Studying Mechanism Of Pathogenicity Regulation Related To Development And Differentiation In Type 7 Long QT Syndrome Using Patient-Specific Induced Pluripotent Stem Cell-Derived Cardiomyocytes

BACKGROUND:Andersen-Tawil syndrome(ATS)is a rare hereditary multisystem disorder usually associated with heterozygous mutations in the gene encoding KCNJ2.Ventricular arrhythmias,periodic paralysis and dysmorphic features constitute the classic triad of ATS symptoms.KCNJ2 gene knockout mouse cannot survive for long periods of time with a clinical severe presentation.Misexpression of KCNJ2 changed the normal pattern of voltage potential regionalization in developing facial membrane,disturbed the expression of important craniofacial pattern genes,and revealed the endogenous control of bioelectric cell status on craniofacial abnormalities.It is therefore speculated that the disease-causing gene will affect nonmal organs development.The development and differentiation of cardiomyocytes and electrophysiological maturity are essential for cardiac function,and their changes may promote the pathop:hysiology of ATS disease.Thus far,the effect of KCNJ2 gene on the normal development and differentiation of cardiomyocytes is only poorly understood.OBJECTIVES:This study intends to establish a model of human cardiomyocytes that can specifically reproduce the phenotype of ATS disease.It will help us better to understand the pathogenic regulatory mechanism of KCNJ2 gene on the development and differentiation of cardiomyocytes,and to explore potential transcription factors and related target genes.METHODSWe used the peripheral blood reprogramming of ATS patients to obtain induced pluripotent stem cells(iPSCs),and at the same time,with the repaired CRISPR group iPSC with Crispr mutation site,they were differentiated into cardiomyocytes(iPSC-CM).The iPSC was identified by morphology,karyotype analysis,teratoma experiment,flow cytometry,immunoconfocal staining,and qPCR experiment.iPSC-CM was identified by morphological spontaneous pulsation recording,immunoconfocal staining,patch clamp action potential and Kir2.1 channel current changes.According to the different stages of development and differentiation,gene and chromatin opening were detected.Using weighted gene co-expression network analysis(WGCNA)to classify and weight the co-expressed gene groups at specific stages of development and differentiation,and compare the top 100 genes with weights to the human transcription factor JASPAR database to obtain potentially regulated transcription factor.Afterwards,the simultaneous detection of ATAC-seq chromatin openness epigenetic data was used to verify these potential transcription factors at specific stages.Finally,by newly collecting two sets of mature cardiomyocytes that are closest to the clinical phenotype,combined with transcriptome and proteome data,a single-sample gene set enrichment analysis(ssGSEA)was used to evaluate the overall regulation status of potassium-related pathways from a pathway perspective.At the same time,key target genes related to diseases that may be regulated by transcription factors can be obtained and verified in association analysis.RESULTS:We established stable passage,uniform shape mutation and repair group induced pluripotent stem cell lines,which not only retains the patient specific genetic information,but also has the characteristics of inducing directional differentiation of stem cells.iPSCs in both groups were successfully differentiated into cardiomyocytes,which could pulsate autonomously and regularly.The differentiation rate was more than 90%.The pulsing rate in the mutation group was significantly lower than that in the repair group,and the myocardial specific markers(TNNT2 and NKX2.5)were all positive.The action potential duration of the mutant group was prolonged,and the inward rectifier potassium channel Kir2.1 current showed a negative effect characteristic,which was consistent with the clinical phenotype of the patient,while the electrophysiological characteristics of the repair group and the healthy control group were not significantly different.The results of WCGNA analysis showed that there were 5 transcription factors with increased access to chromatin that were consistent with development and differentiation:mesoderm(Day2,MIXL1);cardiac mesoderm precursor cells(Day4,GBX1);early pulsatile cardiomyocytes(Day8,DLX5);electrophysiological mature cardiomyocytes(Day15,HOXB4);advanced cardiomyocytes(Day30,ZEB1).The only transcription factor related to pathogenicity is ZNF528,and it continues to be lowly expressed in the mutant group.All the above transcription factors have been verified by ATAC-seq data.Subsequently,transcriptome and proteome expression levels were simultaneouslv detected in newly collected advanced cardiomyocytes,and ssGSEA was used to assess the overall regulatory status of potassium channel related pathways at the gene and protein levels.The results showed that the enrichment scores of 7 pathways in the two groups were significantly reduced(All P<0.05).The three pathways of them involved in the mutant gene KCNJ2 were related to the inward rectifier potassium channel ’s entry of potassium ions into the cell,including induction from the voltage-gated potassium channel complex(GO:0008076),followed by potassium ions entering the cell(GO:1990573),and finally to the pathway that maintains the steady state of potassium ions in the cell(GO:0030007),they were all suppressed.The other four affected pathways were related to the regulation of potassium ion transmembrane pathway and sodium:potassium exchange ATPase(all P<0.05).There are 19 differential genes in these down-regulated potassium channel-related pathways,and 4 differential proteins.After correlation analysis,there were 3 differentially expressed genes with proteins consistent trends(KCNJ2,CTTN,and ATP1B1),which were key target proteins that were potentially significantly down-regulated by the transcription factor ZNF528 reduced.CONCLUSION:We have established a specific human cardiomyocyte model to reproduce ATS disease phenotype,which can provide a reliable platform for exploring disease mechanism or drug treatment.The transcription factors(MIXL1,GBX1,DLX5,HOXB4 and ZEB1)in different stages of ATS early myocardial development and differentiation,as well as the potential transcription factors ZNF528 related to pathogenicity were verified.The whole process of potassium influx and energy consumption related pathways in mature cardiomyocytes were significantly inhibited,and were directly or indirectly affected by the mutation of KCNJ2 gene.Three key down regulated proteins(KCNJ2,CTTN and ATP1B1)by ZNF528 were identified by correlation analysis.The low expression of transcription factors and key target proteins may lead to or aggravate the abnormality of cardiac development and electrophysiological maturation in ATS patients.In the future,it is expected to intervene key regulatory transcription factors to achieve the role of regulating target genes,so as to improve the symptoms of cardiac electrophysiological disorders in ATS patients and provide new ideas for the treatment of rare diseases.