Skeletal muscle is a highly plastic tissue that rapidly adapts to changes in mechanical loading and metabolic activity. Periods of inactivity, including bed rest, limb immobilization or microgravity, induce a pronounced loss of muscle mass and function. This review examines the mechanistic role of myostatin (growth differentiation factor-8; GDF-8), a member of the transforming growth factor-β superfamily, in mediating inactivity-induced skeletal muscle atrophy. Accumulating evidence from human and rodent studies demonstrates that physical inactivity upregulates myostatin expression and signalling, shifting muscle protein turnover toward net protein degradation. Mechanistically, myostatin binds to the activin type IIB receptor (ActRIIB) and activates Smad2/3 signalling, which suppresses Akt phosphorylation and downstream mTOR activity, resulting in reduced protein translation. Diminished Akt signalling activates FoxO transcription factors, promoting ubiquitin-proteasome-mediated proteolysis. In parallel, myostatin maintains satellite cells in a quiescent state, impairing MyoD-driven activation and limiting myogenesis, thereby reducing regenerative capacity during and after physical inactivity. We provide a narrative mini-review on the time course of gene expression of myostatin during inactivity. Finally, these mechanistic insights have stimulated therapeutic strategies targeting the myostatin-ActRIIB axis, notably bimagrumab, a monoclonal antibody against ActRIIB and inhibitor of downstream myostatin signalling. Evidence from human and rodent studies suggests that myostatin inhibition may represent a promising strategy to counteract skeletal muscle disuse atrophy caused by inactivity. Collectively, the current evidence highlights myostatin as a central molecular integrator of mechanical unloading-induced muscle atrophy.

Abstract Patients with long COVID and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) suffer from post-exertional malaise. The accompanying physical inactivity may contribute to a lower aerobic capacity and may explain skeletal muscle adaptations in these patients. Here, we compare whole-body exercise responses and skeletal muscle adaptations after strict 60-day bed rest in healthy people with those in long COVID and ME/CFS patients, and healthy age- and sex-matched controls. Bed rest alters respiratory and cardiovascular responses to maximal exercise, which are dissimilar in patients. Bed rest causes muscle atrophy without altering fiber type. Both patient groups have more glycolytic fibers, and ME/CFS patients display type I-specific atrophy. Only after bed rest is oxidative phosphorylation capacity associated with maximal oxygen uptake. As skeletal muscle characteristics differ between patients and healthy individuals after bed rest, physical inactivity cannot solely explain the lower exercise capacity and skeletal muscle adaptations in long COVID and ME/CFS patients.

Metformin is the first-line therapy for type 2 diabetes mellitus and is commonly co-administered with statins for cardiovascular risk reduction. However, statins can cause statin-associated muscle symptoms, while metformin itself exerts complex effects on skeletal muscle. Because both drugs influence cellular energy metabolism and stress-response pathways in skeletal muscle, their combined effects on muscle cells warrant investigation. C2C12 myotubes were treated with metformin (50 or 1000 μM) in the absence or presence of simvastatin (10 μM) for 24 h. Myotube morphology, differentiation, and fusion indices, myoblast proliferation, and expression of atrophy-, stress-, and metabolism-related genes were assessed. Phosphorylation of key metabolic and anabolic signaling proteins (AMPK/ACC and Akt/mTOR-p70S6K) was analyzed. Mitochondrial respiration was measured using Seahorse respirometry, and mitochondrial network organization was quantified by live-cell imaging. Simvastatin significantly reduced myotube diameter (p < 0.0001), impaired myogenic progression in differentiated myotubes (differentiation index, p < 0.0001; fusion index, p = 0.0152), and inhibited myoblast proliferation (p = 0.003). Simvastatin increased the atrophy markers (Trim63, Fbxo32), stress marker (Perk), and concurrently suppressed myogenic (Myod) and anabolic (p-p70s6k/p70s6k) activity. Simvastatin also induced a broad suppression of mitochondrial and glycolytic metabolism, accompanied by reduced expression of the metabolic genes (Glut4, Hk2) and disruption of mitochondrial network connectivity. Co-exposure with metformin significantly attenuated simvastatin-induced effects, increasing myotube diameter (1.43-fold at low dose, p = 0.0223, and 1.48-fold at high dose, p = 0.0131), differentiation index (low dose: 1.63-fold; high dose: 1.80-fold; both p < 0.0001), and fusion index (low dose: 1.35; high dose: 1.50-fold; both p < 0.01). Compared with simvastatin alone, co-treatment with high-dose metformin increased AMPK and ACC phosphorylation and further suppressed mTOR signaling without amplifying atrophy-related gene expression. Despite deeper suppression of metabolic parameters (routine respiration, ATP production, Hk2 expression), metformin preserved mitochondrial network structure, increased Ppargc1a expression, and reduced cellular stress markers (Hri, Perk, Atf4). Simvastatin induced metabolic suppression, mitochondrial dysfunction, and atrophy-related responses in skeletal muscle cells. Metformin partially attenuated these alterations by preserving myotube structural integrity and reducing cellular stress signaling despite further metabolic suppression. These findings suggest that metformin may promote adaptive metabolic responses that enhance cellular resilience during simvastatin-induced metabolic stress.

