Disrupting KATP channels diminishes the estrogen-mediated protection in female mutant mice during ischemia-reperfusion

Clinical Proteomics

Dong Xu⁴  Wei Xu²  Jianjiong Gao⁵  Hector Valdivia¹  Grzegorz Sabat⁶  Nian-Qing Shi³ 

[1] Department of Internal Medicine, University of Michigan, Ann Arbor, USADepartment of Internal Medicine, University of Michigan, Ann Arbor, USADepartment of Internal Medicine, University of Michigan, Ann Arbor, USA;McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, USAMcArdle Laboratory for Cancer Research, University of Wisconsin, Madison, USAMcArdle Laboratory for Cancer Research, University of Wisconsin, Madison, USA;Department of Medicine, University of Wisconsin, Madison, USADepartment of Medicine, University of Wisconsin, Madison, USADepartment of Medicine, University of Wisconsin, Madison, USA;Department of Computer Science and CS Bond Life Sciences Center, University of Missouri, Columbia, USADepartment of Computer Science and CS Bond Life Sciences Center, University of Missouri, Columbia, USADepartment of Computer Science and CS Bond Life Sciences Center, University of Missouri, Columbia, USA;Computational Biology Center and Center for Molecular Oncology, Memorial Sloan-Kettering Cancer Center, New York, USAComputational Biology Center and Center for Molecular Oncology, Memorial Sloan-Kettering Cancer Center, New York, USAComputational Biology Center and Center for Molecular Oncology, Memorial Sloan-Kettering Cancer Center, New York, USA;Biotechnology Center, Mass Spectrometry Facility, University of Wisconsin, Madison, USABiotechnology Center, Mass Spectrometry Facility, University of Wisconsin, Madison, USABiotechnology Center, Mass Spectrometry Facility, University of Wisconsin, Madison, USA

关键词: KATP channel;    Sulfonylurea receptor;    Myocardial infarction;    Gender difference;    Estrogen;    Estrogen receptor;    Glycosylation;   

DOI  :  10.1186/1559-0275-11-19

来源: Humana Press Inc

【 摘 要 】

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.

Materials and 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.

Results and discussion

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.

【 授权许可】

Unknown