The interactive effect of sustained sleep restriction and resistance exercise on skeletal muscle transcriptomics in young females.

Both sleep loss and exercise regulate gene expression in skeletal muscle, yet little is known about how the interaction of these stressors affects the transcriptome. The aim of this study was to investigate the effect of nine nights of sleep restriction (SR), with repeated resistance exercise (REx) sessions, on the skeletal muscle transcriptome of young, trained females. Ten healthy females aged 18-35 yr old undertook a randomized cross-over study of nine nights of SR (5 h time in bed) and normal sleep (NS; ≥7 h time in bed) with a minimum 6-wk washout. Participants completed four REx sessions per condition (days 3, 5, 7, and 9). Muscle biopsies were collected both pre- and post-REx on days 3 and 9. Gene and protein expression were assessed by RNA sequencing and Western blot, respectively. Three or nine nights of SR had no effect on the muscle transcriptome independently of exercise. However, close to 3,000 transcripts were differentially regulated (false discovery rate < 0.05) 48 h after the completion of three resistance exercise sessions in both NS and SR conditions. Only 39% of downregulated genes and 18% of upregulated genes were common between both conditions, indicating a moderating effect of SR on the response to exercise. SR and REx interacted to alter the enrichment of skeletal muscle transcriptomic pathways in young, resistance-trained females. Performing exercise when sleep restricted may not provide the same adaptive response for individuals as if they were fully rested.NEW & NOTEWORTHY This study investigated the effect of nine nights of sleep restriction, with repeated resistance exercise sessions, on the skeletal muscle transcriptome of young, trained females. Sleep restriction and resistance exercise interacted to alter the enrichment of skeletal muscle transcriptomic pathways in young, resistance-trained females. Performing exercise when sleep restricted may not provide the same adaptive response for individuals as if they were fully rested.

Single-session measures of quadriceps neuromuscular function are reliable in healthy females and unaffected by age.

PURPOSE: This study aimed to determine the inter-session reliability of quadriceps neuromuscular function measurements in healthy young and older females. METHODS: Twenty-six females aged 19-74 years completed two identical experimental sessions on different days. Quadriceps neuromuscular function measurements included isometric maximal voluntary force, high- and low-frequency twitch force, voluntary and evoked (H-reflex, M-wave) electromyography (EMG), and estimated maximal torque, velocity and power derived from torque-velocity relationships. Intra-class correlation coefficients (ICCs), coefficients of variation (CoV) and Bland-Altman plots assessed inter-session reliability. The effect of age on reliability was assessed by linear regression. RESULTS: Excellent reliability (ICC > 0.8) was shown for all voluntary and evoked mechanical outcomes. Vastus lateralis EMG outcomes showed excellent reliability (ICC > 0.8) with CoVs < 12%, which were better than those of vastus medialis and rectus femoris. Age was not associated with reliability for 27/28 outcomes (P > 0.05). CONCLUSION: Excellent reliability of voluntary and evoked force and vastus lateralis EMG outcomes measured in healthy females can be attained in one experimental session, irrespective of age. Female neuromuscular function can be accurately assessed across the lifespan with minimal inconvenience, increasing feasibility for future research. The random error should however be considered when quantifying age-related differences in neuromuscular function.

Muscle miRNAs are influenced by sex at baseline and in response to exercise.

