Mitochondrial Approaches to Protect Against Cardiac Ischemia and Reperfusion Injury

The mitochondrion is a vital component in cellular energy metabolism and intracellular signaling processes. Mitochondria are involved in a myriad of complex signaling cascades regulating cell death vs. survival. Importantly, mitochondrial dysfunction and the resulting oxidative and nitrosative stress are central in the pathogenesis of numerous human maladies including cardiovascular diseases, neurodegenerative diseases, diabetes, and retinal diseases, many of which are related. This review will examine the emerging understanding of the role of mitochondria in the etiology and progression of cardiovascular diseases and will explore potential therapeutic benefits of targeting the organelle in attenuating the disease process. Indeed, recent advances in mitochondrial biology have led to selective targeting of drugs designed to modulate or manipulate mitochondrial function, to the use of light therapy directed to the mitochondrial function, and to modification of the mitochondrial genome for potential therapeutic benefit. The approach to rationally treat mitochondrial dysfunction could lead to more effective interventions in cardiovascular diseases that to date have remained elusive. The central premise of this review is that if mitochondrial abnormalities contribute to the etiology of cardiovascular diseases (e.g., ischemic heart disease), alleviating the mitochondrial dysfunction will contribute to mitigating the severity or progression of the disease. To this end, this review will provide an overview of our current understanding of mitochondria function in cardiovascular diseases as well as the potential role for targeting mitochondria with potential drugs or other interventions that lead to protection against cell injury

Mitochondrial Handling of Excess Ca\u3csup\u3e2+\u3c/sup\u3e is Substrate-dependent with Implications for Reactive Oxygen Species Generation

The mitochondrial electron transport chain is the major source of reactive oxygen species (ROS) during cardiac ischemia. Several mechanisms modulate ROS production; one is mitochondrial Ca2+ uptake. Here we sought to elucidate the effects of extramitochondrial Ca2+ (e[Ca2+]) on ROS production (measured as H2O2 release) from complexes I and III. Mitochondria isolated from guinea pig hearts were preincubated with increasing concentrations of CaCl2 and then energized with the complex I substrate Na+ pyruvate or the complex II substrate Na+ succinate. Mitochondrial H2O2 release rates were assessed after giving either rotenone or antimycin A to inhibit complex I or III, respectively. After pyruvate, mitochondria maintained a fully polarized membrane potential (ΔΨ; assessed using rhodamine 123) and were able to generate NADH (assessed using autofluorescence) even with excess e[Ca2+] (assessed using CaGreen-5N), whereas they remained partially depolarized and did not generate NADH after succinate. This partial ΔΨ depolarization with succinate was accompanied by a large release in H2O2 (assessed using Amplex red/horseradish peroxidase) with later addition of antimycin A. In the presence of excess e[Ca2+], adding cyclosporin A to inhibit mitochondrial permeability transition pore opening restored ΔΨ and significantly decreased antimycin A-induced H2O2 release. Succinate accumulates during ischemia to become the major substrate utilized by cardiac mitochondria. The inability of mitochondria to maintain a fully polarized ΔΨ under excess e[Ca2+] when succinate, but not pyruvate, is the substrate may indicate a permeabilization of the mitochondrial membrane, which enhances H2O2 emission from complex III during ischemia

Ranolazine Reduces Ca\u3csup\u3e2+\u3c/sup\u3e 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-Ringer’s solution; mitochondrial (m) 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 1min 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 the control group. Mitochondria isolated from ranolazinetreated 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

Enhanced Na\u3csup\u3e+\u3c/sup\u3e/H\u3csup\u3e+\u3c/sup\u3e Exchange During Ischemia and Reperfusion Impairs Mitochondrial Bioenergetics and Myocardial Function

Inhibition of Na+/H+ exchange (NHE) during ischemia reduces cardiac injury due to reduced reverse mode Na+/Ca2+ exchange. We hypothesized that activating NHE-1 at buffer pH 8 during ischemia increases mitochondrial oxidation, Ca2+ overload, and reactive O2 species (ROS) levels and worsens functional recovery in isolated hearts and that NHE inhibition reverses these effects. Guinea pig hearts were perfused with buffer at pH 7.4 (control) or pH 8 +/- NHE inhibitor eniporide for 10 minutes before and for 10 minutes after 35- minute ischemia and then for 110 minutes with pH 7.4 buffer alone. Mitochondrial NADH and FAD, [Ca2+], and superoxide were measured by spectrophotofluorometry. NADH and FAD were more oxidized, and cardiac function was worse throughout reperfusion after pH 8 versus pH 7.4, Ca2+ overload was greater at 10-minute reperfusion, and superoxide generation was higher at 30-minute reperfusion. The pH 7.4 and eniporide groups exhibited similar mitochondrial function, and cardiac performance was most improved after pH 7.4+eniporide. Cardiac function on reperfusion after pH 8+eniporide was better than after pH 8. Percent infarction was largest after pH 8 and smallest after pH 7.4+eniporide. Activation of NHE with pH 8 buffer and the subsequent decline in redox state with greater ROS and Ca2+ loading underlie the poor functional recovery after ischemia and reperfusion

Enhanced charge-independent Mitochondrial Free Ca\u3csup\u3e2+\u3c/sup\u3e and Attenuated ADP-induced NADH Oxidation by Isoflurane: Implications for Cardioprotection

