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Propagation of Cell Death During Myocardial Reperfusion

David Garcia-Dorado and Marisol Ruiz-Meana

D. Garcia-Dorado and M. Ruiz-Meana are in the Servicio de Cardiología, Hospital General Universitari Vall d'Hebron, Pg Vall d'Hebron 119-129, 08035 Barcelona, Spain.

	Abstract

 
During myocardial reperfusion, increased cytosolic Ca²⁺ concentration may cause hypercontracture and cell death. Hypercontracture can propagate to adjacent cells by a gap junction-dependent mechanism. This propagation explains infarct geometry and increases the final extent of necrosis. Its prevention may represent a new therapeutic strategy for treating patients with myocardial infarction.

	Introduction

Top Introduction The continuity of contraction... Communication through GJ Cardiomyocyte coupling during... Cell-to-cell propagation of... Cell-to-cell propagation of... References

 
The most relevant consequence of myocardial ischemia-reperfusion is necrosis. In the absence of reperfusion, severe ischemia inevitably leads to myocardial necrosis involving all of the ischemic area (area at risk). Early reperfusion may prevent the death of ischemic myocytes and is the therapeutic strategy of choice in patients with acute myocardial infarction. In most patients, reperfusion salvages only some of the ischemic myocytes, thus limiting, but not preventing, myocardial infarction. The main mechanism of myocyte death in reperfused infarcts is myocyte hypercontracture, which occurs during the first minutes after flow restoration. During ischemia, ATP free energy falls in myocytes, cytosolic H⁺, Na⁺, and Ca²⁺ concentrations rise, and cells develop cytoskeletal and sarcolemmal fragility due to changes in the phosphorylation status of proteins and to other incompletely understood mechanisms. Reperfusion restores ATP synthesis and normalizes cytosolic H⁺, Na⁺, and Ca²⁺ concentrations. However, restoration of ATP free energy occurs immediately on restoration of oxygen availability, whereas normalization of cytosolic Ca²⁺ concentration, mainly by sequestration into the sarcoplasmic reticulum, requires some time. During this time, the availability of energy in the presence of elevated Ca²⁺ concentration results in the generation of excessive contractile force, which, in the presence of cytoskeletal fragility, may lead to hypercontracture (11). In in situ myocytes, the mechanical stress imposed by hypercontracture is aggravated by swelling and mechanical interaction with surrounding tissue and results in immediate sarcolemmal disruption (contraction band necrosis) (6, 7).

	The continuity of contraction band necrosis

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When coronary reperfusion is performed quickly enough to limit myocardial necrosis, infarcts are composed almost exclusively of areas of contraction band necrosis. The spatial distribution of contraction band necrosis in reperfused infarcts presents remarkable features. Hypercontracted, dead myocytes are not found scattered across the reperfused area but are connected to other hypercontracted cells to form clusters. The limits of these clusters are often very irregular and cannot be explained by gradients in collateral or residual flow or by anatomic clues such as fiber orientation or vascular distribution (Fig. 1). This spatial distribution of contraction band necrosis can be reproduced by computer simulation programs only if cell-to-cell interaction is included in the program algorithm, that is, if the probability of cell death is influenced by the survival or death of adjacent cells. In the absence of this condition, simulated infarcts calculated for a given duration of coronary occlusion reproduce the size (number of dead cells) and the transmural distribution of real infarcts produced by a transient coronary occlusion but not its continuity, and dead cells are scattered across the area at risk. These observations predict some kind of propagation of cell death in reperfused infarcts (7).

View larger version (78K): [in this window] [in a new window]   FIGURE 1. Myocardial necrosis secondary to 48 min of coronary occlusion of the left anterior descendent artery and 6 h of reperfusion in the pig heart. A: image of a midventricular myocardial slice obtained under ultraviolet light to outline the area at risk (dark area). Fluorescein had been injected for this purpose during coronary reocclusion immediately before obtaining the heart. B: the same slice illuminated with white light after incubation in triphenyltetrazolium chloride to identify the areas of myocardial necrosis (white areas). C: histochemically detected areas of necrosis sharply correlated with areas of contraction band necrosis at histology and show a very irregular contour (inset).

