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Aim: The aim of this observational study was to determine the immune status and function in young adults with cerebral palsy (CP) in comparison to typically developing individuals. Method: Blood samples from 12 individuals with CP (five males, seven females; mean age: 25 years 1 month (5 years 9 months); age range: 19–38 years) and 17 typically developing individuals (eight males, nine females; mean age: 31 years 4 months (6 years 2 months); age range: 20–40 years) were collected before, immediately after, and 1 hour after 45 minutes of frame running or running respectively. Independent t‐tests were used to compare heart rate, level of exertion, and baseline cell proportions between groups. Mixed model analysis of variance was utilized to investigate immune cell responses to exercise across groups. Results: Baseline levels of gamma delta (TCRγδ+) T‐cells were significantly higher (absolute percentage: +2.65, p = 0.028) in the individuals with CP. Several cell populations showed similar significant changes after exercise in both CP and typically developing groups. Cytotoxic (CD8+) T‐cells were only significantly elevated immediately after exercise in the typically developing participants (p < 0.01). Individuals with CP exhibited significantly lower heart rates (−11.1%, p < 0.01), despite similar ratings of perceived exertion. Interpretation: Elevated baseline TCRγδ+ T‐cells may indicate low‐grade inflammation in adults with CP. Although most of the cell populations showed typical responses to endurance exercise, the absence of response in CD8+ T‐cells in individuals with CP may indicate the need for higher intensity during exercise. What this paper adds: TCRγδ+ T‐cell baseline levels are elevated in adults with cerebral palsy (CP).The CD8+ T‐cell response to exercise was blunted in adults with CP.Exercise intensity is decisive for CD8+ T‐cell responses in individuals with CP. [ABSTRACT FROM AUTHOR]

Pulsatile Myc induction, through epigenetic alterations, mediates several aspects of the exercise response and may restore muscle plasticity with age. Of the "Yamanaka factors" Oct3/4 , Sox2 , Klf4 , and c-Myc (OSKM), the transcription factor c-Myc (Myc) is the most responsive to exercise in skeletal muscle and is enriched within the muscle fiber. We hypothesize that the pulsatile induction of MYC protein after bouts of exercise can serve to epigenetically reprogram skeletal muscle toward a more resilient and functional state. [ABSTRACT FROM AUTHOR]

The central dogma of molecular biology dictates the general flow of molecular information from DNA that leads to a functional cellular outcome. In skeletal muscle fibers, the extent to which global myonuclear transcriptional alterations, accounting for epigenetic and post-transcriptional influences, contribute to an adaptive stress response is not clearly defined. In this investigation, we leveraged an integrated analysis of the myonucleus-specific DNA methylome and transcriptome, as well as myonuclear small RNA profiling to molecularly define the early phase of skeletal muscle fiber hypertrophy. The analysis of myonucleus-specific mature microRNA and other small RNA species provides new directions for exploring muscle adaptation and complemented the methylation and transcriptional information. Our integrated multi-omics interrogation revealed a coordinated myonuclear molecular landscape during muscle loading that coincides with an acute and rapid reduction of oxidative metabolism. This response may favor a biosynthesis-oriented metabolic program that supports rapid hypertrophic growth. Graphical Abstract [ABSTRACT FROM AUTHOR]

MicroRNAs (miRs) control stem cell biology and fate. Ubiquitously expressed and conserved miR-16 was the first miR implicated in tumorigenesis. miR-16 is low in muscle during developmental hypertrophy and regeneration. It is enriched in proliferating myogenic progenitor cells but is repressed during differentiation. The induction of miR-16 blocks myoblast differentiation and myotube formation, whereas knockdown enhances these processes. Despite a central role for miR-16 in myogenic cell biology, how it mediates its potent effects is incompletely defined. In this investigation, global transcriptomic and proteomic analyses after miR-16 knockdown in proliferating C2C12 myoblasts revealed how miR-16 influences myogenic cell fate. Eighteen hours after miR-16 inhibition, ribosomal protein gene expression levels were higher relative to control myoblasts and p53 pathway-related gene abundance was lower. At the protein level at this same time point, miR-16 knockdown globally upregulated tricarboxylic acid (TCA) cycle proteins while downregulating RNA metabolism-related proteins. miR-16 inhibition induced specific proteins associated with myogenic differentiation such as ACTA2, EEF1A2, and OPA1. We extend prior work in hypertrophic muscle tissue and show that miR-16 is lower in mechanically overloaded muscle in vivo. Our data collectively point to how miR-16 is implicated in aspects of myogenic cell differentiation. A deeper understanding of the role of miR-16 in myogenic cells has consequences for muscle developmental growth, exercise-induced hypertrophy, and regenerative repair after injury, all of which involve myogenic progenitors. [ABSTRACT FROM AUTHOR]

