Does a broad-spectrum cannabidiol supplement improve performance in a 10-min cycle ergometer performance-test?

Cannabidiol (CBD) is a non-intoxicating phytocannabinoid which has been proposed to possess anti-inflammatory and analgesic properties. Given the potential for perceptions of pain to limit exercise performance, the aim of the present study was to investigate if 3 weeks of daily CBD supplementation (150 mg day-1) improved performance in a 10-min performance-trial on a cycle ergometer. In a randomized, double-blind and placebo-controlled study, 22 healthy participants (n = 11 male and n = 11 female) completed two 10-min performance trials on a WattBike cycle ergometer interspersed with a 3-week supplementation period. Supplementation involved either 150 mg day-1 oral CBD or 150 mg day-1 of a visually identical placebo (PLA). During trials, ratings of perceived exertion (RPE [6-20]), heart rate (HR) and blood lactate (BLa) were collected every 2 min. Mean power (W) was also taken throughout the exercise at each time point. All data were analyzed using two-way ANOVAs. There were no significant differences (P > 0.05) between CBD or PLA groups for mean power (W) during the 10-min performance trial. There were also no significant differences (P > 0.05) in any of the physiological or perceptual parameters (HR, BLa and RPE) between conditions. Three weeks supplementation of a broad-spectrum CBD supplement did not improve performance via any change in RPE during a 10-min time trial on a cycle ergometer, and as such, this evidence does not support the claim that broad-spectrum CBD supplements could be performance-enhancing in this exercise modality.

Implications of Heat Stress-induced Metabolic Alterations for Endurance Training.

Inducing a heat-acclimated phenotype via repeated heat stress improves exercise capacity and reduces athletes̓ risk of hyperthermia and heat illness. Given the increased number of international sporting events hosted in countries with warmer climates, heat acclimation strategies are increasingly popular among endurance athletes to optimize performance in hot environments. At the tissue level, completing endurance exercise under heat stress may augment endurance training adaptation, including mitochondrial and cardiovascular remodeling due to increased perturbations to cellular homeostasis as a consequence of metabolic and cardiovascular load, and this may improve endurance training adaptation and subsequent performance. This review provides an up-to-date overview of the metabolic impact of heat stress during endurance exercise, including proposed underlying mechanisms of altered substrate utilization. Against this metabolic backdrop, the current literature highlighting the role of heat stress in augmenting training adaptation and subsequent endurance performance will be presented with practical implications and opportunities for future research.

An exercise physiologist's guide to metabolomics.

The field of exercise physiology has undergone significant technological advancements since the pioneering works of exercise physiologists in the early to mid-20th century. Historically, the ability to detect metabolites in biofluids from exercising participants was limited to single-metabolite analyses. However, the rise of metabolomics, a discipline focused on the comprehensive analysis of metabolites within a biological system, has facilitated a more intricate understanding of metabolic pathways and networks in exercise. This review explores some of the pivotal technological and bioinformatic advancements that have propelled metabolomics to the forefront of exercise physiology research. Metabolomics offers a unique 'fingerprint' of cellular activity, offering a broader spectrum than traditional single-metabolite assays. Techniques, including mass spectrometry and nuclear magnetic resonance spectroscopy, have significantly improved the speed and sensitivity of metabolite analysis. Nonetheless, challenges persist, including study design and data interpretation issues. This review aims to serve as a guide for exercise physiologists to facilitate better research design, data analysis and interpretation within metabolomics. The potential of metabolomics in bridging the gap between genotype and phenotype is emphasised, underscoring the critical importance of careful study design and the selection of appropriate metabolomics techniques. Furthermore, the paper highlights the need to deeply understand the broader scientific context to discern meaningful metabolic changes. The emerging field of fluxomics, which seeks to quantify metabolic reaction rates, is also introduced as a promising avenue for future research.

Patellar Tendon Adaptations to Downhill Running Training and Their Relationships With Changes in Mechanical Stress and Loading History.

