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Ann Thorac Surg 1995;60:760-766
© 1995 The Society of Thoracic Surgeons

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I: Pathophysiology of Ischemic-Reperfusion Injury

Free Radicals, Calcium Homeostasis, Heat Shock Proteins, and Myocardial Stunning

Division of Cardiology, Medical College of Virginia, Richmond, Virginia

Abstract

Repeated brief ischemic episodes result in prolonged depression of contractile function despite the absence of irreversible damage, a phenomenon called myocardial stunning. Considerable evidence exists to suggest that oxygen radicals, particularly the hydroxyl radical formed as a result of Fenton reaction or nitric oxide-peroxynitrite pathway, may contribute to the pathogenesis of myocardial stunning. The generation of free radicals may cause sarcoplasmic reticulum dysfunction, and both of these mechanisms may lead to calcium overload, which in turn could exacerbate the damage initiated by oxygen radicals. Antioxidant therapy has been shown to effectively attenuate or even prevent the development of prolonged depression of contractility in many studies. In addition, preconditioning with brief ischemic insults is able to trigger protection, which appears to attenuate stunning 24 to 48 hours later. The mechanism of this protection is not known, although one or more members of the heat shock protein family may have a role in protection against stunning.

Reperfusion of ischemic myocardium during the early phase of myocardial infarction has become the treatment of choice in the management of acute myocardial infarction. Extensive investigations in animals and humans have shown that infarct size can be reduced by very early reperfusion after coronary occlusion with the resultant preservation of ventricular function. Myocardial stunning is defined as mechanical dysfunction that persists after reperfusion of previously ischemic tissue in the absence of irreversible damage including myocardial necrosis. The duration of dysfunction greatly exceeds that of the antecedent ischemia [1]. After 15 minutes of ischemia in dogs, myocardial function remains depressed for 24 hours. Interventions such as inotropic agents can override stunning, and other interventions can prevent its occurrence. Mechanical stunning is associated with a constellation of other reversible derangements, including adenosine triphosphate depletion, collagen damage, cell swelling, increased capillary permeability, and impaired microvascular responsiveness [2]. It is unknown whether these defects have the same pathogenesis as the depression of contractility. This fully reversible injury can result from a variety of pathologic processes as well as iatrogenically from clinical procedures. Unstable angina or acute myocardial infarction with early reperfusion, cardiac operation with cardioplegic arrest, and cardiac transplantation all subject the myocardium to transient ischemia and therefore may be associated with myocardial stunning [3, 4].

Preconditioning and Stunning

These are two distinct entities: stunning is an unfavorable phenomenon, manifest during reperfusion, and is caused largely by events associated with the period of reperfusion where occurrence of myocardial stunning and accompanying contractile abnormalities delay the benefits of reperfusion therapy. Preconditioning is a protective phenomenon that is caused by events associated with an episode of ischemia/reperfusion, but is not manifest until a subsequent ischemic episode. Murry and associates [5] were the first ones to describe the phenomenon of preconditioning. They found that the heart could be protected by a series of four 5-minute coronary branch occlusions, each separated by 5 minutes of reperfusion. After preconditioning they exposed the dogs to 40 minutes of continuous occlusion to induce infarction. After 96 hours of reperfusion, the hearts were removed and the infarct size was determined histologically, and significantly smaller infarcts were noted in the hearts that were ``preconditioned'' as compared with control hearts. Protection was not the result of improved collateral flow, but rather represented a true improvement in the heart's ability to tolerate ischemia. Since then others have confirmed that ischemic preconditioning dramatically limits infarct size in dogs [6–8], pigs [9], rabbits [10], and rats [11]. Thus it appears that preconditioning is universally accepted as a powerful cardioprotectant.

The ability of preconditioning to prevent stunning is less clear, in part due to the fact that preconditioning itself causes some level of stunning. However, there are data to suggest that preconditioning may render the heart more resistant to stunning after a major ischemic insult. Cohen and Downey [12] exposed dog hearts to a series of 12 cycles of 5 minutes of coronary branch occlusion each followed by 10 minutes of reperfusion. It was found that function was very depressed during the first cycle's 10-minute reperfusion period. However, little additional deterioration of function occurred with subsequent cycles; thus it appeared that hearts had been protected against further stunning. On the other hand, Ovize and colleagues [13], using a classic model of myocardial stunning, demonstrated that preconditioning neither preserved contractile function during a 15-minute coronary occlusion nor attenuated myocardial stunning during the initial hours of reperfusion.

