Muscle Biopsy Samples Research Articles

Remodeling the Skeletal Muscle Extracellular Matrix in Older Age-Effects of Acute Exercise Stimuli on Gene Expression.

With advancing age, the skeletal muscle extracellular matrix (ECM) undergoes fibrotic changes that may lead to increased muscle stiffness, injury susceptibility and strength loss. This study tested the potential of different exercises to counter these changes by stimulating the activity of genes associated with ECM remodeling. Twenty-six healthy men (66.9 ± 3.9 years) were stratified to two of four groups, performing unilateral (i) conventional resistance exercise, (ii) conventional resistance exercise followed by self-myofascial release (CEBR), (iii) eccentric-only exercise (ECC) or (iv) plyometric jumps (PLY). The non-trained leg served as control. Six hours post-exercise, vastus lateralis muscle biopsy samples were analyzed for the expression of genes associated with ECM collagen synthesis (COL1A1), matrix metallopeptidases (collagen degradation; MMPs) and peptidase inhibitors (TIMP1). Significant between-group differences were found for MMP3, MMP15 and TIMP1, with the greatest responses in MMP3 and TIMP1 seen in CEBR and in MMP15 in ECC. MMP9 (3.24–3.81-fold change) and COL1A1 (1.47–2.40-fold change) were increased in CEBR and PLY, although between-group differences were non-significant. The expression of ECM-related genes is exercise-specific, with CEBR and PLY triggering either earlier or stronger remodeling than other stimuli. Training studies will test whether execution of such exercises may help counter age-associated muscle fibrosis.

Manufacturing autologous myoblast for regenerative medicine applications.

Autologous myoblasts have been tested in the treatment of muscle-related diseases. However, the standardization of manufacturing myoblasts is still not established. Here we report a flask and animal-free medium-based method of manufacturing clinical-grade myoblast together with establishing releasing criteria for myoblast products under Good Manufacturing Practice (GMP). Quadriceps muscle biopsy samples were donated from three patients with myogenic ptosis. After biopsy samples were digested through enzymatic dissociation, the cells were grown in T175 flasks (passage 0) and hyperflasks (passage 1) in the animal-free SkGMTM-2 skeletal muscle cell growth medium containing 5% human platelet lysate for 15-17 days. The harvested cells were released based on cell morphology, cell dose, viability, sterility, endotoxin, mycoplasma and immunophenotype. Myotube differentiation was also evaluated. 400 to 500 million myoblast cells were manufactured within 15 to 17 days by the end of passage 1, which met pre-determined releasing criteria. The manufactured myoblast cells could differentiate and fuse into myotubes in vitro, with the possible trend that the donor age may impact the differentiation ability of myoblasts. The present study establishes a flask-based method of manufacturing myoblast in the animal-free medium together with releasing criteria, which is simple, robust, inexpensive and easily reproducible. This study will serve as the validation for a planned phase 1 clinical trial to assess the use of autologous myoblast transplants for the treatment of myogenic ptosis and other myogenic diseases.

Effect of Capsinoids Supplementation on Fat Oxidation and Muscle Glycogen Restoration During Post-exercise Recovery in Humans.

Capsinoids (CSN), the novel non-pungent capsaicin analogs have been reported to promote metabolic health and exercise tolerance. However, the effect of CSN on fat oxidation and changes in skeletal muscle glycogen levels during post-exercise recovery has not been investigated in humans. We examined the effect of CSN supplementation on energy reliance, glycogen resynthesis and molecular proteins in the skeletal muscle of young adults during post-exercise recovery. In this crossover-designed study, nine healthy adult male volunteers (aged 21.4±0.2 years, BMI 21.9±1.3 kg/m2) completed a 60-min cycling exercise at 70% VO2max. Participants consumed either CSN (12 mg, single dosage) or placebo capsules with a high-carbohydrate meal (2 g carb/kg bodyweight) immediately after exercise. Biopsied muscle samples (vastus lateralis), blood, and gaseous samples were obtained during 3h postexercise recovery period. We found that oral CSN supplementation right after exercise significantly altered the energy reliance on fat oxidation during recovery. This was evidenced by lower respiratory exchange ratio (RER) and higher fat oxidation rate in CSN trial. Despite this, acute CSN dosage does not contribute in enhancing the glycogen replenishment in skeletal muscle during 3h recovery. We identified no significant differences in postprandial glucose and insulin area under the curve in both trials. Western blot data showed an increased muscle GLUT4 expression, but no significant response of p-Akt/Akt ratio with CSN during post-exercise recovery. Our findings conclude that acute CSN intake could change energy reliance on fat oxidation but is unable to enhance muscle glycogen resynthesis during post-exercise recovery. Thus, ergogenic properties of CSN in relevance to muscle glycogen restoration following exercise needs to be further investigated in young adults.

Muscle biopsy in anti-neutrophil cytoplasmic antibody-associated vasculitis: diagnostic yield depends on anti-neutrophil cytoplasmic antibody type, sex and neutrophil count.

