Mechanism And Impact Factors Of HERG Gene Nonsense Mutation Drug Salvation

BackgroundThe human ether-a-go-go-related gene (HERG) gene encodes the a subunit of rapid delayed rectifier potassium current channel (Ikr) which is important for cardiac repolarization. Mutations of HERG gene which cause the dysfunction of Ikr may lead to type2long QT syndrome (LQT2). About10%of mutations are nonsense mutations which introduce the premature termination codons (PTC) into HERG gene.Aminoglycosides showed the readthrough effect on nonsense mutations to produce full-lengh functional proteins in genetic disease models, such as cystic fibrosis and Duchenne muscular dystrophy (DMD). Our previous studies demonstrated that gentamicin could partially restore the HERG-like currents in HEK293cells expressing R1014X mutant. However, whether other aminoglycosides had the similar effect and what was the most effective concentration as well as whether the sites of mutation would influence in rescue effect have not been studied.PTC124(3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid; C15H9FN2O3) is considered to be a new choice for many genetic diseases related to nonsense mutations. The chemical structure of PTC124is different from aminoglycosides. It can act as a PTC suppressor to overcome the PTC and allow the synthesis of a mature protein similar as gentamicin but without serious toxic side effects.Amino acid sequence analysis indicated that the HERG channel was consisted of four HERG protein subunits. Each subunit contained four parts:the N-terminal; the C-terminal, the six transmembrane helical regions (S1-S6) and a pore region. It is known that about30nonsense mutations existed in different regions. However, reactions of different nonsense mutations on drug rescuing are unclear.ObjectivesThe aim of this study was to evaluate the ability of three aminoglycosides (G418, gentamicin and tobramycin) which share a4,6-disubstituted deoxystreptamine ring and PTC124on rescuing nonsense mutations of HERG gene. To clarify the reaction of different nonsense mutations on drug rescuing, we examined the susceptibility of HERG nonsense mutants located in different regions after G418rescuing.Methods(1) HERG nonsense mutations were generated in the pcDNA3.1WT HERG plasmid using polymerase chain reaction-based mutagenesis strategy and sequence verified.(2) The WT or mutants gene were transiently transfected in HEK293cells. Readthrough effect was studied by adding drugs into the culture medium for24hours.(3) The mRNA levels were identified by real-time PCR.(4) Western blot was performed to evaluate the expression of genes.(5) Patch clamping was performed to evaluate the function of genes.(6) We also used immunofluorescence method to observe the expression HERG protein before and after drug treatment.(7) Mass Spectrometry (MS) was used to analyse the composition of purified proteins.ResultsThe WT protein could express two band about135KDa and155KDa, respectively. There were also two bands in cells expressing R1014X and W927X mutants with molecular weights less than those of WT channels, representing their mature and immature form. The expression levels did not differ from those of WT. By contrast, the cells expressing R863X or E698X mutants, whose mutation sites approached the N-terminal, only showed a single band with a molecular weight of<100kDa and the protein expression levels were decreased signifcantly compared with those in cells expressing WT cDNA. The mRNA levels, however, did not differ between mutants and WT gene. G418, gentamicin and PTC124significantly increased the protein expression of R1014X mutant in a dose-dependent manner and produced full-length protein. Tobramycin had no readthrough effect. The leftward shift of activation curve was corrected only by G418and gentamicin. As the mutation site more close to N-terminal, the rescue efficiency was diminished. MS analysis indicated that HERG protein was expressed in HEK293cells after transfected with His-R1014X and added G418for24hours. However, it could not be verified which amino acid was added in the mutation site.ConclusionsAminoglycosides and PTC124showed different effects on rescue nonsense mutations of HERG gene. The site of mutations might be an import factor for the pharmacological rescue efficiency. BackgroundAminoglycosides were revealed to have the ability of readthrough on nonsense mutations to produce full-lengh functional proteins in genetic disease models. Howard et al. found that G418could suppress PTCs to restore the function of mutant protein in transiently expressed cDNAs which contained mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in mammalian cells for the first time. Our previous studies also demonstrated that G418and gentamicin could partially restore the HERG protein in a dose