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Coronary artery disease is a significant global health issue contributing to a massive burden of morbidity and mortality. One of the manifestations of the condition is acute myocardial infarction (AMI), whereby there is a sudden onset episode of loss of blood supply to an area of the myocardium, resulting most commonly from thromboembolic sequelae. The prompt treatment of AMI requires accurate diagnosis and revolves around rapid re-establishment of coronary artery blood flow to the myocardium. Techniques such as pharmacological thrombolysis and percutaneous coronary intervention (PCI) have become the cornerstones of effective treatment and occupy a sacred position in a multitude of clinical guidelines to that effect. However, when blood flow is re-established in the myocardium there can be inadvertent injury caused to tissue which may be detrimental to the therapeutic process. The term myocardial perfusion injury is correctly used to describe this process, whereby previously viable myocytes die following reperfusion therapy. It is undoubtedly a significant factor in the efficacy of therapy and might help to explain the poor survival rate in individuals who have had an AMI. Indeed, animal studies have suggested that up to 50% of the total final infarct size can be attributed to this phenomenon.
Further research from animal studies has suggested that there may be specific ways to ameliorate damage caused by the process of reperfusion, although there has been significant difficulty in translating this research to effective clinical tools. Some intriguing research suggests that 'preconditioning' of the myocardium to ischaemic episode may be of clinical benefit, following studies in mice where short intervals of ischaemia prior to a larger event demonstrated a significantly smaller infarct size (Hausenloy et al., 2005). The various mechanisms that underlie the nature of the cell death following cellular reperfusion can be attributed to radical oxygen species (ROS), calcium, coronary blood flow and changes in mitochondrial permeability. In addition cell death may be propagated by intercellular gap junction signalling, increasing the extent of apoptosis. The discovery of cardioprotective molecules in the cell, such as members of the protein kinase C (PKC) family, has led to exciting research whereby ischaemic preconditioning may be mimicked by activating specific proteins on a temporal basis, reducing the extent of reperfusion injury. Other protein kinases have been identified as being important in cardioprotection during or following reperfusion injury, which perhaps hold greater therapeutic potential, including members of the reperfusion injury salvage kinase (RISK) pathway (Murphy & Steenbergen, 2008).
The aim of this paper is to consider the mechanisms underlying the initial reperfusion injury and utilise such knowledge as a basis for understanding potential cardioprotective cellular mechanisms. These mechanisms will be discussed in detail, in the context of contemporary understanding of the pathological basis for the condition and then future directions for research will be considered, with an emphasis on the clinical utility of these mechanisms.
Mechanisms underlying myocardial reperfusion injury
Mode of cell death: apoptosis, necrosis and autophagy
A large amount of research has been conducted into he mode of cell death that is responsible for the damage that occurs during reperfusion injury and there is clear evidence that apoptotic, necrotic and mechanisms of autophagy are at work in varying contributions (Murphy & Steenbergen, 2008). Some of this variation may be associated with the experimental model utilised (in vivo versus in vitro systems), however there is increasing evidence that these usually distinct mechanisms may be inter-related in reperfusion injury (Golstein & Kroemer, 2007). For instance, the caspase family of proteins are involved in the process of apoptosis and it has been found that caspases 8 and 9 are activated during myocardial reperfusion injury. They work by cleaving DNA, thus promoting cell death, although this is unlikely to be the cause of cell death in reperfusion injury which happens over the course of several hours. It is proposed that caspases may cleave other cytoskeletal proteins during reperfusion, thus compromising cellular membranes and inducing other forms of cellular death. This would explain the inflammatory component of reperfusion injury, which is not characteristic of apoptosis as cell contents are not extruded and therefore no inflammation occurs (Stephanou et al., 2001; Murphy & Steenbergen, 2008).
