Myocardial injury that has developed through a

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1 I. PATHOPHYSIOLOGY OF ISCHEMIC REPERFUSION INJURY Cellular Mechanisms of Ischemia-Reperfusion Injury H. Michael Piper, MD, PhD, Karsten Meuter, MD, and Claudia Schäfer, PhD Physiologisches Institut, Justus-Liebig-Universität, Giessen, Germany As of yet, only a few strategies to prevent myocardial reperfusion injury have been tested clinically. In the first minutes of reperfusion, the myocardium can be damaged by contracture development, causing mechanical stiffness, tissue necrosis, and the stone heart phenomenon. Reperfusion-induced contracture can have two different causes, namely, Ca 2 overload induced contracture or rigor-type contracture. Ca 2 contracture results from rapid re-energization of contractile cells with a persistent Ca 2 overload. Strategies to prevent this type of injury are directed at cytosolic Ca 2 control or myofibrillar Ca 2 sensitivity. Rigor-contracture occurs when reenergization proceeds very slowly. It does not depend on Ca 2 overload. It may be prevented by strategies improving early mitochondrial reactivation (Ann Thorac Surg 2003;75:S644 8) 2003 by The Society of Thoracic Surgeons Myocardial injury that has developed through a period of ischemia-reperfusion may have many causes. In the past, most research has concentrated on the mechanisms causing cellular injury during ischemia and on protective procedures designed to reduce development of ischemic injury. Potential causes of injury that develop during reperfusion have been difficult to analyze, as these must be clearly differentiated from ischemic causes. The identification of a cause of true reperfusion injury requires that a therapeutic interference at the time of reperfusion attenuates the injury. Only a few strategies directed against reperfusion injury have been tested under clinical conditions. Most of these attempts were made in cardiac surgery, since operations using extracorporeal circulation easily allow varying myocardial reperfusion. In the field of cardiology, specific therapies for reperfusion injury are not yet in clinical use, although interventional catheter techniques also permit application of therapeutic agents at the onset of myocardial reperfusion. In general, research on the principles of reperfusion injury opens an entirely new approach to clinical cardiac protection. Reperfusion injury of the myocardium is a complex phenomenon consisting of several independent etiologies. During the earliest phase (ie, minutes) of reperfusion, development of cardiomyocyte contracture seems to be the primary cause for necrotic cardiomyocyte injury. Thereafter (ie, minutes to hours), various additional causes can lead to a further increment of cell death either by necrosis or apoptosis, and vascular failure may further Presented at the 3 rd International Symposium on Myocardial Protection From Surgical Ischemic-Reperfusion Injury, Asheville, NC, June 2 6, Address reprint requests to Dr Piper, Physiologisches Institut, Justus- Liebig-Universität, Aulweg 129, D Giessen, Germany; michael.piper@physiologie.med.uni-giessen.de. aggravate cardiomyocyte injury. The present review is focused on cellular causes of myocardial contracture developing during the early phase of reperfusion. When contracture affects the entire heart, as may occur after global ischemia, it has been termed as the stone heart phenomenon. Stone hearts are known to cardiac surgeons as a result of prolonged ischemia or unsatisfactory attempts to apply cardioplegic protection. Stone hearts may be caused by contracture developing either during ischemia or reperfusion, as explained below. Two Causes of Reoxygenation-Induced Contracture Contracture (ie, a sustained shortening and stiffening of myocardium) can have several causes. In ischemic myocardium contracture develops by means of a rigor-type mechanism. Studies on skinned cardiac cells or muscle fibers have shown that a force-generating cross bridge cycling is initiated when cytosolic adenosine triphosphate (ATP) is reduced to a low ( 100 mol/l) but nonzero level [1, 2]. In ischemia, this window of low cytosolic ATP