Scholarly article on topic 'Novel therapeutic strategies for ischemic heart disease'

Novel therapeutic strategies for ischemic heart disease Academic research paper on "Basic medicine"

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Pharmacological Research
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{"Myocardial infarction" / Cardioprotection / "Reperfusion injury" / Ischemia–reperfusion / "Cytoskeletal signaling" / "Ischemic preconditioning"}

Abstract of research paper on Basic medicine, author of scientific article — Adam J. Perricone, Richard S. Vander Heide

Abstract Despite significant advances in the physician's ability to initiate myocardial reperfusion and salvage heart tissue, ischemic heart disease remains one of the leading causes of death in the United States. Consequently, alternative therapeutic strategies have been intensively investigated, especially methods of enhancing the heart's resistance to ischemic cell death – so called “cardioprotective” interventions. However, although a great deal has been learned regarding the methods and mechanisms of cardioprotective interventions, an efficacious therapy has yet to be successfully implemented in the clinical arena. This review discusses the current understanding of cardioprotection in the context of ischemic heart disease pathophysiology, highlighting those elements of ischemic heart disease pathophysiology that have received less attention as potential targets of cardioprotective intervention.

Academic research paper on topic "Novel therapeutic strategies for ischemic heart disease"


Pharmacological Research xxx (2014) xxx-xxx


Contents lists available at ScienceDirect

Pharmacological Research

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1 Invited Review

2 Novel therapeutic strategies for ischemic heart disease

3 qi Adam J. Perricone, Richard S. Vander Heide *

4 Louisiana State University Health Sciences Center, New Orleans, LA 70112, United States


Article history:

Received 5June 2014

Received in revised form 21 August 2014

Accepted 22 August 2014

Available online xxx


Myocardial infarction Cardioprotection Reperfusion injury Ischemia-reperfusion Cytoskeletal signaling Ischemic preconditioning


Despite significant advances in the physician's ability to initiate myocardial reperfusion and salvage heart tissue, ischemic heart disease remains one of the leading causes of death in the United States. Consequently, alternative therapeutic strategies have been intensively investigated, especially methods of enhancing the heart's resistance to ischemic cell death - so called "cardioprotective" interventions. However, although a great deal has been learned regarding the methods and mechanisms of cardioprotective interventions, an efficacious therapy has yet to be successfully implemented in the clinical arena. This review discusses the current understanding of cardioprotection in the context of ischemic heart disease pathophysiology, highlighting those elements of ischemic heart disease pathophysiology that have received less attention as potential targets of cardioprotective intervention.

© 2014 Published by Elsevier Ltd.

22 Contents

23 Impact of ischemic heart disease..............................................................................................................................................................................................................................................00

24 Pathology of myocardial infarction........................................................................................................................................................................................................................................00

25 Ischemic cell death................................................................................................................................................................................................................................................................00

26 Reperfusion injury................................................................................................................................................................................................................................................................00

27 Reperfusion injury: correction of intracellular pH..............................................................................................................................................................................00

28 Reperfusion injury: calcium overload........................................................................................................................................................................................................00

29 Reperfusion injury: oxidative stress............................................................................................................................................................................................................00

30 Mechanisms of cardioprotection..............................................................................................................................................................................................................................................00

31 Ischemic preconditioning and cardioprotective signaling..............................................................................................................................................................................00

32 Targeting ischemia-reperfusion injury-clinical trials ........................................................................................................................................................................................00

33 Cytoskeletal signaling and cardioprotection......................................................................................................................................................................................................................00

34 Conclusion ............................................................................................................................................................................................................................................................................................00

35 References ........................................................................................................................................................................................................................................................................................00

37 Impact of ischemic heart disease

38 Heart disease is the leading cause of death in the United States,

39 and ischemia/reperfusion-induced cell death (IR), such as seen

40 during myocardial infarction (MI), is a major cause of morbid-

41 ity and mortality, as about 1.5 million Americans suffer from an

42 MI annually [1-3]. Because terminally differentiated myocytes do

43 not regenerate, the loss of myocardial tissue due to MI forces

* Corresponding author at: Department of Pathology, 1901 Perdido Street, New Orleans, LA 70112, United States. Tel.: +1 504 568 6033; fax: +1 504 568 6037. E-mail address: (R.S. Vander Heide). 1043-6618/© 2014 Published by Elsevier Ltd.

the remaining viable myocytes to work harder to maintain suffi- 44

cient cardiac output. To accomplish this, these remaining myocytes 45

hypertrophy to increase their strength of contraction. While this 46

remodeling is initially adaptive, in severe cases this remodeling 47

can decompensate and become pathological, ultimately leading 48

to heart failure [3]. Thus, the best strategy to improve both sur- 49

vival and quality of life in patients suffering from MI is to minimize 50

myocardial death that occurs due to IR. Clinically, this is achieved 51

through arterial reperfusion of the ischemic myocardium which, 52

in most cases, is accomplished through active dissolution and/or 53

physical obliteration of an occlusive intracoronary lesion. Since 54

the therapeutic importance of prompt myocardial reperfusion has 55

been emphasized and implemented into today's standard of care, 56


2 A.J. Perricone, R.S. Vander Heide / Pharmacological Research xxx (2014) xxx-xxx

the morbidity and mortality of MI has decreased. Aiding that decrease has been refinement in the techniques physicians employ to re-establish coronary blood flow, including the development of percutaneous coronary angioplasty and coronary stenting, as well as the administration of pharmacological adjuvants, such as antiplatelet therapeutics, that help maintain vessel patency [4]. However, as the prevalence of major risk factors for ischemic heart disease, most notably diabetes, hyperlipidemia and hypertension, continues to be substantial, the burden of disease for MI will remain significant.

Because further advances in methods to provide prompt myocardial reperfusion in patients suffering from MI are unlikely to yield significant benefits in morbidity and mortality, there is a great need for the development of novel ischemic heart disease therapies [4]. Consequently, many researchers have investigated strategies that can make the heart more resistant to ischemic death - so called "cardioprotective" interventions. While many cardio-protective strategies have been identified in the laboratory setting, attempts to translate these protective laboratory interventions into a successful clinical therapy have been largely unsuccessful. While reasons for this lack of success may be due to the inherent difficulty of translating results generated in tightly controlled animal models into a heterogeneous patient population [5], the lack of success may also be attributed, in part, to an incomplete understanding of how cardioprotective signaling may be initiated at the level of the cardiac myocyte in response to myocardial stress. Thus, there is great interest in elucidating the mechanisms by which IR induces lethal cellular injury and how cardioprotection may be elicited in the myocardium to allow for the identification of novel targets for ischemic heart disease therapy. Accordingly, the goal of this review is to highlight these potential avenues of cardioprotection in the context of MI pathophysiology.

Pathology of myocardial infarction

Ischemic cell death

Myocardial ischemia results in numerous deleterious consequences at the level of the cardiac myocyte that, if left uncorrected, culminate in necrotic cell death. A major consequence of myocardial ischemia is the depletion of adenosine triphosphate (ATP) and other high energy phosphates due to cessation of aerobic metabolism and oxidative phosphorylation. Because the continually contracting myocardium is highly dependent on aerobic metabolism, ATP depletion occurs rapidly in the ischemic heart and contractility is halted within 60 s. ATP depletion has numerous detrimental effects on myocyte biochemistry and metabolism, including relaxation of myofilaments, glycogen depletion, disruption of ionic equilibrium and cell swelling. Nevertheless, these effects can be reversed and normal myocyte contractile function restored if the duration of ischemia is sufficiently brief (generally considered to be less than 20min of severe ischemia). However, if the ischemia is prolonged, irreversible injury will develop, which is characterized by damage and/or disruption of the myocyte sar-colemmal membrane. Plasma membrane damage leads to loss of osmotic balance and the leakage of cellular metabolites into the extracellular space. Damage to the mitochondrial membranes compromises the cell's ability to generate ATP upon reperfusion, as well as results in release of mitochondrial proteins that can directly stimulate the apoptotic cell death pathway. Disruption of lysoso-mal membranes is especially dire, as this can lead to the release of degradative enzymes capable of digesting essentially all cellular constituents, invariably leading to cellular necrosis [6].

In addition to necrosis, apoptosis also contributes significantly to myocyte death during IR, although the exact contributions of

each during the sustained ischemic episode and during myocar-dial reperfusion remain unclear. While apoptotic myocyte death is most pronounced in the reperfused myocardium [7,8], apoptosis has also been shown to contribute to cell death in ischemic-only hearts [9]. Some studies suggest there may be overlap between the early signaling events that lead to either necrosis or apoptosis, as interventions known to inhibit apoptosis, such as Bcl-2 overexpression, have also been demonstrated to reduce cellular necrosis [10,11]. Other investigators propose that, during myocardial IR, the distinction between apoptosis and necrosis becomes blurred, as injured cells attempting to undergo apoptosis may be unable to maintain plasma membrane integrity, resulting in necrosis instead [12]. In addition, a more recently defined form of cell death known as necroptosis or "programmed necrosis," a form of cell death with characteristics of both necrosis and apoptosis, has been suggested to contribute to myocyte death during IR [13-15].

