Muscle Biopsy Samples Research Articles

Effect of dietary n-3 PUFA supplementation on the muscle transcriptome in older adults.

Dietary fish oil‐derived n‐3 PUFA supplementation can increase muscle mass, reduce oxygen demand during physical activity, and improve physical function (muscle strength and power, and endurance) in people. The results from several studies conducted in animals suggest that the anabolic and performance‐enhancing effects of n‐3 PUFA are at least in part transcriptionally regulated. The effect of n‐3 PUFA therapy on the muscle transcriptome in people is unknown. In this study, we used muscle biopsy samples collected during a recently completed randomized controlled trial that found that n‐3 PUFA therapy increased muscle mass and function in older adults to provide a comprehensive assessment of the effect of n‐3 PUFA therapy on the skeletal muscle gene expression profile in these people. Using the microarray technique, we found that several pathways involved in regulating mitochondrial function and extracellular matrix organization were increased and pathways related to calpain‐ and ubiquitin‐mediated proteolysis and inhibition of the key anabolic regulator mTOR were decreased by n‐3 PUFA therapy. However, the effect of n‐3 PUFA therapy on the expression of individual genes involved in regulating mitochondrial function and muscle growth, assessed by quantitative RT‐PCR, was very small. These data suggest that n‐3 PUFA therapy results in small but coordinated changes in the muscle transcriptome that may help explain the n‐3 PUFA‐induced improvements in muscle mass and function.

Diminished Postprandial Muscle Protein Synthetic Response To Protein Ingestion In Obese Adults

Excess body fat blunts muscle protein synthesis rates under hyperinsulinemic, hyperaminoacidemic clamp conditions. However, the efficacy of the ingestion of single meal containing ample amounts of protein to augment postprandial muscle protein synthesis rates has not been studied in obese and overweight adults. PURPOSE: We aimed to compare myofibrillar protein synthesis after protein ingestion in healthy weight, overweight, and obese adults. METHODS: 10 healthy-weight (24±1 y, BMI 22.7±0.4 kg/m2, HOMA-IR 1.4±0.2), 10 overweight (26±2 y, BMI 27.1±0.5 kg/m2, HOMA-IR 1.25±0.11), and 10 obese (27±3 y, BMI 35.9±1.3 kg/m2, HOMA-IR 5.8±0.8) men and women underwent a primed continuous L-[ring-13C6]phenylalanine infusion. Blood and muscle biopsy samples were collected at rest and after ingestion of 36 g of protein to assess plasma amino acid and insulin concentrations and myofibrillar protein synthesis rates. RESULTS: Protein ingestion increased plasma essential amino acid concentrations similarly in all participants (time effect: P<0.05) with concentrations peaked at 2 h and returning to baseline values at 5 h. The obese participants had a greater peak postprandial plasma insulin response to protein ingestion than the overweight and healthy-weight participants (obese 56.7±8.0 compared to overweight 11.9±1.6 and healthy-weight 14.3±1.7 μIU/L, P<0.001). Protein ingestion increased myofibrillar protein synthesis in both the healthy-weight (rest: 0.057±0.006 %/h, fed: 0.084±0.014 %/h) and overweight groups (rest: 0.061±0.007, fed: 0.087±0.019 %/h) (P<0.05) with no increase in the obese group (rest: 0.055±0.005 %/h, fed: 0.067±0.005 %/h, P=0.45). CONCLUSIONS: Increased adiposity led to an impaired postprandial muscle protein synthetic response to protein ingestion in obese adults when compared to healthy-weight and overweight individuals. These data suggest that poor skeletal muscle remodeling may underlie early metabolic impairments in apparently “healthy” obese adults. Funding provided by the National Pork Board

Strength-Training Induces Skeletal Muscle Adaptations in Patients with Myotonic Dystrophy Type I