Oxygen Radicals and Stunning

Direct electron paramagnetic resonance measurement of free radical production using the spin trap phenyl N-tert-butyl nitrone confirms that free radicals are produced in the stunned myocardium [14, 15], that the univalent pathway of oxygen reduction is the source of free radicals, and that postischemic dysfunction occurs only when the free radicals are not inhibited [16, 17]. The hydroxyl radical is considered to be responsible for much of the ischemia/reperfusion injury; this radical species is known to be generated by the iron catalyzed Fenton's reaction or via Haber Weiss reaction:

2•O₂ ^- + 2H⁺ H₂O₂ + O₂

•O₂^- + Fe³⁺ O₂ + Fe²⁺

Fe²⁺ + H₂O₂ •OH + OH^- + Fe³⁺

Because the •OH radical is extremely unstable, it reacts at diffusion limited rates with the first molecule with which it comes in contact. Beckman's group [18] argued that contribution of •O₂^- to •OH by iron-catalyzed Haber Weiss reaction may be of limited significance in vivo and proposed another pathway, which involves interaction of •O₂^- anion and NO in aqueous solution (k - 3.7 x 10⁷ M^-1 s^-1) to produce the potentially cytotoxic substance peroxynitrite (OONO^-) [19]. The homolytic decay of OONO^- after protonation generates •OH radical and nitrogen dioxide:

NO+•O₂^- ONOO^- (peroxynitrite anion)

ONOO^- + H ONOOH (peroxynitrous acid)

ONOOH •OH + NO₂ (nitrogen dioxide)

It has been postulated [18] that peroxynitrite is considerably more toxic than extracellularly generated •OH radical. This reaction process ultimately produces nitrites and also nitrates. Peroxynitrous acid and nitrites have been shown to initiate the process of lipid peroxidation in vitro [19].

Matheis and colleagues [20] examined the role of •OH generated through peroxynitrite pathway in a hypoxic piglet on cardiopulmonary bypass. A significant decrease in systemic venous and coronary sinus blood content of NO was observed during hypoxia, which increased substantially above prehypoxic levels during reoxygenation on cardiopulmonary bypass. Administration of either the antioxidants mercaptopropionyl glycine and catalase or the nitric oxide synthase inhibitor N^G-nitro-L-arginine methyl ester to the extracorporeal circuit afforded similar and nearly complete protection against myocardial reoxygenation injury. Patel and associates [21] reported reduction in infarct size in a rabbit model of ischemia/reperfusion after treatment with N^G-nitro-L-arginine methyl ester. Because N^G-nitro-L-arginine methyl ester was given as a bolus in these studies, the protective effect was interpreted as similar to ischemic preconditioning. In a Langendorff isolated heart model of ischemia and reperfusion, Naseem and co-workers [22] noted a significant reduction of reperfusion-induced arrhythmia and preservation of myocardial contractility by sustained inhibition of NO generation with N^G-nitro-L-arginine. Thus it appears that NO may have beneficial as well as injurious effects on myocardial function after ischemia and reperfusion. The exact reason for this discrepancy is not clear, although it appears to be dependent on the model and end-point for the assessment of ischemia/reperfusion injury. The friendly role of NO is attributable to the preservation of endothelial function, inactivation of •O₂^- and attenuation in neutrophil accumulation after ischemia/reperfusion. The injurious effect of NO seems to be due to •OH radical generation via peroxynitrite pathway, although, to date, the production of peroxynitrous acid in vivo during ischemia/reperfusion has not been demonstrated.

