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Low-load blood flow-restricted resistance exercise (BFRRE) constitutes an effective means to produce skeletal muscle hypertrophy. Nonetheless, its applicability to counteract the age-related skeletal muscle decay at a cellular level, is not clear. Therefore, we investigated the effect of BFRRE on muscle fiber morphology, integrated muscle protein synthesis, muscle stem cells (MuSCs), myonuclear content, and muscle functional capacity in healthy older individuals. Twenty-three participants with a mean age of 66 yr (56-75 yr) were randomized to 6 wk of supervised BFRRE (3 sessions per week) or non-exercise control (CON). Biopsies were collected from the vastus lateralis before and after the intervention. Immunofluorescent microscopy was utilized to assess muscle fiber type-specific cross-sectional area (CSA) as well as MuSC and myonuclear content. Deuterium oxide was orally administered throughout the intervention period, enabling assessment of integrated myofibrillar and connective tissue protein fractional synthesis rate (FSR). BFRRE produced uniform ∼20% increases in the fiber CSA of both type I and type II fibers (P < 0.05). This occurred concomitantly with improvements in both maximal muscle strength and strength-endurance capacity but in the absence of increased MuSC content and myonuclear addition. The observed muscle fiber hypertrophy was not mirrored by increases in either myofibrillar or connective tissue FSR. In conclusion, BFRRE proved effective in stimulating skeletal muscle growth and increased muscle function in older individuals, which advocates for the use of BFRRE as a countermeasure of age-related deterioration of skeletal muscle mass and function.NEW & NOTEWORTHY We provide novel insight, that as little as 6 wk of low-load blood flow-restricted resistance exercise (BFRRE) produces pronounced fiber type-independent hypertrophy, alongside improvements across a broad range of muscle functional capacity in older individuals. Notably, since th

Funding was provided by the A.P. Møller Foundation; Riisfort Foundation; Toyota Foundation; Independent Research Fund Denmark (DFF–5053-00195 to J.F.); Steno Diabetes Center Aarhus, which is partially funded by an unrestricted donation from the Novo Nordisk Foundation (NNF17OC0027242 to N.J. and NNF16OC0021496 to T.H.P.); Lundbeck Foundation (R190-2014-3904 to T.H.P.); Roche per la Ricerca 2019 to L.M.; and NIH/NIAMS (R01AR076247-01 to P.L.P.)., Type 2 diabetes mellitus (T2DM) is associated with impaired skeletal muscle function and degeneration of the skeletal muscles. However, the mechanisms underlying the degeneration are not well described in human skeletal muscle. Here we show that skeletal muscle of T2DM patients exhibit degenerative remodeling of the extracellular matrix that is associated with a selective increase of a subpopulation of fibro-adipogenic progenitors (FAPs) marked by expression of THY1 (CD90)—the FAPCD90+. We identify platelet-derived growth factor (PDGF) as a key FAP regulator, as it promotes proliferation and collagen production at the expense of adipogenesis. FAPsCD90+ display a PDGF-mimetic phenotype, with high proliferative activity, clonogenicity, and production of extracellular matrix. FAPCD90+ proliferation was reduced by in vitro treatment with metformin. Furthermore, metformin treatment reduced FAP content in T2DM patients. These data identify a PDGF-driven conversion of a subpopulation of FAPs as a key event in the fibrosis development in T2DM muscle., Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Type 2 diabetes mellitus (T2DM) is associated with impaired skeletal muscle function and degeneration of the skeletal muscles. However, the mechanisms underlying the degeneration are not well described in human skeletal muscle. Here we show that skeletal muscle of T2DM patients exhibit degenerative remodeling of the extracellular matrix that is associated with a selective increase of a subpopulation of fibro-adipogenic progenitors (FAPs) marked by expression of THY1 (CD90)—the FAPCD90+. We identify platelet-derived growth factor (PDGF) as a key FAP regulator, as it promotes proliferation and collagen production at the expense of adipogenesis. FAPsCD90+ display a PDGF-mimetic phenotype, with high proliferative activity, clonogenicity, and production of extracellular matrix. FAPCD90+ proliferation was reduced by in vitro treatment with metformin. Furthermore, metformin treatment reduced FAP content in T2DM patients. These data identify a PDGF-driven conversion of a subpopulation of FAPs as a key event in the fibrosis development in T2DM muscle.

