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.
Skeletal muscle immobilization leads to atrophy, decreased metabolic health, and substantial losses in function. Animal models suggest that heat stress can provide protection against atrophy in ...skeletal muscle. This study investigated the effects of daily heat therapy on human skeletal muscle subjected to 10 days of immobilization. Muscle biopsies were collected, and MRIs were analyzed from the vastus lateralis of 23 healthy volunteers (11 women, 12 men) before and after either 10 days of immobilization with a daily sham treatment (Imm) or with a targeted, daily 2-h heat treatment using pulsed shortwave diathermy (Imm + H). Diathermy increased intramuscular temperature 4.2 ± 0.29°C (
< 0.0001), with no change during sham treatment. As a result, heat shock protein (HSP)70 and HSP90 increased (
< 0.05) following Imm + H (25 ± 6.6 and 20 ± 7.4%, respectively) but were unaltered with Imm only. Heat treatment prevented the immobilization-induced loss of coupled (-27 ± 5.2% vs. -8 ± 6.0%,
= 0.0041) and uncoupled (-25 ± 7.0% vs. -10 ± 3.9%,
= 0.0302) myofiber respiratory capacity. Likewise, heat treatment prevented the immobilization-induced loss of proteins associated with all five mitochondrial respiratory complexes (
< 0.05). Furthermore, decreases in muscle cross-sectional area following Imm were greater than Imm + H at both the level of the whole muscle (-7.6 ± 0.96% vs. -4.5 ± 1.09%,
= 0.0374) and myofiber (-10.8 ± 1.52% vs. -5.8 ± 1.49%,
= 0.0322). Our findings demonstrate that daily heat treatments, applied during 10 days of immobilization, prevent the loss of mitochondrial function and attenuate atrophy in human skeletal muscle.
Limb immobilization results in substantial decreases in skeletal muscle size, function, and metabolic capacity. To date, there are few, if any, interventions to prevent the deleterious effects of limb immobilization on skeletal muscle health. Heat stress has been shown to elicit a stress response, resulting in increased heat shock protein expression and improved mitochondrial function. We show that during 10 days of lower-limb immobilization in humans, daily exposure to heat stress maintains mitochondrial respiratory capacity and attenuates atrophy in skeletal muscle. Our findings suggest that heat stress may serve as an effective therapeutic strategy to attenuate the decreases of muscle mass and metabolic function that accompany periods of disuse.
•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.
The Heat Shock Connection: Skeletal Muscle Hypertrophy and Atrophy Fennel, Zachary James; Amorim, Fabiano Trigueiro; Deyhle, Michael R ...
American journal of physiology. Regulatory, integrative and comparative physiology,
07/2022, Volume:
323, Issue:
1
Journal Article
Peer reviewed
Skeletal muscle is an integral tissue system that plays a crucial role in the physical function of all vertebrates and is a key target for maintaining or improving health and performance across the ...lifespan. Based largely on cellular and animal models, there is some evidence that various forms of heat stress with or without resistance exercise may enhance skeletal muscle growth or reduce its loss. It is not clear whether these stimuli are similarly effective in humans or meaningful in comparison to exercise alone across various heating methodologies. Furthermore, the magnitude by which heat stress may influence whole body thermoregulatory responses and the connection to skeletal muscle adaptation remains ambiguous. Finally, the underlying mechanisms, which may include interaction between relevant heat shock proteins and intracellular hypertrophy and atrophy related factors, remain unclear. In this narrative mini-review we examine the relevant literature regarding heat stress alone or in combination with resistance exercise emphasizing skeletal muscle hypertrophy and atrophy across cellular and animal models, as well as human investigations. Additionally, we present working mechanistic theories for heat shock protein mediated signaling effects regarding hypertrophy and atrophy related signaling processes. Importantly, continued research is necessary to determine the practical effects and mechanisms of heat stress with and without resistance exercise on skeletal muscle function via growth and maintenance.
