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The HIF prolyl hydroxylase EglN1 mediates cellular responses to hypoxia, including alterations in cellular metabolism. We hypothesized that EglN1 inhibition might provide protection against ischemia-reperfusion (I/R) injury.
In vitro experimental models included mouse embryonic fibroblasts (EglN1 WT vs. KO MEFs), HL-1 cardiomyocytes (control vs. shRNA-mediated EglN1 knockdown), and pharmacologic EglN inactivation (FG-4497). Cell viability, oxygen consumption, and intracellular ROS were compared at baseline and following in vitro ischemia (1% 02; no serum media) or H2O2-mediated cytotoxicity. In vivo studies compared cardiac I/R injury responses among control animals, mice with a conditional deletion of EglN1 [EglN1 fl/fl;Cre-ER (tamoxifen-inducible, systemic promoter)], and mice treated with the EglN inhibitor FG-4497. Infarct size was assessed by TTC staining and normalized to the “area-at-risk.”
EglN1 KO MEFs showed improved viability following exposure to in vitro ischemia (60.4% vs. 47.5%, KO vs. WT, p<0.05), or H2O2 (55.6% vs. 24.8%). HL-1 cardiomyocytes with EglN1 knockdown (KD) were protected from H2O2 (72% more viable in KD vs. control, p<0.05). Genetic or pharmacologic EglN1 inactivation in mice reduced infarct size by 48% and 42% (p<0.05), respectively, compared to control mice. O2 consumption in EglN1 KO MEFs was 42% less than WT (p<0.05), and intracellular ROS was 28% less than WT (p<0.05). Primary cardiomyocytes from mice with systemic EglN1 inactivation had 44% less O2 consumption (p<0.05) than controls. PDK1 mRNA expression in EglN1 KO MEFs was 8.2-fold higher than WT (p<0.05). Dicholoroacetate (DCA) treatment restored the ROS reduction in WT MEFs after acute EglN inhibition with FG-4497 from 85% to 97% (p<0.05). Preliminary data suggest reversal of in vivo protection seen with FG-4497 after DCA treatment.
EglN1 inactivation protects MEFs, cardiomyocytes, and the murine heart from I/R injury. This protection is at least partially governed by PDK1, which limits glucose oxidation and ROS production. These data suggest that EglN1 inhibition might have clinical applications by preconditioning the heart prior to I/R injury.
Poster Sessions, Expo North
Saturday, March 09, 2013, 10:00 a.m.-10:45 a.m.
Session Title: Acute Coronary Syndromes: Basic I
Abstract Category: 2. Acute Coronary Syndromes: Basic
Presentation Number: 1127-191
- 2013 American College of Cardiology Foundation