Moving Forwards by Blocking Back-Flow

Abstract

Mitochondrial reactive oxygen species production has emerged as an important pathological mechanism in myocardial ischemia/reperfusion injury. Attempts at targeting reactive oxygen species by scavenging using antioxidants have, however, been clinically disappointing. This review will provide an overview of the current understanding of mitochondrial reactive oxygen species in ischemia/reperfusion injury. We will outline novel therapeutic approaches designed to directly target the mitochondrial respiratory chain and prevent excessive reactive oxygen species production and its associated pathology. This approach could lead to more effective interventions in an area where there is an urgent need for new treatments.

Mitochondria are an important source of reactive oxygen species (ROS) in mammalian cells and play a critical role in cardiac function. Under physiological conditions, low levels of ROS are produced as a by-product of mitochondrial respiration and act as essential cellular mediators in a variety of biological processes, including regulation of the immune response and autophagy.^1–3 Stress or injury can, however, cause ROS to increase significantly, overwhelming endogenous antioxidant mechanisms, and resulting in severe oxidative damage to cellular components, such as lipids, proteins, and DNA.⁴ Mitochondrial ROS are now known to be key mediators of mitochondrial dysfunction and disease pathology in a range of cardiovascular conditions, including atherosclerosis, cardiac hypertrophy, chronic heart failure, ventricular remodeling, and ischemia/reperfusion (IR) injury.^5–7 On reperfusion of ischemic myocardium, the rapid reintroduction of oxygen into the cell leads to a burst of ROS generation that triggers opening of the mitochondrial permeability transition (mPTP) pore and myocardial cell death. Significant progress has been made in the field of inhibiting or scavenging ROS in an attempt to preserve mitochondrial and cardiomyocyte function. However, despite the large body of evidence supporting the inhibition of oxidative stress as a valuable therapeutic strategy, treatment with antioxidants has failed to deliver clinically significant benefits.⁸ In the present review, we will discuss the role of mitochondrial ROS in cardiac IR injury, describing the current mechanisms that are thought to drive its production. Furthermore we will highlight current methods at targeting mitochondria ROS production with a particular focus on interventions that inhibit complexes I and II.

IR Injury

IR injury remains a leading cause of death worldwide and the primary cause of chronic heart failure. Although the past few decades have seen a marked improvement in outcomes in patients treated with early reperfusion therapy, currently 1 in 4 patients will die or present with heart failure within 1 year postinjury.⁹ Reperfusion of the ischemic myocardium is essential to salvage viable tissue but paradoxically the rapid restoration of blood flow can induce injury beyond that of the initial ischemic insult. Known as reperfusion injury, studies have shown that it can account for ≤50% of the total tissue damage⁷ for which there is currently no effective therapy available in the clinic. The mechanisms underlying IR injury are multifactorial and have been extensively reviewed elsewhere.^7,10 However, it is generally accepted that mitochondrial dysfunction is central to the pathology of both IR injury and chronic heart failure, with the mitochondrion not only being the main producer of ROS but also a primary target of ROS damage.

Cardiac metabolism is predominantly aerobic. As such, the maintenance of normal cardiac function and viability is highly dependent on the constant delivery of oxygen. During periods of severe myocardial ischemia, profound disturbances in metabolism occur resulting in a shift toward anaerobic glycolysis. ATP depletion and lactic acidosis drive cytosolic sodium accumulation via the sodium/hydrogen exchanger and as a consequence excess Na⁺ is extruded through the reverse action of the plasma membrane sodium/calcium exchanger.¹¹ Typical calcium (Ca²⁺) management by the sarcoplasmic reticulum Ca²⁺-ATPase is prevented because of depletion of mitochondrially derived ATP, resulting in cytosolic Ca²⁺ overload. Furthermore, there is an accumulation of metabolic end-products, including hypoxanthine, xanthine, and succinate,^12–14 and the formation of proinflammatory mediators that promote the infiltration and activation of neutrophils. All these events are thought to prime the heart for the large burst of ROS generation on reperfusion. Reoxygenation of the cell at reperfusion and rapid restoration of the mitochondrial membrane potential (ΔΨ_m) result in a large Ca²⁺ influx into mitochondria. Together with a burst of ROS production¹⁵ and normalization of pH,¹⁶ opening of the mPTP is induced.¹⁷ The prolonged opening of the mPTP is now generally agreed to be decisive in committing cells to death on reperfusion. The mPTP is a highly conducting channel in the mitochondrial inner membrane. Although the exact nature of the pore is under debate, recent evidence suggests that the F_oF₁-ATP synthase is a major component.^18,19 Although the low pH present during ischemia prevents formation of the mPTP, the normalization of pH at reperfusion results in mPTP formation and subsequent collapse of ΔΨ_m, cytochrome c release, ATP depletion, and cellular death. Opening of the mPTP is therefore a critical component in reperfusion injury pathology.^10,19–21