Although the determining factors by which IR will lead to either necrotic or apoptotic cell death are not completely understood, it is known that both ischemia and reperfusion result in substantial ionic changes capable of predisposing the cardiac myocyte to both forms of cell death. Ischemia causes a cessation of aerobic respiration and a shift to of anaerobic respiration, resulting in depletion of glycogen stores and a resulting accumulation of hydrogen ions and tissue acidosis [5,16-18]. To counterbalance this acidosis, Na+/H+ exchange occurs at the sarcolemmal membrane, with the efflux of H+ balanced by the influx of Na+ and resulting in an accumulation of intracellular ions relative to the extracellular environment. As a result of the accumulation of Na+ ions, reverse action of the Na+/Ca2+ exchanger (NCX) is stimulated, which leads to an accumulation of intracellular Ca2+ as the accumulated Na+ is extruded from the cell in exchange for extracellular Ca2+. Compounding these ionic disturbances is the depletion of high energy phosphates that occurs rapidly in the ischemic myocardium, preventing the normal activity of the Na+/K+ ATPase, ATP-dependent reuptake of calcium at the sarcoplasmic reticulum and active Ca2+ excretion [16,19], resulting in further exacerbation of intracellular Na+ and Ca2+ accumulation. As the calcium concentration within the cytosol continues to rise, mitochondria begin to passively uptake calcium into the mitochondrial matrix via the mitochondrial calcium uniporter [18]. If the ischemia is severe and these ionic imbalances are sustained, this cytosolic and mitochondrial calcium overload will ultimately lead to irreversible cellular injury as described below.

Loss of myocyte calcium homeostasis results in a number of cellular changes that predispose the myocyte to irreversible injury. Mitochondrial calcium overload is the primary stimulus for mitochondrial permeability transition (MPT), a stress response mediated by the opening of a high conductance pore located on the inner mitochondrial membrane (discussed further in Section "Reperfusion injury: calcium overload"). While enhanced intra-cellular calcium concentration is capable of directly stimulating apoptosis [20], elevated calcium levels can also stimulate the activation of numerous intracellular degradative enzymes with the potential to damage several different cellular structures and precipitate cell death, including phospholipases, proteases and endonucleases. Activation of phospholipases can lead to the damage of cellular membranes which, as described above, can lead to necrotic cell death as a consequence of disruption of cellular osmotic balance and the release of lysosomal enzymes in the cytoplasm [6]. The ionic imbalances engendered by ischemia and the consequent causes of ischemic myocyte death are summarized in

Fig. 1.

Reperfusion injury

Studies conducted by Jennings and Reimer in the late 1960s and 1970s were pivotal for demonstrating the time-dependent


A.J. Perricone, R.S. Vander Heide / Pharmacological Research xxx (2014) xxx-xxx

Ionic Imbalances: Ischemia

Anaerobic Metabolism i

Acidosis iNHE _Reverse NCX

Calcium Overload

Activation of degradative enzymes

Fig. 1. Causes and consequences of ionic imbalances during ischemia. Ischemia results in a cessation of aerobic metabolism and a reliance on anaerobic metabolism, resulting in cellular and tissue acidosis. Accumulation of intracellular H+ stimulates NHE, resulting in accumulation of intracellular Na+. Accumulation of intracellular Na+, in turn, stimulates reverse activity of the NCX, resulting in intracellular Ca2+ accumulation. If ischemia is sustained, cellular Ca2+ overload may develop, resulting in activation of degradative enzymes (e.g., proteases, phosphatases and endonucleases), and stimulation of MPT, culminating in myocyte death. NHE, Na+/H+ exchanger; NCX, Na+/Ca2+ exchanger; MPT, mitochondrial permeability transition.

Fig. 2. Primary mediators of lethal reperfusion injury. Oxidative stress results in sensitization of mitochondria to MPT as well as direct damage to membrane phospholipids. The abrupt correction of acidosis relieves an inhibitory influence on MPT and degradative enzymes and serves as an important impetus for calcium overload. Calcium overload can culminate in cell death through varying mechanisms, including activation of calpain proteases, MPT and hypercontracture of fragile myocytes. Activation of calpains can result in additional damage to the myocyte cytoskeleton, predisposing myocytes to rupture of the sarcolemma following imposed mechanical stress, such as cell swelling or hypercontracture.

183 progression of myocardial necrosis as the index ischemia was

184 extended and the critical importance of prompt reperfusion for sal-

185 vaging myocardium [21,22]. Interestingly, in an even earlier study

186 conducted by Jennings et al. in 1960, it was observed that reper-

187 fused myocardium displayed a more advanced injury pattern than

188 myocardium subjected to the same ischemic duration that had not

189 been reperfused [23]. Specifically, it was observed that reperfused

190 myocardium displayed myocyte swelling, plasma membrane dis-

191 ruption and dense mitochondrial bodies - histological features that

192 do not manifest in the absence of reperfusion unless ischemia is

193 extended for a much longer duration. This observation led others

194 to later hypothesize that reperfusion could result in lethal myocar-

195 dial injury above and beyond that due to the ischemic insult alone

196 - a concept known as lethal reperfusion injury.

197 The mechanisms of lethal reperfusion injury have been well

198 studied in an effort to discern if the conditions of myocardial reper-

199 fusion could be modified to maximize the salvage of ischemic

200 myocytes. Chief among the primary mediators of reperfusion injury

201 are the rapid correction of intracellular acidosis, myocyte calcium

202 overload and oxidative stress [5,24]. The primary mediators of

203 lethal reperfusion injury are depicted in Fig. 2.

204 Reperfusion injury: correction of intracellular pH

205 While the acidotic conditions of the ischemic myocyte pre-

206 cipitate ionic imbalance and cell swelling, intracellular myocyte

207 acidosis also inhibits several subcellular processes which can

208 potentially culminate in irreversible injury. Acidosis reduces the

209 reverse activity of the NCX, thereby attenuating calcium overload

210 [19]. Furthermore, H+ ion accumulation directly inhibits MPT [25],

211 ostensibly by interfering with the binding of calcium to the MPT

212 trigger site. The acidotic cytosolic environment of the ischemic

213 myocyte also inhibits the activation of proteases called calpains

214 - calcium-dependent enzymes which are capable of digesting

215 and weakening the myocyte's cytoskeletal support [26,27]. How-

216 ever, upon reperfusion there is rapid washout of accumulated H+

ions, alleviating the acidotic inhibition and predisposing reperfused 217

myocytes to cellular injury. 218

Reperfusion injury: calcium overload 219

As discussed above, the accumulation of calcium during 220

ischemia occurs as a result of the reverse mode of the NCX. 221

In addition, ATP depletion and subsequent cessation of active 222

calcium extrusion and ATP-dependent reuptake into the sarcoplas- 223

mic reticulum further contribute to myocyte calcium overload. 224

Unfortunately, upon reperfusion, this myocyte calcium overload 225

is not corrected, but rather is further exacerbated. At reperfu- 226

sion, extracellular washout of accumulated H+ ions establishes a 227

large gradient greatly favoring the influx of sodium via the Na+/H+ 228

exchanger and leading to the rapid correction of cellular pH. This 229

sudden and robust influx of sodium strongly stimulates the reverse 230

action of the NCX, especially now that the acidotic inhibition of 231

the NCX has been relieved, resulting in even greater elevations 232

of intracellular calcium concentration [16]. Enhanced cytosolic 233

calcium levels favor the electrophoretic uptake of calcium into 234

mitochondria via the mitochondrial calcium uniporter [20]. The 235

end result is cytosolic and mitochondrial calcium overload, cul- 236

minating in irreversible myocyte injury through many different 237

potential mechanisms including induction of MPT, activation of cal- 238

pains and myocyte hypercontracture. Each of these mechanisms is 239

discussed further below. Fig. 3 summarizes the establishment and 240

consequences of calcium overload during reperfusion. 241

MPT is a prominent cause of irreversible cell injury elicited by 242

IR and is characterized by the opening of a high conductance, non- 243

specific pore located within the inner mitochondrial membrane. 244

While calcium overload is the primary determinant of MPT, there 245

are other cellular conditions that can influence the calcium con- 246

centration threshold necessary to induce MPT. For example, both 247

oxidative stress and high energy phosphate depletion sensitize 248

mitochondria to permeability transition, lowering the calcium con- 249

centration threshold necessary to induce MPT [28]. Importantly, 250

in addition to the robust influx of calcium that may occur upon 251

yphrs 27361-10_ARTICLE IN PRESS

A.J. Perricone, R.S. Vander Heide / Pharmacological Research xxx (2014) xxx-xxx

Ionic Imbalances:

Washout of extracellular H+ ttt NHE - —»ttt Reverse NCX

Exacerbation of Calcium Overload calpains l^pT* contracture

Fig. 3. Causes and consequences of ionic imbalances during reperfusion. Reperfusion results in an abrupt alleviation of tissue acidosis and a washout of extracellular H+, establishing a highly favorable gradient for Na+ influx via the NHE. The resulting Na+ accumulation potently stimulates reverse NCX activity, resulting in Ca2+ overload. Ca2+ overload in the setting of reperfusion results in activation of the calcium dependent proteases calpains, the activity of which is attenuated in the acidotic conditions of ischemia. The cellular milieu during reperfusion (e.g., Ca2+ overload, oxidative stress and alleviation of acidosis) is particularly well-suited for the induction of MPT. Finally, resumption of contractile activity in the presence of Ca2+ overload results in hypercontracture, imposing a significant physical strain on a weakened myocyte cellular architecture. NHE, Na+/H+ exchanger; NCX, Na+/Ca2+ exchanger; MPT, mitochondrial permeability transition.

reperfusion, oxidative stress and high energy phosphate depletion are both present at reperfusion, highlighting the fact that the cellular milieu is particularly well-suited for mitochondria to undergo MPT following reperfusion. Indeed, many studies have demonstrated that MPT occurs upon reperfusion, primarily in ex vivo models of myocardial IR [29,30].

