BackgroundMyogenesis is susceptible to the availability of nutrients and humoral factors and suboptimal fetal environments affect the number of myofibers and muscle mass.AimWe examined the mechanisms regulating cell cycle progression and arrest in skeletal myoblasts.Materials and methodsMouse C2C12 myoblasts were subjected to proliferation or induction of differentiation in the presence of high glucose and high insulin (HGHI glucose 15 mmol/l, insulin 50 nmol/l), and these effects were compared with the influence of anabolic factor for skeletal muscle, insulin-like growth factor-I (IGF-I 30 nmol/l).ResultsHigh glucose and high insulin, similarly to IGF-I, increased the intracellular level of cyclin A, cyclin B1 and cyclin D1 during myoblast proliferation. In HGHI-treated myoblasts, these cyclins were localized mostly in the nuclei, and the level of cdk4-bound cyclin D1 was augmented. HGHI significantly stimulated the expression of cyclin D3, total level of p21 and cdk-bound fraction of p21 in differentiating cells. The cellular level of MyoD was augmented by HGHI both in proliferating and differentiating myogenic cells.ConclusionsHigh glucose and insulin modify the mechanisms controlling cell cycle progression and the onset of myogenesis by: (1) increase of cyclin A, cyclin B1 and cyclin D1 in myoblast nuclei, and stimulation of cyclin D1-cdk4 binding; (2) increase in cyclin D3 and MyoD levels, and the p21-cdk4 complexes after induction of differentiation. Hyperglycemia/hyperinsulinemia during fetal or postnatal life could exert effects similar to IGF-I and can be, therefore, favourable for skeletal muscle growth and regeneration.

We have examined the progression and regulation of myogenesis, cellular levels of IGFBP-4, -5, -6, and several extracellular matrix (ECM) proteins (fibronectin, integrin α5, β1 subunits and a disintegrin metalloprotease ADAM12) in murine C2C12 myoblasts during 3-day differentiation under high glucose alone or combined with high insulin, factors characteristic for type 1 and 2 diabetes. High ambient glucose inhibited myogenesis of C2C12 myoblasts, an effect manifested by a twofold decrease in myoblast fusion, a drop in intracellular MyoD, myogenin and MHC levels, and increased cellular content of active myostatin isoform. Reduction in myogenesis by high glucose is accompanied by increase in cellular levels of IGFBP-4 and -6 and decrease in IGFBP-5. High glucose could modify ECM components assembly, by the increase in fibronectin levels and the decrease in metalloprotease ADAM12, without the effect on integrin α5 and β1 subunits. In contrast, high glucose and high insulin activate myoblast differentiation, manifested by an increase in fusion index and myogenin, as well as a drop in myostatin levels. The presence of high insulin prevented high-glucose-dependent changes in IGFBPs and ECM proteins. The data indicate the potential mechanisms of the influence of extracellular environment associated with maternal diabetes and insulin resistance on foetal myogenesis.

Skeletal muscle tissue makes up approximately 40% of the total body mass in adult mammals. Contractile muscle fibers building skeletal muscle tissue are coated by an extracellular matrix material (ECM), accounting for 1-10% of the muscle mass. The ECM in skeletal muscle was initially considered as a structure, providing mechanical support for bearing force transmission. Now it is evident that muscle cells adhere to and connect with the ECM, also for signaling, and the ECM provides an appropriate and permissive environment for muscle development and functioning. This chapter summarizes current knowledge on the role of ECM components in skeletal muscle growth and regeneration, which is of great importance for potential therapeutic interventions. It also focuses on the contribution of ECM in the motor function of skeletal muscle as well as on mechanisms mediating muscle ECM remodeling during adaptation to physical activity. The role of the ECM in the metabolic function of skeletal muscle tissue and the ECM disturbances associated with insulin resistance are described. Finally, the attention is paid on potential implications of changes in skeletal muscle ECM assembly and function in health and disease.

The purpose of the study was to examine the mechanisms important for early myogenesis in mouse C2C12 myogenic cells exposed to interleukin-1β. Cyclin A and cyclin B1 were increased by interleukin-1β (1 ng/ml), but the level of cyclin D1 and total DNA content was unaffected. Fusion index and the rate of protein synthesis was increased in the presence of IL-1β, but these effects were limited to 3-day-treatment. IL-1β increased the level of MyoD, myogenin and MHC on the 3 rd day of differentiation, without altering the content of the active form of myostatin, as well as it augmented the level of fibronectin, integrin β1 and full length 100 kDa form of ADAM12. IL-1β caused a decrease in IGFBP-4 and IGFBP-6 levels and a marked increase in IGFBP-5. The phosphorylation of PKB and ERK1/2 and the cellular content of p38 were elevated by IL-1β. We conclude that the myogenic effect of IL-1β was limited to the onset of myoblast fusion and was associated with: i) increase in the level of myogenic transcription factors i.e. MyoD and myogenin expression, ii) modification of extracellular matrix assembly and signaling, manifested by an increase in fibronectin, integrin-β1 and ADAM12 content, iii) drop in IGFBP-4 and IGFBP-6, and an increase in IGFBP-5, that could alter the local IGF-1 bioavailability, and iv) increase in phosphorylation of PKB and ERK1/2, and the expression of p38 kinase, leading to activation of intracellular pathways essential for myogenic differentiation.