ABSTRACT Background Muscle atrophy and weakness are among the most detrimental consequences of disuse, microgravity, hospitalisation and ageing. Oxidative modifications of myofibrillar proteins generated by oxidative stress may contribute to the reduced force‐ and power‐generating capacity of skeletal muscles. As part of the 60‐day AGBRESA bed rest (BR) study, we studied (1) how microgravity‐induced disuse affected markers of systemic and muscle oxidative stress, (2) how these related to muscle function and (3) to what extent artificial gravity (AG) attenuated these changes. Since the myokine irisin may protect against muscle deterioration in disuse, we additionally assessed serum irisin levels. Methods Sixteen men and eight women (33 ± 9 years) participated in the AGBRESA study. Participants were pseudorandomly assigned to a control group (BR only), or a continuous or intermittent centrifugation group (n = 8 in each group) to assess the efficacy of daily 30‐min AG in attenuating the adverse effects of BR‐induced disuse. Muscle function, muscle protein carbonyls, serum irisin and key modulators of oxidative stress and cell protection in muscle and blood were assessed before, on Day 6, and at the end of BR. Results BR caused a reduction in peak torque during maximal voluntary isometric knee extension and knee flexion (p < 0.001) that was greater in women than in men (knee extension, w: −39.7 ± 3.5%, m: −25.1 ± 2.4%; knee flexion, w: −32.9 ± 4.5%, m: −10.2 ± 3.5%, p ≤ 0.002) and faster electrically evoked twitch muscle contractions of plantar flexor and knee extensor muscles (half relaxation time and % peak rate of relaxation, p ≤ 0.003). AG attenuated the BR‐induced increase in evoked twitch contraction speed in the knee extensors (group × time interactions: half relaxation time, p = 0.009; % peak rate of relaxation, p = 0.030), and the loss of evoked twitch peak torque of plantar flexors (AG − 25%, Controls −48%, group × time interactions, p = 0.020). Neither BR nor AG affected the circulating levels of systemic oxidative stress and muscle carbonyl concentration and serum irisin levels. However, participants with the highest serum irisin and brain‐derived neurotrophic factor levels showed lower levels of 8‐iso‐PGF2α, a marker of systemic oxidative stress (r = −0.486, p = 0.019; r = −0.512, p = 0.012, respectively) and circulating levels of the C‐terminal agrin fragment, a biomarker of neuromuscular junction fragmentation. Conclusions AG exposure attenuated some of the BR‐induced changes in twitch contractile properties. Neither BR nor AG induced significant alterations in systemic oxidative stress, or muscle protein carbonylation, suggesting that the main contribution to the BR‐induced loss of muscle strength during the AGBRESA study was not oxidative stress.

