Leucine elicits myotube hypertrophy and enhances maximal contractile force in tissue engineered skeletal muscle in vitro

The amino acid leucine is thought to be important for skeletal muscle growth by virtue of its ability to acutely activate mTORC1 and enhance muscle protein synthesis, yet little data exist regarding its impact on skeletal muscle size and its ability to produce force. We utilized a tissue engineering approach in order to test whether supplementing culture medium with leucine could enhance mTORC1 signaling, myotube growth, and muscle function. Phosphorylation of the mTORC1 target proteins 4EBP‐1 and rpS6 and myotube hypertrophy appeared to occur in a dose dependent manner, with 5 and 20 mM of leucine inducing similar effects, which were greater than those seen with 1 mM. Maximal contractile force was also elevated with leucine supplementation; however, although this did not appear to be enhanced with increasing leucine doses, this effect was completely ablated by co‐incubation with the mTOR inhibitor rapamycin, showing that the augmented force production in the presence of leucine was mTOR sensitive. Finally, by using electrical stimulation to induce chronic (24 hr) contraction of engineered skeletal muscle constructs, we were able to show that the effects of leucine and muscle contraction are additive, since the two stimuli had cumulative effects on maximal contractile force production. These results extend our current knowledge of the efficacy of leucine as an anabolic nutritional aid showing for the first time that leucine supplementation may augment skeletal muscle functional capacity, and furthermore validates the use of engineered skeletal muscle for highly‐controlled investigations into nutritional regulation of muscle physiology.     

Methylarginine metabolites are associated with attenuated muscle protein synthesis in cancer-associated muscle wasting

Cancer cachexia is characterized by reductions in peripheral lean muscle mass. Prior studies have primarily focused on increased protein breakdown as the driver of cancer-associated muscle wasting. Therapeutic interventions targeting catabolic pathways have, however, largely failed to preserve muscle mass in cachexia, suggesting that other mechanisms might be involved. In pursuit of novel pathways, we used untargeted metabolomics to search for metabolite signatures that may be linked with muscle atrophy. We injected 7-week–old C57/BL6 mice with LLC1 tumor cells or vehicle. After 21 days, tumor-bearing mice exhibited reduced body and muscle mass and impaired grip strength compared with controls, which was accompanied by lower synthesis rates of mixed muscle protein and the myofibrillar and sarcoplasmic muscle fractions. Reductions in protein synthesis were accompanied by mitochondrial enlargement and reduced coupling efficiency in tumor-bearing mice. To generate mechanistic insights into impaired protein synthesis, we performed untargeted metabolomic analyses of plasma and muscle and found increased concentrations of two methylarginines, asymmetric dimethylarginine (ADMA) and N^G-monomethyl-l-arginine, in tumor-bearing mice compared with control mice. Compared with healthy controls, human cancer patients were also found to have higher levels of ADMA in the skeletal muscle. Treatment of C2C12 myotubes with ADMA impaired protein synthesis and reduced mitochondrial protein quality. These results suggest that increased levels of ADMA and mitochondrial changes may contribute to impaired muscle protein synthesis in cancer cachexia and could point to novel therapeutic targets by which to mitigate cancer cachexia.     

Mechanical loading stimulates hypertrophy in tissue-engineered skeletal muscle: Molecular and phenotypic responses

Mechanical loading of skeletal muscle results in molecular and phenotypic adaptations typified by enhanced muscle size. Studies on humans are limited by the need for repeated sampling, and studies on animals have methodological and ethical limitations. In this investigation, three-dimensional skeletal muscle was tissue-engineered utilizing the murine cell line C2C12, which bears resemblance to native tissue and benefits from the advantages of conventional in vitro experiments. The work aimed to determine if mechanical loading induced an anabolic hypertrophic response, akin to that described in vivo after mechanical loading in the form of resistance exercise. Specifically, we temporally investigated candidate gene expression and Akt-mechanistic target of rapamycin 1 signalling along with myotube growth and tissue function. Mechanical loading (construct length increase of 15%) significantly increased insulin-like growth factor-1 and MMP-2 messenger RNA expression 21 hr after overload, and the levels of the atrophic gene MAFbx were significantly downregulated 45 hr after mechanical overload. In addition, p70S6 kinase and 4EBP-1 phosphorylation were upregulated immediately after mechanical overload. Maximal contractile force was augmented 45 hr after load with a 265% increase in force, alongside significant hypertrophy of the myotubes within the engineered muscle. Overall, mechanical loading of tissue-engineered skeletal muscle induced hypertrophy and improved force production.     

