E2-induced increase in phosphorylation of Akt, ERK1/2 and GSK-3β is dependent on Gper1 gene expression and disappears with time for Akt and ERK1/2.

<p>A. Protocol used in panels B,C,D. Isolated hearts were either perfused 40 min with oxygenated KH solution or perfused with oxygenated KH for 35 min and for 5 min with KH plus 40 nM E2. B-D. Immunoblots and corresponding bar graphs show that 5 min “pre-ischemic” treatment with E2 provoked pAkt/Akt, pERK1/2/ERK1/2 and pGSK-3β/GSK-3β ratios to increase in WT but not in Gper1-/- samples. Control ratios were set to 100%. Values are mean±S.E.M.; *, P<0.05 control versus E2-treated group.</p

MEK1/2/ERK1/2 –but not PI-3K/Akt- signaling supports E2-mediated protection from I/R via GSK-3β and mPTP.

<p>A. I/R protocol. Arrows mark the time of sample preparation. B. Immunoblots show that addition of the inhibitor of MEK1/2/ERK1,2 pathway, U0126 (1 mM) abolishes E2-induced up-regulation of pERK and the inhibitor of PI3-K/Akt pathway, LY294002 (10 mM) abolishes the increase in pAkt induced by 40 nM E2. C,D. Immunoblots and corresponding bar graphs show that the inhibitor of MEK1/2/ERK1,2 pathway, U0126 (1 mM) -but not the inhibitor of PI3-K/Akt pathway, LY294002 (10 mM)- abolishes the increase in pGSK-3β/GSK-3β ratio induced by 40 nM E2. E. Calcium load measurements were as indicated in Fig. 5. Arrow, addition of mitochondria. Arrowheads, mark the massive Ca2+ release (an index of mPTP opening) in mitochondria from control (black), E2 (40 nM)-treated (open), and treated with E2 (40 nM) + U0126 (1 µM) (grey) hearts. Dashed lines mark the time of Ca2+ addition to the E2+U0126 treated sample. F. Mean values of calcium retention capacity (CRC) demonstrate that 1 µM U0126 prevents the beneficial effect of 40 nM E2. Note that vinculin was used as a loading housekeeping protein. All values were obtained from WT male mice and expressed as mean±SEM; * P<0.05 E2-treated group versus control; and # P<0.05 E2+U0126 versus E2-treated group.</p

ER Western Analysis

<p>E2 triggers ERK1/2 and Akt parallel pathways but only ERK1/2 pathway converges on GSK-3β.</p

Mitochondrial Calcium Retention Capacity

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

Heart Function

<p>Gper1-but not Esr1 nor Esr2- activation is essential for acute E2-mediated heart protection from ischemic/reperfusion (I/R) injury.</p

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

<p>Spectrofluorometric recordings of Ca2+ overload in mitochondria isolated from WT (A) Esr1-/- (B), Esr2-/- (C) and Gper1-/- (D) hearts subjected to I/R (E, inset) in presence of vehicle (Ctrl, black traces) or E2 (gray traces). Arrowheads mark the time of mitochondria addition and the initial mitochondrial Ca2+ uptake. Subsequent 10 nmol Ca2+ pulses (dashed lines; only shown for E2 treated) were delivered until a spontaneous massive release was observed presumably to the opening of mPTP (arrows). Only mitochondria from Gper1-/- lost their ability to endure higher Ca2+ overload by E2 treatment (D). E. Mean Ca2+ retention capacity values (amount of Ca2+ load needed to induce mPTP opening). Inset. I/R protocol marking the time of mitochondria isolation (10 min after reperfusion begun). Values are expressed as mean±SEM; * P<0.05 control versus E2-treated groups (n=5 hearts/group).</p

Table 2. Heart functional recovery parameters by E2 and in presence of MEK1/2, PKC translocation and PI-3K inhibitors in WT mice.

<p>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).</p

E2-induced cardiac protection against I/R injury is prevented by inhibitors of PKC translocation and MEK1/2 but not by a PI-3K inhibitor.

<p>A. Time course of LVDP changes by E2 +/- drugs during the I/R protocol (scheme at top). E2-induced protection during reperfusion was diminished by cotreatment during the whole protocol with MEK1/2 inhibitor, U0126 and with PKC translocation inhibitor, chelerythrine chloride (CC) but not with PI-3K inhibitor, LY294002. B. Heart sections of same heart in each condition stained at the end of the reperfusion period. White areas are infarcted zones. LY294002 co-treatment failed to inhibit E2-mediated prevention of heart infarct. C,D,E. Mean values of Rate Pressure Product (RPP), and % infarct size in control hearts (ctrl, perfused with vehicle) and hearts treated with E2 (40 nM), E2 (40 nM)+U0126 (1 mM), E2 (40 nM)+LY294002 (10 mM), and E2 (40 nM)+CC (1 mM). RPP and dP/dt max were obtained by averaging the last 2 min of reperfusion. Values are expressed as mean±SEM; *, P<0.05 E2-treated group versus control (n=7-8/group); #, P<0.05 E2+U0126 versus E2-treated group; +, P<0.05 E2+CC versus E2-treated group (n=4-7/group).</p

Heart functional recovery parameters by E2 and in presence of MEK_1/2, PKC translocation and PI-3K inhibitors in WT mice.

<p><b>Cardiac functional parameters in WT male mice.</b> 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><p>* P<0.05 control <i>versus</i> E2 group (n = 6-7/ group)</p><p>⁺ P<0.05 E2+CC <i>versus</i> E2 group (n = 4-6/ group)</p><p>^# P<0.05 E2+U0126 <i>versus</i> E2 group (n = 4–6 hearts/ group)</p><p>Heart functional recovery parameters by E2 and in presence of MEK_1/2, PKC translocation and PI-3K inhibitors in WT mice.</p

Heart recovery parameters by E2 in WT, Esr1^-/-, Esr2^-/- and Gper1^-/-.

<p>Cardiac functional parameters, 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 ischemia with and without E2 treatment in WT, Esr1^-/-, Esr2^-/- and Gper1. Values are mean±SEM.</p><p>* P<0.05 WT-control <i>versus</i> WT+E2-treated group (n = 4–6 hearts/ group)</p><p>^# P<0.05 Esr2^-/--control <i>versus</i> Esr1^-/-+E2-treated group (n = 4–6 hearts/ group)</p><p>⁺ P<0.05 Esr2^-/--control <i>versus</i> Esr2^-/-+E2-treated group (n = 4–6 hearts/ group)</p><p>Heart recovery parameters by E2 in WT, Esr1^-/-, Esr2^-/- and Gper1^-/-.</p