Physiological assessment of a 16 day, 4385 km ultra-endurance mountain bike race: A case study.

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 (2 H2 18 O), 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.

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.

Effect of heat stress on heat shock protein expression and hypertrophy-related signaling in the skeletal muscle of trained individuals.

Muscle mass is balanced between hypertrophy and atrophy by cellular processes, including activation of the protein kinase B-mechanistic target of rapamycin (Akt-mTOR) signaling cascade. Stressors apart from exercise and nutrition, such as heat stress, can stimulate the heat shock protein A (HSPA) and C (HSPC) families alongside hypertrophic signaling factors and muscle growth. The effects of heat stress on HSP expression and Akt-mTOR activation in human skeletal muscle and their magnitude of activation compared with known hypertrophic stimuli are unclear. Here, we show a single session of whole body heat stress following resistance exercise increases the expression of HSPA and activation of the Akt-mTOR cascade in skeletal muscle compared with resistance exercise in a healthy, resistance-trained population. Heat stress alone may also exert similar effects, though the responses are notably variable and require further investigation. In addition, acute heat stress in C2C12 muscle cells enhanced myotube growth and myogenic fusion, albeit to a lesser degree than growth factor-mediated hypertrophy. Though the mechanisms by which heat stress stimulates hypertrophy-related signaling and the potential mechanistic role of HSPs remain unclear, these findings provide additional evidence implicating heat stress as a novel growth stimulus when combined with resistance exercise in human skeletal muscle and alone in isolated murine muscle cells. We believe these findings will help drive further applied and mechanistic investigation into how heat stress influences muscular hypertrophy and atrophy.NEW & NOTEWORTHY We show that acute resistance exercise followed by whole body heat stress increases the expression of HSPA and increases activation of the Akt-mTOR cascade in a physically active and resistance-trained population.

CaMKK2 is not involved in contraction-stimulated AMPK activation and glucose uptake in skeletal muscle.

OBJECTIVE: The AMP-activated protein kinase (AMPK) gets activated in response to energetic stress such as contractions and plays a vital role in regulating various metabolic processes such as insulin-independent glucose uptake in skeletal muscle. The main upstream kinase that activates AMPK through phosphorylation of α-AMPK Thr172 in skeletal muscle is LKB1, however some studies have suggested that Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) acts as an alternative kinase to activate AMPK. We aimed to establish whether CaMKK2 is involved in activation of AMPK and promotion of glucose uptake following contractions in skeletal muscle. METHODS: A recently developed CaMKK2 inhibitor (SGC-CAMKK2-1) alongside a structurally related but inactive compound (SGC-CAMKK2-1N), as well as CaMKK2 knock-out (KO) mice were used. In vitro kinase inhibition selectivity and efficacy assays, as well as cellular inhibition efficacy analyses of CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) were performed. Phosphorylation and activity of AMPK following contractions (ex vivo) in mouse skeletal muscles treated with/without CaMKK inhibitors or isolated from wild-type (WT)/CaMKK2 KO mice were assessed. Camkk2 mRNA in mouse tissues was measured by qPCR. CaMKK2 protein expression was assessed by immunoblotting with or without prior enrichment of calmodulin-binding proteins from skeletal muscle extracts, as well as by mass spectrometry-based proteomics of mouse skeletal muscle and C2C12 myotubes. RESULTS: STO-609 and SGC-CAMKK2-1 were equally potent and effective in inhibiting CaMKK2 in cell-free and cell-based assays, but SGC-CAMKK2-1 was much more selective. Contraction-stimulated phosphorylation and activation of AMPK were not affected with CaMKK inhibitors or in CaMKK2 null muscles. Contraction-stimulated glucose uptake was comparable between WT and CaMKK2 KO muscle. Both CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) and the inactive compound (SGC-CAMKK2-1N) significantly inhibited contraction-stimulated glucose uptake. SGC-CAMKK2-1 also inhibited glucose uptake induced by a pharmacological AMPK activator or insulin. Relatively low levels of Camkk2 mRNA were detected in mouse skeletal muscle, but neither CaMKK2 protein nor its derived peptides were detectable in mouse skeletal muscle tissue. CONCLUSIONS: We demonstrate that pharmacological inhibition or genetic loss of CaMKK2 does not affect contraction-stimulated AMPK phosphorylation and activation, as well as glucose uptake in skeletal muscle. Previously observed inhibitory effect of STO-609 on AMPK activity and glucose uptake is likely due to off-target effects. CaMKK2 protein is either absent from adult murine skeletal muscle or below the detection limit of currently available methods.

