—  SOCIETY FOR CARDIOVASCULAR PATHOLOGY   —

Myocardial Ischemia and Reperfusion Injury


L. Maximilian Buja
University of Texas Health Science Center
Houston, TX


Myocardial ischemic injury results from severe impairment of the coronary blood supply usually produced by thrombosis or other acute alterations of coronary atherosclerotic plaques. Sustained investigation has produced considerable insight into the pathobiology of myocardial ischemic injury [1, 2, 3] . It is now recognized that a number of processes can modulate the response of the myocardium to ischemic injury, particularly reperfusion and preconditioning [1, 2, 3] .

Mechanisms of Myocardial Ischemic Injury
The anatomic and biochemical substrates for ischemic myocardial cell injury and death have been well characterized [1, 2, 3] . With loss of oxygen, mitochondrial oxidative phosphorylation rapidly stops, with resultant loss of the major source of ATP production. A compensatory increase in anaerobic glycolysis leads to accumulation of hydrogen ions and lactate, resulting in intracellular acidosis and inhibition of glycolysis and also mitochrondrial fatty acid and residual energy metabolism. Excitation and contraction uncoupling develops in association with alterations in ion transport systems in the sarcolemma and organellar membranes [4, 5, 6] . This establishes a milieu for ventricular arrhythmias.

Ultrastructural features of the ischemic myocytes with metabolic derangements include swelling of mitochrondria and sarcoplasmic reticulum, swelling of the cytoplasm and margination and clumping of nuclear chromatin. Ultrastructural features of irreversible injury include flocculent (amorphous matrix) densities and linear densities in mitochrondria and physical defects (holes) in the sarcolemma (Figure 1). Mitochrondria may also exhibit calcium phosphate deposits [1, 2, 3] . The progression from reversible to irreversible myocardial cell injury is accompanied by changes in the myocardial interstitium and microvasculature. The necrosis of myocytes and non-myocytes triggers an inflammatory reaction with subsequent organization and healing.



FIGURE 1. Schematic diagram comparing pathobiologic features of a transmural infarct produced by permanent coronary occlusion and a subendocardial infarct produced by temporary coronary occlusion followed by reperfusion.

N = nucleus
MC = marginated chromatin
SD = sarcolemmal defect
FD = flocculent (amorphous) density
NMG = normal matrix granule
CB = contraction band
CaD = calcium deposit

The progression to an advanced stage of cardiomyocyte injury results from progressive membrane damage involving several mechanisms (Figure 2) [1, 2, 3, 4, 5, 6] . The altered metabolic milieu, including a sustained increase in cytosolic Ca2+, leads to activation of phospholipases and proteases. This leads to phospholipid degradation with release of lysophospholipids and free fatty acids and cleavage of cytoskeletal filaments. Impaired mitochrondrial fatty acid metabolism results in accumulation of free fatty acids, long-chain acyl CoA and acyl carnitine, and these amphiphilic molecules, together with products of phospholipid degradation, can incorporate into membranes and impair their function. Toxic oxygen species and free radicals are generated from ischemic myocytes, ischemic endothelial cells and activated leukocytes, and they induce peroxidative damage to the fatty acids of membrane phospholipids. These changes collectively lead to progressive increase in membrane permeability, progressively severe derangements of intracellular electrolytes, and ATP exhaustion. The terminal event is physical disruption of the sarcolemma of the swollen myocyte.



FIGURE 2. Postulated sequence of alterations involved in the pathogenesis of irreversible myocardial ischemic injury.

Oncosis and Apoptosis
It is now known that there are two basic patterns of cell injury progressing to cell death: cell injury with swelling, known as oncosis; and cell injury with shrinkage, known as apoptosis [7]. Apoptosis can be initiated by activation of a death receptor pathway or a mitochondrial pathway [8]. Apoptosis is characterized by a series of molecular, biochemical and morphological events including:

  1. gene activation (programmed cell death)

  2. activation of a cascade of cytosolic aspartate-specific cysteine proteases (caspases)

  3. mitochondrial alterations, including loss of membrane potential, initiation of the membrane permeability transition and cytochrome C release

  4. endonuclease activation leading to double-stranded DNA fragmentation

  5. selective alteration of cell membranes with increased expression of phosphatidylserine in the outer leaflet and preservation of selective membrane permeability

  6. cell and nuclear shrinkage and fragmentation.

