Cardioprotective Effects Of Simvastatin And Anisodamine On Preventing Arrhythmias And Reversing Electrical Remodeling Induced By Myocardial Ischemia-reperfusion In Rabbits

Percutaneous coronary intervention (PCI) is frequently associated with the potential ischemic-reperfusion injury. Especially, ventricular tachyarrhythmias, including ventricular tachycardia (VT) and ventricular fibrillation (VF), induced by coronary artery occlusion / reperfusion, are the major direct causes of sudden cardiac death in patients with coronary artery disease, and sudden cardiac death is a leading cause in increasing cardiovascular mortality. However, there is little information regarding the ionic mechanism and prevention of reperfusion arrhythmia. After coronary artery occlusion / reperfusion, surviving myocardium in and around the infarct zone plays an important role in arrhythmogenesis , which is thought to be strongly associated with the alterations of electrophysiological characteristics ,called as"electrical remodeling". So preventing or reversing electrical remodeling induced by ischemia and reperfusion should also be a clinical therapeutic target.Hydroxymethylglutary coenzyme A reductase inhibitors (statins) have been shown to have effects independent of their cholesterol- lowering effects, referred to as pleiotropic effects. It has been shown that pretreatment with statin is effective in preventing reperfusion arrhythmia after ischemia- reperfusion in the experimental model and clinical study. Therefore, it is important to investigate possible anti-arrhythmic effects of statins, but its electrophysiological mechanism is unclear. Anisodamine is a alkaloid isolated from nightshade henbane in China. Being a M-choline receptor blocker, anisodamine have been shown by previous studies that it can improve microcirculation and protect against myocardial damage by ischemia-reperfusion. Clinical studies have also suggested that anisodamine is effective in preventing arrhythmia, but its electrophysiological mechanism is unclear.Based on these observations, the present study was designed to examine the effect of pretreatment with simvastatin as well as anisodamine on the changes in membrane ionic currents, including sodium channel current (INa), L-type calcium channel current (ICa-L) and transient outward potassium channel current (Ito) in left ventricular myocytes of normocholesterolemic rabbits undergoing ischemia and reperfusion, by the whole cell patch-clamp recording technique, so as to explore the ionic mechanism responsible for the anti-arrhythmic effect of statin . The study is comprised of four parts described as follows:Part I:Cardioprotective effects of simvastatin on reversing electrical remodeling induced by myocardial ischemia-reperfusion in normocholesterolemic rabbitsObjective: Recent studies have revealed that pretreatment with statin is effective in preventing arrhythmia, but its electrophysiological mechanism is unclear. This study was conducted to investigate the cardioprotective effects of simvastatin on reversing electrical remodeling in left ventricular myocytes of rabbit heart undergoing ischemia- reperfusion ,so as to explore the ionic mechanisms responsible for the anti-arrhythmic effect of statin .Methods: Forty-five rabbits were randomly divided into three groups : ischemic-reperfusion group (I-R), simvastatin intervention group (Statin) and sham-operated control group (CON). Anesthetized rabbits were subjected to 30-min ischemia by ligating left anterior descending coronary artery and 60-min reperfusion after administration of oral simvastatin (5 mg·kg-1·d-1 ) (Statin group) or placebo (I-R group) for 3 days.The incidence of ventricular arrhythmia of premature ventricular contraction (PVC), ventricular tachycardia (VT) and ventricular fibrillation (VF) were observed. Single ventricular myocytes were isolated enzymatically from the epicardial zone of the infarcted region derived from the hearts in I-R , statin group and the same anatomy region in CON. Whole cell patch clamp technique was used to record membrane ionic currents, including sodium current (INa), L-type calcium current (ICa-L) and transient outward potassium current (Ito). Simultaneously, the level of serum cholesterol was examined.Results:①The concentration of serum cholesterol: there was not significant difference in serum cholesterol concentration among three groups.②The incidence of ventricular arrhythmia: compared with I-R group, Statin decreased the incidence and duration of ventricular arrhythmia by reperfusion, resulting in significant decrease of the scores of arrhythmia(2.3±0.6 VS 3.6±0.8,P<0.05).③Effect of simvastatin on INa in rabbit ischemic myocytes: the peak INa current density (at–30 mV) was significantly decreased in I-R(–22.46±5.32 pA/pF, n=12) compared with CON (–42.78±5.48 pA/pF ,n=16 ,P<0.01) and Statin (–40.66±5.89 pA/pF,n=15 ,P<0.01), while the peak INa current density in Statin group was no different from CON( P>0.05).