Division-Independent Differentiation of Muscle Stem Cells During a Growth Stimulus.

Adult muscle stem cells (MuSCs) are known to replicate upon activation before differentiating and fusing to regenerate myofibers. It is unclear whether MuSC differentiation is intrinsically linked to cell division, which has implications for stem cell population maintenance. We use single-cell RNA-sequencing to identify transcriptionally diverse subpopulations of MuSCs after 5 days of a growth stimulus in adult muscle. Trajectory inference in combination with a novel mouse model for tracking MuSC-derived myonuclei and in vivo labeling of DNA replication revealed an MuSC population that exhibited division-independent differentiation and fusion. These findings demonstrate that in response to a growth stimulus in the presence of intact myofibers, MuSC division is not obligatory.

The effects of resistance training on denervated myofibers, senescent cells, and associated protein markers in middle-aged adults.

Denervated myofibers and senescent cells are hallmarks of skeletal muscle aging. However, sparse research has examined how resistance training affects these outcomes. We investigated the effects of unilateral leg extensor resistance training (2 days/week for 8 weeks) on denervated myofibers, senescent cells, and associated protein markers in apparently healthy middle-aged participants (MA, 55 ± 8 years old, 17 females, 9 males). We obtained dual-leg vastus lateralis (VL) muscle cross-sectional area (mCSA), VL biopsies, and strength assessments before and after training. Fiber cross-sectional area (fCSA), satellite cells (Pax7+), denervated myofibers (NCAM+), senescent cells (p16+ or p21+), proteins associated with denervation and senescence, and senescence-associated secretory phenotype (SASP) proteins were analyzed from biopsy specimens. Leg extensor peak torque increased after training (p < .001), while VL mCSA trended upward (interaction p = .082). No significant changes were observed for Type I/II fCSAs, NCAM+ myofibers, or senescent (p16+ or p21+) cells, albeit satellite cells increased after training (p = .037). While >90% satellite cells were not p16+ or p21+, most p16+ and p21+ cells were Pax7+ (>90% on average). Training altered 13 out of 46 proteins related to muscle-nerve communication (all upregulated, p < .05) and 10 out of 19 proteins related to cellular senescence (9 upregulated, p < .05). Only 1 out of 17 SASP protein increased with training (IGFBP-3, p = .031). In conclusion, resistance training upregulates proteins associated with muscle-nerve communication in MA participants but does not alter NCAM+ myofibers. Moreover, while training increased senescence-related proteins, this coincided with an increase in satellite cells but not alterations in senescent cell content or SASP proteins. These latter findings suggest shorter term resistance training is an unlikely inducer of cellular senescence in apparently healthy middle-aged participants. However, similar study designs are needed in older and diseased populations before definitive conclusions can be drawn.

Relative rDNA copy number is not associated with resistance training-induced skeletal muscle hypertrophy and does not affect myotube anabolism in vitro.

Ribosomal DNA (rDNA) copies exist across multiple chromosomes, and interindividual variation in copy number is speculated to influence the hypertrophic response to resistance training. Thus, we examined if rDNA copy number was associated with resistance training-induced skeletal muscle hypertrophy. Participants (n = 53 male, 21 ± 1 yr old; n = 29 female, 21 ± 2 yr old) performed 10-12 wk of full-body resistance training. Hypertrophy outcomes were determined, as was relative rDNA copy number from preintervention vastus lateralis (VL) biopsies. Pre- and postintervention VL biopsy total RNA was assayed in all participants, and mRNA/rRNA markers of ribosome content and biogenesis were also assayed in the 29 female participants before training, 24 h following training bout 1, and in the basal state after 10 wk of training. Across all participants, no significant associations were evident between relative rDNA copy number and training-induced changes in whole body lean mass (r = -0.034, P = 0.764), vastus lateralis thickness (r = 0.093, P = 0.408), mean myofiber cross-sectional area (r = -0.128, P = 0.259), or changes in muscle RNA concentrations (r = 0.026, P = 0.818), and these trends were similar when examining each gender. However, all Pol-I regulon mRNAs as well as 45S pre-rRNA, 28S rRNA, and 18S rRNA increased 24 h following the first training bout in female participants. Follow-up studies using LHCN-M2 myotubes demonstrated that a reduction in relative rDNA copy number induced by bisphenol A did not significantly affect insulin-like-growth factor-induced myotube hypertrophy. These findings suggest that relative rDNA copy number is not associated with myofiber hypertrophy.NEW & NOTEWORTHY We examined ribosomal DNA (rDNA) copy numbers in men and women who resistance trained for 10-12 wk and found no significant associations with skeletal muscle hypertrophy outcomes. These data, along with in vitro data in immortalized human myotubes whereby rDNA copy number was reduced, provide strong evidence that relative rDNA copy number is not associated with anabolism.

