Systems-based Discovery of Tomatidine as a Natural Small Molecule Inhibitor of Skeletal Muscle Atrophy

Skeletal muscle atrophy is a common and debilitating condition that lacks an effective therapy. To address this problem, we used a systems-based discovery strategy to search for a small molecule whose mRNA expression signature negatively correlates to mRNA expression signatures of human skeletal muscle atrophy. This strategy identified a natural small molecule from tomato plants, tomatidine. Using cultured skeletal myotubes from both humans and mice, we found that tomatidine stimulated mTORC1 signaling and anabolism, leading to accumulation of protein and mitochondria, and ultimately, cell growth. Furthermore, in mice, tomatidine increased skeletal muscle mTORC1 signaling, reduced skeletal muscle atrophy, enhanced recovery from skeletal muscle atrophy, stimulated skeletal muscle hypertrophy, and increased strength and exercise capacity. Collectively, these results identify tomatidine as a novel small molecule inhibitor of muscle atrophy. Tomatidine may have utility as a therapeutic agent or lead compound for skeletal muscle atrophy.     

Muscle progenitor cell proliferation during passive stretch of unweighted soleus in dystrophin deficient mice

Dystrophin, subsarcolemmal protein communicating muscle fiber cytoskeleton to extracellular matrix, is believed to be involved in mechanical signal transduction. The experiment was carried out to assess the role of dystrophin in passive stretch-induced preventing unloaded muscle fiber atrophy and possible linkage between this protein and muscle progenitor (satellite cells) proliferation activity. The study was performed on two months old C57 black and mdx (dystrophin-deficient) mice. Passive stretch resulted in attenuating atrophy development in two fiber types of both C57 black and mdx mice. Altered dystrophin synthesis in mdx mice had virtually no effect on passive stretch preventive action. Thus the hypothesis about dystrophin key role in mediating stretch-induced hypertrophy effects didn't find its confirmation concerning gravitational unloading atrophy. Chronic hindlimb unloading downregulated SC proliferative activity in soleus muscle, passive stretch drastically increased proliferation both in C57 and mdx mice. Thus we observed no relationship between altered dystrophin synthesis and satellite cell proliferation activity in soleus muscle under conditions of simulated microgravity and concurrent passive stretch.     

Insight into skeletal muscle mechanotransduction: MAPK activation is quantitatively related to tension.

The mechanism by which mechanical forces acting through skeletal muscle cells generate intracellular signaling, known as mechanotransduction, and the details of how gene expression and cell size are regulated by this signaling are poorly understood. Mitogen-activated protein kinases (MAPKs) are known to be involved in mechanically induced signaling in various cell types, including skeletal muscle where MAPK activation has been reported in response to contraction and passive stretch. Therefore, the investigation of MAPK activation in response to mechanical stress in skeletal muscle may yield important information about the mechanotransduction process. With the use of a rat plantaris in situ preparation, a wide range of peak tensions was generated through passive stretch and concentric, isometric, and eccentric contractile protocols, and the resulting phosphorylation of c-Jun NH(2)-terminal kinase (JNK), extracellular regulated kinase (ERK), and p38 MAPKs was assessed. Isoforms of JNK and ERK MAPKs were found to be phosphorylated in a tension-dependent manner, such that eccentric > isometric > concentric > passive stretch. Peak tension was found to be a better predictor of MAPK phosphorylation than time-tension integral or rate of tension development. Differences in maximal response amplitude and sensitivity between JNK and ERK MAPKs suggest different roles for these two kinase families in mechanically induced signaling. A strong linear relationship between p54 JNK phosphorylation and peak tension over a 15-fold range in tension (r(2) = 0.89, n = 32) was observed, supporting the fact that contraction-type differences can be explained in terms of tension and demonstrating that MAPK activation is a quantitative reflection of the magnitude of mechanical stress applied to muscle. Thus the measurement of MAPK activation, as an assay of skeletal muscle mechanotransduction, may help elucidate mechanically induced hypertrophy.     

Role of muscle progenitor cells in maintaining morphological characteristics of rat soleus muscle during gravitational unloading by means of passive stretch

Working hypertrophy of skeletal muscle is usually coupled with activation of satellite cells with subsequent incorporation of their nuclei into muscle fibers. Earlier, it has been repeatedly shown that muscle stretching prevents the development of atrophic alterations and is accompanied by an intensification of protein synthesis. We suggested that the elimination of the proliferative abilities of progenitor cells by γ-irradiation would lead to a partial loss of the ability of muscle fibers to maintain their size. To evaluate the role of progenitor cells in the development of the preventive effect of passive stretching, an experiment was carried out with the 2500 rad local irradiation of a rat shin and subsequent hind-limb suspension or hind-limb suspension with stretch. Passive stretching during hind-limb suspension completely prevented atrophy, the transformation of fibers, and a decrease in the myonuclear number observed in the hind-limb-suspension group. Irradiation produced no action of the preventive effect of passive stretch. The conclusion is made that passive stretch preventive action is also realized in the absence of proliferating satellite cells.     

Stimulation of glucose transport in L6 muscle cells by long-term intermittent stretch-relaxation.

Skeletal muscle stretch increases resting metabolism and causes hypertrophy. We have examined the effect of mechanical stretch in vitro on glucose transport activity and transporter contents in L6 muscle cells. Long-term (24-48 h) stretch-relaxation (25% maximal elongation at 30 cycles per min) of cell monolayers significantly increased glucose uptake by 1.6- to 2-fold in myotubes but not in myoblasts. The presence of serum was required for the stretch-relaxation induced increase in glucose uptake. Cycloheximide inhibited the mechanical stimulation of glucose uptake, and the latter response was not additive to the stimulatory effect of long-term exposure to insulin. GLUT1 and GLUT4 glucose transporter contents were not changed in total cell membranes from mechanically stimulated cells relative to controls. These results indicate that mechanical stimulation through passive stretch may be an important regulation of nutrient uptake in fetal myotubes independent of innervation.     

