Mitochondrial function: the heart of myocardial preservation

REVIEW ARTICLE Mitochondrial function: The heart of myocardial preservation EDWARD O. MCFALLS, DAVID LIEM, KEES SCHOONDERWOERD, JOS LAMERS, WIM SLUITER, and DIRK DUNCKER MINNEAPOLIS, MINNESOTA, and ROTTERDAM, THE NETHERLANDS Over the past several decades, it has become widely recognized that the mitochondria serve an important role in energy production and transfer to myocardial cells. More recently, mitochondria have been shown to play a key role in cell-death pathways by activating mitochondrial permeability transition pore formation and causing the release of several proteins, such as cytochrome c and proapoptotic factors. Although the molecular mechanisms for this process are still under investigation, important mitochondrial adaptations have been identified that can inhibit permeability transition activation and prevent apoptosis and necrosis. Specifically, myocardial preconditioning is an intriguing adaptation by which brief ischemia and reperfusion prime the mitochondria so that a subsequent prolonged period of ischemia is better tolerated. Mitochondrial ATP– dependent potassium channels seem to play a critical role in triggering or mediating this protection. The objective of this review is to outline the role of the mitochondria in supporting myocardial energy under normal and ischemic conditions. It will also provide insight into the mechanisms by which mitochondrial signaling in preconditioning can protect the myocyte during subsequent prolonged ischemic periods. (J Lab Clin Med 2003;142: 141-9) Abbreviations: ANT ⫽ adenine nucleotide translocase; CsA ⫽ cyclosporin A; FADH2 ⫽ reduced form of flavin adenine dinucleotide; MPTP ⫽ mitochondrial permeability transition pore; NADH ⫽ reduced form of nicotinamide adenine dinucleotide; NADP ⫽ nicotinamide adenine dinucleotide phosphate; PKC ⫽ protein kinase C; ROS ⫽ reactive oxygen species; SOD ⫽ superoxide dismutase; VDAC ⫽ voltage-dependent ion channel T raditionally it has been thought that the primary role of mitochondria in preserving myocyte integrity during a supply-demand mismatch is From the Department of Medicine, Veterans Affairs Medical Center, University of Minnesota; and the Departments of Experimental Cardiology and Biochemistry, Erasmus MC. Supported in part by a Veterans Affairs Merit Review Grant. Submitted for publication March 10, 2003; accepted May 16, 2003. Reprint requests: Edward O. McFalls, MD, PhD, Cardiology (111C), Veterans Affairs Medical Center, 1 Veterans Drive, Minneapolis, MN 55417; e-mail: mcfal001@tc.umn.edu. Copyright © 2003 by Mosby, Inc. All rights reserved. 0022-2143/2003/$30.00 ⫹ 0 doi:10.1016/S0022-2143(03)00109-4 limited to the production and transfer of high-energy nucleotides. Over the past decade, however, important experimental observations have broadened the therapeutic potential for targeting myocardial mitochondria, particularly with regard to the generation of oxygen free radicals and release of apoptosisinducing factors. It has become apparent that brief periods of oxygen deprivation can prime the mitochondria so that a subsequent prolonged period of oxygen deprivation can be better tolerated. Termed “ischemic myocardial preconditioning,” this phenomenon has stimulated interest in the molecular mechanisms that underlie mitochondrial adaptations during chronic reductions in myocardial blood flow. This review will provide an overview of mitochon141 142 McFalls et al drial function under normal conditions and during ischemia and reperfusion. It will also address the experimental models that might be used to study mitochondrial adaptations that occur in response to sustained periods of myocardial ischemia. MITOCHONDRIAL FUNCTION UNDER NORMOXIC CONDITIONS Under normal conditions, myocardial oxygen supply and expenditure are tightly coupled, from the point of energy production in the inner membrane of the mitochondria to the point of energy use within cellular sites. Reducing equivalents are generated from the sequential breakdown of substrates and transferred in the form of NADH and FADH2. Oxygen serves as the ultimate electron recipient within the electron transport chain, and the overall reaction results in 2.5 mol of ATP per mole of NADH and 1.5 mol of ATP per mole of FADH2 when transferred to O2. The biochemical reactions within the electron-transport chain located in the inner membrane of the mitochondrion result in the transfer of electrons from reducing agents, by virtue of differences in oxidation-reduction potentials (Fig 1, A). As