The goals of this project are to elucidate signaling mechanisms involved in cardioprotection. To determine whether erythropoietin (EPO) is cardioprotective, isolated rat hearts were perfused for 10 minutes in the Langendorff-mode with Krebs-Henseleit buffer in the absence or presence of brief recombinant EPO treatment while left-ventricular-developed pressure (LVDP) was measured. The hearts were then subjected to 20 minutes of normothermic global ischemia followed by 25 minutes of reperfusion. The post-ischemic recovery of LVDP in the untreated control hearts was 26+/- 5% of their baseline LVDP, whereas hearts pre-treated with EPO exhibited significantly improved post-ischemic recovery to 57+/- 7. EPO treatment also significantly reduced infarct size compared to control hearts. Perfusion with the mitogen/extracellular signal-regulated kinase (MEK) inhibitor U0126 just prior to and concomitant with EPO treatment abolished EPO-induced phosphorylation of the MEK substrate extracellular signal-regulated kinase (ERK) but had no effect of EPO-mediated cardioprotection. EPO treatment of the perfused hearts induced translocation of protein kinase C (PKC) epsilon; isoform to the membrane fraction of the hearts and the protective effect of EPO was significantly inhibited by the PKC catalytic inhibitor chelerythrine added prior to and concomitant with EPO. These data demonstrate that EPO-mediated activation of the PKC signaling pathway prior to ischemia is required for the cardioprotective effect of EPO during ischemia-reperfusion injury. Perfusion with the phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 or wortmannin just prior to and concomitant with EPO treatment attenuated EPO-induced phosphorylation of the PI3K substrate Akt but had no effect on EPO-mediated cardioprotection. However, when wortmannin was added during EPO treatment and continued during reperfusion, EPO-mediated cardioprotection was significantly inhibited. We also show that post-ischemia EPO treatment at the onset of reperfusion significantly improved recovery of LVDP and reduced infarct size. Post-ischemia cardioprotection by EPO required the PI3K pathway but was not affected by inhibition of PKC at the time of EPO treatment. To investigate the mechanisms involved in PKC mediated protection we performed additional studies. As PKC has been well documented to modulate sarcoplasmic reticulum (SR) Ca2+, and since altered SR Ca2+ handling during ischemia is involved in cardioprotection, we examined the role of PKC mediated alterations of SR Ca2+ in cardioprotection. Using isolated adult rat ventricular myocytes we found that addition of 1,2-dioctanoyl-sn-glycerol (DOG), to activate PKC under conditions that reduced myocyte death following simulated ischemia and reperfusion, also reduced SR Ca2+. Cell death was 57.9 +/- 2.9 % in untreated vs. 47.3 +/- 1.8 % in DOG treated myocytes. We examined the effect of DOG on SR Ca2+ using caffeine-releasable SR Ca2+. Caffeine-releasable SR Ca2+ was significantly reduced after 10 min of DOG treatment compared to untreated myocytes. We examined the mechanism by which PKC alters SR Ca2+ and present the novel finding that DOG treatment reduced the phosphorylation of phospholamban (PLB) at Ser16. This effect is mediated by PKC-epsilon, since a PKC-epsilon selective inhibitory peptide blocked the DOG mediated decrease in phosphorylation of PLB. Using immunoprecipitiation, we further demonstrated that DOG increased the association between protein phosphatase-1 (PP-1) and PLB. These data suggest that activated PKC-epsilon reduces SR Ca2+ content through PLB dephosphorylation, and that reduced SR Ca2+ may be important in cardioprotection We also examined the role of G protein-coupled receptors (GPCRs) in the protection afforded by ischemic preconditioning (PC). We found that transgenic mice with cardiac-specific overexpression of a Gbeta-gamma-sequestering peptide, BARKct (TG bARKct mice) were not protected by PC, suggesting the protection of PC is Gbeta-gamma -dependent. Recent studies suggest a role for Gbeta-gamma in GPCR internalization. We found that bARKct, a specific PI3K inhibitor wortmannin, and bafilomycin A1, which all block the receptor recycling, all blocked the protective effects of PC. We also examined the role of the Beta2-adrenergic receptor (Beta2-AR) in cardioprotection. To test the role of Beta2-AR in PC, we studied mice lacking Beta2-AR (Beta2-AR-/-), and found that PC had no effect on postischemic LVDP or infarct size in Beta2-AR-/-. These data show an important role for Beta2-AR in cardioprotection and support the novel hypothesis that preconditioning involves switching of beta2-AR coupling from Gs to Gi.. Because of the central role of mitochodria in energy metabolism and in regulating apoptosis, we examined the role of mitochondria in preconditioning. The anti-apoptotic protein Bcl-2 is targeted to the mitochondria, but it is uncertain whether Bcl-2 affects only myocyte survival following ischemia, or whether it also affects metabolic functions of mitochondria during ischemia. Hearts from mice overexpressing human Bcl-2 (Bcl-2) and from their wild-type littermates (WT) were subjected to 24 minutes of global ischemia followed by reperfusion. During ischemia, the fall in pHi and the initial rate of decline in ATP were significantly reduced in Bcl-2 hearts compared with WT hearts. The reduced acidification during ischemia was dependent on the activity of mitochondrial F1F0-ATPase. In the presence of oligomycin (Oligo), an F1F0-ATPase inhibitor, the fall in pHi was attenuated in WT hearts, but in Bcl-2 hearts Oligo had no additional effect on pHi during ischemia. Likewise, addition of Oligo to WT hearts slowed the rate of decline in ATP during ischemia to a level similar to that observed in Bcl-2 hearts, but addition of Oligo had no significant effect on the rate of decline in ATP in Bcl-2 hearts during ischemia. These data are consistent with Bcl-2 mediated inhibition of consumption of glycolytic ATP. This could be accomplished by limiting ATP entry into the mitochondria through the voltage-dependent anion channel (VDAC), an outer mitochondrial membrane channel responsible for providing ATP to F1F0-ATPase. Immunoprecipitation showed greater interaction between Bcl-2 and VDAC during ischemia. Thus, the modulation of VDAC by Bcl-2 during ischemia may be a target in the mechanism for Bcl-2-induced cardioprotection. We also examined the mechanism responsible for reduced injury in female hearts. We previously showed that beta-adrenergic stimulation revealed male/female differences in susceptibility to ischemia/reperfusion (I/R) injury. To explore whether altered [Na+]i regulation is involved in the mechanism of this sex difference, we measured [Na+]i by 23Na-NMR spectroscopy in isolated perfused mouse hearts. [Na+]i increased to 195 +/- 3% of the pre-ischemic level at 20 minutes of ischemia in male hearts, whereas [Na+]i accumulation was slightly less in female hearts (176 +/- 2%, p<0.05). There was no significant difference in the recovery of contractile function after reperfusion (male: 30.6 +/- 3.8%, female: 35.0 +/- 1.9%). If hearts were treated with isoproterenol (ISO, 10nmol/L), males exhibited significantly poorer recovery of post-ischemic contractile function than females (male: 13.0 +/- 1.9%, female: 28.1 +/- 1.2%), and a significantly higher [Na+]i accumulation during ischemia (male: 218 +/- 8%, female: 171 +/- 2%). This ISO-induced male/female difference in [Na+]i accumulation or contractile function was blocked by the NO synthase inhibitor, L-NAME (1 umol/L). Thus the data show that the sex difference in the [Na+]i regulation is mediated through a NO-dependent mechanism.
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