| BackgroundAs one of the key signal pathway proteins involved in the regulation of cardiac function,Calcium/calmodulin-dependent protein kinase II(Ca2+/calmodulin dependent protein kinase II,CaMKⅡ)gets involved in the calcium cycle,hypertrophy and apoptosis of the myocardium.Some researchers believe that in the case of pathological conditions（such as myocardial ischemia,myocardial hypertrophy,heart failure,etc.）excessive CaMKⅡ activation will cause calcium overload in cardiomyocytes and result in life-threatening arrhythmia and even sudden death.Meanwhile,it will also induce cardiomyocytes apoptosis and result in dysfunction or inhibition of systolic and diastolic function.Therefore,inhibition of CaMKⅡ activity may prevent the occurrence of ventricular arrhythmia in the case of pathological conditions by inhibiting the cardiomyocytes apoptosis to improve the myocardial ischemia/reperfusion（ischemia/reperfusion,I/R）injury after myocardial reperfusion therapy.According to the researches,autophagy plays a role in inhibiting myocardial injury and preventing ventricular arrhythmia during the process of myocardial ischemia/reperfusion（I/R）injury.However,the effect of inhibiting CaMKⅡ activity on cardiomyocytes autophagy is still unclear.According to current researches,CaMKⅡ can activate Ry R2 and PLN through phosphoprotein to participate in the occurrence of the ischemia/reperfusion（I/R）injury and ventricular arrhythmia.Therefore,according to the studies on myocardial ischemia/reperfusion（I/R）injury in isolated rat heart and the application of CaMKⅡ specific inhibitor KN93,this thesis explores the effect of changes in CaMKⅡ phosphorylation level on PLN and Ry R2 as well as its relationship with the occurrence of ventricular arrhythmia.Meanwhile,this thesis also explores the effect on cardiomyocytes apoptosis and autophagy during the process of ischemia reperfusion,so as to provide feasible d I/Rection for the protection of myocardial reperfusion injury in the future.Part 1:Effect of CaMKⅡ Phosphorylation Level on Ischemia Reperfusion ArrhythmiaMethods:A total of 40 female SD rats（250-280g）with normal electrocardiogram were selected to establish the myocardial I/R injury model by Langendorff perfusion system,which were divided into 5 groups:I/R group,I/R+isoproterenol group,I/R+KN92 group（KN92 structure is similar to that of KN93,no inhibition of CaMKⅡ activity）,I/R+KN93 group（CaMKⅡ specific inhibitor）and no-treatment control group.The multi-channel physiological recorder was used to detect the electrocardiogram of these rats,observe the incidence of ventricular arrhythmia in different groups,calculate the arrhythmia scores（scoring system criterion modified from Curtis and Walker）and test the levels of CaMKⅡ,p-CaMKⅡ,PLN,p-PLN,Ry R2,p-Ry R2（S2814）in myocardial tissues by means of western blot.Results:According to the analysis on arrhythmia scores,it can be seen that the scores in other groups（P<0.05）are significantly higher than that in the control group（0.500±0.71）.The arrhythmia score difference in I/R group（2.750±0.77）and I/R+KN92 group（2.625±0.99）has no statistical significance（P>0.05）.The score in I/R+isoproterenol group（3.000±1.00）is significantly higher than that in I/R group（P<0.05）while the score in I/R+KN93 group（1.500±0.87）is significantly lower than that in I/R group（P<0.05）although it’s still higher than that of the control group（P<0.05）.According to the results of isolated perfused myocardium calculated by western blot,the expression difference in CaMKⅡ,Ry R2 and PLN groups has no statistical difference（P>0.05）.Compared with the control group（p-CaMKⅡ/CaMKⅡ0.125±0.023、p-Ry R2/Ry R2 0.070±0.030、p-PLN/PLN 0.064±0.020）,the levels in p-CaMKⅡ/CaMKⅡ,p-Ry R2/Ry R2 and p-PLN/PLN groups are significantly higher（P<0.05）.Wherein,the difference in I/R+KN92 group（p-CaMKⅡ/CaMKⅡ0.414±0.053、p-Ry R2/Ry R2 0.438±0.055、p-PLN/PLN 0.360±0.040）and I/R group（p-CaMKⅡ/CaMKⅡ 0.411±0.032、p-Ry R2/Ry R2 0.424±0.040、p-PLN/PLN0.318±0.039）has no statistical significance（P>0.05）.The score in I/R+KN93 group（p-CaMKⅡ/CaMKⅡ 0.230±0.075、p-Ry R2/Ry R2 0.242±0.065、p-PLN/PLN0.201±0.077）is lower than that in I/R group but higher than that in the control group while the score in I/R+isoproterenol