Ranolazine Reduces Mitochondrial Tyrosine Nitration During Cardiac Ischemia and Reperfusion Injury

Abstract

Excess superoxide (O2•-) and nitric oxide (NO•) generate peroxynitrite (OONO-) during cardiac ischemia-reperfusion (IR) injury. NO• alone may be cardioprotective whereas OONO- has deleterious effects. Tyrosine nitration by OONO- may lead to dysfunctional mitochondrial proteins. Ranolazine (RAN), a slow Na+ channel blocker and anti-ischemic drug, may also attenuate mitochondrial complex I respiratory activity. We tested if the tyrosine nitration of mitochondrial proteins that occurred during IR was reduced when RAN was given just before ischemia. Method: Guinea pig hearts were perfused with Krebs -Ringer solution and subjected to one of six treatments: (i) control (no ischemia), (ii) 30 min global ischemia alone, (iii) 30 min ischemia + 10 min reperfusion, (iv) ischemia reperfusion plus RAN given for 10 min before, but not during ischemia, (v) ischemia plus RAN (no reperfusion), (vi) RAN control perfusion (no ischemia). Mitochondria were isolated immediately after each treatment. Tyrosine nitration was measured by Western blotting using 3nitro-tyrosine (3-NT) antibody. Result: RAN markedly improved cardiac function. Two bands positioned at about 25 kDa and 15 kDa were 3-NT immunopositive in all experiment groups. Compared to the control, mitochondria after ischemia reperfusion displayed increased 3-NT immunopositivity at the 25 kDa and 15 kDa positions by approximately 100% and 28%, respectively. Treating hearts with RAN before ischemia reperfusion decreased the 3-NT immunopositive 25 kDa band density to non-ischemia levels and the 15 kDa band density to 10% of the ischemia reperfusion alone level. The nitrated proteins require further identification. Conclusion: Cardiac injury increases the tyrosine nitration of selected mitochondrial proteins. Inhibition of complex I may underlie the cardiac injury-induced increase in mitochondrial protein tyrosine nitration. This reduction in mitochondrial protein nitration may correlate with the improved cardiac function we observed previously with RAN.