Diabetes Abolishes Morphine-Induced Cardioprotection via Multiple Pathways Upstream of Glycogen Synthase Kinase-3β Free

The experimental procedures and protocols used in this study were reviewed and approved by the animal care and use committee of the Medical College of Wisconsin and conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Streptozotocin (Sigma) was used to induce diabetes. The opioid agonist, morphine sulfate (Research Biochemicals International), and the GSK inhibitor, SB216763 (Tocris), were also used for this study. Streptozotocin and morphine sulfate were solubilized in water, while SB216763 was dissolved in DMSO. Streptozotocin was administered to rats via tail vein injection, while all other agents were administered intravenously via the right jugular vein.

The primary antibodies used were for P-GSK3β Ser⁹ and Tyr¹¹⁶, P-JAK-2 Tyr^1,007/1,008, P-STAT1 Tyr⁷⁰¹, P-STAT3 Tyr⁷⁰⁵, P-Akt Ser⁴⁷³ and Thr³⁰⁸, P-p70s6 Thr³⁵⁹ and Thr⁴²¹/Ser⁴²⁴, P-ERK1/P-ERK2 Thr²⁰²/Tyr²⁰⁴, GSK3β, JAK2, STAT1, STAT3, Akt, and p70s6. All primary antibodies were purchased from Cell Signaling, except for GSK3β Tyr¹¹⁶ and JAK2, which were purchased from Upstate, and P-Akt Thr³⁰⁸, purchased from Santa Cruz Biotechnologies. Antibodies were diluted 1:1,000 in Tris-buffered solution containing 3% BSA. The secondary antibodies used were anti-rabbit (purchased from Bio-Rad and diluted 1:5,000), anti-mouse for GSK3β Tyr¹¹⁶ (purchased from Upstate and diluted 1:10,000), and anti-goat for Akt Thr³⁰⁸ (purchased from Santa Cruz Biotechnologies and diluted 1:10,000).

Male Sprague-Dawley rats (250–300 g) were obtained from Charles River Laboratories (Wilmington, MA). Diabetes was induced by streptozotocin (65 mg/kg) given via tail vein injection 2 weeks before study in an in vivo anesthetized rat model. Rats were given unlimited food and water and were not supplemented with insulin or antihyperglycemic agents. Blood glucose was assessed by a Prestige Smart System glucometer (Home Diagnostics). Rats were selected if blood glucose levels after 2 weeks were >500 mg/dl, which correlates to a poorly controlled human diabetic patient with moderately to severely elevated blood glucose levels. Animals were used for an in vivo anesthetized intact rat model of ischemia and reperfusion and prepared for experimentation each morning between 8:00 and 9:00 a.m. to avoid differences in circadian hormone release. Briefly, thiobutabarbital sodium (100 mg/kg i.p.; Inactin) was used for anesthesia, followed by a tracheotomy and artificial ventilation. The left common carotid artery was cannulated for blood pressure, heart rate, and blood gas measurements. A fifth–intercostal space thoracotomy was performed, the pericardium excised, and a ligature placed around the area of the left anterior descending coronary artery. Following surgical intervention and stabilization, rats were separated into groups.

To validate the rat diabetes model, a subset of DBR and NDBR rats (n = 6/group) were subjected to one cycle of ischemic preconditioning, consisting of 5 min of ischemia followed by 5 min of reperfusion. Ischemia was achieved by placing the two ends of the ligature around the left anterior descending coronary artery through a polypropylene tube and fixing the tube to the epicardial surface with a hemostat. Removal of the hemostat allowed for reperfusion of the area at risk (AAR). Rats were then subjected to 30 min of ischemia and 2 h of reperfusion. After 2 h of reperfusion, the ligature was again re-occluded and the AAR was determined by patent blue negative staining. The left ventricle was excised and cross-sectioned into four to five slices and further separated into the normal zone and AAR. Slices were incubated in 1% TTC (2,3,5-triphenylterazolium chloride) to determine infarct size. The heart was incubated overnight in 10% formaldehyde, and the infarcted tissue was dissected from the AAR and gravimetrically measured. Infarct size was expressed as a percent of the AAR.

