B

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Cytosol

Figure 3, Ion homeostasis under nonnoxia (A) and hypoxia or ischemia (B). Ion gradients for Na* and K+ are maintained by the operation of Na+/K~-ATPase. The increased intracellular Ca!+ following triggered Ca2+ release from the sarcoplasmic reticulum (SR) is reduced from Ca1' reuptake by the SR, and outward Ca2+ transport by the NaVCsP exchanger and C'a2+ pump, (B). lit hypoxia or ischcmia, NaVK'-ATPase activity declines and intracellular sodium increases whereas potassium decreases. A further increase in sodium occurs due to the operation of NaVHH exchanger aiming to correct acidosis. Sodium is removed in exchange to Ca;* by the operation of Na"/Ca2+ exchanger in reverse mode. Ca;+ reuptake by SR and outward Ca;' transport by Ca2+ pump is inhibited and intracellular calcium increases. Sodium and calcium overload induces cell damage. Opening of the sarcolemmal and mitoehontrial ATP dependent potassium channels occurs and potassium leakage is increased.

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Cytosol

Figure 3, Ion homeostasis under nonnoxia (A) and hypoxia or ischemia (B). Ion gradients for Na* and K+ are maintained by the operation of Na+/K~-ATPase. The increased intracellular Ca!+ following triggered Ca2+ release from the sarcoplasmic reticulum (SR) is reduced from Ca1' reuptake by the SR, and outward Ca2+ transport by the NaVCsP exchanger and C'a2+ pump, (B). lit hypoxia or ischcmia, NaVK'-ATPase activity declines and intracellular sodium increases whereas potassium decreases. A further increase in sodium occurs due to the operation of NaVHH exchanger aiming to correct acidosis. Sodium is removed in exchange to Ca;* by the operation of Na"/Ca2+ exchanger in reverse mode. Ca;+ reuptake by SR and outward Ca;' transport by Ca2+ pump is inhibited and intracellular calcium increases. Sodium and calcium overload induces cell damage. Opening of the sarcolemmal and mitoehontrial ATP dependent potassium channels occurs and potassium leakage is increased.

"RyR"=Ryanodine receptor, Increase, #= Dccrcasc particularly in the setting of zero-flow global ischemia. The time of the development of contracture seems to coincide with the decrease in ATP availability and corresponds to fully depleted glycogen. Glycogen-derived ATP is present mainly at the myofibrils, and possibly at the sarcoplasmic reticulum (SR), thereby influencing ischemic contracture. Furthermore, glycogen and glycogen-metabolizing enzymes appear to co-locate with the sarcoplasmic reticulum, the main site of Ca2+ regulating proteins.59 Ischemic contracture occurs earlier in glycogen depleted hearts such as the hyperthyroid or ischemic preconditioned hearts and is delayed in hearts in which myocardial glycogen content is increased.60,61 The profile of ischemic contracture is not necessarily related to the postischemic recovery of function. A dissociation between ischemic contracture and cardioprotection has been observed. Interventions such as ischemic preconditioning or chronic thyroid hormone treatment although reduce preischemic glycogen content and exacerbate contracture, increase postischemic recovery of function.60,61 Figure 25.

1.5.2 Hypercontracture

Hypercontracture develops immediately upon reperfusion. Figure 5. Reperfusion-induced hypercontracture might either originate from calcium overload or it is of rigor type. Figure 4. Following the ischemic period, cells are calcium overloaded and this is aggravated upon reperfusion by the persistence of the reverse mode of action of the sodium-calcium exchanger. Furthermore, re-oxygenation causes re-energizing of the sarcoplasmic reticulum which in turn starts to accumulate calcium and once full, releases calcium. Calcium movements lead to oscillatory cytosolic calcium elevations that provoke an uncontrolled myofibrilar activation fuelled by the resupply of ATP, reviewed by Piper.62 Figure 4.

