There are a number of physiologic processes and pharmacologic agents known to confer protection against ischemia-reperfusion injury in the experimental setting. Although the current evidence in support of the efficacy of these novel approaches is largely limited to preclinical studies, several trials are underway to determine the clinical relevance of these new approaches. The purpose of this section is to review emerging cardioprotective strategies that hold the most promise.
“Precondition with ischemia” is an endogenous adaptive phenomenon whereby the heart becomes more tolerant to a period of prolonged ischemia if first exposed to brief episodes of coronary artery occlusion. This adaptation to ischemia was first described by Murry and colleagues and is referred to as classic,first window, or early phase ischemic preconditioning (IPC).75 IPC is associated with a reduction in infarct size, apoptosis, and reperfusion-associated arrhythmias.117 IPC has been demonstrated in every animal species studied and appears to persist as long as 1 to 2 hours after the ischemic preconditioning stimulus.118,119 It becomes ineffective when the sustained ischemic insult exceeds 3 hours. This suggests that the protection is conferred only when prolonged ischemia is followed by timely reperfusion.
Subsequent studies have revealed that this endogenous defense mechanism can manifest itself in multiple ways. After the acute phase of preconditioning disappears, a second phase of protection appears 24 hours later and is sustained for up to 72 hours. This has been referred to as the second window of protection, late-phase preconditioning, or delayed preconditioning. Unlike classic IPC, which protects only against infarction, the late phase protects against both infarction and myocardial stunning.120,121
Cellular Mechanisms of IPC
These reports of adaptation to ischemia have resulted in major investigative efforts to elucidate the intracellular mechanism(s) that underlie the heart's endogenous defenses against ischemia-reperfusion injury. The assumption is that a better understanding of these mechanism(s) could lead to the development of potent new therapeutic modalities that are more effective in treating or preventing the deleterious consequences of ischemia-reperfusion injury. One of the earliest hypotheses was that stimulation of cardiomyocyte adenosine A1 and/or A3 receptors was the primary mediator of acute ischemic preconditioning.119,122 Subsequent studies have revealed that in addition to adenosine, there are multiple guanine nucleotide-binding (G) protein–coupled receptors (GPCRs) that, once activated, can mimic the infarct-reducing effect of ischemic preconditioning, eg, bradykinin, endothelin, and α1 -adrenergic, muscarinic, angiotensin II, and delta-opioid receptors (Fig. 15-4). The infusion of exogenous agents that mimic ischemic preconditioning is referred to as pharmacologic preconditioning. Exactly which of these receptors is the most important in mediating endogenous preconditioning is unknown because there appear to be species differences and redundant signaling pathways. Regardless, it is now thought that these triggers of IPC result in alterations in certain enzymes, such as tyrosine kinases, isoforms of PKC, and mitogen-activated protein kinases (p38 and extracellular signal regulated kinase [ERK]) that, in turn, confer protection against irreversible injury before the onset of prolonged ischemia.
Signaling pathways of ischemic preconditioning. Numerous triggers (opioids, bradykinin, and adenosine) and intracellular signaling pathways are involved in the cardioprotection conferred by IPC. The signal transduction pathways are complex, interactive, and include the HB-EGF receptor, PI3K, Akt, ERK1/2, eNOS, PKG, the opening of the mKATP channel, ROS production, PKC activation, p70 S6 kinase, and GSK-3β. Possible end effectors of IPC include the opening of the mKATP channel and inhibition of the mitochondrial permeability transition pore opening. If only a few mitochondria are affected, cytochrome c may be released and induce apoptosis and cause cell death at a later time. Recent evidence suggests a unique role for the adenosine A2b receptor when activated at the time of reperfusion. Although the process of autophagy has been implicated in IPC induced cardioprotection, where and how this process interacts with the signaling pathways remains to be determined. eNOS = endothelial NOS; ERK = extracellular-signal regulated kinase; GC = guanylyl cyclase; GSK-3β = glycogen synthase kinase; HB-EGF = heparin-binding epidermal growth factor; IPC = ischemic preconditioning; MEK = mitogen activated protein kinase; mKATP = mitochondrial ATP-dependent potassium channel; MMP = matrix metalloproteinases; mPTP = mitochondrial permeability transition pore; NO = nitric oxide; NOS = NO synthase; PDK = phosphoinositide-dependent kinase; PI3 = phosphatidylinositol 3-kinase; PI4,5 P2 = phosphatidylinositol bisphosphate; PI3,4,5 P3 = phosphatidylinositol trisphosphate; PKC = protein kinase C; Pro = Pro-HB-EGF; PKG = protein kinase G; P70S6K = p70S6 kinase; ROS = reactive oxygen species;. (Adapted from: Cohen MV, Downey JM: Adenosine: trigger and mediator of cardioprotection. Basic Res Cardiol 2008; 103:203-215. Epub 2007 Nov 12. Review. PubMed[PubMed: 17999026].)
