INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

METABOLIC INHIBITION is one of the consequences of myocardial ischemia. A previous study in our laboratory (29) showed that preconditioning with metabolic inhibition (MIP) produces delayed or second-window cardioprotection in cultured ventricular myocytes. The effects are mimicked by pretreatment with trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U-50488H), a selective -opioid receptor (-OR) agonist, and antagonized by nor-binaltorphimine (nor-BNI), a selective -OR antagonist (29). This indicates that the delayed cardioprotection of MIP is mediated at least partially via the -OR, which is the predominant type of opioid receptor in the rat heart (25, 26). Apart from the fact that protein kinase C (PKC) may be involved as shown in our previous study (29), the signaling mechanisms responsible for delayed cardioprotection of prior -OR stimulation are not fully understood.

Heat-shock proteins (HSPs) are cardioprotective proteins that are induced in response to a variety of stressful stimuli including heat (19), ischemia, and metabolic inhibition (8, 12). There is compelling evidence that HSPs are involved in cardioprotection of ischemic preconditioning or MIP (17, 18, 22, 23). Although the precise mechanism for the action of HSPs is not well understood, it is believed that the biological property of "molecular chaperones" assists in the assembly and/or repair of newly synthesized or damaged proteins. Potential functions for molecular chaperones in the ischemic heart may include: 1) protein folding of newly synthesized polypeptides essential for maintaining oxidative metabolism after myocardial injury, 2) protection of mitochondria from reactive oxygen species and repair of critical structural proteins after ischemia-induced cytoskeletal alterations, 3) transport of potential toxic byproducts for degradation by the proteasome, 4) repair of ion channels, and 5) modulation of immune-mediated ischemic injury (1, 10). Of the various members of the HSP family, HSP70s are strongly induced in the heart in response to various forms of stress. It is therefore likely that HSP70s are involved in delayed cardioprotection of prior stimulation of -OR with a selective -OR agonist.

The purpose of this study was first to determine whether HSPs mediate the delayed cardioprotection of prior -OR stimulation and second to identify the specific HSP involved. An isolated ventricular myocyte preparation was used as described previously (29). An experimental procedure used previously (29) to induce cardiac injury and preconditioning for the production of delayed cardioprotection was adopted with slight modification. Cell injury and viability and the expression of HSP70s were determined in ventricular myocytes subjected to pretreatment with U-50488H or MIP in the absence or presence of nor-BNI and subsequent lethal simulated ischemia (LSI) 20 h later. Lactate dehydrogenase (LDH) released from the myocytes was used as an index of injury, and trypan blue exclusion was used to determine viability. The expression of both the stress-inducible (HSP) and constitutive (HSC) forms of HSP70 were determined as preliminary experiments showed that only these two HSPs (and not HSP90 or HSP32) exhibited increased expression in response to prior -OR stimulation. Second, experiments were also performed after pretreatment with U-50488H and subsequent LSI when the production of HSP70 and HSC70 was blocked with the respective antisense oligonucleotides (AS). Results from the study showed for the first time that the stress-inducible HSP70 mediates the delayed cardioprotection of prior stimulation of -OR with its agonist, U-50488H.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolation of myocytes and experimental protocol. Ventricular myocytes were isolated from the heart of male Sprague-Dawley rats (200-250 g body wt) according to a previously published procedure (4, 24). After ventricular myocytes were separated, the cells were allowed to stabilize for 30 min before the experiment started. We adopted a ventricular myocytes preparation and procedure series that has been described by Wu and colleagues (29). As shown in Fig. 1, cells were first subjected either to MIP with a glucose-free HEPES buffer (pH 6.5) that contained 20 mM lactate and 10 mM 2-deoxy-D-glucose (2-DOG), an inhibitor of glycolysis, or to pretreatment with U-50488H for 30 min in the presence or absence of 5 µM nor-BNI (for 5 min before pretreatment and throughout the pretreatment period). In the control groups, the cells were subjected to pretreatment with 0.9% saline (vehicle pretreatment, VP) for 30 min or 5 µM nor-BNI for 35 min. Cells were then incubated in culture medium for 20 h before being subjected to LSI with a buffer solution containing a high K⁺ concentration at low pH which mimics myocardial ischemia (see Lethal simulated ischemia for details). When cells were pretreated with U-50488H or metabolic inhibition, there was no change in cellular injury as indicated by LDH release and the expression of HSP70s 20 h later. However, there was a slight but not significant increase in cell viability as indicated by trypan blue exclusion in cells subjected to MIP. To examine the effect of AS oligonucleotides against HSP70s, ventricular myocytes were incubated with sense or AS oligonucleotides (10 µM) for the 20-h period after preconditioning and before LSI.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Experiment design. After 30 min of isolation and stabilization, ventricular myocytes were subjected to preconditioning with trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide (U-50488H) or metabolic inhibition (MIP) in the absence or presence of nor-binaltorphimine (nor-BNI) for 30 min. Cells were washed several times before being incubated in drug-free culture medium for 20 h and subjected to lethal simulated ischemia (LSI) for 2 h then normal incubation (reperfusion, RE) for 2 h. Vehicle pretreatment (VP) cells were incubated with normal HEPES buffer that contained 50 µl of saline; MIP cells were subjected to glucose-free HEPES buffer (pH 6.5) that contained 20 mM lactate and 10 mM 2-deoxy-D-glucose, an inhibitor of glycolysis.

