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Diabetes 56:127-136, 2007
DOI: 10.2337/db06-0907
© 2007 by the American Diabetes Association
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Diabetes Abolishes Morphine-Induced Cardioprotection via Multiple Pathways Upstream of Glycogen Synthase Kinase-3ß

Eric R. Gross, Anna K. Hsu, and Garrett J. Gross

From the Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin

Address correspondence and reprint requests to Garrett J. Gross, PhD, Medical College of Wisconsin, Department of Pharmacology and Toxicology, 8701 Watertown Plank Rd., Milwaukee, WI 53226. E-mail: ggross{at}mcw.edu

Abbreviations: AAR, area at risk; DMEM, Dulbecco’s modified Eagle’s medium; GSK, glycogen synthase kinase; IPC, ischemic preconditioning; JAK, janus-activated kinase; MAPK, mitogen-activated protein kinase; P-, phospho-; PI3k, phosphatidylinositol-3 kinase; STAT, signal transducer and activator of transcription


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cardioprotective effect of opioids or glycogen synthase kinase (GSK) inhibitors given at reperfusion has not been investigated in diabetes models. Therefore, nondiabetic (NDBR) or streptozotocin-induced diabetic (DBR) rat hearts were subjected to 30 min of ischemia and 2 h of reperfusion. Groups of NDBR or DBR were administered either vehicle, morphine (0.3 mg/kg), or the GSK inhibitor SB216763 (0.6 mg/kg) 5 min before reperfusion. SB216763 (but not morphine) reduced infarct size in DBRs (44 ± 1* and 55 ± 2%, respectively), while both agents reduced infarct size in NDBRs versus untreated NDBRs or DBRs (44 ± 3*, 42 ± 3*, 60 ± 2, and 56 ± 2%, respectively, *P < 0.001). Morphine-induced phospho- (P-)GSK3ß was reduced 5 min after reperfusion in DBRs compared with NDBRs (0.83 ± 0.29 and 1.94 ± 0.12 [P < 0.05] pg/µg tissue, respectively). The GSK3ß mediators, P-Akt, P–extracellular signal–related kinase (ERK)1, and P–signal transducer and activator of transcription (STAT)3, were also significantly reduced in untreated DBR compared with NDBR rats. Morphine-induced elevations of P-Akt, P-ERK1, P-p70s6, P–janus-activated kinase-2, and P-STAT3 in NDBRs were also blunted in DBRs. H9C2 cells raised in 25 mmol/l compared with 5.56 mmol/l glucose media also demonstrated reduced morphine-induced P-GSK3ß, P-Akt, P-STAT3, and P-ERK1 after 15 min. Hence, acute GSK inhibition may provide a novel therapeutic strategy for diabetic patients during an acute myocardial infarction, whereas morphine is less effective due to signaling events that adversely affect GSK3ß.

Two of three diabetic patients experience a stroke or a heart attack. The relative risk of myocardial infarction also correlates with the level of hyperglycemia, even in nondiabetic patients (1,2). The ability to reduce myocardial injury by brief ischemic periods, known as ischemic preconditioning (IPC), is also abolished in human diabetic patients (3). Experimental animal models also indicate that diabetes abrogates the ability for IPC and pharmacological agents to reduce infarct size (4–8).

The mechanism of how diabetes abrogates cardioprotection is not clear. No studies have quantified the expression and/or phosphorylation of proteins in diabetic patients that are essential for acute cardioprotection resulting from timely reperfusion. One possible target is proteins that modulate mitochondrial function, since a reversal of superoxide production generated by the mitochondria reduces multiple pathological features of diabetes (9). Scavenging of hyperglycemia-induced myocardial oxygen-derived free radical production can also reverse hyperglycemia-induced shear stress reduction (10). Therefore, it is likely that upstream changes in myocardial signaling occurs during diabetes and hyperglycemia, which affects the mitochondria.

Two mitochondrial sites of action important in acute cardioprotection include the mitochondria permeability transition pore and the mitochondria ATP-regulated potassium channel. Both sites interact with the multifunctional protein, glycogen synthase kinase (GSK)3ß, which is essential for acute cardioprotection at the time of reperfusion by improving cellular protection from free radicals (11). In this regard, diabetes alters insulin signaling and reduces the ability of insulin to phosphorylate and, hence, inactivate GSK3ß at Ser9 in rat hearts (12). GSK3ß is a pivotal convergence point of multiple cellular pathways including tyrosine kinase, janus-activated kinase (JAK) and signal transducer and activator of transcription (STAT), phosphatidylinositol-3 kinase (PI3k), protein kinases A and C, mitogen-activated protein kinase (MAPK), and the ATP-sensitive K+ channel (11,13,14).

