The rRNA epitranscriptome and myonuclear SNORD landscape in skeletal muscle fibers contributes to ribosome heterogeneity and is altered by hypertrophic stimulus.

In cell biology, ribosomal RNA (rRNA) 2'O-methyl (2'-O-Me) is the most prevalent post-transcriptional 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. 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 rRNAand 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.

MicroRNA control of the myogenic cell transcriptome and proteome: the role of miR-16.

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.

Multi-transcriptome analysis following an acute skeletal muscle growth stimulus yields tools for discerning global and MYC regulatory networks.

Myc is a powerful transcription factor implicated in epigenetic reprogramming, cellular plasticity, and rapid growth as well as tumorigenesis. Cancer in skeletal muscle is extremely rare despite marked and sustained Myc induction during loading-induced hypertrophy. Here, we investigated global, actively transcribed, stable, and myonucleus-specific transcriptomes following an acute hypertrophic stimulus in mouse plantaris. With these datasets, we define global and Myc-specific dynamics at the onset of mechanical overload-induced muscle fiber growth. Data collation across analyses reveals an under-appreciated role for the muscle fiber in extracellular matrix remodeling during adaptation, along with the contribution of mRNA stability to epigenetic-related transcript levels in muscle. We also identify Runx1 and Ankrd1 (Marp1) as abundant myonucleus-enriched loading-induced genes. We observed that a strong induction of cell cycle regulators including Myc occurs with mechanical overload in myonuclei. Additionally, in vivo Myc-controlled gene expression in the plantaris was defined using a genetic muscle fiber-specific doxycycline-inducible Myc-overexpression model. We determined Myc is implicated in numerous aspects of gene expression during early-phase muscle fiber growth. Specifically, brief induction of Myc protein in muscle represses Reverbα, Reverbß, and Myh2 while increasing Rpl3, recapitulating gene expression in myonuclei during acute overload. Experimental, comparative, and in silico analyses place Myc at the center of a stable and actively transcribed, loading-responsive, muscle fiber-localized regulatory hub. Collectively, our experiments are a roadmap for understanding global and Myc-mediated transcriptional networks that regulate rapid remodeling in postmitotic cells. We provide open webtools for exploring the five RNA-seq datasets as a resource to the field.

Early transcriptomic signatures and biomarkers of renal damage due to prolonged exposure to embedded metal.

BACKGROUND: Prolonged exposure to toxic heavy metals leads to deleterious health outcomes including kidney injury. Metal exposure occurs through both environmental pathways including contamination of drinking water sources and from occupational hazards, including the military-unique risks from battlefield injuries resulting in retained metal fragments from bullets and blast debris. One of the key challenges to mitigate health effects in these scenarios is to detect early insult to target organs, such as the kidney, before irreversible damage occurs. METHODS: High-throughput transcriptomics (HTT) has been recently demonstrated to have high sensitivity and specificity as a rapid and cost-effective assay for detecting tissue toxicity. To better understand the molecular signature of early kidney damage, we performed RNA sequencing (RNA-seq) on renal tissue using a rat model of soft tissue-embedded metal exposure. We then performed small RNA-seq analysis on serum samples from the same animals to identify potential miRNA biomarkers of kidney damage. RESULTS: We found that metals, especially lead and depleted uranium, induce oxidative damage that mainly cause dysregulated mitochondrial gene expression. Utilizing publicly available single-cell RNA-seq datasets, we demonstrate that deep learning-based cell type decomposition effectively identified cells within the kidney that were affected by metal exposure. By combining random forest feature selection and statistical methods, we further identify miRNA-423 as a promising early systemic marker of kidney injury. CONCLUSION: Our data suggest that combining HTT and deep learning is a promising approach for identifying cell injury in kidney tissue. We propose miRNA-423 as a potential serum biomarker for early detection of kidney injury.

Mechanical overload-induced muscle-derived extracellular vesicles promote adipose tissue lipolysis.

How regular physical activity is able to improve health remains poorly understood. The release of factors from skeletal muscle following exercise has been proposed as a possible mechanism mediating such systemic benefits. We describe a mechanism wherein skeletal muscle, in response to a hypertrophic stimulus induced by mechanical overload (MOV), released extracellular vesicles (EVs) containing muscle-specific miR-1 that were preferentially taken up by epidydimal white adipose tissue (eWAT). In eWAT, miR-1 promoted adrenergic signaling and lipolysis by targeting Tfap2α, a known repressor of Adrß3 expression. Inhibiting EV release prevented the MOV-induced increase in eWAT miR-1 abundance and expression of lipolytic genes. Resistance exercise decreased skeletal muscle miR-1 expression with a concomitant increase in plasma EV miR-1 abundance, suggesting a similar mechanism may be operative in humans. Altogether, these findings demonstrate that skeletal muscle promotes metabolic adaptations in adipose tissue in response to MOV via EV-mediated delivery of miR-1.