BACKGROUND: Sex differences in microRNA (miRNA) expression profiles have been found across multiple tissues. Skeletal muscle is one of the most sex-biased tissues of the body. MiRNAs are necessary for development and have regulatory roles in determining skeletal muscle phenotype and have important roles in the response to exercise in muscle. Yet there is limited research into the role and regulation of miRNAs in the skeletal muscle at baseline and in response to exercise, a well-known modulator of miRNA expression. The aim of this study was to investigate the effect of sex on miRNA expression in the skeletal muscle at baseline and after an acute bout of high-intensity interval exercise. A total of 758 miRNAs were measured using Taqman®miRNA arrays in the skeletal muscle of 42 healthy participants from the Gene SMART study (23 males and 19 females of comparable fitness levels and aged 18-45 years), of which 308 were detected. MiRNAs that differed by sex at baseline and whose change in expression following high-intensity interval exercise differed between the sexes were identified using mixed linear models adjusted for BMI and Wpeak. We performed in silico analyses to identify the putative gene targets of the exercise-induced, sex-specific miRNAs and overrepresentation analyses to identify enriched biological pathways. We performed functional assays by overexpressing two sex-biased miRNAs in human primary muscle cells derived from male and female donors to understand their downstream effects on the transcriptome. RESULTS: At baseline, 148 miRNAs were differentially expressed in the skeletal muscle between the sexes. Interaction analysis identified 111 miRNAs whose response to an acute bout of high-intensity interval exercise differed between the sexes. Sex-biased miRNA gene targets were enriched for muscle-related processes including proliferation and differentiation of muscle cells and numerous metabolic pathways, suggesting that miRNAs participate in programming sex differences in skeletal muscle function. Overexpression of sex-biased miRNA-30a and miRNA-30c resulted in profound changes in gene expression profiles that were specific to the sex of the cell donor in human primary skeletal muscle cells. CONCLUSIONS: We uncovered sex differences in the expression levels of muscle miRNAs at baseline and in response to acute high-intensity interval exercise. These miRNAs target regulatory pathways essential to skeletal muscle development and metabolism. Our findings highlight that miRNAs play an important role in programming sex differences in the skeletal muscle phenotype.

Regulation of mitochondrial calcium uniporter expression and calcium-dependent cell signaling by lncRNA Tug1 in cardiomyocytes.

Cardiomyocyte calcium homeostasis is a tightly regulated process. The mitochondrial calcium uniporter (MCU) complex can buffer elevated cytosolic Ca2+ levels and consists of pore-forming proteins including MCU, and various regulatory proteins such as mitochondrial calcium uptake proteins 1 and 2 (MICU1/2). The stoichiometry of these proteins influences the sensitivity to Ca2+ and the activity of the complex. However, the factors that regulate their gene expression remain incompletely understood. Long noncoding RNAs (lncRNAs) regulate gene expression through various mechanisms, and we recently found that the lncRNA Tug1 increased the expression of Mcu and associated genes. To further explore this, we performed antisense LNA knockdown of Tug1 (Tug1 KD) in H9c2 rat cardiomyocytes. Tug1 KD increased MCU protein expression, yet pyruvate dehydrogenase dephosphorylation, which is indicative of mitochondrial Ca2+ uptake, was not enhanced. However, RNA-seq revealed that Tug1 KD increased Mcu along with differential expression of >1,000 genes including many related to Ca2+ regulation pathways in the heart. To understand the effect of this on Ca2+ signaling, we measured phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and its downstream target cAMP Response Element-Binding protein (CREB), a transcription factor known to drive Mcu gene expression. In response to a Ca2+ stimulus, the increase in CaMKII and CREB phosphorylation was attenuated by Tug1 KD. Inhibition of CaMKII, but not CREB, partially prevented the Tug1 KD-mediated increase in Mcu. Together, these data suggest that Tug1 modulates MCU expression via a mechanism involving CaMKII and regulates cardiomyocyte Ca2+ signaling, which could have important implications for cardiac function.NEW & NOTEWORTHY Calcium is essential for signaling, excitation contraction, and energy homeostasis in the heart. Despite this, molecular regulators of these processes are not completely understood. We report that knockdown of lncRNA Tug1 alters the calcium handling transcriptome and increases mitochondrial calcium uniporter expression via a mechanism involving CaMKII. As overexpression of MCU is known to be protective against pathological cardiac remodeling, targeting Tug1 may be a potential strategy for treating cardiovascular disease.

Physiological and molecular sex differences in human skeletal muscle in response to exercise training.