Lethal simulated ischemia. Twenty hours after pretreatment, the ventricular myocytes were transferred to an ischemic buffer medium supplemented with (in mM) 10 2-DOG, 0.75 sodium hydrosulfate, 12 KCl, and 20 lactate (pH 6.5). Cells were then incubated for 2 h in a CO₂ incubator as described previously (5, 20). Finally the cells were transferred back to normal medium for 2 h of additional incubation.

LDH assay. LDH was used as an index of cellular injury (20, 22): the activity in the cultured medium represented LDH release from the cultured ventricular myocytes. Supernatant as well as cell lysates (prepared by treating cells with 1% Triton X-100) were used for determination of LDH at the end of the experiments. Spectrophotometric enzyme activity assay (Beckman DU-650) was performed with a Sigma assay kit [UV kinetic assay, according to the method of Wroblewski and Ladue (28a)]. This test relies on the reduction of pyruvate to lactate catalyzed by LDH, which results in an equimolar amount of NADH oxidized to NAD. The oxidation of NADH results in a decrease in the absorbance at 340 nm. The rate of decrease in absorbance at 340 nm is directly proportional to LDH activity in the sample. The mean absorbance change per minute was determined from each experimental group. The amount of enzyme released in the nonstressed control group was subtracted as background from the values obtained from the groups subjected to LSI. The cellular injury index was also used to normalize LDH release. It was calculated according to the formula A/(A + B) × 100, where A is LDH activity in the supernatant and B is LDH activity in cells lysed with Triton X-100.

Trypan blue exclusion. Trypan blue exclusion was used to determine the viability of the myocytes (29). The cells were incubated with 0.4% trypan blue dye for 2 min, and ~100 cells in each group were examined in a hemocytometer chamber under a light microscope. Cells that were able to exclude the stain were considered viable and the percentage of nonblue cells over total cells was used as an index of viability.

PAGE and Western blotting. At the end of the experiments, the cells were washed three times with normal HEPES buffer to remove dead cells and were then harvested for analysis of HSP levels. Cells were lysed in 0.5 ml of lysis buffer [50 mM Tris · HCl (pH 7.4), 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml of aprotinin, and 1 µg/ml of leupeptin] under cold conditions and then sonicated (Sonic and Materials; Danbury, CT) with three 15-s bursts while on ice. The cytosolic fraction was obtained by centrifugation at 12,000 g for 5 min at 4°C. Protein concentration was measured using the Bio-Rad protein assay based on the Bradfold dye-binding procedure with BSA as the standard. Equal amounts of protein (20 µg) were separated by SDS-PAGE (12% acrylamide) (13). Verification of equivalent total protein loads was confirmed visually against the actin band by Coomassie blue staining of the gels in parallel (9, 20, 22). Proteins were transferred to nitrocellulose membrane at 100 V for 90 min, and blots were probed with monoclonal antibody specific for both HSP70 and HSC70 (the inducible and constitutive isoforms of the HSP70s), respectively, at a ratio of 1:1,000. The blots were subsequently washed in Tris-buffered saline/0.5% Tween 20, and were incubated with a peroxidase-conjugated second antibody at a dilution of 1:5,000. Detection was then performed using an enhanced chemiluminescence kit. All gels were run in duplicate to confirm satisfactory sample separation.