An intron variant of the GLI family zinc finger 3 (GLI3) gene differentiates resistance training-induced muscle fiber hypertrophy in younger men.

We examined the association between genotype and resistance training-induced changes (12 wk) in dual x-ray energy absorptiometry (DXA)-derived lean soft tissue mass (LSTM) as well as muscle fiber cross-sectional area (fCSA; vastus lateralis; n = 109; age = 22 ± 2 y, BMI = 24.7 ± 3.1 kg/m2 ). Over 315 000 genetic polymorphisms were interrogated from muscle using DNA microarrays. First, a targeted investigation was performed where single nucleotide polymorphisms (SNP) identified from a systematic literature review were related to changes in LSTM and fCSA. Next, genome-wide association (GWA) studies were performed to reveal associations between novel SNP targets with pre- to post-training change scores in mean fCSA and LSTM. Our targeted investigation revealed no genotype-by-time interactions for 12 common polymorphisms regarding the change in mean fCSA or change in LSTM. Our first GWA study indicated no SNP were associated with the change in LSTM. However, the second GWA study indicated two SNP exceeded the significance level with the change in mean fCSA (P = 6.9 × 10-7 for rs4675569, 1.7 × 10-6 for rs10263647). While the former target is not annotated (chr2:205936846 (GRCh38.p12)), the latter target (chr7:41971865 (GRCh38.p12)) is an intron variant of the GLI Family Zinc Finger 3 (GLI3) gene. Follow-up analyses indicated fCSA increases were greater in the T/C and C/C GLI3 genotypes than the T/T GLI3 genotype (P < .05). Data from the Auburn cohort also revealed participants with the T/C and C/C genotypes exhibited increases in satellite cell number with training (P < .05), whereas T/T participants did not. Additionally, those with the T/C and C/C genotypes achieved myonuclear addition in response to training (P < .05), whereas the T/T participants did not. In summary, this is the first GWA study to examine how polymorphisms associate with the change in hypertrophy measures following resistance training. Future studies are needed to determine if the GLI3 variant differentiates hypertrophic responses to resistance training given the potential link between this gene and satellite cell physiology.

The role of extracellular vesicles in skeletal muscle and systematic adaptation to exercise.

Regular exercise has a central role in human health by reducing the risk of type 2 diabetes, obesity, stroke and cancer. How exercise is able to promote such systemic benefits has remained somewhat of a mystery but has been thought to be in part mediated by the release of myokines, skeletal muscle-specific cytokines, in response to exercise. Recent studies have revealed skeletal muscle can also release extracellular vesicles (EVs) into circulation following a bout of exercise. EVs are small membrane-bound vesicles capable of delivering biomolecules to recipient cells and subsequently altering their metabolism. The notion that EVs may have a role in both skeletal muscle and systemic adaptation to exercise has generated a great deal of excitement within a number of different fields including exercise physiology, neuroscience and metabolism. The purpose of this review is to provide an introduction to EV biology and what is currently known about skeletal muscle EVs and their potential role in the response of muscle and other tissues to exercise.

Dysbiosis of the gut microbiome impairs mouse skeletal muscle adaptation to exercise.

There is emerging evidence of a gut microbiome-skeletal muscle axis. The purpose of this study was to determine if an intact gut microbiome was necessary for skeletal muscle adaptation to exercise. Forty-two 4-month-old female C57BL/6J mice were randomly assigned to untreated (U) or antibiotic-treated (T) non-running controls (CU or CT, respectively) or progressive weighted wheel running (PoWeR, P) untreated (PU) or antibiotic-treated (PT) groups. Antibiotic treatment resulted in disruption of the gut microbiome as indicated by a significant depletion of gut microbiome bacterial species in both CT and PT groups. The training stimulus was the same between PU and PT groups as assessed by weekly (12.35 ± 2.06 vs. 11.09 ± 1.76 km/week, respectively) and total (778.9 ± 130.5 vs. 703.8 ± 112.9 km, respectively) running activity. In response to PoWeR, PT showed less hypertrophy of soleus type 1 and 2a fibres and plantaris type 2b/x fibres compared to PU. The higher satellite cell and myonuclei abundance of PU plantaris muscle after PoWeR was not observed in PT. The fibre-type shift of PU plantaris muscle to a more oxidative type 2a fibre composition following PoWeR was blunted in PT. There was no difference in serum cytokine levels among all groups suggesting disruption of the gut microbiome did not induce systemic inflammation. The results of this study provide the first evidence that an intact gut microbiome is necessary for skeletal muscle adaptation to exercise. KEY POINTS: Dysbiosis of the gut microbiome caused by continuous antibiotic treatment did not affect running activity. Continuous treatment with antibiotics did not result in systemic inflammation as indicated by serum cytokine levels. Gut microbiome dysbiosis was associated with blunted fibre type-specific hypertrophy in the soleus and plantaris muscles in response to progressive weighted wheel running (PoWeR). Gut microbiome dysbiosis was associated with impaired PoWeR-induced fibre-type shift in the plantaris muscle. Gut microbiome dysbiosis was associated with a loss of PoWeR-induced myonuclei accretion in the plantaris muscle.

