Transcriptome Analysis Of CTnI R193H Mutation Mice With Restrictive Cardiomyopathy Based On RNA-Seq

Restrictive cardiomyopathy(RCM)is a form of childhood cardiomyopathy that accounts for 2-5% of pediatric cardiomyopathy.It is a kind of cardiomyopathy characterized by severe diastolic function damage caused by increased myocardial stiffness.In children,restrictive cardiomyopathy is mostly idiopathic with unknown etiology,and about 30% of cases have a familial tendency,most of which are autosomal dominant inheritance.Cardiac troponin I(cTnI)is an important cardiac structural protein,whose mutation is closely related to primary cardiomyopathy.Our research group has the restrictive cardiomyopathy mouse model successfully constructed by professor Huang xupei with human cTnI gene mutation(cTnI R193H in mouse line 193),whose diastolic function can be detected by ultrasound at 6-8 weeks,but the specific pathogenesis is still unclear.In this study,high-throughput sequencing was used to analyze the differentially expressed genes of restrictive cardiomyopathy mice and normal mice,and to screen out the molecules and signaling pathways that may be involved in the pathogenesis,providing new ideas and directions for the study of the pathogenesis of restrictive cardiomyopathy.PART I SCREENING AND IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN CTNI R193H RESTRICTIVE CARDIOMYOPATHY MICEObjective: The differentially expressed genes in cTnI R193H mutant restricted cardiomyopathy mice were isolated and identified.Methods: R193H and WT mice heart tissues at 3 months of age were taken and total RNA was extracted.The total RNA was reversely transcribed into cDNA and amplified.After the amplification,cDNA library was established to identify the differentially expressed genes in the myocardial tissues of the two groups of mice using RNA-seq technology.Some genes were selected for PCR verification to determine the reliability of the results of differentially expressed genes.Results: A total of 52 genes with differentially expressed myocardial tissue were analyzed in restricted cardiomyopathy model mice and wild mice,including 28 genes with high expression and 24 genes with low expression(P<0.05),Conclusion: There are multiple gene expression changes in myocardial cells of restrictive cardiomyopathy mice,and these gene expression changes may play a role in the development of restrictive cardiomyopathy.PART II BIOINFORMATICS ANALYSIS OF DIFFERENTIALLY EXPRESSED GENESObjective: The function of differentially expressed genes and the biological processes involved were analyzed to further understand the pathogenesis of cTnI R193H mutation in restrictive cardiomyopathy.Methods: GO enrichment analysis was conducted on differentially expressed genes using bioinformatics software,and regulatory functional relationship network,KEGG Pathway enrichment analysis and signal Pathway interaction analysis were constructed.Results: GO significant enrichment showed that most of the differentially expressed genes were distributed on the surface of mitochondria,cytoskeleton and cells,most of them had regional binding of DNA at the molecular level,triple codon-amino acid activity,etc.,and were mainly enriched in translation extension,innate immune response,and ATP synthesis coupled electron transport in biological process groups.It involves 16 related KEGG pathways,which are mainly involved in biological processes related to immunity and metabolism.The up-regulated genes and products are mostly involved in the process of innate immune response,including Cxcr3,Fcgr1,Icam1,Clec12 a,Ctss and Lbp.The biological processes involved in down-regulated genes and products are mainly translation prolongation and ATP synthesis coupled electron transport,involving genes such as D2 hgdh,ND5,Myom1,Ubr4,Spta1,Lrpprc and Arhgap21.Conclusion: cTnI mutation may be accompanied by up-regulation of immune-related molecules and inhibition of energy metabolism,suggesting that changes in immune function may play an important role in the pathogenesis of restrictive cardiomyopathy,and impaired energy metabolism may be the potential mechanism of restrictive cardiomyopathy.