group（p-CaMKⅡ/CaMKⅡ 0.713±0.093、p-Ry R2/Ry R2 0.603±0.103、p-PLN/PLN 0.532±0.090）is higher than that in I/R group（P<0.05）.Conclusion:The occurrence of myocardial ischemia reperfusion ventricular arrhythmia may be related to the CaMKⅡ hyper-phosphorylation level of calmodulin Ry R2 and PLN.The inhibition of CaMKⅡ phosphorylation may reduce the occurrence of ventricular arrhythmia due to the possible mechanism of reducing calcium overload to effectively prevent myocardial ischemia/reperfusion（I/R）injury.Part 2:Effect of CaMKⅡ Phosphorylation Level on Autophagy and Apoptosis of Cardiomyocytes During Ischemia-Reperfusion InjuryMethods:A total of 40 female SD rats（250-280g）with normal electrocardiogram were selected to establish the myocardial I/R injury model by Langendorff perfusion system,which were divided into 5 groups:I/R group,I/R+isoproterenol group,I/R+KN92 group,I/R+KN93 group and no-treatment control group.Western blot was adopted to detect the changes of expression level of autophagy-related proteins LC3B and beclin-1 as well as apoptosis-related proteins bax,Bcl-2 and Caspase3 in the myocardial tissue.The immunofluorescence method was adopted to detect the expression of LC3B and the TUNEL method was adopted to detect the apoptosis of cardiomyocytes.Results:According to the result calculated by western blot method,the protein expression difference in I/R group and I/R+KN92 group has no statistical difference（P>0.05）.The expressions of Beclin-1,LC3B and Bcl-2 in I/R+KN93 group（Beclin-1 0.274±0.053、LC3B 0.753±0.101、Bcl-2 0.331±0.042）are higher than those in I/R group（Beclin-1 0.489±0.021,LC3B 1.234±0.037 and Bcl-20.484±0.058）but lower than those in the control（Beclin-1 0.659±0.063、LC3B 1.779±0.023、Bcl-2 0.768±0.034）group（P<0.05）.The expressions of Beclin-1,LC3B and Bcl-2 in I/R+isoproterenol group（Beclin-1 0.116±0.015、LC3B0.479±0.025、Bcl-2 0.130±0.023）are lower than those in I/R group（P<0.05）.The expressions of Bax,Cleaved caspase3 and the Bax/Bcl-2 ratio in I/R+KN93 group（Bax 0.370±0.040、Cleaved caspase3 0.163±0.235、Bax/Bcl-2 0.765±0.088）are lower than those in I/R group（Bax 0.485±0.037、Cleaved caspase3 0.341±0.089、Bax/Bcl-2 1.465±0.271）but higher than those in the control（Bax 0.147±0.067、Cleaved caspase3 0.102±0.066、Bax/Bcl-2 0.191±0.161）group（P<0.05）.The expressions of Bax,Cleaved caspase3 and the Bax/Bcl-2 ratio in I/R+isoproterenol group（Bax 0.590±0.050、Cleaved caspase3 0.666±0.070、Bax/Bcl-2 4,525±0.221）are higher than those in I/R group（P<0.05）.According to the analysis on western blot results,the apoptosis level rises along with the p-CaMKⅡ level while the autophagy level is inhibited.In other words,the inhibition of p-CaMKⅡ expression can inhibit the cell apoptosis and promote the occurrence of autophagy.According to the result of LC3B fluorescent labelling,the LC3B expression difference in I/R group（1613.36±253.93）and I/R+KN92 group（1830.01±303.29）has no statistical significance（P>0.05）,the luminosity in I/R+KN93 group（3401.22±612.07）is higher than that in I/R group and lower than that the control group（7666.89±1297.2）（P<0.05）;the luminosity in I/R+isoproterenol group（696.27±116.12）is lower than that in I/R group（P<0.05）while the luminosity in the control group is higher than that in other groups（P<0.05）.According to the result calculated by TUNEL method,the apoptosis level in I/R+isoproterenol group（25.58±3.93%）is the highest and the percentage of apoptotic myocardium is higher than that in the other groups（P<0.05）.The difference of apoptotic myocardium percentage in I/R group（18.17±3.29%）and I/R+KN92group（17.17±3.07%）has no statistical significance（P>0.05）.The percentage of apoptotic myocardium in I/R+KN93 group（12.68±1.39%）is lower than that in I/R group（P<0.05）while the percentage of apoptotic myocardium in control group（9.07±1.06%）is lower than that in other groups（P<0.05）.Conclusion:The level of CaMKⅡ phosphorylation is related to the autophagy and apoptosis of myocardial ischemia/reperfusion（I/R）injury.The inhibition of CaMKⅡ phosphorylation activity can reduce the cardiomyocytes apoptosis and promote the occurrence of autophagy so as to prevent the occurrence of myocardial ischemia/reperfusion（I/R）injury. |
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