Modulation of mitochondrial free Ca2 + ([Ca2 +]m) is implicated as one of the possible upstream factors that initiates anesthetic-mediated cardioprotection against ischemia–reperfusion (IR) injury. To unravel possible mechanisms by which volatile anesthetics modulate [Ca2 +]m and mitochondrial bioenergetics, with implications for cardioprotection, experiments were conducted to spectrofluorometrically measure concentration-dependent effects of isoflurane (0.5, 1, 1.5, 2 mM) on the magnitudes and time-courses of [Ca2 +]m and mitochondrial redox state (NADH), membrane potential (ΔΨm), respiration, and matrix volume. Isolated mitochondria from rat hearts were energized with 10 mM Na+- or K+-pyruvate/malate (NaPM or KPM) or Na+-succinate (NaSuc) followed by additions of isoflurane, 0.5 mM CaCl2 (≈ 200 nM free Ca2 + with 1 mM EGTA buffer), and 250 μM ADP. Isoflurane stepwise: (a) increased [Ca2 +]m in state 2 with NaPM, but not with KPM substrate, despite an isoflurane-induced slight fall in ΔΨm and a mild matrix expansion, and (b) decreased NADH oxidation, respiration, ΔΨm, and matrix volume in state 3, while prolonging the duration of state 3 NADH oxidation, respiration, ΔΨm, and matrix contraction with PM substrates. These findings suggest that isoflurane\u27s effects are mediated in part at the mitochondrial level: (1) to enhance the net rate of state 2 Ca2 + uptake by inhibiting the Na+/Ca2 + exchanger (NCE), independent of changes in ΔΨm and matrix volume, and (2) to decrease the rates of state 3 electron transfer and ADP phosphorylation by inhibiting complex I. These direct effects of isoflurane to increase [Ca2 +]m, while depressing NCE activity and oxidative phosphorylation, could underlie the mechanisms by which isoflurane provides cardioprotection against IR injury at the mitochondrial level

Isoflurane Modulates Cardiac Mitochondrial Bioenergetics by Selectively Attenuating Respiratory Complexes

Mitochondrial dysfunction contributes to cardiac ischemia–reperfusion (IR) injury but volatile anesthetics (VA) may alter mitochondrial function to trigger cardioprotection. We hypothesized that the VA isoflurane (ISO) mediates cardioprotection in part by altering the function of several respiratory and transport proteins involved in oxidative phosphorylation (OxPhos). To test this we used fluorescence spectrophotometry to measure the effects of ISO (0, 0.5, 1, 2 mM) on the time-course of interlinked mitochondrial bioenergetic variables during states 2, 3 and 4 respiration in the presence of either complex I substrate K+-pyruvate/malate (PM) or complex II substrate K+-succinate (SUC) at physiological levels of extra-matrix free Ca2 + (~ 200 nM) and Na+ (10 mM). To mimic ISO effects on mitochondrial functions and to clearly delineate the possible ISO targets, the observed actions of ISO were interpreted by comparing effects of ISO to those elicited by low concentrations of inhibitors that act at each respiratory complex, e.g. rotenone (ROT) at complex I or antimycin A (AA) at complex III. Our conclusions are based primarily on the similar responses of ISO and titrated concentrations of ETC. inhibitors during state 3. We found that with the substrate PM, ISO and ROT similarly decreased the magnitude of state 3 NADH oxidation and increased the duration of state 3 NADH oxidation, ΔΨm depolarization, and respiration in a concentration-dependent manner, whereas with substrate SUC, ISO and ROT decreased the duration of state 3 NADH oxidation, ΔΨm depolarization and respiration. Unlike AA, ISO reduced the magnitude of state 3 NADH oxidation with PM or SUC as substrate. With substrate SUC, after complete block of complex I with ROT, ISO and AA similarly increased the duration of state 3 ΔΨm depolarization and respiration. This study provides a mechanistic understanding in how ISO alters mitochondrial function in a way that may lead to cardioprotection

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

Reversible Blockade of Complex I or Inhibition of PKCβ Reduces Activation and Mitochondria Translocation of p66\u3csup\u3eShc\u3c/sup\u3e 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

Adding ROS Scavengers to Cold K\u3csup\u3e+\u3c/sup\u3e Cardioplegia Reduces Superoxide Emission During 2 h Global Cold Cardiac Ischemia

We reported that the combination of reactive oxygen species (ROS) quenchers Mn(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), catalase, and glutathione (MCG) given before 2 hours cold ischemia better protected cardiac mitochondria against cold ischemia and warm reperfusion (IR)-induced damage than MnTBAP alone. Here, we hypothesize that high K+ cardioplegia (CP) plus MCG would provide added protection of mitochondrial bioenergetics and cardiac function against IR injury. Using fluorescence spectrophotometry, we monitored redox balance, ie reduced nicotinamide adenine dinucleotide and flavin adenine dinucleotide (NADH/FAD), superoxide (O2 •−), and mitochondrial Ca2+ (m[Ca2+]) in the left ventricular free wall. Guinea pig isolated hearts were perfused with either Krebs Ringer’s (KR) solution, CP, or CP + MCG, before and during 27°C perfusion followed immediately by 2 hours of global ischemia at 27°C. Drugs were washed out with KR at the onset of 2 hours 37°C reperfusion. After 120 minutes warm reperfusion, myocardial infarction was lowest in the CP + MCG group and highest in the KR group. Developed left ventricular pressure recovery was similar in CP and CP + MCG and was better than in the KR group. O2 •−, m[Ca2+], and NADH/FAD were significantly different between the treatment and KR groups. O2 •− was lower in CP + MCG than in the CP group. This study suggests that CP and ROS quenchers act in parallel to improve mitochondrial function and to provide protection against IR injury at 27°C

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 ranolazine\u27s 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