reperfusion injury

Pharmacology & Therapeutics 140 (2013) 258–266

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Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/pharmthera

Associate editor: L. Lash

Mitochondrial inner membrane lipids and proteins as targets for decreasing cardiac ischemia/reperfusion injury David A. Brown a,c,⁎, Hani N. Sabbah d, Saame Raza Shaikh b,c a

Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, NC, USA Department of Biochemistry and Molecular Biology, Brody School of Medicine, East Carolina University, Greenville, NC, USA East Carolina Diabetes and Obesity Institute, Brody School of Medicine, East Carolina University, Greenville, NC, USA d Department of Internal Medicine, Henry Ford Hospital, Detroit, MI, USA b c

a r t i c l e

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a b s t r a c t A major manifestation of coronary artery disease is cardiac ischemia/reperfusion injury, with the extent of injury directly relating to short- and long-term prognosis. Emerging evidence suggests that mitochondrial dysfunction is centrally involved in ischemia/reperfusion injury. In this review, we summarize the role of mitochondria in the etiology of ischemia/reperfusion injury. We attempt to highlight emerging areas for cardiac mitochondrial medicine and discuss recent advances regarding the molecular composition of inner membrane channels. We discuss interactions between lipids and proteins in the inner mitochondrial membrane, and the ever-evolving nature of this relationship during ischemia/reperfusion. Potentially novel treatments influencing the biophysical properties of inner membrane lipids (such as cardiolipin) are presented. Finally, we point to areas where future study is needed to expand and improve our understanding of mitochondrial pathophysiology during cardiac ischemia/reperfusion. © 2013 Elsevier Inc. All rights reserved.

Keywords: Cardioprotection Cardiolipin Mitochondria Heart

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The bioenergetics of acute coronary syndromes (acute myocardial infarction) . . . . . . 3. Reperfusion injury: Killing cardiac tissue by trying to save it . . . . . . . . . . . . . . 4. Reducing injury by targeting mitochondria . . . . . . . . . . . . . . . . . . . . . . 5. Mitochondrial calcium fluxes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Reactive oxygen species: Everybody's favorite mechanism, nobody's effective drug (yet?) 7. Mitochondria-targeting peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Mitochondrial membranes are unique molecular targets for therapeutic intervention . . 9. The role of cardiolipin in mitochondrial membrane organization . . . . . . . . . . . . . 10. Biophysical consequences of changes in cardiolipin composition, content and peroxidation 11. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Ischemic heart disease continues to ravage the globe, exacting monetary and human costs that are unmatched by any other disease (AHA, 2007). Clinical manifestations include acute coronary syndromes (ACS) ⁎ Corresponding author at: Brody School of Medicine, 6N-98, 600 Moye Blvd, Greenville, NC 27834, USA. Tel.: 252 744 2862. E-mail address: [email protected] (D.A. Brown). 0163-7258/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pharmthera.2013.07.005

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in the short-term, and progression to heart failure over the long-term. Because the extent of ischemic injury is a good prognosticator of short- and long-term morbidity and mortality (Herlitz et al., 1984, 1988; Pfeffer & Braunwald, 1990; Miller et al., 1995), interventions that can reduce myocardial injury have enormous potential to improve short and long-term outcomes of ACS patients. Pharmacological interventions that reduce the burden of ischemic heart disease are at an interesting crossroads. From pre-clinical studies, there are hundreds of interventions that are known to reduce

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the extent of ischemic injury, but few of these have successfully translated into cardioprotective therapies for ACS patients. Putative reasons for the lack of efficacy in the clinic have been excellently described elsewhere (Downey & Cohen, 2009; Ludman et al., 2010), and are generally attributed to inappropriate animal models, ineffective molecular targets, dosing issues, poor timing (many compounds administered after the onset of reperfusion are not as effective as pre-ischemic administration), or questionable patient selection. Successful treatments that have shown clinical improvements include adenosine, hypothermia, and blocking the sodium–hydrogen exchanger with cariporide (Bolli et al., 2004; Kloner et al., 2006). Among the most recent successful interventions, targeting mitochondria has shown promise in reducing the burden of ischemic heart disease in patients (Piot et al., 2008). In this review, we seek to highlight the potential of targeting mitochondria, particularly the mitochondrial inner membrane lipids and the proteins within, during acute coronary syndromes. Our goal is to focus on recent insights regarding mitochondria-targeting strategies, with an emphasis on potential for translation into the clinic.

