Mitochondria as a therapeutic target in ischemia



by D. J . Hausenloy, United Kingdom

Derek J. HAUSENLOY, MD, PhD Cardiovascular and Metabolic Disorders Program, Duke-NUS Graduate Medical School SINGAPORE National Heart Research Institute Singapore, National Heart Centre Singapore, SINGAPORE The Hatter Cardiovascular Institute Institute of Cardiovascular Science University College London London, UNITED KINGDOM National Institute of Health Research University College London Hospitals Biomedical Research Centre London, UNITED KINGDOM

I schemic heart disease is the leading cause of death and disability worldwide. As such, novel therapeutic targets are urgently required to protect the heart against the detrimental effects of acute ischemia/reperfusion injury in order to preserve cardiac function and improve clinical outcomes in patients with ischemic heart disease. In this regard, mitochondria, which are the powerhouses of the cell and which make up one-third of the volume of a cardiomyocyte, are an important target for cardioprotection. Elucidation of the signaling pathways underlying the endogenous cardioprotective phenomenon of ischemic conditioning, in which the heart can be protected by brief nonlethal episodes of ischemia and reperfusion, has identified mitochondria to be the end-effector in many of the signal transduction pathways. In this article, we review the role of mitochondria as targets for protecting the heart against acute ischemia/reperfusion injury, the therapeutic application of which should help improve clinical outcomes in patients with ischemic heart disease.

Ischemic heart disease remains the leading cause of death and disability both in Europe specifically and worldwide. As such, there remains an urgent need to discover novel therapies that can protect the heart against the detrimental effects of acute ischemia/reperfusion injury (IRI) in order to preserve left ventricular (LV) systolic function and prevent the onset of heart failure. In this article, we review the role of mitochondria as a therapeutic target for protecting the heart against acute IRI. In the heart, mitochondria occupy nearly one-third the volume of a cardiomyocyte, highlighting their importance as the “powerhouses” of the cell, providing the energy required for normal cardiac contractile function.

Metabolic and biochemical consequences of acute ischemia/reperfusion

An acute coronary artery occlusion results in a critical reduction in coronary blood flow and deprivation of oxygen and nutrients to the affected area of myocardium, and it impairs the clearance of waste metabolites, subjecting cardiomyocytes to the abrupt metabolic and biochemical changes associated with acute myocardial ischemia. If blood flow is restored to the affected area by the removal of the coronary artery occlusion, the ischemic myocytes are then exposed to the further metabolic and biochemical changes associated with the reperfusion process. The combined injury sustained by the myocardium during these processes is termed acute myocardial IRI, and the sequential metabolic and biochemical perturbations that occur during this process are reviewed below and presented in Figure 1. Deprivation of oxygen during acute myocardial ischemia impairs oxidative phosphorylation by reducing electron flow through the mitochondrial electron transport chain, leading to an accumulation of NADH and FADH and cessation of adenosine triphosphate (ATP) production.1 The mitochondrial membrane potential collapses as it is no longer maintained by the electrochemical gradient across the inner mitochondrial membrane.2 Intracellular creatine phosphate is depleted with a concomitant rise in intracellular inorganic phosphate (Pi), resulting in mitochondria accumulating Pi.3 Residual reserves of ATP are hydrolyzed by F0F1-ATPase in an attempt to restore the mitochondrial membrane potential,4 resulting in catalytic metabolites such as hypoxanthine, which are oxidized to release free radicals. The activity of the adenine nucleotide translocase is reduced, impairing oxidative phosphorylation further still.5