PURPOSE: Myotonic dystrophy type 1 (DM1) is the most prevalent inherited neuromuscular disease in adults. This multisystemic disease is characterized by skeletal muscle impairments including muscle wasting. Slowing muscle wasting in this population using strength training seems a promising strategy, but it remains unknown if it would trigger cellular and molecular responses similar to the ones observed in healthy people. The objective of this case study is to evaluate the effect of a strength-training program on skeletal muscle adaptations in a DM1 patient. METHODS: One male with DM1 (age = 36) underwent a 12-week strength-training program, twice a week, consisting of 2 sets of 6 exercises at 6 RM supplemented by functional tasks. Vastus lateralis muscle biopsy samples were obtained pre- and post-training program. The proportion of type I and II myofibers and the cross sectional area (CSA) of each type were determined by immunohistochemistry. The percentage of centrally nucleated fibers (CNF) was obtained following staining with hematoxyline/eosine Two blinded evaluators analyzed the data. RESULTS: Following the 12-week strength-training program, the patient showed an increase in the CSA of type I myofibers evaluated at 46% (p<0.05) by evaluator #1 and 51% (p<0.05) by evaluator #2. For type II myofibers, the increase in CSA was evaluated to 24% (p<0.05) and 29% (p<0.05) by evaluator #1 and #2, respectively. A muscle fiber-type switching was also induced by the 12-week strength-training program as shown by the increase in the proportion of type II myofibers from 29% to 71% (p<0.05) noted by the evaluator #1 and the similar observation noted by evaluator #2 (28% to 72%, p<0.05). No change was observed in the percentage of CNF by both evaluators. CONCLUSIONS: Our results suggest that skeletal muscle of patients with DM1 could undergo adaptations linked to muscle growth as demonstrated by the increase in the CSA of type I and type II myofibers. It also seems that strength-training parameters used in this study could also influence the distribution of myofibers, in favour of type II. Further studies comprising a higher number of participants are needed to validate our findings and determine to which extent and how skeletal muscles of patients with DM1 adapt to strength training stimulus.

Analysis of Human Vastus Lateralis Skeletal Muscle Biopsy Samples Changes in Response to High and Low Inflation Pressures during Peristaltic Pulse External Pneumatic Compression Via Transcriptome‐wide RNA Sequencing

We employed next generation RNA sequencing (RNA‐seq) technology to determine the impact of peristaltic pulse external pneumatic compression (EPC) at high and low target inflation pressures on global gene expression in human vastus lateralis skeletal muscle biopsy samples.MethodsEighteen (N=18) apparently healthy male subjects participated in this study and were randomly assigned to one of three groups: 1) sham (n=6), 2) EPC with target inflation pressures of 30 mmHg (n=6) and 3) EPC with target inflation pressures of 70–80 mmHg (n=6). Daily treatment with sham or EPC was given for 7 consecutive days with each treatment lasting 1 h. Vastus lateralis skeletal muscle biopsy samples were harvested at baseline (48 hours before the first treatment; T1), 1 h following the first treatment (T2), and 24 hours following the last (7th) treatment (T3). 3,613 annotated genes with an RPKM value of ≥ 1.0 were selected for analysis. Change from baseline (T1) in gene expression was analyzed via paired comparisons within each group (i.e. T2 vs. T1 and T3 vs. T1). The 3,613 genes were then further filtered to include only those that had a fold‐change (FC) of ≥ 1.5 and a paired t‐test value of &lt;0.01.ResultsFor the sham condition, only 2 genes at T2 (SF3B5, FIS1) and 1 gene at T1 (LYPLA2) were identified as being significantly altered compared to baseline with time. For the 30 mmHg EPC condition, 9 genes were found to be significantly upregulated at T2 and 39 genes were found to be significantly upregulated at T3 compared to baseline. Two of these genes were significantly upregulated at both T2 and T3 (ARRDC2 and DYNLL2). Only one gene was found to be significantly downregulated compared to baseline in the 30 mmHg EPC group (TIMM10) and this was observed at T3. For the 70–80 mmHg EPC condition, 2 genes were found to be significantly upregulated (HBA1, HBA2) and 21 genes were found to be significantly downregulated at T2 compared to baseline. At T3, only 1 gene was found to be significantly altered (downregulation of DNAJB5) in the 70–80 mmHg EPC group. Interestingly, VEGFA was found to be differentially regulated as its expression was significantly upregulated with 30 mmHg EPC at T3 and significantly downregulated with 70–80 mmHg EPC at T3. Follow up analyses respective to gene clusters of interest are presented herein.ConclusionsIn summary, a more dynamic alteration of skeletal muscle gene expression was observed with a lower (i.e. 30 mmHg) EPC target inflation pressure, particularly after 7 consecutive days of treatment. Moreover, altered genes tended to be upregulated with 30 mmHg EPC whereas genes tended to be downregulated with 70 mmHg.Support or Funding InformationThe authors would like to thank NormaTec (Newton Center, MA) for their partial financial support (50%) of this project through a contract awarded to J.S.M.

