Mitochondria as a Drug Target in Ischemic Heart Disease and Cardiomyopathy

Abstract

Ischemic heart disease (IHD) is a significant cause of morbidity and mortality in Western society. Although interventions such as thrombolysis and percutaneous coronary intervention (PCI) have proven efficacious in ischemia and reperfusion (IR) injury, the underlying pathologic process of IHD, laboratory studies suggest further protection is possible, and an expansive research effort is aimed at bringing new therapeutic options to the clinic.

Mitochondrial dysfunction plays a key role in the pathogenesis of IR injury and cardiomyopathy (CM). However, despite promising mitochondria-targeted drugs emerging from the lab, very few have successfully completed clinical trials. As such, the mitochondrion is a potential untapped target for new IHD and CM therapies. Notably, there are a number of overlapping therapies for both these diseases, and as such novel therapeutic options for one condition may find use in the other. This review summarizes efforts to date in targeting mitochondria for IHD and CM therapy, and outlines emerging drug targets in this field.

1. Introduction

Ischemic heart disease (IHD) is a significant cause of morbidity and mortality in Western society.¹ Although interventions such as thrombolysis and percutaneous coronary intervention (PCI) have proven efficacious in ischemia and reperfusion (IR) injury, the underlying pathologic process of IHD, laboratory studies suggest further protection is possible, and an expansive research effort is aimed at bringing new therapeutic options to the clinic.

Mitochondrial dysfunction plays a key role in the pathogenesis of IR injury and cardiomyopathy (CM).^2,3 However, despite promising mitochondria-targeted drugs emerging from the lab, very few have successfully completed clinical trials. As such, the mitochondrion is a potential untapped target for new IHD and CM therapies. Notably, there are a number of overlapping therapies for both these diseases, and as such novel therapeutic options for one condition may find use in the other. This review summarizes efforts to date in targeting mitochondria for IHD and CM therapy, and outlines emerging drug targets in this field.^*

1.1 Mitochondrial energetics in normal cardiac function

The main function of cardiac mitochondria is ATP synthesis via oxidative phosphorylation (Ox-Phos). Following substrate oxidation, reducing equivalents generated by the TCA cycle are utilized by the respiratory chain (RC) to generate a trans-membrane potential (ΔΨ_m) which drives ATP synthesis. Under normal conditions the adult heart relies mostly on fatty acids to fuel Ox-Phos, with 10–30% of total ATP derived from glucose.⁴ Substrate utilization does vary under normal conditions, with increased aerobic glycolysis found postprandially and during exercise, and in relation to substrate availability and circadian rhythm.^5–8 In contrast, the immature heart relies predominately on glucose or lactate to provide carbon to the TCA cycle, with a transition to the mature fat-burning phenotype around 1 week of age.⁹

1.2 Mitochondrial dysfunction in cardiomyopathy

The major cause of acute CM, the precursor to heart failure, is myocardial ischemia (see section 1.3), while less acute forms of CM are idiopathic, inherited, or caused by chronic diseases such as hypertension and diabetes. Regardless of the cause, altered mitochondrial bioenergetics appear to play a substantial role in CM.¹⁰

Under pathologic conditions such as CM, the normal metabolic flexibility of the heart is superseded by activation of a fetal metabolic program, with a preference for glucose over fat as the substrate for Ox-Phos.¹¹ Although such changes lower the O₂ consumed per ATP produced, the yield of ATP per substrate also decreases. Such inefficient metabolism lowers ATP and phosphocreatine (PCr) levels, and decreases metabolic reserve and flexibility.^5,6,8 Interestingly, adult hearts which adopt this neonatal metabolism are refractory to protection by ischemic preconditioning (IPC, see section 3)¹² like the neonatal heart.^13,14 However, IPC itself comprises metabolic remodeling, suggesting that altered metabolism may be an adaptive response to ischemia, becoming pathologic if the stimulus persists.

Metabolic alterations are also an important component of inherited CMs.^6,15 Hypertrophic CM (the most common form with a prevalence of about 0.2% in the US population) is generally caused by contractile protein mutations which raise the energetic costs of contraction, leading to secondary energetic failure. However, around 27% of hypertrophic CM cases are caused by inborn errors of metabolism, leading to primary energetic failure.¹⁶ In addition, about 7% of dilated CM is caused by mutations in metabolism genes. Primary mitochondrial genetic disorders are also the most common etiology in non-compaction CM,¹⁷ and two recent reports suggest restrictive CM and arrhythmogenic right ventricular dysplasia may be plagued by metabolic abnormalities.^18,19

1.3 Mitochondrial dysfunction in ischemic heart disease

The primary pathologic event in IHD is acute myocardial infarction (AMI), caused by coronary artery obstruction. While permanent occlusion often results in cardiac remodeling and hypertrophy, transient occlusion is also detrimental to cardiac function, and the reperfusion phase of IR injury is particularly injurious to mitochondria.

A wide spectrum of mitochondrial derangements occurs in the post-IR heart, including: (i) Inhibition of respiratory complexes²⁰ and the adenine nucleotide translocase (ANT),²¹ (ii) Increased proton leak of the inner membrane,²² (iii) Oxidation of the phospholipid cardiolipin and associated membrane protein dysfunction,²³ (iv) Excessive generation of reactive oxygen species (ROS),²⁴ (v) Opening of the permeability transition (PT) pore, and release of cell-death inducing proteins including cytochrome c,²⁵ (vi) Catastrophic nucleotide depletion,²⁶ (vii) Mitochondrial Ca²⁺ overload.²⁷ These phenomena are summarized in Figure 1, and while a brief understanding is helpful to understand the mechanisms of protection by mitochondrial therapeutics, in-depth discussions are available elsewhere.²⁸

Figure 1. Mitochondrial pathologic events in IR injury.

