Proteomics-based investigation in C2C12 myoblast differentiation

Skeletal muscle cell differentiation is a multistage process extensively studied over the years. Even if great improvements have been achieved in defining biological process underlying myogenesis, many molecular mechanisms need still to be clarified

Myoblast models of skeletal muscle hypertrophy and atrophy

Purpose of review: To highlight recent breakthroughs and controversies in the use of myoblast models to uncover cellular and molecular mechanisms regulating skeletal muscle hypertrophy and atrophy. Recent findings: Myoblast cultures provide key mechanistic models of the signalling and molecular pathways potentially employed by skeletal muscle in-vivo to regulate hypertrophy and atrophy. Recently the controversy as to whether insulin-like growth factor (IGF)-I is important in hypertrophy following mechanical stimuli vs. alternative pathways has been hotly debated and is discussed. The role of myostatin in myoblast models of atrophy and interactions between protein synthetic pathways including Akt/mTOR and the ‘atrogenes’ are explored. Summary: Targeted in-vivo experimentation directed by skeletal muscle cell culture and bioengineering (three-dimensional skeletal muscle cell culture models) will provide key biomimetic and mechanistic data regarding hypertrophy and atrophy and thus enable the development of important strategies for tackling muscle wasting associated with ageing and disease processes

Tadalafil improves lean mass and endothelial function in nonobese men with mild ED/LUTS: in vivo and in vitro characterization

PURPOSE: Phosphodiesterase type-5 inhibitor administration in diabetic men with erectile dysfunction (ED) is associated with reduced waist circumference. We evaluated potential effects of daily tadalafil administration on body composition and investigated its possible mechanism(s) of action in C2C12 skeletal muscle cells in vitro. METHODS: Forty-three men on stable caloric intake (mean age 48.5 ± 7; BMI 25.5 ± 0.9 kg/m2) complaining mild ED and/or low urinary tract symptoms (LUTS) were randomly assigned to receive tadalafil (TAD) 5 mg/daily (once-a-day=OAD-TAD; n = 23) or 20 mg on-demand (on-demand=OD-TAD; n = 20) for 2 months. Primary outcomes were variations of body composition measured by Dual-energy X-ray absorptiometry; secondary outcomes were ED/LUTS questionnaire scores along with hormone (testosterone, estradiol, insulin) and endothelial function (Endopat2000) variations. RESULTS: OAD-TAD increased abdominal lean mass (p < 0.01) that returned to baseline after 2 months withdrawal. LUTS scores improved (p<0.01) in OD-TAD while ED scores improved (p < 0.01) in both groups. We found significant improvements in endothelial function (p < 0.05) that directly correlated with serum insulin (p < 0.01; r = 0.3641) and inversely correlated with estradiol levels (p < 0.01; r = 0.3655) even when corrected for potential confounders. Exposure of C2C12 cells upon increasing tadalafil concentrations (10-7 to 10-6 M) increased total androgen receptor mRNA and protein expression as well as myogenin protein expression after 24 and 72 h (2.8 ± 0.4-fold and 1.4 ± 0.02-fold vs. control, respectively, p < 0.05). CONCLUSIONS: Daily tadalafil improved lean mass content in non-obese men probably via enhanced insulin secretion, estradiol reduction, and improvement of endothelial function in vivo. The in vitro increased myogenin and androgen receptor protein expression in skeletal muscle cells suggests a translational action of phosphodiesterase type-5 on this receptor

A comparison of melatonin and α-lipoic acid in the induction of antioxidant defences in L6 rat skeletal muscle cells.

