起搏电流在老年阵发性房颤启动中的作用及其分子机制
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摘要
心房颤动(AF)是临床上最常见的一种持续性心律失常,其发病率随年龄而增加。综合结果显示,90%以上的PAF起源于肺静脉肌袖,作为诱发房颤的重要机制之一,静脉肌袖自律性增高在启动和维持PAF中也发挥着不可替代的作用。
本实验我们首先选择健康的老龄犬(7-9岁)进行快速起搏刺激,10周后进行房颤诱发刺激,获得老龄犬房颤模型,老龄犬经快速起搏后房颤发生率明显高于非起搏组和成年对照组,而应用起搏通道特异阻滞剂后房颤发生率降低。快速起搏肺静脉肌袖细胞中约82.6%呈现出自发性除极;未经起搏处理肌袖细胞约仅有60%的细胞出现自律性。未经起搏处理的老龄犬肺静脉肌袖细胞上记录到I_f电流幅值较小,且激活较慢。而经快速起搏的老龄犬肺静脉肌袖细胞上I_f电流明显增大,与未经处理组相比有显著性差异(P＜0.01,n=15)。应用β受体激动剂isoproterenol(ISO)可增加I_f电流密度,使动作电位趋于类似窦房结样有自律性特征。而起搏电流特异抑制剂Ivabradine(Iva)可以减少老龄房颤犬肺静脉肌袖细胞I_f电流,动作电位特征更像工作细胞的特征,从而房颤发生率也减低。为了进一步探讨I_f电流的发生机制,应用RT-PCR,Western Bloting、瞬时转染的通道蛋白异源性表达技术和膜片钳技术确定了HCN4亚型是老龄犬肺静脉肌袖细胞膜上起搏通道主要的存在形式。明确了HCN2和HCN4对老龄犬肺静脉肌袖细胞I_f电流的构成比,确定导致I_f电流改变的主要亚型,从一个侧面解释了老年房颤发生的分子和离子机制,对于深入揭示老年PAF的启动和今后特异性药物的研发将有重要意义。
Atrial fibrillation is the most common persistent arrhythmia in clinic,and it's incident increases with age.According to the research results,90%paroxysmal atrial fibrillation(PAF) originate in pulmonary vein sleeve(PVs).As an important mechanism for the trigger of atrial fibrillation,rise of pulmonary vein sleeve automaticity plays an irreplaceable role in the occurence and maintenance of PAF.
The aim of the present study is to investigate the characteristics of ectopic automaticity and I_f of cardiomyocytes from pulmonary veins in older canine with atrial fibrillation.The older canines(7-9 years) were subjected to long-term rapidly atrial pacing for 10 weeks,which induced atrial fibrillation models. Disassociation of PVs of canines yielded single cardiomyocytes from a Landengorff column.I_f current was measured with the patch-clamp technique. Compared with the control group,cardiomyocytes from the rapidly atrial pacing canine PVs had spontaneous diastolic depolarization,shorter action potential duration,and larger I_f densities.Furthermore,I_f currents in the RAP were reduced by Ivabradine,a selective inhibitor of the I_f current.As contrast,I_f current densities were increased significantly by beta-adrenergic receptor stimulation with isoproterenol.Either mRNA or protein expression of HCN4 in cardiomyocytes from PVs in older canine is larger than that of HCN2.Our study revealed an increased I_f in the PVs from older canines with atrial fibrillation. Our results suggested that I_f of PVs with RAP contributed partly to ectopy in atrial fibrillation.
本实验我们首先选择健康的老龄犬(7-9岁)进行快速起搏刺激,10周后进行房颤诱发刺激,获得老龄犬房颤模型,老龄犬经快速起搏后房颤发生率明显高于非起搏组和成年对照组,而应用起搏通道特异阻滞剂后房颤发生率降低。快速起搏肺静脉肌袖细胞中约82.6%呈现出自发性除极;未经起搏处理肌袖细胞约仅有60%的细胞出现自律性。未经起搏处理的老龄犬肺静脉肌袖细胞上记录到I_f电流幅值较小,且激活较慢。而经快速起搏的老龄犬肺静脉肌袖细胞上I_f电流明显增大,与未经处理组相比有显著性差异(P＜0.01,n=15)。应用β受体激动剂isoproterenol(ISO)可增加I_f电流密度,使动作电位趋于类似窦房结样有自律性特征。而起搏电流特异抑制剂Ivabradine(Iva)可以减少老龄房颤犬肺静脉肌袖细胞I_f电流,动作电位特征更像工作细胞的特征,从而房颤发生率也减低。为了进一步探讨I_f电流的发生机制,应用RT-PCR,Western Bloting、瞬时转染的通道蛋白异源性表达技术和膜片钳技术确定了HCN4亚型是老龄犬肺静脉肌袖细胞膜上起搏通道主要的存在形式。明确了HCN2和HCN4对老龄犬肺静脉肌袖细胞I_f电流的构成比,确定导致I_f电流改变的主要亚型,从一个侧面解释了老年房颤发生的分子和离子机制,对于深入揭示老年PAF的启动和今后特异性药物的研发将有重要意义。
Atrial fibrillation is the most common persistent arrhythmia in clinic,and it's incident increases with age.According to the research results,90%paroxysmal atrial fibrillation(PAF) originate in pulmonary vein sleeve(PVs).As an important mechanism for the trigger of atrial fibrillation,rise of pulmonary vein sleeve automaticity plays an irreplaceable role in the occurence and maintenance of PAF.
The aim of the present study is to investigate the characteristics of ectopic automaticity and I_f of cardiomyocytes from pulmonary veins in older canine with atrial fibrillation.The older canines(7-9 years) were subjected to long-term rapidly atrial pacing for 10 weeks,which induced atrial fibrillation models. Disassociation of PVs of canines yielded single cardiomyocytes from a Landengorff column.I_f current was measured with the patch-clamp technique. Compared with the control group,cardiomyocytes from the rapidly atrial pacing canine PVs had spontaneous diastolic depolarization,shorter action potential duration,and larger I_f densities.Furthermore,I_f currents in the RAP were reduced by Ivabradine,a selective inhibitor of the I_f current.As contrast,I_f current densities were increased significantly by beta-adrenergic receptor stimulation with isoproterenol.Either mRNA or protein expression of HCN4 in cardiomyocytes from PVs in older canine is larger than that of HCN2.Our study revealed an increased I_f in the PVs from older canines with atrial fibrillation. Our results suggested that I_f of PVs with RAP contributed partly to ectopy in atrial fibrillation.
引文
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