After validation, DBR and NDBR rats were subjected to 30 min of ischemia and 2 h of reperfusion and subsets treated with either the nonselective opioid agonist morphine (0.3 mg/kg) or the putative GSK inhibitor SB216763 (0.6 mg/kg). These agents were given as a bolus 5 min before reperfusion. After 2 h of reperfusion, infarct size was assessed.

Hemodynamics, including heart rate, mean arterial pressure, and rate pressure product, were quantified during baseline, 15 min into ischemia, and at 2 h of reperfusion.

Additional experiments were performed to quantify the phosphorylation and total densitometry of myocardial proteins. Five minutes following reperfusion, hearts were excised and separated into normal and ischemic zones by patent blue negative staining. Tissue samples were prepared, and 30 μg of each ischemic zone tissue were used for immunoblotting and densitometry quantification, which was performed as previously described (13).

An immunoassay EIA kit (Assay Designs) was used to assess P-GSK3β Ser⁹ in the ischemic zone tissue (200 μg) 5 min after reperfusion, as per the manufacturer’s protocol. The amount of protein chosen achieved a value to preserve measurement accuracy within the calibration curve for the assay to preserve measurement accuracy. The amount was calculated as picograms of P-GSK3β per microgram of myocardial tissue.

The H9C2 cell line was obtained from American Type Culture Collection. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing either 5.56 mmol/l glucose (low glucose) or 25 mmol/l glucose (high glucose) with l-glutamine, pyridoxine HCl, and 1 mmol/l sodium pyruvate (Invitrogen). Both DMEM media were supplemented with 10% fetal bovine serum, 30 U/ml penicillin, 0.01 μg/ml streptomycin, and 2 μg/ml fungizone.

H9C2 cells were maintained in either a low-glucose DMEM or a high-glucose DMEM between three and six passages. Cells were grown to be ∼80% confluent on 60-mm dishes and were placed in respective serum-free low- or high-glucose DMEM for 24 h. Cells were either treated with vehicle (water) or with morphine (0.1 μmol/l) for 1–15 min. H9C2 cells were rapidly placed on ice, the media was removed, and the cells were washed twice in chilled PBS. Lysis buffer (50 mmol/l Tris, pH 7.5, 5 mmol/l EDTA, 10 mmol/l EGTA, 0.05 μl/mg Protease Inhibitor Cocktail-Sigma, 200 μmol/l sodium orthovanadate, 1 mmol/l phenylmethylsulfonyl fluoride, and 0.3% β-mercaptoethanol) was applied to cells and lysates centrifuged at 10,000g for 10 min to remove cellular debris, with 30 μg supernatants used for Western analysis. Results were tabulated as a percent change from the respective unstimulated cells.

All values were denoted as means ± SE. For animal studies and tissue experiments, statistical significance was determined by performing a one-way ANOVA with Bonferroni’s correction for multiplicity. For cell culture experiments, a two-tailed unpaired t test was used to assess significance. Values significantly different from vehicle were indicated by *P < 0.001 or #P < 0.05.

Eighty rats were used to obtain 67 successful experiments. In total, 13 rats were excluded: 3 due to ventricular fibrillation during reperfusion, 1 to acidosis, 1 to alkalosis, and 4 to marked hypotension and 2 to hypertensive status at baseline. In the diabetic group, rats were excluded if baseline blood glucose was <500 mg/dl (n = 2).

A decrease in myocardial protection was found in the diabetes model, since IPC-induced cardioprotection was significantly reduced in DBRs compared with NDBRs (49.2 ± 1.6 vs. 10.2 ± 1.3%*, respectively). DBRs showed a significant elevation of blood glucose compared with NDBRs (>500* vs. 174 ± 10 mg/dl, respectively). No differences were seen in left ventricular weight–to–body weight ratio for DBRs compared with NDBRs (2.57 ± 0.03 × 10⁻³ vs. 2.53 ± 0.03 × 10⁻³, respectively). No significant differences at baseline were found in arterial blood gases, including pH, pCO₂, and pO₂ between diabetic and nondiabetic rats.