Myers and associates [23] have presented considerable evidence supporting an oxygen free radical-mediated mechanism for myocardial stunning. Many of the agents shown to protect against other forms of reperfusion injuries due to free radical injury have also been used in this model. Experiments with open-chest dogs showed protection against myocardial stunning with superoxide dismutase (SOD) and catalase [23], dimethylthiourea [24], N-2-mercatopropionyl [25], and the iron chelator deferoxamine [26, 27]. Other investigators using anesthetized, open-chest dog models have also shown that stunning was attenuated by administering SOD and catalase [28, 29]. Experiments using a model of closed-chest, unsedated dogs subjected to 15 minutes of coronary occlusion confirmed that the protective effects of SOD and catalase were due to free radical scavenging and not to artifacts from anesthesia, operation, or other variables incurred with open-chest models [30]. In these studies, dogs underwent 15 minutes of left anterior descending coronary artery occlusion followed by 2 to 3 hours of reperfusion. Contractile function recovered to a mean of 60% to 70% of baseline values in the treated groups compared with 6% to 30% recovery in the untreated group. In other models, Ambrosio and colleagues [31] showed that when SOD-catalase was given at the time of reperfusion to a previously globally ischemic nonworking isolated rabbit heart, the left ventricular functional recovery was significantly improved. Furthermore, SOD and catalase given together have been shown to improve left ventricular function after reperfusion [23, 28, 29], but when SOD was used alone, no significant changes were noted [32].

Other agents able to attenuate experimental myocardial stunning include methylprednisolone [33], the calcium antagonists verapamil, nifedipine, and diltiazem [34–36], and the angiotensin-converting enzyme inhibitors captopril and zefenopril administered either before occlusion [37] or upon reperfusion [37, 38]. Similarly, glutathione has been shown to provide protection against myocardial dysfunction after short periods of ischemia in isolated rat hearts [39]. It is hypothesized that all of these agents act via an oxygen free radical-related mechanism.

In evaluating the likelihood that a compound acts as an antioxidant, it is essential to know whether the rate at which it reacts with biologically important reactive species would allow the compound to compete with biological molecules for such species in vivo. Therefore, for any one of these agents there is no clear consensus as to a true clinical benefit. Although several studies may demonstrate preservation of myocardial function by reducing the presence of reduced-oxygen intermediates, others will refute this. Further, there has never been a clinical trial in humans that has clearly and conclusively demonstrated a protective effect on myocardial function based on protection from free radicals. Since 1984 at least 40 studies of infarct size limitation by antioxidant therapy have been published, with 20 studies claiming reduction of infarct size and 20 failing to confirm this finding. Failure of SOD to limit infarct size has been attributed to several possibilities, including inappropriate dosing, the scheduling of SOD administration, or premature withdrawal during the reperfusion period.

Several sources of free radical generation including xanthine oxidase, catecholamine oxidation, cyclooxygenase, neutrophils, and mitochondria have been proposed. However, the relative importance of these in the stunned myocardium remains unclear. Although activated neutrophils have the ability to release reactive oxygen metabolites and contribute to contractile dysfunction, the evidence that the latter occurs in the absence of irreversible myocardial damage remains uncertain. Several studies have failed to find a role of the neutrophil in myocardial stunning [2, 40]. Methods used in these studies included filters or antiserum to deplete neutrophils, administration of inhibitors of leukotriene synthesis, and inhibiting leukocyte adhesion with dextran or antibodies to Mo1 glycoprotein [1, 2]. No intramyocardial accumulation of leukocytes is observed after 12 minutes of ischemia and 90 minutes of reperfusion [41]. These results were in sharp contrast to two studies using Leukopak filters in dogs, which prevented or reduced postischemic dysfunction after a 15-minute occlusion [42, 43]. Juneau and co-workers [44] recently demonstrated that severe neutrophil depletion achieved either by leukocyte filtration or cytotoxic drugs did not improve the postischemic recovery of function. Another notable observation by these authors was Leukopak filters caused persistent coronary vasodilation and showed antiarrhythmic properties. Thus it appears that neutrophils do not have any role in the pathogenesis of myocardial stunning.