Fructose Coingestion Does Not Accelerate Postexercise Muscle Glycogen Repletion.

Postexercise muscle glycogen repletion is largely determined by the systemic availability of exogenous carbohydrate provided. This study aimed to assess the effect of the combined ingestion of fructose and glucose on postexercise muscle glycogen repletion when optimal amounts of carbohydrate are ingested. Fourteen male cyclists (age: 28 ± 6 yr; Wmax: 4.8 ± 0.4 W·kg⁻¹) were studied on three different occasions. Each test day started with a glycogen-depleting exercise session. This was followed by a 5-h recovery period, during which subjects ingested 1.5 g·kg⁻¹·h⁻¹ glucose (GLU), 1.2 g·kg⁻¹·h⁻¹ glucose + 0.3 g·kg⁻¹·h⁻¹ fructose (GLU + FRU), or 0.9 g·kg⁻¹·h⁻¹ glucose + 0.6 g·kg⁻¹·h⁻¹ sucrose (GLU + SUC). Blood samples and gastrointestinal distress questionnaires were collected frequently, and muscle biopsy samples were taken at 0, 120, and 300 min after cessation of exercise to measure muscle glycogen content. Plasma glucose responses did not differ between treatments (ANOVA, P = 0.096), but plasma insulin and lactate concentrations were elevated during GLU + FRU and GLU + SUC when compared with GLU (P < 0.01). Muscle glycogen content immediately after exercise averaged 207 ± 112, 219 ± 107, and 236 ± 118 mmol·kg⁻¹ dry weight in the GLU, GLU + FRU, and GLU + SUC treatments, respectively (P = 0.362). Carbohydrate ingestion increased muscle glycogen concentrations during 5 h of postexercise recovery to 261 ± 98, 289 ± 130, and 315 ± 103 mmol·kg⁻¹ dry weight in the GLU, GLU + FRU, and GLU + SUC treatments, respectively (P < 0.001), with no differences between treatments (time × treatment, P = 0.757). Combined ingestion of glucose plus fructose does not further accelerate postexercise muscle glycogen repletion in trained cyclists when ample carbohydrate is ingested. Combined ingestion of glucose (polymers) plus fructose or sucrose reduces gastrointestinal complaints when ingesting large amounts of carbohydrate.

Effect of acute high-intensity exercise in normobaric hypoxia on Thoroughbred skeletal muscle.

We examined the effect of transient hypoxic exposure during high-intensity exercise on satellite cell activation and angiogenesis in Thoroughbred skeletal muscle. Six Thoroughbreds horses (6.3±0.8 years old) ran on a treadmill in normoxia (N; FIO2=21%) and hypoxia (H; FIO2=16%) at the same speed for the same duration, and oxygen consumption and plasma lactate and hemoglobin concentrations were measured. In addition, muscle biopsy samples were obtained from gluteus medius muscle before exercise and immediately, 4 hours, 24 hours, 3 days and 7 days after exercise, and immunohistochemical analysis and relative quantitative analysis of mRNA expression using real-time RT-PCR were performed. Oxygen consumption during exercise in H was 29% lower than that in N, and plasma lactate concentration in H was 47% higher than that in N. The mRNA expressions of satellite cell activation-related factors slightly increased, but the mRNA expressions of factors related to angiogenesis and mitochondrial biogenesis slightly decreased. Fluorescence-stained basal lamina evaluation in stacked images 7 days after exercise showed no difference in capillary density between the two groups. These results suggest that transient hypoxic exposure during exercise increases the contribution of the glycolytic energy supply and promotes satellite cell activation, but does not facilitate angiogenesis and mitochondrial biogenesis.

Heightened TWEAK-NF-κB signaling and inflammation-associated fibrosis in paralyzed muscles of men with chronic spinal cord injury.