During ischemia, lack of O₂ inhibits Ox-Phos, diverting the glycolytic end product pyruvate to lactate, resulting in cellular acidification. Disposal of acid via the Na⁺/H⁺ exchanger (NHE) brings Na⁺ into the cytosol. This Na⁺ is exported via the Na⁺/Ca²⁺ exchanger (NCX) causing cytosolic Ca²⁺ overload, which is exacerbated by low ATP disabling Ca²⁺ export by ATPases. Inhibited Ox-Phos is the primary cause of low ATP. Mitochondrial Ca²⁺ uptake is minimal due to low ΔΨ_m, and both acidic pH and accumulation of NADH keeps the PT pore closed. At reperfusion, rapid re-establishment of Ox-Phos and ΔΨ_m results in mitochondrial Ca²⁺ overload and ROS generation. Together with normalization of pH, this triggers PT pore opening, leading to cell death. In surviving cells, ROS activates mitochondrial uncoupling (H⁺ leak), which inhibits ATP synthesis, exacerbating the metabolic crisis. Oxidation of cardiolipin (CL) also occurs, along with nucleotide loss (both NADH from mitochondria and ATP from the cell). ROS damage to ANT contributes to low ATP levels. For full explanation see text.(Illustration Credit: Ben Smith).

2. Current mitochondrial treatments for cardiomyopathy

Several studies have attempted to treat heart failure associated with CM by targeting bioenergetic dysfunction. The general strategy for primary mitochondrial genetic disorders is supplementation with cofactors such as co-enzyme Q (Co-Q), carnitine, riboflavin, and thiamine, or antioxidants such as ascorbate. The efficacy of such treatments for CM is unclear,^16,17,29 since most mitochondrial disease trials have focused on neurologic complications of these disorders rather than CM. Several groups have tested the Co-Q analog idebenone, and in some studies this molecule slowed or prevented progression of hypertrophic CM in patients with Friedreich’s ataxia (a disease of iron-sulfur cluster assembly that affects the RC), however a more recent trial found no improvement in left ventricle (LV) mass or function.^29,30

Notably, even in CMs of unknown or non-mitochondrial etiology, the mainstay clinical agents are Co-Q and carnitine,³¹ particularly if carnitine deficiency is evident upon blood testing. Idebenone has also been used for non-mitochondrial CM, and it improved cardiac function in a mouse model of Duchenne muscular dystrophy (DMD).³² While a subsequent phase II trial in DMD patients failed to show significant improvement in cardiac function, a promising trend was seen and a larger trial is underway.³³

Another potential therapy for mitochondrial disorders is dichloroacetate (DCA), which stimulates pyruvate dehydrogenase (PDH) to shunt pyruvate into Ox-Phos. DCA has yielded mixed results in treating neurologic symptoms of mitochondrial disorders, leading to suggestions that it may only be useful in patients with PDH deficiency.^29,34 The ability of DCA to treat mitochondrial CM has not been tested, but in patients with a mixture of non-ischemic and ischemic dilated CM, DCA showed mixed effects on myocardial O₂ consumption and mechanical efficiency.^35,36

The amino acid L-arginine has also been proposed to treat mitochondrial disease, and in patients with mitochondrial CM, arginine increased aerobic metabolism and myocardial efficiency. The mechanism appeared to be independent of myocardial blood flow (i.e. arginine as a substrate for NO^• production), possibly ocurring via supplying the TCA cycle with 2-oxyglutarate.³⁷ Another drug of interest is perhexiline, which significantly increased PCr/ATP ratio, corrected diastolic dysfunction, and increased exercise capacity in CM patients, by inhibiting fatty acid oxidation (FAO) and increasing glucose utilization.³⁸ Other potential metabolic modulators, as well as non-pharmacologic therapies such as exercise training, may prove efficacious in treating CM.^5,8 Pharmacologic options are discussed further in section 4.8.

3 Current mitochondrial treatments for ischemic heart disease

Many therapies are aimed at slowing the progression of IHD, including statins to slow atherosclerotic lesion formation, anti-anginals to improve flow to transiently ischemic tissue, and anti-platelet agents to impede clotting at plaque rupture. While these agents do impact the incidence of ischemic events, there are limited therapies to protect myocardial function once an ischemic insult occurs.

In some situations cardiac ischemia can be anticipated in advance, such as during cardiac surgery or balloon inflation during PCI. Careful quantification of cardiac damage using biomarkers can allow clinical trials to identify protective strategies. As such, in cardiac surgery, cardioplegia (controlled arrest of the heart, dropping myocardial energy requirements),³⁹ hypothermia,⁴⁰ beta blockers,⁴¹ and tight glycemic control⁴² are all currently used for cardioprotection.