ABSTRACT: Bontemps, B, Gruet, M, Louis, J, Owens, DJ, Miríc, S, Vercruyssen, F, and Erskine, RM. Patellar tendon adaptations to downhill running training and their relationships with changes in mechanical stress and loading history. J Strength Cond Res 38(1): 21-29, 2024-It is unclear whether human tendon adapts to moderate-intensity, high-volume long-term eccentric exercise, e.g., downhill running (DR) training. This study aimed to investigate the time course of patellar tendon (PT) adaptation to short-term DR training and to determine whether changes in PT properties were related to changes in mechanical stress or loading history. Twelve untrained, young, healthy adults (5 women and 7 men) took part in 4 weeks' DR training, comprising 10 sessions. Running speed was equivalent to 60-65% V̇O2max, and session duration increased gradually (15-30 minutes) throughout training. Isometric knee extensor maximal voluntary torque (MVT), vastus lateralis (VL) muscle physiological cross-sectional area (PCSA) and volume, and PT CSA, stiffness, and Young's modulus were assessed at weeks 0, 2, and 4 using ultrasound and isokinetic dynamometry. Patellar tendon stiffness (+6.4 ± 7.4%), Young's modulus (+6.9 ± 8.8%), isometric MVT (+7.5 ± 12.3%), VL volume (+6.6 ± 3.2%), and PCSA (+3.8 ± 3.3%) increased after 4 weeks' DR (p < 0.05), with no change in PT CSA. Changes in VL PCSA correlated with changes in PT stiffness (r = 0.70; p = 0.02) and Young's modulus (r = 0.63; p = 0.04) from 0 to 4 weeks, whereas changes in MVT did not correlate with changes in PT stiffness and Young's modulus at any time point (p > 0.05). To conclude, 4 weeks' DR training promoted substantial changes in PT stiffness and Young's modulus that are typically observed after high-intensity, low-volume resistance training. These tendon adaptations seemed to be driven primarily by loading history (represented by VL muscle hypertrophy), whereas increased mechanical stress throughout the training period did not seem to contribute to changes in PT stiffness or Young's modulus.

Fluorescent characterization of differentiated myotubes using flow cytometry.

Flow cytometry is routinely used in the assessment of skeletal muscle progenitor cell (myoblast) populations. However, a full gating strategy, inclusive of difficult to interpret forward and side scatter data, which documents cytometric analysis of differentiated myoblasts (myotubes) has not been reported. Beyond changes in size and shape, there are substantial metabolic and protein changes in myotubes allowing for their potential identification within heterogenous cell suspensions. To establish the utility of flow cytometry for determination of myoblasts and myotubes, C2C12 murine cell populations were assessed for cell morphology and metabolic reprogramming. Laser scatter, both forward (FSC; size) and side (SSC; granularity), measured cell morphology, while mitochondrial mass, reactive oxygen species (ROS) generation and DNA content were quantified using the fluorescent probes, MitoTracker green, CM-H2 DCFDA and Vybrant DyeCycle, respectively. Immunophenotyping for myosin heavy chain (MyHC) was utilized to confirm myotube differentiation. Cellular viability was determined using Annexin V/propidium iodide dual labelling. Fluorescent microscopy was employed to visualize fluorescence and morphology. Myotube and myoblast populations were resolvable through non-intuitive interpretation of laser scatter-based morphology assessment and mitochondrial mass and activity assessment. Myotubes appeared to have similar sizes to the myoblasts based on laser scatter but exhibited greater mitochondrial mass (159%, p < 0.0001), ROS production (303%, p < 0.0001), DNA content (18%, p < 0.001) and expression of MyHC (147%, p < 0.001) compared to myoblasts. Myotube sub-populations contained a larger viable cluster of cells which were unable to be fractionated from myoblast populations and a smaller population cluster which likely contains apoptotic bodies. Imaging of differentiated myoblasts that had transited through the flow cytometer revealed the presence of intact, 'rolled-up' myotubes, which would alter laser scatter properties and potential transit through the laser beam. Our results indicate that myotubes can be analyzed successfully using flow cytometry. Increased mitochondrial mass, ROS and DNA content are key features that correlate with MyHC expression but due to myotubes 'rolling up' during flow cytometric analysis, laser scatter determination of size is not positively correlated; a phenomenon observed with some size determination particles and related to surface properties of said particles. We also note a greater heterogeneity of myotubes compared to myoblasts as evidenced by the 2 distinct sub-populations. We suggest that acoustic focussing may prove effective in identifying myotube sub populations compared to traditional hydrodynamic focussing.

Environmental heat stress offsets adaptation associated with carbohydrate periodization in trained male triathletes.

