L-carnitine supplementation attenuates NAFLD progression and cardiac dysfunction in a mouse model fed with methionine and choline-deficient diet.

Non-alcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disorder. NAFLD, associated lipotoxicity, fibrosis, oxidative stress, and altered mitochondrial metabolism, is responsible for systemic inflammation, which contributes to organ dysfunction in extrahepatic tissues, including the heart. We investigated the ability of L-carnitine (LC) to oppose the pathogenic mechanisms underlying NAFLD progression and associated heart dysfunction, in a mouse model of methionine-choline-deficient diet (MCDD). Mice were divided into three groups: namely, the control group (CONTR) fed with a regular diet and two groups fed with MCDD for 6 weeks. In the last 3 weeks, one of the MCDD groups received LC (200 mg/kg each day) through drinking water (MCDD + LC). The hepatic lipid accumulation and oxidative stress decreased after LC supplementation, which also reduced hepatic fibrosis via modulation of α-smooth muscle actin (αSMA), peroxisome-activated receptor gamma (PPARγ), and nuclear factor kappa B (NfƙB) expression. LC ameliorated systemic inflammation, mitigated cardiac reactive oxygen species (ROS) production, and prevented fibrosis progression by acting on signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated kinase 1-2 (ERK1-2), and αSMA. This study confirms the existence of a relationship between fatty liver disease and cardiac abnormalities and highlights the role of LC in controlling liver oxidative stress, steatosis, fibrosis, and NAFLD-associated cardiac dysfunction.

Metformin Counteracts HCC Progression and Metastasis Enhancing KLF6/p21 Expression and Downregulating the IGF Axis.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is the common tumor of the liver. Unfortunately, most HCC seem to be resistant to conventional chemotherapy and radiotherapy. The poor efficacy of antitumor agents is also due, at least in part, to the inefficient drug delivery and metabolism exerted by the steatotic/cirrhotic liver that hosts the tumor. Thus, novel approaches in chemotherapy may be needed to improve the survival rate in patients with HCC. Metformin (METF) has been found to lower HCC risk; however, the mechanisms by which METF performs its anticancer activity are not completely elucidated. Previous studies have showed METF action on growth inhibition in the liver in a dose/time-dependent manner and its antitumor role by targeting multiple pathways. We investigated molecular effects of METF in an in vitro human hepatoma model (HepG2), studying cell cycle regulators, tumorigenesis markers, and insulin-like growth factor (IGF) axis regulation. MATERIALS AND METHODS: HepG2 cells were treated with METF (400 µM) for 24, 48, and 72 hours. METF action on cell cycle progression and cellular pathways involved in metabolism regulation was evaluated by gene expression analysis, immunofluorescence, and Western blot assay. RESULTS: By assessing HepG2 cell viability, METF significantly decreased growth cell capacity raising KLF6/p21 protein content. Moreover, METF ameliorated the cancer microenvironment reducing cellular lipid drop accumulation and promoting AMPK activity. The overexpression of IGF-II molecule and the IGF-I receptor that plays a main role in HCC progression was counteracted by METF. Furthermore, the protein content of HCC principal tumor markers, CK19 and OPN, linked to the metastasis process was significantly reduced by METF stimulus. CONCLUSION: Our data show that METF could suppress HepG2 proliferation, through induction of cell cycle arrest at the G0/G1 phase. In addition, METF effect on the cancer microenvironment and on the IGF axis leads to the development of new METF therapeutic use in HCC treatment.

L-Carnitine Reduces Oxidative Stress and Promotes Cells Differentiation and Bone Matrix Proteins Expression in Human Osteoblast-Like Cells.

Bone fragility and associated fracture risk are major problems in aging. Oxidative stress and mitochondrial dysfunction play a key role in the development of bone fragility. Mitochondrial dysfunction is closely associated with excessive production of reactive oxygen species (ROS). L-Carnitine (L-C), a fundamental cofactor in lipid metabolism, has an important antioxidant property. Several studies have shown how L-C enhances osteoblastic proliferation and activity. In the current study, we investigated the potential effects of L-C on mitochondrial activity, ROS production, and gene expression involved in osteoblastic differentiation using osteoblast-like cells (hOBs) derived from elderly patients. The effect of 5mM L-C treatment on mitochondrial activity and L-C antioxidant activity was studied by ROS production evaluation and cell-based antioxidant activity assay. The possible effects of L-C on hOBs differentiation were assessed by analyzing gene and protein expression by Real Time PCR and western blotting, respectively. L-C enhanced mitochondrial activity and improved antioxidant defense of hOBs. Furthermore, L-C increased the phosphorylation of Ca2+/calmodulin-dependent protein kinase II. Additionally, L-C induced the phosphorylation of ERK1/2 and AKT and the main kinases involved in osteoblastic differentiation and upregulated the expression of osteogenic related genes, RUNX2, osterix (OSX), bone sialoprotein (BSP), and osteopontin (OPN) as well as OPN protein synthesis, suggesting that L-C exerts a positive modulation of key osteogenic factors. In conclusion, L-C supplementation could represent a possible adjuvant in the treatment of bone fragility, counteracting oxidative phenomena and promoting bone quality maintenance.