Drugs and chemicals. Joklik's modified Eagle's medium, U-50488H [the selective -OR agonist (14, 28, 28)], type I collagenase, insulin, HEPES, BSA, 2-DOG, lactate acid, sodium hydrosulfate, Ponceau stain, and the LDH assay kit were purchased from Sigma. Nor-BNI, the selective antagonist of -OR (2), was from Tocris Cookson. Concentrations of U-50488H and nor-BNI used were based on previous studies (27, 29, 32). The peroxidase-conjugated goat anti-mouse IgG antibody was from DAKO. The nitrocellulose membrane and enhanced chemiluminescence kit were from Amersham International. The primary antibody for the HSP70s used in the study was mouse antiserum raised against human (HeLa cell extracts) HSP70-related proteins. This antibody has a wide specificity and apparently recognizes both HSP70 and HSC70, the inducible and constitutively expressed HSP70-related proteins, respectively (Transduction Labs and Pharmingen H53220) (3). The anti-inducible HSP70 (SPA-810; C92F3A-5) and anti-constitutive HSC70 (SPA-815; 1B5) antibodies were from StressGen. HSP70 antibody (C92F3A-5) is specific for the inducible form of HSP70 and does not cross react with constitutive HSC70. The HSC70 antibody (SPA-815; 1B5) is specific for constitutively expressed HSC70 and does not cross react with inducible HSP70. We also confirmed in our lab that there was no cross reaction between HSP70 and the antibodies of HSC70, and vice versa. HSP70 AS oligonucleotides (TGT TTT CTT GGC CAT), HSP70 sense oligonucleotides (ATG GCC AAG AAA ACA), HSC70 antisense oligonucleotides (AGG TCC CTT AGA CAT), and HSC70 sense oligonucleotides (ATG TCT AAG GGA CCT) were synthesized from sequences complementary to the initiation codon and four downstream codons of rat HSP70 mRNA and rat HSC70 mRNA (Life Tech) as described previously (11). All other chemicals were purchased from Bio-Rad, unless indicated otherwise.

Statistical analysis. All data were expressed as means ± SE. One-way ANOVA was first carried out to test for any differences between the mean values within the same study. When a significant P value was obtained, comparisons between individual means of groups were performed by a two-tailed unpaired Student's t-test. A difference of P < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

LDH Release and Percentage of Nonblue Cells of Ventricular Myocytes Subjected to LSI: Effects of Pretreatment with U-50488H or MIP in the Presence or Absence of nor-BNI

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Lactase dehydrogenase (LDH) activity in the culture medium and cellular injury index of ventricular myocytes subjected to preconditioning with U-50488H and exposure to LSI. Isolated ventricular myocytes were exposed to different concentrations of U-50488H for 30 min and then incubated in fresh drug-free medium for 20 h before being exposed to LSI for 2 h. Data represent means ± SE of four independent experiments in four different rats. #P < 0.05; *P < 0.05; ##P < 0.01; **P < 0.01 vs. vehicle group.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   LDH activity in culture medium (A) and cellular injury index (B) of ventricular myocytes subjected to preconditioning with U-50488H (UP) or metabolic inhibition and LSI in the absence or presence of nor-BNI. LDH activity in the culture medium was measured at the end of the experiments (see Fig. 1) at 25°C for 2 min. Data are represented as means ± SE of six independent measurements. LDH values are expressed after correction of background from nonstressed control cells. ##P < 0.01 vs. VP; **P < 0.01 vs. corresponding groups without nor-BNI.

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Effects of U-50488H and MIP on percentage of nonblue cells in the absence and presence of nor-BNI. The experimental protocol was the same as described in Fig. 1. Values, presented as nonblue cells per total myocytes counted, are means ± SE; n = 6. ##P < 0.01 vs. VP; **P < 0.01 vs. corresponding groups without nor-BNI.

Results obtained from the cellular injury index (normalized LDH activity) were the same as those from LDH release (see Fig. 3).

Expression of HSP70s in Ventricular Myocytes Subjected to LSI: Effects of Pretreatment with U-50488H or MIP in the Presence or Absence of nor-BNI

