Abstract 121: Identification of Mitochondrial Protein Regulation by 2D-DIGE and Mass Spectroscopy During G-Protein--Coupled Estrogen Receptor1 Activation
Introduction: Several studies have demonstrated that G-protein coupled estrogen receptor1 (GPER1) can directly bind to estrogen (E2) and mediate its action. We previously demonstrated that GPER1 activation with G1 a specific agonist is cardioprotective in wild-type (WT) mice subjected to ischemia/reperfusion (I/R) injury.
Hypothesis: We investigated the regulation of mitochondrial proteins associated with rapid E2 treatment by comparing 2D-Differential in Gel Electrophoresis (2D-DIGE) in WT and GPER1-/- mice in control and E2-treated groups after I/R.
Methods: Isolated hearts from male WT (C57BL/6NCrL) or GPER1-/- mice (2-3 months old) were perfused using the Langendorff technique with Krebs Henseleit buffer (control), and with the addition of E2 (40 nM). Hearts were subjected to 18 min global normothermic ischemia followed by 60 min reperfusion. Cardiac function was recorded during the entire experiment and myocardial infarct size was measured by TTC staining at the end of the reperfusion. 2D-Differential Gel Electrophoresis (2D-DIGE) followed by mass spectrometry (LC/MS/MS) was performed in isolated mitochondria after 10 min of reperfusion. Proteins of interest were the ones (up- or down- regulated) in WT+E2 vs. WT-control that remained unchanged in GPER1-/-+E2 vs. GPER1-/--control and WT-control vs. GPER1-/--control.
Results: In WT hearts, E2 treatment significantly improved cardiac functional recovery and reduced infarct size. However, E2-effects were completely absent in GPER1-/- hearts. Robust changes of proteins were observed in 45 spots, out of which 14 were down regulated and 31 up regulated. In these 45 spots, 52 unique proteins were identified. In particular, E2 treatment induced down regulation of the mitochondrial inner membrane protein (Mitofilin), which is known to control cristae morphology in WT heart mitochondria but not in GPER1-/-. Suggesting that rapid E2 effects are associated with mitochondria integrity.
Conclusion: These results suggest that GPER1 plays an important role in mediating E2-induced cardioprotection after I/R. E2 effects through GPER1 are associated with regulation of mitochondrial proteins.
- © 2011 by American Heart Association, Inc.