Interestingly, IPC-induced cardioprotection appears to require repopulation of receptors and activation (or, in some instances, reactivation) of “prosurvival” kinases upon relief of sustained ischemia. In this regard, Hausenloy and Yellon introduced the term Reperfusion Injury Salvage Kinase (RISK) pathway to represent the PI3K–Akt and ERK 1/2 pro-survival kinases activated at the time of reperfusion and proposed that manipulation and upregulation of the RISK pathway may represent another approach to myocardial protection.123
Although the identity of the end effector(s) of IPC remain speculative, significant evidence has accumulated indicating that the cardiomyocyte mitochondria are key targets of conditioning-induced protection (Fig. 15-4).124,125 Specifically, inhibition of mPTP and the opening of the mitochondrial KATP (mKATP) channel have been implicated as the effectors of IPC.126,127 Under normal conditions the mitochondrial inner membrane is impermeable to most metabolites and ions and the mPTP is closed. Although the molecular structure of the pore has yet to be determined, it is characterized by the formation of a large conductance megachannel that is regulated by cyclophilin D in the matrix. Although early investigations implicated the voltage-dependent anion channel in the outer mitochondrial membrane and the adenine nucleotide translocator (ANT) in the inner membrane in addition to cyclophilin D, genetic studies have refuted that model. Mouse models in which all ANT isoforms were deleted still exhibited mPTP opening; this was also the case for deletion of VDAC isoforms. However, deletion of cyclophilin resulted in hearts that were much more resistant to I/R injury, and further studies revealed that although the threshold for mPTP opening was greatly increased, it was still possible to trigger pore opening. It was concluded that cyclophilin D plays an important regulatory role in mPTP opening, but the molecular composition remains uncertain. Under conditions of stress, the mPTP may open, resulting in depolarization of the inner mitochondrial membrane and an influx of water and ions into the matrix because of its high oncotic pressure. Matrix swelling expands the highly folded inner membrane, but ultimately ruptures the outer membrane, resulting in release of cytochrome c and other pro-apoptotic factors. Even in the absence of the outer membrane rupture, loss of mitochondrial membrane potential results in ATP hydrolysis by the F0F1 ATP synthase in an effort to restore membrane potential. This futile cycling accelerates energy depletion.
An ATP-sensitive potassium channel in the mitochondrial inner membrane (mKATP) has been implicated on the basis of pharmacologic effects of diazoxide and pinacidil (channel openers) and 5-hydroxydecanoate and glibenclamide (channel closers). Many pharmacologic studies have demonstrated a protective role for the putative mKATP, although its molecular composition remains unknown. Garg and Hu have proposed that PKC activation enhances the import of plasma membrane KATP channels into mitochondria. This was based on their observation that in COS-7 cells, Kir6.2 protein (a subunit of KATP channels) and channel activity increased in mitochondria after PMA treatment, and this increase was inhibited by the selective PKC inhibitor chelerythrine. Pharmacologically triggered opening of the mKATP channel has been shown to reduce calcium overload, mitochondrial free-radical production, and swelling and to preserve ATP levels after ischemia/reperfusion.128
Although early-phase preconditioning shares many of the same signaling mechanisms with late-phase preconditioning, the most obvious difference between the two is the apparent requirement for protein synthesis in the latter. Late-phase IPC has been shown to be associated with the upregulation of various proteins, including, but not limited to, heat-shock proteins, inducible NOS (iNOS), cyclooxygenase 2, heme oxygenase, and manganese superoxide dismutase.129,130 There are, however, conflicting reports on what specific proteins are upregulated during late-phase preconditioning, which may be because of species differences as well as stimulus-specific responses.
