This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.
Ischemic heart disease is one of the clinical problems causing myocardial damage, arrhythmias and stunning. Reperfusion through thrombolysis or percutaneous coronary angioplasty is a standard treatment in impending acute myocardial infarction . Growing evidence from both animal experiments and clinical observations indicates that apoptosis plays a key role in myocardial I/R injury [2-4]. Recent studies demonstrated that blocking the signal transduction leading to apoptosis significantly reduces myocardial infarct size and improves cardiac function, suggesting that apoptosis contributes to I/R injury, and that anti-apoptotic treatment may improve clinical outcomes in patients with ischemic heart disease [5, 6].
Various intracellular signaling pathways are thought to play a critical role in the myocardial response to ischemia and remodelling. Multiple mitogen-activated protein kinase (MAPK) are activated during ischemia and may contribute to the structural and functional changes. Three of the five major MAPK cascades have been extensively studied in the heart: extracellular signal-regulated kinase (ERK1 and ERK2), c-Jun N-terminal kinases (JNK1 and JNK2) and p38 kinases. It has been shown that JNK and p38 contribute to, whereas ERK1/ERK2 protect against, apoptotic cell death. The intracellular signals following infarction lead to diminished contractility, apoptosis, fibrosis and ultimately heart failure. Although inhibition of p38 has been shown to be cardioprotection when given prior to myocardial ischemia, its effect given after ischemia is unclear. Thus, it is of interest to investigate the effect of p38 inhibition of various time points both before and after coronary occlusion. Therefore, the aim of this study is to investigate whether inhibition of p38 at different times of I/R injury could protect the heart from I/R damage in rats. Rats will be divided into 11 groups that mimic the real situations in patients with acute myocardial infarction that go to the hospital at various times after the onset of acute myocardial infarction. The physiological, biochemical and mitochondrial functions of the heart will be studied in a rat model of myocardial I/R injury. ECG limb leads and pressure-conductance catheter will be used to determine the cardiac physiological functions after receiving p38 inhibitor. Furthermore, area at risk and infarct size will be assessed. Cardiac biochemical functions will be studied using western blot analysis to determine the effect of p38 inhibitor on myocardial apoptosis and p38 phosphorylation. The effects of p38 inhibitor on cardiac mitochondria in I/R injured hearts will also be investigated. The knowledge from this study should provide the basis for p38 inhibitor as a possible therapeutic benefit in acute myocardial infarction.
Myocardial ischemia/reperfusion injury is a major risk factor for patients undergoing treatment for many cardiac diseases, including percutaneous coronary angioplasty, coronary artery bypass grafting, heart and lung transplantation, and other cardiac procedures that require cardiopulmonary bypass [8-11]. Although the pathophysiology of myocardial I/R injury remain largely unknown, activation of mitogen-activated protein kinase (MAPKs), including p38 MAPK, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), are indicated as early events. It has been shown that JNK and p38 are associated with cellular response to stress, whereas ERK activity is associated with growth and development process in the cell. Furthermore, it has evidence that increasing p38-MAPK (or p38) activation during prolong ischemia accelerate injury and when inhibition of p38 with pharmacokinetic or genetic means can decrease rate of infarction and death [12, 13].
p38-mitogen-activated protein kinase
The activation of p38-MAPKs in myocytes has not been as fully characterized, but these MAPKs are activated by the cellular stresses that have been examined, including radiation, ultraviolet light, heat shock, osmotic stress, proinflammatory cytokines such as interleukin-1, tumor necrosis factor, and certain mitogens  in addition to myocardial ischemia [12, 15-17]. Activation of p38-MAPK by cellular stresses is also phosphorylate two homologous protein kinases, MAPK-activated protein kinases 2 and 3 (MAPKAPK2 and MAPKAPK3) . The presence and activation of MAPKAPK2 has been clearly demonstrated in the heart [19, 20]. MAPKAPK2 and MAPKAPK3 have overlapping substrate specificities, and both phosphorylate the small heat-shock protein Hsp25/2735 to increase its cytoprotective activity, an action that involves stabilization of the actin cytoskeleton . MAPKAPK2 may also directly regulate the activity of transcription factors (eg, CRE binding protein) . Furthermore, p38-MAPKs also phosphorylates other second transcription factor, ATF2, myocyte enhancer factor 2C (MEF2C) and C/EBP homologous protein (CHOP) (Figure 1) . The regulation of these transcription factors has not yet been studied in cardiac myocytes.
