The Human Muscle Proteome in Aging Cecilia Gelfi,*,† Agnese Vigano` ,†,‡ Marilena Ripamonti,† Alessandro Pontoglio,†,‡ Shajna Begum,§ Maria Antonietta Pellegrino,| Bruno Grassi,‡ Roberto Bottinelli,| Robin Wait,§ and Paolo Cerretelli‡ Institute of Molecular Bioimaging and Physiology, CNR, Segrate (MI), Italy, Department of Sciences and Biomedical Technologies, University of Milano, Milano, Italy, Kennedy Institute of Rheumatology, Imperial College London, London, United Kingdom, and Department of Experimental Medicine, Human Physiology Unit, University of Pavia, Pavia, Italy Received November 22, 2005
The aim of the present study was to assess age-dependent changes of proteins in the vastus lateralis muscle of physically active elderly and young subjects by a combination of two-dimensional difference gel electrophoresis, SDS-PAGE and ESI-MS/MS. The differences observed in the elderly group included down-regulation of regulatory myosin light chains, particularly the phosphorylated isoforms, a higher proportion of myosin heavy chain isoforms 1 and 2A, and enhanced oxidative and reduced glycolytic capacity. Keywords: aging • myosin • phosphorylation • 2-D DIGE • mass spectrometry
Introduction When compared to young adults (age 20-30), moderately active elderly (75 year old) male subjects have lost approximately 25% of lower limb muscle mass (sarcopenia), with concomitant quantitative alterations in muscle maximal cross sectional and physiological areas, fascicle length, fiber pennation angle, maximum voluntary force and relaxation rate.1-3 Qualitative changes at the fiber level include a loss of specific force both in vivo4 and in vitro5-7 Reported changes in fiber type distribution are contradictory, but have featured either a shift toward a slower muscle phenotype following preferential denervation of fast motor units,8,9 no change at all10,11 or even a shift toward a faster phenotype.7,12 Age-associated molecular changes include reduction in myosin heavy chain (MHC),7,13 though the distribution of myosin light chains (MLC) is unchanged, reduced protein synthesis,14 accumulation of lipofuscin,15 impairment of excitation-contraction coupling,16 and decline in respiratory chain function.17 Given the complex nature of the aging process, considerable inter-subject and inter-group variability in age-related sarcopenia is not surprising. Since the amount of tissue obtainable at biopsy is necessarily limited, assessment of all relevant parameters may not be possible. Thus results (e.g., fiber type distribution, single muscle fiber properties, enzyme activity) from subject groups investigated in different laboratories may appear inconsistent. Moreover, until recently, techniques were not available to determine features including myosin regulatory * To whom correspondence should be addressed. CNR-IBFM, Via F.lli Cervi 93, 20090 Segrate-Milano. Tel: +390221717503. Fax:+390221717558. E-mail:
[email protected]. † CNR. ‡ University of Milano. § Imperial College London. | University of Pavia.
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Journal of Proteome Research 2006, 5, 1344-1353
Published on Web 05/03/2006
light chain (MLC) concentrations and, in particular, the phosphorylated isoforms which modulate the rate of force development and twitch force at different initial tension levels,18-20 both of which are relevant to muscle functional impairment. The aim of the present study was to apply a proteomic approach to age-dependent changes in contractile and metabolic proteins of the vastus lateralis muscle in two groups of physically active elderlyand young subjects. This large scale analysis of multiple proteins from the same samples should reduce the confounding effects of inter subject and group variation. In a development of our previous studies of muscle proteins in humans,21-24 we performed a quantitative differential analysis of muscle protein expression in elderly and young subjects using 2-D DIGE (two-dimensional difference gel electrophoresis),25,26 a technique for differential display proteomics characterized by high sensitivity, reproducibility and reliability. We found that the protein profiles of skeletal muscle from young adult and elderly subjects exhibited differences in regulatory myosin light chain distribution, (particularly the phosphorylated isoforms), in myosin heavy chains, and in patterns of metabolic enzymes which were consistent with the functional changes observed in aging.
Materials and Methods Subjects. Twelve subjects were studied, six in the age range 70-76 and six between 20 and 25 yr. All were in good health, four of the elderly walking and cycling between 30 and 60 min a day, while the other two were more active and could be considered as trained in moderate aerobic exercise. The young subjects were moderately active but not involved in any specific training program. Samples (about 15 mg) were obtained by surgical biopsy from the mid part of the vastus lateralis muscle 10.1021/pr050414x CCC: $33.50
2006 American Chemical Society
The Human Muscle Proteome in Aging
Figure 1. Vastus lateralis protein profiling by 2-D DIGE. Typical 2-D pattern gel image of vastus lateralis protein extract in a NL pH 3-10 IPG strip in the first dimension and SDS gel (12% T, 2.5% C) in the second. Automated image analysis by DeCyder software detected and matched 2700 protein spots over all 36 single-gel images. The 52 arrowed and circled spots differ in average ratios, (t-test value was
