The increased Ca(2+)-responsiveness of the contractile apparatus in end-stage failing human hearts cannot be explained by a shift in contractile protein isoforms, but results from the complex interplay between changes in the phosphorylation status of MLC-2 and TnI.

Rationale High-myofilament Ca2+-sensitivity has been proposed as trigger of disease pathogenesis in familial hypertrophic cardiomyopathy (HCM) based on in vitro and transgenic mice studies. However, myofilament Ca2+-sensitivity depends on protein phosphorylation and muscle length, and at present, data in human are scarce. Objective To investigate whether high-myofilament Ca2+-sensitivity and perturbed length-dependent activation are characteristics for human HCM with mutations in thick- and thin-filament proteins. Methods and Results 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 Ca2+-sensitivity in all HCM samples and low phosphorylation of protein kinase A (PKA)-targets compared with donors. After exogenous PKA treatment, myofilament Ca2+-sensitivity was either 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. Conclusions High-myofilament Ca2+-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 post-translational modifications other than PKA-hypophosphorylation or altered protein–protein interactions, and represents a common pathomechanism in HCM.

Myocardial infarction (MI) initiates cardiac remodeling, depresses pump function, and predisposes to heart failure. This study was designed to identify early alterations in Ca2+ handling and myofilament proteins, which may contribute to contractile dysfunction and reduced beta-adrenergic responsiveness in postinfarct remodeled myocardium. Protein composition and contractile function of skinned cardiomyocytes were studied in remote, noninfarcted left ventricular (LV) subendocardium from pigs 3 weeks after MI caused by permanent left circumflex artery (LCx) ligation and in sham-operated pigs. LCx ligation induced a 19% increase in LV weight, a 69% increase in LV end-diastolic area, and a decrease in ejection fraction from 54+/-5% to 35+/-4% (all P<0.05), whereas cardiac responsiveness to exercise-induced increases in circulating noradrenaline levels was blunted. Endogenous protein kinase A (PKA) was significantly reduced in remote myocardium of MI animals, and a negative correlation (R=0.62; P<0.05) was found between cAMP levels and LV weight-to-body weight ratio. Furthermore, SERCA2a expression was 23% lower after MI compared with sham. Maximal isometric force generated by isolated skinned myocytes was significantly lower after MI than in sham (15.4+/-1.5 versus 19.2+/-0.9 kN/m2; P<0.05), which might be attributable to a small degree of troponin I (TnI) degradation observed in remodeled postinfarct myocardium. An increase in Ca2+ sensitivity of force (pCa50) was observed after MI compared with sham (DeltapCa50=0.17), which was abolished by incubating myocytes with exogenous PKA, indicating that the increased Ca2+ sensitivity resulted from reduced TnI phosphorylation. In conclusion, remodeling of noninfarcted pig myocardium is associated with decreased SERCA2a and myofilament function, which may contribute to depressed LV function. The full text of this article is available online at http://circres.ahajournals.org.

Abstract-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 2ϩ -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 8Ϯ1 to 12Ϯ1%, and LVdP/dt P30 from 5295Ϯ207 to 5794Ϯ207 mm Hg/s (both PϽ0.05), and reduced pulmonary congestion. These global effects of exercise were associated with normalization of the MI-induced increase in myofilament Ca 2ϩ -sensitivity (⌬pCa 50 ϭ0.037). This effect of exercise was PKA-mediated and likely because of improved ␤ 1 -adrenergic signaling, as suggested by the increased ␤ 1 -adrenoceptor protein (48%) and cAMP levels (36%; all PϽ0.05). Exercise prevented the MI-induced decreased maximum force generating capacity of skinned cardiomyocytes (F max increased from 14.3Ϯ0.7 to 18.3Ϯ0.8 kN/m 2 PϽ0.05), which was associated with enhanced shortening of unloaded intact cardiomyocytes (from 4.1Ϯ0.3 to 7.0Ϯ0.6%; PϽ0.05). Furthermore, exercise reduced diastolic Ca 2ϩ -concentrations (by ϳ30%, PϽ0.05) despite the unchanged SERCA2a and PLB expression and PLB phosphorylation status. Importantly, exercise had no effect on Ca 2ϩ -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. (Circ Res. 2007;100:1079-1088.)Key Words: cardiac function Ⅲ cardiomyocytes Ⅲ exercise training Ⅲ heart failure L eft ventricular (LV) remodeling after myocardial infarction (MI) is a compensatory mechanism that serves to restore LV pump function. Despite the apparent appropriateness of LV remodeling to maintain cardiac pump function early after MI, remodeling is an independent risk factor for the development of congestive heart failure. 1 The mechanism underlying the progression from LV remodeling to overt heart failure remains incompletely understood, but recent evidence indicates that abnormalities in myofilament function and Ca 2ϩ -handling contribute to the LV dysfunction in the porcine heart, early after MI. 2 In contrast to pathological LV remodeling after MI, LV remodeling produced by regular dynamic exercise is associated with a decreased risk for coronary artery disease and heart failure. 3 Exercise training is associated with an increased myocardial perfusion capacity and with normal or even increased contractile function in the normal heart. 4,5 There is also clinical evidence that exercise after MI has a beneficial effect on disease progression and survival. 6,7 For example, physical conditioning in patients with L...

Surprisingly, the contractile response to MLC-2 dephosphorylation is enhanced in failing hearts, despite the reduced level of basal MLC-2 phosphorylation. The enhanced response to MLC-2 dephosphorylation in failing myocytes might result from differences in basal phosphorylation of other thin and thick filament proteins between donor and failing hearts. Regulation of Ca(2+)-sensitivity via MLC-2 phosphorylation may be a potential compensatory mechanism to reverse the detrimental effects of increased Ca(2+)-sensitivity and impaired Ca(2+)-handling on diastolic function in human heart failure.

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.

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...

Background-During ischemia, the intracellular calcium and inorganic phosphate (P i ) concentrations rise and pH falls. We investigated the effects of these changes on force development in donor and failing human hearts to determine if altered contractile protein composition during heart failure changes the myocardial response to Ca 2ϩ , P i , and pH. Methods and Results-Isometric force was studied in mechanically isolated Triton-skinned single myocytes from left ventricular myocardium. Force declined with added P i to 0.33Ϯ0.02 of the control force (pH 7.1, 0 mmol/L P i ) at 30 mmol/L P i and increased with pH from 0.64Ϯ0.03 at pH 6.2 to 1.27Ϯ0.02 at pH 7.4. Force dependency on P i and pH did not differ between donor and failing hearts. Incubation of myocytes in a P i -containing activating solution caused a potentiation of force, which was larger at submaximal than at maximal [Ca 2ϩ ]. Ca 2ϩ sensitivity of force was similar in donor hearts and hearts with moderate cardiac disease, but in end-stage failing myocardium it was significantly increased. The degree of myosin light chain 2 phosphorylation was significantly decreased in end-stage failing compared with donor myocardium, resulting in an inverse correlation between Ca 2ϩ responsiveness of force and myosin light chain 2 phosphorylation. Conclusions-Our results indicate that contractile protein alterations in human end-stage heart failure alter Ca