Animal models clearly illustrate that the maintenance of skeletal muscle mass depends on the function and interaction of a heterogeneous population of resident and infiltrating mononuclear cells. Several lines of evidence suggest that mononuclear cells also play a role in muscle wasting in humans, and targeting these cells may open new treatment options for intervention or prevention in sarcopenia. Methodological and ethical constraints have perturbed exploration of the cellular characteristics and function of mononuclear cells in human skeletal muscle. Thus, investigations of cellular phenotypes often depend on immunohistochemical analysis of small tissue samples obtained by needle biopsies, which do not match the deep phenotyping of mononuclear cells obtained from animal models. Here, we have developed a protocol for fluorescence-activated cell sorting (FACS), based on single-cell RNA-sequencing data, for quantifying and characterizing mononuclear cell populations in human skeletal muscle. Muscle stem cells, fibro-adipogenic progenitors, and two subsets of macrophages (CD11cþ /–) are present in needle biopsies in comparable quantities per milligram tissue to open surgical biopsies. We find that direct cell isolation is preferable due to a substantial shift in transcriptome when using preculture before the FACS procedure. Finally, in vitro validation of the cellular phenotype of muscle stem cells, fibro-adipogenic progenitors, and macrophages confirms population-specific traits. This study demonstrates that mononuclear cell populations can be quantified and subsequently analyzed from needle biopsy material and opens the perspective for future clinical studies of cellular mechanisms in muscle wasting.

Mitochondrial dysfunction has been implicated as a central mechanism in the metabolic myopathy accompanying critical limb ischemia (CLI). However, whether mitochondrial dysfunction is directly related to lower extremity ischemia and the structural and molecular mechanisms underpinning mitochondrial dysfunction in CLI patients is not understood. Here, we aimed to study whether mitochondrial dysfunction is a distinctive characteristic of CLI myopathy by assessing mitochondrial respiration in gastrocnemius muscle from 14 CLI patients (65.3 ± 7.8 y) and 15 matched control patients (CON) with a similar comorbidity risk profile and medication regimen but without peripheral ischemia (67.4 ± 7.4 y). Furthermore, we studied potential structural and molecular mechanisms of mitochondrial dysfunction by measuring total, sub-population, and fiber-type-specific mitochondrial volumetric content and cristae density with transmission electron microscopy and by assessing mitophagy and fission/fusion-related protein expression. Finally, we asked whether commonly used biomarkers of mitochondrial content are valid in patients with cardiovascular disease. CLI patients exhibited inferior mitochondrial respiration compared to CON. This respiratory deficit was not related to lower whole-muscle mitochondrial content or cristae density. However, stratification for fiber types revealed ultrastructural mitochondrial alterations in CLI patients compared to CON. CLI patients exhibited an altered expression of mitophagy-related proteins but not fission/fusion-related proteins compared to CON. Citrate synthase, cytochrome c oxidase subunit IV (COXIV), and 3-hydroxyacyl-CoA dehydrogenase (β-HAD) could not predict mitochondrial content. Mitochondrial dysfunction is a distinctive characteristic of CLI myopathy and is not related to altered organelle content or cristae density. Our results link this intrinsic mitochondrial deficit to dysregulation of the mitochondrial quality control system, which h