Effect of Hazelnut Oil on Muscle Cell Signalling and Differentiation.

Nuts-enriched diets were shown to bear beneficial effects for human's health. Among nuts, hazelnut plays a major role in human nutrition and health because of its unique fatty acid composition (predominantly MUFA), fat soluble bioactives (tocopherols and phytosterols), vitamins (vitamin E), essential minerals (selenium), essential amino acids, antioxidant phenolics (caffeic acid), dietary fiber (soluble form), and bioactive phtytochemicals. The current study was designed to explore the cellular effects of two particular hazelnut strains (Ordu and Tonda).Four hazelnut oils were obtained from 2 common strains (Ordu hazelnut oil, Ordu cuticle oil, Tonda "gentile" hazelnut oil, Tonda "gentile" cuticle oil). The metabolic and nutritional effects of the four hazelnut oils were assessed using an in vitro model of mouse myoblasts, identifying the intracellular mechanisms involved in muscle differentiation and in the modulation of specific muscle genes.We demonstrated that hazelnut oils induced morphological changes in neo-formed myotubes increasing myotubes size. In particular, the diversified effects of the hazelnuts and cuticle oils on muscle fibres shape (on length and diameter respectively) determine a diversified pattern of action on elongation or hypertrophy of the muscle fibres. Furthermore, hazelnut oils regulate different pathways associated with myoblasts growth and development, stimulate signal transduction, and activate cell commitment and differentiation. The present results provide evidence that hazelnut oils may affect skeletal muscle growth and differentiation, constituting the proof of principle for the future development of novel foods and integrators.

L-Carnitine: An Antioxidant Remedy for the Survival of Cardiomyocytes under Hyperglycemic Condition.

BACKGROUND: Metabolic alterations as hyperglycemia and inflammation induce myocardial molecular events enhancing oxidative stress and mitochondrial dysfunction. Those alterations are responsible for a progressive loss of cardiomyocytes, cardiac stem cells, and consequent cardiovascular complications. Currently, there are no effective pharmacological measures to protect the heart from these metabolic modifications, and the development of new therapeutic approaches, focused on improvement of the oxidative stress condition, is pivotal. The protective effects of levocarnitine (LC) in patients with ischemic heart disease are related to the attenuation of oxidative stress, but LC mechanisms have yet to be fully understood. OBJECTIVE: The aim of this work was to investigate LC's role in oxidative stress condition, on ROS production and mitochondrial detoxifying function in H9c2 rat cardiomyocytes during hyperglycemia. METHODS: H9c2 cells in the hyperglycemic state (25 mmol/L glucose) were exposed to 0.5 or 5 mM LC for 48 and 72 h: LC effects on signaling pathways involved in oxidative stress condition were studied by Western blot and immunofluorescence analysis. To evaluate ROS production, H9c2 cells were exposed to H2O2 after LC pretreatment. RESULTS: Our in vitro study indicates how LC supplementation might protect cardiomyocytes from oxidative stress-related damage, preventing ROS formation and activating antioxidant signaling pathways in hyperglycemic conditions. In particular, LC promotes STAT3 activation and significantly increases the expression of antioxidant protein SOD2. Hyperglycemic cardiac cells are characterized by impairment in mitochondrial dysfunction and the CaMKII signal: LC promotes CaMKII expression and activation and enhancement of AMPK protein synthesis. Our results suggest that LC might ameliorate metabolic aspects of hyperglycemic cardiac cells. Finally, LC doses herein used did not modify H9c2 growth rate and viability. CONCLUSIONS: Our novel study demonstrates that LC improves the microenvironment damaged by oxidative stress (induced by hyperglycemia), thus proposing this nutraceutical compound as an adjuvant in diabetic cardiac regenerative medicine.

