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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.)

Aim: To provide a detailed gene and protein expression analysis related to mitochondrial biogenesis and assess mitochondrial content in skeletal muscle of children with cerebral palsy (CP)., Method: Biceps brachii muscle samples were collected from 19 children with CP (mean [SD] age 15y 4mo [2y 6mo], range 9-18y, 16 males, three females) and 10 typically developing comparison children (mean [SD] age 15y [4y], range 7-21y, eight males, two females). Gene expression (quantitative reverse transcription polymerase chain reaction [PCR]), mitochondrial DNA (mtDNA) to genomic DNA ratio (quantitative PCR), and protein abundance (western blotting) were analyzed. Microarray data sets (CP/aging/bed rest) were analyzed with a focused query investigating metabolism- and mitochondria-related gene networks., Results: The mtDNA to genomic DNA ratio was lower in the children with CP compared to the typically developing group (-23%, p=0.002). Out of five investigated complexes in the mitochondrial respiratory chain, we observed lower protein levels of all complexes (I, III, IV, V, -20% to -37%; p<0.05) except complex II. Total peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) messenger RNA (p<0.004), isoforms PGC1α1 (p=0.05), and PGC1α4 (p<0.001) were reduced in CP. Transcriptional similarities were observed between CP, aging, and 90 days' bed rest., Interpretation: Mitochondrial biogenesis, mtDNA, and oxidative phosphorylation protein content are reduced in CP muscle compared with typically developing muscle. Transcriptional pathways shared between aging and long-term unloading suggests metabolic dysregulation in CP, which may guide therapeutic strategies for combatting CP muscle pathology. What this paper adds Cerebral palsy (CP) muscle contains fewer energy-generating organelles than typically developing muscle. Gene expression in CP muscle is similar to aging and long-term bed rest., (© 2021 The Authors. Developmental Medicine & Child Neurology published by John Wiley & Sons Ltd on behalf of Mac Keith Press.)

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 V̇O 2max ), RE ( n = 10, 4 sets × 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 pre-rRNA, PGC-1α-total, and PGC-1α-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 [V̇O 2max , 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. NEW & NOTEWORTHY In this manuscript, we report for the first time, to our knowledge, the response of circulating levels of mitochondrial-derived peptides humanin and MOTS-c to acute resistance and endurance exercise. Our data support that acute endurance exercise stimulates MDP levels in plasma, whereas acute resistance exercise does not.

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.)

Background: Translational capacity (i.e. ribosomal mass) is a key determinant of protein synthesis and has been associated with skeletal muscle hypertrophy. The role of translational capacity in muscle atrophy and regrowth from disuse is largely unknown. Therefore, we investigated the effect of muscle disuse and reloading on translational capacity in middle-aged men (Study 1) and in rats (Study 2)., Methods: In Study 1, 28 male participants (age 50.03 ± 3.54 years) underwent 2 weeks of knee immobilization followed by 2 weeks of ambulatory recovery and a further 2 weeks of resistance training. Muscle biopsies were obtained for measurement of total RNA and pre-ribosomal (r)RNA expression, and vastus lateralis cross-sectional area (CSA) was determined via peripheral quantitative computed tomography. In Study 2, male rats underwent hindlimb suspension (HS) for either 24 h (HS 24 h, n = 4) or 7 days (HS 7d, n = 5), HS for 7 days followed by 7 days of reloading (Rel, n = 5) or remained as ambulatory weight bearing (WB, n = 5) controls. Rats received deuterium oxide throughout the study to determine RNA synthesis and degradation, and mTORC1 signalling pathway was assessed., Results: Two weeks of immobilization reduced total RNA concentration (20%) and CSA (4%) in men (both P ≤ 0.05). Ambulatory recovery restored total RNA concentration to baseline levels and partially restored muscle CSA. Total RNA concentration and 47S pre-rRNA expression increased above basal levels after resistance training (P ≤ 0.05). In rats, RNA synthesis was 30% lower while degradation was ~400% higher in HS 7d in soleus and plantaris muscles compared with WB (P ≤ 0.05). mTORC1 signalling was lower in HS compared with WB as was 47S pre-rRNA (P ≤ 0.05). With reloading, the aforementioned parameters were restored to WB levels while RNA degradation was suppressed (P ≤ 0.05)., Conclusions: Changes in RNA concentration following muscle disuse and reloading were associated with changes in ribosome biogenesis and degradation, indicating that both processes are important determinants of translational capacity. The pre-clinical data help explain the reduced translational capacity after muscle immobilization in humans and demonstrate that ribosome biogenesis and degradation might be valuable therapeutic targets to maintain muscle mass during disuse., (© 2020 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

Satellite cells are required for postnatal development, skeletal muscle regeneration across the lifespan, and skeletal muscle hypertrophy prior to maturity. Our group has aimed to address whether satellite cells are required for hypertrophic growth in mature skeletal muscle. Here, we generated a comprehensive characterization and transcriptome-wide profiling of skeletal muscle during adaptation to exercise in the presence or absence of satellite cells in order to identify distinct phenotypes and gene networks influenced by satellite cell content. We administered vehicle or tamoxifen to adult Pax7-DTA mice and subjected them to progressive weighted wheel running (PoWeR). We then performed immunohistochemical analysis and whole-muscle RNA-seq of vehicle (SC+) and tamoxifen-treated (SC-) mice. Further, we performed single myonuclear RNA-seq to provide detailed information on how satellite cell fusion affects myonuclear transcription. We show that while skeletal muscle can mount a robust hypertrophic response to PoWeR in the absence of satellite cells, growth, and adaptation are ultimately blunted. Transcriptional profiling reveals several gene networks key to muscle adaptation are altered in the absence of satellite cells., (© The Author(s) 2020. Published by Oxford University Press on behalf of American Physiological Society.)

