Restoration of blood flow is the definitive therapy to salvage myocardium after ischemic injury. Sudden restoration of blood flow to the ischemic myocardium may, however, also cause reperfusion injury (1). Ischemic postconditioning (I-postC) is a newly discovered endogenous protective mechanism capable of protecting the myocardium from I/R injury. Ischemic postconditioning can be evoked by applying cycles of brief intermittent interruption of blood flow to the myocardium during the early reperfusion after a prolonged period of ischemia (2). This will protect the heart against myocardial infarction and reduce coronary endothelium dysfunction to an extent comparable to ischemic preconditioning (IPC) (2). The fact that I-postC can be applied after a prolonged period of ischemia offers a novel approach to cardioprotection (3). However, the cardioprotective mechanism of I-postC is not completely understood. There is some evidence suggesting that preventing an increase in cytosolic calcium (4) and synthesis of proteins (5) are involved.
The endoplasmic reticulum (ER) is a multifunctional signaling organelle (6) and a principal site for secretory protein synthesis and folding, calcium storage, and signaling (7, 8). Alterations in the ER environment such as perturbation of Ca2+ homeostasis, elevated protein synthesis, deprivation of glucose, altered glycosylation, and the accumulation of misfolded proteins cause ER stress (9). Endoplasmic reticulum stress induces the degradation of misfolding proteins and the activation of a highly conserved transcriptional program to increase ER's capacity of protein folding and calcium storage (10). By coordinating the suppression of protein synthesis and up-regulating ER-resident chaperones such as glucose-regulated proteins and calreticulin (CRT), moderate ER stress is often able to restore cellular homeostasis, that is, to exert a cytoprotective effect. When ER stress is intense or persistent, however, production of proapoptotic factors (caspase 12 and C/EBP homologous protein) may cause ER stress-induced apoptosis, which is a feature of I/R injury (10). We recently reported that hypoxic preconditioning (HPC) attenuated ER stress-related apoptosis induced by sustained hypoxia/reoxygenation (H/R) in neonatal cardiomyocytes (11). Conceivably, the cardioprotection conferred by hypoxic postconditioning (H-postC) can involve suppression of I/R-induced intense ER stress.
The cellular mechanisms underlying the I-postC-induced cardioprotection through suppression of ER stress remain to be defined. Mitogen-activated protein kinases (MAPKs) play important roles in the regulation of cellular responses occurring during cell proliferation and stress, which include extracellular signal-regulated protein kinases, c-Jun N-terminal kinase (JNK), and p38 MAPK (12). Among the three distinct MAPK families, p38 MAPK and JNK are known to be regulated by extracellular stresses, including oxidative stress, heat shock, and ultraviolet radiation (13). Most studies have confirmed the role of p38 MAPK as a potential mediator of IPC-induced protection (14). The role of JNK in cardioprotection has been controversial, with most studies supporting its detrimental role, although several studies have demonstrated that JNK mediates the protective effect of IPC (15). The balance between JNK and p38 MAPK may therefore be of central importance for maintaining cellular structure and function during stress (15). We recently found that p38 MAPK activation mediated HPC-induced suppression of ER stress and antiapoptosis effect in neonatal cardiomyocytes subjected to sustained H/R (11). It is also reported that JNK activation mediates ER stress-induced apoptosis (16). We hypothesized that p38 MAPK and JNK might be involved in I-postC-induced suppression of ER stress.
The goals of the present study were as follows: (1) to show whether the cardioprotection conferred by I-postC involves suppression of I/R-induced intense ER stress and (2) to demonstrate whether p38 MAPK and JNK mediate the suppression of ER stress and cardioprotection induced by I-postC. Our results suggest that I-postC protected the myocardium from I/R injury through suppression of ER stress, and that p38 MAPK and JNK pathways are associated with the I-postC-induced suppression of ER stress.
MATERIALS AND METHODS
Animals and Chemicals
Male Wistar rats weighing 220 to 260 g were used for in vivo studies. Rats were housed four per cage at 23°C, with luminosity cycles of 12-h light/12-h dark, fed Purina rat ration, and allowed free access to water. All experimental procedures were performed in accordance with the guidelines of the Council for Animal Research, Health Center, Peking University. All animals were from the Experimental Animal Center, PLA General Hospital, Beijing, China.
