HYPOXIC PRECONDITIONING INDUCES DELAYED CARDIOPROTECTION THROUGH P38 MAPK-MEDIATED CALRETICULIN UPREGULATION

Abstract

The protective mechanisms of hypoxic preconditioning (HPC) involve the mitigation of cellular calcium overload in cardiomyocytes. The sarcoplasmic reticulum (SR) chaperone calreticulin (CRT) plays an important role in regulating calcium homeostasis and is upregulated by HPC. The goal of this study was to show whether the late cardioprotection of HPC is mediated by calreticulin upregulation and to demonstrate whether the calreticulin induction is mediated by p38 MAPK phosphorylation. Hypoxic preconditioning was induced by hypoxemic hypoxic exposure by a 24-h period of normoxic reoxygenation before undergoing LAD occlusion in rats or hypoxia/reoxygenation (H/R) in cardiomyocytes. Ca²⁺ uptake and release of the SR vesicles was determined by use of ⁴⁵Ca²⁺ and the Millipore filtration technique. Western blotting analysis was used to detect calreticulin expression and activity of p38 MAPK. Hypoxic preconditioning induced calreticulin upregulation and attenuated H/R injury in neonatal cardiomyocytes and myocardial ischemia injury by increasing calcium uptake and reducing calcium release in SR. Hearts from the HPC group were more resistant to sustained ischemia and had much stronger phosphorylation of p38 MAPK than sham operation. Inhibition of p38 MAPK with SB202190 (a selective p38 MAPK inhibitor) abolished the calreticulin upregulation and cardioprotection by HPC. Hypoxic preconditioning upregulates calreticulin expression through a p38 MAPK signaling pathway and protects cardiomyocytes from H/R (and ischemia) injury.

INTRODUCTION

The phenomenon whereby brief ischemic episodes can attenuate tissue injury caused by subsequent sustained ischemia/reperfusion (I/R) is defined as ischemic preconditioning (IPC) (¹). Accumulating evidence shows that IPC confers 2 distinct phases of cardioprotection against I/R: an early protective phase which appears rapidly after IPC and lasts approximately 1 to 3 h and delayed protection which develops 12 to 24 h later and seems to last at least 3 days (²). The delayed protection of IPC can protect the heart against myocardial infarction, stunning, and ventricular arrhythmia in animal species (³). The protective mechanisms underlying the delayed protection of hypoxic preconditioning (HPC) is complicated and at least involves the mitigation of calcium overload in cardiomyocytes.

The sarcoplasmic reticulum (SR), a subcellular compartment in myocytes, plays a pivotal role in the control of vital calcium-related cell functions, including calcium storage and signaling. The calcium activity in the SR is therefore high, several orders of magnitude above that of the cytoplasm. Depending on the extent and duration of the disturbance, an isolated impaired SR function is sufficient to induce cell injury (³). Recent investigations indicate that the preinduced SR chaperone GRP78 protects cardiomyocytes from lethal injury, at least in part, by preventing an increase in cytosolic Ca²⁺ (⁴). Our recent investigation indicated that HPC can upregulate the SR chaperone calreticulin (CRT) and protect the myocardium from ischemia injury.

However, the cellular mechanisms underlying the regulation of CRT-induced cardioprotection of IPC, particularly the relation between CRT upregulation and calcium homeostasis in SR, remains to be defined. Oyadomaris et al. (⁵) found that overexpression of CRT increased the Ca²⁺ content of the endoplasmic reticulum (ER) and afforded protection to mouse insulinoma MIN6 cells against NO-mediated apoptosis. In Xenopus laevis oocytes, overexpression of CRT suppressed inositol 1,4,5-trisphosphate-induced Ca²⁺ oscillations in a manner consistent with inhibition of Ca²⁺ uptake into the ER. Aunaudeau et al. (⁶) found that induction of CRT increased free Ca²⁺ content within the ER lumen and doubled the rate of ER refilling. We investigated the relation between CRT expression and calcium uptake and release in SR during HPC.

