We recently found that estrogen treatment delays the mitochondrial permeability transition pore opening and reduces ROS production after ischemia/reperfusion, suggesting that estrogen promotes mitochondrial integrity. As mitochondrial inner membrane protein (mitofilin) has been found to control mitochondrial cristae morphology and function, we investigated whether estrogen effect on mitochondrial integrity after ischemia/reperfusion involved regulation of mitofilin via activation of G-Protein Coupled Estrogen Receptor1 (GPER1).
Isolated hearts from male WT (C57BL/6NCrL), and GPER1-/- mice were perfused using Langendorff technique, with and without estrogen (40 nM). Hearts were subjected to 20 min global ischemia followed by 10 min reperfusion. Mitochondria were isolated, and 2D-DIGE followed by mass spectrometry was performed. Mitofilin expression level was confirmed using Western blot analysis in mitochondrial fractions. Mitofilin distribution was visualized using confocal microscopy in isolated cardiomyocytes, and its spatial organization in mitochondria was imaged using high resolution fluorescence microscopy. Electron microscopy was used to observe mitochondrial cristae morphology.
We obtained 52 unique proteins of interest, in which mitofilin was identified. Immunoblot analysis confirmed the increased in mitofilin level with estrogen treatment as compared to control in WT but not in GPER1 KO. Cardiomyocyte images revealed as observed in non-ischemic myocytes that estrogen treatment conserved mitofilin distribution in the peri-membrane and T-tubules, while only peri-membrane mitofilin was more visible in control group. High resolution microscopy showed a better spatial organization of mitofilin in single mitochondria with estrogen treatment compared to control, in which mitofilin was almost absent. Electron microscopy showed that mitochondrial morphology was conserved in estrogen-treated group as cristae were well organized compared to control, in which cristae were disrupted.
These data indicate that estrogen action induces regulation of mitofilin expression during ischemia/reperfusion. Estrogen effect on mitofilin may contribute to improved mitochondrial integrity and function.