The Study Of Rho-kinase On Myocardial Apoptosis In Ischemia/Reperfusion Injury And Myocardial Ischemic Preconditioning

BackgroundWith the improvement of people's standard of living, cardiovascular disease has now become a great threat to human life and health. The main cause of death of cardiovascular diseases is coronary artery disease. The best treatment of coronary heart disease is the resumption of blood perfusion of myocardium as soon as possible. However, both animal experiments and clinical studies have found that the damage of myocardial cell function and structural became worse with the restoration of blood supply. We call the pathophysiological state ischemia-reperfusion injury (I/R). Reperfusion involved in the clinical practice such as reperfusion myocardial infarction after coronary artery bypass grafting, heart transplants, heart resuscitation of cardiac arrest and cardiopulmonary bypass heart surgery. Ischemic preconditioning (IPC) has been exploited as a powerful endogenous form of cardioprotection. IPC was first discovered by Murry and associates, who demonstrated that a brief period of repetitive cardiac I/R exerts a protective effect against subsequent lethal periods of ischemia. IPC was found to similarly reduce cytosolic and mitochondrial Ca2+ overloading, to augment postischemic functional recovery, and to decrease infarct size. And IPC is known to protect cardiomyocyte apoptosis during reperfusion.The ubiquitously expressed Rho-kinase, a serinethreonine kinase, has been identified as one of the effectors of the small GTP-binding protein Rho. Rho-kinase plays crucial roles in various cellular functions, and mediates cellular events such as changes in cell morphology, cell motility, focal adhesions, and cytokinesis. The Rho-kinase family contains two members:ROCK1 (also called ROKβor p160ROCK) and ROCK2 (also known as ROKa), which share 65% overall identity and 92% identity in the kinase domain. Two relatively selective Rho-kinase inhibitors, Y27632 and fasudil, bind to the kinase domain and inhibit ROCK1 and ROCK2 with similar potency. Rho-kinase phosphorylates a variety of protein substrates at serine or threonine residues. More than 20 Rho-kinase substrates have been identified. The first characterized targets of ROCK are myosin light chain (MLC) and the myosin binding subunit of MLC phosphatase (MYPT1). Rho-kinase can increase MLC phosphorylation through direct effect on MLC or indirectly by inactivating MLC phosphatase. The increased MLC phosphorylation results in stimulation of actomyosin contractility.Accumulating evidences have demonstrated that Rho-kinase plays an important role in many major cardiovascular diseases such as hypertension, heart failure, myocardial infarction and atherosclerosis. Recent animal studies suggest that inhibition of Rho-kinase protects the heart against I/R injury. Previous studies have also demonstrated IPC caused a substantial decrease of Rho-kinase activation during sustained ischemia and reduced infarct size. However, little is known about the mechanism of Rho-kinase increased in I/R and reversed in IPC. Therefore, the aim of this study was to elucidate the mechanism of decreased Rho-kinase activity in IPC. We established IPC and I/R model in vivo and vitro. The activation of Rho-kinase, cardiomyocyte apoptosis, area of myocardial infarction and related signaling pathway were detected.This study includes three parts:PartⅠThe effect of Rho-kinase on I/R and IPCPartⅡThe effect of Rho-kinase on AIF signaling pathway in I/RPartⅢThe effect of ERK1/2 on Rho-kinase in IPC Part I The effect of Rho-kinase on cell apoptosis in I/R and IPCObjectiveTo investigate the effect of Rho-kinase on I/R and IPCMethods1. Animal preparation:The left anterior descending branch (LAD) of the left coronary artery was occluded and loosened to establish rat heart IPC and I/R model. The following experimental groups were studied. (1) control group (n=18); (2) I/R group (n=18); (3) I/R+fasudil(inhibitor of Rho-kinase) group (n=18); (4) IPC group (n=18).2. Rho-kinase activity was assessed by examining phosphorylation of MYPT-1, a well established Rho-kinase specific substrate. Western blot analysis was performed to evaluate Rho-kinase activity.3. Determination of myocardial infarct size:Evans blue and Nitro blue tetrazolium was used to evaluate the infracted and noninfarcted areas. Infarct size was expressed as the percentage of the area at risk(AAR).4. To evaluate apoptotic activity, the TUNEL technique was used. TUNEL-positive cells were determined by randomly counting 10 fields of the section and were expressed as a percentage of normal nuclei.5. Activation of Caspase-3 is a hallmark of apoptotic cell death and Caspase-3 cleavage is indicative of its activation. Caspase-3 cleavage was determined by Western blot analysis.Results1. Phospho-MYPT-1 increased during the I/R protocol. This increase in MYPT-1 phosphorylation was reversed in IPC protocol and I/R+fasudil