Protein synthesis in skeletal muscle measured at different times during a 24 hour period

Six groups of 5 male rats (starting body weight 109 g) were allowed free access to a conventional rat diet. At 4 hourly intervals, starting at 10.00 h muscle protein synthesis was measured. By relating the weights of the gastrocnemius and soleus muscles to the initial body weights of the animals (i.e., at 09.30, day 1), a linear increase in muscle weight throughout the day was demonstrated. The fractional rate of muscle protein synthesis varied from 16.8% per day to 20.3% per day in gastrocnemius muscle and from 17.9% per day and 22.1% per day in the soleus. It was calculated that the maximum error incurred in estimating daily muscle protein synthesis by extrapolation of the value at any one time was 6% in gastrocnemius and 9% in soleus. It is concluded that calculations of the average rate of muscle protein degradation based on the difference between the rates of synthesis and deposition are generally valid in rats allowed free access to an adequate diet.     

Hyperplasia and hypertrophy in the growth of skeletal muscle in juvenile Atlantic salmon, Salmo salar L.

Both red and white muscle fibre numbers in juvenile Atlantic salmon increased gradually with fish length throughout the freshwater growth period. Mean fibre area increased as fish grew to 6.5 cm f.l. , but thereafter was unrelated to fish length. Hyperplasia was most obvious when fish were growing fastest, and was the dominant growth process in fish over 6.5 cm f.l. Hypertrophy was most important when growth was slow, as in autumn and winter.
Mean white fibre area was significantly smaller in deep muscle than at medial and superficial sites. Total cross-sectional area of red, white and total trunk muscle increased with fish length. The ratio of red: white cross-sectional area increased with fish length to a plateau at about 10% after 6.5 cm f.l.     

推拿对失神经骨骼肌萎缩大鼠的治疗作用及其机制*

目的:探讨推拿对失神经骨骼肌萎缩大鼠的治疗作用及其机制。方法:48只雄性SD大鼠随机分为模型组(n=24)和推拿组(n=24),通过切断右侧胫神经制备腓肠肌萎缩大鼠模型。术后第2日开始给推拿组大鼠手术侧腓肠肌给予手法干预,模型组不予干预。两组分别在0 d、7 d、14 d、21 d四个时间点各处死6只大鼠,取大鼠双侧腓肠肌,称重后计算各组大鼠腓肠肌湿重比;HE染色测定肌纤维截面积和直径,实时荧光定量PCR检测腓肠肌中miR-23a、Akt、MuRF1、MAFbx基因相对表达量。结果:与0 d比较,模型组和推拿组大鼠腓肠肌湿重比、肌纤维截面积和直径呈现进行性下降的趋势,其中7 d、14 d、21 d推拿组腓肠肌湿重比、肌纤维截面积和直径均显著高于模型组(P＜0.05,P＜0.01);与0 d比较,模型组和推拿组MuRF1、MAFbx、Akt mRNA表达均呈现先升后降的趋势,其中7 d、21 d推拿组MuRF1 mRNA表达均显著低于模型组(P＜0.05,P＜0.01),7 d、14 d、21 d推拿组MAFbx mRNA表达均显著低于模型组(P＜0.01,P＜0.05,P＜0.01),7 d、14 d、21 d推拿组Akt mRNA表达均显著高于模型组(P＜0.05,P＜0.01);与0 d比较,模型组和推拿组21 d时miR-23a mRNA表达升高,推拿组miR-23a mRNA表达显著高于模型组(P＜0.05)。结论:推拿能延缓失神经骨骼肌的萎缩,其机制可能与上调miR-23a、Akt基因的表达,下调 MuRF1、MAFbx基因的表达,使蛋白降解速度受到抑制,从而减轻骨骼肌蛋白的降解程度有关。     