Reoxygenated cardiomyocytes are in acute jeopardy of the calcium overloaded contracture as long as mitochondrial energy production recovers rapidly upon reperfusion. However, after prolonged ischemia, this mechanism of contracture development is less likely to occur. With the progression of ischemic cellular damage, the ability of the mitochondria to rapidly restore a normal cellular state of energy upon reoxygenation is reduced. Cardiomyocytes, during the early phase of reoxygenation, may contain very low ATP concentrations that provoke rigor contracture. In cases where rigor contracture prevails, therapeutic actions aiming at cytosolic calcium overload are not effective since rigor contracture is essentially calcium independent, reviewed by Piper.62

There are several lines of evidence suggesting that postischemic necrosis and hypercontracture are causally related phenomena. Reperfusion injury of the myocardium is a complex phenomenon consisting of several independent etiologies. During the earliest phase of reperfusion (minutes), the development of cardiomyocyte hypercontracture seems to be the primary cause of cardiomyocyte necrosis. Thereafter, lasting for hours, various additional causes can lead to cell death by necrosis or apoptosis. Furthermore, vascular failure may aggravate cardiomyocyte injury. In cardiac surgery, when hearts are reperfused after prolonged ischemia or unsatisfactory intra-operative cardioplegia, reperfusion provokes the "stone heart" phenomenon i.e. a stiff and pale heart resulting from massive muscle contracture. Histologically, stone hearts present hypercontracted myofibrils and ruptured cellular membranes. A similar pattern of contracture and necrotic cell injury, termed 'contraction band necrosis" is also observed after regional ischemia and reperfusion.4 In the heart in situ, exposed to transient coronary occlusion, the area of necrosis is shown to be composed almost exclusively of contraction band necrosis.63'64

-1 Inhibition

Figure 4. Contraction results when the heads of the thick myosin filaments interact with the thin actin filaments (strong cross-bridge). This process is initiated by triggered release of calcium from SR at the expense of ATP. Reduced calcium and ATP resupply result in myosin-actin detachement (weak cross-bridge) and relaxation. Upon ischemia, the strong cross-bridge state is maintained due to the low levels of ATP and ischemic contracture of rigor type develops. At the time of reperfusion, despite ATP resupply, a state of a strong cross-bridge between actin and myosin can occur due to calcium oscillations (calcium overload hypercontracture). Hypercontracture of a rigor type can develop at reperfusion following prolonged ischemia where severe ATP depletion occurs.

"RyR'-Ryanodinc receptor, *= Increase, *= Decrease"SR"= Sarcoplasmic Reticulum

The extent of contraction band necrosis is shown to correlate well with the magnitude of macroscopic myocardial shrinkage during the first minutes of reperfusion, and with the magnitude of the enzyme release occurring during the initial minutes of reflow.65

1.5.3 Myocardial Stunning

Myocardial stunning is defined as transient contractile dysfunction that appears after reperfusion despite the absence of irreversible damage and restoration of normal or near normal coronary flow.66 In rat models, stunning has been induced by global ischemia in isolated heart preparations. In rabbit models, multiple, completely reversible episodes of regional ischemia result in stunning. In large animals, a single or multiple, completely reversible episode(s) of regional ischemia or prolonged coronary stenosis (without necrosis) were shown to induce myocardial stunning, reviewed by Kim.67 Although the pathogenesis of myocardial stunning has not been definitively established, the two major hypotheses are that it is caused by the generation of oxygen derived free radicals and by calcium overload during reperfusion. These two hypotheses are not mutually exclusive and are likely to represent different facets of the same pathophysiological cascade.68

The first hypothesis was primarily tested in large animals and has been proved by experimental evidence such as the increase in reactive oxygen species (ROS) production in stunned myocardium,69 the protection against stunning by antioxidants70 and the contractile dysfunction induced by direct exposure to ROS.71 Calcium overload is thought to be the possible mechanism through which ROS can induce stunning.72 The calcium hypothesis postulates that stunning is due to calcium overload that occurs during the early phase of reperfusion secondary to intracellular sodium overload following metabolic inhibition of the sodium-potassium ATPase. In fact, NMR measurements show an increase in intracellular sodium during ischemia and reperfusion73 and reperfusion with perfusates containing low calcium concentration resulted in attenuation of stunning.74 Another possible mechanism through which calcium can be implicated in stunning is the activation of calcium dependent proteases. These proteins, known as calpains are enzymes that cleave other proteins when calcium is elevated. This might lead to proteolysis of the troponin I (Tnl) that together with the damage to other contractile proteins (a-actinin, myosin light chain-1) result in decrease in calcium responsiveness.75 Direct exposure of cardiac myofilaments to activated calpain I is shown to reproduce the phe-notype of stunned myocardium with blunted sensitivity and depressed maximal force. Furthermore, these effects were prevented by coincubation with excess calpastatin, the natural inhibitor of calpain.76 A phenotype of stunning, characterized by reduced myofilament calcium sensitivity has been also produced in transgenic mice expressing the major degradation product of Tnl induced by calpain.77 However, in vivo studies in large animals do not confirm the presence of Tnl degradation, indicating that this is not a universal feature of myocardial stunning.78 It is likely that the proteolysis of Tnl observed in the isolated rat heart preparations might be the effect of the increased diastolic pressure and not that of the calcium mediated proteolysis.

