SEPN1-related myopathy depends on the oxidoreductase ERO1A and is druggable with the chemical chaperone TUDCA.

Selenoprotein N (SEPN1) is a protein of the endoplasmic reticulum (ER) whose inherited defects originate SEPN1-related myopathy (SEPN1-RM). Here, we identify an interaction between SEPN1 and the ER-stress-induced oxidoreductase ERO1A. SEPN1 and ERO1A, both enriched in mitochondria-associated membranes (MAMs), are involved in the redox regulation of proteins. ERO1A depletion in SEPN1 knockout cells restores ER redox, re-equilibrates short-range MAMs, and rescues mitochondrial bioenergetics. ERO1A knockout in a mouse background of SEPN1 loss blunts ER stress and improves multiple MAM functions, including Ca2+ levels and bioenergetics, thus reversing diaphragmatic weakness. The treatment of SEPN1 knockout mice with the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) mirrors the results of ERO1A loss. Importantly, muscle biopsies from patients with SEPN1-RM exhibit ERO1A overexpression, and TUDCA-treated SEPN1-RM patient-derived primary myoblasts show improvement in bioenergetics. These findings point to ERO1A as a biomarker and a viable target for intervention and to TUDCA as a pharmacological treatment for SEPN1-RM.

A combination of metformin and galantamine exhibits synergistic benefits in the treatment of sarcopenia.

Age-associated sarcopenia, characterized by a progressive loss in muscle mass and strength, is the largest cause of frailty and disability in the elderly worldwide. Current treatments involve nonpharmacological guidelines that few subjects can abide by, highlighting the need for effective drugs. Preclinical models were employed to test the benefits of RJx-01, a combination drug composed of metformin and galantamine, on sarcopenia. In worms, RJx-01 treatment improved lifespan, locomotion, pharyngeal pumping, and muscle fiber organization. The synergistic effects of RJx-01 were recapitulated in a transgenic mouse model that displays an exacerbated aging phenotype (Opa1-/-). In these mice, RJx-01 ameliorated physical performance, muscle mass and force, neuromuscular junction stability, and systemic inflammation. RJx-01 also improved physical performance and muscle strength in 22-month-old WT mice and also improved skeletal muscle ultrastructure, mitochondrial morphology, autophagy, lysosomal function, and satellite cell content. Denervation and myofiber damage were decreased in RJx-01-treated animals compared with controls. RJx-01 improved muscle quality rather than quantity, indicating that the improvement in quality underlies the beneficial effects of the combination drug. The studies herein indicate synergistic beneficial effects of RJx-01 in the treatment of sarcopenia and support the pursuit of RJx-01 in a human clinical trial as a therapeutic intervention for sarcopenia.

Defective excitation-contraction coupling and mitochondrial respiration precede mitochondrial Ca2+ accumulation in spinobulbar muscular atrophy skeletal muscle.

Polyglutamine expansion in the androgen receptor (AR) causes spinobulbar muscular atrophy (SBMA). Skeletal muscle is a primary site of toxicity; however, the current understanding of the early pathological processes that occur and how they unfold during disease progression remains limited. Using transgenic and knock-in mice and patient-derived muscle biopsies, we show that SBMA mice in the presymptomatic stage develop a respiratory defect matching defective expression of genes involved in excitation-contraction coupling (ECC), altered contraction dynamics, and increased fatigue. These processes are followed by stimulus-dependent accumulation of calcium into mitochondria and structural disorganization of the muscle triads. Deregulation of expression of ECC genes is concomitant with sexual maturity and androgen raise in the serum. Consistent with the androgen-dependent nature of these alterations, surgical castration and AR silencing alleviate the early and late pathological processes. These observations show that ECC deregulation and defective mitochondrial respiration are early but reversible events followed by altered muscle force, calcium dyshomeostasis, and dismantling of triad structure.

