The increased myofilament responsiveness in MI may depend on the preserved cMyBP-C phosphorylation possibly resulting from increased CaMKII-δC activity and may help to maintain proper diastolic performance during exercise.

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 Ca(2+) sensitivity was higher in untreated MI compared with sham (both P<0.05). The difference in Ca(2+) sensitivity was abolished by treatment of cells with the beta-adrenergic kinase, protein kinase A. beta-blocker therapy partially reversed F(max) and Ca(2+) sensitivity to sham values and significantly reduced passive force. Despite the lower myofilament Ca(2+) sensitivity in MI+beta compared with untreated myocardium, the protein kinase A induced reduction in Ca(2+) 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.

Altered myocardial substrate metabolism is associated with myocardial dysfunction in early diabetic cardiomyopathy in rats: studies using positron emission tomography.

BACKGROUND: In vitro data suggest that changes in myocardial substrate metabolism may contribute to impaired myocardial function in diabetic cardiomyopathy (DCM). The purpose of the present study was to study in a rat model of early DCM, in vivo changes in myocardial substrate metabolism and their association with myocardial function. METHODS: Zucker diabetic fatty (ZDF) and Zucker lean (ZL) rats underwent echocardiography followed by [11C]palmitate positron emission tomography (PET) under fasting, and [18F]-2-fluoro-2-deoxy-D-glucose PET under hyperinsulinaemic euglycaemic clamp conditions. Isolated cardiomyocytes were used to determine isometric force development. RESULTS: PET data showed a 66% decrease in insulin-mediated myocardial glucose utilisation and a 41% increase in fatty acid (FA) oxidation in ZDF vs. ZL rats (both p < 0.05). Echocardiography showed diastolic and systolic dysfunction in ZDF vs. ZL rats, which was paralleled by a significantly decreased maximal force (68%) and maximal rate of force redevelopment (69%) of single cardiomyocytes. Myocardial functional changes were significantly associated with whole-body insulin sensitivity and decreased myocardial glucose utilisation. ZDF hearts showed a 68% decrease in glucose transporter-4 mRNA expression (p < 0.05), a 22% decrease in glucose transporter-4 protein expression (p = 0.10), unchanged levels of pyruvate dehydrogenase kinase-4 protein expression, a 57% decreased phosphorylation of AMP activated protein kinase alpha1/2 (p < 0.05) and a 2.4-fold increased abundance of the FA transporter CD36 to the sarcolemma (p < 0.01) vs. ZL hearts, which are compatible with changes in substrate metabolism. In ZDF vs. ZL hearts a 2.4-fold reduced insulin-mediated phosphorylation of Akt was found (p < 0.05). CONCLUSION: Using PET and echocardiography, we found increases in myocardial FA oxidation with a concomitant decrease of insulin-mediated myocardial glucose utilisation in early DCM. In addition, the latter was associated with impaired myocardial function. These in vivo data expand previous in vitro findings showing that early alterations in myocardial substrate metabolism contribute to myocardial dysfunction.

Distinct myocardial effects of beta-blocker therapy in heart failure with normal and reduced left ventricular ejection fraction.