MPT results in the uncoupling of the mitochondrial membrane potential, as the enhanced inner membrane permeability dissipates the proton gradient necessary for oxidative phosphorylation. Furthermore, because the mitochondrial matrix exerts a positive colloidal osmotic pressure due to its higher protein concentration than either the cytosol or intermembranous space, MPT results in marked mitochondrial swelling [6,28]. While the inner mitochon-drial membrane can withstand this swelling without rupturing by virtue of "unfolding" of the cristae of the inner membrane, mito-chondrial swelling is capable of rupturing the outer mitochondrial membrane, which leads to the release of cytochrome c and the stimulation of the intrinsic apoptotic pathway [31]. As mitochondria undergo MPT, swelling, and outer membrane rupture, more calcium is released from these "open" mitochondria back into the cytosol which, in turn, predisposes mitochondria that have so far remained "closed" to undergo MPT. If a critical number of mitochondria undergo MPT, the most likely outcome is bioenergetic failure of the myocyte and necrotic cell death [28].

The presence of myocyte calcium overload upon reperfusion can also lead to the activation of members of the calcium-dependent family of cysteine proteases known as calpains. ^-Calpain and m-calpain are the most abundant calpain isoforms, both of which are expressed in the heart [27]. Calpains can become activated as a result of the calcium accumulation that occurs during ischemia as well as the more robust increase in calcium influx that occurs during reperfusion [5]. Calpain activation results in the hydrolysis of a number of proteins that predispose the myocyte to lethal reperfusion injury. Chief among these calpain substrates are

cytoskeletal proteins, the enzymatic cleavage of which damages myocyte contractile machinery and weakens myocyte structural support [32,33]. This latter consequence is particularly troublesome as it can potentially lead to the rupture of the sarcolemmal membrane upon the resumption of contractile activity [26].

Studies have supported a role for cytoskeletal lesions as important mediators of irreversible IR injury. Seminal studies conducted by Vander Heide and Ganote demonstrated using a Langendorff perfused rat heart model that anoxic perfusion results in enhanced myocardial fragility and decreased resistance to applied mechanical force. Electron microscopy of hearts subjected to anoxic perfusion prior to mechanical stress displayed rupture of the sar-colemmal membrane and the formation of large subsarcolemmal blebs caused by detachment of the sarcolemmal membrane from the Z-disk [34]. A similar study conducted by Steenbergen et al. at nearly the same time correlated the appearance of subsarcolem-mal blebs and plasma membrane rupture induced by ischemia with the breakdown of vinculin, a focal adhesion cytoskeletal scaffolding protein, at the plasma membrane [35]. Thus, while the exact series of events that underlie the transition from reversible to irreversible injury during ischemia are not completely understood, studies support a role of cytoskeletal lesions as critical determinants of irreversible ischemic injury that manifests upon reperfusion.

Further compounding the problem of enhanced myocyte fragility during IRis the development of hypercontracture, a state of nonphysiologic myocyte sarcomere shortening due to ATP generation in a setting of intracellular calcium overload [24,36,37]. The concept that myocyte contracture can lead to irreversible injury and sarcolemmal membrane rupture in the reperfused ischemic myocyte by imposing a great physical stress on an already fragile sarcolemmal membrane was originally postulated by Ganote and Kaltenbach in 1979 [38]. Subsequent studies supported this hypothesis by demonstrating that the calcium paradox, a phenomenon by which the readmission of calcium to calcium depleted hearts results in contraction band necrosis, was reproduced simply with caffeine-stimulated sarcoplasmic reticulum calcium release and resulting myocyte contracture and occurred independently of extracellular calcium repletion [39,40]. Furthermore, a study by Garcia-Dorado et al. demonstrated that attenuation of myocyte contractility at reperfusion significantly reduced infarct size in an in vivo porcine model of MI [41].

Reperfusion injury: oxidative stress

While reactive oxygen species (ROS) are generated during normal cellular metabolism, the reperfusion of ischemic myocytes results in a sudden burst of ROS which overwhelms the cell's capacity to scavenge these radicals, especially since the activity of scavenging enzymes is attenuated as a result of the ischemic insult [42]. Consequently, the reperfused myocyte enters a state of oxidative stress, resulting in a number of deleterious consequences that can culminate in lethal reperfusion injury. First, in addition to mitochondrial calcium overload, this sudden onset of oxidative stress sensitizes mitochondria to permeability transition [28]. Mitochondria that undergo permeability transition can, in turn, release additional free oxygen radicals that can sensitize other mitochondria to MPT; this phenomenon is termed "ROS-induced ROS release" [43]. Furthermore, ROS can directly damage calcium handling proteins, thereby contributing to calcium overload at reperfusion [5,42]. Importantly, ROS are capable of peroxidizing membrane lipids, contributing to the disruption of cellular membranes that occurs during IR [42]. Finally, ROS are also capable of inflicting further cellular damage by cross-linking proteins and creating DNA breaks [6].

There are multiple sources of increased ROS in the reperfused myocyte. Xanthine oxidase is one prominent contributor of oxygen radicals at reperfusion [16,44]. Normally, this enzyme exists in


A.J. Perricone, R.S. Vander Heide / Pharmacological Research xxx (2014) xxx-xxx 5

its dehydrogenase form, which oxidizes hypoxanthine while using NAD+ as its electron acceptor. However, during ischemia, a calcium-dependent protease converts xanthine dehydrogenase to xanthine oxidase, an enzyme which oxidizes hypoxanthine while reducing O2 to O2- [44,45]. During ischemia, the degradation of adenine nucleotides results in a buildup of hypoxanthine, the primary substrate for xanthine oxidase. Thus, the sudden reintroduction of oxygen at reperfusion results in an abundance of xanthine oxidase substrates, hypoxanthine and O2, largely contributing to the observed oxidative burst at reperfusion [44]. Damage to mitochondria during the ischemic episode may elicit increased ROS generation directly from the electron transport chain as a result of incomplete reduction of oxygen [6,42]. Finally, ischemic myocar-dial injury results in the influx of leukocytes into the ischemic risk region upon reperfusion. These inflammatory cells are subsequently activated and become another important source of ROS generation through the action of the enzyme NADPH oxidase [42].

Mechanisms of cardioprotection

Because the best strategy for improving the morbidity and mortality of MI is to minimize the ischemic death of myocardial tissue, there has been great research interest in learning how the heart can be protected from ischemic death. Various laboratory interventions have been shown to be capable of reducing infarct size including hypothermia [46], heat stress [47] and ischemic preconditioning (IP) [48], as well as a myriad of pharmacologic agents, including calcium channel blockers [49], adenosine [50], alpha 1 adrenergic agonists [51] and delta 1 opioid agonists [52]. However, among these interventions, IP has demonstrated the most consistent ability to confer robust cardioprotection in several different mammalian models, including rats, rabbits, dogs, pigs, sheep and monkeys [53]. Importantly, the ability of IP to confer protection has also been supported in humans undergoing coronary artery bypass surgery, as patients subjected to an IP protocol displayed reduced ATP depletion following a controlled ischemic stress produced during the normal course of the surgery [54]. Although the clinical value of IP is limited by the fact that patients suffering from MI do not present until after the onset of ischemia, the discovery of IP provided definitive proof that the heart can be protected from ischemic death, spawning intensive research focused on elucidating these protective mechanisms.

Ischemic preconditioning and cardioprotective signaling

The phenomenon of IP was originally described by Murry et al. in 1986 [48]. Following the original description of IP, it was discovered that the protection afforded by IP is not monophasic, but rather IP produces two temporally distinct windows of cardioprotection. The first window of protection begins within minutes following the cycles of preconditioning. This early window of protection is lost if the time between the cycles of preconditioning and the sustained myocardial ischemia is extended beyond 1-2 h. The second window of protection develops 6-12 h following IP and lasts for 3-4 days. The mechanisms mediating early and late preconditioning are distinct: the protection within the early window is associated with post-translational modification of pre-existing proteins, while the late window of protection is thought to be produced through changes in gene expression and the synthesis of new proteins, notably inducible nitric oxide synthase (iNOS) and cyclo-oxygenase 2 (COX-2) [55]. This review focuses on cardioprotective signaling cascades elicited by acute myocardial stress and therefore will discuss IP in context of the early window of cardioprotection as originally described by Murry et al.

The mechanism of IP's protective effect has been extensively investigated, and the research leading up to the present understanding of IP's protective mechanism is briefly summarized below. IP's cardioprotection is currently thought to be triggered through the release of ischemic metabolites during the index ischemia and the subsequent binding to their respective transmembrane receptors. Adenosine was the first such metabolite to be recognized for having cardioprotective potential [50]. Later, other metabolite triggers were described, including bradykinin [56] and opioids [57]. This ability of multiple triggers to mimic the protective effect of IP led to the conclusion that the respective signaling pathways converge downstream at a common point. Protein kinase C (PKC) was one of the first proposed points of convergence, as PKC inhibitors are not only capable of abolishing the protective effect of IP [58], but also the protection afforded by pharmacological mimickers of IP [59]. PKC has been postulated to elicit cardioprotection, in part, by activating mitochondrial ATP-sensitive K+ channels (mitoKATP) [60]. The cardioprotection associated with mitoKATP is not completely understood, but activation of mitoKATP has been suggested to enhance the resistance of mitochondria to MPT. mitoKATP channel opening results in an influx of K+ into the mitochondrial matrix, thereby depolarizing the mitochondrial membrane potential. This depolarization, in turn, mitigates the driving force of calcium into the mitochondrial matrix during the sustained ischemic episode, thereby attenuating mitochondrial calcium overload and decreasing the likelihood of MPT occurring upon reperfusion [61].

In addition to PKC, several other downstream effector proteins have been implicated as mediators of cardioprotective signaling elicited by IP. Because phosphatidylinositol-3-kinase (PI3K), a lipid kinase implicated in cell survival signaling [62], had been reported to activate PKC, Tong et al. investigated the role of PI3K in IP-elicited cardioprotection. Using a Langendorff perfused adult rat heart model, they found that IP induced the activation of the cardioprotective proteins Akt and PKC-e and that the activation of these proteins was blocked by pharmacological inhibition of PI3K. PI3K inhibition also abolished the protective effect of IP on ventricular functional recovery, further implicating PI3K as an important mediator of IP-elicited cardioprotection [63].