Recently, protecting the heart with ischemic preconditioning (IPC) has begun to translate into the clinic.⁴³ In this method, short intermittent IR cycles are administered prior to index ischemia, yielding reduced infarct size and improved post-ischemic function. IPC has completed successful clinical trials in both PCI and cardiac surgery, however it is usually accomplished by repeatedly cross-clamping the aorta, which may increase the risk of cerebral emboli or aortic vascular damage.⁴⁴ In this regard, it is notable that transient occlusion at sites remote from the heart (remote ischemic preconditioning, RIPC) is also cardioprotective,⁴⁵ with clinical studies showing RIPC efficacy in cardiac and non-cardiac surgery^46,47 and PCI.⁴⁸ However, failure of other RIPC trials suggests protocol optimization may be required.⁴⁹ As shown in Figure 2, the mechanism of IPC protection is incredibly complex, with mitochondria (and particularly mitochondrial K⁺ channels, see section 4.3) playing a key role.^50,51

Figure 2. Cardioprotective signaling: all roads lead to mitochondria.

IPC either directly or indirectly (via G-protein coupled receptors or growth factor receptors) activates numerous protein kinases and other cell signaling molecules, including the generation of ROS for signaling purposes. These signals filter through a limited number of downstream mediators including: (i) eNOS generation of NO^•, (ii) phosphorylation and inhibition of GSK-3β, (iii) changes in metabolism (iv) activation of mitophagy. At the mitochondrial level, many of these signals converge on the opening of mK_ATP and mBK channels, and the activation of H⁺ channels, both of which inhibit the PT pore directly, or inhibit the events leading up to its opening. Volatile anesthetics also trigger mitochondrial K⁺ channel opening and recruit many of the same kinase signals as IPC. Other drugs such as statins can also activate kinases in the RISK pathway. Together, these events inhibit cell death during subsequent IR injury. See text for more details. (Illustration Credit: Ben Smith).

Related to IPC, and more clinically tractable, is volatile anesthetic preconditioning (APC), in which halogenated anesthetics (iso-/des-/sevo-flurane) are known to activate many of the same intracellular signaling events as IPC. ^52,53 APC is also thought to open mitochondrial K⁺ channels (see section 4.3).

Unfortunately, the unanticipated nature of AMI makes it difficult to offer protection by preconditioning, and the current standard of care is early reperfusion via PCI or thrombolytics. Luckily it appears that intermittent reperfusion (ischemic postconditioning, IPoC) can confer some of the protective benefits of IPC.⁵⁴ Clinical trials have confirmed that IPoC during PCI can decrease infarct size and improve post MI heart failure.⁵⁵ Remote ischemic postconditioning (RIPoC) has also shown success in clinical trials.⁵⁶ The mechanism of IPoC is thought to involve opening of mitochondrial K⁺ channels⁵⁷ and inhibition of the mitochondrial PT pore.⁵⁸

Targeting metabolism is another strategy to protect the heart in AMI. Simply reducing cardiac mitochondrial workload with β-blockers has proven effective in reducing mortality after AMI⁵⁹ and coronary artery bypass graft (CABG) surgery,⁶⁰ although how well this extends to non-cardiac surgery patients with IHD is debated.^41,61 Another metabolic therapy is adenosine, which reduces infarct size when delivered at reperfusion in animal models.⁶² Early adenosine clinical trials were successful,⁶³ but later trials (AMISTAD) showed infarct reduction only in patient subsets, with a trend toward more adverse events and no improvement in hospital outcomes.⁶⁴ A large trial (AMISTAD-II) in patients undergoing PCI reported a 60% drop in infarct size, but no change in outcomes at 6 month follow-up.⁶⁵ Further trials using adenosine and analogs, such as AMP579, were disappointing, and meta-analysis revealed no change in overall outcomes.^66,67

Somewhat related to adenosine is acadesine (AICA riboside), an ATP metabolite which was originally thought to replenish post-IR adenine nucleotides. Although acadesine failed to elevate cellular ATP,⁶⁸ it still elicited cardioprotection,⁶⁹ leading to suggestions that its cardioprotective effects may result from activation of AMP dependent protein kinase (AMPK), an important regulator of metabolism (see section 4.8).⁷⁰ Further study showed that acadesine increased extracellular adenosine levels and decreased myocardial stunning,^71,72 leading to successful clinical trials in CABG⁷³ and a large phase II trial (Acadesine1024), which yielded lower 2 year mortality in patients with perioperative MI.⁷⁴ However, a recent multi-center phase III trial (NCT00872001^†) was terminated early (Merck Inc. 2010 2^nd quarter SEC filing) due to futility.

Another class of drugs that have largely failed to alleviate IR injury in the clinic, are antioxidants. Early reports on the involvement of ROS in IR pathogenesis⁷⁵ (Figure 1) were shortly followed by animal studies demonstrating exogenous superoxide dismutase (SOD) and/or catalase could elicit cardioprotection.⁷⁶ However, these reagents failed in large animal⁷⁷ and human trials.⁷⁸ Consistent with xanthine oxidase (XO) as a source of ROS in IR injury,⁷⁹ the XO inhibitor allopurinol was found to be cardioprotective in animal models,⁸⁰ but also failed in clinical trials. Other small molecule antioxidants (including Co-Q,⁸¹ mercaptopropionyl glycine (MPG)⁸² and others⁸³) have also been tested in IR injury, but all yielded mixed results in large animal or human studies.^84,85 Despite this, antioxidants remain key ingredients in cardioplegia.