	Communication through GJ

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In multicellular organisms, virtually all cells are connected to their neighboring cells through GJ. This intercellular communication is essential for coordinating processes such as embryogenesis, growth, electric activity, and cellular response to injury in many tissues. Each GJ channel is formed through the docking of two hemichannels (connexons) located in the membranes of two contacting cells. Typically, several hundreds of these channels are packed together to form plaques in which the membranes of both cells are in close contact. Each connexon is formed by six transmembrane monomeric proteins known as connexins (14). Connexins range in molecular mass from 20 to 56 kDa and, in mammals, are encoded by a highly conserved multigene family of at least 13 related genes. Individual connexins differ in both function and pattern of expression, and each cell type expresses only a single connexin or a limited set of them. The expression of four different connexins, Cx43, Cx40, Cx45, and Cx37, has been identified in the adult mammalian myocardium. GJ connecting ventricular cardiomyocytes are mainly formed by Cx43, whereas Cx40 is more restricted to the atrium and conduction system. Channels may be formed by hemichannels composed of different types of connexins (heterodimeric channels). For example, hemichannels of cardiomyocytes (Cx43) may contact with hemichannels of Purkinje fibers (Cx40), linking the cytoplasmic compartments of two different cell types. Alternatively, different types of connexins expressed by a single cell may assemble into a hemichannel. The genes encoding many of the connexins have been cloned, and an ample array of genetically engineered animals is now available for research. The mechanisms regulating translocation of connexins to the membrane, assembly into hemichannels, displacement, and docking to complementary hemichannels are incompletely understood.

GJ are not fixed channels but may open and close in response to various stimuli. Many of these stimuli act through protein kinases that modify the phosphorylation status of connexins (14). Other stimuli, such as acidosis, may act through different mechanisms (5). When in the open state, GJ behave much like pores and allow the free diffusion of molecules smaller than a critical size (~1000 Da), including various important cell messengers, such as Ca²⁺, inositol trisphosphate, and cAMP (15). The size of the opening and the response to regulating mechanisms differ among connexins. Different stimuli may also differentially affect GJ intercellular communication by modifying either the probability of the open state, the size of the opening, or both. Accordingly, the effect of different stimuli on GJ communication may not only be quantitative but qualitative. Stimuli may also influence GJ communication by modifying the number of channels, either by altering the synthesis/degradation of connexins or their assembly into functional connexons.

Myocardium behaves as a functional syncytium. Adequate synchronization of contraction is possible because GJ allow fast propagation of cell depolarization to adjacent cells. Adult ventricular cardiomyocytes are connected to an average of nine neighboring cells through GJ, mainly distributed in the intercalated disks. Although the role of GJ in the propagation of electric depolarization (electric coupling) has been extensively investigated, the potential importance of chemical exchange unrelated to propagation of the action potential has been much less studied.

Ca²⁺ waves, which are local increases in cytosolic Ca²⁺ concentration that occur spontaneously in cultured quiescent cardiomyocytes and progress across the cell, have been shown to propagate to adjacent cells in a GJ-dependent manner (2). GJ-mediated cell-to-cell propagation of elevation in cytosolic Ca²⁺ concentration, induced by mechanical stimulation or by other mechanisms, has been demonstrated in many other cell types (1, 8, 9). Although Ca²⁺ may cross GJ, there is strong evidence that, at least under certain conditions, propagation of Ca²⁺ waves is not due to the direct passage of Ca²⁺ through GJ but to the passage of other messengers able to induce an increase in cytosolic Ca²⁺ concentration in the adjacent cell (15). Cytosolic Na⁺ concentration is a critical variable in cardiomyocyte homeostasis and function. GJ have been found particularly efficient in equilibrating Na⁺ concentration among neighboring cells.