④Effect of simvastatin on ICa-L in rabbit ischemic myocytes: the peak ICa-L current density (at 0 mV) was significantly increased in I-R (–4.34±0.92 pA/pF, n=15) compared with CON (–3.13±1.22 pA/pF, n= 16, P<0.05) and Statin (–3.46±0.85 pA/pF, n= 16,P<0.05), while the Peak ICa-L current density in Statin was no different from CON (P>0.05).⑤Effect of simvastatin on Ito in rabbit ischemic myocytes: the Ito current density (at +60 mV) was significantly decreased in I-R (9.49±1.91 pA/pF, n=11) compared with CON (17.41±3.13 pA/pF ,n=15 ,P<0.01) and Statin (15.24±2.41 pA/pF, n=11, P<0.01), although there was slight reduction in Statin group compared with CON( P<0.05).Conclusions: Our study showed that simvastatin has the ability to increase electrical stability of the cardiomyocytes and thereby reduce the occurrence of ventricular arrhythmia. Our study also showed that ischemia-reperfusion induced significant down-regulation of INa and Ito, and up-regulation of ICa-L, which may underlie the altered electrical activity and long abnormal transmembrane APD of the surviving ventricular myocytes, thus contributing to ventricular arrhythmias in the infarcted heart. Pretreatment with simvastatin could attenuate these changes, suggesting that simvastatin could reverse this electrical remodeling and attenuate inhomogeneity without lowering the serum cholesterol level, thus contributing to the ionic mechanism responsible for the anti-arrhythmic effect of statin. It might imply that the ionic mechanism of statin for anti-arrhythmia is a pharmacological effect independent on decreasing cholesterol. Accordingly, simvastatin, may contribute to reducing cardiovascular mortality, through its anti-arrhythmic effects. So preventing or reversing electrical remodeling induced by ischemia and reperfusion should also be a clinical therapeutic target. Our findings expand the pleiotropic spectrum of the statins'favorable effects on cardiovascular diseases. Part II: Effects of pretreatment with simvastatin on the area of myocardial infarction in reperfusion injury rabbits after acute myocardial infarctionObjective: To investigate the effects and mechanism of the pretreatment with simvastatin on the area of myocardial infarction in reperfusion injury rabbits after acute myocardial infarction(AMI) .Method: Twenty New Zealand white rabbits were randomly divided into four groups : group A, AMI/ reperfusion; group B, pretreated with simvastatin( 5 mg/kg) for 3 days before AMI ;group C, treated with glibenclamide (KATP channel blocker) (5mg/Kg ) before AMI, group D, treated with glibenclamide and simvastatin before AMI . Models of AMI/ reperfusion were established by 180- minute of coronary occlusion and 60- minute of reperfusion. At the end of reperfusion,the coronary artery was reoccluded ,and the risk zone was delineated with Evan'blue. Hearts were sectioned (2mm) and incubated in 1% TTC in phosphate buffer for 20 min to define white necrotic tissue when fixed in 10% formalin for 24 h, and the level of plasma creatine kinase-MB (CK-MB) was assessed and evaluated.Result:①The content of CK-MB was significantly decreased in group B than that in group A and group C (P < 0. 01) , however, it was markedly decreased in group D than that of group A (P<0.05), and significantly increased than that of group B (P<0.05).②The risk zone sizes were similar among all the groups. The infarct size was (43.6±4.6)% in group A. Simvastatin treatment resulted in a significant limitation of infarct size in group B (23.6±2.8% VS group A, P<0.01), and the infarct size was similar in group C (45.1±4.5%) compared with that in group A (P>0.05). However, it was markedly decreased in group D (36.8±3.4%) than that of group A(P<0.05), and significantly increased than that of group B (P<0.05).Conclusion: Simvastatin significantly reduce myocardial infarct size and the level of of plasma myocardial enzyme during AMI and reperfusion in rabbits, which has protective action against ischemia-reperfusion injury , and the activation of ATP-sensitive K channels might be involved in this protective mechanism.Part III: Effects of anisodamine on multiple ion channels in isolated ventricular myocytes from normal and ischemia-reperfusion myocardiumObjective :Previous studies have shown that anisodamine can protect myocardium against damage by ischemia-reperfusion. Clinical studies have also suggested that anisodamine is effective in preventing arrhythmia, but its electrophysiological mechanism is unclear. This study was conducted to investigate the cardioprotective effects of anisodamine on reversing electrical remodeling in left ventricular myocytes of rabbit heart undergoing ischemia-reperfusion , and to explore the ionic mechanism responsible for the anti-arrhythmic effect of anisodamine .Methods: Forty-five rabbits were randomly divided into three groups : ischemic-reperfusion group (I-R), anisodamine intervention group (Ani) and sham-operated control group (CON). Anesthetized rabbits were subjected to 30-min ischemia by ligation of the left