Supraphysiologic Administration of GDF11 Induces Cachexia in Part by Upregulating GDF15

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低氧暴露所致大鼠骨骼肌萎缩的蛋白转化调节机制

目的 对比低氧暴露和常氧下配对低氧摄食干预(半饥饿状态)下大鼠骨骼肌蛋白质合成和分解相关基因表达的差异,以探讨低氧暴露诱导骨骼肌萎缩发生的可能机制。方法 SD大鼠分为:①常氧正常饮食组(C组);②低氧正常饮食组(H组),氧气浓度为12.4%;③常氧配对饮食组(P组),投食量即为H组前一天摄食量。4周干预后测量大鼠体成分,取比目鱼肌(SOL)和趾长伸肌(EDL),称量湿重;HE染色观察肌纤维形态,计算肌纤维横截面积(FCSA);WB测试骨骼肌中HIF1α、Akt、p-Akt及骨骼肌蛋白合成和分解相关基因蛋白含量。结果 1)H组大鼠体重较C组持续下降,P组与C组间无显著性差异;干预初期H组(P组同)摄食量较C组显著下降,后期两组间无差异;(2)干预后,H组大鼠体质量和肌肉总量较C组和P组显著性降低,P组与C组间无差异;H组两肌肉湿重较C组显著下降;H组EDL的FCSA显著低于C组和P组;(3)H组EDL中HIF1α蛋白含量显著高于C组;H组和P组SOL中p-Akt/Akt比值显著低于C组;H组EDL中mTOR、4EBP1蛋白含量显著低于C组,atrogin1、MuRF1、beclin1蛋白含量及LC3Ⅱ/Ⅰ比值显著高于C组,H组SOL中MuRF1蛋白含量显著高于C组和P组。结论 低氧所致的骨骼肌萎缩由低氧特异性因素诱发,表现为以快肌为主的骨骼肌蛋白合成减少和分解增加,而非低氧下摄食量减少引起。     

Effects of different doses of leucine ingestion following eight weeks of resistance exercise on protein synthesis and hypertrophy of skeletal muscle in rats

[Purpose]

This study was designed to determine the appropriate Leucine intake volume to obtain the effects of restoring damaged muscle through the synthesis of muscle proteins to increase skeletal muscle and improve exercise performance, and to achieve enhanced muscle hypertrophy.

[Methods]

To clarify the effects of leucine on skeletal muscle hypertrophy of SD rats, following eight weeks of resistance exercise (climbing ladder), the mass of the FHL (Flexor hallucis longus) was measured after extraction, after which change in the activity of muscle signaling proteins (PKB/Akt, mTOR, p70S6K, 4EBP1) was analyzed.

[Results]

The expressions of PKB/Akt, mTOR and p70S6K were increased in L5 (Leucine 50% administration group) compared with the control group (CON) and exercise group (Ex, exercise training group); EL1 (exercise + 10% leucine administration group) and EL5 (exercise + 50% Leucine administration) also exhibited increased expressions of PKB/Akt, mTOR, and p70S6K, while no difference between EL1 and EL5 were observed. No significant differences in 4EBP1 were found among any of the groups. In addition, there were no differences in FHL mass, while relative mass (FHL/body mass) was increased in the exercise group (Ex, EL1, EL5) compared with the control group. No differences were observed among the exercise groups.

[Conclusion]

The present study demonstrated that the relative body mass was increased in the EX group compared with the CON group, while no significant differences in muscle mass could be found among the groups. Even though some signaling proteins were increased, or some differences existed among groups, there were no differences in muscle mass between the leucine administration and exercise training combined with leucine administration groups in the present study.     