electrons are transferred between complex I and complex IV, protons are translocated across the inner membrane of the mitochondrion and a proton gradient is established. Ten protons are “pumped” across the membrane in the transfer of 2 electrons from NADH to O2. It is by way of this series of proton pumps that the matrix becomes more alkaline than the external surface of the inner membrane, creating an electrochemical gradient (⫺⌬⌼). This electrochemical energy is sufficient to drive ATP synthesis from ADP in complex V (Fig 1, B). MITOCHONDRIAL FUNCTION DURING ISCHEMIA AND REPERFUSION Among patients with coronary artery disease, the major vessels on the surface of the heart may become narrowed. When coronary-artery perfusion pressure is reduced below a critical threshold, coronary autoregulatory reserve becomes exhausted, and myocardial ischemia ensues. As a result of the decreased supply of oxygen to working myocardial tissue, a supply-demand mismatch occurs, with a rapid decline in creatine phosphate and increased content of intracellular phosphate. Glycolysis is increased, leading to the accumulation of harmful metabolic byproducts such as lactate and hydrogen ion (protons). In the absence of oxygen, mitochondrial ATP levels are rapidly decreased for two reasons. First, production of energy is decreased because of the arrest of the oxidative phosphorylation by a shortage of oxygen. Moreover, depression of complex I of the respiratory chain and reduction in the activity of J Lab Clin Med September 2003 ANT has been reported.1 Second, consumption of highenergy phosphates by F1F0-ATPase is increased by way of the reverse catalytic reaction of ATP to ADP.2 If hypoxia continues indefinitely, myocyte ATP sources are reduced to a critical level and intracellular sodium and calcium are increased, with the ultimate progression to necrosis. Although prolonged myocardial ischemia has deleterious effects on mitochondrial function, emerging evidence suggests that the generation of ROS during reperfusion is even more harmful. Clinically, this is a relevant area of research because patients with active myocardial ischemia receive aggressive mechanical and pharmacologic therapies to restore coronary-artery blood flow and oxygen delivery to the myocardium. Within the inner membrane of the mitochondria of the myocyte, ischemia induces a high concentration of the partially reduced intermediate ubisemiquinone. This reacts rapidly with oxygen during early reperfusion, producing high concentrations of superoxide.3 Normally superoxide is converted into hydrogen peroxide by the manganese-containing SOD present in the mitochondrial matrix. Hydrogen peroxide is a relatively inert oxidant. However, if reduced iron becomes available from iron-sulfur clusters in the electron-transport chain, the tricarboxylic acid– cycle enzyme aconitase, or ferritin, a dangerous situation occurs by way of the generation of the extremely reactive hydroxyl radical from hydrogen peroxide.4 We have found that in normal rat hearts, a progressive 30-fold increase occurs in socalled low molecular weight iron during 45 minutes of ischemia, whereas no increase is observed during 45 minutes of anoxic perfusion. This means that the reductive release of iron from its storage pool, ferritin, is greatly enhanced by the acidification that occurs during ischemia.5 If the iron chelator desferrioxamine is administered during reperfusion, cardiac function is nearly completely restored.6 On the basis of these findings, it was concluded that oxygen deprivation enhances the susceptibility of the heart to ROS by increasing the amount of catalytic ferrous iron in the low molecular weight pool. When the usual defenses in the matrix, such as the antioxidant enzymes SOD; glutathione peroxidase, which inactivates hydrogen peroxide; and the antioxidants glutathione and vitamin E are overwhelmed, hydroxyl radicals target proteins that lie close to the site of formation, particularly thiol groups from enzymes in the respiratory chain. Ultimately, for mitochondria to regain function after ischemia and reperfusion, it is critical that the inner membrane remain impermeable to protons, and the membrane potential will be restored so that the driving force for complex V of oxidative phosphorylation can be sustained. J Lab Clin Med Volume 142, Number 3 McFalls et al 143 Fig 1. Oxidative phosphorylation within the inner mitochondrial membrane under normal conditions. By virtue of differences in oxidation-reduction potentials, electrons are sequentially transferred from reducing agents