Overall, numerous drugs have shown promise in the lab for protecting the heart in IHD via mitochondrial mechanisms, but none have passed clinical trials. The reasons for these failures have been discussed extensively elsewhere^86,87, and may include unforeseen drug interactions, the inappropriate nature of animal models, confounding risk factors such as diabetes and aging, or poor drug delivery to ischemic tissue. Furthermore, many cardioprotective drugs may actually block endogenous cardioprotective signals – for example IPC requires ROS, such that antioxidants block IPC.⁸⁸

4 Emerging mitochondrial therapeutic targets

Despite the wave of failed CM and IHD candidate therapeutics to date, the mitochondrial research community has made significant strides in deciphering mechanisms of cardioprotection (Figure 2), resulting in development of more specific and targeted molecules. Some of these newer candidates are discussed below, categorized by the mitochondrial phenomena which they target.

4.1 Mitochondrial permeability transition pore

Opening of the mitochondrial PT pore⁸⁹ is a major driver of necrotic cell death in IR injury. The identity of the molecules which assemble to form the PT pore remains somewhat controversial, however transgenic animal models have demonstrated pore regulation by the ANT, the phosphate carrier,⁹⁰ and cyclophilin D (CypD),^91,92 with the role of the voltage dependent anion channel (VDAC) less clear.⁹³ Other proteins implicated in the PT pore or its regulation include hexokinase II, which links the pore to cellular metabolism,^94,95 and mitochondrial translocator protein (TSPO) which interacts with VDAC.⁹⁶ Drugs targeting these PT pore components are candidates for cardioprotective therapeutics.

The first drug shown to inhibit PT pore opening was cyclosporine A (CsA),⁹⁷ which binds to CypD. CsA protects against IR injury in myocytes⁹⁸ and intact hearts,⁹⁹ and a pilot clinical trial demonstrated 20% infarct reduction with CsA administration prior to PCI in MI patients,¹⁰⁰ with benefits still evident 6 months later.¹⁰¹ A phase III trial (NCT01502774) is currently underway (NCT01502774). Notably, CsA is also used for immuno-suppression after organ transplant, mediated by its binding to calcineurin. Calcineurin is also a well-known hypertrophic signaling molecule, and hypertrophy due to CsA usage has been reported in transplant patients.^102,103 Thus, CypD-specific CsA analogs have been developed, including sanglifehrin A,¹⁰⁴ NIM811,¹⁰⁵ and Debio025.¹⁰⁶ These molecules are cardioprotective in animal models of IR injury, although none has yet progressed to clinical trials.^‡ A mitochondria-targeted CsA has also been developed, exhibiting higher potency and improved CypD affinity.¹⁰⁷ The TSPO ligands SSR180575, 4′-chlorodiazepam (Ro5-4684), and TRO40303 were also shown to reduce IR injury in various cell and animal models,^108–110 and TRO40303 is entering clinical trials (NCT01374321).

4.2 Nitric oxide based mitochondrial therapies

The role of NO^• in both endogenous cardioprotection and in mediating protective effects of drugs, has been reviewed extensively elsewhere.^111,112 In addition to classical NO^• signaling via cGMP/PKG, which feeds into RISK signaling (see section 4.6), non-classical functions of NO^• are also implicated in cardioprotection, including protein S-nitrosation^113,114 and the generation of nitro-lipids.^115,116 Mitochondria are a critical site of action for these signals, in particular the S-nitrosation of respiratory complex I resulting in reversible inhibition.¹¹⁷ Furthermore cyclophilin D has been identified as a target of S-nitrosation leading to attenuation of PT pore opening.¹¹⁸

A series of molecules have been developed to deliver NO^• to mitochondria. The first such agent was SNO-MPG, derived from the antioxidant MPG.¹¹³ SNO-MPG was actively sequestered into mitochondria, and exhibited cardioprotection in animal models of IR injury.¹¹⁹ Protection was associated with complex I S-nitrosation, and was lost in a complex I mutant mouse. A more recent development is MitoSNO1, an NO^• donor based on the triphenylphosphonium (TPP⁺) mitochondrial targeting moiety, which exhibits cardioprotection when delivered at reperfusion.¹²⁰ A related molecule is 2-hydroxylamine-vinyl-TPP⁺ (HVTP), which requires metabolism by intra-mitochondrial peroxidases to release NO^•,¹²¹ although its efficacy in IR injury is not yet known.

Another set of NO^•-derived molecules that target mitochondria are the nitro-lipids. These molecules are generated endogenously inside mitochondria during IPC, and when added exogenously they protect in various models of IR injury.^115,116 Such protection occurred on a timescale too fast for gene-regulatory effects usually attributed to nitro-lipids (e.g., PPARγ, NFκB, Nrf2/Keap1).¹²² Instead, nitro-lipid protection occurred via covalent modification of ANT, eliciting a mild increase in mitochondrial uncoupling,¹²³ which is known to confer cardioprotection.¹²⁴ Nitro-lipid derivatives are currently in early stage clinical development.

Finally, significant attention has recently focused on the anti-ischemic properties of nitrite (NO₂⁻).^125,126 Several studies have implicated mitochondria, and in particular complex I, as a downstream target of nitrite in cardoprotection.^126,127 Injectable sodium nitrite is FDA approved as a cyanide poisoning antidote, and several nitrite clinical trials are underway for cardiac ischemia (NCT01401517, NCT00924118, NCT01098409).

