| In reperfusion injury, mitochondrial damage by outbreak of the reactive oxygen species (ROS) is a determining factor in causing loss of cardiomyocyte function and viability. In particular, regulation of mitochondrial oxidative stress balance is the key to prevention of myocardial injury at the beginning of myocardial reperfusion. So far ischemic postconditioning (IPC) is known the strongest endogenous protection methods in ischemic myocardium, in which brief cycles of reperfusion and ischemia at reperfusion reduced infarct size similar to preconditioning. As prior knowledge of the onset of ischemia (as in preconditioning) is no longer a prerequisite, the therapeutic potential for IPC is significant. However, the mechanism of IPC is not known clearly. Here, we hypothesed that connexin (Cx)43in cardiac sarcolemma and mitochondria played an important role in IPC’s cardioprotection by regulation of mitochondrial oxidative stress balance. The study was divided into three parts.Firstly, to investigate the effect of postconditioning on cardiomyocytes apoptosis, the model of cardiomyocytes with hypoxia/reoxygenation(H/R) and rats with ischemia/reperfusion (I/R) were established. Results showed that the hypoxic postconditioning (HPC) and IPC significantly reduced cardiomyocytes apoptosis and myocardial injury induced by H/R and I/R.Secondly, we explored the anti-apoptotic mechanisms of HPC and IPC on cardiomyocytes. Results showed both mitochondrial ROS and the number of apoptotic cardiomyocytes reduced significantly in HPC and IPC groups compared to H/R and I/R groups. Bcl-2levels increased while Bax levels decreased in cardiac mitochondria and sarcolemma of HPC and IPC groups. Further, we investigated the influence of mitochondrial oxidative stress on cardiomyocyte apoptosis and the expression of Bcl-2and Bax proteins in cardiac sarcolemma and mitochondria after application of free radical scavengers superoxide dismutase (SOD), catalase (CAT), and heat shock protein90(Hsp90) inhibitor geldanamycin (GA) during HPC and IPC. We found that SOD or CAT alone did not but SOD plus CAT attenuate the anti-apoptotic effect of HPC and IPC, so did the GA. Mitochondrial ROS played an important role in postconditioning’s cardioprotection.Finally, in order to clarify the mechanism of ROS generation during the HPC and IPC, we used RNA interference technology to silence Cx43mRNA at the transcriptional level, and established Cx43low expression in vitro and in vivo in cardiomyocytes with H/R and rats with I/R. The results showed that Cx43low expression significantly reduced mitochondria ROS and increased cardiomyocytes apoptosis of HPC and IPC, suggesting that Cx43low expression canceled or diminished the myocardial protection of HPC and IPC. Thus, it revealed that the balance of ROS regulated by Cx43in cardiac mitochondria and sarcolemma play an important role in postconditioning’s cardioprotection.Part I The effect of postconditioning on cardiomyocytes apoptosis induced by hypoxia/reoxygenation and ischemia/reperfusionObjective To investigate the effects of postconditioning on cardiomyocytes apoptosis induced by H/R and I/R.Methods1.The model of cardiomyocytes with hypoxia/reoxygenation was established and the cardiomyocytes were divided into3groups, including control group, H/R group (hypoxia3h and reoxygenation6h), and hypoxic postconditioning (HPC) group (3intermittent cycles of5min H/R immediately after hypoxia3h, then reoxygenation for6h). Cardiomyocytes apoptosis was detected with Hoechst33342staining and flow cytometry.2. Twenty four SD rats were randomly divided into3groups(n=8):sham operation group(sham group), I/R group [the left anterior descending artery(LAD) was occluded for30min and reperfused for2h], and isehemic postconditioning group(IPC group,3intermittent cycles of30s I/R immediately after occlussion30min, then reperfusion for2h). Cardiomyocyte apoptosis was detected with in situ end labeling. Electrocardiogram and myocardial enzyme spectrum were used to access the efficiency of postconditioning. Results Cardiomyocytes apoptosis decreased significantly in HPC group [(26.99±3.35)%]and IPC group[(12.3±2.4)/vision] compared to H/R group [(45.86±3.29)%] and I/R group(22.1±3.1)/vision], P<0.01. Plasma LDH, CK-MB activities were significantly reduced in IPC group compared to I/R group.Conclusion Postconditioning attenuated cardiomyocytes apoptosis induced by H/R and I/R. Part II The anti-apoptotic mechanisms of hypoxic postconditioning and ischemic postconditioning on cardiomyocytesObjective1.To explore the anti-apoptotic