RXRα Regulates AMI Rat Cardiac Remodeling And Myofibroblast Proliferation In Hypoxia By TGF-β/Smads Signaling Pathway

Object: To observe the effect of the RXRa agonist Bexarotene on the change of the heart structure and function, and the expression of the TGF-β1/Smad2 level and collagen synthesis in AMI rats. To investigate the phosphorylation of Smad2 induced by TGF-β1 in hypoxia, as well as the Type I and III collagen synthesis in myofibroblast. Furthermore, we discuss the possible mechanism of RXRa in myocardiac remodeling after infarction, myofibroblast proliferation and collagen synthesis induced by TGF-β1 in hypoxia.Methods: Animal experiments: Acute myocardial infarction(AMI) model was established by ligating left anterior descending coronary artery. Rats were randomly divived into 4 groups(8 rats per group). Group A was for control with normal, group B was for sham-operated rats. Group C was for AMI and rats were treated with oral gavage once daily for 8 weeks. Group D was for AMI-Bex and rats were treated with the synthetic agonist Bexarotene obtained from Eisai.Inc at the doses of 10 mg/kg body weight, or mpk. Echocardiography was performed to measure left ventricular end diastolic or systolic inner diameter(LVIDd, LVIDs), the end-systolic left ventricular anterior wall and posterior wall(LVAWs, LVPWs), and ejection fractions(EF). Cardiac hemodynamics were evaluated with left cardiac catherization. Body weight(BW), heart weight(HW) and left ventricle weight(LVW) were monitored. Hematoxylin-eosin(HE) and Sirus red staining were used to determine myocardial structure and collagen desiposition. Enzyme Linked Immunosorbent Assay(ELISA) was used to assay the levels of TGF-β1 in serum and tissue homogenate, RXRα and P-Smad2 level were measured by Western-blotting. Cell experiments: Myofibroblast was cultured by tissue dry method and identified by immunocytochemitry and immunoflurescence. An hypoxia environment for myofibroblast was created with continual suppling with nitrogen to evaluate the effect of the RXRa agonist 9-cis-retinoid acid(9-cis-RA) and TGF-β1 on the myofibroblast proliferation and collagen synthesis. Brd U was performed to determine cell proliferation. Cell metabolic activity was assayed by MTT. ELISA was performed to assay the level of collagen. Reverse transcription and real-time PCR were performed to determine the expression of RXRa m RNA. The protein expression and the level of phosphorylation were estimated by Western-blotting. Regulation of RXRα expression was demonstrated using transfection of small interfering RNA(Si RNA) and plasmid DNA.Resultes: Animal experiments: Compared with sham-operation groups: 1. Less cardiomyocytes and more fibrous tissue deposited in AMI rats left ventricular anterior wall, and Bexarotene significantly inhibited it. 2. LVAWs, EF were significantly decreased but LVIDd, LVIDs and LVPWs were increased in AMI group. Compare with the AMI groups, the above indexes were obviously improved in Bex group. 3. LVESP, LV±dp/dtmax were significantly decreased and LVEDP was significantly increased in AMI group, and there were significantly improved in Bex group. 4. The level of TGF-β1 and P-Smad2 in AMI heart tissue was significantly increased and remarkably reduced by Bex. 5. The expression of RXRa in heart infarction area was greatly decreased while Bex significantly increased the level of RXRα expression. Cell experiments: 1. TGF-β1 induced myofibroblasts proliferation in does-dependence, increased myofibroblasts metabolic activities and promoted Type I and III collagen systhesis. 2. RXRα agonist 9-cis-RA could inhibit myofibroblast proliferation and collagen synthesis induced by TGF-β1 in hypoxia. 3. 9-cis-RA could elevate the level of P-Smad2 in fibroblast induced by TGF-β in hypoxia, but inhibit P-Smad2 nucleus translocation. 4. The transfection of RXRα’s Si RNA greatly decreased the level of RXRa and inhibited the role of 9-cis-RA in anti-proliferation and collagen synthesis in hypoxia. Meanwhile, it also reduced the role of 9-cis-RA during TGF-β’s induction in the Smad2 phosphorylation and nucleus translocation. While the up-regulation of RXRα expression in the transfected DNA plasmid helped 9-cis-RA to inhibit the myofibroblast proliferation and collagen synthesis, and also enhanced its effect on the Smad2 phosphorylation and nucleus translocation.Conclusions: 1. RXRa agonist Bexarotene inhibits myocardial collagen deposition and compensatory hypertrophy, reverses left ventricular expansion and improve cardiac function. 2. RXRa agonist Bexarotene decreases the level of TGF-β1 and P-Smad2 in heart infarction area. 3. RXRa agonist 9-cis-RA inhibits TGF-β induced myofibroblast proliferation and collagen synthesis, and blunts the P-Smad2 nuclear localization. 4. RXRa inhibits myocardial infarction remodeling and myofibroblast proliferation and collagen synthesis, possibly through the regulation of TGF-β/Smad2 signaling pathway.