The reduced availability of ATP and oxygen drives anaerobic glycolysis, which results in lactic acid accumulation, leading to intracellular acidification. The fall in pH activates the sodium (Na+)/hydrogen (H+) exchanger in an effort to remove cytosolic protons, which causes the entry of Na+. The Na+/potassium (K+)-ATPase, which normally removes excess Na+, is inhibited because of the reduced availability of ATP and the increase in intracellular Pi. This results in a rise in intracellular Na+, which triggers the Na+/calcium (Ca2+) exchanger to function in reverse in order to remove cytosolic Na+.6 However, this occurs at the expense of an increase in intracellular Ca2+. The rise in cytosolic Ca2+ results in the mitochondrial accumulation of Ca2+ via the mitochondrial Na+/Ca2+ exchanger.7 The onset of rigor contracture coincides with the depletion of ATP,8 and is followed by cellular Ca2+ overload.9

Therefore, after an episode of acute myocardial ischemia, the metabolic and biochemical derangements include a low intracellular pH (<7.0), high intracellular [Ca2+] and [Pi ], and ATP depletion (Figure 1), conditions which are exacerbated once the acutely ischemic myocardium is reperfused.

Figure 1. The metabolic and biochemical changes that occur during acute myocardial ischemia and  reperfusion. During acute myocardial ischemia, there is an increase in intracellular calcium (Ca2+), inorganic phosphate (Pi), sodium (Na+), reactive oxygen species (ROS), NADH, and hydrogen (H+), a fall in adenosine triphosphate (ATP) levels, and collapse of the mitochondrial membrane potential (Δψm). At reperfusion, there is repolarization of the Δψm and restoration of mitochondrial ATP production; a further increase in Ca2+, Pi, and ROS; oxidation of NADH; and restoration of neutral pH—factors that mediate cell death by inducing mitochondrial permeability transition pore (MPTP) opening and cardiomyocyte hypercontracture.

Reperfusion of acutely ischemic myocardium has several important consequences: (i) re-energization of the cardiomyocyte, causing repolarization of the mitochondrial membrane potential; (ii) reoxygenation of a reduced mitochondrial respiratory chain, resulting in the production of reactive oxygen species (ROS) and oxidation of NADH/FADH; (iii) a drop in intracellular Ca2+, but a further influx of Ca2+ into mitochondria via the Ca2+-uniporter driven by the restored mitochondrial membrane potential9; and (iv) the wash-out of lactic acid, which in combination with the reactivation of the Na+/H+ exchange acts to restore a neutral pH. Many of the biochemical and metabolic changes that take place during the first few minutes of reperfusion can mediate cardiomyocyte death by inducing the opening of the mitochondrial permeability transition pore (MPTP), a nonselective channel of the inner mitochondrial membrane whose opening uncouples oxidative phosphorylation, resulting in ATP depletion and cell death by necrosis.10

Therefore, the ultimate aim of any cardioprotective strategy designed to protect the cardiomyocyte against acute IRI is to preserve cellular energetic and ionic homeostasis during this insult. In this regard, protecting mitochondrial function to preserve energy production in response to acute myocardial IRI is an important strategy of cardioprotection, which has been investigated in both experimental and clinical studies (see Figure 2).