It is unknown whether morphine, an opiate commonly administered during an acute myocardial infarction, or a pharmacological GSK inhibitor, such as SB216763, are effective acute therapeutic strategies to reduce the extent of a myocardial infarction in diabetic patients when administered at the time of reperfusion. Therefore, this study characterized the cardioprotective ability of morphine and SB216763 in streptozotocin-induced diabetic rats (DBRs) compared with nondiabetic rats (NDBRs). Furthermore, this study examined the protein status during early reperfusion of the myocardial signaling pathways previously found to contribute to acute cardioprotection, including JAK/STAT, PI3k, and MAPK, in morphine- or vehicle-treated DBRs or NDBRs (13–15). The H9C2 cell line, which has a similar glucose transport system as rat and mouse hearts, was also used to examine the effects of hyperglycemia on morphine signaling (16,17).


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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.

Pharmacological agents.
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.

Antibodies.
The primary antibodies used were for P-GSK3ß Ser9 and Tyr116, P-JAK-2 Tyr1,007/1,008, P-STAT1 Tyr701, P-STAT3 Tyr705, P-Akt Ser473 and Thr308, P-p70s6 Thr359 and Thr421/Ser424, P-ERK1/P-ERK2 Thr202/Tyr204, GSK3ß, JAK2, STAT1, STAT3, Akt, and p70s6. All primary antibodies were purchased from Cell Signaling, except for GSK3ß 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 GSK3ß 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).

Experimental protocol for animal experiments.
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.

Infarct size studies.
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.
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.

Protein analysis of diabetic and nondiabetic tissue.
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-GSK3ß 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-GSK3ß per microgram of myocardial tissue.

H9C2 cell hyperglycemia model.
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% ß-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.

Statistical measurements.
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.


   RESULTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
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 x 10–3 vs. 2.53 ± 0.03 x 10–3, respectively). No significant differences at baseline were found in arterial blood gases, including pH, pCO2, and pO2 between diabetic and nondiabetic rats.

Hemodynamics.
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.


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  		TABLE 1 Hemodynamics for acute opioid and SB216763 experiments at baseline, at 15 min of ischemia, and at 2 h of reperfusion in nondiabetic and diabetic rats

 
Infarct size studies.
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).


Figure 1
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  		FIG. 1. A: AAR as a percent of left ventricular (LV) weight. B: Infarct (IF) size expressed as a percent of the AAR. C: Representative digital images of infracted tissue (white/unstained) within the AAR. n values are in parentheses. D, diabetic; M, morphine; SB, SB216763; V, nondiabetic vehicle. *P < 0.001.

 
Analysis of diabetic and nondiabetic tissue
GSK3ß.
Immunoblots of GSK3ß revealed that morphine-induced P-GSK3ß Ser9 in NDBRs was abrogated in DBRs (173 ± 7* vs. 121 ± 12, respectively) without differences of P-GSK3ß Ser9 between vehicle-treated NDBRs and DBRs (114 ± 9 vs. 133 ± 11, respectively) (Fig. 2). Enzyme immunosorbent assay showed morphine elevated P-GSK3ß 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-GSK3ß/µg tissue, respectively). No significant differences between groups were identified for P-GSK3ß Tyr116 or total GSK3ß (P-GSK3ß Tyr116: 165 ± 20, 152 ± 26, 153 ± 17, and 152 ± 26, respectively; GSK3ß: 101 ± 9, 137 ± 24, 149 ± 18, and 131 ± 26, respectively).


Figure 2
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  		FIG. 2. Representative blots and relative densitometry of P-GSK3ß and GSK3ß in nondiabetic (V), diabetic (D), morphine (M), and diabetic plus morphine (M+D) groups for P-GSK3ß Ser9 (n = 7/group), P-GSK3ß Tyr116 (n = 3/group), and GSK3ß (n = 6/group). Amount of P-GSK3ß Ser9 in pg/µg of tissue (n = 4–5/group; bottom left). *P < 0.001; #P < 0.05.

 
JAK/STAT pathway.
Immunoblots of P-JAK2 Tyr1,007/1,008 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 Tyr1,007/1,008 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).