Individuals with long-standing spinal cord injury (SCI) often present with extreme muscle atrophy and impaired glucose metabolism at both the skeletal muscle and whole body level. Persistent inflammation and increased levels of proinflammatory cytokines in the skeletal muscle are potential contributors to dysregulation of glucose metabolism and atrophy; however, to date no study has assessed the effects of long-standing SCI on their expression or intracellular signaling in the paralyzed muscle. In the present study, we assessed the expression of genes (TNFαR, TNFα, IL-6R, IL-6, TWEAK, TWEAK R, atrogin-1, and MuRF1) and abundance of intracellular signaling proteins (TWEAK, TWEAK R, NF-κB, and p-p65/p-50/105) that are known to mediate inflammation and atrophy in skeletal muscle. In addition, based on the effects of muscle inflammation on promotion of skeletal muscle fibrosis, we assessed the degree of fibrosis between myofibers and fascicles in both groups. For further insight into the distribution and variability of muscle fiber size, we also analyzed the frequency distribution of SCI fiber size. Resting vastus lateralis (VL) muscle biopsy samples were taken from 11 men with long-standing SCI (≈22 yr) and compared with VL samples from 11 able-bodied men of similar age. Our results demonstrated that chronic SCI muscle has heightened TNFαR and TWEAK R gene expression and NF-κB signaling (higher TWEAK R and phospho-NF-κB p65) and fibrosis, along with substantial myofiber size heterogeneity, compared with able-bodied individuals. Our data suggest that the TWEAK/TWEAK R/NF-κB signaling pathway may be an important mediator of chronic inflammation and fibrotic adaptation in SCI muscle.

Reversible immortalisation, human artificial chromosomes, and induced pluripotency: new gene and cell therapy technologies for Duchenne muscular dystrophy

BackgroundDuchenne muscular dystrophy is caused by mutations in the gene that encodes dystrophin, a major component of muscle fibres. The combination of gene therapy with cell therapy to treat the disorder is encouraging, but it is challenging because dystrophin is the largest human gene, and skeletal muscle the most abundant human tissue. We aimed to assess use of autologous stem cells with human artificial chromosomes (HACs) containing the entire dystrophin locus (DYS-HACs) as a solution to these challenges. MethodsWe describe two complementary strategies for the generation of genetically corrected myogenic stem cells from patients with Duchenne muscular dystrophy, one from muscle biopsy samples and the other from induced pluripotent stem (iPS) cells. We also describe the generation of a multifunctional DYS-HAC applicable in both strategies. Notably, these technologies are also being developed in genome-integration-free platforms. FindingsReversible immortalisation with lentivirally delivered excisable telomerase and BMI1 complementary DNAs allowed bypassing of replicative senescence and DYS-HAC transfer in muscle-derived cells. When isolation of tissue-derived progenitors proved challenging, we successfully derived skeletal myogenic cells from Duchenne muscular dystrophy iPS cells reprogrammed with non-integrating technologies and corrected them with the DYS-HAC. Finally we describe the engineering of a next-generation, multifunctional DYS-HAC that can provide complete genetic correction, enhanced proliferation, controllable cell death (as a safety system), and inducible myogenesis in both tissue-derived or iPS cell-derived stem cells. InterpretationThis project provides the foundation for preclinical and clinical development of an autologous ex-vivo gene therapy protocol for Duchenne muscular dystrophy based on HACs and myogenic cells. FundingNational Institute for Health Research, Medical Research Council, Takeda New Frontier Science, Muscular Dystrophy UK, Duchenne Children's Trust, Duchenne Research Fund, Duchenne Parent Project Onlus.

Effect of High-Intensity Training in Normobaric Hypoxia on Thoroughbred Skeletal Muscle.

Hypoxic training is believed to increase endurance capacity in association with hypoxia inducible factor-1α (HIF-1α), a modulator of vascular endothelial growth factor-A (VEGF-A), and to influence activation of satellite cells (SCs). However, the effect of hypoxic training on SC activation and its relation to angiogenesis has not been thoroughly investigated. Eight Thoroughbred horses were subjected to normoxic (FIO2 = 21%) or hypoxic (FIO2 = 15%) training for 3 days/week (100% ) for 4 weeks. Incremental exercise tests (IET) were conducted on a treadmill under normoxia and the maximal oxygen consumption () and running distance were measured before and after each training session. Muscle biopsy samples were obtained from the gluteus medius muscle at 6 scheduled times before, during, and one week after IET for immunohistochemical analysis and real-time RT-PCR analysis. Running distance and , measured during IET, increased significantly after hypoxic training compared with normoxic training. Capillary density and mRNA expression related to SC activation (e.g., myogenin and hepatocyte growth factor) and angiogenesis (VEGF-A) increased only after hypoxic training. These results suggest that increases in mRNA expression after training enhance and prolong SC activation and angiogenesis and that nitric oxide plays an important role in these hypoxia-induced training effects.