Significant differences in baseline heart rate were present between the untreated DBRs and SB216763-treated DBRs compared with the vehicle-treated group (Table 1). A significant difference in heart rate was also evident between vehicle-treated DBRs and NDBRs during occlusion. Rate pressure product was also significantly different at reperfusion between the morphine-treated DBRs and vehicle-treated NDBRs.

No differences between groups were observed between the AAR to left ventricle weight (Fig. 1). The infarct size of DBRs was not significantly different than vehicle-treated NDBRs (55.6 ± 1.6 vs. 59.7 ± 1.7%, respectively). Morphine treatment in NDBRs reduced infarct size compared with vehicle-treated NDBRs (44.1 ± 1.4%*); however, it failed to significantly reduce infarct size in DBRs (54.7 ± 4.9%). Interestingly, the GSK inhibitor SB216763 reduced infarct size in both NDBRs and DBRs (43.6 ± 3.2* vs. 42.0 ± 2.6%*, respectively).

Immunoblots of GSK3β revealed that morphine-induced P-GSK3β Ser⁹ in NDBRs was abrogated in DBRs (173 ± 7* vs. 121 ± 12, respectively) without differences of P-GSK3β Ser⁹ between vehicle-treated NDBRs and DBRs (114 ± 9 vs. 133 ± 11, respectively) (Fig. 2). Enzyme immunosorbent assay showed morphine elevated P-GSK3β Ser⁹ in NDBRs, with no changes observed for morphine-treated DBRs or untreated NDBRs or DBRs (1.94 ± 0.12#, 0.83 ± 0.29, 1.04 ± 0.10, and 0.89 ± 0.23 pg P-GSK3β/μg tissue, respectively). No significant differences between groups were identified for P-GSK3β Tyr¹¹⁶ or total GSK3β (P-GSK3β Tyr¹¹⁶: 165 ± 20, 152 ± 26, 153 ± 17, and 152 ± 26, respectively; GSK3β: 101 ± 9, 137 ± 24, 149 ± 18, and 131 ± 26, respectively).

Immunoblots of P-JAK2 Tyr^1,007/1,008 displayed no differences between vehicle-treated NDBRs and DBRs (140 ± 8 vs. 132 ± 9, respectively) (Fig. 3). Morphine treatment in NDBRs significantly increased P-JAK2 Tyr^1,007/1,008 with this effect diminished in DBRs (189 ± 10# vs. 108 ± 15, respectively). No differences were present in total JAK2 (168 ± 12, 153 ± 24, 153 ± 14, and 162 ± 14, respectively).

A significant reduction of P-STAT3 Tyr⁷⁰⁵ occurred in vehicle-treated DBRs versus NDBRs (61 ± 12# vs. 127 ± 14, respectively). Morphine also significantly induced P-STAT3 Tyr⁷⁰⁵ in NDBRs, an effect significantly abrogated in DBRs (190 ± 15# vs. 84 ± 8, respectively). Measurement of total STAT3 showed no differences between groups (213 ± 1, 187 ± 20, 205 ± 7, and 155 ± 36, respectively).

Analysis of P-STAT1 Tyr⁷⁰¹ showed no significant differences between vehicle- or morphine-treated NDBRs and DBRs (118 ± 6, 93 ± 7, 123 ± 11, and 113 ± 6, respectively). However, total STAT1 was reduced in DBRs and morphine-treated DBRs compared with vehicle- and morphine-treated NDBRs (87 ± 12#, 83 ± 10#, 159 ± 16, and 164 ± 20, respectively).