Inducible deletion of raptor and mTOR from adult skeletal muscle impairs muscle contractility and relaxation.

Skeletal muscle weakness has been associated with different pathological conditions, including sarcopenia and muscular dystrophy, and is accompanied by altered mammalian target of rapamycin (mTOR) signalling. We wanted to elucidate the functional role of mTOR in muscle contractility. Most loss-of-function studies for mTOR signalling have used the drug rapamycin to inhibit some of the signalling downstream of mTOR. However, given that rapamycin does not inhibit all mTOR signalling completely, we generated a double knockout for mTOR and for the scaffold protein of mTORC1, raptor, in skeletal muscle. We found that double knockout in mice results in a more severe phenotype compared with deletion of raptor or mTOR alone. Indeed, these animals display muscle weakness, increased fibre denervation and a slower muscle relaxation following tetanic stimulation. This is accompanied by a shift towards slow-twitch fibres and changes in the expression levels of calcium-related genes, such as Serca1 and Casq1. Double knockout mice show a decrease in calcium decay kinetics after tetanus in vivo, suggestive of a reduced calcium reuptake. In addition, RNA sequencing analysis revealed that many downregulated genes, such as Tcap and Fhod3, are linked to sarcomere organization. These results suggest a key role for mTOR signalling in maintaining proper fibre relaxation in skeletal muscle. KEY POINTS: Skeletal muscle wasting and weakness have been associated with different pathological conditions, including sarcopenia and muscular dystrophy, and are accompanied by altered mammalian target of rapamycin (mTOR) signalling. Mammalian target of rapamycin plays a crucial role in the maintenance of muscle mass and functionality. We found that the loss of both mTOR and raptor results in contractile abnormalities, with severe muscle weakness and delayed relaxation following tetanic stimulation. These results are associated with alterations in the expression of genes involved in sarcomere organization and calcium handling and with an impairment in calcium reuptake after contraction. Taken together, these results provide a mechanistic insight into the role of mTOR in muscle contractility.

CFTR corrector C17 is effective in muscular dystrophy, in vivo proof of concept in LGMDR3.

Limb-girdle muscular dystrophy R3 (LGMDR3) is caused by mutations in the SGCA gene coding for α-sarcoglycan (SG). Together with ß- γ- and δ-SG, α-SG forms a tetramer embedded in the dystrophin associated protein complex crucial for protecting the sarcolemma from mechanical stresses elicited by muscle contraction. Most LGMDR3 cases are due to missense mutations, which result in non-properly folded, even though potentially functional α-SG. These mutants are prematurely discarded by the cell quality control. Lacking one subunit, the SG-complex is disrupted. The resulting loss of function leads to sarcolemma instability, muscle fiber damage and progressive limb muscle weakness. LGMDR3 is severely disabling and, unfortunately, still incurable. Here, we propose the use of small molecules, belonging to the class of cystic fibrosis transmembrane regulator (CFTR) correctors, for recovering mutants of α-SG defective in folding and trafficking. Specifically, CFTR corrector C17 successfully rerouted the SG-complex containing the human R98H-α-SG to the sarcolemma of hind-limb muscles of a novel LGMDR3 murine model. Notably, the muscle force of the treated model animals was fully recovered. To our knowledge, this is the first time that a compound designated for cystic fibrosis is successfully tested in a muscular dystrophy and may represent a novel paradigm of treatment for LGMDR3 as well as different other indications in which a potentially functional protein is prematurely discarded as folding-defective. Furthermore, the use of small molecules for recovering the endogenous mutated SG has an evident advantage over complex procedures such as gene or cell transfer.

Activation of Akt-mTORC1 signalling reverts cancer-dependent muscle wasting.

