| Objective: 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. Therefore, reperfusion arrhythmia after ischemia- reperfusion is one of existing warm spots ot syudy and research.Recently, it has benn shown that pretreatment with statin is effective in preventing reperfusion arrhythmia after ischemia- reperfusion, but its electrophysiological mechanism is unclear. Based on these observations, the present study was designed firstly to examine the effect of pretreatment with simvastatin on the changes in membrane ionic currents ,including sodium channel current(INa), L-type calcium channel curren(ICa-L) and transient outward potassium channel current(Ito) in left ventricular myocytes of rabbit from infracted and non-infarcted zone undergoing ischemia and reperfusion ,by the whole cell patch-clamp recording technique, so as to explore the cellular(ionic ) basis of statin treatment for antiarrhythmia .Material and methods:1 Experimental animals: Adult healthy New Zeland rabbits of either sex weighting 2.0-2.5 kg were provided by the Experimental Animal Center of Hebei Medical University, China. 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 ligation of the left anterior descending coronary artery and 120-min reperfusion after administration of oral simvastatin 5 mg·kg-1·d-1 (Statin group) or placebo (I-R group) for 3 days.2 Arrythmia point scale: (1) 0 points, non-arrythmia; (2) 1 point, the accidental premature ventricular beats; (3) 2 points, the frequent premature ventricular beats; (4) 3 points, accidental ventricular tachycardia; (5) 4 points, the frequent ventricular tachycardia; (6) 5 points, the frequent ventricular fibrillation.3 Cell separation: Single rabbit ventricular myocytes were isolated enzymatically(collagenase, Sigma Corporation),myocytes were obtained from the epicardial zone of the infracted area (I-R and Statin group), and the same anatomy region of control noninfarcted hearts (CON).4 Current record: Transmembrane currents (INa,ICa-L and Ito) were recorded with the whole-cell patch clamp technique, using EPC-9 patch clamp amplifier. Data acquisition and processing were performed by the pulse+ pulsefit software.Then,the currents wered compared among statin,I-R and control groups.5 Organization pathology research: Myocardial infarction area TTC dyes.6 Statistics:Data were expressed as mean value±standard deviation ( x±s). The statistical analysis was performed using ANOVY . The variance within groups was analyzed by q test(SPSS,versin 13.0), A value of P < 0.05 was considered statistically significant.Result1 Antriarrhythmic effect of Statin is ischemic-reperfusion in rabbitsThe sham-operation control group's rabbit only occasionally presents the premature ventricular beats , no ventricular tachycardia and ventricular fibrillation. In I-R group, rabbit may present the premature ventricular beats and so on arrythmias when the electrocardiogram ST section raises obviously during ischemic, and severe ventricular tachycardia and ventricular fibrillation at the moment of reperfusion.With the extension of reperfusion again , the ST section of electrocardiogram restores obviously, the cardiac muscle ischemia is improved , the occurrence of the arrythmia reduces. Compares with the control group, the arrhythmia of I-R group grading increases (P <0.01). Simvastatin cuts down the occurrence of arrythmia in I-R period. Comparing with the I-R group, its duration reduces obviously, the formation rate and arrythmia grading reduces (P <0.01).2 Effect of Simvastatin on ionic channels2.1 Peak INa current density(pA/pF) from infarction to non-infarc regions between I-R group and simvastatin groupAt test potential of–30 mV, the peak INa current density(pA/pF) in infracted and non-infarcted regions of I-R group was–22.46±5.32 (n=12),,-41.89±2.84(pA/pF)(n=14) respectively (infarction vs non-infarct, P<0.01). The difference from infarction to non-infarc regions between I-R group and simvastatin group was -19.43,-1.60(pA/pF)respectively (Statin vs I-R , P<0.01).2.2 Effect of simvastatin on INa in rabbit infarcted myocytes2.2.1 INa was generated by applying a series depolarizing pulses over the range -80 mV to +60 mV with a 10 mV increment from a holing potential of -90 mV . INa were elicited in response to depolarizing pulses positive to–60 mV, reaching a maximal value around–30 mV and reversing at about +40 mV. Figure 2 showes representative INa current traces recorded from myocytes of CON ,I-R and Statin group. At test potential of–30 mV, the peak INa current density(pA/pF) in CON ,I-R and Statin group was–42.78±5.48 (n=16),–22.46±5.32 (n=12),–40.66±5.89 (n=15), respectively (I-R vs CON and Statin, both P<0.01; Statin vs CON, P>0.05).The mean current density -voltage relationships curve (I-V curve) for INa was illustratsd in Figure 3. Simvastatin increased INa current density without changing the threshold, peak and reversal potentials. The peak INa current density was down-regulated in ventricular myocytes from I-R group compared with CON, simvastatin could ameliorate this change. This was not accompanied by a shift in the INa current density - voltage relation .2.2.2 Fig.4 shows INa current steady-inactivation curves.Half-inactivation potential(V0.5) of the availability curve(I/Imax curve)was shifted significantly in the hyperpolarizing