The Study Of The Protective Role Of Pim-3 Against Acute Cardiomyocyte Injury And Involved Mechanisms

It was reported in the middle of the last century that under certain circumstances , reperfusion of blood to ischemia tissues and organs not only fail to alleviate the damage but also led to further aggravate the injury and dysfunction. The phenomenon that the ultrastructure, metabolism, functions and electrophysiological of ischemia organizations got further injury after reperfusion was named ischemia / reperfusion injury. Recently, clinicians have found that IRI occured in shock therapy, cardiopulmonary-cerebral resuscitation, coronary spasm angina pectoris, cardiovascular and cerebrovascular embolization recanalization, off-pump open heart surgery, percutaneous transluminal coronary angioplasty, organ and limb transplants, skin burns and other surgical cases, and it could be one of the important reasons why post-operative patients got condition deteriorated. How to protecte organs from IRI has now become the focus of clinical research, in particular to cardiothoracic Surgery. Through transfection certain gene into body, gene therapy can confer cadiocyte the ability to resist acute myocardial injury, meanwhile avoid the second-hit of surgery. Gene therapy could be a new and hopeful way to prevent and treat the acute myocardial injury.Proto-oncogene is a kind of normal cell gene that encodes a key regulatory protein. When proto-oncogene is activated, it will not only promote cell proliferation but also inhibit cell apoptosis. Apoptosis is an important contributory factor in the loss and damage of cardiomyocytes when the myocardium is subjected to acute anoxia or ischemic injury. The fundamental cellular mechanism behind apoptosis can best be described as a balance between anti-apoptotic and pro-apoptotic activities. Activation of the endogenous proto-oncogene or introduction of the exogenous one may fundamentally repair myocardial injury, through stimulation of anti-apoptotic factors. Some studies have shown that myocardial ischemic injury can be repaired by the proto-oncogene src, ras, vav-mediated pathway, futher to prevent the occurrence of heart failure. Therefore, proto-oncogene plays an important role in the treatment of myocardial damage, which inspires people to put the role of the proto-oncogene into the treatment of acute myocardial injury.A proto-oncogene Pim kinase belongs to a serine/threoine protein kinase family consisting of Pim-1, -2 and -3. The three members are well conserved in vertebrates and show high degrees of sequence and structural similarities. There is also overlap and redundancy in their biological functions. Such other serine/threoine protein kinases as PI3K-Akt or mTOR, Pim kinases can phosphorylate serine/threonine residue that regulate apoptosis and play important roles in the regulation of cell growth and surviva by other pathway differented from PI3K-Akt-mTOR. The expression of Pim kinase can be mediated by many growth factors, hormone and cell growth pathway such as JAK/STAT, PI3K and Akt, so it has been implicated in stimulating cell growth, inhibiting cell apoptosis and promoting cell cycle progressioN. Pim-3 was found to be overexpressed in ischemic brain tissue, which indicated that Pim-3 may play an important role in cell survival and functional recovery. Given that Pim-3 could prevent apoptosis and promote cell survival, we assumed that Pim-3 might play a protective effect on the myocardium subjected to I/R injury. Based on the above hypothesis, we design the experiment as follows. Firstly, the A/R and anoxia preconditioning (APC) models of primary cultured neonatal rat myocytes were established, AND we examined the expressions of mRNA and protein of Pim-3. WE then cloned Pim-3 expression vector and transfected it into rat cardiomyocytes and examined Pim-3 expression in rat cardiomyocytes, especially in the cells subjected to A/R injury in order to explore the protective role of Pim-3 in acute cardiomyocyte injury.Secondly, we studied the role of three major MAPK pathways, p38 MAPK, JNK, and ERK1/2, in order to evaluate the molecular mechanism underlying Pim-3 overexpression. Thirdly, we also investigate