4.3 Mitochondrial potassium channels

The mitochondrial inner membrane contains numerous ion channels,¹²⁸ and many studies have suggested a role for mitochondrial K⁺ channels in cardioprotection. The mechanism of such protection is unclear, and may involve mild uncoupling, diminishing the ΔΨ_m driving force for Ca²⁺ overload and ROS generation (see section 1.3). Mild swelling associated with mitochondrial K⁺ influx may also regulate the PT pore.⁵²

A mitochondrial K_ATP channel (mK_ATP) is reported to play a critical role in cardioprotection by IPC. ^129,130 One of the most widely tested mK_ATP openers is diazoxide, which has demonstrated protection from IR injury in multiple cell and animal models. Two small clinical trials in CABG patients reported cardioprotection in patients pretreated with diazoxide or undergoing surgery supported by diazoxide-supplemented cardioplegia.^131,132 Other mK_ATP openers including BMS-191095, pinacidil, cromakalim, minoxidil, and nicorandil^§ are cardioprotective in animal models.^134–137

Although the molecular identity of mK_ATP is unclear, a link exists between respiratory complex II and mK_ATP, such that mK_ATP openers inhibit complex II, and complex II inhibitors open mK_ATP.^138–140 It is hoped that definitive identification of this channel will aid in the design of mK_ATP specific drugs. Notably, many pharmaceuticals including fluoxetine and some sulfonylureas inhibit mK_ATP, abrogating cardioprotection by IPC.^140,141

In addition to mK_ATP, mitochondria are also thought to contain a large conductance K⁺ channel (“BK”) encoded by the SLO gene family,¹⁴² which is implicated in cardioprotection by anesthetic preconditioning (APC).¹⁴³ Until recently, consensus held that the mitochondrial BK channel (mBK) was the SLO1 isoform, based on cardioprotection by the SLO1 opener NS1619 in animal models of IR injury.¹⁴⁴ However, NS1619 acts on several ion channels^145,146 and other mitochondrial targets.¹⁴⁷ A more specific SLO1 activator (NS11021) is also cardioprotective,¹⁴³ but controversy remains as it was recently shown in C. elegans and mice that SLO1 is dispensable for APC, and SLO2 is necessary for APC in the worm.¹³⁹ There are currently no SLO2 specific activators, but SLO2 may be a future therapeutic target.

4.4 Mitochondrial antioxidants

As discussed in section 3, multiple generic non-targeted antioxidants have been clinically unsuccessful in preventing IR injury, although several newer antioxidants are in clinical trials, including melatonin (NCT01172171), mangafodipir (NCT00966563), and edaravone (NCT00265239). In addition, a potentially novel therapeutic approach is the targeting of antioxidants to mitochondria.

A key chemical strategy for mitochondrial targeting is the TPP⁺ moiety (see section 4.2). First exploited in the 1970s as a method to measure ΔΨ_m,¹⁴⁸ several TPP⁺-conjugated antioxidants were developed the 1990s. The most prominent of these, MitoQ, was cardioprotective in a rat model of IR injury,¹⁴⁹ and is being developed clinically for other indications (NCT00433108). Other antioxidants conjugated to TPP⁺ include α-tocopherol (Mito-E), and nitroxides.¹⁵⁰ As discussed in section 4.2, a TPP⁺-conjugated NO^• donor confers potent cardioprotection.

Somewhat related to TPP⁺ conjugates are the SS peptides, which contain positively charged amino acids and a dimethyl tyrosine (DMT) residue. Originally developed as opioid peptide analgesics, following the discovery they were cardioprotective¹⁵¹ their mechanism was assigned to a mitochondrial antioxidant activity.¹⁵² The lead compound in this series, SS-31 (D-Arg-DMT-Asn-Phe-NH₂) has been renamed “Bendavia”, and is in clinical trials for AMI (NCT01572909).

Bioactivation is another strategy to target drugs to mitochondria, an example being the omega-(1-methyl-1H-imidazol-2-ylthio)alkanoic acids (Imz-S_NAs). These inactive pro-drugs are metabolized by fatty acid β-oxidation in the mitochondrial matrix to reveal a methimizole antioxidant.¹⁵³ Imz-S_NAs were shown to protect isolated cardiomyocytes from IR injury, in a manner inhibited by the β-oxidation blocker etomoxir. Such molecules may be particularly efficacious in the heart, given its preference for fatty acids as a metabolic substrate (see section 1).

4.5 Respiratory Chain inhibitors

Ischemia inhibits Ox-Phos due to lack of O₂ as the terminal electron acceptor for the RC. Thus, it is somewhat paradoxical that many chemical RC inhibitors are protective in ischemia. This includes inhibitors of complex I (rotenone,¹⁵⁴ amobarbital¹⁵⁵, S-nitrosothiols, nitrite), complex II (diazoxide,¹³¹ atpenin A5¹⁴⁰), complex III (antimycin A¹⁵⁶), and complex IV (CO,¹⁵⁷ H₂S,¹⁵⁸ NO^•). The mechanism of protection may involve inhibition of the large burst of ROS generation and Ca²⁺ overload that occurs at reperfusion, with inhibitor washout allowing a more gradual re-introduction of electron flux to the RC, somewhat akin to IPoC or slow reperfusion.¹⁵⁹ Although neurological side effects associated with RC inhibitors (e.g., Parkinson’s disease for rotenone, Huntington’s disease for 3-NP) may preclude their clinical applicability, amobarbital is clinically available, and is protective when delivered at reperfusion.¹⁵⁵

Related to RC inhibitors, mild uncoupling of Ox-Phos by chemicals such as FCCP can also elicit cardioprotection. The mechanism underlying this is unclear, but may include inhibition of ROS generation and Ca²⁺ overload.^124,160 As discussed in section 4.2, cardioprotection by nitro-lipids may also proceed via mild uncoupling.

