压力超负荷大鼠心房颤动易感性及基于基因测序的分子机制

Susceptibility of atrial fibrillation in pressure overload rats and molecular mechanism based on gene sequencing

目的通过压力超负荷模型,探讨高血压条件下心房颤动易感性变化及分子机制。方法12周龄雄性SD大鼠32只,随机分为对照组(n=15)和腹主动脉缩窄(AAC)组(简称模型组,n=17)。模型组采用7号针头(外径0.7mm)制备AAC模型,对照组不进行AAC,其余步骤相同。比较2组大鼠术后4周心脏超声学、血流动力学指标(每组取5只)和心脏电生理指标(P波离散度、心房间传导时间和左心房传导时间、心房有效不应期,每组取6只)及心房颤动易感性(心房颤动诱发率、持续时间、非自行终止性心房颤动发生率)的变化。提取对照组、模型组动物左心房组织mRNA,利用基因组测序方法筛选差异表达基因,并通过富集分析、蛋白互作网络分析,探索关键的分子靶点。结果超声指标中,模型组左心房直径[(4.08±0.43)比(3.57±0.42)mm,t=2.376,P=0.030]、舒张期和收缩期室间隔厚度[(2.44±0.40)比(2.05±0.24)mm,t=2.167,P=0.046;(3.44±0.42)比(2.98±0.35)mm,t=2.304,P=0.035]高于对照组,左心室射血分数低于对照组[(61.36±8.62)%比(71.70±8.53)%,t=2.406,P=0.029]。模型组左心室收缩压[162.5(159.4~164.2)比132.6(130.8~136.1)mm Hg,Z=-2.611,P=0.009]、主动脉收缩压[157.2(154.7~162.1)比127.1(126.1~129.9)mm Hg,Z=-2.617,P=0.009]、外周动脉收缩压[151.0(149.0~155.0)比124.0(120.0~127.0)mm Hg,Z=-3.134,P=0.002]高于对照组。模型组左心房传导时间、心房间传导时间高于对照组(均P<0.05)。模型组心房颤动持续时间长于对照组[15.00(12.75~18.00)比0(0~2.25)s,Z=-2.945,P=0.003]。模型组在不同频率Burst刺激下,右心房刺激时心房颤动诱发率高于左心房刺激(13/60比3/60,χ^(2)=7.212,P=0.007)。模型组非自行终止性心房颤动发生率高于对照组(10%比0%,Fisher确切概率检验,P<0.001)。2组间P波离散度差异无统计学意义(P>0.05)。对照组、模型组左心房组织基因组测序筛选出5829个差异表达基因。京都基因与基因组百科全书(KEGG)、基因本体(GO)富集分析显示模型组线粒体呼吸链酶复合体Ⅰ、Ⅱ多重亚基组分和兰诺丁受体2(RyR2)、一氧化氮合酶1(NOS1)、磷酸二酯酶4D(PDE4D)、小窝蛋白1(Cav1)均显著下调(P_(校正)<0.05且log_(2)差异倍数<1);其中涉及线粒体、氧化应激的基因是Ndufs8、Ndufa12。蛋白互作网络分析提示酶复合体Ⅰ的还原型烟酰胺腺嘌呤二核苷酸(NADH)泛醌氧化还原酶铁-硫蛋白1、4、7、8及NADH泛醌氧化还原酶亚基a5、a12为关键节点。结论0.7mm ACC 4周能制备高血压相关的大鼠心房颤动易感模型;呼吸链酶复合体Ⅰ、Ⅱ关键组分和钙调控分子mRNA的低表达是模型组心房颤动易感性增加的主要分子机制。

Objective To explore the change and molecular mechanism of atrial fibrillation(AF)susceptibility in pressure-overload hypertension rats.Methods Thirty two 12-week-old male SD rats were randomly divided into control group(n=15)and abdominal aorta constriction(AAC)group(model group,n=17).ACC model was established by 0.7mm needles.In control group other steps were the same except ACC.Four weeks after operation,the changes of echocardiological,hemodynamic indexes(5rats in each group),cardiac electrophysiological indexes[P wave dispersion,left atrial condution time(LACT),interatrial conduction time(IACT),effective refractory pe-riod(ERP),6rats in each group]and atrial fibrillation susceptibility(atrial fibrillation rate,duration,onset of nonself-terminating AF)were compared between the 2groups.The mRNA of left atrial samples from control and model groups were extracted,and differently expressed genes(DEG)were selected by genome sequencing,and potential molecular targets were explored by enrichment analysis and protein-protein interaction(PPI)network analysis.Results The left atrial diameter[(4.08±0.43)vs(3.57±0.42)mm,t=2.376,P=0.030],diastolic and systolic inter-ventricular thickness[(2.44±0.40)vs(2.05±0.24)mm,t=2.167,P=0.046;(3.44±0.42)vs(2.98±0.35)mm,t=2.304,P=0.035]in model group were higher,and left ventricular ejection fraction[(61.36±8.62)%vs(71.70±8.53)%,t=2.406,P=0.029]was lower than those of control group.The left ventricular systolic pressure[162.5(159.4-164.2)vs 132.6(130.8-136.1)mm Hg,Z=-2.611,P=0.009],aortic systolic pressure[157.2(154.7-162.1)vs 127.1(126.1-129.9)mm Hg,Z=-2.617,P=0.009]and peripheral arterial systolic blood pressure[151.0(149.0-155.0)vs 124.0(120.0-127.0)mm Hg,Z=-3.134,P=0.002]in model group were higher than those in the control group.The LACT and IACT in model group were also higher than those in the control group(all P<0.05).The duration of AF in the two groups were 15.00(12.75-18.00)and 0(0-2.25)s,which was higher in model group than in control group(Z=-2.945,P=0.003).In the model group,the onset of non-self-terminating AF was higher than that in the control group(10%vs 0%,Fisher exact probability test,P<0.001).There was no difference in P wave dispersion between 2groups(P>0.05).A total of 5829DEGs were identified in left atrial samples separated from control and model group.The enrichment analysis of Kyoto Encyclopedia of genes and genomes(KEGG)and gene ontology(GO)showed that Ryanodine receptor 2(RyR2),nitric oxide synthase 1(NOS1),phosphodiesterase 4D(PDE4D),caveolin 1(Cav1)and a series of structural/functional components of multiple subunit components of mitochondrial electron transport chain(ETC)complexⅠorⅡwas significantly down regulated in model group(P_(corrected)<0.05and log_(2) difference multiple<1).Among these components,Ndufs8,ndufa12involved mitochondria,oxidative stress,and Ndufs1,Ndufs4,Ndufs7,Ndufs8,Ndufa5,Ndufa12were identified as central nodes in PPI network.Conclusions Hypertensive related AF susceptible models can be established by 0.7mm AAC operation.Dysfunction characterized by down-regulation of crucial components of ETC complexⅠorⅡand calcium homeostasis may play crucial roles in enhanced susceptiblity of AF.