Effect of suppressing calcium-sensing receptor on rat myocardial H9c2 cell hypertrophy induced by angiotensin Ⅱ
10.3760/cma.j.issn.2095-4255.2015.04.009
- VernacularTitle:抑制钙敏感受体表达对血管紧张素Ⅱ诱导大鼠心肌H9c2细胞肥大的影响
- Author:
Lei LIU
;
Chao WANG
;
Yuhui XI
;
Hong LI
;
Sa SHI
;
Ye TIAN
;
Lina WANG
- Publication Type:Journal Article
- Keywords:
Calcium sensing receptor;
Autophagy;
Angiotensin Ⅱ;
Rats;
Cardiac hypertrophy
- From:
Chinese Journal of Endemiology
2015;34(4):265-269
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore the effects and possible mechanism of calcium-sensing receptor(CaSR) in rat myocardial H9c2 cells hypertrophy model using angiotensin Ⅱ (Ang Ⅱ).Methods Cardiac hypertrophy model was established by treating cultured H9c2 cells with Ang Ⅱ in vitro.Hypertrophic H9c2 cells were treated with gadolinium chloride (GdCl3,a specific agonist of CaSR) and/or with Calhex231 (a specific inhibitor of CaSR) and 3-methyladenine (3-MA,a specific inhibitor of autophagy) to divided into 5 groups (six in each group):control,Ang Ⅱ,GdCl3 + Ang lⅡ,GdCl3 + Calhex231 + Ang Ⅱ,GdCl3 + 3-MA + Ang Ⅱ groups.To evaluate the status of H9c2 cells hypertrophy,protein content was determined through a coomassie brilliant blue protein kit and the expression of Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) and the phosphorylation form (pCaMK Ⅱ/CaMK Ⅱ) was analyzed by Western blotting.The protein expression of CaSR,autophagy maker [Beclin-1,micmtubule-associated protein 1 light chain 3(LC3)Ⅱ/LC3 Ⅰ,P62] and Ca2+/calmodulin-dependent-protein kinase-kinase-β (CaMKKβ)-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway was analyzed by Western blotting.Results ①GdCl3 further increased H9c2 cells protein content [control group:(2.52 ± 0.84) g/L,Ang Ⅱ group:(8.72 ± 3.60) g/L GdCl3 + Ang Ⅱ group:(14.17 ± 4.49) g/L,all P ＜ 0.05] and the expression of CaSR (control group:0.22 ± 0.04,Ang Ⅱ group:0.43 ± 0.02,GdCl3 + Ang Ⅱ group:0.63 ± 0.08,all P ＜ 0.05) and pCaMK Ⅱ/CaMKⅡ (control group:0.25 ± 0.05,AngⅡ group:0.51 ± 0.03,GdCl3 + AngⅡ group:0.77 ± 0.06,all P＜ 0.05) induced by Ang Ⅱ.Calhex231 suppressed the increasing of hypertrophy indicators induced by GdCl3 [GdCl3 + Calhex231 + AngⅡ group,CaSR:0.41 ± 0.16,protein content:(9.92 ± 2.54) g/L,pCaMK Ⅱ/CaMKⅡ:0.58 ± 0.08,all P ＜ 0.05].②GdCl3 promoted the effect of Ang Ⅱ in regulation of autophagy such as Beclin-1 protein increased (control group:0.31 ± 0.06,AngⅡ group:0.55 ± 0.09,GdCl3 + AngⅡ group:0.74 ± 0.08,all P ＜ 0.05),LC3 Ⅱ/LC3 Ⅰ increased (control group:0.28 ± 0.06,Ang Ⅱ group:0.56 ± 0.10,GdCl3 + Ang Ⅱ group:1.00 ± 0.15,all P ＜ 0.05) and P62 protein decreased (control group:0.54 ± 0.03,AngⅡ group:0.34 ± 0.02,GdCl3 + AngⅡ group:0.15 ± 0.03,all P ＜ 0.05).Moreover,Calhex231 suppressed autophagy induced by GdCl3 (GdCl3 + Calhex231 + Ang Ⅱ group,Beclin-1:0.53 ± 0.14,LC3 Ⅱ/LC3 Ⅰ:0.57 ± 0.12,P62:0.28 ± 0.05,all P ＜ 0.05).③GdCl3 increased pCaMKKβ/CaMKKβ (control group:0.43 ± 0.09,AngⅡ group:0.76 ± 0.12,GdCl3 + AngⅡ group:1.19 ± 0.21,all P ＜ 0.05),pAMPK/AMPK (control group:0.38 ± 0.11,AngⅡ group:0.68 ± 0.08,GdCl3 + AngⅡ group:1.18 ± 0.08,all P ＜ 0.05) and decreased pmTOR/mTOR (control group:0.90 ± 0.10,Ang Ⅱ group:0.54 ± 0.04,GdCl3 + AngⅡ group:0.29 ± 0.09,all P ＜ 0.05).Furthermore,Calhex231 blocked the effect of GdCl3 on the above-mentioned proteins changes (GdCl3 + Calhex231 + Ang Ⅱ group,pCaMKKβ/CaMKKβ:0.75 ± 0.06,pAMPK/AMPK:0.57 ± 0.05,pmTOR/mTOR:0.51 ± 0.08,all P ＜ 0.05).Conclusion Inhibiting calcium-sensing receptor expression has reversed H9c2 cell hypertrophy induced by Ang Ⅱ,which may be related to suppressing autophagy and suppressing CaMKKβ-AMPK-mTOR pathway.
