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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 14, 2005 as doi:10.1096/fj.04-3084fje. |
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* Human Nutrition Research Center, Nutrition and Protein Metabolism Laboratory, INRA UR551, Ceyrat, France;
Growth and Muscle Metabolism Laboratory, INRA UR1213, Ceyrat, France;
Laboratory of Neuroendocrinology of Aging, Blaise Pascal University, Aubiere, France; and
Meat Research Station, INRA UR370, Ceyrat, France
1 Correspondence: Human Nutrition Research Center, Nutrition and Protein Metabolism Laboratory, INRA UR551, Theix, Ceyrat 63122, France. E-mail: daniel.bechet{at}clermont.inra.fr
SPECIFIC AIMS
The progressive decline in skeletal muscle mass and function due to aging (sarcopenia) is a key feature of age-related frailty and contributes significantly to greater morbidity. Age-related degenerative changes are reflected in alterations of muscle morphology, function, and biochemical properties. To gain more information on the molecular mechanisms underlying aging of skeletal muscle, we have undertaken a high resolution differential proteomic analysis in young adult, mature adult, and old rats. The present study describes the first systematic identification of aging-induced changes in skeletal muscle proteins.
PRINCIPAL FINDINGS
1. Identification of differentially expressed proteins during maturation and/or aging
Five Lou/c/jall male rats were used for each age group (7, 18, and 30 -months of age). Total protein extracts were prepared from gastrocnemius muscles and gels were made in triplicate for each animal. Forty-five gels were analyzed with PDQuest software and 564 spots were detected in the Coomassie blue-stained gels. Statistical analysis revealed 114 spots that were differentially expressed. Only 25 spots were differentially expressed during maturation (7–18 month period); among them, 12 varied in an opposite manner during aging (18–30 month period). In contrast, 78 spots were differentially expressed specifically during aging without being modified during maturation. Therefore, most differentially expressed proteins characterized old (30 months) animals. Preparative 2-dimensional gel electrophoresis and subsequent mass spectrometry analyses led to the identification of 53 differentially expressed spots corresponding to 40 proteins (Table 1
).
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2. Aging is associated with differential expression of myofibrillar regulatory proteins
In striated muscle, force is generated by interactions between myosin thick filaments and actin thin filaments. Several differentially expressed proteins belong to the actomyosin complex. Aging of gastrocnemius muscle was associated with lower levels of several isoforms of fast and slow myosin light chains. In addition to myosin as its major component, the thick filament contains important regulatory proteins. Two, myosin binding proteins (MyBP) C and H, were selectively down-regulated during aging.
Muscle aging was associated with the down-regulation of skeletal and cardiac 
-actins and with the differential expression of several isoforms of troponin T, which controls the position of tropomyosin near the interface between actin and myosin. Sarcomeric actin filament ends continuously undergo monomer association and dissociation. Muscle aging was notably associated with a 2.4-fold down-regulation of an actin-capping protein (CapZ-ß), which blocks the exchange of actin monomers and anchors the thin filament to the Z-line.
3. Up-regulation of cytoskeletal proteins during aging
Highly specialized cytoskeletal structures maintain the integrity of skeletal muscle fibers. A feature of muscle aging was the selective up-regulation of several proteins of the intermediate filament, microtubule, and microfilament cytoskeletons. These include desmin, a major constituent of the perisarcomeric and intermyofibrillar intermediate filaments, and ß-tubulin, which constitutes the protomer for microtubule assembly and participates in the mechanical integration of various organelles. The actin microfilament is the third component of the cytoskeletal lattice, and aging was characterized by a 2.5-fold increase in gelsolin, which is critical for the remodeling of microfilaments. Therefore, up-regulation of cytoskeletal structures appears to compensate for disorganization of sarcomeric myofibrillar proteins.
4. Perturbations in the energy metabolism of old muscle
Several features emphasized important perturbations in the energy metabolism of old skeletal muscle. In rat gastrocnemius, the glycolytic enzymes triosephosphate isomerase-1 and ß-enolase were selectively down-regulated during aging. Creatine kinase, which catalyzes the transphosphorylation between phosphocreatine and ADP, is central to the regulation of muscle bioenergetics and was selectively down-regulated during muscle aging. Alterations in mitochondrial energy metabolism were revealed by the age-dependent reductions in (NAD+) isocitrate dehydrogenase and in cytochrome c oxidase (polypeptide Va). Finally, down-regulation of glycerol 3-phosphate dehydrogenase suggests reduced cytosolic-to-mitochondrial shuttle of NADH in old skeletal muscle.
5. Detoxification of cytotoxic products in the old muscle
Proteomic analysis of maturation and aging in muscle identified enzymes implicated in scavenging of reactive oxygen, as well as enzymes challenged with the detoxification of cytotoxic compounds produced by oxidative stress. Evidence for increased detoxification of cytotoxic products in old muscle was provided by the differential up-regulations of mitochondrial aldehyde dehydrogenase and glutathione transferase. A feature of muscle aging was also up-regulation of the molecular chaperones Hsp20, Hsp27, and ER60, which detect, refold, and eventually eliminate abnormal proteins. However, differential expressions of antioxidative Cu/Zn SOD and H-ferritin occurred both during maturation and aging; thus, these two proteins were not specifically regulated during muscle aging.
6. Miscellaneous
Age-related perturbations in signal transduction pathways were indicated by the down-regulation of phosphohistidine phosphatase and by up-regulation of the phosphopeptide binding 14-3-3 protein and of guanine deaminase, which interferes with G-protein signaling. Several proteins implicated in the regulation of RNA metabolism were selectively up-regulated during aging. They included mRNA-capping enzyme, an essential processing enzyme of nascent mRNA, and APOBEC-2, which mediates (post)transcriptional editing of RNA. Another important observation was the overexpression of galectin-1, which regulates essential functions such as neuronal path finding and myoblast spreading and fusion.
CONCLUSIONS AND SIGNIFICANCE
The present study underlines 4 major observations. 1) Aging alters the organization of sarcomeric thin and thick filaments. This disorganization of myofibrillar proteins is compensated for by the rearrangement of the cytoskeletal proteins in the old gastrocnemius. Our study points to the differential expression of regulatory proteins directly implicated in the remodeling of myofibrillar and cytoskeletal structures. 2) The analogous decline of glycolytic, Krebs cycle, and respiratory chain proteins supports the concept that profound perturbations in energy metabolism exist in the old skeletal muscle. We identify new differentially expressed proteins that are central for energy production. 3) Aging of skeletal muscle is associated with the up-regulation of several proteins that play a universal role in maintaining cellular homeostasis. In particular, the old gastrocnemius is challenged with the detoxification of cytotoxic aldehydes and overexpresses molecular chaperones that counteract protein misfolding and prevent protein aggregation. 4) The majority of the candidate proteins identified herein by differential proteomics were previously unrecognized in aging of skeletal muscle. They represent novel starting points for elucidating the mechanisms of muscle aging.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3084fje; doi: 10.1096/fj.04-3084fje
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