Dataset Information

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.

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 17?-estradiol (E2) or placebo pellets (0.1 ?g/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.

SUBMITTER: Gao J

PROVIDER: S-EPMC4047774 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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

Gao Jianjiong J Xu Dong D Sabat Grzegorz G Valdivia Hector H Xu Wei W Shi Nian-Qing NQ

Clinical proteomics 20140506 1

<h4>Background</h4>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 a ...[more]

PMID: 24936167

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Project description:Mitochondrial potassium channels have been implicated in myocardial protection mediated through pre-/postconditioning. Compounds that open the Ca2+- and voltage-activated potassium channel of big-conductance (BK) have a pre-conditioning-like effect on survival of cardiomyocytes after ischemia/reperfusion injury. Recently, mitochondrial BK channels (mitoBKs) in cardiomyocytes were implicated as infarct-limiting factors that derive directly from the KCNMA1 gene encoding for canonical BKs usually present at the plasma membrane of cells. However, some studies challenged these cardio-protective roles of mitoBKs. Herein, we present electrophysiological evidence for paxilline- and NS11021-sensitive BK-mediated currents of 190 pS conductance in mitoplasts from wild-type but not BK-/- cardiomyocytes. Transmission electron microscopy of BK-/- ventricular muscles fibres showed normal ultra-structures and matrix dimension, but oxidative phosphorylation capacities at normoxia and upon re-oxygenation after anoxia were significantly attenuated in BK-/- permeabilized cardiomyocytes. In the absence of BK, post-anoxic reactive oxygen species (ROS) production from cardiomyocyte mitochondria was elevated indicating that mitoBK fine-tune the oxidative state at hypoxia and re-oxygenation. Because ROS and the capacity of the myocardium for oxidative metabolism are important determinants of cellular survival, we tested BK-/- hearts for their response in an ex-vivo model of ischemia/reperfusion (I/R) injury. Infarct areas, coronary flow and heart rates were not different between wild-type and BK-/- hearts upon I/R injury in the absence of ischemic pre-conditioning (IP), but differed upon IP. While the area of infarction comprised 28±3% of the area at risk in wild-type, it was increased to 58±5% in BK-/- hearts suggesting that BK mediates the beneficial effects of IP. These findings suggest that cardiac BK channels are important for proper oxidative energy supply of cardiomyocytes at normoxia and upon re-oxygenation after prolonged anoxia and that IP might indeed favor survival of the myocardium upon I/R injury in a BK-dependent mode stemming from both mitochondrial post-anoxic ROS modulation and non-mitochondrial localizations.

Project description:Preserving mitochondrial activity is crucial in rescuing cardiac function following acute myocardial ischemia/reperfusion (I/R). The sex difference in myocardial functional recovery has been observed after I/R. Given the key role of mitochondrial connexin43 (Cx43) in cardiac protection initiated by ischemic preconditioning, we aimed to determine the implication of mitochondrial Cx43 in sex-related myocardial responses and to examine the effect of estrogen (17β-estradiol, E2) on Cx43, particularly mitochondrial Cx43-involved cardiac protection following I/R. Mouse primary cardiomyocytes and isolated mouse hearts (from males, females, ovariectomized females, and doxycycline-inducible Tnnt2-controlled Cx43 knockout without or with acute post-ischemic E2 treatment) were subjected to simulated I/R in culture or Langendorff I/R (25-min warm ischemia/40-min reperfusion), respectively. Mitochondrial membrane potential and mitochondrial superoxide production were measured in cardiomyocytes. Myocardial function and infarct size were determined. Cx43 and its isoform, Gja1-20k, were assessed in mitochondria. Immunoelectron microscopy and co-immunoprecipitation were also used to examine mitochondrial Cx43 and its interaction with estrogen receptor-α by E2 in mitochondria, respectively. There were sex disparities in stress-induced cardiomyocyte mitochondrial function. E2 partially restored mitochondrial activity in cardiomyocytes following acute injury. Post-ischemia infusion of E2 improved functional recovery and reduced infarct size with increased Cx43 content and phosphorylation in mitochondria. Ablation of cardiac Cx43 aggravated mitochondrial damage and abolished E2-mediated cardiac protection during I/R. Female mice were more resistant to myocardial I/R than age-matched males with greater protective role of mitochondrial Cx43 in female hearts. Post-ischemic E2 usage augmented mitochondrial Cx43 content and phosphorylation, increased mitochondrial Gja1-20k, and showed cardiac protection.

Dataset Information

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

Background

Materials and methods

Results and discussion

Conclusions

Publications

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

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