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[Cardioprotective mechanism of calreticulin up-regulation induced by hypoxic postconditioning.] [Ariticle in Chinese]

XU Fei-Fei, LIU Xiu-Hua^*, ZHANG Zhen-Ying, CAI Li-Rong

Department of Pathophysiology, Chinese PLA General Hospital, Beijing, 100853, China

Abstract

Calreticulin (CRT) is an essential Ca2+-binding chaperone existing in endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR), and is involved in intracellular Ca2+ homeostasis and protein folding. Ischemic postconditioning (I-postC), a newly discovered endogenous protective phenomenon, induces CRT up-regulation. The present study aimed to investigate the cardioprotective mechanism of CRT up-regulation induced by hypoxic postconditioning (H-postC). Primary cultured neonatal rat cardiomyocytes were exposed to 2 h of hypoxia followed by 24 h of reoxygenation. Postconditioning was carried out by two cycles of 10 min of reoxygenation and 20 min of rehypoxia after 2 h of hypoxia. Antisense oligodeoxynucleotides (AS-ODNs) were used to inhibit CRT expression 36 h before hypoxia. Cardiomyocytes were randomly divided into 6 groups as follows (n=4): control, hypoxia/reoxygenation (H/R), HpostC, AS, AS + H/R, and AS + H-postC. Morphological studies, lactate dehydrogenase (LDH) activity assay in culture medium, and flow cytometry were used to detect cardiomyocyte necrosis and apoptosis. Intracellular Ca2+ concentration was detected by fluorescent Fluo-3/AM staining through laser confocal microscope, and p-nitrophenyl phosphate (PNPP) was used as substrate to measure calcineurin (CaN) activity. The expression of CRT, CaN, nuclear factor kappa B (NFκB) and apoptosis-related proteins, such as Bcl-2, Bax and C/EBP homologous protein (CHOP) were detected by Western blot. The results were as follows. (1) H-postC protected neonatal cardiomyocytes from H/R injury. Compared with H/R group, cell survival rate increased by 17.1%, apoptotic rate and LDH leakage decreased by 6.67% and 27.9% in H-postC group, respectively (P<0.05). (2) H-postC induced mild up-regulation of CRT expression. Inhibition of CRT by AS-ODNs attenuated the cardioprotection of H-postC partly. Compared with H-postC group, cell survival rate decreased by 8.98%, and apoptotic rate and LDH leakage increased by 1.74% and 13.6% in AS + H-postC group, respectively (P<0.05), but intracellular Ca2+ concentration, CaN activity, and expression of CaN and NFκB did not change significantly (P>0.05), suggesting that CRT participates in endogenous protection, not through Ca2+-CaN pathway. (3) H-postC inhibited the expression of pro-apoptosis proteins such as Bax and CHOP, but induced up-regulation of anti-apoptosis protein Bcl-2. Inhibition of CRT by AS-ODNs partly inhibited the changes in apoptosis-related proteins expression induced by H-postC, suggesting that CRT participates in the anti-apoptosis effect of H-postC through regulating expression of apoptosis-related proteins. These results indicate that CRT up-regulation induced by H-postC is involved in the cardioprotection through regulating expression of apoptosis-related proteins, not through Ca2+-CaN pathway in neonatal cardiomyocytes.

Key words: calreticulin; calcineurin; calcium; iron overload; ischemic postconditioning; hypoxia

Received: 2008-07-18　　Accepted: 2008-11-20

Corresponding author: 刘秀华　　E-mail: xiuhualiu98@yahoo.com.cn

Citing This Article：

XU Fei-Fei, LIU Xiu-Hua, ZHANG Zhen-Ying, CAI Li-Rong. [Cardioprotective mechanism of calreticulin up-regulation induced by hypoxic postconditioning.] [Ariticle in Chinese] . Acta Physiol Sin 2009; 61 (1): 35-42 (in Chinese with English abstract).