低温缺血再灌注心律失常大鼠心室肌microRNA表达的变化

Changes in expression of microRNAs in ventricular myocardium in a rat model of hypothermic ischemia-reperfusion

目的观察低温缺血再灌注心律失常大鼠心室肌microRNA表达的变化。方法清洁级健康雄性SD大鼠,2~3月龄,体重300~400 g,成功制备离体心脏灌注模型16个,采用随机数字表法分为2组(n=8):对照组(C组)和低温缺血再灌注损伤组(I/R组)。采用全心停灌60 min再灌注30 min的方法制备低温心脏缺血再灌注损伤模型。记录再灌注期间心律失常的类型、持续时间和心脏复跳时间,并将I/R组大鼠分为低危组(I/R-L组)和高危组(I/R-H组)。再灌注结束后取左心室心肌组织,进行高通量测序,筛选差异表达的microRNA,并用qRT-PCR验证测序结果可靠性,通过Gene Ontology、KEGG等数据库分析差异基因所在的生物调控通路。结果与C组比较,I/R-L组表达上调的microRNA有437个,表达下调的microRNA有242个,I/R-H组表达上调的microRNA有419个,表达下调的microRNA有260个。与I/R-L组比较,I/R-H组表达上调的microRNA有392个,表达下调的microRNA有287个。各组间表达量变化绝对值≥2倍且显著性差异表达(P<0.01)的micro-RNAs有84个,随机选取4个行qRT-PCR验证,证实测序结果真实可靠。差异表达microRNA的靶基因参与调控的与再灌注心律失常相关的生物学过程有11个,KEGG通路有6个,且靶基因富集程度最高的生物学过程和KEGG通路分别为钾离子跨膜转运和心肌细胞肾上腺素能受体信号通路。结论低温心肌缺血再灌注后,大鼠心室肌microRNA的表达发生显著变化。这些差异表达的micro-RNA可能主要通过心肌细胞肾上腺素能受体信号通路调控钾离子跨膜转运,参与低温缺血再灌注心律失常的发生发展。

Objective To observe the changes in the expression of microRNAs in ventricular myocardium in a rat model of hypothermic ischemia-reperfusion(I/R).Methods Healthy clean-grade male Sprague-Dawley rats,aged 2-3 months,weighing 300-400 g,were anesthetized with intraperitoneal chloral hydrate.Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95%O2-5%CO2.Sixteen Langendorff-perfused hearts were prepared and divided into 2 groups(n=8 each)by a random number table method:control group(group C)and hypothermic I/R group(group I/R).The hearts were made globally ischemic for 60 min followed by 30-min hypothermic(4℃)reperfusion to establish the model of hypothermic I/R injury.The type and duration of arrhythmia and time of recovery of spontaneous heartbeats were recorded during reperfusion.The rats in group I/R were further divided into low-risk group(I/R-L group)and high-risk group(I/R-H group).The left ventricular myocardium was collected after the end of perfusion for high throughput sequencing to screen the differentially expressed microRNAs,and the reliability of the sequencing results was verified by quantitative real-time polymerase chain reaction.Gene Ontology and KEGG databases were used to analyze the biological regulatory pathways of differentially expressed target genes.Results Compared with group C,there were 437 up-regulated microRNAs and 242 down-regulated microRNAs in group I/R-L and 419 up-regulated microRNAs and 260 down-regulated microRNAs in group I/R-H.Compared with group I/R-L,392 microRNAs were up-regulated,and 287 microRNAs were down-regulated in group I/R-H.There were 84 microRNAs with absolute value of fold change≥2 and significantly differential expression(P<0.01)among the three groups.Subsequently,4 microRNAs were randomly selected for validation using quantitative real-time polymerase chain reaction,confirming that the sequencing results were reliable.These differentially expressed target genes were involved in 11 biological processes and 6 KEGG pathways which were related to reperfusion arrhythmia.Potassium ion transmembrane transport and the adrenergic receptor signaling pathway in cardiomyocytes were enriched by the largest number of target genes.Conclusion The expression of microRNAs in ventricular myocardium changes significantly after heart hypothermic I/R.These differentially expressed microRNAs regulate potassium ion transmembrane transport probably and mainly through the adrenergic receptor signaling pathway in the cardiomyocytes and thus are involved in the occurrence and development of hypothermic I/R arrhythmias.