Antioxidant administration attenuates mechanical ventilation-induced rat diaphragm muscle atrophy independent of protein kinase B (PKB Akt) signalling

Abstract

Oxidative stress promotes controlled mechanical ventilation (MV)-induced diaphragmatic atrophy. Nonetheless, the signalling pathways responsible for oxidative stress-induced muscle atrophy remain unknown. We tested the hypothesis that oxidative stress down-regulates insulin-like growth factor-1-phosphotidylinositol 3-kinase-protein kinase B serine threonine kinase (IGF-1-PI3K-Akt) signalling and activates the forkhead box O (FoxO) class of transcription factors in diaphragm fibres during MV-induced diaphragm inactivity. Sprague-Dawley rats were randomly assigned to one of five experimental groups: (1) control (Con), (2) 6 h of MV, (3) 6 h of MV with infusion of the antioxidant Trolox, (4) 18 h of MV, (5) 18 h of MV with Trolox. Following 6 h and 18 h of MV, diaphragmatic Akt activation decreased in parallel with increased nuclear localization and transcriptional activation of FoxO1 and decreased nuclear localization of FoxO3 and FoxO4, culminating in increased expression of the muscle-specific ubiquitin ligases, muscle atrophy factor (MAFbx) and muscle ring finger-1 (MuRF-1). Interestingly, following 18 h of MV, antioxidant administration was associated with attenuation of MV-induced atrophy in type I, type IIa and type IIb/IIx myofibres. Collectively, these data reveal that the antioxidant Trolox attenuates MV-induced diaphragmatic atrophy independent of alterations in Akt regulation of FoxO transcription factors and expression of MAFbx or MuRF-1. Further, these results also indicate that differential regulation of diaphragmatic IGF-1-PI3K-Akt signalling exists during the early and late stages of MV.

Figure 1

Simplified schematic diagram of protein kinase B (PKB–Akt) signalling pathways shown to be involved in inactivity atrophy Involvement of the pathways shown here is discussed in the Introduction. Signalling represented is one likely component (Sandri et al. 2004; Stitt et al. 2004) of the overall complex signalling pathways involved in atrophy.

Figure 4

Cytosolic and nuclear compartmentalization of Akt in diaphragm skeletal muscle with mechanical ventilation A, cellular fractionation of proteins. Whole skeletal muscle homogenates were fractionated into cytosolic and nuclear proteins for Western blot analysis. Representative Western blot of nuclear histone proteins for verification of cellular fractionation. Cytosolic (Cyt) and nuclear (Nuc) protein fractions were subjected to electrophoresis, transferred to nitrocellulose membranes and subsequently probed for expression of the nuclear compartmentalized histone protein 1 (H2B) or the cytosolic compartmentalized copper zinc superoxide dismutase (CuZnSOD) protein. B, representative Western blot and ponceau- stained nitrocellulose membranes to demonstrate even loading and transfer for the analysis of Akt in the cytosolic and nuclear compartments of skeletal muscle. C, percentage total cytosolic phosphorylated (% phospho) Akt protein. D, percentage total nuclear phosphorylated (% phospho) Akt protein. Values are percentages of total phosphorylated protein abundance and presented as means ± s.e.m.* Significantly (P < 0.05) different from control values. † Significantly (P < 0.05) different from 6 h MV values. ‡ Significantly (P < 0.05) different from 6 h MVT values.

Figure 2

Fibre cross-sectional area (CSA) in diaphragm skeletal muscle myofibres expressing myosin heavy chain (MHC) I (Type I), MHC IIa (Type IIa) and MHC IIb/IIx (Type IIb/IIx) A, representative fluorescence staining of MHC I (DAPI filter/blue), MHC IIa (FITC filter/green), and dystrophin (rhodamine filter/red) proteins in diaphragm from control (Con), 18 h mechanical ventilation (18 h MV) and 18 h mechanical ventilation with Trolox antioxidant administration (18 h MVT). B, mean cross-sectional area (CSA, μm²) of type I, type IIa, and type IIb/IIx diaphragm skeletal muscle myofibres. Scale bar, 30 μm. Values are means ± s.e.m.* Significantly (P < 0.05) different from control values. † Significantly (P < 0.05) different from 6 h MV values. ‡ Significantly (P < 0.05) different from 6 h MVT values. κ Significantly (P < 0.05) different from 18 h MV values.

Figure 3

Phosphoinositidyl-3 kinase (PI3K) signalling in diaphragm skeletal muscle with mechanical ventilation A, representative Western blots and ponceau-stained nitrocellulose membranes to demonstrate even loading and transfer for the analysis of tyrosine (Tyr) phosphorylated p85 PI3K, total PI3K protein, phosphorylated PDK1 (Ser-241), and total PDK1 protein expression. B, percentage total cytosolic phosphorylated (% phospho) p85 PI3K protein. C, percentage total cytosolic phosphorylated (% phospho) PDK1 protein. Values are percentages of total phosphorylated protein abundance and presented as means ± s.e.m.

Figure 5

Forkhead transcription factor (FoxO) protein expression in diaphragm skeletal muscle with mechanical ventilation A, representative Western blot and ponceau-stained nitrocellulose membranes to demonstrate even loading and transfer for the analysis of FoxO1 (Ser-256), phosphorylated FoxO3 (Thr-32), and phosphorylated FoxO4 (Ser-193) and nuclear FoxO1, total FoxO3, and total FoxO4 proteins. B, phosphorylated FoxO1 (Ser-256) protein. C, phosphorylated FoxO3 (Thr-32) protein expression. D, phosphorylated FoxO4 (Ser-256) protein expression. E, total nuclear FoxO1 protein expression. F, total nuclear FoxO3 protein expression. G, total nuclear FoxO4 protein expression. Values are normalized to control values and presented as means ± s.e.m.* Significantly (P < 0.05) different from control values.

Figure 6

FKHR transcriptional activation and skeletal muscle-specific ubiquitin ligase gene expression in diaphragm skeletal muscle after mechanical ventilation A, DNA binding of FoxO1. Values are normalized to control values and presented as means ± s.e.m.* Significantly (P < 0.05) different from control values. B, real-time polymerase chain reaction (RT-PCR) for muscle atrophy transcription factor (MAFbx) mRNA abundance. C, real-time polymerase chain reaction (RT-PCR) for muscle ring finger-1 (MuRF-1) mRNA abundance. Real-time mRNA values are corrected for β-glucuronidase mRNA abundance, normalized to control values and presented as means ± s.e.m.* Significantly (P < 0.05) different from control values.