Aging is characterized by a progressive deterioration in physiological functions and metabolic processes. The loss of cells during aging in vital tissues and organs is related to several factors including oxidative stress and inflammation. Skeletal muscle degeneration is common in elderly people; in fact, this tissue is particularly vulnerable to oxidative stress since it requires large amounts of oxygen, and thus, oxidative damage is abundant and accumulates with increasing age. Melatonin (N-acetyl-5-methoxytryptamine) is a highly efficient scavenger of reactive oxygen species and it also exhibits beneficial anti-inflammatory and anti-aging effects. This study investigated the susceptibility of rat L6 skeletal muscle cells to an induced oxidative stress following their exposure to hydrogen peroxide (50 μM) and evaluating the potential protective effects of pre-treatment with melatonin (10 nM) compared to the known beneficial effect of alpha-lipoic acid (300 μM). Hydrogen peroxide-induced obvious oxidative stress; it increased the expression of tumour necrosis factor-alpha and in turn promoted nuclear factor kappa-B and overrode the endogenous defence mechanisms. Conversely, pre-treatment of the hydrogen peroxide-exposed cells to melatonin or alpha-lipoic acid increased endogenous antioxidant enzymes, including superoxide dismutase-2 and heme oxygenase-1; moreover, they ameliorated significantly oxidative stress damage and partially reduced alterations in the muscle cells, which are typical of aging. In conclusion, melatonin was equally effective as alpha-lipoic acid; it exhibited marked antioxidant and anti-aging effects at the level of skeletal muscle in vitro even when it was given in a much lower dose than alpha-lipoic acid

Further considerations on in vitro skeletal muscle cell death

The present review discusses the apoptotic behavior induced by chemical and physical triggers in C2C12 skeletal muscle cells, comparing myoblast to myotube sensitivity, and investigating it by means of morphological, biochemical and cytofluorimetric analyses. After all treatments, myotubes, differently from myoblasts, showed a poor sensitivity to cell death. Intriguingly, in cells exposed to staurosporine, etoposide and UVB radiation, apoptotic and normal nuclei within the same fibercould be revealed. The presence of nuclear-dependent “territorial” death domains in the syncytium could explain a delayed cell death of myotubes compared to mononucleated cells. Moreover, autophagic granules abundantly appeared in myotubes after each treatment. Autophagy could protect muscle cell integrity against chemical and physical stimuli, making C2C12 myotubes, more resistant to cell death inductio

Disease-associated mutations in KCNE potassium channel subunits (MiRPs) reveal promiscuous disruption of multiple currents and conservation of mechanism.

KCNE genes encode single transmembrane-domain subunits, the MinK-related peptides (MiRPs), which assemble with pore-forming alpha subunits to establish the attributes of potassium channels in vivo. To investigate whether MinK, MiRP1, and MiRP2 operate similarly with their known native alpha subunit partners (KCNQ1, HERG, and Kv3.4, respectively) two conserved residues associated with human disease and influential in channel function were evaluated. As MiRPs assemble with a variety of alpha subunits in experimental cells and may do so in vivo, each peptide was also assessed with the other two alpha subunits. Inherited mutation of aspartate to asparagine (D --&gt; N) to yield D76N-MinK is linked to cardiac arrhythmia and deafness; the analogs D82N-MiRP1 and D90N-MiRP2 were studied. Mutation of arginine to histidine (R --&gt; H) to yield R83H-MiRP2 is associated with periodic paralysis; the analogs K69H-MinK and K75H-MiRP1 were also studied. Macroscopic and single-channel currents showed that D --&gt; N mutations suppressed a subset of functions whereas R/K --&gt; H changes altered the activity of MinK, MiRP1, and MiRP2 with all three alpha subunits. The findings indicate that the KCNE peptides interact similarly with different alpha subunits and suggest a hypothesis: that clinical manifestations of inherited KCNE point mutations result from disruption of multiple native currents via promiscuous interactions

The omega-3 fatty acid, eicosapentaenoic acid (EPA), prevents the damaging effects of tumour necrosis factor (TNF)-alpha during murine skeletal muscle cell differentiation