P-Akt Ser⁴⁷³ in vehicle-treated groups was significantly blunted in DBRs compared with NDBRs (80 ± 7# vs. 130 ± 13, respectively) (Fig. 4). Morphine-treated NDBRs elevated P-Akt Ser⁴⁷³, which did not occur in DBRs (179 ± 14* vs. 70 ± 12#, respectively). No differences were seen in P-Akt Thr³⁰⁸ or total Akt between vehicle- and morphine-treated NDBRs and DBRs (P-Akt Thr³⁰⁸: 154 ± 14, 124 ± 8, 164 ± 9, and 140 ± 18, respectively; Akt: 183 ± 3, 182 ± 2, 180 ± 1, and 186 ± 4, respectively).

No differences in P-p70s6 Thr⁴²¹/Ser⁴²⁴ or P-p70s6 Thr³⁸⁹ were evident between NDBRs, DBRs, and morphine-treated DBRs (P-p70s6 Thr⁴²¹/Ser⁴²⁴: 118 ± 16, 109 ± 11, and 130 ± 10, respectively; P-p70s6 Thr³⁸⁹: 98 ± 3, 98 ± 4, and 101 ± 7, respectively). However, morphine significantly increased P-p70s6 Thr⁴²¹/Ser⁴²⁴ and P-p70s6 Thr³⁸⁹ in NDBRs (163 ± 12# and 127 ± 7#, respectively). No differences were seen between groups for total p70s6 (173 ± 23, 164 ± 25, 157 ± 8, and 146 ± 19, respectively).

A reduction of P-ERK1 Thr²⁰²/Tyr²⁰⁴ was found for untreated DBRs versus NDBRs (65 ± 14# vs. 128 ± 16, respectively) (Fig. 5). Morphine induced P-ERK1 Thr²⁰²/Tyr²⁰⁴ in NDBRs and DBRs, however, P-ERK1 Thr²⁰²/Tyr²⁰⁴ in DBRs did not achieve a level compared with that in NDBRs (194 ± 5# vs. 119 ± 18, respectively). No changes were found in total ERK1, P-ERK2 Thr²⁰²/Tyr²⁰⁴, and total ERK2 between vehicle- and morphine-treated NDBRs and DBRs (ERK1: 80 ± 6, 84 ± 3, 86 ± 3, and 85 ± 2, respectively; P-ERK2 Thr²⁰²/Tyr²⁰⁴: 167 ± 12, 131 ± 26, 185 ± 8, and 178 ± 9, respectively; ERK2: 95 ± 10, 104 ± 3, 102 ± 6, and 99 ± 5, respectively).

No differences in morphology or proliferation rate were present between low glucose–treated and high glucose–treated H9C2 cells (Fig. 6). Morphine stimulated P-GSK3β Ser⁹, P-Akt Ser⁴⁷³, P-STAT3 Tyr⁷⁰⁵, and P-ERK1 Thr²⁰²/Tyr²⁰⁴ in low glucose–treated cells, which was significantly different from the time course response in high glucose–treated cells, particularly 15 min after morphine application (P-GSK3β: 148 ± 18# vs. 87 ± 10; P-Akt: 177 ± 28# vs. 89 ± 16; P-STAT3: 161 ± 14# vs. 57 ± 9; P-ERK1: 235 ± 35# vs. 118 ± 4%, respectively, percent change in densitometry from unstimulated cells). No differences in total protein expression between low- and high-glucose H9C2 cells were evident for the proteins studied.

The present data suggest that pharmacological inhibition of GSK3β may provide a novel therapeutic strategy to reduce infarct size in diabetic patients when administered at the time of reperfusion. Furthermore, morphine-induced cardioprotection is abrogated in diabetic rats with altered components of the JAK/STAT, PI3k, and MAPK signaling pathways that inhibit GSK3β (13,14,18). Results in H9C2 cells also indicate that similar alterations of morphine-induced signaling occur in the presence of hyperglycemia.