Sex differences in exercise physiology, such as substrate metabolism and skeletal muscle fatigability, stem from inherent biological factors, including endogenous hormones and genetics. Studies investigating exercise physiology frequently include only males or do not take sex differences into consideration. Although there is still an underrepresentation of female participants in exercise research, existing studies have identified sex differences in physiological and molecular responses to exercise training. The observed sex differences in exercise physiology are underpinned by the sex chromosome complement, sex hormones and, on a molecular level, the epigenome and transcriptome. Future research in the field should aim to include both sexes, control for menstrual cycle factors, conduct large-scale and ethnically diverse studies, conduct meta-analyses to consolidate findings from various studies, leverage unique cohorts (such as post-menopausal, transgender, and those with sex chromosome abnormalities), as well as integrate tissue and cell-specific -omics data. This knowledge is essential for developing deeper insight into sex-specific physiological responses to exercise training, thus directing future exercise physiology studies and practical application.

The effect of the menstrual cycle on the circulating microRNA pool in human plasma: a pilot study.

STUDY QUESTION: Do ovarian hormone changes influence the levels of cell-free or circulating microRNA (cf-miRNA) across the menstrual cycle? SUMMARY ANSWER: This exploratory study suggests that fluctuations in hormonal levels throughout the menstrual cycle may alter cf-miRNAs levels. WHAT IS KNOWN ALREADY: cf-miRNA levels vary with numerous pathological and physiological conditions in both males and females and are regulated by exogenous and endogenous factors, including hormones. STUDY DESIGN, SIZE, DURATION: A prospective, monocentric study was conducted between March and November 2021. Since this was a pilot study, the sample size was based on feasibility as well as previous similar human studies conducted in different tissues. A total of 20 participants were recruited for the study. PARTICIPANTS/MATERIALS, SETTING, METHODS: We conducted an exploratory study where blood samples were collected from 16 eumenorrheic females in the early follicular phase, the ovulation phase and the mid-luteal phase of the menstrual cycle. The levels of oestrogen, progesterone, LH and FSH were measured in serum by electrochemiluminescence. The levels of 174 plasma-enriched miRNAs were profiled using a PCR-based panel, including stringent internal and external controls to account for the potential differences in RNA extraction and reverse-transcription stemming from low-RNA input samples. MAIN RESULTS AND THE ROLE OF CHANCE: This exploratory study suggests that cf-miRNAs may play an active role in the regulation of the female cycle by mediating the expression of genes during fluctuating hormonal changes. Linear mixed-models, adjusted for the relevant variables, showed associations between phases of the menstrual cycle, ovarian hormones and plasma cf-miRNA levels. Validated gene targets of the cf-miRNAs varying with the menstrual cycle were enriched within female reproductive tissues and are primarily involved in cell proliferation and apoptosis. LARGE SCALE DATA: All relevant data are available from the Mendeley database: LEGER, Bertrand (2022), 'MiRNA and menstrual cycle', Mendeley Data, V1, doi: 10.17632/2br3zp79m3.1. LIMITATIONS, REASONS FOR CAUTION: Our study was conducted on a small participant cohort. However, it was tightly controlled for endogenous and exogenous confounders, which is critical to ensure robust and reproducible cf-miRNA research. Both adjusted and non-adjusted P-values are presented throughout the article. WIDER IMPLICATIONS OF THE FINDINGS: Measures of ovarian hormones should be rigorously included in future studies assessing cf-miRNA levels in females and used as time-varying confounders. Our results reinforce the importance of accounting for female-specific biological processes in physiology research by implementing practical or statistical mitigation strategies during data collection and analysis. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Clinique romande de réadaptation, Sion, Switzerland. S.L. was supported by an Australian Research Council (ARC) Future Fellowship (FT10100278). D.H. was supported by an Executive Dean's Postdoctoral Research Fellowship from Deakin University. The authors declare no competing interests.

Long non-coding RNA Tug1 modulates mitochondrial and myogenic responses to exercise in skeletal muscle.

