The Effect And Mechanism Of Rho-kinase And PARP On Myocardial Apoptosis In Ischemia/Reperfusion Injury

BackgroundCoronary heart disease has been one major cause of disability and death in our country, approximately 700000 people die of coronary heart disease every year, accounting for about one quarter of all death caused by cardiovascular diseases. Reperfusion therapy, which can make the return of blood supply as soon as possible to the ischemic region of the myocardium, has been the chief treatment of acute myocardial ischemia. However, Braunwald and Kloner point out that reperfusion is a "double edged sword", because reperfusion itself can accelerate myocardial damage and can produce additional myocardial injury, which we called the Ischemia/reperfusion (I/R) injury. Ischemia/reperfusion (I/R) injury can be induced by organ transplant dysfunction, stroke, myocardial infarction, shock and so on. Reperfusion is followed by myocardial biochemical, structural, and functional changes and may determine myocardial cell survival and death. Reperfusion can lead to diminished cardiac contractile function and arrhythmia. There is experimental evidence that irreversible myocardial cell injury originated by reperfusion can result in cell necrosis and apoptosis. How to improve myocardial function, decrease the arrhythmogenesis, delay the episode of necrosis, and decrease the infarction size during ischemia/reperfusion injury is of great clinical significance. Therefore, increasingly importance has been attached to ischemia/reperfusion injury.Recently, the research of Rho-kinase and poly (ADP-ribose) polymerase in myocardial cell apoptosis caused by ischemia/reperfusion injury has been more and more popular. Rho-kinases (ROCKs) are serine/threonine kinases with a molecular weight of 160 KD and are downstream effectors of the small GTPase Rho. ROCKs consist of an amino-terminal kinase domain, a mid-coiled-coil-forming lesion, the Pleckstrin homology (PH) motif and a carboxy-terminal cysteine-rich domain. There are two isoforms of ROCK:ROCK1 and ROCK2. ROCKs implicated in many kinds of vital movement, including the regulation of cellular contraction, growth, division, metabolism, migration apoptosis and gene expression. According to scientific survey, Rho-kinase is closely related to the development of a wide range of diseases such as coronary heart disease, atherosclerosis, hypertension, pulmonary hypertension, heart failure, diabetes, stroke, cancer and so on. Rho-kinase can mediate a series of Phosphorylation/phosphorylation reaction, dozens of substrates of ROCK have been identified, including:the myosin phosphatase-targeting subunit 1(MYPT1) and the myosin-binding subunit of myosin light chain phosphatase (MLCP), LIM, PTEN, and so on. The first characterized targets of ROCK are MLC and MYPT1. Evidence from clinical use of ROCK inhibitors, such asY-27632, fasudil and statins, supports the hypothesis that ROCK is a potential therapeutic target.Poly (ADP-ribose) polymerases are resided in eukaryotes, and they are a family of cell signaling enzymes. There are 18 superfamily, including PARP-1,PARP-2,PARP-3, etc. PARP-1 is the most abundant subtype of poly (ADP-ribose) polymerases. Poly (ADP-ribose) polymerase (PARP) is involved in many important cellular pathways, and it can contribute to chromosomal structure and genomic stability, detects and repairs the damage of DNA. DNA repair, gene rearrangements, oxidative stress, DNA binding drugs and protein-protein interactions can cause the activation of Poly (ADP-ribose) polymerase. However, poly (ADP-ribose) polymerase overactivation can exhausts NAD+, steps down the rate of glycolysis, electron transport, and ATP formation, finally leads to functional impairment or death of cells. Clinical data indicates that, poly (ADP-ribose) polymerase plays an important role in many diseases such as myocardial ischemia/reperfusion injury, myocarditis, heart failure, shock, atherosclerosis and so on.In our previous study, we found that both Rho-kinase and PARP play an important role in rat myocardial ischemia/reperfusion injury, and we further study the mechanism of them. We found that both ROCK/JNK/AIF pathway and PARP /JNK/AIF pathway are new pathways of cardiac myocyte injury. But whether there are interactions between Rho-kinase and PARP, what are the mechanisms of their interaction. In our study, we establish myocardial cell I/R model and the rat heart I/R model, investigate the interaction of Rho-kinase and PARP, identify the signaling pathway between them, so that we can provide a new therapeutic approach for ischemic heart disease.This study includes there parts:PartⅠIn vitro:The relationship and effect of Rho-kinase and PARP on cell apoptosis in the primary myocardial cell model of I/RPartⅡIn vitro: The interaction and mechanism of Rho-kinase and PARP on the I/R model of primary cultured myocardial cellPartⅢIn vivo:The interaction and mechanism of Rho-kinase and PARP on the rat I/R injury modelPartⅠIn vitro:The relationship and effect of Rho-kinase and PARP on cell apoptosis in the primary myocardial cell model of I/RObjectiveTo investigate the interaction of Rho-kinase and PARP on I/RMethods1. Primary cultures of myocardial cells:obtain the heart of neonatal wistar rat, shred to 1mm3, and then digest with trypsin and collagenaseⅡ. After differential adhesion, put the myocardial cells in incubator. 