Background Disease-related malnutrition is common among hospitalised and recently discharged older adults. Consequences of malnutrition are physical limitations, negative health outcomes, decreased quality of life and increased healthcare costs. Dietetic care can counteract the effects of malnutrition by increasing protein and energy intake. The ProIntens study aims to evaluate the impact of an intensive dietetic care pathway on dietary protein intake and physical functioning in older adults during hospitalisation and after discharge. Process and economic evaluations will be performed. Methods The ProIntens study is a multicentre two-armed parallel individually randomised trial in five hospitals in the Amsterdam region. In total, 250 hospitalised older adults, aged ≥55 years and at risk of malnutrition will be randomised to receive regular care (control) or intensive dietetic care (intervention). The intervention consists of intensive personalised care which involves transmural guidance by trained dietitians, supportive materials on protein intake in a social context and a mobilization program during hospitalisation until three months after discharge. Controls will receive regular care. The primary outcome will be the between-groups difference in the mean change on the Short Physical Performance Battery three months after discharge. Secondary outcomes include dietary protein intake, body composition, physical activity, activities of daily living, quality of life and healthcare costs. Discussion The ProIntens trial will study the effects and costs of an intensive dietetic treatment on recovery outcomes in hospitalised older adults at risk of malnutrition. The intervention will result in practical tools for care professionals which aim to improve malnutrition treatment for older patients. Trial registration Landelijk Trial Register (NL8041; NL72069.029.19); registered 2019-09-23; covering all WHO Trial Registration Data Set items.

The assessment of skeletal muscle volume is valuable for fundamental research and clinical practice, but remains limited in larger cohorts due to its time-consuming nature. Here, we developed a method to accurately estimate vastus lateralis (VL) muscle volume based on a single measurement of anatomical cross-sectional area (ACSA) or tissue thickness. Sixty-nine healthy participants (20–91 years) volunteered. In a subgroup (n = 34) we measured VL volume and ACSAs at 10% intervals along the muscle length to derive a VL muscle shape factor. We subsequently estimated VL volume by multiplying this muscle shape factor with muscle length and a single measure of ACSA at 50% muscle length (ACSAVL50%) or an estimated ACSAVL50% from a single ultrasound scan of tissue thickness in an independent cohort (n = 35). VL muscle shape factor was determined by integrating a fourth-order polynomial of muscle length and ACSA, and was dependent on muscle size. Estimating muscle volume had a high accuracy (R²=0.976, CCC = 0.987), low bias and error (< 8.5%) in both the main cohort and an independent validation group. Estimating muscle volume from stitching 2D images at 50% muscle length or estimating ACSA with a geometric model explained 91–95% of variance in measured volumes, with high accuracy and concordance correlation coefficients. VL muscle volume can be estimated by multiplying a muscle shape factor with muscle length and ACSAVL50% from a single ultrasound image. We present a novel, cost-effective, rapid, yet accurate assessment of VL muscle mass for (large-scale) studies and clinical practice.

The antibiotic streptomycin is an integral part of cell culture medium. Because streptomycin inhibits bacterial protein synthesis, streptomycin might also have off‐target effects on muscle cell function. Here, we studied the effect of streptomycin on C2C12 myoblasts, myofiber growth, and metabolism. C2C12 myoblasts were cultured with or without streptomycin. The control condition consisted of carbenicillin and ampicillin. Streptomycin did not impair myoblast proliferation rate. Streptomycin exposure led to a ~ 40% reduction in myotube diameter and reduced protein synthesis rate. Myotubes with streptomycin showed a 25% lower differentiation and 60% lower fusion index. Expression of cell stress markers was upregulated by streptomycin. Mitochondrial respiration rate was unaffected by streptomycin, but gene expression levels of Myh3 and Acta1 were lower, as well as the protein content of mitochondrial complex I subunits. Myotubes cultured in the presence of streptomycin showed fragmentation of the mitochondrial network, a smaller mitochondrial footprint (−64%), and shorter branch lengths (−34%). Streptomycin does not alter C2C12 myoblast proliferation but reduces global protein synthesis rates in differentiating myotubes. The routine use of streptomycin in muscle cell cultures should be carefully evaluated, particularly when investigating muscle growth, metabolism, or protein synthesis, where off‐target effects may confound experimental outcomes.

