Study Of Effects Of Doxycycline On Chronic Intermittent Hypoxia-induced Atrial Remodeling And Related Mechanisms Underlying Such Changes In Rats

Objective:The aim of this study was to investigate the effects of doxycycline on atrial remodeling induced by chronic intermittent hypoxia(CIH)and the related mechanisms of these changes in a rat model of atrial fibrillation(AF).Methods:A total of 150 Sprague–Dawley rats were randomized into 3 groups:normal control group(Control),rats with chronic intermittent hypoxia group(CIH),CIH with doxycycline treatment group(CIH-D)(n=50).CIH rats were subjected to CIH 8 h/d for 30 days,CIH-D rats were administrated with doxycycline while they received CIH.As the weighing,echocardiography and hemodynamic examination were fulfilled,in each group,10 rats were randomly sampled for histological experiment,5 rats were randomly selected for isolated heart electrophysiology,5 rats were randomly sampled for RT-q PCR experiment,5 rats were randomly sampled for Western Blotting,20 rats were selected for whole-cell patch clamp experiment,and the remaining 5 rats were selected for confocal microscope experiment.H-E staining was used to observe the general structural of atrial tissue,Masson's trichrome staining was used to evaluate the extent of atrial fibrosis.And atrial epicardial conduction velocity,conduction inhomogeneity,interatrial conduction time(IACT),high right atrial effective refractory period(HRAERP),high left atrial effective refractory period(HLAERP)and AF inducibility were measured by isolated heart electrophysiological experiment.Immunohistochemical staining(IHC)and Western Blotting was used to analyze the protein expression levels of PTEN,PI3K,AKT,p-AKT,TGF-?1,CTGF,Na_v1.5,Ca_v1.2,K_v4.2,K_v4.3,NCX1,Ry R2 in each group.RT-q PCR was used to compare the m RNA expression levels of atrial remodeling related micro RNAs(mi R-1,mi R-21,mi R-29b,mi R-30,mi R-133a,and mi R-328)and their downstream factors in each group.AP,APD,current density of I_Ca,L,I_Na,I_to and ion channels kinetic parameters in rats'atrial myocytes were measured by whole-cell patch clamp experiment.Confocal microscopy experiment was used to analyze intracellular Ca²⁺transients in rats'atrial myocytes.Results:Compared to the Control rats,the CIH rats showed higher heart weight,mean pulmonary artery pressure,mean arterial blood pressure,atrial interstitial collagen deposition,and AF inducibility,longer APD,increased conduction inhomogeneity,increased expression levels of mi R-1,mi R-21,mi R-133a,mi R-328,TGF-?1,CTGF,Ry R2,and decreased atrial epicardial conduction velocity,expression levels of PTEN,PI3K,AKT,p-AKT,Nav1.5,Cav1.2,Kv4.2,Kv4.3,and the current density of INa,ICa,L,I_to were significantly decreased.There were no significant changes in the expression level of NCX1 and ion channel kinetic parameters between the two groups.Whereas treatment with doxycycline attenuated CIH-induced atrial fibrosis,AF inducibility,reduced the expression levels of mi R-21,mi R-133a,TGF-?1,CTGF,and increased the expression levels of PI3K,AKT,p-AKT,Nav1.5,Cav1.2,Kv4.2,Kv4.3,the current density of INa and Ito were also increased.Conclusions:1)CIH induced significant atrial remodeling in our rat model,which is an important platform for the study of AF.2)Increased atrial fibrosis,conduction heterogeneity,expression levels of mi R-1,mi R-21,mi R-133a,mi R-328,TGF-?1,CTGF,Ry R2,and decreased atrial epicardial conduction velocity,expression levels of PI3K,AKT,p-AKT,Nav1.5,Cav1.2,Kv4.2,Kv4.3,current density of INa,ICa,L,Ito,and prolonged APD may be important mechanisms of CIH promoting AF.3)CIH-induced atrial remodeling can be significantly attenuated by doxycycline,those changes may be explained by alterations in the current density of INa and Ito,expression levels of Nav1.5,Kv4.2,Kv4.3,Ry R2,and the expression of mi R-21/PTEN/PI3K/AKT and mi R-133a/TGF-?1/CTGF signaling pathways.