The signals that induce CRT expression are not fully characterized. Preconditioning-induced cardioprotection has been attributed, in part, to the change in activation of intracellular signaling molecules, including mitogen-activated protein kinases (MAPKs), which include extracellular signal-regulated protein kinase, c-Jun N-terminal kinase, and p38 MAPK. Mitogen-activated protein kinases are important regulatory proteins that transduce various extracellular signals into intracellular events (⁷). p38 MAPK and MAPKAP kinase 2 are strongly activated by ischemia in the perfused rat heart (⁸). Direct activation of p38 MAPK by anisomycin mimicked IPC in hearts (⁹) and myocytes (¹⁰) against hypoxic insults. Dana et al. (¹¹) showed that late preconditioning induced by transient activation of adenosine A₁ receptor with 2-chloro-N⁶-cyclopentyladenosine was accompanied by a significant increase in p38 MAPK activity. Zhao et al. (¹²) further demonstrated that 2-chloro-N⁶-cyclopentyladenosine-induced delayed protection was abolished by the p38 MAPK inhibitor SB203580 in the mouse heart, which suggests an essential role of p38 MAPK in protection. Hung et al. (¹³) showed that GRP78 and CRT attenuates H₂O₂-induced cell injury in LLC-PK₁ renal epithelial cells by potentiating extracellular signal-regulated protein kinase activation and decreasing c-Jun N-terminal kinase activation. However, the role of p38 MAPK in CRT-mediated cardioprotection of HPC has not been investigated thoroughly.

The goals of the present study were as follows: (1) to show whether the late cardioprotection of HPC is mediated by CRT upregulation, which regulates the SR calcium homeostasis in rat hearts after ischemia and cardiomyocytes after hypoxia/reoxygenation (H/R); and (2) to demonstrate whether the CRT induction during late preconditioning is mediated by p38 MAPK phosphorylation and activation of transcription factor nuclear factor kappa beta (NF-κB). Our results suggest that phosphorylation of p38 MAPK with HPC leads to the delayed cardioprotective effect, which is mediated by NF-κB upregulation and synthesis of CRT.

MATERIALS AND METHODS

Animals and chemicals

Male Sprague Dawley rats weighing 220 to 260 g were used for in vivo studies. Rats were housed 4 per cage at 23°C, with cycles of 12 h light/12 h dark, fed Purina rat ration, and allowed free access to water. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996).

β-Glycerophosphate, phenylmethylsulfonyl fluoride, sodium orthovanadate, acrylamide, sodium dodecyl sulfate (SDS), N,N′-methylenebisacrylamide, ammonium persulfate, N,N,N′,N′-tetramethylethylenediamine, EDTA, leupeptin and dithiothreitol, β-glycerophosphate, and SB202190 were purchased from Sigma Chemical Co (St Louis, Mo). Rabbit antimouse calreticulin monoantibody was from Stressgen (San Diego, Calif). Phospho-specific antibody against p38 MAPK(p-p38 MAPK) and NF-κB, other monoclonal antibodies, and enhanced chemiluminescence immunodetection kits were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif). Other chemicals were purchased from Sigma unless otherwise noted.

Myocardial infarction model

After a 5-day acclimatization, open-chest surgery was performed on Sprague Dawley rats under anesthesia with pentobarbital sodium (30 mg/kg) and artificial ventilation (¹⁴). The thorax was opened at the fourth or fifth left intercostal space. The left coronary artery was ligated 2-3 mm from its origin with a 6-0 silk suture attached to an atraumatic needle (model K801H, Ethicon). Sham rats underwent the same surgical procedure without ligation. The chest wall was then closed, and the animal was allowed to recover. This protocol resulted in the creation of 3 groups (n = 10): sham-operated rats, myocardial infarct rats (MI group), and hypoxic preconditioned rats followed by myocardial infarct (HPC + MI group). Mean arterial pressure (MAP), heart rate (HR), and ±dp/dt were measured for 3 consecutive minutes in anesthetized rats 24 h after the operation. For measurement of these parameters, the right carotid artery was cannulated and connected to a pressure transducer in-line to a Grass polygraph.

After hemodynamic measurements, the heart was removed and perfused with a Langendorff apparatus for 10 min to wash out the blood, and 2 mL of 2% Even blue was infused into the aortic root. Infarct size was measured by the triphenyltetrazolium chloride staining method (¹⁵). The infarcted and risk zone regions were quantified with use of Imageproplus software (Version 4. 1, Media Cybernetics, Silver Spring, Md). Infarct size is expressed as proportion infarcted in the area at risk.