4.6 RISK Pathway

In IR injury, the PT pore remains closed during ischemia and opens early in reperfusion (Figure 1).²⁵ Opening of the pore at reperfusion is regulated by the “reperfusion injury salvage kinase” (RISK) signaling pathway.¹⁶¹ This pathway is implicated in IPC and IPoC,¹⁶² and involves numerous kinases which converge on phosphorylation and inhibition of glycogen synthase kinase-3β(GSK3-β),¹⁶¹ which is thought to phosphorylate PT pore components (Figure 2).¹⁶³ The GSK3-β inhibitors SB-216763 and lithium improve post-IR cardiac function in animal models¹⁶⁴ (although mK_ATP channels may also be involved¹⁶⁵).

Many extracellular signals (e.g., insulin¹⁶⁶) can activate RISK signaling via receptors, and NO^• is also implicated in RISK signaling.^167,168 Several drugs also exert cardioprotective effects by activating RISK, most notably statins (simvastatin,¹⁶⁹ mevastatin,¹⁷⁰ atorvastatin¹⁷¹) via a mechanism not linked to cholesterol lowering. Multiple clinical trials (ARYMDA, NAPLES) and a meta-analysis¹⁷² have demonstrated cardioprotection with statin administration before emergent PCI. Further clinical trials are ongoing to deliver statins at reperfusion for patients presenting with MI (NCT01050348, NCT01334671, NCT00772564). Many other drugs that activate RISK signaling are reviewed elsewhere.^173,174

4.7 Aldehyde dehydrogenase 2

A relatively new target for mitochondrial protection in IR injury is ALDH2, a mitochondrial enzyme which removes toxic aldehydes such as 4-hydroxy-2-nonenal (HNE). HNE has been implicated in cardiac IR injury, and can inhibit metabolic enzymes and RC complexes, and open the PT pore.¹⁷⁵

Overexpression of ALDH2 decreases HNE levels, yielding decreased infarct size and increased post ischemic function.¹⁷⁶ Furthermore Alda-1, a small molecule ALDH2 activator, decreases infarct size in animal models of IR injury.¹⁷⁶ Recently α-lipoic acid, a cofactor for ALDH2, has shown cardioprotection in an ALDH2 dependent fashion,¹⁷⁷ and is currently in clinical trials for several conditions including diabetes (NCT00398892) and atherosclerosis (NCT00765310). In addition, ALDH2 activation may be capable of delivering cardioprotection in conditions where other drugs have failed, including diabetes and nitrate tolerance.^178,179 The latter is a major clinical problem, as patients taking nitrates are often at risk for MI¹⁸⁰.

4.8 Mitochondrial metabolism

One of the earliest methods to improve mitochondrial metabolism in IR was the use of glucose-insulin-potassium (GIK) supplementation. In addition to GIK, insulin alone can drive both glucose and fatty acids into the cell to support metabolism.¹⁸¹ GIK has shown mixed results in CABG surgery, with a recent meta-analysis showing no improvement in the incidence of arrhythmia or mortality.¹⁸² However, with recent emphasis on tight glucose control during cardiac surgery, many patients receive intraoperative GIK anyway.¹⁸³ Results in AMI have been consistently negative, although a recent trial (NCT00091507) of GIK delivery by emergency personnel in the pre-hospital setting yielded a 50% drop in in-hospital cardiac arrest and mortality.¹⁸⁴

FAO is also an attractive drug target in ischemia, since it uses more O₂ per ATP produced (vs. glucose). During IR injury, catecholamine-induced increases in serum fatty acid levels may enhance FAO,¹⁸⁵ and indeed reperfusion with fatty acid free serum is cardioprotective.¹⁸⁶ Inhibitors of carnitine palmitoyltransferase (CPT1, which transports fatty acids into mitochondria) such as etomoxir have also demonstrated cardioprotection in animal models of IR injury,¹⁸⁷ and improved hemodynamics in clinical trials for heart failure.¹⁸⁸ Another CPT1 inhibitor, perhexiline, corrected diastolic dysfunction, and increased exercise capacity in patients with hypertrophic CM.³⁸ Oxfenicine shows similar effects but is not used in humans.¹⁸⁹

Other drugs that inhibit FAO are the anti-anginals trimetazidine and ranolazine.¹⁹⁰ A meta-analysis of trimetazidine in heart failure demonstrated improved ventricular function and decreased mortality, cardiovascular events and hospitalizations.¹⁹¹ In IR injury it exhibited mixed results in both animal models^192,193 and clinical trials, demonstrating protection when administered before CABG,¹⁹⁴ but no protection in AMI except for a cohort of patients who did not receive thrombolysis.¹⁹⁵ Ranolazine is reported to shift cell metabolism from FAO toward glycolysis,¹⁹⁶ although may also have effects on cellular Na⁺ and Ca²⁺ homeostasis,¹⁹⁷ or protection of complex I.¹⁹⁸ In animal models of heart failure ranolazine reduced diastolic dysfunction and improved LV efficiency,¹⁹⁹ and in IR injury it decreased both infarct size and cardiac enzyme release, and improved post-ischemic ventricular function.²⁰⁰ Clinical trials confirmed its efficacy in treating angina, but showed no protection in AMI.²⁰¹ Further ranolazine trials are in development for PCI (NCT01491061). Despite the consensus that fatty acids are toxic to the ischemic heart, it is notable that carnitine supplementation is paradoxically cardioprotective in animal models of IR injury.²⁰² In clinical trials carnitine prevented left ventricular remodeling after STEMI (CEDIM-I), and reduced 5 day mortality, however this mortality benefit was non-significant at 6 months (CEDIM-II).^203,204