There is considerable circumstantial evidence that ischemic preconditioning occurs in the human. Investigators have reported that patients experiencing angina before an MI have a better in-hospital prognosis and a reduced incidence of cardiogenic shock, fewer and less severe episodes of congestive heart failure, and smaller infarcts as assessed by cardiac enzyme release.131 Moreover, follow-up studies suggest that patients who have had angina before an infarct have better long-term survival rates.132–134 There are also a myriad of reports that patients who undergo percutaneous coronary interventions (PCI) have an enhanced tolerance to ischemia after the first balloon inflation, provided that the first balloon inflation exceeds 60 to 90 seconds.118 Chest pain severity, regional wall motion abnormalities, ST-segment elevation, QT dispersion, lactate production, and CKMB release all have been reported to be attenuated in this setting as well.135,136
In patients undergoing PCI, a preconditioning-like effect has been mimicked by the administration of a variety of pharmacologic agents that are known to induce preconditioning in animal studies. For example, the administration of adenosine before PCI has been reported to attenuate myocardial ischemic indices during the first balloon inflation.137 Administration of other agents, such as bradykinin and nicorandil (a KATP channel opener), also have been reported to produce similar effects.138,139 Conversely, the administration of aminophylline (a nonselective adenosine receptor antagonist), glibenclamide (a KATP channel blocker), or naloxone (an opioid receptor blocker) reportedly abolishes the effects of ischemic preconditioning during PCI.140,141 Additional studies provide evidence of delayed pharmacologic preconditioning in the clinical setting. Leesar and colleagues reported that a 4-hour intravenous infusion of nitroglycerin (an NO donor) 24 hours before PCI decreased ST-segment changes and reduced chest pain during the first balloon occlusion compared with patients treated with saline vehicle.142 An earlier report by this same group indicated that delayed preconditioning with nitroglycerin decreased exercise-induced ST-segment changes and improved exercise tolerance. Thus, there are observational studies that support the hypothesis that myocardial protection conferred by ischemic preconditioning and its possible mediators in animal studies is translatable to humans. It is important to note, however, that classic or early ischemic preconditioning observed in animals is associated with a reduction in infarct size, but not protection against stunning, and that many of the clinical studies are either retrospective in nature or have used surrogate markers of injury as end points.
With respect to a role for IPC during cardiac surgery, numerous small trials have been conducted.143 One of the first studies was conducted by Yellon and colleagues in patients undergoing CABG surgery.144 Patients were subjected to a protocol that involved two cycles of 3 minutes of global ischemia. The aorta was cross-clamped intermittently and the heart was paced at 90 beats per minute to induce ischemia. This was followed by 2 minutes of reperfusion before a 10-minute period of global ischemia and ventricular fibrillation. Myocardial biopsies were obtained during the 10-minute period of global ischemia, and ATP tissue content was measured. The results showed that the ATP levels in the biopsies obtained from patients subjected to the preconditioning-like protocol were higher. However, because ATP content is not a marker of necrosis, a follow-up study was performed, and troponin T serum levels were measured. In this study, the investigators reported that troponin release was attenuated in the patients subjected to the preconditioning protocol. In 2002, Teoh and colleagues reported that IPC conferred myocardial protection beyond that provided by intermittent cross-clamp fibrillation in patients undergoing CABG.79 Other investigators have reported similar findings.
Thus, a number of cardiac surgical studies suggest IPC may be effective in the setting of aortic cross-clamping and administration of cardioplegia. It is important to note, however, that to date the total number of patients studied has been relatively small, and the outcomes have been limited to surrogate markers of myocardial necrosis, viz., CK-MB levels and troponin release, and not clinical endpoints. This explains in part why IPC has not been adopted as an adjuvant approach among the myocardial protection techniques employed to date. A more promising strategy may be to develop a better understanding of the intracellular events and effectors that confer protection and then design the appropriate pharmacologic agent to mimic the phenomenon.
The phenomenon of ischemic postconditioning (PostCond) was first reported by Zhao et al. in the canine model.145 The term refers to rapid intermittent interruptions of blood flow in the early phase of reperfusion, ie, relief of ischemia in a stuttered or staccato manner. Although the cellular mechanisms underlying PostCond are poorly defined, they appear to involve many of the same signal transduction pathways that are involved in IPC, including cell surface receptor signaling, pro-survival kinases, the mPTP, and the mKATP channel. Although the duration and frequency of reperfusion may be variable, for the most part, the cycles that induce PostCond are measured in seconds in smaller species, and slightly longer in larger animals and humans, justifying the name stuttering reperfusion. The reduction in infarct size appears to be comparable with that observed with IPC. Preclinical studies conducted in multiple models and species (including dog, rat, rabbit, mouse, and pig) have demonstrated a reduction in infarct size that ranges from 20 to 70%. The restoration of blood flow in a stuttering manner during early reperfusion is of major interest to clinicians because it holds particular promise for patients presenting with an acute MI. In the surgical setting, PostCond could be applied in the operating room after release of the aortic cross-clamp.
Evidence that PostCond exists in humans was first reported in patients undergoing PCI. Patients receiving brief balloon inflations/deflations in the initial minutes of reperfusion during PCI exhibited smaller ST-segment changes and lower levels of total creatine kinase release compared with patients that were not subjected to stuttering reperfusion. More recently, Darling et al. conducted a retrospective chart review in patients undergoing emergent cardiac catheterization for ST-segment elevation MI (STEMI).146 The hypothesis was that outcome would be better in patients undergoing multiple balloon inflations after primary angioplasty. Patients were divided into two cohorts: those who, at the discretion of the interventional cardiologist, received one to three balloon inflations, and those in whom four or more inflations were applied. In this retrospective analysis, peak CK release was less in patients requiring ≥four inflations. In a separate study by Lønborg et al., the cardioprotective effects of PostCond in patients treated with PCI was evaluated using MRI.147 These investigators reported that mechanical PostCond appeared to be independent of the size of myocardium at risk. The findings were consistent with the concept that stuttering reperfusion confers cardioprotection during percutaneous interventions.
