Myostatin Activates the Ubiquitin-Proteasome and Autophagy-Lysosome Systems Contributing to Muscle Wasting in Chronic Kidney Disease

Figure 5

Overexpression of myostatin activates the ubiquitin-proteasome and autophagy-lysosome systems in myotubes via phosphorylation of PI3k/Akt/FoxO3a signaling pathway. C2C12 cells were transfected with myostatin plasmid versus plasmid expressing green fluorescent protein (GFP). After differentiating into myotubes, cells were treated with 100 ng/mL TNF-α for 24 h or LY294002 (50 μM) for 1 h. (a) Left: representative immunoblotting of Atg3, Atg7, Atg12, Beclin-1, LC3-I/II, and GAPDH. Right: the ratio of Atg3, Atg7, Atg12, Beclin-1, LC3-II, and GAPDH normalized to control. (b) Representative electron micrograph of the autophagosomes. Autophagosomes were identified in the images as shown by arrows. Scale bar = 1 μm. (c) Upper: representative immunoblotting of MAFbx, MuRF1 and GAPDH. Lower: The ratio of MAFbx, MuRF1, and GAPDH normalized to control. (d) Upper: representative immunoblotting of PAMA4, Rpt5, Rpn11, and GAPDH. Lower: the ratio of PAMA4, Rpt5, Rpn11, and GAPDH normalized to control. (e) Left: representative immunoblotting of p-PI3K, p-Akt, p-FoxO3a, p-FoxO1, and GAPDH. Right: the ratio of p-PI3K, p-Akt, p-FoxO3a, p-FoxO1, and GAPDH normalized to control. (f) Left: representative immunoblotting of p-FoxO3a, LC3-I/II, MAFbx, MuRF1, and GAPDH. Right: the ratio of p-FoxO3a, LC3-II, MAFbx, MuRF1, and GAPDH normalized to control. Values were described means, with SD represented by vertical bars. Significantly different () from control group, (^†, independent experiments) from TNF-α or myostatin (plasmid) groups.

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