Concurrent to the research investigating the mechanism by which IP induces cardioprotection, other animal research emerged that demonstrated that various interventions initiated at the onset of reperfusion, notably the administration of insulin [64], insulin-like growth factor-1 (IGF-1) [65], bradykinin [66] and the hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor atorvastatin [67], also protected the myocardium from lethal IR injury. Importantly, research into the mechanism of these protective interventions revealed that in each case either PI3K/Akt and/or ERK 1/2 signaling was involved in mediating the conferred cardioprotection. Because PI3K/Akt signaling had been previously implicated in mediating the protective effect of IP when stimulated during the preconditioning phase [63], Hausenloy et al. postulated that these survival kinases could stimulate cardioprotection not only when activated during the cycles of preconditioning, but also when activated at the onset of reperfusion [68]. Indeed, a subsequent study by Hausenloy et al. found that activated Akt and ERK 1/2 expression displayed a biphasic pattern with expression significantly enhanced both immediately following preconditioning and again at the onset of reperfusion [69]. Because activation of both PI3K/Akt and ERK 1/2 at reperfusion was demonstrated to reduce infarct size, these signaling kinases were termed "reperfusion injury salvage kinases" or RISKs. Importantly, this study demonstrated for the first time that IP may elicit cardioprotection by mitigating reperfusion injury. This conclusion led to the formulation of the hypothesis that the robust protective effect of IP can be triggered at the onset of reperfusion, a viable target for clinical intervention.


6 A.J. Perricone, R.S. Vander Heide / Pharmacological Research xxx (2014) xxx-xxx

The mechanisms responsible for the cardioprotection associated with RISK activation have been well studied. Although strong evidence consistently supports a cardioprotective role of P13K/Akt signaling, the evidence surrounding ERK signaling is somewhat conflicting. While most data indicate that ERK is important in cardioprotective signaling [69,70], other studies have not supported a causal role of ERK signaling in mediating 1P-elicited cardioprotection [71,72]. The cardioprotective phenotype associated with ERK signaling is not completely understood, but thought to be due to its ability to inhibit pro-apoptotic proteins such as BAD [68,73]. The protective effect of P13K is largely attributed to its ability to stimulate the downstream activation of the serine/threonine protein kinase Akt, also known as protein kinase B, a well-established car-dioprotective signaling molecule [74,75]. Akt is thought to provide protection from 1R injury through multiple different mechanisms, including inhibition of apoptosis [74], stimulation of nitric oxide synthesis [76] and prevention of MPT through phosphorylation and inhibition of glycogen synthase kinase 3P (GSK-3P) [77,78].

In addition to the well characterized cardioprotective role of RISK signaling, recent evidence has implicated reversible cysteine redox-based mechanisms of post-translational modifications, such as S-nitrosylation (SNO) and S-glutathionylation, in mediating cardioprotection. Strong evidence supports SNO in particular as an important contributor to preconditioning-elicited cardioprotection. SNO, a process in which NO forms a covalent bond with the thiol groups of cysteine residues, is thought to mediate protection from 1R injury by preventing the irreversible oxidation of protein thiols during periods of oxidative stress, such as during the respiratory burst of early myocardial reperfusion [79]. Furthermore, SNO of the L-type calcium channel [80] and SR/ER calcium-ATPase [81] results in decreased cytosolic influx of calcium and increased uptake of calcium into the sarcoplasmic reticulum respectively, thereby attenuating cellular calcium overload during 1R [79]. Importantly, studies have shown that SNO is enhanced in the preconditioned heart and that induction of SNO during 1R results in reduced ischemic cell death [81,82].

Targeting ischemia-reperfusion injury-clinical trials

The discovery by Hausenloy and 'ellon that 1P-elicited cardioprotection could be aborted by inhibition of R1SK signaling at reperfusion, along with numerous studies demonstrating that therapies initiated at reperfusion onset are protective, strongly renewed interest in investigating the pathology of lethal reperfusion injury and how it could be prevented or attenuated. Consequently, the therapeutic potential of methods and therapies capable of mimicking 1P and eliciting cardioprotective signaling at reperfusion has been intensively evaluated by numerous animal studies and clinical trials. The findings of these studies have been thoroughly discussed by several excellent reviews to which the reader is directed [4,5,73,83], and these studies will not be reviewed here. 1nstead, this review will focus on studies examining the car-dioprotective potential of interventions capable of interfering with mediators of reperfusion injury, including correction of acidosis, calcium overload, oxidative stress and MPT. The results of these studies are summarized in Table 1.

Perhaps the best demonstration that modification of the conditions of reperfusion can result in significant cardioprotection is a phenomenon known as ischemic postconditioning (PostC). In an effort to enhance the clinical value of IP, Zhao et al. investigated whether subjecting the heart to cycles of preconditioning following the sustained coronary occlusion could result in infarct size reduction. Using an open chest canine model of MI, they found that PostC, performed by subjecting the heart to three cycles of 30 s of reperfusion and 30 s of coronary re-occlusion immediately following a 60 min coronary occlusion, resulted in significant

reduction in infarct size, comparable to the protection afforded by IP [84].

Shortly after Zhao and colleague's description of PostC, clinical trials were underway to determine the clinical efficacy of PostC. The results of these clinical trials have been variable, with some trials showing improved patient outcomes [85,86] and others showing no difference in patient outcomes between the PostC and placebo groups [87,88]. Reasons for the variability of results between trials may be due to the small number of patients enrolled in each of the trials and/or differences in inclusion criteria and comorbidi-ties of the enrolled patient populations. Regardless, PostC may offer a therapeutic benefit in specific patient populations undergoing angioplasty for the treatment of acute MI. However, it should be noted that the therapeutic utility of PostC is limited in the clinical arena, as patients must be undergoing angioplasty in a catheteriza-tion lab to receive the therapy. Patients who require thrombolytic therapy for restoration of coronary blood flow cannot benefit from PostC. Thus, a therapeutic strategy that could be employed in all patients regardless of how blood flow is restored to the ischemic myocardium would be preferable to PostC [4].

As discussed above, the rapid correction of ischemic acido-sis at reperfusion can result in irreversible injury to myocytes by stimulating calcium overload and cellular swelling while concomi-tantly alleviating the acidotic inhibition of MPT and calpains. Thus, investigators hypothesized that by curbing the effects of ischemic acidosis through inhibition of Na+-H+ exchange myocytes could be protected from reperfusion injury. An early study by Karmazyn demonstrated that amiloride, an inhibitor of the Na+-H+ exchanger (NHE), administered at reperfusion improved post-ischemic ventricular recovery [89]. Later, Wang et al. demonstrated that NHE-1 knockout (KO) mice were resistant to lethal IR injury [90].

Based on these salutary animal studies, clinical trials were conducted to assess NHE inhibition as a potential therapy for IR injury. Overall, these trials did not demonstrate a benefit of NHE inhibition in patients suffering from acute MI. The ESCAMI (Evaluation of the Safety and Cardioprotective Effects of Eniporide in Acute Myocardial Infarction) trial found no differences in clinical outcomes between patients receiving eniporide, a specific inhibitor of the NHE-1 isoform (the predominant NHE expressed in the heart) or placebo [91]. The EXPEDITION trial examined the therapeutic potential of cariporide, a specific NHE-1 inhibitor, in high risk patients undergoing coronary artery bypass graft (CABG) surgery [92]. While the EXPEDITION trial found that NHE inhibition reduced mortality associated with MI, all-cause mortality was paradoxically increased in the NHE inhibition group due to an increased incidence of cerebrovascular events. Therefore, the EXPEDITION investigators concluded that the trial provided proof of concept for the cardiopro-tective potential of NHE inhibition, but that cariporide was likely not suitable for clinical use due to unacceptable adverse effects.

As myocyte calcium overload is responsible for much of the irreversible injury that occurs during both ischemia and at reperfusion, inhibition of the Na+-Ca2+ exchanger (NCX), has been proposed to be cardioprotective. Imahashi et al. found that Langendorff-perfused hearts of NCX KO mice sustained less necrosis following IR than control hearts [93]. Furthermore, a study conducted by Kawa-sumi et al. found that a 30min intravenous infusion of caldaret, an NCX inhibitor, at reperfusion significantly attenuated ischemic death in the canine heart [94]. Unfortunately, a clinical trial examining the therapeutic efficacy of caldaret, the CASTEMI trial, provided no evidence of cardioprotection in patients presenting with large acute ST-segment elevation MI [95].

It has been well known for many years that scavengers of oxygen-derived free radicals could enhance contractile function and/or reduce infarct size in animal models of myocardial ischemia. Recombinant human superoxide dismutase administered at re-flow in the Langendorff perfused rabbit heart has been documented


A.J. Perricone, R.S. Vander Heide / Pharmacological Research xxx (2014) xxx-xxx 7

Table 1

Summary of animal studies and clinical trials assessing cardioprotective potential of interventions against known mediators of lethal reperfusion injury. IR, ischemia-reperfusion; NHE, Na+-H+ exchanger; KO, knockout; CABG, coronary artery bypass graft; CVA, cerebrovascular accident; NCX, Na+-Ca2+ exchanger; MI, myocardial infarction; SOD, superoxide dismutase; MPT; mitochondrial permeability transition.

