Key points:

• How defects in muscle contractile function contribute to weakness in amyotrophic lateral sclerosis (ALS) were systematically investigated.
• Weakness in whole muscles from late stage SOD1^G93A mice was explained by muscle atrophy as seen by reduced mass and maximal force.
• On the other hand, surviving single muscle fibres in late stage SOD1^G93A have preserved intracellular Ca²⁺ handling, normal force-generating capacity and increased fatigue resistance.
• These intriguing findings provide a substrate for therapeutic interventions to potentiate muscular capacity and delay the progression of the ALS phenotype.

Amyotrophic lateral sclerosis (ALS) is a motor neuron disease characterized by degeneration and loss of motor neurons, leading to severe muscle weakness and paralysis. The SOD1^G93A mouse model of ALS displays motor neuron degeneration and a phenotype consistent with human ALS. The purpose of this study was to determine whether muscle weakness in ALS can be attributed to impaired intrinsic force generation in skeletal muscles. In the current study, motor neuron loss and decreased force were evident in whole flexor digitorum brevis (FDB) muscles of mice in the late stage of disease (125–150 days of age). However, in intact single muscle fibres, specific force, tetanic myoplasmic free [Ca²⁺] ([Ca²⁺]_i), and resting [Ca²⁺]_i remained unchanged with disease. Fibre-type distribution was maintained in late-stage SOD1^G93A FDB muscles, but remaining muscle fibres displayed greater fatigue resistance compared to control and showed increased expression of myoglobin and mitochondrial respiratory chain proteins that are important determinants of fatigue resistance. Expression of genes central to both mitochondrial biogenesis and muscle atrophy where increased, suggesting that atrophic and compensatory adaptive signalling occurs simultaneously within the muscle tissue. These results support the hypothesis that muscle weakness in SOD1^G93A is primarily attributed to neuromuscular degeneration and not intrinsic muscle fibre defects. In fact, surviving muscle fibres displayed maintained adaptive capacity with an exercise training-like phenotype, which suggests that compensatory mechanisms are activated that can function to delay disease progression.

We generated an inducible, skeletal muscle-specific Dicer knockout mouse to deplete microRNAs in adult skeletal muscle. Following tamoxifen treatment, Dicer mRNA expression was significantly decreased by 87%. Wild-type (WT) and Dicer knockout (KO) mice were subjected to either synergist ablation or hind limb suspension for two weeks. There was no difference in muscle weight with hypertrophy or atrophy between WT and KO groups; however, even with the significant loss of Dicer expression, myomiR (miR-1, -133a and -206) expression was only reduced by 38% on average. We next aged WT and KO mice for ~22 months following Dicer inactivation to determine if myomiR expression would be further reduced over a prolonged timeframe and assess the effects of myomiR depletion on skeletal muscle phenotype. Skeletal muscle Dicer mRNA expression remained significantly decreased by 80% in old KO mice and sequencing of cloned Dicer mRNA revealed the complete absence of the floxed exons in KO skeletal muscle. Despite a further reduction of myomiR expression to ~50% of WT, no change was observed in muscle morphology between WT and KO groups. These results indicate the life-long reduction in myomiR levels did not adversely affect skeletal muscle phenotype and suggest the possibility that microRNA expression is uniquely regulated in skeletal muscle.

BACKGROUND: Skeletal muscle mass and strength are crucial determinants of health. Muscle mass loss is associated with weakness, fatigue, and insulin resistance. In fact, it is predicted that controlling muscle atrophy can reduce morbidity and mortality associated with diseases such as cancer cachexia and sarcopenia.

METHODS: release in individual fibers. Finally, to explore the relationship between LMCD1 and calcineurin, we ectopically expressed Lmcd1 in the gastrocnemius muscle and treated those mice with cyclosporine A (calcineurin inhibitor). In addition, we used a luciferase reporter construct containing the myoregulin gene promoter to confirm the role of a LMCD1-calcineurin-myoregulin axis in skeletal muscle mass control and calcium handling.

RESULTS: Here, we identify LIM and cysteine-rich domains 1 (LMCD1) as a positive regulator of muscle mass, that increases muscle protein synthesis and fiber size. LMCD1 expression in vivo was sufficient to increase specific force with lower requirement for calcium handling and to reduce muscle fatigue. Conversely, silencing LMCD1 expression impairs calcium handling and force, and induces muscle fatigue without overt atrophy. The actions of LMCD1 were dependent on calcineurin, as its inhibition using cyclosporine A reverted the observed hypertrophic phenotype. Finally, we determined that LMCD1 represses the expression of myoregulin, a known negative regulator of muscle performance. Interestingly, we observed that skeletal muscle LMCD1 expression is reduced in patients with skeletal muscle disease.

