Comparative Proteomic Profile Of Rat Gastronemius In Adaptation Eccentric Exercise By 2-D DIGE And MS

Objective:Skeletal muscle plays a major role in whole body protein metabolism, We hypothesized that relatively intense aerobic conditioning of eccentric exercise would increase the activity of metabolic enzymes associated with muscle oxidative capacity, and characterization of cytoskeleton proteins response to resistance exercise would come into being adaptation.Methods:Sprague-Dawley male healthy rats(220-250g) were divided randomly into four groups:one week control(C1), eccentric exercise for one week(E1W,-16°,16m/min, 1h), four weeks control (C4) and progressive intense eccentric exercise for four weeks(E4W, lstweek:-16°,16m/min, 1h; 2ndweek:-16°,20m/min, 1h; 3rdweek:-16°,20m/min,90min; 4thweek:-16°,20m/min,120min). Gastrocnemius muscle tissue were dissected from the anaesthetized animals, immediately frozen in liquid nitrogen and stored at -80℃. Small aliquots of specimen were then ground and precipitation with TCA/acetone before exposed to the lysis solution (8 M urea,2M thiourea,4% CHAPS,1 mM PMSF,50μg/ml leupeptin,10μg/ml pepstatin,50 mM DTT). Determination of protein concentration using a Coomassie Plus assay. Immobilized pH gradients (IPG) of 3-10 (non-linear, NL) and 4-7 (linear, L) were used in the first dimension isoelectric separation, while the second dimension separation was performed using laboratory cast SDS-PAGE gels (12%T). After completion of the second dimension the gels were submitted to silver staining. Scanned gels were analyzed with PDQuest software. The normalized spot volumes of individual proteins were determined and only spots significantly changed in all replicate gels were included. Statistically significant differences spots were digested in gel and aliquots were removed and analyzed by MALDI-TOF-TOF mass spectrometry.Results:More than 500 protein spots were detected on each gel, gel analysis and protein characterization have identified a number of proteins whose abundance is significantly altered in eccentric exercise. 199 proteins are of decreased abundance in one week eccentric exercise tissue, whilst 182 proteins are of increased abundance in the E1W state. In four weeks progressive intense eccentric exercise group,159 proteins increased and 203 decreased. MHC isoform has a significant increment in MHCI in eccentric exercise muscle. The precise change in MHC isoforms composition is mandatory to determine the change in muscle functional properties.Conclusion:This research established 2-D reference maps of rat gastrocnemius response to eccentric exercise. We have also evaluated eccentric exercise-induced changes in differential functional proteins expression and assessed differential expression of MHC isoforms in gastrocemius muscle.