Activation of G protein‐coupled oestrogen receptor 1 at the onset of reperfusion protects the myocardium against ischemia/reperfusion injury by reducing mitochondrial dysfunction and mitophagy

Figures

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  Figure 1. Post‐ischaemic GPER activation induces cardioprotective effects against I/R injury in male and OVX‐female rats. (A) Protocol showing administration of a single bolus of saline (0.9%) for control, E2 or G15. (B) Example of images showing post‐ischaemic E2‐induced reduction in myocardial infarct size (IS) prevented by pre‐administration of G15 in male and OVX‐female rats. (C, F) Graphs showing the % of area at risk (AAR) in the LV. (D, G) Graph showing the percentage of myocardial IS in the AAR and in the LV. (E, H) *P < 0.05 E2‐treated versus control. + P < 0.05 E2 versus E2 + G15‐treated group (n = 6 hearts per group). Note that while the areas at risk are similar in all groups, myocardial IS as a function of the LV and AAR is reduced in E2‐treated group versus both control and E2 + G15‐treated groups indicating a GPER‐dependent mechanism.

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  Figure 2. Post‐ischaemic GPER activation decreases mitochondrial sensitivity to Ca2+ overload in male and OVX‐female rats. In (A), the data were obtained after 35 min ischaemia followed by 30 min reperfusion, and in (B), the results were obtained after only 10 min reperfusion. Left: spectrofluorometric recordings of Ca2+ overload in mitochondria isolated from hearts subjected to I/R in vehicle group (Ctrl), E2, E2 + G15 and sham group in male (top) and female (bottom) rats. Arrowheads mark the time of mitochondria addition and the initial mitochondrial Ca2+ uptake. Subsequent 20 nmol·mg−1 of protein Ca2+ pulses were delivered until a spontaneous massive release was observed, presumably to the opening of mPTP (arrows). Only mitochondria from the E2‐treated group kept their ability to endure higher Ca2+ overload similar to non‐ischaemic group (sham) much more than both control and E2 + G15‐treated groups indicating a GPER‐dependent mechanism. Right: graphs of mean Ca2+ retention capacity values (amount of Ca2+ load needed to induce mPTP opening) in male (top) and female (bottom) rats. Values are expressed as mean ± SEM; *P < 0.05 control versus sham group, + P < 0.05 control versus…

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  Figure 3. Post‐ischaemic GPER activation reduces mitophagy after I/R injury. Top: flow cytometry analysis of mitophagy with Mitodye (green) and mitochondria fused to lysosomes with Lysodye (blue) in all the groups. Bottom: graph showing reduction in mitophagy in E2‐treated group similar to sham versus control and E2 + G15‐treated groups. Note that the number of mitochondria fused to lysosomes is significantly reduced in the E2‐treated group versus control and E2 + G15‐treated groups suggesting a GPER‐dependent mechanism. Values are expressed as mean ± SEM; *P < 0.05 control versus sham groups, + P < 0.05 control versus E2‐treated group and # P < 0.05 E2 versus E2 + G15‐treated group (n = 5 per group).

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  Figure 4. Post‐ischaemic GPER activation reduces mitophagy via the PINK1/Parkin mechanism. In the whole‐cell lysate, post‐ischaemic E2 treatment decreased the protein levels of LC3I and LC3II and increased p62 and PINK1 expression versus control. In mitochondria, the Parkin level is reduced, while it is increased in the cytosol in E2 group versus control group indicating a reduction of Parkin translocation into mitochondrial. Note that all these E2 effects were prevented by addition of G15 suggesting a GPER‐dependent mechanism. Values are expressed as mean ± SEM; *P < 0.05 control versus sham groups, + P < 0.05 control versus E2‐treated group and # P < 0.05 E2 versus E2 + G15‐treated group (n = 5 per group).

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  Figure 5. GPER activation preserves mitochondrial integrity. Electron microscopy images of mitochondria in tissue (magnification 10 000×, 25 000× and 50 000×) from non‐ischaemic (sham), after I/R control, E2 and E2 + G15 treatment hearts showing a better mitochondrial structure integrity in E2‐treated and sham groups in which mitochondrial cristae are intact versus control and E2 + G15‐treated groups in which mitochondrial cristae morphology is drastically disrupted and damaged. Note that post‐ischaemic E2‐induced protection of mitochondrial structural integrity and decrease in the number of mito‐phagosomes are abolished by the addition of G15 suggesting a GPER‐dependent mechanism.