The mitochondrial electron transport chain is an important source of ROS during IR injury but several other sources can also contribute. They include monoamine oxidase on the surface of the mitochondrial outer membrane, xanthine oxidases, NAD(P)H oxidases, and uncoupled nitric oxide synthases.^22–24 The contribution of these enzymes to total IR-induced ROS production is, however, thought to be lower than that of mitochondria and to occur later in the IR injury process, so will not be discussed further in this review.

Mitochondrial Respiratory Chain

Because this review is aimed at a general audience, a brief primer on mitochondrial respiratory activity is provided here, and is illustrated in Figure 1. Substrates (pyruvate from glycolysis or acetyl-CoA from β-oxidation) are decarboxylated in the tricarboxylic acid cycle to yield reducing equivalents NADH and FADH₂. Electrons are then passed onto complexes I or II, respectively, and then to the mobile electron carrier coenzyme Q₁₀ (ubiquinone), reducing it to ubiquinol. Ubiquinol is reoxidized by complex III, passing electrons to cytochrome c, then cytochrome oxidase, and finally oxygen, generating H₂O. The respiratory complexes are electron-driven proton pumps, such that this passage of electrons is coupled to the generation of a transmembrane proton electrochemical potential gradient (positive outside). The electrochemical energy in this gradient is then used by the F_oF₁-ATP synthase to generate ATP. It is important to note that the sharing of Co-Q as a common electron carrier by complexes I, II, and III is what permits these complexes to exist in multiple configurations, such that electrons can flow from I to III, II to III, I to II, and II to I, as shown in Figure 2.

Figure 1. The mitochondrial electron transport chain.

Electrons derived from the oxidation of NADH and FADH₂ enter the electron transport chain at complexes I (NADH ubiquinone oxidoreductase) and II (Succinate dehydrogenase). They are then funneled through the electron carriers, Coenzyme Q, and complex III (Ubiquinol cytochrome c oxidoreductase), until they reach complex IV (cytochrome c oxidase) where they are used to reduce molecular oxygen to water. This transfer of electrons is coupled to the extrusion of protons at complexes I, III, and IV generating an electrochemical gradient across the mitochondrial membrane. Protons in the intermembrane space are then used to drive the synthesis of ATP at complex V (ATP synthase). C indicates cytochrome c; and TCA, tricarboxylic acid. Dashed arrows indicate path of electrons.

Figure 2. Respiratory Complex I and II Yin-Yang during ischemia and reperfusion.

Under normoxic conditions, both complex I (red) and complex II (blue) work in the forward direction (dashed gray line indicates direction of electron flow), taking electrons from NADH and succinate, respectively, and reducing ubiquinone (Q) to ubiquinol (QH₂). Electrons are eventually passed down the respiratory chain to O₂, and complex I pumps protons to generate a transmembrane ΔpH. During ischemia, QH₂ generated by complex I working forward, is oxidized by complex II working in reverse. In this Yin-Yang formation, fumarate acts as an electron acceptor, resulting in accumulation of succinate. This process allows complex I to continue pumping protons during ischemia. At reperfusion, the rapid consumption of accumulated succinate generates too much QH₂ for the reoxygenated terminal respiratory chain to handle (dotted line). Coupled with residual acidic pH from ischemia, this drives reverse electron transfer in complex I, resulting in the generation of significant amounts of reactive oxygen species (ROS).