the protective effect of proto-oncogene Pim-3 on cardiomyocytes aganist anoxia–reoxygenation(A/R) injury and the relation of this process with THE release of cytochrome c mediated by mitochondrial signal transduction pathways. PartⅠThe Expression and significance of Pim-3 in cardiomyocytes subjected to acute damage.Objective:We established the A/R and anoxia preconditioning (APC) models of primary cultured neonatal rat myocytes and detected expression of Pim-3 by RT-PCR and Western blotting, and then we cloned Pim-3 expression vector and transfected it into rat cardiomyocytes in order to study the protective effect of proto-oncogene Pim-3 on cardiomyocytes aganist anoxia–reoxygenation(A/R) injury.Methods:The primarily neonatal rat ventricular cardiomyocytes were randomly divided into 5 groups: (1) control group; (2) A/R group; (3) APC+A/R group; (4) pcDNA3.1+A/R group; (5) pcDNA3.1/Pim-3+A/R group. Expression of Pim-3 was detected by RT-PCR and Western blotting. LDH and CK-MB activity, MTT and TUNEL were detected after treatment.Results:1. The expression of Pim-3 mRNAThe expression level of Pim-3 mRNA showed a significant increase in A/R group compared with that of the control group (p<0.01). The expression level of Pim-3 mRNA was further increased in APC+A/R group, compared with that of A/R group (p<0.01). In pcDNA3.1/Pim-3+A/R group, the expression level of Pim-3 mRNA was remarkably elevated compared to that of A/R group(p<0.01). However, there was little change of the expression level in pcDNA3.1+A/R group(p>0.05).2. The expression of Pim-3 proteinThe expression level of Pim-3 protein showed a significant increase in A/R group compared with that of the control group (p<0.01). The expression level of Pim-3 protein was further increased in APC+A/R group, compared with that of A/R group (p<0.01). In pcDNA3.1/Pim-3+A/R group, the expression level of Pim-3 protein was remarkably elevated compared to that of A/R group(p<0.01). However, there was little change of the expression levels in pcDNA3.1+A/R group(p>0.05).3. Protective Effects of Pim-3 against A/R on Cell Viability and LDH ActivityThe viabilities of cardiomyocytes in the control and A/R groups were 92.4±8.8 % and 36.7±6.1%, respectively. Consistent with viability, LDH activity levels in the cultured medium were 4.0±0.7 for the control cells and 27.1±5.0 for the A/R-treated cells with a significant difference between them (p<0.01). In both groups of APC+A/R and pcDNA3.1/Pim-3+A/R, the levels of LDH activity were noticeably lower than those of A/R group; while the viability of ventricular myocytes was significantly increased compared with those of A/R group. The transfection of pcDNA3.1 into cardiomyocytes, however, showed little protective effect on the cells. This suggests that APC could induce cardioprotection and transfection of Pim-3 gene could mimic APC induced cardioprotection.4. Protective Effects of Pim-3 against A/R on Cardiomyocyte ApoptosisVery few TUNEL-positive cells were detected among cardiomyocytes from the control group. Numerous cardiomyocytes from A/R group presented as positive for TUNEL(p<0.01). In contrast, the number of TUNEL-positive cells was significantly reduced among cardiomyocytes of APC+A/R group; meanwhile, pcDNA3.1/Pim-3+A/R group showed an effect similar to that of APC+A/R group. There were 30.4±4.5% of cells that presented apoptosis in A/R group, whereas only 14.7±2.1% and 17.0±3.2% cardiomyocytes underwent apoptosis in APC+A/R and pcDNA3.1/Pim-3+A/R groups, respectively.Discussion and Conclusion:Cardiomyocyte apoptosis is the character for A/R injury. It is well known that APC is the most powerful means against A/R injury. APC has been investigated for many years; yet its physiological mechanisms of action are still not completely understood. There are growing evidences indicating that the protection is dependent on de novo protein synthesis. These proteins can inhibit apoptosis of myocardial cells, which play a protective role. Pim family is composed of at least three members: Pim-1, Pim-2, and Pim-3. Anti-apoptotic effects of Pim-1 and Pim-2 have been demonstrated earlier in several independent experimental systems. Because both Pim-1 and Pim-2 can induce