dependent manner in HEK293cells expressing R1014X mutant.The importance of the local sequence context in determining the efficiency of aminoglycosides on rescuing nonsense mutation was established in some disease models. Different stop codons appear to facilitate the termination process with different efficiencies, besides, the efficiency of termination suppressing can also be influenced by the local sequence context surrounding the stop codon. The strongest bias was found at the base immediately following the stop codon in most experiments. However, little is currently known about how the sequence context influences the efficiency of aminoglycosides on rescuing HERG channels in mammalian cells.ObjectivesWe set up this study to examine the susceptibility of different termination codons and+4nucleotides of HERG gene to suppression by aminoglycoside G418in HEK293cells.Methods(1) HERG nonsense mutations were generated in the pcDNA3.1WT HERG plasmid using polymerase chain reaction-based mutagenesis strategy and sequence verified.(2) The WT or mutants gene were transiently transfected in HEK293cells. Readthrough effect was studied by adding drugs into culture medium for24hours.(3) Immunofluorescence method was used to observe the expression HERG protein before and after drug treatment.(4) Patch clamping was performed to evaluate the function of genes.ResultsWe transfected R1014-TGA, R1014X-TAG and R1014X-TAA into HEK293cells respectively. N-terminal antibody and C-terminal antibody were used befor and after adding G418into the medium. HERG protein was detected before and after G418treatment by immunofluorescence. The stop codon TGA、TAA is more susceptible to G418. Similar results were also observed on W927X-TGA and W927X-TAA mutants. Then, we constructed R1014X-TGAC, R1014X-TGAG and R1014X-TGAA mutants on the basis of R1014X-TGAT mutant. Red fluorescence was observed before and after the administration of G418by using the N-terminal and C-terminal antibodies. However, the tail current density increased only on R1014X-TGAT mutant after G418treatment. The+4nucleocide could influence the function of HERG-R1014X after rescuing by G418.ConclusionThe type of stop codons and the context around the stop codons might determine the pharmacological rescue efficiency on HERG gene. BackgroundEvidence has been accumulating that autoimmunity plays a role in the occurrence and progression of CHF. For example, β1-adrenergic receptor autoantibodies (functioning as receptor agonists) were detected in the serum of CHF patients and removal of these autoantibodies has been shown to improve hemodynamic parameters. In addition, antibodies against Na-K-ATPase exerted arrhythmogenic effects and correlated with SCD in certain DCM patients. Immunization of rabbits with sarcolemmal Na-K-ATPase resulted in myocardial hypertrophy due to left ventricular pressure overload and myocardial fibrosis. Therefore, characterizing these antibodies may be helpful in the understanding of CHF pathogenesis.The L-type calcium channel plays an important role in cardiac excitation-contraction coupling. Dysfunction of the channel often correlates with ventricular arrhythmias (VAs). Current studies suggest that autoantibodies are directly linked to SCD in DCM patients and calcium channel dysfunction contributes to pathogenesis. Xiao et al detected the presence of calcium channel autoantibodies (CC-AAbs) in patients with DCM and found that CC-AAbs could prolong action potential duration (APD) and ultimately lead to VT in animal models. They believe that CC-AAbs could serve as a new biomarker for autoimmunity.ObjectiveWe set out to evaluate whether CC-AAbs could predict prognosis and SCD in CHF patients.MethodsA total of2096patients with CHF (841DCM patients,1255ICM patients) and834 control subjects were recruited. CC-AAbs were detected and the relationship between CC-AAbs and patient prognosis was analyzed.ResultsDuring a median follow-up time of52months, there were578deaths (248DCM and330ICM patients). Of these, sudden cardiac death (SCD) occurred in102cases of DCM and121cases of ICM. The presence of CC-AAbs in patients were significantly higher than that of controls (both P<0.001). Multivariate analysis revealed that positive CC-AAbs could predicte SCD (HR3.191,95%CI1.598-6.369for DCM; HR2.805,95%CI1.488-5.288for ICM) and all-cause mortality (HR1.733,95%CI1.042-2.883for DCM; HR2.219,95%CI1.461-3.371for ICM) in CHF patients. A significant association between CC-AAbs and non-SCD (NSCD) was found in ICM patients (HR=1.887,95%CI1.081-3.293).ConclusionOur results demonstrated that presence of CC-AAbs was higher in CHF patients versus controls and corresponds to a higher incidence of all-cause death and SCD. Positive CC-AAbs may serve as an independent predictor for SCD and all-cause death in these patients.