The cellular effects of functional unloading and passive stretch on m. soleus of dystrophin-deficient mdx mice

Dystrophin, subsarcolemmal protein communicating muscle fiber cytoskeleton to extracellular matrix, is believed to participate in mechanical signal transduction. Recent works testify possible signaling role of this protein to prevent development ofproteolytic processes accompanying muscle fiber atrophy and to stimulate the passive stretch anabolic effects. The experiment was carried out to assess the role of dystrophin in these processes. The study was performed on two months old C57 black and mdx (dystrophin-deficient) mice. Passive stretch resulted in attenuating atrophy development in two fiber types of both C57 black and mdx mice, at the same time fiber type slow-to-fast transformation did not occur in mdx soleus. We established ablatitious effect of chronic hindlimb unloading on SC proliferative activity in soleus muscle and drastic increase of proliferation under effect of passive stretch. We observed no relationship between altered dystrophin synthesis and satellite cell proliferation activity in soleus muscle under conditions of simulated microgravity and concurrent passive stretch. It is concluded that altered dystrophin synthesis partly retarded slow myofibers atrophy and had virtually no effect on passive stretch preventive action. Thus, the hypothesis about dystrophin key role in downregulation of atrophy signaling mechanisms has not found its confirmation concerning gravitational unloading atrophy.     

Heat stress facilitates stretch-induced hypertrophy of cultured muscle cells

Increased mechanical stress induced by stretch is an important growth stimulus in skeletal muscle. Heat shock proteins (HSPs) are an important family of endogenous, protective proteins. HSP90 and HSP70 families show elevated levels under beat stress. Mechanical stress, such as physical exercise, is known to induce not only muscular hypertrophy but also the elevation of HSPs expression in skeletal muscle. The purpose of this study was to determine whether heat stress facilitates the stretch-induced hypertrophy of skeletal muscle cells. Cultured rat myotubes (L6) were plated on collagenized Silastic membranes and incubated at 41 degrees C for 60 and 75 minutes (heat shock). Following the incubation, the cells were subjected two-second stretching and four-second releasing for 4 days at 37 degrees C. Protein concentrations in the homogenates and pellets of the cultured skeletal muscle cells increased under heat shock and/or mechanical stretching. The protein concentration of cells following mechanical stretching following heat shock was significantly higher than that following either heat shock or mechanical stretching alone. HSP72 in supernatants and HSP90 in pellets increased under heat shock and/or mechanical stretching. HSP90 in supernatants decreased following heat shock and/or mechanical stretching. Changes in HSPs and cellular protein concentrations in stressed cells suggest that the expression of HSPs may be closely related with muscular hypertrophy.     

A proteome map of murine heart and skeletal muscle

The balance of hypertrophy and atrophy is critical for the adaptation of cardiac and skeletal muscle mass to the demands of the environment and when deregulated can cause disease. Here we have used a proteomics approach to generate protein reference maps for the mouse heart and skeletal muscle, which provide a molecular basis for future functional and pathophysiological studies. The reference map provides information on molecular mass, pI, and literature data on function and localization, to facilitate the identification of proteins based on their migration in 2-D gels. In total, we have identified 351 cardiac and 284 skeletal muscle protein spots, representing 249 and 214 different proteins, respectively. In addition, we have visualized the protein pattern of mouse heart and skeletal muscle at defined conditions comparing knockout (KO) animals deficient in the sarcomeric protein titin (a genetic atrophy model) and control littermates. We found 20 proteins that were differently expressed linking titin's kinase region to the heat-shock- and proteasomal stress response. Taken together, the established reference maps should provide a suitable tool to relate protein expression and PTM to cardiovascular and skeletal muscle disease using the mouse as an animal model.     

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.     

Satellite cells are increasingly refractory to activation by nitric oxide and stretch in aged mouse-muscle cultures

Age-related muscle atrophy or sarcopenia results in progressive loss of muscle function and satellite cells in aging muscle are increasingly refractory to activation that could mitigate atrophy. We know that nitric oxide release triggered by mechanical stretch of skeletal muscle, initiates satellite cell activation in vitro in single fiber, single cell and whole-muscle cultures, and in vivo in animals. This study examined muscle cell activation using tritiated-thymidine incorporation into the DNA of muscle cells in cultured muscles from female mice between 6 weeks and 18 months-of-age. Experiments examined age-related changes in activation by mechanical stretch and/or NO treatments (with the substrate of nitric oxide synthase (l-arginine), a nitric oxide donor (isosorbide dinitrate) and/or nitric oxide synthase inhibition). Activation without stretch was highest at 8 months. Stretching muscles by 10% more than doubled activation in muscles at 6 weeks of age and only a 20% stretch similarly activated cells in cultured 6-month-old muscles. Only treatment with ISDN in combination with a 20% stretch activated cell proliferation in muscles from 8-month-old mice. A nitric-oxide donor drug rescued muscle satellite cells in adult, 8-month-old mice from being refractory to mechanical stretch, apparently by overcoming an ineffective release of nitric oxide during stretch. Results suggest that treatment with nitric oxide has the potential to enhance the effectiveness of exercise in preventing the onset of age-related muscle atrophy in adult muscle.