PURPOSE: This study aimed to explore the feasibility of using time-matched uniaxial accelerometers for measuring movement in daily life in children with cerebral palsy (CP) before and after botulinum toxin injections. METHODS: This observational study of clinical care with a pre-post design was set in the home and school environment. Participants included eleven children (4–13 years of age) with CP (GMFCS I-III). The children wore uniaxial accelerometers (ActiGraph, model GT1M) for 4 days on both wrists, the right ankle and around the waist before, 3 weeks and 3 months after BoNT-A injections in the legs. Five children also got BoNT-A in the most affected arm. All injections were given according to clinical indications and routine. The accelerometers were all time-matched to define ambulation, arm swing, voluntary activity of arms, and bimanual activity. The feasibility of wearing accelerometers with this setup was evaluated. A linear mixed model was used for analysis of the percentage time and at which intensity the different activities were performed. The confidence interval demonstrated any difference between the dominant and non-dominant arm. RESULTS: Time-matching of accelerometers placed on both wrists, the waist, and one ankle is a feasible method of registering ambulation, arm swing during gait, and arm movements while not ambulating. Before injections, the children spent 5.6% of their time ambulating. This value declined to 3.9% at 3 months. Contrary to clinical goals, arm movement did not increase after injecting the most affected arm with BoNT-A, however, injections may have decreased mirror movements, which are often bothersome for the child. CONCLUSION: A time-matched 4-accelerometer set-up is feasible in children with cerebral palsy. A future study including time-matched multi-axial accelerometers on all four limbs, could provide important information on the effect of BoNT-A in daily life. [ABSTRACT FROM AUTHOR]

In this study, the properties of circulating extracellular vesicles (EVs) were examined in cerebral palsy (CP) and typically developed (TD) individuals at rest and after aerobic exercise, focusing on the size, concentration, and microRNA cargo of EVs. Nine adult individuals with CP performed a single exercise bout consisting of 45 min of Frame Running, and TD participants completed either 45min of cycling (n = 10; TD EX) or were enrolled as controls with no exercise (n = 10; TD CON). Blood was drawn before and 30min after exercise and analyzed for EV concentration, size, and microRNA content. The size of EVs was similar in CP vs. TD, and exercise had no effect. Individuals with CP had an overall lower concentration (~25%, p < 0.05) of EVs. At baseline, let-7a, let-7b and let-7e were downregulated in individuals with CP compared to TD (p < 0.05), while miR-100 expression was higher, and miR-877 and miR-4433 lower in CP compared to TD after exercise (p < 0.05). Interestingly, miR-486 was upregulated ~2-fold in the EVs of CP vs. TD both at baseline and after exercise. We then performed an in silico analysis of miR-486 targets and identified the satellite cell stemness factor Pax7 as a target of miR-486. C2C12 myoblasts were cultured with amiR-486 mimetic and RNA-sequencing was performed. Gene enrichment analysis revealed that several genes involved in sarcomerogenesis and extracellular matrix (ECM) were downregulated. Our data suggest that circulating miR-486 transported by EVs is elevated in individuals with CP and that miR-486 alters the transcriptome of myoblasts affecting both ECM- and sarcomerogenesis-related genes, providing a link to the skeletal muscle alterations observed in individuals with CP. [ABSTRACT FROM AUTHOR]

Circulating microRNAs (miRNAs) have become increasingly popular biomarker candidates in various diseases. However, heparin-based anticoagulants might affect the detection of target miRNAs in blood samples during quantitative polymerase chain reaction (qPCR)-based analysis of miRNAs involving RNA extraction, cDNA synthesis and the polymerase catalyzed reaction. Because low-molecular-weight heparins (LMWH) are widely used in routine healthcare, we aimed to investigate whether a prophylactic dose of the LMWH tinzaparin influences qPCR-based quantification of circulating miRNAs. A total of 30 subjects were included: 16 fracture patients with tinzaparin treatment and 14 non-fracture controls without anticoagulation therapy. To control for the effect of tinzaparin on miRNA analysis an identical concentration of synthetic miRNAs was added to plasma, isolated RNA and prepared complementary DNA (cDNA) from all samples in both groups. No significant difference was observed for cDNA synthesis or qPCR when comparing tinzaparin-treated patients with untreated controls. Among the tinzaparin-treated patients, plasma levels of six endogenous miRNAs (hsa-let-7i-5p, hsa-miR-30e-5p, hsa-miR-222-3p, hsa-miR-1-3p, hsa-miR-133a-3p, hsa-miR-133b) were measured before and one to six hours after a subcutaneous injection of tinzaparin 4500IU. No significant effect was observed for any of the investigated miRNAs. A prophylactic dose of 4500IU tinzaparin does not seem to affect cDNA synthesis or qRT-PCR-based quantification of circulating miRNAs. [ABSTRACT FROM AUTHOR]

Introduction and aims: Accurate determination of skeletal muscle size is of great importance in multiple settings including resistance exercise, aging, disease, and disuse. Ultrasound (US) measurement of muscle thickness (MT) is a method of relatively high availability and low cost. The present study aims to evaluate a multisite ultrasonographic protocol for measurement of MT with respect to reproducibility and correlation to gold-standard measurements of muscle volume (MV) with magnetic resonance imaging (MRI) in children. Material and methods: 15 children completed the study (11 ± 1 year, 41 ± 8 kg, 137 ± 35 cm). Following 20 min supine rest, two investigators performed US MT measurements of all four heads of the m. quadriceps femoris , at pre-determined sites. Subsequently, MRI scanning was performed and MV was estimated by manual contouring of individual muscle heads. Results: Ultrasound measurement of MT had an intra-rater reliability of ICC = 0.985–0.998 (CI 95% = 0.972–0.998) and inter-rater reliability of ICC = 0.868–0.964 (CI 95% = 0.637–0.983). The US examinations took less than 15 min, per investigator. Muscle thickness of all individual quadriceps muscles correlated significantly with their corresponding MV as measured by MRI (overall r = 0.789, p < 0.001). Conclusion: The results of this study indicate that US measurement of MT using a multisite protocol is a competitive alternative to MRI scanning, especially with respect to availability and time consumption. Therefore, US MT could allow for wider clinical and scientific implementation. [ABSTRACT FROM AUTHOR]