	Cardiomyocyte coupling during ischemia and reperfusion

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Ischemia induces ATP depletion, intracellular acidosis, increased cytosolic Ca²⁺ concentration, and accumulation of amphipathic catabolites, changes that have been shown to reduce GJ intercellular communication. Electric uncoupling occurs in acidotic, ATP-depleted myocardium shortly after development of rigor contracture and Ca²⁺ rise and has been interpreted as the consequence of an abrupt reduction in GJ conductance (4). The onset of electric uncoupling is associated with the onset of a period of marked electric instability manifested as frequent premature ventricular contractions and a high incidence of ventricular fibrillation (phase Ib of ischemic arrhythmias). This electric instability is thought to be caused by spatial nonhomogeneities resulting from the coexistence of coupled and uncoupled zones within the ischemic area. However, whether GJ may allow synchronization of rigor contracture, a key event in the progression of ischemic injury, is not known. Moreover, the changes in GJ-mediated biochemical coupling between ischemic cardiomyocytes need to be established. It has been suggested that GJ may remain open during ischemic conditions in other cell types (3). Although it is obvious that electric coupling must be restored after reperfusion in surviving myocardium rapidly recovering normal electrophysiological behavior, the time course of recoupling has not yet been characterized.

	Cell-to-cell propagation of reperfusion-induced hypercontracture

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The hypothesis of cell-to-cell transmission of hypercontracture has been tested in end-to-end connected pairs of isolated cardiomyocytes resulting from incomplete dissociation during the cell isolation procedure. In this model, hypercontracture induced by microinjection of extracellular buffer in one of the cells, simulating sarcolemmal rupture, induces a rapid rise in cytosolic Ca²⁺ in the adjacent cell and its hypercontracture within a few seconds (Fig. 2). GJ closure with heptanol, demonstrated by blockade of intercellular diffusion of a GJ-permeant dye (Lucifer yellow), prevents propagation of hypercontracture to adjacent cells (6). The Ca²⁺ rise and hypercontracture in the adjacent cell is not due to the direct passage of Ca²⁺ from the microinjected cell, since removal of extracellular Ca²⁺ prevents it despite the fact that GJ remain open and allow diffusion of the permeant dye. This indicates that Ca²⁺ enters the adjacent cell from the extracellular space. Blockade of L-type Ca²⁺ channels did not modify propagation, but inhibition of Na⁺/Ca²⁺ exchange in its reverse mode prevented it. Thus extracellular Ca²⁺ enters the adjacent cell via reverse Na⁺/Ca²⁺ exchange in response to passage of Na⁺ from the primarily injured cell, in which cytosolic Na⁺ concentration is dramatically increased (13). The question arises as to why this cell-to-cell propagation of Na⁺ overload and subsequent Na⁺/Ca²⁺ exchange does not propagate to the whole area at risk. There are several potential mechanisms by which propagation of hypercontracture can be arrested (Fig. 3). Less fragile cells could undergo hypercontracture without developing dramatic sarcolemmal disruption and massive Na⁺ and Ca²⁺ influx. Thus cells with less mechanical fragility could withstand hypercontracture without developing sarcolemmal rupture and interrupt cell-to-cell propagation of hypercontracture. On the other hand, cells with less severe ischemic injury can be more efficient in normalizing cytosolic Ca²⁺ concentration and thus less susceptible to Ca²⁺ overload.

View larger version (94K): [in this window] [in a new window]   FIGURE 2. Cell-to-cell propagation of hypercontracture in a pair of end-to-end connected adult rat cardiomyocytes. The intercalated disk connecting both cells is clearly visible as a transversal line. The cells had been submitted to metabolic inhibition and were "reoxygenated" by exposure to inhibitor-free buffer only 5 min after development of rigor contracture to avoid reoxygenation-induced spontaneous hypercontracture. Immediately after reoxygenation, hypercontracture was induced in one of the cells by microinjection of extracellular buffer (arrow denotes position of the micropipette during injection). Hypercontracture propagated to the adjacent cell, which completely rounded up in <30 s.