The heat stress response is associated with several beneficial adaptations that promote cell health and survival. Specifically, in vitro and animal investigations suggest that repeated exposures to a ...mild heat stress (~40°C) elicit positive mitochondrial adaptations in skeletal muscle comparable to those observed with exercise. To assess whether such adaptations translate to human skeletal muscle, we produced local, deep tissue heating of the vastus lateralis via pulsed shortwave diathermy in 20 men and women ( n = 10 men; n = 10 women). Diathermy increased muscle temperature by 3.9°C within 30 min of application. Immediately following a single 2-h heating session, we observed increased phosphorylation of AMP-activated protein kinase and ERK1/2 but not of p38 MAPK or JNK. Following repeated heat exposures (2 h daily for 6 consecutive days), we observed a significant cellular heat stress response, as heat shock protein 70 and 90 increased 45% and 38%, respectively. In addition, peroxisome proliferator-activated receptor gamma, coactivator-1 alpha and mitochondrial electron transport protein complexes I and V expression were increased after heating. These increases were accompanied by augmentation of maximal coupled and uncoupled respiratory capacity, measured via high-resolution respirometry. Our data provide the first evidence that mitochondrial adaptation can be elicited in human skeletal muscle in response to repeated exposures to mild heat stress. NEW & NOTEWORTHY Heat stress has been shown to elicit mitochondrial adaptations in cell culture and animal research. We used pulsed shortwave diathermy to produce deep tissue heating and explore whether beneficial mitochondrial adaptations would translate to human skeletal muscle in vivo. We report, for the first time, positive mitochondrial adaptations in human skeletal muscle following recurrent heat stress. The results of this study have clinical implications for many conditions characterized by diminished skeletal muscle mitochondrial function.
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.
AMP deaminase 1 (AMPD1; AMP → IMP + NH
) 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 (
= 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.
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.
The Tour Divide (TD) is a 4385 km ultra‐endurance bicycle race that follows the continental divide from Canada to Mexico. In this case study, we performed a comprehensive molecular and physiological ...profile before and after the completion of the TD. Assessments were performed 35 days before the start (Pre‐TD) and ∼36 h after the finish (Post‐TD). Total energy expenditure was assessed during the first 9 days by doubly labelled water (2H218O), abdominal and leg tissue volumes via MRI, and graded exercise tests to quantify fitness and substrate preference. Vastus lateralis muscle biopsies were taken to measure mitochondrial function via respirometry, and vascular function was assessed using Doppler ultrasound. The 47‐year‐old male subject took 16 days 7 h 45 min to complete the route. He rode an average of 16.8 h/day. Neither maximal O2 uptake nor maximal power output changed pre‐ to post‐TD. Measurement of total energy expenditure and dietary recall records suggested maintenance of energy balance, which was supported by the lack of change in body weight. The subject lost both appendicular and trunk fat mass and gained leg lean mass pre‐ to post‐TD. Skeletal muscle mitochondrial and vascular endothelial function decreased pre‐ to post‐TD. Overall, exercise performance was maintained despite reductions in muscle mitochondrial and vascular endothelial function post‐TD, suggesting a metabolic reserve in our highly trained athlete.
What is the main observation in this case?
This paper provides a unique multi‐parameter physiological analysis of a male subject who completed a 4385 km ultra‐endurance bicycle race. Although no specific hypotheses were tested, several intriguing observations were made. First, measurements of energy intake and expenditure indicated that energy balance was maintained, as was body mass. Second, after completion of the race, exercise performance was maintained despite reductions in skeletal muscle mitochondrial and vascular endothelial function.
What insights does it reveal?
Overall, the data provide a unique insight into the plasticity and tolerability of multiple physiological systems for the extreme physical stress imposed by multi‐day ultra‐endurance events in a trained athlete.