The role of autophagy in reperfusion injury remains unclear. Autophagy is usually initiated following cell damage and involves the phagocytosis of organelles, such as mitochondria in order to protect the cell from further physiological damage. Thus, some authors have suggested the phenomenon in the context of reperfusion as protective of further cellular death. However, other studies have demonstrated that inhibition of autophagy activators can result in a reduction of infarct size, seemingly contradicting this hypothesis (Takagi et al., 2007). The story gets more complex, as it has been shown that calpain, a calcium-activated protease, may promote autophagy but also interacts with pro-apoptotic molecules. Thus the two processes may be interlinked, complicating the analysis of the effects of autophagy alone (Hamacher-Brady et al., 2006; Murphy & Steenbergen, 2008).
Necrotic cell death involves extrusion of cellular contents and a significant inflammatory response as intracellular components become exposed to the extracellular milieu. This is a characteristic of myocyte injury, as demonstrable in the clinical detection of creatine kinase following myocardial injury. The regulatory mechanisms of this process are only now being elucidated, however and remain shrouded in mystery (Zong & Thomson, 2006). Whether cytoskeletal protein cleavage results in membrane rupture or loss of ATP during reperfusion injury causes mitochondrial membrane channels to open, leading to destruction of cellular integrity is uncertain. But it is clear that the mechanisms leading to membrane rupture are a core component of reperfusion injury pathophysiology (Murphy & Steenbergen, 2008). Several of these will now be discussed in brief, followed by identification of the key therapeutic targets that emerge from such research.
Instigators of cell death
Cell death in myocardial infarction is often attributed to the initial ischaemic episode, whereby myocyte blood flow is interrupted. However, the clinical importance of what happens after this cannot be disputed: following therapeutic tissue reperfusion there is significant additional tissue death, which has been shown to contribute up to 50% of the total infarct size in experimental models (Boll et al., 2004). The mechanical dysfunction was noted in the early 1960s in the canine heart, including features such as intramitochondrial calcium deposition, contracture of myofibrils and disruption of the sarcolemma (Jennings et al., 1960). Since then it has been recognised that there are four types of injury that occur following reperfusion: myocardial stunning; no-reflow phenomenon; reperfusion arrhythmias; and lethal reperfusion injury (Yellon & Hausenloy, 2007). The first three mechanisms are either transient or have suitable therapeutic modalities to control their impact to the patient. However, lethal reperfusion injury is unique in this respect as it poses a problem to the clinician to this day.
There are numerous mechanisms proposed to account for this injury. The first is the 'oxygen paradox'. Although re-oxygenation of the damaged myocardial tissue is effective in rescuing around 40% of the infarction area, reperfusion can cause oxidative stress which is damaging to myocytes in itself (Zweier, 1988). However, the exact contribution of oxidative stress to this process has been questioned by a variety of researchers, owing to the lack of amelioration with antioxidant therapy (Ly et al., 2001). Additional support for the negative effects of oxygen come from data suggesting that increased levels of oxygen reduce the availability of nitric oxide within the cell, which is an intracellular molecule which potentially cardioprotective effects (Zweier & Talukder, 2006). However, it is not certain whether or not the role of nitric oxide is in the facilitation of effective reperfusion itself or in subsequent cardioprotective signalling (Ishii et al., 2005).
It has also been noted that the level of intracellular calcium increases greatly during cardiac reperfusion as a result of sarcolemma damage. This increased calcium results in hypercontracture of the myocyte and subsequent cell death. In addition the level of calcium in the mitochondria increases to such a degree that proton leakage may be induced, which further adds to the myocyte destruction process. The PTP channel on the mitochondrial membrane is a non-selective channel that allows for ATP depletion when opened by diminishing the transmembrane potential, thus leading to cell death. It has been observed that this channel remains closed during myocardial ischaemia, but opens momentarily during reperfusion, when high calcium levels, oxidative stress and ATP depletion occur. Therefore this channel presents itself as an interesting target for future research (Hausenloy & Yellon, 2003). Several other events have been postulated to induce increased permeability to protons in the mitochondrial membrane, including uncoupling proteins and the cycle of non-esterified fatty acids (Jezek et al., 2004). On a related note, the restoration of physiological pH that occurs following reperfusion has been shown to have profound effects on lethal reperfusion injury through activation of sodium transporters, among other transmembrane proteins (Lemasters et al., 1996).
