Effects Of Rapid Atrial Pacing On Atrial PGC、GSK-3β Activity And ANP Secretion

Objectices:In this study,we prepared a rapid atrial pacing model to simulate the occurrence of human atrial fibrillation in New Zealand large-eared white rabbits,and observed the effects of rapid atrial pacing on p GC activity,Akt/GSK-3βprotein expression and ANP secretion in atrial tissue.To investigate the mechanism of c GMP level alteration in vivo during atrial fibrillation and the downstream signaling pathways that regulate ANP secretion in the atria,we aimed to elucidate the pathophysiological mechanism of endocrine function alteration in the atria due to atrial fibrillation.Methods:1.Preparation of in vivo rapid atrial pacing model in New Zealand large-eared rabbits:New Zealand large-eared white rabbits were divided into two groups,the control group and the group with rapid atrial pacing at 8 hours.A rabbit in vivo rapid atrial pacing model was prepared by inserting a J-type polyethylene catheter through the right internal jugular vein into the right atrium of the rabbit and performing electrical stimulation.The atrial effective refractory period（AERP）and atrial effective refractory period dispersion（AERPd）were recorded;changes in atrial structure were observed by H&E staining;Western blotting assay was performed to detect the expression levels of NPR-A,Akt,GSK-3β,p-Akt,p-GSK-3βin atrial tissue;ELISA assay to detect changes in c GMP concentration in rabbit blood and indirect determination of p GC activity by c GMP concentration;radioimmunoassay to detect ANP concentration in serum.2.Left atrium was taken for perfusion,and the experiment was divided into four groups:low-frequency excitation group（1.5 Hz）,high-frequency excitation group（4.0Hz）,high-frequency excitation plus LY294002 group,and high-frequency excitation plus SB216763 group.The first 36 min of perfusate was the blank control group,and the 24 min of perfusate continued to be collected after the addition of c GMP from 36min onwards was the experimental group.High-frequency excitation（4.0 Hz）plus LY294002/SB216763 group was collected for the first 12 min of perfusate as a blank control group,after 12 min the inhibitor LY294002/SB216763 was added and continued to be collected for 24 min,and after 36 min LY294002 and c GMP/SB216736 and c GMP were added and collected after 24min.The changes in atrial pulse pressure and atrial beat volume were also recorded.c GMP and ANP concentrations in the perfusion fluid were measured by ELISA and radioimmunoassay,respectively.Akt,p-Akt,GSK-3β,and p-GSK-3βprotein expression were detected in the atrial tissue removed after perfusion.Results:1.In the in vivo rapid atrial pacing model of New Zealand Large-eared White rabbits,AERP₁₅₀ and AERP₂₀₀ were shortened and AERPd was prolonged in the model group compared with the control group.H&E staining revealed a disturbed arrangement of myocytes in the atrial myocytes of the rapid pacing group and a significant edematous portion between the interstitial myocytes of the cardiac myocytes.It was also observed that the mitochondria in the atrial myocytes of the rapid pacing group were abnormally altered,the nuclear membrane was broken and depressed,and the gap between the myocytes was widened,and the myofilaments were no longer found.These results indicate that the rabbit atrial fibrillation model has been successfully established.2.In a rapid atrial pacing model in vivo in rabbits,ELISA and radioimmunoassay results showed that ANP concentrations increased and c GMP concentrations decreased in the rapid pacing group（P<0.05）;NPR-A protein expression did not differ between the model and control groups,but p GC activity was significantly reduced in the model group（P<0.05）;and p-Akt and p-GSK-3βproteins were reduced（P<0.05）.3.In the isolated perfused atrial model of New Zealand large-eared white rabbits,ANP secretion was significantly increased under high-frequency stimulation compared with the low-frequency stimulation group,and atrial pulse pressure and atrial beat volume were instead attenuated under high-frequency stimulation;after the addition of c GMP,ANP,atrial pulse pressure and atrial beat volume were attenuated on the original basis regardless of high or low frequency stimulation（P<0.05）.4.In an isolated perfused atrial model,after we added both c GMP and Akt and GSK-3βinhibitors under high frequency stimulation,we found that Akt and GSK-3βinhibitors attenuated the inhibitory effect of c GMP on ANP secretion;however,the inhibitors further enhanced the inhibitory effect of c GMP on atrial pulse pressure and atrial beat volume（P<0.05）.5.The levels of p-Akt and p-GSK-3βwere attenuated by high frequency stimulation;their phosphorylation levels increased instead after the addition of c GMP;p-Akt and p-GSK-3βlevels were attenuated after the addition of Akt inhibitor;p-Akt levels were unchanged but p-GSK-3βlevels were attenuated after the addition of GSK-3βinhibitor（P<0.05）.Conclusions:1.Rapid atrial pacing stimulation leads to increased ANP secretion and decreased c GMP concentration in vivo.2.Rapid atrial pacing stimulation leads to decreased sensitivity of NPR-A/p GC to ANP,which in turn leads to decreased c GMP production.3.Rapid atrial pacing stimulation leads to decreased Akt and GSK-3βactivity in the atria,while c GMP regulates ANP secretion through the Akt/GSK-3βsignaling pathway during the progression of atrial fibrillation.