Brief periods of stress have been shown to initiate protective signaling mechanisms, that reduce injury during subsequent more severe or sustained injury. The goals of this project are to elucidate signaling mechanisms involved in this protection. Brief intermittent stress (termed preconditioning (PC)) can lead to release of cytokines and hormones such as adenosine, opioids, bradykinin, and other peptides which through activation of G protein coupled receptors (GPCR) leads to protection during a subsequent longer period of stress. To examine the role of G protein-coupled receptors (GPCRs) in the protection afforded by ischemic preconditioning (PC), we used transgenic mice with cardiac-specific overexpression of a Gbeta-gamma-sequestering peptide, BARKct (TG bARKct mice), to test whether the protection of PC is Gbeta-gamma -dependent. To test the role of Gi protein, we used wild type (WT) mice pretreated with the Gi inhibitor pertussis toxin (PTX). Before 20 minutes of global ischemia, mouse hearts were perfused for 30 minutes (control) or preconditioned with 4 cycles of 5-minute ischemia and 5-minute reflow (PC). Recovery of left ventricular developed pressure and infarct size were measured after 1 hour and 2 hours of reflow respectively. PC induced a protective effect in WT mice, but this protection was blocked by PTX treatment and was also blocked in TG BARKct mice. To determine the mechanism of Gbeta-gamma -induced protection in PC, we investigated one of the downstream targets of G Gbeta-gamma, the PI3K/p70S6K pathway in these mice. We found that PC-induced phosphorylation of p70S6K was not blocked in TG bARKct hearts; therefore, we investigated other targets of Gbeta-gamma. 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. To further test whether PI3K is involved in PC-activated receptor internalization and endosomal signaling, we used transgenic mice with cardiac-specific overexpression of a catalytically inactive mutant PI3K-gamma (PI3K-gammainact), which disrupts the recruitment of functional PI3K to agonist-activated GPCRs in vivo. We found that PI3Kgammainact blocks the protective effect of PC. In summary, these data suggest the novel findings that the cardioprotective effect of PC requires receptor internalization. Erythropoietin (EPO), the principal hematopoietic cytokine that regulates mammalian erythropoiesis, exhibits diverse cellular effects in non-hematopoietic tissues. The physiologic functions of EPO are mediated by its specific cell surface receptor EPOR. We demonstrated EPOR expression in adult rat cardiac myocytes and examined the direct effects of EPO on the heart to investigate whether recombinant EPO exerts an acute cardioprotective effect during ischemia-reperfusion injury. To determine whether 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 continuously to assess contractile function. 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%. We used 31P nuclear magnetic resonance (NMR) spectroscopy to determine whether modulation of intracellular pH and/or high energy phosphate levels during ischemia contributed to EPO-mediated cardioprotection. These experiments revealed that the rapid cardioprotective effect of EPO during ischemia-reperfusion injury was associated with preservation of ATP levels in the ischemic myocardium. 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 (p<0.05). 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 ?-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% (M ? SE) 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%; p>0.05). 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%, p<0.05), and a significantly higher [Na+]i accumulation during ischemia (male: 218 ? 8%, female: 171 ? 2%, p<0.05). 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). Furthermore, in ISO-treated hearts, the Na+/K+-ATPase inhibitor, ouabain (200umol/L) did not abolish the male/female difference in [Na+]i accumulation during I/R or functional protection. Thus the data show that the sex difference in the [Na+]i regulation is mediated through a NO-dependent mechanism, and the difference in susceptibility to I/R injury appears to result from a difference in Na+ influx.
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