Following reperfusion of cardiac myocytes an acute inflammatory response is initiated within the first 6 hours following insult. Neutrophils are chemoattracted to the site of injury and infiltrate cardiac tissue in the 24 hours following injury, a process mediated through cell adhesion molecules such as the cadherin family (Vinten-Johansen, 2004). Neutrophils then work by vascular plugging and the release of degradative enzymes along with reactive oxygen species (ROS) to promote endothelial dysfunction and other characteristics of reperfusion injury (Jordan et al., 1999). Therefore it is attractive to suppose that intervention might be targeted against the chemokine signalling or cell adhesion molecules following injury to prevent neutrophil action and accumulation. Experimental models have demonstrated this fact to some extent, however the clinical utility of such effects is open to debate. Indeed, when combined with percutaneous coronary intervention, anti-inflammatory therapy reduced infarct size by 11%, but clinical benefits were perceived only if therapy was delivered within 3 hours following injury (Kloner et al., 2006; Yellon & Hausenloy, 2007).
There is increasing research being conducted on the effects of metabolic modulation through treatment with insulin and glucose on myocyte survival. There is evidence that suggests that glucose metabolism rather then fatty acid metabolism may abrogate injury during reperfusion. However, the cellular mechanisms are not well understood and therapeutic interventions have not demonstrated any significant reduction in clinical characteristics or infarct size following myocardial infarction (Mehta et al., 2005). Modulation of metabolism through therapeutic hypothermia is another area that has attracted some attention in recent years, following evidence that for every 1C that body temperature is reduced, infarct size is reduced by 10% which has been demonstrated in human-sized adult pig hearts. However, the clinical utility of this mechanism has not produced substantial benefit in human studies (Dae et al., 2002; Murphy & Steenbergen, 2008).
Although various lines of research have revealed many potential mechanisms for the modulation of reperfusion injury, there are only a few hypotheses that are backed up with robust evidence. While therapeutic examples of infarct size reduction using metabolic and thermoregulatory measures may be of clinical benefit, their cellular basis is poorly understood. As such the remainder of this paper will focus on those theories which predominate the scientific literature, as a result of concrete evidence in their favour in conjunction with their therapeutic potential. Preconditioning, the RISK pathway, the mitochondrial PTP and postconditioning mechanisms for cardioprotection in acute reperfusion injury shall be discussed in turn.
Cardioprotective mechanisms and targets
The term preconditioning refers to short periods of ischaemia followed by reperfusion prior to sustained ischaemia as in myocardial infarction. When preconditioning occurs infarct size is reduced, lactate production (indicative of anaerobic metabolism) is decreased and levels of ATP fall at a slower rate, suggesting improvements in aerobic metabolism (Stein et al., 2004). These effects are less pronounced if the period of time between short ischaemic episodes and he larger event is increased to approximately 1 hour, suggesting that there is a time-dependent mechanism at work, most likely involving degradation of protective factors or synthesis of damaging agents (Miura et al., 1991; Murphy & Steenbergen, 2008). Hence, there has been a great deal of emphasis placed on the intracellular signalling pathways, which permit synthesis and degradation, and interactions that occur during preconditioning in an attempt to elucidate the protective mechanism(s).
The intracellular signalling cascade is triggered by molecules such as adenosine, bradykinin and opioids which act through G-protein coupled receptors to induce phosphorylation of a number of substrates (Murphy & Steenbergen, 2008). One of the main pathways involved is the phosphoinositide-3 kinase (PI3K) pathway, which is directly activated by such molecules and results in widespread intracellular activity. Indeed, inhibition of this pathway results in a loss of cardioprotective effects of preconditioning (Steenbergen et al., 1987). However, the intracellular molecules involved are more difficult to pin down as there are a great number of phosphorylation targets including Akt, p70S6K, SGK and ERK which can be activated by PI3K either in vitro or in vivo. Although it is beyond the scope of this review to discuss all possible targets at length, there are some candidates that warrant further attention.