PURPOSE: Carbohydrate (CHO) intake periodization via the sleep low train low (SL-TL) diet-exercise model increases fat oxidation during exercise and may enhance endurance-training adaptation and performance. Conversely, training under environmental heat stress increases CHO oxidation, but the potential of combined SL-TL and heat stress to enhance metabolic and performance outcomes is unknown. METHODS: Twenty-three endurance-trained males were randomly assigned to either control (n = 7, CON), SL-TL (n = 8, SLTemp ) or SL-TL + heat stress (n = 8, SLHeat ) groups and prescribed identical 2-week cycling training interventions. CON and SLTemp completed all sessions at 20°C, but SLHeat at 35°C. All groups consumed matched CHO intake (6 g·kg-1 ·day-1 ) but timed differently to promote low CHO availability overnight and during morning exercise in both SL groups. Submaximal substrate utilization was assessed (at 20°C), and 30-min performance tests (at 20 and 35°C) were performed Pre-, Post-, and 1-week post-intervention (Post+1). RESULTS: SLTemp improved fat oxidation rates at 60% MAP (~66% VO2peak ) at Post+1 compared with CON (p < 0.01). Compared with SLTemp , fat oxidation rates were significantly lower in SLHeat at Post (p = 0.02) and Post+1 (p < 0.05). Compared with CON, performance was improved at Post in SLTemp in temperate conditions. Performance was not different between any groups or time points in hot conditions. CONCLUSION: SL-TL enhanced metabolic adaptation and performance compared with CON and combined SL-TL and heat stress. Additional environmental heat stress may impair positive adaptations associated with SL-TL.

Acute heat stress amplifies exercise-induced metabolomic perturbations and reveals variation in circulating amino acids in endurance-trained males.

NEW FINDINGS: What is the central question of this study? Whole-body substrate utilisation is altered during exercise in hot environments, characterised by increased glycolytic metabolism: does heat stress alter the serum metabolome in response to high intensity exercise? What are the main finding and its importance? Alongside increases in glycolytic metabolite abundance, circulating amino acid concentrations are reduced following exercise under heat stress. Prior research has overlooked the impact of heat stress on protein metabolism during exercise, raising important practical implications for protein intake recommendations in the heat. ABSTRACT: Using untargeted metabolomics, we aimed to characterise the systemic impact of environmental heat stress during exercise. Twenty-three trained male triathletes ( V ̇ O 2 peak ${\dot V_{{{\rm{O}}_2}{\rm{peak}}}}$  = 64.8 ± 9.2 ml kg min-1 ) completed a 30-min exercise test in hot (35°C) and temperate (21°C) conditions. Venous blood samples were collected immediately pre- and post-exercise, and the serum fraction was assessed via untargeted 1 H-NMR metabolomics. Data were analysed via uni- and multivariate analyses to identify differences between conditions. Mean power output was higher in temperate (231 ± 36 W) versus hot (223 ± 31 W) conditions (P < 0.001). Mean heart rate (temperate, 162 ± 10 beats min-1 , hot, 167 ± 9 beats min-1 , P < 0.001), peak core temperature (Trec ), core temperature change (ΔTrec ) (P < 0.001) and peak rating of perceived exertion (P = 0.005) were higher in hot versus temperate conditions. Change in metabolite abundance following exercise revealed distinct clustering following multivariate analysis. Six metabolites increased (2-hydroxyvaleric acid, acetate, alanine, glucarate, glucose, lactate) in hot relative to temperate (P < 0.05) conditions. Leucine and lysine decreased in both conditions but to a greater extent in temperate conditions (P < 0.05). Citrate (P = 0.04) was greater in temperate conditions whilst creatinine decreased in hot conditions only (P > 0.05). Environmental heat stress increased glycolytic metabolite abundance and led to distinct alterations in the circulating amino acid availability, including increased alanine, glutamine, leucine and isoleucine. The data highlight the need for additional exercise nutrition and metabolism research, specifically focusing on protein requirements for exercise under heat stress.

PCM1 labeling reveals myonuclear and nuclear dynamics in skeletal muscle across species.

Myonuclei transcriptionally regulate muscle fibers during homeostasis and adaptation to exercise. Their subcellular location and quantity are important when characterizing phenotypes of myopathies, the effect of treatments, and understanding the roles of satellite cells in muscle adaptation and muscle "memory." Difficulties arise in identifying myonuclei due to their proximity to the sarcolemma and closely residing interstitial cell neighbors. We aimed to determine to what extent (pericentriolar material-1) PCM1 is a specific marker of myonuclei in vitro and in vivo. Single isolated myofibers and cross sections from mice and humans were studied from several models including wild-type and Lamin A/C mutant mice after functional overload and damage and recovery in humans following forced eccentric contractions. Fibers were immunolabeled for PCM1, Pax7, and DNA. C2C12 myoblasts were also studied to investigate changes in PCM1 localization during myogenesis. PCM1 was detected at not only the nuclear envelope of myonuclei in mature myofibers and in newly formed myotubes but also centrosomes in proliferating myogenic precursors, which may or may not fuse to join the myofiber syncytium. PCM1 was also detected in nonmyogenic nuclei near the sarcolemma, especially in regenerating areas of the Lmna+/ΔK32 mouse and damaged human muscle. Although PCM1 is not completely specific to myonuclei, the impact that PCM1+ macrophages and interstitial cells have on myonuclei counts would be small in healthy muscle. PCM1 may prove useful as a marker of satellite cell dynamics due to the distinct change in localization during differentiation, revealing satellite cells in their quiescent (PCM1-), proliferating (PCM1+ centrosome), and prefusion states (PCM1+ nuclear envelope).