Skeletal muscle myosin heavy chain fragmentation as a potential marker of protein degradation in response to resistance training and disuse atrophy.

We examined how resistance exercise (RE), cycling exercise and disuse atrophy affect myosin heavy chain (MyHC) protein fragmentation. The 1boutRE study involved younger men (n = 8; 5 ± 2 years of RE experience) performing a lower body RE bout with vastus lateralis (VL) biopsies being obtained prior to and acutely following exercise. With the 10weekRT study, VL biopsies were obtained in 36 younger adults before and 24 h after their first/naïve RE bout. Participants also engaged in 10 weeks of resistance training and donated VL biopsies before and 24 h after their last RE bout. VL biopsies were also examined in an acute cycling study (n = 7) and a study involving 2 weeks of leg immobilization (n = 20). In the 1boutRE study, fragmentation of all MyHC isoforms (MyHCTotal) increased 3 h post-RE (∼200%, P = 0.018) and returned to pre-exercise levels by 6 h post-RE. Interestingly, a greater magnitude increase in MyHC type IIa versus I isoform fragmentation occurred 3 h post-RE (8.6 ± 6.3-fold vs. 2.1 ± 0.7-fold, P = 0.018). In 10weekRT participants, the first/naïve and last RE bouts increased MyHCTotal fragmentation 24 h post-RE (+65% and +36%, P < 0.001); however, the last RE bout response was attenuated compared to the first bout (P = 0.045). Although cycling exercise did not alter MyHCTotal fragmentation, ∼8% VL atrophy with 2 weeks of leg immobilization increased MyHCTotal fragmentation (∼108%, P < 0.001). Mechanistic C2C12 myotube experiments indicated that MyHCTotal fragmentation is likely due to calpain proteases. In summary, RE and disuse atrophy increase MyHC protein fragmentation. Research into how ageing and disease-associated muscle atrophy affect these outcomes is needed. HIGHLIGHTS: What is the central question of this study? How different exercise stressors and disuse affect skeletal muscle myosin heavy chain fragmentation. What is the main finding and its importance? This investigation is the first to demonstrate that resistance exercise and disuse atrophy lead to skeletal muscle myosin heavy chain protein fragmentation in humans. Mechanistic in vitro experiments provide additional evidence that MyHC fragmentation occurs through calpain proteases.

Resistance training diminishes mitochondrial adaptations to subsequent endurance training in healthy untrained men.

We investigated the effects of performing a period of resistance training (RT) on the performance and molecular adaptations to a subsequent period of endurance training (ET). Twenty-five young adults were divided into an RT+ET group (n = 13), which underwent 7 weeks of RT followed by 7 weeks of ET, and an ET-only group (n = 12), which performed 7 weeks of ET. Body composition, endurance performance and muscle biopsies were collected before RT (T1, baseline for RT+ET), before ET (T2, after RT for RT+ET and baseline for ET) and after ET (T3). Immunohistochemistry was performed to determine fibre cross-sectional area (fCSA), myonuclear content, myonuclear domain size, satellite cell number and mitochondrial content. Western blots were used to quantify markers of mitochondrial remodelling. Citrate synthase activity and markers of ribosome content were also investigated. RT improved body composition and strength, increased vastus lateralis thickness, mixed and type II fCSA, myonuclear number, markers of ribosome content, and satellite cell content (P < 0.050). In response to ET, both groups similarly decreased body fat percentage (P < 0.0001) and improved endurance performance (e.g. V ̇ O 2 max ${\dot V_{{{\mathrm{O}}_2}\max }}$ , and speed at which the onset of blood lactate accumulation occurred, P < 0.0001). Levels of mitochondrial complexes I-IV in the ET-only group increased 32-66%, while those in the RT+ET group increased 1-11% (time, P < 0.050). Additionally, mixed fibre relative mitochondrial content increased 15% in the ET-only group but decreased 13% in the RT+ET group (interaction, P = 0.043). In conclusion, RT performed prior to ET had no additional benefits to ET adaptations. Moreover, prior RT seemed to impair mitochondrial adaptations to ET. KEY POINTS: Resistance training is largely underappreciated as a method to improve endurance performance, despite reports showing it may improve mitochondrial function. Although several concurrent training studies are available, in this study we investigated the effects of performing a period of resistance training on the performance and molecular adaptations to subsequent endurance training. Prior resistance training did not improve endurance performance and impaired most mitochondrial adaptations to subsequent endurance training, but this effect may have been a result of detraining from resistance training.

