Exercise-Induced 4-Hydroxynonenal Alters Myotube Cellular Redox Homeostasis and Mitochondrial Metabolism

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

Lipid peroxidation is involved in calcium dependent upregulation of mitochondrial metabolism in skeletal muscle

BACKGROUND: Skeletal muscle cells continuously generate reactive oxygen species (ROS). Excessive ROS can affect lipids resulting in lipid peroxidation (LPO). Here we investigated the effects of myotube intracellular calcium-induced signaling eliciting contractions on the LPO induction and the impact of LPO-product 4-hydroxynonenal (4-HNE) on physiology/pathology of myotubes using C2C12 myoblasts. METHODS: C2C12 myoblasts were differentiated into myotubes, stimulated with caffeine and analyzed for the induction of LPO and formation of 4-HNE protein adducts. Further effects of 4-HNE on mitochondrial bioenergetics, NADH level, mitochondrial density and expression of mitochondrial metabolism genes were determined. RESULTS: Short and long-term caffeine stimulation of myotubes promoted superoxide production, LPO and formation of 4-HNE protein adducts. Furthermore, low 4-HNE concentrations had no effect on myotube viability and cellular redox homeostasis, while concentrations from 10 μM and above reduced myotube viability and significantly disrupted homeostasis. A time and dose-dependent 4-HNE effect on superoxide production and mitochondrial NADH-autofluorescence was observed. Finally, 4-HNE had strong impact on maximal respiration, spare respiratory capacity, ATP production, coupling efficiency of mitochondria and mitochondrial density. CONCLUSION: Data presented in this work make evident for the first time that pathological 4-HNE levels elicit damaging effects on skeletal muscle cells while acute exposure to physiological 4-HNE induces transient adaptation. GENERAL SIGNIFICANCE: This work suggests an important role of 4-HNE on the regulation of myotube's mitochondrial metabolism and cellular energy production. It further signifies the importance of skeletal muscle cells hormesis in response to acute stress in order to maintain essential biological functions

Proteomic Analysis of Cardiac Adaptation to Exercise by High Resolution Mass Spectrometry

Regular exercise has many health benefits among which is a significant reduction of cardiovascular risk. Although many beneficial effects of exercise are well described, the exact mechanisms by which exercise confers cardiovascular benefits remain to be fully understood. In the current study, we have used high resolution mass spectrometry to determine the proteomic responses of the heart to exercise training in mice. The impact of exercise-induced oxidative stress on modifications of cardiomyocyte proteins with lipid peroxidation biomarker 4-hydroxynonenal (4-HNE) was examined as well. Fourteen male mice were randomized into the control (sedentary) group and exercise group subjected to a swim exercise training program for 5 days a week for 5 months. Proteins were isolated from the left ventricular tissue, fractionated and digested for shotgun proteomics. Peptides were separated by nanoliquid chromatography and analyzed on Orbitrap Fusion mass spectrometer using higher energy collision-induced dissociation and electron transfer dissociation fragmentation. We identified distinct ventricular protein signatures established in response to exercise training. Comparative proteomics identified 23 proteins upregulated and 37 proteins downregulated with exercise in addition to 65 proteins identified only in ventricular tissue samples of exercised mice. Most of the proteins specific for exercised mice are involved in respiratory electron transport, and/or implicated in glutathione conjugation. Additionally, 10 proteins were found to be modified with 4-HNE. This study provides new data on the effects of exercise on the cardiac proteome and contributes to our understanding of the molecular mechanisms underlying the beneficial effects of exercise on the heart

Whole blood storage in CPDA1 blood bags alters erythrocyte membrane proteome

Autologous blood transfusion (ABT) has been frequently abused in endurance sport and is prohibited since the mid-1980s by the International Olympic Committee. Apart from any significant performance-enhancing effects, the ABT may pose a serious health issue due to aging erythrocyte-derived "red cell storage lesions." The current study investigated the effect of blood storage in citrate phosphate dextrose adenine (CPDA1) on the red blood cell (RBC) membrane proteome. One unit of blood was collected in CPDA1 blood bags from 6 healthy female volunteers. RBC membrane protein samples were prepared on days 0, 14, and 35 of storage. Proteins were digested in gel and peptides separated by nanoliquid chromatography coupled to tandem mass spectrometry resulting in the confident identification of 33 proteins that quantitatively change during storage. Comparative proteomics suggested storage-induced translocation of cytoplasmic proteins to the membrane while redox proteomics analysis identified 14 proteins prone to storage-induced oxidation. The affected proteins are implicated in the RBC energy metabolism and membrane vesiculation and could contribute to the adverse posttransfusion outcomes. Spectrin alpha chain, band 3 protein, glyceraldehyde-3-phosphate dehydrogenase, and ankyrin-1 were the main proteins affected by storage. Although potential biomarkers of stored RBCs were identified, the stability and lifetime of these markers posttransfusion remain unknown. In summary, the study demonstrated the importance of studying storage-induced alterations in the erythrocyte membrane proteome and the need to understand the clearance kinetics of transfused erythrocytes and identified protein markers