Muscle fibers are syncytial postmitotic cells that can acquire exogenous nuclei from resident muscle stem cells, called satellite cells. Myonuclei are added to muscle fibers by satellite cells during conditions such as load-induced hypertrophy. It is difficult to dissect the molecular contributions of resident versus satellite cell-derived myonuclei during adaptation due to the complexity of labeling distinct nuclear populations in multinuclear cells without label transference between nuclei. To sidestep this barrier, we used a genetic mouse model where myonuclear DNA can be specifically and stably labeled via nonconstitutive H2B-GFP at any point in the lifespan. Resident myonuclei (Mn) were GFP-tagged in vivo before 8 wk of progressive weighted wheel running (PoWeR) in adult mice (>4-mo-old). Resident þ satellite cell-derived myonuclei (MnþSC Mn) were labeled at the end of PoWeR in a separate cohort. Following myonuclear isolation, promoter DNA methylation profiles acquired with low-input reduced representation bisulfite sequencing (RRBS) were compared to deduce epigenetic contributions of satellite cell-derived myonuclei during adaptation. Resident myonuclear DNA has hypomethylated promoters in genes related to protein turnover, whereas the addition of satellite cell-derived myonuclei shifts myonuclear methylation profiles to favor transcription factor regulation and cellcell signaling. By comparing myonucleus-specific methylation profiling to previously published single-nucleus transcriptional analysis in the absence (Mn) versus the presence of satellite cells (MnþSC Mn) with PoWeR, we provide evidence that satellite cellderived myonuclei may preferentially supply specific ribosomal proteins to growing myofibers and retain an epigenetic "memory" of prior stem cell identity. These data offer insights on distinct epigenetic myonuclear characteristics and contributions during adult muscle growth. [ABSTRACT FROM AUTHOR]

Using a mouse model of conditional and inducible in vivo fluorescent myonuclear labeling (HSA-GFP), sorting purification of nuclei, low-input reduced representation bisulfite sequencing (RRBS), and a translatable and reversible model of exercise (progressive weighted wheel running, PoWeR), we provide the first nucleus type-specific epigenetic information on skeletal muscle adaptation and detraining. Adult (>4 mo) HSA-GFP mice performed PoWeR for 8 wk then detrained for 12 wk; age-matched untrained mice were used to control for the long duration of the study. Myonuclei and interstitial nuclei from plantaris muscles were isolated for RRBS. Relative to untrained, PoWeR caused similar myonuclear CpG hypo- and hyper-methylation of promoter regions and substantial hypomethylation in interstitial nuclear promoters. Over-representation analysis of promoters revealed a larger number of hyper- versus hypo-methylated pathways in both nuclear populations after training and evidence for reciprocal regulation of methylation between nucleus types, with hypomethylation of promoter regions in Wnt signaling-related genes in myonuclei and hypermethylation in interstitial nuclei. After 12 wk of detraining, promoter CpGs in documented muscle remodeling-associated genes and pathways that were differentially methylated immediately after PoWeR were persistently differentially methylated in myonuclei, along with long-term promoter hypomethylation in interstitial nuclei. No enduring gene expression changes in muscle tissue were observed using RNA-sequencing. Upon 4 wk of retraining, mice that trained previously grew more at the whole muscle and fiber type-specific cellular level than training naïve mice, with no difference in myonuclear number. Muscle nuclei have a methylation epi-memory of prior training that may augment muscle adaptability to retraining. Graphical Abstract [ABSTRACT FROM AUTHOR]

Skeletal muscle in individuals with CP also contains lower amounts of mtDNA, potentially indicating fewer mitochondria in CP skeletal muscle compared with typically developing muscle. We compared skeletal muscle samples from children with cerebral palsy (CP) and typically developing children and observed evidence of reduced mtDNA and OXPHOS protein content in CP skeletal muscle, indicating reduced mitochondrial abundance. Cerebral palsy (CP) muscle contains fewer energy-generating organelles than typically developing muscle. [Extracted from the article]

Mitochondrial-derived peptides (MDPs) humanin (HN) and mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) are involved in cell survival, suppression of apoptosis, and metabolism. Circulating levels of MDPs are altered in chronic diseases such as diabetes type 2 and chronic kidney disease. Whether acute resistance (RE) or endurance (EE) exercise modulates circulating levels of HN and MOTS-c in humans is unknown. Following familiarization, subjects were randomized to EE (n = 10, 45 min cycling at 70% of estimated VO2max), RE (n = 10, 4 sets x 7RM, leg press and knee extension), or control (CON, n = 10). Skeletal muscle biopsies and blood samples were collected before and at 30 min and 3 h following exercise. Plasma concentration of HN and MOTS-c, skeletal muscle MOTS-c as well as gene expression of exercise-related genes were analyzed. Acute EE and RE promoted changes in skeletal muscle gene expression typically seen in response to each exercise modality (c-Myc, 45S prerRNA, PGC-1a-total, and PGC-1a-ex1b). At rest, circulating levels of HN were positively correlated to MOTS-c levels and age. Plasma levels of MDPs were not correlated to fitness outcomes [VO2max, leg strength, or muscle mitochondrial (mt) DNA copy number]. Circulating levels of HN were significantly elevated by acute EE but not RE. MOTS-C levels showed a trend to increase after EE. These results indicate that plasma MDP levels are not related to fitness status but that acute EE increases circulating levels of MDPs, in particular HN. [ABSTRACT FROM AUTHOR]

