Damage to mitochondrial complex I during cardiac ischemia reperfusion injury is reduced indirectly by anti-anginal drug ranolazine

Abstract

Ranolazine, an anti-anginal drug, is a late Na(+) channel current blocker that is also believed to attenuate fatty acid oxidation and mitochondrial respiratory complex I activity, especially during ischemia. In this study, we investigated if ranolazine's protective effect against cardiac ischemia/reperfusion (IR) injury is mediated at the mitochondrial level and specifically if respiratory complex I (NADH Ubiquinone oxidoreductase) function is protected. We treated isolated and perfused guinea pig hearts with ranolazine just before 30 min ischemia and then isolated cardiac mitochondria at the end of 30 min ischemia and/or 30 min ischemia followed by 10 min reperfusion. We utilized spectrophotometric and histochemical techniques to assay complex I activity, Western blot analysis for complex I subunit NDUFA9, electron paramagnetic resonance for activity of complex I Fe-S clusters, enzyme linked immuno sorbent assay (ELISA) for determination of protein acetylation, native gel histochemical staining for respiratory supercomplex assemblies, and high pressure liquid chromatography for cardiolipin integrity; cardiac function was measured during IR. Ranolazine treated hearts showed higher complex I activity and greater detectable complex I protein levels compared to untreated IR hearts. Ranolazine treatment also led to more normalized electron transfer via Fe-S centers, supercomplex assembly and cardiolipin integrity. These improvements in complex I structure and function with ranolazine were associated with improved cardiac function after IR. However, these protective effects of ranolazine are not mediated by a direct action on mitochondria, but rather indirectly via cytosolic mechanisms that lead to less oxidation and better structural integrity of complex I.

Fig. 1

A: Representative spectrophotometric assay of mitochondrial complex I activity during cardiac ischemia reperfusion depicting the time points of addition of substrate, enzyme and the inhibitor. B: Summary data shows ischemia alone reduced the activity of the enzyme, which was restored by treatment with ranolazine. Reperfusion itself corrected the decrease in activity, but this was not as pronounced as with ranolazine (Ran) on reperfusion. Note that the activities depicted in B have been corrected for rotenone sensitivity and normalized to citrate synthase levels. * indicates p<0.05 for I30/IR vs. TC; # indicates p<0.05 for RanI30/RanIR vs. I30/IR

Fig. 1

A: Representative spectrophotometric assay of mitochondrial complex I activity during cardiac ischemia reperfusion depicting the time points of addition of substrate, enzyme and the inhibitor. B: Summary data shows ischemia alone reduced the activity of the enzyme, which was restored by treatment with ranolazine. Reperfusion itself corrected the decrease in activity, but this was not as pronounced as with ranolazine (Ran) on reperfusion. Note that the activities depicted in B have been corrected for rotenone sensitivity and normalized to citrate synthase levels. * indicates p<0.05 for I30/IR vs. TC; # indicates p<0.05 for RanI30/RanIR vs. I30/IR

Fig. 2

Upper panel: Representative histochemical gel staining of complex I activity, measured as NBT-oxidoreductase, during cardiac ischemia reperfusion. Lower panel: Summary data shows ischemia alone resulted in lower staining than in time controls and reperfusion. Ran treatment increased staining, indicating improved complex I function. * indicates p<0.05 for I30/IR vs. TC; # indicates p<0.05 for RanI30/RanIR vs. I30/IR

Fig. 3

Determination of supercomplex assemblies using native PAGE. Panel A: Coomassie stained gel after native PAGE. SC1 and SC2 indicate supercomplexes composed of complexes I, III and IV, with varying copies of each complex. Components of supercomplex determined by histochemical staining for complex I (panel B), complexes III and IV (panel C) and by Western blot analysis of SC1 and SC2 against respiratory complex subunits (panel D). Summary data of supercomplex assemblies from Coomassie stained gels (panel E) shows reduction in supercomplex assemblies following ischemia and reperfusion, and restoration by ranolazine treatment. * indicates p<0.05 for I30/IR vs. TC; # indicates p<0.05 for RanI30/RanIR vs. I30/IR

Fig. 3

Determination of supercomplex assemblies using native PAGE. Panel A: Coomassie stained gel after native PAGE. SC1 and SC2 indicate supercomplexes composed of complexes I, III and IV, with varying copies of each complex. Components of supercomplex determined by histochemical staining for complex I (panel B), complexes III and IV (panel C) and by Western blot analysis of SC1 and SC2 against respiratory complex subunits (panel D). Summary data of supercomplex assemblies from Coomassie stained gels (panel E) shows reduction in supercomplex assemblies following ischemia and reperfusion, and restoration by ranolazine treatment. * indicates p<0.05 for I30/IR vs. TC; # indicates p<0.05 for RanI30/RanIR vs. I30/IR

Fig. 4

Upper panel: Representative western blot detection of complex I subunit NDUFA9 during cardiac ischemia reperfusion. Lower panel: Summary data shows that ischemia reduced the amount of detectable subunit, indicating a loss of protein or protein damage. Both reperfusion and Ran treatment during ischemia restored protein levels to time control levels. * indicates p<0.05 for I30/IR vs. TC; # indicates p<0.05 for RanI30/RanIR vs. I30/IR

Fig. 5

A: Representative traces of EPR signals during cardiac ischemia reperfusion denoting changes in three mitochondrial Fe-S clusters and semiubiquinone. B: Summary data shows the changes in spectral magnitudes of the Fe-S clusters and semiubiquinone. Ischemia increased electron transfer within complex I and semiubiquinone; this was reversed on reperfusion. Ran had a small effect to decrease electron transfer during reperfusion. I, II, III = respiratory complexes; N and Rieske = FeS clusters. * indicates p<0.05 for I30/IR vs. TC; # indicates p<0.05 for RanI30/RanIR vs. I30/IR

Fig. 5

A: Representative traces of EPR signals during cardiac ischemia reperfusion denoting changes in three mitochondrial Fe-S clusters and semiubiquinone. B: Summary data shows the changes in spectral magnitudes of the Fe-S clusters and semiubiquinone. Ischemia increased electron transfer within complex I and semiubiquinone; this was reversed on reperfusion. Ran had a small effect to decrease electron transfer during reperfusion. I, II, III = respiratory complexes; N and Rieske = FeS clusters. * indicates p<0.05 for I30/IR vs. TC; # indicates p<0.05 for RanI30/RanIR vs. I30/IR

Fig. 6

A: Representative traces of cardiolipin integrity by HPLC analysis during cardiac ischemia reperfusion. B: Summary data shows that ischemia alone reduced the area under the major peak of cardiolipin, and reperfusion decreased it further. The additional peaks seen in the spectra could be modified/breakdown products of cardiolipin. Treatment with ranolazine restored peak heights and also decreased the number of peaks, indicating that ranolazine treatment preserves cardiolipin. * indicates p<0.05 for I30/IR vs. TC; # indicates p<0.05 for RanI30/RanIR vs. I30/IR

Fig. 6

A: Representative traces of cardiolipin integrity by HPLC analysis during cardiac ischemia reperfusion. B: Summary data shows that ischemia alone reduced the area under the major peak of cardiolipin, and reperfusion decreased it further. The additional peaks seen in the spectra could be modified/breakdown products of cardiolipin. Treatment with ranolazine restored peak heights and also decreased the number of peaks, indicating that ranolazine treatment preserves cardiolipin. * indicates p<0.05 for I30/IR vs. TC; # indicates p<0.05 for RanI30/RanIR vs. I30/IR