anterior descending coronary artery and 60-min reperfusion . Ani group was injected with anisodamine at a dose of 5mg/kg via femoral vein 1 min before operation.The incidence of ventricular arrhythmia of premature ventricular contraction (PVC), ventricular tachycardia (VT) and ventricular fibrillation (VF) was observed. Single ventricular myocytes were isolated enzymatically from the epicardial zone of the infracted region derived from the hearts in I-R , Ani group and the same anatomy region in CON .Whole cell patch clamp technique was used to record membrane ionic currents, including sodium current (INa), L-type calcium current (ICa-L) and transient outward potassium current (Ito).Results:①The incidence of ventricular arrhythmia: compared with I-R group , Ani decreased the incidence and duration of ventricular arrhythmia by reperfusion, resulting in significant decrease the scores of arrhythmia (2.6±0.7 VS 3.6±0.8,P<0.05).②Effects of anisodamine on membrane ionic currents of normal rabbit ventricular myocytes: Ani of 10 nmol/L did not affect the ICa-L, Ani of 100, 1000 nmol/ L inhibited ICa-L by 31.3% and 41.8% respectively( borh P < 0.01). Ani of 10 nmol/L did not affect the INa, Ani of 100, 1000 nmol/ L inhibited INa by 22.3% and 31.5% respectively( borh P < 0.01). Ani of 10 , 100, 1000 nmol/ L inhibited Ito by 7.3%,13.2% and 14.1% respectively(P all >0.05).③Effect of Ani on membrane ionic currents (ICa-L ,INa and Ito)of rabbit ischemic/reperfusion myocytes : ICa-L—the peak ICa-L current density(at 0 mV) was significantly increased in I-R (–4.34±0.92 pA/pF, n=15) compared with CON (–3.13±1.22 pA/pF, n= 16, P<0.05),while it was significantly decreased in Ani group (–3.25±0.79 pA/pF,n= 12) compared with I-R group(P<0.05); INa—the peak INa current density (at–30 mV) was significantly decreased in I-R(–22.46±5.32 pA/pF, n=12) compared with CON (–42.78±5.48 pA/pF ,n=16 ,P<0.01), while it was significantly increased in Ani group(–38.89±5.24 pA/pF, n=13 ) compared with I-R group(P<0.01); Ito—the Ito current density (at +60 mV) was significantly decreased in I-R(9.49±1.91 pA/pF, n=11) compared with CON (17.41±3.13 pA/pF, n=15, P<0.01), while it was significantly increased in Ani group(16.55±2.86 pA/pF,n=10) compared with I-R group (P<0.01).Conclusions:①Anisodamine have the ability to reduce the occurrence of ventricular arrhythmia.②Anisodamine can inhibit ICa-L and INa from isolated normal cardiac myocytes in a concentration dependent manner and has calcium antagonistic effect, so as to decrease the development of reperfusion arrhythmias .③Our study also showed that ischemia- reperfusion induced significant down-regulation of INa and Ito, and up-regulation of ICa-L, while pretreatment with anisodamine could attenuate this change, suggesting that anisodamine could reverse this electrical remodeling, which may be partly responsible for its antiarrhythmia effects.④Anisodamine , may be expected to have a wide spectrum of antiarrhythmic effects through multiple target with fewer proarrhythmic side effects.Part IV:Influence of anisodamine on QT dispersion and ventricular fibrillation threshold in rabbits during ischemia-reperfusionObjective: This study was conducted to investigate the influence of anisodamine on QT dispersion(QTcd),effective refractory period (ERP) and ventricular fibrillation thr eshold (VFT) in rabbits during ischemia- reperfusion , and to explore the physiological mechanism responsible for the anti-arrhythmic effect of anisodamine .Methods: Forty-five rabbits were randomly divided into three groups : ischemic-reperfusion (I-R) group, anisodamine intervention (Ani) group and sham-operated control group (CON). Anesthetized rabbits were subjected to 30-min ischemia by ligation of the left anterior descending coronary artery and 60-min reperfusion . Ani group was injected with anisodamine at a dose of 5mg/Kg via femoral vein 1 minute before operation.Surface 12-lead ECG was recorded before and after operation to measure the QTd. ERP and VFT were measured simultaneously using S1-S2 programmed electrical stimulation method.Results:①There was not significant difference in QTcd among three groups before operation. After reperfusion ,QTcd was increased significantly in I-R group compared with CON (64.6±7.2 VS 37.5±3.8 ms,P<0.01), however , QTcd was decreased significantly in Ani group compared with I-R group (40.3±5.3 VS 64.6±7.2 ms ,P<0.01).②The ERP of I-R group was shortened compared with CON ( 126.2±11.8 VS 154.6±13.2 ms ,P<0.05),and it was prolonged in Ani group compared with I-R group(148.5±12.3 VS 126.2±11.8 ms,P<0.05).③VFT of I-R group was decreased compared with CON(0.26±0.11 VS 3.51±0.56 mJ , P<0.01),which increased significantly in Ani group compared with I-R group(2.06±0.48 VS 0.26±0.11 mJ,P<0.01)Conclusions: Anisodamine could reduce rabbit's QTcd , prolong ERP and increase VFT, suggesting that anisodamine have the ability to increase electrical stability of the cardiac muscle and reduce the occurrence of ventricular arrhythmia.