Regular physical exercise is associated with numerous health benefits and physiological adaptations. Mild and reversible oxidative stress induced by skeletal muscle contractions during exercise can result in long-term systemic redox adaptations. However, strenuous exercise can alter redox homeostasis and induce oxidative damage to macromolecules triggering a chain reaction of lipid peroxidation. Lipid peroxidation, yields formation of reactive aldehydes among which is biologically active 4-hydroxynonenal (4-HNE). 4-HNE can easily diffuse through membranes and covalently binds to macromolecules, such as proteins, altering cellular functions. Still little is known about the possible pathophysiological role of exercise induced 4-HNE on mitochondrial performance. In this work the effect of a caffeine, frequently used as an effective ergogenic aid by athletes, on the myotube mitochondrial metabolism, signaling and cellular redox homeostasis was studied using mouse C2C12, rat L6 and human HSMM skeletal muscle cells. Furthermore, the role of 4-HNE on myotube redox homeostasis, mitochondrial energy metabolism, mitochondrial transcriptome and mitochondrial density was investigated. Finally, the effect of physical exercise on 4-HNE post-translational modifications of proteins was studied using skeletal muscle samples from exercised animals by genuine mass spectrometry method. Obtained data indicate that short and long-term stimulation of myotubes with caffeine promotes reactive oxygen species formation and peroxidation of lipids leading to formation of 4-HNE protein adducts. 4-HNE further altered cellular redox homeostasis, mitochondrial metabolism and significantly increased mitochondrial density. Both caffeine and 4-HNE were found to regulate mitochondrial respiration and biogenesis gene expression. Finally, proteomics analysis of tissue samples from exercised mice revealed modifications of proteins susceptible to 6 oxidative stress. In conclusion, the findings signify the importance of skeletal muscle cells hormesis in response to acute stress and also suggest an important role of caffeine and 4-HNE on regulation of myotube’s metabolism and cellular energy production