Key signalling factors and pathways in the molecular determination of skeletal muscle phenotype

The molecular basis and control of the biochemical and biophysical properties of skeletal muscle, regarded as muscle phenotype, are examined in terms of fibre number, fibre size and fibre types. A host of external factors or stimuli, such as ligand binding and contractile activity, are transduced in muscle into signalling pathways that lead to protein modifications and changes in gene expression which ultimately result in the establishment of the specified phenotype. In skeletal muscle, the key signalling cascades include the Ras-extracellular signal regulated kinase-mitogen activated protein kinase (Erk-MAPK), the phosphatidylinositol 3'-kinase (PI3K)-Akt1, p38 MAPK, and calcineurin pathways. The molecular effects of external factors on these pathways revealed complex interactions and functional overlap. A major challenge in the manipulation of muscle of farm animals lies in the identification of regulatory and target genes that could effect defined and desirable changes in muscle quality and quantity. To this end, recent advances in functional genomics that involve the use of micro-array technology and proteomics are increasingly breaking new ground in furthering our understanding of the molecular determinants of muscle phenotype.     

Support for a trimeric collar of myosin binding protein C in cardiac and fast skeletal muscle, but not in slow skeletal muscle

Myosin-binding protein C (MyBPC) is proposed to take on a trimeric collar arrangement around the thick filament backbone in cardiac muscle, based on interactions between cardiac MyBPC domains C5 and C8. We have now determined, using yeast two-hybrid and in vitro binding assays, that the C5:C8 interaction is not dependent on the 28-residue cardiac-specific insert in C5. Furthermore, an interaction of similar affinity occurs between domains C5 and C8 of fast skeletal muscle MyBPC, but not between these domains of the slow skeletal muscle protein. These data have implications for the role and quaternary structure of MyBPC in skeletal muscle.     

A multiplexed in vitro assay system for evaluating human skeletal muscle functionality in response to drug treatment

In vitro systems that mimic organ functionality have become increasingly important tools in drug development studies. Systems that measure the functional properties of skeletal muscle are beneficial to compound screening studies and also for integration into multiorgan devices. To date, no studies have investigated human skeletal muscle responses to drug treatments at the single myotube level in vitro. This report details a microscale cantilever chip-based assay system for culturing individual human myotubes. The cantilevers, along with a laser and photo-detector system, enable measurement of myotube contractions in response to broad-field electrical stimulation. This system was used to obtain baseline functional parameters for untreated human myotubes, including peak contractile force and time-to-fatigue data. The cultured myotubes were then treated with known myotoxic compounds and the resulting functional changes were compared to baseline measurements as well as known physiological responses in vivo. The collected data demonstrate the system's capacity for screening direct effects of compound action on individual human skeletal myotubes in a reliable, reproducible, and noninvasive manner. Furthermore, it has the potential to be utilized for high-content screening, disease modeling, and exercise studies of human skeletal muscle performance utilizing iPSCs derived from specific patient populations such as the muscular dystrophies.     

Adaptation of the ubiquitin-proteasome proteolytic pathway in cancer cachexia

The ubiquitin-proteasome proteolytic pathway is of major importance in the breakdown of skeletal muscle proteins. The first step in this pathway is the covalent attachment of polyubiquitin chains to the targeted protein. Polyubiquitinylated proteins are then recognized and degraded by the 26S proteasome complex. In this review, we critically analyze recent findings in the regulation of ubiquitinylation of protein substrates and of their subsequent proteasome-dependent degradation in animal models of cancer cachexia. In particular, we discuss the influence of various mediators (anorexia, hormones, prostaglandins, cytokines, and proteolysis-inducing factor) in signaling the activation of ubiquitin-proteasome proteolysis in skeletal muscle. These findings have lead to new concepts that are starting to be used for preventing cachexia in cancer and other wasting diseases.     