Influence of muscle fibre composition on muscle oxygenation during maximal running

AimsTo investigate the relationship between muscle oxygenation (specifically, the levels of oxygenated haemoglobin and myoglobin [oxyHb/Mb]) during maximal running and muscle fibre composition, and to determine whether muscle fibre composition...

Differences in postprandial protein handling after beef compared with milk ingestion during postexercise recovery: a randomized controlled trial

Protein consumed after resistance exercise increases postexercise muscle protein synthesis rates. To date, dairy protein has been studied extensively, with little known about the capacity of other protein-dense foods to augment postexercise muscle protein synthesis rates. We aimed to compare protein digestion and absorption kinetics, postprandial amino acid availability, anabolic signaling, and the subsequent myofibrillar protein synthetic response after the ingestion of milk compared with beef during recovery from resistance-type exercise. In crossover trials, 12 healthy young men performed a single bout of resistance exercise. Immediately after cessation of exercise, participants ingested 30 g protein by consuming isonitrogenous amounts of intrinsically l-[1-(13)C]phenylalanine-labeled beef or milk. Blood and muscle biopsy samples were collected at rest and after exercise during primed continuous infusions of l-[ring-(2)H5]phenylalanine and l-[ring-3,5-(2)H2]tyrosine to assess protein digestion and absorption kinetics, plasma amino acid availability, anabolic signaling, and subsequent myofibrillar protein synthesis rates in vivo in young men. Beef protein-derived phenylalanine appeared more rapidly in circulation compared with milk ingestion (P < 0.001). The availability of phenylalanine during the 5-h postexercise period tended to be higher after beef (64% ± 3%) ingestion than after milk ingestion (57% ± 3%; P = 0.08). Both beef and milk ingestion were followed by an increase in the phosphorylation of mammalian target of rapamycin complex 1 and 70-kDa S6 protein kinase 1 during postexercise recovery. Milk ingestion increased myofibrillar protein synthesis rates to a greater extent than did beef ingestion during the 0- to 2-h postexercise phase (P = 0.013). However, the increase in myofibrillar protein synthesis rates did not differ between milk and beef ingestion during the entire 0- to 5-h postexercise phase (P = 0.114). Both milk and beef ingestion augment the postexercise myofibrillar protein synthetic response in young men, with a stronger stimulation of myofibrillar protein synthesis during the early postprandial stage after milk ingestion. This trial was registered at www.clinicaltrials.gov as NCT01578590.

Exertional rhabdomyolysis: recent advances in knowledge

Our understanding of exertional rhabdomyolysis in the horse has advanced considerably over the past 10 years. Not long ago, this syndrome was considered to be a single disease entity, also known as azoturia or Monday morning disease. Now, several distinct and very different conditions that result in this common clinical presentation have been recognised. The most documented of these conditions are recurrent exertional rhabdomyolysis (RER) and polysaccharide storage myopathy (PSSM), the latter having been recently classified into type 1 and type 2 PSSM. The availability of genetic testing and widespread use of muscle biopsy sampling have facilitated clinical diagnosis of underlying myopathies in the field.

Impaired Muscle Mitochondrial Biogenesis and Myogenesis in Spinal Muscular Atrophy.