In the context of heart surgery, Luo reported a beneficial effect of surgical PostCond in 24 patients undergoing repair for Tetralogy of Fallot (TOF) at the time of aortic declamping. The postconditioning protocol consisted of aortic reclamping for 30 seconds and declamping for 30 seconds. The process was repeated twice. The intervention was reported to reduce perioperative troponin T and CK-MB release and decreased the need for inotropic support after surgery.148 A similar finding was reported by the same investigator in a study of adult patients undergoing valve surgery and children undergoing corrective surgery using cardioplegia.
Thus, there is evidence that a PostCond protocol may be of benefit to patients undergoing heart surgery. Although PostCond may offer more promise than IPC in terms of clinical application, it is important to note that both are invasive in nature. Ultimately, the elucidation of mechanisms underlying PostCond may hold the most promise for development of new therapeutic approaches to cardioprotection.
Remote Ischemic Preconditioning
Remote ischemic preconditioning is a phenomenon whereby brief ischemia of one organ or tissue confers protection on a distant naive organ or tissue against a sustained ischemia-reperfusion injury. Remote ischemic preconditioning (RIPC) was first described by Przyklenk and colleagues in 1993.149 In the original study, the investigators questioned whether IPC protected only the heart cells exposed to brief coronary artery occlusions or was it possible for repetitive or stuttering occlusions in a remote naive vascular bed to reduce infarct size in the area subjected to prolonged ischemia. They used a canine preparation in which a branch of the circumflex coronary artery was subjected to four episodes of 5-minute occlusion and reperfusion; this was followed by 1 hour occlusion of the left anterior descending (LAD) coronary artery. After 4.5 hours of reflow, infarct size in the distribution of the LAD was measured. A marked reduction in infarct size was observed. Since then, numerous other investigators have confirmed these findings, and the phenomenon has been observed in various species and with different organs. Brief occlusions of the renal and mesenteric arteries and brief restriction of blood flow to the skeletal muscle of the lower limb have been shown to reduce myocardial infarct size by up to 65%.150 As a consequence, RIPC is now also referred to as interorgan preconditioning.
Not surprisingly, multiple mechanisms have been implicated to play a role in triggering and mediating remote preconditioning, including both humoral factors (such as adenosine, bradykinin, and calcitonin gene-related peptide), and neuronal factors, followed by activation of one or more kinases (including p38MAPK, ERK1/2 and JNK). To date the mechanism(s) underlying RIPC remain largely speculative. As with many other cardioprotective interventions, lack of a clear understanding of the molecular basis of the phenomenon has not dampened enthusiasm to apply the approach clinically as a possible new approach to cardioprotection in humans.
One of the first studies involving patients was conducted in 17 children undergoing congenital heart surgery with cardiopulmonary bypass.151 Brief intermittent lower limb ischemia was associated with attenuated troponin release and a reduction in the need for postoperative inotropic support. Other investigators have reported similar findings in patients undergoing both adult heart surgery and resection of abdominal aortic aneurysms. For example, in one study involving 23 adult patients undergoing on-pump CABG surgery in which cold blood cardioplegia was used, RIPC was associated with a 42% reduction in total troponin T release. RIPC was induced by three 5-minute cycles of right forearm ischemia by inflating a blood pressure cuff on the upper arm to 200 mm Hg with an intervening 5-minute reperfusion period. The control group had a deflated cuff placed on the upper arm for 30 minutes. The outcome was similar to another study by Hausenloy et al., in which 57 patients undergoing elective CABG surgery were randomly assigned to RIPC or control groups. During the CABG operation, either intermittent cross-clamping or cardioplegia was used. RIPC was associated with a reduction in perioperative troponin T release by 43%.152 Whether this mode of cardioprotection will become a standard of care will depend on whether or not RIPC can be shown to have a salutary effect on more robust clinical endpoints such as perioperative MI, stroke, and death.
Autophagy is the process whereby a double-membrane structure called the autophagosome sequesters cytoplasmic components such as ubiquitinated protein aggregates or organelles including mitochondria, peroxisomes, and endoplasmic reticulum. It is involved in degradation of long-lived proteins and the removal of excess or damaged organelles. The outer autophagosomal membrane fuses with a lysosomal membrane to deliver its contents into an autophagolysosome where the cargo is degraded by lysosomal hydrolases and the resulting macromolecules recycled153 (Fig. 15-5).