CONCLUSIONS: handling, and fatigue resistance. This work uncovers a novel role for LMCD1 in the regulation of skeletal muscle mass and function with potential therapeutic implications.

The specific impact of reduced temperature on skeletal muscle adaptation has been poorly investigated. Cold water immersion, one situation leading to decreased skeletal muscle temperature, is commonly proposed to reduce the perception of fatigue and muscle soreness after strenuous exercise. In contrast, it may impair long-term benefits of resistance exercise training on muscle strength and hypertrophy. To date, the physiological factors responsible for this blunted muscle adaptation remain unclear. Here, we used a cell culture model of human primary myotubes to specifically investigate the intrinsic behavior of muscle cells during mild hypothermia (MH). Newly formed myotubes were exposed to either 37°C or 32°C to evaluate the effect of MH on myotube size and morphology, protein synthesis and anabolic signaling. We also compared the glutamine (GLUT)-induced hypertrophic response between myotubes incubated at 32°C or 37°C. We showed that 48 h exposure to MH altered the cellular morphology (greater myotube area, shorter myosegments, myotubes with irregular shape), and impaired GLUT-induced myotube hypertrophy. Moreover, MH specifically reduced protein synthesis at 8 h. This result may be explained by an altered regulation of ribosome biogenesis, as evidenced by a lower expression of 45S pre-rRNA and MYC protein, and a lower total RNA concentration. Furthermore, MH blunted GLUT-induced increase in protein synthesis at 8 h, a finding consistent with an impaired activation of the mechanistic target of rapamycin (mTOR) pathway. In conclusion, this study demonstrates that MH impairs the morphology of human myotubes and alters the hypertrophic response to GLUT.

The ribosome is typically viewed as a supramolecular complex with constitutive and invariant capacity in mediating translation of mRNA into protein. This view has been challenged by recent research revealing that ribosome composition could be heterogeneous, and this heterogeneity leads to functional ribosome specialization. This review presents the idea that ribosome heterogeneity results from changes in its various components, including variations in ribosomal protein (RP) composition, post-translational modifications of RPs, changes in ribosomal-associated proteins, alternative forms of rRNA and post-transcriptional modifications of rRNAs. Ribosome heterogeneity could be orchestrated at several levels and may depend on numerous factors, such as the subcellular location, cell type and tissue specificity, the development state, cell state, ribosome biogenesis, RP turnover, physiological stimuli and circadian rhythm. Ribosome specialization represents a completely new concept for the regulation of gene expression. Specialized ribosomes could modulate several aspects of translational control, such as mRNA translation selectivity, translation initiation, translational fidelity and translation elongation. Recent research indicates that the expression of Rpl3 is markedly increased, while that of Rpl3l is highly reduced during mouse skeletal muscle hypertrophy. Moreover, Rpl3l overexpression impairs the growth and myogenic fusion of myotubes. Although the function of Rpl3 and Rpl3l in the ribosome remains to be clarified, these findings suggest that ribosome specialization may be potentially involved in the control of protein translation and skeletal muscle size. Limited data concerning ribosome specialization are currently available in skeletal muscle. Future investigations have the potential to delineate the function of specialized ribosomes in skeletal muscle.

Introduction: Patients with breast cancer experience muscle dysfunction, which is a clinical challenge that is not restricted to advanced stage patients, but also observed in newly diagnosed weight-stable patients with low tumor burden. Recent data indicate that physical activity can reduce breast cancerassociated mortality, suggesting that improved muscle performance per secan have positive impact on survival. Here, the transgenic PyMT mouse model of breast cancer was used to elucidate molecular mechanisms underlying breast cancer-induced muscle impairments.

Materials and Methods: PyMT mice and wildtype (WT) littermates w/wo access to an in-cage running wheel for four weeks (week 8-12). Functional readouts included Ca2+imaging; isometric force measurement on single fibers and intact fast-and slow-twitchmuscles. Intramuscular signaling was assessed using immunofluorescence, immunoblotting and enzymatic assays.