Proteolytic markers associated with a gain and loss of leg muscle mass with resistance training followed by high-intensity interval training.

We recently reported that vastus lateralis (VL) cross-sectional area (CSA) increases after 7 weeks of resistance training (RT, 2 days/week), with declines occurring following 7 weeks of subsequent treadmill high-intensity interval training (HIIT) (3 days/week). Herein, we examined the effects of this training paradigm on skeletal muscle proteolytic markers. VL biopsies were obtained from 11 untrained college-aged males at baseline (PRE), after 7 weeks of RT (MID), and after 7 weeks of HIIT (POST). Tissues were analysed for proteolysis markers, and in vitro experiments were performed to provide additional insights. Atrogene mRNAs (TRIM63, FBXO32, FOXO3A) were upregulated at POST versus both PRE and MID (P < 0.05). 20S proteasome core protein abundance increased at POST versus PRE (P = 0.031) and MID (P = 0.049). 20S proteasome activity, and protein levels for calpain-2 and Beclin-1 increased at MID and POST versus PRE (P < 0.05). Ubiquitinated proteins showed model significance (P = 0.019) with non-significant increases at MID and POST (P > 0.05). in vitro experiments recapitulated the training phenotype when stimulated with a hypertrophic stimulus (insulin-like growth factor 1; IGF1) followed by a subsequent AMP-activated protein kinase activator (5-aminoimidazole-4-carboxamide ribonucleotide; AICAR), as demonstrated by larger myotube diameter in IGF1-treated cells versus IGF1 followed by AICAR treatments (I+A; P = 0.017). Muscle protein synthesis (MPS) levels were also greater in IGF1-treated versus I+A myotubes (P < 0.001). In summary, the loss in RT-induced VL CSA with HIIT coincided with increases in several proteolytic markers, and sustained proteolysis may have driven this response. Moreover, while not measured in humans, we interpret our in vitro data to suggest that (unlike RT) HIIT does not stimulate MPS. NEW FINDINGS: What is the central question of this study? Determining if HIIT-induced reductions in muscle hypertrophy following a period of resistance training coincided with increases in proteolytic markers. What is the main finding and its importance? Several proteolytic markers were elevated during the HIIT training period implying that increases in muscle proteolysis may have played a role in HIIT-induced reductions in muscle hypertrophy.

The role of extracellular vesicles in skeletal muscle and systematic adaptation to exercise.

Regular exercise has a central role in human health by reducing the risk of type 2 diabetes, obesity, stroke and cancer. How exercise is able to promote such systemic benefits has remained somewhat of a mystery but has been thought to be in part mediated by the release of myokines, skeletal muscle-specific cytokines, in response to exercise. Recent studies have revealed skeletal muscle can also release extracellular vesicles (EVs) into circulation following a bout of exercise. EVs are small membrane-bound vesicles capable of delivering biomolecules to recipient cells and subsequently altering their metabolism. The notion that EVs may have a role in both skeletal muscle and systemic adaptation to exercise has generated a great deal of excitement within a number of different fields including exercise physiology, neuroscience and metabolism. The purpose of this review is to provide an introduction to EV biology and what is currently known about skeletal muscle EVs and their potential role in the response of muscle and other tissues to exercise.

Dysbiosis of the gut microbiome impairs mouse skeletal muscle adaptation to exercise.