Calcium Overload and Stunning

The mechanism(s) by which free radicals depress contractile function resulting in stunned myocardium are not fully understood. Weisel and associates [45] reported the release of conjugated dienes into the coronary sinus after cardioplegic arrest during bypass operations, and Romaschin and colleagues [46] noted a similar increase in conjugated dienes after global normothermic ischemia in dogs. Several studies [47–49] suggest that injury of the sarcoplasmic reticulum or the sarcolemma by oxygen free radicals, perhaps via lipid peroxidation, may be the mechanism(s) for impairment of contractile function. These injuries could result in uncoupling of excitation-contraction and in cellular calcium overload. It is, however, not clear how free radicals perturb calcium homeostasis. It is believed that glycolysis plays a favored role in maintaining transmembrane ion gradients in general [50, 51]. Krause and associates [52] reported that reperfusion with the glycolytic inhibitor iodoacetate prevented functional recovery and restoration of calcium homeostasis after 20 minutes of ischemia in rabbit hearts. Also, it is known that free radicals inactivate glyceraldehyde-3-phosphate dehydrogenase, the key glycolytic enzyme [53]. Thus it may be possible that inhibition of glycolysis and disruption of calcium homeostasis may have a cause-and-effect relationship when free radicals are generated during reperfusion. Corretti and colleagues [54] tested this hypothesis with simultaneous measurement of intracellular [Ca²⁺] and high-energy phosphates. They found that •OH radical caused inhibition of glycolytic enzymes and an increase in intracellular [Ca²⁺]. Thus they hypothesized that radicals inhibit glycolysis, glycolytic inhibition in turn leads to deranged Ca²⁺ homeostasis, and the resultant calcium overload produces dysfunction.

Several reports indicate that intracellular [Ca²⁺] increases during ischemia [55–59], remains elevated during early reperfusion [57] and returns to normal levels during late reperfusion without causing permanent damage [60]. Time-averaged measurements of [Ca²⁺] have shown that such an increase occurred as early as 10 to 15 minutes of total ischemia [57, 58, 61] and persisted during the early moments of reflow. Reperfusion with solutions of low [Ca²⁺] or induction of intracellular acidosis [62] improved functional recovery. Kitakaze and associates [63] reported that transient Ca²⁺ overload in the absence of ischemia mimicked stunning physiologically, metabolically, and histologically. Similarly, calcium overload induced by ventricular fibrillation also left behind contractile dysfunction [64]. Elevated [Ca²⁺] may cause activation of protein kinase [61, 65] and alter Ca²⁺ sensitivity or maximal Ca²⁺-activated force through phosphorylation of one or more of the contractile proteins.

Krause and associates [52] measured Ca²⁺ uptake and Ca²⁺-ATPase activity in sarcoplasmic reticulum vesicles from regionally stunned myocardium. The maximal oxalate-supported Ca²⁺ transport rate was reduced by 17%, paralleled by a similar decrease in maximal Ca²⁺-adenosine triphosphatase activity. The reduction in sarcoplasmic reticulum Ca²⁺ uptake was even greater when the free Mg²⁺ concentration was increased from 0.6 to 1.5 mmol/L [66] to simulate the recently detected elevation of free [Mg²⁺] in reperfused myocardium [67, 68]. The physiologic implications of the decrease in maximal Ca²⁺ uptake rate and in the Ca²⁺-adenosine triphosphatase activity are difficult to predict. Less Ca²⁺ sequestration would lead to an increase in cytoplasmic Ca²⁺ (if less is taken up by the sarcoplasmic reticulum, more remains in the cytosol). In the longer term, reduced Ca²⁺ sequestration by the sarcoplasmic reticulum means that less Ca²⁺ will be available to be released from the sarcoplasmic reticulum during each beat; thus Ca²⁺ transients might be reduced. The only parameter of excitation-contraction coupling that directly reflects the rate of Ca²⁺ uptake by sarcoplasmic reticulum is the rate of decline of the Ca²⁺ transient, which, in turn, could influence the rate of mechanical relaxation [69]. Thus the changes in Ca²⁺ uptake by sarcoplasmic reticulum may be manifested most strikingly in the impaired relaxation that is evident in stunned myocardium rather than in the decline of systolic force.