p38-MAPKs consist of 4 isoforms (α, β, δ and γ) which have preserved structure but variable sensitivity to pharmacological inhibition. All 4 isoforms have a Thr180-Gly181-Tyr182 (TGY) dual phosphorylation motif which is used by investigators to infer activation. Two relative specific chemical inhibitors of p38α and p38β, SB203580 and SB202190 have been identified [18, 23-26]. They found that p38α and β have high sequence homology and share sensitivity to pharmacological inhibition by prydinyl imidazole molecules (such as SB203580) but have only 60% homology with p38γ and δ, which are resistant to SB203580 (SB) inhibition . Of the SB-sensitive isoforms, p38α is the predominant form in human and rodent myocardium [28-31]. Studies with knockout mice and cells have shown that p38α is essential for embryonic development as knockout of the α isoform results in embryonal lethality, but mice lacking p38β, p38γ, and p38δ are viable [32-35]. Furthermore, they found that SB203580 at concentration normally used (³10 µmol/l) inhibits recombinant JNK2 in vitro38 and at least two JNK isoforms in the heart .
p38 and ischemia/reperfusion injury
Ischemic heart disease is the main cause of death in the United Kingdom. In the present, the most effective method of reducing mortality in patients with myocardial infarction (MI) is to achieve rapid reperfusion by lysis or mechanical disruption of the occlusive coronary thrombus and plaque. The mortality from acute MI under these events is related to the amount of myocardial salvage achieved by reperfusion. Therefore, it has an importance for verify the exactly mechanisms of I/R injury for decreasing the rate of injury and mortality after I/R treatment.
It was fist demonstrated as early as 1996 that p38α and β are activated in response to ischemia and reperfusion in the heart  and it is clear that these stresses powerfully activate the stress responsive MAPKs in the intact isolated rat heart. p38s phosphorylate a number of known transcription factors to alter their transactivating potential influencing gene expression. However, the immediate downstream targets of p38 that aggravate myocardial injury are still largely unknown. One downstream substrate of p38α is MAPKAP2 which are activated during ischemia, and their activation is sustained or increased during reperfusion [15, 37]. MAPKAP2 can phosphorylate HSP27, a heat shock protein, which is thought to confer a number of protective effects . In addition, phosphorylation of MAPKAP2 can also result in phosphorylation of factors that transactivate cytokine genes, such as TNF-α, a cytokine implicated in chronic heart failure. The activation of MAPKAPK2 is completely inhibited by SB203580, implicating particularly p38α and/or p38β in its activation in the heart .
There is numerous studies from preclinical investigation demonstrated the effects of p38 inhibitor during I/R injury in both in vitro, ex vivo and in vivo models. In in vitro model that simulated I/R in rat neonatal cardiomyocytes, found that p38 inhibitor, SB203580, administration during ischemia at concentration 10 µM protect cell death by inhibition of p38 phosphorylation, decreasing creatine kinase (CK) and lactate dehydrogenase (LDH) releasing [12, 29, 38-42]. In working isolated perfused heart, inhibition of p38 during prolonged ischemia improve cardiac functions, slow the rate of infarction/death and inhibits the production of inflammatory cytokines, such as TNF-α, IL-1 and IL-8, which aggravate ischemic injury [43-47]. Furthermore, in in vivo myocardial ischemia model, administration of p38 inhibitor attenuate cardiac remodeling, improve cardiac functions, decrease infarct size and delays cell death.
p38-MAPKs and apoptosis
The role of p38α and/or p38β on proapoptotic during myocardial ischemia have been shown by protection of cardiac myocytes from ischemic damage using a selective p38α/p38β isoform inhibitor, SB203580 . Previous studies in adenoviral-mediated expression of p38α and p38β in rat neonatal cardiomyocytes have shown that after simulated ischemia p38α was activated, whereas p38β activation was significantly inhibited, resulted in an increase in cell viability . This event have been supported by Wang et al. who suggested that p38α activation in cardiac myocytes is sufficient to cause apoptosis whereas activation of the β isoforms leads to protection and hypertrophy . However, there is some evidence to suggest that p38 isoforms may have potential protective function and suggest a possible adverse effect of prolonged p38 inhibition in the heart.
There are several studies shown the effect of p38 inhibitor on apoptosis. In rat neonatal cardiomyocytes that simulated hypoxia/reoxygenation demonstrated that SB203580 abrogated activation of p38-MAPK, translocation of HSP27, prevented cytochrome c release, caspase-3 activation and DNA fragmentation [42, 49]. As in the isolated rat heart and in in vivo myocardial I/R shown that SB203580 inhibit p38-MAPK activation during ischemia resulted in less MAPKAPK2, caspase-1, caspase-3, caspase-11 activation and DNA fragmentation after myocardial ischemia as well as increasing functional recovery of the heart [50, 51].
p38-MAPKs and cardiac electrophysiological alterations
In the heart, Connexin 43 (Cx43) is the most prominent connexin in the mammalian ventricular myocardium and form gap junctions that mediate electrical coupling between cardiomyocytes, forming the intercellular pathways that allow orderly spread of the wave of electrical excitation responsible for synchronous contraction. Therefore, the normal cardiac rhythm largely depends on the coupling of cardiomyocytes by gap junctions. Previous studies showed that reduced expression of Cx43 during acute MI results in significant reduction in conduction velocity and increased the incidence of ischemia-induced ventricular arrhythmias [52, 53]. These results suggest that the alteration of Cx43 expression may contributes to the genesis of ventricular arrhythmias during MI. Therefore, the influence of abnormal gap junction function on myocardial electrical behavior has been extensively investigated.