Activation of skeletal muscle contractions require that action potentials can be excited and propagated along the muscle fibers. Recent studies have revealed that muscle fiber excitability is regulated during repeated firing of action potentials by cellular signaling systems that control the function of ion channel that determine the resting membrane conductance (G ). In fast-twitch muscle, prolonged firing of action potentials triggers a marked increase in G , reducing muscle fiber excitability and causing action potential failure. Both ClC-1 and K ion channels contribute to this G rise, but the exact molecular regulation underlying their activation remains unclear. Studies in expression systems have revealed that ClC-1 is able to bind adenosine nucleotides, and that low adenosine nucleotide levels result in ClC-1 activation. In three series of experiments, this study aimed to explore whether ClC-1 is also regulated by adenosine nucleotides in native skeletal muscle fibers, and whether the adenosine nucleotide sensitivity of ClC-1 could explain the rise in G muscle fibers during prolonged action potential firing. First, whole cell patch clamping of mouse muscle fibers demonstrated that ClC-1 activation shifted in the hyperpolarized direction when clamping pipette solution contained 0 mM ATP compared with 5 mM ATP. Second, three-electrode G measurement during muscle fiber stimulation showed that glycolysis inhibition, with 2-deoxy-glucose or iodoacetate, resulted in an accelerated and rapid >400% G rise during short periods of repeated action potential firing in both fast-twitch and slow-twitch rat, and in human muscle fibers. Moreover, ClC-1 inhibition with 9-anthracenecarboxylic acid resulted in either an absence or blunted G rise during action potential firing in human muscle fibers. Third, G measurement during repeated action potential firing in muscle fibers from a murine McArdle disease model suggest that the rise in G was accelerated in a subset of fibers. To

Activation of skeletal muscle contractions require that action potentials can be excited and propagated along the muscle fibers. Recent studies have revealed that muscle fiber excitability is regulated during repeated firing of action potentials by cellular signaling systems that control the function of ion channel that determine the resting membrane conductance (Gm). In fast-twitch muscle, prolonged firing of action potentials triggers a marked increase in Gm, reducing muscle fiber excitability and causing action potential failure. Both ClC-1 and KATP ion channels contribute to this Gm rise, but the exact molecular regulation underlying their activation remains unclear. Studies in expression systems have revealed that ClC-1 is able to bind adenosine nucleotides, and that low adenosine nucleotide levels result in ClC-1 activation. In three series of experiments, this study aimed to explore whether ClC-1 is also regulated by adenosine nucleotides in native skeletal muscle fibers, and whether the adenosine nucleotide sensitivity of ClC-1 could explain the rise in Gm muscle fibers during prolonged action potential firing. First, whole cell patch clamping of mouse muscle fibers demonstrated that ClC-1 activation shifted in the hyperpolarized direction when clamping pipette solution contained 0 mM ATP compared with 5 mM ATP. Second, three-electrode Gm measurement during muscle fiber stimulation showed that glycolysis inhibition, with 2-deoxy-glucose or iodoacetate, resulted in an accelerated and rapid >400% Gm rise during short periods of repeated action potential firing in both fast-twitch and slow-twitch rat, and in human muscle fibers. Moreover, ClC-1 inhibition with 9-anthracenecarboxylic acid resulted in either an absence or blunted Gm rise during action potential firing in human muscle fibers. Third, Gm measurement during repeated action potential firing in musc

Background: Protein supplementation has been suggested to augment endurance training adaptations by increasing mixed muscle and myofibrillar protein synthesis and lean body mass. However, a potential beneficial effect on mitochondrial adaptations is yet to be clarified. The aim of the present study was to investigate the effect of consuming whey protein hydrolysate before and whey protein hydrolysate plus carbohydrate (PRO-CHO) after each exercise session during a six-week training period compared to similarly timed intake of isocaloric CHO supplements on biomarkers of mitochondrial biogenesis, VO2max and performance in trained runners.Methods: Twenty-four trained runners (VO2max 60.7 ± 3.7 ml O2 kg- 1 min1) completed a six-week block randomized controlled intervention period, consisting of progressive running training. Subjects were randomly assigned to either PRO-CHO or CHO and matched in pairs for gender, age, VO2max, training and performance status. The PRO-CHO group ingested a protein beverage (0.3 g kg- 1) before and protein-carbohydrate beverage (0.3 g protein kg- 1 and 1 g carbohydrate kg- 1) after each exercise session. The CHO group ingested an energy matched carbohydrate beverage. Resting muscle biopsies obtained pre and post intervention were analyzed for mitochondrial specific enzyme activity and mitochondrial protein content. Subjects completed a 6 K time trial (6 K TT) and a VO2max test pre, midway (only 6 K TT) and post intervention.Results: Following six weeks of endurance training Cytochrome C (Cyt C) protein content was significantly higher in the PRO-CHO group compared to the CHO group (p < 0.05), with several other mitochondrial proteins (Succinate dehydrogenase (SDHA), Cytochrome C oxidase (COX-IV), Voltage-dependent anion channel (VDAC), Heat shock protein 60 (HSP60), and Prohibitin (PHB1)) foll

