Role of ca lc ium - sen sing receptor in apoptosis of ra t card iom yocytes induced by anox ia - reoxygena tion

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1 Chinese Journal of Pathophysiology 2007, 23 (7) : [ ] (2007) ,,,,,,,,, (,, ) [ ] : (CaSR) /, : Lagendorff - ( anoxia - reoxygenation, A /R) ; / CaSR CaSR ; (LSCM ) / [ Ca 2 + ] i ; HE ; TUNEL ; W estern blotting caspase 3 caspase 9 : A /R CaSR CaSR, HE A /R, TUNEL 2, A /R caspase 3 caspase 9 : CaSR A /R, A /R [ ], ; ; ; [ ] R363 [ ] A Role of ca lc ium - sen sing receptor in apoptosis of ra t card iom yocytes induced by anox ia - reoxygena tion WU Bo, ZHANGW ei - hua, L IQuan - feng, GAO Xiu - xiang, ZHANG L i, YANG J in - xia, WANG Xiu - li, ZHANG M ing - shuang, ZHANG Zhuo - ran, XU Chang - qing (D epartm ent of Pathophysiology, Harbin M edical U niversity, Harbin , China. E - m ail: xucq@ em s. hrbm u. edu. cn) [ ABSTRACT] A IM : To observe the exp ression of calcium - sensing recep tor (CaSR) of rat cardiomyocytes in an2 oxia - reoxygenation (A /R) injury and CaSR - induced changes of intracellular calcium; involvement of CaSR in apop tosis and relevant signaling transduction pathway. M ETHOD S: The A /R injury was remodeled in vitro; CaSR, caspase 3 and caspase 9 were deteced byw estern blotting. LSCM was used to observe changes of intracellular calcium during reperfusion with or without CaSR agonist. Morphological changes in different group s were observed by light m icroscopes. cells were measured by TUNEL assay. Apop totic RESUL TS: By LSCM, it was found that the intracellular calcium was significantly increased during reperfusion both in A /R and activator group. Severe injury was found by HE staining in the above two group s, the number of apop totic cellswas significantly increased according to TUNEL assay. The exp ression of CaSR, caspase 3 and caspase 9 was significantly increased in A /R group and activator group compared with control. CO NCL US IO N: CaSR is involved in intracellular calcium overload in A /R cardiomyocyte, which can accelerate apop tosis during A /R. [ KEY WO RD S] Recep tors, calcium sensing; Calcium; Apop tosis; Anoxia, ( calcium - sensing recep ter, CaSR ),, [ 1 ] CaSR, Na + - Ca 2 + L - [ 5 ], 7 G Ca 2 + [ 2, 3 ], 1993, B rown [ 4 ], [ 6, 7 ] [ ] [ ] [ ] (No ; No ) ; Tel: ; E - mail: xucq@ em s. hrbmu. edu. cn

2 ,W ang [ 7 ] CaSR, PLC CaSR, A /R, CaSR A / R, pha D iagnostic International, USA ), anti - caspase 3 (Neomarker Corporation), anti - caspase 9 (Neomarker Corporation), W estern IP (Beyotime Corporation), Fluo - 3 /AM ( Sigma), anti - actin ( Boster) 3 (Meridian), (Beckman), (B io - Rad),M ini - PROTEAN g 3 System (B io - Rad), US (Beckman), (O lympus) 4 / 4. 1 Lagendorff [ 8 ] W istar, 2% ( 2 mg/kg) (1 000 U /kg),, 4-5 mm KH (mmol/l: NaCl 11814; KCl 417; MgSO 4 111; KH 2 PO ; NaHCO ; CaCl ; glucose 1111; ph 714),, ( 1 ) : 2 h; ( 2 ) ( an2 oxia group, A ) : 20 m in, 40 m in; (3) / ( anoxia / reperfusion, A /R) 20 m in, 40 m in, 2 h; (4) ( blocker group ) : 20 m in, 40 m in, 10 mmol/l N icl mmol/l CdCl 2 40 min, 10 mmol/l NiCl mmol/l CdCl mmol/l GdCl 3, 2 h 013 mmol/l GdCl 3 CaSR 5 ( L SCM ) 5. 1 Tyrode (mmol/l) : NaCl 143, KCl 514, MgCl 2 015, NaH 2 PO , HEPES 5, CaCl 2 118, glucose 515, ph NaOH Tyrode : Tyrode W istar, g,, CaCl 2 Tyrode : Tyrode 013 g/l : Tyrode 2 glucose, ph 618, N icl 2 CdCl 2 GdCl 3 ( Promega), anti - CaSR ( A l2 1 h KB (mmol/l) : 70, 15, KCl 30, KH 2 PO 4 10, HEPES 10, MgCl 2 015, EGTA 015, glucose 10, ph KOH Langendorff, Tyrode 3 m in,, Tyrode,, Tyrode 50 ml 30 m in,, 2-5 m in, 37 KB, KB 513 LSCM Fluo - 3 / AM :, Tyrode 1, Fluo - 3 /AM ( 10 M ), 37 30m in, Tyrode LSCM [ Ca 2 + ] i, 488 nm : Fluo - 3 / AM 3 : ( 1) / (A /R) ( n = 6) : Tyrode 8 m in, 30 m in, Ty2 rode 20 m in, 3 ; (2) ( n = 6) : Tyrode 8 m in, 30 m in, 10 mmol/l N icl mmol/l CdCl 2 3, Tyrode 20 m in; (3) ( n = 6) : Tyrode 8 m in, , 8 30 m in, 10 mmol/l N icl mmol/l CdCl mmol/l GdCl 3 Tyrode 20 m in 96 s 90 s 30 s, HE 015 cm 2, 4% 2 h 10 mmol/l N icl 2 Na + /Ca 2 +, h, 012 mmol/l CdCl 2 L ; (5) HE, ( activator group) : 20 m in,