The important depletion of mitochondrial DNA (mtDNA) and the general depression of mitochondrial respiratory chain complex levels (including complex II) have been confirmed, implying an increasing paucity of mitochondria in the muscle from patients with types I, II, and III spinal muscular atrophy (SMA-I, -II, and -III, respectively). To investigate mitochondrial dysfunction in a large series of muscle biopsy samples from patients with SMA. We studied quadriceps muscle samples from 24 patients with genetically documented SMA and paraspinal muscle samples from 3 patients with SMA-II undergoing surgery for scoliosis correction. Postmortem muscle samples were obtained from 1 additional patient. Age-matched controls consisted of muscle biopsy specimens from healthy children aged 1 to 3 years who had undergone analysis for suspected myopathy. Analyses were performed at the Neuromuscular Unit, Istituto di Ricovero e Cura a Carattere Scientifico Foundation Ca' Granda Ospedale Maggiore Policlinico-Milano, from April 2011 through January 2015. We used histochemical, biochemical, and molecular techniques to examine the muscle samples. Respiratory chain activity and mitochondrial content. Results of histochemical analysis revealed that cytochrome-c oxidase (COX) deficiency was more evident in muscle samples from patients with SMA-I and SMA-II. Residual activities for complexes I, II, and IV in muscles from patients with SMA-I were 41%, 27%, and 30%, respectively, compared with control samples (P < .005). Muscle mtDNA content and cytrate synthase activity were also reduced in all 3 SMA types (P < .05). We linked these alterations to downregulation of peroxisome proliferator-activated receptor coactivator 1α, the transcriptional activators nuclear respiratory factor 1 and nuclear respiratory factor 2, mitochondrial transcription factor A, and their downstream targets, implying depression of the entire mitochondrial biogenesis. Results of Western blot analysis confirmed the reduced levels of the respiratory chain subunits that included mitochondrially encoded COX1 (47.5%; P = .004), COX2 (32.4%; P < .001), COX4 (26.6%; P < .001), and succinate dehydrogenase complex subunit A (65.8%; P = .03) as well as the structural outer membrane mitochondrial porin (33.1%; P < .001). Conversely, the levels of expression of 3 myogenic regulatory factors-muscle-specific myogenic factor 5, myoblast determination 1, and myogenin-were higher in muscles from patients with SMA compared with muscles from age-matched controls (P < .05). Our results strongly support the conclusion that an altered regulation of myogenesis and a downregulated mitochondrial biogenesis contribute to pathologic change in the muscle of patients with SMA. Therapeutic strategies should aim at counteracting these changes.

Activation of autophagy in human skeletal muscle is dependent on exercise intensity and AMPK activation.

In humans, nutrient deprivation and extreme endurance exercise both activate autophagy. We hypothesized that cumulating fasting and cycling exercise would potentiate activation of autophagy in skeletal muscle. Well-trained athletes were divided into control (n = 8), low-intensity (LI, n = 8), and high-intensity (HI, n = 7) exercise groups and submitted to fed and fasting sessions. Muscle biopsy samples were obtained from the vastus lateralis before, at the end, and 1 h after a 2 h LI or HI bout of exercise. Phosphorylation of ULK1(Ser317) was higher after exercise (P < 0.001). In both the fed and the fasted states, LC3bII protein level and LC3bII/I were decreased after LI and HI (P < 0.05), while p62/SQSTM1 was decreased only 1 h after HI (P < 0.05), indicating an increased autophagic flux after HI. The autophagic transcriptional program was also activated, as evidenced by the increased level of LC3b, p62/SQSTM1, GabarapL1, and Cathepsin L mRNAs observed after HI but not after LI. The increased autophagic flux after HI exercise could be due to increased AMP-activated protein kinase α (AMPKα) activity, as both AMPKα(Thr172) and ACC(Ser79) had a higher phosphorylation state after HI (P < 0.001). In summary, the most effective strategy to activate autophagy in human skeletal muscle seems to rely on exercise intensity more than diet.

Visualization and quantitation of GLUT4 translocation in human skeletal muscle following glucose ingestion and exercise.

Insulin- and contraction-stimulated increases in glucose uptake into skeletal muscle occur in part as a result of the translocation of glucose transporter 4 (GLUT4) from intracellular stores to the plasma membrane (PM). This study aimed to use immunofluorescence microscopy in human skeletal muscle to quantify GLUT4 redistribution from intracellular stores to the PM in response to glucose feeding and exercise. Percutaneous muscle biopsy samples were taken from the m. vastus lateralis of ten insulin-sensitive men in the basal state and following 30 min of cycling exercise (65% VO2 max). Muscle biopsy samples were also taken from a second cohort of ten age-, BMI- and VO2 max-matched insulin-sensitive men in the basal state and 30 and 60 min following glucose feeding (75 g glucose). GLUT4 and dystrophin colocalization, measured using the Pearson's correlation coefficient, was increased following 30 min of cycling exercise (baseline r = 0.47 ± 0.01; post exercise r = 0.58 ± 0.02; P < 0.001) and 30 min after glucose ingestion (baseline r = 0.42 ± 0.02; 30 min r = 0.46 ± 0.02; P < 0.05). Large and small GLUT4 clusters were partially depleted following 30 min cycling exercise, but not 30 min after glucose feeding. This study has, for the first time, used immunofluorescence microscopy in human skeletal muscle to quantify increases in GLUT4 and dystrophin colocalization and depletion of GLUT4 from large and smaller clusters as evidence of net GLUT4 translocation to the PM.