There is emerging evidence of a gut microbiome-skeletal muscle axis. The purpose of this study was to determine if an intact gut microbiome was necessary for skeletal muscle adaptation to exercise. Forty-two 4-month-old female C57BL/6J mice were randomly assigned to untreated (U) or antibiotic-treated (T) non-running controls (CU or CT, respectively) or progressive weighted wheel running (PoWeR, P) untreated (PU) or antibiotic-treated (PT) groups. Antibiotic treatment resulted in disruption of the gut microbiome as indicated by a significant depletion of gut microbiome bacterial species in both CT and PT groups. The training stimulus was the same between PU and PT groups as assessed by weekly (12.35 ± 2.06 vs. 11.09 ± 1.76 km/week, respectively) and total (778.9 ± 130.5 vs. 703.8 ± 112.9 km, respectively) running activity. In response to PoWeR, PT showed less hypertrophy of soleus type 1 and 2a fibres and plantaris type 2b/x fibres compared to PU. The higher satellite cell and myonuclei abundance of PU plantaris muscle after PoWeR was not observed in PT. The fibre-type shift of PU plantaris muscle to a more oxidative type 2a fibre composition following PoWeR was blunted in PT. There was no difference in serum cytokine levels among all groups suggesting disruption of the gut microbiome did not induce systemic inflammation. The results of this study provide the first evidence that an intact gut microbiome is necessary for skeletal muscle adaptation to exercise. KEY POINTS: Dysbiosis of the gut microbiome caused by continuous antibiotic treatment did not affect running activity. Continuous treatment with antibiotics did not result in systemic inflammation as indicated by serum cytokine levels. Gut microbiome dysbiosis was associated with blunted fibre type-specific hypertrophy in the soleus and plantaris muscles in response to progressive weighted wheel running (PoWeR). Gut microbiome dysbiosis was associated with impaired PoWeR-induced fibre-type shift in the plantaris muscle. Gut microbiome dysbiosis was associated with a loss of PoWeR-induced myonuclei accretion in the plantaris muscle.

Genetic and epigenetic regulation of skeletal muscle ribosome biogenesis with exercise.

KEY POINTS: Ribosome biogenesis and MYC transcription are associated with acute resistance exercise (RE) and are distinct from endurance exercise in human skeletal muscle throughout a 24 h time course of recovery. A PCR-based method for relative ribosomal DNA (rDNA) copy number estimation was validated by whole genome sequencing and revealed that rDNA dosage is positively correlated with ribosome biogenesis in response to RE. Acute RE modifies rDNA methylation patterns in enhancer, intergenic spacer and non-canonical MYC-associated regions, but not the promoter. Myonuclear-specific rDNA methylation patterns with acute mechanical overload in mice corroborate and expand on rDNA findings with RE in humans. A genetic predisposition for hypertrophic responsiveness may exist based on rDNA gene dosage. ABSTRACT: Ribosomes are the macromolecular engines of protein synthesis. Skeletal muscle ribosome biogenesis is stimulated by exercise, although the contribution of ribosomal DNA (rDNA) copy number and methylation to exercise-induced rDNA transcription is unclear. To investigate the genetic and epigenetic regulation of ribosome biogenesis with exercise, a time course of skeletal muscle biopsies was obtained from 30 participants (18 men and 12 women; 31 ± 8 years, 25 ± 4 kg m-2 ) at rest and 30 min, 3 h, 8 h and 24 h after acute endurance (n = 10, 45 min cycling, 70% V̇O2max ) or resistance exercise (n = 10, 4 × 7 × 2 exercises); 10 control participants underwent biopsies without exercise. rDNA transcription and dosage were assessed using quantitative PCR and whole genome sequencing. rDNA promoter methylation was investigated using massARRAY EpiTYPER and global rDNA CpG methylation was assessed using reduced-representation bisulphite sequencing. Ribosome biogenesis and MYC transcription were associated primarily with resistance but not endurance exercise, indicating preferential up-regulation during hypertrophic processes. With resistance exercise, ribosome biogenesis was associated with rDNA gene dosage, as well as epigenetic changes in enhancer and non-canonical MYC-associated areas in rDNA, but not the promoter. A mouse model of in vivo metabolic RNA labelling and genetic myonuclear fluorescence labelling validated the effects of an acute hypertrophic stimulus on ribosome biogenesis and Myc transcription, and also corroborated rDNA enhancer and Myc-associated methylation alterations specifically in myonuclei. The present study provides the first information on skeletal muscle genetic and rDNA gene-wide epigenetic regulation of ribosome biogenesis in response to exercise, revealing novel roles for rDNA dosage and CpG methylation.

Molecular Differences in Skeletal Muscle After 1 Week of Active vs. Passive Recovery From High-Volume Resistance Training.

