Myocardial Ischemia Basic Concepts

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Constantinos Pantos*, Iordanis Mourouzis*, Dennis V. Cokkinos**

1. THE PATHOPHYSIOLOGY OF ISCHEMIA AND REPERFUSION INJURY 1.1. Cellular injury

An imbalance between oxygen supply and demand due to compromised coronary flow results in myocardial ischemia. In theory, the process is very simple; lack of adequate oxygen and metabolic substrates rapidly decreases the energy available to the cell and leads to cell injury that is of reversible or irreversible nature. In practice, the process is very complex. The extent of injury is determined by various factors; the severity of ischemia (low-flow vs zero-flow ischemia), the duration of ischemia, the temporal sequence of ischemia (short ischemia followed by long ischemia), changes in metabolic and physical environment (hypothermia vs normothermia, preischemic myocardial glycogen content, perfusate composition) as well as the inflammatory response. Reperfusion generally pre-requisite for tissue survival may also increase injury over and above that sustained during ischemia. This phenomenon named reperfusion injury leads in turn to myocardial cell death.

Two major forms of cell death are recognized in the pathology of myocardial injury; the necrotic cell death and the apoptotic cell death. The exact contributions of the necrotic and apoptotic cell death in myocardial cell injury is unclear. Both forms of cell death occur in experimental settings of ischemia and reperfusion. Necrotic cell death was shown to peak after 24h of reperfusion and apoptotic cell death was increased up to 72 h of reperfusion, in a canine model of ischemia and reperfusion.1 Furthermore, apoptotic cell death can evolve into necrotic cell death and pharmacological inhibition of the apoptotic signaling cascade during the reperfusion phase is able to attenuate both the apoptotic and necrotic components of cell death.2,3 Apoptosis and necrosis seem to share common

*Dcpartment of Pharmacology, University of Athens, 75 Mikras Asias Ave. 11527 Goudi, Athens, Grecce """Professor of Cardiology, Medical School, University of Athens Greece and Chairman, department of cardiology, Onassis Cardiac Surgery Center

Correspondence: Ass. Professor Constantinos Pantos, Department of Pharmacology, University of Athens, 75 Mikras Asias Ave. 11527 Goudi, Athens, Greece, Tel: (+30210) 7462560, Fax: (+30210) 7705185, Email: [email protected]

mechanisms in the early stages of cell death. The intensity of the stimulus is likely to determine the apoptosis or necrosis.

Necrosis is characterized by membrane disruption, massive cell swelling, cell lysis and fragmentation, and triggers the inflammatory response. The primary site of irreversible injury has been a subject of intense investigation and several hypotheses are postulated. These include the lysosomal, the mitochondrial, the metabolic end-product, calcium overload, the phospholipase, the lipid peroxidation and the cytoskeleton hypotheses, reviewed by Ganote.4

