Disease Modeling And Drug Screening Using Short QT Syndrome Patient-specific Induced Pluripotent Stem Cell-derived Cardiomyocytes

Objectives:Short QT syndrome?SQTs?is an extremely rare and heterogenic myocardial ion channel disease,which is characterized by the significant shortening of QT interval,ventricular and atrial refractory period on the electrocardiogram,and can cause malignant arrhythmias leading to sudden cardiac death.SQTs has familial heritability,and its pathogenesis is still unclear.In clinical practice,there is a lack of specific drugs,which seriously endangers the health of patients.Six subtypes of SQTs have been identified,of which types 1,2 and 3 are associated with functional changes in potassium channels.KCNH2?Potassium duct-gated channel subfamily H member 2?gene is the first mutation site of the pathogenic gene identified.However,no complete studies have been conducted to explain why mutations of KCNH2 can induce SQTs,and drug studies on such mutations have been conducted.Therefore,based on previous studies and literature reports,the purpose of this study was to establish a patient-specific induced pluripotent stem cells?i PSCs?model for SQTs to study the pathogenesis of the disease,and to screen targeted drugs through i PSC-derived cardiomyocytes.Methods:This project enrolled one SQTs patient with KCNH2 T618I mutation and two healthy subjects,established SQTs patient-specific i PSCs and normal i PSCs of healthy subjects,and obtained induced pluripotent stem cell-derived cardiomyocytes?i PSCs-CMs?derived from patient-specific i PSCs.In order to have a more rigorous control in subsequent experiments,this project adopted CRISPR/Cas9 gene editing technology to conduct gene correction of T618I mutation site at the i PSCs level of SQTs patients,expecting to obtain the same allele i PSCs with the genotype of the patient cells.By comparing the functional differences of healthy subjects?normal group?,SQTs patient group?patient group?and allele correction group?gene-corrected group?in detail,such as cell morphology and electrophysiological characteristics,the functional phenotype of cardiomyocytes in the patient group was revealed,and the SQTs disease model was established.The changes of ion channels in the patient group were examined in detail,including Action potential?AP?and potassium,sodium and calcium channels,to further study the pathogenesis of the disease.Through molecular experiments,the protein expression of the three groups at the molecular level was detected to explain the change of ion channel.Later,transcriptome sequencing analysis?RNA-seq analysis?was used to find the genes related to SQTs disease,so as to determine whether the changes of ion channels were related to the changes of genes.Finally,based on the laboratory test results of common drugs for clinical treatment of SQTs,targeted drugs were screened by applying patient-specific i PSCs-CMs.Results:After skin samples of SQTs patients were obtained,the i PSCs of the normal group and the patient group were established by somatic reprogramming technology,and the T618I gene locus of the i PSCs of the patient group were fixed by CRISPR/Cas9gene editing technology,so as to obtain the i PSCs of the gene-corrected group.Immunostaining with i PSCs specific markers SOX2,NANOG,OCT4 and SSEA4showed significant dryness in all three groups of i PSCs.IPSCs in the control group,the patient group and the gene-corrected group were differentiated into cardiomyocytes by the method of directed differentiation of small molecule compounds,and then the specific markers of cardiomyocytes TNNT2 and?-actinin were used for immune staining of cardiomyocytes.MLC2v,a specific marker of ventricular myocytes,and MLC2a,a specific marker of atrial myocytes,were used to conduct immune staining on the cardiomyocytes of the three groups.The results showed that there was no significant difference in cell size,proportion of ventricular and atrial myocytes,and texture arrangement of myofilaments in different groups.The whole-cell patch-clamp technique was used to detect the action potential of i PSCs-CMs in three groups,respectively.The results showed that compared with the normal group and the gene-corrected group,the cardiomyocytes in the patient group showed significant arrhythmia,increased heart rate,and significantly shortened AP duration?APD?.At the same time,in order to determine whether the abnormal action potential phenotype found in i PSCs-CMs of the patient group was caused by the functional enhancement caused by KCNH2 mutation,specific potassium channel agonist PD-118057 was applied to conduct the transient loading experiment of i PSCs-CMs of the control group,and the APD shortening degree of i PSCs-CMs of the control group was similar to that of i PSCs-CMs of the patient group.Whole-cell patch clamp technique was used to detect potassium,sodium and calcium channels of i PSCs-CMs in the gene-corrected group and the patient group,respectively.The results showed that the density of fast activation delayed rectifier potassium current(I_Kr)and slow activation delayed rectifier potassium current(I_Ks)increased significantly,while the sodium and calcium ion current increased significantly.The membrane protein expression test was applied to detect the membrane protein expression of potassium and sodium ions of i PSCs-CMs in the gene-corrected group and the patient group.The results showed that the membrane protein expression of potassium ions?KCNH2?and sodium ions in the patient group increased significantly,indicating that the changes of potassium current and sodium current might be related to the changes of membrane protein.Application of real-time fluorescent quantitative PCR?q PCR?technology and RNA-Seq analysis of gene-corrected group and patient group 14 may affect ion channel expression change gene m RNA level of testing,the results showed that influence of potassium,sodium and calcium ions understanding to the simple expression of multiple genes have obvious changes,the electrophysiological and membrane protein detection before and the experimental results are identical with each other.The whole-cell patch-clamp technique was used to perform the instantaneous dosing experiment of quinidine and Bm KKx2 with i PSCs-CMs in the patient group.The results showed that quinidine and Bm KKx2 could effectively prolong the APD in the patient group.Application of molecular biology experiment technology to construct KCNH2 mutation plasmid and T618I specific locus mutation plasmid,and the cell line HEK 293 after transient transfection,using whole cell patch clamp technique record I_Krcurrent of the two groups of cells,and the short-peptide T618I specific locus mutation groups scorpion toxin Bm KKx2 instantaneous dosing experiments,the results showed Bm KKx2 can significantly increase the current density,therefore,Bm KKx2 might be for T618I locus mutation of targeted drugs.Conclusions:This research proves that SQTs due to functional acquisition of mutant KCNH2 T618I can generalize the disease phenotype by patient-specific and gene-corrected i PSCs-CMs,and these experimental results will help clarify the pathogenesis of SQTs.At the same time,short-peptide toxin can be found as lead compounds to develop SQTs targeted drugs.