Figure 2. Mitochondrial targets for cardioprotection. The diagram provides a simplified scheme of some of the potential mitochondrial targets for cardioprotection, many of which have been elucidated from studies investigating the signaling pathways underlying ischemic conditioning. These signal transduction pathways include the RISK (involving PI3K-Akt and MEK1/2-Erk1/2), SAFE (involving JAK-STAT), and NO-cGMP pathways, all of which terminate at the mitochondria and, particularly, the mitochondrial permeability transition pore (MPTP). These reperfusion salvage pathways have been shown to activate downstream mediators, such as eNOS, GSK3b, HKII, PKCe, and KATP, which then mediate the inhibitory effect on MPTP opening. The modulation of mitochondrial energetics by actions on the electron transport chain and adenosine triphosphate production can also indirectly prevent MPTP opening in response to acute ischemia/reperfusion injury (IRI). Cyclosporin A protects against acute IRI by inhibiting MPTP opening via inhibition of CypD. TRO40303 is believed to protect the heart by inhibiting MPTP opening via attenuation of reactive oxygen species (ROS) production in response to acute IRI. MTP-131 protects the heart against acute IRI by improving mitochondrial energetics via targeting of cardiolipin in the inner mitochondrial membrane. Trimetazidine protects the heart against acute IRI by inhibiting fatty acid oxidation, thereby promoting glucose oxidation, the result of which is improved mitochondrial energetics. Ivabradine protects the heart against acute IRI by lowering the heart rate and through pleiotropic effects, which include attenuation of ROS production and inhibition of MPTP opening. Abbreviations: cGMP, cyclic guanosine monophosphate; Cardio, cardiolipin; CypD, cyclophilin D; eNOS, endothelial nitric oxide synthase; Erk1/2, extracellular signal–regulated kinase 1/2; ETC, electron transport chain; GSK3β, glycogen synthase kinase 3b; HKII, Hexokinase II; IRI, ischemia/reperfusion injury; JAK, Janus kinase; KATP, mitochondrial adenosine-triphosphate–dependent potassium channel; MEK1/2, mitogen-activated protein kinase (MAP)/extracellular signal–regulated kinase (ERK)1/2; MPTP, mitochon-drial permeability transition pore; NO, nitric oxide; PI3K, phosphatidylinositol 3-kinase; PKCε, protein kinase C epsilon; PKG, protein kinase G; ROS, reactive oxygen species; SAFE, survivor activating factor enhancement; STAT, signal transducer and activator of transcription; RISK, reperfusion injury salvage kinase.
Modified from reference 64: Hausenloy et al. Basic Res Cardiol. 2009;104(2):189-202. © 2009, Springer-Verlag.

Inhibiting MPTP opening to protect the heart against acute IRI

The MPTP is a nonselective channel of the inner mitochondrial membrane, the opening of which mediates cell death by uncoupling oxidative phosphorylation and inducing mitochondrial swelling, resulting in ATP depletion and necrotic cell death.10 The molecular composition of the MPTP is not clear, although it has been suggested that ATP synthase11-13 and mitochondrial cyclophilin D14,15 are important components. In the setting of an acute myocardial infarction (AMI), it has been shown to remain closed during acute myocardial ischemia and to be open in only the first few minutes of reperfusion. Therefore, preventing its opening at the onset of reperfusion is an important therapeutic strategy for reducing myocardial infarct (MI) size after an AMI.16,17 Preventing MPTP opening at the onset of reperfusion can be achieved in various ways as follows10,18,19: (i) directlybypharmacological MPTP inhibition; (ii) indirectly through the activation of signaling pathways that converge on the MPTP; or (iii) indirectly by modifying factors such as mitochondrial Ca2+ overload and ROS production, which are known to induce MPTP opening (see Figure 2).

Signaling pathways underlying ischemic conditioning that target the MPTP
The heart can be protected from the detrimental effects of acute IRI by subjecting it to brief nonlethal episodes of ischemia and reperfusion, a phenomenon that has been termed “ischemic conditioning.” 20,21 Importantly, the protective stimulus can be applied directly to the heart before the index ischemic event (ischemic preconditioning) 22 or at the onset of reperfusion (ischemic postconditioning) 23 or it can be applied to an organ or tissue remote from the heart (remote ischemic conditioning). 24 A number of different signal transduction pathways have been shown to mediate ischemic conditioning, including the reperfusion injury salvage kinase (RISK) pathway (comprising the PI3K- Akt and MEK1/2- Erk1/2),25,26 the survival activating factor enhancement (SAFE) pathway (comprising tumor necrosis factor a, JAK-STAT3),27,28 and the nitric oxide [NO]- cyclic guanosine monophosphate [cGMP] pathway.29 These signaling cascades mediate the ischemic conditioning stimulus from the cell surface receptor to the mitochondria where they mediate cardioprotection by inhibiting MPTP opening (see Figure 2). The elucidation of these signaling pathways underlying ischemic conditioning has made it possible to use pharmacological agents to activate these signal mediators and recapitulate cardioprotection (Figure 2). Examples of RISK, SAFE, and NO-cGMP pathway activators that have been shown to protect the heart against acute IRI include growth factors and cytokines, such as atrial natriuretic peptide, insulin, erythropoietin, and glucagon-like peptide-1.25-29

