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.
Muscle atrophy occurs in many pathological states and results primarily from accelerated protein degradation and activation of the ubiquitin-proteasome pathway. However, the importance of lysosomes ...in muscle atrophy has received little attention. Activation of FoxO transcription factors is essential for the atrophy induced by denervation or fasting, and activated FoxO3 by itself causes marked atrophy of muscles and myotubes. Here, we report that FoxO3 does so by stimulating overall protein degradation and coordinately activating both lysosomal and proteasomal pathways. Surprisingly, in C2C12 myotubes, most of this increased proteolysis is mediated by lysosomes. Activated FoxO3 stimulates lysosomal proteolysis in muscle (and other cell types) by activating autophagy. FoxO3 also induces the expression of many autophagy-related genes, which are induced similarly in mouse muscles atrophying due to denervation or fasting. These studies indicate that decreased IGF-1-PI3K-Akt signaling activates autophagy not only through mTOR but also more slowly by a transcription-dependent mechanism involving FoxO3.
•Baseline separation of ATP and its 9 degradation products with reverse phase UPLC.•Volatile buffers allow simultaneous detection by UV–Vis and MS.•Phosphate acid wash and iron chelator are required ...to limit peak tailing.•Applicable to complex matrix of contracted skeletal muscle extracts.
ATP and its degradation products are essential metabolic and signaling molecules. Traditionally, they have been quantified via high-performance liquid chromatography (HPLC) with UV–Vis detection while utilizing phosphate buffer mobile phase, but this approach is incompatible with modern mass detection. The goal of this study was to develop an ultra-performance liquid chromatography (UPLC) method free of phosphate buffer, to allow for analysis of adenine nucleotides with UV–Vis and mass spectrometry (MS) simultaneously. The final conditions used an Acquity HSS T3 premier column with a volatile ammonium acetate buffer to successfully separate and quantify ATP-related analytes in a standard mixture and in extracts from non-contracted and contracted mouse hindlimb muscles. Baseline resolution was achieved with all 10 metabolites, and a lower limit of quantification down to 1 pmol per inject was observed for most metabolites using UV–Vis. Therefore, this method allows for the reliable quantification of adenine nucleotides and their degradation products via UV–Vis and their confirmation and/or identification of unknown peaks via MS.
Overexpression of the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), like exercise, increases mitochondrial content and inhibits muscle atrophy. To ...understand these actions, we tested whether PGC-1α or its close homolog, PGC-1β, influences muscle protein turnover. In myotubes, overexpression of either coactivator increased protein content by decreasing overall protein degradation without altering protein synthesis rates. Elevated PGC-1α or PGC-1β also prevented the acceleration of proteolysis induced by starvation or FoxO transcription factors and prevented the induction of autophagy and atrophy-specific ubiquitin ligases by a constitutively active FoxO3. In mouse muscles, overexpression of PGC-1β (like PGC-1α) inhibited denervation atrophy, ubiquitin ligase induction, and transcription by NFκB. However, increasing muscle PGC-1α levels pharmacologically by treatment of mice with 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside failed to block loss of muscle mass or induction of ubiquitin ligases upon denervation atrophy, although it prevented loss of mitochondria. This capacity of PGC-1α and PGC-1β to inhibit FoxO3 and NFκB actions and proteolysis helps explain how exercise prevents muscle atrophy.
Loss of myofibrillar proteins is a hallmark of atrophying muscle. Expression of muscle RING-finger 1 (MuRF1), a ubiquitin ligase, is markedly induced during atrophy, and MuRF1 deletion attenuates ...muscle wasting. We generated mice expressing a Ring-deletion mutant MuRF1, which binds but cannot ubiquitylate substrates. Mass spectrometry of the bound proteins in denervated muscle identified many myofibrillar components. Upon denervation or fasting, atrophying muscles show a loss of myosin-binding protein C (MyBP-C) and myosin light chains 1 and 2 (MyLC1 and MyLC2) from the myofibril, before any measurable decrease in myosin heavy chain (MyHC). Their selective loss requires MuRF1. MyHC is protected from ubiquitylation in myofibrils by associated proteins, but eventually undergoes MuRF1-dependent degradation. In contrast, MuRF1 ubiquitylates MyBP-C, MyLC1, and MyLC2, even in myofibrils. Because these proteins stabilize the thick filament, their selective ubiquitylation may facilitate thick filament disassembly. However, the thin filament components decreased by a mechanism not requiring MuRF1.