mechanisms of hypoxic postconditioning and ischemic postconditioning on cardiomyocytes.2. To investigate the effect mitochondrial oxidative stress on cardiomyocytes apoptosis and the expression of Bcl-2and Bax proteins in cardiac mitochondria and sarcolemma after application of free radical scavengers and inhibitor of heat shock protein90.Methods1.The model of cardiomyocytes with H/R was established and divided into3groups, including control group, H/R group, and HPC group. Cardiac mitochondria and sarcolemma were isolated by differential centrifugation. Mitochondrial reactive oxygen species (ROS) of cardiomyocytes was detected with fluorescent probes. The expressions of Bcl-2and Bax proteins in the cardiac mitochondria and sarcolemma were measured by Western blot.2. The model of rats with I/R was established and twenty four SD rats were randomly divided into3groups(n=8):sham operation group(sham group), I/R group and IPC group. Cardiac mitochondria and sarcolemma were isolated by differential centrifugation. Mitochondrial ROS of myocardial tissue was detected with fluorescent probes. The expressions of Bcl-2and Bax proteins in the cardiac mitochondria and sarcolemma were measured by Western blot.3. Cardiomyocytes were exposed to3h hypoxia followed by (1)3intermittent cycles of5min H/R before6h of reoxygenation (HPC),(2) application of SOD before HPC (SOD+HPC),(3) application of CAT before HPC (CAT+HPC),(4) application of SOD plus CAT before HPC (SOD+CAT+HPC) and (5) application of GA before HPC (GA+HPC). Mitochondrial ROS was detected with fluorescent probes and cardiomyocyte apoptosis was detected with Hoechst33342staining and flow cytometry. The expressions of Bcl-2and Bax proteins in cardiac sarcolemma and mitochondria were measured by Western blot.4. LAD of forty SD rats was occluded for30min followed by (1)3intermittent cycles of30s I/R before2h of reperfusion (IPC),(2) intraperitoneal injection of SOD before IPC (SOD+IPC),(3) intraperitoneal injection of CAT before IPC (CAT+IPC),(4) intraperitoneal injection of SOD plus CAT before IPC (SOD+CAT+IPC) and (5) intraperitoneal injection of GA before IPC (GA+IPC). Mitochondrial ROS was detected with fluorescent probes and cardiomyocyte apoptosis was detected with in situ end labeling. The expressions of Bcl-2and Bax proteins in cardiac sarcolemma and mitochondria were measured by Western blot.Results1. Mitochondrial ROS of cardiomyocytes increased significantly in H/R (61.53±4.73), while reduced significantly in HPC(32.72±2.86), SOD+HPC(23.05±1.13), CAT+HPC (23.82+1.88), GA+HPC groups (17.52+1.02) and especially in SOD+CAT+HPC group (16.58+0.74, all P <0.01). The rate of apoptotic cardiomyocytes reduced significantly in HPC [(26.42+2.96)%], SOD+HPC [(26.01+4.24)%] and CAT+HPC [(26.98+3.66)%] but not in SOD+CAT+HPC [(44.60+3.12)%] and GA+HPC groups [(45.65+3.71)%](all P<0.01). Bcl-2levels increased while Bax levels decreased in cardiac sarcolemma and mitochondria in HPC, SOD+HPC and CAT+HPC groups (all P<0.01), while in H/R, SOD+CAT+HPC and GA+HPC groups Bcl-2levels decreased and Bax levels increased (all P<0.01).2. Mitochondrial ROS of myocardial tissues increased significantly in I/R (72.26±4.01), while reduced significantly in IPC (35.59±3.49), SOD+IPC (23.26±2.03), CAT+IPC (25.03±2.59), GA+IPC (17.77±1.42) groups and especially in SOD+CAT+IPC groups (16.21±1.62, all P<0.01). The number of apoptotic cardiomyocytes reduced significantly in IPC [(12.3±2.4)/vision], SOD+IPC [(13.3±2.3)/vision] and CAT+IPC [(13.0±1.6)/vision] but not in SOD+CAT+IPC [(20.6±2.5)/vision] and GA+HPC groups [(23.4±2.3)/vision](all P<0.01). Bcl-2levels increased while Bax levels decreased in cardiac sarcolemma and mitochondria in IPC, SOD+IPC and CAT+IPC groups (all P <0.01), while in I/R, SOD+CAT+IPC and GA+IPC groups Bcl-2levels decreased and Bax levels increased (all P<0.01).Conclusions Postconditioning attenuated H/R and I/R induced ROS and cardiomyocytes apoptosis, which potentially mediated by upregulating the expression of Bcl-2and downregulating the Bax in mitochondria and sarcolemma; SOD or CAT alone did not but SOD plus CAT attenuate the anti-apoptotic effect of HPC and IPC, so did the GA; mitochondrial ROS played an important role in postconditioning’s cardioprotection. Part III The effect of reactive oxygen species regulated by connexin43in cardiac mitochondria and sarcolemma on postconditioning’s cardioprotectionObjective1.To investigate the effect of reactive oxygen species on cardiac protection regulated by connexin43in cardiac mitochondria and sarcolemma during HPC