BACKGROUND: Mitochondria have an essential role in regulating metabolism and integrate environmental and physiological signals to affect processes such as cellular bioenergetics and response to stress. In the metabolically active skeletal muscle, mitochondrial biogenesis is one important component contributing to a broad set of mitochondrial adaptations occurring in response to signals, which converge on the biogenesis transcriptional regulator peroxisome proliferator-activated receptor coactivator 1-alpha (PGC-1α), and is central to the beneficial effects of exercise in skeletal muscle. We investigated the role of long non-coding RNA (lncRNA) taurine-upregulated gene 1 (TUG1), which interacts with PGC-1α in regulating transcriptional responses to exercise in skeletal muscle. RESULTS: In human skeletal muscle, TUG1 gene expression was upregulated post-exercise and was also positively correlated with the increase in PGC-1α gene expression (PPARGC1A). Tug1 knockdown (KD) in differentiating mouse myotubes led to decreased Ppargc1a gene expression, impaired mitochondrial respiration and morphology, and enhanced myosin heavy chain slow isoform protein expression. In response to a Ca2+-mediated stimulus, Tug1 KD prevented an increase in Ppargc1a expression. RNA sequencing revealed that Tug1 KD impacted mitochondrial Ca2+ transport genes and several downstream PGC-1α targets. Finally, Tug1 KD modulated the expression of ~300 genes that were upregulated in response to an in vitro model of exercise in myotubes, including genes involved in regulating myogenesis. CONCLUSIONS: We found that TUG1 is upregulated in human skeletal muscle after a single session of exercise, and mechanistically, Tug1 regulates transcriptional networks associated with mitochondrial calcium handling, muscle differentiation and myogenesis. These data demonstrate that lncRNA Tug1 exerts regulation over fundamental aspects of skeletal muscle biology and response to exercise stimuli.

Gendered perspectives on women's anabolic-androgenic steroid (AAS) usage practices.

BACKGROUND: The masculinizing effects from anabolic-androgenic steroid (AAS) appear to be different between men and women, leading to calls for more gender-specific information regarding women and AAS use. This study sought to gather perspectives from both men and women on the unique challenges surrounding women's use of AAS, irrespective of their personal use. Secondly, the study interrogated how women's AAS practices differ from those of men specifically. METHODS: The data presented in this paper come from a subsample of participants who participated in a larger study investigating women and performance and image enhancing drug (PIED) use in Australia. Participants were included in the current analysis if they were: (i) males or females who competed with or coached female strength athletes using AAS and (ii) female and male strength athletes who used AAS. The final sample comprised 21 participants of which there was a proportion of males (n = 7) and females (n = 7) using AAS. RESULTS: Women's choices in AAS selection were predominantly around oral compounds (e.g. Oxandrolone) as well as other PIEDs (e.g. Clenbuterol). Some women report the use of injectable AAS represents a change in the profile of the typical female user as it reportedly comes alongside drastic physical and psychological changes. CONCLUSIONS: The unique challenges facing women who use AAS are largely isolation and stigma, with little evidence-based practice or education being available to them online or through peer-groups. Future work may consider piloting harm reduction strategies that may be co-designed with this group.

Striated muscle activator of Rho signalling (STARS) overexpression in the mdx mouse enhances muscle functional capacity and regulates the actin cytoskeleton and oxidative phosphorylation pathways.

NEW FINDINGS: What is the central question of this study? Striated muscle activator of rho signalling (STARS) is an actin-binding protein that regulates transcriptional pathways controlling muscle function, growth and myogenesis, processes that are impaired in dystrophic muscle: what is the regulation of the STARS pathway in Duchenne muscular dystrophy (DMD)? What is the main finding and its importance? Members of the STARS signalling pathway are reduced in the quadriceps of patients with DMD and in mouse models of muscular dystrophy. Overexpression of STARS in the dystrophic deficient mdx mouse model increased maximal isometric specific force and upregulated members of the actin cytoskeleton and oxidative phosphorylation pathways. Regulating STARS may be a therapeutic approach to enhance muscle health. ABSTRACT: Duchenne muscular dystrophy (DMD) is characterised by impaired cytoskeleton organisation, cytosolic calcium handling, oxidative stress and mitochondrial dysfunction. This results in progressive muscle damage, wasting and weakness and premature death. The striated muscle activator of rho signalling (STARS) is an actin-binding protein that activates the myocardin-related transcription factor-A (MRTFA)/serum response factor (SRF) transcriptional pathway, a pathway regulating cytoskeletal structure and muscle function, growth and repair. We investigated the regulation of the STARS pathway in the quadriceps muscle from patients with DMD and in the tibialis anterior (TA) muscle from the dystrophin-deficient mdx and dko (utrophin and dystrophin null) mice. Protein levels of STARS, SRF and RHOA were reduced in patients with DMD. STARS, SRF and MRTFA mRNA levels were also decreased in DMD muscle, while Stars mRNA levels were decreased in the mdx mice and Srf and Mrtfa mRNAs decreased in the dko mice. Overexpressing human STARS (hSTARS) in the TA muscles of mdx mice increased maximal isometric specific force by 13% (P < 0.05). This was not associated with changes in muscle mass, fibre cross-sectional area, fibre type, centralised nuclei or collagen deposition. Proteomics screening followed by pathway enrichment analysis identified that hSTARS overexpression resulted in 31 upregulated and 22 downregulated proteins belonging to the actin cytoskeleton and oxidative phosphorylation pathways. These pathways are impaired in dystrophic muscle and regulate processes that are vital for muscle function. Increasing the STARS protein in dystrophic muscle improves muscle force production, potentially via synergistic regulation of cytoskeletal structure and energy production.