Genetic and epigenetic regulation of skeletal muscle ribosome biogenesis with exercise.

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% V̇O2max ) 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.

Reduced mitochondrial DNA and OXPHOS protein content in skeletal muscle of children with cerebral palsy.

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.

Depletion of resident muscle stem cells negatively impacts running volume, physical function, and muscle fiber hypertrophy in response to lifelong physical activity.

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.

Serum extracellular vesicle miR-203a-3p content is associated with skeletal muscle mass and protein turnover during disuse atrophy and regrowth.

Small noncoding microRNAs (miRNAs) are important regulators of skeletal muscle size, and circulating miRNAs within extracellular vesicles (EVs) may contribute to atrophy and its associated systemic effects. The purpose of this study was to understand how muscle atrophy and regrowth alter in vivo serum EV miRNA content. We also associated changes in serum EV miRNA with protein synthesis, protein degradation, and miRNA within muscle, kidney, and liver. We subjected adult (10 mo) F344/BN rats to three conditions: weight bearing (WB), hindlimb suspension (HS) for 7 days to induce muscle atrophy, and HS for 7 days followed by 7 days of reloading (HSR). Microarray analysis of EV miRNA content showed that the overall changes in serum EV miRNA were predicted to target major anabolic, catabolic, and mechanosensitive pathways. MiR-203a-3p was the only miRNA demonstrating substantial differences in HS EVs compared with WB. There was a limited association of EV miRNA content to the corresponding miRNA content within the muscle, kidney, or liver. Stepwise linear regression demonstrated that EV miR-203a-3p was correlated with muscle mass and muscle protein synthesis and degradation across all conditions. Finally, EV miR-203a-3p expression was significantly decreased in human subjects who underwent unilateral lower limb suspension (ULLS) to induce muscle atrophy. Altogether, we show that serum EV miR-203a-3p expression is related to skeletal muscle protein turnover and atrophy. We suggest that serum EV miR-203a-3p content may be a useful biomarker and future work should investigate whether serum EV miR-203a-3p content is mechanistically linked to protein synthesis and degradation.

Time-course analysis of the effect of embedded metal on skeletal muscle gene expression.

As a consequence of military operations, many veterans suffer from penetrating wounds and long-term retention of military-grade heavy metal fragments. Fragments vary in size and location, and complete surgical removal may not be feasible or beneficial in all cases. Increasing evidence suggests retention of heavy metal fragments may have serious biological implications, including increased risks for malignant transformation. Previous studies assessed the tumorigenic effects of metal alloys in rats, demonstrating combinations of metals are sufficient to induce tumor formation after prolonged retention in skeletal muscle tissue. In this study, we analyzed transcriptional changes in skeletal muscle tissue in response to eight different military-relevant pure metals over 12 mo. We found that most transcriptional changes occur at 1 and 3 mo after metal pellets are embedded in skeletal muscle and these effects resolve at 6 and 12 mo. We also report significant immunogenic effects of nickel and cobalt and suppressive effects of lead and depleted uranium on gene expression. Overall, skeletal muscle exhibits a remarkable capacity to adapt to and recover from internalized metal fragments; however, the cellular response to chronic exposure may be restricted to the metal-tissue interface. These data suggest that unless affected regions are specifically captured by biopsy, it would be difficult to reliably detect changes in muscle gene expression that would be indicative of long-term adverse health outcomes.

Exercise-mediated alteration of hippocampal Dicer mRNA and miRNAs is associated with lower BACE1 gene expression and Aß1-42 in female 3xTg-AD mice.

