Aging skeletal muscle shows a drastic increase in the small heat shock proteins αB-crystallin/HspB5 and cvHsp/HspB7

Abstract

Most heat shock proteins operate as molecular chaperones and play a central role in the maintenance of normal cellular function. In skeletal muscle, members of the a-crystallin domain-containing family of small heat shock proteins are believed to form a cohort of essential stress proteins. Since aB-crystallin (aBC/HspB5) and the cardiovascular heat shock protein (cvHsp/HspB7) are both implicated in the molecular response to fibre transformation and muscle wasting, it was of interest to investigate the fate of these stress proteins in young adult versus aged muscle. The agerelated loss of skeletal muscle mass and strength, now generally referred to as sarcopenia, is one of the most striking features of the senescent organism. In order to better understand the molecular pathogenesis of age-related muscle wasting, we have performed a two-dimensional gel electrophoretic analysis, immunoblotting and confocal microscopy study of aged rat gastrocnemius muscle. Fluorescent labelling of the electrophoretically separated soluble muscle proteome revealed an overall relatively comparable protein expression pattern of young adult versus aged fibres, but clearly an up-regulation of aBC and cvHsp. This was confirmed by immunofluorescence microscopy and immunoblot analysis, which showed a dramatic age-induced increase in these small heat shock proteins. Immunodecoration of other major stress proteins showed that they were not affected or less drastically changed in their expression in aged muscle. These findings indicate that the increase in muscle-specific small heat shock proteins constitutes an essential cellular response to fibre aging and might therefore be a novel therapeutic option to treat sarcopenia of old age.

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

“…Chemiluminescence substrate was purchased from Roche Diagnostics (Mannheim, Germany). Primary and secondary antibodies were obtained from various commercial sources, as listed in previous studies [16][17][18]. Ultrapure lysine for quenching the DIGE labeling reaction and all other analytical-grade chemicals were purchased from Sigma Chemical Company (Dorset, UK).…”

“…Several mass spectrometry-based proteomic studies have investigated changes in the protein complement of aging skeletal muscles over the last few years, as reviewed by Doran et al [13]. These proteomic profiling studies have focused on crude soluble extracts and have shown that muscle aging is associated with altered expression levels of many key contractile proteins, regulatory elements, ion handling proteins, metabolic enzymes and mediators of the cellular stress response [14][15][16][17][18][19][20]. The proteomic finding of a drastically decreased density and reduced enzymatic activity of certain glycolytic enzymes in senescent fibres [21] is in agreement with the idea of a fast-to-slow transformation process during aging [13].…”

Reduced expression of sarcalumenin and related Ca2+-regulatory proteins in aged rat skeletal muscle

“…Comparative studies were performed with young adult (3-month-old), adult (6-month-old) and senescent (30-monthold) tibialis anterior, gastrocnemius and soleus preparations. Muscle specimens (100 mg wet weight) were pulverized in liquid nitrogen using a mortar and pestle, and the resulting powder was placed into 1 ml of homogenisation buffer consisting of 0.5 M HEPES, pH 7.4, 200 mM EGTA, 10% (w/v) sucrose, 3 mM MgCl 2 solution and 0.1% (w/v) NaN 3 , as well as a protease inhibitor cocktail (Doran et al, 2007). Tissue suspensions were sonicated for 10 s, subsequently incubated on ice with vortexing every 10 min for 10 s over a 4 h period, and then centrifuged for 20 min at 20,000g.…”

“…Gels were stained with Coomassie Blue or transferred to nitrocellulose membranes at 100 V for 70 min using a Transblot Cell from BioRad Laboratories (HemelHempstead, Herts., UK). Blocking, washing, incubation with antibodies and enhanced chemiluminescence detection of immuno-decorated proteins was carried out by optimized methodology (Doran et al, 2007).…”

“…Fixation, washing and immunolabelling with sufficiently diluted primary antibodies were carried out as previously described in detail (Doran et al, 2007). If Ca 2+ -handling proteins of interest showed internal labelling patterns, fluorescent staining of laminin was employed to outline the fibre periphery.…”