BACKGROUND:Inadequate protein intake is associated with poor physical functioning and suboptimal recovery in hospitalised older adults. Despite standard dietetic care, dietary protein intakes falls well below the recommended levels. To address this problem, we developed an intensified trans-sectorial dietetic intervention that targets hospitalised older adults. This study aims to evaluate its impact on physical functioning and dietary protein intake during and post hospitalisation. METHODS:This multicentre individually randomised controlled trial was conducted in five hospitals from January 2021 until December 2022. Hospitalised older adults, aged ≥55 years and at risk of malnutrition were randomised to receive regular care (CON) or intensive dietetic intervention (INT). The intervention consisted of personalized, intensive care, including trans-sectorial guidance by trained dietitians, increased consultations, and supportive materials focused on protein intake. Additionally, the intervention emphasized engagement in dietary behaviour and physical activity during hospitalisation and continued for three months post-discharge. The primary outcome was change in physical functioning measured by the Short Physical Performance Battery (SPPB) from admission to three months post-discharge, analysed with linear mixed models for repeated measures. Secondary outcomes included protein intake, body composition, muscle strength, physical activity, activities of daily living, fear of falling, pain, fatigue, appetite and quality of life. RESULTS:A total of 76 hospitalised older adults were included in the study of which 38 were in CON and 38 received INT. The overall drop-out was 30 % (CON 26 %; INT 34 %). The participants had a median age 73 y (Inter Quartile Range: 62-78 y) with 50 % females. Overall, Physical functioning improved from 6 points (IQR: 1-9 points) at baseline to 9 points (IQR: 7-11) at three months post-discharge (p < 0.0001). Likewise, protein intake increased from 0.8 g/kg bodyweight (IQR: 0.6-1.0) to 1.0 g/kg bodyweight (IQR: 0.8-1.2) (p < 0.0001). There were no significant differences between intervention and control group. All secondary outcomes improved over time, except for fear of falling, leg extension strength, and body composition, with no significant differences between intervention and control group. CONCLUSIONS:Hospitalised older patients improved their physical functioning and protein intake after three months post-discharge, although the majority not to recommended levels. No effects of the intensive dietetic treatment could be detected due to low intervention adherence and a small sample size. Future research should be conducted with an intervention consisting of a strong combination of nutritional support and exercise with a successful implementation and a flexible study design catered to the needs of the older patient. TRIAL REGISTRATION:Landelijk Trial Register (NL8041; NL72069.029.19) www.onderzoekmetmensen.nl; registered 2019-09-23; covering all WHO Trial Registration Data Set items.

AIMS:Active cigarette smoking is a major risk factor for chronic obstructive pulmonary disease that remains elevated after cessation. Skeletal muscle dysfunction has been well documented after smoking, but little is known about cardiac adaptations to cigarette smoking. The underlying cellular and molecular cardiac adaptations, independent of confounding lifestyle factors, and time course of reversibility by smoking cessation remain unclear. We hypothesized that smoking negatively affects cardiac metabolism and induces local inflammation in mice, which do not readily reverse upon 2-week smoking cessation. METHODS:Mice were exposed to air or cigarette smoke for 14 weeks with or without 1- or 2-week smoke cessation. We measured cardiac mitochondrial respiration by high-resolution respirometry, cardiac mitochondrial density, abundance of mitochondrial supercomplexes by electrophoresis, and capillarization, fibrosis, and macrophage infiltration by immunohistology, and performed cardiac metabolome and lipidome analysis by mass spectrometry. RESULTS:Mitochondrial protein, supercomplex content, and respiration (all p < 0.03) were lower after smoking, which were largely reversed within 2-week smoking cessation. Metabolome and lipidome analyses revealed alterations in mitochondrial metabolism, a shift from fatty acid to glucose metabolism, which did not revert to control upon smoking cessation. Capillary density was not different after smoking but increased after smoking cessation (p = 0.02). Macrophage infiltration and fibrosis (p < 0.04) were higher after smoking but did not revert to control upon smoking cessation. CONCLUSIONS:While cigarette-impaired smoking-induced cardiac mitochondrial function was reversed by smoking cessation, the remaining fibrosis and macrophage infiltration may contribute to the increased risk of cardiovascular events after smoking cessation.