The current study explored whether the marked hypertrophic response noted with a short-term unilateral concurrent exercise paradigm was associated with more prominent changes in myonuclei accretion, ribosome biogenesis, and capillarization compared with resistance exercise alone (RE). Ten men (age 25 ± 4 yr) performed aerobic and resistance exercise (AE + RE) for one leg while the other leg did RE. Muscle biopsies were obtained before and after 5 wk of training and subjected to fiber-type specific immunohistochemical analysis, and quantification of total RNA content and mRNA/rRNA transcript abundance. Type II fiber cross-sectional area (CSA) increased with both AE + RE (22%) and RE (16%), while type I fiber CSA increased mainly with AE + RE (16%). The change score tended to differ between legs for type I CSA (P = 0.099), and the increase in smallest fiber diameter was greater in AE + RE than RE (P = 0.029). The number of nuclei per fiber increased after AE + RE in both fiber types, and this increase was greater (P = 0.027) than after RE. A strong correlation was observed between changes in number of nuclei per fiber and fiber CSA in both fiber types, for both AE + RE and RE (r > 0.8, P < 0.004). RNA content increased after AE + RE (24%, P = 0.019), but the change-scores did not differ across legs. The capillary variables generally increased in both fiber types, with no difference across legs. In conclusion, the accentuated hypertrophic response to AE + RE was associated with more pronounced myonuclear accretion, which was strongly correlated with the degree of fiber hypertrophy. This suggests that myonuclear accretion could play a role in facilitating muscle hypertrophy also during very short training periods. [ABSTRACT FROM AUTHOR]

Advanced chronic kidney disease (CKD) is characterized by a premature aging phenotype of multifactorial origin. Mitochondrial dysfunction is prevalent in CKD and has been proposed as a major contributor to poor muscle function. Although the mitochondria-derived peptides (MDPs) humanin and mitochondrial open reading frame of 12S rRNA-c (MOTS-c) are involved in cell survival, suppression of apoptosis, and glucose control, the implications of MDP in CKD are unknown. We investigated humanin and MOTS-c protein expression in skeletal muscle and serum levels in CKD at stage 5 (glomerular filtration rate: < 15 ml/min) patients and age-matched controls with normal renal function. Whereas circulating levels of humanin were increased in CKD, local muscle expression was reduced. In contrast, MOTS-c levels were reduced in both skeletal muscle and serum in CKD. Humanin in serum correlated positively to circulating TNF levels. Reduced MDP levels in skeletal muscle were associated with lower mitochondrial density and evidence of oxidative stress. These results indicate a differential regulation of MDPs in CKD and suggest an alternative site for humanin production than skeletal muscle in the uremic milieu. MDP levels were linked to systemic inflammation and evidence of oxidative stress in the muscle, two hallmark features of premature aging and uremia. [ABSTRACT FROM AUTHOR]

Skeletal muscle mass responds in a remarkable manner to alterations in loading and use. It has long been clear that skeletal muscle hypertrophy can be prevented by inhibiting RNA synthesis. Since 80% of the cell's total RNA has been estimated to be rRNA, this finding indicates that de novo production of rRNA via transcription of the corresponding genes is important for such hypertrophy to occur. Transcription of rDNA by RNA Pol I is the rate-limiting step in ribosome biogenesis, indicating in turn that this biogenesis strongly influences the hypertrophic response. The present minireview focuses on 1) a brief description of the key steps in ribosome biogenesis and the relationship of this process to skeletal muscle mass and 2) the coordination of ribosome biogenesis and protein synthesis for growth or atrophy, as exemplified by the intracellular AMPK and mTOR pathways. [ABSTRACT FROM AUTHOR]