between complexes I and IV (A). In the process, protons are translocated across the inner membrane of the mitochondria, and a proton gradient is established. Ten protons are “pumped” across the membrane in the transfer of 2 electrons from NADH to O2. It is by way of this series of proton pumps that the matrix becomes more alkaline than the external surface of the inner membrane, creating an electrochemical gradient (⫺⌬⌼) (B). APOPTOSIS AND THE MPTP After prolonged myocardial ischemia, a critical depletion of energy leads to loss of ionic homeostasis, with cell swelling, rupture, and necrotic death. Over the past decade, a distinctly different process of myocyte death has been observed that is programmed by stressinduced events within the mitochondria. Termed “apoptosis,” this process is characterized by cell shrinkage without release of intracellular material, combined with nuclear condensation and expression of phosphatidylserine at the cell surface. The phenomenon is triggered by the release of 1 or more proteins from the mitochondria, including cytochrome c, apoptosis-inducing factor, endonuclease G, second mitochondria-derived activator of caspases/direct inhibitors of apoptosis protien (IAP)-binding protein (Smac/Diablo), and procaspases 144 McFalls et al (2,3,8, and 9). Once released into the cytosol through the outer membrane, these proteins activate a family of proteases that can account for the structural changes within apoptotic myocytes. In several models of myocardial ischemia and reperfusion, necrosis and apoptosis can coexist, though it is unclear what factors determine the ultimate fate of a cell. It has been proposed that myocardial segments exposed to the most severe areas of ischemia and reperfusion undergo necrosis, whereas cells on the peripheral regions, exposed to a less severe insult, undergo apoptosis.7,8 This is consistent with the notion that cells exposed to severe stimuli undergo necrosis, whereas cells exposed to less hypoxia become apoptotic and sustain partial recovery. Whether the apoptotic stimulus during the phase of oxygen deprivation is greater than that during the phase of reoxygenation with reperfusion is still under investigation. On the basis of data from hearts from patients with recent myocardial infarctions,9 as well as in vivo rat hearts exposed to prolonged ischemia and reperfusion,10 the generation of free radicals during reoxygenation appears to be the most potent stimulus for apoptosis. This area remains controversial, however, because myocardial ischemia in the absence of reperfusion can also lead to apoptosis11 and raises the question of whether the generation of ROS is the cause or consequence of mitochondrially mediated apoptosis. The precise molecular mechanisms that underlie apoptosis are under intense investigation. An important concept in the initiation of necrosis or apoptosis is the opening of a nonspecific pore within the inner membrane of the mitochondria, the MPTP. Current evidence suggests that any number of stress responses signal the pore to open, with release of proapoptotic proteins from the matrix into the cytosol. For decades it has been known that opening of the MPTP results in swelling and uncoupling of mitochondria exposed to high concentrations of calcium.12,13 To activate the MPTP, the matrix enzyme peptidyl-prolyl cis-trans isomerase (PPIase), or cyclophilin D, binds to ANT on the inner membrane of the mitochondria. Under physiologic conditions, ANT plays an integral role in the translocation of ATP and ADP across the inner membrane. With the binding of cyclophilin D to ANT, a conformational change in the peptide bond around proline residues converts the specific transporter into a nonspecific pore.14 The reaction is calcium-dependent and is facilitated under conditions of increased inorganic phosphate and free radicals and decreased ATP (Fig 2, A). Once the ANT transporter is converted into the nonspecific pore, molecules of less than 1.5 kD can be translocated from the matrix across the inner membrane and into the intermembrane space. J Lab Clin Med September 2003 Fig 2. Activation of the MPTP is a nonspecific pore formation between proteins in the inner and outer membranes of the mitochondria (A). The MPTP is activated during conditions of ischemia and reperfusion, when ATP stores are reduced and inorganic phosphate and free radicals are increased. During activation of the MPTP, cyclophyilin D or the matrix enzyme peptidyl-prolyl cis-trans isomerase binds to ANT in a calcium-dependent reaction and alters the conformation