Insulin sensitivity and metabolic flexibility decrease in response to bed rest, but the temporal and causal adaptations in human skeletal muscle metabolism are not fully defined. Here, we use an integrative approach to assess human skeletal muscle metabolism during bed rest and provide a multi-system analysis of how skeletal muscle and the circulatory system adapt to short- and long-term bed rest (German Clinical Trials: DRKS00015677). We uncover that intracellular glycogen accumulation after short-term bed rest accompanies a rapid reduction in systemic insulin sensitivity and less GLUT4 localization at the muscle cell membrane, preventing further intracellular glycogen deposition after long-term bed rest. We provide evidence of a temporal link between the accumulation of intracellular triglycerides, lipotoxic ceramides, and sphingomyelins and an altered skeletal muscle mitochondrial structure and function after long-term bed rest. An intracellular nutrient overload therefore represents a crucial determinant for rapid skeletal muscle insulin insensitivity and mitochondrial alterations after prolonged bed rest.

AIMS:Genetic hypertrophic cardiomyopathy (HCM) is caused by mutations in sarcomere protein-encoding genes (i.e. genotype-positive HCM). In an increasing number of patients, HCM occurs in the absence of a mutation (i.e. genotype-negative HCM). Mitochondrial dysfunction is thought to be a key driver of pathological remodelling in HCM. Reports of mitochondrial respiratory function and specific disease-modifying treatment options in patients with HCM are scarce.METHODS AND RESULTS:Respirometry was performed on septal myectomy tissue from patients with HCM (n = 59) to evaluate oxidative phosphorylation and fatty acid oxidation. Mitochondrial dysfunction was most notably reflected by impaired NADH-linked respiration. In genotype-negative patients, but not genotype-positive patients, NADH-linked respiration was markedly depressed in patients with an indexed septal thickness ≥10 compared with <10. Mitochondrial dysfunction was not explained by reduced abundance or fragmentation of mitochondria, as evaluated by transmission electron microscopy. Rather, improper organization of mitochondria relative to myofibrils (expressed as a percentage of disorganized mitochondria) was strongly associated with mitochondrial dysfunction. Pre-incubation with the cardiolipin-stabilizing drug elamipretide and raising mitochondrial NAD+ levels both boosted NADH-linked respiration.CONCLUSION:Mitochondrial dysfunction is explained by cardiomyocyte architecture disruption and is linked to septal hypertrophy in genotype-negative HCM. Despite severe myocardial remodelling mitochondria were responsive to treatments aimed at restoring respiratory function, eliciting the mitochondria as a drug target to prevent and ameliorate cardiac disease in HCM. Mitochondria-targeting therapy may particularly benefit genotype-negative patients with HCM, given the tight link between mitochondrial impairment and septal thickening in this subpopulation.