Constructing 3D Macroporous Microfibrous Scaffolds with a Featured Surface by Heat Welding and Embossing.

Three-dimensional (3D) microfibrous scaffolds hold great promise for biomedical applications due to their good mechanical properties and biomimetic structure similar to that of the fibrous natural extracellular matrix. However, the large diameter and smooth surface of microfibers provide limited cues for regulating cell activity and behaviors. In this work, we report a facile heat-welding-and-embossing strategy to develop 3D macroporous microfibrous scaffolds with a featured surface topography. Here, solid monosodium glutamate (MSG) particles with crystalline ridge-like surface features play a key role as templates in both the formation of scaffold pores and the surface embossing of scaffold fibers when short thermoplastic polypropylene microfibers were heat-welded. The embossing process can be programmed by adjusting heating temperatures and MSG/fiber ratios. Compared to traditional 3D microfibrous scaffolds, the as-welded 3D scaffolds show higher compressive strength and modulus. Taking mouse C2C12 myoblasts as a model cell line, the scaffolds with embossed surface features significantly promoted the growth of cells, interactions of cells and scaffolds, and formation of myotubes. The findings indicate that the as-prepared 3D scaffolds are a good platform for cell culture study. The facile strategy can be applied to fabricate different fibrous scaffolds by changing the combination of templates and thermoplastic polymer fibers with a melting temperature lower than that of the template. The obtained insights in this work could provide a guide and inspiration for the design and fabrication of functional 3D fibrous scaffolds.

Identifying and Assessing Inter-Muscular Fat at the Distal Diaphyseal Femur Measured by Peripheral Quantitative Computed Tomography (pQCT).

INTRODUCTION: Inter-/intramuscular fat can be assessed with peripheral Quantitative Computed Tomography (pQCT) and is of interest as an indicator of "muscle quality." Typical pQCT scan sites (forearm, lower leg) have a low amount of inter-/intramuscular fat, however distal diaphyseal femur scan sites with conspicuous inter-/intramuscular fat have been identified as potentially more prudent scan sites, even in healthy adolescents. However, current state of the art analysis methods require labor-intensive manual segmentation of the scan. The purpose of the present study was to evaluate the reliability of a novel open source automated enclosing convex polygon approach (source code https://github.com/tjrantal/pQCT, commit cec9bce) to quantify inter-/intramuscular fat from femoral pQCT scans in healthy adults. METHODOLOGY: The distal diaphyseal femur (25% of tibial length from the knee joint towards the hip) of 27 adults aged 18-50 yr were scanned twice, 1 wk apart, using pQCT. Subcutaneous fat, muscle, inter-/intramuscular fat, and marrow areas, and corresponding densities were evaluated using a method we have reported previously, as well as the novel enclosing convex polygon method. RESULTS: The session-to-session reliability of the assessments was fair to excellent using the previously reported method as indicated by intraclass correlation coefficient (ICC2,1) ranging from 0.45 to 1.00, while the novel method produced excellent reliability (ICC2,1 0.78-1.00). CONCLUSION: Distal diaphyseal femur appears to be a potentially informative and prudent scan site for inter-/intramuscular fat evaluation with pQCT.

