Bioinformatics Analysis Of Nuclear-encoded Mitochondrial Genes And The Role Of CLIC1 In Cardiac Hypertrophy

Background:Hypertrophic cardiomyopathy（HCM）is a hereditary disease characterized by asymmetrical left ventricular myocardial hypertrophy with unexplained secondary causes.It is inherited in an autosomal dominant manner and is usually associated with mutations in genes encoding sarcomere proteins.Histological characteristics include:cardiomyocyte hypertrophy,muscle fiber disorder,and obvious interstitial fibrosis,which are the main causes of sudden death（SCD）in adolescents and athletes.The mechanism of HCM is complex,involving abnormal excitation and contraction coupling,autophagy disorders,mitochondrial dysfunction,and abnormal energy metabolism.Mitochondria are involved in the regulation of cardiomyocyte synthesis and catabolism,intracellular Ca²⁺homeostasis,initiation of inflammation,and cell death.Sarcomeric gene mutations might cause mitochondrial dysfunction and damage to myocardial energy,thereby increasing the formation of reactive oxygen species（ROS）,impairing myocardial cell contractility,and then promoting the development of HCM.The functional proteins of mitochondria are mainly encoded by nuclear genes,which are called nuclear-encoded mitochondrial genes（NMGenes）.Recent studies have shown that aging and stress stimulation can induce abnormal expression or function of NMGenes,resulting in reduced ATP production and excessive accumulation of ROS,leading to the development of cardiovascular diseases.With the deepening of molecular mechanism research,people have begun to explore the essence of HCM from epigenetic,genome,transcriptome and other aspects.Bioinformatics analysis based on gene expression microarrays has been widely used to identify various cardiovascular disease-related genes or non-coding RNAs,which helps to explore the potential molecular mechanisms of disease occurrence and development.At present,the systematic research of NMGenes in HCM expression profile is still lacking,and its specific role in the development of HCM is still largely unknown.Therefore,integrating multi-platform data,screening out the key mitochondrial genes and corresponding regulatory factors involved in HCM,and further studying the expression of key genes,as well as functional and molecular mechanisms,will help to explore new therapeutic targets for HCM.The results of bioinformatics study indicate that intracellular chloride ion channel 1（Intracellular chloride ion channel 1,CLIC1）is up-regulated in the sample of HCM patients.Based on this,we infer that CLIC1 may play an important role in pathological myocardial hypertrophy.Research on its function and mechanism will help to provide a new perspective for exploring the treatment of HCM.Objective:By bioinformatics analysis,this study explore NMGenes,of which expression has changed significantly in HCM.A interaction network of differentially expressed gene was constructed,aiming to screen key genes and potential upstream regulatory factors,and discover important signal pathways.The expression and role of the key genes CLIC1 in the mouse TAC model were verified through molecular biology experiments to clarify that CLIC1 may affect the process of cardiac hypertrophy by regulating autophagy flux,which is expected to provide a new strategy for the treatment of HCM.Methods:1)Standardized gene chip expression data set（GSE36961）including 107 HCM samples and 40 control samples was downloaded from GEO database.The differentially expressed genes（DEGs）between the two was analyzed by using the limma software package,and nuclear-encoded mitochondrial genes were collected from Mito Carta,Mito Miner,IMPI and Uni Prot databases,and the differential expression NMGenes between HCM and the control sample NMGenes were filtered out;2)Gene Onotology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）were applied pathway enrichment analysis to annotate and functionally enrich DE-NMGenes;3)DE-NMGenes interaction network was constructed based on STRING,the key hubs genes of HCM were screened.Combining the MCODE plug-in to analyze the hotspot modules of the interaction network,and the GO Biological process（BP）enrichment analysis of the module was performed;4)Cytoscape’s plug-in"i Regulon"was used to predict possible transcription factors（TFs）that regulate DE-NMGenes;5)A mouse cardiac hypertrophy model was established by Thoracic Aorta Coarctation（TAC）,and the key DE-NMGenes screened out by biosynthesis analysis were verified by fluorescence quantitative PCR in myocardial tissue of TAC mouse;6)The key gene CLIC1 was select for function and mechanism research.The primary neonatal rat cardiomyocytes（NRCMs）hypertrophy model and mice TAC model were used to explore the role of CLIC1 in myocardial hypertrophy and its