Identification of Mitochondrial Proteins Involved in Acute Estrogen‐Induced Cardioprotection Via GPER1

We recently found that activation of G‐protein coupled estrogen receptor (GPER1) with G1, its agonist, induces cardioprotective effect against ischemia/reperfusion injury by the inhibition of the mitochondrial permeability transition pore. Here, we identified regulated mitochondrial proteins involved in estrogen action after ischemia/reperfusion. Isolated hearts from male WT (C57BL/6NCrL) or GPER1‐KO mice hearts were perfused using the Langendorff technique with Krebs Henseleit buffer (control) or with the addition of estrogen (40nM). Hearts were subjected to 18min ischemia followed by 10min reperfusion. Mitochondria were isolated and 2D‐DIGE followed by mass spectrometry was performed. Proteins of interest were the ones (up‐ or down‐ regulated) in WT+E2 vs. WT‐control that remained unchanged in GPER1‐KO+estrogen vs. GPER1‐KO‐control and WT‐control vs. GPER1‐KO‐control. Robust changes of proteins were observed in 45 spots, out of which 14 were downregulated and 31 upregulated. In these spots, 52 unique proteins were identified. Estrogen treatment down‐regulated filament proteins (filamin A,B,C) and the mitofilin, which controls mitochondrial cristae morphology, upregulated activating transcription proteins, and contractile proteins (tropomyosin and myosin), and regulated proteins acting in stress (stress‐70 protein, and 60 kDa heat shock protein), in cell communication (glial fibrillary acidic protein), in electron transfer chain and ATP production, and in signaling pathways (membrane‐associated phosphatidylinositol transfer protein). In conclusion, acute estrogen effects via GPER1 activation are associated with the down‐ or up‐regulation of mitochondrial and non‐mitochondrial proteins likely in close contact to mitochondria.