Preparation of SR vesicles

The SR vesicles were prepared as described by Osada et al. (¹⁶). The ventricular tissue was weighed, minced, and transferred to a tube containing a solution [(in mmol/L) NaHCO₃, 10; NaN₃, 5; and Tris HCl, 15; pH 6.8]. The tissue was homogenized and centrifuged at 9500 rpm for 20 min to remove cellular debris, and the supernatant was transferred to a new tube and centrifuged at 19,000 rpm for 45 min. This pellet was suspended in 8 mL of 600 mmol/L KCl and 10 mmol/L Tris HCl, pH 6.8, and centrifuged at 19,000 rpm for 45 min. The pellet was resuspended in 1 mL of 250 mmol/L sucrose and 10 mmol/L histidine buffer. The concentration of the SR proteins was determined with use of a bicinchoninic acid protein assay kit (Pierce, Rockford, Ill) according to the manufacturer's protocol. The activities of glucose-6-phosphatase, ouabain-sensitive Na⁺-K⁺-adenosine triphosphatase (ATPase) (a maker enzyme in sarcolemma which is located especially in sarcolemma and shows the sarcolemma contamination in SR preparation), and cytochrome c oxidase (a maker enzyme in mitochondria which is located especially in mitochondrial membrane and shows the mitochondria membrane contamination in SR preparation) in SR preparations were measured to assess cross-contamination as described by Osada et al. (¹⁶).

Measurement of Ca²⁺ uptake and release

Ca²⁺ uptake activity of the SR vesicles was determined by use of ⁴⁵Ca²⁺ and the Millipore filtration technique as detailed elsewhere (¹⁶). The reaction was initiated by the addition of SR (0.03-0.07 mg protein) to the standard incubation medium [Tris-ATP, 5 mmol/L; NaN₃, 5 mol/L; KCl, 100 mmol/L; MgCl₂, 25 mmol/L; potassium oxalate, 5 mmol/L; EGTA, 0.5 mmol/L; Tris-HCl, 20 mmol/L; pH 6.8] and reacted for 3 min in 37°C. The reaction was terminated after 1 min by filtering 200-μL aliquots of the incubation mixture through a Millipore filter (0.45 μm). The radioactivity was counted by use of the standard liquid scintillation counting technique.

Ca²⁺ release from the ⁴⁵Ca²⁺-loaded SR vesicles was studied according to the method of Ganguly et al. (¹⁷). The SR preparation (62.5 μg of SR protein) was suspended in 625 μL of a loading buffer containing (in mmol/L) KCl, 100; MgCl₂, 5; potassium oxalate, 5; NaN₃, 5; and Tris-HCl, 20 (pH 6.8), and the samples were incubated with 10 μmol/L ⁴⁵CaCl₂ (20 mCi/l) and 5 mmol/L ATP for 45 min at room temperature. To measure EGTA-induced Ca²⁺ release, 1 mmol/L EGTA was added to the medium, and the reaction was stopped at 15 s by use of the Millipore filtration technique. Radioactivity in the filter was counted in 10 mL of the scintillation fluid.

Culture of the neonatal cardiomyocytes

For each experiment, primary cultures of cardiomyocytes were prepared from 6 liters of neonatal Sprague Dawley rats and pooled as described previously (¹⁸). Briefly, cardiomyocytes were isolated from the ventricles of 1-day-old Sprague Dawley rats by a trypsin dispersion procedure. The dispersed cells were preplated for 30 min to minimize fibroblast contamination, thus producing cultures that were in general more than 95% cardiomyocytes. Cardiomyocytes were plated at a density of 1 × 10⁶ cells per well on 6-well plates for cell viability studies or of 2 × 10⁶ cells per well on 60-mm dishes for SDS-PAGE in Dulbecco modified Eagle medium supplemented with 10% (vol/vol) fetal calf serum and 1% penicillin/streptomycin (100 U/mL) in a culture incubator (Napco 302; Precision & Napco, Winchester, Va) maintained at normal atmosphere oxygen (with 5% carbon dioxide). After 24 h in culture, cells were transferred to low-serum maintenance medium (Dulbecco modified Eagle medium containing 1% fetal calf serum and 1% penicillin/streptomycin) for 24 h before experimentation.