Central Role of Mitochondria

Mitochondria are essential organelles for normal cellular function. Occupying ≤30% of total cardiomyocyte volume, they are the main source of ATP for the contracting cell through oxidative phosphorylation.²⁵ Mitochondria are also a key source of cellular ROS production with oxygen being converted by mitochondria to superoxide at complexes I and III.²⁶ However, although the amounts of superoxide produced by isolated mitochondria can be readily estimated, the amount produced in vivo and the factors that regulate this production remain obscure. The superoxide produced in the mitochondrial matrix is then largely dismutated to hydrogen peroxide (H₂O₂) by manganese superoxide dismutase (Mn-SOD).²⁷ Although several other sources of ROS within mitochondria have been documented (eg, α-ketoglutarate dehydrogenase, monoamine oxidase, ETF-QOR of β-oxidation, α-glycoerophosphate dehydrogenase),^28,29 their relative importance under in vivo conditions is poorly understood. Thus, the rest of this review will focus primarily on complex I because this seems to be quantitatively the most important source of ROS in the setting of IR injury.⁴

In the context of IR pathology, elevated mitochondrial ROS levels drive oxidative damage to mitochondria, which results in disruption of the respiratory machinery and ATP generation. In addition, in conjunction with dysregulated calcium levels, mitochondrial ROS lead to induction of the mPTP, contributing to both apoptotic and necrotic cell death caused by IR. Because of the central role of mitochondrial ROS in IR pathology, many investigations have focused on characterizing the pathways that underlie their generation. In the past decade, such studies have increasingly highlighted a central role for mitochondrial complex I as the most significant superoxide source during IR.^30,31 More recently, it has been shown that generation of superoxide from complex I during IR is dependent on electron supply from the mitochondrial citric acid cycle intermediate succinate.¹⁴ Succinate, which accumulates significantly during ischemia through the reverse action of complex II, is rapidly oxidized in the first minutes of reperfusion. This rapid oxidation drives reverse electron transport (RET) at complex I, in which electrons are forced from reduced Coenzyme Q (CoQ) back to complex I generating large amounts of superoxide. This process can be described as a yin-yang formation in which during ischemia, QH₂ generated by complex I working forward is oxidized by complex II working in reverse. At reperfusion, complex II acting in forward mode consumes the accumulated succinate driving RET at complex I (Figure 2). Most interestingly, from a therapeutic perspective, it has been shown that this generation of damaging ROS on reperfusion can be inhibited, either by preventing the accumulation of succinate during ischemia or by inhibiting the succinate-dependent superoxide production by transient inactivation of complex I.^14,32 Both approaches will be discussed further below.

Good Versus Bad ROS

An intriguing aspect to ROS production in the heart is that depending on the circumstances and context, it can be considered either good or bad. That is, not all amounts of ROS are damaging and only when levels reach beyond the capacity of endogenous antioxidant mechanisms will ROS become detrimental to cell function and contribute to IR pathology. Conversely, ROS production has also been found to be a trigger for protection against IR injury particularly through the activation of survival programs during ischemic preconditioning (IPC) and ischemic postconditioning (IPost).^33,34 IPC, first demonstrated by Murray in 1986,³⁵ is a phenomenon in which brief cycles of IR protect the heart from reperfusion injury after a prolonged ischemic insult. ROS generated from these IR cycles are recognized as triggers for a cascade of signaling events that result in reduced tissue damage with the mitochondrion considered as a primary source.³⁶ Pre-treating isolated rabbit hearts with oxygen radicals can reproduce the beneficial effect of IPC on infarct size,³⁷ whereas giving ROS scavengers before ischemia abolishes IPC-induced protection.³⁴ The most straightforward interpretation of this intriguing observation is that although low levels of ROS can be beneficial by upregulating protective mechanisms, a larger amount of ROS has detrimental effects. This counterbalance between good and bad levels, known as mitohormesis,² is supported by an increasing body of work in which low levels of ROS are thought to act as signaling molecules to promote health and extend lifespan.² In the context of IR injury, a small increase in ROS, sufficient to lead to transient mPTP opening, has been shown to be protective against subsequent IR injury.³⁸ On the contrary, prolonged ROS exposure leading to sustained mPTP opening inevitably leads to irreversible mitochondrial damage and ultimately cell death. The threshold at which ROS production transitions from being protective to becoming harmful may be modulated by a variety of factors, such as diabetes mellitus, sex, and age; and risk factors which are already established to affect the efficacy of cardioprotective strategies.³⁹ For example, one way in which sex may determine the mPTP response to ROS is in the levels of nitric oxide. It is known that endothelial nitric oxide synthase is regulated by estrogen,⁴⁰ and this may directly impact ROS levels, in addition to nitric oxide being a direct inhibitor of the pore.⁴¹ Similarly for aging, the sensitivity of the Keap1/Nrf2 signaling axis, a key genetic response to oxidative stress, is known to decline with age.⁴²