BACKGROUND: Cancer-related muscle wasting occurs in most cancer patients. An important regulator of adult muscle mass and function is the Akt-mTORC1 pathway. While Akt-mTORC1 signalling is important for adult muscle homeostasis, it is also a major target of numerous cancer treatments. Which role Akt-mTORC1 signalling plays during cancer cachexia in muscle is currently not known. Here, we aimed to determine how activation or inactivation of the pathway affects skeletal muscle during cancer cachexia. METHODS: We used inducible, muscle-specific Raptor ko (mTORC1) mice to determine the effect of reduced mTOR signalling during cancer cachexia. On the contrary, in order to understand if skeletal muscles maintain their anabolic capacity and if activation of Akt-mTORC1 signalling can reverse cancer cachexia, we generated mice in which we can inducibly activate Akt specifically in skeletal muscles. RESULTS: We found that mTORC1 signalling is impaired during cancer cachexia, using the Lewis lung carcinoma and C26 colon cancer model, and is accompanied by a reduction in protein synthesis rates of 57% (P < 0.01). Further reduction of mTOR signalling, as seen in Raptor ko animals, leads to a 1.5-fold increase in autophagic flux (P > 0.001), but does not further increase muscle wasting. On the other hand, activation of Akt-mTORC1 signalling in already cachectic animals completely reverses the 15-20% loss in muscle mass and force (P < 0.001). Interestingly, Akt activation only in skeletal muscle completely normalizes the transcriptional deregulation observed in cachectic muscle, despite having no effect on tumour size or spleen mass. In addition to stimulating muscle growth, it is also sufficient to prevent the increase in protein degradation normally observed in muscles from tumour-bearing animals. CONCLUSIONS: Here, we show that activation of Akt-mTORC1 signalling is sufficient to completely revert cancer-dependent muscle wasting. Intriguingly, these results show that skeletal muscle maintains its anabolic capacities also during cancer cachexia, possibly giving a rationale behind some of the beneficial effects observed in exercise in cancer patients.

Raptor is critical for increasing the mitochondrial proteome and skeletal muscle force during hypertrophy.

Loss of skeletal muscle mass and force is of critical importance in numerous pathologies, like age-related sarcopenia or cancer. It has been shown that the Akt-mTORC1 pathway is critical for stimulating adult muscle mass and function, however, it is unknown if mTORC1 is the only mediator downstream of Akt and which intracellular processes are required for functional muscle growth. Here, we show that loss of Raptor reduces muscle hypertrophy after Akt activation and completely prevents increases in muscle force. Interestingly, the residual hypertrophy after Raptor deletion can be completely prevented by administration of the mTORC1 inhibitor rapamycin. Using a quantitative proteomics approach we find that loss of Raptor affects the increases in mitochondrial proteins, while rapamycin mainly affects ribosomal proteins. Taken together, these results suggest that mTORC1 is the key mediator of Akt-dependent muscle growth and its regulation of the mitochondrial proteome is critical for increasing muscle force.

Protein profile of fiber types in human skeletal muscle: a single-fiber proteomics study.

BACKGROUND: Human skeletal muscle is composed of three major fiber types, referred to as type 1, 2A, and 2X fibers. This heterogeneous cellular composition complicates the interpretation of studies based on whole skeletal muscle lysate. A single-fiber proteomics approach is required to obtain a fiber-type resolved quantitative information on skeletal muscle pathophysiology. METHODS: Single fibers were dissected from vastus lateralis muscle biopsies of young adult males and processed for mass spectrometry-based single-fiber proteomics. We provide and analyze a resource dataset based on relatively pure fibers, containing at least 80% of either MYH7 (marker of slow type 1 fibers), MYH2 (marker of fast 2A fibers), or MYH1 (marker of fast 2X fibers). RESULTS: In a dataset of more than 3800 proteins detected by single-fiber proteomics, we selected 404 proteins showing a statistically significant difference among fiber types. We identified numerous type 1 or 2X fiber type-specific protein markers, defined as proteins present at 3-fold or higher levels in these compared to other fiber types. In contrast, we could detect only two 2A-specific protein markers in addition to MYH2. We observed three other major patterns: proteins showing a differential distribution according to the sequence 1 > 2A > 2X or 2X > 2A > 1 and type 2-specific proteins expressed in 2A and 2X fibers at levels 3 times greater than in type 1 fibers. In addition to precisely quantifying known fiber type-specific protein patterns, our study revealed several novel features of fiber type specificity, including the selective enrichment of components of the dystrophin and integrin complexes, as well as microtubular proteins, in type 2X fibers. The fiber type-specific distribution of some selected proteins revealed by proteomics was validated by immunofluorescence analyses with specific antibodies. CONCLUSION: We here show that numerous muscle proteins, including proteins whose function is unknown, are selectively enriched in specific fiber types, pointing to potential implications in muscle pathophysiology. This reinforces the notion that single-fiber proteomics, together with recently developed approaches to single-cell proteomics, will be instrumental to explore and quantify muscle cell heterogeneity.