2. The bioenergetics of acute coronary syndromes (acute myocardial infarction) Mounting evidence suggests that both short- and long-term manifestations of ACS have a bioenergetics component. Specifically, a decline in mitochondrial energetics is noted in acute and chronic manifestations of ischemia/reperfusion. Because a major focus of this review is on therapies that can sustain mitochondrial function, we will provide a brief overview of mitochondrial bioenergetics in health and disease. As they resembled granular threads upon microscopic investigation, Carl Benda named “mitochondria” at the turn of the 20th century. The early function of mitochondria was assigned to the processing of genetic information, synthesis of proteins and lipids, and cellular respiration (Tzagoloff, 1983; Scheffler, 2008). Of these, the latter function is known to be the primary function of mitochondria: utilizing substrates

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and atmospheric oxygen to create a transmembrane electrochemical gradient, which is in turn used to regenerate ATP. Under normoxic conditions, electrons are passed to the electron transport system in the inner mitochondrial membrane, and the energy liberated by electrons flowing down the electron transport chain ‘vector’ creates an electrochemical proton gradient (Mitchell, 1961). This gradient is predominantly reflected in the mitochondrial membrane potential, ΔΨm. As protons flow down their gradient back into the mitochondrial matrix, the liberated energy has three major fates: 1) The regeneration of ATP through complex V (Fo–F1ATPase; complex V). This ATP is quickly transported out of mitochondria and hydrolyzed to sustain myocardial contraction and maintain intracellular ion gradients; 2) The replenishment of cellular NADPH levels through the transhydrogenase, a process essential for cellular redox homeostasis; or 3) The release of the energy as heat through the function of uncoupling proteins (see Fig. 1). Importantly, many of the proteins involved in mitochondrial transport processes depend on the presence of cardiolipin (CL) in the inner membrane (these CL “micro-domains” will be discussed in detail below). As the final electron acceptor, oxygen plays a central role in the establishment and maintenance of mitochondrial energetics. The abolishment of oxygen and substrate delivery by interrupting coronary flow, as is the case during myocardial ischemia, results in a rapid decline in cellular ATP levels. Despite temporary buffering by anaerobic catabolism and the creatine kinase system, global declines in ATP levels are evident as early as 15 min after the onset of ischemia (Brennan et al., 2006; Murphy & Steenbergen, 2008a). The consequences of this decline in bioenergetics are the onset of ischemic contracture and abnormalities in cellular ion gradients (Frasier et al., 2011; Walters et al., 2012). As cellular ion gradients decline, cellular membrane potential becomes more positive and action potentials shorten due to opening of ATP-sensitive potassium channels (Akar et al., 2005). Eventually, the propagation of electrical signals through the ischemic region is impaired due to sustained tissue depolarization and electrical “uncoupling” (not to be confused with mitochondrial uncoupling) through closure of gap junctions (Cascio et al., 2005). If uncorrected, this loss of energetics results in

Fig. 1. The proton circuit in healthy bioenergetics. Respiratory complexes are tightly arranged in functional “supercomplexes” or “respirasomes.” Cardiolipin-enriched microdomains, depicted in light blue, are necessary for optimal protein organization and function. The proton-motive force consists primarily of the mitochondrial membrane potential, Δψm. The potential energy in Δψm can be utilized to regenerate ATP, replenish cellular redox pools through the nicotinamide nucleotide transhydrogenase (NNT), generate heat through uncoupling proteins (UCP), or promote trans-membrane transport (not depicted).