Apoptosis is a programmed, energy dependent process that results in chromatin condensation, DNA fragmentation and apoptotic body formation, preserved cell membrane integrity and does not involve the inflammatory response. Apoptosis occurs during the ischemic phase and can be accelerated during reperfiision5 or can be triggered at reperfusion.6 Apoptosis can progress to necrosis by the loss of ATP in severely ischemic tissue. The cellular processes by which the apoptotic signal is transduced are divided into two basic pathways; the extrinsic and the intrinsic pathway. Figure 1. Both pathways are executed by proteases known as caspases. The extrinsic pathway is a receptor-mediated system activated by tumor necrosis factor-a (TNF-a) and Fas receptors and executed through the activation of caspase-8 and caspase-3.7 Figure 1. Cardiomyocytes have Fas and TNF-a receptors, and cardiac cells produce Fas ligand and TNF-a, which can activate the death receptor mediated pathway.8 Fas ligand and TNF-a are involved in late apoptosis after reperfusion. In hearts from mice lacking functional Fas, apoptosis was reduced 24h later following 30 min of ischemia.9 Fas ligand and TNF-a have not been implicated in apoptosis induced by hypoxia alone. The intrinsic pathway of apoptosis signaling is mediated through the mitochondria and is activated by stimuli such as hypoxia, ischemia and reperfusion and oxidative stress.10 Figure 1. These pro-apoptotic signals induce mitochondrial permeability transition, which is characterized by increased permeability of the outer and inner mitochondrial membranes. The mitochondrial permeability transition pore (MPTP) is a protein complex that spans both membranes and consists of the voltage anion channel (VDAC) in the outer membrane, the adenine nucleotide translocase (ANT) in the inner membrane and cyclophilin-D in the matrix." MPTP opening occurs by mitochondrial calcium overload particularly in the presence of oxidative stress, depletion of adenine nucleotides, increase in phosphate levels and mitochondrial depolarization while low pH is a restraining factor for MPTP opening. In fact, with correction of acidosis at reperfusion this restrain is removed and MPTP opens. MPTP opening occurs mainly at reperfusion but there is an increasing evidence that can also occur during ischemia.12 Opening of the MPTP leads to the release of cytochrome c, Smac/DIABLO, endonuclease G (EndoG) and apoptosis-inducing factor (AIF) all of which facilitate the apoptosis signaling.13,14 Figure 1. Cytochrome c is a catalytic scavenger for the mitochondrial superoxide and loss of cytochrome c results in inactivation of mitochondrial respiratory chain, reactive oxygen species (ROS) production and initiation of apoptosis. Cytochrome c binds to the cytosolic protein Apaf -1 and results in caspase-9 and caspase-3 activation.15 Figure 1. This process can only be executed when sufficient ATP is available. Therefore, cytochrome c release may have little or no consequences on apoptosis with severe ischemia as ATP depletion will limit caspase activation and cause necrosis. Smac/DIABLO indirectly activates caspases by sequestering caspase-inhibitory proteins while EndoG and AIF translocate to nucleus where they facilitate DNA fragmentation. Figure 1. It appears that MPTP converts the mitochondrion from an organelle that provides ATP to sustain cell life to an instrument of programmed cell death if the insult is mild and necrosis if the insult is severe. Interventions which inhibit MPTJP opening and enhance pore closure, either directly in the form of cyclosporin A or sanglifebrin A, or indirectly, in the form of propofol, pyruvate, or ischemic preconditioning are shown to provide protection against ischemia and rcperfusion injury.12

Extracellular Space

Extrinsic Pathway Intrinsic Pathway

Extrinsic Pathway Intrinsic Pathway

Ngf Signal

Figure 1. Schema lie of the apoptotic signaling pathways. The intrinsic apoptotic pathway consists of the mitochondrial pathway, The extrinsic pathway mediates apoptosis through activation of the death receptors, TNF-o/Fas receptors. Apoptosis is executed by activation of proteases, known as caspascs. ATP is essential for apoptosis. Bcl-2 family proteins are apoptosis regulating proteins; Bcl-2 inhibits while Bid, Bax, Bad facilitate apoptosis. An interaction between these two apoptotic pathways exists. See text for a more detailed explanation.

Figure 1. Schema lie of the apoptotic signaling pathways. The intrinsic apoptotic pathway consists of the mitochondrial pathway, The extrinsic pathway mediates apoptosis through activation of the death receptors, TNF-o/Fas receptors. Apoptosis is executed by activation of proteases, known as caspascs. ATP is essential for apoptosis. Bcl-2 family proteins are apoptosis regulating proteins; Bcl-2 inhibits while Bid, Bax, Bad facilitate apoptosis. An interaction between these two apoptotic pathways exists. See text for a more detailed explanation.

The Bcl-2 family of proteins are considered as apoptosis regulating proteins. Members of this family are the Bcl-2 and BcI-xL which are ami-apoptotic while Bax, Bad, Bid, Bini are pro-apoptotic. Pro-apoptotic and a nti-apoptotic Bcl-2 proteins can bind directly to the components of mitochondrial pore, leading to either its opening or closure respectively.16 Figure 1. Alternatively, pro-apoptotic members, such as Bale or Bax, insert into the outer mitochondrial membrane where they oligomerke to form a permeable pore.17 Furthermore, an interaction between the intrinsic and the extrinsic pathway can occur through Bcl-2 proteins. Bid is cleaved by caspase-8 and translocates to the mitochondria and induces permeability pore transition.18 Figure 1. Bcl-2 proteins are regulated through various processes. For instance, phosphorylation of Bad by kinases results to its inactivation while phosphorylaton of Bim leads to its proteosomal degradation.'9'20