direction in I-R group (-91.66±0.42mV n=12) compared with CON(-76.71±0.48 mV,n=16), P<0.05. V0.5 was also shifted in the same direction in statin group (-80.53±0.75mV,n=15) compared with CON ,although this shift was not significant, P>0.05. the rate of INa current steady-inactivation curves among three groups (Con,I-R and statin) is 8.25±0.42 mV ( n=16 ) , 8.71±0.36mV(n=12),9.11±0.64mV(n=15)respectively,there was not significant difference (P>0.05).2.2.3 Fig 5 shows representative INa recovery traces from inactivation,Fig 6 shows INa recovery curves from inactivation. the time-constant (τ) of INa recovery curves from inactivation among three groups (Con,I-R and statin) is 76.03±16.72ms (n=16), 102.19±41.29ms (n=12), 83.16±34.41ms (n=15) respectively (I-R vs CON and Statin, both P<0.01; Statin vs CON, P>0.05). Ischemic-reperfusion prolong INa recovery curves from inactivation,simvastatin reverse the tendency.2.3 Effect of simvastatin on ICa-L in rabbit infarcted myocytes2.3.1 ICa-L was elicited by depolarization from the holding potential of -50mV to +40mV with a 10 mV increment.The amplitude of the ICa-L was taken as the difference between the inward peak and the current level at the end of the test pulse (100 ms). ICa-L were elicited in response to depolarizing pulses positive to–40 mV, reaching a maximal value around 0 mV and reversing at about +50 mV.Figure 7 showes representative ICa-L current traces recorded from myocytes of CON,I-R and Statin group . The peak ICa-L current density(at 0mV) 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) and Statin (-3.46±0.85 pA/pF, n= 13),both P<0.05,while the peak ICa-L current density in Statin was no different from CON, P>0.05.The mean current density -voltage relationships for ICa-L was illustratsd in Fig 8. Simvastatin lifted the I-V curve of without changing the threshold, peak and reversal potentials.The peak ICa-L current density was up-regulated in ventricular myocytes from I-R compared with CON, simvastatin could ameliorate this change. This was not accompanied by a shift in the ICa-L current- voltage relation .2.3.2 Fig.9 shows ICa-L current steady-inactivation curves.Half-inactivation potential(V0.5) of the availability curve(I/Imax curve)was shifted significantly in the hyperpolarizing direction in I-R group (-28.56±0.92mV,n=15) compared with CON(-16.27±0.92mV,n=16), P<0.01. V0.5 was also shifted in the same direction in statin group (-22.40±0.81,n=13) compared with CON , P<0.05. the rate of ICa-L current steady-inactivation curves among three groups (Con,I-R and statin) is 6.34±0.80 mV(n=16),11.42±0.75mV(n=15),10.67±0.68mV(n=13)respectively (I-R and Statin vs CON, both P<0.01; Statin vs CON, P>0.05).2.4 Effect of simvastatin on Ito in rabbit infarcted myocytes2.4.1 Ito was elicited by 300 ms voltages steps over the range -40 mV to +60 mV in 10 mV increment from a holding potential of -60 mV. Figure 10 shows the representative Ito current traces recorded from myocytes of CON,I-R and Statin group. At test potential of +60 mV, the current density of Ito (pA/ pF) in CON ,I-R and Statin group was 17.41±3.13 (n=15), 9.49±1.91 (n=11), 14.54±2.41 (n=11), respectively (I-R vs CON and Statin, both P<0.01; Statin vs CON, P<0.05). The mean current density -voltage relationships for Ito was illustratsd in Fig 11.The peak Ito current density was down-regulated in ventricular myocytes from I-R group compared with control group, simvastatin could ameliorate this change, although there was slight reduction in Statin group compared with CON.2.4.2 Fig.12 shows Ito current steady-inactivation curves.Half-inactivation potential(V0.5) of the availability curve(I/Imax curve)was shifted significantly in the hyperpolarizing direction in I-R group (-55.41±1.58mV,n=11) compared with CON(-45.98±1.94mV,n=15), P<0.01. V0.5 was also shifted in the same direction in statin group (-47.91±2.17,n=11) compared with CON , although this shift was not significant, P>0.05. the rate of Ito current steady-inactivation curves among three groups (Con,I-R and statin) is 16.73±1.46 mV(n=15),16.31±1.07mV(n=11),17.05±1.58mV(n=11)6.34±0.80 mV(n=16)respectively ,there was not significant difference (P>0.05).3.TTC dyeing resultFigure 13 shows the percentage of the infarcted region occupying traverses area in each group of pathology: The I-R group, the Glib-simvas group, simvastatin group respectively are 28.17±2.31% (n=5), 17.16±0.35% (n=5),5.34±0.72 (n=5)%; (Statin vs I-R and Glib-simvas group,P<0.01).Conclusions1 The animal experimentation indicated that simvastatin reduces the incidence rate of the arrythmia obviously during ischemic-reperfusion.2 It is shown that ischemia-reperfusion induces significant down-regulation of INa and Ito, 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 attenuated inhomogeneous without lowering the serum cholesterol level, thus contributing to the cellular basis of statin treatment for antiarrhythmia . Accordingly, simvastatin, through the antiarrhythmic effects, may contribute to reducing cardiovascular mortality. 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.3 This research proved that simvastatin may reduce the infarcted area, extrapolated that its mechanism is concerned with the ATP sensitive potassium channel opening...
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