Background Cardioprotection by volatile anesthetic-induced preconditioning (APC) involves activation of protein kinase C (PKC). The current study investigated the importance of APC-activated PKC in delaying mitochondrial permeability transition pore (mPTP) opening. Methods Rat ventricular myocytes were exposed to isoflurane in the presence or absence of nonselective PKC inhibitor chelerythrine or isoform-specific inhibitors of PKC-δ (rottlerin) and PKC-ε (myristoylated PKC-ε V1-2 peptide), and the mPTP opening time was measured using confocal microscopy. Ca2+-induced mPTP opening was measured in mitochondria isolated from rats exposed to isoflurane in the presence and absence of chelerythrine, or in mitochondria directly treated with isoflurane after isolation. Translocation of PKC-ε was assessed in APC and control cardiomyocytes by Western blotting. Results In cardiomyocytes, APC prolonged time necessary to induce mPTP opening (261±26 s APC vs. 216±27 s control; P<0.05), while chelerythrine abolished this delay to 213±22 s. The effect of isoflurane was also abolished when PKC-ε inhibitor was applied (210±22 s), but not in the presence of PKC-δ inhibitor (269±31 s). Western blotting revealed translocation of PKC-ε toward mitochondria in APC cells. The Ca2+ concentration required for mPTP opening was significantly higher in mitochondria from APC rats (45±8 μM mg-1 control vs. 64±8 μM mg-1 APC), and APC effect was reversed with chelerythrine. In contrast, isoflurane did not protect directly treated mitochondria. Conclusion APC induces delay of mPTP opening through PKC-ε-mediated inhibition of mPTP opening, but not through PKC-δ. These results point to the connection between cytosolic and mitochondrial components of cardioprotection by isoflurane.

Background Endothelial nitric oxide synthase activity is regulated by tetrahydrobiopterin (BH4) and heat shock protein 90. We tested the hypothesis that hyperglycemia abolishes anesthetic preconditioning (APC) through BH4- and heat shock protein 90-dependent pathways. Methods Myocardial infarct size was measured in rabbits in the absence or presence of APC (30 min of isoflurane), with or without hyperglycemia, and in the presence or absence of the BH4 precursor sepiapterin. Isoflurane-dependent nitric oxide production was measured (ozone chemiluminescence) in human coronary artery endothelial cells cultured in normal (5.5 mM) or high (20 mM) glucose conditions, with or without sepiapterin (10 or 100 µM). Results APC decreased myocardial infarct size compared with control experiments (26±6 vs 46±3%, respectively; P<0.05), and this action was blocked by hyperglycemia (43±4%). Sepiapterin alone had no effect on infarct size (46±3%) but restored APC during hyperglycemia (21±3%). The beneficial actions of sepiapterin to restore APC were blocked by the nitric oxide synthase inhibitior N (G)-nitro-L-arginine methyl ester (47±2%) and the BH4 synthesis inhibitor N-acetylserotonin (46±3%). Isoflurane increased nitric oxide production to 177±13% of baseline, and this action was attenuated by high glucose concentrations (125±6%). Isoflurane increased, whereas high glucose attenuated, intracellular BH4/BH2 (high-performance liquid chromatography), heat shock protein 90-endothelial nitric oxide synthase co-localization (confocal microscopy), and endothelial nitric oxide synthase activation (immunoblotting). Sepiapterin increased BH4/BH2 and dose-dependently restored nitric oxide production during hyperglycemic conditions (149±12 and 175±9%; 10 and 100 µM, respectively). Conclusion The results indicate that tetrahydrobiopterin and heat shock protein 90-regulated endothelial nitric oxide synthase activity play a central role in cardioprotection that is favorably modulated by volatile anesthetics and dysregulated by hyperglycemia. Enhancing the production of BH4 may represent a potential therapeutic strategy.