Another hypothesis explaining the protean roles of ROS could be ascribed to the spatial distribution of its sites of production. It is well established that for many signaling pathways, the intracellular location of the signal plays a crucial role; for example, the compartmentalization of the cGMP—guanylate cyclase pathway.⁴³ Unfortunately, the details of the localization of ROS signaling are difficult to assess in vivo. However, given that different classes of mitochondria exist in the heart (subsarcolemmal versus intrafibrillar populations) behave differently during IPC and IPost,^44,45 it seems likely that the spatial distribution of mitochondrial ROS generation may also be a key variable. Finally, the timing of ROS generation could be important during IR with ROS being beneficial as a trigger of preconditioning-like signaling before a prolonged period of ischemia, whereas the large ROS burst at reperfusion induces many detrimental downstream effects.

Therapeutic Implications: Preventing Excessive ROS Generation

The compelling body of evidence linking reperfusion-induced ROS production to cardiac pathology has not surprisingly led to the testing of a wide range of antioxidant approaches to mitigate the detrimental effects of oxidative stress on reperfusion. Although many antioxidant strategies have shown benefit when applied to in vitro and in vivo model systems, only a tiny fraction has translated to improvements in major clinical end-points in human trials.⁴⁶ For example, antioxidants including Vitamin C, Vitamin E, Edavarone, and Coenzyme Q10 have shown disappointing or conflicting outcomes in patients.⁸ Many possible reasons for these poor results have been considered; the dosage of drug may not be optimal to achieve sufficient myocardial levels at reperfusion; the timing of the intervention in relation to the onset of ischemia or point of reperfusion may be incorrect and preclinical models used may not be appropriate for screening new compounds for human use.^47,48 The development of mitochondria-targeted antioxidants in which compounds are localized to the mitochondrion by conjugation to a triphenylphosphonium (TPP⁺) cation may address some difficulties inherent in using untargeted antioxidants that do not accumulate in mitochondria.⁴⁹ MitoQ is a TPP⁺ modified ubiquinol which on delivery to mitochondria decreases oxidative damage, and has been shown to be protective against both cardiac⁵⁰ and liver IR injury⁵¹ in vivo, as well as protecting against oxidative damage in a murine model of heart transplantation.⁵² The potential benefit of these targeted compounds against IR injury in humans has yet to be determined. A further consideration is that a more effective strategy may be to block the excessive ROS production that occurs on reperfusion at its source, rather than scavenge it after it has been produced. Moreover, this approach could in principle allow the blockade of ROS production only when it becomes pathological, avoiding the potential disruption to cellular homeostasis by altering physiologically important cellular signals by good ROS through chronic antioxidant treatment. Given that the mitochondrial respiratory chain is a critical source of ROS on reperfusion, it has become a major target for novel compounds aimed at ameliorating IR injury, and this strategy will be discussed in the next section.

Pharmacological Inhibitors of the Respiratory Chain as Therapeutics for IR Injury