This study aimed to examine the sensitivity of muscle biopsy (MB) in ANCA-associated vasculitis (AAV), identify factors predicting MB positivity and assess the prognostic value of a positive MB. We conducted a single-centre retrospective study of AAV with an MB performed at diagnosis. AAV classification [granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), eosinophilic granulomatosis with polyangiitis (EGPA)] followed the European Medicines Agency algorithm. A logistic regression model was used to identify the factors associated with MB positivity. Survival curves were generated using the Kaplan-Meier method. Among 276 AAV patients (1995-2018), 101 had an MB. Seventy-eight patients were included: 33 with GPA, 25 with MPA and 20 with EGPA. MB samples were positive in 45 cases (58%): 17 GPA, 16 MPA and 12 EGPA. Univariate analysis focussed on GPA and MPA, revealed that the MB yield was higher in females [22/31 (71%) vs 11/27 (41%); P = 0.02] and in anti-MPO patients [25/37 (68%) vs 6/19 (32%) for anti-PR3; P = 0.01]. By multivariate analysis, three factors predicted MB positivity: anti-MPO ANCA [odds ratio (OR) 10.67 (CI 2.09, 81.68)], female sex [OR 5.3 (CI 1.16, 32.35)] and neutrophil count [OR 1.33 (CI 1.07, 1.8)]. MB positivity had no impact on relapse, death or end-stage renal disease-free survival. MB is a safe and efficient diagnostic tool for AAV. Predictors of MB yield include ANCA type, sex and neutrophil count. MB cannot substitute for kidney biopsy when indicated, but should be considered in other cases.

Effect of carbohydrate\u2013protein supplementation on endurance training adaptations

PurposeTo examine the influence of post-exercise protein feeding upon the adaptive response to endurance exercise training.MethodsIn a randomised parallel group design, 25 healthy men and women completed 6 weeks of endurance exercise training by running on a treadmill for 30–60 min at 70–75% maximal oxygen uptake (VO2max) 4 times/week. Participants ingested 1.6 g per kilogram of body mass (g kg BM−1) of carbohydrate (CHO) or an isocaloric carbohydrate–protein solution (CHO-P; 0.8 g carbohydrate kg BM−1 + 0.8 g protein kg BM−1) immediately and 1 h post-exercise. Expired gas, blood and muscle biopsy samples were taken at baseline and follow-up.ResultsExercise training improved VO2max in both groups (p ≤ 0.001), but this increment was not different between groups either in absolute terms or relative to body mass (0.2 ± 0.2 L min−1 and 3.0 ± 2 mL kg−1 min−1, respectively). No change occurred in plasma albumin concentration from baseline to follow-up with CHO-P (4.18 ± 0.18 to 4.23 ± 0.17 g dL−1) or CHO (4.17 ± 0.17 to 4.12 ± 0.22 g dL−1; interaction: p > 0.05). Mechanistic target of rapamycin (mTOR) gene expression was up-regulated in CHO-P (+ 46%; p = 0.025) relative to CHO (+ 4%) following exercise training.ConclusionPost-exercise protein supplementation up-regulated the expression of mTOR in skeletal muscle over 6 weeks of endurance exercise training. However, the magnitude of improvement in VO2max was similar between groups.

Passive Transfer of Sera from ALS Patients with Identified Mutations Evokes an Increased Synaptic Vesicle Number and Elevation of Calcium Levels in Motor Axon Terminals, Similar to Sera from Sporadic Patients.

Previously, we demonstrated increased calcium levels and synaptic vesicle densities in the motor axon terminals (MATs) of sporadic amyotrophic lateral sclerosis (ALS) patients. Such alterations could be conferred to mice with an intraperitoneal injection of sera from these patients or with purified immunoglobulin G. Later, we confirmed the presence of similar alterations in the superoxide dismutase 1 G93A transgenic mouse strain model of familial ALS. These consistent observations suggested that calcium plays a central role in the pathomechanism of ALS. This may be further reinforced by completing a similar analytical study of the MATs of ALS patients with identified mutations. However, due to the low yield of muscle biopsy samples containing MATs, and the low incidence of ALS patients with the identified mutations, these examinations are not technically feasible. Alternatively, a passive transfer of sera from ALS patients with known mutations was used, and the MATs of the inoculated mice were tested for alterations in their calcium homeostasis and synaptic activity. Patients with 11 different ALS-related mutations participated in the study. Intraperitoneal injection of sera from these patients on two consecutive days resulted in elevated intracellular calcium levels and increased vesicle densities in the MATs of mice, which is comparable to the effect of the passive transfer from sporadic patients. Our results support the idea that the pathomechanism underlying the identical manifestation of the disease with or without identified mutations is based on a common final pathway, in which increasing calcium levels play a central role.