The current study examined the effects of a preceding bout of aerobic exercise (AE) on subsequent molecular signaling to resistance exercise (RE) of the elbow extensors. Eleven men performed unilateral elbow-extensor AE (~45 min at 70% peak workload) followed by unilateral RE (4 × 7 maximal repetitions) for both arms. Thus, one arm performed AE+RE interspersed with 15 min recovery, whereas the other arm conducted RE alone. Muscle biopsies were taken from the triceps brachii of each arm immediately before (PRE) and 15 min (POST1) and 3 h (POST2) after RE. Molecular markers involved in translation initiation, protein breakdown, mechanosignaling, and ribosome biogenesis were analyzed. Peak power during RE was reduced by 24% (±19%) when preceded by AE (P < 0.05). Increases in PGC1a and MuRF1 expression were greater from PRE to POST2 in AE+RE compared with RE (18-vs. 3.5- and 4- vs. 2-fold, respectively, interaction, P < 0.05). Myostatin mRNA decreased in both arms (P < 0.05). Phosphorylation of AMPK (Thr172) increased (2.5-fold), and 4E-BP1 (Thr37/46) decreased (2.0-fold), after AE (interactions, P < 0.05). p70 S6K, yesassociated protein, and c-Jun NH2-terminal kinase phosphorylation were unaltered, whereas focal adhesion kinase decreased ~1.5-fold, and β1- integrin increased ~1.3- to 1.5-fold, (time effect, P < 0.05). Abundance of 45S pre-ribosomal (r)RNA (internally transcribed spacer, ITS) decreased (~30%) after AE (interaction, P < 0.05), whereas CMYC mRNA was greater in AE+RE compared with RE (12-fold, P < 0.05). POLR1B abundance increased after both AE+RE and RE. All together, our results suggest that a single bout of AE leads to an immediate decrease in signaling for translation initiation and ribosome biogenesis. Yet, this did not translate into altered RE-induced signaling during the 3-h postexercise recovery period. [ABSTRACT FROM AUTHOR]

Introduction: Children with cerebral palsy (CP) and acquired brain injury (ABI) commonly develop muscle contractures with advancing age. An underlying growth defect contributing to skeletal muscle contracture formation in CP/ABI has been suggested.Methods: The biceps muscles of children and adolescents with CP/ABI (n = 20) and typically developing controls (n = 10) were investigated. We used immunohistochemistry, quantitative real-time polymerase chain reaction, and Western blotting to assess gene expression relevant to growth and size homeostasis.Results: Classical pro-inflammatory cytokines and genes involved in extracellular matrix (ECM) production were elevated in skeletal muscle of children with CP/ABI. Intramuscular collagen content was increased and satellite cell number decreased and this was associated with reduced levels of RNA polymerase I transcription factors, 45s pre-rRNA and 28S rRNA.Discussion: The present study provides novel data suggesting a role for pro-inflammatory cytokines and reduced ribosomal production in the development/maintenance of muscle contractures, possibly underlying stunted growth and perimysial ECM expansion. Muscle Nerve 58: 277-285, 2018. [ABSTRACT FROM AUTHOR]

Children with cerebral palsy (CP) often develop reduced passive range of motion with age. The determining factor underlying this process is believed to be progressive development of contracture in skeletal muscle that likely changes the biomechanics of the joints. Consequently, to identify the underlying mechanisms, we modeled the mechanical characteristics of the forearm flexors acting across the wrist joint. We investigated skeletal muscle strength (Grippit®) and passive stiffness and viscosity of the forearm flexors in 15 typically developing (TD) children (10 boys/5 girls, mean age 12 years, range 8-18 yrs) and nine children with CP Nine children (6 boys/3 girls, mean age 11 ± 3 years (yrs), range 7-15 yrs) using the NeuroFlexor® apparatus. The muscle stiffness we estimate and report is the instantaneous mechanical response of the tissue that is independent of reflex activity. Furthermore, we assessed cross-sectional area of the flexor carpi radialis (FCR) muscle using ultrasound. Age and body weight did not differ significantly between the two groups. Children with CP had a significantly weaker (-65%, p < 0.01) grip and had smaller cross-sectional area (-43%, p < 0.01) of the FCR muscle. Passive stiffness of the forearm muscles in children with CP was increased 2-fold (p < 0.05) whereas viscosity did not differ significantly between CP and TD children. FCR cross-sectional area correlated to age (R² = 0.58, p < 0.01), body weight (R² = 0.92, p < 0.0001) and grip strength (R² = 0.82, p < 0.0001) in TD children but only to grip strength (R² = 0.60, p < 0.05) in children with CP. We conclude that children with CP have weaker, thinner, and stiffer forearm flexors as compared to typically developing children. [ABSTRACT FROM AUTHOR]

Ribosome production is an early event during skeletal muscle hypertrophy and precedes muscle protein accretion. Signaling via mTOR is crucial for ribosome production and hypertrophy; however, the mechanisms by which it regulates these processes remain to be identified. Herein, we investigated the activation of mTOR signaling in hypertrophying myotubes and determined that mTOR coordinates various aspects of gene expression important for ribosome production. First, inhibition of translation with cycloheximide had a more potent effect on protein synthesis than rapamycin indicating that mTOR function during hypertrophy is not on general, but rather on specific protein synthesis. Second, blocking Pol II transcription had a similar effect as Rapamycin and, unexpectedly, revealed the necessity of Pol II transcription for Pol I transcription, suggesting that mTOR may regulate ribosome production also by controlling Class II genes at the transcriptional level. Third, Pol I activity is essential for rDNA transcription and, surprisingly, for protein synthesis as selective Pol I inhibition blunted rDNA transcription, protein synthesis, and the hypertrophic response of myotubes. Finally, mTOR has nuclear localization in muscle, which is not sensitive to rapamycin. Inhibition of mTOR signaling by rapamycin disrupted mTOR-rDNA promoter interaction and resulted in altered histone marks indicative of repressed transcription and formation of higher-order chromatin structure. Thus mTOR signaling appears to regulate muscle hypertrophy by affecting protein synthesis, Class I and II gene expression, and chromatin remodeling. [ABSTRACT FROM AUTHOR]

