Proteomic Analysis Of Rat Gastrocnemius Muscle During Denervation-reinnervation

Objective: To investigate the differentially expressed proteins in rat gastrocnemius muscle during denervation-reinnervation by proteomics technique, and to provide the scientific basis for further elucidating the molecuar mechanism of denervated skeletal muscle atrophy as well as searching for better method of prevention and treatment of skeletal muscle atrophy.Methods: Establish the model of rat sciatic nerve crush, extract the total proteins from the sciatic nerve crush gastrocnemius tissue and normal gastrocnemius tissue, and run isoelectric focusing (IEF) electrophores as the first dimension, the sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) as the second dimension to separate the total proteins. Protein spots were visualized by Bio-Safe^TM Commassie and the image were obtained through the GS-800 scanning. Measure the position deviation of matched spots in IEF and SDS-PAGE. The protein spots were analyzed by PDQuest software and find differences between gastrocnemius proteins of sciatic nerve crush rats and control, then the differentially expressed proteins were digested in-gel by enzyme and the generated peptides were measured by matrix assistant lasar desorption ionization-time of flight-mass spectrum (MALDI-TOF-MS). The proteins were identified through database searching.Results: The average position deviation of matched spots was (0.49±0.44) mm in IEF direction, and (0.51±0.44) mm in SDS-PAGE direction. 61 proteins in the gastrocnemius muscle of the operated groups were noted to have qualitative or quantitative (p＜0.05) differences compared with the control muscle. Among the 61 proteins, the 16 proteins were unique (p＜0.01) and the 16 proteins in the rat gastrocnemius muscle exhibited two distinct types of change pattern in their relative abundance: (1) The relative expression levels of 11 proteins were decreased either within one week or two weeks post sciatic nerve injury, followed by restoration during the ensuing days until four weeks. (2) The other 5 proteins displayed an up-regulation in their relative expression levels within one week following sciatic nerve injury, and a subsequent gradual decrease in their relative expression levels until four weeks. These 16 proteins were used for further analysis. They were identified and grouped into different classes including metabolic proteins such as creatine kinase, beta enolase etc.; contractile apparatus proteins such as myosin heavy chainⅡB/ⅡX etc.; chaperone proteins such as alpha B crystallin; scaffolding proteins such as lamins C etc.; and other proteins such as signal peptide peptidase-like 3 etc.Conclusions: Two-dimensional gel electrophoresis with rapid high resolution was a very powerful way to analyze skeletal muscle protein. Some proteins in rat gastrocnemius muscle exhibited a temporal change pattern during denervation-reinnervation, the proteins whose expressed levels reduced may be related to keeping the normal function of muscle, the proteins increased may participate in the process of denervated skeletal muscle atrophy. These results provide the scientific basis for further elucidating the molecuar mechanism of denervated skeletal muscle atrophy.