Betaine promotes cell differentiation of human osteoblasts in primary culture.

BACKGROUND: Betaine (BET), a component of many foods, is an essential osmolyte and a source of methyl groups; it also shows an antioxidant activity. Moreover, BET stimulates muscle differentiation via insulin like growth factor I (IGF-I). The processes of myogenesis and osteogenesis involve common mechanisms with skeletal muscle cells and osteoblasts sharing the same precursor. Therefore, we have hypothesized that BET might be effective on osteoblast cell differentiation. METHODS: The effect of BET was tested in human osteoblasts (hObs) derived from trabecular bone samples obtained from waste material of orthopedic surgery. Cells were treated with 10 mM BET at 5, 15, 60 min and 3, 6 and 24 h. The possible effects of BET on hObs differentiation were evaluated by real time PCR, western blot and immunofluorescence analysis. Calcium imaging was used to monitor intracellular calcium changes. RESULTS: Real time PCR results showed that BET stimulated significantly the expression of RUNX2, osterix, bone sialoprotein and osteopontin. Western blot and immunofluorescence confirmed BET stimulation of osteopontin protein synthesis. BET stimulated ERK signaling, key pathway involved in osteoblastogenesis and calcium signaling. BET induced a rise of intracellular calcium by means of the calcium ions influx from the extracellular milieu through the L-type calcium channels and CaMKII signaling activation. A significant rise in IGF-I mRNA at 3 and 6 h and a significant increase of IGF-I protein at 6 and 24 h after BET stimulus was detected. Furthermore, BET was able to increase significantly both SOD2 gene expression and protein content. CONCLUSIONS: Our study showed that three signaling pathways, i.e. cytosolic calcium influx, ERK activation and IGF-I production, are enhanced by BET in human osteoblasts. These pathways could have synergistic effects on osteogenic gene expression and protein synthesis, thus potentially leading to enhanced bone formation. Taken together, these results suggest that BET could be a promising nutraceutical therapeutic agent in the strategy to counteract the concomitant and interacting impact of sarcopenia and osteoporosis, i.e. the major determinants of senile frailty and related mortality.

Ranolazine promotes muscle differentiation and reduces oxidative stress in C2C12 skeletal muscle cells.

PURPOSE: The purpose of this study is to investigate Ranolazine action on skeletal muscle differentiation and mitochondrial oxidative phenomena. Ranolazine, an antianginal drug, which acts blocking the late INaL current, was shown to lower hemoglobin A1c in patients with diabetes. In the present study, we hypothesized an action of Ranolazine on skeletal muscle cells regeneration and oxidative process, leading to a reduction of insulin resistance. METHODS: 10 µM Ranolazine was added to C2C12 murine myoblastic cells during proliferation, differentiation and newly formed myotubes. RESULTS: Ranolazine promoted the development of a specific myogenic phenotype: increasing the expression of myogenic regulator factors and inhibiting cell cycle progression factor (p21). Ranolazine stimulated calcium signaling (calmodulin-dependent kinases) and reduced reactive oxygen species levels. Furthermore, Ranolazine maintained mitochondrial homeostasis. During the differentiation phase, Ranolazine promoted myotubes formation. Ranolazine did not modify kinases involved in skeletal muscle differentiation and glucose uptake (extracellular signal-regulated kinases 1/2 and AKT pathways), but activated calcium signaling pathways. During proliferation, Ranolazine did not modify the number of mitochondria while decreasing osteopontin protein levels. Lastly, neo-formed myotubes treated with Ranolazine showed typical hypertrophic phenotype. CONCLUSION: In conclusion, our results indicate that Ranolazine stimulates myogenesis and reduces a pro-oxidant inflammation/oxidative condition, activating a calcium signaling pathway. These newly described mechanisms may partially explain the glucose lowering effect of the drug.

Metformin Treatment Prevents Sedentariness Related Damages in Mice.

Metformin (METF), historical antihyperglycemic drug, is a likely candidate for lifespan extension, treatment and prevention of sedentariness damages, insulin resistance, and obesity. Skeletal muscle is a highly adaptable tissue, capable of hypertrophy response to resistance training and of regeneration after damage. Aims of this work were to investigate METF ability to prevent sedentariness damage and to enhance skeletal muscle function. Sedentary 12-week-old C57BL/6 mice were treated with METF (250 mg/kg per day, in drinking water) for 60 days. METF role on skeletal muscle differentiation was studied in vitro using murine C2C12 myoblasts. Muscular performance evaluation revealed that METF enhanced mice physical performance (Estimated VO2max). Biochemical analyses of hepatic and muscular tissues indicated that in liver METF increased AMPK and CAMKII signaling. In contrast, METF inactivated ERKs, the principal kinases involved in hepatic stress. In skeletal muscle, METF activated AKT, key kinase in skeletal muscle mass maintenance. In in vitro studies, METF did not modify the C2C12 proliferation capacity, while it positively influenced the differentiation process and myotube maturation. In conclusion, our novel results suggest that METF has a positive action not only on the promotion of healthy aging but also on the prevention of sedentariness damages.

