Development of a gene doping detection method to detect overexpressed human follistatin using an adenovirus vector in mice

Background Gene doping is the misuse of genome editing and gene therapy technologies for the purpose of manipulating specific genes or gene functions in order to improve athletic performance. However, a non-invasive detection method for gene doping using recombinant adenoviral (rAdV) vectors containing human follistatin (hFST) genes (rAdV<hFST>) has not yet been developed. Therefore, the aim of this study was to develop a method to detect gene doping using rAdV<hFST>. Methods First, we generated rAdV<hFST> and evaluated the overexpression of the hFST gene, FST protein, and muscle protein synthesis signaling using cell lines. Next, rAdV<hFST> was injected intravenously or intramuscularly into mice, and whole blood was collected, and hFST and cytomegalovirus promoter (CMVp) gene fragments were detected using TaqMan-quantitative polymerase chain reaction (qPCR). Finally, to confirm the specificity of the primers and the TaqMan probes, samples from each experiment were pooled, amplified using TaqMan-qPCR, and sequenced using the Sanger sequencing. Results The expression of hFST and FST proteins and muscle protein synthesis signaling significantly increased in C2C12 cells. In long-term, transgene fragments could be detected until 4 days after intravenous injection and 3 days after intramuscular injection. Finally, the Sanger sequencing confirmed that the primers and TaqMan probe specifically amplified the gene sequence of interest. Conclusions These results indicate the possibility of detecting gene doping using rAdV<hFST> using TaqMan-qPCR in blood samples. This study may contribute to the development of detection methods for gene doping using rAdV<hFST>.

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Notch Inhibition via GSI Treatment Elevates Protein Synthesis in C2C12 Myotubes

Biology ◽

10.3390/biology9060115 ◽

2020 ◽

Vol 9 (6) ◽

pp. 115

Author(s):

Joshua R. Huot ◽

Joseph S. Marino ◽

Michael J. Turner ◽

Susan T. Arthur

Keyword(s):

Skeletal Muscle ◽

Protein Synthesis ◽

Notch Signaling ◽

Muscle Protein ◽

Muscle Protein Synthesis ◽

C2c12 Cells ◽

Skeletal Muscle Regeneration ◽

C2c12 Myotubes ◽

Myonuclear Domain ◽

Myotube Hypertrophy

The role of Notch signaling is widely studied in skeletal muscle regeneration but little is known about its influences on muscle protein synthesis (MPS). The purpose of this study was to investigate whether Notch signaling is involved in the regulation of MPS. C2C12 cells were treated with a γ-secretase inhibitor (GSI), to determine the effect of reduced Notch signaling on MPS and anabolic signaling markers. GSI treatment increased myotube hypertrophy by increasing myonuclear accretion (nuclei/myotube: p = 0.01) and myonuclear domain (myotube area per fusing nuclei: p < 0.001) in differentiating C2C12 cells. GSI treatment also elevated myotube hypertrophy in differentiated C2C12s (area/myotube; p = 0.01). In concert, GSI treatment augmented pmTOR Ser2448 (p = 0.01) and protein synthesis (using SUnSET method) in myotubes (p < 0.001). Examining protein expression upstream of mTOR revealed reductions in PTEN (p = 0.04), with subsequent elevations in pAKT Thr308 (p < 0.001) and pAKT Ser473 (p = 0.05). These findings reveal that GSI treatment elevates myotube hypertrophy through both augmentation of fusion and MPS. This study sheds light on the potential multifaceted roles of Notch within skeletal muscle. Furthermore, we have demonstrated that Notch may modulate the PTEN/AKT/mTOR pathway.

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Syncytin-1 Expression is Increased in Skeletal Muscle of Humans with Obesity and is Inversely Correlated to Muscle Protein Synthesis

10.21203/rs.3.rs-147911/v1 ◽

2021 ◽

Author(s):

Jayachandran Ravichandran ◽

Lori R Roust ◽

Christos Katsanos

Keyword(s):