Fast Food Results In Similar Post-exercise Glycogen Recovery And Exercise Performance Compared To Sport Supplements

A variety of dietary choices are marketed to enhance recovery after physical activity. These include traditional sports supplements (SS) and even chocolate milk. Previous research indicates that rates of recovery are influenced by macronutrient composition, amount, and timing of ingestion. PURPOSE: This study investigated the effects of SS vs fast food (FF) feedings during a 4-hour recovery period on glycogen recovery and subsequent 20k time-trial performance (TT). METHODS: In a randomized-cross over design, 11 male subjects (4.1±0.62 L/min VO2 max, 78.8±13.6 kg, 31.5±11.5 y), completed a 90-minute glycogen depletion ride on a cycle ergometer. Following 4 hours of recovery a TT was performed. Subjects were fed the same absolute amounts of macronutrients (1.54±0.27 g/kg carbohydrate, 0.24±0.04 g/kg fat, and 0.18±0.03g/kg protein) as either SS or FF items at 0 and 2 hours during recovery with 7 days between trials. Blood samples for glucose and insulin were obtained at 0, 30, 60, 120, 150, 180, and 240 minutes post exercise. Muscle biopsy samples from the vastus lateralis were obtained immediately post exercise and after the 4-hour recovery period for determination of glycogen concentration. A visual analogue gastrointestinal discomfort questionnaire assessing feelings of hunger, fullness, sickness, discomfort, satisfaction, taste, and meal acceptability were administered at 0, 1, 2, 3, and 4 hours of recovery. RESULTS: A feeding effect was observed at time points 30 and 150 minutes for blood glucose and insulin but there were no differences between trials. Rates of glycogen recovery were not different across the diets (6.9±1.7 and 7.9±2.4 mmol.kg− 1 wet weight.hr-1 for SS and FF, respectively). TT results were also not significantly different between trials (34.1±1.8 and 34.3±1.7 minutes for SS and FF, respectively. The questionnaire demonstrated an increase in satiety between 0 and 2 hour feedings for FF (p<0.05). CONCLUSIONS: These data indicate that short-term food options to initiate glycogen recovery can include a wide range of dietary options if total macronutrient composition is considered.

Ultrasonic assessment of exercise-induced change in skeletal muscle glycogen content

BackgroundUltrasound imaging is a valuable tool in exercise and sport science research, and has been used to visualize and track real-time movement of muscles and tendons, estimate hydration status in body tissues, and most recently, quantify skeletal muscle glycogen content. In this validation study, direct glycogen quantification from pre-and post-exercise muscle biopsy samples was compared with glycogen content estimates made through a portable, diagnostic high-frequency ultrasound and cloud-based software system (MuscleSound®, Denver, CO).MethodsWell-trained cyclists (N = 20, age 38.4 ± 6.0 y, 351 ± 57.6 wattsmax) participated in a 75-km cycling time trial on their own bicycles using CompuTrainer Pro Model 8001 trainers (RacerMate, Seattle, WA). Muscle biopsy samples and ultrasound measurements were acquired pre- and post-exercise. Specific locations on the vastus lateralis were marked, and a trained technician used a 12 MHz linear transducer and a standard diagnostic high resolution GE LOGIQ-e ultrasound machine (GE Healthcare, Milwaukee, WI) to make three ultrasound measurements. Ultrasound images were pre-processed to isolate the muscle area under analysis, with the mean pixel intensity averaged from the three scans and scaled (0 to 100 scale) to create the glycogen score. Pre- and post-exercise muscle biopsy samples were acquired at the vastus lateralis location (2 cm apart) using the suction-modified percutaneous needle biopsy procedure, and analyzed for glycogen content.ResultsThe 20 cyclists completed the 75-km cycling time trial in 168 ± 26.0 minutes at a power output of 193 ± 57.8 watts (54.2 ± 9.6% wattsmax). Muscle glycogen decreased 77.2 ± 17.4%, with an absolute change of 71.4 ± 23.1 mmol glycogen per kilogram of muscle. The MuscleSound® change score at the vastus lateralis site correlated highly with change in measured muscle glycogen content (R = 0.92, P < 0.001).ConclusionsMuscleSound® change scores acquired from an average of three ultrasound scans at the vastus lateralis site correlated significantly with change in vastus lateralis muscle glycogen content. These data support the use of the MuscleSound® system for accurately and non-invasively estimating exercise-induced decreases in vastus lateralis skeletal muscle glycogen content.