Despite the lack of oxygen during ischemia or hypoxia leading to inhibition of the respiratory chain, a wide variety of respiratory inhibitors have been demonstrated to afford protection against IR injury. The Table lists several such inhibitors and their sites of action within the respiratory chain. Until recently, it was thought the mechanism of action for these respiratory inhibitors centered on the gradual wake up hypothesis of reperfusion therapy.⁸⁴ In this paradigm, a rapid reestablishment of respiratory activity at reperfusion leads to a surge of mitochondrial Ca²⁺ uptake and ROS generation which contribute to mPTP opening. It was hypothesized that the washout of a respiratory inhibitor present at reperfusion would permit a more gradual wake up of metabolism, thus avoiding these pathogenic effects. However, the recent identification of the source of ROS at reperfusion, namely the reverse electron transfer at complex I, forces a further focusing of this paradigm.¹⁴ Namely, it cannot go unnoticed that ≈85% of the protective respiratory inhibitors listed in the Table act at the level of complex I or II. Although the prevalence of agents hitting a given pharmacological target cannot be taken as evidence of the central biological importance of the target, it is notable that there are well-known inhibitors of other parts of the respiratory chain (eg, cyanide for complex IV, myxothiazol for complex III) that have not been found useful in a therapeutic setting. Furthermore, although many of the molecules in the Table act at a pleiotropic level, there are some exquisitely specific drugs targeted at complexes I and II (eg, rotenone and atpenin A5), which are most likely mediating their effects via these complexes and not through off-target mechanisms. We will now discuss these complexes in turn and the current evidence for their modulation in protecting the myocardium during IR injury.

Table.

Mitochondrial Respiratory Inhibitors That Have Been Shown to Protect the Heart or Brain From Ischemia/Reperfusion Injury, and Their Sites of Action

Site of Action	Inhibitor	References
Complex I	Rotenone	Lesnefsky et al31
	Amobarbital	Stewart et al53
	S-nitrosothiols	Nadtochiy et al,54 Prime et al,55 Burwell et al,56 and Nadtochiy et al57
	Nitrite	Shiva et al58 and Dezfulian et al59
	Metformin	Owen et al,60 Legtenberg et al,61 Bhamra et al,62 and Matsuzaki et al63
	Capsaicin	D’Alonzo et al64 and Satoh et al65
	Isoflurane	Hanley et al66 and Cope et al67
	Ranolazine	Wyatt et al68
Complex II	Atpenin A5	Wojtovich et al69
	Diazoxide	Anastacio et al70 and Wang et al71
	Malonate	Chouchani et al,14 Boylston et al,72 and Wojtovich et al73
	Nitroxyl	Pagliaro et al74 and Shiva et al75
	3-Nitropropionate	Riepe et al76
	Nitro-alkenes	Nadtochiy et al77
Complex III	Antimycin A	Kabir et al78
Complex IV	Nitric oxide	Zhao et al79
	Carbon monoxide	Clark et al80
	Hydrogen sulfide	Khan et al,81 Pan et al,82 and Elrod et al83

Note: Some references are paired such that the phenomenon of a molecule inhibiting a respiratory complex and the phenomenon of it being protective in ischemia/reperfusion injury are not necessarily co-observed in the same experimental system. Inclusion of a molecule in this table should not be misconstrued as claiming that the mechanism of its protection is dependent on its effects on a given respiratory complex.

Complex I

Complex I (NADH ubiquinone oxidoreductase) is the primary point of electron entry within mitochondria responsible for the oxidation of NADH, derived from glycolysis, the citric acid cycle, and the β-oxidation of fatty acids. Complex I transfers electrons to CoQ, and protons are transported across the inner membrane contributing to the mitochondrial proton motive force. In addition, it is an important site for ROS generation with complex I producing large amounts of superoxide in the presence of a high NADH/NAD⁺ ratio, where oxygen reacts with a fully reduced flavin mononucleotide site.⁴ Complex I can also produce a large amount of ROS during RET, where in the presence of highly reduced CoQ pool and close to maximal proton motive force, electrons are pushed backward from CoQH₂ through complex I reducing NAD⁺ to NADH and also producing superoxide.^85,86 Although the physiological relevance of RET in vivo is only now being elucidated, it produces the largest rate of mitochondrial ROS production known to occur within mitochondria. Furthermore, this process of superoxide production by RET at complex I seems to be the major source of ROS early during IR injury.¹⁴