AbstractBackgroundSystemic inflammation is associated with skeletal muscle atrophy and metabolic dysfunction. Although the nucleotide‐binding oligomerization domain‐like receptor family pyrin domain containing 3 (NLRP3) inflammasome contributes to cytokine production in immune cells, its role in skeletal muscle is poorly understood. Here, we studied the link between inflammation, NLRP3, muscle morphology, and metabolism in in vitro cultured C2C12 myotubes, independent of immune cell involvement.MethodsDifferentiated C2C12 myotubes were treated with lipopolysaccharide (LPS; 0, 10, and 100–200 ng/mL) to induce activation of the NLRP3 inflammasome with and without MCC950, a pharmacological inhibitor of NLRP3‐induced IL‐1β production. We assessed markers of the NLRP3 inflammasome, cell diameter, reactive oxygen species, and mitochondrial function.ResultsNLRP3 gene expression and protein concentrations increased in a time‐dependent and dose‐dependent manner. Intracellular IL‐1β concentration significantly increased (P < 0.0001), but significantly less with MCC950 (P = 0.03), suggestive of moderate activation of the NLRP3 inflammasome in cultured myotubes upon LPS stimulation. LPS suppressed myotube growth after 24 h (P = 0.03), and myotubes remained smaller up to 72 h (P = 0.0009). Exposure of myotubes to IL‐1β caused similar alterations in cell morphology, and MCC950 mitigated these LPS‐induced differences in cell diameter. NLRP3 appeared to co‐localize with mitochondria, more so upon exposure to LPS. Mitochondrial reactive oxygen species were higher after LPS (P = 0.03), but not after addition of MCC950. Myotubes had higher glycolytic rates, and mitochondria were more fragmented upon LPS exposure, which was not altered by MCC950 supplementation.ConclusionsLPS‐induced activation of the NLRP3 inflammasome in cultured myotubes contributes to morphological and metabolic alterations, likely due to its mitochondrial association.

Skeletal muscle-related symptoms are common in both acute coronavirus disease (Covid)-19 and post-acute sequelae of Covid-19 (PASC). In this narrative review, we discuss cellular and molecular pathways that are affected and consider these in regard to skeletal muscle involvement in other conditions, such as acute respiratory distress syndrome, critical illness myopathy, and post-viral fatigue syndrome. Patients with severe Covid-19 and PASC suffer from skeletal muscle weakness and exercise intolerance. Histological sections present muscle fibre atrophy, metabolic alterations, and immune cell infiltration. Contributing factors to weakness and fatigue in patients with severe Covid-19 include systemic inflammation, disuse, hypoxaemia, and malnutrition. These factors also contribute to post-intensive care unit (ICU) syndrome and ICU-acquired weakness and likely explain a substantial part of Covid-19-acquired weakness. The skeletal muscle weakness and exercise intolerance associated with PASC are more obscure. Direct severe acute respiratory syndrome coronavirus (SARS-CoV)-2 viral infiltration into skeletal muscle or an aberrant immune system likely contribute. Similarities between skeletal muscle alterations in PASC and chronic fatigue syndrome deserve further study. Both SARS-CoV-2-specific factors and generic consequences of acute disease likely underlie the observed skeletal muscle alterations in both acute Covid-19 and PASC.

This study aimed to investigate the mediating role of perceived motor competence in the relationship between motor competence and physical activity in children as hypothesized by Stodden and colleagues (2008) in their conceptual model of motor competence. A total of 207 children aged 8-13 years (58.9% girls) took part in the study. Actual and perceived motor competence (i.e., overall competence, and competence in locomotor and object control skills) were measured using the Test of Gross Motor Development, 3rd Edition, and the Perceived Motor Competence questionnaire in Childhood, respectively. Physical activity was assessed using a self-report questionnaire. Mediation analyses revealed that the relationship between actual motor competence and physical activity was mediated by perceived motor competence. Specifically, a significant indirect effect of actual competence on physical activity through perceived competence was observed for overall competence (β = 0.168) and object control skills (β = 0.178). The present study provides evidence towards mechanisms underlying the relationship between motor competence and physical activity as postulated by Stodden et al. (2008). Moreover, findings indicate that perceived motor competence is important for physical activity promotion during childhood. As such, fostering perceived motor competence should be considered a key outcome in physical education and youth sport programs.

Pennation angle is an important architecture parameter to understand muscle functioning. It is commonly measured using 2D ultrasound. However, it is difficult to infer 3D muscle architecture from 2D imaging. Therefore, we compare the pennation angle measurements obtained with 3D-DTI fiber-tractography and 3D-ultrasound (3D-US). We acquired data of the Vastus Lateralis muscle in 9 healthy subjects. The mean pennation angle with 3D-US was 18.9°± 5.9°, whereas we found 33.3°±6.7° (straight fiber approximation) and 34.5°±4.8°(curved fiber fit) for DTI fiber-tractography. These differences between 3D-US and DTI could be of technical or physiological origin.