Cellular process of autophagy. Autophagy is a dynamic adaptive process in the setting of I/R injury. The process involves the synthesis of a cup-shaped pre-autophagosomal double-membrane structure that surrounds cytoplasmic material and closes to form an autophagosome. This process is regulated by the autophagy proteins Atg 4, Atg7, LC3, and the complex of Atg12-Atg5-Atg16L. The process is activated by a number of stimuli including ROS or RNS. Induction by Beclin1 and Vps34 in conjunction with other Atg proteins results in the formation of an isolation membrane to which Atg proteins are recruited. Atg12-Atg5 and LC3 proteins are involved in the expansion of the membrane. This allows the phagophore to surround and engulf damaged organelles or protein aggregates that may accumulate as a result of I/R injury. The result is the formation of an autophagosome. The green insert shows autophagosomes (green puncta). This photo was obtained in a cell expressing a fusion protein of green fluorescent protein (GFP) fused to the N-terminus of LC3; the GFP-LC3 was incorporated into the double membrane structure of the phagophore. Wortmannin and 3 MA are agents that can inhibit the initiation phase of autophagy; bafilomycin and chloroquine can inhibit the degradation phase. AMPK = AMP-activated protein kinase; Atg1,Atg4, Atg7, Atg12, Atg16L = autophagy regulating proteins; I/R = ischemia/reperfusion; LC3 = light chain 3; mTOR = mammalian target of rapamycin; PIP3 = phosphatidylinositol 3,4,5-trisphosphate; 3MA = 3-methyladenine; RNS = reactive nitrogen species; ROS = reactive oxygen species; Vps34 = a class III PI3 kinase involved in vesicular trafficking, nutrient signaling, and autophagy. (Adapted from Gottlieb RA, Finley KD, Mentzer RM Jr: Cardioprotection requires taking out the trash. Basic Res Cardiol 2009; 104:169-180.)
One of the first studies to suggest autophagy is an adaptive process responsive to stress in the heart was the report by Decker et al. in which they described an association between the formation of autophagosomes and an increase in degenerating mitochondria in rabbit hearts exposed to hypoxia and reperfusion. Reperfusion restored contractility and injured myocytes underwent a cellular repair process that involved a marked increase in lysosomal autophagy. These investigators concluded that this process is important in the efforts to repair cardiac cells during and after hypoxia.154
Autophagy has now been reported to be upregulated in isolated cells subjected to simulated ischemia and reperfusion and rodent models of ex vivo and in vivo ischemia reperfusion injury. Shimomura showed that upregulation of autophagy in HL-1 myocytes protected against cell death induced by simulated ischemia reperfusion (sI/R) whereas inhibition of autophagy enhanced cell death.155 Dosenko et al. was one of the first to report a more direct linkage between autophagy and cytoprotection and observed that autophagy may have a protective effect during anoxia-reoxygenation.156 Subsequently, upregulation of autophagy induced by sI/R in HL-1 myocytes was reported to be protective, whereas inhibition of autophagy enhanced cell death. Hamacher-Brady and colleagues subjected HL-1 cells to sI/R as an in vitro model of ischemia reperfusion. Using three-dimensional high-resolution fluorescence imaging, they analyzed the autophagic response to sI/R. They observed that autophagy is an important underlying protective response against sI/R injury.157 Matsui et al. found that glucose deprivation increased the number of autophagosomes in neonatal cardiac myocytes, and that inhibiting autophagy with 3-methyladenine (3-MA) enhanced cell death induced by glucose deprivation.158 Yan et al. reported that cardiac myocytes with enhanced autophagy were negative for apoptosis, whereas apoptotic cells were negative for autophagy in a porcine model of chronic myocardial ischemia and hibernating myocardium.159
Gurusamy et al. investigated the role of autophagy during ischemia-reperfusion injury and reported that increased BAG-1 expression in the heart correlated with the onset of protection in an in vivo model of myocardial stunning.160 Collectively, these studies suggest that upregulation of autophagy promotes survival during stress such as ischemia-reperfusion.
There is now direct evidence that autophagy plays an important role in mediating ischemic and pharmacologic preconditioning. Yitzhaki et al. investigated the effect of the adenosine preconditioning agent 2-chloro-N(6)- cyclopentyladenosine (CCPA) on autophagy and cell survival after sI/R and GFP-LC3 infected HL-1 cells and neonatal rat cardiomyocytes. Autophagy was induced within 10 minutes of adenosine treatment and increased autophagy was evident when examined 24 hours later. Inhibition of autophagy resulted in significant loss of both immediate and delayed cytoprotection against sI/R as measured by release of lactate dehydrogenase. To assess autophagy in vivo, transgenic mice expressing the red fluorescent autophagy marker mCherry-LC3 were treated with CCPA. Treated hearts revealed a large increase in the number of autophagosomes. Subsequent ex vivo and in vivo studies examining the role of autophagy in pharmacologic and ischemic preconditioning have provided strong evidence that autophagy is required for the protective effect. Based on these findings, it appears that autophagy may serve as an important mediator of protection of the preconditioning agent CCPA.161 A more detailed understanding of the role of autophagy in myocardial protection may lead to a new therapeutic approach to the management and treatment of ischemia-reperfusion injury.