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the formation of rigor, cell death through necrotic and apoptotic pathways, inflammation, and ultimately myocardial infarction. 3. Reperfusion injury: Killing cardiac tissue by trying to save it The most important intervention to salvage ischemic myocardial tissue is the re-establishment of coronary perfusion. Important advancements have been made in the clinical realm over the last 50 years to improve tissue reperfusion (Kloner & Schwartz, 2011; Schwartz Longacre et al., 2011). However, the re-establishment of coronary perfusion comes with a different set of problems, collectively termed “reperfusion injury.” Reperfusion injury results in increased infarct size, further impairment of myocardial contraction, and the development of reperfusion arrhythmia. Furthermore, even after removal of the coronary obstruction, cardiac perfusion can still be greatly impaired through the “coronary no-reflow” phenomenon, whereby microvascular damage precludes restoration of flow (Schwartz & Kloner, 2012). Although each of these indices of reperfusion injury is multi-factorial, there is a clear (and potentially reversible) bioenergetic component, suggesting that targeting mitochondria under these circumstances can be helpful. Several recent reviews have comprehensively addressed the cellular mechanisms involved in reperfusion injury (Kloner et al., 2006; Murphy & Steenbergen, 2008b; Walters et al., 2012). In general, cellular overload of calcium and heightened production of reactive oxygen species (ROS) causes the opening of energy-dissipating proteins along the inner membrane, ultimately diminishing the ability of the mitochondrial network to sustain ATP production to meet demand. The loss of ATP further hinders the ability of the heart to sustain ion gradients, resulting in rhythm disturbances, contractile dysfunction, and infarction. In the sections below, we will discuss these mechanisms in greater depth and elucidate the potential to mitigate myocardial reperfusion injury through mitochondria-dependent pathways. Although pharmacological interventions seeking to reduce reperfusion injury are the major focus of our review, it is important to note that the intrinsic ability of the heart to protect itself through endogenous mechanisms is robust, and has already shown translational relevance. It is well known that gradually bringing heart tissue in and out of ischemia (“pre- or post-conditioning”) reduces injury (Murry et al., 1986; Kin et al., 2004; Vinten-Johansen et al., 2007; Ovize et al., 2010). Post-conditioning in particular appears to show promise in the clinic, as sequential balloon inflations during angioplasty provided short- and long-term benefits to patients (Staat et al., 2005; Darling et al., 2007; Thibault et al., 2008). Exercise is another intervention where the endogenous ability of the heart to protect itself is clear. Exercise provides potent and sustainable cardioprotection in animals and humans (reviewed in Brown & Moore, 2007; Powers et al., 2008; Frasier et al., 2011). There is also a role for mitochondria in endogenous protection against injury, suggesting that targeting mitochondria has significant potential to reduce the burden of ischemic heart disease. 4. Reducing injury by targeting mitochondria In early reperfusion, a number of cellular changes take place that impair proper restoration of cardiac energetics, and subsequently cardiac function. Cellular overload of calcium and ROS has been well characterized, and have important implications for mitochondria. With the initiation of reperfusion, restoration of ΔΨm returns the potential to regenerate ATP. However, the capacity to restore ATP is extremely fragile, and ΔΨm often collapses after the initiation of reperfusion (Aon et al., 2003; Matsumoto-Ida et al., 2006; Brown et al., 2010). A number of strategies have the potential to stabilize energetics by targeting mitochondria during this vulnerable early reperfusion period.

Many excellent review articles have covered in detail the ability to reduce cardiac injury by targeting the permeability transition pore (PTP) (Baines, 2009; Halestrap, 2009; Baines, 2010, 2011). Opening of the PTP results in collapse of ΔΨm, leading to apoptotic and necrotic cell death. Increased ROS in early reperfusion primes the PTP for opening, which can be triggered by mitochondrial calcium overload. The intracellular pH also has an important effect on the PTP. Acidic pH, as is observed in ischemia, is thought to reduce PTP opening probability. The alkaline shift of pH in reperfusion promotes PTP opening, and indeed maintenance of an acidic intracellular pH in early reperfusion has been shown to be cardioprotective (Heusch, 2004; Cohen et al., 2007). Compounds that directly block the PTP (by inhibiting the association of the isomerase cyclophilin-D) are currently being tested in clinical trials, having shown some success in small trials of coronary reperfusion in ACS patients (Piot et al., 2008; Mewton et al., 2010). There is also a role for the mitochondrial ATP-sensitive potassium channel in many models of cardioprotection (Gross & Fryer, 1999; Gross & Fryer, 2000; O'Rourke, 2000, 2004; O'Rourke et al., 2005). The opening of these channels is believed to be beneficial, although there is some debate regarding the pharmacological specificity of compounds commonly used to activate or block these channels (Hanley et al., 2002, 2003, 2005). Although the molecular identity of this complex has been somewhat elusive over the years (Hanley et al., 2002, 2003, 2005; O'Rourke et al., 2005), very recent findings by Foster et al. (2012) suggest that functional channels contain renal outer medullary potassium channel subunits. This identification of channel subunits may open the door for novel therapies designed to reduce myocardial injury by targeting this channel. 5. Mitochondrial calcium fluxes The restoration of ΔΨm can have harmful consequences with regard to mitochondrial calcium handling. Intracellular and intramitochondrial free calcium concentrations are roughly similar during the cardiac cycle (in the high nM to low μM range; Bernardi, 1999), making ΔΨm the major driving force for calcium entry into the mitochondrial matrix. Calcium entry into the matrix exerts hormesis, where a small increase in matrix concentrations can stimulate oxidative phosphorylation (Balaban, 2009; Glancy & Balaban, 2012), but significant calcium overload in the matrix promotes the collapse of ΔΨm. Several studies have noted that blocking mitochondrial calcium influx decreases the extent of injury (Scarabelli et al., 2004; GarciaRivas Gde et al., 2006; Zhang et al., 2006; Romero-Perez et al., 2008), although specificity and cell permeability concerns have been noted with several of the experimental compounds used (Gupta et al., 1988, 1989; Griffiths, 2000). With the recent molecular identification of the ion channel components responsible for calcium influx (Baughman et al., 2011; De Stefani et al., 2011), continued investigation seems likely to foster new therapies to reduce cardiac injury by lowering mitochondrial calcium overload. Targeting the major route for calcium extrusion from the matrix, namely the recently identified sodium–calcium–lithium exchanger (NCLX; Palty et al., 2010), may also decrease the extent of myocardial injury. Although much more work is needed in this area, recent studies suggest that sustaining mitochondrial calcium levels by inhibiting matrix calcium efflux may be promising. Liu et al. (2010) found that impaired bioenergetics with ouabain (which induces cellular sodium overload similar to that observed during ischemia/reperfusion) were reversed in the presence of the NCLX inhibitor CGP-37157, implying that the sustained activation of bioenergetics by calcium can be cardioprotective. These data are consistent with other reports in which CGP-37157 prevented mitochondrial depolarization during conditions of calcium overload (Nicolau et al., 2009). Finding means to ensure appropriate balance between maintaining calcium to