ABSTRACT: Vann, CG, Haun, CT, Osburn, SC, Romero, MA, Roberson, PA, Mumford, PW, Mobley, CB, Holmes, HM, Fox, CD, Young, KC, and Roberts, MD. Molecular differences in skeletal muscle after 1 week of active vs. passive recovery from high-volume resistance training. J Strength Cond Res 35(8): 2102-2113, 2021-Numerous studies have evaluated how deloading after resistance training (RT) affects strength and power outcomes. However, the molecular adaptations that occur after deload periods remain understudied. Trained, college-aged men (n = 30) performed 6 weeks of whole-body RT starting at 10 sets of 10 repetitions per exercise per week and finishing at 32 sets of 10 repetitions per exercise per week. After this period, subjects performed either active (AR; n = 16) or passive recovery (PR; n = 14) for 1 week where AR completed ∼15% of the week 6 training volume and PR ceased training. Variables related to body composition and recovery examined before RT (PRE), after 6 weeks of RT (POST), and after the 1-week recovery period (DL). Vastus lateralis (VL) muscle biopsies and blood samples were collected at each timepoint, and various biochemical and histological assays were performed. Group × time interactions (p < 0.05) existed for skeletal muscle myosin heavy chain (MHC)-IIa mRNA (AR > PR at POST and DL) and 20S proteasome activity (post-hoc tests revealed no significance in groups over time). Time effects (P < 0.05) existed for total mood disturbance and serum creatine kinase and mechano growth factor mRNA (POST > PRE &D L), VL pressure to pain threshold and MHC-IIx mRNA (PRE&DL > POST), Atrogin-1 and MuRF-1 mRNA (PRE < POST < DL), MHC-I mRNA (PRE < POST & DL), myostatin mRNA (PRE & POST < DL), and mechanistic target of rapamycin (PRE > POST & DL). No interactions or time effects were observed for barbell squat velocity, various hormones, histological metrics, polyubiquitinated proteins, or phosphorylated/pan protein levels of 4E-BP1, p70S6k, and AMPK. One week of AR after a high-volume training block instigates marginal molecular differences in skeletal muscle relative to PR. From a practical standpoint, however, both paradigms elicited largely similar responses.

Acute and chronic resistance training downregulates select LINE-1 retrotransposon activity markers in human skeletal muscle.

Herein, we examined if acute or chronic resistance exercise affected markers of skeletal muscle long interspersed nuclear element-1 (LINE-1) retrotransposon activity. In study 1, 10 resistance-trained college-aged men performed three consecutive daily back squat sessions, and vastus lateralis biopsies were taken before (Pre), 2 h following session 1 (Post1), and 3 days following session 3 (Post2). In study 2, 13 untrained college-aged men performed a full-body resistance training program (3 days/wk), and vastus lateralis biopsies were taken before ( week 0) and ~72 h following training cessation ( week 12). In study 1, LINE-1 mRNA decreased 42-48% at Post1 and 2 ( P < 0.05), and reverse transcriptase (RT) activity trended downward at Post2 (-37%, P = 0.067). In study 2, LINE-1 mRNA trended downward at week 12 (-17%, P = 0.056) while LINE-1 promoter methylation increased (+142%, P = 0.041). Open reading frame (ORF)2p protein expression (-24%, P = 0.059) and RT activity (-26%, P = 0.063) also trended downward by week 12. Additionally, changes in RT activity versus satellite cell number were inversely associated ( r = -0.725, P = 0.008). Follow-up in vitro experiments demonstrated that 48-h treatments with lower doses (1 µM and 10 µM) of efavirenz and nevirapine (non-nucleoside RT inhibitors) increased myoblast proliferation ( P < 0.05). However, we observed a paradoxical decrease in myoblast proliferation with higher doses (50 µM) of efavirenz and delavirdine. This is the first report suggesting that resistance exercise downregulates markers of skeletal muscle LINE-1 activity. Given our discordant in vitro findings, future research is needed to thoroughly assess whether LINE-1-mediated RT activity enhances or blunts myoblast, or primary satellite cell, proliferative capacity.

Skeletal muscle amino acid transporter and BCAT2 expression prior to and following interval running or resistance exercise in mode-specific trained males.

Endurance (END)- and resistance (RES)-trained males performed interval running or resistance exercise during three consecutive days (bouts 1-3). Muscle biopsies were obtained at baseline, 2 h post-bout 1, and 72 h post-bout 3. Amino acid transporter SNAT2 mRNA was 75% greater in END (group p = 0.008), and increased ~ 70% 2 h post in both groups (time p = 0.023). Amino acid transporter PAT1 mRNA was 2.7-fold greater in RES (group p = 0.002). Baseline protein levels of the mitochondrial aminotransferase BCAT2 were 79% greater in END (p = 0.015).

Correction to: Ten weeks of branched-chain amino acid supplementation improves select performance and immunological variables in trained cyclists.

For the author R. Mac Thompson, the first name should be R. Mac and the last name should be Thompson. On SpringerLink the name is listed correctly, but on PubMed he is listed as Mac Thompson R.

Effect of 1-week betalain-rich beetroot concentrate supplementation on cycling performance and select physiological parameters.

