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Since the initial description of the phenomenon by Jennings et al 50 years ago, our understanding of the underlying mechanisms of reperfusion injury has grown significantly. Its pathogenesis reflects the confluence of multiple pathways, including ion channels, reactive oxygen species, inflammation, and endothelial dysfunction. The purposes of this review are to examine the current state of understanding of ischemia-reperfusion injury, as well as to highlight recent interventions aimed at this heretofore elusive target. In conclusion, despite its complexity our ongoing efforts to mitigate this form of injury should not be deterred, because nearly 2 million patients annually undergo either spontaneous (in the form of acute myocardial infarction) or iatrogenic (in the context of cardioplegic arrest) ischemia-reperfusion.
Each year in the United States, there are approximately 1 million myocardial infarctions (MI), and 700,000 patients undergo cardioplegic arrest for various cardiac surgeries. Minimizing ischemic time in these clinical scenarios has appropriately received a great deal of attention because of the long-established relation between the duration of ischemia and the extent of myocardial injury. Once coronary flow is restored, however, the myocardium is susceptible to another form of insult stemming from reperfusion of the previously ischemic tissue. Given that cardiac ischemia is either unpredictable (MI) or inevitable (in the operating room), there is great interest in developing strategies to minimize reperfusion-mediated injury.
Historical Perspective
The seminal observation that reperfusion after ischemia was associated with myocardial injury was made in 1960 by Jennings et al. Their report was based on experiments with canine hearts subjected to coronary ligation in which reperfusion appeared to accelerate the development of necrosis. For example, those investigators noted that the histologic changes seen after only 30 to 60 minutes of ischemia-reperfusion (IR) were comparable to the degree of necrosis normally seen after 24 hours of permanent coronary occlusion.
Whether reperfusion is independently responsible for tissue injury or simply hastens the demise of cells otherwise destined for necrosis remained a matter of debate for some years. Evidence for direct myocardial reperfusion–dependent injury was summarized in 1985 in the classic editorial by Braunwald and Kloner. However, it was not until the discovery of ischemic preconditioning that the independent effects of ischemia and reperfusion began to be unraveled from each other.
In 1986, Murry et al described a process whereby repetitive short bouts of ischemia preceding prolonged periods of ischemia with reperfusion resulted in significantly decreased infarct sizes in dogs. Subsequently, this “ischemic preconditioning” was confirmed in a number of animal models, including humans, highlighting it as an evolutionarily conserved mechanism. Subsequent experiments revealed that the reperfusion event is key to the initiation of a molecular cascade leading to cardioprotection, thereby serving to solidify the important distinction between ischemia and subsequent reperfusion.
Mechanisms of Ischemia-Reperfusion Injury
Molecular and cellular events underlying IR injury are complex, representing the confluence of divergent biologic pathways. Furthermore, the extent to which each of these pathways is relevant to human disease remains unclear, as animal models do not always faithfully recapitulate the IR disease process in humans. These limitations notwithstanding, several key pathophysiologic features of clinically relevant IR have emerged.
Ischemia induces the accumulation of intracellular sodium, hydrogen, and calcium ions, culminating in tissue acidosis. Reperfusion, in turn, elicits rapid alterations in ion flux, and some evidence suggests that rapid renormalization of pH paradoxically leads to enhanced cytotoxicity. Sodium-dependent pH regulatory mechanisms, including the Na + -H + exchanger and the Na + -HCO 3 − transporter, are activated, which consequently leads to intracellular sodium accumulation. High sodium concentrations, in turn, drive increases in sarcoplasmic reticular Ca 2+ by the Na + -Ca 2+ exchange. Enhanced Ca 2+ entry by sarcolemmal L-type Ca 2+ channels and a deficient import of cytosolic Ca 2+ into the sarcoplasmic reticulum by sarcoplasmic/endoplasmic reticulum Ca 2+ –adenosine triphosphatase further promote Ca 2+ overload. The result is myofibrillar hypercontractility, adenosine triphosphate depletion, ultrastructural damage to mitochondria, and myocardial stunning.
Cardiac myocytes consume large quantities of energy. To accommodate this requirement, these cells host a high density of mitochondria. Thus, it is not surprising that these complex, energy-generating organelles, filled with reactive intermediates and proapoptotic signals, are intimately involved in IR injury. As part of this, the mitochondrial permeability transition pore (mPTP) has been the center of a growing amount of attention. The inner mitochondrial membrane, responsible for maintaining mitochondrial transmembrane potential, is normally impermeable to ions and proteins. Dissipation of the electrical potential across this membrane is termed “permeability transition,” a process thought to be mediated through the mPTP. Although the constituent protein components of the pore remain unknown, formation of the pore creates a nonselective channel between the inner membrane of the mitochondrion and the sarcoplasm. This results in loss of the electrochemical gradient, the release of reactive oxygen species (ROS), and apoptosome formation. Triggers for mPTP include Ca 2+ overload, rapid normalization of pH, and oxidative stress.
