This study sought to characterize the relation between the oxidation of protein sulfhydryl (SH) groups and Ca2+-activated force production in the human myocardium. Triton-permeabilized left ventricular cardiomyocytes from donor hearts were exposed to an oxidative (2,2'-dithiodipyridine, DTDP) agent in vitro, and the changes in isometric force, its Ca2+ sensitivity, the cross-bridge-sensitive rate constant of force redevelopment at saturating [Ca2+] (k(tr,max)), and protein SH oxidation were monitored. DTDP (0.1-10 mM for 2 min) oxidized the myocardial proteins and diminished the Ca2+-activated force with different concentration dependences (EC(50,SH) = 0.17 +/- 0.02 mM and EC(50,force) = 2.46 +/- 0.22 mM; mean +/- SEM). The application of 2.5 mM DTDP decreased the maximal Ca2+-activated force (to 64%), its Ca2+ sensitivity (DeltapCa(50) = 0.22 +/- 0.02), and the steepness of the Ca2+-force relation (n(Hill), from 2.01 +/- 0.08 to 1.76 +/- 0.08). These changes were paralleled by reductions in the free SH content of the proteins (to 15%) and in k(tr,max) (to 75%). SH-specific labeling identified SH oxidation of myosin light chain 1 and actin at DTDP concentrations at which the changes in the contractile parameters occurred. Our data suggest that SH oxidation in selected myofilament proteins diminishes the Ca2+-activated force and its Ca2+ sensitivity through an impaired Ca2+ regulation of the actin-myosin cycle in the human heart.
The lack of any major SEA0400-induced shift in Ca(2+) transients or contractility of myocytes can well be explained by its limited inhibitory effect on NCX (further attenuated by elevated [Ca(2+)](i) levels) and a concomitant reduction in Ca(2+) influx due to the predominantly reverse mode blockade of NCX and suppression of L-type Ca(2+) current.
In order to clarify the mechanisms of the positive inotropic actions of levosimendan and its optical isomer, dextrosimendan, we compared their concentration-dependent effects in intact papillary muscles, permeabilized cardiomyocytes and in purified phosphodiesterase enzyme preparations of guinea-pig hearts. In papillary muscles twitch tension increased with EC 50 values of 60 nM and 2.8 mM for levosimendan and dextrosimendan, respectively. Hence, the two enantiomers exhibited a 47 times potency difference in their positive inotropic effects in a preparation where theoretically Ca 2π -sensitization and phosphodiesterase inhibition could both contribute to the positive inotropic effects. In guinea-pig cardiomyocytes, levosimendan and dextrosimendan increased isometric force production (at pCa 6.2) due to Ca 2π -sensitization with EC 50 values of 8.4 nM and 0.64 mM, respectively, with a similar relative potency difference of 76. A major difference appeared in their relative pharmacological potencies, however, when the inhibitory effects of the two enantiomers were assayed on phosphodiesterase III, purified from guinea pig left ventricle (i.e. the phosphodiesterase isoenzyme which is dominant in that tissue). Levosimendan was a 427 times more potent phosphodiesterase inhibitor than dextrosimendan, with IC 50 values of 7.5 nM, and 3.2 mM, respectively. Taken together, our data support the hypothesis that levosimendan and dextrosimendan exert their positive inotropic effects via a stereoselective Ca 2π -sensitizing mechanism and not via stereoselective inhibition of phosphodiesterase III in the myocardium.Levosimendan is a positive inotropic and vasodilatory drug developed for the treatment of acute de novo or decompensated congestive heart failure (Follath et al. 2002). The drug binds to the Ca 2π saturated cardiac isoform of troponin C (Pollesello et al. 1994;Sorsa et al. 2001Sorsa et al. & 2003 and this binding is stereoselective (Sorsa et al. 2004). A correlation between the binding to troponin C and the positive inotropic effect of levosimendan has been established (Haikala et al. 1995a; Levijoki et al. 2000). However, the molecule displays structural similarities with a family of inhibitors of phosphodiesterases, which leads to an alternative hypothesis for its cardiotonic effects that signifies a possible phosphodiesterase inhibitory action (Boknik et al. 1997) and consequent augmentation of the Ca 2π transient. In contrast to this hypothesis, however, several reports show that levosimendan need not increase the intracellular Ca 2π concentration for the development of a sensible positive inotropic effect (Lancaster & Cook 1997;Hasenfuss et al. 1998;Brixius et al. 2002). To elucidate the pharmacological background of levosimendan-induced positive inotropy, the effects of levosimendan and of its active metabolite, OR-1896 as positive inotropes, Ca terase inhibitors were recently critically compared (Szilagyi et al. 2004). This study demonstrated well-pronounced similarities in the concentration dependen...