Dose-response effects of dietary protein on muscle protein synthesis during recovery from endurance exercise in young men: a double-blind randomized trial

ABSTRACTBackgroundProtein ingestion increases skeletal muscle protein synthesis rates during recovery from endurance exercise.ObjectivesWe aimed to determine the effect of graded doses of dietary protein co-ingested with carbohydrate on whole-body protein metabolism, and skeletal muscle myofibrillar (MyoPS) and mitochondrial (MitoPS) protein synthesis rates during recovery from endurance exercise.MethodsIn a randomized, double-blind, parallel-group design, 48 healthy, young, endurance-trained men (mean ± SEM age: 27 ± 1 y) received a primed continuous infusion of l-[ring-2H5]-phenylalanine, l-[ring-3,5-2H2]-tyrosine, and l-[1-13C]-leucine and ingested 45 g carbohydrate with either 0 (0 g PRO), 15 (15 g PRO), 30 (30 g PRO), or 45 (45 g PRO) g intrinsically l-[1-13C]-phenylalanine and l-[1-13C]-leucine labeled milk protein after endurance exercise. Blood and muscle biopsy samples were collected over 360 min of postexercise recovery to assess whole-body protein metabolism and both MyoPS and MitoPS rates.ResultsProtein intake resulted in ∼70%–74% of the ingested protein-derived phenylalanine appearing in the circulation. Whole-body net protein balance increased dose-dependently after ingestion of 0, 15, 30, or 45 g protein (mean ± SEM: −0.31± 0.16, 5.08 ± 0.21, 10.04 ± 0.30, and 13.49 ± 0.55 μmol phenylalanine · kg−1 · h−1, respectively; P < 0.001). 30 g PRO stimulated a ∼46% increase in MyoPS rates (%/h) compared with 0 g PRO and was sufficient to maximize MyoPS rates after endurance exercise. MitoPS rates were not increased after protein ingestion; however, incorporation of dietary protein–derived l-[1-13C]-phenylalanine into de novo mitochondrial protein increased dose-dependently after ingestion of 15, 30, and 45 g protein at 360 min postexercise (0.018 ± 0.002, 0.034 ± 0.002, and 0.046 ± 0.003 mole percentage excess, respectively; P < 0.001).ConclusionsProtein ingested after endurance exercise is efficiently digested and absorbed into the circulation. Whole-body net protein balance and dietary protein–derived amino acid incorporation into mitochondrial protein respond to increasing protein intake in a dose-dependent manner. Ingestion of 30 g protein is sufficient to maximize MyoPS rates during recovery from a single bout of endurance exercise.This trial was registered at trialregister.nl as NTR5111.

Preserved Capacity for Adaptations in Strength and Muscle Regulatory Factors in Elderly in Response to Resistance Exercise Training and Deconditioning.

Aging is related to an inevitable loss of muscle mass and strength. The mechanisms behind age-related loss of muscle tissue are not fully understood but may, among other things, be induced by age-related differences in myogenic regulatory factors. Resistance exercise training and deconditioning offers a model to investigate differences in myogenic regulatory factors that may be important for age-related loss of muscle mass and strength. Nine elderly (82 ± 7 years old) and nine young, healthy persons (22 ± 2 years old) participated in the study. Exercise consisted of six weeks of resistance training of the quadriceps muscle followed by eight weeks of deconditioning. Muscle biopsy samples before and after training and during the deconditioning period were analyzed for MyoD, myogenin, insulin-like growth-factor I receptor, activin receptor IIB, smad2, porin, and citrate synthase. Muscle strength improved with resistance training by 78% (95.0 ± 22.0 kg) in the elderly to a similar extent as in the young participants (83.5%; 178.2 ± 44.2 kg) and returned to baseline in both groups after eight weeks of deconditioning. No difference was seen in expression of muscle regulatory factors between elderly and young in response to exercise training and deconditioning. In conclusion, the capacity to gain muscle strength with resistance exercise training in elderly was not impaired, highlighting this as a potent tool to combat age-related loss of muscle function, possibly due to preserved regulation of myogenic factors in elderly compared with young muscle.

A Twist2 Expressing Progenitor Cell Type Population Exists In Human Skeletal Muscle

