Differential expression of the fast skeletal muscle proteome following chronic low-frequency stimulation

Abstract

Physiological and biochemical responses of skeletal muscle fibres to enhanced neuromuscular activity under conditions of maximum activation can be studied experimentally by chronic low-frequency stimulation of fast muscles. Stimulation-induced changes in the expression pattern of the rabbit fast skeletal muscle proteome were evaluated by two-dimensional gel electrophoresis and compared to the altered isoform expression profile of established transformation markers such as the Ca 2+ -ATPase, calsequestrin and the myosin heavy chain. Sixteen muscle proteins exhibited a marked change in their expression level. This included albumin with a 4-fold increase in abundance. In contrast, glycolytic enzymes, such as enolase and aldolase, showed a decreased expression. Concomitant changes were observed with marker elements of the contractile apparatus. While the fast isoforms of troponin T and myosin light chain 2 were drastically down-regulated, their slow counterparts exhibited increased expression. Interestingly, mitochondrial creatine kinase expression increased while the cytosolic isoform of this key muscle enzyme decreased. The expression of the small heat shock protein HSP-B5/aB-crystallin and the oxygen carrier protein myoglobin were both increased 2-fold following stimulation. The observed changes indicate that the conversion into fatigue-resistant red fibres depends on: (i) the optimum utilization of free fatty acids via albumin transportation, (ii) a rearrangement of the creatine kinase isozyme pattern for enhanced mitochondrial activity, (iii) an increased availability of oxygen for aerobic metabolism via myoglobin transport, (iv) the conversion of the contractile apparatus to isoforms with slower twitch characteristics and (v) the up-regulation of chaperone-like proteins for stabilising myofibrillar components during the fast-to-slow transition process. D

Lectin-based proteomic profiling of aged skeletal muscle: Decreased pyruvate kinase isozyme M1 exhibits drastically increased levels of N-glycosylation

“…The drastic increase in the oxygen transporter myoglobin and the fatty acid-binding protein also agrees with this idea , and was previously established as a reliable indicator of fiber type transformation (Donoghue et al, 2005(Donoghue et al, , 2007. Reduced expression levels of the pyruvate kinase M1 isozyme and its altered glycosylation pattern, as revealed in this report by a combination of colloidal Coomassie and WGA lectin labeling, supports the hypothesis that an altered glycolytic flux rate is associated with sarcopenia of old age.…”

“…For proteomic analysis, gel plugs containing a protein spot of interest were placed in 1.5-ml pre-siliconized plastic tubes and then desalted, destained, washed, dried, digested and mixed with matrix by an optimized procedure previously described in detail by our laboratory (Donoghue et al, 2005;Doran et al, 2004Doran et al, , 2006. Glycoproteins were digested with 1 mg trypsin per 20 ml resuspension buffer and 300 ml 50 mM NH 4 HCO 3 for 1 h at 37 1C, and then incubated overnight at 37 1C with 15ml 50mM NH 4 HCO 3 after the removal of excess trypsin solution.…”

Drastic increase of myosin light chain MLC-2 in senescent skeletal muscle indicates fast-to-slow fibre transition in sarcopenia of old age

“…The question of whether it is feasible to determine subtle changes in the density and/or isoform expression pattern of contractile proteins by proteomic techniques has been recently addressed by the mass spectrometric analysis of chronic low-frequency stimulated fast muscles (Donoghue et al, 2005(Donoghue et al, , 2007. This is not a trivial point, since the biomolecules forming the thick and thin filaments of the basic contractile units in skeletal muscles exist in numerous fibre type-specific isoforms (Pette and Staron, 1990).…”

Subcellular proteomics of mice gastrocnemius and soleus muscles

“…In this regard, the soleus and the white portion of gastrocnemius of mice are composed mainly of fast-and slow-twitch muscle fibers, respectively [5][6][7], and these two muscles have been used as typical models in proteomics. During the past few years, several studies have been performed using two-dimensional gel electrophoresis (2-DE) 1 combined with mass spectrometry (MS) to characterize skeletal muscle protein composition of typical slow-and fast-twitch skeletal muscles [8][9][10][11][12][13][14][15][16][17]. In a recent proteomics study on murine gastrocnemius and soleus muscle extracts, performed by Gelfi and coworkers [9], more than 800 spots on each 2-DE were detected by silver staining, leading to the identification of 85 different proteins belonging to the most abundant structural and metabolic protein classes.…”