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Analysis by two-dimensional Blue Native/SDS-PAGE of membrane protein alterations in rat soleus muscle after hindlimb unloading

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Abstract

Muscle atrophy occurring in several pathophysiological conditions determines decreases in muscle protein synthesis, increases in the rate of proteolysis and changes in muscle fiber composition. To determine the effect of muscle atrophy induced by hindlimb unloading (HU) on membrane proteins from rat soleus, a proteomic approach based on two-dimensional Blue Native/SDS-PAGE was performed. Proteomic analysis of normal and HU soleus muscle demonstrates statistically significant changes in the relative level of 36 proteins. Among the proteins identified by mass spectrometry, most are involved in pathways associated with muscle fuel utilization, indicating a shift in metabolism from oxidative to glycolytic. Moreover, immunoblotting analysis revealed an increase in aquaporin-4 (AQP4) water channel and an alteration of proteins belonging to the dystrophin–glycoprotein complex (DGC). AQP4 and DGC are regulated in soleus muscle subjected to simulated microgravity in response to compensatory mechanisms induced by muscle atrophy, and they parallel the slow-to-fast twitch conversion that occurs in soleus fibers during HU. In conclusion, the alterations of soleus muscle membrane proteome may play a pivotal role in the mechanisms involved in disuse-induced muscle atrophy.

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Abbreviations

α-syn:

Alpha-syntrophin

β-DG:

Beta-dystroglycan

ACN:

Acetonitrile

AQP4:

Aquaporin-4

ADP:

Adenosine diphosphate

ATP:

Adenosine triphosphate

BN:

Blue Native

CALR:

Calreticulin

Cav-3:

Caveolin-3

CBB G-250:

Coomassie brilliant blue G-250

COX4I1:

Cytochrome-c oxidase subunit 4 isoform 1

DGC:

Dystrophin–glycoprotein complex

EDTA:

Ethylenediaminetetraacetic acid

ER:

Endoplasmic reticulum

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

HM:

Heavy microsomes

HU:

Hindlimb unloading

MALDI-TOF:

Matrix-assisted laser desorption ionization-time of flight

MHC:

Myosin heavy chain

MPC:

Multi-protein complex

MS:

Mass spectrometry

MTP:

Mitochondrial trifunctional protein

NADH:

Nicotinamide adenine dinucleotide

NDUFS1:

NADH-ubiquinone oxidoreductase 75-kDa subunit

OAPs:

Orthogonal arrays of particles

PAGE:

Polyacrylamide gel electrophoresis

PDI:

Protein disulfide isomerase

ROS:

Reactive oxygen species

SDS:

Sodium dodecyl sulfate

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Acknowledgments

Financial support from the Italian Space Agency (WP1B12-5 OsMA) and FIRB-Rete Nazionale di Proteomica (RBRN07BMCT_009) are gratefully acknowledged. We are grateful to Mauro Mastrototaro and Gaetano De Vito for their excellent technical assistance and Francesco Pisani for many helpful discussions. We would like to thank Anthony Green for proofreading the manuscript and for suggesting language improvements to the paper.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of General and Environmental Physiology and Centre of Excellence in Comparative Genomics (CEGBA), University of Bari, 70126, Bari, Italy

    Davide Basco, Grazia Paola Nicchia, Antonio Frigeri & Maria Svelto

  2. Section of Pharmacology, Department of Pharmacobiology, Faculty of Pharmacy, University of Bari, Bari, Italy

    Jean-François Desaphy & Diana Conte Camerino

Authors
  1. Davide Basco
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  2. Grazia Paola Nicchia
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  3. Jean-François Desaphy
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  4. Diana Conte Camerino
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  5. Antonio Frigeri
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  6. Maria Svelto
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Corresponding author

Correspondence to Antonio Frigeri.

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Communicated by Susan Ward.

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Basco, D., Nicchia, G.P., Desaphy, JF. et al. Analysis by two-dimensional Blue Native/SDS-PAGE of membrane protein alterations in rat soleus muscle after hindlimb unloading. Eur J Appl Physiol 110, 1215–1224 (2010). https://doi.org/10.1007/s00421-010-1592-6

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  • DOI: https://doi.org/10.1007/s00421-010-1592-6

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