β-Glycerophosphate, phenylmethylsulfonyl fluoride, sodium orthovanadate, leupeptin, acrylamide, sodium dodecyl sulfate, N,N,-methylenebisacrylamide, ammonium persulfate, Triton X-100, N,N,N,N-tetramethyl ethylenediamine, EDTA, and dithiothreitol were purchased from Sigma (St. Louis, Mo). Rabbit antimouse CRT monoantibody was from Stressgen (San Diego, Calif). Phosphospecific antibody against p38 MAPK and JNK, rabbit antihuman β-actin monoantibody, and enhanced chemiluminescence immunodetection kits were purchased from Santa Cruz (Santa Cruz, Calif). Rabbit antimouse caspase 12 polyclonal antibody was purchased from Biovision (Mountain View, Calif). Other chemicals were purchased from Sigma unless otherwise noted.
Myocardial I/R Model
After a 5-day acclimatization, male Wistar rats underwent open chest surgery under anesthesia with pentobarbital sodium (30 mg/kg) and artificial ventilation. The thorax was opened at the fourth or fifth left intercostal space. The animals were randomly divided into four groups as follows: (1) I/R group (n = 10): the left anterior descending coronary artery (LAD) was reversibly occluded for 45 min followed by 2 h of reperfusion as described previously (17). (2) Ischemic postconditioning group (n = 10): after 45 min of LAD occlusion, reperfusion was initiated for 30 s of reperfusion followed by 30 s of reocclusion, repeated for three cycles, then subjected to 2 h reperfusion. (3) Ischemic preconditioning group (n = 10): the LAD was occluded for 5 min followed by 5 min of reperfusion, repeated for three cycles before I/R. (4) Sham group (n = 8): rats underwent the same surgical procedure without ligation.
Upon completion of the experimental periods, MAP, heart rate (HR), and ±dp/dt were measured for 3 consecutive minutes in anesthetized rats. For measurement of these parameters, the right carotid artery was cannulated and connected to a pressure transducer inline to a Grass polygraph. After hemodynamic measurements, blood samples were taken for measurement of plasma lactate dehydrogenase (LDH). The heart was then excised and washed in saline solution. Myocardium was taken from the sites of risk area (infarct margin) for Western blotting analysis.
For triphenyltetrazolium chloride staining, the same operation was performed on another animal. After hemodynamic measurements, the heart was excised, and the aorta was cannulated and perfused with 0.9% saline to remove blood from the vascular tree. The LAD was then reoccluded with a ligature at the same site previously chosen for occlusion, and 2 mL of 2% Evans blue was infused into the aortic root. Infarct size was measured by triphenyltetrazolium chloride staining method (17) and expressed as percentage that was infarcted of the area at risk.
Culture of the Neonatal Cardiomyocytes
For each experiment, primary cultures of cardiomyocytes were prepared from three litters of neonatal Wistar rats and pooled as described previously (18). Briefly, cardiomyocytes were isolated from the ventricles of 1-day-old Wistar rats by a trypsin dispersion procedure. The dispersed cells were preplated for 30 min to minimize fibroblast contamination, thus giving cultures generally containing greater than 95% cardiomyocytes. Cardiomyocytes were plated at a density of 5 × 106 cells on a 75-mm culture flask in Dulbecco's Modified Eagle's Medium supplemented with 10% (vol/vol) fetal calf serum and 1% penicillin/streptomycin (100 U/mL) in culture incubator (Napco 302) maintained at normal-atmosphere oxygen (with 5% CO2). After a 24-h culture, cells were transferred to serum-free maintenance medium (Dulbecco's Modified Eagle's Medium containing 1% penicillin/streptomycin) for 24 h before experimentation. A known number of cardiomyocytes were randomly and homogeneously distributed into different experimental groups as follows: (1) H/R group: cardiomyocytes were placed into the hypoxia chamber (Anero-Pack series, Mitsubishi Gas Chemical, Co. Inc., Tokyo, Japan) for 2 h at 37°C to induce hypoxia by exposing to a gas mixture (5% CO2/N2/air) to obtain 1% final fraction of oxygen, followed by reoxygenation for 14 h at 37°C in an atmosphere containing 95% air/5% CO2 (resulting in 19.5% O2) (19). (2) Hypoxic postconditioning group: cardiomyocytes were directly exposed to hypoxia for 2 h followed by 3 cycles of brief reoxygenation (5 min) and hypoxia (5 min) to induce H-postC as detailed elsewhere (4). Then cardiomyocytes were incubated in a cell incubator for 14 h to induce sustained reoxygenation. (3) Hypoxic preconditioning group: cardiomyocytes were placed into the hypoxia chamber for 20 min to induce HPC as described previously (19). After the transient hypoxia, the cultures were subjected to H/R as described above. (4) SB202190 + H-postC (SB + H-postC) group: cardiomyocytes were preincubated with SB202190 (a selective inhibitor of p38 MAPK; 5 μM) for 10 min before H-postC. (5) SP600125 + H-postC (SP + H-postC) group: cardiomyocytes were preincubated with SP600125 (a selective inhibitor of JNK; 10 μM) for 10 min before H-postC. (6) Control group: cardiomyocytes were incubated for 16 h in a humidified incubator at 37°C in an atmosphere containing 95% air/5% CO2 (resulting in 19.5% O2).