Purpose: The present study investigated muscle metabolism and fatigue during simulated elite male ice hockey match-play.Methods: Thirty U20 male national team players completed an experimental game comprising three periods of 8x1-min shifts separated by 2-min recovery intervals. Two vastus lateralis biopsies were obtained either during the game (n = 7) or pre- and post-game (n = 6). Venous blood samples were drawn pre-game and at the end of the first and last period (n = 14). Activity pattern and physiological responses were continuously monitored using local positioning system and heart rate recordings. Further, repeated-sprint ability was tested pre-game and after each period.Results: Total distance covered was 5980±199 m with almost half the distance covered at high skating speeds (>17 km·h-1). Average and peak on-ice heart rate was 84±2 and 97±2% of maximum heart rate, respectively. Muscle lactate increased (P≤0.05) more than 5- and 3-fold, while muscle pH decreased (P≤0.05) from 7.31±0.04 pre-game to 6.99±0.07 and 7.13±0.11 during the first and last period, respectively. Muscle glycogen decreased by 53% post-game (P≤0.05) with ~65% of fast- and slow-twitch fibers depleted of glycogen. Blood lactate increased 6-fold (P≤0.05), while plasma free fatty acid levels increased 1.5- and 3-fold (P≤0.05) after the first and last period. Repeated-sprint ability was impaired (~3%; P≤0.05) post-game concomitant with a ~10% decrease in the number of accelerations and decelerations during the second and last period (P≤0.05).Conclusion: Our findings demonstrate that a simulated ice hockey match-play scenario encompasses a high on-ice heart rate response and glycolytic loading resulting in a marked degradation of muscle glycogen, particularly in specific sub-groups of fibers. This may be of importance both for fatigue in the final stages of a g

Activation of skeletal muscle contractions require that action potentials can be excited and propagated along the muscle fibers. Recent studies have revealed that muscle fiber excitability is regulated during repeated firing of action potentials by cellular signaling systems that control the function of ion channel that determine the resting membrane conductance (Gm). In fast-twitch muscle, prolonged firing of action potentials triggers a marked increase in Gm, reducing muscle fiber excitability and causing action potential failure. Both ClC-1 and KATP ion channels contribute to this Gm rise, but the exact molecular regulation underlying their activation remains unclear. Studies in expression systems have revealed that ClC-1 is able to bind adenosine nucleotides, and that low adenosine nucleotide levels result in ClC-1 activation. In three series of experiments, this study aimed to explore whether ClC-1 is also regulated by adenosine nucleotides in native skeletal muscle fibers, and whether the adenosine nucleotide sensitivity of ClC-1 could explain the rise in Gm muscle fibers during prolonged action potential firing. First, whole cell patch clamping of mouse muscle fibers demonstrated that ClC-1 activation shifted in the hyperpolarized direction when clamping pipette solution contained 0 mM ATP compared with 5 mM ATP. Second, three-electrode Gm measurement during muscle fiber stimulation showed that glycolysis inhibition, with 2-deoxy-glucose or iodoacetate, resulted in an accelerated and rapid >400% Gm rise during short periods of repeated action potential firing in both fast-twitch and slow-twitch rat, and in human muscle fibers. Moreover, ClC-1 inhibition with 9-anthracenecarboxylic acid resulted in either an absence or blunted Gm rise during action potential firing in human muscle fibers. Third, Gm measurement during repeated action potential firing in musc