protocol, demonstraying that Rho-kinase activity decreased in IPC.2. Rho-kinase inhibition decreased the infarct size of the heart. The infarct size of the heart was 59.89±3.89% in I/R group. Administration of fasudil, the infarct size was 38.62±2.66%, demonstraying that inhibition of Rho-kinase activity reduces myocardial infarct size in rat heart I/R injury. In IPC, the myocardial infarct size was 29.16±1.08% (P<0.05 vs I/R group). These data suggest that myocardial infarct size was attenuated in IPC.3. Inhibition of Rho-kinase reduced cardiomyocyte apoptosis. No TUNEL positive cells were found in the control group. The number of TUNEL positive cells were significantly increased in I/R group (32.78±5.1%). After fasudil was used, the TUNEL positive cells reduced to 17.05±4.2% (P<0.05 vs I/R group). The TUNEL positive cells were significantly reduced to 17.29±0.84% in IPC group (P<0.05 vs I/R group).Conclusion1. Rho-kinase activity increased in I/R. The overexpression of Rho-kinase in rat heart I/R protocol can aggravate the heart damage.2. Rho-kinase inhibition in I/R has the cardiacprotective effect via reduced the apoptosis of cardiomyocytes and infarct size.3. Rho-kinase activity increased in I/R and reversed in IPC. IPC has the cardiacprotective effect via reduced the apoptosis of cardiomyocytes and infarct size.PartⅡThe effect of Rho-kinase on AIF signaling pathway in I/RObjectiveTo investigate the effect of Rho-kinase on AIF signaling pathway in I/RMethods1. Animal preparation:The left anterior descending branch (LAD) of the left coronary artery was occluded and loosened to establish rat heart IPC and I/R model. The following experimental groups were studied. (1) control group (n=18); (2) I/R+NS group (n=18); (3) I/R+fasudil (inhibitor of Rho-kinase) group (n=18); (4) IPC+SP600125 (inhibitor of JNK) group (n=18); (5) I/R+DMSO group (n=18). 2. Determination of myocardial infarct size:Evans blue and Nitro blue tetrazolium was used to evaluate the infracted and noninfarcted areas. Infarct size was expressed as the percentage of the area at risk(AAR).3. To evaluate apoptotic activity, the TUNEL technique was used. TUNEL-positive cells were determined by randomly counting 10 fields of the section and were expressed as a percentage of normal nuclei.4. Western blot analysis was performed to evaluate Rho-kinase, JNK, and AIF activity.Results1. Rho kinase, JNK and AIF were activated in the rat I/R model. Western blot analysis was performed to evaluate whether Rho-kinase, JNK and AIF can be activated in the myocardial I/R rat model. Rho-kinase activity was assessed by examining phosphorylation of MYPT-1, a well established Rho-kinase specific substrate. JNK activity was assessed by examining phosphorylation of JNK. Both phospho-MYPT1 and phospho-JNK increased in I/R. AIF activity was assessed by nuclear translocation of AIF. The activation of AIF increased in nuclear fractions while it decreased in mitochondria fractions. The results indicate that AIF translocated to the nucleus in the I/R rat model.2. Inhibition of Rho-kinase or JNK activity reduced myocardial infarct size. Administration of fasudil or SP600125 caused significant reduction of AAR and infarct size. The infarct sizes of the heart were 38.62±2.66% in I/R+fasudil group and 41.1±2.57% in I/R+SP600125 group respectively. These data suggest that inhibition of Rho-kinase or JNK activity reduces myocardial infarct size in rat heart I/R injury.3. Inhibition of Rho-kinase or JNK activity reduced cell apoptosis of the heart. The number of TUNEL positive cells was significantly increased in I/R+NS group (32.78±5.1%). The TUNEL positive cells were significantly reduced to 17.05±4.2% in I/R+fasudil group. The number of TUNEL positive cells was significantly increased in I/R+DMSO group (34.45±3.73%). The TUNEL positive cells were significantly reduced to 16.25±3.29% in I/R+SP600125 group. These data suggest that inhibition of Rho-kinase or JNK activity activity reduces cell apoptosis.4. Inhibition of Rho-kinase activity reduced the activation of JNK. Phospho-JNK increased after reperfusion in I/R group. The activation of phospho-JNK was significantly attenuated in I/R+fasudil group.5. Inhibition of Rho-kinase or JNK activity reduces the AIF translocation: AIF increased after reperfusion in nuclear fractions. The activation of AIF was significantly attenuated by inhibition of Rho-kinase or JNK activity. On the contrary, AIF decreased in mitochondria fractions after reperfusion. Inhibition of Rho-kinase or JNK activity could increase the activation of AIF in mitochondria fractions. These data suggest that inhibition of Rho-kinase or JNK activity was able to reduce the mitochondrial-nuclear translocation of AIF.Conclusion1. JNK may be downstream of Rho-kinase activation and JNK may be required for Rho-kinase mediates AIF translocation in a rat model of myocardial ischemia and reperfusion.2. Rho-kinase/JNK/AIF pathway may be a new pathway of cardiac