Muscle wasting (or sarcopenia) is a common feature of the uremic phenotype and predisposes this vulnerable patient population to increased risk of comorbid complications, poor quality of life, frailty and premature death. The old age of dialysis patients is in addition a likely contributor to loss ofmuscle mass. As recent evidence suggests that assessment of muscle strength (i.e. function) is a better predictor of outcome and comorbidities than muscle mass, this opens new screening, assessment and therapeutic opportunities. Among established treatment strategies, the benefit of resistance exercise and endurance training are increasingly recognized among nephrologists as being effective and should be promoted in sedentary chronic kidney disease patients. Testosterone and growth hormone replacement appear as the most promising among emerging treatments strategies for muscle wasting. As treatment of muscle wasting is difficult and seldom successful in this often old, frail, sedentary and exercisehesitant patient group, novel treatment strategies are urgently needed. In this review, we summarize recent studies on stimulation of mitochondrial biogenesis, myogenic stem (satellite) cells andmanipulation of transforming growth factor familymembers, all of which hold promise for more effective therapies to target muscle mass loss and function in the future. [ABSTRACT FROM AUTHOR]

The main goal of the present study was to investigate the regulation of ribosomal DNA (rDNA) gene transcription at the onset of skeletal muscle hypertrophy. Mice were subjected to functional overload of the plantaris by bilateral removal of the synergist muscles. Mechanical loading resulted in muscle hypertrophy with an increase in rRNA content. rDNA transcription, as determined by 45S pre-rRNA abundance, paralleled the increase in rRNA content and was consistent with the onset of the hypertrophic response. Increased transcription and protein expression of c-Myc and its downstream polymerase I (Pol I) regulon (POL1RB, TIF-1A, PAF53, TTF1, TAF1C) was also consistent with the increase in rRNA. Similarly, factors involved in rDNA transcription, such as the upstream binding factor and the Williams syndrome transcription factor, were induced by mechanical loading in a corresponding temporal fashion. Chromatin immunoprecipitation revealed that these factors, together with Pol I, were enriched at the rDNA promoter. This, in addition to an increase in histone H3 lysine 9 acetylation, demonstrates that mechanical loading regulates rRNA synthesis by inducing a gene expression program consisting of a Pol I regulon, together with accessory factors involved in transcription and chromatin remodeling at the rDNA promoter. Altogether, these data indicate that transcriptional and epigenetic mechanisms take place in the regulation of ribosome production at the onset of muscle hypertrophy. [ABSTRACT FROM AUTHOR]

α-Actinins are structural proteins of the Z-line. Human skeletal muscle expresses two α-actinin isoforms, α-actinin-2 and α-actinin-3, encoded by their respective genes ACTN2 and ACTN3. ACTN2 is expressed in all muscle fiber types, while only type II fibers, and particularly the type lib fibers, express ACTN3. ACTN3 (R577X) polymorphism results in loss of α-actinin-3 and has been suggested to influence skeletal muscle function. The X allele is less common in elite sprint and power athletes than in the general population and has been suggested to be detrimental for performance requiring high power. The present study investigated the association of ACTN3 genotype with muscle power during 30-s Wingate cycling in 120 moderately to well-trained men and women and with knee extensor strength and fatigability in a subset of 21 men performing isokinetic exercise. Muscle biopsies were obtained from the vastus lateralis muscle to determine fiber-type composition and ACTN2 and ACTN3 mRNA levels. Peak and mean power and the torque-velocity relationship and fatigability output showed no difference across ACTN3 genotypes. Thus this study suggests that R577X polymorphism in ACTN3 is not associated with differences in power output, fatigability, or force-velocity characteristics in moderately trained individuals. However, repeated exercise bouts prompted an increase in peak torque in RR but not in XX genotypes, suggesting that ACTN3 genotype may modulate responsiveness to training. Our data further suggest that α-actinins do not play a significant role in determining muscle fiber-type composition. Finally, we show that ACTN2 expression is affected by the content of α-actinin-3, which implies that α-actinin-2 may compensate for the lack of α-actinin-3 and hence counteract the phenotypic consequences of the deficiency. [ABSTRACT FROM AUTHOR]

Abstract: Body fluid redistribution occurs in astronauts traveling in space, potentially altering interstitial water content and hence impedance. This in turn may impact the features of electromyographic (EMG) signals measured to compare in-flight muscle function with pre- and post-flight conditions. Thus, the current study aimed at investigating the influence of similar fluid shifts on EMG spectral variables during muscle contractile activity. Ten men performed sustained isometric actions (120s) at 20% and 60% of maximum voluntary contraction (MVC) following 1-h rest in the vertical or supine position. From single differential EMG signals, recorded from the soleus (SOL), the medial (MG) and lateral (LG) gastrocnemius muscles, initial value and rate of change over time (slope) of mean power frequency (MNF) and average rectified value (ARV) were assessed. MNF initial value showed dependence on muscle (P <0.01), but was unaffected by body tilt. MNF rate of change increased (P <0.001) with increased force and differed across muscles (P <0.05), but was not influenced (P =0.85) by altered body position. Thus, fluid shift resulting from vertical to supine tilt had no impact on myoelectrical manifestations of muscle fatigue. Furthermore, since such alteration of body fluid distribution resembles that occurring in microgravity, our findings suggest this may not be a methodological limitation, when comparing EMG fatigue indices on Earth versus in space. [Copyright &y& Elsevier]

A response to a letter to the editor about the measures of 3-methylhistidine in muscle interstitium as a marker of muscle proteolysis is presented.