AIMS: Left ventricular (LV) myocardial structure and function differ in heart failure (HF) with normal (N) and reduced (R) LV ejection fraction (EF). This difference could underlie an unequal outcome of trials with beta-blockers in heart failure with normal LVEF (HFNEF) and heart failure with reduced LVEF (HFREF) with mixed results observed in HFNEF and positive results in HFREF. To investigate whether beta-blockers have distinct myocardial effects in HFNEF and HFREF, myocardial structure, cardiomyocyte function, and myocardial protein composition were compared in HFNEF and HFREF patients without or with beta-blockers. METHODS AND RESULTS: Patients, free of coronary artery disease, were divided into beta-(HFNEF) (n = 16), beta+(HFNEF) (n = 16), beta-(HFREF) (n = 17), and beta+(HFREF) (n = 22) groups. Using LV endomyocardial biopsies, we assessed collagen volume fraction (CVF) and cardiomyocyte diameter (MyD) by histomorphometry, phosphorylation of myofilamentary proteins by ProQ-Diamond phosphostained 1D-gels, and expression of beta-adrenergic signalling and calcium handling proteins by western immunoblotting. Cardiomyocytes were also isolated from the biopsies to measure active force (F(active)), resting force (F(passive)), and calcium sensitivity (pCa(50)). Myocardial effects of beta-blocker therapy were either shared by HFNEF and HFREF, unique to HFNEF or unique to HFREF. Higher F(active), higher pCa(50), lower phosphorylation of troponin I and myosin-binding protein C, and lower beta(2) adrenergic receptor expression were shared. Higher F(passive), lower CVF, lower MyD, and lower expression of stimulatory G protein were unique to HFNEF and lower expression of inhibitory G protein was unique to HFREF. CONCLUSION: Myocardial effects unique to either HFNEF or HFREF could contribute to the dissimilar outcome of beta-blocker therapy in both HF phenotypes.

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

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 Ca(2+)-sensitivity and sarco(endo)plasmic reticulum Ca(2+)-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 Ca(2+) homeostasis, as myofilament F(max) 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 F(max). 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."

Myofilament degradation and dysfunction of human cardiomyocytes in Fabry disease.

Early detection of myocardial dysfunction in Fabry disease (FD) cardiomyopathy suggests the contribution of myofilament structural alterations. Six males with untreated FD cardiomyopathy submitted to cardiac studies, including tissue Doppler imaging and left ventricular endomyocardial biopsy. Active and resting tensions before and after treatment with protein kinase A (PKA) were determined in isolated Triton-permeabilized cardiomyocytes. Cardiomyocyte cross-sectional area, glycosphingolipid vacuole area, myofibrillolysis, and extent of fibrosis were also determined. Biopsies of mitral stenosis in patients with normal left ventricles served as controls. Active tension was four times lower in FD cardiomyocytes and correlated with extent of myofibrillolysis. Resting tension was six times higher in FD cardiomyocytes than in controls. PKA treatment decreased resting tension but did not affect active force. Protein analysis revealed troponin I and desmin degradation products. FD cardiomyocytes were significantly larger and filled with glycosphingolipids. Fibrosis was mildly increased compared with controls. Tissue Doppler imaging lengthening and shortening velocities were reduced in FD cardiomyocytes compared with controls, correlating with resting and active tensions, respectively, but not with cardiomyocyte area, percentage of glycosphingolipids, or extent of fibrosis. In conclusion, myofilament degradation and dysfunction contribute to FD cardiomyopathy. Partial reversal of high resting tension after pharmacological PKA treatment of cardiomyocytes suggests potential benefits from enzyme replacement therapy and/or energy-releasing agents.

Myofilament dysfunction in cardiac disease from mice to men.

In healthy human myocardium a tight balance exists between receptor-mediated kinases and phosphatases coordinating phosphorylation of regulatory proteins involved in cardiomyocyte contractility. During heart failure, when neurohumoral stimulation increases to compensate for reduced cardiac pump function, this balance is perturbed. The imbalance between kinases and phosphatases upon chronic neurohumoral stimulation is detrimental and initiates cardiac remodelling, and phosphorylation changes of regulatory proteins, which impair cardiomyocyte function. The main signalling pathway involved in enhanced cardiomyocyte contractility during increased cardiac load is the beta-adrenergic signalling route, which becomes desensitized upon chronic stimulation. At the myofilament level, activation of protein kinase A (PKA), the down-stream kinase of the beta-adrenergic receptors (beta-AR), phosphorylates troponin I, myosin binding protein C and titin, which all exert differential effects on myofilament function. As a consequence of beta-AR down-regulation and desensitization, phosphorylation of the PKA-target proteins within the cardiomyocyte may be decreased and alter myofilament function. Here we discuss involvement of altered PKA-mediated myofilament protein phosphorylation in different animal and human studies, and discuss the roles of troponin I, myosin binding protein C and titin in regulating myofilament dysfunction in cardiac disease. Data from the different animal and human studies emphasize the importance of careful biopsy procurement, and the need to investigate localization of kinases and phosphatases within the cardiomyocyte, in particular their co-localization with cardiac myofilaments upon receptor stimulation.