In normal myocardium, most of Cx43 is phosphorylated , whereas during myocardial ischemia, Cx43 is dephosphorylated and resulted in increasing of qconductance and permeability of connexons and hemichannels [55, 56]. Cx43 is phosphorylated by protein kinase C (PKC, α and ε) [57, 58] and protein tyrosine kinases (PTK, such as src) [59, 60]. More recently, mitogen activated protein kinases (MAPK), such as p42 and p44 ERK [61-64], p38 , and JNK [65, 66] have been implicated in the regulation of Cx43 expression and phosphorylation. The role of p38-MAPK in the regulation of gap junctions in the heart underphysiological and pathophysiological conditions is still unclear. In the study of Polontchouk et al. found that stimulation of the ERK and p38 signal pathways via endothelin receptor (ETA) and angiotensin-II receptors (AT1) may participate in the regulation of cardiac gap junctions that was studied in ventricular cardiomyocytes . However, the effect of p38-MAPK on the ventricular arrhythmias via the alteration of Cx43 expression has yet to be proved, but it remains an interesting possibility.
p38-MAPKs and mitochondrial functions
Over the past few years, mitochondria have emerged as central regulators of cell death in a variety of disease states. Cell death can occur by either necrosis or apoptosis, and mitochondria are intimately involved in both processes. In recent years, numerous studies indicate that each of the major kinase signaling pathways can be stimulated to target the mitochondrion, one is the p38-MAPK. Many studies indicate a role for p38 MAPK signaling in regulating events associated with cell death, including translocation of Bax from cytosolic to mitochondrial compartments [67, 68], caspase-independent potassium efflux , and transcriptional regulation of TR3, a steroid receptor-like protein that translocates from the nucleus to the mitochondria to initiate the intrinsic pathway of apoptosis . Activation of p38-MAPK induces translocation of Bax from the cytosol to mitochondria, reducing mitochondrial membrane potential. This leads to cytochrome c release, which results in caspase-9 and -3 activation and, eventually, apoptotic cell death  (see Figure 2).
It is known that mitochondria, a vital source of energetic pool in the cell during oxidative phosphorylation, are the main source of reactive oxygen species (ROS) production . In cardiac cell, the population of mitochondria in cytoplasm is greater than 50% of the cell volume. A small amount (1-5%) of electrons that flows through the electron transport chain in the mitochondria and leak into the cytoplasm and mitochondrial matrix contributes to ROS production [72, 73]. ROS has been shown to play an important role as the determinant of cell survival in ischemia [74-76]. It has been shown that ROS release during ischemic preconditioning can act as signaling molecules that may activate PKC and mitochondrial ATP-sensitive potassium channels (mitoKATP). This results in the protection of cardiomyocytes against injury [74, 75, 77-79]. Despite that, an increase in ROS production in postischemic hearts shows different effects. Under physiological condition, there is a balance between ROS production and the intracellular ROS scavenging capacity . However, such balance is upset under pathological condition. It has been shown that oxidative stress is induced by an increase in ROS production or a depletion of ROS scavengers or the depletion of cytosolic reduced glutathione (GSH) . These ROS may lead to mitochondrial dysfunction, apoptotic cell death and persistence contractile dysfunction [71, 72, 81, 82].
On the other hand, Clerk et al.  have explored the redox regulation of MAPK by comparing the influence of H2O2 to that of ischemia and I/R on JNK and p38 activation. H2O2 appears to mimic the effect of I/R, and free-radical-trapping agents inhibit the activation of MAPK by I/R, demonstrating the role of ROS in the process and introducing the paradigm that antioxidants might play a regulatory role in the activation of MAPK during I/R. It is interesting to note that mitochondrial ROS and the permeability transition have both been implicated upstream of JNK activation under both physiologic and pathologic conditions [84, 85]. Likewise, p38 MAPK is activated by mitochondrial derived ROS . Thus, these kinases, like ERK, may signal in both directions to influence mitochondrial functions as well as to communicate mitochondrial messages to the rest of the cell (Figure 3).