PURPOSE: Betalains are indole-derived pigments found in beet root, and recent studies suggest that they may exert ergogenic effects. Herein, we examined if supplementation for 7 days with betalain-rich beetroot concentrate (BLN) improved cycling performance or altered hemodynamic and serum analytes prior to, during and following a cycling time trial (TT). METHODS: Twenty-eight trained male cyclists (29 ± 10 years, 77.3 ± 13.3 kg, and 3.03 ± 0.62 W/kg) performed a counterbalanced crossover study whereby BLN (100 mg/day) or placebo (PLA) supplementation occurred over 7 days with a 1-week washout between conditions. On the morning of day seven of each supplementation condition, participants consumed one final serving of BLN or PLA and performed a 30-min cycling TT with concurrent assessment of several physiological variables and blood markers. RESULTS: BLN supplementation improved average absolute power compared to PLA (231.6 ± 36.2 vs. 225.3 ± 35.8 W, p = 0.050, d = 0.02). Average relative power, distance traveled, blood parameters (e.g., pH, lactate, glucose, NOx) and inflammatory markers (e.g., IL-6, IL-8, IL-10, TNFα) were not significantly different between conditions. BLN supplementation significantly improved exercise efficiency (W/ml/kg/min) in the last 5 min of the TT compared to PLA (p = 0.029, d = 0.45). Brachial artery blood flow in the BLN condition, immediately post-exercise, tended to be greater compared to PLA (p = 0.065, d = 0.32). CONCLUSIONS: We report that 7 days of BLN supplementation modestly improves 30-min TT power output, exercise efficiency as well as post-exercise blood flow without increasing plasma NOx levels or altering blood markers of inflammation, oxidative stress, and/or hematopoiesis.

Progressive resistance-loaded voluntary wheel running increases hypertrophy and differentially affects muscle protein synthesis, ribosome biogenesis, and proteolytic markers in rat muscle.

We examined if 6 weeks of progressive resistance-loaded voluntary wheel running in rats induced plantaris, soleus, and/or gastrocnemius hypertrophy and/or affected markers of translational efficiency, ribosome biogenesis, and markers of proteolysis. For 6 weeks, 8 male Sprague-Dawley rats (~9-10 weeks of age, ~300-325 g) rats were assigned to the progressive resistance-loaded voluntary wheel running model (EX), and ten rats were not trained (SED). For EX rats, the wheel-loading paradigm was as follows - days 1-7: free-wheel resistance, days 8-15: wheel resistance set to 20%-25% body mass, days 16-24: 40% body mass, days 25-32: 60% body mass, days 33-42: 40% body mass. Following the intervention, muscles were analysed for markers of translational efficiency, ribosome biogenesis, and muscle proteolysis. Raw gastrocnemius mass (+13%, p < .01), relative (body mass-corrected) gastrocnemius mass (+16%, p < .001), raw plantaris mass (+13%, p < .05), and relative plantaris mass (+15%, p < .01) were greater in EX vs. SED rats. In spite of gastrocnemius hypertrophy, EX animals presented a 54% decrease in basal muscle protein synthesis levels (p < .01), a 125% increase in pan 4EBP1 levels (p < .001) and a 31% decrease in pan eIF4E levels (p < .05). However, in relation to SED animals, EX animals presented a 70% increase in gastrocnemius c-Myc protein levels (p < .05). Most markers of translational efficiency and ribosome biogenesis were not altered in the plantaris or soleus muscles of EX vs. SED animals. Gastrocnemius F-box protein 32 and poly-ubiquinated protein levels were approximately 150% and 200% greater in SED vs. EX rats (p < .001). These data suggest that the employed resistance training model increases hind limb muscle hypertrophy, and this may be mainly facilitated through reductions in skeletal muscle proteolysis, rather than alterations in ribosome biogenesis or translational efficiency.

Whey protein-derived exosomes increase protein synthesis and hypertrophy in C2-C12 myotubes.