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Expression of heat-shock protein 70 (HSP70) in the culture of ventricular myocytes subjected to preconditioning with U-50488H and exposure to LSI. The primary antibody recognizes both the inducible HSP70 and the constitutive HSP (HSC70). Each lane was loaded with 20 µg of total protein. A: representative Western blot. From left to right: lane 1, VP; lane 2, 30 µM U-50488H pretreatment; lane 3, 30 µM U-50488H pretreatment in the presence of 5 µM nor-BNI; and lane 4, 5 µM nor-BNI pretreatment. Protein bands representing HSP70 and HSC70 are indicated by arrows. B and C: relative levels of HSP70 and HSC70 as assessed by densitometry (Multi-Analyst/PC; Bio-Rad) normalizing to actin band on a duplicate Coomassie brilliant blue R250-stained gel. Quantitations were normalized to value obtained for VP, which was given an arbitrary value of 1.0. Each point is an average of triplicate samples. Error bars represent SE; ##P < 0.01 vs. VP; **P < 0.01 vs. corresponding groups without nor-BNI.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Expression of HSP70 and HSC70 in the culture of ventricular myocytes subjected to pretreatment with MIP and exposure to LSI. Western blot (A), from left to right: lane 1, VP; lanes 2 and 3, MIP; lane 4, MIP in the presence of nor-BNI. Relative levels of HSP70 and HSC70 (B and C) as assessed by densitometry. Quantitations were normalized to value obtained for VP, which was given an arbitrary value of 1.0. Values are averages of triplicate samples. Error bars represent SE; ##P < 0.01 vs. VP; **P < 0.01 vs. corresponding groups without nor-BNI.

LDH Release, Percentage of Nonblue Cells, and Expression of HSP70s in Ventricular Myocytes Subjected to LSI

Effects of pretreatment with U-50488H in the presence or absence of AS oligonucleotides to HSP70s. As shown in Fig. 5, pretreatment with U-50488H enhanced the expression of HSP70, which was accompanied by a reduction in LDH release and an increase in the percentage of nonblue cells. In the group with AS but not sense oligonucleotides to HSP70 during the 20-h incubation period, the effects of pretreatment with U-50488H on LDH activity, the percentage of nonblue cells, and the expression of HSP70 were completely abolished (see Fig. 7). It should be noted that the LDH activity in the culture medium was even higher than in the control group (see Fig. 7C). On the other hand, in the group with AS oligonucleotides to HSC70 during the 20-h incubation period, the increased expression of HSC70 was abolished, whereas LDH activity and the percentage of nonblue cells were the same as in the group with U-50488H pretreatment but no AS oligonucleotides (see Fig. 8).

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Expression of HSP70 (A), LDH activity in culture medium (B), and percentage of nonblue cells (C) in ventricular myocytes subjected to preconditioning with 30 µM U-50488H and LSI in the presence of 10 µM antisense (AS) oligonucletides to HSP70 during the incubation period (between U-50488H pretreatment and LSI). Values were measured as described in MATERIALS AND METHODS. Primary antibody recognizes HSP70. Data represent means ± SE of four independent experiments using four rats. ##P < 0.01 vs. VP group; **P < 0.01 vs. corresponding groups without AS oligonucleotides to HSP70.

View larger version (31K): [in this window] [in a new window]   Fig. 8.   Expression of HSC70 (A), LDH activity in culture medium (B), and percentage of nonblue cells (C) in ventricular myocytes subjected to preconditioning with 30 µM U-50488H and LSI in the presence of 10 µM AS oligonucletides to HSC70 during the incubation period (between U-50488H pretreatment and LSI). Primary antibody recognizes HSC70. Data represent means ± SE of eight independent experiments for eight rats; ##P < 0.01 vs. VP group.

Effects of pretreatment with MIP in the presence or absence of AS oligonucleotides to HSP70s. In the group with AS oligonucleotides to HSP70 during the 20-h incubation period, the effects of pretreatment with MIP on LDH activity, the percentage of nonblue cells, and the expression of HSP70 were completely abolished (see Fig. 9). However, in the group with AS oligonucleotides to HSC70, the increased expression of HSC70 was abolished, whereas the responses of the LDH activity and the percentage of nonblue cells were also the same as in the group with MIP pretreatment only (see Fig. 10, B and C).

View larger version (32K): [in this window] [in a new window]   Fig. 9.   HSP70 protein expression (A), LDH activity in culture medium (B), and percentage of nonblue cells (C) in ventricular myocytes subjected to preconditioning with MIP and LSI in the presence of 10 µM AS oligonucletides to HSP70 during the incubation period (between MIP and LSI). Primary antibody recognizes HSP70. Data represent means ± SE of four independent experiments with four rats. ##P < 0.01 vs. VP group; **P < 0.01 vs. corresponding groups without AS oligonucleotides to HSP70.