Immunohistological and Morphological Analysis from a Skeletal Muscle Microbiopsy

ABSTRACTThe study of skeletal muscle often requires invasive muscle biopsies, traditionally with the Bergstrom technique. Microbiopsies, or fine‐needle biopsies are used with many biochemistry applications but are scarcely used for histology due to inconsistencies with sample weight and integrity. Thus, the aim of the current study was to develop procedures for muscle fiber morphology and immunohistological analysis from a microbiopsy technique.METHODS1,366 muscle fibers were analyzed from 8 muscle biopsy samples obtained from four healthy men (26.8 ± 5.4y, 175.9 ± 4.3 cm, 91.9 ± 5.3kg). Percutaneous microbiopsies were taken of the vastus lateralis with a 14‐gauge microbiopsy needle on two separate occasions. Samples were removed from the needle and promptly weighed. The tissue was oriented in a cryomold, embedded in Tissue‐Tek® then frozen in liquid nitrogen cooled isopentane. The muscle sections were stained with haematoxylin and eosin or anti‐laminin and counterstained with AlexaFlour 488® and viewed under 4x magnification. Fiber number and mean fiber area was determined by Image J.RESULTSThe mean ± SD (range) microbiopsy sample weight was 18.3 ± 2.9 mg (14‐22mg). Fiber number within the microbiopsy specimens was 150.4 ± 120.6 (64–366). All viable fibers were measured in each sample and the mean fiber area was 4385.1 ± 1265.8 µm2 (977.0 – 10,132.93 µm2). The intra‐subject coefficient of variation for mean fiber area was 7.2%.CONCLUSIONResults show the potential of a minimally invasive muscle biopsy technique for use in immunohistological and morphological analysis. This could allow future advances in the study of skeletal muscle by allowing for smaller muscle groups to be examined.

Gene expression and fiber type variations in repeated vastus lateralis biopsies.

Muscle sample collection can introduce variation in any measured variable due to inter- and intramuscle variation. We investigated the variation in gene expression and fiber type composition after repeated biopsy sampling from the vastus lateralis muscle. Six subjects donated 3 tissue samples each. One hour after baseline sampling from 1 vastus lateralis muscle, samples from both vastus lateralis muscles were obtained. The fiber type composition differed between biopsies taken from the same leg. There were no within-subject differences in gene expression between the 3 biopsies. Multivariate analysis supports a model in which gene expression differs significantly between individuals but is not affected by repeated muscle biopsy sampling from the same subject. One vastus lateralis muscle sample per subject is sufficient to establish a reliable baseline for comparing gene expression representing selected pathways over time within the same individual.

Combined training enhances skeletal muscle mitochondrial oxidative capacity independent of age.

Skeletal muscle from sedentary older adults exhibits reduced mitochondrial abundance and oxidative capacity. The primary objective was to determine whether 8 weeks of combined training (CT) has a more robust effect than endurance training (ET) or resistance training (RT) on mitochondrial physiology in healthy young (18-30 years) and older (≥ 65 years) adults. Thirty-four young and 31 older adults were randomly assigned to 8 weeks of ET, RT, and control/CT. Control subjects completed 8 weeks of no exercise (control) followed by 8 weeks of CT. Body composition, skeletal muscle strength, and peak oxygen uptake were measured before and after the intervention. Vastus lateralis muscle biopsy samples were obtained before and 48 hours after the intervention. Mitochondrial physiology was evaluated by high-resolution respirometry and expression of mitochondrial proteins and transcription factors by quantitative PCR and immunoblotting. ET and CT significantly increased oxidative capacity and expression of mitochondrial proteins and transcription factors. All training modalities improved body composition, cardiorespiratory fitness, and skeletal muscle strength. CT induced the most robust improvements in mitochondria-related outcomes and physical characteristics despite lower training volumes for the ET and RT components. Importantly, most of the adaptations to training occurred independent of age. Collectively, these results demonstrate that both ET and CT increase muscle mitochondrial abundance and capacity although CT induced the most robust improvements in the outcomes measured. In conclusion, CT provides a robust exercise regimen to improve muscle mitochondrial outcomes and physical characteristics independent of age.