We have investigated whether treatment with an estrogen receptor (ER)-selectiveagonist (2,3-bis(4-hydroxyphenyl)-propionitrile, DPN) can provide cardioprotection in female mice lacking endogenous estrogen. To study the effect of ER- stimulation in ischemia-reperfusion injury, we treated ovariectomized (ovx) female mice with 0.1 mg/kg/day of 17β-estradiol, 0.8 mg/kg/day of DPN, or vehicle for two weeks. Isolated hearts were Langendorff perfused for 25 minutes prior to a one minute treatment with isoproterenol, followed by 20 minutes of normothermic global ischemia and 40 minutes of reperfusion. Left ventricular developed pressure (LVDP) and heart rate were measured. Recovery of function at the end of 40 minutes 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 , and cyclooxygenase 2.? Although estrogen has effects on the heart, little is known regarding which genes in the heart are directly responsive to estrogen. We have shown previously that lipoprotein lipase (LPL) expression was increased in female hearts compared to male hearts. To test whether LPL gene expression in heart is regulated by estrogen, we perfused mouse hearts from ovariectomized females (OVX) with 100nM 17-etradiol or vehicle for 2 hours after which hearts were frozen and RNA was isolated. The SYBR green real-time PCR method was used to detect LPL gene expression. We found that addition of 17 estradiol to hearts from OVX females resulted in a significant increase in LPL mRNA. This estrogen effect on LPL gene expression in mouse heart can be blocked by the estrogen receptor antagonist ICI 182,780 or by progesterone. We also identified a potential estrogen receptor element (ERE) enhancer sequence located in the first intron of the mouse LPL gene. The potential ERE sequence was linked to a TATA-LUC reporter plasmid in HeLa cells. Both estrogen receptor (ER)α and ERβ stimulated strong activity on the heterologous promoter reporter in Hela cells upon estrogen addition. Both ERα and ERβ activities on the LPL ERE reporter were abrogated by the estrogen receptor antagonist ICI182,780. Progesterone also dose dependently inhibited the estrogen mediated increase in LPL ERE reporter activity. These results show that heart LPL is an estrogen responsive gene exhibiting an intronic regulatory sequence.? We also did studies to examine the role of nitric oxide (NO)in cardioprotection. NO has been shown to be an important signaling messenger in ischemic preconditioning (IPC). Accordingly, we investigated whether protein S-nitrosylation occurs in IPC hearts and whether S-nitrosoglutathione (GSNO) elicits similar effects on S-nitrosylation and cardioprotection. Hearts from C57BL/6J mice were perfused in the Langendorff mode and subjected to: (1) control perfusion; (2) IPC; or (3) GSNO treatment (GSNO), followed by 20 min of no-flow ischemia and reperfusion. Compared with control, IPC and GSNO significantly improved post-ischemic recovery of left ventricular developed pressure and reduced infarct size. IPC and GSNO both significantly increased the S-nitrosothiol (SNO) contents and S-nitrosylation level of the L-type Ca2+ channel 1 subunit in heart membrane fractions. We identified several candidate S-nitrosylated proteins by proteomic analysis following the biotin switch method, including, the cardiac sarcoplasmic reticulum Ca2+-ATPase, α-ketoglutarate dehydrogenase, and the mitochondrial F1-ATPase  subunit. The activities of these enzymes were altered in a concentration dependent manner by GSNO treatment. We further developed a 2D proteomic method that used DyLight fluors and a modified biotin switch method to identify S-nitrosylated proteins. IPC and GSNO produced a similar pattern of protein S-nitrosylation modification and similar protection suggesting that protein S-nitrosylation may play an important cardioprotective role in heart.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Intramural Research (Z01)
Project #
1Z01ES010004-28
Application #
7593853
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
28
Fiscal Year
2007
Total Cost
$1,565,680
Indirect Cost
City
State
Country
United States
Zip Code
Huss, Janice M; Imahashi, Ken-ichi; Dufour, Catherine R et al. (2007) The nuclear receptor ERRalpha is required for the bioenergetic and functional adaptation to cardiac pressure overload. Cell Metab 6:25-37
Delozier, Tracy C; Kissling, Grace E; Coulter, Sherry J et al. (2007) Detection of human CYP2C8, CYP2C9, and CYP2J2 in cardiovascular tissues. Drug Metab Dispos 35:682-8
Murphy, Elizabeth; Steenbergen, Charles (2007) Cardioprotection in females: a role for nitric oxide and altered gene expression. Heart Fail Rev 12:293-300
Murphy, Elizabeth; Steenbergen, Charles (2007) Gender-based differences in mechanisms of protection in myocardial ischemia-reperfusion injury. Cardiovasc Res 75:478-86
Murphy, Elizabeth; Steenbergen, Charles (2007) Preconditioning: the mitochondrial connection. Annu Rev Physiol 69:51-67
Nikolic, Ivana; Liu, Dianxin; Bell, Jamie A et al. (2007) Treatment with an estrogen receptor-beta-selective agonist is cardioprotective. J Mol Cell Cardiol 42:769-80
Shiva, Sruti; Huang, Zhi; Grubina, Rozalina et al. (2007) Deoxymyoglobin is a nitrite reductase that generates nitric oxide and regulates mitochondrial respiration. Circ Res 100:654-61
Seubert, John M; Sinal, Christopher J; Graves, Joan et al. (2006) Role of soluble epoxide hydrolase in postischemic recovery of heart contractile function. Circ Res 99:442-50
Sun, Junhui; Picht, Eckard; Ginsburg, Kenneth S et al. (2006) Hypercontractile female hearts exhibit increased S-nitrosylation of the L-type Ca2+ channel alpha1 subunit and reduced ischemia/reperfusion injury. Circ Res 98:403-11
Sun, Junhui; Steenbergen, Charles; Murphy, Elizabeth (2006) S-nitrosylation: NO-related redox signaling to protect against oxidative stress. Antioxid Redox Signal 8:1693-705

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