Considerable progress has been made towards limiting myocardial ischemia-reperfusion injury in the setting of global ischemia associated with surgical procedures. Despite considerable efforts to translate promising experimental results into clinical therapies, interventions to date have yielded few successes with the exceptions of adenosine and glucose-insulin-potassium. These disappointments led the National Heart Lung and Blood Institute to convene a working group to discuss the reasons for the failure to translate potential therapies for protecting the heart from ischemia and reperfusion. The working group concluded that cardioprotection in the setting of acute myocardial infarction, cardiac surgery, and cardiac arrest requires more relevant animal models corresponding to the human conditions of atherosclerosis, hypercholesterolemia, hypertension, diabetes, and advanced age, all of which are increasingly recognized to interfere with cardioprotective strategies.162 At the same time, a number of recent studies provided a sense of optimism regarding the encouraging clinical data for the following cardioprotective interventions.
There is considerable experimental evidence that activation of various adenosine receptor subtypes results in cardioprotection similar to that induced by IPC. Preischemic administration of the nucleoside adenosine retards the rate of ischemia-induced ATP depletion, prolongs the time to onset of ischemic contracture, attenuates myocardial stunning, enhances postischemic myocardial energetics, and reduces infarct size.163
There are at least four distinct adenosine receptor subtypes, which are designated A1, A2a, A2b, and A3. They couple to a variety of guanine nucleotide-binding (G) proteins (Go, Giα2, Giα3, Gq, and Gs) depending on the receptor subtype and tissue studied. There is definitive evidence that two, and possibly three, of these receptors are expressed in the adult human heart. Recent preclinical reports suggest that an adenosine A2b receptor agonist confers cardioprotection when administered before the onset of ischemia (preconditioning) and at reperfusion (postconditioning).164
With respect to cardiac surgery, there have been a limited number of clinical trials performed. Fremes and colleagues reported the results of an open-label, nonrandomized CABG surgery study in which adenosine administration was combined with antegrade warm blood cardioplegia. The adenosine concentrations studied were 15, 20, and 25 mol/L. These investigators reported that adenosine could be added safely as a supplement to cardioplegic solutions, but it had no effect on myocardial function at the doses studied.165
Cohen and colleagues observed a similar lack of efficacy in a phase II double-blind, placebo-controlled trial performed in patients undergoing CABG surgery. Patients were treated with placebo (saline) or warm blood cardioplegia supplemented with 15, 50, or 100 μM adenosine. These investigators found that adenosine had no effect on survival, the incidence of MI or the incidence of low cardiac output syndrome. A limitation of this study was the use of low concentrations of adenosine in the setting of warm blood cardioplegia. The nucleoside is metabolized rapidly to inosine and hypoxanthine, and the half-life in blood is measured in seconds, thus limiting its potential effect.166
Mentzer and colleagues reported a beneficial effect in an open-label, single-center study in which the safety, tolerance, and efficacy of high doses of adenosine were studied when added to cold blood cardioplegia in CABG surgery patients. Sixty one patients were randomized to receive standard cold blood cardioplegia or cold blood cardioplegia containing one of five adenosine doses ranging between 100 μM and 2 mM. Invasive and noninvasive studies of myocardial function were obtained sequentially after bypass. Parameters included the recording of inotropic utilization rates for the postoperative treatment of low cardiac output. Blood samples were collected for the measurement of nucleoside levels. High-dose adenosine treatment was associated with a 249-fold increase in the plasma adenosine concentration and was associated with a reduction in postbypass inotropic drug utilization, improved regional wall motion, and global function measured by transthoracic echocardiography.167
Subsequently, Mentzer and colleagues examined the effects of high-dose adenosine treatment in 253 patients randomized to one of three treatment arms. This was a double-blind, placebo-controlled multicenter trial using cold blood cardioplegia; adenosine was administered in three different concentrations and rates. Invasive and noninvasive measurements of ventricular performance were obtained before, during, and after surgery. The results of this study revealed a trend toward a decrease in high-dose inotropic agent utilization rates and a lower incidence of perioperative MI. A posthoc composite outcome analysis revealed that patients who received the high-dose adenosine were less likely to experience high-dose dopamine and epinephrine use, insertion of an intra aortic balloon pump, MI, or death.168
In summary, there is preclinical and clinical evidence that adenosine is a cardioprotective agent. Its clinical use is limited because large doses are associated with marked hypotension in patients not on cardiopulmonary bypass. The identification of a selective adenosine receptor subtype, eg, an adenosine A2b agonist that confers protection without systemic hypotension could lead to the development of a drug that is effective in preventing perioperative MI and stunning similar to that observed with late-phase preconditioning.