Experimental models suggest the pathologic state of diabetes is related to elevated expression of GSK in both humans and animals. Muscle biopsies taken from human non–insulin-dependent diabetic patients (type 2 diabetes) and nondiabetic patients show that elevated GSK activity occurs in type 2 diabetes biopsies compared with nondiabetes (19). Chronic pharmacological inhibition of GSK lowers elevated blood glucose in diabetes-prone Zucker diabetic fatty rats and db/db mice (20,21). Acute application of the GSK inhibitor SB216763 in the present study did not effect blood glucose levels up to 2 h after reperfusion, perhaps since the dose of GSK inhibitor used in our study was 50 times less.

IPC fails to induce cardioprotection in many diabetes models, including Zucker fatty diabetic rats, Goto-Kakizaki lean diabetic rats, and streptozotocin-induced diabetic animals (4,8,22). Indeed, possible alterations in GSK3β signaling within different animal models may be the cause of altered IPC or pharmacological-induced cardioprotection, since, recently, a cardiac-specific mouse with a constitutively active GSK3β failed to respond to hypoxic preconditioning and pharmacological-induced cardioprotection (11).

Previous studies suggest that ≤2 weeks duration of streptozotocin-induced diabetes alone is protective in rats (8,23), while others have shown deleterious or no effects after 1 week (7). The differences found in cardioprotection may be due to the duration of diabetes, dose of streptozotocin used, serum glucose before initiation of ischemia, and, in isolated heart experiments, the concentration of glucose used in the perfusion buffer. The present data suggest that streptozotocin-induced diabetes at 2 weeks does not effect infarct size compared with vehicle-treated rats in vivo.

The infarct size reduction afforded by morphine and the selective GSK inhibitor SB216763 were equivalent in nondiabetic rats, whereas only the GSK inhibitor reduced infarct size in diabetic rats. The baseline decreases in heart rate for the diabetic groups compared with the vehicle-treated nondiabetic rats is due to less tolerance of the diabetic rats to barbiturate anesthetic. However, the decreased heart rate did not effect infarct size since DBR and NDBR infarct size is equivocal. In addition, there are also marked differences in infarct size between the diabetic and diabetic with GSK inhibitor groups, where mean baseline heart rates were comparable yet significantly lower than the nondiabetic groups. These findings indicate that GSK and its upstream mediators could be altered in diabetes, which abrogates the ability of morphine to reduce infarct size. Indeed, reduced phosphorylation of GSK3β Ser⁹ by morphine, which is required for its inactivation, was found at 5 min of reperfusion in diabetic rats, as assessed by both semiquantitative Western analysis and quantitative immunoassay analysis. Furthermore, the Tyr¹¹⁶ site of GSK3β, normally required for GSK3β to be active, was found unchanged in the present study, as well as levels of total GSK3β. These findings indicate that the alteration involved in diabetes causes reduced inactivation of GSK3β, at the Ser⁹ site rather than enhanced activation via phosphorylation at the Tyr¹¹⁶ site. Analysis of the coding region of GSK3β in type 2 diabetic patients found no causal link between GSKβ mutations and the development of type 2 diabetes (24). These findings interpreted in the realm of our present findings may suggest that both posttranscriptional modification of GSK3β and proteins that target GSK3β may contribute to the diabetic state and resistance to myocardial ischemia. Although the kinase activity of GSK3β was not measured in this study, a previous report has demonstrated that changes in phosphorylation at Ser⁹ for GSK3β produces a decreased activity in isolated rat hearts, with only a 25% reduction of GSK3β needed for IPC-induced infarct size reduction to occur due to the normally high basal activity of GSK3β (25).

The JAK/STAT signaling pathway (specifically, activation by phosphorylation of JAK2/STAT3) is an upstream mediator of GSK3β in opioid-induced cardioprotection at reperfusion (14). STAT1 activation, detrimental to cardioprotection, has also been demonstrated (26). These present data suggest an alteration in the balance between JAK2, STAT3, and STAT1 in the diabetic myocardium, where levels of phosphorylated STAT3 and total STAT1 at reperfusion were significantly reduced in DBRs compared with NDBRs. Previous findings indicate that genetic manipulation of STAT3 directly affects the diabetic state (27,28). More definitive studies will be needed to discern whether overexpression of JAK2 or STAT3 or inhibition of STAT1 can reverse the diabetes-induced blockade of cardioprotection.