PERM1 interacts with the MICOS-MIB complex to connect the mitochondria and sarcolemma via ankyrin B.

Skeletal muscle subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria subpopulations have distinct metabolic activity and sensitivity, though the mechanisms that localize SSM to peripheral areas of muscle fibers are poorly understood. A protein interaction study and complexome profiling identifies PERM1 interacts with the MICOS-MIB complex. Ablation of Perm1 in mice reduces muscle force, decreases mitochondrial membrane potential and complex I activity, and reduces the numbers of SSM in skeletal muscle. We demonstrate PERM1 interacts with the intracellular adaptor protein ankyrin B (ANKB) that connects the cytoskeleton to the plasma membrane. Moreover, we identify a C-terminal transmembrane helix that anchors PERM1 into the outer mitochondrial membrane. We conclude PERM1 functions in the MICOS-MIB complex and acts as an adapter to connect the mitochondria with the sarcolemma via ANKB.

Neuromuscular junction instability and altered intracellular calcium handling as early determinants of force loss during unloading in humans.

KEY POINTS: Few days of unloading are sufficient to induce a decline of skeletal muscle mass and function; notably, contractile force is lost at a faster rate than muscle mass. The reasons behind this disproportionate loss of muscle force are still poorly understood. We provide strong evidence of two mechanisms only hypothesized until now for the rapid muscle force loss in only 10 days of bed rest. Our results show that an initial neuromuscular junction instability, accompanied by alterations in the innervation status and impairment of single fibre sarcoplasmic reticulum function contribute to the loss of contractile force in front of a preserved myofibrillar function and central activation capacity. Early onset of neuromuscular junction instability and impairment in calcium dynamics involved in excitation-contraction coupling are proposed as eligible determinants to the greater decline in muscle force than in muscle size during unloading. ABSTRACT: Unloading induces rapid skeletal muscle atrophy and functional decline. Importantly, force is lost at a much higher rate than muscle mass. We aimed to investigate the early determinants of the disproportionate loss of force compared to that of muscle mass in response to unloading. Ten young participants underwent 10 days of bed rest (BR). At baseline (BR0) and at 10 days (BR10), quadriceps femoris (QF) volume (VOL) and isometric maximum voluntary contraction (MVC) were assessed. At BR0 and BR10 blood samples and biopsies of vastus lateralis (VL) muscle were collected. Neuromuscular junction (NMJ) stability and myofibre innervation status were assessed, together with single fibre mechanical properties and sarcoplasmic reticulum (SR) calcium handling. From BR0 to BR10, QFVOL and MVC decreased by 5.2% (P = 0.003) and 14.3% (P < 0.001), respectively. Initial and partial denervation was detected from increased neural cell adhesion molecule (NCAM)-positive myofibres at BR10 compared with BR0 (+3.4%, P = 0.016). NMJ instability was further inferred from increased C-terminal agrin fragment concentration in serum (+19.2% at BR10, P = 0.031). Fast fibre cross-sectional area (CSA) showed a trend to decrease by 15% (P = 0.055) at BR10, while single fibre maximal tension (force/CSA) was unchanged. However, at BR10 SR Ca2+ release in response to caffeine decreased by 35.1% (P < 0.002) and 30.2% (P < 0.001) in fast and slow fibres, respectively, pointing to an impaired excitation-contraction coupling. These findings support the view that the early onset of NMJ instability and impairment in SR function are eligible mechanisms contributing to the greater decline in muscle force than in muscle size during unloading.