specific molecular mechanisms for regulating autophagy from various aspects in vivo and in vitro.Result:1.Bioinformatics analysis1)A total of 2927 differentially expressed genes（DEGs）were found among all the genes detected by the microarray,of which 316 DE-NMGenes were found.Among the top 10 up-regulated and down-regulated DE-NMGenes,4 up-regulated genes（PDK4,STAT3,HCLS1,FKBP11）and 4 down-regulated genes（GATM,ATPIF1,CPT1B,GJA1）have been shown to play an important role in pathological myocardial hypertrophy.The remaining 6 up-regulated genes DDIT4,TKT,CLIC1,ACTB,DDOST,TUBB and the 6 down-regulated genes SNCA,CASQ1,LYPLAL1,SDSL,KLHDC9,and DPYSL4 in myocardial hypertrophy has not yet been reported and are available for further study;2)Among the 316 DE-NMGenes in the HCM sample,175 down-regulated DE-NMGenes are more functionally rich than 141 up-regulated DE-NMGenes,and are significantly involved in 16 GO biological processes,such as redox,branched chain amino acid catabolism and fatty acidβoxidation,and are significantly enriched in 17energy metabolism KEGG pathways such as carbon,pyruvate,fatty acid metabolism and citric acid cycle;3)316 DE-NMGenes was analyzed by using STRING,and a protein-protein interaction（PPI）network including 440 interactions and 215 protein nodes was obtained.13 nodes（DE-NMGenes）with the top 5%connectivity were selected as hub genes,among which DLD,ACLY,CAT genes have been reported to be related to HCM,and the remaining 10 hub genes ACADM,HADH,MRPL46,MRPL53,MRPL1,MRPL40,MRPS16,ACAT1,RPLP0,OXA1L may play an important role in the biological mechanism of HCM;4)By the MCODE plug-in,12 hotspot modules in the PPI network was detected,and modules with more than 5 nodes were screened out and named 1-5.KEGG enrichment analysis shows the associated biological process with modules 1,2,4,and5 separately are"mitochondrial respiratory chain complex I assembly","mitochondrial translation","asparagine N-catenin glycosylation"and"folic acid and its derivatives biosynthesis process";5)17 TFs were predicted by using i Regulon that may regulate 316 DE-NMGenes,among which 8 TFs,NFYA,NFYC,PBX3,GATA1,IRF2,MYBL2,GATA5,RARA have not been reported in HCM;6)In the mouse TAC model,it was verified that the expression of key up-regulated genes CLIC1,DDIT4,TKT,and DDOST identified by bioinformatics analysis increased.2.Experimental study1)The expression of CLIC1 is increased in hypertrophic NRCMs,and the expression of CLIC1 is increased in the myocardial tissue of TAC mice;2)Interfering with CLIC1 further promotes the expression of hypertrophic markers ANP and BNP;3)Overexpression of CLIC1 inhibits the expression of hypertrophic markers ANP and BNP;4)Interference with CLIC1 increases the overall mortality of TAC mice,promotes ventricular wall thicken,myocardial interstitial fibrosis,increases the cross-sectional area of myocardial cells,and promotes the expression of ANP and BNP;5)Interference with CLIC1 inhibits the ROS production of NRCMs,inhibits the expression of SOD2 and HO-1,while overexpression of CLIC1 promotes the ROS production of NRCMs,promotes the expression of SOD2 and HO-1;6)Interference with CLIC1 inhibits the expression of p62,PI3K III,and makes LC3II/I and the amount of autophagic vesicles decrease,and promotes mitochondrial swelling and vacuolation,while overexpression of CLIC1 promotes the expression of p62,PI3KIII,and makes LC3II/I and the amount of autophagic vesicles increase,and reduces the mitochondrial swelling and vacuolation;7)Compared with si RNA-Control+PBS,the LC3 II/I ratio of the si RNA-CLIC1+PBS group decreased,and there was no statistical difference in LC3 II/I between the si RNA-CLIC1+RAPA and si RNA-Control+RAPA groups;8)Compared with the si RNA-Control+PE+RAPA group,the expression of ANP and BNP in the si RNA-CLIC1+PE+RAPA group were significantly decreased.Conclusion:1)In the HCM sample,6 up-regulated DE-NMGenes DDIT4,TKT,CLIC1,ACTB,DDOST,TUBB and 6 down-regulated DE-NMGenes SNCA,CASQ1,LYPLAL1,SDSL,KLHDC9,DPYSL4 were screened out.These genes might play an important role in the occurrence of HCM;2)The 175 down-regulated DE-NMGenes are more functionally rich,and are significantly involved in the biological processes of redox,branched-chain amino acid catabolism and fatty acidβ-oxidation,and are significantly enriched in the energy metabolism pathway;3)STRING analysis showed that the unreported 10 hub genes ACADM,HADH,MRPL46,MRPL53,MRPL1,MRPL40,MRPS16,ACAT1,RPLP0,OXA1L may play an important role in the biological mechanism of HCM;4)i Regulon predicts that 8 unreported TFs,NFYA,NFYC,PBX3,GATA1,IRF2,MYBL2,GATA5,RARA may be involved in regulating the expression of DE-NMGene in HCM samples;5)Overexpression of CLIC1 plays a protective role in cardiac hypertrophy through activation of autophagy mediated by"ROS-m TORC1".