of the protein. Bax is a proapoptic protein in the cytosol that can translocate to porin or the VDAC in the outer membrane. These proteins can then form a complex, which creates a large pore, allowing translocation of proteins of less than 1.5 kD to pass into the cytoplasm (B). The release of cytochrome c is one of the important initiators of this process. Important insight into the molecular mechanisms of the MPTP have been obtained through the observation that CsA can inhibit the reaction of cyclophilin D with ANT and that certain proteins such as Bcl-2 and hexokinase can prevent the releases of cytochrome c through the VDAC (C). J Lab Clin Med Volume 142, Number 3 Although the MPTP is primarily a pore formation in the inner membrane, the pore complex also requires permeabilization of the outer membrane. This requires the formation of a complex in the outer membrane, which is believed to be a VDAC, or porin. This protein comprises a pore that allows translocation of low molecular weight solutes across the outer membrane. Antibodies to VDAC have been shown to inhibit activation of MPTP and loss of membrane potential, providing evidence that rupture of the outer membrane in apoptosis is not just a mechanical stress related to swelling.15 The juxtaposition of ANT on the inner membrane with VDAC is most evident where the mitochondrial folds, or cristae, come into close contact with the outer membrane (Fig 2, A). Once the complex is activated, the MPTP is opened and cytochrome c can be released from the intermembrane space into the cytosol. Cytochrome c is an essential electron carrier between complexes III and IV; depletion of cytochrome c results in depressed state III respiration. If the process of MPTP persists, additional proteins such as apoptosis-inducing factor are released from the intermembrane space, resulting in apoptosis and irreversible loss of membrane potential (⫺⌬⌼) (Fig 2, B). Important insight into the molecular mechanisms of MPTP has been provided by the observation that the immunosuppressive agent CsA inhibits the cascade of events in the opening of the MPTP.16 CsA binds to cyclophilin D in the matrix of the mitochondria and prevents pore opening by interacting with the conformational change of ANT. Other regulators of MPTP can also alter the complex between ANT and the outer membrane protein. For instance, within the Bcl family, antiapoptotic proteins called Bcl-2 prevent VDAC from forming a permeable pore in the presence of proapototic factors from the Bcl family such as Bax. These proteins modify VDAC on the outer membrane and prevent the release of cytochrome c in the proapoptotic milieu.17 In addition, glycolytic enzymes such as hexokinase can play a role in preventing permeabilization of the outer membrane, possibly by stabilizing VDAC during ischemia (Fig 2, C).18 Although the primary trigger for MPTP is an increase in calcium in the matrix, low pH inhibits this process as a result of proton competition for the binding site of calcium on the cyclophilin D–ANT complex. This is particularly germane to the observation that apoptosis may be more evident during the reperfusion phase than during the ischemic phase in myocardial tissue. During reperfusion, pH is rapidly normalized as lactate is washed out and protons are pumped out of the cell by way of the Na⫹/H⫹ exchange. The decreased proton formation, together with the increased superoxide generated during reoxygenation, produces conditions that McFalls et al 145 favor activation of the MPTP during reperfusion. In an attempt to address the timing of MPTP relative to ischemia and reperfusion, the release of preloaded mitochondrial 2-deoxyglucose has been used as a marker of MPTP in isolated rat heart preparations.19 Using this method, investigators have shown that activation of the MPTP occurs more frequently during the early reperfusion phase than during the prolonged ischemic phase. Despite the transient opening of the pore in this model, in the presence of CsA, mechanical function can recover.20 These data are intriguing and provide evidence that activation of the MPTP may not necessarily be an irreversible process, committing the myocyte to either apoptosis or necrosis. MITOCHONDRIA AND ISCHEMIC PRECONDITIONING A seminal study in the anesthetized-dog model of regional myocardial ischemia demonstrated that a brief period of coronary artery occlusion before the onset of rolonged ischemia reduced the degree of necrosis after reperfusion by 75%.21 This observation, termed “ischemic preconditioning,” has widespread implications for