Muscle functional recovery is driven by extracellular vesicles combined with muscle extracellular matrix in a volumetric muscle loss murine model.

Biological scaffolds derived from decellularized tissues are being investigated as a promising approach to repair volumetric muscle losses (VML). Indeed, extracellular matrix (ECM) from decellularized tissues is highly biocompatible and mimics the original tissue. However, the development of fibrosis and the muscle stiffness still represents a major problem. Intercellular signals mediating tissue repair are conveyed via extracellular vesicles (EVs), biologically active nanoparticles secreted by the cells. This work aimed at using muscle ECM and human EVs derived from Wharton Jelly mesenchymal stromal cells (MSC EVs) to boost tissue regeneration in a VML murine model. Mice transplanted with muscle ECM and treated with PBS or MSC EVs were analyzed after 7 and 30 days. Flow cytometry, tissue analysis, qRT-PCR and physiology test were performed. We demonstrated that angiogenesis and myogenesis were enhanced while fibrosis was reduced after EV treatment. Moreover, the inflammation was directed toward tissue repair. M2-like, pro-regenerative macrophages were significantly increased in the MSC EVs treated group compared to control. Strikingly, the histological improvements were associated with enhanced functional recovery. These results suggest that human MSC EVs can be a naturally-derived boost able to ameliorate the efficacy of tissue-specific ECM in muscle regeneration up to the restored tissue function.

Exercise-dependent increases in protein synthesis are accompanied by chromatin modifications and increased MRTF-SRF signalling.

AIM: Resistance exercise increases muscle mass over time. However, the early signalling events leading to muscle growth are not yet well-defined. Here, we aim to identify new signalling pathways important for muscle remodelling after exercise. METHODS: We performed a phosphoproteomics screen after a single bout of exercise in mice. As an exercise model we used unilateral electrical stimulation in vivo and treadmill running. We analysed muscle biopsies from human subjects to verify if our findings in murine muscle also translate to exercise in humans. RESULTS: We identified a new phosphorylation site on Myocardin-Related Transcription Factor B (MRTF-B), a co-activator of serum response factor (SRF). Phosphorylation of MRTF-B is required for its nuclear translocation after exercise and is accompanied by the transcription of the SRF target gene Fos. In addition, high-intensity exercise also remodels chromatin at specific SRF target gene loci through the phosphorylation of histone 3 on serine 10 in myonuclei of both mice and humans. Ablation of the MAP kinase member MSK1/2 is sufficient to prevent this histone phosphorylation, reduce induction of SRF-target genes, and prevent increases in protein synthesis after exercise. CONCLUSION: Our results identify a new exercise signalling fingerprint in vivo, instrumental for exercise-induced protein synthesis and potentially muscle growth.

High-throughput proteomics fiber typing (ProFiT) for comprehensive characterization of single skeletal muscle fibers.

