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

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.

[1]  V. Edgerton,et al.  Fiber type and fiber size of cat ankle, knee, and hip extensors and flexors following low thoracic spinal cord transection at an early age , 1986, Experimental Neurology.

[2]  P. Gardiner,et al.  Myosin heavy chains in fibers of TTX-paralyzed rat soleus and medial gastrocnemius muscles. , 2000, Journal of applied physiology.

[3]  G. Yancopoulos,et al.  Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo , 2001, Nature Cell Biology.

[4]  Jon R Lorsch,et al.  The molecular mechanics of eukaryotic translation. , 2003, Annual review of biochemistry.

[5]  A. Gingras,et al.  eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. , 1999, Annual review of biochemistry.

[6]  J. Blenis,et al.  Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression , 2004, Oncogene.

[7]  P. Fuller,et al.  Insulin-independent pathways mediating glucose uptake in hindlimb-suspended skeletal muscle. , 2005, Journal of applied physiology.

[8]  E. Sundström,et al.  Motor Performance Score: A New Algorithm for Accurate Behavioral Testing of Spinal Cord Injury in Rats , 1996, Experimental Neurology.

[9]  K M Baldwin,et al.  Effects of zero gravity on myofibril content and isomyosin distribution in rodent skeletal muscle , 1990, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[10]  D. Desplanches,et al.  Skeletal muscle adaptation in rats flown on Cosmos 1667. , 1990, Journal of applied physiology.

[11]  D. Hardie,et al.  Analysis of the role of the AMP-activated protein kinase in the response to cellular stress. , 2000, Methods in molecular biology.

[12]  K. Esser,et al.  Regulation of translation factors during hindlimb unloading and denervation of skeletal muscle in rats. , 2001, American journal of physiology. Cell physiology.

[13]  V. Edgerton,et al.  Atrophy responses to muscle inactivity. I. Cellular markers of protein deficits. , 2003, Journal of applied physiology.

[14]  B. Viollet,et al.  AMPK-Mediated AS160 Phosphorylation in Skeletal Muscle Is Dependent on AMPK Catalytic and Regulatory Subunits , 2006, Diabetes.

[15]  C. Proud,et al.  The mTOR pathway in the control of protein synthesis. , 2006, Physiology.

[16]  V. Edgerton,et al.  Atrophy responses to muscle inactivity. II. Molecular markers of protein deficits. , 2003, Journal of applied physiology.

[17]  K. Esser,et al.  Phosphorylation of p70(S6k) correlates with increased skeletal muscle mass following resistance exercise. , 1999, The American journal of physiology.

[18]  S. Lotta,et al.  Muscle fiber type morphology and distribution in paraplegic patients with traumatic cord lesion , 1982, Acta Neuropathologica.

[19]  S. Kimball,et al.  Signaling pathways and molecular mechanisms through which branched-chain amino acids mediate translational control of protein synthesis. , 2006, The Journal of nutrition.

[20]  S. DiCarlo,et al.  TENS attenuates response to colon distension in paraplegic and quadriplegic rats. , 2002, American journal of physiology. Heart and circulatory physiology.

[21]  Laurent Malisoux,et al.  Effect of long-term muscle paralysis on human single fiber mechanics. , 2007, Journal of applied physiology.

[22]  G. Diffee,et al.  Contractile and biochemical properties of rat soleus and plantaris after hindlimb suspension. , 1991, The American journal of physiology.

[23]  David Carling,et al.  Thr2446 Is a Novel Mammalian Target of Rapamycin (mTOR) Phosphorylation Site Regulated by Nutrient Status* , 2004, Journal of Biological Chemistry.

[24]  S. Kimball.  Interaction between the AMP-activated protein kinase and mTOR signaling pathways. , 2006, Medicine and science in sports and exercise.

[25]  M. Devivo,et al.  A cross-sectional study of the relationship between age and current health status for persons with spinal cord injuries , 1992, Paraplegia.

[26]  D. Bolster,et al.  AMP-activated Protein Kinase Suppresses Protein Synthesis in Rat Skeletal Muscle through Down-regulated Mammalian Target of Rapamycin (mTOR) Signaling* , 2002, The Journal of Biological Chemistry.

[27]  X. Han,et al.  Reciprocal GLUT-1 and GLUT-4 expression and glucose transport in denervated muscles. , 1996, The American journal of physiology.

[28]  S. Gygi,et al.  SKAR Is a Specific Target of S6 Kinase 1 in Cell Growth Control , 2004, Current Biology.

[29]  Peter Nowotny,et al.  Overactivation of S6 kinase 1 as a cause of human insulin resistance during increased amino acid availability. , 2005, Diabetes.

[30]  P. Thompson,et al.  Analysis of human skeletal muscle after 48 h immobilization reveals alterations in mRNA and protein for extracellular matrix components. , 2006, Journal of applied physiology.

[31]  Vincent J Caiozzo,et al.  Skeletal muscle unweighting: spaceflight and ground-based models. , 2003, Journal of applied physiology.

[32]  F. Booth,et al.  Protein metabolism and beta-myosin heavy-chain mRNA in unweighted soleus muscle. , 1989, The American journal of physiology.

[33]  C. Peterson,et al.  Early changes in muscle fiber size and gene expression in response to spinal cord transection and exercise. , 1998, The American journal of physiology.

[34]  N. Fujii,et al.  Distinct Signals Regulate AS160 Phosphorylation in Response to Insulin, AICAR, and Contraction in Mouse Skeletal Muscle , 2006, Diabetes.

[35]  S. Kandarian,et al.  Intracellular signaling during skeletal muscle atrophy , 2006, Muscle & nerve.

[36]  Glen M. Davis,et al.  Effects of electrical stimulation leg training during the acute phase of spinal cord injury: a pilot study , 2000, European Journal of Applied Physiology.

[37]  Jiahuai Han,et al.  Stimulation of Glucose Transport by AMP-activated Protein Kinase via Activation of p38 Mitogen-activated Protein Kinase* , 2001, The Journal of Biological Chemistry.

[38]  S. Kimball,et al.  Eukaryotic initiation factor 2B and its role in alterations in mRNA translation that occur under a number of pathophysiological and physiological conditions. , 2006, Progress in nucleic acid research and molecular biology.

[39]  M. Rennie,et al.  Decrease in human quadriceps muscle protein turnover consequent upon leg immobilization. , 1987, Clinical science.

[40]  D. Chinkes,et al.  Resistance exercise increases AMPK activity and reduces 4E‐BP1 phosphorylation and protein synthesis in human skeletal muscle , 2006, The Journal of physiology.

[41]  B. Walmsley,et al.  The effect of spinal cord transection on motor units in cat medial gastrocnemius muscles , 1984, Muscle & nerve.

[42]  D. Hardie,et al.  Inactivation of acetyl-CoA carboxylase and activation of AMP-activated protein kinase in muscle during exercise. , 1996, The American journal of physiology.

[43]  GLUT4 translocation precedes the stimulation of glucose uptake by insulin in muscle cells: potential activation of GLUT4 via p38 mitogen-activated protein kinase. , 2001, The Biochemical journal.

[44]  D. Glass.  Signalling pathways that mediate skeletal muscle hypertrophy and atrophy , 2003, Nature Cell Biology.

[45]  S. DiCarlo,et al.  Acute exercise reduces the response to colon distension in T(5) spinal rats. , 2002, American journal of physiology. Heart and circulatory physiology.

[46]  M. Devivo,et al.  Cause of death for patients with spinal cord injuries. , 1990, Archives of internal medicine.