Results: The specific force (i.e. force/cross-sectional area) was significantly decreased by ~ 35% in slow-twitch soleus muscles from breast cancermice as compared to WT muscles, which was the result of reduced Ca2+release and impaired myofibrillar function. There were no difference in muscle size or fiber type between the two groups. However, higher intramuscular stress (e.g. p38 activation and carbonylation (DNP)) was observed in PyMT than in WT. Intriguingly, voluntary running for four weeks reversed the weakness and PyMT soleus muscles generated similar forces as muscles of exercised WT mice. The running induced higher SOD2 expression and normalized levels of p38 and DNP.

Conclusion: Intrinsic contractile dysfunction and higher intramuscular stress was present in mice with breast cancer, which was counteracted with voluntary running.

Cold water immersion and other strategies are extensively used by athletes to reduce muscle temperature (TEMP) and are believed to limit muscle soreness and improve recovery after strenuous exercise. However, it remains unclear whether low TEMP affects skeletal muscle hypertrophy and protein synthesis in response to anabolic stimuli. In this study, we used an in vitro model to investigate whether a reduced TEMP (32°C, TEMP in skeletal muscle during cold water immersion vs. 37°C, core TEMP) affects the growth and impairs glutamine (GLUT)-induced hypertrophic response in human myotubes. Myoblasts from human muscle biopsies (n=8) were first differentiated into myotubes during 48h at 37°C. Then, myotubes were treated with GLUT to induce hypertrophy and were incubated at either 37 or 32°C during 1h (T1), 8h (T8) or 48h (T48). Myotube area (a marker of myotube size) was assessed at T48 by using immunocytology. Protein synthesis (puromycin incorporation) and phosphorylation levels of two components of the mechanistic target of rapamycin (mTOR) pathway [P70 S6 kinase (P70S6K) and eukaryotic translation initiation factor 4E (4E-BP1] were assessed at T1 and T8 from western blot analysis. Ribosome biogenesis was evaluated at T8 by determining total RNA concentration, and by assessing the levels of 18S ribosomal RNA (18S rRNA), precursor 45S rRNA (pre-45S rRNA) and MYC mRNA from quantitative polymerase chain reaction. Data were analysed by a two-way ANOVA for repeated measures. A first major observation was that low TEMP induced morphological alterations (short myotube segments and larger myotube area). Importantly, GLUT-induced myotube hypertrophy was only observed at 37°C (+40%, P<0,01 at 37°C; +4%, NS at 32°C). Similarly, GLUT stimulated protein synthesis at T1 (main effect, P<0,01), with a 53% increase at 37°C (P<0,05) and a 23% increase at 32°C (NS). Protein synthesis was reduced at 32°C at T8 (main effect, P<0,01), while GLUT increased protein synthesis at 37°C (+17%, P<0,01) but not at 32°C (-7%, NS). GLUT-induced 4E-BP1 phosphorylation at T1 was found at 37°C (+16%, P<0,01) but not at 32°C (-1%, NS). Moreover, GLUT increased the phosphorylation of P70S6K at T8 by 50% at 37°C (P<0,01) and by 26% at 32°C (P<0,05). Overall, total RNA concentration, levels of MYC mRNA, 18S rRNA and pre-45S rRNA were reduced at 32°C. In conclusion, this study indicates that low TEMP affects myotube morphology. In addition, we demonstrate that low TEMP impairs GLUT-induced myotube hypertrophy and protein synthesis, a result accompanied by a minored activation of the mTOR pathway. The impaired ribosome biogenesis observed at 32°C at T8 may also contribute to the reduced protein synthesis at this time point. These findings suggest that reducing muscle TEMP after resistance exercise may be detrimental for muscle hypertrophy and training adaptations.

Breast cancer accounts for ~25% of diagnosed cancer types in woman [1]. Decreased physical fitness and muscle weakness are common complications in patients with breast cancer. In cancer, muscle weakness has traditionally been linked to muscle wasting and significant weight loss (cachexia) [2]. However, muscle weakness is present in non-cachectic, weight-stable patients with breast cancer [3]. In fact, cancer-induced muscle dysfunction is a broad clinical challenge that is not restricted to palliative or advanced stage patients, but also observed in newly diagnosed patients with low tumor burden [4]. Further, with the breast cancer treatment improving, it is important to take a look on the patients quality of life [5]. However, little is known about the features underlying breast cancer-induced muscle impairments and no drug preventing cancer-induced muscle weakness is clinically proven. Here we aim at characterizing the metabolic status and the muscle function in mice with breast cancer.

