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 (EC50,peroxynitrite = 49 mu M; EC50,DTDP = 2.75 mM). However, k(tr,max) was decreased only by 2.5-mM DTDP, but not by 50 mu M 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 (delta F-o,F-:similar to 13%; P < 0.05) despite full reversion in protein SH content after 50 mu M 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.