The goals of this study are to understand the mechanism responsible for the male-female differences in ischemia-reperfusion injury and cardioprotection. To determine the role of membrane bound estrogen receptors in cardioprotection we used an estrogen that was attached to a dendimer (EDC). We treated ovariectomized female mice with estrogen, the EDC conjugated estrogen and vehicle. We found that EDC-conjugated estrogen showed a reduction in infarct size following ischemia and reperfusion similar to that observed in estrogen treated mice. We were also interested in examining signaling pathways involved in the protection in females and we used a resin-assisted capture method (SNO-RAC) to measure protein S-nitrosylation. We observed a number of proteins that were differentially S-nitrosylated between males and females. In addition we also 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 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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National Heart, Lung, and Blood Institute
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Shao, Qin; Casin, Kevin M; Mackowski, Nathan et al. (2017) Adenosine A1 receptor activation increases myocardial protein S-nitrosothiols and elicits protection from ischemia-reperfusion injury in male and female hearts. PLoS One 12:e0177315
Menazza, Sara; Sun, Junhui; Appachi, Swathi et al. (2017) Non-nuclear estrogen receptor alpha activation in endothelium reduces cardiac ischemia-reperfusion injury in mice. J Mol Cell Cardiol 107:41-51
Murphy, Elizabeth; Amanakis, Georgios; Fillmore, Natasha et al. (2017) Sex Differences in Metabolic Cardiomyopathy. Cardiovasc Res :
Shao, Qin; Fallica, Jonathan; Casin, Kevin M et al. (2016) Characterization of the sex-dependent myocardial S-nitrosothiol proteome. Am J Physiol Heart Circ Physiol 310:H505-15
Murphy, Elizabeth; Ardehali, Hossein; Balaban, Robert S et al. (2016) Mitochondrial Function, Biology, and Role in Disease: A Scientific Statement From the American Heart Association. Circ Res 118:1960-91
Menazza, Sara; Murphy, Elizabeth (2016) The Expanding Complexity of Estrogen Receptor Signaling in the Cardiovascular System. Circ Res 118:994-1007
Menazza, Sara; Aponte, Angel; Sun, Junhui et al. (2015) Molecular Signature of Nitroso-Redox Balance in Idiopathic Dilated Cardiomyopathies. J Am Heart Assoc 4:e002251
Harrington, Josephine L; Murphy, Elizabeth (2015) The mitochondrial calcium uniporter: mice can live and die without it. J Mol Cell Cardiol 78:46-53
Murphy, Elizabeth; Steenbergen, Charles (2014) Estrogen regulation of protein expression and signaling pathways in the heart. Biol Sex Differ 5:6
Murphy, Elizabeth; Steenbergen, Charles (2014) Sex, drugs, and trial design: sex influences the heart and drug responses. J Clin Invest 124:2375-7

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