Mechanistic Study Of TMEM43 Regulating The Arrhythmogenic Right Ventricular Cardiomyopathy Using Induced Pluripotent Stem Cell-derived Cardiomyocyte Model

Objectives:Arrhythnogenic right ventricular cardiomyopathy(ARVC)is a cardiomyopathy in which the right ventricular myocardium is replaced by progressive fibrous adipose tissue.Its clinieal manifestations are right ventricular enlargement,ventricular arrhythmia and sudden cardiac death,which eventually leads to heart failure and seriously endangers peopled health.ARVC is hereditary.At present,at least nine ARVC-related genes are known,including desmosome protein gene and non-desmosome protein gene.TMEM43 is a non-desmosome protein gene associated with ARVC.However,little is known about the function of TMEM43,and how the mutation of TMEM43 causes ARVC disease is unclear.Therefore,based on the existing literature and previous work,the purpose of this research is to establish an iPSC model of ARVC disease carrying TMEM43 gene mutation,reveal the cell phenotype of ARVC disease,and elucidate the molecular mechanism of ARVC disease caused by TMEM43 gene mutation.Methods:We successfully enrolled one ARVC patient with TMEM43 gene mutation and 3 healthy volunteers.Then we established a patient-specific cell line of ARVC and obtained the patient-specific iPSC-CMs of ARVC.The genome editing technique CRISPR/Cas9 was used to carry out TMEM43 gene mutation at the level of iPSC of ARVC patients.Isogenic iPS cell line with the same genotype as the patient’s was obtained as a stricter control in the follow-up study.The functional differences of cardiomyocytes between normal control group,patient group and gene correction group were compared in detail to reveal the functional phenotype of cardiomyocytes in patient group and establish AR.Cell model of VC disease;electrophysiology of cardiomyocytes in patients and fat accumulation of cardiomyocytes in patients;expression of desmosome-related and calcium-regulated genes in cardiac myocytes of patients by q-PCR and RNAseq;and overexpression of mutant TMEM43 in normal cardiac myocytes The change of the site;the signal pathway was analyzed by RNAseq and other data,and the interaction target sites were further searched by protein mass spectrometry.To elucidate the molecular mechanism of ARVC disease caused by TMEM43 gene mutation.Results:The TMEM43 gene was repaired by CRISPR/Cas9 technique,and the successfully repaired ARVC-Gene-corrected cell(ARVC-GC)line was identified by sequencing.Cells in normal,patient and ARVC-GC groups were successfully differentiated into cardiomyocytes by small molecule compounds-based methodology.Cardiac specific markers TNNT2 and alpha-actinin were used for immunostaining of cardiomyocytes.The results showed that there were no significant differences in cell size,proportion of multinucleated cells and texture arrangement of myofibrils between different groups.Nile red staining and transmission electron microscopy were used to analyze the adipose deposition in cardiomyocytes.The data showed that there was a significant increase in adipose deposition phenotype in the patients compared with the control group and the correction group.Apoptosis of cardiomyocytes was detected by TUNEL staining.The results figured out that there was no significant difference in apoptosis between different groups.Patch clamp technique was used to record the action potential and sodium channel currents of cardiomyocytes.The results showed that compared with the control group and the correction group,the cells in the patient group showed significant arrhythmia phenotype,the sodium channel currents of cardiac myocytes in the patient group decreased significantly,and the steady-state activation and inactivation curves shifted significantly to the right,presenting a sodium channel function deficiency phenotype.Overexpression of wild type TMEM43 and mutant type TMEM43 in normal cardiomyocytes showed significant arrhythmias,which verified the electrophysiological phenotype of cardiomyocytes in patient group and proved that TMEM43 mutation was pathogenie mutation.The results of RNAseq and bioinformatics analysis show that differentially expressed genes are enriched in signal pathways such as NF-κB,calcium regulation,TGF-beta,ARVC and PPAR.To verify the NF-κB signaling pathway,Western Blot results showed that the phosphorylation levels of IκBα and P65 in patients are significantly increased compared with those in corrected cardiomyocytes,suggesting that the NF-κB signaling pathway was involved in the occurrence of ARVC disease.Western Blot also figured out that the protein expression of RYR2 and SERCA 2A decreased significantly,the protein phosphorylation level of PLN increased significantly,while the protein expression of NCX1 and Cavl.2 did not change significantly.Fluo-4 and Fura-2 calcium imaging techniques were used to record the calcium signal of cardiomyocytes.The data suggested that compared with the correction group,the calcium signal disorder phenotype appeared in the patient cells,which was consistent with the observed electrophysiological phenotype,suggesting that the disorder of calcium signal regulation may cause the electrophysiological phenotype of cardiomyocytes in the patient group and participate in the occurrence of ARVC disease.Conclusions:We successfully established a patient-specific ARVC model and a pluripotent stem cell line repaired by TMEM43.Patient-specific iPSC-CMs of ARVC exhibit disease phenotypes such as fat deposition,arrhythmia and loss of sodium channel function.RNA-Seq analysis and subsequent functional verification suggest that abnormal NF-κB and calcium regulatory signaling pathways may be the key mechanism of ARVC disease.