Increased Adenine Nucleotide Degradation in Skeletal Muscle Atrophy

Adenine nucleotides (AdNs: ATP, ADP, AMP) are essential biological compounds that facilitate many necessary cellular processes by providing chemical energy, mediating intracellular signaling, and regulating protein metabolism and solubilization. A dramatic reduction in total AdNs is observed in atrophic skeletal muscle across numerous disease states and conditions, such as cancer, diabetes, chronic kidney disease, heart failure, COPD, sepsis, muscular dystrophy, denervation, disuse, and sarcopenia. The reduced AdNs in atrophic skeletal muscle are accompanied by increased expression/activities of AdN degrading enzymes and the accumulation of degradation products (IMP, hypoxanthine, xanthine, uric acid), suggesting that the lower AdN content is largely the result of increased nucleotide degradation. Furthermore, this characteristic decrease of AdNs suggests that increased nucleotide degradation contributes to the general pathophysiology of skeletal muscle atrophy. In view of the numerous energetic, and non-energetic, roles of AdNs in skeletal muscle, investigations into the physiological consequences of AdN degradation may provide valuable insight into the mechanisms of muscle atrophy

Sex-Related Differences in the Maximal Lactate Steady State

The maximal lactate steady state (MLSS) is one of the factors that differentiates performance in aerobic events. The purpose of this study was to investigate the sex differences in oxygen consumption (VO2), heart rate (HR), and the respiratory exchange ratio (RER) at the MLSS in well-trained distance runners. Twenty-two (12 female, 10 male) well-trained distance runners (23 ± 5.0 years) performed multiple 30-min steady-state runs to determine their MLSS, during which blood lactate and respiratory gas exchange measures were taken. To interpret the MLSS intensity as a training tool, runners completed a time-to-exhaustion (TTE) run at their MLSS. The relative intensity at which the MLSS occurred was identical between males and females according to both oxygen consumption (83 ± 5 %O2max) and heart rate (89 ± 7 %HRmax). However, female runners displayed a significantly lower RER at MLSS compared to male runners (p < 0.0001; 0.84 ± 0.02 vs. 0.88 ± 0.04, respectively). There was not a significant difference in TTE at MLSS between males (79 ± 17 min) and females (80 ± 25 min). Due to the observed difference in the RER at the MLSS, it is suggested that RER derived estimates of MLSS be sex-specific. While the RER data suggest that the MLSS represents different metabolic intensities for males and females, the relative training load of MLSS appears to be similar in males and female runners

Skeletal muscle contraction kinetics and AMPK responses are modulated by the adenine nucleotide degrading enzyme AMPD1

AMP deaminase 1 (AMPD1; AMP → IMP + NH3) deficiency in skeletal muscle results in an inordinate accumulation of AMP during strenuous exercise, with some but not all studies reporting premature fatigue and reduced work capacity. To further explore these inconsistencies, we investigated the extent to which AMPD1 deficiency impacts skeletal muscle contractile function of different muscles and the [AMP]/AMPK responses to different intensities of fatiguing contractions. To reduce AMPD1 protein, we electroporated either an inhibitory AMPD1-specific miRNA encoding plasmid or a control plasmid, into contralateral EDL and SOL muscles of C57BL/6J mice (n = 48 males, 24 females). After 10 days, isolated muscles were assessed for isometric twitch, tetanic, and repeated fatiguing contraction characteristics using one of four (None, LOW, MOD, and HIGH) duty cycles. AMPD1 knockdown (∼35%) had no effect on twitch force or twitch contraction/relaxation kinetics. However, during maximal tetanic contractions, AMPD1 knockdown impaired both time-to-peak tension (TPT) and half-relaxation time (½ RT) in EDL, but not SOL muscle. In addition, AMPD1 knockdown in EDL exaggerated the AMP response to contractions at LOW (+100%) and MOD (+54%) duty cycles, but not at HIGH duty cycle. This accumulation of AMP was accompanied by increased AMPK phosphorylation (Thr-172; LOW +25%, MOD +34%) and downstream substrate phosphorylation (LOW +15%, MOD +17%). These responses to AMPD1 knockdown were not different between males and females. Our findings demonstrate that AMPD1 plays a role in maintaining skeletal muscle contractile function and regulating the energetic responses associated with repeated contractions in a muscle- but not sex-specific manner. NEW & NOTEWORTHY: AMP deaminase 1 (AMPD1) deficiency has been associated with premature muscle fatigue and reduced work capacity, but this finding has been inconsistent. Herein, we report that although AMPD1 knockdown in mouse skeletal muscle does not change maximal isometric force, it negatively impacts muscle function by slowing contraction and relaxation kinetics in EDL muscle but not SOL muscle. Furthermore, AMPD1 knockdown differentially affects the [AMP]/AMPK responses to fatiguing contractions in an intensity-dependent manner in EDL muscle