Prolonged Ca2؉ stimulations often result in a decrease in contractile force of isolated, demembranated human ventricular cardiomyocytes, whereas intact cells are likely to be protected from this deterioration. We hypothesized that cytosolic protein kinase C (PKC) contributes to this protection. Prolonged contracture (10 min) of demembranated human cardiomyocytes at half-maximal Ca 2؉ resulted in a 37 ؎ 5% reduction of active force (p < 0.01), whereas no decrease (2 ؎ 3% increase) was observed in the presence of the cytosol (reconstituted myocytes). The PKC inhibitors GF 109203X and Gö 6976 (10 mol/liter) partially antagonized the cytosol-mediated protection (15 ؎ 5 and 9 ؎ 2% decrease in active force, p < 0.05). Quantitation of PKC isoform expression revealed the dominance of the Ca 2؉ -dependent PKC␣ over PKC␦ and PKC⑀ (189 ؎ 31, 7 ؎ 3, and 7 ؎ 2 ng/mg protein, respectively). Ca 2؉ stimulations of reconstituted human cardiomyocytes resulted in the translocation of endogenous PKC␣, but not PKC1, ␦, and ⑀ from the cytosol to the contractile system (PKC␣ association: control, 5 ؎ 3 arbitrary units; ؉Ca ). Our data suggest that PKC␣ translocates to the contractile system and anchors to TnI in a Ca 2؉ -dependent manner in the human heart, contributing to the maintenance of contractile force. Protein kinase C (PKC)3 is a family of serine/threonine kinases (1). Multiple PKC isozymes are often expressed in the same cell, mediating specific functions. Conventional and novel PKCs can be activated by lipids, like the endogenous diacylglycerol (DAG) or the exogenous phorbol ester PMA. It was reported decades ago that PMA activation of PKC leads to the translocation of PKC from the soluble to the particulate fraction (2). This observation has been confirmed by later works, and some of the binding proteins for activated PKC isozymes were identified (receptors for activated C kinases) (3, 4). Binding to its respective receptors for activated C kinase localizes each PKC isozyme in the vicinity of a subset of substrates and away from others, and hence this spatial organization may well explain the specificity of PKC isozymes in their intracellular signaling.It is of interest that from the many PKC isozymes expressed in the heart (5), PKC␣ is the single isozyme that translocates to the contractile system upon Ca 2ϩ stimulation in the rat heart (6), suggesting a unique physiological role for PKC␣ in the Ca 2ϩ -dependent regulation of myofibrillar contractility. As a matter of fact, PKC␣ has been implicated in models of ischemic heart failure, myocardial hypertrophy, hypertension, and atherosclerosis (7). In addition, PKC-dependent phosphorylation of myofibrillar proteins such as desmin (8), myosin light chain (9), troponin I (TnI), and troponin T (TnT) (10) has been documented with suggested functional consequences ranging from changes in mechanical integrity of the cardiac sarcomere to decreased actin-myosin ATPase activity and force generation.Long term activation of PKC is an essential step in ischemic preconditioning (11), a...
IntroductionOxidative and nitrosative radicals have been associated with the development of myocardial tissue injury during chronic heart failure, reperfusion that follows ischaemia and in response to inflammatory cytokines or cardiotoxic drugs [1][2][3][4][5][6][7][8][9]. Under these pathologic conditions, various protein and lipid molecules serve as targets for the accumulating oxidative and/or nitrosative agents [10,11]. Proteins of the sarcomere are of special interest because their alterations will influence the structure and/or the regulation of the interaction between the thin and the thick myofilaments, and thereby will affect directly the conversion of chemical energy into force and shortening [12,13].Peroxynitrite, a metabolite of nitric oxide, is frequently cited as one of the most damaging nitrosative agents [2,4,14] Abstract In this study, we aimed to determine the contribution of peroxynitrite-dependent sulfhydryl group (SH) oxidation to the contractile dysfunction in permeabilized left ventricular human cardiomyocytes using a comparative approach with the SH-oxidant 2,2Ј-dithiodipyridine (DTDP). Additionally, different antioxidants: dithiothreitol (DTT), reduced glutathione (GSH) or N-acetyl-L-cysteine (NAC) were employed to test reversibility. Maximal isometric active force production (Fo) and the maximal turnover rate of the cross-bridge cycle (ktr,max) illustrated cardiomyocyte mechanics. SH oxidation was monitored by a semi-quantitative Ellman's assay and by SH-specific protein biotinylation. Both peroxynitrite and DTDP diminished Fo in a concentration-dependent manner (EC50,peroxynitrite
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