Background: Protein supplementation has been suggested to augment endurance training adaptations by increasing mixed muscle and myofibrillar protein synthesis and lean body mass. However, a potential beneficial effect on mitochondrial adaptations is yet to be clarified. The aim of the present study was to investigate the effect of consuming whey protein hydrolysate before and whey protein hydrolysate plus carbohydrate (PRO-CHO) after each exercise session during a six-week training period compared to similarly timed intake of isocaloric CHO supplements on biomarkers of mitochondrial biogenesis, VO2max and performance in trained runners.Methods: Twenty-four trained runners (VO2max 60.7 ± 3.7 ml O2 kg- 1 min1) completed a six-week block randomized controlled intervention period, consisting of progressive running training. Subjects were randomly assigned to either PRO-CHO or CHO and matched in pairs for gender, age, VO2max, training and performance status. The PRO-CHO group ingested a protein beverage (0.3 g kg- 1) before and protein-carbohydrate beverage (0.3 g protein kg- 1 and 1 g carbohydrate kg- 1) after each exercise session. The CHO group ingested an energy matched carbohydrate beverage. Resting muscle biopsies obtained pre and post intervention were analyzed for mitochondrial specific enzyme activity and mitochondrial protein content. Subjects completed a 6 K time trial (6 K TT) and a VO2max test pre, midway (only 6 K TT) and post intervention.Results: Following six weeks of endurance training Cytochrome C (Cyt C) protein content was significantly higher in the PRO-CHO group compared to the CHO group (p < 0.05), with several other mitochondrial proteins (Succinate dehydrogenase (SDHA), Cytochrome C oxidase (COX-IV), Voltage-dependent anion channel (VDAC), Heat shock protein 60 (HSP60), and Prohibitin (PHB1)) foll

Electrical membrane properties of skeletal muscle fibers have been thoroughly studied over the last five to six decades. This has shown that muscle fibers from a wide range of species, including fish, amphibians, reptiles, birds, and mammals, are all characterized by high resting membrane permeability for Cl(-) ions. Thus, in resting human muscle, ClC-1 Cl(-) ion channels account for ∼80% of the membrane conductance, and because active Cl(-) transport is limited in muscle fibers, the equilibrium potential for Cl(-) lies close to the resting membrane potential. These conditions-high membrane conductance and passive distribution-enable ClC-1 to conduct membrane current that inhibits muscle excitability. This depressing effect of ClC-1 current on muscle excitability has mostly been associated with skeletal muscle hyperexcitability in myotonia congenita, which arises from loss-of-function mutations in the CLCN1 gene. However, given that ClC-1 must be drastically inhibited (∼80%) before myotonia develops, more recent studies have explored whether acute and more subtle ClC-1 regulation contributes to controlling the excitability of working muscle. Methods were developed to measure ClC-1 function with subsecond temporal resolution in action potential firing muscle fibers. These and other techniques have revealed that ClC-1 function is controlled by multiple cellular signals during muscle activity. Thus, onset of muscle activity triggers ClC-1 inhibition via protein kinase C, intracellular acidosis, and lactate ions. This inhibition is important for preserving excitability of working muscle in the face of activity-induced elevation of extracellular K(+) and accumulating inactivation of voltage-gated sodium channels. Furthermore, during prolonged activity, a marked ClC-1 activation can develop that compromises muscle excitability. Data from ClC-1 expression systems suggest that this ClC-1 activation may arise from loss of regulation by adenosine nucleotides and/or oxidation.

Electrical membrane properties of skeletal muscle fibers have been thoroughly studied over the last five to six decades. This has shown that muscle fibers from a wide range of species, including fish, amphibians, reptiles, birds, and mammals, are all characterized by high resting membrane permeability for Cl(-) ions. Thus, in resting human muscle, ClC-1 Cl(-) ion channels account for ∼80% of the membrane conductance, and because active Cl(-) transport is limited in muscle fibers, the equilibrium potential for Cl(-) lies close to the resting membrane potential. These conditions-high membrane conductance and passive distribution-enable ClC-1 to conduct membrane current that inhibits muscle excitability. This depressing effect of ClC-1 current on muscle excitability has mostly been associated with skeletal muscle hyperexcitability in myotonia congenita, which arises from loss-of-function mutations in the CLCN1 gene. However, given that ClC-1 must be drastically inhibited (∼80%) before myotonia develops, more recent studies have explored whether acute and more subtle ClC-1 regulation contributes to controlling the excitability of working muscle. Methods were developed to measure ClC-1 function with subsecond temporal resolution in action potential firing muscle fibers. These and other techniques have revealed that ClC-1 function is controlled by multiple cellular signals during muscle activity. Thus, onset of muscle activity triggers ClC-1 inhibition via protein kinase C, intracellular acidosis, and lactate ions. This inhibition is important for preserving excitability of working muscle in the face of activity-induced elevation of extracellular K(+) and accumulating inactivation of voltage-gated sodium channels. Furthermore, during prolonged activity, a marked ClC-1 activation can develop that compromises muscle excitability. Data from ClC-1 expression systems suggest that this ClC-1 activation may arise from loss of regulation by adenosine nucleotides and/or oxidation.