Skeletal muscle atrophy is evident after muscle disuse, unloading, or spaceflight and results from decreased protein content as a consequence of decreased protein synthesis, increased protein breakdown or both. At this time, there are essentially no human data describing proteolysis in skeletal muscle undergoing atrophy on Earth or in space, primarily due to lack of valid and accurate methodology. This particular study aimed at assessing the effects of short-term unloading on the muscle contractile proteolysis rate. Eight men were subjected to 72-h unilateral lower limb suspension (ULLS) and intramuscular interstitial levels of the naturally occurring proteolytic tracer 3-methyihistidine (3MH) were measured by means of microdialysis before and on completion of this intervention. The 3MH concentration following 72-h ULLS (2.01 ± 0.22 nmol/ml) was 44% higher (P < 0.05) than before ULLS (1.56 ± 0.20 nmol/ml). The present experimental model and the employed method determining 3MH in microdialysates present a promising tool for monitoring skeletal muscle proteolysis or metabolism of specific muscles during conditions resulting in atrophy caused by, e.g., disuse and real or simulated microgravity. This study provides evidence that the atrophic processes are evoked rapidly and within 72 h of unloading and suggests that countermeasures should be employed in the early stages of space missions to offset or prevent muscle loss during the period when the rate of muscle atrophy is the highest. [ABSTRACT FROM AUTHOR]

Roberts et al. have provided an insightful counterpoint to our review article on the utility of the synergist ablation model. The purpose of this review is to provide some further dialogue regarding the strengths and weaknesses of the synergist ablation model. Specifically, we highlight that the robustness of the model overshadows surgical limitations. We also compare the transcriptomic responses to synergist ablation in mice and resistance exercise in humans to identify common pathways. We conclude that "cell growth is cell growth" and that the mechanisms available to cells to accumulate biomass and increase in size are similar across cell types and independent of the rate of growth.

In cell biology, ribosomal RNA (rRNA) 2' O -methyl (2'- O -Me) is the most prevalent posttranscriptional chemical modification contributing to ribosome heterogeneity. The modification involves a family of small nucleolar RNAs (snoRNAs) and is specified by box C/D snoRNAs (SNORDs). Given the importance of ribosome biogenesis for skeletal muscle growth, we asked if rRNA 2'- O -Me in nascent ribosomes synthesized in response to a growth stimulus is an unrecognized mode of ribosome heterogeneity in muscle. To determine the pattern and dynamics of 2'- O -Me rRNA, we used a sequencing-based profiling method called RiboMeth-seq (RMS). We applied this method to tissue-derived rRNA of skeletal muscle and rRNA specifically from the muscle fiber using an inducible myofiber-specific RiboTag mouse in sedentary and mechanically overloaded conditions. These analyses were complemented by myonuclear-specific small RNA sequencing to profile SNORDs and link the rRNA epitranscriptome to known regulatory elements generated within the muscle fiber. We demonstrate for the first time that mechanical overload of skeletal muscle 1 ) induces decreased 2'- O -Me at a subset of skeletal muscle rRNA and 2 ) alters the SNORD profile in isolated myonuclei. These findings point to a transient diversification of the ribosome pool via 2'- O -Me during growth and adaptation in skeletal muscle. These findings suggest changes in ribosome heterogeneity at the 2'- O -Me level during muscle hypertrophy and lay the foundation for studies investigating the functional implications of these newly identified "growth-induced" ribosomes. NEW & NOTEWORTHY Ribosomal RNAs (rRNAs) are posttranscriptionally modified by 2' O -methyl (2'- O -Me). This study applied RiboMeth-seq (RMS) to detect changes in 2'- O -Me levels during skeletal muscle hypertrophy, uncovering transient diversification of the ribosome pool in skeletal muscle fibers. This work implies a role for ribosome heterogeneity in skeletal muscle growth and adaptation.

The central dogma of molecular biology dictates the general flow of molecular information from DNA that leads to a functional cellular outcome. In skeletal muscle fibers, the extent to which global myonuclear transcriptional alterations, accounting for epigenetic and post-transcriptional influences, contribute to an adaptive stress response is not clearly defined. In this investigation, we leveraged an integrated analysis of the myonucleus-specific DNA methylome and transcriptome, as well as myonuclear small RNA profiling to molecularly define the early phase of skeletal muscle fiber hypertrophy. The analysis of myonucleus-specific mature microRNA and other small RNA species provides new directions for exploring muscle adaptation and complemented the methylation and transcriptional information. Our integrated multi-omics interrogation revealed a coordinated myonuclear molecular landscape during muscle loading that coincides with an acute and rapid reduction of oxidative metabolism. This response may favor a biosynthesis-oriented metabolic program that supports rapid hypertrophic growth., Competing Interests: None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Physiological Society.)