Global regulators of metabolism may also be therapeutic targets in IHD. An example is AMPK, which up-regulates both FAO and glucose utilization in response to decreased ATP levels. Whether increased AMPK is helpful or harmful to myocardium after IR injury is still unclear.²⁰⁵ The AMPK specific activator A-769662 is cardioprotective during IR injury, ²⁰⁶ but activators such as AICAR or metformin may have non-specific effects. Another important metabolic regulator is SIRT1, which is required for cardioprotection by acute IPC.²⁰⁷ Despite controversy regarding the SIRT1 activator resveratrol,²⁰⁸ SIRT1 activators are in clinical development (NCT00933530).

Similarly SIRT3, which is located in the mitochondria, has also emerged as an important metabolic regulator known to deacetylate numerous mitochondrial proteins involved in FAO and Ox-Phos.²⁰⁹ Notably a mitochondrial acetyltransferase (GCN5L1) that is counterpart to SIRT3 has recently been described, although from a pharmacologic standpoint, specific drugs that target mitochondrial SIRT3 (vs. other SIRTs) have not yet been developed.²¹⁰ Finally, it has recently emerged that mitophagy, the process by which damaged mitochondria are removed and recycled, is an important player not only in metabolic homeostasis, but is also critical for IPC.^211,212

5 Conclusions & Future directions

In summary, despite numerous failures in clinical development of drugs to treat IHD and CM, the pipelines of several companies still contain IHD and CM drugs, and many of these therapies appear targeted at mitochondria. Coupled with novel compounds which have not yet reached clinical trials, these are exciting times to be involved in drug development for these debilitating conditions. However, it should be emphasized that, despite the large number of mitochondrial drugs discussed in section 4, there is no guarantee that these molecule will be more efficacious in the clinic than those tested to date. Many of the same reasons that have been invoked to explain previous clinical failures also apply to these therapeutic candidates.⁸⁷

In addition to the novel compounds discussed, other significant developments also deserve brief mention. The first is CAESAR, an NIH sponsored consortium which aims to bridge the gap between pre-clinical and clinical trials for promising cardioprotective therapies.²¹³ By adopting standardized IR protocols in several animal models, and using practices common to clinical trials (double blinding, multiple centers, placebo controls), the consortium aims to put only the most promising therapies forward into human clinical trials. This will clearly be a valuable tool for researchers in the field.

Secondly, with a few exceptions, high-throughput screening (HTS) has been under-utilized as a drug development approach for IHD and CM. This may be because clinically relevant models of IR injury often use animals and are time consuming and expensive (i.e., not amenable to HTS), while HTS-ready IR models may not be physiologically relevant. In a recent screen for molecules that alter cell metabolism, it was hypothesized that hits which promote glycolysis would be beneficial during ischemia, and indeed one such molecule (meclizine) was protective in heart and neuronal models of IR injury.²¹⁴ In another recent screen, we developed an IR injury model using a 24-well metabolic phenotyping device, with post-IR cell death and metabolism as endpoints. One hit from the screen was cloxyquin, which is closely related to the mitophagy-inducing drug clioquinol, currently in clinical trials for cancer (NCT00963495). Another was ipiriflavone, which was first proposed as an anti-ischemic drug 30 years ago.²¹⁵ Further phenotypic screens of this type may yield novel cardioprotective drugs which engage the mitochondrial targets discussed herein.

Table 1.

Clinical development of mitochondrial therapies for cardiomyopathy and ischemic heart disease.

