| Background:Atrial fibrillation is a kind of arrhythmia characterized by rapid and irregular atrial electrical activity.Its treatment is very important because it can increase the mortality risk of patients.But nowadays there are limitations of the therapeutic measures for atrial fibrillation.It is found that the tension imbalance of autonomic nervous system is closely related to the occurrence and maintenance of atrial fibrillation.Stimulating and ablating the autonomic nervous can change its balance state so as to promote or suppress the onset and maintenance of atrial fibrillation.Spinal cord stimulation can regulate the activity of sympathetic nerves and parasympathetic nerves to correct the tension imbalance of autonomic nervous system.Spinal cord stimulation has been successfully applied in the treatment of refractory angina pectoris,arrhythmia storm,refractory heart failure,endless ventricular tachycardia and other critical diseases.But whether it can be used for curing atrial fibrillation has not been studied systematically at home and abroad.Objectives:The purposes of this study were to give the canine atrial fibrillation models cervical and thoracic spinal cord stimulation,and to observe the changes in tissue cell structure,expression changes of markers related to atrial remodeling and the changes in the expression profile of microRNA in their left atriums.Compared with the blank control group and the non-spinal cord stimulation canine atrial fibrillation models,this study’s purposes were to analyze the therapeutic effect of spinal cord stimulation on atrial fibrillation and its influence on left atrial remodeling and miRNA expression profile of canine atrial fibrillation models,and finally to explore the mechanism and targets of spinal cord stimulation in treating atrial fibrillation.Methods:1、Eighteen healthy adult beagles were randomly and equally divided into three groups:the blank control group(Ctrl group),the atrial fibrillation model control group(AF group)and the atrial fibrillation model with spinal cord stimulation group(SCS group).(1)Implanted high frequency pacemakers into the experimental dogs in AF group and SCS group.And then established atrial fibrillation models by rapid pacing to right atrium in AOO mode.(2)Implanted spinal cord stimulators into the canine atrial fibrillation models in SCS group.The stimulating electrode was located at the dorsal T1-T4 level of spinal cord.Adjusted the parameters and gradually increased the stimulating voltage.Stimulated the experimental dogs in SCS group for a total of 12 weeks(each time with 2 to 6 hours’ constant stimulation).(3)Implanted miniaturized insertable cardiac monitors subcutaneously in the anterior chest wall of dogs in AF group and SCS group to observe and analyze their ECG activity and the variation of atrial fibrillation burden.(4)After the completion of model making and spinal cord stimulation treatment executed the experimental dogs and collected their left atriums for microscopic observing the changes of organization structure and fibrosis by HE staining section and Masson staining section.And observed the changes of cell ultrastructure of left atriums of dogs in three groups under electron microscope.2、Tested the expression levels of the mRNA and protein of L-type calcium channel protein alpha subunit,connexin 40,connexin 43,tyrosine hydroxylase,choline acetyltransferase and growth associated protein 43 by qRT-PCR and western blot methods and statistically analyzed the differences among three groups.Immunohistochemistry and immunofluorescence methods were used to observe the expression and distribution of the above six target proteins in the left atrial muscle of three groups of experimental dogs.Statistically analyzed the differences of average optical density among three groups.3、Tested the miRNA expression profile of left atriums of the experimental dogs in three groups by miScript miRNA PCR Array.And screened out the miRNAs which expressed differently obviously among three groups.Made cluster analysis of these miRNAs and verified them in the left atriums of experimental dogs in three groups by qRT-PCR method.Forecasted the target genes and binding sites of miR-33a and miR-219-3p which expressed differently obviously among three groups and searched the target gene’s downstream signaling pathway in KEGG database.Results:1、Of the eighteen dogs selected,three died in the atrial fibrillation modeling’s process and the remaining fifteen dogs completed the experiment.(1)The nine survival dogs in AF group and SCS group established atrial fibrillation models successfully by rapid right atrial pacing in AOO mode.The time of the appearance of atrial fibrillation was 10.45±2.32 weeks and the modeling’s success rate was 75%.The pacemaker frequency was 587.82±13.35(times/min)when atrial fibrillation models established successfully.