The breast cancer mouse-model MMTV-PyMT (PyMT) used here, is characterized by an early onset of mammary cancer (from 5 weeks of age) and follows a similar progression pattern as the one observed in human patients [6]. Soleus muscle from PyMT mice exhibited ~30% lower specific force (kN/m²) than soleus muscle from wildtype (WT) mice (n=28-29, p ≤ 0.05, mice were 12 week old at sacrifice). There were no significant differences in muscle mass, fiber size or fiber type distribution between PyMT and WT muscle. Furthermore, there were no differences in glycogen content (μg/g muscle) in soleus muscle from PyMT and WT mice. Simultaneous measurement of numerous parameters (e.g. oxygen consumption (VO₂), carbon dioxide production (VCO₂), and food and water intake) was carried out using comprehensive lab animal monitoring system (CLAMS) to gain insight into the metabolic status of the mice. The mice were monitored over a week and the average respiratory exchange ratio (RER = CO₂production: O₂ uptake) were significantly differed between PyMT and WT mice, with mean PyMT RER of 0.95±0.01 and WT RER of 1.0±0.01 (mean data +/-SEM, n=8, p<0.001). Thus, indicative that PyMT have an altered metabolism towards fatty acid utilization.

In summary, soleus muscles are weaker and the whole-body metabolism appears altered in mice with breast cancer as compared with healthy control mice. Gene and molecular analysis are currently being performed to further assess mitochondrial and glucose metabolism. Nevertheless, further studies are needed to gain insight into cancer-derived factors that contributes to skeletal muscle dysfunction and altered metabolism.

1. Jemal, A., et al., Cancer statistics, 2008. CA Cancer J Clin, 2008. 58(2): p. 71-96.

2. Johns, N., N.A. Stephens, and K.C. Fearon, Muscle wasting in cancer. Int J Biochem Cell Biol, 2013. 45(10): p. 2215-29.

3. Klassen, O., et al., Muscle strength in breast cancer patients receiving different treatment regimes. Journal of Cachexia, Sarcopenia and Muscle, 2017. 8(2): p. 305-316.

4. Villasenor, A., et al., Prevalence and prognostic effect of sarcopenia in breast cancer survivors: the HEAL Study. J Cancer Surviv, 2012. 6(4): p. 398-406.

5. Perry, S., T.L. Kowalski, and C.H. Chang, Quality of life assessment in women with breast cancer: benefits, acceptability and utilization. Health Qual Life Outcomes, 2007. 5: p. 24.

6. Fantozzi, A. and G. Christofori, Mouse models of breast cancer metastasis. Breast Cancer Res, 2006. 8(4): p. 212.

Skeletal muscle is able to modify its size, and its metabolic/contractile properties in response to a variety of stimuli, such as mechanical stress, neuronal activity, metabolic and hormonal influences, and environmental factors. A reduced oxygen availability, called hypoxia, has been proposed to inducemetabolic adaptations and loss ofmass in skeletal muscle. In addition, several evidences indicate that muscle fiber-type composition could be affected by hypoxia. The main purpose of this review is to explore the adaptation of skeletal muscle fiber-type composition to exposure to high altitude (ambient hypoxia) and under conditions of pathological hypoxia, including chronic obstructive pulmonary disease (COPD), chronic heart failure (CHF) and obstructive sleep apnea syndrome (OSAS). The muscle fiber-type composition of both adult animals and humans is not markedly altered during chronic exposure to high altitude. However, the fast-to-slow fiber-type transition observed in hind limb muscles during post-natal development is impaired in growing rats exposed to severe altitude. A slow-to-fast transition in fiber type is commonly found in lower limb muscles from patients with COPD and CHF, whereas a transition toward a slower fiber-type profile is often found in the diaphragm muscle in these two pathologies. A slow-to-fast transformation in fiber type is generally observed in the upper airway muscles in rodent models of OSAS. The factors potentially responsible for the adaptation of fiber type under these hypoxic conditions are also discussed in this review. The impaired locomotor activity most likely explains the changes in fiber type composition in growing rats exposed to severe altitude. Furthermore, chronic inactivity and muscle deconditioning could result in the slow-to-fast fiber-type conversion in lower limb muscles during COPD and CHF, while the factors responsible for the adaptation of muscle fiber type during OSAS remain hypothetical. Finally, the role played by cellular hypoxia, hypoxia-inducible factor-1 alpha (HIF-1 alpha), and other molecular regulators in the adaptation of muscle fiber-type composition is described in response to high altitude exposure and conditions of pathological hypoxia.