Skeletal Muscle ◽

Protein Synthesis ◽

Protein Metabolism ◽

Muscle Protein ◽

Muscle Protein Synthesis ◽

C2c12 Cell ◽

C2c12 Cells ◽

Anabolic Hormone ◽

Lower Protein ◽

Potential Link

Abstract Background: Various pathophysiological conditions alter protein metabolism in skeletal muscle, with obesity being one of them. Obesity impairs regeneration of skeletal muscle, and the same biological mechanism(s) may adversely affect protein metabolism in the muscle of these individuals. Methods: We used C2C12 cell line to evaluate the effects of the anabolic hormone insulin on the expression of protein syncytin-1, which regulates regeneration of muscle, and in the presence of fatty acids whose metabolism is altered in obesity. We used muscle biopsy samples from obese humans with lower muscle protein synthesis and lean controls to evaluate expression of syncytin-1 in obesity and its correlation with protein synthesis in muscle. Results: Insulin upregulated syncytin-1 expression in C2C12 cells and this response was impaired in the presence of the fatty acid palmitate, but not oleate. Expression of the protein 4E-BP1, which signals increase in protein synthesis in muscle, showed response similar to that of syncytin-1. Humans with obesity characterized by lower muscle protein synthesis had higher expression of syncytin-1 in muscle compared to lean humans (P < 0.01). The rate of synthesis of protein in skeletal muscle across humans subjects correlated inversely (r = -0.51; P = 0.03) with the expression of syncytin-1 in muscle. Conclusions: Our studies provide novel insights in the regulation of syncytin-1 in skeletal muscle, and describe potential link between syncytin-1 expression and protein metabolism in skeletal muscle of humans. Altered syncytin-1 expression in muscle may mediate lower protein turnover in muscle of humans with obesity.

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GSI Treatment Preserves Protein Synthesis in C2C12 Myotubes

Cells ◽

10.3390/cells10071786 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1786

Author(s):

Joshua R. Huot ◽

Brian Thompson ◽

Charlotte McMullen ◽

Joseph S. Marino ◽

Susan T. Arthur

Keyword(s):

Protein Synthesis ◽

Notch Signaling ◽

Muscle Protein ◽

Muscle Protein Synthesis ◽

Mtor Signaling ◽

C2c12 Cells ◽

C2c12 Myotubes ◽

Myotube Formation ◽

Mediated Effects ◽

Secretase Inhibitor

It has been demonstrated that inhibiting Notch signaling through γ-secretase inhibitor (GSI) treatment increases myogenesis, AKT/mTOR signaling, and muscle protein synthesis (MPS) in C2C12 myotubes. The purpose of this study was to determine if GSI-mediated effects on myogenesis and MPS are dependent on AKT/mTOR signaling. C2C12 cells were assessed for indices of myotube formation, anabolic signaling, and MPS following GSI treatment in combination with rapamycin and API-1, inhibitors of mTOR and AKT, respectively. GSI treatment increased several indices of myotube fusion and MPS in C2C12 myotubes. GSI-mediated effects on myotube formation and fusion were completely negated by treatment with rapamycin and API-1. Meanwhile, GSI treatment was able to rescue MPS in C2C12 myotubes exposed to rapamycin or rapamycin combined with API-1. Examination of protein expression revealed that GSI treatment was able to rescue pGSK3β Ser9 despite AKT inhibition by API-1. These findings demonstrate that GSI treatment is able to rescue MPS independent of AKT/mTOR signaling, possibly via GSK3β modulation.

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L-Arginine Enhances Protein Synthesis by Phosphorylating mTOR (Thr 2446) in a Nitric Oxide-Dependent Manner in C2C12 Cells

Oxidative Medicine and Cellular Longevity ◽

10.1155/2018/7569127 ◽

2018 ◽

Vol 2018 ◽

pp. 1-13 ◽

Cited By ~ 8

Author(s):

Ruxia Wang ◽

Hongchao Jiao ◽

Jingpeng Zhao ◽

Xiaojuan Wang ◽

Hai Lin

Keyword(s):

Nitric Oxide ◽

Protein Synthesis ◽

Culture Media ◽

Muscle Protein ◽

Muscle Protein Synthesis ◽

C2c12 Cells ◽

Dependent Manner ◽

No Donor ◽

Nos Inhibitor ◽

Oxide Synthase

Muscle atrophy may arise from many factors such as inactivity, malnutrition, and inflammation. In the present study, we investigated the stimulatory effect of nitric oxide (NO) on muscle protein synthesis. Primarily, C2C12 cells were supplied with extra L-arginine (L-Arg) in the culture media. L-Arg supplementation increased the activity of inducible nitric oxide synthase (iNOS), the rate of protein synthesis, and the phosphorylation of mTOR (Thr 2446) and p70S6K (Thr 389). L-NAME, an NOS inhibitor, decreased NO concentrations within cells and abolished the stimulatory effect of L-Arg on protein synthesis and the phosphorylation of mTOR and p70S6K. In contrast, SNP (sodium nitroprusside), an NO donor, increased NO concentrations, enhanced protein synthesis, and upregulated mTOR and p70S6K phosphorylation, regardless of L-NAME treatment. Blocking mTOR with rapamycin abolished the stimulatory effect of both L-Arg and SNP on protein synthesis and p70S6K phosphorylation. These results indicate that L-Arg stimulates protein synthesis via the activation of the mTOR (Thr 2446)/p70S6K signaling pathway in an NO-dependent manner.

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