3 TUNEL TUNEL ( 2C) ;, DNA 3π- OH,, ( 2D) ; A /R ( 2E), 20 L K m in, 3% H 2 O 2 10 m in, 50 L TUNEL, m in 50 L POD, m in DAB 20 m in, PBS, ( TUNEL ), ( TUNEL ) 5, 500, : A I(% ) = ( /500) 100% 7 CaSR ca spa se 9 ca spa se 3 [ 9 ],, PMSF, 4 40 m in, r/m in 40 m in, BSA, B radford 20 g, 10% SDS - PAGE, 100 V 1 h, 37 1 h; anti - CaSR ( ) anti - caspase 3 ( ) anti - caspase 9 ( ) - actin ( 1 500), 4, 1, TBS - T,, (AP) IgG ( ), 1h,, W estern blotting (A ) 8 gx s,,, t - test, : (22% 5% ), A /R (48% 1 L SCM ( 1) A /R, ( P < 0101), ( P < 0105) ( P < 0105) 2 ( 2),, ( 2A ) ;, ( 2B ) ; A /R, Fig 1 Changes of intracellular Ca 2 + in cardiomyocytes. A: a, b and c rep resented p re - perfusion, anoxia and A /R; B: The changes of fluorescent intensities in Ca 2 + treatments. intracellular were recorded continuously with LSCM in different Intracellular Ca 2 + was recorded for 53 m in in all, including 30 m in of ischem ia, 8 m in of p re - perfu2 sion, 15 m in of reperfusion. 1 3 ( 3),, 6% ), ( 44% 8% ), ( 24% 7% ) ( P < 0105), A /R ( P < 0105), A /R 4 CaSR ca spa se 9 ca spa se CaSR ( 4) W estern blotting CaSR, CaSR 130 kd 150 kd,, CaSR ; A /R CaSR ( P < 0105), A /R ( P < 0101) 4. 2 Caspase 9 caspase 3 A /R caspase 9 ( P < 0105 ) ( 5) caspase 3 17kD A /R ( P < 0105) ( 6)

4 1252 Fig 2 Morphological changes of cardiac tissue in different group s ( 400). A: control; B: anoxia; C: A /R; D: N icl 2 + CdCl 2 + A /R; E: GdCl 3 +N icl 2 + CdCl 2 +A /R. 2 Fig 3 Rep resentative illustration of TUNEL staining in cardiomyocytes( 400). Nuclei with brown staining indicated TUNEL positive cells ( upper panel). A: control; B: anoxia; C: A /R; D: N icl 2 + CdCl 2 +A /R; E: GdCl 3 +N icl 2 + CdCl 2 +A /R. control. 3 TUNEL # P < vs A /R CaSR CaSR 3, kd kd kd ; kd, Fig 4 W estern blotting analysis for CaSR in rat cardiac tissue of different group s. A: control; B: anoxia; C: A /R; D: N icl 2 + CdCl 2 + A /R; E: GdCl 3 + N icl 2 + CdCl 2 + A /R. # P < 0101 vs control. gx s. n = 6. 4 CaSR W estern blotting W estern blotting A /R kd kd CaSR, GdCl 3 Fig 5 Exp ression of activated caspase 9 determ ined byw estern blotting analysis in cardiac tissue. A: control; B: A /R; C: N icl 2 + CdCl 2 + A /R; D: GdCl 3 + N icl 2 + CdCl 2 + A /R. # P < 0105 vs control. gx s. n = 6. 5 ca spa se 9 W estern blotting