IR injury mediator Study



Rapid correction of acidosis

Calcium overload

Oxidative stress

Karmazyn [89] Wang et al. [90] ESCAMI [91] EXPEDITION [92]

Imahashi et al. [93] Kawasumi et al. [94] CASTEMI [95]

Ambrosio et al. [96] Guanet al. [97] Ye et al. [98]

Hausenloyet al. [101] Piotet al. [102]

Cardioprotective potential of NHE inhibitor amiloride assessed in isolated-perfused rat heart

Resistance to IR injury assessed in NHE1 KO mice using isolated-perfused heart model

Clinical trial examining therapeutic potential ofNHE1 inhibitor eniporide when administered 10 min before reperfusion Clinical trial examining therapeutic potential ofNHE1 inhibitor cariporide in high risk patients undergoing CABG

Resistance to ischemic death assessed in isolated-perfused hearts of NCX KO mice

Cardioprotective potential of NCX inhibitor caldaret assessed in in vivo canine model of MI

Clinical trial examining therapeutic potential of NCX inhibitor caldaret in patients presenting with ST-elevation MI

Ability of SOD to protect against IR injury assessed in isolated-perfused rabbit hearts

Small clinical trial assessing therapeutic potential of xanthine oxidase inhibitor allopurinol

Meta-analysis of clinical trials assessing effect of antioxidant vitamin supplementation on cardiac outcomes

Cardioprotective potential of MPT inhibitor cyclosporin A assessed in isolated-perfused rat heart Small clinical trial assessing therapeutic potential of cyclosporin A when administered at reperfusion

Enhanced ventricular recovery in

amiloride-treated hearts

Improved ventricular function in NHE1 KO mice

No reduction in infarct size or adverse outcomes

Reduced MI mortality, but increased CVA mortality

Reduced infarct size in NCX KO hearts Reduced infarct size in caldaret-infused hearts No reduction in infarct size

Improved post-ischemic function in SOD hearts

Enhanced ventricular recovery in patients receiving allopurinol

No effect on incidence of adverse cardiovascular events

Infarct size reduction when cyclosporin A administered at reperfusion Infarct size reduction

to improve post-ischemic contractile function [96]. Clinical trials examining the protective potential of attenuating oxidative stress as a treatment for MI have yielded conflicting results but, in general, have not supported a cardioprotective role. In a small trial enrolling 38 patients, allopurinol administered orally just after admission (approximately 60 min prior to reperfusion) assisted ventricular recovery in patients undergoing angioplasty for acute MI [97]. However, a meta-analysis of clinical trials examining the role of antioxidant vitamin supplementation in providing protection from cardiovascular disease found no benefit of antioxidant supplementation in improving patient outcomes [98].

Because MPT has been widely implicated as a central mediator of reperfusion injury, and because many known cardioprotec-tive interventions, such as inhibition of NHE and NCX, have been shown to exert their protective effects partly through inhibition of MPT [28], many studies have focused on investigating methods by which MPT may be directly prevented. The immunosuppres-sant cyclosporin A (CsA) has been demonstrated to prevent MPT [99], and this prevention of MPT has been attributed to CsA's ability to inhibit the MPT pore component cyclophilin D [28]. Animal models of myocardial IR have demonstrated that CsA administration is cardioprotective [100,101]. In a small pilot clinical trial, CsA administered at time of re-flow to patients suffering from acute MI was demonstrated to reduce infarct size as assessed through release of creatine kinase and MRI, making CsA an attractive candidate for MI therapy [102]. However, concerns have been raised regarding the results of the trial, as, in some patients, reperfusion may not have been initiated up to 12 h following onset of chest pain, a time point at which few myocytes within the risk region would remain salvageable [103]. Furthermore, there are some notable limitations in regard to CsA's cardioprotective effect. First, although CsA administration was well-tolerated in the pilot clinical trial, CsA administration can potentially elicit some adverse effects at the level of the heart as a consequence of its interaction with calcineurin [31]. Secondly, the therapeutic range of CsA may be somewhat narrow, as a study conducted by Griffiths and Halestrap found that higher doses of CsA were not protective [104]. The protective effect of CsA can also be overcome by enhancing the

stimulus of MPT, further limiting the therapeutic efficacy of CsA [28]. Thus, more work is required to establish the clinical utility of CsA as a treatment for acute MI.

Interestingly, a recent study by Pan et al. demonstrated that knockout of the mitochondrial calcium uniporter (MCU), which was demonstrated to abolish both mitochondrial calcium uptake and calcium-induced MPT, does not confer protection from IR injury [105]. This result is surprising in light of the many studies demonstrating that drugs and protocols that inhibit MPT, including administration of CsA at reperfusion, elicit cardioprotection [28]. One potential explanation as to why MCU knockout failed to confer cardioprotection may be due to the indirect effect that MCU knockout has on the cellular handling of calcium. Although calcium-induced MPT greatly predisposes myocytes to cell death during IR, the ability of mitochondria to buffer increases in cytoso-lic calcium concentration through passive uptake of calcium via the MCU may help protect cardiac myocytes from other calcium-mediated mechanisms of irreversible injury, such as the activation of calcium-dependent degradative enzymes and hypercontrac-ture (discussed in Section "Reperfusion injury: calcium overload"). However, MCU knockout eliminates the ability of mitochondria to uptake calcium, thereby abolishing the ability of mitochondria to buffer rising cytosolic calcium concentrations and potentially rendering MCU-deficient cardiomyocytes susceptible to these other forms of calcium overload-mediated irreversible injury. In contrast to MCU knockout, CsA, which reversibly inhibits MPT by binding to the permeability transition pore component cyclophilin D, does not absolutely abolish the ability of mitochondria to uptake calcium but rather increases the calcium load required for MPT to occur [28,106]. This qualitative difference in the mechanism of MPT inhibition may partly explain why CsA administration has been shown to confer protection and MCU knockout has not.

While it is clear from the above discussion that a great many promising cardioprotective interventions have been described, the fact that none of these have become implemented into standard clinical practice underlines the importance of enhancing our understanding of how the heart can be protected from ischemic death, allowing for the identification of novel targets for MI


8 A.J. Perricone, R.S. Vander Heide / Pharmacological Research xxx (2014) xxx-xxx

therapy. Although the cytoskeleton has been demonstrated to play an important role in lethal reperfusion injury [26,35], the role ofthe cytoskeleton in mediating cardioprotective signaling has received little attention and is largely unknown. However, it is known that cytoskeletal signaling has been shown to play a prominent role in maintaining cell viability in both non-muscle cells and in cardiac myocytes [107-109]. Furthermore, the cytosolic tyrosine kinase focal adhesion kinase (FAK), an essential mediator of cytoskeletal signaling [110], has been shown by numerous studies to play an important role in cell proliferation, survival and hypertrophy signaling cascades [111-113]. Based on this evidence, studies were conducted to assess the cardioprotective potential of cytoskeletal signaling; the results of these studies are reviewed below in Section "Cytoskeletal signaling and cardioprotection".

Cytoskeletal signaling and cardioprotection

Because previous studies had shown that cytoskeletal lesions are a critical determinant of irreversible cellular injury during IR [26,41], it was hypothesized that interventions that protect the cytoskeleton from ischemic stress would be cardioprotective. Wei and Vander Heide demonstrated that heat stress (HS) significantly enhanced the expression of heat shock proteins (HSPs), proteins capable of associating with and stabilizing the actin cytoskeleton [114,115], thereby enhancing the cell's resistance to stress. Both HS and the dual expression of HSP27 and HSP70 protected neonatal rat ventricular myocytes (NRVM) from cell death induced by metabolic inhibition (simulated IR). Furthermore, HS was associated with the assembly of an integrin-paxillin-FAK cytoskeletal signaling complex, which was abolished through targeted inhibition of FAK via overexpression of the endogenous FAK competitive inhibitor FRNK (FAK-related non-kinase). Interference with FAK activity also enhanced NRVM cell death in response to simulated IR [47].

In subsequent studies, Wei and Vander Heide hypothesized that FAK and cytoskeletal signaling may represent a unique cardiopro-tective signaling pathway elicited by myocardial stress. To test this hypothesis, the role of FAK was directly interrogated in HS and IP-elicited signaling pathways. In cultured NRVM, HS significantly enhanced the FAK activation. HS also enhanced the interaction between FAK and PI3K, and this interaction was associated with enhanced expression of activated Akt. Overexpression of FRNK in NRVM reduced the expression of activated Akt following HS both at baseline and following 10 min of simulated ischemia, implicating Akt as a mediator of cardioprotection downstream of FAK [116]. In Langendorff perfused adult mouse hearts, IP also significantly enhanced the expression of activated FAK and Akt and protected the hearts from ischemic death, suggesting that cytoskeletal signaling may mediate a common pathway by which myocardial stress leads to downstream cardioprotective signaling [117].

Whereas these earlier studies utilized in vitro and ex vivo model systems to demonstrate activation and protection resulting from a cytoskeletal-based cardioprotective signaling pathway, a later study conducted by Perricone et al. utilized a novel myocyte-restricted, inducible FAK KO mouse model to assess the importance of FAK and cytoskeletal signaling in an in vivo model of MI. This study found that, while IP elicited significant protection from lethal IR injury in control mice, the protective effect of IP was abrogated in FAK KO mice. Furthermore, the expression of activated PI3K and Akt was enhanced in preconditioned control hearts but not in preconditioned FAK KO hearts [118], in accordance with the findings of the results of the earlier studies conducted by Wei and Vander Heide discussed above [116,117].

Because cytoskeletal lesions predispose cardiomyocytes to sar-colemmal membrane rupture and cell death, mechanisms that

either protect cellular membranes during IR or enhance the repair of damaged membranes should be cardioprotective. Accordingly, a study by Wang et al. assessed the cardioprotective potential of the striated muscle-specific tripartite motif family protein MG53, a known mediator of skeletal muscle membrane repair. This study found that cardiomyocyte membrane damage stimulates the localization of MG53 exclusively to the damaged membrane, and that membrane healing is impaired in MG53 deficient cardiomyocytes. Importantly, genetic ablation of MG53 exacerbated IR injury in the Langendorff-perfused mouse heart, supporting a cardioprotective role ofMG53 and membrane repair [119]. Interestingly, a study by Cao et al. found that MG53 knockout abolished the protective effect of IP in the Langendorff-perfused mouse heart and that overexpression of MG53 in cultured myocytes conferred protection from hypoxia and oxidative stress-induced cell death. This study determined that the protective mechanism of MG53 was due to its ability to stimulate downstream activation of PI3K-Akt and Erk1/2 signaling pathways [120]. Therefore, MG53 may exert its cardioprotective influence through multiple mechanisms.