Background: Eicosapentaenoic acid (EPA) is a -3 polyunsaturated fatty acid with antiinflammatory and anti-cachetic properties that may have potential benefits with regards to skeletal muscle atrophy conditions where inflammation is present. It is also reported that pathologic levels of the pro-inflammatory cytokine tumour necrosis factor (TNF)-α are associated with muscle wasting, exerted through inhibition of myogenic differentiation and enhanced apoptosis. These findings led us to hypothesize that EPA may have a protective effect against skeletal muscle damage induced by the actions of TNF-α. Results: The deleterious effects of TNF-α on C2C12 myogenesis were completely inhibited by co-treatment with EPA. Thus, EPA prevented the TNF-mediated loss of MyHC expression and significantly increased myogenic fusion (p < 0.05) and myotube diameter (p < 0.05) indices back to control levels. EPA protective activity was associated with blocking cell death pathways as EPA completely attenuated TNF-mediated increases in caspase-8 activity (p < 0.05) and cellular necrosis (p < 0.05) back to their respective control levels. EPA alone significantly reduced spontaneous apoptosis and necrosis of differentiating myotubes (p < 0.001 and p < 0.05, respectively). A 2 hour pre-treatment with EPA, prior to treatment with TNF alone, gave similar results. Conclusion: In conclusion, EPA has a protective action against the damaging effects of TNF-α on C2C12 myogenesis. These findings support further investigations of EPA as a potential therapeutic agent during skeletal muscle regeneration following injury

Reduction in BACE1 decreases body weight, protects against diet-induced obesity and enhances insulin sensitivity in mice

Insulin resistance and impaired glucose homoeostasis are important indicators of Type 2 diabetes and are early risk factors of AD (Alzheimer's disease). An essential feature of AD pathology is the presence of BACE1 (β-site amyloid precursor protein-cleaving enzyme 1), which regulates production of toxic amyloid peptides. However, whether BACE1 also plays a role in glucose homoeostasis is presently unknown. We have used transgenic mice to analyse the effects of loss of BACE1 on body weight, and lipid and glucose homoeostasis. BACE1−/− mice are lean, with decreased adiposity, higher energy expenditure, and improved glucose disposal and peripheral insulin sensitivity than wild-type littermates. BACE1−/− mice are also protected from diet-induced obesity. BACE1-deficient skeletal muscle and liver exhibit improved insulin sensitivity. In a skeletal muscle cell line, BACE1 inhibition increased glucose uptake and enhanced insulin sensitivity. The loss of BACE1 is associated with increased levels of UCP1 (uncoupling protein 1) in BAT (brown adipose tissue) and UCP2 and UCP3 mRNA in skeletal muscle, indicative of increased uncoupled respiration and metabolic inefficiency. Thus BACE1 levels may play a critical role in glucose and lipid homoeostasis in conditions of chronic nutrient excess. Therefore strategies that ameliorate BACE1 activity may be important novel approaches for the treatment of diabetes

Trichinella inflammatory myopathy: host or parasite strategy?

The parasitic nematode Trichinella has a special relation with muscle, because of its unique intracellular localization in the skeletal muscle cell, completely devoted in morphology and biochemistry to become the parasite protective niche, otherwise called the nurse cell. The long-lasting muscle infection of Trichinella exhibits a strong interplay with the host immune response, mainly characterized by a Th2 phenotype

Receptor Expression in Rat Skeletal Muscle Cell Cultures

One on the most persistent problems with long-term space flight is atrophy of skeletal muscles. Skeletal muscle is unique as a tissue in the body in that its ability to undergo atrophy or hypertrophy is controlled exclusively by cues from the extracellular environment. The mechanism of communication between muscle cells and their environment is through a group of membrane-bound and soluble receptors, each of which carries out unique, but often interrelated, functions. The primary receptors include acetyl choline receptors, beta-adrenergic receptors, glucocorticoid receptors, insulin receptors, growth hormone (i.e., somatotropin) receptors, insulin-like growth factor receptors, and steroid receptors. This project has been initiated to develop an integrated approach toward muscle atrophy and hypertrophy that takes into account information on the populations of the entire group of receptors (and their respective hormone concentrations), and it is hypothesized that this information can form the basis for a predictive computer model for muscle atrophy and hypertrophy. The conceptual basis for this project is illustrated in the figure below. The individual receptors are shown as membrane-bound, with the exception of the glucocorticoid receptor which is a soluble intracellular receptor. Each of these receptors has an extracellular signalling component (e.g., innervation, glucocorticoids, epinephrine, etc.), and following the interaction of the extracellular component with the receptor itself, an intracellular signal is generated. Each of these intracellular signals is unique in its own way; however, they are often interrelated