The goals of this study are to understand the mechanism responsible for the male-female differences in ischemia-reperfusion injury and cardioprotection. We tested the hypothesis that cardioprotection in females is mediated by altered mitochondrial protein levels and/or posttranslational modifications. Using both an in vivo and an isolated heart model of ischemia and reperfusion (I/R), we found that females had less injury than males. Using proteomic methods we found that female hearts had increased phosphorylation and activity of aldehyde dehydrogenase (ALDH)2, an enzyme that detoxifies reactive oxygen species (ROS)-generated aldehyde adducts, and that an activator of ALDH2 reduced I/R injury in males but had no significant effect in females. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, blocked the protection and the increased phosphorylation of ALDH2 in females, but had no effect in males. Furthermore, we found an increase in phosphorylation of alpha-ketoglutarate dehydrogenase (alpha-KGDH) in female hearts. Alpha-KGDH is a major source of ROS generation particularly with a high NADH/NAD ratio which occurs during I/R. We found decreased ROS generation in permeabilized female mitochondria given alpha-KGDH substrates and NADH, suggesting that increased phosphorylation of alpha-KGDH might reduce ROS generation by alpha-KGDH. 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+. We 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. In support of this hypothesis, we found that protein kinase C dependent phosphorylation of purified alpha-KGDH reduced ROS generation. Additionally, myocytes from female hearts had less ROS generation following I/R than males and addition of wortmannin increased ROS generation in females to the same levels as in males. These data suggest that posttranslational modifications can modify ROS handling and play an important role in female cardioprotection. We further 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. We have also begun to examine whether there are male-female differences in microRNAs. One mechanism of gene regulation is through the expression of microRNAs. MicroRNAs are small (20-24 nucleotides) single stranded pieces of non-coding RNA that bind to mRNA and disrupt translation. Accordingly, the hypothesis of this study was that microRNA expression would differ between male and female mice hearts. Using an Affymetrix microRNA array, we demonstrated a significant difference in the principle component analysis between male and female microRNA.
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