myocyte injury.PartⅢThe effect of ERK1/2 on Rho-kinase in IPCObjectiveTo investigate the mechanism of decreased Rho-kinase activity in IPCMethods1. Animal preparation:(1) I/R group (n=18); (2) PC group (n=18); (3) IPC+PD98059 group (n=18); (4) IPC+fasudil (n=18); (5) IPC+PD98059+fasudil group (n=18); (6) IPC+DMSO (n=18).2. Determination of myocardial infarct size:Evans blue and Nitro blue tetrazolium was used to evaluate the infracted and noninfarcted areas. Infarct size was expressed as the percentage of the area at risk(AAR).3. To evaluate apoptotic activity, the TUNEL technique was used. TUNEL-positive cells were determined by randomly counting 10 fields of the section and were expressed as a percentage of normal nuclei.4. Western blot analysis was performed to evaluate ERK, Rho-kinase, Caspase-3 activity and ROCK1, ROCK2 expression.5. Immunohistochemistry Staining was performed to evaluate phospho-ERK1/2.6. Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate ROCK1 mRNA.Results1. Effect of IPC on cardiomyocyte apoptosis, Caspase-3 cleavage and infarct size. TUNEL positive cells were expressed as a percentage of normal nuclei. The number of TUNEL positive cells was 33.87±1.57% in I/R group. The TUNEL positive cells were significantly reduced to 17.29±0.84% in IPC group. Activation of Caspase-3 is a hallmark of apoptotic cell death and Caspase-3 cleavage is indicative of its activation. IPC resulted in a 65% reduction in the amount of Capsase-3 cleavage (P<0.05 vs I/R group). Capsase-3 activity attenuated in IPC group. The infarct size of the heart was 60.53±4.08% in I/R group. In IPC, the myocardial infarct size was 29.16±1.08%. These values showed no significant differences between IPC and IPC+DMSO groups.2. Effect of IPC on activity of ERK1/2. Phospho-ERK1/2 expression increased clearly in the heart in IPC. IPC resulted in an immediate increase in ERK1/2 phosphorylation. The increase in the phosphorylation of ERK1/2 was about 1.8-fold (P<0.05 vs I/R group).3. Effect of IPC on activity of RhoA, ROCK1, ROCK2 and Rho-kinase. RhoA protein was determined by immunoblotting of cytosolic and membrane fractions. In all groups, RhoA protein was detected in both cytosolic and membrane fractions. An enhanced RhoA translocation to the membrane was detected in the I/R rat model. But no difference was observed between the I/R and IPC groups. ROCK1 and ROCK2 were also determined by western blot analysis. ROCK1 increased in the I/R group and attenuated in IPC group. ROCK2 increased in the I/R group, but there were no significant differences between the I/R and IPC groups. RT-PCR was also used to evaluate ROCK1 activity. A significant increase in mRNA expression of ROCK1 was detected in the I/R group, and the expression was decreased by IPC. Rho-kinase activity was assessed by examining phosphorylation of MYPT-1, a well established Rho-kinase specific substrate. IPC resulted in a 49% reduction in MYPT-1 phosphorylation and a 54% reduction in ROCK1 (P<0.05 vs I/R group).4. Inhibition of ERK1/2 increased cardiomyocyte apoptosis, Caspase-3 activity and infarct size during IPC. The TUNEL positive cells were significantly increased with administration of PD98059 (29.83±0.7%) in IPC+PD98059 group (P<0.05 vs IPC group). The increase in the amount of cleaved Caspase-3 in IPC+PD98059 group was about 2.1-fold (P<0.05 vs IPC group). The infarct size of the heart was 44.88±0.7% in IPC+PD98059 group (P<0.05 vs IPC group).5. ERK1/2 opposed Rho-kinase during IPC. ERK1/2 was strongly activated and Rho-kinase activity decreased in IPC. Inhibition of ERK1/2 in IPC could lead to upregulation of MYPT-1 phosphorylation and the amount of ROCK1. Inhibition of ERK1/2 in IPC also could lead to upregulation of ROCK1 mRNA. In the IPC+fasudil group, phospho-ERK1/2 expression was similar to the IPC group. In the IPC+PD98059 group, phospho-ERK1/2 expression significantly decreased. And phospho-ERK1/2 expression was not recovered in the IPC+PD98059+fasudil group.6. Inhibition of Rho-kinase rescued the effect of inhibition of ERK1/2 on apoptosis. Fasudil treatment reversed cell apoptosis caused by treatment with PD98059 in IPC. The number of TUNEL positive cells was 23.3±0.67% in IPC+PD98059+fasudil group. The amount of cleaved Caspase-3 was also significantly reduced in IPC+PD98059+fasudil group. Fasudil resulted in a 22% reduction in cardiomyocyte apoptosis and a 30% reduction in cleaved Caspase-3 in IPC+PD98059+fasudil group (p<0.05 vs IPC+PD98059 group). Additionally, the infarct size of the heart was 39.44±0.92% in IPC+PD98059+fasudil group. The combination of PD98059 and fasudil also resulted in a 12% reduction in myocardial infarct size (p<0.05 vs IPC+PD98059 group).Conclusion1. ERK-MAPK signaling is required in IPC to oppose the Rho-kinase signaling on cardiomyocyte apoptosis.2. Overpression of RhoA in I/R might not be changed by preconditioning.3. ROCK1 may be the major Rho-kinase which is opposed by ERK-MAPK signaling in IPC.