The peroxynitrite evoked contractile depression can be partially reversed by antioxidants in human cardiomyocytes.

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 2,2'-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 (F(o)) and the maximal turnover rate of the cross-bridge cycle (k(tr,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 F(o) in a concentration-dependent manner (EC(50,peroxynitrite) = 49 microM; EC(50,DTDP) = 2.75 mM). However, k(tr,max) was decreased only by 2.5-mM DTDP, but not by 50 microM peroxynitrite. The diminution of F(o) to zero by DTDP was paralleled by the complete elimination of the free SH groups, while the peroxynitrite-induced maximal reduction in free SH groups was only to 58 +/- 6% of the control (100%). The diminutions in F(o) and free SH groups evoked by 2.5-mM DTDP were completely reverted by DTT. In contrast, DTT induced only a partial restoration in F(o) (DeltaF(o,): approximately 13%; P < 0.05) despite full reversion in protein SH content after 50 microM peroxynitrite. Although, NAC or DTT were equally effective on F(o) after peroxynitrite exposures, NAC or GSH did not restore F(o) or k(tr,max) after DTDP treatments. Our results revealed that the peroxynitrite-evoked cardiomyocyte dysfunction has a small, but significant component resulting from reversible SH oxidation, and thereby illustrated the potential benefit of antioxidants during cardiac pathologies with excess peroxynitrite production.

Protein kinase C contributes to the maintenance of contractile force in human ventricular cardiomyocytes.

Prolonged Ca(2+) 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 micromol/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 PKCalpha over PKCdelta and PKCepsilon (189 +/- 31, 7 +/- 3, and 7 +/- 2 ng/mg protein, respectively). Ca(2+) stimulations of reconstituted human cardiomyocytes resulted in the translocation of endogenous PKCalpha, but not PKCbeta1, delta, and epsilon from the cytosol to the contractile system (PKCalpha association: control, 5 +/- 3 arbitrary units; +Ca(2+), 39 +/- 8 arbitrary units; p < 0.01, EC(50,Ca) = 645 nmol/liter). One of the PKCalpha-binding proteins were identified as the thin filament regulatory protein cardiac troponin I (TnI). Finally, the Ca(2+)-dependent interaction between PKCalpha and TnI was confirmed using purified recombinant proteins (binding without Ca(2+) was only 28 +/- 18% of that with Ca(2+)). Our data suggest that PKCalpha 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.

Oxidation of myofilament protein sulfhydryl groups reduces the contractile force and its Ca2+ sensitivity in human cardiomyocytes.

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 Na+/Ca2+ exchange blocker SEA0400 fails to enhance cytosolic Ca2+ transient and contractility in canine ventricular cardiomyocytes.

AIMS: This study was designed to evaluate the effects of the Na(+)/Ca(2+) exchange (NCX) inhibitor SEA0400 on Ca(2+) handling in isolated canine ventricular myocytes. METHODS AND RESULTS: Intracellular Ca(2+) ([Ca(2+)](i)) transients, induced by either field stimulation or caffeine flush, were monitored using Ca(2+) indicator dyes. [Ca(2+)](i)-dependent modulation of the inhibitory effect of SEA0400 on NCX was characterized by the changes in Ni(2+)-sensitive current in voltage-clamped myocytes. Sarcoplasmic reticulum (SR) Ca(2+) release and uptake were studied in SR membrane vesicles. Gating properties of single-ryanodine receptors were analysed in lipid bilayers. Ca(2+) sensitivity of the contractile machinery was evaluated in chemically skinned myocytes. In myocytes paced at 1 Hz, neither diastolic [Ca(2+)](i) nor the amplitude of [Ca(2+)](i) transients was significantly altered by SEA0400 up to the concentration of 1 microM, which was shown to inhibit the exchange current. The blocking effect of SEA0400 on NCX decreased with increasing [Ca(2+)](i), and it was more pronounced in reverse than in forward mode operation at every [Ca(2+)](i) examined. The rate of decay of the caffeine-induced [Ca(2+)](i) transients was decreased significantly by 1 microM SEA0400; however, this effect was only a fraction of that observed with 10 mM NiCl(2). Neither SR Ca(2+) release and uptake nor cell shortening and Ca(2+) sensitivity of the contractile proteins were influenced by SEA0400. CONCLUSION: 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.

Positive inotropic effect of levosimendan is correlated to its stereoselective Ca2+-sensitizing effect but not to stereoselective phosphodiesterase inhibition.

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 EC50 values of 60 nM and 2.8 microM 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 Ca2+-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 Ca2+-sensitization with EC50 values of 8.4 nM and 0.64 microM, 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 IC50 values of 7.5 nM, and 3.2 microM, respectively. Taken together, our data support the hypothesis that levosimendan and dextrosimendan exert their positive inotropic effects via a stereoselective Ca2+-sensitizing mechanism and not via stereoselective inhibition of phosphodiesterase III in the myocardium.