INTRODUCTION. Satellite cells are muscle stem cells that function to support long-term muscle homeostasis, repair and exercise adaptations. Recent evidence in rodents has revealed the existence of an additional muscle progenitor cell population with the capacity to specifically regulate the repair and maintenance of type-IIb skeletal muscle fibres. These cells are typified by the expression of the transcription factor Twist2 (Tw2) and represent a district, non-satellite cell population found within the myofibre interstitium. However, the presence and function of Tw2-positive cells within human skeletal muscle is currently unknown. Therefore, the PURPOSE of this investigation was to identify and characterize Tw2-positive cells within skeletal muscle under basal conditions. METHODS: Muscle biopsy samples were obtained from the hamstrings muscle of young healthy males and females undergoing anterior cruciate ligament repair (n=8, 3 male, 4 female, mean age ~25years), for immunohistochemical (IHC) analysis of muscle cross-sections and immunocytochemical (ICC)analysis of cytospun mononuclear cells enzymatically digested from muscle biopsy samples. RESULTS: ICC staining revealed numerous TW2-positive cells in the isolated mononuclear cell fraction suggesting they originated from the myofibre interstitium. This was confirmed through IHC staining for Tw2 and laminin in tissue cross-sections which revealed that Tw2 expression was localized to a population of cells outside the myofibre membrane at a density of 0.014/mm2. In agreement with previous reports, Tw2 protein expression was localized within both the cytosol and the nucleus ofTw2-positive cells. Importantly, IHC analysis of the satellite cell marker pax7 and Tw2 demonstrated that cells expressing these markers were mutually exclusive demonstrated that Tw2-positive cells represent a unique cell type, independent of satellite cells. Ongoing analysis is examining the response to Tw2-positive cells to acute and chronic exercise stimuli. CONCLUSION: These findings identify a novel non-satellite cell population typified by Twist-2 expression in human skeletal muscle, the function of which currently remains unknown.

Human Skeletal Muscle Mrna Expression In Response To Treadmill-based Endurance Training And Post-exercise Protein Supplementation

Nutrient availability is known to influence the exercise response. However, there is a paucity of information regarding the role of post-exercise protein ingestion in modulating intra-muscular adaptation to treadmill-based endurance exercise training. PURPOSE: To examine the influence of post-exercise protein ingestion on mRNA gene expression in response to six-weeks treadmill running. METHODS: In a randomized parallel group design, 15 individuals (VO2max 55 ± 6 ml·kg-1·min-1) completed six weeks of treadmill running (4 sessions per week), progressively increasing in both duration (30-60 min) and intensity (70-75 % VO2max). Participants were randomly assigned to a group receiving a supplement containing carbohydrate (CHO; 1.6 g sucrose·kg-1; n= 7) or carbohydrate-protein (CHO-P; 0.8 g sucrose·kg-1 and 0.8 g whey protein hydrolysate·kg-1; n=8) ingested immediately post-exercise and then 1 h later. To determine mRNA expression of several mitochondrial, mitogenic signaling, protein synthesis and lipid/carbohydrate metabolism genes, muscle biopsy samples were collected at baseline and follow-up, with 48 h of lifestyle standardization to exclude any acute effects on transcriptional changes. RESULTS: An up-regulation in mammalian target of rapamycin (mTOR) gene expression was shown in CHO-P (+46%; p= 0.025) relative to CHO (+4%) following the intervention. Mitochondrial transcription factor A (TFAM) up-regulation was shown in CHO-P group (26%) when compared with CHO (13%), albeit this did not reach statistical significance (p=0.07). No changes in the expression of other mitochondrial, lipid/carbohydrate metabolism, mitogenic signaling genes were observed between groups (p>0.05). CONCLUSION: post-exercise protein supplementation up-regulates the expression of mTOR in skeletal muscle over six-weeks of treadmill-based endurance exercise training, indicating that post-exercise protein supplementation may have a potential role in accentuating skeletal muscle adaptations to endurance exercise training. Funder: Health Sciences Research Center, Lifestyle & Health Research Center (LHRC), Princess Nourah bint Abdulrahman University.

Gene Expression Differences In Three-dimensional Myobundles Compared To Two-dimensional Myocultures

PURPOSE: Traditional two-dimensional (2D) in vitro models of human skeletal muscle are limited in their ability to fully mimic in vivo muscle, as in vivo muscle exists in a complex three-dimensional (3D) structure. We have developed a novel engineered three-dimensional (3D) myobundle in vitro model that we believe more closely models skeletal muscle behavior. Here, we determined baseline gene expression differences among three models: the 3D myobundles, 2D cell cultures, and explant biopsies. METHODS: Previously collected skeletal muscle (vastus lateralis) biopsy samples from adult men and women (n = 6) were used. Each sample was used to generate the following groups: explant (RNA from biopsy), 2D (RNA from differentiated myotubes) and 3D (RNA from 3D myobundles seeded from each primary sample). 200ng of isolated RNA for each sample was used to generate global gene expression profiles (HumanHT-12 v4.0 Gene Expression BeadChip Arrays). Data were processed using Illumina Genome Studio and imported into Partek Genomics Suite for statistical analysis. Differential gene expression was assessed via 2-way ANOVA (group*ID) with the following post-hoc comparisons: 2D/biopsy, 3D/biopsy and 3D/2D. Resultant lists were filtered at p<0.01 and fold change >|1.5|. Biological pathway analyses of differentially regulated gene sets were done using Ingenuity Pathway Analysis. RESULTS: ANOVA detected 3754 genes different between 2D/biopsy, 3273 genes different between 3D/biopsy and 488 genes different between 3D/2D cultures. Biological pathway analysis identified representation of the following canonical pathways in our gene set: calcium signaling (26 genes; z-score=1.508, -log p-value=13.9), integrin signaling (28 genes; z-score=-2.711, -log p-value=13.5), and actin cytoskeleton signaling (29 genes; -log p-value=12.3). CONCLUSION: The 3D cell myobundle system produced relatively fewer differences from biopsies as compared to 2D cell cultures, but some significant differences from biopsy samples remain. Comparison of 3D to 2D culture systems shows transcriptional changes that align with increases in calcium signaling, while downregulations in the actin cytoskeleton and integrin signaling demonstrate significant structural differences between the two in vitro models tested.