The time course of different neuromuscular adaptations to short-term downhill running training and their specific relationships with strength gains.

PURPOSE: Due to its eccentric nature, downhill running (DR) training has been suggested to promote strength gains through neuromuscular adaptations. However, it is unknown whether short-term chronic DR can elicit such adaptations. METHODS: Twelve untrained, young, healthy adults (5 women, 7 men) took part in 4 weeks' DR, comprising 10 sessions, with running speed equivalent to 60-65% maximal oxygen uptake ([Formula: see text]O2max, assessed at weeks 0 and 4). Isometric and isokinetic knee-extensor maximal voluntary torque (MVT), vastus lateralis (VL) muscle morphology/architecture (anatomical cross-sectional area, ACSA; physiological CSA, PCSA; volume; fascicle length, Lf; pennation angle, PA) and neuromuscular activation (VL EMG) were assessed at weeks 0, 2 and 4. RESULTS: MVT increased by 9.7-15.2% after 4 weeks (p < 0.01). VL EMG during isometric MVT increased by 35.6 ± 46.1% after 4 weeks (p < 0.05) and correlated with changes in isometric MVT after 2 weeks (r = 0.86, p = 0.001). VL ACSA (+2.9 ± 2.7% and +7.1 ± 3.5%) and volume (+2.5 ± 2.5% and +6.6 ± 3.2%) increased after 2 and 4 weeks, respectively (p < 0.05). PCSA (+3.8 ± 3.3%), PA (+5.8 ± 3.8%) and Lf (+2.7 ± 2.2%) increased after 4 weeks (p < 0.01). Changes in VL volume (r = 0.67, p = 0.03) and PCSA (r = 0.71, p = 0.01) correlated with changes in concentric MVT from 2 to 4 weeks. [Formula: see text]O2max (49.4 ± 6.2 vs. 49.7 ± 6.3 mL·kg-1·min-1) did not change after 4 weeks (p = 0.73). CONCLUSION: Just 4 weeks' moderate-intensity DR promoted neuromuscular adaptations in young, healthy adults, typically observed after high-intensity eccentric resistance training. Neural adaptations appeared to contribute to most of the strength gains at 2 and 4 weeks, while muscle hypertrophy seemed to contribute to MVT changes from 2 to 4 weeks only.

Three weeks of a home-based "sleep low-train low" intervention improves functional threshold power in trained cyclists: A feasibility study.

BACKGROUND: "Sleep Low-Train Low" is a training-nutrition strategy intended to purposefully reduce muscle glycogen availability around specific exercise sessions, potentially amplifying the training stimulus via augmented cell signalling. The aim of this study was to assess the feasibility of a 3-week home-based "sleep low-train low" programme and its effects on cycling performance in trained athletes. METHODS: Fifty-five trained athletes (Functional Threshold Power [FTP]: 258 ± 52W) completed a home-based cycling training program consisting of evening high-intensity training (6 × 5 min at 105% FTP), followed by low-intensity training (1 hr at 75% FTP) the next morning, three times weekly for three consecutive weeks. Participant's daily carbohydrate (CHO) intake (6 g·kg-1·d-1) was matched but timed differently to manipulate CHO availability around exercise: no CHO consumption post- HIT until post-LIT sessions [Sleep Low (SL), n = 28] or CHO consumption evenly distributed throughout the day [Control (CON), n = 27]. Sessions were monitored remotely via power data uploaded to an online training platform, with performance tests conducted pre-, post-intervention. RESULTS: LIT exercise intensity reduced by 3% across week 1, 3 and 2% in week 2 (P < 0.01) with elevated RPE in SL vs. CON (P < 0.01). SL enhanced FTP by +5.5% vs. +1.2% in CON (P < 0.01). Comparable increases in 5-min peak power output (PPO) were observed between groups (P < 0.01) with +2.3% and +2.7% in SL and CON, respectively (P = 0.77). SL 1-min PPO was unchanged (+0.8%) whilst CON improved by +3.9% (P = 0.0144). CONCLUSION: Despite reduced relative training intensity, our data demonstrate short-term "sleep low-train low" intervention improves FTP compared with typically "normal" CHO availability during exercise. Importantly, training was completed unsupervised at home (during the COVID-19 pandemic), thus demonstrating the feasibility of completing a "sleep low-train low" protocol under non-laboratory conditions.