5 1253 Fig 6 Exp ression of activated caspase 3 determ ined byw estern blotting analysis in cardiac tissue. A: control; B: A /R; C: N icl 2 + CdCl 2 + A /R; D: GdCl 3 + N icl 2 + CdCl 2 + A /R. # P < vs control. gx s. n = 6. 6 ca spa se 3 W estern blotting, [ 10, 11 ] CaSR G - PLC - IP 3, A /R, CaSR LSCM A /R, ;,, CaSR, A /R, A /R CaSR CaSR A /R, HE TUNEL A /R HE A /R, TUNEL 2, Ca 2 + DNA, Ca 2 + A /R CaSR, caspase 3 caspase 9 caspase 9 A /R caspase 3 17 kd 12 kd 2, 17 kd, A /R,,, CaSR A /R, cyt C, cyt C caspase 9, caspase 3, [ 12, 13 ] CaSR A /R,, cyt C, [ ] [ 1 ] Martin SJ, Green DR. Apop tosis and cancer: the failure of controls on cell death and cell survival[ J ]. Crit Rev Oncol Hematol, 1995, 18 ( 2 ) : , 103 (15) : [ 2 ] Tao J, Xu H, Yang C, et al. Effect of urocortin on L - type calcium currents in adult rat ventricular myocytes [ J ]. Pharmacol Res, 2004, 50 ( 5) : [ 3 ] Sun HY, W ang NP, Halkos ME, et al. Involvement of Na + /H + exchanger in hypoxia / re - oxygenation - in2 duced neonatal rat cardiomyocyte apop tosis [ J ]. Pharmacol, 2004, 486 ( 2) : Eur J [ 4 ] B rown EM, Gamba G, R iccardi D, et al. Cloning and characterization of an extracellular Ca sensing recep2 tor from bovine parathyroid [ J ]. Nature, 1993, 366 (6455) : [ 5 ] B rown EM, MacLeod RJ. Extracellular calcium sensing and extracellular calcium singaling [ J ]. Physiol Rev, 2001, 81 (1) : [ 6 ] Squires PE, Harris TE, Persaud SJ, et al. The extracel2 lular calcium - sensing recep tor on human beta - cell neg2 atively modulates insulin secretion [ J ]. D iabetes, 2000, 49 (3) : [ 7 ] W ang R, Xu CQ, Zhao WM, et al. Calcium and poly2 am ine regulated calcium - sensing recep tor in cardiac tis2 sues [ J ]. Eur J B iochem, 2003, 270 ( 12 ) : [ 8 ],,,. / [ J ]., 2002, 18 (10) : [ 9 ] Quaini F, Cigola E, Lagrasta C, et al. End - stage cardi2 ac failure in humans is coup led with the induction of p ro2 liferating cell nuclear antigen and nuclear m itotic division in ventricular myocytes [ J ]. Circ Res, 1994, 75 ( 6 ) : [ 10 ] de Jonge N, vanw ichen DF, van Kuik J, et al. Cardiomyo2 cyte death in patients with end - stage heart failure before and after supportwith a left ventricular assist device: low in2 cidence of apop tosis despite ubiquitous mediators [ J ]. Heart Lung Transp lant, 2003, 22 (9) : [ 11 ] Bergmann MW, Loser P, D ietz R, et al. Effect of NF - kappa B inhibition on TNF - alpha - induced apop tosis and downstream pathways in cardiomyocytes [ J ]. Cell Cardiol, 2001, 33 ( 6) : J J Mol [ 12 ] de Moissac D, Gurevich RM, Zheng H, et al. Caspase activation and m itochondrial cytochrome C release during hypoxia - mediated apop tosis of adult ventricular myocytes [ J ]. J Mol Cell Cardiol, 2000, 32 (1) : [ 13 ] Piper H. M icheal K, SchasferMC, et al. Cellular mech2 anism s of ischem ia - reperfusion injury[ J ]. Surg, 2003, 75 (2) : S644 - S648. Ann Thorac