Figure 3
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  		FIG. 3. Reperesentative blots and relative densitometry values for JAK2, STAT1, and STAT3 in nondiabetic (V), diabetic (D), morphine (M), and diabetic plus morphine (M+D) groups for P-JAK2 Tyr1,007/1,008 (n = 6/group), JAK2 (n = 3/group), P-STAT3 Tyr705 (n = 3–5/group), STAT3 (n = 3/group), P-STAT1 Tyr701 (n = 3–7/group), and STAT1 (n = 5–6/group). #P < 0.05.

 
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).

PI3k pathway.
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).


Figure 4
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  		FIG. 4. Representative blots and relative densitometry levels in nondiabetic (V), diabetic (D), morphine (M), and diabetic plus morphine (M+D) groups for P-Akt Ser473 (n = 6/group), P-Akt Thr308 (n = 3/group), Akt (n = 3/group), P-p70s6 Thr421/Ser424 (n = 5/group), P-p70s6 Thr359 (n = 3/group), and p70s6 (n = 3/group). #P < 0.05.

 
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).

MAPK pathway.
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).


Figure 5
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  		FIG. 5. Representative blots and relative densitometry levels at 5 min of reperfusion in nondiabetic (V), diabetic (D), morphine (M), and diabetic plus morphine (M+D) groups for P-ERK1 (n = 6/group), P-ERK2 (n = 6/group), ERK1 (n = 3/group), and ERK2 (n = 3/group). #P < 0.05.

 
H9C2 cell hyperglycemia model.
No differences in morphology or proliferation rate were present between low glucose–treated and high glucose–treated H9C2 cells (Fig. 6). Morphine stimulated P-GSK3ß 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-GSK3ß: 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.


Figure 6
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  		FIG. 6. Representative blots and percent change in phosphorylation from unstimulated cells versus the time-course response of morphine stimulation (0.1 µmol/l) from 1 to 15 min in low-glucose (LG; black lines) or high-glucose (HG; gray lines) incubated H9C2 cells for P-GSKß Ser9 (n = 7 time points/group), P-Akt Ser473 (n = 3 time points/group), P-STAT3 Tyr705 (n = 2 time points/group), and P-ERK1 (n = 3 time points/group). Significant difference compared with low- and high-glucose cells at a time point, #P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present data suggest that pharmacological inhibition of GSK3ß 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 GSK3ß (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 GSK3ß 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 GSK3ß 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 GSK3ß 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 GSK3ß, normally required for GSK3ß to be active, was found unchanged in the present study, as well as levels of total GSK3ß. These findings indicate that the alteration involved in diabetes causes reduced inactivation of GSK3ß, at the Ser9 site rather than enhanced activation via phosphorylation at the Tyr116 site. Analysis of the coding region of GSK3ß in type 2 diabetic patients found no causal link between GSKß 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 GSK3ß and proteins that target GSK3ß may contribute to the diabetic state and resistance to myocardial ischemia. Although the kinase activity of GSK3ß was not measured in this study, a previous report has demonstrated that changes in phosphorylation at Ser9 for GSK3ß produces a decreased activity in isolated rat hearts, with only a 25% reduction of GSK3ß needed for IPC-induced infarct size reduction to occur due to the normally high basal activity of GSK3ß (25).

The JAK/STAT signaling pathway (specifically, activation by phosphorylation of JAK2/STAT3) is an upstream mediator of GSK3ß 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 GSK3ß 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 GSK3ß 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.


Figure 7
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  		FIG. 7. Differences between morphine-induced cardioprotective signaling pathways in nondiabetic rats (A) compared with diabetic (B) rats. Phosphorylation leading to activation (+) of proteins upstream of GSK3ß leads to phosphorylation of GSK3ß (-) and inactivation, which ultimately leads to cardioprotection. STAT3, Akt, and ERK1 (double arrows) showed reduced phosphorylation in DBRs at reperfusion regardless of morphine administration. JAK2, p70s6 kinase, and GSK3ß proteins had decreased morphine-induced phosphorylation at reperfusion, without evident differences in phosphorylation for untreated diabetic and nondiabetic rats (single arrow). These alterations in morphine-induced signaling events leads to a continued activation of GSK3ß and failed morphine-induced cardioprotection in diabetic rats.

 


   ACKNOWLEDGMENTS
 
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.).


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received for publication July 3, 2006 and accepted in revised form October 13, 2006


   REFERENCES
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
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