During prolonged ischemia, when complex I is not oxidizing NADH because of the lack of oxygen, the protein converts to a deactive state.^87,88 Reperfusion of the tissue results in the rapid reactivation of complex I and the generation of large amounts of cytotoxic ROS by RET.¹⁴ Inhibitors of complex I including rotenone³¹ and amobarbital⁵³ have found to be protective when given during cardiac IR injury, indicating that preventing the reactivation of complex I on reperfusion is a promising potential therapeutic strategy. Of course, the use of irreversible complex I inhibitors is not viable as a therapy, but interestingly when complex I undergoes the deactive transition a critical cysteine, cysteine 39, on the ND3 subunit becomes exposed to modification.^87,88 This residue can be reversibly inhibited by its S-nitrosation by S-nitrosothiols such as SNO-MPG⁵⁴ or MitoSNO.³² Further supporting a role for this cysteine residue in cardioprotection, recent work has shown that damage protection during IR, IPC, and IPost correlates highly with the persistent S-nitrosation of mitochondrial protein thiols, with complex I as a chief target.^32,89–91 One example of this protective mechanism is MitoSNO, a mitochondria-targeted drug that prevents ROS production from complex I during early reperfusion after IR injury.³² MitoSNO is a mitochondria-targeted S-nitrosothiol based on the NO donor S-nitroso-N-acetylpenicillamine coupled to the TPP⁺ cation which leads to its rapid, several hundred-fold accumulation, driven by both the plasma and mitochondrial membrane potentials, into the mitochondrial matrix where it accumulates within minutes of intravenous injection.^55,92 On uptake into mitochondria, MitoSNO reacts rapidly with intramitochondrial thiols and S-nitrosates cysteine 39 on subunit ND3 of complex I locking the enzyme in its deactive form at reperfusion, and thereby preventing the excessive burst of ROS on reperfusion.³² The modification is reversed with a half-life of ≈5 minutes by the endogenous mitochondrial glutathione and thioredoxin systems, allowing complex I to return to full levels of activity a few minutes after reperfusion.¹⁴ Our studies have shown that MitoSNO not only protected against IR injury in vivo³² but also greatly enhanced long-term cardiac function post-IR injury.⁹³

Complex II

Complex II (succinate dehydrogenase) catalyzes the oxidation of succinate to fumarate, resulting in the donation of electrons to the respiratory chain via the reduction of FAD to FADH₂. Unlike the other respiratory complexes, it does not pump protons across the inner membrane but instead acts to maintain a reduced CoQ pool which has been largely considered to be its primary function.⁹⁴ This sequence also creates a direct link between 2 major mitochondrial pathways, the citric acid cycle and the respiratory chain. Several roles for complex II have, however, also been recently proposed that expand beyond this with evidence now for direct complex II–mediated ROS generation,⁹⁵ as well as a mechanistic link with the putative mitochondrial ATP-sensitive potassium channel (mtK_ATP).⁹⁶ Complex II is also now recognized as a key modulator of mitochondrial ROS production by other respiratory complexes, particularly complex I.

The accumulation of excessive ischemic succinate, via the reverse action of complex II, is considered a critical driver of ROS formation at reperfusion. Preventing either its build-up during ischemia or its rapid oxidation at reperfusion are therefore potential valuable therapeutic strategies to reduce detrimental ROS generation and protect against IR injury. In agreement with this, an extensive body of work exists demonstrating the inhibition of respiration at complex II can decrease ROS production.⁹⁷ Inhibitors such as dimethyl malonate, diazoxide, and atpenin A5 all protect against IR injury when given before ischemia.^14,69,70,72 Moreover, protection afforded by dimethyl malonate in vivo was attributed directly to the attenuation of ischemic levels of succinate and inhibition of mitochondrial ROS generation at reperfusion.¹⁴ There is also some evidence that malonate itself may act as an endogenous protector against IR with the compound being generated endogenously in mitochondria under conditions mimicking IPC.⁷³ However, whether these compounds exert cardioprotective effects solely via complex II inhibition and ROS generation or whether the mtK_ATP channel is involved remains a controversially and actively discussed issue. Moreover, although these strategies may be highly useful in situations of predictable ischemia, including elective surgery and organ transplantation, they are not clinically appropriate, given patients undergoing myocardial infarction (MI) arrive at hospital with an already occluded artery. Succinate accumulation during ischemia becomes pathological only on its rapid oxidation at reperfusion in which it drives RET-mediated ROS production through complex I. By suppressing succinate oxidation at the point of reperfusion through complex II inhibition, compounds such as dimethyl malonate could be potentially cardioprotective. It is therefore essential to determine whether complex II inhibitors are equally effective at ameliorating cardiac injury when used later in IR, such as just before reperfusion. Indeed, recent work in the isolated mouse heart has demonstrated that the administration of malonate during the first 15 minutes of reperfusion was cardioprotective only through the inhibition of succinate reoxidation and the reduction in ROS production and mPTP opening.⁹⁸ Whether this important result can be translated to in vivo models, however, remains to be determined.