Despite the great multitude of interventions implicated as having cardioprotective potential that have been identified over the past several years, an efficacious therapy based on what has been learned about cardioprotection is still lacking. The fact that so many different interventions are capable of reducing ischemic death underlines the fact that there are likely several different pathways within a robust cardiac signaling network that can ultimately elicit a cardioprotective state. The pathophysiology of ischemic heart disease is complex, and a therapeutic strategy capable of activating multiple arms of the cardioprotective signaling network and/or simultaneously counteracting multiple mediators of IR injury would likely have the greatest efficacy in mitigating ischemic death and improving the morbidity and mortality associated with MI. While the cardioprotective potential of therapies targeting many of the mediators of IR injury have been well studied in both animal models and clinical trials, the therapeutic potential of other mediators, especially cytoskeletal lesions and the role of cytoskeletal signaling, have received less attention and are not completely understood. Future studies will be important for enhancing both our knowledge of MI pathophysiology and mechanisms of cardioprotection, thereby providing additional avenues of cardioprotective signaling that may be potentially harnessed toward the ultimate goal of establishing a highly efficacious therapeutic strategy for enhancing the heart's resistance to myocardial IR injury.


[1] Go AS, Mozaffarian D, Roger VL, Benjamin EJ, Berry JD, Borden WB, et al. Heart disease and stroke statistics - 2013 update: a report from the American Heart Association. Circulation 2013;127:e6-245.

[2] Heron M. Deaths: leading causes for 2008. Natl Vital Stat Rep 2012;60: 1-94.

[3] Schoen FJ, Mitchell RN. The heart. In: Kumar V, Abbas AK, Fausto N, Aster JC, editors. Robbins and Cotran pathologic basis of disease. Philadelphia: Saun-ders; 2010. p. 529-87.

[4] Gerczuk PZ, Kloner RA. An update on cardioprotection: a review of the latest adjunctive therapies to limit myocardial infarction size in clinical trials. J Am Coll Cardiol 2012;59:969-78.

[5] Vander Heide RS, Steenbergen C. Cardioprotection and myocardial reperfusion: pitfalls to clinical application. Circ Res 2013;113:464-77.

[6] Kumar V, Abbas AK, Fausto N, Aster JC. Cellular responses to stress and toxic insults: adaptation, injury, and death. In: Kumar V, Abbas AK, Fausto N, Aster JC, editors. Robbins and Cotran pathologic basis ofdisease. Philadelphia: Saunders; 2010. p. 3-42.

[7] Gottlieb RA, Burleson KO, Kloner RA, Babior BM, Engler RL. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest 1994;94:1621-8.


A.]. Perricone, R.S. Vander Heide /Pha

[8] Zhao ZQ, Velez DA, Wang NP, Hewan-Lowe KO, Nakamura M, Guyton RA, et al. Progressively developed myocardial apoptotic cell death during late phase of reperfusion. Apoptosis 2001;6:279-90.

[9] Fliss H, Gattinger D. Apoptosis in ischemic and reperfused rat myocardium. Circ Res 1996;79:949-56.

[10] Imahashi K, Schneider MD, Steenbergen C, Murphy E. Transgenic expression of Bcl-2 modulates energy metabolism, prevents cytosolic acidification during ischemia, and reduces ischemia/reperfusion injury. Circ Res 2004;95:734-41.

[11] Shimizu S, Eguchi Y, Kamiike W, Waguri S, Uchiyama Y, Matsuda H, et al. Retardation of chemical hypoxia-induced necrotic cell death by Bcl-2 and ICE inhibitors: possible involvement of common mediators in apoptotic and necrotic signal transductions. Oncogene 1996;12:2045-50.

[12] Gottlieb RA, Engler RL. Apoptosis in myocardial ischemia-reperfusion. Ann N YAcad Sci 1999;874:412-26.

[13] Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, et al. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol 2005;1:112-9.

[14] Smith CC, Yellon DM. Necroptosis, necrostatins and tissue injury. J Cell Mol Med 2011;15:1797-806.

[15] Smith CC, Davidson SM, Lim SY, Simpkin JC, Hothersall JS, Yellon DM. Necro-statin: a potentially novel cardioprotective agent. Cardiovasc Drugs Ther 2007;21:227-33.

[16] Sanada S, Komuro I, Kitakaze M. Pathophysiology of myocardial reperfusion injury: preconditioning, postconditioning, and translational aspects of protective measures. Am J Physiol Heart Circ Physiol 2011;301:H1723-41.

[17] TurerAT, Hill JA. Pathogenesis of myocardial ischemia-reperfusion injury and rationale for therapy. Am J Cardiol 2010;106:360-8.

[18] Chiong M, Wang ZV, Pedrozo Z, Cao DJ, Troncoso R, Ibacache M, et al. Car-diomyocyte death: mechanisms and translational implications. Cell Death Dis 2011;2:e244.

[19] ChenS, Li S. The Na+/Ca(2)+ exchanger in cardiac ischemia/reperfusion injury. Med Sci Monit 2012;18:RA161-5.

[20] Orrenius S, Zhivotovsky B, Nicotera P. Regulation of cell death: the calcium-apoptosis link. Nat Rev Mol Cell Biol 2003;4:552-65.

[21] Jennings RB, Sommers HM, Herdson PB, Kaltenbach JP. Ischemic injury of myocardium. Ann N Y Acad Sci 1969;156:61-78.

[22] Reimer KA, Jennings RB. The wavefront phenomenon of myocardial ischemic cell death. II. Transmural progression of necrosis within the framework of ischemic bed size (myocardium at risk) and collateral flow. Lab Invest 1979;40:633-44.

[23] Jennings RB, Sommers HM, Smyth GA, Flack HA, Linn H. Myocardial necrosis induced by temporary occlusion of a coronary artery in the dog. Arch Pathol 1960;70:68-78.

[24] Yellon DM, Hausenloy DJ. Myocardial reperfusion injury. N Engl J Med 2007;357:1121-35.

[25] Szabo I, Bernardi P, Zoratti M. Modulation of the mitochondrial megachannel by divalent cations and protons. J Biol Chem 1992;267:2940-6.

[26] Ganote CE, Vander Heide RS. Cytoskeletal lesions in anoxic myocardial injury. A conventional and high-voltage electron-microscopic and immunofluorescence study. Am J Pathol 1987;129:327-44.

[27] Inserte J, Hernando V, Garcia-Dorado D. Contribution of calpains to myocar-dial ischaemia/reperfusion injury. Cardiovasc Res 2012;96:23-31.

[28] Halestrap AP. What is the mitochondrial permeability transition pore. J Mol Cell Cardiol 2009;46:821-31.

[29] Di Lisa F, Menabo R, Canton M, Barile M, Bernardi P. Opening of the mito-chondrial permeability transition pore causes depletion of mitochondrial and cytosolic NAD+ and is a causative event in the death of myocytes in postis-chemic reperfusion of the heart. J Biol Chem 2001;276:2571-5.

[30] Griffiths EJ, Halestrap AP. Mitochondrial non-specific pores remain closed during cardiac ischaemia, but open upon reperfusion. Biochem J 1995;307(Pt 1):93-8.

[31] Halestrap AP, Clarke SJ, Javadov SA. Mitochondrial permeability transition pore opening during myocardial reperfusion - a target for cardioprotection. Cardiovasc Res 2004;61:372-85.

[32] Iizuka K, Kawaguchi H, Kitabatake A. Effects of thiol protease inhibitors on fodrin degradation during hypoxia in cultured myocytes. J Mol Cell Cardiol 1993;25:1101-9.

[33] Liu X, Schnellmann RG. Calpain mediates progressive plasma membrane permeability and proteolysis of cytoskeleton-associated paxillin, talin, and vinculin during renal cell death. J Pharmacol Exp Ther 2003;304: 63-70.

[34] Vander Heide RS, Ganote CE. Increased myocyte fragility following anoxic injury. J Mol Cell Cardiol 1987;19:1085-103.

[35] Steenbergen C, Hill ML, Jennings RB. Cytoskeletal damage during myocar-dial ischemia: changes in vinculin immunofluorescence staining during total in vitro ischemia in canine heart. Circ Res 1987;60:478-86.

[36] Vander Heide RS, Angelo JP, Altschuld RA, Ganote CE. Energy dependence of contraction band formation in perfused hearts and isolated adult myocytes. Am J Pathol 1986;125:55-68.

[37] Piper HM, Garcia-Dorado D, Ovize M. A fresh look at reperfusion injury. Cardiovasc Res 1998;38:291-300.

[38] Ganote CE, Kaltenbach JP. Oxygen-induced enzyme release: early events and a proposed mechanism. J Mol Cell Cardiol 1979;11:389-406.

[39] Vander Heide RS, Ganote CE. Caffeine-induced myocardial injury in calcium-free perfused rat hearts. Am J Pathol 1985;118:55-65.

logical Research xxx (2014) xxx-xxx 9

[40] Vander Heide RS, Altschuld RA, Lamka KG, Ganote CE. Modification of caffeine-induced injury in Ca2+-free perfused rat hearts. Relationship to the calcium paradox. AmJ Pathol 1986;123:351-64.

