Establishment Of IPSC-CMs Model Of Restrictive Cardiomyopathy And Study Of Pathogenic Mechanism Of SLC4A3 Gene Mutation

BackgroundRestrictive cardiomyopathy is the rarest type of cardiomyopathies.Due to reduced myocardial compliance,the patient's ventricular diastolic function is impaired.There are very limited reports of hereditary restrictive cardiomyopathy and the related genes,meanwhile the pathogenicity of mutated genes,and underlying molecular pathogenic mechanisms still need discussion.SLC4A3 encodes a chloride-bicarbonate exchanger AE3 which is highly expressed in heart.Mutations of SLC4A3 can cause abnormal ion transport in cell lines and animal cardiomyocytes,and affect intracellular pH homeostasis.AE3 plays a key role in intracellular pH regulation when cardiomyocytes are subjected to ischemia,hypoxia and extracellular acidosis.This study is the first to use the iPSC-CMs model to study the phenotype and mechanism of restrictive cardiomyopathy patients with novel SLC4A3 gene mutation.ObjectiveTo establish a rare restrictive cardiomyopathy-specific iPSC-CMs model,verify the phenotypic characteristics of the restrictive cardiomyopathy,study the relationship between the novel SLC4A3 gene mutation and the occurrence of RCM disease,and explore its pathogenic mechanism.MethodsPeripheral blood mononuclear cells of a patient diagnosed of restricted cardiomyopathy carrying a novel mutation of SLC4A3 was collected and a patient-specific induced pluripotent stem cell(iPSCs)line was successfully built using episome plasmid transfection.The point mutation of SLC4A3 was corrected using CRISPR/Cas9.Cell lines of pluripopent stem cell derived cardiomyocytes(iPSC-CMs)was induced from the patient-specific and correct iPSCs.Patch clamp and microelectrode array(MEA)technology were used to study the changes of iPSC-CMs ion channels,action potentials and contraction,thus establish a disease model of restrictive cardiomyopathy.The functional changes of mutant AE3 protein were evaluated by intracellular pH measurement.Based on transcriptome and proteomics,changes in the expression profile of diseased cardiomyocytes was evaluated to explore the pathogenic mechanism of SLC4A3 mutation.ResultsPeripheral blood PBMCs of patients with restricted cardiomyopathy carrying SLC4A3 gene mutations were successfully reprogrammed to obtain patient-specific iPSCs,and the characteristics of pluripotent stem cells and the identity of the patient's origin were confirmed.CRISPR/Cas9 point mutation repairing was performed on the SLC4A3 heterozygous mutation carried by the patient.IPSC-CMs were successfully induced and verified.Based on single-cell patch clamp recording,the spontaneous action potential duration of RCM iPSC-CMs was significantly shortened and diastolic membrane potential was reduced.The L-type calcium channel(LTCC)current was significantly weakened,with the inactivation curve left-shifted and the inactivation-recovery delayed.Point-mutation-repairing rescued the abnormal performance of action potentials and LTCC.Based on MEA data,the duration from complete repolarization to the next excitement was shortened in the disease group,and the excitement-contraction delay was shorter,suggesting that the RCM iPSC-CMs model has restrictive cardiomyopathy characteristics.RT-QPCR and indirect immunofluorescence staining imaging showed that the expression of mutant AE3 protein was basically the same as that of the control.The steady pHi of cardiomyocyte was not altered,but the transmembrane base equivalent transportation rate is significantly reduced.Transcriptomics combined with proteomics analysis found that the hypoxia related metabolism,extracellular matrix-related pathways of iPSC-CMs changed significantly,and the HCM,DCM,and sarcomere structure-related pathways were significantly downregulated in RCM cardiomyocytes compared to the rescued group.ConclusionWe identified a novel heterozygous mutation of SLC4A3(c.3629C>A,p.S1210X)in a patient diagnosed of RCM.After phenotypic and functional verification based on iPSC-CMs model,it was inferred that SLC4A3 mutation led to RCM disease phenotype.This heterozygous mutation harmed the transmembrane anion transport function of AE3 protein.Cardiomyocytes carrying mutations have significant altered metabolic pathways related to hypoxia stimulation,cardiac excitation-contraction coupling pathways,cell adhesion and extracellular matrix homeostasis pathways.Point-mutation-correcting ameliorated the RCM phenotype and altered expression profile of mutation carrying iPSC-CMs.