concentrations is open only during a brief period, because cellular ATP reserves are quickly exhausted. The myofibrillar shortening then stays fixed, as all cross bridges between actin and myosin remain in an attached state. The contracture developed by this ischemic mechanism does not actually cause major structural damage but leads to cytoskeletal defects. These defects render cardiomyocytes more fragile and thus susceptible to mechanical damage [3]. When energy depletion is rapidly relieved, ischemic rigor contracture is usually reversible. After prolonged ischemia myocardial cells may develop severe contracture, which can lead to cytoskeletal defects that consequently increase the fragility of cardiomyocytes upon reperfusion. As a consequence, end by The Society of Thoracic Surgeons /03/$30.00 Published by Elsevier Science Inc PII S (02)

2 Ann Thorac Surg MYOCARDIAL PROTECTION PIPER ET AL 2003;75:S644 8 CELLULAR MECHANISMS OF I/R INJURY S645 Fig 1. Reversible changes in cytosolic cation control in ischemiareperfusion. In ischemia, cardiomyocytes accumulate Na by means of (1) the Na /H exchanger, (2) the Na /HCO 3 symporter, and (3) other routes. With reduction of the Na gradient and membrane depolarization, the Na /Ca 2 exchanger is turned into its reverse mode, which leads to cytosolic accumulation of Ca 2. In reperfusion, energy recovery reactivates Na -K -ATPase (4) and restores the Na gradient and the membrane potential. The forward mode of the Na /Ca 2 exchanger eventually extrudes excess cytosolic Ca 2. ATP adenosine triphosphate; NCE Na /H exchanger. diastolic ventricular pressure increases and ventricular compliance decreases. Substantial contracture is accompanied by a specific form of tissue necrosis, the so-called Fig 3. Characteristic changes in cytosolic Ca 2 and cell length in a single cardiomyocyte under simulated ischemia-reperfusion conditions. (Left panel) Cells are elongated in normoxia, become rigorshortened in ischemia, and become hypercontracted upon reperfusion. (Upper panel) Rise of cytosolic Ca 2 (monitored by fluorescence of the indicator Fura-2, continuous trace) and cell shortening (open circles). During simulated ischemia, Ca 2 rises and rigor contracture develops. Upon reoxygenation Ca 2 declines and the cell hypercontracts. (Lower panel) Early reperfusion, in higher time resolution. Ca 2 level declines but starts to oscillate. During oscillations, extensive contracture develops. Fig 2. Cause of cytosolic Ca 2 oscillations and Ca 2 -induced contracture in cardiomyocytes. After ischemia, cardiomyocytes contain an excessive cytosolic Ca 2 overload. In the early phase of reoxygenation, this may still be aggravated by a reverse mode action of the Na /Ca 2 exchanger. Reoxygenation causes a reenergization of the sarcoplasmic reticulum (SR). This starts to accumulate Ca 2 and, once full, releases Ca 2. These Ca 2 movements lead to oscillatory cytosolic Ca 2 elevations, which provoke uncontrolled myofibrillar activation. ATP adenosine triphosphate; NCE Na /H exchanger. contraction band necrosis [4]. The histologic picture is characterized by coexistence of supercontracted sarcomeres, overextension of spaces in between, and sarcolemmal disruptions, all in the same cells. This picture results from strong and inhomogeneous mechanical forces. In a number of studies we have shown that pathogenesis of reperfusion-induced contracture can be analyzed on the cellular level. This analysis revealed two independent causes of reperfusion-induced contracture: (1) Ca 2 overload induced contracture, and (2) rigor contracture. Ca 2 overload induced contracture is elicited in a cardiomyocyte, if it develops Ca 2 overload during ischemia and is then rapidly reenergized. High cytosolic Ca 2 plus energy leads to uncontrolled activation of the contractile machinery. Rigor-contracture may be activated during reoxygenation, if reenergization of the ischemic cardiomyocytes occurs at a very low rate. It may, therefore, be observed after prolonged or severe ischemia. Rigor-contracture is not essentially dependent on Ca 2 overload. These two causal mechanisms for reperfusion-induced contracture are described separately below.

