PDE3-mediated CGMP And CAMP Signail Transduction Changes In Rabbit Rapid Atrial Pacing

Objective:Cyclic guanosine monophosphate(cGMP)is a second intracellular messenger that regulates cardiac function through the cGMP-gated ion channels,cGMP-dependent phosphodiesterase(PDE),protein kinase G(PKG),cAMP cross regulation present on the cell membrane which are involved in myocardial contraction and ion current regulation.The activity of PDE3 is controlled by cGMP concentration in the cells,and the combination of atrial peptide and its receptor results in increased cGMP production,so it is possible to change the cAMP concentration in the cell to regulate the mechanical activity of the atrium.In this study,the rapid atrial pacing in rabbits was studied,and the cGMP-PDE3-cAMP signaling pathway was used as the cut-in point to explore the PDE3 mediated cGMP and cAMP cross-effect.Thus,it is further clarified that the cGMP-PDE3-cAMP signaling pathway in atrial fibrillation provides a new target for the mechanism of atrium dynamic changes.Method:1.Animal model preparation:New Zealand's big-eared white rabbits with weight of 2.0 to 2.5 kg were selected irrespective of gender,then divided into P0 control group,which were intubated without stimulation and P8 experimental group,in which stimulation time was 8 hours,each group of 8.Using RM6240 physiological and experimental system,the rabbit atria were electrically stimulated by right external jugular vein catheterization and the ECG of body limb lead was recorded.2.Morphological observation:after electrical stimulation,the right atrium of the animal was extracted,fixed,embedded and sectioned.The changes of atrial muscle structure were observed by light microscope and transmission electron microscope.The effects of rapid pacing on the ultrastructure of the atrial muscle sarcoplasmic reticulum and mitochondria were observed and confirmed.3.ANP concentration determination:after the stimulation is completed,blood is collected,centrifuged and the supematant is taken.Radioimmunoassay was used to detect the level of ANP in serum and further observe the effect of rapid pacing on atrial secretion of ANP.4.ELISA assay,cGMP and cAMP content:to observe the effect of cGMP and cAMP content of atrial myocytes.5.Western blot was used to detect the expression of PDE3A in atrial tissue,and the change of PDE3 activity was determined by external standard method.Results:1.Using the right external jugular vein catheterization,rapid atrial pacing in a rabbit model was established,which indicated ease to stimulate the occurrence of atrial fibrillation.2.By means of light microscopy and transmission electron microscopy,it was observed that hematoxylin-eosin staining showed that the fibers of the atria were branched and connected to a reticulate,long ribbon band.Light and dark stripes of atrial fibers could be seen.The nucleus is oval and resides in the middle of the muscle fiber.Compared with the control group,there was no significant change of myocardium in the 8-hour stimulation group.The ultrastructure of the control group and the 8-hour stimulation group were observed by transmission electron microscope.The mitochondria were deformed and swollen,ridge arrangement irregular,the sarcoplasmic reticulum was dilated,a large number of vacuoles were formed and the myofilament was dissolved,visible glycogen particles,nuclear membrane was broken and sunken.These changes were more obvious in the 8-hour stimulation group.3.The blood ANP concentrations of PO and P8 rabbits were determined by radioimmunoassay.Results of PO group was 1.6760±0.113 ?g/L and P8 group was 2.142±0.156 ?g/L,compared with PO group the difference was statistically significant(P<0.01).4.The results of ELISA showed that the content of cGMP and cAMP decreased gradually with the prolongation of stimulation time.The cGMP and cAMP levels of atrial tissue in 8-hour stimulation group were lower than those in control group,the difference was statistically significant(P<0.01).5.The rapid atrial pacing model of atrial fibrillation on PDE3 expression and activity:the results can be observed after 8 hours pacing,atrial PDE3 expression was not significantly changed compared with the control group.Compared with the control group,the PDE3 activity after increase in atrial pressure due to rapid pacing of the atrium was significantly lower than that in the control group.Conclusion:1.Rapid atrial pacing induces changes in the sarcoplasmic reticulum and mitochondrial structure;2.Decreased PDE3 activity interferes with the cGMP-PDE3-cAMP signaling pathway,which may serve as a pharmacological target to prevent atrial remodeling caused by atrial fibrillation.