1.2. Spread of cell injury

1.2.1 Gap junctions; cell to cell communication

While much has been learned about mechanisms of cell death in cultured cardiomyo-cytes, heart muscle cells in vivo form a functional syncytium and do not exist in isolation. The communication between cells occurs through Gap junctions (GJ). Gap junctions are specialized membrane areas containing a tightly packed array of channels. Each channel is formed by the two end to end connected hemichannels (also known as connexons) contributed by each of the two adjacent cells. Hemichannels are formed by six connexins. Gap junctions are not connected to other cytoskeletal filaments and are not considered part of the cytoskeletal system.21 GJ are now recognized to play an important role in progression and spread of cell injury and death during myocardial ischemia and reperfusion.22 Closure of gap junctions during ischemia was initially thought to occur as a protective mechanism preventing spreading of injury across the cardiomyocytes. However, it is now realized that persistent cell to cell communication can exist during ischemia and reperfusion. In fact, gap junction communication allows cell to cell propagation of rigor contracture and equalization of calcium overload in ischemic myocardium. Although the mechanism of propagation of ischemic contracture is not clear, it can be speculated that cells developing rigor contracture and consuming ATP in an accelerated way may steal gap junction permeable ATP from adjacent cells, decreasing their ATP levels to the critical values at which rigor contracture develops, reviewed by Garcia-Dorado.22

The role of GJ in ischemia and reperfusion injury has been shown in studies using GJ blockers; reduction of necrosis after ischemia and reperfusion was observed in in situ rabbit hearts and in isolated rat hearts with administration of halothane (presumed to be a G J uncoupling agent).23,24 Furthermore, regulation of the phosphorylation of connexin 43 (non phosphorylated Cx43 increases the opening of G J) resulted in modification of ischemic injury. In fact, ischemic preconditioning (brief episodes of ischemia and reperfusion) induced cardioprotection was associated with preservation of connexin 43 phosphorylation.25

GJ opening may also contribute to cardioprotection. Survival signals can be transferred from one cell to another. In fact, cell to cell interaction through GJ has been described to prevent apoptosis in neonatal rat ventricular myocytes.26 The intensity of the stimulus is likely to determine the beneficial or detrimental role of GJ communication. See also chapter 4.

1.2.2 The inflammatory response

Myocardial ischemia is associated with an inflammatory response that further contributes to myocardial injury and ultimately leads to myocardial healing and scar formation. Myocardial necrosis has been associated with complement activation and free radical generation that trigger cytokine cascades and upregulate chemokines expression. Mononuclear cell chemoattractants, such as the CC chemokines CCL2/Monocyte

Chemoattractant Protein (MCP)-l, CCL3/Macrophage Inflammatory Protein (MIP)-l alpha, and CCL4/MIP-1 beta are expressed in the ischemic area, and regulate monocyte and lymphocyte recruitment. Chemokines have also additional effects on healing infarcts beyond their leukotactic properties. The CXC chemokine CXCL 10/Interferon-y inducible Protein (IP)-10, a potent angiostatic factor with antifibrotic properties, is induced in the infarct and may prevent premature angiogenesis and fibrous tissue deposition. Chemokine induction in the infarct is transient, suggesting that inhibitory mediators, such as transforming growth factor (TGF)-beta may be activated suppressing chemokine synthesis and leading to resolution of inflammation and fibrosis, reviewed by Frangogiannis.27 Daily repetitive episodes of brief ischemia and reperfusion in mice resulted in chemokine upregulation followed by suppression of chemokine synthesis and interstitial fibrosis, in the absence of myocardial infarction.28

Interleukin-8 and C5a are released in the ischemic myocardium and may have a crucial role in neutrophil recruitment.29 Neutrophils are cells rich in oxidant species and proteolytic enzymes and can cause cell injury. In fact, annexin 1, a potent inhibitor of neutrophil extravasation in vivo was shown to protect the heart against ischemia and reperfusion injury.30 However, the importance of neutrophil in causing myocardial damage in the context of ischemia and reperfusion is now questioned. Experimental evidence shows that the time course of neutrophil accumulation in postischemic myocardium seems to be different from the time course of injury, myocardial injury is observed in neutrophil free conditions and anti-inflammatory interventions do not consistently limit infarct size, reviewed by Baxter.31

Cytokines also exert direct negative inotropic effects via paracrine and autocrine modulation. This negative inotropic effect appears early (2-5min) and at later stages.32 Tumor necrosis factor (TNF-a), interleukin (IL-6) and (IL-1 ) are all shown to reduce myocardial contractility acting in synergistic and cascade-like reactions.