MicroRNAs (miRs) control stem cell biology and fate. Ubiquitously expressed and conserved miR-16 was the first miR implicated in tumorigenesis. miR-16 is low in muscle during developmental hypertrophy and regeneration. It is enriched in proliferating myogenic progenitor cells but is repressed during differentiation. The induction of miR-16 blocks myoblast differentiation and myotube formation, whereas knockdown enhances these processes. Despite a central role for miR-16 in myogenic cell biology, how it mediates its potent effects is incompletely defined. In this investigation, global transcriptomic and proteomic analyses after miR-16 knockdown in proliferating C2C12 myoblasts revealed how miR-16 influences myogenic cell fate. Eighteen hours after miR-16 inhibition, ribosomal protein gene expression levels were higher relative to control myoblasts and p53 pathway-related gene abundance was lower. At the protein level at this same time point, miR-16 knockdown globally upregulated tricarboxylic acid (TCA) cycle proteins while downregulating RNA metabolism-related proteins. miR-16 inhibition induced specific proteins associated with myogenic differentiation such as ACTA2, EEF1A2, and OPA1. We extend prior work in hypertrophic muscle tissue and show that miR-16 is lower in mechanically overloaded muscle in vivo. Our data collectively point to how miR-16 is implicated in aspects of myogenic cell differentiation. A deeper understanding of the role of miR-16 in myogenic cells has consequences for muscle developmental growth, exercise-induced hypertrophy, and regenerative repair after injury, all of which involve myogenic progenitors.

Introduction: The ischaemic pain of acute compartment syndrome (ACS) can be difficult to discriminate from the pain linked to an associated fracture. Lacking objective measures, the decision to perform fasciotomy is based on clinical findings and performed at a low level of suspicion. Biomarkers of muscle cell damage may help to identify and monitor patients at risk, similar to current routines for patients with acute myocardial infarction. This study will test the hypothesis that biomarkers of muscle cell damage can predict ACS in patients with tibial fractures., Methods and Analysis: Patients aged 15-65 years who have suffered a tibial fracture will be included. Plasma (P)-myoglobin and P-creatine phosphokinase will be analysed at 6-hourly intervals after admission to the hospital (for 48 hours) and-if applicable-after surgical fixation or fasciotomy (for 24 hours). In addition, if ACS is suspected at any other point in time, blood samples will be collected at 6-hourly intervals. An independent expert panel will assess the study data and will classify those patients who had undergone fasciotomy into those with ACS and those without ACS. All primary comparisons will be performed between fracture patients with and without ACS. The area under the receiver operator characteristics curves will be used to identify the success of the biomarkers in discriminating between fracture patients who develop ACS and those who do not. Logistic regression analyses will be used to assess the discriminative abilities of the biomarkers to predict ACS corrected for prespecified covariates., Ethics and Dissemination: The study has been approved by the Regional Ethical Review Boards in Linköping (2017/514-31) and Helsinki/Uusimaa (HUS/2500/2000). The BioFACTS study will be reported in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology recommendations., Trial Registration Number: NCT04674592., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Key Points: Ribosome biogenesis and MYC transcription are associated with acute resistance exercise (RE) and are distinct from endurance exercise in human skeletal muscle throughout a 24 h time course of recovery. A PCR-based method for relative ribosomal DNA (rDNA) copy number estimation was validated by whole genome sequencing and revealed that rDNA dosage is positively correlated with ribosome biogenesis in response to RE. Acute RE modifies rDNA methylation patterns in enhancer, intergenic spacer and non-canonical MYC-associated regions, but not the promoter. Myonuclear-specific rDNA methylation patterns with acute mechanical overload in mice corroborate and expand on rDNA findings with RE in humans. A genetic predisposition for hypertrophic responsiveness may exist based on rDNA gene dosage., Abstract: Ribosomes are the macromolecular engines of protein synthesis. Skeletal muscle ribosome biogenesis is stimulated by exercise, although the contribution of ribosomal DNA (rDNA) copy number and methylation to exercise-induced rDNA transcription is unclear. To investigate the genetic and epigenetic regulation of ribosome biogenesis with exercise, a time course of skeletal muscle biopsies was obtained from 30 participants (18 men and 12 women; 31 ± 8 years, 25 ± 4 kg m -2 ) at rest and 30 min, 3 h, 8 h and 24 h after acute endurance (n = 10, 45 min cycling, 70% V ̇ O 2 max ) or resistance exercise (n = 10, 4 × 7 × 2 exercises); 10 control participants underwent biopsies without exercise. rDNA transcription and dosage were assessed using quantitative PCR and whole genome sequencing. rDNA promoter methylation was investigated using massARRAY EpiTYPER and global rDNA CpG methylation was assessed using reduced-representation bisulphite sequencing. Ribosome biogenesis and MYC transcription were associated primarily with resistance but not endurance exercise, indicating preferential up-regulation during hypertrophic processes. With resistance exercise, ribosome biogenesis was associated with rDNA gene dosage, as well as epigenetic changes in enhancer and non-canonical MYC-associated areas in rDNA, but not the promoter. A mouse model of in vivo metabolic RNA labelling and genetic myonuclear fluorescence labelling validated the effects of an acute hypertrophic stimulus on ribosome biogenesis and Myc transcription, and also corroborated rDNA enhancer and Myc-associated methylation alterations specifically in myonuclei. The present study provides the first information on skeletal muscle genetic and rDNA gene-wide epigenetic regulation of ribosome biogenesis in response to exercise, revealing novel roles for rDNA dosage and CpG methylation., (© 2021 The Authors. The Journal of Physiology © 2021 The Physiological Society.)