LDH Activity and Trypan Blue Assay
At the end of the reoxygenation period, LDH activity in cardiomyocyte supernatant was determined by use of an LDH assay kit (Sigma) according to the manufacturer's instructions. For the trypan blue assay (20), cardiomyocytes were harvested with 0.125% trypsin, rinsed with ice-cold phosphate-buffered saline (PBS), and pelleted by centrifugation at 1,850 × g for 5 min. A 15-µL aliquot of resuspended cardiomyocytes from each group was withdrawn, resuspended in 150 µL of hypotonic buffer (85 mOsm) containing 3 mM amytal sodium as a mitochondrial inhibitor, and allowed to equilibrate for 3 to 4 min. On a microscope slide, a 15-µL sample of this solution was then mixed with an equal volume of trypan blue solution (0.5% glutaraldehyde in 85 mOsm NaCl-deficient Tyrode solution containing 1% trypan blue). Three widely separated fields at ×100 magnification were then examined to determine cell morphology and permeability (blue vs. not blue), and the results were averaged for each group. Cardiomyocytes that were not able to exclude trypan blue were considered nonviable.
Annexin V and Propidium Iodide Fluorescence-Activated Cell Sorter Analysis
Cardiomyocytes were labeled with annexin V and propidium iodide (PI) (Becton-Dickinson company) according to the manufacturer's instructions. Briefly, 1 × 105 cells were washed with cold PBS, resuspended with binding buffer, then transferred to a 5-mL tube. Annexin V and PI were added to the cell preparations and incubated for 25 min in the dark. Binding buffer (400 μL) was added to tubes, and the samples were analyzed by flow cytometry (21).
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 1,850 × g for 5 min. Each cell pellet was washed with and resuspended in extraction buffer containing (in mM) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 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 performed at 4°C. Protein concentration in the detergent-soluble supernatant was determined by using a bicinchoninic acid protein assay kit (Pierce, Rockford, Ill) according to the manufacturer's protocol.
The supernatant was mixed with Laemmli buffer (22) and heated for 5 min at 95°C. Soluble extracts (100 μg) were loaded in each lane and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, proteins were electrophoretically transferred to a polyvinylidene difluoride filter membrane (0.45 μm; Amersham, Buckinghamshire, UK) blocked with 10% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) ([in mM]: Tris-HCl, 20 (pH 7.6); NaCl, 137; and 0.1% Tween 20) (19). The blots were then incubated with the phosphospecific antibody against p38 MAPK or JNK, anti-CRT, p38 MAPK, and JNK at 1:500 dilution, or anticaspase 12 (1:200) and β-actin (1:1000) in TBS-T and incubated with antirabbit immunoglobulin G conjugated to horseradish peroxidase 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 was determined with the use of Image-Pro software.
Values are expressed as mean ± SD. Differences in means between groups were tested by one-way ANOVA. P values less than 0.05 were considered statistically significant. All statistical analyses were performed using Stata version 7.0.
Effects of I-postC on I/R Injury of Rat Myocardium
Hemodynamic parameters are shown in Table 1. MAP and HR did not significantly differ among groups. The I/R group showed markedly impaired cardiac systolic (+LVdp/dtmax) and diastolic (−LVdp/dtmax) function compared with the sham group. Ischemic postconditioning significantly improved cardiac systolic and diastolic dysfunction induced by I/R as compared with the I/R group. The values of +LVdp/dtmax and −LVdp/dtmax increased by 62% and 67%, respectively (P < 0.05) and were similar to those from the IPC group (P > 0.05).
Infarct size as measured by proportion of area at risk averaged 72.30 ± 7.82% in the I/R group. In the I-postC and IPC groups, infarct size was substantially smaller, 39.78 ± 9.51% and 33.90 ± 4.63% on average, respectively (P < 0.05 vs. I/R group). No significant differences were found in areas at risk among I/R, I-postC, and IPC groups.