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stimulate oxidative phosphorylation whilst avoiding matrix calcium overload, is essential when targeting the mitochondrial calcium circuit to treat reperfusion injury. 6. Reactive oxygen species: Everybody's favorite mechanism, nobody's effective drug (yet?) There is arguably no mechanistic player in myocardial reperfusion injury that has received more attention over the last 30 years than ROS. Spin-trap resonance studies in the 1980's detected high levels of ROS in the coronary effluent, most notable in the first 30 min of reperfusion (Zweier et al., 1987; Bolli, 1988; Zweier, 1988; Zweier et al., 1989). During this time, tissue production of reactive intermediates exceeds scavenging capacity, resulting in oxidation of cellular redox pools. This shift can directly damage proteins and lipids, or indirectly alter function vis-a-vis post-translational modifications. Administration of exogenous ROS scavengers have shown cardioprotective efficacy in many experimental studies (reviewed in Murphy & Steenbergen, 2008b). Experimental approaches have shown some efficacy with exogenous ROS ‘scavengers’ (such as MPG), superoxide mimetics (Bognar et al., 2006), glutathione precursors such as Nacetyl-cysteine (NAC) (Kingma & Rouleau, 1989; Alberola et al., 1991; Wang et al., 2011), or by tethering scavengers to molecules that hone to mitochondria. To the best of our knowledge, the translation of NAC with regard to infarct size reduction has not yet been shown in the clinic, although NAC showed efficacy in reducing post-operative arrhythmic burden in a small clinical trial (Ozaydin et al., 2008). Although the use of ROS scavengers in clinical studies in attempts to corroborate pre-clinical findings appeared to be a promising approach to limit coronary reperfusion injury, studies have been fraught with negative results (Flaherty et al., 1994; Tsujita et al., 2004). Further, effective concentrations for some of these compounds are very high, and can result in various adverse clinical events including anaphylaxis (Lynch & Robertson, 2004). A class of mitochondria-targeting compounds has been developed in which molecules are tethered to triphenylphosphonium, a cationic lipophilic compound that is drawn to the highly negative ΔΨm. These compounds showed promise (Ross et al., 2005), although it should be noted that due to their cationic charge, their uptake may be self-limiting and may preferentially target mitochondria that are essentially intact or well on their way to recovery (Szeto, 2008). 7. Mitochondria-targeting peptides Very recent studies support the development of cell-permeable peptides, some of which localize to mitochondria (Horton et al., 2008; Yousif et al., 2009; Horton et al., 2012). These peptides are typically low-molecular-weight amphiphiles that penetrate plasma membranes without the need for specific transport proteins (Lindgren et al., 2000; Richard et al., 2003; Zhao et al., 2003). Proposed mechanisms for cellular penetration involve inverse micelle formation, electroporation, and bonding interactions between arginine residues and membrane phosphate groups (Derossi et al., 1998; Futaki et al., 2001; Lundberg & Langel, 2003; Cahill, 2010), which are energy-independent processes. Many membrane-permeable peptides are water-soluble cations (Wender et al., 2000; Futaki et al., 2001; Wadia & Dowdy, 2002; Heitz et al., 2009; Kelley et al., 2011), and typically have aromatic motifs in their structure, resulting in hydrostatic interactions with anionic phospholipids (Wadhwani et al., 2012). In 2004, Szeto and Schiller reported the discovery of a new class of mitochondria-targeting peptides, collectively termed “SS-peptides,” and were the first to show the efficacy of these peptides in preserving contractile force after cardiac ischemia/reperfusion (Zhao et al., 2004). Subsequent studies confirmed the cardioprotective efficacy of one such peptide known as Bendavia (SS-31; MTP-131). In models spanning isolated mitochondria, ventricular cardiomyocytes, isolated