MicroRNA-99b-5p downregulates protein synthesis in human primary myotubes.

microRNAs (miRNAs) are important regulators of cellular homeostasis and exert their effect by directly controlling protein expression. We have previously reported an age-dependent negative association between microRNA-99b (miR-99b-5p) expression and muscle protein synthesis in human muscle in vivo. Here we investigated the role of miR-99b-5p as a potential negative regulator of protein synthesis via inhibition of mammalian target for rapamycin (MTOR) signaling in human primary myocytes. Overexpressing miR-99b-5p in human primary myotubes from young and old subjects significantly decreased protein synthesis with no effect of donor age. A binding interaction between miR-99b-5p and its putative binding site within the MTOR 3'-untranslated region (UTR) was confirmed in C2C12 myoblasts. The observed decline in protein synthesis was, however, not associated with a suppression of the MTOR protein but of its regulatory associated protein of mTOR complex 1 (RPTOR). These results demonstrate that modulating the expression levels of a miRNA can regulate protein synthesis in human muscle cells and provide a potential mechanism for muscle wasting in vivo.

An obesogenic maternal environment impairs mouse growth patterns, satellite cell activation, and markers of postnatal myogenesis.

Skeletal muscle is sensitive to environmental cues that are first present in utero. Maternal overnutrition is a model of impaired muscle development leading to structural and metabolic dysfunction in adult life. In this study, we investigated the effect of an obesogenic maternal environment on growth and postnatal myogenesis in the offspring. Male C57BL/6J mice born to chow- or high-fat-diet-fed mothers were allocated to four different groups at the end of weaning. For the following 10 wk, half of the pups were maintained on the same diet as their mother and half of the pups were switched to the other diet (chow or high-fat). At 12 wk of age, muscle injury was induced using an intramuscular injection of barium chloride. Seven days later, mice were humanely killed and muscle tissue was harvested. A high-fat maternal diet impaired offspring growth patterns and downregulated satellite cell activation and markers of postnatal myogenesis 7 days after injury without altering the number of newly synthetized fibers over the whole 7-day period. Importantly, a healthy postnatal diet could not reverse any of these effects. In addition, we demonstrated that postnatal myogenesis was associated with a diet-independent upregulation of three miRNAs, mmu-miR-31-5p, mmu-miR-136-5p, and mmu-miR-296-5p. Furthermore, in vitro analysis confirmed the role of these miRNAs in myocyte proliferation. Our findings are the first to demonstrate that maternal overnutrition impairs markers of postnatal myogenesis in the offspring and are particularly relevant to today's society where the incidence of overweight/obesity in women of childbearing age is increasing.

Mitochondrial regulation in skeletal muscle: A role for non-coding RNAs?

NEW FINDINGS: What is the topic of this review? This article draws evidence from the current literature to support the hypothesis that non-coding RNA-mediated gene regulation takes place in the mitochondria. An emphasis is put on skeletal muscle. What advances does it highlight? This review highlights the potential role of microRNAs and long non-coding RNAs in mitochondrial gene regulation. The discovery of a new level of skeletal muscle mitochondria-controlled gene regulation presents exciting perspectives for our understanding of skeletal muscle physiology in health and disease. ABSTRACT: Skeletal muscle is a highly metabolic tissue characterized by high mitochondrial abundance. As such, skeletal muscle homeostasis relies on the tight control of mitochondrial gene expression to ensure efficient mitochondrial function. Mitochondria retain a conserved genome from prokaryotic ancestors, and mitochondrial gene regulation relies on communication between mitochondrial- and nuclear-encoded transcripts. Small and long non-coding RNAs (ncRNAs) have regulatory roles in the modulation of gene expression. Emerging evidence demonstrates that regulatory ncRNAs, particularly microRNAs (miRNAs) and long ncRNAs (lncRNAs), localize within the mitochondria in diverse physiological and pathological states. These molecules present intriguing possibilities for the regulation of mitochondrial gene expression. Current research suggests that all known miRNAs are encoded by the nuclear genome but can target mitochondrial genes. Initial investigations demonstrate direct interactions between the muscle-enriched miR-1 and miR-181c and mitochondrial transcripts, suggesting advanced roles of miRNAs in mitochondrial gene regulation. This review draws evidence from the current literature to discuss the hypothesis that a level of ncRNA-mediated gene regulation, in particular miRNA-mediated gene regulation, takes place in the mitochondria. Although ncRNA-mediated regulation of the mitochondrial genome is a relatively unexplored field, it presents exciting possibilities to further our understanding of mitochondrial metabolism and human muscle physiology.