Cyclosporin A: a direct inhibitor of the MPTP
The immunosuppressant drug cyclosporin A (CsA) is a potent inhibitor of MPTP opening that has been demonstrated to reduce MI size in a number of experimental animal studies, 16,30,31 but not all.32 This therapeutic strategy has been translated into the clinical setting in several phase 2 clinical trials in AMI, coronary artery bypass graft (CABG) surgery, and stroke, but the results have been mixed.33-37 The recently completed CYCLE trial (CYCLosporinE A in reperfused acute myocardial infarction [NCT01650662]), which included 410 ST-segment elevation MI (STEMI) patients, also failed to demonstrate any benefits with CsA administered before primary percutaneous coronary intervention (PPCI), in terms of ST-segment resolution and enzymatically estimated MI size.38 Whether MPTP inhibition can improve clinical outcomes has been recently tested in the CIRCUS trial (does Cyclosporine ImpRove Clinical oUtcome in ST-elevation myocardial infarction patients), which involved 970 patients. In that trial, it was shown that the administration of CsA immediately before PPCI failed to improve clinical outcomes at one year (all-cause death, heart failure hospitalization, and adverse LV remodeling) in anterior STEMI patients.39 Why this large phase 3 trial did not confirm the positive results reported in previous phase 2 studies remains unclear, but potential reasons include the following40: (i) a possible type I error observed in small-size clinical studies; (ii) off-target effects of CsA, as CsA is known to inhibit cyclophilin A and calcineurin, the results of which may have counteracted the benefit of inhibiting MPTP opening41; and, perhaps, (iii) changes in STEMI patients since the initial phase 2 trial, including a greater use of the new P2Y12 platelet inhibitors (prasugrel, ticagrelor), which are known to reduce MI size per se.42

TRO40303: an indirect inhibitor of the MPTP
TRO40303 binds to the translocator protein TSPO in the outer mitochondrial membrane and is believed to inhibit MPTP opening by attenuating ROS production. It has been reported in small animal experimental studies to reduce MI size,43 but the cardioprotective effect was not replicated in a clinically relevant porcine MI model.44 In the 163–STEMI-patient MITOCARE study (which investigated the efficacy and safety of TRO40303 for reduction in reperfusion injury in patients undergoing revascularization for STEMI),45 this agent failed to reduce MI size when administered at the time of PPCI, despite careful patient selection (completely occluded infarct-related artery, large area at risk [AAR]). The neutral findings of the MITOCARE study may be due in part to ambiguous cardioprotective effects previously revealed in experimental studies and the fact that the formulation and dosage of TRO40303 used in the clinical study differed from that in experimental studies. Finally, more adverse events were reported in patients administered TRO40303 than in the placebo arm,45 thereby limiting the clinical application of this therapeutic approach.

MTP-131 and myocardial energetics
MTP-131, a mitochondria-targeting peptide, has been shown to optimize mitochondrial energetics and attenuate the production of ROS by selectively targeting cardiolipin in the inner mitochondrial membrane. It has been reported in both small and large animal experimental studies to reduce MI size when administered at the onset of reperfusion and to prevent adverse LV remodeling after MI.46,47 However, in the EMBRACE STEMI clinical trial (Evaluation of Myocardial effects of Bendavia for reducing Reperfusion injury in patients with Acute Coronary Events),48 intravenous MTP-131 administered before PPCI failed to reduce enzymatically estimated MI size in a carefully selected population of anterior STEMI patients (ischemic time <4 hours, no collateral vessels, and fully occluded coronary artery). The reasons for the neutral results of this study are not known, but potential reasons may include a single-targeted approach to cardioprotection, or pharmacokinetic or pharmacodynamic difficulties in targeting mitochondria in STEMI patients.

