Role Of NGF/TrKA Signaling Pathway In The Occurrence Of Valvular Atrial Fibrillation

BackgroundAtrial fibrillation(AF)is a clinical common arrhythmia that can increase in prevalence with aging.Cardiac dysfunction and embolism are its common complications,especially in patients with valvular heart disease(VHD).Even now it is known that valvular AF is associated with atrial structural changes and electrophysiological abnormalities.But the exact mechanism is still unclear.According to some recent research,autonomic stimulation can lead to AF,and beta-blocker can reduce it.Therefore,we are interested in how the autonomic nervous system participates in AF.Nerve growth factor(NGF)is a typical member of the neuro-trophic factor family and an endogenous survival factor of cardiac myocytes.It is essential for the differentiation,survival and synaptic activity of autonomic nervous systems.The expression level of NGF in the innervation tissue is proportional to the innervation density.Tropomyosin-related kinase receptor A(TrkA)is a specific receptor of NGF.After NGF combines with TrKA,phosphatidylinositol 3-kinase(PI3K),the well-known protein in the growth and survival of cells,is activated.PI3K and its downstream molecule serine threonine kinase Akt(or protein kinase B)play an important role in myocardial protection.Meanwhile,Akt is a potent upstream regulator of glycogen synthase kinase 3?(GSK3?).Activated Akt(phosphorylation of Akt at Ser-473)can induce the phosphorylation of the S9 residue of GSK-3?,which results in inactivation of GSK3?.Inactivated GSK3? increases the accumulation of Nuclear Factor of Activated T-cells(NFATs)in the nucleus through NFATs phosphorylation,which leads to myocardial hypertrophy.Our previous studies have found that Calcineurin(CaN)-NFATs signaling pathway has been involved in the pathogenesis of valvular AF.As NFATs are effector of GSK3 ?,we hypothesized NGF-TrkA-Akt-GSK3? signaling pathway may be also involved in the development of valvular AF.However,the clinical study on whether NGF-TrkA-Akt-GSK3 ? signaling pathway regulates the pathogenesis of valvular atrial fibrillation is very limited,especially in human.Therefore,this experiment was designed to investigate whether NGF/TrkA signaling pathway is involved in the occurrence of human valvular atrial fibrillation.ObjectiveIn this study,we analyzed the different protein and gene expressions of NGF/TrKA and its downstream protein Akt,GSK3 ? in different structures of normal human heart,to observe their distribution in heart tissue,in order to provide a possible clue for exploring the role of NGF/TrkA pathway in different parts of the heart.At the same time,to study the difference of protein and gene expression in patients with valvular heart disease with sinus rhythm and atrial fibrillation rhythm,in order to explore whether NGF/TrkA signaling pathway is involved in the occurrence of atrial fibrillation in patients with valvular heart disease,and to provide a new direction for clinical treatment of valvular atrial fibrillation1.Object1.1 Three donor hearts from non cardiac death donors were dissected into eight parts:left ventricle,right ventricle,left atrium,right atrium,left atrial appendage,right atrial appendage,interventricular septum and apex.1.2 Thirty adult patients with valvular heart disease requiring open heart surgery,patients treated with class I or III antiarrhythmic drugs,and patients with familial paroxysmal atrial fibrillation,pulmonary artery disease,sick sinus syndrome,kidney disease or cardiomyopathy were excluded from the study.According to the preoperative medical history and ECG examination,the patients were divided into two groups:atrial fibrillation group(n=15)and sinus rhythm group(n=15).Data of medical history,physical examination and auxiliary examination were collected before operation.At the same time,5ml of peripheral blood was extracted in the morning of the second day after admission.Human peripheral blood lymphocytes were extracted with lymphocyte extract,and the haemorrhagic plasma was separated,and protein and RNA were extracted from peripheral blood lymphocytes.Part of the right atrial appendage was obtained before the establishment of three-dimensional external circulation,and the right atrial appendage was quickly stored in liquid nitrogen.2.Experimental methods2.1 Nerve growth factor(NGF),tropomyosin receptor A(TrkA),total protein kinase B(total Akt),phosphorylated protein kinase B(p-s473 Akt)and total glycogen synthesis kinase 3 ?(total)were detected by immunohistochemistry and Western blot,the expression and distribution of GSK3 ? and p-s9 GSK3 ? in different myocardial structures were studied.2.2 Human peripheral blood lymphocytes and plasma were extracted with human lymphocyte extract.Total protein and RNA were extracted rapidly and stored in-80 °refrigerator.2.3 Western blot and qt-PCR were used to detect NGF,TrkA,total Akt,p-s473 Akt,total GSK3 ? and p-s9 GSK3 ? in right atrial appendage myocardial tissue and peripheral blood lymphocytes.The protein and gene expression levels of GSK3 ?were determined by using glyceraldehyde phosphate dehydrogenase(GAPDH)as internal reference.2.4 The levels of NGF-? in plasma of patients with valvular heart disease were measured by enzyme-linked immunosorbent assay(ELISA).The levels of NGF-? in patients with sinus rhythm and atrial fibrillation rhythm were statistically analyzed.Result1.The distribution of NGF,TrkA,GSK3 ?,p-ser9,GSK3 ?,Akt and p-ser473 Akt in normal cardiac cells.In normal heart,there are not only cardiac