anti-apoptotic effects, as a member of the Pim family, Pim-3 has been intriguing to investigators who tried to investigate if Pim-3 is involved in cell cycle regulation or anti-apoptosis through phosphorylating some substrates that regulate apoptosis. Pim-3 was found to be overexpressed in ischemic brain tissue, which indicated that Pim-3 may play an important role in cell survival and functional recovery. In this study, we confirmed that APC induced an over-expression of Pim-3 in parallel with the protection of cardiomyocytes against A/R injury. Furthermore, we demonstrated that transfection of Pim-3 into cardiomyocytes was able to mimic myocardial protection similar to that of APC. PartⅡThe Expression of Pim-3 in Acute Myocardial Injury Mediated by p38 MAPK Signal PathwayObjective: we established the A/R and anoxia preconditioning (APC) models of primary cultured neonatal rat myocytes and treated cardiomyocytes with U0126, SP600125 and SB203580 separately at 30 minutes prior to APC to inhibit the activities of ERK1/2, JNK and p38 MAPK, respectively. The aim is to investigate the role of mitogen-activated protein kinases (MAPKs) pathways and the molecular mechanism by which the proto-oncogene Pim-3 would protect cardiomyocyte against anoxia/reoxygenation(A/R) injury.Methods:The primarily neonatal rat ventricular cardiomyocytes were randomly divided into 6 groups: control group; A/R group; APC+A/R group; SB203850+APC+A/R group; U0126+APC+A/R group; SP600125+APC+A/R group. The cells were pre-incubated with U0126(ERK1/2 inhibitor), SP600125(SAPK/JNK inhibitor), or SB203580(p38 MAPK) at 10μmol/L concentration for 30 min before the APC. The activities of p38 MAPK, JNK and ERK1/2 were detected by Western blotting. The viability of cardiomyocytes was assayed by MTT and the apoptosis of cardiomyocyte was performed by TUNEL.Results:1. The Effects of MAPKs Signal Pathway on the Expression of Pim-3Alterations in the activity of MAPKs in cardiomyocytes were detected by Western blotting using phospho-specific antibodies against ERK1/2, p38 MAPK, and JNK respectively. At the same time, levels of total ERK1/2, p38 MAPK, and JNK proteins were also examined in parallel with anti-ERK1/2 antibody, anti-p38 MAPK antibody, and anti-JNK antibody. As seen in Figure 4, the cardiomyocytes showed 2.3- and 3.2-fold increases in ERK1/2 and p38 MAPK activities as compared to those of the control group (p<0.01) at 3 h after A/R. Meanwhile, in APC+A/R group, ERK1/2 and p38 MAPK activities showed further increases up to 6.7- and 10.1-fold compared to those of the control group (p<0.01). At the same time, cardiomyocytes of A/R group, compared to the control group, showed an approximately 1.4-fold increase in JNK activity (p<0.05). However, APC did not induce any further increase in JNK activity as compared with A/R group. U0126, SB203580, and SP600125, given at 30 min before APC, abolished the increased expression of ERK1/2, p38 MAPK, and JNK proteins induced by APC or A/R, respectively. Subsequently, we detected the expression level of Pim-3 protein after treating cardiomyotes with U0126, SB203580, and SP600125 respectively. The results showed that the expression level of Pim-3 protein significantly decreased when the p38 MAPK signal pathway was inhibited.2. The Effects of SB203850 on Cell Viability and LDH ActivityThe viability of cardiomyocytes in the A/R group was remarkably decreases compared to the control group, while LDH in the A/R group was significant increases compared to the control group(p<0.01). Once administrated with p38 MAPK inhibitor SB203850, the protective effects of APC was abolished.3. The Effects of SB203850 on Cardiomyocyte ApoptosisVery few TUNEL-positive cells were detected among cardiomyocytes from the control group. Numerous cardiomyocytes from A/R group presented as positive for TUNEL(p<0.01). In contrast, the number of TUNEL-positive cells was significantly reduced among cardiomyocytes of APC+A/R group. There were 32.4±4.5% of cells that presented apoptosis in A/R group, whereas only 13.8±2.6% cardiomyocytes underwent apoptosis in APC+A/R group(p<0.01). Once administrated with p38 MAPK inhibitor SB203850, 26.7±4.1% cardiomyocytes underwent apoptosis (p<0.01).Discussion and Conclusion:We have confirmed that the expression of Pim-3 could be significantly increased induced by APC and then play a protective role in cardiomyocytes against A/R injury in the part 1 of the research project. In this part, we studied the cellular signal pathway involved in the expression of Pim-3 induced by APC. In cardiomyocytes, the intracellular signaling mechanisms that mediate anoxic preconditioning require the presence of one or more members of MAPK cascades. These members are involved in the activation of genes that play important roles in the regulation of cellular responses occurring during cell proliferation and stress. A variety of extracellular stimuli (ischemia, stress, hormones, cytokines, growth factors, etc.), whether it is through the G protein or tyrosine kinase activation, can activate the Raf→MAPK kinase (MAPK kinase)→MAPK phosphorylation chain reaction. The nuclear translocation of MAPK can cause phosphorylation of transcription factors to regulate the expression of proto-oncogenes, stress protein gene and then promote protein synthesis induced by the extracellular stimuli. The principal MAPKs investigated in cardiac myocytes are ERK1/2, JNK and p38 MAPK. ERK1/2 is potently activated by hypertrophic stimuli, whereas JNK and p38 MAPK are activated by cellular stresses such as oxidative stress. However, there is a cross talk between JNK and p38 MAPK that results in the activation of cellular stresses when they are activated by hypertrophic stimuli and ERK1/2. The contribution of each pathway to the overall cardiac myocyte response is currently not entirely clear. In the present study, we treated cardiomyocytes with U0126, SP600125 and SB203580 separately at 30 minutes prior to APC to inhibit the activities of ERK1/2, JNK and p38 MAPK, respectively. We found that the inhibition of p38 MAPKs resulted in the abolishment of Pim-3 up-regulation in cardiomyocytes, which is induced by APC; while inhibition of either ERK1/2 or JNK did not show any effect on the Pim-3 expression. These findings indicate that over-expression of Pim-3 gene induced by APC may be regulated through the p38 MAPK signaling pathway. Part III The Role of Mitochondrial Apoptosis Signaling pathway in Pim-3 Protecting Against Acute Myocardial InjuryObjective: we established the A/R and anoxia preconditioning (APC) models of primary cultured neonatal rat myocytes and cloned Pim-3 expression vector and transfected it into rat cardiomyocytes in order to investigate the protective effect of proto-oncogene Pim-3 on cardiomyocytes aganist anoxia–reoxygenation(A/R) injury and the relation of this process with release of cytochrome c mediated by mitochondrial signal transduction pathways.Methods:The primarily neonatal rat ventricular cardiomyocytes were randomly divided into 4 groups: control group; A/R group; pcDNA3.1+A/R group; pcDNA3.1/Pim-3+A/R group. LDH activity, MTT and TUNEL were detected after treatment. Mitochondrial swelling was assayed by spectrophotometer. The expressions of Pim-3 and caspase-3 and the release of cytochrome c from mitochondria to cytoplasm were detected by Western blotting.Results:1. The expression of Pim-3 proteinThe expression level of Pim-3 protein showed a significant increase in A/R group compared with that of the control group (p<0.01). The expression level of Pim-3 protein was further increased in pcDNA3.1/Pim-3+A/R group, compared with that of A/R group (p<0.01).2. The Effects of Pim-3 on Cell Viability and LDH ActivityThe viability of cardiomyocytes in the A/R group was remarkably decreased and LDH activity was remarkably increased compared with those of control group (p<0.01). In the group of pcDNA3.1/Pim-3+A/R, the levels of LDH activity were noticeably lower than those of A/R group; while the viability of ventricular myocytes was significantly increased compared with those of A/R group (p<0.01). The transfection of pcDNA3.1 into cardiomyocytes, however, showed little protective effect on the cells.3. The Effects of Pim-3 on Cardiomyocyte ApoptosisThere were about 31.2±5.2% cardiomyocytes from A/R group presented as positive for TUNEL. Very few TUNEL-positive cells were detected among cardiomyocytes from the