Study The Molecular Mechanism Of Ventricular Arrhythmia And Regulatory Effect Of Yangxin Dingji Capsule Based On Calcium/Calmodulin-Dependent Kinase Ⅱ Pathway

Part One Isoprenaline-induced ventricular arrhythmia in SD rats and the antagonistic effect of Yangxin Dingji capsulesObjective:To observe the general condition,VA incidence,the degree of myocardial damage and the pathological changes of myocardial tissue in rats with ventricular arrhythmias（VA）,and to explore the effect of Yangxin Dingji Capsule（YXDJ）on rats with VA,and provide experimental evidence for YXDJ’s application in clinic.Methods:1.Animal grouping:Sixty healthy male SD rats were randomly divided into control group and intervention groups such as model group,western medicine group,YXDJ capsule low,middle and high dose group.Each group had 10 rats.2.Drug intervention:Normal saline was administered orally at daily dose of 0.5 g/kg to rats in the control and model group.Propranolol Hydrochloride was administered orally at daily dose of 15 mg/kg to rats in the western medicine group.Chinese medicine was administered orally at daily dose of 0.5g/kg,1.0g/kg,2.0g/kg to rats in the YXDJ capsule low dose,middle and high dose group,respectively.Each group was treated by intragastric administration for 7 consecutive days.3.Model preparation:Isoproterenol hydrochloride（ISO）was administered subcutaneously at daily dose of 5mg/kg for 6 consecutive days in the intervention groups at 2 hours after intragastric administration.On the seventh day,the rats were accurately weighed（BW）,anesthetized,and ISO3mg/kg intraperitoneally injected to induce ventricular arrhythmias.The death of rats during the model preparation was recorded and the mortality was calculated.4.Test indicators and methods:The BL-420F physiological function experiment system was used to monitor the ECG for 1 hour to calculate VA,ventricular tachycardia（VT）and ventricular premature beats（VP）Incidence.When the ECG monitoring was completed,blood from the femoral artery and the heart tissue was taken.The heart weight（HW）was accurately weighed,and the heart weight index（HWI）was calculated,HWI=HW/BW.Automatic biochemical analyzer was used to detect serum creatine kinase isoenzyme（CK-MB）.Enzyme-linked immunosorbent assay（ELISA）detected serum troponin（cTnI）.Hematoxylin-eosin staining（HE）were used to observe the pathological changes of myocardial tissue.2,3,5-triphenyltetrazolium chloride staining（TTC）were used to observe degree of myocardial injury.5.Statistical analysis:The experimental data were expressed as mean±SD.SPSS 21.0 statistical software was used for data analysis.One-way analysis of variance was performd to multiple comparison.Tukey was used for comparison among groups.Chi-square test was used for count data and rate comparison.All results were considered statistically significant at P<0.05.Results:1.The general condition and mortality:Rats in the control group were quick in movement,with stable food and water intake as well as body weight growth.The rats in the model group became slow in action,with decreased food and water intake and body weight growth,whereas those in the western medicine group and the traditional Chinese medicine group were quick in movement,with stable food and water intake as well as body weight growth.Compared with the control group,the mortality of rats in the model group did not increase significantly（P>0.05）.The mortality of rats in the western medicine group and the traditional Chinese medicine group did not significantly decrease,as compared with the model group（P>0.05）.2.HW/BW:Compared with the control group,HWI was increased significantly（P<0.01）in the model group.Compared with the model group,rats HWI in the western medicine group and YXDJ capsule high-dose group was decreased significantly（P<0.01）;Rats HWI in the YXDJ capsule low and middle-dose group was significantly reduced（P<0.01 or P<0.05）.3.Comparison of VA in rats in each group:The electrocardiogram of the rats in the control group was normal.The ECG in the model group was mainly VT and VP.The electrocardiogram of rats in the western medicine group and the traditional Chinese medicine group showed significant decrease in VT and VP.Some rats behaved normally.Compared with the control group,VA,VT and VP incidence were increased significantly（P<0.01）.Compared with the model group,VT and VP incidence in the western medicine group were decreased significantly（P<0.05）,while VA incidence was desreased and did not have significance.VT incidence in the YXDJ capsule high-dose group was decreased significantly（P<0.05）,while VA and VP incidence was desreased and did not have