ABSTRACTThe purpose of this study was to test the hypothesis that macrophage polarization is altered in old compared to young skeletal muscle, possibly contributing to the poor satellite cell ...response observed in older muscle tissue. Muscle biopsies were collected prior to and at 3, 24, and 72 h following a muscle‐damaging exercise in young and old individuals. Immunohistochemistry was used to measure i.m. macrophage content and phenotype, and cell culture experiments tested macrophage behavior and influence on primary myoblasts from older individuals. We found that macrophage infiltration was similar between groups at 24 (young: 3712 ± 2407 vs. old: 5035 ± 2978 cells/mm3) and 72 (young: 4326 ± 2622 vs. old: 5287 ± 2248 cells/mm3) hours postdamage, yet the proportion of macrophages that expressed the proinflammatory marker CD11b were markedly lower in the older subjects (young: 74.5 ± 15 vs. old: 52.6 ± 17%). This finding was coupled with a greater overall proportion of CD206+, anti‐inflammatory macrophages in the old (group: P = 0.0005). We further demonstrate in vitro that proliferation, and in some cases differentiation, of old primary human myoblasts increase as much as 30% when exposed to a young macrophage‐conditioned environment. Collectively, the data suggest that old macrophages appear less capable of adapting and maintaining inflammatory function, which may contribute to poor satellite cell activation and delayed recovery from muscle damage.—Sorensen, J. R., Kaluhiokalani, J. P., Hafen, P. S., Deyhle, M. R., Parcell, A. C., Hyldahl, R. D. An altered response in macrophage phenotype following damage in aged human skeletal muscle: implications for skeletal muscle repair. FASEB J. 33,10353–10368 (2019). www.fasebj.org
Limb disuse has profound negative consequences on both vascular and skeletal muscle health. The purpose of this investigation was to determine whether repeated application of passive heat, applied to ...the knee extensor muscles, could mitigate the detrimental effects of limb disuse on vascular function. This was a randomized, single‐blinded placebo controlled trial. Twenty‐one healthy volunteers (10 women, 11 men) underwent 10 days of unilateral lower limb immobilization and were randomized to receive either a daily 2 h sham (Imm) or heat treatment (Imm+H) using pulsed shortwave diathermy. Vascular function was assessed with Doppler ultrasound of the femoral artery and the passive leg movement technique. Biopsies of the vastus lateralis were also collected before and after the intervention. In Imm, femoral artery diameter (FAD) and PLM‐induced hyperaemia (HYP) were reduced by 7.3% and 34.3%, respectively. Changes in both FAD (4% decrease; P = 0.0006) and HYP (7.8% increase; P = 0.003) were significantly attenuated in Imm+H. Vastus lateralis capillary density was not altered in either group. Immobilization significantly decreased expression of vascular endothelial growth factor (P = 0.006) and Akt (P = 0.001), and increased expression of angiopoietin 2 (P = 0.0004) over time, with no differences found between groups. Immobilization also upregulated elements associated with remodelling of the extracellular matrix, including matrix metalloproteinase 2 (P = 0.0046) and fibronectin (P = 0.0163), with no differences found between groups. In conclusion, limb immobilization impairs vascular endothelial function, but daily muscle heating via diathermy is sufficient to counteract this adverse effect. These are the first data to indicate that passive muscle heating mitigates disuse‐induced vascular dysfunction.
Key points
Limb disuse can be unavoidable for many of reasons (i.e. injury, bed rest, post‐surgery), and can have significant adverse consequences for muscular and vascular health.
We tested the hypothesis that declines in vascular function that result from lower limb immobilization could be mitigated by application of passive heat therapy.
This report shows that 10 days of limb immobilization significantly decreases resistance artery diameter and vascular function, and that application of passive heat to the knee extensor muscle group each day for 2 h per day is sufficient to attenuate these declines.
Additionally, muscle biopsy analyses showed that 10 days of heat therapy does not alter capillary density of the muscle, but upregulates multiple factors indicative of a vascular remodelling response.
Our data demonstrate the utility of passive heat as a therapeutic tool to mitigate losses in lower limb vascular function that occur from disuse.
figure legend A schematic of the primary study findings. Passive muscle heating attenuated the loss of vascular function that occurred due to 10 days of limb disuse.
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