To date, studies that have aimed to investigate the role of satellite cells during adult skeletal muscle adaptation and hypertrophy have utilized a nontranslational stimulus and/or have been performed over a relatively short time frame. Although it has been shown that satellite cell depletion throughout adulthood does not drive skeletal muscle loss in sedentary mice, it remains unknown how satellite cells participate in skeletal muscle adaptation to long-term physical activity. The current study was designed to determine whether reduced satellite cell content throughout adulthood would influence the transcriptome-wide response to physical activity and diminish the adaptive response of skeletal muscle. We administered vehicle or tamoxifen to adult Pax7-diphtheria toxin A (DTA) mice to deplete satellite cells and assigned them to sedentary or wheel-running conditions for 13 mo. Satellite cell depletion throughout adulthood reduced balance and coordination, overall running volume, and the size of muscle proprioceptors (spindle fibers). Furthermore, satellite cell participation was necessary for optimal muscle fiber hypertrophy but not adaptations in fiber type distribution in response to lifelong physical activity. Transcriptome-wide analysis of the plantaris and soleus revealed that satellite cell function is muscle type specific; satellite cell-dependent myonuclear accretion was apparent in oxidative muscles, whereas initiation of G protein-coupled receptor (GPCR) signaling in the glycolytic plantaris may require satellite cells to induce optimal adaptations to long-term physical activity. These findings suggest that satellite cells play a role in preserving physical function during aging and influence muscle adaptation during sustained periods of physical activity.

Considering that thiol-containing enzymes like kinases are critical for several metabolic pathways and energy homeostasis, we investigated the effects of cystine dimethyl ester and/or cysteamine administration on kinases crucial for energy metabolism in the kidney of Wistar rats. Animals were injected twice a day with 1.6 µmol/g body weight cystine dimethyl ester and/or 0.26 µmol/g body weight cysteamine from the 16th to the 20th postpartum day and euthanized after 12 hours. Pyruvate kinase, adenylate kinase, creatine kinase activities and thiol/disulfide ratio were determined. Cystine dimethyl ester administration reduced thiol/disulfide ratio and inhibited the kinases activities. Cysteamine administration increased the thiol/disulfide ratio and co-administration with cystine dimethyl ester prevented the inhibition of the enzymes. Regression between the thiol/disulfide ratio, and the kinases activities were significant. These results suggest that redox status may regulate energy metabolism in the rat kidney. If thiol-containing enzymes inhibition and oxidative stress occur in patients with cystinosis, it is possible that lysosomal cystine depletion may not be the only beneficial effect of cysteamine administration, but also its antioxidant and thiol-protector effect.

Muscle hypertrophy occurs following increased protein synthesis, which requires activation of the ribosomal complex. Additionally, increased translational capacity via elevated ribosomal RNA (rRNA) synthesis has also been implicated in resistance training-induced skeletal muscle hypertrophy. The time course of ribosome biogenesis following resistance exercise (RE) and the impact exerted by differing recovery strategies remains unknown. In the present study, the activation of transcriptional regulators, the expression levels of pre-rRNA, and mature rRNA components were measured through 48 h after a single-bout RE. In addition, the effects of either low-intensity cycling (active recovery, ACT) or a cold-water immersion (CWI) recovery strategy were compared. Nine male subjects performed two bouts of high-load RE randomized to be followed by 10 min of either ACT or CWI. Muscle biopsies were collected before RE and at 2, 24, and 48 h after RE. RE increased the phosphorylation of the p38-MNK1-eIF4E axis, an effect only evident with ACT recovery. Downstream, cyclin D1 protein, total eIF4E, upstream binding factor 1 (UBF1), and c-Myc proteins were all increased only after RE with ACT. This corresponded with elevated abundance of the pre-rRNAs (45S, ITS-28S, ITS-5.8S, and ETS-18S) from 24 h after RE with ACT. In conclusion, coordinated upstream signaling and activation of transcriptional factors stimulated pre-rRNA expression after RE. CWI, as a recovery strategy, markedly blunted these events, suggesting that suppressed ribosome biogenesis may be one factor contributing to the impaired hypertrophic response observed when CWI is used regularly after exercise., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

This study aimed to determine through a questionnaire applied to interviewers, the current or past use of anabolic androgenic steroids (AAS), as well as other hormones (OH), and other medicines (OM), food supplement and illicit drugs among strength training apprentices in the city of Porto Alegre, RS. We interviewed 288 subjects draw from a sample of 13 gyms. The prevalence of current and past use of AAS was about 11.1% (32/288), OH 5.2% (16/288) and OM 4.2% (12/288). The most used AAS were nandrolone and stanozolol; the OH were gonadotropin, triiodothyronine (T3) and OM, like lipostabil, diuretics and veterinary medicines (Monovin E). The most frequent side-effects were behavioral such as humor oscillation, irritability and hostility, and endocrine disturbances such as acne and increased or decreased libido. When analyzed together with other hormones in a variable named "hormonal agents" (AH), AAS presented a statistical difference (p< 0.05) among genders considering that the most frequent use of AH occurred among men and those who consume food supplements. The comparison of these findings to other national and international results is difficult due to the epidemiological design. Even if it is considered, the observed prevalence suggests that preventive attitudes as well as special care in the orientation and education of this population must be taken.