Calreticulin (CRT), an important Ca(2+)-binding
molecular chaperone in the endoplasmic reticulum (ER), and
caspase-12, a pivotal molecule mediating ER-initiated apoptosis, are involved in the ER stress (ERS). Using primary cultured neonatal cardiomyocytes, CRT and
caspase-12 expression and activation during hypoxic preconditioning (HPC) and
hypoxia/reoxygenation (H/R) were studied to explore the role of ERS in cardioprotection by HPC. And by using
SB203580 and
SP600125 [the specific inhibitors of
p38 mitogen-activated protein kinase (MAPK) and
c-Jun N-terminal kinase (JNK)] separately, the role of
p38 MAPK in HPC-induced ERS was also detected. Neonatal cardiomyocytes were prepared from Sprague-Dawley rats aged 24 h, and cultured in DMEM medium containing 10%
fetal bovine serum, and then randomly divided into six groups as follows: H/R, HPC+H/R,
SB203580+HPC+H/R,
SP600125+HPC+H/R, HPC and control groups. H/R was produced by 2-hour
hypoxia/14-hour reoxygenation, and HPC by 20-minute
hypoxia/24-hour reoxygenation. Morphological studies, estimation of
lactate dehydrogenase (LDH) leakage and flow cytometry were employed to assess cell apoptosis and
necrosis. CRT and
caspase-12 expression and activation, levels of phospho-p38 MAPK and phospho-JNK were detected by Western blot. All experiments were repeated at least four separate times. The results obtained are as follows: (1) HPC relieved the cell injury caused by H/R. Compared with that in H/R group, cellso survival rate in HPC+H/R group increased by 6.4%, and the apoptosis rate and LDH leakage in the cell culture medium decreased by 6.6% and 70.0%, respectively. (2) H/R induced
caspase-12 activation (33.2-fold increase in comparison with control) and CRT expression (8.1-fold increase in comparison with control). HPC itself resulted in mild CRT up-regulation (2.6-fold increase in comparison with control), but the extent of up-regulation was lower than that induced by H/R. HPC before H/R was found to relieve the over-expression of CRT induced by H/R (72.4% decrease), and to inhibit the activation of
caspase-12 (59.6% decrease). (3) The protection of HPC and HPC-induced up-expression of CRT and inhibition of
caspase-12 activation were almost eliminated when the inhibitor of
p38 MAPK, not of JNK, was present before HPC. These results suggest that HPC protects the neonatal cardiomyocytes from severe ERS-induced apoptosis during sustained H/R through pre-invoking proper ERS response. Mild up-expression of CRT and inhibition of
caspase-12 activation induced by HPC, which are important protection factors, are mediated by
p38 MAPK, not by JNK.