Meiying Yang

Ranolazine reduces Ca2+ overload and oxidative stress and improves mitochondrial integrity to protect against ischemia reperfusion injury in isolated hearts

Ranolazine is a clinically approved drug for treating cardiac ventricular dysrhythmias and angina. Its mechanism(s) of protection is not clearly understood but evidence points to blocking the late Na+ current that arises during ischemia, blocking mitochondrial complex I activity, or modulating mitochondrial metabolism. Here we tested the effect of ranolazine treatment before ischemia at the mitochondrial level in intact isolated hearts and in mitochondria isolated from hearts at different times of reperfusion. Left ventricular (LV) pressure (LVP), coronary flow (CF), and O2 metabolism were measured in guinea pig isolated hearts perfused with Krebs-Ringers solution; mitochondrial (m) superoxide (O2·-), Ca2+, NADH/FAD (redox state), and cytosolic (c) Ca2+ were assessed on-line in the LV free wall by fluorescence spectrophotometry. Ranolazine (5 μM), infused for 1 min just before 30 min of global ischemia, itself did not change O2·-, cCa2+, mCa2+ or redox state. During late ischemia and reperfusion (IR) O2·- emission and m[Ca2+] increased less in the ranolazine group vs. the control group. Ranolazine decreased c[Ca2+] only during ischemia while NADH and FAD were not different during IR in the ranolazine vs. control groups. Throughout reperfusion LVP and CF were higher, and ventricular fibrillation was less frequent. Infarct size was smaller in the ranolazine group than in the control group. Mitochondria isolated from ranolazine-treated hearts had mild resistance to permeability transition pore (mPTP) opening and less cytochrome c release than control hearts. Ranolazine may provide functional protection of the heart during IR injury by reducing cCa2+ and mCa2+ loading secondary to its effect to block the late Na+ current. Subsequently it indirectly reduces O2·- emission, preserves bioenergetics, delays mPTP opening, and restricts loss of cytochrome c, thereby reducing necrosis and apoptosis.

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

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 ranolazines 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.

Tyrosine nitration of voltage-dependent anion channels in cardiac ischemia-reperfusion: reduction by peroxynitrite scavenging

Excess superoxide (O(2)(-)) and nitric oxide (NO) forms peroxynitrite (ONOO(-)) during cardiac ischemia reperfusion (IR) injury, which in turn induces protein tyrosine nitration (tyr-N). Mitochondria are both a source of and target for ONOO(-). Our aim was to identify specific mitochondrial proteins that display enhanced tyr-N after cardiac IR injury, and to explore whether inhibiting O(2)(-)/ONOO(-) during IR decreases mitochondrial protein tyr-N and consequently improves cardiac function. We show here that IR increased tyr-N of 35 and 15kDa mitochondrial proteins using Western blot analysis with 3-nitrotyrosine antibody. Immunoprecipitation (IP) followed by LC-MS/MS identified 13 protein candidates for tyr-N. IP and Western blot identified and confirmed that the 35kDa tyr-N protein is the voltage-dependent anion channel (VDAC). Tyr-N of native cardiac VDAC with IR was verified on recombinant (r) VDAC with exogenous ONOO(-). We also found that ONOO(-) directly enhanced rVDAC channel activity, and rVDAC tyr-N induced by ONOO(-) formed oligomers. Resveratrol (RES), a scavenger of O(2)(-)/ONOO(-), reduced the tyr-N levels of both native and recombinant VDAC, while L-NAME, which inhibits NO generation, only reduced tyr-N levels of native VDAC. O(2)(-) and ONOO(-) levels were reduced in perfused hearts during IR by RES and L-NAME and this was accompanied by improved cardiac function. These results identify tyr-N of VDAC and show that reducing ONOO(-) during cardiac IR injury can attenuate tyr-N of VDAC and improve cardiac function.

Reversible Blockade of Complex I or Inhibition of PKCβ Reduces Activation and Mitochondria Translocation of p66Shc to Preserve Cardiac Function after Ischemia

Aim Excess mitochondrial reactive oxygen species (mROS) play a vital role in cardiac ischemia reperfusion (IR) injury. P66Shc, a splice variant of the ShcA adaptor protein family, enhances mROS production by oxidizing reduced cytochrome c to yield H2O2. Ablation of p66Shc protects against IR injury, but it is unknown if and when p66Shc is activated during cardiac ischemia and/or reperfusion and if attenuating complex I electron transfer or deactivating PKCβ alters p66Shc activation during IR is associated with cardioprotection. Methods Isolated guinea pig hearts were perfused and subjected to increasing periods of ischemia and reperfusion with or without amobarbital, a complex I blocker, or hispidin, a PKCβ inhibitor. Phosphorylation of p66Shc at serine 36 and levels of p66Shc in mitochondria and cytosol were measured. Cardiac functional variables and redox states were monitored online before, during and after ischemia. Infarct size was assessed in some hearts after 120 min reperfusion. Results Phosphorylation of p66Shc and its translocation into mitochondria increased during reperfusion after 20 and 30 min ischemia, but not during ischemia only, or during 5 or 10 min ischemia followed by 20 min reperfusion. Correspondingly, cytosolic p66Shc levels decreased during these ischemia and reperfusion periods. Amobarbital or hispidin reduced phosphorylation of p66Shc and its mitochondrial translocation induced by 30 min ischemia and 20 min reperfusion. Decreased phosphorylation of p66Shc by amobarbital or hispidin led to better functional recovery and less infarction during reperfusion. Conclusion Our results show that IR activates p66Shc and that reversible blockade of electron transfer from complex I, or inhibition of PKCβ activation, decreases p66Shc activation and translocation and reduces IR damage. These observations support a novel potential therapeutic intervention against cardiac IR injury.

