title: Disrupting KATP channels diminishes the estrogen-mediated protection in female mutant mice during ischemia-reperfusion.
abstract: BACKGROUND
  Estrogen has been shown to mediate protection in female hearts against ischemia-reperfusion (I-R) stress. Composed by a Kir6.2 pore and an SUR2 regulatory subunit, cardiac ATP-sensitive potassium channels (KATP) remain quiescent under normal physiological conditions but they are activated by stress stimuli to confer protection to the heart. It remains unclear whether KATP is a regulatory target of estrogen in the female-specific I-R signaling pathway. In this study, we aimed at delineating the molecular mechanism underlying estrogen modulation on KATP channel activity during I-R.
  
  
  METHODS
  We employed KATP knockout mice in which SUR2 is disrupted (SUR2KO) to characterize their I-R response using an in vivo occlusion model. To test the protective effects of estrogen, female mice were ovariectomized and implanted with 17β-estradiol (E2) or placebo pellets (0.1 μg/g/day, 21-day release) before receiving an I-R treatment. Comparative proteomic analyses were performed to assess pathway-level alterations between KO-IR and WT-IR hearts.
  
  
  CONCLUSIONS
  Echocardiographic results indicated that KO females were pre-disposed to cardiac dysfunction at baseline. The mutant mice were more susceptible to I-R stress by having bigger infarcts (46%) than WT controls (31%). The observation was confirmed using ovariectomized mice implanted with E2 or placebo. However, the estrogen-mediated protection was diminished in KO hearts. Expression studies showed that the SUR2 protein level, but not RNA level, was up-regulated in WT-IR mice relative to untreated controls possibly via PTMs. Our antibodies detected different glycosylated SUR2 receptor species after the PNGase F treatment, suggesting that SUR2 could be modified by N-glycosylation. We subsequently showed that E2 could further induce the formation of complex-glycosylated SUR2. Additional time-point experiments revealed that I-R hearts had increased levels of N-glycosylated SUR2; and DPM1, the first committed step enzyme in the N-glycosylation pathway. Comparative proteomic profiling identified 41 differentially altered protein hits between KO-IR and WT-IR mice encompassing those related to estrogen biosynthesis.
  
  
  CONCLUSIONS
  Our findings suggest that KATP is likely a downstream regulatory target of estrogen and it is indispensable in female I-R signaling. Increasing SUR2 expression by N-glycosylation mediated by estrogen may be effective to enhance KATP channel subunit expression in I-R.
authors.affiliation: Computational Biology Center and Center for Molecular Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.
authors.forename: Jianjiong
authors.initials: J
authors.lastname: Gao
authors.affiliation: Department of Computer Science and CS Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA.
authors.forename: Dong
authors.initials: D
authors.lastname: Xu
authors.affiliation: Biotechnology Center, Mass Spectrometry Facility, University of Wisconsin, Madison, WI 53706, USA.
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Sabat
authors.affiliation: Department of Internal Medicine, University of Michigan, 2800 Plymouth Ave., 26-235 N, Ann Arbor, MI 48105, USA.
authors.forename: Hector
authors.initials: H
authors.lastname: Valdivia
authors.affiliation: McArdle Laboratory for Cancer Research, University of Wisconsin, 1400 University Ave., Madison, WI 53706, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Xu
authors.affiliation: Department of Medicine, University of Wisconsin, Room 8418, WIMR II, 1111 Highland Ave., Madison, WI 53705, USA.
authors.forename: Nian-Qing
authors.initials: NQ
authors.lastname: Shi
keywords: Estrogen; Estrogen receptor; Gender difference; Glycosylation; KATP channel; Myocardial infarction; Sulfonylurea receptor