Dysregulation of microRNA biogenesis machinery and microRNA/RNA ratio in skeletal muscle of amyotrophic lateral sclerosis mice.

INTRODUCTION: The pathology of amyotrophic lateral sclerosis (ALS) is associated with impaired RNA processing and microRNA (miRNA) dysregulation. Here we investigate the regulation of the members of the miRNA biogenesis pathways and total miRNA levels at different stages of the disease. METHODS: Muscle, brain, and spinal cord tissue were obtained from presymptomatic, symptomatic, and end-stage superoxide dismutase 1 (SOD1)G93A mice. miRNA and transcript levels were measured by quantitative polymerase chain reaction. RESULTS: As the diseases progresses, several genes involved in miRNA biogenesis as well as the miRNA/total RNA (totRNA) ratio increased in the tibialis anterior (TA) muscle but not in the soleus or in neural tissue. DISCUSSION: We propose that a dysregulation in the miRNA/totRNA ratio in the TA muscle from SOD1G93A mice reflects a pathological increase in miRNA biogenesis machinery. Alterations in the miRNA/totRNA ratio influence the levels of reference noncoding RNAs and may therefore potentially compromise the accuracy of commonly used miRNA normalization strategies. Muscle Nerve 57: 838-847, 2018.

Hsp72 preserves muscle function and slows progression of severe muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder caused by mutations in the dystrophin gene that result in the absence of the membrane-stabilizing protein dystrophin. Dystrophin-deficient muscle fibres are fragile and susceptible to an influx of Ca(2+), which activates inflammatory and muscle degenerative pathways. At present there is no cure for DMD, and existing therapies are ineffective. Here we show that increasing the expression of intramuscular heat shock protein 72 (Hsp72) preserves muscle strength and ameliorates the dystrophic pathology in two mouse models of muscular dystrophy. Treatment with BGP-15 (a pharmacological inducer of Hsp72 currently in clinical trials for diabetes) improved muscle architecture, strength and contractile function in severely affected diaphragm muscles in mdx dystrophic mice. In dko mice, a phenocopy of DMD that results in severe spinal curvature (kyphosis), muscle weakness and premature death, BGP-15 decreased kyphosis, improved the dystrophic pathophysiology in limb and diaphragm muscles and extended lifespan. We found that the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA, the main protein responsible for the removal of intracellular Ca(2+)) is dysfunctional in severely affected muscles of mdx and dko mice, and that Hsp72 interacts with SERCA to preserve its function under conditions of stress, ultimately contributing to the decreased muscle degeneration seen with Hsp72 upregulation. Treatment with BGP-15 similarly increased SERCA activity in dystrophic skeletal muscles. Our results provide evidence that increasing the expression of Hsp72 in muscle (through the administration of BGP-15) has significant therapeutic potential for DMD and related conditions, either as a self-contained therapy or as an adjuvant with other potential treatments, including gene, cell and pharmacological therapies.

MicroRNA expression patterns in post-natal mouse skeletal muscle development.

