Targeted Gene-based Inhibition Of Gα_i2 C-terminal Peptide Decreases Vagal Atrial Fibrillation By Selective Parasympathetic Attenuation

Objectives: The parasympathetic nervous system is known to be involved in the genesis and maintain of atrial fibrillation(AF). Vagal dennervation ablation is a hotspot in the field of AF therapy, it can obviously improve the success rate of the ablation of AF. Parasympathetic signalling in the heart is initiated upon presynaptic release of acetylcholine(ACh), which activates an inward rectifying k+ current, IK-ACh through G-protein and induces the occurrence of AF.Our initial research demonstrated the feasibility of using injection of Gαi2 C-terminal peptide(Gαi2ctp) to achieve selective parasympathetic denervation in the left atrial, with a resulting decrease vagal-induced atrial fibrillaiton. However, the utility of such a peptide-based pharmacotherapy requires sustained and controlled intracellular expression of peptide in the atrial myocardium, so restricts the application by certain degree. Accordingly, in the present study, we establish the rapid pacing atrial fibrillation model of canine, construct the plasmid DNA vector expressing Gαi2ctp gene, and targeted transduction to the left atrium through the method of open-heart local injection and electroporation.The Gαi2ctp gene attenuates parasympathetic activity and reduces vagal-induced AF. And verify the feasibility from the electrophysiology, histology, molecular biology etc.To investigate the validity and feasibility of Gene targeting intervention treatment, provide new ideas and theoretical basis for gene therapy of AF.Methods: 1) Established the adenovirus vector expressing Gαi2ctp: the amount of r Ad-Gαi2ctp was amplified with 293 cell. Polymerase chain reaction(PCR) assay was used to identify the augmented of r Ad-Gαi2ctp; 2) Adult beagle dogs were randomly assigned to 2 groups: a fast left atrial appendage 6-hr pacing group(n=6), and a 6-hrpacing+vagosympathetic nerve stimulation(VNS) group(n=7). All dogs underwent tests for their pulmonary vein and atrial effective refractory period(ERP),monophasic action potential(MAP) and AF induction rate at various time points, after which they were sacrificed, and samples of left and right atrial tissue were removed and microscopically examined. Additionally, the atrial anterior right ganglionated plexi(ARGP) were collected and examined for expression of nerve factor tyrosine hydroxylase(TH) and choline acetyl transferase(CHAT); 3) Adult beagle dogs were randomly assigned to 3 groups: a sham operation group(Group A, n=5), a fast left atrial appendage 6-hr pacing + r Ad-EGFP injection group(Group B, n=6), and a 6-hr pacing + r Ad-Gαi2ctp injection group(Group C, n=6). All dogs underwent tests for their atrial ERP at various time points, after which they were sacrificed, and samples of left and right atrial tissue were removed and microscopically examined. Field action potential duration(FAPD) of left and right atrial tissue from each group were measured by microelectrode arrays chip(MEA). Additionally, the atrial anterior right ganglionated plexi(ARGP) were collected and examined for expression of nerve factor; 4) Adult beagle dogs were randomly assigned to 3 groups: a sham operation + rapid atrial pacing(RAP) group(Group A, n=6), RAP + r Ad-Gαi2ctp group(Group B, n=7), and RAP + vagal dennervation ablation group(Group C, n=6). All dogs underwent tests for their pulmonary vein and atrial ERP、MAP at various time points, after which they were sacrificed, and samples of left and right atrial tissue were removed and microscopically examined for expression of TH and CHAT.Results: 1) The titre of r Ad-Gαi2ctp was 1×1011 pfu/ml post augmentation. Gene vector r Ad-Gαi2ctp transfect ion best time was 7 days. The method via chest atrial muscle injection r Ad-Gαi2ctp gene transfection was success and safety; 2) As pacing times increased, the mean ERP and MAP in group B became significantly shortened, however, groups A and B did show significant differences(P<0.05). There was no difference in atrial myocardial fibrosis among the two groups. ARGP expressions of TH and CHAT in groups A was lower than expression in group B(P<0.05); 3) As pacing times increased, the mean ERP in group B became significantly shortened. Groups A and C showed no significant differences in ERP at baseline and 6 hours(P>0.05); however, groups B and C did show significant differences(P<0.05). There was significant difference in atrial FAPD and periodic acid-Schiff stain among the three groups(P < 0.05).ARGP expressions of TH and CHAT in groups A and C were lower than expression in group B(P<0.05); 4) Inter-group comparisons of MAP and ERP demonstrated that Group A wassignificantly different from Groups B and C(P<0.05), while the difference between Groups B and C was not significant(P>0.05). When compared to Group A, both Groups B and C had reduced TH and CHAT expression.Conclusion: 1) Successful build Gαi2ctp adenovirus expression vector and determine the optimal transfect ion time of r Ad-Gαi2ctp and for the safety of other organs; 2) RAP+VNS model are stable and good repeatability. 3) r Ad-Gαi2ctp does not only reverse the effect of fast pacing atrial electrical remodeling in dogs, but also affects autonomic nervous remodeling; 4) Vagal dennervation ablation and r Ad-Gαi2ctp injection are both equivalent to reverse electrical remodeling and autonomic nervous remodeling.