| ObjectiveMyocardial ischemia which could induce cardiomyocyte apoptosis, plays an important role in the progress of coronary heart disease and heart dysfunction. Cardiac Shock Wave Therapy (CSWT) is a new technology for severe coronary heart disease. Clinical studies have shown the data about angiogenesis, alleviation of inflammation and oxidative stress inhibition in ischemic areas after CSWT, the improvement of myocardial perfusion, the amelioration of left ventricular function were also reported. But the effects of apoptosis on ischemia tissue after CSWT is still not clear. According to our early studies, we demonstrated that shock wave (SW) could effectively attenuate apoptosis of H9c2 cells in condition of ischemia and hypoxia. For further study, we made the rat model of acute myocardial infarction (AMI) by coronary artery ligation to explore the effects of CSWT on cardiomyocyte apoptosis and possible regulation pathway. The safety of CSWT was also assessed. It is helpfully to provide a new idea for the treatment of end-stage ischemic heart disease patients.Methods12 month old of male Wistar rats was used for the AMI model in this study by ligation of left anterior descending coronary artery (LAD).2 Western blotting was used for detection of those protein associated with apoptosis (Bax、Bcl-2 and Cleaved-Caspase-3) after coronary ligation of 6h、12h、24h、 36h and 48h respectively. Apoptosis rate of myocardial cells was measured by TUNEL staining.3 The rats after 12h of AMI were received SW(600,800,1000 shots respectively). Western blot analysis was used to detect the expression of Bax、Bcl-2 and Cleaved-Caspase-3 in the border zone of infarcted myocardium.4 The rats in SW group were treated with SW(energy:0.1mJ/mm2, time:800shots, frequancy:4Hz) and were killed after 36h of AMI. Heart tissue were observed by hematoxylin and eosin staining. Cell apoptosis rate was detected by TUNEL staining. The level of cytochrome C and the expression of apoptosis related proteins(Bcl-2, Bax, Caspase-3) and the phosphorylation of signaling pathway proteins (JNK, p38MAPK, Akt) were analyzed by western blotting.5 Normal Wistar rats were set up for normal control (NC) group and treated with SW (energy:0.1mJ/mm2, time:800shots, frequency:4Hz). Electrocardiogram, blood pressure (BP) and heart rate (HR) was recorded before and after SW. The rats were killed after 24h of AMI and heart tissue were observed histologically by hematoxylin and eosin staining. Cell apoptosis rate was detected by TUNEL staining. The level of cytochrome C and the expression of apoptosis related proteins (Bcl-2, Bax, Caspase-3) and the phosphorylation of signaling pathway proteins (JNK, p38MAPK, Akt) were analyzed by western blotting.Results1 Methodology of CSWT in AMI rat model1.1 AMI rats model was successfully established with the survival rate 75.4%, mortality 24.6% and the success rate of survival model was 93.9%.1.2 The exploration of the opportunity of CSWTThe tissue from AMI border zone was extracted for the detection of apoptosis rate at 61h、12h、24h、36h and 48h after ligation procedures respectively. The apoptosis rate by TUNEL of NC group and SW group of 6h、12h、24h、36h and 48h groups post ligation were 4.31%、8.16%、19.96%、30.96%、48.59%、69.28% respectively. Apoptosis rate at 12h after AMI increased significantly compared with NC group (P< 0.01) while decreased significantly compared with 24h and 36h after AMI (P<0.01). The expression of Bax (2.69±0.44) increased significantly as well compared with NC group (1.00±0.34). It suggested that apoptosis would obviously occurred at 12h after AMI but not achieve the extensive amount and the pro-apoptototic signaling pathways were activated remarkably at 12h as well. So we set up SW therapy on 12h after AMI.1.3 Exploration of the dose of shock waveThe rats were treated with SW 600,800 and 1000 shots respectively at 12h after AMI. The expression of Bcl-2 increased significantly and the expression of Bax and Cleaved-Caspase-3 decreased significantly in all SW groups compared with AMI without SW group. In 800 shots protocol, the expression of Bcl-2 most increased while the expression of Bax and Cleaved-Caspase-3 most decreased.2 The effects of CSWT on apoptosis of cardiomyocyte from AMI ratsRats were divided into