Plasma LDH activity
Plasma LDH activities are shown in Table 1. I/R increased LDH leakage by 2.4-fold more than sham (P < 0.01). Ischemic postconditioning decreased the LDH leakage from myocardium subjected to I/R by 36.3% (P < 0.05), which is similar to the result from the IPC group.
Cardiomyocyte viability in all groups before the experiment was greater than 95%. The cellular viability detected by trypan blue assay and LDH activity in culture medium at the end of the experiment are shown in Table 2. Compared with controls, viability of cardiomyocytes subjected to H/R decreased by 32.6% (P < 0.05), and LDH release increased by 3.3-fold (P < 0.05). Both H-postC and HPC significantly attenuated the H/R injury compared with the H/R group. The cardiomyocytes showed a 10.3% and 23.2% increase in survival rate after lethal H/R in H-postC and HPC groups, respectively, compared with the H/R group (P < 0.05). Lactate dehydrogenase release from cardiomyocytes was decreased by 62.2% and 58.2% in the H-postC and HPC groups, respectively, as compared with that in the H/R group (P < 0.05). p38 MAPK inhibitor SB202190 given 10 min before H-postC blocked the cardioprotection (P < 0.05 vs. H-postC group). By contrast, the JNK inhibitor SP600125 given 10 min before H-postC had no effect on cardioprotection induced by H-postC (P > 0.05).
Apoptosis detected by flow cytometry (double staining with Annexin V and PI) at the end of the experiment is shown in Table 2. The apoptosis rate in control cardiomyocytes is 4.2%. Compared with controls, apoptosis rate from cardiomyocytes subjected to H/R increased by 127% (P < 0.05). The cardiomyocytes showed a 30% and 38.6% decrease in apoptosis rate in the H-postC and HPC groups, respectively (P < 0.05, vs. H/R group). p38 MAPK inhibitor SB202190 given 10 min before H-postC blocked the cardioprotection (P < 0.05 vs. H-postC group). The JNK inhibitor SP600125 given 10 min before H-postC did not alter the rate of apoptosis compared with the H-postC group (P > 0.05).
Effects of I-postC (H-postC) on Expression of ER Stress Molecules
Alterations in protein expression of CRT in myocardium were detected by Western blotting. Positive results were followed-up with blots from myocardial proteins subjected to I/R (Fig. 1), which showed a 139% increase in CRT expression as compared with that in the sham group (P > 0.05). Ischemic postconditioning induced a 56.8% decrease in CRT expression as compared with the I/R group (P < 0.05), and no significant difference was found in CRT expression between the I-postC and IPC groups (P > 0.05). As seen in Figure 2, blotting of whole cell extracts from cardiomyocytes subjected to H/R resulted in a 3-fold increase in CRT expression as compared with that in the control group (P < 0.05). Hypoxic postconditioning induced a 38.6% decrease in CRT expression as compared with that in the H/R group (P < 0.05), and no significant difference was found in CRT expression between the H-postC and HPC groups (P > 0.05). p38 MAPK inhibitor SB202190 given 10 min before H-postC abolished H-postC-induced down-regulation of CRT expression in cardiomyocytes (P < 0.05 vs. H-postC group), whereas JNK inhibitor SP600125 given 10 min before H-postC further down-regulated the CRT expression by 34.4% (P > 0.05) as compared with the H-postC group (P < 0.05).
Caspase 12 activity in cardiomyocytes
Alterations in caspase 12 activity and expression in cardiomyocytes were detected by Western blotting. As seen in Figure 3, The procaspase form of caspase 12 (55 kd) showed no change in cardiomyocytes among groups (P > 0.05). The level of 35-kd proteolytic fragment of caspase, which showed the activation of caspase 12, increased by 7.9-fold in the H/R group compared with the control group (P < 0.05). Hypoxic postconditioning reduced activation of caspase 12 by 50.1% as compared with the H/R group (P < 0.05), as shown by the level of the 35-kd proteolytic fragment. p38 MAPK inhibitor SB202190 given 10 min before H-postC did not alter the caspase 12 activation compared with the H-postC group (P > 0.05), whereas JNK inhibitor SP600125 reduced the activation of caspase 12 by 68.4% in cardiomyocytes compared with the H-postC group (P < 0.05).
