Mutation In Kv1.5 Auxiliary Subunit Leads To Sudden Cardiac Death

Objective: To screen and collect peripheral blood from patients with sudden cardiac death(SCD),excavate new disease-causing mutant genes.A number of biological predictive analysis software and experiments have verified the pathogenicity of the new genetic pathogenic gene KCNAB3 mutation,which is a functional deletion mutation.In ex vivo cardiomyocyte(AC16)silencing KCNAB3 mimics KCNAB3 loss of function,combined with ordinary transcriptome sequencing techniques to discover the activation of intracardiomyocytic apoptosis signaling pathway and identify the relevant mechanisms.Methods: Patients with malignant arrhythmia,syncope and family history of sudden death were collected since 2013,and informed consent was signed with the patients or their families to collect peripheral blood samples and clinical history data.The patients’ peripheral blood DNA was extracted,and the mutation was screened by PCR amplification combined with Sanger sequencing method.Two methods,HRM and Sna PShot,were used for sequencing verification.The physicochemical analysis of the protein was carried out by Ex PASy software,and the three-dimensional spatial conformation of the protein was predicted by the Swiss Model software.Mutation Taster,Polyphen-2 and SIFT software jointly predict gene pathogenicity,and STRING predicts interacting proteins.KCNAB3 normal and mutant KCNAB3(p.A233 Gfs X22 and p.L93)plasmids were constructed using pc DNA3.1as empty vector and incorporating His tag,and KCNA5 normal plasmid incorporating Flag tag.After grouping the transfected cells,the current changes were detected by patch clamp,and the localization of proteins in the cells was scanned by confocal laser scanning.Construct KCNAB3 target targeted si RNA silencing AC16 cardiomyocytes KCNAB3,q RT-PCR and Western Blot to detect transcription and protein expression of KCNAB3 after silencing for RNA sequencing.Apoptosis was verified by flow cytometry(Annexin V/PI staining)and TUNEL staining.Apoptosis family-related proteins Caspase-7,Cleaved-caspase-7,Caspase-3,Cleaved-caspase-3,Caspase-9,Cleaved-caspase-9,Parp,Cleaved-parp,Bcl-2 and Bax protein levels were detected by Western Blot.Results: A proband with a clear family was collected with nev gene mutation(p.A233 Gfs X22).Meanwhile,a sporadic patient with SCD,also was collected with a new gene mutations KCNAB3(p.L93).By genetic screening analysis,WES and Sanger sequencing,we found a new SCD pathogenic gene KCNAB3 mutation.The physicochemical analysis of the protein showed that the hydrophobicity of normal KCNAB3 was enhanced than normal,and the three-dimensional spatial conformation of the protein was changed after mutation.Mutation Taster,Polyphen-2,and SIFT software predict mutations that are all highly pathogenic.The normal KCNA5 and KCNAB3 plasmids were co-transfected into HEK293 cells,and the current change process of the normal Kv1.5-gated channel was simulated by patch clamp.Transfected with KCNA5 alone,patch clamp revealed slow inactivation of Kv1.5-gated channels.Co-transfection of normal KCNA5 and mutant KCNAB3(p.A233 Gfs X22and p.L93)showed that the Kv1.5-gated channel was delayed inactivation by patch clamp,which was similar to KCNA5 transfection alone,indicating that the mutation may cause loss of function.Normal KCNAB3 and mutant KCNAB3(p.A233 Gfs X22)were transfected into HEK293 cells,respectively,and the cytoskeletal protein phalloidin and nucleus were simultaneously labeled.Confocal laser scanning showed that normal KCNAB3 was distributed near the cell membrane,at the outer edge of the cytoskeleton,and KCNAB3(p.A233 Gfs X22)clustered on the side of the nucleus,at the inner edge of the cytoskeleton.To observe the relationship between mutant KCNAB3(p.A233 Gfs X22)and wild-type KCNAB3 in cell sub-localization changes and the α subunit KCNA5 of Kv1.5.Confocal laser scanning showed that normal KCNAB3 was distributed near the cell membrane,and at the outer edge of the cell,the overlap rate with normal KCNA5 cells was greater than 70%.The mutant KCNAB3(p.A233 Gfs X22)aggregated in the nucleus on the side of the nucleus,and the sublocalization rate of the normal KCNAB3 cells was significantly reduced at the inner edge of the cell.The m RNA and protein expressions of KCNAB3 were significantly decreased after silencing.RNA sequencing showed that there were 613 significantly differentially expressed genes in the experimental group,including 308up-regulated genes and 305 down-regulated genes.Both GO and KEGG enrichment analysis showed activation of apoptosis signaling pathway.Checking the differential gene heat map found that the apoptosis family factor Caspase-7 was highly expressed in the experimental group.q RT-PCR showed that the m RNA expression of KCNAB3 in the experimental group increased(P<0.01).Western Blot showed that the expression of KCNAB3 protein in the experimental group increased(P<0.05).q RT-PCR showed that the m RNA expression of Caspase-7 in the experimental group increased(P<0.05).Western Blot showed that the expression of Caspase-7 protein in the experimental group increased(P<0.01).Flow cytometry showed that the apoptosis rate of the si KCNAB3 group was about 36.7%,while the control group was only about 7.56%.The apoptosis rate was significantly increased(P<0.05).Immunofluorescence analysis of apoptotic cells was performed by TUNEL staining,green marked TUNEL positive,blue marked cell nucleus.Confocal showed that the number of TUNEL positive cells increased significantly compared with the control group(P<0.05).The apoptosis-related signaling pathways and apoptosis-related protein expression levels detected.Western Blot shows the expression protein levels of Cleaved-caspase-7,Caspase-3,Parp and Cleaved-parp increased(P<0.01).The expression of Cleaved-caspase-3 increased significantly(P<0.001).The expression level of Cleaved-caspase-9 and Caspase-7 protein were all increased in the si KCNAB3 group(P<0.05).There was no significant difference in the expressions of Caspase-9 and Bax between two groups.The expression of anti-apoptotic protein Bcl-2in the si KCNAB3 group decreased significantly(P<0.001).Conclusion:1.By collecting family medical history and samples of SCD patients,we found a new SCD pathogenic gene KCNAB3(β subunit of potassium ion voltage-gated channel Kv1.5).The mutation of the proband has been shown to cause loss of function of KCNAB3.2.Silencing KCNAB3 in cardiomyocytes mimics the loss-of-function model of KCNAB3,indicating that silencing KCNAB3 in cardiomyocytes induces cardiomyocyte apoptosis by regulating Caspase-7.3.Both flow cytometry and TUNEL staining indicated that apoptosis was activated.The apoptosis signaling pathway was activated by up-regulating the protein levels of Caspase-7,Cleaved-caspase-7,Caspase-3,Cleaved-caspase-3,Cleaved-caspase-9,Parp and Cleaved-parp and down-regulating the protein level of Bcl-2.This study broadens the pathogenic map of SCD,it is beneficial to early molecular detection in SCD disease for early diagnosis,early prevention and early genetic counseling.Clarifying the pathophysiological mechanism of KCNAB3 is conducive to mining new clinical drug treatment targets and providing new method for SCD treatment.