The generation of free radicals through incomplete reduction of oxygen during IR has been well described. These oxygen species are highly reactive and can quickly overwhelm a cell’s endogenous free radical scavenging system. This in turn triggers cellular injury by reactions with lipids, proteins, and nucleic acids. The enzyme xanthine oxidase has been particularly implicated as a generator of free radicals in the reperfused heart, as its substrates (xanthine and hypoxanthine) accumulate during ischemia. In addition to damaging nuclear and cytosolic elements, ROS can trigger the opening of the mPTP. This results in a positive feedback loop of additional free radical release from the mitochondria (“ROS-induced ROS release”).
Not only is IR injury dependent on events occurring within cardiomyocytes, but the endothelium is an active participant as well. The endothelium is the major source of the evanescent molecule, nitric oxide (NO). Under normal conditions, NO generation elicits vasodilation, which has beneficial, protective effects during IR, likely by influencing oxygen consumption, platelet aggregation, leukocyte adhesion, and free radical scavenging. Paradoxically, in high concentrations, NO may potentiate ROS-mediated toxicity by promoting the formation of highly reactive species, such as peroxynitrite. Beyond NO, the coronary endothelium has several other pathophysiologic roles in IR, such as serving as a source of vasoactive substances and by activating the immune system through expression of cytokines, chemokines, and adhesion molecules.
Recent work has implicated autophagy, an evolutionarily ancient mechanism of controlled cellular cannibalism, in the pathogenesis of IR. Time will tell whether this mechanism is a suitable target for therapeutic manipulation in this and other heart disease–related contexts.
Endothelial activation and injury increase vascular permeability and recruitment of inflammatory cells. Cellular adhesion molecules elicited by the injured endothelium (e.g., intercellular cell adhesion molecule–1, vascular cell adhesion molecule–1, E-selectin) promote tissue invasion by inflammatory cells. These infiltrating cells, including (and in particular) neutrophils, are directly toxic to the myocardium by secreting proteases, generating ROS, and occluding the microvasculature. Other components of the innate immune system, such as Toll-like receptors, mannose-binding lectin, and the complement cascade, also appear to participate in the pathogenesis of IR stress. Additionally, there is a growing appreciation of the role of cell-mediated immunity (i.e., T-cells and macrophages) in the pathogenesis of myocardial damage after reperfusion.
Mechanisms of Ischemia-Reperfusion Injury
Molecular and cellular events underlying IR injury are complex, representing the confluence of divergent biologic pathways. Furthermore, the extent to which each of these pathways is relevant to human disease remains unclear, as animal models do not always faithfully recapitulate the IR disease process in humans. These limitations notwithstanding, several key pathophysiologic features of clinically relevant IR have emerged.
Ischemia induces the accumulation of intracellular sodium, hydrogen, and calcium ions, culminating in tissue acidosis. Reperfusion, in turn, elicits rapid alterations in ion flux, and some evidence suggests that rapid renormalization of pH paradoxically leads to enhanced cytotoxicity. Sodium-dependent pH regulatory mechanisms, including the Na + -H + exchanger and the Na + -HCO 3 − transporter, are activated, which consequently leads to intracellular sodium accumulation. High sodium concentrations, in turn, drive increases in sarcoplasmic reticular Ca 2+ by the Na + -Ca 2+ exchange. Enhanced Ca 2+ entry by sarcolemmal L-type Ca 2+ channels and a deficient import of cytosolic Ca 2+ into the sarcoplasmic reticulum by sarcoplasmic/endoplasmic reticulum Ca 2+ –adenosine triphosphatase further promote Ca 2+ overload. The result is myofibrillar hypercontractility, adenosine triphosphate depletion, ultrastructural damage to mitochondria, and myocardial stunning.
Cardiac myocytes consume large quantities of energy. To accommodate this requirement, these cells host a high density of mitochondria. Thus, it is not surprising that these complex, energy-generating organelles, filled with reactive intermediates and proapoptotic signals, are intimately involved in IR injury. As part of this, the mitochondrial permeability transition pore (mPTP) has been the center of a growing amount of attention. The inner mitochondrial membrane, responsible for maintaining mitochondrial transmembrane potential, is normally impermeable to ions and proteins. Dissipation of the electrical potential across this membrane is termed “permeability transition,” a process thought to be mediated through the mPTP. Although the constituent protein components of the pore remain unknown, formation of the pore creates a nonselective channel between the inner membrane of the mitochondrion and the sarcoplasm. This results in loss of the electrochemical gradient, the release of reactive oxygen species (ROS), and apoptosome formation. Triggers for mPTP include Ca 2+ overload, rapid normalization of pH, and oxidative stress.
The generation of free radicals through incomplete reduction of oxygen during IR has been well described. These oxygen species are highly reactive and can quickly overwhelm a cell’s endogenous free radical scavenging system. This in turn triggers cellular injury by reactions with lipids, proteins, and nucleic acids. The enzyme xanthine oxidase has been particularly implicated as a generator of free radicals in the reperfused heart, as its substrates (xanthine and hypoxanthine) accumulate during ischemia. In addition to damaging nuclear and cytosolic elements, ROS can trigger the opening of the mPTP. This results in a positive feedback loop of additional free radical release from the mitochondria (“ROS-induced ROS release”).