Metabolic modulation to protect the heart against acute IRI

Improving myocardial energetics by modulating mitochondrial metabolism is an important strategy for cardioprotection that has been extensively investigated over the last 30 to 40 years. In 1970, Lionel Opie first used insulin to promote glucose oxidation to protect the heart against acute myocardial ischemia. 49 This metabolic approach to protecting the ischemic heart underlies the cardioprotective effects of trimetazidine.

Trimetazidine and metabolic modulation
Trimetazidine is known to improve myocardial glucose utilization by inhibiting fatty acid metabolism. It does this by inhibiting long-chain 3-ketoacyl-coenzyme A thiolase, thereby blocking b-oxidation of fatty acids and promoting glucose oxidation.50 In the ischemic heart, where oxygen is scarce, glucose oxidation is more beneficial than fatty acid oxidation as the former requires less oxygen consumption than the latter. This metabolic effect of trimetazidine is central to its antianginal effects in patients with stable coronary artery disease (CAD).51

A number of experimental studies have shown that trimetazidine can protect the heart against acute IRI, as evidenced by reductions in MI size when administered as a pretreatment and when administered at the onset of reperfusion.52 This therapeutic approach has been investigated in patients presenting with an AMI in the large EMIP-FR clinical trial (European Myocardial Infarction Project – Free Radicals), and although it did not improve clinical outcomes in AMI patients reperfused by thrombolysis, it appeared to have a beneficial effect in nonreperfused patients, underscoring its anti-ischemic effect.53

Recent meta-analyses have shown that it can decrease perioperative and periprocedural myocardial injury in patients undergoing coronary revascularization by CABG surgery54 and PCI,55 respectively, suggesting it has a cardioprotective effect in these clinical settings. The beneficial effects from modulating mitochondrial metabolism may also be helpful in heart failure, another condition in which disturbances in mitochondrial metabolism play an important role.56

Ivabradine and myocardial energetics
Another effective approach to cardioprotection is to reduce the myocardial energy requirements of the heart during acute IRI. This can be achieved with little hemodynamic consequence by the drug ivabradine, which by inhibiting the If current in the sinus node can induce a selective lowering of heart rate.57 This drug has been demonstrated to lower heart rate and reduce myocardial ischemia in several experimental studies. 58-60 However, whether the anti-ischemic effect of ivabradine is secondary to heart rate lowering is not clear, and recent experimental studies have found that this drug can protect the heart against acute IRI in paced animal hearts,58,60 suggesting beneficial pleiotropic effects of this drug, which may include attenuation of ROS production and inhibition of MPTP opening in response to acute IRI.60 Further experimental studies are required to elucidate the mechanisms underlying the cardioprotective effect of ivabradine.

In the clinical setting, by lowering heart rate to reduce myocardial oxygen consumption and increase coronary blood flow, ivabradine has been shown to be an effective antianginal agent in patients with stable CAD.61 However, this therapeutic approach did not improve clinical outcomes in a group of such patients without heart failure.62 In contrast, it has been shown to improve clinical outcomes (less cardiovascular mortality or heart failure hospitalization) in stable CAD patients with heart failure and heart rates above 70 beats per minute.63

Summary and conclusions

Mitochondria lie at the heart of a number of cardioprotective signaling pathways underlying ischemic conditioning. As such, a variety of pharmacological treatment strategies aimed at protecting mitochondria against the detrimental effects of acute myocardial IRI have been investigated in both experimental and clinical studies, with mixed results. Of these strategies, the current treatments that are already in clinical practice include trimetazidine and ivabradine—further studies are required to elucidate the benefit of these agents in acute myocardial IRI and in improvement of clinical outcomes in patients with ischemic heart disease.

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