Frequency-dependent myofilament Ca2+ desensitization in failing rat myocardium.

The positive force-frequency relation, one of the key factors modulating performance of healthy myocardium, has been attributed to an increased Ca(2+) influx per unit of time. In failing hearts, a blunted, flat or negative force-frequency relation has been found. In healthy and failing hearts frequency-dependent alterations in Ca(2+) sensitivity of the myofilaments, related to different phosphorylation levels of contractile proteins, could contribute to this process. Therefore, the frequency dependency of force, intracellular free Ca(2+) ([Ca(2+)](i)), Ca(2+) sensitivity and contractile protein phosphorylation were determined in control and monocrotaline-treated, failing rat hearts. An increase in frequency from 0.5 to 6 Hz resulted in an increase in force in control (14.3 +/- 3.0 mN mm(-2)) and a decrease in force in failing trabeculae (9.4 +/- 3.2 mN mm(-2)), whereas in both groups the amplitude of [Ca(2+)](i) transient increased. In permeabilized cardiomyocytes, isolated from control hearts paced at 0 and 9 Hz, Ca(2+) sensitivity remained constant with frequency (pCa(50): 5.55 +/- 0.02 and 5.58 +/- 0.01, respectively, P>0.05), whereas in cardiomyocytes from failing hearts Ca(2+) sensitivity decreased with frequency (pCa(50): 5.62 +/- 0.01 and 5.57 +/- 0.01, respectively, P<0.05). After incubation of the cardiomyocytes with protein kinase A (PKA) this frequency dependency of Ca(2+) sensitivity was abolished. Troponin I (TnI) and myosin light chain 2 (MLC2) phosphorylation remained constant in control hearts but both increased with frequency in failing hearts. In conclusion, in heart failure frequency-dependent myofilament Ca(2+) desensitization, through increased TnI phosphorylation, contributes to the negative force-frequency relation and is counteracted by a frequency-dependent MLC2 phosphorylation. We propose a novel role for PKC-mediated TnI phosphorylation in modulating the force-frequency relation.

Early exercise training normalizes myofilament function and attenuates left ventricular pump dysfunction in mice with a large myocardial infarction.

The extent and mechanism of the cardiac benefit of early exercise training following myocardial infarction (MI) is incompletely understood, but may involve blunting of abnormalities in Ca(2+)-handling and myofilament function. Consequently, we investigated the effects of 8-weeks of voluntary exercise, started early after a large MI, on left ventricular (LV) remodeling and dysfunction in the mouse. Exercise had no effect on survival, MI size or LV dimensions, but improved LV fractional shortening from 8+/-1 to 12+/-1%, and LVdP/dt(P30) from 5295+/-207 to 5794+/-207 mm Hg/s (both P<0.05), and reduced pulmonary congestion. These global effects of exercise were associated with normalization of the MI-induced increase in myofilament Ca(2+)-sensitivity (DeltapCa(50)=0.037). This effect of exercise was PKA-mediated and likely because of improved beta(1)-adrenergic signaling, as suggested by the increased beta(1)-adrenoceptor protein (48%) and cAMP levels (36%; all P<0.05). Exercise prevented the MI-induced decreased maximum force generating capacity of skinned cardiomyocytes (F(max) increased from 14.3+/-0.7 to 18.3+/-0.8 kN/m(2) P<0.05), which was associated with enhanced shortening of unloaded intact cardiomyocytes (from 4.1+/-0.3 to 7.0+/-0.6%; P<0.05). Furthermore, exercise reduced diastolic Ca(2+)-concentrations (by approximately 30%, P<0.05) despite the unchanged SERCA2a and PLB expression and PLB phosphorylation status. Importantly, exercise had no effect on Ca(2+)-transient amplitude, indicating that the improved LV and cardiomyocyte shortening were principally because of improved myofilament function. In conclusion, early exercise in mice after a large MI has no effect on LV remodeling, but attenuates global LV dysfunction. The latter can be explained by the exercise-induced improvement of myofilament function.