We sought to examine potential amino acid independent mechanisms whereby hydrolyzed whey protein (WP) affects muscle protein synthesis (MPS) and anabolism in vitro. Specifically, we tested (1) whether 3-h and 6-h treatments of WP, essential amino acids, or l-leucine (Leu) affected MPS, and whether 6-h treatments with low-, medium-, or high doses of WP versus Leu affected MPS; (2) whether knockdown of the primary Leu transporter affected WP- and Leu-mediated changes in MPS, mammalian target of rapamycin (mTOR) signaling responses, or both, following 6-h treatments; (3) whether exosomes isolated from WP (WP-EXO) affected MPS, mTOR signaling responses, or both, compared with untreated (control) myotubes, following 6-h, 12-h, and 24-h treatments, and whether they affected myotube diameter following 24-h and 48-h treatments. For all treatments, 7-d post-differentiated C2C12 myotubes were examined. In experiment 1, 6-h WP treatments increased MPS compared with control (+46%), Leu (+24%), and essential amino acids (+25%). Moreover, the 6-h low-, medium-, and high WP treatments increased MPS by approximately 40 to 50% more than corresponding Leu treatments. In experiment 2 (LAT short hairpin RNA-transfected myotubes), 6-h WP treatments increased MPS compared with control (+18%) and Leu (+19%). In experiment 3, WP-EXO treatments increased MPS over controls at 12h (+18%) and 24h (+45%), and myotube diameters increased with 24-h (+24%) and 48-h (+40%) WP-EXO treatments compared with controls. The WP-EXO treatments did not appear to operate through mTOR signaling; instead, they increased mRNA and protein levels o eukaryotic initiation factor 4A. Bovine-specific microRNA following 24-h WP-EXO treatments were enriched in myotubes (chiefly miR-149-3p, miR-2881), but were not related to hypertrophic gene targets. To summarize, hydrolyzed WP-EXO increased skeletal MPS and anabolism in vitro, and this may be related to an unknown mechanism that increases translation initiation factors rather than enhancing mTOR signaling or the involvement of bovine-specific microRNA.

Testosterone and trenbolone enanthate increase mature myostatin protein expression despite increasing skeletal muscle hypertrophy and satellite cell number in rodent muscle.

The androgen-induced alterations in adult rodent skeletal muscle fibre cross-sectional area (fCSA), satellite cell content and myostatin (Mstn) were examined in 10-month-old Fisher 344 rats (n = 41) assigned to Sham surgery, orchiectomy (ORX), ORX + testosterone (TEST; 7.0 mg week-1 ) or ORX + trenbolone (TREN; 1.0 mg week-1 ). After 29 days, animals were euthanised and the levator ani/bulbocavernosus (LABC) muscle complex was harvested for analyses. LABC muscle fCSA was 102% and 94% higher in ORX + TEST and ORX + TREN compared to ORX (p < .001). ORX + TEST and ORX + TREN increased satellite cell numbers by 181% and 178% compared to ORX, respectively (p < .01), with no differences between conditions for myonuclear number per muscle fibre (p = .948). Mstn protein was increased 159% and 169% in the ORX + TEST and ORX + TREN compared to ORX (p < .01). pan-SMAD2/3 protein was ~30-50% greater in ORX compared to SHAM (p = .006), ORX + TEST (p = .037) and ORX + TREN (p = .043), although there were no between-treatment effects regarding phosphorylated SMAD2/3. Mstn, ActrIIb and Mighty mRNAs were lower in ORX, ORX + TEST and ORX + TREN compared to SHAM (p < .05). Testosterone and trenbolone administration increased muscle fCSA and satellite cell number without increasing myonuclei number, and increased Mstn protein levels. Several genes and signalling proteins related to myostatin signalling were differentially regulated by ORX or androgen therapy.

Comparative effects of whey protein versus L-leucine on skeletal muscle protein synthesis and markers of ribosome biogenesis following resistance exercise.

We compared immediate post-exercise whey protein (WP, 500 mg) versus L-leucine (LEU, 54 mg) feedings on skeletal muscle protein synthesis (MPS) mechanisms and ribosome biogenesis markers 3 h following unilateral plantarflexor resistance exercise in male, Wistar rats (~250 g). Additionally, in vitro experiments were performed on differentiated C2C12 myotubes to compare nutrient (i.e., WP, LEU) and 'exercise-like' treatments (i.e., caffeine, hydrogen peroxide, and AICAR) on ribosome biogenesis markers. LEU and WP significantly increased phosphorylated-rpS6 (Ser235/236) in the exercised (EX) leg 2.4-fold (P < 0.01) and 2.7-fold (P < 0.001) compared to the non-EX leg, respectively, whereas vehicle-fed control (CTL) did not (+65 %, P > 0.05). Compared to the non-EX leg, MPS levels increased 32 % and 52 % in the EX leg of CTL (P < 0.01) and WP rats (P < 0.001), respectively, but not in LEU rats (+15 %, P > 0.05). Several genes associated with ribosome biogenesis robustly increased in the EX versus non-EX legs of all treatments; specifically, c-Myc mRNA, Nop56 mRNA, Bop1 mRNA, Ncl mRNA, Npm1 mRNA, Fb1 mRNA, and Xpo-5 mRNA. However, only LEU significantly increased 45S pre-rRNA levels in the EX leg (63 %, P < 0.001). In vitro findings confirmed that 'exercise-like' treatments similarly altered markers of ribosome biogenesis, but only LEU increased 47S pre-rRNA levels (P < 0.01). Collectively, our data suggests that resistance exercise, as well as 'exercise-like' signals in vitro, acutely increase the expression of genes associated with ribosome biogenesis independent of nutrient provision. Moreover, while EX with or without WP appears superior for enhancing translational efficiency (i.e., increasing MPS per unit of RNA), LEU administration (or co-administration) may further enhance ribosome biogenesis over prolonged periods with resistance exercise.

