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OBJECTIVEAlteration in endoplasmic reticulum (ER) stress in diabetic hearts and its effect on cytoprotective signaling are unclear. Here, we examine the hypothesis that ER stress in diabetic hearts impairs phospho–glycogen synthase kinase (GSK)-3β–mediated suppression of mitochondrial permeability transition pore (mPTP) opening, compromising myocardial response to cytoprotective signaling.RESEARCH DESIGN AND METHODSA rat model of type 2 diabetes (OLETF) and its control (LETO) were treated with tauroursodeoxycholic acid (TUDCA) (100 mg · kg−1 · day−1 for 7 days), an ER stress modulator. Infarction was induced by 20-min coronary occlusion and 2-h reperfusion.RESULTSLevels of ER chaperones (GRP78 and GRP94) in the myocardium and level of nonphoshopho–GSK-3β in the mitochondria were significantly higher in OLETF than in LETO rats. TUDCA normalized levels of GRP78 and GRP94 and mitochondrial GSK-3β in OLETF rats. Administration of erythropoietin (EPO) induced phosphorylation of Akt and GSK-3β and reduced infarct size (% risk area) from 47.4 ± 5.2% to 23.9 ± 3.5% in LETO hearts. However, neither phosphorylation of Akt and GSK-3β nor infarct size limitation was induced by EPO in OLETF rats. The threshold for mPTP opening was significantly lower in mitochondria from EPO-treated OLETF rats than in those from EPO-treated LETO rats. TUDCA restored responses of GSK-3β, mPTP opening threshold, and infarct size to EPO receptor activation in OLETF rats. There was a significant correlation between mPTP opening threshold and phospho–GSK-3β–to–total GSK-3β ratio in the mitochondrial fraction.CONCLUSIONSDisruption of protective signals leading to GSK-3β phosphorylation and increase in mitochondrial GSK-3β are dual mechanisms by which increased ER stress inhibits EPO-induced suppression of mPTP opening and cardioprotection in diabetic hearts.

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Rationale: The diabetic heart is resistant to ischemic preconditioning because of diabetes-associated impairment of phosphatidylinositol 3-kinase (PI3K)-Akt signaling. The mechanism by which PI3K-Akt signaling is impaired by diabetes remains unclear. Objective: Here, we examined the hypothesis that phosphorylation of iabetes mellitus not only accelerates atherosclerosis of the coronary artery but also induces functional and structural abnormalities in the myocardium. In addition, recent studies have shown that myocardial response to ischemic preconditioning (IPC) and its mimetics is blunted or lost in diabetic hearts. 1-3 Protection afforded by IPC is triggered by activation of G protein-coupled receptors 5 and impaired activation of phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) in models of diabetes mellitus have been reported. 1-3 Our recent study 3 showed impaired activation of Jak2 and endoplasmic reticulum (ER) stress-mediated disruption of signaling from ERK to glycogen synthase kinase (GSK)-3␤ in diabetic hearts. However, the mechanism by which Jak2-mediated signaling is disabled in diabetic hearts remains unclear. In the present study, we tested the hypothesis that Jak2-mediated protection is impaired in diabetic myocardium by an angiotensin II type 1 (AT 1 ) receptor-mediated mechanism via upregulation of SOCS3 (suppressor of cytokine signaling 3)-Jak2 interaction or calcineurin. MethodsMale Otsuka-Long-Evans-Tokushima fatty (OLETF) rats, which spontaneously develop obesity and type 2 diabetes, and their controls (Long-Evans-Tokushima-Otsuka [LETO] rats) were used. Preparation of myocardial infarction, immunoblotting, quantitative real-time RT-PCR, calcineurin activity assay, and determination of insulin sensitivity were performed by standard methods (see the expanded Methods section in the Online Data Supplement, available at

Ischemic preconditioning (PC) suppresses chemical coupling of cardiomyocytes via gap junctions (GJs) during ischemia, which is an adjunct mechanism of protection. The aim of this study was to characterize roles of protein kinases in PC-induced GJ modulation. In isolated rat hearts, ventricular tissues were sampled before and after ischemia with or without PC, and intercalated disc-rich fractions were separated for immunoprecipitation and immunoblotting. Levels of protein kinase C (PKC)-ε, p38mitogen-activated protein kinase (MAPK)-α, and Src coimmunoprecipitated with connexin-43 (Cx43) were increased after ischemia, whereas p38MAPKβ was not detected in the Cx43 immunoprecipitates. PC did not modify the level of Cx43-Src complex after ischemia. However, PC enhanced Cx43-PKCε complex formation, which was abolished by PKCε translocation inhibitory peptide (TIP). In contrast, PC reduced Cx43-p38MAPKα complex level and p38MAPK activity in the Cx43 immunoprecipitates after ischemia. The effect of PC on Cx43-p38MAPKα interaction was mimicked by SB-203580, a p38MAPK inhibitor. PC reduced permeability of GJs to Lucifer yellow in the myocardium at 25 min after ischemia, and this effect was abolished by PKCε-TIP. SB-203580 increased the GJ permeability at 15 min after ischemia compared with that in untreated controls, but the difference became insignificant 25 min after ischemia. In conclusion, PC has distinct effects on interaction of GJ Cx43 with PKCε, p38MAPKα, and Src during ischemia. Suppression of GJ permeability during ischemia by PC is primarily achieved by enhanced interaction of Cx43 with PKCε, which overwhelms the counterbalancing effect of reduced Cx43-p38MAPKα interaction.