Ribosome biogenesis during skeletal muscle hypertrophy

Muscle adaptation to chronic resistance exercise (RE) is the result of a cumulative effect on gene expression and protein content. Following a bout of RE, muscle protein synthesis increases and, if followed by consecutive bouts (training), protein accretion and muscle hypertrophy develops. The protein synthetic capacity of the muscle is dictated by ribosome content. Therefore, the general aim of this thesis is to investigate the regulation of ribosome biogenesis during skeletal muscle hypertrophy. To begin addressing this question, we employed a prevalent rodent model of skeletal muscle hypertrophy, synergist ablation (SA) of plantar flexor muscles. SA resulted in muscle hypertrophy with a concomitant increase in total RNA content. We observed a marked re- induction of c-Myc in overloaded skeletal muscle correlating with the expression of Pol I specific factors and 45S pre-rRNA levels. UBF and WSTF were increased at the protein level in myonuclei and enriched at the rDNA promoter following mechanical loading. This was associated with increased Pol I loading and epigenetic marks of active, de-condensed chromatin at the rDNA promoter. Similarly, acute mechanical loading of human skeletal muscle resulted increased mTOR signalling, a re-induction of c-Myc and increased 45s pre- rRNA abundance. Once the hypertrophic phenotype was evident in both mouse and human, rDNA transcription had returned to baseline levels. A conditional, skeletal muscle specific c- Myc knockout model was generated to investigate the mechanistic importance of c-Myc in ribosome biogenesis and hypertrophy. Animals lacking c-Myc in skeletal muscle displayed normal post-natal development with respect to body weight, muscle size, rDNA transcription and RNA content. To further challenge the growth machinery in skeletal muscle lacking c- Myc, animals were subjected to SA-imposed overload. No difference with respect to RNA accumulation or hypertrophic response was detected, indicating that c-Myc is dispensable for cellular hypertrophy in terminally differentiated muscle cells. These results were verified in C2C12 myotubes with compromised c-Myc function (Myra-A). On the contrary, c-Myc inactivation in proliferating C2C12 myoblasts severely compromised rDNA transcription, DNA synthesis as well as cell proliferation. Thus, our data suggests cell stage-specific effects of ablated c-Myc function in cells of the myogenic lineage. Moreover, the importance of the mTOR network for rDNA gene regulation during skeletal muscle hypertrophy was investigated. mTOR inhibition with rapamycin prevented the development of hypertrophy, and decreased mTOR binding to rDNA correlated with decreased 45S pre-rRNA synthesis and perturbed rRNA accumulation. Selective inhibition of RNA Pol I with CX-5461 efficiently prevented skeletal muscle hypertrophy in a similar fashion. Collectively, the data presented in this thesis proposes an important role for ribosome biogenesis at the onset of skeletal muscle hypertrophy, which, if blocked, prevents the development of the hypertrophic phenotype. During the time in which measurable hypertrophy is evident, rRNA synthesis rates have normalized. In addition, our results indicate that mTOR regulates this process via numerous different mechanisms, including direct binding to the rDNA promoter but likely not via p70S6K1-dependent functions. c-Myc proved dispensable for Pol I transcription and skeletal muscle hypertrophy in the differentiated state both in vivo and in vitro, instead controlling cell proliferation in myoblasts, likely via a Pol I-dependent mechanism

BioFACTS : biomarkers of rhabdomyolysis in the diagnosis of acute compartment syndrome - protocol for a prospective multinational, multicentre study involving patients with tibial fractures

Introduction The ischaemic pain of acute compartment syndrome (ACS) can be difficult to discriminate from the pain linked to an associated fracture. Lacking objective measures, the decision to perform fasciotomy is based on clinical findings and performed at a low level of suspicion. Biomarkers of muscle cell damage may help to identify and monitor patients at risk, similar to current routines for patients with acute myocardial infarction. This study will test the hypothesis that biomarkers of muscle cell damage can predict ACS in patients with tibial fractures. Methods and analysis Patients aged 15-65 years who have suffered a tibial fracture will be included. Plasma (P)-myoglobin and P-creatine phosphokinase will be analysed at 6-hourly intervals after admission to the hospital (for 48 hours) and-if applicable-after surgical fixation or fasciotomy (for 24 hours). In addition, if ACS is suspected at any other point in time, blood samples will be collected at 6-hourly intervals. An independent expert panel will assess the study data and will classify those patients who had undergone fasciotomy into those with ACS and those without ACS. All primary comparisons will be perforated between fracture patients with and without ACS. The area under the receiver operator characteristics curves will be used to identify the success of the biomarkers in discriminating between fracture patients who develop ACS and those who do not. Logistic regression analyses will be used to assess the discriminative abilities of the biomarkers to predict ACS corrected for prespecified covariates. Ethics and dissemination The study has been approved by the Regional Ethical Review Boards in Linkoping (2017/514-31) and Helsinki/Uusimaa (HUS/2500/2000). The BioFACTS study will be reported in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology recommendations.Peer reviewe

A prophylactic subcutaneous dose of the anticoagulant tinzaparin does not influence qPCR-based assessment of circulating levels of miRNA in humans

