Research On The Treatment Of Atrial Fibrillation Via Atrial Vagal-selective Denervation Using Targeted Ultrasound Contrast Agent Mediated Gα_i2 C-terminal Peptide

Objectives:1) To study the feasibility of preparing the targeted ultrasound contrast agents incorporated with Gαi2C-terminal peptide (Gαi2ctp);2) To establish an animal model of paroxysmal atrial fibrillaton induced by canine parasympathetic nerve; To explore the effect of parasympathetic nerve stimulation both on the electrophysiological properties in different parts of atrium and the inducibility as well maintenance of AF; To investigate electrophysiological mechanism of AF vagus denervated therapy, through the corresponding probe in the changes of electrophysiological properties and the inducibility as well maintenance in different parts of atrium after vagal denervation via targeted transfered Gαi2ctp;3) To discuss the potential molecular mechanism of vagal denervation via targeted transfered Gαi2ctp. Methods:1) Sulfonated rhodamine-labeled Gαi2ctp were divided into three groups according to the magnitude of dose, the targeted-ultrasound microbubbles contrast agents incorporated with Gαi2ctp was prepared using direct connection methods, and the carrier was SonoVue in this study; The size, binding rate, stability and targeted ability of these three groups were measured and analyzed.2) Fourteen adult mongrel dogs weighing from14to23kg were randomly divided into two groups:control group (n=7), were given the simple ultrasound microbubble contrast agent; experimental group (n=7), were given the targeted-ultrasound microbubbles contrast agents incorporated with Gαi2ctp. Through the method of stimulating cervical vagal trunk and rapid atrial pacing, then, the vagal-mediated acute canine model of atrial fibrillation was established, The rate of AF inducibility was measured in different parts of atrium and pulmonary veins, to explore the effect of parasympathetic nerve stimulation on inducibility and maintenance of AF; The AF inducibility, ERP, dERP, ERP frequency adaptability were respectively measured in LA, RA and PVs, through the cardiac electrophysiology mapping, to investigate electrophysiological mechanism of AF vagus denervated therapy.3) Employing the technique of immunohistochemistry and immunofluorescence to detect the expression of Gαi2ctp in LA, RA and PVs; Employing Western blot method to measure the expression level of Gαi2ctp protein in LA, RA and PVs; Employing RT-PCR technique to assay the expression of Gαi2ctp’s mRNA in LA, RA and PVs of both experimental group and the control group dogs, Employing ELISA in the experimental group and the control group dogs to analyse the variation of cAMP content in LA, to discuss the potential molecular mechanism of vagus denervation via targeted transfered Gαi2ctp. Results:1) SonoVue and sulfonated rhodamine-labeled Gαi2ctp could stably bound together, especially the dose of1.5mg was optimun. prepared contrast agent could emit salmon fluorescence in the fluorescence microscope; flow cytometry detection results showed:the carriage ratio of the group with1.5mg/1Gαi2ctp microbubbles contrast agent are steadily the highest among the different standing time. When the standing time were0.5hours,1hour,2hours, the carriage ratios were93.07±4.59%,93.60±7.35%, and96.52±3.35%respectively, and the carriage ratios would not wane as time elongated. In vivo evaluation, it found that in LA and PV of experimental group could detect the salmon fluorescence under a fluorescence microscope, However, there were no fluorescent material gathered in the control group.2) Through the method of stimulating cervical vagal trunk and rapid atrial pacing, the vagal-mediated acute canine model of atrial fibrillation was successfully established, the stimulation of vagus nerve would significantly shorten the ERP in different parts of atrium and PV, increase the dERP, thereby increasing the ratio of AF inducibility; compared with the control group, ERP was significantly prolonged in the LA, LSPV, LIPV after trasfering targeted Gαi2ctp in the LA of experimental group0.5h,1h,2h later,the difference was statistically significant (P<0.05), ERP was slightly lengthened or shortened in the RA, RSPV, RIPV, but the difference was not statistically significant; the dERP of all parts of atrium was significantly reduced (P<0.05), ERP frequency adaptability was increased (P<0.05), AF inducibility was decreased after trasfering targeted Gαi2ctp in the LA of experimental group0.5h,1h,2h later.3) The expression of Gαi2ctp was presented in different parts of canine atrium and PVs; targeted transduction of exogenous Gαi2ctp could prevent Gai-mediated signal transduction pathways, thereby affecting cAMP content of the downstream pathway, and reducing the occurrence of vagal-mediated atrial fibrillation. Conclusion:the targeted-ultrasound microbubbles contrast agents incorporated with Gαi2ctp could be successfully produced using direct connection methods. Through the method of stimulating cervical vagal trunk and rapid atrial pacing, the vagal-mediated acute canine model of atrial fibrillation could be successfully established. The electrical stimulation of vagus nerve could significantly increase the ratio of AF inducibility in atrium and PV. The ultrasound microbubbles contrast agents incorporated with Gαi2ctp prepared by the direct connection method could have even better targeted ability, it could successfully rupture at the targeted position in LA, acting on Gαi protein C-terminal peptide structure to barricade the Gai-mediated signal transduction pathways, thereby selectively interfering the excitability of vagus nerve, finally in order to achieve " the role of drugs to vagal denervation", as well to reduce the occurrence of vagal-mediated atrial fibrillation.