Moderate Intensity Training Impact on the Inflammatory Status and Glycemic Profiles in NOD Mice.

The nonobese diabetic (NOD) mouse represents a well-established experimental model analogous to human type 1 diabetes mellitus (T1D) as it is characterized by progressive autoimmune destruction of pancreatic ß-cells. Experiments were designed to investigate the impact of moderate-intensity training on T1D immunomodulation and inflammation. Under a chronic exercise regime, NOD mice were trained on a treadmill for 12 weeks (12 m/min for 30 min, 5 d/wk) while age-matched, control animals were left untrained. Prior to and upon completion of the training period, fed plasma glucose and immunological soluble factors were monitored. Both groups showed deteriorated glycemic profiles throughout the study although trained mice tended to be more compensated than controls after 10 weeks of training. An exercise-induced weight loss was detected in the trained mice with respect to the controls from week 6. After 12 weeks, IL-6 and MIP-1ß were decreased in the trained animals compared to their baseline values and versus controls, although not significantly. Morphometric analysis of pancreata revealed the presence of larger infiltrates along with decreased α-cells areas in the control mice compared to trained mice. Exercise may exert positive immunomodulation of systemic functions with respect to both T1D and inflammation, but only in a stringent therapeutic window.

Potential therapeutic role of L-carnitine in skeletal muscle oxidative stress and atrophy conditions.

The targeting of nutraceutical treatment to skeletal muscle damage is an emerging area of research, driven by the need for new therapies for a range of muscle-associated diseases. L-Carnitine (CARN) is an essential nutrient and plays a key role in mitochondrial ß-oxidation and in the ubiquitin-proteasome system regulation. As a dietary supplement to improve athletic performance, CARN has been studied for its potential to enhance ß-oxidation. However, CARN effects on myogenesis, mitochondrial activity, and hypertrophy process are not completely elucidated. This in vitro study aims to investigate CARN role on skeletal muscle remodeling, differentiation process, and myotubes formation. We analyzed muscle differentiation and morphological features in C2C12 myoblasts exposed to 5 mM CARN. Our results showed that CARN was able to accelerate C2C12 myotubes formation and induce morphological changes, characterizing the start of hypertrophy process. In addition, CARN improved AKT activation and downstream cellular signaling pathways involved in skeletal muscle atrophy process prevention. Also, CARN positively regulated the pathways involved in oxidative stress defense. In this work, we provide an interesting novel mechanism of the potential therapeutic use of CARN to treat pathological conditions characterized by skeletal muscle morphological and functional impairment, oxidative stress production, and atrophy process in aging.

DNA demethylation enhances myoblasts hypertrophy during the late phase of myogenesis activating the IGF-I pathway.

Skeletal muscle regeneration and hypertrophy are important adaptive responses to both physical activity and pathological stimuli. This research was performed to investigate DNA demethylation action on the late phase of muscle differentiation and early stage of hypertrophy. The epigenetic process involved in myogenesis was studied with the DNA-demethylating agent 5-azacytidine (AZA). We induced muscle differentiation in C2C12 mouse myoblasts in the presence of 5 µM AZA and growth or differentiation medium for 48, 72, and 96 h. To study a potential AZA hypertrophic effect, we stimulated 72 h differentiated myotubes with AZA for 24 h. Unstimulated cells were used as control. By western blot and immunofluorescence analysis, we examined AZA action on myogenic regulatory factors expression, hypertrophic signaling pathway and myotube morphology. During differentiation, protein levels of myogenic markers, Myf6 and Myosin Heavy Chain (MyHC), were higher in AZA stimulated cells compared to control. Myostatin and p21 analysis revealed morphological changes which reflect a tendency to hypertrophy in myotubes. In AZA stimulated neo formed myotubes, we observed that IGF-I pathway, kinases p70 S6, 4E-BP1, and ERK1/2 were activated. Furthermore, AZA treatment increased MyHC protein content in stimulated neo myotubes. Our work demonstrates that DNA demethylation could plays an important role in promoting the late phase of myogenesis, activating endocellular pathways involved in protein increment and stimulating the hypertrophic process.