Interleukin (IL)-8 is released both in visceral adipose tissue and in contracting skeletal muscles. In this study, we examined cellular pathways associated with muscle hypertrophy, chosen on the basis of microRNA profiling, in differentiating rat primary skeletal muscle cells (RSkMC) treated with IL-8 (1 ng/ml) for 11 days. IL-8 increased myocilin expression, Akt phosphorylation, FoxO3 dispersion throughout the cytoplasm, and reduced FoxO3 level. IL-8 decreased the expression of atrogin and MuRF1 and increased myotube length and diameter. We concluded that IL-8 present in extracellular environment of myoblasts induced to differentiation stimulates expression of myocilin, a protein important for skeletal muscle hypertrophy. This phenomenon was associated with: (a) activation of myogenic transcription, (b) increased phosphorylation and activation of PKB/Akt, leading to (c) cytoplasm distribution and degradation of a transcription factor FoxO3, (d) decreased expression of gene markers of proteolysis, atrogin and Murf1, and (e) increased myotube length and diameter. In this regard, IL-8 affects skeletal muscle cells similarly to IGF-I and can be considered as a potent anticatabolic factor for skeletal muscle.

The purpose of the study was to examine mechanisms controlling cell cycle progression/arrest and differentiation of mouse C2C12 myoblasts exposed to long-chain saturated fatty acid salt, palmitate. Treatment of proliferating myoblasts with palmitate (0.1 mmol/l) markedly decreased myoblast number. Cyclin A and cyclin D1 levels decreased, whereas total p21 and p21 complexed with cyclin-dependent kinase-4 (cdk4) increased in myoblasts treated with palmitate. In cells induced to differentiation addition of palmitate augmented the level of cyclin D3, the early (myogenin) and late (α-actinin, myosin heavy chain) markers of myogenesis, and caused an increase of myotube diameter. In conclusion, exposure to palmitate inhibits proliferation of myoblasts through a decrease in cyclin A and cyclin D1 levels and an increase of p21-cdk4 complex formation; however, it promotes cell cycle exit, myogenic differentiation and myotube growth.

High-fat diet, exposure to saturated fatty acids, or the presence of adipocytes in myoblast microenvironment affects skeletal muscle growth and function. The aim of the present study was to investigate the effect of palmitate supplementation on transcriptomic profile of mouse C2C12 myoblasts. Global gene expression was evaluated using whole mouse genome oligonucleotide microarrays, and the results were validated through qPCR. A total of 4047 genes were identified as differentially expressed, including 3492 downregulated and 555 upregulated genes, during a 48-h exposure to palmitate (0.1 mmol/l). Functional classification showed the involvement of these genes in several processes which regulate cell growth. In conclusion, the addition of palmitate modifies the expression of genes associated with (1) myoblast responsiveness to hormones and growth factors, (2) cytokine and growth factor expression, and (3) regulation of cell-cell and cell-matrix communication. Such alterations can affect myoblast growth and differentiation; however, further studies in this field are required.

Cachexia is a multifactorial syndrome of atrophy of skeletal muscle and adipose tissue, resulting in progressive loss of body weight associated with low quality of life and poor prognosis in cancer. Studies on experimental animal models and observations on patients have shown that the soluble factors secreted by tumor cells and tissues of the patient can participate in regulation of the wasting process. Cachexia is often accompanied by anorexia, which is caused by predominance of signals inhibiting appetite in the hypothalamus, such as release of proopiomelanocortin and anorexigenic action of proinflammatory cytokines (IL-1α, IL-1β, IL-6, TNF-α). Cachexia is also accompanied by extensive metabolic changes consisting of increase of resting energy expenditure and disturbance of carbohydrate, protein and lipid metabolism. Increased expression of protein uncoupling phosphorylation leads to increased thermogenesis in skeletal muscle. Tumor tissue hypoxia caused by its growth beyond blood vessels activates the transcription factor HIF-1, which results in increase in glycolysis, and leads to lactic acid accumulation and activation of the energy inefficient Cori cycle. Loss of fat tissue is caused by increase of lipolysis induced by lipid-mobilizing factor (LMF) and proinflammatory cytokines. Skeletal muscle wasting in cachexia is caused by a reduction of protein synthesis at the stage of initiation and elongation of translation and the simultaneous increase of protein degradation via ubiquitin-dependent and lysosomal pathways. The main mediators of skeletal muscle wasting in cancer are proteolysis-inducing factor (PIF), proinflammatory cytokines, and angiotensin II acting through increased levels of reactive oxygen species (ROS) and nuclear factor NF-κB activation, as well as glucocorticoid activated FOXO transcription factors and myostatin. Understanding of the complexity of the interaction of factors produced by the tumor and the patient's body may form the basis for the development of effective treatments for cachexia in cancer and other pathological conditions.