Loss of mitochondrial MnSOD in acute cardiac rejection: synergistic protection by low-dose cyclosporine plus vitamin C

Abstract

Background: Tyrosine nitration (NO-Tyr) of proteins may occur via peroxynitrite (ONOO−), the product of nitric oxide (NO) and superoxide, or via secondary radicals formed after reaction of ONOO− and endogenous bicarbonate to form nitrosoperoxocarboxylate. Tyrosine nitration of purified manganese superoxide dismutase (MnSOD) by ONOO− leads to decreased enzyme activity, thereby increasing oxidative stress. Purpose: We examined the role of NO derived from inducible nitric oxide synthase (iNOS) on MnSOD protein and activity in acute cardiac rejection, and the benefits of treatment with vitamin C (VitC). Methods: Lewis (isograft) or Wistar-Furth (allograft) donor hearts were transplanted into Lewis recipient rats. Rats were untreated or treated daily with low-dose cyclosporine (CsA) 2.5 mg/kg i.m., VitC 120 mg/100 g gavage or VitC+CsA. We determined histological rejections scores, MnSOD activity, MnSOD and iNOS protein (Western blots), plasma NO metabolites and NO-Tyr (slot blot). Results: Rejection scores (untreated: 4.6 ± 0.2; VitC: 4.1 ± 0.4; CsA: 2.6 ± 0.3, P < 0.001 vs. untreated; VitC + CsA: 3.4 ± 0.4, P < 0.05 vs. untreated) as well as myocardial fractional shortening determined by sonomicrometry were improved by treatment (untreated: 0.77 ± 0.17; VitC: 1.45 ± 0.09; CsA: 2.24 ± 0.22, P < 0.01 vs. untreated; VitC + CsA: 1.77 ± 0.19, P < 0.05 vs. untreated). MnSOD activity was decreased on POD6 (5.8 ± 1.3 U/mg/min) but not POD4 (12.1 ± 2.1 U/mg/min) in untreated allografts vs. isografts (10.4 ± 2.5 U/mg/min). On POD6, MnSOD protein was decreased by 50% and was prevented by treatment with an iNOS dimerization inhibitor, L-N6-(1-iminoethyl) lysine (LNIL), (2.7 ± 0.3, 1.5 ± 0.4, 3.5 ± 0.4 arbitrary units for isografts, allografts and treated allografts, respectively). Loss of MnSOD protein was partially protected by CsA or VitC alone, but fully protected by VitC + CsA. VitC alone had no effect on iNOS protein while CsA alone or VitC + CsA decreased iNOS to a similar level. NO-Tyr was elevated by 3-fold in allografts at POD6. In contrast to effects of treatments on iNOS, VitC or CsA alone decreased NO-Tyr (untreated: 45 ± 9; VitC: 32 ± 1; CsA: 26 ± 6, ng NO-Tyr-BSA equivalents). Conclusions: Decreases in MnSOD protein occur via an iNOS-dependent pathway. As VitC alone did not change iNOS protein or NO production (i.e., NO metabolites) and the published rate constant of reaction of VitC with superoxide is modest, it is unlikely that VitC preserves MnSOD protein simply by limiting ONOO− formation. Rather, VitC may act to decrease NO-Tyr by quenching tyrosyl radicals produced secondary to ONOO−formation.