iTRAQ-based proteomic analysis of myofibrillar contents and relevant synthesis and proteolytic proteins in soleus muscle of hibernating Daurian ground squirrels (Spermophilus dauricus)

Abstract

BackgroundDaurian ground squirrels (Spermophilus dauricus) deviate from significant increase of protein catabolism and loss of myofibrillar contents during long period of hibernation inactivity.MethodsHere we use iTRAQ based quantitative analysis to examine proteomic changes in the soleus of squirrels in pre-hibernation, hibernation and post-hibernation states. The total proteolysis rate of soleus was measured by the release of the essential amino acid tyrosine from isolated muscles. Immunofluorescent analysis was used to determine muscle fiber cross-sectional area. Western blot was used for the validation of the quantitative proteomic analysis.ResultsThe proteomic responses to hibernation had a 0.4- to 0.8-fold decrease in the myofibrillar contractile protein levels of myosin-3, myosin-13 and actin, but a 2.1-fold increase in myosin-2 compared to pre-hibernation group. Regulatory proteins such as troponin C and tropomodulin-1 were 1.4-fold up-regulated and 0.7-fold down-regulated, respectively, in hibernation compared to pre-hibernation group. Moreover, 10 proteins with proteolytic function in hibernation, which was less than 14 proteins in the post-hibernation group, were up-regulated relative to the pre-hibernation group. The total proteolysis rates of soleus in hibernation and post-hibernation groups were significantly inhibited as compared with pre-hibernation group.ConclusionThese findings suggest that the myofibrillar remodeling and partial suppression of myofibrillar proteolysis were likely responsible for preventing skeletal muscle atrophy during prolonged disuse in hibernation. This is the first study where the myofibrillar contents and relevant synthesis and proteolytic proteins in slow soleus was discussed based on proteomic investigation performed on wild Daurian ground squirrels. Our results lay the foundation for further research in preventing disuse-induced skeletal muscle atrophy in mammals.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-016-0105-x) contains supplementary material, which is available to authorized users.

Preliminary Observation about Alteration of Proteins and Their Potential Functions in Spinal Cord of SOD1 G93A Transgenic Mice

“…These supernatants were transferred to a new tube and quantified. These proteins in supernatants were kept at -80 °C for further analysis 70 .…”

“…Elution was monitored by measuring the absorbance at 214 nm, and fractions were collected every 1 minute. These eluted peptides were pooled into 20 fractions, desalted with a Strata X C18 column (Phenomenex) and vacuum-dried 70 .…”

“…A sweeping collision energy setting of 35 ± 5 eV coupled with iTRAQ adjust rolling collision energy was applied to all precursor ions for collision-induced dissociation. Dynamic exclusion was set for 1/2 of peak width (15 seconds), and then the precursor was refreshed off the exclusion list 70 .…”

“…The quantitative protein ratios were weighted and normalized by the median ratio in Mascot. We only used the fold changes of > 1.2 and < 0.833 with p values < 0.05 were considered as significant 70 .…”

“…GO has 3 ontologies which can describe the molecular function, the cellular component and the biological process respectively. The cluster of orthologous KEGG pathway is a collection of manually drawn pathway maps representing our knowledge on the molecular interaction and reaction networks, among them, molecules are represented as nodes, and the biological relationship between two nodes is indicated by a line 70 .…”