Agent	Target/MOA	Clinical development/usage	Status of cardiac clinical development	Clinical trial	Ref’s
PT pore inhibitors
4′-chlorodiazepam (Ro5-4684)	TSPO				109
CsA	CypD	In-use (immunosupression)	Phase III (AMI)	NCT01502774	100
Debio025	CypD	Phase II (Hepatitis C)			106
NIM811	CypD	Phase II (Hepatitis C)			216
Sanglifehrin A	CypD				104
TRO40303	TSPO		Phase II (AMI)	NCT01374321	110
NO analogs
Mito-SNO1					120
Nitrolipids					123
Nitrite		In-use (CN antidote)	Phase II (AMI)	NCT01584453	127
SNO-MPG					119
Antioxidants
Edaravone		In-use (Stroke, Japan)	Phase IV (AMI)	NCT00265239
Glutathione		Nutriceutical	Cardioplegia additive		217
HVTP					121
Imz-SNAs					153
Mangafodipir			Phase II (AMI)	NCT00265239
Melatonin		Nutriceutical	Phase II (AMI)	NCT01172171
MitoE					150
MitoQ		Phase II (Hepatitis C, NASH)			149
MPG					82
SOD, Catalase		Various clinical trials			78
SS31 (Bendavia)			Phase II (AMI)	NCT01572909
Potassium Channel Openers
3-NP	mKATP				152
Atpenin A5	mKATP				140
Cromakalim	mKATP				134
BMS-191095	mKATP				135
Diazoxide	mKATP	Various clinical trials			132
Malonate	mKATP	Phase II (Osteoporosis)			139
Minoxidil	mKATP	In-use (Hypertension, Alopecia)			136
NS11021	mBK				143
NS1619	mBK				145
Pinacidil	mKATP	In-use (Hypertension)			134
Respiratory Chain Inhibitors
Amobarbital	Complex I	In-use (Anxiety, sedation)			155
Antimycin A	Complex III				156
H2S	Complex IV	Phase I (Renal function)			158
Rotenone	Complex I				154
RISK Pathway Modulators
Lithium		In-use (Bipolar disorder)			164
SB216763	GSK3-β				165
Statins	HMG-CoA reductase	In-use (Hyperlipidemia)	Completed phase IV (AMI)	ARYMDA and ARYMDA-ACS	172
Aldehyde Dehydrogenase 2
α-lipoic acid		Nutriceutical			177
Alda1					176
Metabolic modulators
A-769662	AMPK				206
Acadesine (AICAR)	AMPK, nonspecific		Failed phase III (MI)	NCT00872001	74
Co-factors (carnitine, Co-Q etc.)		Nutriceutical	Various clinical trials (CM)
DCA	Pyruvate dehydrogenase	Failed clinical trials for lactic acidosis, MELAS, Multiple trials in cancer.			36
Etomoxir	CPT1				188
GIK			Multiple trials (CABG, non-cardiac surgery, AMI)		182
Idebenone	Respiratory chain	In-use (mito’ myopathies)	Phase III (DMD)	NCT01027884	30
L-Arginine	NOS & TCA cycle substrate	In-use (mito’ myopathies)	Failed phase II (MI)	NCT00051376	37
Oxfenicine	CPT1				189
Perhexiline	CPT1		Clinical (AMI, Aus/NZ)		38
Ranolazine	Late Na+ channel, specific target unclear	In-use (Angina)	Failed phase III (MI) Phase IV (PCI)	NCT00099788 NCT01491061	200
Trimetazidine	Late Na+ channel, specific target unclear	In-use (Angina)	Multiple clinical trials (CM)		191
Other
Adenosine	Adenosine receptors		Failed clinical trials (MI, CABG)		65
AMP579	Adenosine receptors		Failed phase III (MI)	ADMIRE I & II	66
Anesthetic preconditioning		In-use	Phase IV (cardiac surgery)	NCT00364637	52,53
β-blockers	β-adrenergic receptors	In-use (patients at risk of MI)			61
Cloxyquin/clioquinol		In-use (anti-fungal/-protozoal)			170
Hypothermia		In use	Phase III (AMI)	COOL-MI NCT01379261	40
Meclizine	Histamine receptors	In-use (Antihistamine)			214
Preconditioning (IPC, IPoC, RIPC)			Multiple clinical trials

Abbreviations as per master list at start of paper

Abbreviations Used

*Note – many drug names contain proprietary abbreviations relating to company names (e.g. NS1619 from NeuroSearch A/S). Such abbreviations are not defined herein

ALDH2

Aldehyde dehydrogenase isoform 2

AMI

Acute myocardial infarction

AMPK

Adenosine monophosphate (AMP) dependent protein kinase

ANT

Adenine nucleotide translocase

APC

Anesthetic preconditioning

CABG

Coronary artery bypass graft

CAESAR

NIH consortium for preclinicAl AssESsment of CARdioprotective therapies

cGMP

Cyclic guanosine monophosphate

Co-Q

C-enzyme Q₁₀ (ubiquinone)

CM

Cardiomyopathy

CPT1

Carnitine palmitoyltransferase 1

CsA

Cyclosporin A

CypD

Cyclophilin D

DCA

Dichloroacetate

DMD

Duchenne muscular dystrophy

ΔΨ_m

Mitochondrial membrane potential

FAO

Fatty acid oxidation

FCCP

Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone

FDA

Federal drug administration

GIK

Glucose-insulin-potassium

GSK3-β

Glycogen synthase kinase-3β

HTS

High throughput screening

IHD

Ischemic heart disease

Imz-S_NA

omega-(1-methyl-1H-imidazol-2-ylthio)alkanoic acid

IPC

Ischemic preconditioning

IPoC

Ischemic postconditioning

IR

Ischemia and reperfusion

Keap-1

Kelch-like ECH-associated protein 1

LV

Left ventricle

MELAS

Mitochondrial encephalopathy lactic acidosis and stroke-like episodes

MI

Myocardial infarction

MitoQ

Mitochondrially targeted co-enzyme Q

mK_ATP

Mitochondrial ATP sensitive K⁺ channel

MOA

Mechanism of action

MPG

Mercaptopropionyl glycine

NASH

Non alcoholic steatohepatitis

NCT #

National clinical trials registry #

NFκB

Nuclear factor κB

NOS

Nitric oxide synthase

3-NP

3-nitropropionic acid

Nrf2

Nuclear factor erythroid 2-related factor 2

Ox-Phos

Oxidative phosphorylation

PCI

Percutaneous coronary intervention

PDH

Pyruvate dehydrogenase

PCr

Phosphocreatine

PKG

cGMP dependent protein kinase

PPARγ

Peroxisome proliferator activated receptor isoform gamma

PT

Permeability transition

RC

Respiratory chain

RISK

Reperfusion injury salvage kinase

ROS

Reactive oxygen species

RIPC

Remote ischemic preconditioning

SIRT

Silent information regulator two P homolog

SNO-MPG

S-nitroso MPG

SS

Szeto Schiller

STEMI

ST segment elevation myocardial infarction

TCA

Tricarboxylic acid

TPP⁺

Triphenylphosphonium

TSPO

mitochondrial translocator protein

VDAC

Voltage dependent anion channel

XO

Xanthine oxidase