A known number of cultured neonatal rat cardiomyocytes were randomly and homogeneously distributed into different experimental groups as follows: (1) HPC group: under sterile conditions, the cultured plates (or dishes) were placed into the hypoxia chamber (Anero-Pack series, Mitsubishi Gas Chemical Co Inc) for 20 min to induce HPC as described previously (¹⁸). After the transient hypoxia, the medium was washed off, and the cultures were reperfused with maintenance medium. Hypoxic preconditioning cardiomyocytes were exposed to lethal hypoxia by incubating them in the same hypoxic condition for 3 h at 37°C 24 h later. After 3 h of lethal hypoxia, cardiomyocytes were returned to the maintenance medium (0.5 mL) and incubated for a further 3 h. (2) H/R group: cardiomyocytes were directly exposed to hypoxia (for 3 h) followed by reoxygenation (for 3 h) as described above. (3) SB202190 + HPC (SBA + HPC) group: cardiomyocytes were preincubated with SB202190 (a selective inhibitor of p38 MAPK, 50 μmol/L) for 10 min before HPC. (4) Control group: cardiomyocytes were incubated in cell incubator for 30 h.

At the end of the reoxygenation period, lactate dehydrogenase (LDH) activity in cardiomyocyte supernatant was determined by use of an LDH assay kit (Sigma) according to the manufacturer's instructions to assess cell membrane integrity. For the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), assay cells were washed with phosphate-buffered saline (PBS) at 37°C and incubated with 0.5 mg/mL MTT in 0.5 mL of PBS for 20 min at 37°C. The reaction was stopped, and cells were lysed by the addition of an equal volume of 10% Triton X-100/0.1 M HCl in propan-2-ol., and the absorbance was read at 570 nm (¹⁹).

Preparation of whole cell extracts from cardiomyocytes (myocardium) and Western blotting analysis

Upon completion of the experimental period, the cardiomyocytes were rinsed and harvested with ice-cold PBS and pelleted by centrifugation at 1850 × g for 5 min. Each cell pellet was washed with and resuspended in extraction buffer containing (mmol/L) HEPES, 20 (pH 7.7); MgC1₂, 2.5; EDTA, 0.1; β-glycerophosphate, 20; dithiothreitol, 0.5; sodium orthovanadate, 0.1; NaCl, 75; leupeptin, 4 (μg/mL); phenylmethylsulfonyl fluoride, 20 (μg/mL); and Triton X-100 0.05% (vol/vol). The myocardium was washed and homogenized in the same extraction buffer. The homogenate was incubated and centrifuged. The detergent soluble supernatant was frozen with liquid N₂, and stored at −70°C. All centrifugation was carried out at 4°C. Protein concentration in the detergent soluble supernatant was determined involved use of a bicinchoninic acid protein assay kit (Pierce) according to the manufacturer's protocol.

The supernatant was mixed with Laemmli buffer and heated for 5 min at 95°C. Soluble extracts (50 μg) were loaded in each lane and separated by SDS-polyacrylamide gel electrophoresis. After electrophoresis, proteins were electrophoretically transferred to a polyvinylidene difluoride filter membrane [0.45 μm, Amersham, Oakville, Ontario, Canada) blocked with 10% nonfat dry milk in Tris-buffered saline Tween 20 (TBS-T) (mmol/L)]: Tris-HCl, 20 (pH 7.6); NaCl, 137; and 0.1% Tween (²⁰). The blots were then incubated with the phospho-specific antibody against p38 MAPK or anti-NF-κB at 1:1000 dilution in TBS-T and incubated with antirabbit IgG conjugated to horseradish peroxidase (Amersham) in TBS-T. Membranes were exposed to x-ray film for between 30 s and 2 min. The staining was quantified by scanning the films and the band density determined with use of Image-Pro software.

Statistical analysis

Values are expressed as mean ± SD from at least 6 independent experiments. Each treatment in each experiment was performed in triplicate wells. In each experiment, cardiomyocytes were pooled from 6 different liters. The data from the 6 experiments were then pooled (n = 6). Differences in means between groups were tested by one-way ANOVA followed by the Student-Newman-Keul test (comparisons between multiple groups). All P values corresponded to 2-tailed tests, and P values < 0.05 were considered statistically significant.