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  Figure 6. Post‐ischaemic GPER activation increases the phosphorylation of ERK1/2 and GSK‐3β. Immunoblots and corresponding bar graphs show post‐ischaemic E2 treatment increasing pERK1/2/ERK1/2 and pGSK‐3β/GSK‐3β ratios versus control. These E2 effects were prevented by addition of G15 (A) suggesting a GPER‐dependent mechanism and abolished by addition of U0126, a MEK/ERK pathway inhibitor (B). Values are expressed as mean ± SEM; *P < 0.05 E2‐treated versus control group and + P < 0.05 E2 versus E2 + G15‐treated groups (n = 5 per group).

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  Figure 7. Inhibition of mitophagy prevents post‐ischaemic GPER activation reduction of myocardial infarct size (IS). (A) Protocol showing administration of a single bolus of saline (0.9%) for control, E2, U0126 or 3‐MA as well as co‐addition of E2 + U0126 and E2 + 3‐MA. (B) Images showing post‐ischaemic E2‐induced reduction in myocardial IS prevented by pre‐administration of U0126 but not 3‐MA in male rats. (C) Graphs showing % AAR in the LV. (D) Graph showing the percentage of myocardial IS in the AAR and in the LV. (E) *P < 0.05 E2‐treated versus control. + P < 0.05 E2 versus E2 + U0126‐treated group (n = 5 hearts per group). # P < 0.05 E2 versus E2 + 3‐MA‐treated group (n = 5 hearts per group). Note that while the areas at risk are similar in all groups, myocardial IS as a function of the LV and AAR is reduced in the E2‐treated group (red) versus both control and E2 + U0126‐treated (green) groups but not in the E2 + 3‐MA group (purple) indicating a MEK/ERK‐dependent mechanism of mitophagy.

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  Figure 8. Post‐ischaemic GPER activation reduces mitochondrial protein ubiquitination including that of cyclophilin D (CypD). (A) Immunoblot and graph showing post‐ischaemic E2 treatment reduced mitochondrial ubiquitinated proteins versus control. Note that this E2 effect is prevented by addition of G15 suggesting a GPER‐dependent mechanism. Values are expressed as mean ± SEM; *P < 0.05 control versus sham groups, + P < 0.05 control versus E2‐treated group and # P < 0.05 E2 versus E2 + G15‐treated group (n = 5 per group). (B) To determine whether cyclophilin D is ubiquinated, IP was performed with anti‐ubiquitin polyclonal antibody and Western blot analysis with anti‐cyclophilin monoclonal antibody. The result indicates that post‐ischaemic E2 treatment reduces the cyclophilin D ubiquitination level versus control, and this effect is abolished by G15, a GPER antagonist.

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  Figure 9. Post‐ischaemic GPER activation preserves the mitochondrial membrane potential (MMP) and reduces ROS production. (A) MMP was measured spectrofluorometrically. Left: recordings of fluorescence in the presence of TMRM in mitochondria isolated from hearts subjected to I/R in vehicle group (Ctrl), E2, E2 + G15 and sham group. Arrowheads mark the time of mitochondria and CCCP addition. Note that addition of CCCP induces mitochondrial uncoupling, which attenuates the MMP. Right: the graph represents mean MMP values measured after CCCP addition in all the groups showing increased MMP in mitochondria treated with E2 versus both control and E2 + G15‐treated groups. Values are expressed as mean ± SEM; *P < 0.05 control versus sham group, + P < 0.05 control versus E2‐treated group and # P < 0.05 E2 versus E2 + G15‐treated group (n = 5 hearts per group). (B) Left: recording of mitochondrial ROS production using amplex red in the presence of horseradish peroxidase after stimulation of complex I with glutamate/malate. Right: graph showing reduction in ROS production in post‐ischaemic E2‐treated group versus control and E2 + G15‐treated groups. Values are expressed as mean ± SEM; *P < 0.05…