Proteomic profiling of chronic low‐frequency stimulated fast muscle

Abstract

Skeletal muscle fibre transitions occur in many biological processes, in response to alterations in neuromuscular activity, in muscular disorders, during age-induced muscle wasting and in myogenesis. It was therefore of interest to perform a comprehensive proteomic profiling of muscle transformation. Chronic low-frequency stimulation of the rabbit tibialis anterior muscle represents an established model system for studying the response of fast fibres to enhanced neuromuscular activity under conditions of maximum activation. We have conducted a DIGE analysis of unstimulated control specimens versus 14-and 60-day conditioned muscles. A differential expression pattern was observed for 41 protein species with 29 increased and 12 decreased muscle proteins. Identified classes of proteins that are changed during the fast-to-slow transition process belong to the contractile machinery, ion homeostasis, excitation-contraction coupling, capillarization, metabolism and stress response. Results from immunoblotting agreed with the conversion of the metabolic, regulatory and contractile molecular apparatus to support muscle fibres with slower twitch characteristics. Besides confirming established muscle elements as reliable transition markers, this proteomics-based study has established the actin-binding protein cofilin-2 and the endothelial marker transgelin as novel biomarkers for evaluating muscle transformation.

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.…”

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

“…During electrostimulation-induced muscle transformation, the various light and heavy chains of myosin undergo a stepwise replacement from fast to slow isoforms, which has also been shown to occur in the case of regulatory elements such as the individual subunits of troponin (Pette and Staron, 2001). Proteomic investigations have clearly confirmed these changes in the isoform expression pattern of the contractile apparatus (Donoghue et al, 2007), demonstrating that two-dimensional gel electrophoresis in combination with mass spectrometric analysis is capable of detecting delicate alterations in the skeletal muscle proteome.…”

“…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).…”

DIGE analysis of rat skeletal muscle proteins using nonionic detergent phase extraction of young adult versus aged gastrocnemius tissue

“…For the comparative proteomic analysis of proteins fractionated into AQ phase and DT phase, minimal fluorescent dye labeling was performed at a ratio of 25 µg of protein per 200 pmol of CyDye as previously described [30], following the recommendations of Karp and co-workers for optimized DIGE analysis [31,32]. Labeled samples were incubated on ice for 30 min in the dark.…”