RESULTS

Effects of HPC on ischemia (or H/R) injury of rat myocardium

Hemodynamics-

Results of hemodynamic parameters are shown in Table 1. MAP and HR did not significantly differ among groups. Compared with the sham group, the MI group showed markedly impaired cardiac systolic (+LVdp/dt_max) and diastolic (−LVdp/dt_max) function. However, HPC significantly improved cardiac systolic and diastolic dysfunction induced by MI as compared with MI alone. The values of +LVdp/dt_max and −LVdp/dt_max increased by 43% and 59% (P < 0.05) respectively.

Table 1: Hemodynamic parameters (mean ± SD, n = 6 in sham group, n= 9 in MI and HPC groups)

Infarct size-

In the MI group, infarct size as measured by proportion of area at risk was 65.13 ± 6.20% on average. In the HPC group, infarct size was substantially smaller, 38.12 ± 3.81%, on average (P < 0.05 vs. MI group). No significant differences were found in areas at risk between the MI and HPC groups.

Cellular viability-

Cellular viability in all groups before the experiment was greater than 95%. The cellular viability and LDH activity in the culture medium at the end of the experiment are shown in Table 2. Compared with viability of controls, that of cardiomyocytes subjected to H/R decreased by 44% (P < 0.05), and LDH release increased by 178% (P < 0.05). Hypoxic preconditioning significantly attenuated the H/R injury compared with H/R alone. The cardiomyocytes showed a 26% increase of survival after lethal H/R when preceded by HPC, as determined by preservation of MTT bioreductive capacity (P < 0.05). Lactate dehydrogenase release from cardiomyocytes was decreased by 44% as compared with that in the H/R-alone group (P < 0.05). p38 MAPK inhibitor SB202190 given 10 min before HPC blocked the delayed cardioprotection (P < 0.05 vs. HPC group).

Table 2: Effects of HPC on viability and LDH release from neonatal cardiomyocytes subjected to H/R Injury (n = mean ± SD)

Effects of HPC on calcium homeostasis in ER from rat heart

Identification of the SR preparation-

Sarcoplasmic reticulum yield rate and marker enzyme activity (Table 3) did not significantly differ among groups. The activity of glucose-6-phosphatase was similar in the myocardial SR preparation from sham, MI, and HPC rats. Contamination by other organelles was low. As judged by ouabain-sensitive Na⁺-K⁺-ATPase activity (a maker enzyme in sarcolemma), contamination by the sarcolemma membrane was 6.8% to 9.6%. Cytochrome c oxidase activity (a maker enzyme in mitochondria) results showed a maximum of 1.2% to 2.3% contamination in by mitochondrial membranes.

Table 3: SR protein yield, marker enzyme activities, Ca²⁺ uptake, and Ca²⁺ release in rat myocardium from different groups (n = 12, mean ± SD)

SR Ca²⁺ release and uptake activities-

Adenosine triphosphate-dependent Ca²⁺ uptake and EGTA-induced Ca²⁺ release were determined in SR preparations from control, MI, and HPC hearts (Table 3). In SR preparations from MI hearts, Ca²⁺ uptake decreased by 63.9%, and Ca²⁺ release increased by 73.2% compared with that from sham-treated rats (P < 0.05). These changes were significantly attenuated in the HPC ischemic hearts. The SR Ca²⁺ uptake increased by 94.1%, and Ca²⁺ release decreased by 18.9% in the HPC group as compared with the MI group (P < 0.05).

Effects of HPC on CRT expression-

Alteration in protein expression of CRT in myocardium was detected by Western blotting. Positive results were followed-up with blots from myocardial proteins subjected to MI (Fig. 1), which showed an 8% increase in CRT expression as compared with that in the sham group (P = 0.071). Hypoxic preconditioning induced a 142% increase in CRT expression as compared with MI group (P < 0.05).