Resveratrol promotes myogenesis and hypertrophy in murine myoblasts.

BACKGROUND: Nutrigenomics elucidate the ability of bioactive food components to influence gene expression, protein synthesis, degradation and post-translational modifications.Resveratrol (RSV), natural polyphenol found in grapes and in other fruits, has a plethora of health benefits in a variety of human diseases: cardio- and neuroprotection, immune regulation, cancer chemoprevention, DNA repair, prevention of mitochondrial disorder, avoidance of obesity-related diseases. In skeletal muscle, RSV acts on protein catabolism and muscle function, conferring resistance against oxidative stress, injury and cell death, but its action mechanisms and protein targets in myogenesis process are not completely known. Myogenesis is a dynamic multistep process regulated by Myogenic Regulator Factors (MRFs), responsible of the commitment of myogenic cell into skeletal muscle: mononucleated undifferentiated myoblasts break free from cell cycle, elongate and fuse to form multinucleated myotubes. Skeletal muscle hypertrophy can be defined as a result of an increase in the size of pre-existing skeletal muscle fibers accompanied by increased protein synthesis, mainly regulated by Insulin Like Growth Factor 1 (IGF-1), PI3-K/AKT signaling pathways.Aim of this work was the study of RSV effects on proliferation, differentiation process and hypertrophy in C2C12 murine cells. METHODS: To study proliferative phase, cells were incubated in growth medium with/without RSV (0.1 or 25 µM) until reaching sub confluence condition (24, 48, 72 h). To examine differentiation, at 70% confluence, cells were transferred in differentiation medium both with/without RSV (0.1 or 25 µM) for 24, 48, 72, 96 hours. After 72 hours of differentiation, the genesis of hypertrophy in neo-formed myotubes was analyzed. RESULTS: Data showed that RSV regulates cell cycle exit and induces C2C12 muscle differentiation. Furthermore, RSV might control MRFs and muscle-specific proteins synthesis. In late differentiation, RSV has positive effects on hypertrophy: RSV stimulates IGF-1 signaling pathway, in particular AKT and ERK 1/2 protein activation, AMPK protein level and induces hypertrophic morphological changes in neo-formed myotubes modulating cytoskeletal proteins expression. CONCLUSIONS: RSV might control cell cycle promoting myogenesis and hypertrophy in vitro, opening a novel field of application of RSV in clinical conditions characterized by chronic functional and morphological muscle impairment.

Betaine supplement enhances skeletal muscle differentiation in murine myoblasts via IGF-1 signaling activation.

BACKGROUND: Betaine (BET) is a component of many foods, including spinach and wheat. It is an essential osmolyte and a source of methyl groups. Recent studies have hypothesized that BET might play a role in athletic performance. However, BET effects on skeletal muscle differentiation and hypertrophy are still poorly understood. METHODS: We examined BET action on neo myotubes maturation and on differentiation process, using C2C12 murine myoblastic cells. We used RT2-PCR array, Western blot and immunofluorescence analysis to study the BET effects on morphological features of C2C12 and on signaling pathways involved in muscle differentiation and hypertrophy. RESULTS: We performed a dose-response study, establishing that 10 mM BET was the dose able to stimulate morphological changes and hypertrophic process in neo myotubes. RT2-PCR array methodology was used to identify the expression profile of genes encoding proteins involved in IGF-1 pathway. A dose of 10 mM BET was found to promote IGF-1 receptor (IGF-1 R) expression. Western blot and immunofluorescence analysis, performed in neo myotubes, pointed out that 10 mM BET improved IGF-1 signaling, synthesis of Myosin Heavy Chain (MyHC) and neo myotubes length. CONCLUSIONS: Our findings provide the first evidence that BET could promote muscle fibers differentiation and increase myotubes size by IGF-1 pathway activation, suggesting that BET might represent a possible new drug/integrator strategy, not only in sport performance but also in clinical conditions characterized by muscle function impairment.

Modulation of cell cycle progression by 5-azacytidine is associated with early myogenesis induction in murine myoblasts.