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hearts, and in vivo sheep and rabbits, Bendavia was shown to reduce ROS-dependent cell death, to reduce infarct size, and to improve post-ischemic coronary perfusion when administered prior to reperfusion (Kloner et al., 2012; Sloan et al., 2012). Treatment of diabetic mitochondria with Bendavia reduced PTP opening in diabetic hearts, although there was no discernible effect of Bendavia in healthy mitochondria (Sloan et al., 2012). Two therapeutic benefits of mitochondria-targeting peptides are worth noting. First, Bendavia appears to target mitochondria because of the anionic phospholipid CL (Birk et al., 2013), and not necessarily because of the highly negative ΔΨm. Whether in isolated cells exposed to the uncoupler FCCP, or in intact hearts during early reperfusion (when ΔΨm often collapses), mitochondrial uptake of Bendavia does not appear to be dependent on ΔΨm (Kloner et al., 2012 and unpublished data, DA Brown and SR Shaikh). As discussed above, this approach would ensure targeting mitochondria whose ΔΨm is collapsing or oscillating, as commonly seen in acute coronary syndromes. Second, mitochondria-targeting peptides can penetrate the mitochondria at very low concentrations. NMR studies have shown mitochondrial uptake of these peptides at peptide:lipid ratios as low as 1:10 (Marbella et al., 1828). This suggests that such peptides would show efficacy at very low concentrations, thus reducing the chances of undesirable off-target treatment effects. The National Heart Lung and Blood Institute has emphasized that more pre-clinical compounds should be tested across laboratories to increase the translational relevance of basic science studies (Bolli et al., 2004; Kloner & Schwartz, 2011; Schwartz Longacre et al., 2011). The cardioprotective effects of Bendavia observed across many different laboratories (Wu et al., 2002; Zhao et al., 2004; Cho et al., 2007; Szeto, 2008; Kloner et al., 2012; Sloan et al., 2012; Frasier et al., 2013) are consistent with this directive, and serves as a foundation for translation into clinical trials. A clinical trial with Bendavia is currently underway in ACS patients to assess the safety and efficacy of this peptide in limiting reperfusion injury and resulting infarct size (Chakrabarti et al., 2013). Daily administration of any pharmacotherapy should, at a minimum, not interfere with endogenous cardiac protective mechanisms. Even though Bendavia reduces ROS-dependent cell death, it has no discernible effect on endogenous cardiac preconditioning (Frasier et al., 2013 and Fig. 2). This is important in light of a wide number of cardioprotective interventions that are abolished by prototypical ROS scavengers (Sun et al., 1996; Baines et al., 1997; Arnaud et al., 2002; Das et al., 2006; Hirata et al., 2011), supporting a beneficial role for ROS-dependent cardioprotective signaling, and arguing against Bendavia as a ROS-scavenging compound. Although originally described as an anti-oxidant peptide, recent studies have advanced our understanding of Bendavia's mechanism of action. Studies in cell-free systems have recently compared the ROS-scavenging capacity of Bendavia to known ROS scavengers, and showed no discernible ROS-scavenging activity with Bendavia (Brown et al., manuscript in review). 8. Mitochondrial membranes are unique molecular targets for therapeutic intervention One emerging area in the field of mitochondrial bioenergetics is the regulatory role of the membrane bilayer in mitochondrial function. It is well accepted that lipid bilayers are complex structures that have many functions beyond serving as a semi-permeable barrier (Escribá et al., 2008). The mitochondrial inner and outer membranes have highly unique features relative to other membranes, which make them a potential target for a variety of pharmacological and dietary interventions. The exclusive structural features of mitochondrial membranes are presented below, with a description of how changes in these structures can impact mitochondrial function in the setting of heart disease.

