大鼠心肌梗死后HCN表达的变化及普伐他汀干预研究
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摘要
目的:急性心肌梗死(AMI)后由于一系列复杂的分子和细胞机制可能导致心肌细胞膜离子通道异常活动形成电重构,可能是心肌梗死后心律失常发生的重要机制。心肌细胞离子通道分子结构的研究表明超极化激活环核苷酸门控(HCN)阳离子通道亚单位为编码心脏起搏电流(If)的通道基因,包括HCN1～4共4个异构型,HCN主要存在于起博细胞中,有少量存在工作心肌细胞中.成年大鼠心室肌细胞中,HCN2是最主要的异构型,有极少量HCN4表达。生理情况下超极化电流是起搏细胞舒张期去极化的主要离子流,有研究发现病理情况下HCN2和HCN4通道表达及激活异常,这可能引起心肌细胞自律性增高而诱发心律失常。近年来随着一系列大规模随机临床试验的揭晓,他汀类药物的非降脂作用逐渐被认识,其中抗心律失常作用是研究的热点之一。但其抗心律失常的具体机制尚不明确。本研究通过检测急性心肌梗死后右室心肌HCN2,HCN4mRNA和蛋白质表达的变化趋势,以及普伐他汀对HCN2,HCN4表达的影响来探讨其抗心律失常的可能机制。
方法:结扎Sprague-Dawley大鼠左冠状动脉前降支建立心肌梗死模型。将成功建立模型的20只大鼠随机分为24小时组、1周组、4周组和普伐他汀干预组(普伐他汀20mg.kg-1.d-1,灌胃给药4周),每组5只,同时采用左冠状动脉只穿线不打结的方法于24小时、1周、4周三个时间点设立假手术组,每个时间点5只。于各时间点取右室心肌组织样本,用Real Time-PCR检测HCN2、HCN4mRNA的表达,用Western-Blot检测HCN2、HCN4蛋白质表达。
结果:心肌梗死后HCN通道表达增高,与假手术组相比,4周时HCN2 mRNA及蛋白质表达显著升高(P<0.01),HCN4 mRNA表达升高(P<0.05);运用普伐他汀后HCN2 mRNA及蛋白质表达明显低于四周组(P<0.05),HCN4mRNA无明显变化。各组均未检测到HCN4蛋白质表达。
结论:急性心肌梗死后心室肌HCN2、HCN4通道表达水平上调,其可能原因为1)、急性心肌梗死后RAS系统激活,AngⅡ水平增高,刺激醛固酮分泌增加,通过激活盐皮质激素受体上调HCN表达;2)、心肌梗死后肥厚的心室肌HCN表达增加;3)、AMI后大量内皮素释放,刺激HCN2mRNA表达水平增高。普伐他汀能抑制心肌梗死后HCN2表达的上调,其机制可能为一方面干扰RAS系统,降低醛固酮水平,另一方面降低ET-1水平以及对抗心肌肥厚,从而减弱了心梗后引起HCN升高的因素。
Backgroud and objective: The reasons of ventricular cells electric remodeling post myocardial infarction may be a series of complex molecular and celluar mechanisms,and this may be the reason of arrhythmogenesis.Research on molecular structure of cardiac ion channel indicated that it is the hyperpolarization-activated cyclic nucleotide-gated cation channel subunit which encoded If current.The HCN family includes 4 isoforms. HCN channels are highly expressed in pacemaker cells, but in working cardial cell,the expression are low.In adult rat ventricular cells,HCN2 is the mainly subunit, there is a small quantity of HCN4. In sinus node and Purkinje cells, the hyperpolarization-activated inward current (If) is considered to contribute significantly to the spontaneous diastolic depolarization phase.In pacemaker cells, If is believed to be the major current determining the diastolic depolarization phase.Researches has shown that change of expression or function of HCN channel may have a potential proarrhythmic under pathophysiological conditions. Recent research of large scale randomized clinical trial has shown additional beneficial pleiotropic effects independent of the simple cholesterol lowering effect, especially antiarrhythmic effect.At present,the mechanisms of anti-arrhythmias are unclear.Therefore,the purpose of this study was to investigate variation of expressions of HCN channels post myocardial infarction and to evaluate the effects of pravastatin on expressions of HCN channels,we try to find the mechanism of antiarrhythmic effect.
Methods: The AMI rat model was established by ligation of the left anterior descending coronary artery,the Sham-operated rats underwent the same procedure except for the ligation.20 infarcted rats were randomly divided into 4 groups(n=5 in each group): AMI24h group、AMI1w group、AMI4w group and pravastatin treated group(20mg.kg-1.d-1,drug was orally given to AMI rats by gastric gavate once a day for 4 weeks.)and meanwhile, Sham-operated group of each time point was established(n=5).Harvest right ventricular myocardial tissues at each time point,the expressions of HCN2、HCN4mRNA were evaluated by Real Time PCR and the expressions of HCN2、HCN4 protein were evaluated by Western blot.
Results: The expressions of HCN2 mRNA and protein were increased significantly at 4week after AMI compared with sham-operated group,and the expression of HCN4 mRNA was increased also. The expressions of HCN2 mRNA and protein were decreased in pravastatin treated group compare with AMI4week group,there was no significant difference between pravastatin treated group and AMI 4-week group.We were failed to detect the expression of HCN4 protein in each group.