[41] Garcia-Dorado D, Theroux P, DuranJM, Solares J, AlonsoJ, Sanz E, et al. Selective inhibition ofthe contractile apparatus. A new approach to modification of infarct size, infarct composition, and infarct geometry during coronary artery occlusion and reperfusion. Circulation 1992;85:1160-74.

[42] Zweier JL, Talukder MA. The role of oxidants and free radicals in reperfusion injury. Cardiovasc Res 2006;70:181-90.

[43] Zorov DB, Filburn CR, Klotz LO, Zweier JL, Sollott SJ. Reactive oxygen species (ROS)-induced ROS release: a new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes. J Exp Med 2000;192:1001-14.

[44] Thompson-Gorman SL, Zweier JL. Evaluation of the role of xanthine oxidase in myocardial reperfusion injury. J Biol Chem 1990;265:6656-63.

[45] Crompton M, Virji S, Doyle V, Johnson N, Ward JM. The mitochondrial permeability transition pore. Biochem Soc Symp 1999;66:167-79.

[46] Jones RN, Reimer KA, Hill ML, Jennings RB. Effect of hypothermia on changes in high-energy phosphate production and utilization in total ischemia. J Mol Cell Cardiol 1982;14(Suppl. 3):123-30.

[47] Wei H, Campbell W, Vander Heide RS. Heat shock-induced cardioprotection activates cytoskeletal-based cell survival pathways. Am J Physiol Heart Circ Physiol 2006;291:H638-47.

[48] Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 1986;74:1124-36.

[49] Vander Heide RS, Schwartz LM, Reimer KA. The novel calcium antagonist Ro 40-5967 limits myocardial infarct size in the dog. Cardiovasc Res 1994;28:1526-32.

[50] Liu GS, Thornton J, Van Winkle DM, Stanley AW, Olsson RA, Downey JM. Protection against infarction afforded by preconditioning is mediated by A1 adenosine receptors in rabbit heart. Circulation 1991;84:350-6.

[51] Bankwala Z, Hale SL, Kloner RA. Alpha-adrenoceptor stimulation with exogenous norepinephrine or release of endogenous catecholamines mimics ischemic preconditioning. Circulation 1994;90:1023-8.

[52] Schultz Je J, Hsu AK, Nagase H, Gross GJ. TAN-67, a delta 1-opioid receptor agonist, reduces infarct size via activation of Gi/o proteins and KATP channels. AmJ Physiol 1998;274:H909-14.

[53] Cohen MV, Downey JM. Myocardial preconditioning promises to be a novel approach to the treatment of ischemic heart disease. Annu Rev Med 1996;47:21-9.

[54] Yellon DM, Alkhulaifi AM, Pugsley WB. Preconditioning the human myocardium. Lancet 1993;342:276-7.

[55] Bolli R, Li QH, Tang XL, Guo Y, Xuan YT, Rokosh G, et al. The late phase of preconditioning and its natural clinical application - gene therapy. Heart Fail Rev 2007;12:189-99.

[56] Goto M, Liu Y, Yang XM, Ardell JL, Cohen MV, Downey JM. Role of bradykinin in protection of ischemic preconditioning in rabbit hearts. Circ Res 1995;77:611-21.

[57] Schultz JE, Hsu AK, Gross GJ. Morphine mimics the cardioprotective effect of ischemic preconditioning via a glibenclamide-sensitive mechanism in the rat heart. Circ Res 1996;78:1100-4.

[58] Ytrehus K, Liu Y, DowneyJM. Preconditioning protects ischemic rabbit heart by protein kinase C activation. Am J Physiol 1994;266:H1145-52.

[59] Yang X, Cohen MV, Downey JM. Mechanism of cardioprotection by early ischemic preconditioning. Cardiovasc Drugs Ther 2010;24:225-34.

[60] Liu Y, Gao WD, O'Rourke B, Marban E. Synergistic modulation of ATP-sensitive K+ currents by protein kinase C and adenosine, Implications for ischemic preconditioning. Circ Res 1996;78:443-54.

[61] Murata M, Akao M, O'Rourke B, Marban E. Mitochondrial ATP-sensitive potassium channels attenuate matrix Ca(2+) overload during simulated ischemia and reperfusion: possible mechanism of cardioprotection. Circ Res 2001;89:891-8.

[62] Matsui T, Li L, del Monte F, Fukui Y, Franke TF, Hajjar RJ, et al. Adenoviral gene transfer of activated phosphatidylinositol 3'-kinase and Akt inhibits apoptosis of hypoxic cardiomyocytes in vitro. Circulation 1999;100:


[63] Tong H, Chen W, Steenbergen C, Murphy E. Ischemic preconditioning activates phosphatidylinositol-3-kinase upstream of protein kinase C. Circ Res 2000;87:309-15.

[64] Baines CP, Wang L, Cohen MV, Downey JM. Myocardial protection by insulin is dependent on phospatidylinositol 3-kinase but not protein kinase C or KATP channels in the isolated rabbit heart. Basic Res Cardiol 1999;94: 188-98.

[65] Otani H, Yamamura T, Nakao Y, Hattori R, Kawaguchi H, Osako M, et al. Insulin-like growth factor-I improves recovery of cardiac performance during reperfusion in isolated rat heart by a wortmannin-sensitive mechanism. J Cardiovasc Pharmacol 2000;35:275-81.

[66] Bell RM, Yellon DM. Bradykinin limits infarction when administered as an adjunct to reperfusion in mouse heart: the role of PI3K, Akt and eNOS. J Mol Cell Cardiol 2003;35:185-93.

[67] Bell RM, Yellon DM. Atorvastatin, administered at the onset of reperfusion, and independent of lipid lowering, protects the myocardium by up-regulating a pro-survival pathway. J Am Coll Cardiol 2003;41:508-15.

[68] Hausenloy DJ, Yellon DM. New directions for protecting the heart against ischaemia-reperfusion injury: targeting the reperfusion injury salvage kinase (RISK)-pathway. Cardiovasc Res 2004;61:448-60.


10 A.J. Perricone, R.S. Vander Heide / Pharmacological Research xxx (2014) xxx-xxx

983 [69] Hausenloy DJ, Tsang A, Mocanu MM, Yellon DM. Ischemic preconditioning in patients undergoing primary percutaneous coronary intervention for ST- 1062

984 protects by activating prosurvival kinases at reperfusion. Am J Physiol Heart elevation myocardial infarction: the randomized multicentre CASTEMI study. 1063

985 Circ Physiol 2005:288:H971-6. Eur Heart J 2006;27:2516-23. 1064

986 [70] Ping P, Zhang J, Cao X, Li RC, Kong D, Tang XL, et al. PKC-dependent activa- [96] Ambrosio G, Weisfeldt ML, Jacobus WE, Flaherty JT. Evidence for a reversible 1065

987 tion of p44/p42 MAPKs during myocardial ischemia-reperfusion in conscious oxygen radical-mediated component of reperfusion injury: reduction by 1066

988 rabbits. Am J Physiol 1999;276:H1468-81. recombinant human superoxide dismutase administered at the time of 1067

989 [71] Behrends M, Schulz R, Post H, Alexandrov A, Belosjorow S, Michel MC, et al. reflow. Circulation 1987;75:282-91. 1068

990 Inconsistent relation of MAPK activation to infarct size reduction by ischemic [97] Guan W, Osanai T, Kamada T, Hanada H, Ishizaka H, Onodera H, et al. Effect 1069

991 preconditioning in pigs. Am J Physiol Heart Circ Physiol 2000;279:H1111-9. of allopurinol pretreatment on free radical generation after primary coro- 1070

992 [72] Mocanu MM, Bell RM, Yellon DM. P13 kinase and not p42/p44 appears to be nary angioplasty for acute myocardial infarction. J Cardiovasc Pharmacol 1071

993 implicated inthe protection conferred by ischemic preconditioning. J Mol Cell 2003;41:699-705. 1072

994 Cardiol 2002;34:661-8. [98] Ye Y, Li J, Yuan Z. Effect of antioxidant vitamin supplementation on cardiovas- 1073

995 [73] Murphy E, Steenbergen C. Mechanisms underlying acute protection from car- cular outcomes: a meta-analysis of randomized controlled trials. PLOS ONE 1074

996 diac ischemia-reperfusion injury. Physiol Rev 2008;88:581-609. 2013;8:e56803. 1075

997 [74] SussmanMA,VolkersM,FischerK,BaileyB,CottageCT,DinS,etal.Myocardial [99] Crompton M, Ellinger H, Costi A. Inhibition by cyclosporin A of a Ca2+- 1076

998 AKT: the omnipresent nexus. Physiol Rev 2011;91:1023-70. dependent pore in heart mitochondria activated by inorganic phosphate and 1077

999 [75] Fujio Y, Nguyen T, Wencker D, Kitsis RN, Walsh K. Akt promotes survival of oxidative stress. Biochem J 1988;255:357-60. 1078

1000 cardiomyocytes in vitro and protects against ischemia-reperfusion injury in [100] Halestrap AP, Connern CP, Griffiths EJ, Kerr PM. Cyclosporin A bind- 1079

1001 mouse heart. Circulation 2000;101:660-7. ing to mitochondrial cyclophilin inhibits the permeability transition pore 1080

1002 [76] Fulton D, GrattonJP, McCabe TJ, Fontana J, Fujio Y, Walsh K, et al. Regulation and protects hearts from ischaemia/reperfusion injury. Mol Cell Biochem 1081

1003 of endothelium-derived nitric oxide production by the protein kinase Akt. 1997;174:167-72. 1082

1004 Nature 1999;399:597-601. [101] Hausenloy DJ, Maddock HL, Baxter GF, Yellon DM. Inhibiting mitochondrial 1083

1005 [77] Miura T, Tanno M. The mPTP and its regulatory proteins: final common tar- permeability transition pore opening: a new paradigm for myocardial pre- 1084