BACKGROUND: Skeletal muscles are composed of a heterogeneous collection of fiber types with different physiological adaption in response to a stimulus and disease-related conditions. Each fiber has a specific molecular expression of myosin heavy chain molecules (MyHC). So far, MyHCs are currently the best marker proteins for characterization of individual fiber types, and several proteome profiling studies have helped to dissect the molecular signature of whole muscles and individual fibers. METHODS: Herein, we describe a mass spectrometric workflow to measure skeletal muscle fiber type-specific proteomes. To bypass the limited quantities of protein in single fibers, we developed a Proteomics high-throughput fiber typing (ProFiT) approach enabling profiling of MyHC in single fibers. Aliquots of protein extracts from separated muscle fibers were subjected to capillary LC-MS gradients to profile MyHC isoforms in a 96-well format. Muscle fibers with the same MyHC protein expression were pooled and subjected to proteomic, pulsed-SILAC, and phosphoproteomic analysis. RESULTS: Our fiber type-specific quantitative proteome analysis confirmed the distribution of fiber types in the soleus muscle, substantiates metabolic adaptions in oxidative and glycolytic fibers, and highlighted significant differences between the proteomes of type IIb fibers from different muscle groups, including a differential expression of desmin and actinin-3. A detailed map of the Lys-6 incorporation rates in muscle fibers showed an increased turnover of slow fibers compared to fast fibers. In addition, labeling of mitochondrial respiratory chain complexes revealed a broad range of Lys-6 incorporation rates, depending on the localization of the subunits within distinct complexes. CONCLUSION: Overall, the ProFiT approach provides a versatile tool to rapidly characterize muscle fibers and obtain fiber-specific proteomes for different muscle groups.

KBTBD13 is an actin-binding protein that modulates muscle kinetics.

The mechanisms that modulate the kinetics of muscle relaxation are critically important for muscle function. A prime example of the impact of impaired relaxation kinetics is nemaline myopathy caused by mutations in KBTBD13 (NEM6). In addition to weakness, NEM6 patients have slow muscle relaxation, compromising contractility and daily life activities. The role of KBTBD13 in muscle is unknown, and the pathomechanism underlying NEM6 is undetermined. A combination of transcranial magnetic stimulation-induced muscle relaxation, muscle fiber- and sarcomere-contractility assays, low-angle x-ray diffraction, and superresolution microscopy revealed that the impaired muscle-relaxation kinetics in NEM6 patients are caused by structural changes in the thin filament, a sarcomeric microstructure. Using homology modeling and binding and contractility assays with recombinant KBTBD13, Kbtbd13-knockout and Kbtbd13R408C-knockin mouse models, and a GFP-labeled Kbtbd13-transgenic zebrafish model, we discovered that KBTBD13 binds to actin - a major constituent of the thin filament - and that mutations in KBTBD13 cause structural changes impairing muscle-relaxation kinetics. We propose that this actin-based impaired relaxation is central to NEM6 pathology.

Skeletal muscle mTORC1 regulates neuromuscular junction stability.

BACKGROUND: Skeletal muscle is a plastic tissue that can adapt to different stimuli. It is well established that Mammalian Target of Rapamycin Complex 1 (mTORC1) signalling is a key modulator in mediating increases in skeletal muscle mass and function. However, the role of mTORC1 signalling in adult skeletal muscle homeostasis is still not well defined. METHODS: Inducible, muscle-specific Raptor and mTOR k.o. mice were generated. Muscles at 1 and 7 months after deletion were analysed to assess muscle histology and muscle force. RESULTS: We found no change in muscle size or contractile properties 1 month after deletion. Prolonging deletion of Raptor to 7 months, however, leads to a very marked phenotype characterized by weakness, muscle regeneration, mitochondrial dysfunction, and autophagy impairment. Unexpectedly, reduced mTOR signalling in muscle fibres is accompanied by the appearance of markers of fibre denervation, like the increased expression of the neural cell adhesion molecule (NCAM). Both muscle-specific deletion of mTOR or Raptor, or the use of rapamycin, was sufficient to induce 3-8% of NCAM-positive fibres (P < 0.01), muscle fibrillation, and neuromuscular junction (NMJ) fragmentation in 24% of examined fibres (P < 0.001). Mechanistically, reactivation of autophagy with the small peptide Tat-beclin1 is sufficient to prevent mitochondrial dysfunction and the appearance of NCAM-positive fibres in Raptor k.o. muscles. CONCLUSIONS: Our study shows that mTOR signalling in skeletal muscle fibres is critical for maintaining proper fibre innervation, preserving the NMJ structure in both the muscle fibre and the motor neuron. In addition, considering the beneficial effects of exercise in most pathologies affecting the NMJ, our findings suggest that part of these beneficial effects of exercise are through the well-established activation of mTORC1 in skeletal muscle during and after exercise.