and IPC.Methods1. Cx43normal expression in myocardial protection after HPC. Cardiomyocytes were exposed to3h hypoxia followed by(1)6h of reoxygenation (R)(H/R),(2)3intermittent cycles of5min H/R before6h of reoxygenation (HPC),(3) application of SOD before HPC (SOD+HPC),(4) application of CAT before HPC (CAT+HPC),(5) application of SOD plus CAT before HPC (SOD+CAT+HPC), and (6) application of GA before HPC (GA+HPC). Expression of Cx43mRNA was determined by real time PCR and total Cx43and phospho-Cx43protein in myocardial mitochondria and sarcolemma were detected by Western blot.2. Cx43normal expression in myocardial protection after IPC. LAD of forty-eight SD rats was occluded for30min followed by (1)2h of reperfusion(I/R),(2)3intermittent cycles of30s I/R before2h of reperfusion(IPC),(3) intraperitoneal injection of SOD before IPC (SOD+IPC),(4) intraperitoneal injection of CAT before IPC (CAT+IPC),(5) intraperitoneal injection of SOD plus CAT before IPC (SOD+CAT+IPC) and (6) intraperitoneal injection of GA before IPC (GA+IPC). Expression of Cx43mRNA was determined by real time PCR and total Cx43and phospho-Cx43protein in myocardial mitochondria and sarcolemma were detected by Western blot.3. Effect of RNA interfering expression of Cx43on HPC. The model of cardiomyocytes with H/R with Cx43low expression in vitro was established and divided into4groups, including control group, negative control group (pGCFu-RNAi-NC-LV in vitro, NC), normal expression of Cx43before HPC group (Cx43-HPC), and low expression of Cx43before HPC group(Cx43-RNAi-LV in vitro, iCx43-HPC). Mitochondrial reactive oxygen species (ROS) was detected with fluorescent probes and cardiomyocytes apoptosis was detected with Hoechst33342staining and flow cytometry. The expressions of Bcl-2and Bax proteins in the cardiac mitochondria and sarcolemma were measured by Western blot.4. The effect of RNA interfering expression of Cx43on IPC. Establishment of Cx43low expression in rats in vivo, and thirty two SD rats were randomly divided into4groups (n=8):sham operation group (sham group), normal expression of Cx43before IPC group (Cx43-IPC), low expression of Cx43before IPC group(Cx43-RNAi-LV in vivo, iCx43-IPC), and negative control group (pGCFu-RNAi-NC-LV in vivo, NC). Cardiac mitochondria and sarcolemma were isolated by differential centrifugation. Mitochondrial ROS of myocardial tissues was detected with fluorescent probes and cardiomyocytes apoptosis was detected with in situ end labeling. The expressions of Bcl-2and Bax proteins in the cardiac mitochondria and sarcolemma were measured by Western blot.Results1. The expressions of Cx43mRNA, total Cx43and phospho-Cx43protein in myocardial mitochondria and sarcolemma were significantly increased in HPC, SOD+HPC and CAT+HPC groups (all P<0.01), but significantly decreased in H/R, SOD+CAT+HPC and GA+HPC groups (all P <0.01).2. The expressions of Cx43mRNA, total Cx43and phospho-Cx43protein in myocardial mitochondria and sarcolemma were significantly increased in IPC, SOD+IPC and CAT+IPC groups (all P<0.01), but significantly decreased in1/R, SOD+CAT+IPC and GA+IPC groups (all P <0.01).3.The rate of apoptotic cardiomyocytes in iCx43-HPC group[(31.46±3.35)%] was significantly higher than that of the control group [(4.68±1.45)%], negative control group [(5.51±1.53)%] and Cx43-HPC group [(20.11±2.84)%]; mitochondrial ROS in iCx43-HPC group (16.78±2.05) was significantly higher than the control group (10.95±1.82) and NC group (11.76±2.3), but significantly lower than that of Cx43-HPC group (30.67±2.08)(all P<0.01); Bcl-2levels increased while Bax levels decreased in cardiac sarcolemma and mitochondria in Cx43-HPC,(P<0.01), while in iCx43-HPC group Bcl-2levels decreased and Bax levels increased (P<0.01).4. The number of apoptotic cardiomyocytes in iCx43-HPC group [(23.5±2.8)/vision] was significantly higher than sham group [(1.5±0.6)/vision], the NC group [(1.7±0.5)/vision] and Cx43-IPC group[(12.3±2.4)/vision]; mitochondrial ROS in iCx43-IPC group (15.74±1.00) was significantly higher than the sham group (10.65±0.96) and negative control group (10.82±0.78), but significantly lower than that of Cx43-IPC group (31.99±1.02)(all P<0.01); Bcl-2levels increased while Bax levels decreased in cardiac sarcolemma and mitochondria in Cx43-IPC,(P<0.01), while in iCx43-IPC group Bcl-2levels decreased and Bax levels increased (P<0.01).Conclusions ROS regulated by Cx43in cardiac mitochondria and sarcolemma play an important role in postconditioning’s cardioprotection. |
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