control group(p<0.01). In contrast, the number of TUNEL-positive cells was significantly reduced among cardiomyocytes of pcDNA3.1/Pim-3+A/R group, only 18.7±3.2% cardiomyocytes underwent apoptosis pcDNA3.1/Pim-3+A/R groups (p<0.01).4. The Effects of Pim-3 on Mitochondrial SwellingA/R induced damage to the inner mitochondrial membrane can be assessed by the classic swelling techniques, which monitor the net influx of the osmotic support associated with a non-specific increase in membrane permeability. It was shown that A/R induced mitochondrial swelling as revealed by the large decrease in absorbance of the mitochondrial suspension at 520 nm. However, Pim-3 inhibited the swelling process (P < 0.01).5. The Effects of Pim-3 on Cytochrome c Release from mitochondria to cytoplasmThe overall amount of cytochrome c in whole-cell extracts of cardiomyocytes remained at the same level. In control group, there was a low level of cytochrome c expression observed in the cytoplasmic fraction. However, the cardiomyocytes treated with A/R showed a higher expression level of cytochrome c in the cytosolic fraction when cells were undergoing apoptosis, which was concurrent with a lower level of cytochrome c in the mitochondrial fraction. In pcDNA3.1/Pim-3+A/R group, majority of cytochrome c was found to be retained in the mitochondrial fraction, indicating the low level of mitochondrial apoptosis in these cells(p<0.01).6. The Effects of Pim-3 on the activity of caspase-3The expression level of caspase-3 protein showed a significant increase in A/R group compared with that of the control group (p<0.01). However there was little change of the expression levels between pcDNA3.1+A/R and A/R group. In contrast, the expression levels of caspase-3 in pcDNA3.1/Pim-3+A/R group dramatically declined as compared to that of A/R group (P<0.01).Discussion and Conclusion:In this experiment, we demonstrated that transfection of Pim-3 into cardiomyocytes could effectively protect cardiomyocytes against A/R injury, and further investigated the mechanism of Pim-3 protective effects. Apoptosis is an important contributory factor in the loss and damage of cardiomyocytes when the myocardium is subjected to acute anoxia or ischemic injury. The fundamental cellular mechanism behind apoptosis can best be described as a balance between anti-apoptotic and pro-apoptotic activities. As a member of the Pim family, Pim-3 has been intriguing to investigators who tried to investigate if Pim-3 may fundamentally repair myocardium damage, through its anti-apoptotic effects. The mechanism of apoptosis is very complex. Recently, more investigations showed that Mitochondria should play a critical role in apoptosis in response to many stimuli. Mitochondria release cytochrome c into the cytosol, in response to an excessive open of mPTP. Therefore, cytochrome c release from the intermembrane space is supposed to be the determining factors in the final step to apoptosis. Our data indicated that the cardiomyocytes treated with A/R showed a higher expression level of cytochrome c in the cytosolic fraction when cells were undergoing apoptosis, which was concurrent with a lower level of cytochrome c in the mitochondrial fraction. However, transfection of cardiomyocytes with exogenous Pim-3 significantly lessened the release of cytochrome c from the mitochondria into the cytosol, which confirms that Pim-3 indeed has a cardio-protective effect by anti-apoptosis. The release of cytochrome c acts as an essential cofactor in the activation of the dormant killer proteases. Cytochrome c is released following the increase of mitochondrial membrane permeabilization. Cytochrome c can activate caspase cascade, the whole process is a positive feedback. The activation of caspases can carry out their function, mainly specific substrate cutting, DNA fragmentation, and cell apoptosis. Caspase-3 is the main executors of apoptosis and is known as the "terminator of apoptosis". Our results showed that caspase-3 was activated when cardiomyocytes subjected to A/R injury, while Pim-3 could inhibit the activity of caspase-3. In conclusion, the mechanism of Pim-3 anti-apoptosis is involved in mitochondrial apoptosis signaling pathway.