Identity and function of a cardiac mitochondrial small conductance Ca2+-activated K+ channel splice variant

We provide evidence for location and function of a small conductance, Ca2+-activated K+ (SKCa) channel isoform 3 (SK3) in mitochondria (m) of guinea pig, rat and human ventricular myocytes. SKCa agonists protected isolated hearts and mitochondria against ischemia/reperfusion (IR) injury; SKCa antagonists worsened IR injury. Intravenous infusion of a SKCa channel agonist/antagonist, respectively, in intact rats was effective in reducing/enhancing regional infarct size induced by coronary artery occlusion. Localization of SK3 in mitochondria was evidenced by Western blot of inner mitochondrial membrane, immunocytochemical staining of cardiomyocytes, and immunogold labeling of isolated mitochondria. We identified a SK3 splice variant in guinea pig (SK3.1, aka SK3a) and human ventricular cells (SK3.2) by amplifying mRNA, and show mitochondrial expression in mouse atrial tumor cells (HL-1) by transfection with full length and truncated SK3.1 protein. We found that the N-terminus is not required for mitochondrial trafficking but the C-terminus beyond the Ca2+ calmodulin binding domain is required for Ca2+ sensing to induce mK+ influx and/or promote mitochondrial localization. In isolated guinea pig mitochondria and in SK3 overexpressed HL-1 cells, mK+ influx was driven by adding CaCl2. Moreover, there was a greater fall in membrane potential (ΔΨm), and enhanced cell death with simulated cell injury after silencing SK3.1 with siRNA. Although SKCa channel opening protects the heart and mitochondria against IR injury, the mechanism for favorable bioenergetics effects resulting from SKCa channel opening remains unclear. SKCa channels could play an essential role in restraining cardiac mitochondria from inducing oxidative stress-induced injury resulting from mCa2+ overload.

Reduced Tyrosine Nitration of VDAC and Decreased Apoptosis by Mitochondria-Directed Therapy After Cardiac Ischemia Reperfusion in Isolated Hearts

Superoxide (O2•-) produced during cardiac ischemia-reperfusion (IR) injury reacts with nitric oxide to form peroxynitrite (ONOO-). ONOO- induces protein tyrosine nitration (tyrN) that causes protein structural alteration and dysfunction. The mitochondrial voltage-dependent anion channel (VDAC) plays an important role in regulating the metabolic and energetic functions of mitochondria and contributes to mitochondrial-mediated apoptosis. It is not known if VDAC is nitrated by ONOO- during IR or how this modification might compromise cardiac function after IR. Because of the importance of VDAC modification, we hypothesized that the clinically used anti-anginal drug ranolazine (RAN), which also reduces cardiac IR injury, does so via a mitochondrial mechanism, i.e., in part by decreasing VDAC tyrN. To test this, isolated guinea pig hearts were perfused with KR buffer for 40 min (time control, TC), or for 30 min of ischemia plus 10 min of reperfusion, with or without 10 µM RAN infused before ischemia. Mitochondria were isolated at the end of each treatment. VDAC tyrN was determined by IP with anti-nitrotyrosine antibody (NTab), followed by Western blotting (WB) with anti-VDAC antibody. The effect of RAN on VDAC tyrN was also examined. Cytochrome c release was checked as the marker for apoptosis. We found that enhanced VDAC tyrN was increased by 108% after IR vs. TC and cytochrome c was higher in the cytosol after IR than after TC. RAN treatment decreased VDAC tyrN by 31%, while decreasing cytochrome c release by 38%, compared to IR. These results indicate that VDAC tyrN and a concomitant increase in cytochrome c release occur during IR injury, and importantly, that cardioprotection by RAN occurs in part by reducing VDAC tyrN, which may impede activation of apoptotic pathways during IR injury.

Differential Decreases in Respiratory Complex I and II Expression and Activity after Cardiac Ischemia and Reperfusion and Modulation by Mitochondria-Targeted Therapy

Mitochondrial respiratory complexes are variably susceptible to structural and functional changes during ischemia reperfusion injury (IR). Complex I may be most susceptible to IR and complex II (SQR), both an electron donor and TCA cycle intermediate, relatively less susceptible. We tested for differential expression and activities of complexes I and II during IR +/- a mitochondria-targeted therapy (MTT). Guinea pig hearts were exposed to one of 5 protocols: 40 min time control (TC), 30 min global ischemia (I30), 30 min ischemia with 10 min reperfusion (IR10), and 10 µM ranolazine (Ran), as a MTT, given 1 min before I30 (Ran+I30) or IR (Ran+IR10). Mitochondria were isolated and assayed for complex I (Ndufa9) and complex II (FP) protein expression (western blotting), or complex I and II enzyme activities (absorbance spectrophotometry) after hypotonic freeze-thaw rupture. Complex I expression at I30 and activity at I30 and IR10 were markedly decreased; Ran treatment nearly restored complex I expression and activity after I30 and IR10. Complex II expression was not altered by I or IR, +/- Ran, complex II activity was decreased less than in complex I, and Ran increased activity only with I30. Differences in complex I and II expression and activities may result from more deleterious post-translational modifications triggered by I and IR in complex I than II. A more compromised function in complex I may reduce forward electron flux to complex II, allowing increased electron flux from succinate to ubiquinone because of less compromised SQR activity. Further studies will ascertain the mechanism behind the greater functional impairment of complex I vs. complex II after IR and the partial restoration of complex I and II activity by Ran, a known cardioprotective drug.