2. Establish the hypoxia-reoxygenation injury model of myocardial cell, The following experimental groups were studied. (1) control group; (2) I/R+DMSOgroup; (3) I/R+Y27632group; (4) I/R+3-AB group.3. Detect the activity of Rho-kinase and PARP: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 and PARP activity.4. Evaluate the apoptotis of myocardial cells, the TUNEL technique was used.Results1. Rho-kinase and PARP both play an important role in I/R injury, there are interaction between them:Phospho-MYPT-1 and PARP increased during the hypoxia-reoxygenation injury model. Administration of inhibitors of Rho-kinase can depress the expression of Phospho-MYPT-1 and PARP. Administration of inhibitors of PARP can depress the expression of PARP, but do not effect the expression of Phospho-MYPT-1.This indicate that Rho-kinase and PARP both play an important role in I/R injury, and PARP is the downstream molecules of Rho-kinase.2. The inhibition of Rho-kinase and PARP can both decrease the apoptosis of myocardial cells:The apoptosis ratio of myocardial cells was 46.5±1.2% in I/R +DMSO group. Administration of Y27632, the apoptosis ratio was 16±0.5%(P＜0.05 vs I/R+DMSO group); Administration of 3-AB, the apoptosis ratio was 20±0.3%(P <0.05 vs I/R+DMSO group), demonstrating that inhibition of Rho-kinase and PARP activity reduces myocardial cells apoptosis in rat heart I/R injury.Conclusion1. Rho-kinase and PARP activities increased in the hypoxia-reoxygenation injury model, they can aggravate the apoptosis of myocardial cells.2. Rho-kinase and PARP inhibition in the hypoxia-reoxygenation injury can both reduce the apoptosis of myocardial cells.3. There are interactions between Rho-kinase and PARP in the hypoxia-reoxygenation injury. By regulation the activity of PARP and Rho-kinase can reduce the apoptosis of myocardial cells. Part II In vitro:The interaction and mechanism of Rho-kinase and PARP on the I/R model of primary cultured myocardial cellsObjectiveTo investigate the effect of Rho-kinase, Akt, caspase-3 and PARP in I/RMethods1. Primary cultures of myocardial cells from the neonatal wistar rat2. Establish the hypoxia-reoxygenation injury model of myocardial cell. The following experimental groups were studied. (1) control group; (2) I/R+DMSO group; (3) I/R+Y27632 group; (4) I/R+LY294002 group; (5) I/R+Ac-DEVD-CHO group; (6) I/R+3-AB group.3. Detect the activity of Rho-kinase, Akt, caspase-3 and PARP:Rho-kinase activity was assessed by examining phosphorylation of MYPT-1. Caspase-3 activity was assessed by examining the cleaved-Caspase-3. Western blot analysis was performed to evaluate Akt, p-Akt, cleaved-caspase-3 and PARP activity.4. Evaluate the apoptosis of myocardial cells, the TUNEL technique was used.Results1. The expression of p-MYPT-1.. cleaved-Caspase-3 and PARP were increased in myocardial cell I/R model, but the expression of p-Akt was degraded. This shows that the activity of Rho-kinase,Caspase-3 and PARP increased in myocardial cell I/R model, phosphorylation of Akt was degraded.2. The apoptosis ratio of myocardial cell is 60±1.68% I/R+DMSO group. The apoptosis ratio of myocardial cell is significantly reduced to 24±0.52% in I/R+ Y27632 group (P<0.05 vs I/R+DMSO group). The apoptosis ratio of myocardial cell is reduced to 32±1.24% in I/R+LY294002 group (P<0.05 vs I/R+DMSO group). The apoptosis ratio of myocardial cell is reduced to 36±0.65% in I/R+3-AB group (P <0.05 vs I/R+DMSO group). This suggests that inhibition of Rho-kinase, Caspase-3 and PARP activity reduces cell apoptosis.3. The expression of p-MYPT-1 increased in I/R+DMSO group, but Phospho-Akt decreased in I/R+DMSO group. After administration of Y27632, the expression of Phospho-Akt was increased. After administration Akt inhibitor, the expression of p-MYPT-1 did not change significantly. This indicates Akt is the downstream molecules of Rho-kinase.4. The expression of p-MYPT-1 and cleaved-Caspase-3 were increased in I/R +DMSO group. After administration of Rho-kinase inhibitor, the expression of cleaved-Caspase-3 was decreased. After