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Fig. 2. The mitochondria-targeting peptide, Bendavia, does not abolish intrinsic cardioprotective signaling. (A) Effect of ischemic preconditioning and/or Bendavia perfusion on infarct reduction in isolated, perfused guinea pig hearts. (B) Effect of exercise in the presence or absence of Bendavia in intact Sprague–Dawley rats. Data in (B) are modified from Frazier et al., Cardiovascular Research 2013, reprinted with permission.

Mitochondrial lipid composition is highly similar across cell types, which suggests some universal functions of the organelle. The major phospholipids in the mitochondrial membranes are phosphatidylcholines (PC), phosphatidylethanolamines (PE) and CL, which represent ~40%, 30%, and 15% of the total lipids respectively (Osman et al., 2011). Less abundant phospholipids include phosphatidic acid, phosphatidylinositols and phosphatidylserines. CL appears to be concentrated at select sites in the membrane with specific proteins to facilitate bioenergetic processes (Epand et al., 2002; Kim et al., 2004). PE and CL are cone-shaped lipids that have small head-groups relative to the area occupied by the acyl chains. As a result, both lipids are capable of adopting a non-bilayer structure known as hexagonal phase, particularly under conditions dependent on pH and/or in the presence of divalent cations (Vasilenko et al., 1982; Ortiz et al., 1999). Hexagonal phase for both lipids has been observed in cell-free model membrane systems using a combination of methods including X-ray diffraction, 31P NMR, and differential scanning calorimetry. The propensity to form this phase is known to impart high curvature stress, which may be physiologically relevant by impacting the geometry of the membrane and thereby protein function (van den Brink-van der Laan et al., 2004). Mitochondrial bioenergetics and function at multiple levels are highly regulated by the total content of CL and the composition of the associated acyl chains. For example, CL content and acyl chain composition impact apoptosis, formation of respiratory supercomplexes, fusion and fission events, and the activity of respiratory complexes I, III, IV, and V (Chicco & Sparagna, 2007). Given the diverse roles of CL, understanding its functional role is critical in the development of mitochondriatargeted therapies for ischemic heart disease. One molecular target may be CL synthase, which synthesizes CL. For example, Kiebish et al. (2012) showed that myocyte-specific CL synthase transgene expression increased linoleic acid (18:2) levels but not total CL content, which ultimately led to an increase in bioenergetic efficiency. More importantly, expression of the CL synthase in streptozotocin (STZ)-induced diabetes diminished the alterations reported in CL content and acyl chain remodeling. Another promising aspect of targeting CL in the myocardium is that the cellular turnover for CL is much faster, on the order of days (Wahjudi et al., 2011), than the turnover of essential inner membrane proteins, which is on the order of weeks (Kasumov et al., 2013). Such rapid CL turnover means that a compound that improves CL remodeling