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.
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.
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%).
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.
•AMP deaminase (AMPD) decreases ATP without activating AMPK or its substrates.•AMPD alters the intracellular metabolome similar to atrophic muscle.•AMPD slows mitochondria synthesis and oxygen consumption similar to atrophic muscle.•Metabolome shift is independent of metabolic genes and precede mitochondria changes.
Since its recent discovery, Bourbon virus has been isolated from a human and ticks. To assess exposure of potential vertebrate reservoirs, we assayed banked serum and plasma samples from wildlife and ...domestic animals in Missouri, USA, for Bourbon virus-neutralizing antibodies. We detected high seroprevalence in raccoons (50%) and white-tailed deer (86%).
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
INTRODUCTIONEvidence suggests that the energy efficiency of key ATPases involved in skeletal muscle contractile activity are improved in a hypothermic condition. However, it is unclear how a decrease ...in temperature affects skeletal muscle O2 consumption (mVO2) induced by muscle contraction.
METHODSIsolated mouse extensor digitorum longus (EDL) muscles were incubated in a temperature-controlled (37°C or 25°C) bath that included an O2 probe. EDL muscles from one limb were subjected to the measurement of resting mVO2, and the contralateral EDL muscles were used for the measurement of mVO2 with electrically-stimulated contraction. For the resting protocol, muscles were suspended at resting tension for 15 mins with continuous O2 recordings. For the contraction protocol, EDL muscles underwent ten electrically-stimulated isometric contractions with continuous O2 recordings for 15 mins. The rate of O2 disappearance was quantified as μmole O2 per minute and normalized to the wet weight of the muscle.
RESULTSResting mVO2 was greater at 37°C than at 25°C, consistent with the idea that lower temperature reduces basal metabolic rate. Electrically-stimulated contraction robustly increased mVO2 at both 37°C and 25°C, which was sustained for ~3 min post-contraction. During that period, mVO2 was elevated ~5-fold at both 37°C and 25°C. Greater contraction-induced mVO2 at 37°C compared to 25°C occurred despite lower force generated at 37°C than at 25°C.
CONCLUSIONTogether, O2 cost for muscle contraction (force-time integral per O2 consumed) was greater at 37°C than at 25°C. Levels of high-energy phosphates were consistent with greater energy demand at 37°C compared to 25°C. In conclusion, these results indicate that muscle contraction that occurs at subnormal temperature requires less O2 than at 37°C.
Stored muscle carbohydrate supply and energetic efficiency constrain muscle functional capacity during exercise and are influenced by common physiological variables (e.g. age, diet, and physical ...activity level). Whether these constraints affect overall functional capacity or the timing of muscle energetic failure during acute hypoxia is not known. We interrogated skeletal muscle contractile properties in two anatomically distinct rodent hindlimb muscles that have well characterized differences in energetic efficiency (locomotory- extensor digitorum longus (EDL) and postural- soleus muscles) following a 24 hour fasting period that resulted in substantially reduced muscle carbohydrate supply. 180 mins of acute hypoxia resulted in complete energetic failure in all muscles tested, indicated by: loss of force production, substantial reductions in total adenosine nucleotide pool intermediates, and increased adenosine nucleotide degradation product-inosine monophosphate (IMP). These changes occurred in the absence of apparent myofiber structural damage assessed histologically by both transverse section and whole mount. Fasting and the associated reduction of the available intracellular carbohydrate pool (~50% decrease in skeletal muscle) did not significantly alter the timing to muscle functional impairment or affect the overall force/work capacities of either muscle type. Fasting resulted in greater passive tension development in both muscle types, which may have implications for the design of pre-clinical studies involving optimal timing of reperfusion or administration of precision therapeutics.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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