Four Weeks of Probiotic Supplementation Alters the Metabolic Perturbations Induced by Marathon Running: Insight from Metabolomics.

Few data are available that describe how probiotics influence systemic metabolism during endurance exercise. Metabolomic profiling of endurance athletes will elucidate mechanisms by which probiotics may confer benefits to the athlete. In this study, twenty-four runners (20 male, 4 female) were block randomised into two groups using a double-blind matched-pairs design according to their most recent Marathon performance. Runners were assigned to 28-days of supplementation with a multi-strain probiotic (PRO) or a placebo (PLB). Following 28-days of supplementation, runners performed a competitive track Marathon race. Venous blood samples and muscle biopsies (vastus lateralis) were collected on the morning of the race and immediately post-race. Samples were subsequently analysed by untargeted 1H-NMR metabolomics. Principal component analysis (PCA) identified a greater difference in the post-Marathon serum metabolome in the PLB group vs. PRO. Univariate tests identified 17 non-overlapped metabolites in PLB, whereas only seven were identified in PRO. By building a PLS-DA model of two components, we revealed combinations of metabolites able to discriminate between PLB and PRO post-Marathon. PCA of muscle biopsies demonstrated no discernible difference post-Marathon between treatment groups. In conclusion, 28-days of probiotic supplementation alters the metabolic perturbations induced by a Marathon. Such findings may be related to maintaining the integrity of the gut during endurance exercise.

"Fuel for the Damage Induced": Untargeted Metabolomics in Elite Rugby Union Match Play.

The metabolic perturbations caused by competitive rugby are not well characterized. Our aim is to utilize untargeted metabolomics to develop appropriate interventions, based on the metabolic fluctuations that occur in response to this collision-based team sport. Seven members of an English Premiership rugby squad consented to provide blood, urine, and saliva samples daily, over a competitive week including gameday (GD), with physical demands and dietary intake also recorded. Sample collection, processing and statistical analysis were performed in accordance with best practice set out by the metabolomics standards initiative employing 700 MHz NMR spectroscopy. Univariate and multivariate statistical analysis were employed to reveal the acute energy needs of this high intensity sport are met via glycolysis, the TCA cycle and gluconeogenesis. The recovery period after cessation of match play and prior to training recommencing sees a re-entry to gluconeogenesis, coupled with markers of oxidative stress, structural protein degradation, and reduced fatty acid metabolism. This novel insight leads us to propose that effective recovery from muscle damaging collisions is dependent upon the availability of glucose. An adjustment in the periodisation of carbohydrate to increase GD+1 provision may prevent the oxidation of amino acids which may also be crucial to allay markers of structural tissue degradation. Should we expand the 'Fuel for the work required' paradigm in collision-based team sports to include 'Fuel for the damage induced'?

Carbohydrate improves exercise capacity but does not affect subcellular lipid droplet morphology, AMPK and p53 signalling in human skeletal muscle.

KEY POINTS: Muscle glycogen and intramuscular triglycerides (IMTG, stored in lipid droplets) are important energy substrates during prolonged exercise. Exercise-induced changes in lipid droplet (LD) morphology (i.e. LD size and number) have not yet been studied under nutritional conditions typically adopted by elite endurance athletes, that is, after carbohydrate (CHO) loading and CHO feeding during exercise. We report for the first time that exercise reduces IMTG content in both central and peripheral regions of type I and IIa fibres, reflective of decreased LD number in both fibre types whereas reductions in LD size were exclusive to type I fibres. Additionally, CHO feeding does not alter subcellular IMTG utilisation, LD morphology or muscle glycogen utilisation in type I or IIa/II fibres. In the absence of alterations to muscle fuel selection, CHO feeding does not attenuate cell signalling pathways with regulatory roles in mitochondrial biogenesis. ABSTRACT: We examined the effects of carbohydrate (CHO) feeding on lipid droplet (LD) morphology, muscle glycogen utilisation and exercise-induced skeletal muscle cell signalling. After a 36 h CHO loading protocol and pre-exercise meal (12 and 2 g kg-1 , respectively), eight trained males ingested 0, 45 or 90 g CHO h-1 during 180 min cycling at lactate threshold followed by an exercise capacity test (150% lactate threshold). Muscle biopsies were obtained pre- and post-completion of submaximal exercise. Exercise decreased (P < 0.01) glycogen concentration to comparable levels (∼700 to 250 mmol kg-1 DW), though utilisation was greater in type I (∼40%) versus type II fibres (∼10%) (P < 0.01). LD content decreased in type I (∼50%) and type IIa fibres (∼30%) (P < 0.01), with greater utilisation in type I fibres (P < 0.01). CHO feeding did not affect glycogen or IMTG utilisation in type I or II fibres (all P > 0.05). Exercise decreased LD number within central and peripheral regions of both type I and IIa fibres, though reduced LD size was exclusive to type I fibres. Exercise induced (all P < 0.05) comparable AMPKThr172 (∼4-fold), p53Ser15 (∼2-fold) and CaMKIIThr268 phosphorylation (∼2-fold) with no effects of CHO feeding (all P > 0.05). CHO increased exercise capacity where 90 g h-1 (233 ± 133 s) > 45 g h-1 (156 ± 66 s; P = 0.06) > 0 g h-1 (108 ± 54 s; P = 0.03). In conditions of high pre-exercise CHO availability, we conclude CHO feeding does not influence exercise-induced changes in LD morphology, glycogen utilisation or cell signalling pathways with regulatory roles in mitochondrial biogenesis.