can alter the cellular microenvironment potentially faster than a compound seeking to influence protein levels. 9. The role of cardiolipin in mitochondrial membrane organization CL is a unique phospholipid that is localized mostly to the inner mitochondrial membrane and consists of four acyl chains and three glycerol backbones (Lewis & McElhaney, 2009). Several aspects of CL are dysregulated in a variety of conditions ranging from aging and Barth syndrome to metabolic diseases such as cardiovascular disease and diabetes (Chicco & Sparagna, 2007). One component of CL that is disrupted in several human diseases is the profile of the associated acyl chains. Although the composition of the acyl chains associated with CL varies tremendously between tissues and cell types, they are tightly regulated within a given cell type. In rodent hearts, the predominant fatty acid species is linoleic acid (18:2), with tetralinoleoyl CL as the most abundant species. Using a shotgun lipidomics approach, Han et al. (2007) demonstrated that induction of murine diabetes with STZ resulted in a dramatic increase in the levels of docosahexaenoic acid (22:6) at the expense of 18:2 in CL. The 22:6 acyl chains, due to their high degree of conformational flexibility, are well known to disrupt several aspects of membrane organization (Stillwell & Wassall, 2003; Shaikh & Brown, 2013). Other groups reported that the 18:2 levels were decreased as other fatty acids were increased in humans and experimental models of heart failure (Reibel et al., 1986; O'Rourke & Reibel, 1992; Heerdt et al., 2002; Sparagna et al., 2005; Sparagna et al., 2007; O'Shea et al., 2009). Thus, the total levels of 18:2 in addition to changes in the profile of other fatty acids will impact several biophysical properties of the membrane (Shaikh & Brown, 2013). The biosynthesis and remodeling of CL are a crucial determinant of the acyl side chain composition. CL is synthesized from phosphatidylglycerol and cytidinediphosphate-diacylglycerol by the enzyme CL synthase, and the four acyl side chains are then remodeled into “healthy” CL with a very high density of linoleic acid (18:2). As a part of the remodeling process, acyl chains are cleaved off, predominantly by the phospholipase isoform 2 in the heart. The resultant monolysoCL is then remodeled into CL enriched with unsaturated bonds, and this process appears to be very rapid (Schlame et al., 2012). There are three enzymes that contribute to the remodeling of monolysoCL,

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namely tafazzin, monolysoCL acyl transferase (MLCLAT), and acyl-CoAlysoCL acyltransferase (ALCAT). Among these, tafazzin is the most widely studied. Decreases in tafazzin-1 are seen in human heart failure as well as animal models of heart failure (Saini-Chohan et al., 2009), and knockdown of tafazzin-1 results in a cardiomyopathy phenotype (Schlame, 1831; Phoon et al., 2012). Decrements in tafazzin-1 are associated with the clinical condition Barth Syndrome, and patients with Barth Syndrome have a high incidence of cardiomyopathy. Very recent studies suggested that tafazzin-1 has a tendency to interact with lipids that form hexagonal phase (Schlame et al., 2012), explaining why this enzyme would show some specificity for CL. It is speculated that this may be most important in regions of membrane with curvature, such as the ends of the cristae, implying that tafazzin-1 may be important in specific mitochondrial membrane ‘domains’. The exact role of MLCLAT and ALCAT in CL remodeling warrants further investigation. MLCLAT is increased in animal models of heart failure, suggesting compensatory changes (Saini-Chohan et al., 2009). Future studies examining factors responsible for MLCLAT transcription are needed to further ascertain the role of this enzyme in CL remodeling. ALCAT is highly expressed in heart tissues (Cao et al., 2004), and its role in mitochondrial lipid remodeling is not entirely clear. Despite its name, ALCAT is not specific for CL, and likely has a role in the remodeling process of a number of phospholipids (Zhao et al., 2009). A few studies have noted that ALCAT upregulation was associated with oxidative stress, decreased 18:2 CL, and mitochondrial fragmentation, suggesting that the remodeling catalyzed by ALCAT may be pathophysiological (Li et al., 2010, 2012; Liu et al., 2012). Taken together, the role of CL remodeling in reperfusion injury is an area where future study is needed. Given how rapidly these events occur, interventions that sustain healthy CL remodeling have the potential to quickly act during the window of cardiac reperfusion. Mitochondrial content of CL is another component of CL status that can play a critical role in optimal electron transport. Decrements in CL levels are associated with maladaptations that include increased ROS production (Chen & Lesnefsky, 2006 and Fig. 3). Lesnefsky et al.

