Chronic paraplegia-induced muscle atrophy downregulates the mTOR/S6K1 signaling pathway

Abstract

Ribosomal S6 kinase 1 (S6K1) is a downstream component of the mammalian target of rapamycin (mTOR) signaling pathway and plays a regulatory role in translation initiation, protein synthesis, and muscle hypertrophy. AMP-activated protein kinase (AMPK) is a cellular energy sensor, a negative regulator of mTOR, and an inhibitor of protein synthesis. The purpose of this study was to determine whether the hypertrophy/cell growth-associated mTOR pathway was downregulated during muscle atrophy associated with chronic paraplegia. Soleus muscle was collected from male Sprague-Dawley rats 10 wk following complete T(4)-T(5) spinal cord transection (paraplegic) and from sham-operated (control) rats. We utilized immunoprecipitation and Western blotting techniques to measure upstream [AMPK, Akt/protein kinase B (PKB)] and downstream components of the mTOR signaling pathway [mTOR, S6K1, SKAR, 4E-binding protein 1 (4E-BP1), and eukaryotic initiation factor (eIF) 4G and 2alpha]. Paraplegia was associated with significant soleus muscle atrophy (174 +/- 8 vs. 240 +/- 13 mg; P < 0.05). There was a reduction in phosphorylation of mTOR, S6K1, and eIF4G (P < 0.05) with no change in Akt/PKB or 4E-BP1 (P > 0.05). Total protein abundance of mTOR, S6K1, eIF2alpha, and Akt/PKB was decreased, and increased for SKAR (P < 0.05), whereas 4E-BP1 and eIF4G did not change (P > 0.05). S6K1 activity was significantly reduced in the paraplegic group (P < 0.05); however, AMPKalpha2 activity was not altered (3.5 +/- 0.4 vs. 3.7 +/- 0.5 pmol x mg(-1) x min(-1), control vs. paraplegic rats). We conclude that paraplegia-induced muscle atrophy in rats is associated with a general downregulation of the mTOR signaling pathway. Therefore, in addition to upregulation of atrophy signaling during muscle wasting, downregulation of muscle cell growth/hypertrophy-associated signaling appears to be an important component of long-term muscle loss.

Fig. 1

Rat whole body and soleus wet muscle weight. Body weight is expressed in grams (A) and soleus weight in milligrams (B). Body weight was not different between the control group (open bars) and the paraplegic group (solid bars) but soleus weight was reduced by 28% (B). Values are means ± SE; n = 8 per group. *Significantly different from control, P < 0.05.

Fig. 2

Soleus muscle phosphorylation status (Ser473) (phospho) and total Akt/protein kinase B (PKB) protein abundance (A), and phosphorylation status (Ser2448) and total mTOR protein abundance (B). Data are expressed relative to an internal loading control (Int) and as means ± SE; n = 8 per group. Inset, representative Western blot for duplicate samples for control and paraplegic rats. AU, arbitrary units. *Significantly different from control, P < 0.05.

Fig. 3

Soleus muscle phosphorylation status (Thr37/46) and total 4E-binding protein 1 (4E-BP1) protein abundance (A), and phosphorylation status (Ser1108) and total eukaryotic initiation factor-4G (eIF4G) protein abundance (B). Data are expressed relative to an internal loading control and as means ± SE; n = 8 except total and phospho/total eIF4G, n = 6. Inset, representative Western blot for duplicate samples for control and paraplegic rats. *Significantly different from control, P < 0.05.

Fig. 4

Soleus muscle phosphorylation status (Thr389) and total Ribosomal S6 kinase 1 (S6K1) protein abundance (A) and total SKAR protein abundance (C). Data are expressed relative to an internal loading control and as means ± SE; n = 8 per group. Inset, representative Western Blot for duplicate samples for control and paraplegic rats. Soleus muscle S6K1 activity (B): data are expressed as means ± SE, n = 4 per group. *Significantly different from control, P < 0.05.

Fig. 5

Soleus muscle phosphorylation status (Ser52) and total eukaryotic initiation factor 2α (eIF2α) protein abundance. Data are expressed relative to an internal loading control and as means ± SE; n = 6 per group. Inset, representative Western blot for duplicate samples for control and paraplegic rats. *Significantly different from control, P < 0.05.

Fig. 6

Soleus muscle AMP-activated protein kinaseα2 (AMPKα2) activity. AMPK activity was not different between groups. Open bar, control rats; filled bar, paraplegic rats. Values are means ± SE; n = 8 per group.