Effects of H-postC on activities of p38 MAPK and JNK
Alterations in activities of p38 MAPK in cardiomyocytes were detected by Western blotting with phosphospecific antibody against p38 MAPK. As seen in Figure 4, cardiomyocytes from the H/R group showed no difference from control because the statistics do not support a difference (P > 0.05). Hypoxic postconditioning induced a 38% increase in p38 MAPK activity as compared with the H/R group (P < 0.05). SB202190, a p38 MAPK inhibitor, given 10 min before H-postC blocked the activation of p38 MAPK in cardiomyocytes (P < 0.05 vs. H-postC group), whereas JNK inhibitor SP600125 had no effect on the activation of p38 MAPK induced by I-postC.
As seen in Figure 5, blotting of whole cell extracts from cardiomyocytes subjected to H/R resulted in a 5.5-fold increase in JNK activity as compared with that in the control group (P < 0.05). Hypoxic postconditioning and HPC reduced the JNK activation by 46.9% and 66%, respectively, as compared with H/R group (P < 0.05). c-Jun NH2-terminal kinase inhibitor SP600125 given 10 min before H-postC blocked the activation of JNK in cardiomyocytes (P < 0.05 vs. H-postC group), whereas p38 MAPK inhibitor SB202190 had no effect on JNK activity compared with I-postC group.
In 2003, Zhao et al. (2) first introduced the concept of I-postC in which brief intermittent repetitive interruptions to reperfusion at the onset of reperfusion after a prolonged period of ischemia significantly reduces myocardial I/R injury. In the present study, we show that I-postC causes a marked reduction in myocardial infarct size, LDH release, and improved cardiac systolic and diastolic function in rat heart subjected to I/R. The results from cardiomyocytes also show a reduction in LDH leakage and apoptosis rate and an increase in viability in hypoxic postconditioned cardiomyocytes from neonatal rats. These data harmonize with the concept that I-postC has cardioprotective effects both on adult myocardium subjected to I/R and on neonatal cardiomyocytes subjected to H/R, as previously described by Zhao et al. (2) and Sun et al. (4).
Although the cardioprotective mechanism of I-postC is not completely understood, there is some evidence suggesting that synthesis of proteins (5) and preventing an increase in cytosolic calcium (4) are involved. Endoplasmic reticulum is a principal site for protein synthesis, folding, and calcium storage. I/R causes alterations in ER environment (9) and intense ER stress that induces the activation of proapoptosis factors such as caspase 12 and results in ER-associated apoptosis (23). In this study, we demonstrate that I/R (or H/R) can induce CRT expression, caspase 12 activation, and apoptosis in cardiomyocytes. These results indicate that I/R (H/R) injury involves severe ER stress, in agreement with the findings of Bando et al. (24), Xudong et al. (25), and Shibata et al. (26). We also found that postconditioning suppressed I/R (or H/R)-induced ER stress, as shown in a decrease in CRT expression and caspase 12 activation, and cardiomyocyte apoptosis. This agrees with the findings of Zhang et al. (23), who reported that preinduced ER stress protects cardiomyocytes from lethal oxidative injury.
The signaling pathway underlying cardioprotection induced by I-postC through suppressing severe ER stress is not well characterized. Mitogen-activated protein kinases convey extracellular signals to their intracellular targets and control a diverse array of cellular processes (12). c-Jun NH2-terminal kinase and p38 MAPK, two major members of this superfamily, are activated by cell stresses. Our previous study suggested that p38 MAPK activation mediated HPC-induced cardioprotection through inhibition of intense ER stress (11). Srivastava et al. (27) reported that thapsigargin-induced ER stress activated the JNK pathway that occurs before apoptosis in Jurkat T cells. The c-Jun-negative mutant prevents thapsigargin-induced apoptosis. In the present study, we show that H-postC up-regulates p38 MAPK phosphorylation and down-regulates JNK phosphorylation in cardiomyocytes subjected to sustained H/R. Inhibition of p38 MAPK with SB202190 partly abolishes down-regulation of CRT expression and caspase 12 activation induced by H-postC, suggesting that activation of p38 MAPK plays an important role in H-postC-induced suppression of ER stress during H/R. We also found that H-postC down-regulated H/R-induced JNK activation, and inhibition of JNK with SP600125 further down-regulates CRT expression and caspase 12 activation induced by H-postC, suggesting that JNK might mediate H/R-induced severe ER stress and cardiomyocyte apoptosis, whereas H-postC attenuates H/R-induced apoptosis through suppressing JNK activation-induced ER stress.
This is the first demonstration that I-postC protects myocardium from I/R injury by suppressing intense ER stress, and that p38 MAPK/JNK pathways are associated with the I-postC-induced suppression of ER stress.
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