In evolving myocardial ischemic injury, findings have ranged from apoptosis involving the majority of injured myocytes during the first hours after coronary occlusion to apoptosis occurring only during reperfusion [3, 8, 10] . A major source of variability in the results is the detection of apoptosis based primarily on the pattern of DNA fragmentation using the terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated dVTP nick end-labeling (TUNEL) method. TUNEL staining is not specific for the characteristic DNA fragmentation of apoptosis and also can be seen in oncotic necrosis [3, 9, 10] . Other assays for DNA fragmentation may provide more specificity [11]. Nevertheless, Ohno presented evidence that a minority of ischemic myocytes had evidence of TUNEL positivity by light microscopy and, by electron microscopy, TUNEL positive cells had features of oncotic injury [12]. Other investigators showed that a minority of ischemic myocytes had evidence of phosphatidylserine translocation by annexin V labeling [13]. However, reduction in infarct size has been reported after treatment with caspase inhibitors [3, 9] . Thus, some ischemic myocytes apparently undergo cell death by apoptosis. The rate and magnitude of ATP depletion is a major determinant of whether cell injury progresses via apoptosis or oncosis since oncosis is an ATP-dependent process. It is also possible that ischemically injured myocytes undergo activation of the apoptotic pathway as well as other pathways leading to oncosis as the predominant manifestation of ischemic injury [12]. A synthesis of the evidence indicates that oncotic injury mediated by mechanisms leading to progressive membrane damage is the dominant mode of ischemic myocardial injury.

Determinants of Myocardial Infarct Evolution and Size
Myocardial infarcts evolve as a wavefront of necrosis within an ischemic myocardial bed-at-risk in the distribution of an occluded coronary artery [14]. There is a rather sharp demarcation between ischemic and non-ischemic myocardium at the lateral margins of the bed-at-risk. The onset of irreversible injury begins after about 20 to 30 minutes in the ischemic subendocardium, where the perfusion deficit is most severe compared to the subepicardium which receives some collateral blood flow. Irreversible myocardial injury then progresses in a wavefront movement from the subendocardium into the subepicardium. Most myocardial infarcts are completed within about 3 to 6 hours of onset of severe ischemia. The resulting infarcts have distinctive central and peripheral zones (Figure 1) [15]. The major determinants of ultimate infarct size are the duration and severity of ischemia, the size of the myocardial bed-at-risk and the amount of collateral blood flow available shortly after coronary occlusion.

In response to myocardial infarction, progressive changes occur in viable myocardium in an attempt to normalize increased stress on the ventricle. This process, known as remodeling, involves hypertrophy and apoptosis of myocytes, formation of new myocytes from stem cells, and connective tissue changes [3, 16] . Controlled remodeling can lead to normalization of wall stress. With excessive wall stress, however, remodeling can lead to fixed structural dilatation of the ventricle and heart failure.

Reperfusion and Reperfusion Injury
Once the importance of infarct size in determining prognosis was established, intensive investigation was focused on finding means of reducing infarct size. Pharmacological approaches generally have not proved to have major clinically applicable effects on reducing infarct size [17]. In contrast, reperfusion and preconditioning have been found to have profound effects on limitation of infarct size. Reperfusion clearly can limit the extent of myocardial necrosis with the magnitude of the sparing directly related to the timing of the intervention [18, 19, 20] . However, the effects of reperfusion are complex and include some deleterious effects collectively referred to as reperfusion injury. This reperfusion injury involves activation of an inflammatory cascade and is manifest as functional impairment, arrhythmia, and accelerated progression of cell death in certain critically injured myocytes. The major mediators of reperfusion injury are oxygen radicals, calcium loading and neutrophils. The oxygen radicals are generated by injured myocytes and endothelial cells in the ischemic zone as well as neutrophils that enter the ischemic zone and become activated on reperfusion. These oxygen radicals exacerbate membrane damage which leads to calcium loading. The neutrophils accumulate in the microcirculation, release inflammatory mediators, and contribute to microvascular obstruction and the no reflow phenomenon in the reperfused myocardium [18, 19, 20] .

