Expression And Significance Of MicroRNA In Ventricular Myocardium Of Rats With Hypothermia Ischemia-reperfusion Arrhythmia

Objective:This study aims to investigate the changes of miRNA expression and target genes in myocardium of hypothermic ischemia-reperfusion arrhythmia（RA）rats,so as to provide a new target and direction for the prevention and treatment of hypothermic ischemia-reperfusion arrhythmia（RA）.Methods:Healthy clean-grade male Sprague-Dawley rats,weighing 300±20g,were anesthetized with intraperitoneal chloral hydrate.Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95%0₂-5%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 IR group（group IR）.The hearts were made globally ischemic for 60 min followed by 30-min hypothermic reperfusion to establish the model of hypothermic I/R injury.Group C was continued to be infused with 37℃K-H solution for 105 min;Group IR:K-H fluid was stopped after continuous perfusion for 15 min and cardiac arrest was induced for 60 min with the injection of Thomas solution（4℃,20 ml/kg）while the heart was protected by the low temperature Thomas solution（4℃）around it.Reperfusion of Thomas solution（4℃,10ml/kg）was performed at the middle time during cardiac arrest and the heart was resuscitated and reperfused with K-H fluid for 30 min.The type and duration of arrhythmia and time of recovery of spontaneous heartbeats were recorded during reperfusion.The rats in group IR were further divided into low-risk group（IR-DW group）and high-risk group（IR-GW group）.The left ventricular myocardium was collected after the end of perfusion for high throughput sequencing to screen the differentially expressed micro RNAs.miRNAs expression profiles of ventricular myocardium with global hypothermic ischemia–reperfusion and those of ventricular myocardium with hypothermic ischemia–RA were established through high-throughput sequencing.Furthermore,the aberrantly expressed miRNAs in myocardium with and without hypothermic ischemia–RA were screened and verified.The target genes of these aberrantly expressed miRNAs were predicted using RNAhybrid and Mi Randa software.Based on Gene Ontology（GO）and the Kyoto Encyclopedia of Genes and Genomes（KEGG）databases,we determined the mRNA targets associated with these miRNAs and studied the miRNA–mRNA interaction during the cardiovascular disease progression.The aberrantly expressed miRNAs relatedtohypothermicischemia–RAwerevalidatedby Real-time Quantitative polymerase chain reaction（RT-q PCR）.Results:Eight significantly differentially expressed miRNA were screened by high-throughput sequencing,in which the expression of novel-miR-1,novel-miR-17,novel-miR-19,novel-miR-30,novel-miR-43 and rno-miR-122-5p was up-regulated,while the expression of novel-miR-16 and rno-miR-429 was down-regulated.Moreover,target genes and signaling pathways associated with these aberrantly expressed miRNAs were predicted and analyzed.Mi RNA-mRNA correlation analysis revealed that GJA1gene is the target of novel-miR-17 and may be involved in the electrical conduction of arrhythmia ventricular muscle after hypothermic global ischemia.Conclusion:Aberrantly expressed miRNAs were possibly associated with the formation mechanism of hypothermic ischemia–RA.Specific miRNAs,such as novel-miR-17and rno-miR-429 are probably new potential targets for further functional studies of hypothermic ischemia–RA.