Mice lacking the PI3k pathway protein Akt2 develop insulin resistance and diabetes (29,30). Our current findings indicate a significant reduction of P-Akt at 5 min of reperfusion in untreated diabetic hearts compared with nondiabetic hearts, similar to a previous study in nonischemic hearts (12). The phosphorylation sites investigated traditionally increase activation of Akt and p70s6. Since this was the first study to examine opioid-induced signaling, we did not assume that one specific site of Akt or p70s6 kinase was modified by opioids and investigated both sites. Morphine induced the elevation of P-Akt Ser⁴⁷³ and P-p70s6 Thr⁴²¹/Ser⁴²⁴ or Thr³⁸⁹ at reperfusion, which was also diminished in DBRs compared with NDBRs. A study in nonischemic DBR hearts previously showed that insulin-induced phosphorylation of Akt is abrogated in DBRs, which supports our current findings (12).

The same study found no effects on phosphorylation of the MAPK proteins (ERK1 or ERK2) between untreated or insulin-treated DBRs or NBDRs (12), in contrast to a decrease in ERK1 phosphorylation in untreated DBRs compared with NDBRs for our model. Differences between our current study and previous findings may be due to a variation of the experimental streptozotocin-induced diabetes window. Our data would also suggest that ERK1 is phosphorylated by morphine in the diabetes environment, which causes a recovery of phosphorylation trending toward the level for the vehicle group. This suggests that morphine-induced phosphorylation of ERK1 is still functional, and the mechanism of ERK1 abrogation is different than that of Akt, which showed no degree of phosphorylation recovery after morphine administration.

Findings in H9C2 cells paralleled those found in DBRs and suggest that a reduction of phosphorylation present in DBRs is due to hyperglycemia. Defective activation of Akt by insulin has also been found in cell lines incubated in high glucose (31). Incubation of H9C2 cells with elevated glucose (33 mmol/l) also significantly increased reactive oxygen species compared with H9C2 cells incubated in low concentrations of glucose (5.5 mmol/l) (32).

The results of this study need to be interpreted with potential limitations, including the application to humans. The streptozotocin-induced diabetes model more closely parallels type 1 diabetes, instead of type 2 diabetes; however, cardioprotection afforded by IPC can be abrogated as effectively in type 2 diabetes rat models such as the GK and ZFD rats (22). Hence, although the model of induction differs, these findings may lead to potential targets to further examine in type 2 diabetes models. The glucometer used for this study did not measure >600 mg/dl, and in half of the diabetic rats, the blood glucose value was higher than the sensitivity of the instrument. Short-term inhibition of GSK during a myocardial infarction may be an effective and novel strategy to reduce the extent of myocardial injury in diabetic patients; however, prophylactic or chronic use of a GSK inhibitor for myocardial infarction may be contraindicated since inhibition could potentially increase the incidence of cancer (33). The ability for GSK inhibition to reduce infarct size will need to be further investigated in diabetic female mice, since transgenic skeletal muscle overexpression of human GSK3β results in significant glucose intolerance only in male mice (34). This suggests that GSK inhibition may not be as effective in female mice since a different mechanism may contribute to the diabetic state.

In conclusion, three pathways essential for acute cardioprotection, JAK/STAT, PI3k, and MAPK, are altered during streptozotocin-induced diabetes. Hyperglycemia and diabetes also reduce the ability of morphine to phosphorylate components of these pathways, and morphine-induced infarct size reduction is less effective in diabetic subjects due to deficient upstream signaling alterations that adversely effect GSK3β signaling (Fig. 7). These data also suggest that acute GSK inhibition may provide a novel therapeutic strategy for treating diabetic patients during a myocardial infarction.

This work was supported by National Institutes of Health grants HL08311 and HL074314 (to G.J.G.) and an American Heart Predoctoral Fellowship (Northland; to E.R.G.).