This agent is a member of a class of drugs referred to as adenosine regulating agents. It is a purine nucleoside analog that purportedly raises adenosine tissue levels selectively during ischemic conditions.169 The mechanism by which this agent regulates adenosine levels during ATP catabolism has not been fully established. Early preclinical studies have indicated that acadesine treatment: (1) improves left ventricular wall motion after intermittent ischemia, (2) attenuates frequency of ventricular arrhythmias, and (3) attenuates myocardial stunning and preserves myocardial function after cardiac arrest and cold cardioplegia. The observations led to five large-scale clinical trials in CABG surgery patients in the 1990s. The findings, however, were inconclusive due in part to the fact they were powered to detect only effect sizes of 50% or more.
As a consequence, Mangano combined the data from all five trials and performed a meta-analysis approach. The entire clinical experience of acadesine in more than 4000 CABG patients were analyzed to determine the effects of this agent on prespecified perioperative outcomes of MI, stroke, and cardiac death. The results of the meta-analysis indicated that acadesine given intravenously before and during surgery along with a fixed concentration in cardioplegia solution was effective in reducing perioperative MI, cardiac death, and combined adverse cardiovascular outcomes.170
Subsequently, Mangano et al. examined the two-year all-cause mortality after perioperative MI in a follow-up of the Acadesine 1024 Trial. Although the primary outcome was negative, the findings at two years among patients experiencing postreperfusion MI indicated a significant four-fold reduction in mortality (15/54 patients versus 3/46 patients).171 Based on these findings, a large scale Phase III study was initiated in 2010 (RED CABG). This study was stopped after 30% enrollment, however, due to futility. Whether the apparent lack of efficacy was due to clinical trial design, the characteristics of this class of drugs, or properties unique to agent itself, is unknown. It is important to note, however, that the salutary effects of this agent may not be apparent immediately after the operation.
Sodium-Hydrogen Exchanger Inhibitors
The sodium-hydrogen exchangers (NHEs) are a family of membrane proteins with nine isoforms that are involved in the transport of hydrogen ions in exchange for sodium ions. NHE-1 is the isoform that is expressed in the heart and may play a minor role in the normal excitation-contraction coupling process; however, it has been implicated in the etiology of arrhythmias, stunning, apoptosis, necrosis associated with acute myocardial ischemia-reperfusion injury, postinfarction ventricular remodeling, and heart failure.
The driving forces for Na+/H+ exchange are the relative transmembrane N+ and H+ gradients. The activity of the exchanger is regulated by the interaction of the H+ with a sensor site on the exchanger protein and can be additionally modulated by phosphorylation.172 During ischemia, cytoplasmic pH falls as low as 6.6 because of increased production of H+ from anaerobic glycolysis, but upon reperfusion, NHE-1 is activated to restore intracellular pH by exchange of intracellular protons for extracellular sodium. The resulting accumulation of intracellular Na+ is further exacerbated by diminished activity of Na+/K+ ATPase as a result of ischemia and lowered ATP availability. The increased intracellular Na+ competes for sites on the Na+/Ca++ exchanger and can actually drive it to run in reverse, resulting in cytosolic calcium overload. As noted earlier, calcium overload has numerous adverse consequences including activation of calcium-dependent proteases and phospholipases, gap junction dysfunction, and triggering of the mPTP culminating in membrane rupture and cell death (see Fig. 15-1).
