Doxycycline Attenuates Chronic Intermittent Hypoxia-induced Atrial Fibrosis In Rats

Objective: Atrial structural remodeling in the form of fibrosis contributes to the arrhythmic substrate in atrial fibrillation(AF).Doxycycline,a widely used tetracycline antibiotic,can attenuate Matrix Metalloproteinases(MMPs)activity and It has recently been shown to exert protective effects against ventricular remodeling in an animal model of heart failure.The aim of this study was to investigate the effects of doxycycline on chronic intermittent hypoxia(CIH)-induced atrial fibrosis and the pathophysiological mechanisms underlying such changes.Methods: A total of 30 Sprague-Dawley rats were randomized into three groups: Control group(Con),CIH group,CIH with doxycycline treatment(CIH-D)group.CIH lasted 5 hours per day for 4 weeks.CIH-D rats were administrated doxycycline for 4 weeks while they received CIH(30mg/Kg/d).Transthoracic echocardiography was performed after the 4 weeks CIH.The following parameters were determined: left atrial diameter,left ventricular end-diastolic dimension,interventricular septal thickness,left ventricular end-systolic dimension,right ventricular diameter,mean pulmonary artery pressure and left ventricular ejection fraction.After anaesthetization,median sternotomy was immediately performed.Blood samples were collected to measure the level of hs-CRP and IL-6.And hearts were quickly removed and weighted,and atrial tissue samples was obtained from atrium.HE staining was used to observe the morphology of atrial tissue,while Masson's trichrome staining was used to determine collagen deposit in the atrial myocardium.Immunehistochemical staining was used to analysis the semi quantitative of the protein levels of extracellular signal-regulated kinase 1/2(ERK1/2)and phosphorylated-ERK1/2(p-ERK1/2).Protein and mRNA levels of Matrix Metallo proteinase-2(MMP-2)and Metallo--proteinase-9(MMP-9),microRNA-21(miRNA-21)and its downstream target Sprouty 1(Spry 1),and ERK1/2 were measured using Western blotting or real-time qRT-PCR,respectively.Results: No significant difference in body weight or heart weight was observed between the control,CIH and CIH-D groups.And our experiments showed normal dimensions of left atrial diameter,interventricular septal thickness,left ventricular end diastolic diameter,left ventricular end systolic diameter and right ventricular diameter.Left ventricular ejection fraction was preserved at 72.51�4.32%.None of the above parameters showed differences when compared to the CIH group or CIH-D group(all P > 0.05).By contrast,mean pulmonary artery pressure was higher in CIH group compared to the controls,and that of the CIH-D group fell between these values.CIH induced a significant 63% higher atrial interstitial collagen deposition as compared to the Control group.The collagen deposition was diffuse and homogenous,with no patchy fibrotic infiltrates in any of the analyzed samples.Immunohistochemistry staining showed that all rats in the three groups had positive expression of ERK1/2 and p-ERK1/2 in the atrial tissue.The result of the semi quantitative analysis indicated that the CIH group had elevated level of ERK1/2,and p-ERK1/2,and an increased ratio of p-ERK/ERK,when compared with the Con group,which was attenuated by doxycycline.The mRNA levels of miRNA-21 was upregulated in the CIH group compared to the control group and this upregulation was significantly attenuated by doxycycline when compared to control and CIH groups.Spry 1,a downstream target of miR-21 was downregulated in the CIH group compared to the control group,which was attenuated by doxycycline.MMP-2 and MMP-9 mRNA expression in atria were analyzed,demonstrating opposing changes.The expression of MMP-2 mRNA was reduced whereas that of MMP-9 was increased,with both changes being reversed by doxycycline.Subsequent Western blot experiments confirmed the above mRNA studies.Thus,Spry 1 and MMP-2 protein levels were significantly lower,whereas MMP-9 protein levels were significantly higher,in the CIH group when compared to the control group.Moreover,further experiments demonstrated protein levels of both ERK1/2 and phosphorylated ERK1/2 were significantly increased in the CIH group when compared to control.All of these changes were attenuated by doxycycline.Conclusions: 1.CIH treatment induced significant atrial fibrosis in our rat model.2.doxycycline attenuated atrial fibrosis induced by CIH.3.These changes can be explained by alterations in the MMPs and miR-21/ERK signaling pathways.