Knockdown of the E3 ubiquitin ligase UBR5 and its role in skeletal muscle anabolism.

UBR5 is an E3 ubiquitin ligase positively associated with anabolism, hypertrophy, and recovery from atrophy in skeletal muscle. The precise mechanisms underpinning UBR5's role in the regulation of skeletal muscle mass remain unknown. The present study aimed to elucidate these mechanisms by silencing the UBR5 gene in vivo. To achieve this aim, we electroporated a UBR5-RNAi plasmid into mouse tibialis anterior muscle to investigate the impact of reduced UBR5 on anabolic signaling MEK/ERK/p90RSK and Akt/GSK3ß/p70S6K/4E-BP1/rpS6 pathways. Seven days after UBR5 RNAi electroporation, although reductions in overall muscle mass were not detected, the mean cross-sectional area (CSA) of green fluorescent protein (GFP)-positive fibers were reduced (-9.5%) and the number of large fibers were lower versus the control. Importantly, UBR5-RNAi significantly reduced total RNA, muscle protein synthesis, ERK1/2, Akt, and GSK3ß activity. Although p90RSK phosphorylation significantly increased, total p90RSK protein levels demonstrated a 45% reduction with UBR5-RNAi. Finally, these early events after 7 days of UBR5 knockdown culminated in significant reductions in muscle mass (-4.6%) and larger reductions in fiber CSA (-18.5%) after 30 days. This was associated with increased levels of phosphatase PP2Ac and inappropriate chronic elevation of p70S6K and rpS6 between 7 and 30 days, as well as corresponding reductions in eIF4e. This study demonstrates that UBR5 plays an important role in anabolism/hypertrophy, whereby knockdown of UBR5 culminates in skeletal muscle atrophy.

Achieving energy balance with a high-fat meal does not enhance skeletal muscle adaptation and impairs glycaemic response in a sleep-low training model.

NEW FINDINGS: What is the central question of this study? Does achieving energy balance mainly with ingested fat in a 'sleep-low' model of training with low muscle glycogen affect the early training adaptive response during recovery? What is the main finding and its importance? Replenishing the energy expended during exercise mainly from ingested fat to achieve energy balance in a 'sleep-low' model does not enhance the response of skeletal muscle markers of early adaptation to training and impairs glycaemic control the morning after compared to training with low energy availability. These findings are important for optimizing post-training dietary recommendations in relation to energy balance and macronutrient intake. ABSTRACT: Training with low carbohydrate availability (LCHO) has been shown to acutely enhance endurance training skeletal muscle response, but the concomitant energy deficit (ED) in LCHO interventions has represented a confounding factor in past research. This study aimed at determining if achieving energy balance with high fat (EB-HF) acutely enhances the adaptive response in LCHO compared to ED with low fat (ED-LF). In a crossover design, nine well-trained males completed a 'sleep-low' protocol: on day 1 they cycled to deplete muscle glycogen while reaching a set energy expenditure (30 kcal (kg of fat free mass (FFM))-1 ). Post-exercise, low carbohydrate, protein-matched meals completely (EB-HF, 30 kcal (kg FFM)-1 ) or partially (ED-LF, 9 kcal (kg FFM)-1 ) replaced the energy expended, with the majority of energy derived from fat in EB-HF. In the morning of day 2, participants exercised fasted, and skeletal muscle and blood samples were collected and a carbohydrate-protein drink was ingested at 0.5 h recovery. Muscle glycogen showed no treatment effect (P < 0.001) and decreased from 350 ± 98 to 192 ± 94 mmol (kg dry mass)-1 between rest and 0.5 h recovery. Phosphorylation status of the mechanistic target of rapamycin and AMP-activated protein kinase pathway proteins showed only time effects. mRNA expression of p53 increased after exercise (P = 0.005) and was higher in ED-LF at 3.5 h compared to EB-HF (P = 0.027). Plasma glucose and insulin area under the curve (P < 0.04) and peak values (P ≤ 0.05) were higher in EB-HF after the recovery drink. Achieving energy balance with a high-fat meal in a 'train-low' ('sleep-low') model did not enhance markers of skeletal muscle adaptation and impaired glycaemia in response to a recovery drink following training in the morning.