Future Perspectives and Translational Significance

An important consideration for the potential future use of respiratory inhibition as a therapy for IR injury is the timing of delivery. Clearly, although the inhibition of complex I or II at early reperfusion would be anticipated to minimize ROS generation from RET, it is not immediately clear that inhibition of these complexes during ischemia itself would be beneficial. This is because of the yin-yang nature of complexes I and II during ischemia, in which complex I continues to operate as a proton pump allowing to some extent the ΔΨ_m to be maintained. As such, inhibition of complex I during ischemia may have unforeseen detrimental effects by removing this important function. A further consideration in moving such molecules into a clinical setting is their ease of washout, that is, their tightness of binding to their targets. In the case of rotenone and other tight-binding lipophilic molecules, inhibition would be expected to reverse rather slowly, if at all, whereas the complex II inhibitor, 3-nitropropionate, is a suicide inhibitor that covalently modifies complex II potentially resulting in long-term toxic effects on organ function.⁹⁹ Furthermore, currently available inhibitors of mitochondrial respiratory complexes are not tissue-specific and are therefore present in other important tissues such as the brain. Consequently, the chronic delivery of a respiratory inhibitor would be expected to elicit toxic side-effects such as neurodegenerative disease. Specifically, long-term inhibition of complex I is associated with Parkinson disease, complex II Huntington disease, and complex IV Alzheimer disease.^100,101 With this regard, another advantage to nitric oxide donors and other short-lived species, such as MitoSNO as mitochondrial respiratory inhibitors, is their short time of action and rapid metabolism, which would permit reestablishment of normal mitochondrial function once the initial early-reperfusion danger-period has passed.

Recently, it has been shown that treatment with a P2Y₁₂ inhibitor, such as clopidogrel or ticagrelor, was highly protective in animal models of acute MI as well as in small human studies, and that many conditioning strategies, such as IPost do not offer additional benefits in reducing infarct size.^102,103 This evidence could well be the reason for the failures of many recent clinical trials of either IPost or interventions mimicking conditioning. To translate any of the above-mentioned compounds targeting complex I or III is therefore crucial to test whether they have additive effects on top of an effective treatment with P2Y₁₂ inhibitors.¹⁰⁴ Further aspects on how to translate preclinical findings in patient care and the challenges especially in acute MI have been extensively reviewed elsewhere.¹⁰⁵

Summary

There is a pressing need for therapeutic approaches to be applied in conjunction with reperfusion therapy to reduce infarction injury and long-term outcome in patients with MI. Modulation of the respiratory chain through inhibiting complexes I and II is an important emerging strategy. These interventions can now be considered as potential rational therapies, arising from the view that the initial burst of ROS from complex I on reperfusion is because of the accumulation of succinate by the reversal of complex II during ischemia, which then drives the initial burst of ROS at reperfusion by RET at complex I. The reversible inhibition of complexes I and II would therefore prevent this burst of ROS and protect against infarction. Currently, approaches that prevent the accumulation of succinate during ischemia, such as dimethyl malonate, or stabilize the deactive form of complex I by S-nitrosation, such as MitoSNO, have been shown to be effective in animal models. Whether these results will translate into the clinic remains to be seen. Certainly, the next stages are to see whether it is possible to extend and optimize these targets with new and better drugs. However, the model of succinate-driven ROS production mediated by complexes I and II should facilitate the future development of novel-targeted therapies against the generation of excessive mitochondrial ROS in a range of pathologies, such as MI and stroke.

Nonstandard Abbreviations and Acronyms

I/R

ischemia/reperfusion

IPC

ischemic preconditioning

IPost

ischemic postconditioning

MI

myocardial infarction

mPTP

mitochondrial permeability transition

RET

reverse electron transport

ROS

reactive oxygen species