| Objectices:In our research,we studied the changes of PI3K/Akt/GSK-3? signaling pathway mediated by atrial cGMP and the effect of this signaling pathway on the secretion of ANP in atrium of rabbits,which provides experimental basis for further elucidating the changes of atrial structure and secretory function in atrial fibrillation.Methods:(1)Rapid atrial pacing rabbit model in vivo:Divided into control group and stimulation group.The rabbit atrial fibrillation model was established by stimulating rabbit atrial through right external jugular vein catheterization,meanwhile record limb-lead ECG on surface by using RM6240 physiological signal acquisition system.Atrial morphological changes were observed by HE staining after rapid pacing for 8 hours and using transmission electron microscopy to observe myocardial endoplasmic reticulum,mitochondria,and gap junction ultrastructural changes after rapid pacing for 8 hours.The expression of Akt and GSK-3? in atrial tissue were detected by Western Bolt.The changes of cGMP concentration were detected by ELISA and serum ANP concentration was detected by radioimmunoassay.(2)Isolated perfused beating atrial model:divided into three groups:Low stimulation frequency group,high stimulation frequency group,high frequency stimulation including inhibitor group.The low-frequency stimulation group was the atrial perfused HEPES buffer and paced at 1.5Hz throughout experiment.After 40 minutes of stationary phase,Atrial perfusate was collected at 2-min intervals at 4? for 24min then the fluid was added to CNP.The perfusate was continuously collected for 24 minutes.The high-frequency stimulation group was the atrial perfused HEPES buffer and paced at 4Hz throughout experiment.After 40 minutes of stationary phase,Atrial perfusate was collected at 2-min intervals at 4 ? for 24min then the fluid was added to CNP.The perfusate was continuously collected for 24 minutes.High frequency stimulation including inhibitor group was the atrial perfused HEPES buffer and paced at 4Hz throughout experiment.After 40 minutes of stationary phase,Atrial perfusate was collected at 2-min intervals at 4? for 12min then the fluid was added to LY294002,the perfusate was collected for 24 minutes.Then the perfusion fluid was added to CNP and continuously collected for 24 minutes.The changes of atrial dynamics were recorded by RM6420 physiological signal transduction system.Atrial stroke volume was measured by recording liquid column differences in hyaline cannula between systolic and diastolic period.The concentration changes of ANP and cGMP in perfusate were detected.Results:1.Electrodes was located to right atrium through external jugular vein catheterization and electrophysiological changes were detected by ECG.The rabbit model of rapid atrial pacing is stable and reliable,and successfully simulates the occurrence of atrial fibrillation.2.(1)The changes of atrial tissue structure were detected by light microscope and electron microscope.The observation of light microscope showed that with the muscle fibers visible light and dark stripes,the distribution of atrial fibers was banded,branched and connected to each other in a reticular pattern.The nucleus size uniform,round or oval,located in the center of the muscle fiber.In the stimulation group,the myocardial fibers were arranged in disorder with the interstitial edem.(2)The observation of light microscope showed visible dilatation of sarcoplasmic reticulum,mitochondrial welling,deformation,ridge arranged in disorder,disappear,glycogen particles aggregated significantly,the nuclear membrane broken,the gap junction widened and the myofilament dissolved.3.The results of Western Blot showed that the expression of Akt and GSK-3?and phosphorylation in cardiomyocytes of Stimulation group were significantly decreased compared with control group(p<0.05).4.The results of ELISA showed that the level of CGMP in cardiomyocytes of Stimulation group were significantly decreased compared with the control group(p<0.05).Isolated perfused beating atrial model in vitro showed that high frequency stimulation decreased the concentration of cGMP and CNP increased the concentration of cGMP in perfusate.LY294002,the PI3K/Akt inhibitor,and further enhanced the effect of CNP on cGMP(p<0.05).5.The results of radioimmunoassay showed that the secretion of ANP was increased after 8 hours stimulation in vivo experiment.Isolated perfused beating atrial model in vitro showed that high frequency stimulation increased ANP secretion(p<0.05),decreased atrial stroke volume and CNP inhibited ANP secretion(p<0.05),decreased atrial stroke volume.Pretreatment with PI3K/Akt inhibitor LY294002,the decrease of ANP induced by CNP was blocked(p<0.05)and the inhibitory effect of CNP on atrial motility was enhanced(p<0.05).Conclusion:1.Rapid atrial pacing results in the changes of structural cardiac myocyte.2.Rapid atrial pacing increased the secretion of ANP,but the concentration of cGMP in pacing 8h atrial tissue was decreased.3.The activity of Akt was decreased and the activity of GKS-3? was increased in rapid pacing atrial tissue.4.PI3K/Akt/GSK-3?signaling pathway mediated by cGMP is involved in atrial ANP secretion in atrial fibrillation. |
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