Graded reductions in pre-exercise glycogen concentration do not augment exercise-induced nuclear AMPK and PGC-1α protein content in human muscle.

NEW FINDINGS: What is the central question of this study? What is the absolute level of pre-exercise glycogen concentration required to augment the exercise-induced signalling response regulating mitochondrial biogenesis? What is the main finding and its importance? Commencing high-intensity endurance exercise with reduced pre-exercise muscle glycogen concentrations confers no additional benefit to the early signalling responses that regulate mitochondrial biogenesis. ABSTRACT: We examined the effects of graded muscle glycogen on the subcellular location and protein content of AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and mRNA expression of genes associated with the regulation of mitochondrial biogenesis and substrate utilisation in human skeletal muscle. In a repeated measures design, eight trained male cyclists completed acute high-intensity interval (HIT) cycling (8 × 5 min at 80% peak power output) with graded concentrations of pre-exercise muscle glycogen. Following initial glycogen-depleting exercise, subjects ingested  2 g kg-1  (L-CHO), 6 g kg-1 (M-CHO) or 14 g kg-1 (H-CHO) of carbohydrate during a 36 h period, such that exercise was commenced with graded (P < 0.05) muscle glycogen concentrations (mmol (kg dw)-1 : H-CHO, 531 ± 83; M-CHO, 332 ± 88; L-CHO, 208 ± 79). Exercise depleted muscle glycogen to <300 mmol (kg dw)-1 in all trials (mmol (kg dw)-1 : H-CHO, 270 ± 88; M-CHO, 173 ± 74; L-CHO, 100 ± 42) and induced comparable increases in nuclear AMPK protein content (∼2-fold) and PGC-1α (∼5-fold), p53 (∼1.5-fold) and carnitine palmitoyltransferase 1 (∼2-fold) mRNA between trials (all P < 0.05). The magnitude of increase in PGC-1α mRNA was also positively correlated with post-exercise glycogen concentration (P < 0.05). In contrast, neither exercise nor carbohydrate availability affected the subcellular location of PGC-1α protein or PPAR, SCO2, SIRT1, DRP1, MFN2 or CD36 mRNA. Using a sleep-low, train-low model with a high-intensity endurance exercise stimulus, we conclude that pre-exercise muscle glycogen does not modulate skeletal muscle cell signalling.

Lamin Mutations Cause Increased YAP Nuclear Entry in Muscle Stem Cells.

Mutations in the LMNA gene, encoding the nuclear envelope A-type lamins, are responsible for muscular dystrophies, the most severe form being the LMNA-related congenital muscular dystrophy (L-CMD), with severe defects in myonucleus integrity. We previously reported that L-CMD mutations compromise the ability of muscle stem cells to modulate the yes-associated protein (YAP), a pivotal factor in mechanotransduction and myogenesis. Here, we investigated the intrinsic mechanisms by which lamins influence YAP subcellular distribution, by analyzing different conditions affecting the balance between nuclear import and export of YAP. In contrast to wild type (WT) cells, LMNADK32 mutations failed to exclude YAP from the nucleus and to inactivate its transcriptional activity at high cell density, despite activation of the Hippo pathway. Inhibiting nuclear pore import abolished YAP nuclear accumulation in confluent mutant cells, thus showing persistent nuclear import of YAP at cell confluence. YAP deregulation was also present in congenital myopathy related to nesprin-1KASH mutation, but not in cells expressing the LMNAH222P mutation, the adult form of lamin-related muscle dystrophy with reduced nuclear deformability. In conclusion, our data showed that L-CMD mutations increased YAP nuclear localization via an increased nuclear import and implicated YAP as a pathogenic contributor in muscle dystrophies caused by nuclear envelop defects.

