Effect of suppressing calcium-sensing receptor on rat myocardial H9c2 cell hypertrophy induced by angiotensin Ⅱ / 中华地方病学杂志

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 group0.22 ± 0.04,Ang Ⅱ group0.43 ± 0.02,GdCl3 + Ang Ⅱ group0.63 ± 0.08,all P ＜ 0.05) and pCaMK Ⅱ/CaMKⅡ (control group0.25 ± 0.05,AngⅡ group0.51 ± 0.03,GdCl3 + AngⅡ group0.77 ± 0.06,all P＜ 0.05) induced by Ang Ⅱ.Calhex231 suppressed the increasing of hypertrophy indicators induced by GdCl3 [GdCl3 + Calhex231 + AngⅡ group,CaSR0.41 ± 0.16,protein content(9.92 ± 2.54) g/L,pCaMK Ⅱ/CaMKⅡ0.58 ± 0.08,all P ＜ 0.05].②GdCl3 promoted the effect of Ang Ⅱ in regulation of autophagy such as Beclin-1 protein increased (control group0.31 ± 0.06,AngⅡ group0.55 ± 0.09,GdCl3 + AngⅡ group0.74 ± 0.08,all P ＜ 0.05),LC3 Ⅱ/LC3 Ⅰ increased (control group0.28 ± 0.06,Ang Ⅱ group0.56 ± 0.10,GdCl3 + Ang Ⅱ group1.00 ± 0.15,all P ＜ 0.05) and P62 protein decreased (control group0.54 ± 0.03,AngⅡ group0.34 ± 0.02,GdCl3 + AngⅡ group0.15 ± 0.03,all P ＜ 0.05).Moreover,Calhex231 suppressed autophagy induced by GdCl3 (GdCl3 + Calhex231 + Ang Ⅱ group,Beclin-10.53 ± 0.14,LC3 Ⅱ/LC3 Ⅰ0.57 ± 0.12,P620.28 ± 0.05,all P ＜ 0.05).③GdCl3 increased pCaMKKβ/CaMKKβ (control group0.43 ± 0.09,AngⅡ group0.76 ± 0.12,GdCl3 + AngⅡ group1.19 ± 0.21,all P ＜ 0.05),pAMPK/AMPK (control group0.38 ± 0.11,AngⅡ group0.68 ± 0.08,GdCl3 + AngⅡ group1.18 ± 0.08,all P ＜ 0.05) and decreased pmTOR/mTOR (control group0.90 ± 0.10,Ang Ⅱ group0.54 ± 0.04,GdCl3 + AngⅡ group0.29 ± 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/CaMKKβ0.75 ± 0.06,pAMPK/AMPK0.57 ± 0.05,pmTOR/mTOR0.51 ± 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.