Uric acid formation is driven by crosstalk between skeletal muscle and other cell types

Hyperuricemia is implicated in numerous pathologies, but the mechanisms underlying uric acid production are poorly understood. Using a combination of mouse studies, cell culture studies, and human serum samples, we sought to determine the cellular source of uric acid. In mice, fasting and glucocorticoid treatment increased serum uric acid and uric acid release from ex vivo-incubated skeletal muscle. In vitro, glucocorticoids and the transcription factor FoxO3 increased purine nucleotide degradation and purine release from differentiated muscle cells, which coincided with the transcriptional upregulation of AMP deaminase 3, a rate-limiting enzyme in adenine nucleotide degradation. Heavy isotope tracing during coculture experiments revealed that oxidation of muscle purines to uric acid required their transfer from muscle cells to a cell type that expresses xanthine oxidoreductase, such as endothelial cells. Last, in healthy women, matched for age and body composition, serum uric acid was greater in individuals scoring below average on standard physical function assessments. Together, these studies reveal skeletal muscle purine degradation is an underlying driver of uric acid production, with the final step of uric acid production occurring primarily in a nonmuscle cell type. This suggests that skeletal muscle fiber purine degradation may represent a therapeutic target to reduce serum uric acid and treat numerous pathologies

AMP deamination is sufficient to replicate an atrophy-like metabolic phenotype in skeletal muscle

Background: Skeletal muscle atrophy, whether caused by chronic disease, acute critical illness, disuse or aging, is characterized by tissue-specific decrease in oxidative capacity and broad alterations in metabolism that contribute to functional decline. However, the underlying mechanisms responsible for these metabolic changes are largely unknown. One of the most highly upregulated genes in atrophic muscle is AMP deaminase 3 (AMPD3: AMP → IMP + NH3), which controls the content of intracellular adenine nucleotides (AdN; ATP + ADP + AMP). Given the central role of AdN in signaling mitochondrial gene expression and directly regulating metabolism, we hypothesized that overexpressing AMPD3 in muscle cells would be sufficient to alter their metabolic phenotype similar to that of atrophic muscle. Methods: AMPD3 and GFP (control) were overexpressed in mouse tibialis anterior (TA) muscles via plasmid electroporation and in C2C12 myotubes using adenovirus vectors. TA muscles were excised one week later, and AdN were quantified by UPLC. In myotubes, targeted measures of AdN, AMPK/PGC-1α/mitochondrial protein synthesis rates, unbiased metabolomics, and transcriptomics by RNA sequencing were measured after 24 h of AMPD3 overexpression. Media metabolites were measured as an indicator of net metabolic flux. At 48 h, the AMPK/PGC-1α/mitochondrial protein synthesis rates, and myotube respiratory function/capacity were measured. Results: TA muscles overexpressing AMPD3 had significantly less ATP than contralateral controls (-25%). In myotubes, increasing AMPD3 expression for 24 h was sufficient to significantly decrease ATP concentrations (-16%), increase IMP, and increase efflux of IMP catabolites into the culture media, without decreasing the ATP/ADP or ATP/AMP ratios. When myotubes were treated with dinitrophenol (mitochondrial uncoupler), AMPD3 overexpression blunted decreases in ATP/ADP and ATP/AMP ratios but exacerbated AdN degradation. As such, pAMPK/AMPK, pACC/ACC, and phosphorylation of AMPK substrates, were unchanged by AMPD3 at this timepoint. AMPD3 significantly altered 191 out of 639 detected intracellular metabolites, but only 30 transcripts, none of which encoded metabolic enzymes. The most altered metabolites were those within purine nucleotide, BCAA, glycolysis, and ceramide metabolic pathways. After 48 h, AMPD3 overexpression significantly reduced pAMPK/AMPK (-24%), phosphorylation of AMPK substrates (-14%), and PGC-1α protein (-22%). Moreover, AMPD3 significantly reduced myotube mitochondrial protein synthesis rates (-55%), basal ATP synthase-dependent (-13%), and maximal uncoupled oxygen consumption (-15%). Conclusions: Increased expression of AMPD3 significantly decreased mitochondrial protein synthesis rates and broadly altered cellular metabolites in a manner similar to that of atrophic muscle. Importantly, the changes in metabolites occurred prior to reductions in AMPK signaling, gene expression, and mitochondrial protein synthesis, suggesting metabolism is not dependent on reductions in oxidative capacity, but may be consequence of increased AMP deamination. Therefore, AMP deamination in skeletal muscle may be a mechanism that alters the metabolic phenotype of skeletal muscle during atrophy and could be a target to improve muscle function during muscle wasting