Growth and multiplication of white skeletal muscle fibres in carp larvae in relation to somatic growth rate

A morphometric analysis of white axial muscle of common carp Cyprinus carpio was undertaken in order to quantify increase in fibre size, fibre nuclei and fibre number in relation to somatic growth rate during early life. In fast-growing carp larvae fed zooplankton, length and height of fibres from the central part of dorsolateral muscle increased at the same rate (0.75) relative to the total length of the larvae during the first 2 weeks of feeding. During this period, the number of nuclei per fibre increased threefold while the number of nuclei per unit fibre surface remained constant. In fast-growing larvae fed a formulated diet, the total cross-sectional area of one epaxial quadrant of white muscle and the total area of white fibres increased at almost the same rate (3.15; 3.23) relative to larval total length during the first 28 days of exogenous feeding. The total number of white fibres increased faster (2.07) relative to the total length of larvae than the mean area of white fibres (1.16). Hyperplasia accounted for 64% of muscle growth in these larvae. The proportion of fibres with a width < 10 μm decreased from 72% at first feeding to 14% 28 days later, while the proportion of fibres with a width >20 μm which was 0% at first feeding increased up to 34% in the same time. The recruitment of new white fibres seemed to be almost the same in the whole muscle quadrant at first feeding and 18 or 28 days later but, 8 days after first feeding, a transient significant recruitment of new fibres was shown at the apex of the myotome. Comparisons between fast- and slow-growing groups of larvae showed that for a given larval total length: (1) the mean width of central white fibres was higher and the proportion of central fibres with a width <10 μm was lower in slow-growing larvae than in fast-growing ones; (2) the total number of white fibres was lower for a higher total cross-sectional area of white muscle in slow-growing larvae than in fast-growing ones. These results suggest that, in Cyprinus carpio larvae, slow-growing conditions are related to a decreased contribution of hyperplasia to muscle growth.     

Short-term thermal acclimation induces adaptive changes in the inner mitochondrial membranes of fish skeletal muscle

In the oxidative muscles (musculi laterales superficiales) of crucian carp Carassius carassius acclimated for 6 weeks to either 5 or 25° C, the volume density and the surface density of fibres per tissue did not differ significantly between the control and experimental groups. The correlation ratio (μ²) for these values was below 50, 39·3 and 43·9 respectively. After acclimation to 5° C, the surface density of outer mitochondrial membrane per fibre increased significantly from 0·93 to 1·23m² cm⁻³ in the summer population but dropped from 0·94 to 0·67 m² cm⁻³ in the winter population. The surface density of outer mitochondrial membrane per mitochondrion increased from 3·24 to 4·52 m² cm⁻³ in summer fish. After acclimation to 25° C, the surface density of inner mitochondrial membranes per muscle fibre decreased from 4·04 to 1·79 m² cm⁻³ in summer fish and from 3·86 to 1·07 m² cm⁻³ in winter fish. The surface density of inner mitochondrial membranes per mitochondrion increased from 14·17 to 15·60 m²cm⁻³ in summer fish but dropped from 13·91 to 10·67 m² cm⁻³ in winter fish. Correlation matrices demonstrate a negative correlation of the surface density of outer mitochondrial membrane per mitochondrion with the volume density of mitochondria per fibre and temperature, suggesting cold-induced proliferation of small mitochondria. It was concluded that short-term cold acclimation increased surface area of the inner mitochondrial membranes in summer fish.     

Endurance exercise training in common carp Cyprinus carpio L. induces proliferation of myonuclei in fast muscle fibres and slow muscle fibre hypertrophy

Endurance exercise training (2·4–2·6 body lengths s⁻¹, 16 h day⁻¹ for 28 days) resulted in an increased density of myonuclei in fast muscle fibres relative to tank rested controls and induced slow muscle fibre hypertrophy. The results indicate that exercise is a powerful stimulus for the proliferation of myogenic cells and nuclear accretion.