The heart is a tumor necrosis factor-a producing organ (TNF-a). TNF-a is produced in response to stress. Macrophages and cardiac myocytes themselves synthesize TNF-a and TNF-a is also released by mast cells. TNF-a is an autocrine contributor to myocardial dysfunction and cardiomyocyte death in ischemia and reperfusion injury. Ischemia-reperfusion induced activation of p38 MAPK results in activation of the nuclear factor kappa B (NFkB) and leads to TNF-a production. During reperfusion, TNF-a release occurs early (from mast cell activation) as well as at a later phase as a result of de novo synthesis possibly induced by TNF-a itself and /or intracellular oxidative stress. Antioxidant treatment and mast cell stabilizers have been shown to prevent TNF-a release.33 TNF-a depresses myocardial function by nitric oxide independent (sphingosine dependent) (early effect) and nitric oxide dependent (later effect) mechanisms. Sphingosine is produced by the sphingomyelin pathway which inhibits calcium release from sarcoplasmic reticulum (SR) by blocking the ryanodine receptor. Activation of TNF-a receptor or Fas also induces apoptosis. However, TNF-a at low doses before ischemia and reperfusion is shown to be cardioprotective through a reactive oxygen species dependent signaling pathway.34

IL-1 increases nitric oxide (NO) production by upregulating the synthesis of iNOS.35 This cytokine acts also via an NO-independent mechanism and causes downregulation of calcium regulating genes with subsequent depressed myocardial contractility.36

IL-6 levels are elevated in patients with acute myocardial infarction. IL-6 is secreted by mononuclear cells in the ischemic area and is also produced by cardiac myocytes. IL-6 apart from its inflammatory effect regulates contractile function by its acute effect on calcium transients.37

Complement activation also contributes to ischemic injury. Current evidence indicates that ischemia leads to the expression of neoantigen or ischemia antigen on cellular surfaces, and this induces binding of circulating IgM natural antibody. This immune complex causes CI binding, complement activation and the formation of C3a and C3b. C3b activates the remainder of the complement cascade leading to the formation of the membrane attack complex, which is the principal mediator of injury. Complement inhibition results in less myocardial ischemia and reperfusion injury, reviewed by Chan.38

Platelet-activating factor (PAF) is released during ischemia and reperfusion injury from non cardiac cells and cardiomyocytes. PAF is rapidly synthesized during ischemia and reperfusion from membrane phospholipids after sequential activation of phospholi-pase A2 and acetyl-transferase. The effect of PAF is mediated through specific PAF cell surface receptors that belong to G protein-coupled receptors. It depresses cardiac contractility by negatively regulating calcium handling. Furthermore, PAF stimulates the release of other biologically active mediators such as eicosanoids, superoxide anions and TNF-a that can further enhance myocardial injury. An adverse effect of PAF is also mediated by the induction of vascular constriction and capillary plugging.39

Despite the potential injurious effect, the reperfusion inflammatory response also triggers the healing process. Accumulation of monocyte derived macrophages and mast cells increase expression of growth factors inducing angiogenesis and fibroblast accumulation. Inflammatory mediators may induce recruitment of blood derived primitive stem cells in the healing infarct which may differentiate into endothelial cells and even lead to myocardial regeneration.40

