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(−)‐Epicatechin Stimulates Mitochondrial Biogenesis in C2C12 Myotubes: Involvement of the G‐protein Coupled Estrogen Receptor (GPER)

During the last decade, it has been of great interest to identify natural products with promising effects on skeletal muscle function. In part, this has been fueled by the interest of developing small molecules with the capability to mimic and/or enhance the effects of exercise in order to improve muscle performance and more importantly to mitigate the effects of aging and disease‐induced muscle wasting. In this regard, our group has provided amply evidence as to how a natural small molecule that belongs to the family of flavonoids (subclass of flavanols), (−)‐epicatechin ((−)‐EPI), is capable to augment exercise capacity in mice and humans associated to an increase in mitochondrial function and biogenesis. The aim of this work was to evaluate the role that the GPER has on mediating the effects of (−)‐EPI on mitochondrial biogenesis in skeletal muscle cells. As an in vitro model of skeletal muscle cells we employed C2C12 myotubes. Using confocal microscopy we demonstrated that the GPER is localized in the cell surface and cytoplasm in C2C12 myotubes. The binding of (−)‐EPI to GPER was evaluated using flow cytometry by means of displacement of estradiol conjugated to fluorescein‐bovine serum albumin (E2‐BSA‐FITC) with increasing concentrations of (−)‐EPI and compared to the potent and selective GPER agonist, G1. To evaluate mitochondrial biogenesis we used fluorescence microscopy in order to quantify mitochondria density accompanied by measurement of oxidative phosphorylation complex proteins II and IV densities by using an in‐cell ELISA kit. Results indicate that (−)‐EPI is capable of displacing E2‐BSA‐FITC similarly to G1. In addition, 48 h of (−)‐EPI treatment (10 M) significantly increases mitochondrial biogenesis in C2C12 myotubes with similar efficacy to that observed with G1, effects that were partially and completely blocked by G36 (GPER antagonist), respectively. Altogether, these results indicate that GPER is expressed in muscle cells and appears to mediate to a significant extent the stimulatory effects of (−)‐EPI on mitochondrial biogenesis. Thus, GPER activation may account for the stimulatory effects on skeletal muscle structure/function observed by either cocoa or pure (−)‐EPI treatment in animals and humans.