myocytes,but also myoblasts,smooth muscle cells,endothelial cells and cellulose.We found that NGF,TrkA,GSK3 ?,p-ser9,GSK3 ?,Akt and p-ser473 Akt were expressed in the cytoplasm of cardiomyocytes,but not in vascular endothelial cells and vascular smooth muscle cells.2.The protein expression levels of NGF TrkA and its downstream protein Akt-Gsk3 ? were different in different parts of the heart.Compared with the right atrial appendage,the protein content of NGF was the lowest in the right atrial appendage(P<0.05).The level of TrkA protein was the highest in the right atrium(P<0.05);the expression level of GSK3 ? protein in the left atrium was significantly lower than that in other atrioventricular structures(P<0.05),Akt protein level was the highest in the right ventricle,and the difference was statistically significant(P<0.05)compared with other structures(P<0.05),and its expression content was the lowest in the left atrium,and the difference was statistically significant(P<0.05)compared with other atrioventricular structures;p-s473 Akt was the highest in the right atrium(P<0.05);the protein expression of p-s9 GSK3 ? in left atrium was the lowest(P<0.05).3.Analysis of clinical data between patients with valvular atrial fibrillation and patients with valvular sinus rhythm.3.1 Analysis of clinical characteristics:there was no significant difference in gender,age,course of disease,smoking history,drinking history,systolic blood pressure and BMI between the two groups(all P>0.05).There was no significant difference in the degree of valve disease involving valve disease and NYHA classification between the two groups(P>0.05).However,the heart rate and bun,DBIL,BNP,P,OSM and Ag levels in patients with atrial fibrillation were significantly higher than those in patients with atrial fibrillation The systolic blood pressure in patients with atrial fibrillation was significantly higher than that in patients with sinus rhythm(P<0.05).Ultrasound analysis showed that the diameter of left atrium in patients with atrial fibrillation was significantly larger than that in patients with sinus rhythm(P<0.05),but there was no significant difference in left ventricular ejection fraction(LVEF)between patients with atrial fibrillation and patients with sinus rhythm(P>0.05).3.2 Analysis of medication history:the rate of taking ?-receptor blocker in AF group was higher than that in sinus rhythm group(P<0.05);there was no significant difference in other drugs,such as digoxin,diuretics,ARB and ACEI(all P>0.05).3.3 Analysis of cardiac function:there was no significant difference in NYHA classification of heart function in patients with atrial fibrillation and the same to LVEF measured by ultrasound,but the BNP value was significantly higher than that in patients with sinus rhythm(P<0.05).3.4 Analysis of comorbidities:The incidence rate of liver function impairment and renal function injury in AF group was not significantly different from those in sinus rhythm(P>0.05),and there was no significant difference in EUROSCORE score between the two groups(P>0.05).4.Immunohistochemical analysis of NGF/TrkA in right atrial appendage of patients with valvular atrial fibrillation and valvular sinus rhythm:Akt,GSK3 ?,NGF,p-Ser 473 Akt,p-S9 GSK3 ? and TrkA were expressed in cardiomyocytes and cytoplasm,but not in endocardium or epicardium.Among them,the positive rate of NGF expression in myocardial cells of patients with atrial fibrillation was significantly higher than that of patients with sinus rhythm(P<0.05).5.The protein expression of NGF,TrKA,GSK3 ?,p-S9 GSK3?,AKT and p-S473 AKT in the myocardial tissue of right atrium The protein levels of NGF in atrial samples from patients with AF were higher than those with SR(p<0.05).However,compared to the SR patients,the protein expression levels of TrKA(p<0.05),GSK3?(p<0.05),p-S9 GSK3?(p<0.05)and p-S473 AKT(p<0.05)were significantly decreased in AF patients.There was no difference in the expression of AKT protein between the two groups(p>0.05).6.The mRNA expression of NGF,TrKA,GSK3? and AKT:The mRNA expression of NGF was remarkably increased in patients with AF compared to those with SR.Meanwhile,the mRNA expression of GSK3?(p<0.05)and TrKA(p<0.05)were significantly decreased.In addition,there was no statistical difference in the mRNA level of AKT between these two groups(p>0.05).7.Analysis of the protein expressions of NGF,TrkA,GSK3 ?,p-s9 GSK3 ?,Akt and p-s473 Akt in peripheral blood lymphocytes:The protein expression trend of NGF,TrkA,GSK3 ?,p-s9 GSK3 ?,Akt and p-s473 Akt in peripheral blood lymphocytes of patients with atrial fibrillation and sinus rhythm was consistent with that in right atrial appendage.The level of NGF protein in peripheral blood lymphocytes of patients with atrial fibrillation was significantly higher than that of patients with sinus rhythm(P<0.05).There was no difference in Akt protein expression between the two groups(P=0.789,P>0.05),but the expression of other proteins in lymphocytes of patients with atrial fibrillation decreased significantly.8.Analysis of the difference of NGF-? microprotein expression in plasma:there was no significant difference between atrial fibrillation patients and sinus rhythm patients(P=0.143,P>0.05)Conclusion1.The expression levels of NGF TrkA,Akt,GSK3 ? and their phosphorylated proteins were different in different parts of the heart2.NGF-TrKA signaling pathway was involved in the occurrence of AF in the patients with VHD,inactivation of GSK3? could increase the incidence of AF,but not through phosphorylation.