Conclusion: The level of HCN2 mRNA、protein and HCN4mRNA are upregulated,the reason below may support it:1) Aldosterone level is increased post myocardial infarction, and aldosterone can up-regulates HCN2 mRNA、protein and HCN4mRNA expression by Mineralocorticoid receptor MR activation in cardiac myocytes;2) Pathologic myocyte hypertrophy after AMI accompany with increase of HCN2 and HCN4 level;3) Endothelin-1 level is increased post myocardial infarction, and ET-1 can up-regulate mRNA levels for HCN2 and HCN4.Pravastatin pertly inhibited the up-regulate of expression of HCN2.The reason below may support it :1)Pravastatin interrupt RASS, degrade the level of aldosterone;2) Pravastatin alleviate cardiomyocyte hypertrophy;3) Pravastatin decrease level of ET-1,thus attenuated the factor of that which up-regulate HCN level.
方法:结扎Sprague-Dawley大鼠左冠状动脉前降支建立心肌梗死模型。将成功建立模型的20只大鼠随机分为24小时组、1周组、4周组和普伐他汀干预组(普伐他汀20mg.kg-1.d-1,灌胃给药4周),每组5只,同时采用左冠状动脉只穿线不打结的方法于24小时、1周、4周三个时间点设立假手术组,每个时间点5只。于各时间点取右室心肌组织样本,用Real Time-PCR检测HCN2、HCN4mRNA的表达,用Western-Blot检测HCN2、HCN4蛋白质表达。
结果:心肌梗死后HCN通道表达增高,与假手术组相比,4周时HCN2 mRNA及蛋白质表达显著升高(P<0.01),HCN4 mRNA表达升高(P<0.05);运用普伐他汀后HCN2 mRNA及蛋白质表达明显低于四周组(P<0.05),HCN4mRNA无明显变化。各组均未检测到HCN4蛋白质表达。
结论:急性心肌梗死后心室肌HCN2、HCN4通道表达水平上调,其可能原因为1)、急性心肌梗死后RAS系统激活,AngⅡ水平增高,刺激醛固酮分泌增加,通过激活盐皮质激素受体上调HCN表达;2)、心肌梗死后肥厚的心室肌HCN表达增加;3)、AMI后大量内皮素释放,刺激HCN2mRNA表达水平增高。普伐他汀能抑制心肌梗死后HCN2表达的上调,其机制可能为一方面干扰RAS系统,降低醛固酮水平,另一方面降低ET-1水平以及对抗心肌肥厚,从而减弱了心梗后引起HCN升高的因素。
Backgroud and objective: The reasons of ventricular cells electric remodeling post myocardial infarction may be a series of complex molecular and celluar mechanisms,and this may be the reason of arrhythmogenesis.Research on molecular structure of cardiac ion channel indicated that it is the hyperpolarization-activated cyclic nucleotide-gated cation channel subunit which encoded If current.The HCN family includes 4 isoforms. HCN channels are highly expressed in pacemaker cells, but in working cardial cell,the expression are low.In adult rat ventricular cells,HCN2 is the mainly subunit, there is a small quantity of HCN4. In sinus node and Purkinje cells, the hyperpolarization-activated inward current (If) is considered to contribute significantly to the spontaneous diastolic depolarization phase.In pacemaker cells, If is believed to be the major current determining the diastolic depolarization phase.Researches has shown that change of expression or function of HCN channel may have a potential proarrhythmic under pathophysiological conditions. Recent research of large scale randomized clinical trial has shown additional beneficial pleiotropic effects independent of the simple cholesterol lowering effect, especially antiarrhythmic effect.At present,the mechanisms of anti-arrhythmias are unclear.Therefore,the purpose of this study was to investigate variation of expressions of HCN channels post myocardial infarction and to evaluate the effects of pravastatin on expressions of HCN channels,we try to find the mechanism of antiarrhythmic effect.
Methods: The AMI rat model was established by ligation of the left anterior descending coronary artery,the Sham-operated rats underwent the same procedure except for the ligation.20 infarcted rats were randomly divided into 4 groups(n=5 in each group): AMI24h group、AMI1w group、AMI4w group and pravastatin treated group(20mg.kg-1.d-1,drug was orally given to AMI rats by gastric gavate once a day for 4 weeks.)and meanwhile, Sham-operated group of each time point was established(n=5).Harvest right ventricular myocardial tissues at each time point,the expressions of HCN2、HCN4mRNA were evaluated by Real Time PCR and the expressions of HCN2、HCN4 protein were evaluated by Western blot.
Results: The expressions of HCN2 mRNA and protein were increased significantly at 4week after AMI compared with sham-operated group,and the expression of HCN4 mRNA was increased also. The expressions of HCN2 mRNA and protein were decreased in pravastatin treated group compare with AMI4week group,there was no significant difference between pravastatin treated group and AMI 4-week group.We were failed to detect the expression of HCN4 protein in each group.
Conclusion: The level of HCN2 mRNA、protein and HCN4mRNA are upregulated,the reason below may support it:1) Aldosterone level is increased post myocardial infarction, and aldosterone can up-regulates HCN2 mRNA、protein and HCN4mRNA expression by Mineralocorticoid receptor MR activation in cardiac myocytes;2) Pathologic myocyte hypertrophy after AMI accompany with increase of HCN2 and HCN4 level;3) Endothelin-1 level is increased post myocardial infarction, and ET-1 can up-regulate mRNA levels for HCN2 and HCN4.Pravastatin pertly inhibited the up-regulate of expression of HCN2.The reason below may support it :1)Pravastatin interrupt RASS, degrade the level of aldosterone;2) Pravastatin alleviate cardiomyocyte hypertrophy;3) Pravastatin decrease level of ET-1,thus attenuated the factor of that which up-regulate HCN level.
引文
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