BACKGROUND: MiRNAs are essential regulators of skeletal muscle development and homeostasis. To date, the role and regulation of miRNAs in myogenesis have been mostly studied in tissue culture and during embryogenesis. However, little information relating to miRNA regulation during early post-natal skeletal muscle growth in mammals is available. Using a high-throughput miRNA qPCR-based array, followed by stringent statistical and bioinformatics analysis, we describe the expression pattern and putative role of 768 miRNAs in the quadriceps muscle of mice aged 2 days, 2 weeks, 4 weeks and 12 weeks. RESULTS: Forty-six percent of all measured miRNAs were expressed in mouse quadriceps muscle during the first 12 weeks of life. We report unprecedented changes in miRNA expression levels over time. The expression of a majority of miRNAs significantly decreased with post-natal muscle maturation in vivo. MiRNA clustering identified 2 subsets of miRNAs that are potentially involved in cell proliferation and differentiation, mainly via the regulation of non-muscle specific targets. CONCLUSION: Collective miRNA expression in mouse quadriceps muscle is subjected to substantial levels of regulation during the first 12 weeks of age. This study identified a new suite of highly conserved miRNAs that are predicted to influence early muscle development. As such it provides novel knowledge pertaining to post-natal myogenesis and muscle regeneration in mammals.

Expression of microRNAs and target proteins in skeletal muscle of rats selectively bred for high and low running capacity.

Impairments in mitochondrial function and substrate metabolism are implicated in the etiology of obesity and Type 2 diabetes. MicroRNAs (miRNAs) can degrade mRNA or repress protein translation and have been implicated in the development of such disorders. We used a contrasting rat model system of selectively bred high- (HCR) or low- (LCR) intrinsic running capacity with established differences in metabolic health to investigate the molecular mechanisms through which miRNAs regulate target proteins mediating mitochondrial function and substrate oxidation processes. Quantification of select miRNAs using the rat miFinder miRNA PCR array revealed differential expression of 15 skeletal muscles (musculus tibialis anterior) miRNAs between HCR and LCR rats (14 with higher expression in LCR; P < 0.05). Ingenuity Pathway Analysis predicted these altered miRNAs to collectively target multiple proteins implicated in mitochondrial dysfunction and energy substrate metabolism. Total protein abundance of citrate synthase (CS; miR-19 target) and voltage-dependent anion channel 1 (miR-7a target) were higher in HCR compared with LCR cohorts (~57 and ~26%, respectively; P < 0.05). A negative correlation was observed for miR-19a-3p and CS (r = 0.32, P = 0.015) protein expression. To determine whether miR-19a-3p can regulate CS in vitro, we performed luciferase reporter and transfection assays in C2C12 myotubes. MiR-19a-3p binding to the CS untranslated region did not change luciferase reporter activity; however, miR-19a-3p transfection decreased CS protein expression (∼70%; P < 0.05). The differential miRNA expression targeting proteins implicated in mitochondrial dysfunction and energy substrate metabolism may contribute to the molecular basis, mediating the divergent metabolic health profiles of LCR and HCR rats.

Overexpression of sphingosine kinase 1 in liver reduces triglyceride content in mice fed a low but not high-fat diet.

Hepatic insulin resistance is a major risk factor for the development of type 2 diabetes and is associated with the accumulation of lipids, including diacylglycerol (DAG), triacylglycerols (TAG) and ceramide. There is evidence that enzymes involved in ceramide or sphingolipid metabolism may have a role in regulating concentrations of glycerolipids such as DAG and TAG. Here we have investigated the role of sphingosine kinase (SphK) in regulating hepatic lipid levels. We show that mice on a high-fat high-sucrose diet (HFHS) displayed glucose intolerance, elevated liver TAG and DAG, and a reduction in total hepatic SphK activity. Reduced SphK activity correlated with downregulation of SphK1, but not SphK2 expression, and was not associated with altered ceramide levels. The role of SphK1 was further investigated by overexpressing this isoform in the liver of mice in vivo. On a low-fat diet (LFD) mice overexpressing liver SphK1, displayed reduced hepatic TAG synthesis and total TAG levels, but with no change to DAG or ceramide. These mice also exhibited no change in gluconeogenesis, glycogenolysis or glucose tolerance. Similarly, overexpression of SphK1 had no effect on the pattern of endogenous glucose production determined during a glucose tolerance test. Under HFHS conditions, normalization of liver SphK activity to levels observed in LFD controls did not alter hepatic TAG concentrations. Furthermore, DAG, ceramide and glucose tolerance were also unaffected. In conclusion, our data suggest that SphK1 plays an important role in regulating TAG metabolism under LFD conditions.