Leucine Supplementation Does Not Attenuate Metabolic Or Functional Declines Following 7-days Of Unilateral Lower-limb Immobilisation.

Unavoidable periods of physical inactivity (i.e. illness/injury) lead to muscle atrophy and functional declines, which likely stem from alterations in both anabolic signaling processes and oxidative metabolism. Preventing such declines is important to reduce the risk of re-injury and preserve musculoskeletal health across the lifespan. PURPOSE: To determine the effectiveness of high-dose leucine supplementation to preserve muscle mass, strength, and morphology following 7-days of unilateral knee immobilisation. METHODS: Sixteen healthy, recreationally active males (23±1yrs) underwent 7-days of unilateral knee immobilisation, with (LEU; n=8) or without (PLA; n=8) thrice daily leucine supplementation (15g/d). Strength and compartmental tissue composition were assessed prior to and following immobilisation. Muscle biopsy samples obtained immediately following immobilisation were used to determine muscle fibre morphology as well as key indicators of mitochondrial function between the control (CTL) and immobilised (IMB) limbs. RESULTS: Leg fat-free mass was reduced in the IMB leg following immobilisation (-3.6±0.5%; P<0.05) in both the LEU and PLA conditions (P>0.05), with no such alterations noted in the CTL leg (-0.6±0.5%; P>0.05). Isometric knee extensor strength declined following immobilisation (P<0.01), with a greater (P<0.05) and equivocal decline in the IMB (-27.9±4.4%) vs. CTL (-14.3±4.4%) leg in both the LEU and PLA group (P>0.05). Following immobilisation, type II fibre cross sectional area was significantly lower in the IMB vs. CTL limb (5561.49±465.97 vs. 6319.9±412.9μm2; P<0.05) but not in type I fibre cross sectional area (4805.5±324.3 vs. 5424.95±323.63μm2; P>0.05), with no differences between treatment groups (P>0.05). A significant Group*Leg interaction was identified for mitochondrial complex I phosphorylating respiration (Pi; P<0.05). However, post-hoc analysis revealed no differences in Pi in the IMB vs. CTL limb in either the PLA (FC=1.20±0.19, P=0.06) or LEU (FC=0.87±0.27, P=0.87) group. CONCLUSION: Leucine supplementation, even at a high-dose (15g/d), does not appear to attenuate declines in leg fat-free mass, strength, muscle morphology or mitochondrial respiration following 7-days of unilateral knee immobilisation.

Effects of zinc propionate supplementation on growth performance, skeletal muscle fiber, and receptor characteristics in beef steers.

A randomized complete block design experiment with 32 yearling crossbred steers (average body weight [BW] = 442 ± 17.0 kg) fed a steam-flaked corn-based diet was used to evaluate the effects of dietary Zn (KemTRACE Zn propionate 27; Kemin Industries, Inc., Des Moines, IA) supplementation on live growth performance, skeletal muscle fiber, and beta-adrenergic receptor (β-AR) characteristics during the finishing phase. Steers were blocked by BW (n = 4 blocks; 8 steers/block), assigned to pens (n = 4 steers/pen), and randomly assigned to the following treatments: control (CON; 0.0 g/[head (hd) · d] of additional Zn) or additional dietary Zn (ZnP; 1.0 g/[hd · d] additional Zn). The basal diet contained Zn (60 ppm dry matter basis) from ZnSO4; additional Zn was top-dressed at feeding. Ractopamine hydrochloride (RH; Optaflexx: Elanco Animal Health, Greenfield, IN) was included at 300 mg/(hd · d) for the final 28 d of the 111-d feeding period. Longissimus muscle biopsy samples, BW, and blood were obtained on days 0, 42, 79, and 107. Final BW was collected prior to shipping on day 111. Biopsy samples were used for immunohistochemical (IHC), mRNA, and protein analysis. Serum urea nitrogen (SUN) and nonesterified fatty acid (NEFA) concentrations were measured. Steers fed ZnP had a greater average daily gain (P = 0.02) and gain to feed ratio (G:F; P = 0.03) during the RH feeding period compared with CON. There were no differences (P > 0.05) in other growth performance variables, carcass traits, mRNA abundance, or relative protein concentration for fiber type and β-AR. Fiber types I and IIA had no differences in the cross-sectional area; however, the IIX area was greater for CON (P < 0.04) compared with ZnP and increased (P < 0.02) over time. There were no differences between treatments for the β1-AR density (P > 0.05) in skeletal muscle tissue throughout the study. A treatment × day interaction was observed in β2-AR density (P = 0.02) and β3-AR density (P = 0.02) during the RH feeding period, where the abundance of the receptors increased with ZnP but did not change in CON. Compared with CON, ZnP had greater (P < 0.01) mean NEFA concentrations. Mean SUN concentrations did increase by day (P < 0.01). Additional dietary Zn, supplied as Zn propionate, upregulates β2-AR and β3-AR and improves growth performance in feedlot steers during the RH feeding period, likely through a shift of resource utilization from lipogenesis to muscle maintenance and hypertrophy.