Human skeletal muscle satellite cells (SCs) are essential for muscle regeneration and remodeling processes in healthy and clinical conditions involving muscle breakdown. However, the potential influence of protein supplementation on post-exercise SC regulation in human skeletal muscle has not been well investigated. In a comparative human study, we investigated the effect of hydrolyzed whey protein supplementation following eccentric exercise on fiber type-specific SC accumulation. Twenty-four young healthy subjects received either hydrolyzed whey protein + carbohydrate (whey, n = 12) or iso-caloric carbohydrate (placebo, n = 12) during post-exercise recovery from 150 maximal unilateral eccentric contractions. Prior to and 24, 48 and 168 h post-exercise, muscle biopsies were obtained from the exercise leg and analyzed for fiber type-specific SC content. Maximal voluntary contraction (MVC) and serum creatine kinase (CK) were evaluated as indices of recovery from muscle damage. In type II fiber-associated SCs, the whey group increased SCs/fiber from 0.05 [0.02; 0.07] to 0.09 [0.06; 0.12] (p < 0.05) and 0.11 [0.06; 0.16] (p < 0.001) at 24 and 48 h, respectively, and exhibited a difference from the placebo group (p < 0.05) at 48 h. The whey group increased SCs/myonuclei from 4 % [2; 5] to 10 % [4; 16] (p < 0.05) at 48 h, whereas the placebo group increased from 5 % [2; 7] to 9 % [3; 16] (p < 0.01) at 168 h. MVC decreased (p < 0.001) and muscle soreness and CK increased (p < 0.001), irrespective of supplementation. In conclusion, whey protein supplementation may accelerate SC proliferation as part of the regeneration or remodeling process after high-intensity eccentric exercise.

Human skeletal muscle satellite cells (SCs) are essential for muscle regeneration and remodeling processes in healthy and clinical conditions involving muscle breakdown. However, the potential influence of protein supplementation on post-exercise SC regulation in human skeletal muscle has not been well investigated. In a comparative human study, we investigated the effect of hydrolyzed whey protein supplementation following eccentric exercise on fiber type-specific SC accumulation. Twenty-four young healthy subjects received either hydrolyzed whey protein + carbohydrate (whey, n = 12) or iso-caloric carbohydrate (placebo, n = 12) during post-exercise recovery from 150 maximal unilateral eccentric contractions. Prior to and 24, 48 and 168 h post-exercise, muscle biopsies were obtained from the exercise leg and analyzed for fiber type-specific SC content. Maximal voluntary contraction (MVC) and serum creatine kinase (CK) were evaluated as indices of recovery from muscle damage. In type II fiber-associated SCs, the whey group increased SCs/fiber from 0.05 [0.02; 0.07] to 0.09 [0.06; 0.12] (p < 0.05) and 0.11 [0.06; 0.16] (p < 0.001) at 24 and 48 h, respectively, and exhibited a difference from the placebo group (p < 0.05) at 48 h. The whey group increased SCs/myonuclei from 4 % [2; 5] to 10 % [4; 16] (p < 0.05) at 48 h, whereas the placebo group increased from 5 % [2; 7] to 9 % [3; 16] (p < 0.01) at 168 h. MVC decreased (p < 0.001) and muscle soreness and CK increased (p < 0.001), irrespective of supplementation. In conclusion, whey protein supplementation may accelerate SC proliferation as part of the regeneration or remodeling process after high-intensity eccentric exercise.