administration of Caspase-3inhibitor, the expression of p-MYPT-1 did not change significantly. This indicates that Caspase-3 is the downstream molecules of Rho-kinase.5. The expression of p-Akt was decreased in I/R+DMSO group, and the expression of cleaved-Caspase-3 were increased. After administration of Akt inhibitor, the expression of cleaved-Caspase-3 was increased. After administration of Caspase-3 inhibitor, the expression of p-Akt did not change significantly. This indicates that Caspase-3 is the downstream molecules of Akt.6. The expression of PARP and cleaved-Caspase-3 were increased in I/R+ DMSO group, and the Cleavage fragments (24KD) of PARP was increased too. After administration of Caspase-3-inhibitor, the activity of PARP was decreased, the Cleavage fragments (24KD) of PARP were decreased. After administration of PARP inhibitor, the expression of cleaved-Caspase-3 did not change significantly. This indicates that PARP is the downstream molecules of Caspase-3.Conclusion1. In I/R model, the activity of Rho-kinase,Caspase-3 and PARP were increased, but phosphorylation of Akt was degraded. Inhibition the activity of Rho-kinase,Caspase-3 and PARP can reduce myocardial infarct size and the ratio of myocardial cell apoptosis.2. Rho-kinase/Akt/caspase-3/PARP pathway may be a new pathway of heart I/R injury.PartⅢIn vivo:The interaction and mechanism of Rho-kinase and PARP on the rat I/R injury modelObjectiveTo investigate the effect of Rho-kinase, Akt, caspase-3 and PARP in I/R Methods1. The rat model of I/R:The left anterior descending branch (LAD) of the left coronary artery was occluded and loosened to establish rat heart I/R model. The following experimental groups were studied:(1) control group (n=20) (2) I/R+DMSO group (n=20); (3) I/R+fasudil group (n=20); (4) I/R+LY294002 group (n=20); (5) I/R+Ac-DEVD-CHO group (n=20); (6) I/R+3-AB (n=20).2. Detect the activity of Rho-kinase, Akt, caspase-3 and PARP:Rho-kinase activity was assessed by examining phosphorylation of MYPT-1. Caspase-3 activity was assessed by examining the cleaved-Caspase-3. Western blot analysis was performed to evaluate Akt, p-Akt and PARP 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. Evaluate the apoptosis of myocardial cells, the TUNEL technique was used.Results1. The expression of p-MYPT-1,cleaved-Caspase-3 and PARP were increased in the I/R model, but the expression of p-Akt was degraded. This shows that the activity of Rho-kinase,Caspase-3 and PARP increased in I/R model, phosphorylation of Akt was degraded.2. Inhibition of Rho-kinase, Caspase-3 and PARP activity reduced myocardial infarct size. The infarct sizes of the heart were 60.22±1% in I/R+DMSO group, 30.5±1.2% in I/R+fasudil group,24.9±0.8% in I/R+LY294002 group and 29.1±0.5% in I/R+3-AB group(P<0.05 vs I/R+DMSO group). This indicates that inhibition of Rho-kinase, Caspase-3 and PARP can reduce myocardial infarct size.3. The expression of p-MYPT-1 increased in I/R+DMSO group, but Phospho-Akt decreased in I/R+DMSO group. After administration of Y27632, the expression of Phospho-Akt was increased. After administration Akt inhibitor, the expression of p-MYPT-1 did not change significantly. This indicates Akt is the downstream molecules of Rho-kinase.4. The expression of p-MYPT-1 and cleaved-Caspase-3 were increased in I/R +DMSO group. After administration of Rho-kinase inhibitor, the expression of cleaved-Caspase-3 was decreased. After administration of Caspase-3 inhibitor, the expression of p-MYPT-1 did not change significantly. This indicates that Caspase-3 is the downstream molecules of Rho-kinase.5. The expression of p-Akt was decreased in I/R+DMSO group, and the expression of cleaved-Caspase-3 were increased. After administration of Akt inhibitor, the expression of cleaved-Caspase-3 was increased. After administration of Caspase-3 inhibitor, the expression of p-Akt did not change significantly. This indicates that Caspase-3 is the downstream molecules of Akt.6. The expression of PARP and cleaved-Caspase-3 were increased in I/R+DMSO group, and the Cleavage fragments (24KD) of PARP was increased too. After administration of Caspase-3 inhibitor, the activity of PARP was decreased, the Cleavage fragments (24KD) of PARP were decreased. After administration of PARP inhibitor, the expression of cleaved-Caspase-3 did not change significantly. This indicates that PARP is the downstream molecules of Caspase-3.Conclusion1. In rat I/R model, the activity of Rho-kinase ^ Caspase-3 and PARP were increased, but phosphorylation of Akt was degraded. Inhibition the activity of Rho-kinase-. Caspase-3 and PARP can reduce myocardial infarct size.2. Rho-kinase/Akt/caspase-3 /PARP pathway may be a new pathway of rat heart I/R injury.