Matrix metalloproteinases (MMPs) and their inhibitors regulate extracellular matrix deposition and play an important role in ventricular remodeling. Three MMPs (MMP-1, MMP-2, and MMP-9) appear to be of importance, with each enzyme being generated from different sources and most likely responsible for different aspects of the pathological process of tissue necrosis and healing. MMP-1, which is activated through a p38 MAPK dependent pathway (either directly or indirectly), can induce cardiomyo-cyte death that might contribute to the immediate lethal injury observed within the first few minutes of reperfusion. MMP-2, which could be present intracellularly or possibly released from platelets activated by ischemia, appears to play a very early role following myocardial reperfusion, where, it is involved in the breakdown of the contractile apparatus, resulting in cellular injury and in the functional consequence of impaired myocardial contractility. MMP-9 is most closely associated with neutrophils, which are known to infiltrate injured tissue later at reperfusion, where, it is likely to contribute to the extension of cellular death, reviewed by Wainwright.41 MMP effects can be modulated by the tissue inhibitors of MMP, the TIMPs and the extent of injury seems to be determined by TIMP/MMP balance during ischemia and reperfusion. In fact, angiotensin II is shown to modulate this balance and in an in vivo dog model of regional ischemia and reperfusion, inhibition of angiotensin II type 1 receptor by valsartan resulted in protection by increasing TIMP-3 expression and improving the balance of TIMP-3 /MMP-9.42

1.3. Microvascular injury

Endothelial dysfunction and microvascular injury start at the interphase of the endothelium with the bloodstream. Reperfusion of ischemic vasculature results in production of excessive quantities of vasoconstrictors, oxygen-free radical formation and neutrophil activation and accumulation. Neutrophils and macrophages further increase the formation of reactive oxygen species and act as an amplifier of the ischemic injury. Severe microvascular dysfunction can arrest the microcirculation, a phenomenon known as the "no reflow phenomenon". Capillaries may be occluded by extravascular compression, endothelial swelling or intravascular plugs, such as platelet aggregates and thrombi. In experimental models of temporary coronary artery occlusion, tissue perfusion at the microvascular level remains incomplete even after the patency of the infarct related epicardial coronary artery is established, and distinct perfusion defects develop within the risk zone. A major determinant of the extent of no reflow seems to be the infarct size itself. Reperfusion related expansion of no reflow zones occurs within the first hours following reopening of the coronary artery with a parallel reduction of regional myocardial flow. On a long-term basis, tissue perfusion after ischemia and reperfusion remains markedly compromised for at least 4 weeks, reviewed by Reffemann.43

1.4. Biochemical aspects of ischemia-reperfusion

The heart is a metabolic omnivore that is able to use a variety of substrates to generate energy, including exogenous glucose, fatty acids, lactate, amino acids and ketone bodies as well as endogenous glycogen and triglycerides. Glucose taken up by the myocardium is rapidly phosphorylated and either is catabolized or incorporated into glycogen. Myocardial glucose uptake is facilitated by transport via the glucose transporters GLUT1 (basal glucose uptake) and GLUT4 (insulin or stress mediated glucose uptake). Glucose is converted to pyruvate by sequential series of reactions, termed the glycolytic pathway. Figure 2. The enzymes hexokinase and 6-phospho-fructo-kinase-l are among the major sites regulating flux through this pathway. Pyruvate which may also be derived from glycogen or lactate is converted to acetyl-CoA in the mitochondria, and pyruvate dehydrogenase determines the extent of this reaction. Figure 2. Acetyl-CoA enters the tricarboxylic acid cycle (TCA) for oxidation (i.e. generation of H+) and combustion (i.e. generation of C02). Long chain fatty acids uptake is facilitated by membrane-bound and cytosolic fatty acid-binding proteins. They are esterified with coenzyme-A prior to incorporation into triacylglycerols or transport to mitochondrial matrix (regulated by carnitine palmitoyltransferase-!, CPT-1). Figure 2. In the mitochondria, fatty acid oxidation occurs by means of the p-oxidation spiral (FAO) and tricarboxylic acid cycle (TCA). Figure 2. The NADH2 and FADH2 formed from glycolytic pathway, TCA cycle and fatty acid oxidation are oxidized in the respiratory chain and the energy generated from the transport of electrons to oxygen is the driving force for ATP production. Figure 2. This process is known as oxidative phosphorylation.