Insulin/IGF1 signalling mediates the effects of ß2 -adrenergic agonist on muscle proteostasis and growth.

BACKGROUND: Stimulation of ß2 -adrenoceptors can promote muscle hypertrophy and fibre type shift, and it can counteract atrophy and weakness. The underlying mechanisms remain elusive. METHODS: Fed wild type (WT), 2-day fasted WT, muscle-specific insulin (INS) receptor (IR) knockout (M-IR-/- ), and MKR mice were studied with regard to acute effects of the ß2 -agonist formoterol (FOR) on protein metabolism and signalling events. MKR mice express a dominant negative IGF1 receptor, which blocks both INS/IGF1 signalling. All received one injection of FOR (300 µg kg-1 subcutaneously) or saline. Skeletal muscles and serum samples were analysed from 30 to 240 min. For the study of chronic effects of FOR on muscle plasticity and function as well as intracellular signalling pathways, fed WT and MKR mice were treated with formoterol (300 µg kg-1  day-1 ) for 30 days. RESULTS: In fed and fasted mice, one injection of FOR inhibited autophagosome formation (LC3-II content, 65%, P ≤ 0.05) that was paralleled by an increase in serum INS levels (4-fold to 25-fold, P ≤ 0.05) and the phosphorylation of Akt (4.4-fold to 6.5-fold, P ≤ 0.05) and ERK1/2 (50% to two-fold, P ≤ 0.05). This led to the suppression (40-70%, P ≤ 0.05) of the master regulators of atrophy, FoxOs, and the mRNA levels of their target genes. FOR enhanced (41%, P ≤ 0.05) protein synthesis only in fed condition and stimulated (4.4-fold to 35-fold, P ≤ 0.05) the prosynthetic Akt/mTOR/p70S6K pathway in both fed and fasted states. FOR effects on Akt signalling during fasting were blunted in both M-IR-/- and MKR mice. Inhibition of proteolysis markers by FOR was prevented only in MKR mice. Blockade of PI3K/Akt axis and mTORC1, but not ERK1/2, in fasted mice also suppressed the acute FOR effects on proteolysis and autophagy. Chronic stimulation of ß2 -adrenoceptors in fed WT mice increased body (11%, P ≤ 0.05) and muscle (15%, P ≤ 0.05) growth and downregulated atrophy-related genes (30-40%, P ≤ 0.05), but these effects were abolished in MKR mice. Increases in muscle force caused by FOR (WT, 24%, P ≤ 0.05) were only partially impaired in MKR mice (12%, P ≤ 0.05), and FOR-induced slow-to-fast fibre type shift was not blocked at all in these animals. In MKR mice, FOR also restored the lower levels of muscle SDH activity to basal WT values and caused a marked reduction (57%, P ≤ 0.05) in the number of centrally nucleated fibers. CONCLUSIONS: NS/IGF1 signalling is necessary for the anti-proteolytic and hypertrophic effects of in vivo ß2 -adrenergic stimulation and appears to mediate FOR-induced enhancement of protein synthesis. INS/IGF1 signalling only partially contributes to gain in strength and does not mediate fibre type transition induced by FOR.

SELENON (SEPN1) protects skeletal muscle from saturated fatty acid-induced ER stress and insulin resistance.

