Myocardial Ischemia and Reperfusion Injury
L. Maximilian Buja
University of Texas Health Science Center
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
. It is now recognized that a number of processes can modulate the response of the myocardium to
ischemic injury, particularly reperfusion and preconditioning
Mechanisms of Myocardial Ischemic Injury
The anatomic and biochemical substrates for ischemic myocardial cell injury and death have been well
. 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
. 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
. 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)
. 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 .
Apoptosis can be initiated by activation of a death receptor pathway or a mitochondrial pathway .
Apoptosis is characterized by a series of molecular, biochemical and morphological events including:
- gene activation (programmed cell death)
- activation of a cascade of cytosolic aspartate-specific cysteine proteases (caspases)
- mitochondrial alterations, including loss of membrane potential, initiation of the membrane permeability transition and cytochrome C release
- endonuclease activation leading to double-stranded DNA fragmentation
- selective alteration of cell membranes with increased expression of phosphatidylserine in the outer leaflet and preservation of selective membrane permeability
- 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
. 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
Other assays for DNA fragmentation may provide more specificity . 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 . Other investigators
showed that a minority of ischemic myocytes had evidence of phosphatidylserine translocation by annexin V
labeling . However, reduction in infarct size has been reported after treatment with caspase
. 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 . A synthesis of the evidence indicates that oncotic
injury mediated by mechanisms leading to progressive membrane damage is the dominant mode of ischemic
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 . 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) .
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
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
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 . 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
. 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
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 . Hibernation is a
chronic depression of myocardial function due to chronic moderate reduction of perfusion .
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
. 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 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 . 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 . 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
. These gene products create a protective milieu for the cardiomyocyte, but again the
exact mechanisms of the protective effect are uncertain.
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.
- Reimer KA, Jennings RB. Myocardial ischemia, hypoxia, and infarction. In: Fozzard HA, Haber E, Jennings RB, Katz AM, Morgan HE, eds. The Heart and Cardiovascular System: Scientific Foundations, 2nd Edition. New York: Raven Press; 1991:1875-1973.
- Reimer KA, Ideker RE. Myocardial ischemia and infarction: anatomic and biochemical substrates for ischemic cell death and ventricular arrhythmias. Hum Pathol. 1987;18:462-475.
- Buja LM. Modulation of the myocardial response to ischemia. Lab Invest. 1998;78:1345-1373.
- Buja LM, Hagler HK, Willerson JT. Altered calcium homeostasis in the pathogenesis of myocardial ischemic and hypoxic injury. Cell Calcium. 1988;9:205-217.
- Thandroyen FT, Bellotto D, Katayama A, Hagler HK, Willerson JT, Buja LM. Subcellular electrolyte alterations during hypoxia and following reoxygenation in isolated rat ventricular myocytes. Circ Res. 1992;71:106-119.
- Buja LM. Lipid abnormalities in myocardial cell injury. Trends Cardiovasc Med. 1991;1:40-45.
- Majno G, Joris I. Apoptosis, oncosis, and necrosis: an overview of cell death. Am J Pathol. 1995;146:3-15.
- Reed JC. Mechanisms of apoptosis. Am J Pathol. 2000;157:1415-1430.
- Buja LM, Entman ML. Modes of myocardial cell injury and cell death in ischemic heart disease. Circulation. 1998;98:1355-1357.
- Kajstura J, Cheng W, Reiss K, Clark WA, Sonnenblick EH, Krajewski S, Reed JC, Olivetti G, Anversa P. Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats. Lab Invest. 1996;74:86-107.
- Anversa P, Leri A, Beltrami CA, Guerra S, Kajstura J. Myocyte death and growth in the failing heart. Lab Invest. 1998;78:767-786.
- Ohno M, Takemura G, Ohno A, Misao J, Hayakawa Y, Minatoguchi S, Fujiwara T, Fujiwara H. "Apoptotic" myocytes in infarct area in rabbit hearts may be oncotic myocytes with DNA fragmentation: analysis by immunogold electron microscopy combined with in situ nick end-labeling. Circulation. 1998;98:1422-1430.
- Dumont EAWJ, Hofstra L, van Heerde WL, van den Eijnde S, Doevendans PAF, DeMuinck E, Daemen MARC, Smits JFM, Frederik P, Wellens HJJ, Daemen MJAP, Reutelingsperger CPM. Cardiomyocyte death induced by myocardial ischemia and reperfusion: measurement with recombinant human annexin-V in a mouse model. Circulation. 2000;102:1564-1568.
- Reimer KA, Jennings RB. The "wavefront phenomenon'' of myocardial ischemic cell death: II. Transmural progression of necrosis within the framework of ischemic bed size (myocardium at risk) and collateral flow. Lab Invest. 1979;40:633-644.
- Buja LM, Tofe AJ, Kulkarni PV, Mukherjee A, Parkey RW, Francis MD, Bonte FJ, Willerson JT. Sites and mechanisms of localization of technetium-99m phosphorus radiopharmaceuticals in acute myocardial infarcts and other tissues. J Clin Invest. 1977;60:724-740.
- Nadal-Ginard B, Kajstura J, Leri A, et al. Myocyte death, growth and regeneration in cardiac hypertrophy and failure. Circ Res. 2003;92:139-150.
- Reimer KA, Jennings RB, Cobb FR, Murdock RH, Greenfield JC Jr, Becker LC, Healey Bulkley B, Hutchins GM, Schwartz RP Jr, Bailey KR, Passamani ER. Animal models for protecting ischemic myocardium (AMPIM): Results of the NHLBI Cooperative Study: Comparison of unconscious and conscious dog models. Circ Res. 1985;56:651-665.
- Maxwell SRJ, Lip GYH. Reperfusion injury: a review of the pathophysiology, clinical manifestations and therapeutic options. Internatl J Cardiol. 1997;58:95-117.
- Park JL, Lucchesi BR. Mechanisms of myocardial reperfusion injury. Ann Thoracic Surg. 1998;68:1905-1912.
- Ambrosio G, Tritto I. Reperfusion injury: experimental evidence and clinical implications. Am Heart J. 1999;138:S69-75.
- Kusuoka H, Porterfield JK, Weisman HF, Weisfeldt ML, Marban E. Pathophysiology and pathogenesis of stunned myocardium: depressed Ca2+ activation of contraction as a consequence of reperfusion-induced cellular calcium overload in ferret hearts. J Clin Invest. 1987;79:950-961.
- Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986;74:1124-1136.
- Yellon DM, Downey JM. Preconditioning the myocardium: from cellular physiology to clinical cardiology. Physiol Rev. 2003;83:1113-1151.
- Bolli R. The late phase of preconditioning. Circ Res. 2000;87:972-983.