Role of the Extracellular Matrix in Loss of Muscle Force With Age and Unloading Using Magnetic Resonance Imaging, Biochemical Analysis, and Computational Models.

The focus of this review is the application of advanced MRI to study the effect of aging and disuse related remodeling of the extracellular matrix (ECM) on force transmission in the human musculoskeletal system. Structural MRI includes (i) ultra-low echo times (UTE) maps to visualize and quantify the connective tissue, (ii) diffusion tensor imaging (DTI) modeling to estimate changes in muscle and ECM microstructure, and (iii) magnetization transfer contrast imaging to quantify the macromolecular fraction in muscle. Functional MRI includes dynamic acquisitions during contraction cycles enabling computation of the strain tensor to monitor muscle deformation. Further, shear strain extracted from the strain tensor may be a potential surrogate marker of lateral transmission of force. Biochemical and histological analysis of muscle biopsy samples can provide “gold-standard” validation of some of the MR findings. The review summarizes biochemical studies of ECM adaptations with age and with disuse. A brief summary of animal models is included as they provide experimental confirmation of longitudinal and lateral force transmission pathways. Computational muscle models enable exploration of force generation and force pathways and elucidate the link between structural adaptations and functional consequences. MR image findings integrated in a computational model can explain and predict subject specific functional changes to structural adaptations. Future work includes development and validation of MRI biomarkers using biochemical analysis of muscle tissue as a reference standard and potential translation of the imaging markers to the clinic to noninvasively monitor musculoskeletal disease conditions and changes consequent to rehabilitative interventions.

Concomitant aerobic- and hypertrophy-related skeletal muscle cell signaling following blood flow-restricted walking

This study is aimed to examine the acute responses to a blood flow restriction (BFR) low-intensity aerobic exercise, compared with a similar exercise without BFR, on several aerobic- and hypertrophy-related proteins expression and hormonal responses. On two different occasions, five healthy untrained male subjects were required to perform (i) a BFR low-intensity aerobic exercise consisting of 5 sets of 2-min bouts of walking at an exercise intensity of 63–65% HRmax (equivalent to 40% of VO2max), interspersed by 1-min rest; and (ii) similar exercise bouts without BFR (Ctrl). For each condition, venous blood samples were collected at pre- (baseline), immediately and 2-h post-exercise. Baseline and 3-h post-exercise muscle biopsy samples (vastus lateralis) were also performed for protein expression analysis. HIF-1α, PGC-1α, and VEGF proteins content were significantly higher (P < 0.05) at 3-h post-exercise in BFR compared with baseline values. In reference to baseline, phosphorylation of Akt also increased significantly (P < 0.05) following BFR, and in a greater extent (P < 0.05) than following Ctrl. IGF-1 concentration significantly increased (P = 0.001) immediately following BFR exercise than baseline values and serum GH showed a significant increase (P = 0.046) compared with Ctrl. The addition of blood flow restriction during walking exercise initiate a concomitant cell signaling pathways regulating mitochondrial biogenesis, angiogenesis, and skeletal muscle protein expression. Le but de cette étude est d’examiner les réponses aiguës à un exercice aérobie de faible intensité réalisé avec ou sans occlusion vasculaire (blood flow restriction, [BFR]) sur l’expression de protéines impliquées dans le développement de l’hypertrophie et de l’endurance. Selon un plan croisé, 5 sujets sains non entraînés ont réalisé à deux occasions différentes: (i) 5 séries de 2 min de marche à 63–65 % de la fréquence cardiaque maximale (équivalent à 40 % de la consommation maximale d’oxygène) entrecoupées de 1 min de repos, le tout sous occlusion vasculaire des membres inférieurs (BFR); et (ii) le même exercice sans occlusion (Ctrl). Pour chaque condition, des échantillons sanguins ont été prélevés avant, immédiatement après et 2 h après chaque condition. Des biopsies musculaires (vastus lateralis) ont également été pratiquée au repos (avant exercice) et 3 h après. Le contenu protéique du facteur induit par l’hypoxie 1 alpha (Hypoxic Inducible Factor 1α, HIF-1α), de peroxisome proliferator-activated receptor gamma-coactivator-1alpha (PGC-1α), et du facteur de croissance de l’endothélium vasculaire (Vascular Endothelial Growth Factor, VEGF) ont significativement augmenté (p < 0,05) après l’exercice aérobie BFR. La phosphorylation de la protéine kinase B (Akt) a également augmenté de manière significative (p < 0,05) après l’exercice aérobie BFR et dans une plus grande proportion (p < 0,05) que suite à l’exercice Ctrl. La concentration du facteur de croissance Insulin-like Growth Factor-1 (IGF-1) augmente (p < 0,001) immédiatement après l’exercice aérobie BFR et l’hormone de croissance (sérum GH) augmente significativement (p < 0,05) en comparaison avec Ctrl. L’ajout d’une restriction de la circulation sanguine au cours d’un exercice de marche stimule des voies de signalisation musculaire concomitantes régulant la biogenèse mitochondriale, l’angiogenèse et l’expression des protéines du muscle squelettique.