(2)As measured by the miniaturized insertable cardiac monitors the atrial fibrillation burden of SCS group(7.35±2.42)%was significantly lower than that of AF group(46.45±6.21)%,P<0.05.(3)Under the light microscope of HE staining of the left atrial muscle,the myocardial cells in Ctrl group were arranged neatly and the structure was intact.Myocardial cells in AF group were arranged in disorder.Microscopically,a few cells showed degeneration and necrosis along with interstitial edema and fibrosis.The arrangement of myocardial cells in SCS group was slightly disordered with some interstitial edema and nuclear concentration,but the degree was less than that in AF group.(4)Masson staining of left atriums under light microscope showed that:The degree of left atrial fibrosis in AF group was significantly increased compared with that in Ctrl group,and SCS group also showed left atrial fibrosis,but the degree was lower than that of AF group.(5)Under the electron microscope of the left atrium,the myocardial nuclei in Ctrl group had normal structure and orderly arrangement of myofibril segments.Myocardial cells in AF group showed few expansion of sarcoplasmic reticulum and deformation of mitochondria swelling,but they were not prominent.Myofibril arrangement in SCS group was relatively neat without obvious mitochondrial swelling or sarcoplasmic reticulum expansion.2、Effects of spinal cord stimulation on makers of left atrial remodeling in canine atrial fibrillation models.In this study,four methods including qRT-PCR,Western blot,immunohistochemistry and immunofluorescence were used to detect the expression levels of mRNA and protein of GAP43,TH,CHAT,CX40,CX43 and L-type calcium channel protein alpha subunit in the left atrium tissues of three groups of experimental dogs,and it was found that there were significantly differences of the indicators among the three groups.(1)At the mRNA expression level,compared with Ctrl group,the expression of GAP43,TH and CHAT in AF group were up-regulated and the expression of CX40,CX43 and L-type calcium channel protein alpha subunit in AF group were down-regulated,P<0.05.Compared with Ctrl group,the expression of GAP43 and CHAT in SCS group were up-regulated,P<0.05,and the expression of TH in SCS group was up-regulated,P>0.05,and the expression of CX40 and CX43 in SCS group were down-regulated,P<0.05,and the expression of L-type calcium channel protein alpha subunit in SCS group was down-regulated,P>0.05.Compared with AF group,the expression of GAP43,TH and CHAT in SCS group were down-regulated,P<0.05,and the expression of CX40 and CX43 in SCS group were up-regulated,P>0.05,and the expression of L-type calcium channel protein alpha subunit in SCS group was up-regulated,P<0.05.(2)At the protein expression level,compared with Ctrl group,the expression of GAP43,TH and CHAT in AF group were up-regulated while the expression of CX40,CX43 and L-type calcium channel protein alpha subunit in AF group were down-regulated,P<0.05.Compared with Ctrl group,the expression of GAP43,TH and CHAT in SCS group were up-regulated while the expression of CX40,CX43 and L-type calcium channel protein alpha subunit were down-regulated in SCS group,P<0.05.Compared with AF group,the expression of GAP43,TH and CHAT were down-regulated in SCS group while the expression of CX40,CX43 and L-type calcium channel protein alpha subunit were up-regulated in SCS group,P<0.05.(3)Immunohistochemical results showed that:Compared with Ctrl group,the positive staining reactions of GAP43,TH and CHAT which represented neural remodeling in left atrium samples increased in AF group while the staining of SCS group also increased but to a lesser extent than that of AF group.The marker proteins of CX40,CX43 and L-type calcium channel protein alpha subunit which represented electrical-anatomical remodeling in left atriums showed lighter staining in AF group than Ctrl group,and SCS group showed intermediated staining.The average optical density values of three groups were calculated by image processing software.The statistical results showed that:Compared with Ctrl group,the values of GAP43,TH and CHAT rose while the values of CX40,CX43 and L-type calcium channel protein alpha subunit declined in AF group,P<0.05.Compared with Ctrl group,the values of GAP43 and TH rose in SCS group,P<0.05,and the value of CHAT rose in SCS group,P>0.05,and the values of CX40,CX43 and L-type calcium channel protein alpha subunit declined in SCS group,P<0.05.Compared with AF group,the values of GAP43,TH and CHAT all declined in SCS group,P<0.05,and the values of CX40,CX43 and L-type calcium channel protein alpha subunit all rose,P>0.05.