Akt is a molecule that has increased levels of phosphorylation during preconditioning and it has been demonstrated that when PI3K mediated activation of Akt is disrupted, there is a reduction in this level of phosphorylation and subsequent cardioprotection (Budas et al., 2006). One way in which Akt may facilitate this protection is through phosphorylation of caspase-9. When this occurs the pro-apoptotic role of caspase-9 is blocked and in addition there is reduced activation of caspase-9 and -3 in response to hypoxia (Uchiyama et al., 2004).
GSK also presents itself as a molecule targeted by the PI3K pathway which may affect apoptosis. Phosphorylation of GSK, which may in itself be mediated by Akt among other factors, results in deactivation of the protein and anti-apoptotic effects. Inactivation is increased during preconditioning and PI3K inhibitors block this effect (Tong et al., 2000). One of the substrates of GSK is glycogen synthase, which becomes more active if phosphorylation by GSK is blocked, as during preconditioning (Murphy & Steenbergen, 2008). It has been suggested that glycogen may be a candidate molecule for providing the 'memory' in preconditioning: reduced levels of glycogen following preconditioning, limiting anaerobic glycolysis and lactate production. This would minimise harmful production of elevated calcium and sodium, thus stabilising cell membranes. In addition, glycogen could be re-synthesized, thus accounting for the ineffectiveness of preconditioning over long periods of time (Steenbergen et al., 1993). However, there have been no reported benefits of glycogen depletion in cardiac tissue during reperfusion injury. Other apoptotic proteins, such as BAX have been reported to undergo phosphorylation via GSK, adding to the interest in this molecule.
The reperfusion injury salvage kinase (RISK) pathway is thought to be important when activated during the onset of reperfusion. Activation can reduce the potential difference across the mitochondrial membrane (stabilising process) providing a protective effect (Hausenloy et al., 2006). This is mediated by preventing the opening of the mitochondrial permeability transition pore (PTP) which permits proton and ATP escape during the reperfusion process (see earlier). Activationof the RISK pathway has been demonstrated with a variety of substances such as atorvastatin, erythropoietin and glucagon-like peptide-1, which all reduce infarct size in experimental models (Yellon & Hausenloy, 2007). Atorvastatin has also been shown to have clinical efficacy by reducing myocardial damage when delivered in high dose during PCI (Patti et al., 2007).
Interestingly the role of the RISK pathway in both preconditioning and postconditioning may be achieved in unison if the pathway is activated during the process of reperfusion (Hausenloy et al., 2005). The activation of this pathway seems to be necessary within the first 30 minutes of the reperfusion process initiation, otherwise the effects of downstream effector molecules are significantly abrogated (Solenkova et al., 2005).
Unique to the process of preconditioning, Zhao et al (2003) demonstrated that intermittent reperfusion following the ischaemic episode could reduce the size of the infarction from 47% to 11% in dog hearts. This was termed postconditioning, although it may be viewed as a modified version of cardiac reperfusion. The overall effects of the process seem to provide strong evidence for efficacy: calcium levels are reduced, physiological pH is restored more swiftly, neutrophil activity and accumulation is stunted, oxidative damage is reduced and apoptotic cell death is attenuated (Yellon & Hausenloy, 2007). Activation of the RISK pathway seems to be involved in this process, although the precise mechanisms are poorly understood, however there is a large amount of overlap between molecules associated with postconditioning and preconditioning.
The PI3K pathway is again implicated: inhibition of the pathway resulted in reversal of cardioprotective effects following reperfusion (Tsang et al., 2004). Akt and p70S6K are also implicated in the intracellular processes. Signalling through protein kinase C (PKC) has also been recognised as important, as inhibition of PKC signals blocks postconditioning-associated cardioprotection (Philipp et al., 2006). This includes reduction in infarction size and translocation of PKC to the mitochondrial membrane. Interestingly, by adding an activator of PKC signalling during reperfusion similar effects to postconditioning may be achieved, highlighting the role of this signalling cascade (Philipp et al., 2006).