1006 gets of signalling pathways for protection against necrosis. Cardiovasc Res conditioning. Cardiovasc Res 2002;55:534-43. 1085

1007 2012;94:181-9. [102] Piot C, Croisille P, Staat P, Thibault H, Rioufol G, Mewton N, et al. Effect of 1086

1008 [78] PastorinoJG, HoekJB, Shulga N. Activation of glycogen synthase kinase 3beta cyclosporine on reperfusion injury in acute myocardial infarction. N Engl J 1087

1009 disrupts the binding of hexokinase 11 to mitochondria by phosphorylating Med 2008;359:473-81. 1088

1010 voltage-dependent anion channel and potentiates chemotherapy-induced [103] Jennings RB. Historical perspective on the pathology of myocardial 1089

1011 cytotoxicity. Cancer Res 2005;65:10545-54. ischemia/reperfusion injury. Circ Res 2013;113:428-38. 1090

1012 [79] Murphy E, Kohr M, Sun J, Nguyen T, Steenbergen C. S-nitrosylation: a radical [104] Griffiths EJ, Halestrap AP. Protection by cyclosporin A of 1091

1013 way to protect the heart. J Mol Cell Cardiol 2012;52:568-77. ischemia/reperfusion-induced damage in isolated rat hearts. J Mol Cell 1092

1014 [80] Campbell DL, Stamler JS, Strauss HC. Redox modulation of L-type calcium Cardiol 1993;25:1461-9. 1093

1015 channels in ferret ventricular myocytes. Dual mechanism regulation by nitric [105] Pan X, Liu J, Nguyen T, Liu C, Sun J, Teng Y, et al. The physiological role of 1094

1016 oxide and S-nitrosothiols. J Gen Physiol 1996;108:277-93. mitochondrial calcium revealed by mice lacking the mitochondrial calcium 1095

1017 [81] SunJ, Morgan M,ShenRF, Steenbergen C, Murphy E. Preconditioning results in uniporter. Nat Cell Biol 2013;15:1464-72. 1096

1018 S-nitrosylation of proteins involved in regulation of mitochondrial energetics [106] Basso E, Fante L, FowlkesJ, Petronilli V, Forte MA, Bernardi P. Properties ofthe 1097

1019 and calcium transport. Circ Res 2007;101:1155-63. permeability transition pore in mitochondria devoid of cyclophilin D. J Biol 1098

1020 [82] Nadtochiy SM, Burwell LS, Brookes PS. Cardioprotection and mitochondrial Chem 2005;280:18558-61. 1099

1021 S-nitrosation: effects of S-nitroso-2-mercaptopropionyl glycine (SNO-MPG) [107] van de Water B, Nagelkerke JF, Stevens JL Dephosphorylation of focal 1100

1022 in cardiac ischemia-reperfusion injury. J Mol Cell Cardiol 2007;42:812-25. adhesion kinase (FAK) and loss of focal contacts precede caspase-mediated 1101

1023 [83] Minamino T. Cardioprotection from ischemia/reperfusion injury: basic and cleavage of FAK during apoptosis in renal epithelial cells. J Biol Chem 1102

1024 translational research. CircJ 2012;76:1074-82. 1999;274:13328-37. 1103

1025 [84] Zhao ZQ, CorveraJS, Halkos ME, Kerendi F, Wang NP, Guyton RA, et al. Inhi- [108] Heidkamp MC, Bayer AL, Kalina JA, Eble DM, Samarel AM. GFP-FRNK disrupts 1104

1026 bition of myocardial injury by ischemic postconditioning during reperfusion: focal adhesions and induces anoikis in neonatal rat ventricular myocytes. Circ 1105

1027 comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol Res 2002;90:1282-9. 1106

1028 2003;285:H579-88. [109] Pfister R, Acksteiner C, Baumgarth J, Burst V, Geissler HJ, Margulies KB, et al. 1107

1029 [85] Staat P, Rioufol G, Piot C, Cottin Y, CungTT, L'Huillier 1, et al. Postconditioning Loss of beta1D-integrin function in human ischemic cardiomyopathy. Basic 1108

1030 the human heart. Circulation 2005;112:2143-8. Res Cardiol 2007;102:257-64. 1109

1031 [86] Laskey WIK, Yoon S, Calzada N, Ricciardi MJ. Concordant improvements [110] Parsons JT. Focal adhesion kinase: the first ten years. J Cell Sci 1110

1032 in coronary flow reserve and ST-segment resolution during percutaneous 2003;116:1409-16. 1111

1033 coronary intervention for acute myocardial infarction: a benefit of postcon- [111] Samarel AM. Costameres, focal adhesions, and cardiomyocyte mechanotrans- 1112

1034 ditioning. Catheter Cardiovasc Interv 2008;72:212-20. duction. Am J Physiol Heart Circ Physiol 2005;289:H2291-301. 1113

1035 [87] Sorensson P, Saleh N, Bouvier F, Bohm F, Settergren M, Caidahl K, et al. Effect [112] DavalM,GurloT,CostesS,HuangCJ,ButlerPC.Cyclin-dependentkinase5pro- 1114

1036 of postconditioning on infarct size in patients with ST elevation myocardial motes pancreatic beta-cell survival via Fak-Akt signaling pathways. Diabetes 1115

1037 infarction. Heart 2010;96:1710-5. 2011;60:1186-97. 1116

1038 [88] FreixaX, Bellera N, Ortiz-Perez JT, Jimenez M, Pare C, BoschX, et al. Ischaemic [113] Provenzano PP, Keely PJ. The role of focal adhesion kinase in tumor initiation 1117

1039 postconditioning revisited: lack of effects on infarct size following primary and progression. Cell Adh Migr 2009;3:347-50. 1118

1040 percutaneous coronary intervention. Eur Heart J 2012;33:103-12. [114] Quinta HR, Galigniana NM, Erlejman AG, Lagadari M, Piwien-Pilipuk G, 1119

1041 [89] Karmazyn M. Amiloride enhances postischemic ventricular recovery: possi- GalignianaMD.Managementofcytoskeletonarchitecturebymolecularchap- 1120

1042 ble role of Na+-H+ exchange. Am J Physiol 1988;255:H608-15. erones and immunophilins. Cell Signal 2011;23:1907-20. 1121

1043 [90] Wang Y, MeyerJW,AshrafM,ShullGE. Mice witha null mutation inthe NHE1 [115] Huot J, Lambert H, LavoieJN, Guimond A, Houle F, Landry J. Characterization 1122

1044 Na+-H+ exchanger are resistant to cardiac ischemia-reperfusion injury. Circ of45-kDa/54-kDaHSP27 kinase, a stress-sensitive kinase which may activate 1123

1045 Res 2003;93:776-82. the phosphorylation-dependent protective function of mammalian 27-kDa 1124

1046 [91] ZeymerU, SuryapranataH, MonassierJP, OpolskiG, DaviesJ, Rasmanis G, et al. heat-shock protein HSP27. Eur J Biochem 1995;227:416-27. 1125

1047 The Na(+)/H(+) exchange inhibitor eniporide as an adjunct to early reperfu- [116] Wei H, Vander Heide RS. Heat stress activates AKT via focal adhesion kinase- 1126

1048 sion therapy for acute myocardial infarction: results of the evaluation of the mediated pathway in neonatal rat ventricular myocytes. Am J Physiol Heart 1127

1049 safetyandcardioprotectiveeffectsofeniporideinacutemyocardialinfarction Circ Physiol 2008;295:H561-8. 1128

1050 (ESCAM1) trial. J Am Coll Cardiol 2001;38:1644-50. [117] Wei H, Vander Heide RS. Ischemic preconditioning and heat shock 1129

1051 [92] Mentzer Jr RM, Bartels C, Bolli R, Boyce S, Buckberg GD, Chaitman B, et al. activate Akt via a focal adhesion kinase-mediated pathway in Langendorff- 1130

1052 Sodium-hydrogen exchange inhibition by cariporide to reduce the risk of perfused adult rat hearts. Am J Physiol Heart Circ Physiol 2010;298: 1131

1053 ischemic cardiac events in patients undergoing coronary artery bypass graft- H152-7. 1132

1054 ing: results ofthe EXPED1T1ON study. Ann Thorac Surg 2008;85:1261-70. [118] Perricone AJ, Bivona BJ, Jackson FR, Vander Heide RS. Conditional knockout of 1133

1055 [93] 1mahashi K, Pott C, Goldhaber J1, Steenbergen C, Philipson KD, Murphy myocyte focal adhesion kinase abrogates ischemic preconditioning in adult 1134

1056 E. Cardiac-specific ablation ofthe Na+-Ca2+ exchanger confers protection murine hearts. J Am Heart Assoc 2013;2:e000457. 1135

1057 against ischemia/reperfusion injury. Circ Res 2005;97:916-21. [119] Wang X, Xie W, Zhang Y, Lin P, Han L, Han P, et al. Cardioprotection 1136

1058 [94] Kawasumi H, Satoh N, Kitada Y. Caldaret, an intracellular Ca2+ handling of ischemia/reperfusion injury by cholesterol-dependent MG53-mediated 1137

1059 modulator, limits infarct size of reperfused canine heart. J Pharmacol Sci membrane repair. Circ Res 2010;107:76-83. 1138

1060 2007;103:222-33. [120] Cao CM, Zhang Y, Weisleder N, Ferrante C, Wang X, Lv F, et al. MG53 consti- 1139

1061 [95] BarFW,Tzivoni D, DirksenMT, Fernandez-Ortiz A, HeyndrickxGR, Brachmann tutes a primary determinant of cardiac ischemic preconditioning. Circulation 1140

J, et al. Results ofthe first clinical study of adjunctive CAldaret (MCC-135) 2010;121:2565-74. 1141