Changes to cerebral miRNA expression have been implicated in the progression of Alzheimer's disease (AD), as miRNAs that regulate the expression of gene products involved in amyloid beta (Aß) processing, such as BACE1, are dysregulated in those that suffer from AD. Exercise training improves cognition and reduces BACE1 and Aß-plaque burden; however, the mechanisms are not fully understood. Using our progressive weighted wheel running (PoWeR) exercise program, we assessed the effect of 20 wk of exercise training on changes in hippocampal miRNA expression in female 3xTg-AD (3xTg) mice. PoWeR was sufficient to promote muscle hypertrophy and increase myonuclear abundance. Furthermore, PoWeR elevated hippocampal Dicer gene expression in 3xTg mice, while altering miRNA expression toward a more wild-type profile. Specifically, miR-29, which is validated to target BACE1, was significantly lower in sedentary 3xTg mice when compared with wild-type but was elevated following PoWeR. Accordingly, BACE1 gene expression, along with detergent-soluble Aß1-42, was lower in PoWeR-trained 3xTg mice. Our data suggest that PoWeR training upregulates Dicer gene expression to alter cerebral miRNA expression, which may contribute to reduced Aß accumulation and delay AD progression.NEW & NOTEWORTHY Previous studies have outlined the beneficial effects of exercise on lowering BACE1 expression and reducing Aß plaques. This study extends upon the work of others by outlining a new potential mechanism by which exercise elicits beneficial effects on Alzheimer's disease pathology, specifically through modulation of Dicer and miRNA expression. This is the first study to examine Dicer and miRNA expression in the hippocampus of the 3xTg model within the context of exercise.

Phosphorylation of eukaryotic initiation factor 4E is dispensable for skeletal muscle hypertrophy.

The eukaryotic initiation factor 4E (eIF4E) is a major mRNA cap-binding protein that has a central role in translation initiation. Ser209 is the single phosphorylation site within eIF4E and modulates its activity in response to MAPK pathway activation. It has been reported that phosphorylation of eIF4E at Ser209 promotes translation of key mRNAs, such as cyclin D1, that regulate ribosome biogenesis. We hypothesized that phosphorylation at Ser209 is required for skeletal muscle growth in response to a hypertrophic stimulus by promoting ribosome biogenesis. To test this hypothesis, wild-type (WT) and eIF4E knocked-in (KI) mice were subjected to synergist ablation to induce muscle hypertrophy of the plantaris muscle as the result of mechanical overload; in the KI mouse, Ser209 of eIF4E was replaced with a nonphosphorylatable alanine. Contrary to our hypothesis, we observed no difference in the magnitude of hypertrophy between WT and KI groups in response to 14 days of mechanical overload induced by synergist ablation. Similarly, the increases in cyclin D1 protein levels, ribosome biogenesis, and translational capacity did not differ between WT and KI groups. Based on these findings, we conclude that phosphorylation of eIF4E at Ser209 is dispensable for skeletal muscle hypertrophy in response to mechanical overload.

Elevated myonuclear density during skeletal muscle hypertrophy in response to training is reversed during detraining.

Myonuclei gained during exercise-induced skeletal muscle hypertrophy may be long-lasting and could facilitate future muscle adaptability after deconditioning, a concept colloquially termed "muscle memory." The evidence for this is limited, mostly due to the lack of a murine exercise-training paradigm that is nonsurgical and reversible. To address this limitation, we developed a novel progressive weighted-wheel-running (PoWeR) model of murine exercise training to test whether myonuclei gained during exercise persist after detraining. We hypothesized that myonuclei acquired during training-induced hypertrophy would remain following loss of muscle mass with detraining. Singly housed female C57BL/6J mice performed 8 wk of PoWeR, while another group performed 8 wk of PoWeR followed by 12 wk of detraining. Age-matched sedentary cage-dwelling mice served as untrained controls. Eight weeks of PoWeR yielded significant plantaris muscle fiber hypertrophy, a shift to a more oxidative phenotype, and greater myonuclear density than untrained mice. After 12 wk of detraining, the plantaris muscle returned to an untrained phenotype with fewer myonuclei. A finding of fewer myonuclei simultaneously with plantaris deconditioning argues against a muscle memory mechanism mediated by elevated myonuclear density in primarily fast-twitch muscle. PoWeR is a novel, practical, and easy-to-deploy approach for eliciting robust hypertrophy in mice, and our findings can inform future research on the mechanisms underlying skeletal muscle adaptive potential and muscle memory.

Extracellular vesicle distribution and localization in skeletal muscle at rest and following disuse atrophy.