The use of ischaemic postconditioning in patients has not been tested on a large scale, although several small studies have indicated that infarction size may be reduced by 36% if myocardial reperfusion is modified (Ma et al., 2006). A similar mechanism, known as remote ischaemic postconditioning, involves induction of transient ischaemic episodes in an organ remote from the heart, including upper limb ischaemia. Trials assessing this technique are in their early stages and thus little can be concluded as to the effectiveness of the approach.
Targeting the mitochondrial PTP
One final aspect of cardioprotection focuses specifically on the mitochondrial PTP, as it has a predominant feature in the pathophysiology of reperfusion injury. Simple clinical evidence can be achieved through opening or closing of this channel, followed by assessment of the cellular and clinical consequences. Suppression of PTP is achieved through cyclosporine and if this is delivers prior to PCI, cardioprotection is achieved (Hausenloy et al., 202). The significance of this effect has been shown through cyclosporine use during the reperfusion process, demonstrating that PTP opening may be responsible for 50% of the total infarct size (Hausenloy et al., 2002). In addition if constituents of the PTP channel are disrupted in murine models then infarct size is similarly reduced- such is the case in mice lacking cyclophilin D (Baines et al., 2005). However, although the infarct size is reduced there does not appear to be an increased level of protection offered by postconditioning, therefore inhibition of PTP in postconditioning can be seen as acting separately. In further support of the role for PTP blockade during postconditioning, recent data has shown that postconditioning maintains an acidotic cellular milieu, which inhibits the action of MTP (Cohen et al., 2007). Adding an alkaline buffer inhibited this effect, strengthening the role of the mitochondrial PTP as a therapeutic target in reperfusion injury (Cohen et al., 2007).
In order for the therapeutic benefits of PTP blockade to be truly realised it will be necessary to develop specific and non-harmful inhibitors of PTP activity for use in a set of patients who often have co-morbidities and previous cardiac damage.
Future research directions
There are a number of theories that demonstrate that a variety of mechanisms can influence the effectiveness of reperfusion following myocardial infarction. The strongest candidate and most likely target for future therapies seems to be the mitochondrial PTP. Future research therefore should focus not only on the identification of intracellular signalling pathways that contribute towards PTP activation/deactivation during reperfusion, but also during normal physiology. Little is known about the role of PTP outside of pathology and therefore further work is necessary to establish the extent to which this may influence the cell (Hausenloy & Yellon, 2007).
Clinical trials are likely to focus on treatments that may be given prior to or during reperfusion procedures, such as PCI as these approaches seem the most practical and clinically advantageous. It should be decided therefore what approaches are optimal- whether or not multiple cell death mediators should be targeted at once, as the maximal infarct size decrease in experiments is 50%, or if pursuing one particular pathway is the safer approach.
Damage to he myocardium during reperfusion therapy is an often under-appreciated phenomenon that is nonetheless vital for clinicians and researchers in the field. Reperfusion results in a number of physiological responses that may propagate infarction, including raised intracellular calcium and sodium, deviation from the physiological pH, neutrophil attraction and accumulation, mitochondrial membrane instability and microvascular changes. The mechanism of cell death has been shown to share characteristics of apoptosis, autophagy and necrosis, suggesting that a large range of intracellular mechanisms are at work, with significant overlap in their functions. Further clarification of these signalling pathways have been demonstrated to some degree by research into preconditioning, postconditioning and the role of the RISK pathway in cardioprotection. It is clear that at the different stages following infarction and through to reperfusion that the same cellular molecules have separate but important effects. Clarification of these pathways is necessary to identify the molecules most amenable to therapeutic modification and the ideal timing of therapy in order to minimise the extent or reperfusion infarction.