Utilizing Nonequilibrium Isotope Enrichments to Dramatically Increase Turnover Measurement Ranges in Single Biopsy Samples from Humans.

The synthesis of new proteins and the degradation of old proteins in vivo can be quantified in serial samples using metabolic isotope labeling to measure turnover. Because serial biopsies in humans are impractical, we set out to develop a method to calculate the turnover rates of proteins from single human biopsies. This method involved a new metabolic labeling approach and adjustments to the calculations used in previous work to calculate protein turnover. We demonstrate that using a nonequilibrium isotope enrichment strategy avoids the time dependent bias caused by variable lag in label delivery to different tissues observed in traditional metabolic labeling methods. Turnover rates are consistent for the same subject in biopsies from different labeling periods, and turnover rates calculated in this study are consistent with previously reported values. We also demonstrate that by measuring protein turnover we can determine where proteins are synthesized. In human subjects a significant difference in turnover rates differentiated proteins synthesized in the salivary glands versus those imported from the serum. We also provide a data analysis tool, DeuteRater-H, to calculate protein turnover using this nonequilibrium metabolic 2H2O method.

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.

Liquid chromatography method for simultaneous quantification of ATP and its degradation products compatible with both UV-Vis and mass spectrometry.

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.

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 compared with 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 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. In addition, 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.

Passive muscle heating attenuates the decline in vascular function caused by limb disuse.

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.

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.

Accumulation of Skeletal Muscle T Cells and the Repeated Bout Effect in Rats.

PURPOSE: The purpose of this investigation was to characterize skeletal muscle T-cell accumulation after contraction-induced muscle damage and test the hypothesis that T cells contribute to postdamage muscle protection (i.e., the repeated bout effect) in a way reminiscent of their role in adaptive immunity. METHODS: In vivo lengthening contractions were used to model the repeated bout effect and contralateral repeated bout effect in rats. Intramuscular T-cell subsets were characterized by flow cytometry after single and repeated bouts of lengthening contractions, and an adoptive T-cell transfer experiment was done to test whether T cells from muscle damage-experienced rats can confer protection from injury to damage-naive rats. RESULTS: Electrically stimulated lengthening contractions elicited the repeated bout effect, but not the contralateral repeated bout effect. Although leukocytes (CD45+) were scarce in undamaged muscle (2.1% of all cells), substantially more (63% of all cells) were observed after a single bout of lengthening contractions. Within the leukocyte population were several subsets of T cells, including conventional CD4+, CD8+, memory, and regulatory T cells. In contrast, a minimal increase in T cells was observed after a second bout of lengthening contractions. Conventional CD4+ T cells (FoxP3-) were the most abundant subset in muscle after lengthening contractions. Adoptive T-cell transfer from damage-experienced rats did not confer protection to damage-naive recipient rats. CONCLUSIONS: The robust T-cell accumulation, particularly the CD4 subset, after contraction-induced damage suggests a role for these cells in muscle repair and adaptation to muscle damaging contractions. Moreover, T cells are unlikely to mediate the protective adaptations of the repeated bout effect in a manner similar to their role in adaptive immunity.

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.

An altered response in macrophage phenotype following damage in aged human skeletal muscle: implications for skeletal muscle repair.

The 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.

Daily heat treatment maintains mitochondrial function and attenuates atrophy in human skeletal muscle subjected to immobilization.

