Sulforaphane, Urolithin A, and ZLN005 induce time-dependent alterations in antioxidant capacity, mitophagy, and mitochondrial biogenesis in muscle cells

Abstract

Efficient signal transduction that mediates mitochondrial turnover is a strong determinant of metabolic health in skeletal muscle. Of these pathways, our focus was aimed towards the enhancement of antioxidant capacity, mitophagy, and mitochondrial biogenesis. While physical activity is an excellent inducer of mitochondrial turnover, its ability to ubiquitously activate and enhance mitochondrial turnover prevents definitive differentiation of the contribution made by each pathway. Therefore, we employed three agents, Sulforaphane (SFN), Urolithin A (UroA), and ZLN005 (ZLN), which are activators of important biological markers involved in antioxidant signaling, mitophagy, and biogenesis, respectively. We investigated the time-dependent changes in proteins related to each mechanism in C2C12 myotubes. SFN treatment resulted in increased nuclear localization of the transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) after 4 ​hour (h), with subsequent 2-fold increases in the antioxidant enzymes Nicotinamide Quinone Oxidoreductase 1 (NQO1) and Heme-Oxygenase-1 (HO-1) by 24 ​h and 48 ​h. Mitochondrial respiration and ATP production were significantly increased by both 24 h and 48 ​h. UroA showed a 2-fold increase in AMP-activated Protein Kinase (AMPK) after 4 ​h, which led to a modest 30% increase in whole cell mitophagy markers p62 and LC3, after 48 ​h. This was accompanied by a reduction in cellular Reactive Oxygen Species (ROS), detected with the CellROX Green reagent. Mitophagy flux measurements showed mitophagy activation as both LC3-II and p62 flux increased with UroA at 24-h and 48-h time points, respectively. Finally, AMPK activation was observed by 4 ​h, in addition to a 2-fold increase in Mitochondrial Transcription Factor A (TFAM) promoter activity by 24 ​h of ZLN treatment following transient transfection of a TFAM promoter-luciferase construct. Mitochondrial respiration and ATP production were enhanced by 24 ​h. Our results suggest that early time points of treatment increase upstream pathway activity, whereas later time points represent the increased phenotypic expression of related downstream markers. Our findings suggest that the spatiotemporal progression of these mechanisms following drug treatment is another important factor to consider when examining subcellular changes towards mitochondrial turnover in muscle.