Gper1 activation is essential for E2-mediated cardioprotection.

<p><b>A.</b> Recordings of left ventricular developed pressure (LVDP) comparing heart activity from wild-type (WT) animals not subjected to ischemia/reperfusion (Sham) with the function of hearts subjected to the ischemia reperfusion (I/R) protocol under the continuous presence of vehicle (control) or 40 nM E2 in KH buffer. In sham hearts, function stabilizes at about 30–40 min after mounting and is stable for at least 2 hours. In hearts subjected to I/R (middle and bottom traces), E2 improved recovery from ischemia (during reperfusion) compared to control. <b>B-D.</b> LVDP traces from hearts of Esr1, Esr2 and Gper1 knockouts (-/-) subjected to I/R. Only in Gper1^-/- hearts, E2 lost its ability to improve functional recovery (<b>E</b>). n = 6–8 hearts/ group.</p

E2-induced increase in phosphorylation of Akt, ERK_1/2 and GSK-3β is dependent on Gper1 gene expression and disappears with time for Akt and ERK_1/2.

<p><b>A.</b> Protocol used in panels <b>B,C,D</b>. 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>B-D.</b> Immunoblots and corresponding bar graphs show that 5 min “pre-ischemic” treatment with E2 provoked pAkt/Akt, pERK_1/2/ERK_1/2 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 <i>versus</i> E2-treated group, n = 7 hearts/group.</p

Salvage mechanisms triggered by E2 via Gper1 during I/R.

<p>E2 binding to Gper1 may initially trigger protein kinase C (PKC) translocation. PKC could directly or via activation of MEK_1/2/ERK_1/2 pathway increase phosphorylation of GSK-3β, which in turn would inhibit mPTP opening resulting in cardioprotection. Chelerythrine chloride, inhibitor of PKC translocation; U0126, inhibitor of the MEK_1/2/ERK_1/2-pathway; and LY294002, inhibitor of PI-3K. E2 through Gper1 can also induce a transient activation of PI-3K/Akt pathway, but this activation does not play an important role in the acute E2 induced cardioprotection after I/R. Black arrows, pathways demonstrated in this work. Dashed arrows, putative pathways.?, unknown target.</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

Gper1 activation is essential for E2 to induce increased resistance to Ca²⁺-overload of mitochondria from ischemic-reperfused hearts.

<p><b>A-D.</b> Spectrofluorometric recordings of Ca²⁺ overload in mitochondria isolated from WT (<b>A</b>) Esr1^-/- (<b>B</b>), Esr2^-/- (<b>C</b>) and Gper1^-/- (<b>D</b>) hearts subjected to I/R (<b>E</b>, 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 (<b>D</b>). <b>E.</b> 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 <i>versus</i> E2-treated groups (n = 5 hearts/group).</p

E2-induced cardiac protection against I/R injury is prevented by inhibitors of PKC translocation and MEK_1/2 but not by a PI-3K inhibitor.

<p><b>A.</b> 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 MEK_1/2 inhibitor, U0126 and with PKC translocation inhibitor, chelerythrine chloride (CC) but not with PI-3K inhibitor, LY294002. <b>B.</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. <b>C,D,E.</b> 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 μM), E2 (40 nM)+LY294002 (10 μM), and E2 (40 nM)+CC (1 μM). 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 <i>versus</i> control (n = 6-8/group); #, P<0.05 E2+U0126 <i>versus</i> E2-treated group; +, P<0.05 E2+CC <i>versus</i> E2-treated group (n = 4-7/group). Functional values as a function of time are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135988#pone.0135988.t002" target="_blank">Table 2</a>. All values were obtained using WT male mice.</p

MEK_1/2/ERK_1/2 –but not PI-3K/Akt- signaling supports E2-mediated protection from I/R via GSK-3β and mPTP.

<p><b>A.</b> I/R protocol. Arrows mark the time of sample preparation. <b>B.</b> Immunoblots show that addition of the inhibitor of MEK_1/2/ERK_1,2 pathway, U0126 (1 μM) abolishes E2-induced up-regulation of pERK and the inhibitor of PI3-K/Akt pathway, LY294002 (10 μM) abolishes the increase in pAkt induced by 40 nM E2. <b>C,D.</b> Immunoblots and corresponding bar graphs show that the inhibitor of MEK_1/2/ERK_1,2 pathway, U0126 (1 μM)-but not the inhibitor of PI3-K/Akt pathway, LY294002 (10 μM)- abolishes the increase in pGSK-3β/GSK-3β ratio induced by 40 nM E2. <b>E.</b> Calcium load measurements were as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135988#pone.0135988.g005" target="_blank">Fig 5</a>. 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. <b>F.</b> 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 <i>versus</i> control; and # P<0.05 E2+U0126 <i>versus</i> E2-treated group (n = 5–7 hearts/group).</p

Gper1 activation is required to protect the heart from infarct induced by I/R.

<p>Hearts were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135988#pone.0135988.g002" target="_blank">Fig 2</a> and analyzed at the end of each protocol. <b>A-E.</b> Images are from slices of the same heart in each condition. White areas correspond to the infarcted zone. <b>A.</b> Infarct is minimal in isolated hearts not subjected to I/R. <b>B-D.</b> E2 protected equally well hearts from WT, Esr1 and Esr2 knockouts against infarct induced by I/R. <b>E.</b> Gper1^-/- was the only E2 receptor knockout examined where I/R caused the same degree of infarct in the presence or absence of E2. <b>F.</b> Individual and mean±SEM values of % infarct size. * P<0.05 control <i>versus</i> E2-treated groups (WT, n = 7–8 hearts/group; Esr1^-/-, n = 5 hearts/group; Esr2^-/-, n = 5–6 hearts/group). In Gper1^-/- hearts, E2 treatment did not reduce the infarct size (control = 52±3% <i>vs</i>. E2-treated = 47±4%, n = 6 hearts/group).</p

Gper1 transcripts predominate over Esr1 and Esr2 in male mouse heart.

<p><b>A.</b> An example of relative fluorescence intensity versus PCR cycle number for Esr1, Esr2, Gper1 and β-actin (housekeeping gene) in mouse ventricle. Inset, melting curves for Gper1, Esr1, Esr2, and β-actin reactions. <b>B.</b> Real-time PCR end-products for Gper1, Esr1, Esr2 and β-actin with reverse transcriptase (RT), without RT (-RT) and water instead of total RNA (H₂O). <b>C-E.</b> Standard calibration curves for Gper1, Esr1, and Esr2 DNA plasmids, respectively. Open symbols, known amount of linearized plasmid DNA. Closed symbols, interpolated absolute values of estrogen receptor cDNAs in mouse heart (arrow). <b>F.</b> Mean absolute values of Gper1, Esr1 and Esr2 transcript levels in mouse heart. Quantifications were performed in duplicate from samples of three different animals (n = 3). In this and following figures, data were acquired from male mice.</p