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 (P < 0.0001), with no change during sham treatment. As a result, heat shock protein (HSP)70 and HSP90 increased (P < 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%, P = 0.0041) and uncoupled (-25 ± 7.0% vs. -10 ± 3.9%, P = 0.0302) myofiber respiratory capacity. Likewise, heat treatment prevented the immobilization-induced loss of proteins associated with all five mitochondrial respiratory complexes (P < 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%, P = 0.0374) and myofiber (-10.8 ± 1.52% vs. -5.8 ± 1.49%, P = 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. NEW & NOTEWORTHY 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.

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.

Repeated exposure to heat stress induces mitochondrial adaptation in human skeletal muscle.

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.

CXCL10 increases in human skeletal muscle following damage but is not necessary for muscle regeneration.

CXCL10 is a chemokine for activated and memory T cells with many important immunological functions. We recently found that CXCL10 is upregulated in human muscle following contraction-induced damage. No information is available on the role of CXCL10 in the context of muscle damage or repair. In this study, we confirm that CXCL10 is elevated in human muscle at 2 and 3 days following damage and perform cell culture and animal studies to examine the role of CXCL10 in muscle repair. CXCL10 did not impact proliferation rates of human primary myoblasts but it did promote myogenic differentiation in vitro, suggesting a possible direct impact on muscle regeneration. To test if CXCL10 was dispensable for effective muscle regeneration in vivo, we measured functional and histological markers of muscle repair out to 14 days postmuscle injury caused by a myotoxin in wild-type (WT) mice and CXCL10 knockout (KO) mice. Between genotypes, no significant differences were found in loss or restoration of in situ muscle force, cross-sectional area of newly formed myofibers, or the number of embryonic myosin heavy chain-positive myofibers. In addition, KO animals were not deficient in T-cell accumulation in the damaged muscle following injury. Gene expression of the other two ligands (CXCL9 and 11) that bind to the same receptor as CXCL10 were also elevated in the damaged muscle of KO mice. Thus, other ligands may have compensated for the lack of CXCL10 in the KO mice. We conclude that CXCL10 is not necessary for effective muscle regeneration.

Physiological Responses and Hedonics During Prolonged Physically Interactive Videogame Play.

OBJECTIVE: This study was designed to assess physiologic responses and hedonics (i.e., liking) during prolonged physically interactive videogame play. MATERIALS AND METHODS: Participants (n = 24) completed three 30-minute videogame conditions on separate days in a random order. During two of the conditions participants played physically interactive videogames (Nintendo of America, Inc. [Redmond, WA] "Wii™ Fit" "Basic Run" and "Basic Step"). During the third condition participants played a traditional/sedentary game ("Tanks!"), which required minimal physical movement for gameplay. Oxygen consumption (VO2) was assessed using indirect calorimetry throughout each condition and averaged every 5 minutes. Liking was assessed via visual analog scale at the 15- and 30-minute time points during each condition. RESULTS: Mean VO2 was significantly (P < 0.001) greater during "Basic Run" (16.14 ± 5.8 mL/kg/minute, 4.6 ± 1.7 metabolic equivalents [METs]) than either "Basic Step" (11.4 ± 1.7 mL/kg/minute, 3.3 ± 0.5 METs) or the traditional/sedentary videogame (5.39 ± 1.0 mL/kg/minute, 1.5 ± 0.1 METs). "Basic Step" was also greater (P < 0.001) than the traditional/sedentary game. VO2 did not significantly (P = 0.25) fluctuate across the 30-minute session for any game. In other words, participants maintained a consistent physiologic intensity throughout each 30-minute condition. There were no differences (P ≥ 0.20) across gaming conditions or time for liking. CONCLUSIONS: Participants achieved and maintained moderate-intensity physical activity (≥3.0 METs) during both 30-minute physically interactive videogame conditions. Furthermore, because liking was similar across all gaming conditions, participants may be willing to substitute the physically interactive videogames in place of the traditional/sedentary game.