Not only is IR injury dependent on events occurring within cardiomyocytes, but the endothelium is an active participant as well. The endothelium is the major source of the evanescent molecule, nitric oxide (NO). Under normal conditions, NO generation elicits vasodilation, which has beneficial, protective effects during IR, likely by influencing oxygen consumption, platelet aggregation, leukocyte adhesion, and free radical scavenging. Paradoxically, in high concentrations, NO may potentiate ROS-mediated toxicity by promoting the formation of highly reactive species, such as peroxynitrite. Beyond NO, the coronary endothelium has several other pathophysiologic roles in IR, such as serving as a source of vasoactive substances and by activating the immune system through expression of cytokines, chemokines, and adhesion molecules.
Recent work has implicated autophagy, an evolutionarily ancient mechanism of controlled cellular cannibalism, in the pathogenesis of IR. Time will tell whether this mechanism is a suitable target for therapeutic manipulation in this and other heart disease–related contexts.
Endothelial activation and injury increase vascular permeability and recruitment of inflammatory cells. Cellular adhesion molecules elicited by the injured endothelium (e.g., intercellular cell adhesion molecule–1, vascular cell adhesion molecule–1, E-selectin) promote tissue invasion by inflammatory cells. These infiltrating cells, including (and in particular) neutrophils, are directly toxic to the myocardium by secreting proteases, generating ROS, and occluding the microvasculature. Other components of the innate immune system, such as Toll-like receptors, mannose-binding lectin, and the complement cascade, also appear to participate in the pathogenesis of IR stress. Additionally, there is a growing appreciation of the role of cell-mediated immunity (i.e., T-cells and macrophages) in the pathogenesis of myocardial damage after reperfusion.
Ischemia-Reperfusion in Acute Myocardial Infarction
Although “reperfusion injury” in the most general sense refers to that component of the infarction process related to restoration of epicardial patency and anterograde blood flow, in the catheterization laboratory, “IR injury” is often synonymous with the “no-reflow” phenomenon. The term was first applied to myocardial ischemia after coronary ligation in dogs. Regarded as a dreaded complication of acute MI intervention, it is estimated to occur in >30% of cases and is associated with adverse prognosis. No reflow is thought to be related in part to microvascular plugging by vasoactive debris. Although dramatic, no reflow is probably just the most angiographically apparent form of IR injury in acute MI, and it should be recognized that significant reperfusion injury occurs even without the obvious “hang-up” of contrast dye.
Deciphering the contribution of IR to myocardial infarct size in humans is more challenging than in animal models. Acute MI in humans is generally associated with thrombotic occlusion of an epicardial coronary, and this prothrombotic and proinflammatory event is not well captured in models involving surgical ligation of the artery. This may be particularly important, as microvascular plugging with leukocytes and platelet “debris” has been implicated as an important component of the IR process. Another complexity relates to patient co-morbidities that influence the myocardial substrate during IR. Factors such as left ventricular hypertrophy, diabetes mellitus, and chronic ischemia preceding artery total occlusion (i.e., recapitulating ischemic preconditioning) potentially influence sensitivity to IR injury. The resulting heterogeneity in the human myocardial phenotype renders analyses of experimental models challenging and limits the degree to which the findings can be extrapolated to the human case. Finally, the emergent nature of most acute MIs (ST-segment elevation MIs in particular) makes it challenging, often both ethically and logistically, to study these patients, as issues arise of informed consent and seeming coercion during the race to achieve vessel patency. Nonetheless, these limitations have not precluded the testing of a series of therapeutic interventions in patients over the past 3 decades.
Therapeutic Interventions Targeting Ischemia-Reperfusion Injury in Acute Myocardial Infarction
Despite the substantial progress in understanding mechanisms of IR on the basis of models of acute MI, and the associated enthusiasm for translating these findings into patient care, the results of clinical studies have been largely disappointing. Whether this reflects our still incomplete understanding of the biology of IR or just a naive belief that a single intervention could be protective against a process involving multiple major pathophysiologic components is not clear. Initial pilot successes have been met with subsequent failures in larger confirmatory trials. The results of these trials have been summarized elsewhere, but interventions have included a spectrum of targets, including oxidant, inflammatory, sodium-hydrogen exchange, NO metabolism, and metabolic pathways.
Despite these setbacks, investigation continues in this field. Erythropoietin, for instance, is currently being investigated in clinical studies of acute MI, after the discovery of erythropoietin receptor expression in the myocardium. Erythropoietin has antiapoptotic activity and positive effects on remodeling, and it recruits endothelial progenitor cells. It is hoped that this drug may exert positive effects by more than 1 of these pathways. Other therapeutic strategies currently in clinical trials include the interleukin-1 receptor antagonist anakinra and glucagon-like peptide–1 analogues.