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

Extracellular vesicle characteristics and micro RNA 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 \u3c 0.05) of EVs. At baseline, let-7a, let-7b and let-7e were downregulated in individuals with CP compared to TD (p \u3c 0.05), while miR-100 expression was higher, and miR-877 and miR-4433 lower in CP compared to TD after exercise (p \u3c 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 C

Humanin skeletal muscle protein levels increase after resistance training in men with impaired glucose metabolism

Humanin (HN) is a mitochondrially encoded and secreted peptide linked to glucose metabolism and tissue protecting mechanisms. Whether skeletal muscle HN gene or protein expression is influenced by exercise remains unknown. In this intervention study we show, for the first time, that HN protein levels increase in human skeletal muscle following 12 weeks of resistance training in persons with prediabetes. Male subjects (n = 55) with impaired glucose regulation (IGR) were recruited and randomly assigned to resistance training, Nordic walking or a control group. The exercise interventions were performed three times per week for 12 weeks with progressively increased intensity during the intervention period. Biopsies from the vastus lateralis muscle and venous blood samples were taken before and after the intervention. Skeletal muscle and serum protein levels of HN were analyzed as well as skeletal muscle gene expression of the mitochondrially encoded gene MT-RNR2, containing the open reading frame for HN. To elucidate mitochondrial training adaptation, mtDNA, and nuclear DNA as well as Citrate synthase were measured. Skeletal muscle HN protein levels increased by 35% after 12 weeks of resistance training. No change in humanin protein levels was seen in serum in any of the intervention groups. There was a significant correlation between humanin levels in serum and the improvements in the 2 h glucose loading test in the resistance training group. The increase in HN protein levels in skeletal muscle after regular resistance training in prediabetic males may suggest a role for HN in the regulation of glucose metabolism. Given the preventative effect of exercise on diabetes type 2, the role of HN as a mitochondrially derived peptide and an exercise-responsive mitokine warrants further investigation.</p

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, Reverbβ, 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

Acute hypoxia attenuates resistance exercise-induced ribosome signaling but does not impact satellite cell pool expansion in human skeletal muscle.

Cumulative evidence supports the hypothesis that hypoxia acts as a regulator of muscle mass. However, the underlying molecular mechanisms remain incompletely understood, particularly in human muscle. Here we examined the effect of hypoxia on signaling pathways related to ribosome biogenesis and myogenic activity following an acute bout of resistance exercise. We also investigated whether hypoxia influenced the satellite cell response to resistance exercise. Employing a randomized, crossover design, eight men performed resistance exercise in normoxia (FiO2 21%) or normobaric hypoxia (FiO2 12%). Muscle biopsies were collected in a time-course manner (before, 0, 90, 180 min and 24 h after exercise) and were analyzed with respect to cell signaling, gene expression and satellite cell content using immunoblotting, RT-qPCR and immunofluorescence, respectively. In normoxia, resistance exercise increased the phosphorylation of RPS6, TIF-1A and UBF above resting levels. Hypoxia reduced the phosphorylation of these targets by ~37%, ~43% and ~ 67% throughout the recovery period, respectively (p &lt; .05 vs. normoxia). Resistance exercise also increased 45 S pre-rRNA expression and mRNA expression of c-Myc, Pol I and TAF-1A above resting levels, but no differences were observed between conditions. Similarly, resistance exercise increased mRNA expression of myogenic regulatory factors throughout the recovery period and Pax7+ cells were elevated 24 h following exercise in mixed and type II muscle fibers, with no differences observed between normoxia and hypoxia. In conclusion, acute hypoxia attenuates ribosome signaling, but does not impact satellite cell pool expansion and myogenic gene expression following a bout of resistance exercise in human skeletal muscle

Skeletal muscle proteolysis in response to short-term unloading in humans

Skeletal muscle atrophy is evident after muscle disuse, unloading,or spaceflight and results from decreased protein content asa consequence of decreased protein synthesis, increased proteinbreakdown or both. At this time, there are essentially no humandata describing proteolysis in skeletal muscle undergoing atrophyon Earth or in space, primarily due to lack of valid and accuratemethodology. This particular study aimed at assessing the effectsof short-term unloading on the muscle contractile proteolysisrate. Eight men were subjected to 72-h unilateral lower limbsuspension (ULLS) and intramuscular interstitial levels of thenaturally occurring proteolytic tracer 3-methylhistidine (3MH)were measured by means of microdialysis before and on completionof this intervention. The 3MH concentration following 72-h ULLS(2.01 ± 0.22 nmol/ml) was 44% higher (P &lt; 0.05) thanbefore ULLS (1.56 ± 0.20 nmol/ml). The present experimentalmodel and the employed method determining 3MH in microdialysatespresent a promising tool for monitoring skeletal muscle proteolysisor metabolism of specific muscles during conditions resultingin atrophy caused by, e.g., disuse and real or simulated microgravity.This study provides evidence that the atrophic processes areevoked rapidly and within 72 h of unloading and suggests thatcountermeasures should be employed in the early stages of spacemissions to offset or prevent muscle loss during the periodwhen the rate of muscle atrophy is the highest.