In response to ischemic stress, several changes in the metabolic pathways and energy production are observed in cytosol and mitochondria followed by changes in membrane ion homeostasis and morphological alterations in subcellular organelles (See chapter 1). These changes might contribute to cell survival under anaerobic conditions. Glucose uptake is increased either by the translocation of glucose transporters to the membrane or by the orientation of the transporters within the sarcolemma. Glycogen breakdown is enhanced. The glycolytic rate increases (the key enzyme 6-phospho-fructokinase-l is activated). However, pyruvate cannot entry the TCA cycle because pyruvate dyhydrogenase (PDH) is inhibited. Instead, it is converted to lactate and alanine. Figure 2. Accumulation of NADH2 in cytosol is increased due to its reduced removal by mitochondria (inhibition of the malate-aspartate cycle) and is counterbalanced by NADH2 conversion to NAD through the formation of lactate from the pyruvate. Figure 2. Mild acidosis develops from

ATP hydrolysis and this could be seen as beneficial; competes with calcium and decreases contractility, inhibits nucleotidases and prevents further breakdown of AMP. AMP activates AMP kinase with subsequent increase in the rate of glycolysis and fatty acid oxidation. Figure 2. AMPK is responsible for the activation of glucose uptake and glycolysis during low-flow ischemia and seems to play an important protective role in limiting damage and apoptotic activity associated with ischemia and reperfusion in the heart.44

Glucose derived ATP preserves sarcolemmal pump function and membrane integrity while glycogen breakdown derived ATP (present at myofibrils and possibly at the sarcoplasmic reticulum) supports cell contractile function. Under normoxia these functions are supported by oxidative phosphorylation derived ATP. With more severe ischemia, the progressive accumulation of the end-products of anaerobic metabolism inhibits glucose uptake and glycolysis. Thus, severe ATP depletion and acidosis occur. Fatty acyl-CoA derivatives accumulate resulting in cell damage of irreversible nature, reviewed by Opie.45

Loss of the activity of the respiratory complexes occurs during ischemia. Progression of the ischemic damage is shown to progressively inhibit the respiratory chain with complex I activity to be lower in less severe ischemia and complex IV activity to be reduced in severe ischemia.46 Mitochondrial changes during ischemia and reperfusion result in increased production and accumulation of reactive oxygen species. The energy transport from mitochondria to cytosol is also impaired. Adenine nucleotide translocase and mitochondrial creatine kinase activity (enzymes that are required for transportation of ATP from the mitochondria to the cytosol) is reduced with subsequent impaired ATP transportation into the sites of utilization. ATP in the mitochondrial matrix is hydrolyzed by the reversal of the ATP synthase, reviewed by Opie.45

Selective inhibition of TCA cycle enzymes aconitase and a-ketoglutarate dehydrogenase, both known to be sensitive to in vitro oxidative modification occurs at reperfusion. TCA enzymes activation does not decline with ischemia. As a consequence the production of NADH2 and a rise in reactive oxygen production occurs.47'48 Glucose metabolism is limited to the cytosolic pathway (increased glycolysis and glycogen synthesis) while fatty acids are oxidized at high rates. Malonyl-coenzyme (CoA) production is decreased and facilitates fatty acid transport to mitochondria. Increased fatty acid metabolism by |3-oxidation represses glucose metabolism due to its inhibitory effect on the pyruvate dehydrogenase activation. The imbalance between glucose and fatty acid oxidation leads to the decrease in cardiac efficiency, reviewed by Lopaschuk.49

Energy depletion and acidosis result in several changes in ion homeostasis with important physiological consequences. Sodium enters the cell due to the inhibition of the sodium pump (Na+/K+-ATPase) and enhanced activation of the sodium-proton exchanger (NHE). NHE is important for correcting cell acidosis. NHE activity is regulated by a variety of G-protein coupled receptor systems. An increase in NHE activity occurs in response to the activation of the a,-adrenergic, angiotensin (AT,), endothelin and thrombin receptors. (3, -adrenergic stimulation inhibits NHE activity while stimulation of adenosine A, and angiotensin (AT2) receptors have a modulatory effect and attenuates NHE activation induced by other ligands, reviewed by Avkiran.50 In the setting of ischemia and reperfusion most of these stimulatory and inhibitory systems are operated and ultimately modulate NHE activity with detrimental or protective effects. NHE 1 mRNA is increased after global ischemia (30 min) and apoptosis is induced in a mitochondrial calcium-dependent manner.51,52 However, the expression of NHE1 is found to be decreased in the non infarcted myocardium in a rat model of acute myocardial infarction.53 Furthermore, mice with a null mutation in the NHE1 exchanger are resistant to ischemia and reperfusion injury.54

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