1883-P: Metformin Increases Protein Phosphatase 2A Activity in Primary Skeletal Muscle Cells Derived from Lean Healthy Insulin-Sensitive Human Participants

Skeletal muscle insulin resistance is one of the primary contributors of type 2 diabetes (T2D). Metformin is the first-line drug for the treatment of T2D. Main known effects of metformin include inhibition of glucose production in liver and attenuation of insulin resistance in skeletal muscle. However, molecular mechanism of metformin’s action in skeletal muscle is not well understood. Protein phosphatase 2A (PP2A), a major serine/threonine phosphatase, plays a pivotal role in cellular processes (e.g., signal transduction, apoptosis), and acts through dephosphorylating key signaling molecules such as AKT, AMPK, etc. However, whether or not PP2A plays a role in metformin-induced insulin sensitivity improvement in human skeletal muscle cells remains to be elucidated. In the present study, hyperinsulinemic-euglycemic clamp was performed to assess insulin sensitivity in human subjects and skeletal muscle biopsy samples were obtained. Primary human skeletal muscle cells (shown to retain metabolic characteristics of donors) were cultured from these muscle biopsies from 8 lean insulin sensitive participants (4 Female and 4 male; age: 21.4±0.8 yrs; BMI: 22.1±0.7 kg/m2; 2-hour OGTT: 96.5± 6.3 mg/dl; HbA1c: 5.3±0.1 %; fasting plasma glucose: 87.5±1.4 mg/dl; M-value; 10.9±0.8 mg/kgBW/min). The cells were expanded, differentiated into myotubes, and treated with 50µM metformin (the physiologic concentration) for 24 hours before harvesting. PP2A activity was measured by a phosphatase activity assay (Millipore). The results indicated that metformin increased the activity of PP2A in the myotubes derived from all 8 participants, and the average fold increase is 1.59±0.13 (p&amp;lt;0.05). These results provided the first evidence to suggest that metformin promotes activation of PP2A in human skeletal muscle cells derived from lean insulin sensitive participants, and may help to understand metformin’s action in skeletal muscle in humans. Disclosure A.H. Mestareehi: None. B. Seyoum: None. Z. Msallaty: None. A.P. Mallisho: None. X. Zhang: None. A. Kowluru: None. Z. Yi: None. Funding American Diabetes Association (1-13-TS-27 to Z.Y.); National Institutes of Health (R01DK107666, R01DK081750 to Z.Y.), (T32HL120822 to A.H.M.); DOTS Scholars (to A.H.M., Z.Y.)

243-LB: Enzymes for Dicarbonyl Detoxification Are Attenuated with Obesity by Mechanisms of Acetylation

Glyoxalase I (GLOI) is the primary enzyme for detoxifying the reactive dicarbonyl methylglyoxal. Pre-clinical models demonstrate that loss of GLOI promotes the development of diabetes in obesogenic conditions. To determine if GLOI is attenuated with human obesity, we compared GLOI expression in skeletal muscle biopsy samples from 5 obese (OB) and 15 age-matched lean, healthy individuals (LH). GLOI protein was ~50% lower (p&amp;lt;0.05), whereas GLOI mRNA was ~2-fold higher in OB muscle (p&amp;lt;0.001). The NRF2/Keap1 axis, which regulates GLOI transcription, was unperturbed by obesity. Given the NAD+-dependent deacetylase SIRT1 is capable of deacetylating GLOI which may prevent GLOI’s degradation, we measured the expression of SIRT1 as well as NAMPT and HIF1α which regulate NAD+ bioavailability. HIF1α mRNA was increased 3-fold (p=0.002) and NAMPT protein was ~50% lower (p=0.2) in OB muscle suggesting a pseudo-hypoxic environment which may attenuate NAD+ bioavailability. SIRT1 protein was ~50% lower in OB muscle (p=0.09). SIRT1 protein (r = 0.673, p = 0.003) and HIF1α transcripts (r = -0.487, p = 0.041) correlated with GLO1 protein expression. NAMPT inhibition in human primary myotubes increased GLOI acetylation (~50%) and GLO1 protein (~40%) both of which were rescued by treatment with the NAD+ precursor nicotinamide riboside. Silencing of NAMPT or SIRT1 also reduced GLOI protein by 50%. These data suggest that OB muscle may be in a pseudo-hypoxic state that limits NAD+ bioavailability and SIRT1 activity promoting GLO1 acetylation and degradation. Disclosure E.R. Miranda: None. J. Shadiow: None. D.B. Lombard: None. A.T. Ludlow: None. J.M. Haus: None. Funding National Institutes of Health (R01DK109948)