View larger version (15K): [in this window] [in a new window]   FIGURE 3. Diagram illustrating a proposed mechanism for limiting propagation of hypercontracture in reperfused myocardium

Cell-to-cell propagation of hypercontracture to adjacent myocytes during myocardial reperfusion results in increased infarct size. Keeping the GJ closed during the initial minutes of reoxygenation in the globally hypoxic isolated rat heart with 1 mM heptanol, a concentration lacking significant effects on the sarcolemmal channels, results in less enzyme release, less contraction band necrosis (as assessed by quantitative histology), and better functional recovery. In the in situ pig heart submitted to transient coronary occlusion, intracoronary infusion of 1 mM heptanol during the first 15 min of reperfusion reduced infarct size and modified infarct geometry, with more fractionated areas of contraction band necrosis. Maintaining closed GJ in the area at risk during the first minutes of reperfusion seems not to be associated with an increased incidence of lethal arrhythmias (6).

	Cell-to-cell propagation of injury in other tissues

Top Introduction The continuity of contraction... Communication through GJ Cardiomyocyte coupling during... Cell-to-cell propagation of... Cell-to-cell propagation of... References

 
The role of GJ-mediated intercellular communication in the vulnerability of cells to different types of injury has recently been investigated in cell types other than cardiomyocytes (9). In theory, intercellular communication could help to dilute the intensity of noxious stimuli and increase the ability of cells to survive them. However, although a protective effect of GJ communication against oxidative stress has been described in astrocytes (1), there is more information suggesting that cell injury can in fact propagate through GJ to adjacent cells. This phenomenon may be relevant in the case of cancer treatment, since it allows the amplification of death to cells not directly targeted by antineoplastic agents (bystander effect). Moreover, achieving a concomitant increase in connexin expression and GJ communication has been proposed as a promising strategy to improve the effectiveness of gene therapies (10).

The case of ischemia-reperfusion has also been studied in other tissues. Some observations suggest that, in contrast to what is expected, GJ may remain permeable in glial cells during ischemic conditions (3) and that GJ communication may result in expansion of cell death in cerebral ischemia. Moreover, GJ blockade with octanol has recently been described to reduce cell death during cerebral ischemia (12).

In summary, GJ, essential for the development and function of multicellular systems, can also play an important role in the progression of cell injury. In myocardial tissue, there is evidence that myocyte hypercontracture may propagate to adjacent cells through GJ. Cell-to-cell propagation of hypercontracture during reperfusion results in spreading of contraction band necrosis, can explain infarct geometry, and increases infarct size in myocardium submitted to ischemia-reperfusion. Many details of the regulation of GJ permeability, its behavior during ischemia and reperfusion, and its modification by drugs need to be established.

Prevention of cell-to-cell propagation of hypercontracture and cell death appears as a new therapeutic target to enhance myocardial salvage achieved by reperfusion in patients with coronary occlusion. However, drugs able to selectively block GJ in reperfused myocardium without unacceptable side effects or toxicity are still to be developed. Alternatively, the identification of the messengers and mechanisms of propagation of hypercontracture through GJ may provide new targets for its prevention without the problems associated with interfering with electric cell coupling. In this regard, selective inhibition of the Na⁺/Ca²⁺ exchanger in its reverse mode appears as a promising new therapeutic approach.

	Acknowledgments

 
The ideas on which this manuscript is based were generated during the research supported, in part, by the PL-951254 BIOMED-2 program of the European Union and by CICYT-SAF 99/102.

	References

Top Introduction The continuity of contraction... Communication through GJ Cardiomyocyte coupling during... Cell-to-cell propagation of... Cell-to-cell propagation of... References

 

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