all aspects of tissue preservation. Although the duration of protection is short, lasting just 1 to 2 hours after initiation (the “first window”), it reappears 24 hours later and may last 72 hours (the “late window”). This phenomenon has been observed in every species studied and has important clinical relevance to the preservation of myocardial tissue among patients with coronary-artery disease. The role of mitochondria in this process is recognized and is supported by the initial observation that the rate of ATP loss was reduced during the reference period of ischemia in preconditioned hearts.21 Additional studies have also shown that preconditioned myocardium improves the energy state during ischemia and early reperfusion.22,23 Although several agonists released during ischemia and reperfusion—including adenosine, bradykinin, noradrenaline, and nitric oxide—are known to trigger a preconditioning effect, a common secondary signal in the protective cascade appears to involve PKC-⑀.24 The translocation of PKC-⑀ to the surface of the mitochondria during cardioprotection provides an important clue that the mitochondria play a critical role in the infarction-sparing effects of preconditioning.25 The most widely accepted paradigm for inducing a preconditioning effect involves the opening of ATP-dependent potassium channels, particularly those situated on the inner membrane of the mitochondria. After a protocol of ischemic preconditioning, opening of these channels can be inhibited with glibenclamide or 5-hydroxydecanoate, and the protection during a subsequent prolonged period of ischemia is abolished.26 Likewise, 146 McFalls et al J Lab Clin Med September 2003 Fig 3. Ischemic myocardial preconditioning is characterized as the infarction-sparing effects of brief ischemia and reperfusion before a subsequent prolonged period of ischemia. ATP-dependent potassium channels are found in the inner membrane of the mitochondria and play an important role in this cardioprotection. The protective effects of ischemic preconditioning can be mimicked by administration of diazoxide, which opens the mitochondrial ATP– dependent potassium channels and allows potassium to move into the matrix. This protects the mitochondria by either depolorizing the inner membrane, causing an increase in the potential (⫺⌬⌼), or increasing the matrix volume. Either of these effects can preserve energy production during ischemia (see text). mitochondrial ATP– dependent potassium channels can be pharmacologically opened with diazoxide, providing a level of protection that mimics that of ischemic preconditioning.27,28 ROLE OF MITOCHONDRIAL POTASSIUM ATP CHANNELS IN PRECONDITIONING The mechanism of protection afforded by mitochondrial ATP– dependent potassium channels is under intense scrutiny. In isolated skinned myocytes exposed to simulated ischemia and reperfusion, diazoxide attenuated the degree of apoptosis by inducing a modest level of depolarization in the membrane potential.29 This has the protective effect of minimizing calcium overload, particularly during the reperfusion period, when the MPTP is activated. It is a matter of controversy, however, whether the protection afforded by diazoxide resides in its effects on mitochondrial membrane potential. In isolated rat heart mitochondria, diazoxide in protective doses (⬍50 ␮mol/L) is not sufficient to depolarize the inner membrane and alter membrane potential.30 Enabling the influx of positive (potassium) charge into the matrix helps the electron-transport chain pump protons in return into the intermembrane space. Obviously, in theory, this will not change the net membrane potential, but it does decrease the reductive state of the respiratory complexes and therefore the concentration of the potentially dangerous ubisemiquinone anion. According to the authors, an alternative mechanism of protection is a modest increase in the matrix volume that protects against the loss of ADP and energy stores. Similar findings have been observed in isolated mitochondria from adult rabbit hearts. These protective effects of mitochondrial ATP– dependent potassium channel opening are illustrated in Fig 3. Under ischemic conditions, pharmacologic doses of diazoxide (25–50 ␮mol/L) prevented the MPTP and loss of cytochrome c by way a mechanism that did not appear to alter membrane potential.31 Addition of phorbol myristate acetate mimicked the protection, demonstrating the importance of PKC activation in this model of isolated mitochondria. ROLE OF FREE RADICALS AND CARDIOPROTECTION It is not universally