significance.4.The level of myocardial enzymes and protein in serum:Compared with the control group,the serum cTnI and CK-MB in the model group was increased（P<0.01）.Compared with the model group,the expression of cTnI and CK-MB were decreased in the western medicine group,YXDJ capsule middle and high-dose group（P<0.01 or P<0.05）;CK-MB was decreased significantly（P<0.01）in the YXDJ capsule low-dose group.5.Myocardial injury in each group:TTC staining shows,compared with the control group,myocardial injury degree in the model group was increased（P<0.01）.Myocardial injury degree in the western medicine group and the traditional Chinese medicine group was reduced notably,as compared with model group（P<0.01）.6.Pathological changes of myocardial tissue in each group:HE staining shows,there was no abnormality in the myocardial tissue of rats in the control group.The myocardial tissue of the rats in the model group showed disordered arrangement,unclear borders,and lytic necrosis.The myocardial tissue of rats in the western medicine group and the traditional Chinese medicine group improved significantly.Part Two The role of Isoprenaline-induced oxidative stress and inflammation in the occurrence of ventricular arrhythmia and the effect of Yangxin Dingji capsulesObjective:To Observe the degree of oxidative stress and the expression of inflammatory factors in the myocardium of rats with VA,and to clarify the effect of YXDJ on oxidative stress and inflammation in rats with VA,and explore the mechanism of YXDJ’s myocardial protection.Methods:1.Animal grouping,model preparation,drug intervention and statistical analysis were performed,as previously described in Part One.2.Test indicators and methods:Water-soluble tetrazolium（WST-1）method was used to detect serum SOD activity.Thiobarbituric acid（TBA）method was utilized to detect serum malondialdehyde（MDA）.Microplate method as applied to detect serum glutathione（GSH）.Nitrate reductase method were employed to detect serum nitric oxide（NO）.ELISA method was used to interleukin-6（IL-6）and interleukin 1β（IL-1β）,transforming growth factor-α（TNF-α）in serum.Real time-PCR was used to detect the expression nicotinamide adenine dinucleotide phosphate oxidase 2/4(NOX_2/4).Immunohistochemical method was utilized to detect the expression of NOX₄ in cardiomyocytes.Results:1.The content of SOD and GSH in serum:Compared with the control group,the serum SOD and GSH in the model group were significantly reduced（P<0.01）.Compared with the model group,the expression of SOD and GSH in the western medicine group,YXDJ capsule middle and high-dose groups were significantly increased（P<0.01）;SOD activity did not increase significantly（P>0.05）,while GSH level was increased significantly（P<0.01）in the YXDJ capsule low-dose group.2.The content of MDA in serum:Compared with the control group,the serum MDA in the model group was significantly increased（P<0.01）.MDA level in the western medicine group and YXDJ capsule high-dose was reduced（P<0.05）;serum MDA in the YXDJ capsule low-dose and middle-dose groups did not decrease significantly（P>0.05）,as compared with model group.3.The content of NO in serum:Compared with the control group,serum NO in the model group was significantly increased（P<0.01）.NO level in the western medicine group and the traditional Chinese medicine group was reduced notably（P<0.01）,as compared with model group.4.The expression of NOX_2/4 in myocardial tissue of rats:Compared with the control group,the expression of NOX_2/4 mRNA in the model group were increased（P<0.01）.Compared with the model group,the expression of NOX_2/4 mRNA in the western medicine group,YXDJ capsule middle and high-dose group were decreased（P<0.01 or P<0.05）;NOX_2/4 mRNA in the YXDJ capsule low-dose and middle-dose groups were decreased and did not have significance.5.The expression of NOX₄ molecular protein in heart of rats by SABC:Compared with the control group,NOX₄ in cardiomyocytes of the model group was significantly increased.Compared with the model group,the western medicine group and the traditional Chinese medicine group were significantly weakened.6.The content of inflammatory factors in serum:Compared with the control group,serum IL-6,IL-1βand TNF-αin the model group were increased（P<0.01）.Compared with the model group,the level of IL-6,IL-1βand TNF-αin the western medicine group were decreased（P<0.01 or P<0.05）;the level of IL-6,IL-1βin the YXDJ capsule middle and high-dose group were reduced（P<0.01 or P<0.05）,whereas TNF-αwas reduced slightly.Part Three Expression of CaMKⅡ and SERCA2 in Cardiac Myocytes of Rats with Isoprenaline-induced Ventricular Arrhythmia and