Introduction We recently found that acute pre‐ischemic estrogen (17 b‐estradiol) treatment induces cardioprotection against ischemia/reperfusion (I/R) injury mainly via G protein‐coupled estrogen receptor1 (GPER1) activation. We, furthermore, showed that pre‐ischemic estrogen‐GPER1 effects preserve mitochondrial integrity and function via MEK/ERK/GSK‐3b pathway. Hypothesis We investigated whether acute post‐ischemic estrogen (PI‐E2) treatment can also induce cardio‐protective effects via GPER1 activation and determine whether PI‐E2 action involves the reduction of mitophagy. Methods Male and ovarectomized female Sprague‐Dawley rats were anesthetized with ketamine (80 mg/kg i.p.) and xylazine (8 mg/kg i.p.). Hearts were subjected to 35 min of the left anterior descending (LAD) artery occlusion, followed by 180 min reperfusion. An estrogen bolus (50 mg/kg body weight) or PBS (same volume) was applied via the femoral vein 5 min before reperfusion, and GPER1 antagonist, G15, was given 10 min before estrogen. Area at risk (AAR) was identified using Evans Blue dye and myocardial infarct size assessed by TTC staining method. Mitochondrial calcium retention capacity (CRC) required to induce mitochondrial permeability transition pore (mPTP) opening was assessed after 20 min reperfusion. Expression levels of LC3I LC3II; p62; PINK1; and pERK were detected by western blot in whole cell lysates. Parkin and ubiquitinated protein were measured by Western blot in cytosolic and mitochondrial fractions. Results We found that PI‐E2 treatment reduced myocardial infarct size normalized to the AAR or to the whole LV and increased mitochondrial CRC compared to untreated. We also found that PI‐E2 treatment decreased the level of LC3II, increased the level of p62 and pERK1/2, inhibited the translocation of Parkin to mitochondria and decreased ubiquitinated proteins in mitochondria compared to control. Interestingly, all these PI‐E2 effects were abolished by addition of G15. Conclusion Acute post‐ischemic GPER1 activation by estrogen treatment induces cardioprotective effects against I/R injury. PI‐E2 effects through GPER1 involve the inhibition of mitophagy and reduction of mitochondrial proteins ubiquitination. Together, these post‐ischemic estrogen effects via GPER1 lead to inhibition of the mPTP opening, and subsequently cardioprotection. Support or Funding Information Voelcker Fund

Introduction: We recently showed that pre-ischemic estrogen (17 β-estradiol) treatment induces cardioprotective effects against ischemia/reperfusion (I/R) injury via G-protein coupled estrogen receptor1 (GPER1) activation by preserving mitochondrial integrity and function via MEK/ERK/GSK-3β pathway. Hypothesis: We investigated whether post-ischemic estrogen (PI-E2) treatment can also induce cardioprotective effects via GPER1 activation and determine whether PI-E2 action involves the reduction of mitophagy and the mitochondrial permeability transition pore (mPTP) opening. Methods: Male and ovariectomized female Sprague-Dawley rats were used. Hearts were subjected to 35 min of the left anterior descending artery occlusion followed by 180 min reperfusion. An estrogen bolus (50 mg/kg body weight) or PBS (same volume) was applied via the femoral vein 5 min before reperfusion, and GPER1 antagonist, G15, given 10 min before estrogen. Area at risk (AAR) was identified using Evans Blue dye and myocardial infarct s...

We also examined whether Gper1 expression is required for acute E2-induced improvement of heart viability by determining the myocardial infarct size in sham or hearts subjected to I/R. 3A. Hearts were sectioned and analyzed at the end of the perfusion or reperfusion periods. As expected, acute E2 treatment reduced myocardial infarct size significantly in WT animals. Supporting the functional data, acute E2 treatment had the same protective action (as in the WT animals) when hearts from Esr1and Esr2 knockouts were used but lost its protective effect in the absence of Gper1 as hearts from Gper1-/- animals displayed a robust infarcted area regardless of E2 treatment.

Three types of estrogen receptors (ER) exist in the heart, Esr1, Esr2 and the G protein-coupled estrogen receptor 1, Gper1. However, their relative importance in mediating estrogen protective action is unknown. We found that, in the male mouse ventricle, Gper1 transcripts are three- and seventeen-fold more abundant than Esr1 and Esr2 mRNAs, respectively. Analysis of the three ER knockouts (Esr1-/-, Esr2-/- and Gper1-/-) showed that only the Gper1-/- hearts lost their ability to be protected by 40 nM estrogen as measured by heart function, infarct size and mitochondrial Ca2+ overload, an index of mitochondrial permeability transition pore (mPTP) activity. Analysis of Akt, ERK1/2 and GSK-3β salvage kinases uncovered Akt and ERK1/2 transient activation by estrogen whose phosphorylation increased during the first 5 min of non-ischemic perfusion. All these increase in phosphorylation effects were abrogated in Gper1-/-. Inhibition of MEK1/2/ERK1/2 (1 μM U0126) and PI-3K/Akt (10 μM LY294002) signaling showed that the MEK1/2/ERK1/2 pathway via GSK-3β exclusively was responsible for cardioprotection as an addition of U0126 prevented estrogen-induced GSK-3β increased phosphorylation, resistance to mitochondrial Ca2+-overload, functional recovery and protection against infarction. Further, inhibiting PKC translocation (1 μM chelerythrin-chloride) abolished estrogen-induced cardioprotection. These data indicate that estrogen-Gper1 acute coupling plays a key role in cardioprotection against ischemia/reperfusion injury in male mouse via a cascade involving PKC translocation, ERK1/2/GSK-3β phosphorylation leading to the inhibition of the mPTP opening.

Cardiac functional parameters in WT male mice. Left ventricular systolic pressure (LVSP); left ventricular end-diastolic pressure (LVEDP); left ventricular developed pressure (LVDP) and heart rate (HR) before ischemia (Basal) and at different times of reperfusion after I/R in control, E2-treated, and E2+Inhibitors (U0126, LY294002 and CC: chelerythrine chloride). Values are mean±SEM. * P<0.05 control versus E2 group (n=6-7/ group); + P<0.05 E2+CC versus E2 group (n=4-6/ group); and # P<0.05 E2+U0126 versus E2 group (n=4-6 hearts/ group).

Gper1 activation is required for E2-mediated increase in mitochondrial Ca2+ retention capacity.