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(2004) reported that ischemia in rabbit hearts resulted in decreased CL content in parallel with a decrease in oxidative metabolism. CL levels were not further lowered upon reperfusion, suggesting that the loss of CL was unique to the ischemia period. In contrast, Petrosillo et al. (2003) and Paradies et al. (2004) showed that ischemia and reperfusion in rat hearts lowered CL levels by ~22% and 50%, respectively. The reduction in CL levels in ischemia and reperfusion correlated with a decrease in complex I and III activities. Interestingly, the addition of exogenous CL to the rat heart preparation restored complex activity, and this was not seen with the addition of oxidized CL or other phospholipids (Petrosillo et al., 2003; Paradies et al., 2004). Future studies are needed to explore means by which CL levels could be restored in ischemia/reperfusion injury, possibly by targeting CL biosynthesis. The presence of double bonds in the structure of CL renders the lipid highly susceptible to peroxidation. This susceptibility makes CL highly relevant to many diseases including ischemia/reperfusion injury, whereby the organ is subjected to free radical damage. Increased CL peroxidation is seen concomitant with reperfusion (Paradies et al., 2004), and is associated with increased programmed cell death, possibly driven by increased permeabilization of the plasma membrane, which enhances cytochrome c release (Orrenius & Zhivotovsky, 2005). Several studies support the notion that peroxidized CL does not support optimal protein activity. For example, Paradies et al. (2002) demonstrated that peroxidized CL lowered complex I activity, which could be restored with the addition of non-oxidized CL. Recently, Ji et al. (2012) demonstrated that traumatic brain injury in rats was associated with numerous oxidized species of CL containing polyunsaturated species. One critical aspect for the development of mitochondrial-targeted therapies is the detection and quantification of the numerous CL (and other phospholipid) species abundant in mitochondria of healthy and diseased individuals. Specifically, this entails measuring the amount of phospholipid, associated acyl chains, and peroxidation products. Various chemical methods such as thin-layer chromatography and high-performance liquid chromatography are employed to quantitatively separate CL from other phospholipids (Chicco & Sparagna,

Fig. 3. Aberrant electron flow during reperfusion. Production of reactive oxygen species damages cardiolipin, resulting in aberrant cardiolipin microdomains (depicted in red zones). The resultant impairment in electron flow further exacerbates ROS production and leads to impaired Δψm through the opening of energy-dissipating channels or pores. As a result, the low Δψm leads to poor ATP replenishment, decreased redox buffering capacity, and can ultimately lead to necrotic or apoptotic cell death.

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2007). The isolated lipids can then be analyzed with classical methods such as gas chromatography with flame ionization detection, which can provide the composition of associated acyl chains and relative levels of fatty acids. A major limitation of this approach is that oxidized subspecies cannot be effectively analyzed. The more sophisticated approach, and one that is critical for measuring peroxidation, is mass spectrometry. Several mass spectrometry and tandem mass spectrometry approaches have been developed that provide highly relevant subspecies of CL and other lipids (Han et al., 2006; Domingues et al., 2008; Kim et al., 2011). However, the major limitations toward using the various types of mass spectrometry are the need for advanced instrumentation, technical expertise, standards, and associated costs. 10. Biophysical consequences of changes in cardiolipin composition, content and peroxidation The majority of studies in the field have focused on how changes in CL biosynthesis, acyl chain profile, content, and peroxidation influence protein activity and thereby mitochondrial function. However, an area of investigation that remains in its infancy is the possibility that the aforementioned changes in CL could have indirect effects on several biophysical properties of mitochondrial membranes. These properties include membrane microviscosity (i.e., fluidity), permeability, curvature stress, CL microdomain formation, protein lateral dynamics and protein rotational diffusion. Membrane microviscosity is a commonly measured endpoint in numerous studies of mitochondrial dysfunction (Shaikh & Brown, 2013). For example, mitochondrial membrane fluidity was shown to increase in the myocardium of diabetic rats (Waczulikova et al., 2007). A major limitation of such a measurement, however, is that any type of manipulation in CL in response to changes in the diet or administration of pharmacological agents will generally modify the global parameter of membrane microviscosity. Thus, it is essential to consider more specific endpoints of mitochondrial membrane function. One example of this is the potential role of CL-enriched microdomains that may serve to concentrate proteins in order to enhance vectorial electron flow (see Fig. 1). Membrane microdomains are generally formed by favorable physicochemical properties of lipids. In the case of CL, its propensity to form non-bilayer structures may make it uniquely adapted to form unique lipid microdomains. Indeed, recent imaging data are starting to emerge that show CL forms distinct microdomains in areas of negative curvature (Renner & Weibel, 2011). 11. Conclusions Advances made in recent years that uncovered the molecular composition of inner mitochondrial membrane proteins have made it possible to explore the role of the inner membrane and its constituent lipids in diseases of the heart. Additional studies are needed that more clearly delineate the role of these lipids and proteins in ischemia/reperfusion injury. Nonetheless, current knowledge has led to an emergence of new thinking with respect to the role of mitochondrial membrane lipids, particularly CL, in cardiac reperfusion injury. The development of novel therapies that target mitochondria to alter the lipid microenvironment represents a new therapeutic paradigm with several compounds showing promise in this area.

Conflict of interest statement DAB, HNS, and SRS have received grant support from Stealth Peptides, Inc. DAB and HNS have received consulting income from Stealth Peptides, Inc.

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