(4)Immunofluorescence results showed that:Compared with Ctrl group,GAP43,TH and CHAT which represented atrial neural remodeling were highly expressed in AF group,and CX40,CX43 and L-type calcium channel protein alpha subunit which represented atrial electrical-anatomical remodeling were all low in expression in AF group.Compared with Ctrl group,SCS group showed high expression of GAP43,TH and CHAT and low expression of CX40,CX43 and L-type calcium channel protein alpha subunit.Compared with AF group,GAP43,TH and CHAT in SCS group showed low expression while CX40,CX43 and L-type calcium channel protein alpha subunit in SCS group showed no significant difference.Image analysis software was used to calculate the average optical density values.The statistical results showed that:Compared with Ctrl group,GAP43,TH and CHAT were increased in AF group,and CX40,CX43 and L-type calcium channel protein alpha subunit were decreased in AF group,P<0.05.Compared with Ctrl group,GAP43 was increased in SCS group,P<0.05,and TH and CHAT were increased in SCS group,P>0.05,and CX40,CX43 and L-type calcium channel protein alpha subunit were all decreased in SCS group,P<0.05.Compared with AF group,GAP43,TH and CHAT were decreased in SCS group,P<0.05,and CX40,CX43 and L-type calcium channel protein alpha subunit were increased in SCS group,P>0.05.3、Effect of spinal cord stimulation on miRNA expression profile of left atriums in canine atrial fibrillation models.The miScript miRNA PCR Array was used to select the differentially expressed miRNAs in left atrial tissues of the three groups of experimental dogs.The results were obtained according to the fold change(FC)>2 or<0.5,and P<0.05.Compared with Ctrl group,the up-regulated miRNAs in AF group were let-7a,miR-33a,miR-92b,miR-345,miR-500 and miR-875,and the down-regulated miRNAs were miR-30b,miR-143,miR-196b,miR-216a,miR-219-3p,miR-222,miR-665 and miR-1306.Compared with Ctrl group,the up-regulated miRNAs in SCS group were miR-33a and miR-105b while the down-regulated miRNAs were miR-122,miR-143,miR-216a,miR-219-3p,miR-222,miR-665 and miR-1306.Compared with AF group,the up-regulated miRNAs in SCS group were miR-30b and miR-105b while the down-regulated miRNAs were let-7a,miR-33a,miR-92b,miR-375,miR-500 and miR-875.These differentially expressed miRNAs were verified by the method of qRT-PCR and the results were consistent with the screening results of miScript miRNA PCR Array.These differentially expressed miRNAs were analyzed by clustering,and in TargetScan database we found that TLR4 was the common target gene of miR-33a and miR-219-3p.While miR-33a and TLR4 only had one poorly conserved action site miR-219-3p and TLR4 had one conserved action site and two poorly conserved action sites.And in KEGG database we found that TLR4 played an important role in NF-κB signaling pathway.Conclusions:1、Making canine atrial fibrillation model with high frequency continuous pacing of the right atrium is safe,effective and has a high successful rate.Beginning from 90 times/min and gradually increasing the frequency of pacemaker intermittently or continuously according to the actual situation of experimental dogs to make atrial fibrillation models can reduce the incidence of experimental accidents.And the atrial fibrillation model made by this method is more stable and more similar to the actual situation of occurrence and development of chronic atrial fibrillation.2、Using spinal cord stimulation to intervene atrial fibrillation models has high safety and operability.3、Spinal cord stimulation can effectively inhibit the occurrence and persistence of atrial fibrillation in dogs,and can relieve and reverse left atrial remodeling and fibrosis related with atrial fibrillation,and may be used as one of the means of treating atrial fibrillation.4、Left atrial electrical-anatomical remodeling and neural remodeling existed in atrial fibrillation model dogs,and spinal cord stimulation can alleviate and reverse such remodeling,especially atrial neural remodeling.Spinal cord stimulation may reverse left atrial neural remodeling by regulating cardiac autonomic nerve.The reversal effect of spinal cord stimulation on left atrial remodeling may be beneficial to the treatment of atrial fibrillation.5、Spinal cord stimulation can change the partial differential miRNA expression in the left atrial muscle of atrial fibrillation dogs,and the mainly involving miRNAs included let-7a,miR-30b,miR-92b,miR-375,miR-500 and miR-875.They may be the molecular regulatory mechanism of the intervention of atrial fi brillation by spinal cord stimulation,and may be the targets of the intervention of atrial fibrillation by spinal cord stimulation.6、MiR-33a and miR-219-3p which express differentially in left atriums among three groups may take part in the classic inflammation signaling pathway of TLR4/NF-κB to regulate the inflammatory response of the atrial fibrillation dogs so as to affect the outcome of atrial fibrillation.It may be one of the targets of the treatment of spinal cord stimulation for curing atrial fibrillation. |
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