Heat Shock Proteins and Stunning

The exposure of cells to harmful events or agents creating a noxious stress is met by several different cellular defense mechanisms including detoxifying enzymes such as cytochrome P450 [70] or catalase and SOD-removing oxygen radicals [71, 72]. A frequent consequence of noxious stresses, like hypoxia, is an accumulation of proteins that are not correctly folded [73]. Malfolded proteins do not remain soluble and become denatured proteins. Induction of stress or heat shock proteins (HSPs) protects the cells against harmful consequences of protein denaturation [74, 75]. ``Stress'' or ``heat-stress'' proteins were originally identified because of increased synthesis by many cell types after exposure to elevated temperatures. The major HSPs are divided according to their molecular weight into the small HSPs (26 to 28 kd), HSP 60 family (which are located in the mitochondria), HSP 70, HSP 90, and HSP 100 gene families. HSP 70 was identified in neonatal and adult heart tissue from several species, including dog, rat, and rabbit [76]. The HSP 70 gene family includes HSP 70c (70 to 71 kd), which is always present in cells. HSP 70c serves an important role by associating with nascently formed proteins that have not reached their permanent folding state and preventing their denaturation [77]. In addition, they serve as unfoldases by associating with proteins being incorporated into mitochondria and placing them into a translocation competent configuration [78]. These effects of HSP 70 has been termed their chaperone function [79].

Studies in different species have shown that increased quantities of HSPs may protect the heart against subsequent damage. Similarly a wide variety of other stressful stimuli have been shown to increase HSP 70 synthesis in cardiac tissue. These include ischemia [80], pressure or volume overload [81], and treatment with heavy metals such as cadmium [82], as well as drugs such as vasopressin or angiotensin [83] and isoproterenol [84]. We have recently demonstrated that exposure of isolated perfused heart to free radical generating systems for short periods induces transcription of HSP 70 messenger RNA [85]. In addition, ischemia/reperfusion-induced increase in HSP 70 messenger RNA was significantly decreased by SOD, suggesting that free radicals are partially responsible for the increase in HSPs. Thus free radicals generated by preconditioning may trigger the synthesis of HSPs, which may lead to delayed or perhaps longer lasting protection (second window of protection), as described by Kuzuya and colleagues [86] and Marber and associates [87]. Sun and co-workers [88] recently reported significantly less (almost 50%) stunning when conscious pigs were subjected to two identical ischemic insults (ten 2-minute coronary occlusions) at an interval of 24 hours. They further observed that the effect of preconditioning on attenuation of stunning could be maintained for at least 48 hours. The development of this type of preconditioning was accompanied by a significant increase in HSP 70, which further suggested the role of heat shock proteins in myocardial protection. The attenuation of stunning in their experiments was not blocked by adenosine receptor antagonists, suggesting that adenosine was not the mediator in second window of preconditioning. Because free radicals have been shown to be generated during stunning, further studies are required to demonstrate their role in triggering the mechanisms responsible for second window of preconditioning.

Conclusions

The mechanisms discussed above are not mutually exclusive and in fact may represent different steps of the same pathophysiologic cascade. The generation of free radicals may cause sarcoplasmic reticulum dysfunction, and both of these mechanisms may lead to calcium overload, which in turn could exacerbate the damage initiated by oxygen radicals. Although heat shock proteins are also known to be rapidly expressed during brief ischemia or preconditioning, the cause and effect of its upregulation in stunning or preconditioning is currently a matter of speculation. Recent preliminary studies from two independent groups (Currie and associates [89] and Marber and co-workers [90]) have shown that HSP 70 transgene in the mouse heart protected against ischemia/reperfusion injury. Kukreja and co-workers [91, 92] have shown that quercetin, the inhibitor of heat shock transcription factor, blocked ischemic tolerance and synthesis of HSP 70 in heat-stressed rat hearts, further suggesting the role of HSPs in myocardial protection. Thus it appears that HSP 70 may have a cause-and-effect relationship with myocardial protection, which may be exploited to develop therapy for attenuation of stunning or reducing ischemia-related damage in the myocardium.

Acknowledgments

This work was supported in part by National Institutes of Health grant HL 46763 and the Jeffress Trust Foundation (J-225).

Footnotes

Presented at the International Symposium on Myocardial Protection From Surgical Ischemic-Reperfusion Injury, Asheville, NC, Sep 25-28, 1994.

Address reprint requests to Dr Hess, Cardiopulmonary Research, Medical College of Virginia, Richmond, VA 23298.

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