Lamin-Related Congenital Muscular Dystrophy Alters Mechanical Signaling and Skeletal Muscle Growth.

Laminopathies are a clinically heterogeneous group of disorders caused by mutations in the LMNA gene, which encodes the nuclear envelope proteins lamins A and C. The most frequent diseases associated with LMNA mutations are characterized by skeletal and cardiac involvement, and include autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD), limb-girdle muscular dystrophy type 1B, and LMNA-related congenital muscular dystrophy (LMNA-CMD). Although the exact pathophysiological mechanisms responsible for LMNA-CMD are not yet understood, severe contracture and muscle atrophy suggest that mutations may impair skeletal muscle growth. Using human muscle stem cells (MuSCs) carrying LMNA-CMD mutations, we observe impaired myogenic fusion with disorganized cadherin/ß catenin adhesion complexes. We show that skeletal muscle from Lmna-CMD mice is unable to hypertrophy in response to functional overload, due to defective fusion of activated MuSCs, defective protein synthesis and defective remodeling of the neuromuscular junction. Moreover, stretched myotubes and overloaded muscle fibers with LMNA-CMD mutations display aberrant mechanical regulation of the yes-associated protein (YAP). We also observe defects in MuSC activation and YAP signaling in muscle biopsies from LMNA-CMD patients. These phenotypes are not recapitulated in closely related but less severe EDMD models. In conclusion, combining studies in vitro, in vivo, and patient samples, we find that LMNA-CMD mutations interfere with mechanosignaling pathways in skeletal muscle, implicating A-type lamins in the regulation of skeletal muscle growth.

UBR5 is a novel E3 ubiquitin ligase involved in skeletal muscle hypertrophy and recovery from atrophy.

KEY POINTS: We have recently identified that a HECT domain E3 ubiquitin ligase, named UBR5, is altered epigenetically (via DNA methylation) after human skeletal muscle hypertrophy, where its gene expression is positively correlated with increasing lean leg mass after training and retraining. In the present study we extensively investigate this novel and uncharacterised E3 ubiquitin ligase (UBR5) in skeletal muscle atrophy, recovery from atrophy and injury, anabolism and hypertrophy. We demonstrated that UBR5 was epigenetically altered via DNA methylation during recovery from atrophy. We also determined that UBR5 was alternatively regulated versus well characterised E3 ligases, MuRF1/MAFbx, at the gene expression level during atrophy, recovery from atrophy and hypertrophy. UBR5 also increased at the protein level during recovery from atrophy and injury, hypertrophy and during human muscle cell differentiation. Finally, in humans, genetic variations of the UBR5 gene were strongly associated with larger fast-twitch muscle fibres and strength/power performance versus endurance/untrained phenotypes. ABSTRACT: We aimed to investigate a novel and uncharacterized E3 ubiquitin ligase in skeletal muscle atrophy, recovery from atrophy/injury, anabolism and hypertrophy. We demonstrated an alternate gene expression profile for UBR5 vs. well characterized E3-ligases, MuRF1/MAFbx, where, after atrophy evoked by continuous-low-frequency electrical-stimulation in rats, MuRF1/MAFbx were both elevated, yet UBR5 was unchanged. Furthermore, after recovery of muscle mass post TTX-induced atrophy in rats, UBR5 was hypomethylated and increased at the gene expression level, whereas a suppression of MuRF1/MAFbx was observed. At the protein level, we also demonstrated a significant increase in UBR5 after recovery of muscle mass from hindlimb unloading in both adult and aged rats, as well as after recovery from atrophy evoked by nerve crush injury in mice. During anabolism and hypertrophy, UBR5 gene expression increased following acute loading in three-dimensional bioengineered mouse muscle in vitro, and after chronic electrical stimulation-induced hypertrophy in rats in vivo, without increases in MuRF1/MAFbx. Additionally, UBR5 protein abundance increased following functional overload-induced hypertrophy of the plantaris muscle in mice and during differentiation of primary human muscle cells. Finally, in humans, genetic association studies (>700,000 single nucleotide polymorphisms) demonstrated that the A alleles of rs10505025 and rs4734621 single nucleotide polymorphisms in the UBR5 gene were strongly associated with larger cross-sectional area of fast-twitch muscle fibres and favoured strength/power vs. endurance/untrained phenotypes. Overall, we suggest that: (i) UBR5 comprises a novel E3 ubiquitin ligase that is inversely regulated to MuRF1/MAFbx; (ii) UBR5 is epigenetically regulated; and (iii) UBR5 is elevated at both the gene expression and protein level during recovery from skeletal muscle atrophy and hypertrophy.