Fig. 1: A, Western blot of CRT protein expression in myocardium. Equal protein loading was routinely verified by stripping the blot and reblotting with an antiβ-actin mouse monoclonal antibody. B, Histogram of densitometry of the immunoblots shown in A. Error bars indicate SD (*, P < 0.05); lane 1, sham group; lane 2, MI group; lane 3, HPC group.

As seen in Figure 2, blotting of whole cell extracts from cardiomyocytes subjected to H/R resulted in a 61% increase of CRT expression as compared with that in the control group (P < 0.05). Hypoxic preconditioning induced a further 84% increase of CRT expression as compared with that in the MI group (P < 0.05). SB202190 given 10 min before HPC abolished the induction of CRT by HPC as compared with HPC group (P < 0.05).

Fig. 2: A, Western blot of CRT protein expression in cardiomyocytes. Equal protein loading was routinely verified by stripping the blot and reblotting with an anti-β-actin mouse monoclonal antibody. B, Histogram of densitometry of the immunoblots shown in A. Error bars indicate SD (*, P < 0.05); lane 1, control group; lane 2, H/R group; lane 3, HPC group; lane 4, SB + HPC group.

Effects of HPC on p38 MAPK activity-

Alteration in activity of p38 MAPK in myocardium was detected by Western blotting with phospho-specific antibody against p38 MAPK. As seen in Figure 3, myocardium from the MI group showed a 10% increase in p38 MAPK activity as compared with that from the sham group (P = 0.068), but HPC induced a further 167% increase in activity as compared with MI alone (P < 0.05).

Fig. 3: A, Western blot of p38 MAPK activity in myocardium. Equal protein loading was routinely verified by stripping the blot and reblotting with an anti-β-actin mouse monoclonal antibody. B, Histogram of densitometry of the immunoblots shown in A. Error bars indicate SD (*, P < 0.05); lane 1, sham group; lane 2, MI group; lane 3, HPC group.

Hypoxia/Reoxygenation cardiomyocytes showed a 10% increase in p38 MAPK activity as compared with the control group (P = 0.062) 3 h after H/R. However, compared with H/R alone, HPC induced a further 151% increase in p38 MAPK activity (P < 0.05). SB202190 given 10 min before HPC abolished the activation of p38 MAPK induced by HPC as compared with HPC alone (P < 0.05) (Fig. 4).

Fig. 4: A, Western blot of p38 MAPK activity in cardiomyocytes. Equal protein loading was routinely verified by stripping the blot and reblotting with an anti-β-actin mouse monoclonal antibody. B, Histogram of densitometry of the immunoblots shown in A. Error bars indicate SD (*, P < 0.05); lane 1, control group; lane 2, H/R group; lane 3, HPC group; lane 4, SB + HPC group.

Effects of HPC on NF-κB expression-

Figure 5 shows the alteration in protein expression of NF-κB in myocardium. Myocardium from the MI group showed a 12% increase in NF-κB expression as compared with that in the sham group (P = 0.076). However, compared with MI alone, HPC induced a further 138% increase in NF-κB expression (P < 0.05).

Fig. 5: A, Western blot of NF-κB protein expression in myocardium. Equal protein loading was routinely verified by stripping the blot and reblotting with an anti-β-actin mouse monoclonal antibody. B, Histogram of densitometry of the immunoblots shown in A. Error bars indicate SD (*, P < 0.05); lane 1, sham group; lane 2, MI group; lane 3, HPC group.

Cardiomyocytes in the H/R group showed a 13% increase in NF-κB expression as compared with that in the control group (P = 0.057) 3 h after H/R. However, compared with H/R alone, HPC induced a further 119% increase in NF-κB expression (P < 0.05). SB202190 given 10 min before HPC abolished the NF-κB upregulation induced by HPC, as compared with HPC alone (P < 0.05) (Fig. 6).

Fig. 6: A, Western blot of NF-κB protein expression in cardiomyocytes. Equal protein loading was routinely verified by stripping the blot and reblotting with an anti-β-actin mouse monoclonal antibody. B, Histogram of densitometry of the immunoblots shown in A. Error bars indicate SD (*, P < 0.05); lane 1, control group; lane 2, H/R group; lane 3, HPC group; lane 4, SB + HPC group.