The absence of irreversible cellular damage in stunned myocardium may correspond to an increased resistance of the heart to ischemia. Myocardial stunning may trigger the expression of different sets of genes acting to protect the myocardium against irreversible injury. In fact, it has been recently shown that in a swine model of regional reversible ischemia, stunned and normal areas within the same heart corresponded to different gene expression. Interestingly, more than 30% of the genes which were upreg-ulated in the stunned myocardium are known to be involved in different mechanisms of cell survival including resistance to apoptosis, cytoprotection and cell growth. It seems that gene response matches the flow reduction.79

Stunning resolves spontaneously and it can be viewed as a protective mechanism which should be given sufficient time to recover. However, in clinical settings where stunning impairs myocardial function to the extent that compromises other organ perfusion it requires treatment.

1.5.4 Myocardial Hibernation

Myocardial hibernation is an adaptation caused by chronic or intermittent reduction in coronary flow characterized as reduced regional contractile function that recovers after removal of the artery stenosis. A "subacute downregulation" of contractile function in response to reduced regional myocardial blood flow can occur, which normalizes regional energy and substrate metabolism but does not persist more than 12-24 h. Chronic hibernation develops in response to episodes of myocardial ischemia and reperfusion, progressing from repetitive stunning with normal blood flow to hibernation with reduced blood flow, reviewed by Heusch.80

Salient features of the hibernating myocardium are the increase in glucose uptake out of proportion to coronary flow (metabolism/perfusion mismatch)81 and the increase in myocardial glycogen content with ultrastructural characteristics resembling those of the fetal heart.82

Morphological changes are observed in long-term hibernation. The morphology of hibernating myocardium is characterized by both adaptive and degenerative features. The number of myofibrils is reduced while the number of mitochondria and glycogen deposits are increased after 24 h and these changes are reversed in a week following the release of the coronary stenosis. Other morphological changes include a variable degree of fibrosis and expansion of the interstitium by increased infiltration of macrophages and fibroblasts together with collagen deposition. Extracellular matrix proteins (such as desmin, tubulin and vinculin) are increased, indicating disorganization of the cytoskeleton. Mitochondria are small and doughnut like. Depletion of sarcomeres and sarcoplasmic reticulae is observed and glycogen is seen to fill the place previously occupied by filaments. These changes result in myocytes that appear to be de-differentiated. Apoptosis has also been identified in biopsies taken from hibernating myocardium at the time of surgical revascularization. It is suggested that cellular de-differentiation may lead to apoptosis but this finding has not been documented in humans, reviewed by Heusch.80

The pathophysiology of hibernation remains under intense investigation. In short term hibernation, the only possible mechanism that has been identified is reduced calcium responsiveness.83 In long-term hibernation, changes seem to correspond to a genetic program of cellular survival that is induced probably from repetitive episodes of ischemia and reperfusion. Heat shock protein 70 and hypoxia-inducible factor-la are upregulated in the hibernating myocardium.84 Furthermore, iNOS and cyclooxygenase-2 immunoreactivity are increased in hibernating human myocardium resembling the survival gene program of delayed preconditioning.85 Enhanced expression of chemokines at the level of mRNA accompanied by extensive macrophage infiltration and fibrosis has been observed in a mouse model with repeated brief episodes of ischemia and reperfusion (with absence of infarction and decreased regional contractile function).28 Similarly, in human hibernating myocardium, tumor necrosis factor-a (TNF-a) and iNOS mRNA were higher than in the remote control myocardium.86 Interestingly, these features of inflammation are also found with microembolization where a contractile

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