Myopathic changes associated with psychomotor delay and seizures caused by a novel homozygous mutation in TBCK

Biallelic mutations in TBC1-domain containing kinase (TBCK) lead to hypotonia, global developmental delay with severe cognitive and motor deficits, and variable presentation of dysmorphic facial features and brain malformations. It remains unclear whether hypotonia in these individuals is purely neurogenic, or also caused by progressive muscle disease. Whole exome sequencing was performed on a family diagnosed with nonspecific myopathic changes by means of histological analysis and immunohistochemistry of muscle biopsy samples. A novel homozygous truncation in TBCK was found in two sisters diagnosed with muscle disease and severe psychomotor delay. TBCK was completely absent in these patients. Our findings identify a novel early truncating variant in TBCK associated with a severe presentation and add muscle disease to the variability of phenotypes associated with TBCK mutations. Inconsistent genotype/phenotype correlation could be ascribed to the multiple roles of TBCK in intracellular signaling and endolysosomal function in different tissues.

Immobilization leads to alterations in intracellular phosphagen and creatine transporter content in human skeletal muscle.

The role of dysregulated intracellular creatine (Cr) metabolism in disuse atrophy is unknown. In this study, skeletal muscle biopsy samples were obtained after 7 days of unilateral leg immobilization (IMMOB) and from the nonimmobilized control limb (CTRL) of 15 healthy men (23.1 ± 3.5 yr). Samples were analyzed for fiber type cross-sectional area (CSA) and creatine transporter (CreaT) at the cell membrane periphery (MEM) or intracellular (INT) areas, via immunofluorescence microscopy. Creatine kinase (CK) and AMP-activated protein kinase (AMPK) were determined via immunoblot. Phosphocreatine (PCr), Cr, and ATP were measured via enzymatic analysis. Body composition and maximal isometric knee extensor strength were assessed before and after disuse. Leg strength and fat-free mass were reduced in IMMOB (~32% and 4%, respectively; P < 0.01 for both). Type II fiber CSA was smaller (~12%; P = 0.028) and intramuscular PCr lower (~13%; P = 0.015) in IMMOB vs. CTRL. CreaT protein was greater in type I fibers in both limbs (P < 0.01). CreaT was greater in IMMOB vs. CTRL (P < 0.01) and inversely associated with PCr concentration in both limbs (P < 0.05). MEM CreaT was greater than INT CreaT in type I and II fibers of both limbs (~14% for both; P < 0.01 for both). Type I fiber CreaT tended to be greater in IMMOB vs. CTRL (P = 0.074). CK was greater and phospho-to-total AMPKThr172 tended to be greater, in IMMOB vs. CTRL (P = 0.013 and 0.051, respectively). These findings suggest that modulation of intracellular Cr metabolism is an adaptive response to immobilization in young healthy skeletal muscle.

Altered gene expressions in periferal blood cells of first-degree non-diabetic relatives of type 2 diabetic patients.

Introduction and aim: We were looking for altered gene expression on peripheral blood cells significant to type 2 diabetes causing the world epidemic. Method: Muscle biopsy samples of healthy volunteers with (n = 6) or without (n = 6) first degree type 2 diabetic relatives were analyzed by mRNS microarray. After confirmation of microarrays results by quantitative real-time PCR, the expression of eight differently expressed genes were further investigated on peripheral blood cells of 58 healthy volunteers without diabetic relatives and 58 healthy ones with first-degree type 2 diabetic relatives. Results: The expressions of SERPINF1 gene were significantly lover in blood cells both from females (relative quantification: FC - female: = 0.69, p<6*10-3) and males (FC - male: = 0.65, p<2*10-3) with diabetic relatives. This change may not be the consequence of worsening metabolic state as it was identical in cells of type 2 diabetic patients and in healthy volunteers with diabetic relatives. We suggest that the altered SERPINF1 gene expression in peripheral mononuclear blood cells could be a genetic definiteness. Conclusion: With the help of SERPINF1 gene expression in white blood cells and lipid and biochemical blood parameters we suggest a mathematical formula for the augury of type 2 diabetes that should be checked on a larger population, but we hope it could be used as a diabetic marker. The expression of LAMP2 gene did not differ between the two healthy groups, but it showed a maternal parent of origin effect. In the case of maternal inheritance, we found higher LAMP2 expression suggesting that gene from the mother has a determining effect. Orv Hetil. 2020; 161(18): 738-746.