Abstract 154: Hydrogen Peroxide-Induced Contractile Dysfunction is Mediated Through Oxidation of SERCA on Cysteine-674

Abstract

Background: Reactive oxygen species (ROS) are critical mediators of cardiomyocyte contractile dysfunction. We previously reported that H 2 O 2 induces thiol oxidative post-translational modifications of the calcium handling protein sarco/endoplasmic reticulum calcium ATPase (SERCA), leading to a reduction of its activity and contractile dysfunction. Here, we tested the hypothesis that cysteine-674 of SERCA is critical for H 2 O 2 -induced contractile dysfunction in adult rat ventricular myocytes (ARVM). Methods: Overexpression of wild-type (WT) or mutant SERCA, in which cysteine-674 was mutated to serine (C674S), was accomplished by 36 hours of adenoviral infection. ARVM were paced at 5Hz and superfused with 100μM H 2 O 2 . Cell shortening and calcium transients were measured over 20 minutes using video-edge detection and fura-2 fluorescence. Thiol oxidation of SERCA was assessed using biotinylated iodoacet- amide (BIAM)-labeling. Results: After 14min of H 2 O 2 exposure, cell shortening was markedly reduced in WT-SERCA overexpressing ARVM (−43 ± 21%) but not in C674S-SERCA overex-pressing ARVM (−49% ± 31%, p<0.05 vs WT-SERCA, n=21–24/group). Time of 50% cell relengthening was prolonged by 15 ± 5% in WT-SERCA ARVM (p<0.05 vs time 0) but not in C674S-SERCA cells (+1 ± 4%, NS vs time 0). Compared with WT-SERCA ARVM, expression of C674S-SERCA attenuated the H 2 O 2 -induced reduction in calcium transient amplitude (−51 ± 11% vs −87 ± 6%, p<0.05 C674S-SERCA vs WT-SERCA). The 50% calcium reuptake time was prolonged in WT-SERCA compared with C674S-SERCA ARVM (60 ± 3ms vs 46 ± 3ms, p<0.05), reflecting a decrease in SERCA activity in WT-SERCA but not in C674S-SERCA ARVM. H 2 O 2 decreased BIAM-labeling in ARVM overexpressing WT-SERCA by 40% ± 7% (p<0.01,n=3) but failed to decrease BIAM-labeling in ARVM overexpressing C674S-SERCA. Conclusion: Increased thiol oxidative post-translational modification was correlated with a reduced SERCA activity and calcium handling abnormalities, and these effects were attenuated in ARVM overexpressing mutated SERCA modified at Cys674. These findings suggest that oxidation on Cys674 of SERCA is responsible for H 2 O 2 -induced reduction in its activity leading to contractile dysfunction.