accepted that the opening of mitochondrial ATP– dependent potassium channels is the end-mediator of myocardial preconditioning. Instead, the primary trigger of preconditioning may depend on a burst of free radicals that prime the mitochondria so that a subsequent longer period of ischemia and reperfusion is protective. In isolated rabbit hearts, opening of the mitochondrial ATP– dependent potassium channels triggered a preconditioning state but the mediator of protection was afforded by the secondary release of free radicals.32 This finding has been repro- J Lab Clin Med Volume 142, Number 3 duced in isolated myocytes exposed to hypoxia, where the release of ROS was downstream from the opening of mitochondrial ATP– dependent potassium channels.33 Perhaps the most interesting paradox with regard to free-radical generation in preconditioning is the notion that a small degree of release is required to prevent a large burst during the reference period of ischemia and reperfusion. In chick embryonic ventricular myocytes, hypoxic preconditioning was mimicked by exposure to hydrogen peroxide, leading to less cell death during a subsequent period of hypoxia and reoxygenation.34 Inhibition of the VDAC during ischemia abolished the preconditioning effect, presumably by inhibiting superoxide release by way of the channel. In a similar model, the investigators have shown that preconditioning myocardium demonstrates a reduction in free-radical generation during the reference period of ischemia and reperfusion.35 These observations are intriguing and are consistent with the findings that cardioprotection afforded by diazoxide is mediated by a change in the myocardial redox state.36 The generation of a large burst of oxygen free radicals during early reperfusion is a critical ingredient in the activation of mitochondrial permeability transition and suggests that the mitochondrial redox state is an important modulator. ANT contains 3 cysteine residues, and the thiol groups can react with ROS such as hydroxyl radical and activate MPTP. In support of this theory, activation of MPTP is prevented by thiol reductants such as dithiothreitol and enhanced by thiol oxidants such as diamide and phenylarsine oxide. One important defense against the accumulation of hydroxyl radicals may involve preservation of the NADPH pool after ischemia. Increased concentrations of NADPH relative to NADP⫹ promote the generation of glutathione and thioredoxin, which are substrates for peroxidases, resulting in conversion of hydrogen peroxide to water. In turn, a small degree of mitochondrial uncoupling may be an important protective mechanism in reconstituting this NADPH pool. Mild dissipation of the membrane potential, such as that which occurs during uncoupling, could act to diminish oxygen free radicals while still favoring the membrane potential– sensitive NADP transhydrogenase–mediated reaction of NADP to NADPH.37 This also decreases the concentration of NADH so that fewer reducing equivalents become available to produce superoxide by way of the electron-transport chain component ubisemiquinone. Perhaps future work will be directed at learning how a small degree of mitochondrial uncoupling, regardless of the trigger, can prevent large bursts of superoxide during myocardial ischemia and reoxygenation. McFalls et al 147 CONCLUSION It is apparent that novel approaches to the treatment of chronic heart diseases will evolve from a better understanding of the mechanisms by which mitochondria regulate myocyte integrity. The therapeutic potential for targeting specific aspects of the mitochondria can certainly be explored through various transgenic mouse lines that are now available.38 In the absence of large-animal transgenic lines, however, the role of mitochondrial adaptations in response to chronic reductions in blood flow will need to be identified from specific models of clinical relevance. In our laboratories, we have focused on swine models of acute and chronic regional myocardial ischemia, including preconditioning,39 hibernation,40 and repetitive supply-demand ischemia.41 By correlating in vivo physiologic parameters with ex vivo tissue analysis, we hope to understand the role of mitochondria in modulating freeradical generation and the milieu for permeability transition pore activation. By analyzing temporal changes in submitochondrial proteomics, we hope to identify changes in specific proteins within the important inner membrane that are critical to myocyte survival. REFERENCES 1. 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