Impaired diastolic function after exchange of endogenous troponin I with C-terminal truncated troponin I in human cardiac muscle.

The specific and selective proteolysis of cardiac troponin I (cTnI) has been proposed to play a key role in human ischemic myocardial disease, including stunning and acute pressure overload. In this study, the functional implications of cTnI proteolysis were investigated in human cardiac tissue for the first time. The predominant human cTnI degradation product (cTnI(1-192)) and full-length cTnI were expressed in Escherichia coli, purified, reconstituted with the other cardiac troponin subunits, troponin T and C, and subsequently exchanged into human cardiac myofibrils and permeabilized cardiomyocytes isolated from healthy donor hearts. Maximal isometric force and kinetic parameters were measured in myofibrils, using rapid solution switching, whereas force development was measured in single cardiomyocytes at various calcium concentrations, at sarcomere lengths of 1.9 and 2.2 mum, and after treatment with the catalytic subunit of protein kinase A (PKA) to mimic beta-adrenergic stimulation. One-dimensional gel electrophoresis, Western immunoblotting, and 3D imaging revealed that approximately 50% of endogenous cTnI had been homogeneously replaced by cTnI(1-192) in both myofibrils and cardiomyocytes. Maximal tension was not affected, whereas the rates of force activation and redevelopment as well as relaxation kinetics were slowed down. Ca(2+) sensitivity of the contractile apparatus was increased in preparations containing cTnI(1-192) (pCa(50): 5.73+/-0.03 versus 5.52+/-0.03 for cTnI(1-192) and full-length cTnI, respectively). The sarcomere length dependency of force development and the desensitizing effect of PKA were preserved in cTnI(1-192)-exchanged cardiomyocytes. These results indicate that degradation of cTnI in human myocardium may impair diastolic function, whereas systolic function is largely preserved.

Functional effects of protein kinase C-mediated myofilament phosphorylation in human myocardium.

OBJECTIVE: In human heart failure beta-adrenergic-mediated protein kinase A (PKA) activity is down-regulated, while protein kinase C (PKC) activity is up-regulated. PKC-mediated myofilament protein phosphorylation might be detrimental for contractile function in cardiomyopathy. This study was designed to reveal the effects of PKC on myofilament function in human myocardium under basal conditions and upon modulation of protein phosphorylation by PKA and phosphatases. METHODS: Isometric force was measured at different [Ca(2+)] in single permeabilized cardiomyocytes from non-failing and failing human left ventricular tissue. Basal phosphorylation of myofilament proteins and the influence of PKC, PKA, and phosphatase treatments were analyzed by one- and two-dimensional gel electrophoresis, Western immunoblotting, and ELISA. RESULTS: Troponin I (TnI) phosphorylation at the PKA sites was decreased in failing compared to non-failing hearts and correlated well with myofilament Ca(2+) sensitivity (pCa(50)). Incubation with the catalytic domain of PKC slightly decreased maximal force under basal conditions, but not following PKA and phosphatase pretreatments. PKC reduced Ca(2+) sensitivity to a larger extent in failing (DeltapCa(50)=0.19+/-0.03) than in non-failing (DeltapCa(50)=0.08+/-0.01) cardiomyocytes. This shift was reduced, though still significant, when PKC was preceded by PKA, while PKA following PKC did not further decrease pCa(50). Protein analysis indicated that PKC phosphorylated PKA sites in human TnI and increased phosphorylation of troponin T, while myosin light chain phosphorylation remained unaltered. CONCLUSION: In human myocardium PKC-mediated myofilament protein phosphorylation only has a minor effect on maximal force development. The PKC-mediated decrease in Ca(2+) sensitivity may serve to improve diastolic function in failing human myocardium in which PKA-mediated TnI phosphorylation is decreased.