Selenoprotein N (SELENON) is an endoplasmic reticulum (ER) protein whose loss of function leads to a congenital myopathy associated with insulin resistance (SEPN1-related myopathy). The exact cause of the insulin resistance in patients with SELENON loss of function is not known. Skeletal muscle is the main contributor to insulin-mediated glucose uptake, and a defect in this muscle-related mechanism triggers insulin resistance and glucose intolerance. We have studied the chain of events that connect the loss of SELENON with defects in insulin-mediated glucose uptake in muscle cells and the effects of this on muscle performance. Here, we show that saturated fatty acids are more lipotoxic in SELENON-devoid cells, and blunt the insulin-mediated glucose uptake of SELENON-devoid myotubes by increasing ER stress and mounting a maladaptive ER stress response. Furthermore, the hind limb skeletal muscles of SELENON KO mice fed a high-fat diet mirrors the features of saturated fatty acid-treated myotubes, and show signs of myopathy with a compromised force production. These findings suggest that the absence of SELENON together with a high-fat dietary regimen increases susceptibility to insulin resistance by triggering a chronic ER stress in skeletal muscle and muscle weakness. Importantly, our findings suggest that environmental cues eliciting ER stress in skeletal muscle (such as a high-fat diet) affect the pathological phenotype of SEPN1-related myopathy and can therefore contribute to the assessment of prognosis beyond simple genotype-phenotype correlations.

Changes in the fraction of strongly attached cross bridges in mouse atrophic and hypertrophic muscles as revealed by continuous wave electron paramagnetic resonance.

Electron paramagnetic resonance (EPR), coupled with site-directed spin labeling, has been proven to be a particularly suitable technique to extract information on the fraction of myosin heads strongly bound to actin upon muscle contraction. The approach can be used to investigate possible structural changes occurring in myosin of fiber s altered by diseases and aging. In this work, we labeled myosin at position Cys707, located in the SH1-SH2 helix in the myosin head cleft, with iodoacetamide spin label, a spin label that is sensitive to the reorientational motion of this protein during the ATPase cycle and characterized the biochemical states of the labeled myosin head by means of continuous wave EPR. After checking the sensitivity and the power of the technique on different muscles and species, we investigated whether changes in the fraction of strongly bound myosin heads might explain the contractile alterations observed in atrophic and hypertrophic murine muscles. In both conditions, the difference in contractile force could not be justified simply by the difference in muscle mass. Our results showed that in atrophic muscles the decrease in force generation was attributable to a lower fraction of strongly bound cross bridges during maximal activation. In contrast in hypertrophic muscles, the increase in force generation was likely due to several factors, as pointed out by the comparison of the EPR experiments with the tension measurements on single skinned fibers.

A maladaptive ER stress response triggers dysfunction in highly active muscles of mice with SELENON loss.

Selenoprotein N (SELENON) is an endoplasmic reticulum (ER) protein whose loss of function leads to human SELENON-related myopathies. SelenoN knockout (KO) mouse limb muscles, however, are protected from the disease, and display no major alterations in muscle histology or contractile properties. Interestingly, we find that the highly active diaphragm muscle shows impaired force production, in line with the human phenotype. In addition, after repeated stimulation with a protocol which induces muscle fatigue, also hind limb muscles show altered relaxation times. Mechanistically, muscle SELENON loss alters activity-dependent calcium handling selectively impinging on the Ca2+ uptake of the sarcoplasmic reticulum and elicits an ER stress response, including the expression of the maladaptive CHOP-induced ERO1. In SELENON-devoid models, ERO1 shifts ER redox to a more oxidised poise, and further affects Ca2+ uptake. Importantly, CHOP ablation in SelenoN KO mice completely prevents diaphragm dysfunction, the prolonged limb muscle relaxation after fatigue, and restores Ca2+ uptake by attenuating the induction of ERO1. These findings suggest that SELENON is part of an ER stress-dependent antioxidant response and that the CHOP/ERO1 branch of the ER stress response is a novel pathogenic mechanism underlying SELENON-related myopathies.