3 S646 MYOCARDIAL PROTECTION PIPER ET AL Ann Thorac Surg CELLULAR MECHANISMS OF I/R INJURY 2003;75:S644 8 Ca 2 Overload Induced Contracture Ischemic cells become energy depleted and subsequently develop a Ca 2 overload of the cytosol due to a reversemode operation of the sarcolemmal Na /Ca 2 exchanger (Fig 1). If the ability of mitochondria to resume ATP synthesis is not critically impaired during the ischemic period, reoxygenation leads to a rapid recovery of energy production. Resynthesis of ATP can enable cardiomyocytes to recover from the loss of cytosolic cation balance, but it also reactivates the contractile machinery that had been fixed in rigor contracture after ischemic loss of ATP. The latter effect is normally faster then the former, which leads to an uncontrolled Ca 2 -dependent contraction. When analyzed in detail, it was found that cyclic uptake and release of Ca 2 by the sarcoplasmic reticulum (SR) in the reoxygenated cardiomyocytes triggers a Ca 2 overload induced contracture [5, 6] (Fig 2). These oscillatory Ca 2 shifts lead to high cytosolic peak Ca 2 concentrations (Fig 3). The frequency of these Ca 2 peaks is influenced by an ongoing Ca 2 influx across the sarcolemma during the early phase of reoxygenation [6]. During this period the transsarcolemmal Na gradient is still reduced and the Na /Ca 2 exchanger still operates in reverse mode. Experimentally, various protocols have been shown to interfere with Ca 2 overload induced contracture: First, contracture can be prevented by an initial, time-limited inhibition of the contractile machinery. For this purpose, the chemical phosphatase 2,3 butane dione monoxime has been used [7, 8]. Part of the protective effects of cgmp-mediated effectors (NO, atrial natriuretic peptides) or cytosolic acidosis can also be attributed to contractile inhibition, as these agents reduce Ca 2 sensitivity of myofibrils. Second, contracture can be reduced by reducing SR-dependent Ca 2 oscillations. This can either be achieved by agents interfering Fig 4. Importance of the rapidity of adenosine triphosphate (ATP) recovery for reoxygenation-induced contracture. Cardiomyocytes with rapid ATP recovery quickly pass through the critical window of rigor contracture and may develop Ca 2 contracture if they have a cytosolic Ca 2 overload. Cardiomyocytes with slow ATP recovery creep slowly through the critical window and develop rigor contracture. Fig 5. Relationship between cytosolic Ca 2 overload and reoxygenation-induced contracture. In cells with very slow energy recovery (mitochondrial damage, long ischemic exposure), contracture develops by a rigor-type mechanism that is essentially Ca 2 -independent. In cells with fast energy recovery (intact mitochondria, brief ischemic exposure), contracture develops only at high cytosolic Ca 2 overload. States between these extremes are possible. with SR Ca 2 sequestration or by inhibition of the Ca 2 influx into the cells still occurring during the early phase of reoxygenation. Ca 2 cycling across the SR can be inhibited by specific agents interfering with SR Ca 2 ATPase or SR Ca 2 release [5] or with less specific means such as the anesthetic halothane or intracellular acidosis [9, 10]. Of particular interest is the therapeutic value of proton transport inhibition during the early phase of reperfusion. We showed previously that preservation of ischemic intracellular acidosis during the initial phase of reperfusion protects cardiac cells against reoxygenationinduced contracture [10]. To achieve effective protection, simultaneous inhibition of two acid extruder mechanisms is required, namely, the Na /H exchanger and the Na /HCO 3 symporter. Sole applications of Na /H exchanger inhibitors have failed to provide myocardial protection during reperfusion, both in experimental studies [11, 12] and in clinical studies [13]. When cardiomyocytes are reoxygenated in the constant presence of cytosolic acidosis, they can recover metabolically while contractile activation remains inhibited and SR Ca 2 movements are attenuated. Metabolic recovery drives the renormalization of cellular cation control, thus removing the causes for Ca 2 -induced contracture. Another interesting principle of acute reperfusion protection is use of agents stimulating soluble [14, 15] or particulate guanylyl cyclase in the myocardium [16, 17]. Elevation of cellular cgmp levels activates protein kinase G. Its action on myofibrils (potential target: troponin I) causes a Ca 2 desensitization, which is beneficial in