Ten weeks of branched-chain amino acid supplementation improves select performance and immunological variables in trained cyclists.

We examined if supplementing trained cyclists (32 ± 2 year, 77.8 ± 2.6 kg, and 7.4 ± 1.2 year training) with 12 g/day (6 g/day L-Leucine, 2 g/day L-Isoleucine and 4 g/day L-Valine) of either branched-chain amino acids (BCAAs, n = 9) or a maltodextrin placebo (PLA, n = 9) over a 10-week training season affected select body composition, performance, and/or immune variables. Before and after the 10-week study, the following was assessed: (1) 4-h fasting blood draws; (2) dual X-ray absorptiometry body composition; (3) Wingate peak power tests; and (4) 4 km time-trials. No group × time interactions existed for total lean mass (P = 0.27) or dual-leg lean mass (P = 0.96). A significant interaction existed for body mass-normalized relative peak power (19 % increase in the BCAA group pre- to post-study, P = 0.01), and relative mean power (4 % increase in the BCAA group pre- to post-study, P = 0.01). 4 km time-trial time to completion approached a significant interaction (P = 0.08), as the BCAA group improved in this measure by 11 % pre- to post-study, though this was not significant (P = 0.15). There was a tendency for the BCAA group to present a greater post-study serum BCAA: L-Tryptophan ratio compared to the PLA group (P = 0.08). A significant interaction for neutrophil number existed (P = 0.04), as there was a significant 18 % increase within the PLA group from the pre- to post-study time point (P = 0.01). Chronic BCAA supplementation improves sprint performance variables in endurance cyclists. Additionally, given that BCAA supplementation blunted the neutrophil response to intense cycling training, BCAAs may benefit immune function during a prolonged cycling season.

The Effects of Fortetropin Supplementation on Body Composition, Strength, and Power in Humans and Mechanism of Action in a Rodent Model.

OBJECTIVE: The purpose of this study was to investigate the effects of Fortetropin on skeletal muscle growth and strength in resistance-trained individuals and to investigate the anabolic and catabolic signaling effects using human and rodent models. METHODS: In the rodent model, male Wistar rats (250 g) were gavage fed with either 1.2 ml of tap water control (CTL) or 0.26 g Fortetropin for 8 days. Then rats participated in a unilateral plantarflexion exercise bout. Nonexercised and exercised limbs were harvested at 180 minutes following and analyzed for gene and protein expression relative to mammalian target of rapamycin (mTOR) and ubiquitin signaling. For the human model, 45 (of whom 37 completed the study), resistance-trained college-aged males were divided equally into 3 groups receiving a placebo macronutrient matched control, 6.6 or 19.8 g of Fortetropin supplementation during 12 weeks of resistance training. Lean mass, muscle thickness, and lower and upper body strength were measured before and after 12 weeks of training. RESULTS: The human study results indicated a Group × Time effect (p ≤ 0.05) for lean mass in which the 6.6 g (+1.7 kg) and 19.8 g (+1.68 kg) but not placebo (+0.6 kg) groups increased lean mass. Similarly, there was a Group × Time effect for muscle thickness (p ≤ 0.05), which increased in the experimental groups only. All groups increased equally in bench press and leg press strength. In the rodent model, a main effect for exercise (p ≤ 0.05) in which the control plus exercise but not Fortetropin plus exercise increased both ubiquitin monomer protein expression and polyubiquitination. mTOR signaling was elevated to a greater extent in the Fortetropin exercising conditions as indicated by greater phosphorylation status of 4EBP1, rp6, and p70S6K for both exercising conditions. CONCLUSIONS: Fortetropin supplementation increases lean body mass (LBM) and decreases markers of protein breakdown while simultaneously increasing mTOR signaling.