BACKGROUND: Skeletal muscle (SkM) is a large, secretory organ that produces and releases myokines that can have autocrine, paracrine, and endocrine effects. Whether extracellular vesicles (EVs) also play a role in the SkM adaptive response and ability to communicate with other tissues is not well understood. The purpose of this study was to investigate EV biogenesis factors, marker expression, and localization across cell types in the skeletal muscle. We also aimed to investigate whether EV concentrations are altered by disuse atrophy. METHODS: To identify the potential markers of SkM-derived EVs, EVs were isolated from rat serum using density gradient ultracentrifugation, followed by fluorescence correlation spectroscopy measurements or qPCR. Single-cell RNA sequencing (scRNA-seq) data from rat SkM were analyzed to assess the EV biogenesis factor expression, and cellular localization of tetraspanins was investigated by immunohistochemistry. Finally, to assess the effects of mechanical unloading on EV expression in vivo, EV concentrations were measured in the serum by nanoparticle tracking analysis in both a rat and human model of disuse. RESULTS: In this study, we show that the widely used markers of SkM-derived EVs, α-sarcoglycan and miR-1, are undetectable in serum EVs. We also found that EV biogenesis factors, including the tetraspanins CD63, CD9, and CD81, are expressed by a variety of cell types in SkM. SkM sections showed very low detection of CD63, CD9, and CD81 in myofibers and instead accumulation within the interstitial space. Furthermore, although there were no differences in serum EV concentrations following hindlimb suspension in rats, serum EV concentrations were elevated in human subjects after bed rest. CONCLUSIONS: Our findings provide insight into the distribution and localization of EVs in SkM and demonstrate the importance of methodological guidelines in SkM EV research.

Does a Hypertrophying Muscle Fibre Reprogramme its Metabolism Similar to a Cancer Cell?

In 1924, Otto Warburg asked "How does the metabolism of a growing tissue differ from that of a non-growing tissue?" Currently, we know that proliferating healthy and cancer cells reprogramme their metabolism. This typically includes increased glucose uptake, glycolytic flux and lactate synthesis. A key function of this reprogramming is to channel glycolytic intermediates and other metabolites into anabolic reactions such as nucleotide-RNA/DNA synthesis, amino acid-protein synthesis and the synthesis of, for example, acetyl and methyl groups for epigenetic modification. In this review, we discuss evidence that a hypertrophying muscle similarly takes up more glucose and reprogrammes its metabolism to channel energy metabolites into anabolic pathways. We specifically discuss the functions of the cancer-associated enzymes phosphoglycerate dehydrogenase and pyruvate kinase muscle 2 in skeletal muscle. In addition, we ask whether increased glucose uptake by a hypertrophying muscle explains why muscularity is often negatively associated with type 2 diabetes mellitus and obesity.

Extracellular vesicle characteristics and microRNA content in cerebral palsy and typically developed individuals at rest and in response to aerobic exercise.

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 45 min of cycling (n = 10; TD EX) or were enrolled as controls with no exercise (n = 10; TD CON). Blood was drawn before and 30 min 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 a miR-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.

Skeletal Muscle Cell Growth Alters the Lipid Composition of Extracellular Vesicles.

We sought to characterize the lipid profile of skeletal muscle cell-derived Extracellular Vesicles (EVs) to determine if a hypertrophic stimulus would affect the lipid composition of C2C12 myotube-derived EVs. Analyses included C2C12 murine myoblasts differentiated into myotubes and treated with Insulin-Like Growth Factor 1 (IGF-1) for 24 h to induce hypertrophic growth. EVs were isolated from cell culture media, quantified using Nanoparticle Tracking Analysis (NTA) and analyzed using Transmission Electron Microscopy (TEM). EVs were homogenized and lipids extracted for quantification by Mass Spectrometry followed by downstream lipid class enrichment and lipid chain analysis. IGF-1 treatment elicited an increase in CD63 and CD81 levels (39% and 21%) compared to the controls (16%), respectively. Analysis revealed that skeletal muscle-derived EVs are enriched in bioactive lipids that are likely selectively incorporated into EVs during hypertrophic growth. IGF-1 treatment of myotubes had a significant impact on the levels of diacylglycerol (DG) and ceramide (Cer) in secreted EVs. Specifically, the proportion of unsaturated DG was two- to three-fold higher in EVs derived from IGF-treated cells, as compared to those from control cells. The levels of saturated DG were unaffected. Selective increases were similarly seen in C16- and C24-Cer but not in other species. Levels of free sphingoid bases tended to decrease, while those of sphingosine-1-phosphate was unaffected. Our results suggest that the lipid composition and biogenesis of skeletal muscle-derived EVs, are specific and highly selective during hypertrophic growth.