The EXPEDITION trial was initiated to address the safety and efficacy of NHE-1 inhibition by cariporide in the prevention of death or MI in patients undergoing CABG surgery. High-risk CABG patients (n = 5,770) were randomized to receive either intravenous cariporide or placebo. The composite endpoint was assessed at 5 days, and patients were followed for up to 6 months. The results revealed an 18.3% relative risk reduction (RRR) in the incidence of death or MI at 5 days (p = 0 .0002). The RRR for death or MI at day 30 and month 6 was 16.1% (p = 0.0009) and 15.7% (p = 0.0006), respectively. When analyzed separately, the RRR in the incidence of MI alone at 5 days was 23.8% (p = 0.000005) and at month 6 was 25.6% (p = 0.000001). The mortality rate, however, increased at 5 days from 1.5% in the placebo group to 2.2% in the group with cariporide. This was associated with an overall increase in the incidence of cerebrovascular events. Thus, although cariporide treatment was effective in reducing the incidence of nonfatal MI, its efficacy was associated with toxicity, and the overall assessment of benefits and risks associated with cariporide indicated that the imbalances in the safety profile outweighed the reduction in the observed MI rate. Thus, it is unlikely that cariporide will be used clinically. The importance of the study, however, is that myocardial necrosis after CABG is higher than previously appreciated and it suggested NHE-1 inhibitors represent a new class of drugs that hold promise for reduction of myocardial infarction associated with ischemia-reperfusion injury.81
There are numerous studies that suggest glucose-insulin-potassium (GIK) infusions are effective in reducing perioperative MIs, postischemic myocardial dysfunction, and atrial fibrillation in patients undergoing heart surgery.173 The rationale for this form of treatment is based on the concept that insulin stimulates potassium reuptake through stimulation of the Na+,K+-ATPase while it stimulates glucose uptake for glycolytic energy production. High glucose, insulin, and an increased glycolytic flux also increase pyruvate generation and in turn preserve the citric acid cycle. Additionally, glycolytic ATP protects membranes, drives uptake of Ca2+ by the sarcoplasmic reticulum, and improves sodium homeostasis of ischemic myocardium.
Despite a strong rationale for its application in surgery, the efficacy of GIK in heart surgery remains controversial. This is due in part to the mixed results with the use of GIK to treat patients with acute myocardial infarction. In 1997, Fath-Ordoubadi and Beatt showed a benefit of treatment in a meta-analysis of patients who received GIK reperfusion therapy for acute myocardial infarction.174 Van der Horst reported a benefit of GIK in 940 randomized STEMI patients treated by PCI. It was only in the subgroup of 156 patients without heart failure, however, that a significant reduction in mortality was seen.175 In a follow-up report, Timmer et al. were unable to demonstrate a therapeutic benefit in patients without signs of heart failure. Thus, a role for GIK in the treatment of acute myocardial infarction (STEMI) is yet to be determined.176
With respect to cardiac surgery, Lazar et al. conducted a study to determine whether tight perioperative glycemic control in diabetic CABG patients with a modified GIK solution would optimize myocardial metabolism and improve perioperative outcomes. Patients (n = 141) were randomized to receive GIK or no GIK. The preoperative patient profiles were similar in age, sex, ejection fraction, urgency of surgery, or the type of diabetes. Although the 30-day survival was comparable in both groups, the GIK-treated patients had significantly higher cardiac indices and less need for inotropic support. There was also a lower incidence of atrial fibrillation. Follow-up data two years after surgery were available in 60 of 70 (83.3% GIK) and 60 of 69 (86.9% GIK) patients. Survival was better in the patients who received GIK; the investigators attributed this to long-lasting benefits from perioperative GIK treatment.177
In another single-center study, Quinn et al. reported their results with a prospective, randomized, double-blind, placebo-controlled trial in 280 nondiabetic CABG surgery patients. They found that GIK treatment was associated with fewer episodes of low cardiac output, less inotropic support postoperatively, and a reduction in serum cardiac troponin I levels. The authors concluded that GIK is an effective, inexpensive, and safe adjunctive cardioprotective technique.80
In contrast, S. Bruemmer-Smith et al. reported that GIK infusion during surgery failed to reduce myocardial cellular damage as measured by cTnI levels six hours after cardiopulmonary bypass. It was associated with increased hyperglycemia. In this study, only 42 patients were enrolled although it was a randomized, prospective, double-blinded study in patients undergoing elective CABG surgery. The patients received either GIK or placebo administered via a central line. The groups were well-matched for aged and number of bypassed vessels.178 In another study, Lell et al. reported on 46 patients undergoing elective off-pump CABG surgery; the patients received either normal saline or a GIK infusion for 12 hours. These investigators were unable to demonstrate a beneficial effect on cardiac performance using the clinical measurements of cardiac index and inotropic requirements. They did report the finding of a persistent hyperglycemia, however, despite the use of supplemental insulin.179 Finally, Barcellos et al. reported their experience with 24 patients with type-2 diabetes mellitus who underwent CABG surgery. Patients were administered GIK or subcutaneous insulin from the onset of anesthesia until 12 hours postoperatively. The use of GIK neither improved the cardiac index nor reduced the use inotropic agents albeit it did improve glucose control.180
Thus, the evidence supporting the efficacy of GIK in the setting of heart surgery is still controversial. Although a meta-analysis of randomized studies using GIK suggests that it may improve postoperative recovery of contractile function and reduce atrial arrhythmias, the individual studies have involved small numbers of patients and, therefore, insufficiently powered to detect efficacy. Until a large randomized, multicenter clinical trial has been performed, the use of GIK as a form of myocardial protection will remain controversial.