4 Ann Thorac Surg MYOCARDIAL PROTECTION PIPER ET AL 2003;75:S644 8 CELLULAR MECHANISMS OF I/R INJURY S647 reoxygenated cells overloaded with Ca 2. Its action on SR-Ca 2 ATPase (potential target, phospholamban) inhibits SR-dependent Ca 2 cycling in reoxygenated cardiomyocytes. Both actions attenuate Ca 2 -induced contraction. Using NO donors in reperfused myocardium also has other beneficial effects such as leukocyte inhibition and vasodilatation. It should be noted, however, that NO donors may also induce apoptosis in cardiomyocytes, either by a radical-mediated mechanism or by cgmp signaling. Reoxygenation-Induced Rigor Contracture As long as mitochondrial energy production recovers rapidly upon reperfusion/reoxygenation, reoxygenated cardiomyocytes are in acute jeopardy by Ca 2 overload induced contracture. After prolonged ischemia the ability of mitochondria to rapidly restore a normal cellular state of energy is reduced. However, during the early phase of reoxygenation cardiomyocytes may then contain very low (even though rising) concentrations of ATP which provoke rigor contracture (see above) (Fig 4). In comparison to ischemia, upon reoxygenation cardiomyocytes may spend much more time at the window of low cytosolic ATP suitable to induce rigor-type contracture. Therefore, cell shortening can be much more pronounced than observed in ischemic rigor contracture. In fact, the rigor mechanism may become the major contributor to reoxygenation-induced contracture (Piper HM, unpublished data). In the event that rigor contracture prevails in acute reperfusion injury, therapeutic actions aiming at cytosolic Ca 2 overload are not effective, inasmuch as rigor contracture is essentially Ca 2 independent (Fig 5). It can be shown experimentally that one can reduce rigor contracture by improving the conditions for energy recovery. A first approach is application of mitochondrial energy substrates, eg, succinate, with the aim of accelerating oxidative energy production. Second, one may speculate about means to protect mitochondria and resume respiratory activity in the early phase of reperfusion from compulsory calcium uptake. Spreading of Contracture These cellular mechanisms that contribute to reperfusion-induced contracture seem to represent the major causes for lethal cell injury occurring during the early phase of reperfusion. Model calculations have suggested that they cannot explain, however, the continuous geometry of contraction band necrosis in reperfused myocardium [18]. Cell-to-cell interactions seem to take part in the expansion of early necrosis. Recent studies have shown that gap junction mediated communication between ischemic cells allows spreading of cell injury during myocardial reperfusion [19]. Passage of sodium through gap junctions from hypercontracting cells to adjacent ones and subsequent a change of Ca 2 through a reverse mode of Na /Ca 2 exchange may result in propagation of contracture [20]. It also seems possible that metabolical coupling synchronizes the rate of ATP recovery in reperfused myocardium and therefore synchronizes the development of rigor contracture. Apart from these chemical coupling mechanisms, cells undergoing contracture exchange forces with their neighbors may disrupt these. This also contributes to the spreading of necrosis. Reperfusion Injury: The Second Act The pathologic mechanisms described so far occur during the first minutes of reperfusion. Other mechanisms originating from the vasculature and blood elements can enhance reperfusion injury by mechanisms activated during the subsequent hours. To place the early mechanisms in perspective, these additional causes of injury are briefly summarized. The endothelial lining of blood vessels subjected to ischemia-reperfusion becomes permeable, thus causing interstitial edema with the resumption of blood flow. Endothelial cells in reperfused myocardium assume an activated state in which they express adhesion proteins, release cytokines, and reduce production of NO. This promotes adherence, activation, and accumulation of neutrophils and monocytes in the ischemic-reperfused tissue. The release of reactive oxygen species and proteolytic enzymes from these activated leukocytes can contribute to the damage of myocytes and vascular cells. Vascular plugging by adherent leukocytes can also promote a slow- or no-reflow phenomenon, already favored by tissue contracture and increased pressure of interstitial water. It seems that these additional reperfusion-induced noxes contribute to infarct development predominantly during the first 2 hours of reperfusion, as myocardial necrosis almost reaches its final size during this period. In summary, the early phase of reperfusion represents an important target for strategies protecting ischemicreperfused myocardium. Adaptation of these protective strategies to clinical therapeutic use would represent a major advance in the field of cardiology for treatment of acute myocardial infarction and for myocardial protection in cardiac surgery. References 1. Altschuld RA, Wenger WC, Lamka KG, et al. Structural and functional properties of adult rat heart myocytes lysed with digitonin. J Biol Chem 1985;260: Nichols CG, Lederer WJ. The role of ATP in energydeprivation contractures in unloaded rat ventricular myocytes. Can J Physiol Pharmacol 1990;68: Schlüter KD, Jakob G, Ruiz-Meana M, García-Dorado D, Piper HM. Protection of reoxygenated cardiomyocytes against osmotic fragility by NO donors. Am J Physiol 1996; 271:H Ganote CE. Contraction band necrosis and irreversible myocardial injury. J Mol Cell Cardiol 1983;15: Siegmund B, Schlack W, Ladilov YV, Balser C, Piper HM. Halothane protects cardiomyocytes against reoxygenationinduced hypercontracture. Circulation 1997;96: Schäfer C, Ladlov Y, Inserte J, et al. Role of the reverse mode of the Na /Ca 2 exchanger in reoxygenation-induced cardiomyocyte injury. Cardiovasc Res 2001;51: Siegmund B, Klietz T, Schwartz P, Piper HM. Temporary

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