PF, Kersten JR. Decreased tetrahydrobiopterin and disrupted association of Hsp90 with eNOS by hyperglycemia impair myocardial ischemic preconditioning. Am J Physiol Heart Circ Physiol 301: H2130 -H2139, 2011. First published September 9, 2011 doi:10.1152/ajpheart.01078.2010.-Cardioprotection by ischemic preconditioning (IPC) is impaired during hyperglycemia, but the mechanisms underlying this phenomenon are poorly understood. This study investigated the role of hyperglycemia to adversely modulate tetrahydrobiopterin (BH 4) and heat shock protein 90 (Hsp90) during cardioprotection by IPC. Rabbits or mice underwent 30 min of coronary occlusion followed by reperfusion with or without IPC in the presence or absence of hyperglycemia. IPC significantly (P Ͻ 0.05) decreased myocardial infarct size (46 Ϯ 1 to 19 Ϯ 2% of the area at risk in control and IPC rabbits, respectively) and increased BH 4 concentrations (HPLC; 7.6 Ϯ 0.2 to 10.2 Ϯ 0.3 pmol/mg protein, respectively), Hsp90-endothelial nitric oxide synthase (eNOS) association (coimmunoprecipitation and Western blotting in mice; 4.0 Ϯ 0.3 to 5.4 Ϯ 0.1, respectively), and the ratio of phosphorylated eNOS/total eNOS. These beneficial actions of IPC on infarct size, BH 4, Hsp90/eNOS, and phosphorylated eNOS were eliminated by hyperglycemia. Pretreatment of animals with the Hsp90 inhibitor geldanamycin (0.6 mg/kg) or the BH4 synthesis inhibitor diamino-6-hydroxypyrimidine (1.0 g/kg) also eliminated cardioprotection produced by IPC. In contrast, the BH4 precursor sepiapterin (2 mg/kg iv) restored the beneficial effects of IPC on myocardial BH4 concentrations, eNOS dimerization, and infarct size during hyperglycemia. A-23871 increased Hsp90-eNOS association (0.33 Ϯ 0.06 to 0.59 Ϯ 0.3) and nitric oxide production (184 Ϯ 17%) in human coronary artery endothelial cells cultured in normal (5.5 mM) but not high (20 mM) glucose media. These data indicate that hyperglycemia eliminates protection by IPC via decreases in myocardial BH4 concentration and disruption of the association of Hsp90 with eNOS. The results suggest that eNOS dysregulation may be a central mechanism of impaired cardioprotection during hyperglycemia. hyperglycemia; ischemia reperfusion; tetrahydrobiopterin; heat shock protein; nitric oxide; endothelial nitric oxide synthase HYPERGLYCEMIA (HG) is an independent risk factor for increased cardiovascular morbidity and mortality (9, 12, 13). Previous studies have suggested that HG-associated risk of death is greater in patients with acute myocardial infarction who do not have antecedent diabetes than in those with diabetes, although the responsible mechanisms are not entirely clear (9, 32, 52). Available evidence indicates that endogenous cardioprotective mechanisms such as ischemic preconditioning (IPC) and pharmacological activation of cardioprotective signal transduction pathways are eliminated by HG in animals and in humans (2,16,26,29,31). We tested the hypothesis that regulation of endothelial nitric oxide synthase (eNOS) by its cofactors and protein chape...

Increased BH(4) by cardiomyocyte-specific overexpression of GTPCH-1 preserves the ability of IPC to elicit myocardial and mitochondrial protection that is impaired by HG, and this action appears to be dependent on NO.

Summary Previous studies have suggested that exogenous nitric oxide (NO) and NO-dependent signalling pathways modulate intracellular pH (pHi) in different cell types, but the role of NO in pHi regulation in the heart is poorly understood. Therefore, in this study we investigated the effect of NO donors S-nitroso-N-acetyl-DL-penicillamine, Spermine and PAPA NONOate on pHi in isolated rat ventricular myocytes.The cells were isolated from the hearts of adult Wistar rats, and pHi was monitored using a pH-sensitive fluorescent indicator 5-(and-6)-carboxy SNARF-1 with a confocal microscope. To test the effect of NO donors on sodium-hydrogen exchanger, basal pHi in Na+-free buffer and pHi recovery from intracellular acidosis after an ammonium chloride prepulse were monitored. The role of carbonic anhydrase was tested using acetazolamide. Cl−-OH− and Cl−-HCO3− exchangers were inhibited with 4,4 diisothiocyanatostilbene 2,2' disulfonic acid.All three NO donors acutely decreased pHi. This effect lasted until NO donor was removed. In a Na+-free buffer decrease in basal pHi was increased, while inhibition of carbonic anhydrase and Cl−-OH− and Cl−-HCO3− exchangers did not change the effect of NO donors on pHi. After an ammonium preload, pHi recovery was accelerated in the presence of NO donors.In conclusion, exogenous NO decreased the basal pHi leading to increased activity of sodium-hydrogen exchanger. Carbonic anhydrase and chloride-dependent sarcolemmal HCO3− and OH− transporters are not involved in the NO-induced pHi decrease in isolated rat ventricular myocytes.