Myogenesis is a multistep process, in which myoblasts withdraw from the cell cycle, cease to divide, elongate and fuse to form multinucleated myotubes. Cell cycle transition is controlled by a family of cyclin-dependent protein kinases (CDKs) regulated by association with cyclins, negative regulatory subunits and phosphorylation. Muscle differentiation is orchestrated by myogenic regulatory factors (MRFs), such as MyoD and Myf-5. DNA methylation is crucial in transcriptional control of genes involved in myogenesis. Previous work has indicated that treatment of fibroblasts with the DNA-demethylating agent 5-azacytidine (AZA) promotes MyoD expression. We studied the effects of AZA on cell cycle regulation and MRFs synthesis during myoblast proliferation and early myogenesis phases in C2C12 cells. During the proliferation phase, cells were incubated in growth medium with 5µM AZA (GMAZA) or without AZA (GM) for 24 hours. At 70% confluence, cells were kept in growth medium in order to spontaneously achieve differentiation or transferred to differentiation medium with 5µM AZA (DMAZA) or without AZA (DM) for 12 and 24 hours. Cells used as control were unstimulated. In the proliferation phase, AZA-treated cells seemed to lose their characteristic circular shape and become elongated. The presence of AZA resulted in significant increases in the protein contents of Cyclin-D (FC:1.23 GMAZA vs GM p≤0.05), p21 (FC: 1.23 GMAZA vs GM p≤0.05), Myf-5 (FC: 1.21 GMAZA vs GM p≤0.05) and MyoD (FC: 1.20 GMAZA vs GM p≤0.05). These results propose that AZA could inhibit cell proliferation. During 12 hours of differentiation, AZA decreased the downregulation of genes involved in cell cycle arrest and in restriction point (G1 and G1/S phase) and the expression of several cyclins, E2F Transcription Factors, cyclin-dependent kinase inhibitors, specific genes responsible of cell cycle negative regulation. During 24 hours of differentiation, AZA induced an increment in the protein expression of Myf-5 (FC: 1.57 GMAZA vs GM p≤0.05), MyoD (FC: 1.14 DM vs GM p≤0.05; FC: 1.47 DMAZA vs GM p≤0.05), p21 (FC: 1.36 GMAZA vs GM p≤0.01; FC: 1.49 DM vs GM p≤0.05; FC: 1.82 DMAZA vs GM p≤0.01) and MyHC (FC: 1.40 GMAZA vs GM p≤0.01; FC: 2.39 DM vs GM p≤0.05; FC: 3.51 DMAZA vs GM p≤0.01). Our results suggest that AZA-induced DNA demethylation can modulate cell cycle progression and enhance myogenesis. The effects of AZA may open novel clinical uses in the field of muscle injury research and treatment.

Genetic polymorphisms of the enzymes involved in DNA methylation and synthesis in elite athletes.

Physical exercise induces adaptive changes leading to a muscle phenotype with enhanced performance. We first investigated whether genetic polymorphisms altering enzymes involved in DNA methylation, probably responsible of DNA methylation deficiency, are present in athletes' DNA. We determined the polymorphic variants C667T/A1298C of 5,10-methylenetetrahydrofolate reductase (MTHFR), A2756G of methionine synthase (MTR), A66G of methionine synthase reductase (MTRR), G742A of betaine:homocysteine methyltransferase (BHMT), and 68-bp ins of cystathionine ß-synthase (CBS) genes in 77 athletes and 54 control subjects. The frequency of MTHFR (AC), MTR (AG), and MTRR (AG) heterozygous genotypes was found statistically different in the athletes compared with the control group (P=0.0001, P=0.018, and P=0.0001), suggesting a reduced DNA methylating capacity. We therefore assessed whether DNA hypomethylation might increase the expression of myogenic proteins expressed during early (Myf-5 and MyoD), intermediate (Myf-6), and late-phase (MHC) of myogenesis in a cellular model of hypomethylated or unhypomethylated C2C12 myoblasts. Myogenic proteins are largely induced in hypomethylated cells [fold change (FC)=Myf-5: 1.21, 1.35; MyoD: 0.9, 1.47; Myf-6: 1.39, 1.66; MHC: 1.35, 3.10 in GMA, DMA, respectively] compared with the control groups (FC=Myf-5: 1.0, 1.38; MyoD: 1.0, 1.14; Myf-6: 1.0, 1.44; MHC: 1.0, 2.20 in GM, DM, respectively). Diameters and length of hypomethylated myotubes were greater then their respective controls. Our findings suggest that DNA hypomethylation due to lesser efficiency of polymorphic MTHFR, MS, and MSR enzymes induces the activation of factors determining proliferation and differentiation of myoblasts promoting muscle growth and increase of muscle mass.