Endocrine Abstracts

Inflamed ageing is associated with reduced muscle mass and increased susceptibility to TNF-induced muscle protein degradation. We therefore aimed to elucidate mechanisms supporting reduced hypertrophy and increased atrophy of skeletal myoblasts. C₂ myoblasts display diminished differentiation and increased susceptibility to TNF-α-induced apoptosis versus daughter C₂C₁₂ cells, providing us with relevant models. C₂ and C₂C₁₂ cells were cultured for 72 h±TNF-α (20 ng/ml), to assess differentiation (creatine kinase), proliferation (protein), death (trypan blue) and anabolic/catabolic parameters (RT-PCR). Under basal conditions, larger myotubes were evident in C₂C₁₂ versus C₂ cells. Significantly higher CK activity (320.3±6.8 vs 30.7±2.5, P<0.05; 544.9±27.7 vs 39.4±3.4 μmg/ml, P<0.05), and fold increases in myoD (21.5±3.1 vs 4±1.8, P<0.05; 31.1±3.1 vs 6.8±1.9, P<0.05) and myogenin mRNA (241.8±40 vs 36.80±19.3, P<0.05; 440±100.5 vs 201.1±86, P<0.05) were detected at 48 and 72 h, respectively. Fold increases in IGF1 mRNA (243.1±3.1 vs 105.7±21.9, P<0.05), together with reduced proliferation and significantly lower protein expression (1.21±0.28 vs 1.79±0.29 mg/ml, P<0.05) were evident at 72 h. Significant fold reductions in IGFBP2 mRNA occurred in the C₂C₁₂ cells (15.04±2.91, 8.27±4.27, 4.21±2.09, P<0.05) versus increases in the C₂ cells (1.61±0.803, 11.98±2.26, 18.38±3.56, P<0.05) at 0, 48 and 72 h, respectively. TNF-α increased apoptosis in the C₂ cells (2.67±1.54, 34.42±5.39, 29.71±5.79% (0, 48, 72 h), P<0.05)), was without effect in the C₂C₁₂s at 48 h but caused a small significant increase at 72 h (9.88±4.02% (TNF-α) versus 6.17±0.749% (DM), 72 h)). TNF-α and TNFRI mRNA were unchanged, however, larger reductions in IGF1 (8.2 and 7.5 vs 4.5 and 4.1-fold (48, 72 h)) and IGFBP5 (2.7 versus no reduction (48 h) P<0.05)) mRNA were observed in C₂ versus C₂C₁₂ cells in the presence of TNF-α. This model provides insight into altered hypertrophic/atrophic and myogenic regulators of basal and TNF-induced adaptations of skeletal muscle and may provide insight into therapeutic initiatives for ageing and wasting disorders.