Activation of the erythropoietin system in exhaustively exercise muscle relates to ACE gene polymorphism-modulated metabolic signalling and mitochondrial transcript expression

Introduction Introduction A fall in muscle oxygen saturation is a potent activator of mitochondrial biogenesis in exercised skeletal muscle which is subject to the training-modulated influence of the insertion/deletion polymorphism of the angiotensin converting enzyme gene (ACE-I/D; Gasser et al 2022) and may involve paracrine erythropoietin (EPO) signaling (Desplanches et al 2014, Nijholt et al 2021). We tested the hypothesis that erythropoietin expression and signaling in fatiguingly exercised muscle would correspond to the expression of hypoxia-regulated mitochondrial genes and altered metabolic signaling and would be subject to a genetic influence by ACE-I/D. Methods Methods 22 healthy, male white Caucasian men (27.0 +/- 1.4 years; BMI 23.6 +/- 0.6 kg m-2) completed a session of fatiguing one-legged exercise in the fasted state. Microbiopsies were collected from m. vastus lateralis of the non-exercised leg immediately before exercise, and ½, 3, and 8 hours thereafter from the exercising leg. Levels of the hypoxia-regulated cytochrome c oxidase subunit 4 isoforms 1 and 2 (COX4I1 and COX4I2), ACE and EPO transcript, glycogen concentration in m. vastus lateralis, the ACE-I/D genotype and aerobic fitness state were assessed as described (Desplanches et al 2014; Gasser et al 2022). EPO protein concentration and phosphorylation of intracellular transducers of EPO signaling were quantified in muscle homogenates with validated enzyme-linked immune sorbent and a phospho-kinase assays. Effects and Pearson correlations were assessed with analysis of variance and declared significant at p < 0.05. Results One-legged exercise produced metabolic fatigue as indicated by the voluntary cessation of exercise and a reduced glycogen concentration in m. vastus lateralis in all subjects ½ hour after exercise (-0.044 mg mg-1). Concomitantly, EPO protein levels were 4-fold lowered; and subsequently increased 3-8 hours after cessation of exercise alike EPO transcripts levels. Aerobically fit ACE I-allele carriers demonstrated a sparing of muscle glycogen, exaggerated EPO transcript response, and higher phosphorylation levels of EPO signal transducers [STAT5a-Y694 (+31%), STAT5b-Y699 (+40%)]. The phosphorylation level of the metabolic signal transducer AMPKa2-T172 correlated to EPO transcript levels 3 hours post exercise (r = 0.61). EPO protein levels correlated to ACE and COX4I2 transcript levels (r = -0.79; -0.54). Discussion The findings highlight that a paracrine loop of metabolically-regulated EPO signaling exists in exercised human skeletal muscle which variability is associated with the ACE-I/D gene polymorphism in fair correspondence with a mitochondrial marker of local hypoxia. References Desplanches, D., Amami, M., Mueller, M., Hoppeler, H., Kreis, R., & Flück, M. (2014). Hypoxia refines plasticity of mitochondrial respiration to repeated muscle work. European Journal of Applied Physiology, 114, 405-417. https://doi.org/10.1007/s00421-013-2783-8 Gasser, B., Franchi, M. V., Ruoss, S., Frei, A., Popp, W. L., Niederseer, D., Catuogno, S., Frey, W. O., & Flück, M. (2022). Accelerated muscle deoxygenation in aerobically fit subjects during exhaustive exercise is associated with the ACE insertion allele. Frontiers in Sports and Active Living, 4, Article 814975. https://doi.org/10.3389/fspor.2022.814975 Nijholt, K. T., Meems, L. M. G., Ruifrok, W. P. T., Maass, A. H., Yurista, S. R., Pavez-Giani, M. G., Mahmoud, B., Wolters, A. H. G., van Veldhuisen, D. J., van Gilst, W. H., Silljé, H. H. W., de Boer, R. A., & Westenbrink, B. D. (2021). The erythropoietin receptor expressed in skeletal muscle is essential for mitochondrial biogenesis and physiological exercise. Pflügers Archiv - European Journal of Physiology, 473, 1301-1313. https://doi.org/10.1007/s00424-021-02577-4