the Effect of Yangxin Dingji CapsuleObjective:To observe the expression levels of calcium/calmodulin-dependent kinase II（CaMKⅡ）,sarcoplasmic reticulum calcium ATPase（SERCA2）and phosphoprotein phosphorylation（P-PLB）in rats with VA model,and to explore the effect of YXDJ on the expression of CaMKⅡ,SERCA2 and P-PLB,and further clarify the target of YXDJ on ventricular arrhythmia.Methods:1.Animal grouping,model preparation,drug intervention and statistical analysis were performed,as previously described in Part One.2.Test indicators and methods:The microplate method was used to detect the Ca²⁺content of myocardial tissue.The ELISA method was used to detect the activity of CaMKⅡ in cardiac myocytes.Real time-PCR was used to detect the expression of SERCA2 and CaMKⅡδmRNA in myocardial tissue.Immunohistochemical method was utilized to detect the expression of CaMKⅡ in cardiac myocytes.Western blot was applied to detect the expression of CaMKⅡ,oxidized CaMKⅡ（OX-CaMKⅡ）,P-PLB and sarcoplasmic reticulum calcium pump（SERCA2）in cardiac myocytes.Results:1.The content of Ca²⁺level in heart of SD rats among groups:Compared with the control group,myocardial tissue Ca²⁺in the model group was significantly increased（P<0.01）.The content of Ca²⁺in the western medicine group and YXDJ capsule middle and high dose group was reduced notably（P<0.01）;YXDJ capsule low group doesn,t have significantly decrease（P>0.05）,as compared with model group.2.The expression of CaMKⅡδmRNA in heart of SD rats:Compared with the control group,myocardial tissue CaMKⅡδmRNA in the model group was significantly increased（P<0.01）.Compared with the model group,the expression of CaMKⅡδmRNA in the western medicine group and the traditional Chinese medicine group was reduced notably（P<0.01 or P<0.05）.3.CaMKⅡ activity in heart of SD rats:Compared with the control group,cardiac myocytes CaMKⅡ activity in the model group was significantly increased（P<0.01）.Compared with the model group,CaMKⅡ activity in the western medicine group and YXDJ capsule middle and high dose group was reduced notably（P<0.05）;YXDJ capsule low group did not significantly decrease（P>0.05）.4.The expression of CaMKⅡ molecular protein in heart of rats by SABC:Compared with the control group,CaMKⅡ in cardiac myocytes of the model group was significantly increased（P<0.01）.Compared with the model group,the western medicine group,YXDJ capsule middle and high dose group were significantly weakened（P<0.01）,while YXDJ capsule low did not significantly decreased（P>0.05）.5.The expression of OX-CaMKⅡ,CaMKⅡ protein in heart of rats by Western blot:Compared with the control group,the expression of OX-CaMKⅡ,CaMKⅡ in the model group were significantly increased（P<0.01）.Compared with the model group,OX-CaMKⅡ,CaMKⅡ protein in the western medicine group,YXDJ capsule middle and high dose group were significantly reduced（P<0.05 or P<0.01）;The expression level of OX-CaMKⅡ in the YXDJ capsule low-dose group did not significantly decreased（P>0.05）,while the expression of CaMKⅡ decreased significantly（P<0.05）.6.The expression of SERCA2 mRNA in heart of SD rats:Compared with the control group,myocardial tissue SERCA2 mRNA in the model group was significantly decreased（P<0.01）.Compared with the model group,the expression of SERCA2 mRNA in the western medicine group and the traditional Chinese medicine group was increased notably（P<0.01 or P<0.05）.7.The expression of SERCA2 protein in heart of rats by Western blot:Compared with the control group,the expression of SERCA2 in the model group were significantly decreased（P<0.01）.Compared with the model group,SERCA2 protein in the western medicine group,YXDJ capsule middle and high dose group were significantly increased（P<0.05 or P<0.01）;The expression level of SERCA2 in the YXDJ capsule low-dose group did not significantly increased（P>0.05）.8.The expression of P-PLB protein in heart of rats by Western blot:Compared with the control group,cardiac myocytes P-PLB in the model group was significantly increased（P<0.01）.The expression of P-PLB in the western medicine group and the traditional Chinese medicine group was increased notably（P<0.01 or P<0.05）,as compared with model group.Conclusions:1.ISO can induce cardiomyocyte damage in SD rats and cause obvious ventricular arrhythmias.The mechanism may be related to the induction of oxidative stress and inflammation in cardiomyocytes,which in turn activates CaMKⅡ and inhibits SERCA2 activity.2.Yangxin Dingji Capsules can reduce oxidative stress and inflammation in cardiomyocytes,regulate the CaMKⅡ pathway,inhibit the activity of SERCA2,and antagonize the occurrence of ventricular arrhythmias.