Differential Cysteine Labeling and Global Label-Free Proteomics Reveals an Altered Metabolic State in Skeletal Muscle Aging

Abstract

The molecular mechanisms underlying skeletal muscle aging and associated sarcopenia have been linked to an altered oxidative status of redox-sensitive proteins. Reactive oxygen and reactive nitrogen species (ROS/RNS) generated by contracting skeletal muscle are necessary for optimal protein function, signaling, and adaptation. To investigate the redox proteome of aging gastrocnemius muscles from adult and old male mice, we developed a label-free quantitative proteomic approach that includes a differential cysteine labeling step. The approach allows simultaneous identification of up- and downregulated proteins between samples in addition to the identification and relative quantification of the reversible oxidation state of susceptible redox cysteine residues. Results from muscles of adult and old mice indicate significant changes in the content of chaperone, glucose metabolism, and cytoskeletal regulatory proteins, including Protein DJ-1, cAMP-dependent protein kinase type II, 78 kDa glucose regulated protein, and a reduction in the number of redox-responsive proteins identified in muscle of old mice. Results demonstrate skeletal muscle aging causes a reduction in redox-sensitive proteins involved in the generation of precursor metabolites and energy metabolism, indicating a loss in the flexibility of the redox energy response. Data is available via ProteomeXchange with identifier PXD001054.

Comparative proteomic profiling reveals a role for Cisd2 in skeletal muscle aging

“…Therefore, the phenotypic effect of Cisd2 knockout of skeletal muscle would seem to occur after the postnatal stage rather than during prenatal early development. Regarding the natural aging of WT mice, 18 of the 46 DEPs have also been identified by other groups as involved in aging, including P4hb (McDonagh, Sakellariou, Smith, Brownridge & Jackson, 2014), Casq1 (McDonagh et al., 2014), Pgam2 (Chaves et al., 2013), and Gstm2 (Chaves et al., 2013), suggesting common protein alterations can be found by different experimental approaches.…”

Redox homeostasis and age‐related deficits in neuromuscular integrity and function

“…It is important to recognize that the rate constants within these protein families and isoforms can vary by up to 10 6  M −1  s −1 ,149 suggesting that the concentration of H 2 O 2 and the enzymes that regulate it play a significant role in redox signalling. PRX proteins have been shown to play a role in transmitting redox signals into a dynamic biological response and to have subtle changes in both abundance and oxidative state with age 52, 150, 151. PRXII has recently been found to form an interdisulfide with STAT3 in response to cytokines, suggesting that it plays an important regulatory role 152…”

“…Contraction‐induced RONS by skeletal muscle has shown to contribute to muscle fatigue186; induce oxidative damage including lipid peroxidation, protein oxidation and DNA damage66; and alter the function of redox‐sensitive proteins within myofibres 151. The sensitivity of a particular target is defined by the intrinsic sensitivity of the molecule to oxidation–reduction and the local redox state,103 and evidence has shown that RONS produced by skeletal muscle can alter myofilament structure and function 187.…”

“…The sensitivity of a particular target is defined by the intrinsic sensitivity of the molecule to oxidation–reduction and the local redox state,103 and evidence has shown that RONS produced by skeletal muscle can alter myofilament structure and function 187. Several myofilament proteins including actin, α‐actinin,151, 187 troponin C188 and myosin heavy chains189, 190, 191 are susceptible to RONS‐induced oxidative modifications, thus affecting Ca 2+ dynamics and Ca 2+ sensitivity192 and, inevitably, cross‐bridge kinetics,188 which may result in contractile dysfunction.…”

“…Recent quantitative proteomic approaches have further provided evidence that muscle ageing is associated with altered catalytic activity of regulatory enzymes and a reduction in detection of redox‐sensitive proteins involved in the generation of precursor metabolites and energy metabolism,151, 183 implying age‐related redox changes as an underlying cause of skeletal muscle ageing.…”