View larger version (34K): [in this window] [in a new window]   Fig. 10.   HSC70 protein expression (A), LDH activity (B), and percentage of nonblue cells (C) in ventricular myocytes subjected to preconditioning with MIP and LSI in the presence of 10 µM AS oligonucletides to HSC70 during the incubation period (between MIP and LSI). Primary antibody recognizes HSC70. Data represent means ± SE of four independent experiments with four rats. ##P < 0.01 vs. VP group.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The most important observations in this study were 1) in ventricular myocytes, prior stimulation of the -OR with its selective agonist, U-50488H, reduced LDH activity (an index of cell injury) in the culture medium and increased the percentage of nonblue cells (an index of viability) induced by LSI, which was accompanied by increased expression of HSP70s; and 2) blockade of the production of stress-inducible HSP70 but not constitutive HSC70 with a selective AS oligonucleotide abolished the ameliorating effect of pretreatment with U-50488H on cell injury and viability. The observations confirm that stimulation of -OR produces delayed cardioprotection. The novel finding is that stress-inducible HSP70 mediates the delayed cardioprotection of prior stimulation of -OR.

We also showed in this study that the effects of pretreatment with U-50488H on cell injury, viability, and the expression of HSP70s were similar to those of MIP, and that the ameliorating effects of both U-50488H pretreatment and MIP were antagonized by nor-BNI, the selective -OR antagonist. These results confirm our previous findings (29) that -OR mediates the delayed cardioprotection of MIP. That MIP increased the expression of HSP70s is also in agreement with the previous finding (22) that these proteins are involved in the delayed cardioprotection of MIP.

Cardioprotection of ischemic preconditioning has been well established (21) to be mediated by cardiac receptors to humoral substances including adenosine, opioids, catecholamines, angiotensin II, bradykinin, and endothelin. In the isolated ventricular myocyte preparation, the effect of MIP is not completely blocked by nor-BNI. This suggests that receptors other than -OR may also contribute to cardioprotection of MIP. Although the -opioid receptor (-OR) is reportedly involved in ischemic preconditioning in intact rat hearts and chicken cardiomyocytes (6, 16), in a previous study (29) we failed to demonstrate that -OR mediates cardioprotection of MIP in the isolated adult rat ventricular myocytes preparation. It is unlikely that -OR contributes to the delayed cardioprotection of MIP in this preparation.

In this study we found that U-50488H produced delayed cardioprotection in a concentration-dependent manner with a maximum effect at the 30 µM concentration. This is in agreement with the result of a previous study (29) in the same type of ventricular myocyte preparation. On the other hand, the expression of HSP70s reached a peak at 10 µM U-50488H. A possible explanation for this phenomenon is that pretreatment with U-50488H also induced increased production of other proteins (such as manganese superoxidase dismutase), which also produce cardioprotection (31). Therefore at the 30 µM concentration, U-50488H may produce cardioprotection by activating other cardioprotective proteins as well. Further studies are needed to verify this.

It has been reported that the expression of HSP70 increases markedly at 2-3 h after hyperthermia, although the reduction in the infarct size only occurred 24 h later in the anesthetized rat (30). This observation suggests that there is a time lag between the expression of the HSP and cardioprotection. In this study, we determined the expression of the HSPs and cardioprotection at 2 h after LSI and found that both expression of HSP70 and cardioprotection correlated very well with each other after preconditioning with and without blockade of protein production, which indicates that HSP70 mediates delayed cardioprotection of preconditioning. The experiments, however, did not provide information on whether there was also a time lag between the expression of HSP and cardioprotection. The experimental protocols and preparations of the two experiments are different.

ATP-sensitive K⁺ (K_ATP) channels have been shown to mediate cardioprotection of preconditioning with heat stress, opioids, and adenosine (6, 7, 15). In our previous study (29), PKC (which is known to activate K_ATP channels) was also involved in the delayed protection of pretreatment with U-50488H as well as in cardioprotection in response to heat stress (9). It is likely that both K_ATP channels and PKC may be sequential steps in the signal transduction path that mediates cardioprotection of preconditioning with U-50488H. In the present study, we showed that HSP70 mediates the cardioprotection of preconditioning with U-50488H. On the other hand, it was found that the enhanced expression of HSP70 after preconditioning was not attenuated by blockade of K_ATP channels with K_ATP channel inhibitors (7), suggesting that K_ATP channels and HSP70 may not be on the same signaling cascade in mediating cardioprotection of prior -OR stimulation. Further study is needed to delineate the relationships among these three messengers.

In conclusion, this study has provided unequivocal evidence for the first time that stress-inducible HSP70 mediates the delayed cardioprotection of prior stimulation of -OR. Because previous and present studies also showed that -OR mediates delayed cardioprotection of MIP, the cascade of events arising from MIP involves -OR and subsequently HSP70. Further studies are needed to delineate the signaling mechanisms, in particular those related to PKC and K_ATP channels, which have been shown to be involved in delayed cardioprotection of prior OR stimulation.