The goals of this study are to understand the mechanism responsible for the male-female differences in ischemia-reperfusion injury and cardioprotection. We were interested in determining whether treatment with an estrogen receptor-beta (ER-beta)-selective agonist (2,3-bis(4-hydroxyphenyl)-propionitrile, DPN) can provide cardioprotection in female mice lacking endogenous estrogen. To study the effect of ER-beta stimulation in ischemia-reperfusion injury, we treated ovariectomized (ovx) female mice with 0.1 mg/kg/day of 17beta-estradiol, 0.8 mg/kg/day of DPN, or vehicle for 2 weeks. Isolated hearts were Langendorff perfused for 25 min prior to a 1-min treatment with isoproterenol, followed by 20 min of normothermic global ischemia and 40 min of reperfusion. Left ventricular developed pressure (LVDP) and heart rate were measured. Recovery of function at the end of 40 min of reperfusion was expressed as a percentage of pre-ischemic rate pressure product (RPP=LVDP x heart rate). Hearts from ovx female mice had a significantly lower recovery of LVDP than the hearts from intact female mice (12.4+/-1.6% vs. 19.6+/-1.6%, p<0.05, respectively). Furthermore, hearts from ovx female mice treated with DPN exhibited significantly better functional recovery than hearts from either vehicle-treated ovx female mice (20.1+/-2.2% vs. 12.4+/-1.6%, p<0.05, respectively) or wild type male mice (20.1+/-2.2% vs. 6.4+/-0.6%, p<0.05, respectively). DPN did not increase uterine weight in ovx females compared to vehicle treatment. Gene profiling showed that treatment with DPN resulted in upregulation of a number of protective genes such as heat shock protein 70, the antiapoptotic protein, growth arrest and DNA damage 45 beta, and cyclooxygenase 2. We also tested the hypothesis that estrogen protects by estrogen-receptor-beta (ER-beta) activation which leads to S-nitrosation of key cardioprotective proteins. To test this hypothesis, we treated bilaterally ovariectomized female mice with an ER-beta selective agonist, 2,2-bis(4-hydroxyphenyl)-proprionitile (DPN) (0.8 mg/kg/day), 17-estradiol (E2) (0.1 mg/kg/day) or vehicle for 2 weeks. Isolated hearts were Langendorff perfused for 20 minutes prior to 1 minute of isoproterenol treatment, followed by 20 minutes of global ischemia, and 120 minutes of reperfusion. Compared with vehicle, DPN and 17-estradiol treated hearts had significantly better post-ischemic recovery of left ventricular function as well as decreased infarct size. . To test the specificity of DPN, we treated ER-beta knockout (Beta-ERKO) mice with DPN. However, no cardioprotective effect of DPN was found in Beta-ERKO mice, indicating the DPN-induced cardioprotection occurs through the activation of ER-beta. Using DyLight-maleimide fluors and a modified biotin switch method, we employed a 2D DyLight fluorescence difference gel electrophoresis (DIGE) proteomic method to quantify differences in protein S-nitrosation between our three treatment groups. We identified several cardiac proteins with a significant increase in S-nitrosation in the DPN and 17-estradiol groups, including F1-ATPase 1 subunit, malate dehydrogenase, aconitase, heat shock protein 60, cytochrome c oxidase subunit 5A and creatine kinase. In addition, the DPN-induced cardioprotection and increased SNO were abolished by treatment with a nitric oxide (NO) synthase inhibitor. In summary, the activation of ER- by DPN treatment leads to increased protein S-nitrosylation and cardioprotection against I/R injury, suggesting that chronic estrogen exposure protects hearts largely via activation of ER- and NO/SNO signaling. We propose that S-nitrosation alters the activity of cardiac proteins such as mitochondrial F1-ATPase leading to protection during ischemia-reperfusion. Historically, estrogen has been thought to signal through two nuclear receptors, ER-alpha and ER-beta;however, a third, membrane bound receptor, GPR30, has been identified and shown to bind estrogen with high affinity. To date, there is little information on GPR30 in the heart and no study has looked at the effect of GPR30 activation during myocardial ischemia and reperfusion (IR). Therefore, the goal of this study was to determine whether activation of GPR30 results in a cardioprotective phenotype in rats. A highly specific GPR30 agonist, G-1, was administered to Sprague Dawley (200-350g) rat hearts 10 minutes prior to 20 minutes of ischemic followed by 120 minutes of reperfusion using a Langendorff model. With administration of 110 nM of G-1, post-ischemic contractile dysfunction was significantly reduced compared to untreated controls (43.84.3 vs 26.92.1% of pre-ischemic rate pressure product, p<0.05). Additionally, infarct size was reduced in the G-1 treated animals when compared to control (19.62.9 vs 31.82.3% p<0.05). Through western blot analysis, it was demonstrated that G-1 induces the activation of both Akt and Erk1/2. Furthermore, the protection afforded by G-1 was blocked by co-administration of a PI3K inhibitor (wortmannin 100nM). Taken together, the data show that G-1 activation of GPR30 improves functional recovery and reduces infarct size in isolated rat hearts following ischemia and reperfusion through a PI3K dependent mechanism. To identify protein changes involved in protection in females, isolated mitochondrial proteins from male (M) and female (F) Sprague Dawley rats were analyzed by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE labeling) followed by mass spectrometry for protein identification. Using 2D-DIGE analysis, a comparison between M and F revealed significant differences in 20 proteins that appear to be due to post translational modification. Because of these observations we performed 2D gel electrophoresis followed by a Pro-Q Diamond stain or 2D gel electrophoresis followed by probing with antiphospho antibodies to identify phosphorylation. We observed sex differences in phosphorylation of pyruvate dehydrogenase, -ketoglutarate dehydrogenase and aldehyde dehydrogenase. Consistent with alterations in enzymes involved in ROS homeostasis, female mitochondria have less ROS production from -ketoglutarate dehydrogenase and less ROS following anoxia and reoxygenation. Inhibition of the PI3-kinase pathway blocked the protection observed in females and also blocked the increased phosphorylation of aldehyde dehydrogenase, suggesting that increased basal activation of the PI3-kinase pathway in females alters protein phosphorylation and is involved in the cardioprotection observed in females. We were interested in examining the potential role of the increase in post-translational modifications alpha-ketoglutarate dehydrogenase (a-KGDH) in female rat heart compare to male. Alpha-KGDH is a highly regulated Krebs cycle enzyme, which has been suggested to be one source for generation of reactive oxygen species (ROS), especially under conditions of high NADH/NAD+.
The aim of the present study was to determine whether the post-translational modification of alpha-KGDH might alter ROS production of the enzyme. The production of ROS by alpha-KGDH in permeabilized mitochondria in the absence of NADH was not different between sexes (M: 1.0 0.01 pmols/min/mg of protein vs. F:0.9 0.05, p=ns), however with addition of NADH, M generated significantly more ROS (M: 5.2 0.5;F: 2.6 0.1 p<0.01). We examined the effect of phosphorylation of -KGDH on ROS production by treating purified a-KGDH, with active protein kinase C-epsilon (PKCe). The resulting increase in phosphorylated a-KGDH E1 subunit resulted in a reduction of ROS generated in the presence of NADH compared to non-phosphorylated a-KGDH (p-aKGDH: 47.60.8 vs aKGDH: 64.23.5;p<0.05). ROS generated from phosphorylated and non-phosphorylated a-KGDH in the presence of NAD was similar.
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