替米沙坦降低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

Telmisartan reduces atrial fibrillation susceptibility in pressure overload rats and its molecular mechanism based on gene sequencing

目的评价替米沙坦对压力超负荷大鼠心房颤动(房颤)易感性的影响,探讨内在的分子机制和信号途径。方法12周龄雄性SD大鼠49只,随机分为对照组(假手术组,n=15)、腹主动脉缩窄(AAC)模型组(模型组,n=17)和替米沙坦组(n=17)。模型组和替米沙坦组采用外径0.7 mm针头制备AAC模型,对照组不进行AAC;替米沙坦组于AAC术后予以10 mg/(kg·d)替米沙坦连续灌胃4周。术后4周,观察心脏超声学、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)及房颤易感性的变化。提取三组大鼠左心房组织mRNA,利用基因组测序筛选差异表达基因,并通过富集分析和Venn分析,探索相关的分子靶点。结果模型组左心室收缩压、主动脉收缩压、左心房内径、收缩期室间隔厚度、舒张期室间隔厚度高于对照组,替米沙坦组上述指标较模型组降低(均P<0.05)。三组间P波离散度差异无统计学意义(P>0.05)。模型组左心房传导时间、心房间传导时间大于对照组,替米沙坦组上述指标低于模型组[左心房传导时间:对照组(34.40±4.04)比模型组(51.00±3.24)比替米沙坦组(38.00±1.91)ms,F=37.590,P<0.001;心房间传导时间:对照组(21.2±1.92)比模型组(30.4±4.39)比替米沙坦组(23.3±1.70)ms,F=11.232,P<0.001]。对Langendorff灌流离体心脏行高位右心房Burst刺激,三组诱发的房颤持续时间分别为(1.83±1.33)、(15.17±2.48)、(10.20±1.10)s,三组之间差异有统计学意义(F=75.334,P<0.001),模型组非自行终止房颤发生率分别为8.3%(5/60),而对照组和替米沙坦组均为0。对三组左心房组织基因高通量测序和生信分析显示,在模型组下调(与对照组比较)且替米沙坦组上调(与模型组比较)的mRNA共897个(基因集down/up897),模型组上调(与对照组比较)且替米沙坦组下调(与模型组比较)的mRNA共1542个(基因集up/down1542)。对上述两个基因集的富集分析和Venn分析显示,于down/up897基因集筛选出的5个目标基因均为脂肪酸氧化的关键限速酶;down/up1542基因集筛选的5个目标基因涉及Wnt/β-连环蛋白(β-catenin)信号途径和Ⅰ、Ⅱ型胶原组分。结论替米沙坦能有效改善压力超负荷大鼠的左心房扩张、缩短心房传导时间,且降低房颤易感性;其分子机制与改善左心房细胞的脂肪酸氧化和抑制Wnt/β-catenin途径激活、胶原成分过度表达密切相关。

Objective To assess the effects of telmisartan on susceptibility of atrial fibrillation(AF)in pressure overload rats,and to probe the underlying molecular mechanism and signal pathways.Methods Forty nine 12-week-old male SD rats were randomly divided into control group(sham operation group,n=15),abdominal aorta coarctation(AAC)model group(model group,n=17)and telmisartan group(n=17).AAC model was established using 0.7 mm diameter needles in model group and telmisartan group,while AAC was not performed in the control group.Rats in the telmisartan group were treated with telmisartan 10 mg/(kg·d)by gavage for 4 weeks after AAC.Four weeks after operation,the indexes of echocardiography and hemodynamics(5 rats in each group),as well as cardiac electrophysiological indexes(6 rats in each group)of P wave dispersion,left atrial condution time(LACT),interatrial conduction time(IACT)were observed.Atrial fibrillation susceptibility was analyzed.The mRNA was extracted from left atrial tissues of the above 3 groups and the differentially expressed genes(DEGs)were screened by genome sequencing.The potential molecular targets were explored by enrichment analysis and Venn analysis.Results Systolic left ventricular pressure(LVSP),systolic aortic pressure(ASP),left atrial diameters,systolic interventricular septal thickness and diasolic interventricular septal thickness in model group were higher than those in control group(all P<0.05),while the above indexes in telmisartan group were lower than those in model group(all P<0.05).There was no significant difference in P wave dispersion among three groups(P>0.05).LACT and IACT were significantly increased in model group,which were ameliorated by telmisartan treatment[LACT was(34.40±4.04),(51.00±3.24),(38.00±1.91)ms and IACT was(21.2±1.92),(30.4±4.39),(23.3±1.70)ms respectively in control,model,telmisartan group,F=37.590,11.232;both P<0.001].In isolated Langendorff perfused heart,AF sustained duration induced by Burst-stimulation administered at high-right-atria was(1.83±1.33),(15.17±2.48),(10.20±1.10)s respectively in control,model and telmisrtan group,there was significant difference among three groups(F=75.334,P<0.001);the ratios of non-self-terminating AF was 8.3%(5/60)in model group,while which was 0 in the control group and telmisartan group.Total 897 DEGs down-regulated in model group(compared with control group)and up-regulated in telmisratan group(compared with control group)were clustered into a gene set and named down/up897 gene set,1542 DEGs up-regulated in model group while down-regulated in telmisratan group were clustered into up/down1542 gene set.The intersection genes of the two gene sets were screened by enrichment analysis and Venn analysis.The results showed that the five target genes screened in the down/up897 gene set were the key rate limiting enzymes of fatty acid oxidation,five target genes screened in the up/down1542 gene set involved Wnt/β-catenin signal pathway and typeⅠandⅡcollagen components.Conclusions Telmisartan can effectively lower AF susceptibility via suppressing the left atrial enlargement and shortening atrial conduction time.The molecular mechanism is closely related to the improvement of fatty acid oxidation,down-regulation of the over-activated Wnt/β-catenin pathway and alleviating the over-expression of collagen.