Myocardial stunning refers to the prolonged depression of contractile function of the salvaged myocardium that develops on reperfusion even after relatively brief periods of coronary occlusion, on the order of 15 minutes, which are insufficient to cause myocardial necrosis. After longer intervals of coronary occlusion, of 2 to 4 hours, even more severe and persistent depression of contractile function occurs even though there is significant sparing of subepicardial myocardium by reperfusion. Myocardial stunning is mediated by the effects of reperfusion, including free radicals and calcium loading, on myocytes that retain viability and ultimately recover contractile function [21]. Hibernation is a chronic depression of myocardial function due to chronic moderate reduction of perfusion [3].

Reperfusion alters the pattern of myocardial injury in the core of myocardium already irreversibly injured during the ischemic episode. Reperfusion induces contraction bands and calcium loading in the irreversibly injured myocytes, hemorrhage in the region due to leakage of blood out of damaged blood vessels, and accelerated release of marker proteins used to detect myocardial infarction in laboratory tests. All of these changes occur within the already irreversibly injured myocardium. However, there is evidence that reperfusion can lead to the conversion from reversible to irreversible injury of a population of myocytes that have been severely impaired during the prior period of ischemia. The mechanism of the irreversible injury is massive calcium overloading of the metabolically impaired myocytes with damaged sarcolemma [3, 18, 19, 20] . However, if reperfusion is instituted within 2 to 3 hours of the onset of ischemia, the amount of salvage greatly exceeds the amount of myocardium undergoing irreversible reperfusion injury.

Myocardial Preconditioning
Myocardial preconditioning is the protective effect produced by prior short intervals of coronary occlusion and reperfusion on the rate of progression of myocardial necrosis during a subsequent sustained coronary occlusion. Preconditioning results in a major reduction of infarct size compared to the non-preconditioned state when hearts are subjected to approximately 60 to 90 minutes of coronary occlusion, but the effect is lost if the coronary occlusion is maintained for 2 or more hours [22]. The preconditioning effect is operative for one to two hours, then is lost for several hours (refractory period), but redevelops when sustained coronary occlusion is induced approximately 24 hours after the preconditioning. The first phase is known as early or classical preconditioning and the delayed phase as the second window of protection (SWOP). Early or classical preconditioning is triggered by activation of adenosine and other agonist receptors and is mediated by activation of protein kinase C coupled to G proteins, and opening of ATP-dependent potassium channels in the sarcolemma and mitochondria, with activation of the mitochondrial ATP-dependent potassium channels as the key event [23]. Uncertainty remains as to the immediate effectors of preconditioning that are induced by activation of the ATP-dependent potassium channels. The second window of protection is mediated by ischemia-induced gene activation mediated by a kinase cascade, including mitogen-activated protein (MAP) kinases and nuclear factor kappa B (NFκB). Significant gene products implicated in protection include superoxide dismutase, nitric oxide synthase and its product, nitric oxide, cyclooxygenase 2 (COX2), and heat shock proteins [23, 24] . These gene products create a protective milieu for the cardiomyocyte, but again the exact mechanisms of the protective effect are uncertain.

Future Directions
Work is continuing to understand basic mechanisms of myocardial ischemic injury, the basis for the complex effects of reperfusion on myocardial ischemic injury, and the mechanisms of the powerful but transient effect of preconditioning on ischemic injury. These ongoing studies have promise to provide new pharmacological agents and other approaches that can be effective in the treatment of ischemic heart disease. Now the new approach of using stem cells to repair and repopulate the damaged myocardium is under intense investigation. Success depends on understanding how stem cells can interact with damaged myocardium and possibly modulate the basic pathobiology of myocardial ischemic injury.

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