four groups:sham group, sham with SW (sham+SW) group, AMI group and AMI with SW (AMI+SW) group.2.1 Hematoxylin and Eosin staining revealed that the degeneration, necrosis and infiltration were present in AMI group compared with sham group. Similar changes were observed in AMI+SW group.2.2 Detection of apoptosis rates by TUNEL staining in sham group、AMI group and AMI+SW group were 7.52±1.24%、48.92±5.13%,25.33±3.55% respectively. The apoptosis rate significantly increased in AMI group compared with sham group (P <0.01) while significantly reduced(P<0.01) after SW therapy.2.3 Western blot analysis revealed that the level of mitochondrial cytochrome C in sham group, AMI group and AMI+SW group was 1.00±0.19,0.42±0.06,0.93±0.20 respectively. Cytoplasm cytochrome C was 1.00±0.12、4.91±0.71、2.68±0.70 respectively. Compared with sham, mitochondrial cytochrome C decreased significantly while cytoplasm cytochrome C increased significantly. SW could inhibited the transition of cytochrome C from mitochondria to cytoplasm after AMI. CSWT could improve cardiomyocyte mitochondrial membrane integrity and attenuate cytochrome C release after AMI.2.4 Western blot revealed that the expression of Bcl-2 in sham group、AMI group and AMI+SW group was 1.00±0.11、0.80±0.14、1.09±0.11 respectively; the expression of Bax in three groups above was 1.00±0.16、4.95±0.45、3.09±0.30 respectively; the expression of Cleaved-Caspase-3 in those three groups was 1.00± 0.11、4.36±0.71±1.72±0.33 respectively. Compared with sham group, the expression of Bcl-2 decreased significantly while the expression of Bax and Cleaved-Caspase-3 increased significantly after AMI. Differences were statistically significant. CSWT could upregulate the expression of Bcl-2 and downregulate the expression of Bax and Cleaved-Caspase-3.2.5 The phosphorylation of JNK in sham group, AMI group and AMI+SW group was 1.00±0.08,2.23±0.72 and 0.78±0.21 respectively. The phosphorylation of p38MAPK was 1.00±0.04,1.49±0.16 and 1.25±0.24 respectively. The phosphorylation of Akt was 1.00±0.09,0.41±0.09 and 0.38±0.09 respectively. Compared with sham group, the phosphorylation of JNK, p38MAPK increased significantly in AMI group while phosphorylation of Akt decreased significantly. Compared with AMI group, phosphorylation of JNK decreased significantly in AMI+SW group (P<0.01), but no statistically differences on p38MAPK and Akt. The results suggested that apoptosis, inflammation,and survival related signaling pathways was activated after AMI. JNK-dependent signaling pathway might be inhibited by CSWT through anti-appototic effects.3 Safety studies of CSWT on rat heartThe data of rats heart tissue from NC group and NC+SW groups was showed as below:3.1 There was no obvious apomorphosis, necrosis, or infiltration were observed histologically by Hematoxylin and eosin staining in both groups.3.2 There was no significant difference of cardiomyocyte apoptosis rate by TUNEL staining between the two groups (P=0.71).3.3 The level of mitochondrial and cytoplasmic cytochrome C were similar between those two groups by western blot analysis.3.4 The expression of Bcl-2, Bax and Cleaved-Caspase-3 was no statistically differences between the two groups by western blot analysis (P value was 0.12、0.66、 0.42 respectively). Phosphorylation of JNK、p38MAPK and Akt was no statistically differences neither (P value was 0.07、0.69、0.74 respectively).3.5 No abnormal changes were observed on ECG, BP, HR before and after SW treatment. Differences were not statistically significant.Conclusions1 Cardiomyocyte apoptosis in the border zone could induce the release of cytochrome C from mitochondria to cytoplasm, downregulate the expression of anti-apoptosis protein Bcl-2 and upregulate the expression of pro-apoptosis protein Bax and Cleaved-Caspase-3 after AMI.2 CSWT could reduce the release of cytochrome C from mitochondria to cytoplasm, increase the expression of anti-apoptosis protein Bcl-2 and decrease the expression of pro-apoptosis protein Bax and Cleaved-Caspase-3.3 CSWT may decrease cardiomyocyte apoptosis through inhibition of JNK signal pathway way after AMI and further lead to protect cardiomyocyte.4 CSWT with appropriated protocol is a safe treatment for adult rat. |
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