DISCUSSION

The delayed cardioprotection by ischemic (hypoxic) preconditioning can protect the heart against myocardial infarction, stunning, and ventricular arrhythmia (²). The delayed cardioprotective mechanism of HPC is incompletely understood. Although the upregulation of endogenous protective proteins has been implicated in the delayed cardioprotection of HPC, the role of Ca²⁺ binding chaperones in SR controlling calcium homeostasis and cell injury during H/R remains unclear. Cells that overexpress calreticulin have increased ER Ca²⁺ buffering capacity and/or resist Ca²⁺ toxicity (^{21, 22}). Our recent investigation indicates that HPC can upregulate the SR chaperone CRT and protect myocardium from ischemia injury. Despite efforts of a number of laboratories, however, the mechanism of cytoprotection of HPC due to CRT induction remains unclear. The results from this investigation demonstrate that the protection that HPC affords against H/R and sustained ischemia injury is due to the enhanced CRT expression via p38 MAPK activation.

Calreticulin, a 46-kDa Ca²⁺ binding chaperone found mainly in SR, plays an important role in regulating calcium homeostasis through Ca²⁺ storage, inhibiting repetitive Ca²⁺ waves by interacting selectively with distinct isoforms of SERCA2 (²³) and Ca²⁺ influx (²⁴). Chaperone calreticulin is also a key component of the CRT/calnexin cycle, which is responsible for the folding of newly synthesized proteins and for quality control pathways in the SR (²⁵). Both the chaperoning functions and the regulation of calcium homeostasis of CRT play an important role in determining cellular sensitivity to stress and injury. In addition to its Ca²⁺ binding and protein-folding properties, CRT also interacts with the DNA-binding domain of the glucocorticoid receptor and other nuclear hormone receptors (^{26, 27}), thus inhibiting glucocorticoid-sensitive gene expression and retinoic acid-dependent differentiation. The mechanisms of CRT in regulation of calcium homeostasis and prevention of cells from H/R injury remain unclear. Chaperone calreticulin has been shown to modulate ER Ca²⁺ release. Overexpression of CRT increased the Ca²⁺ content of the ER and afforded protection to mouse insulinoma MIN6 cells against NO-mediated apoptosis (⁵). The results from this investigation demonstrate that HPC induced CRT upregulation, increased SR calcium uptake, and decreased SR calcium release, which suggests that HPC might mitigate calcium overload in cardiomyocytes by controlling calcium storage through the Ca²⁺ regulation ability of CRT. Downregulation of CRT expression by transfecting neonatal cardiomyocytes with phosphothioate oligonucleotide CrtASl (an 18-nucleotide antisense sequence) suppressed antiapoptosis effect induced by HPC (data not shown).

The influence of cell signaling pathways on CRT expression and cell survival during HPC is not well characterized. The present study investigated the signal pathway by which HPC induces CRT expression and revealed that HPC cardiomyocytes have greater p38 MAPK phosphorylation after H/R, which was positively associated with CRT expression. Inhibition of p38 MAPK with SB202190, a selective p38 MAPK inhibitor, abolished the CRT upregulation and cardioprotective effect of HPC. Our findings demonstrate for the first time that CRT induction by HPC acts via the p38 MAPK pathway to modulate cardiomyocyte injury in response to I/R. In the present study, it is also found that HPC induced NFκB upregulation. SB202190, a selective inhibitor of p38 MAPK, inhibited NF-κB expression and abolished the cardioprotection induced by HPC, suggesting that NF-κB might be involved in cardioprotection.

In conclusion, much attention has been directed toward understanding the endogenous protective proteins and the significance in delayed cardioprotection by HPC. However, our knowledge of how SR calcium-binding proteins affects delayed cardioprotection remains incomplete. In this study, we have demonstrated that HPC-induced CRT upregulation attenuates myocardial ischemic and H/R injury in neonatal cardiomyocytes by increasing calcium uptake and reducing calcium release in SR. Induction of CRT expression is mediated by the p38 MAPK pathway during HPC, and inhibition of p38 MAPK with SB202190 abolishes the CRT upregulation and cardioprotective effect of HPC. These results indicate that the p38 MAPK signaling pathway is a critical upstream effector of the protection afforded by HPC-induced CRT expression against ischemia (or H/R) injury in cardiomyocytes.