J Physiol Biochem (2013) 69:697–705 DOI 10.1007/s13105-013-0246-7
ORIGINAL PAPER
Autophagic response to exercise training in skeletal muscle with age Yong An Kim & Young Sang Kim & Seung Lyul Oh & Hee-Jae Kim & Wook Song
Received: 25 October 2012 / Accepted: 25 February 2013 / Published online: 8 March 2013 # University of Navarra 2013
Abstract Autophagy, a highly conserved quality control mechanism, is essential for the maintenance of cellular homeostasis and for the orchestration of an efficient cellular response to stress. During aging, the efficiency of autophagic degradation declines, and intracellular waste products accumulate. Therefore, in this study, we tested the hypothesis that skeletal muscle from old mice would have decreased autophagosome formation when compared to the muscle from young mice. We also examined whether autophagic regulatory events differ between muscle fiber types and in response to exercise in aged male mice. The extensor digitorum longus (EDL) and gastrocnemius muscles were studied Y. A. Kim Institute of Biotechnology, Chungnam National University, Daejeon, Korea Y. S. Kim Department of Biochemistry, Chungnam National University, Daejeon, Korea Y. A. Kim : S. L. Oh : H.-J. Kim : W. Song (*) Health and Exercise Science Laboratory, Institute of Sports Science, Seoul National University, 599 Gwanangno, Gwanak-gu, Seoul 151-742, Korea e-mail:
[email protected] W. Song Institute on Aging, Seoul National University, 599 Gwanangno, Gwanak-gu, Seoul 151-742, Korea
in young and old ICR mice. Exercise was performed by allowing the mice to run on a treadmill with a 5° incline at 16.4 m/min for 40 min/day, 5 days/week for 8 weeks after a 1-week adaptation period. Our results indicated that the levels of microtubule-associated protein 1b light chain 3, a marker of autophagosome formation, were lower in both the EDL and the gastrocnemius muscle of old mice compared to those young mice. To identify the factors related to the changes observed, the expression of autophagy regulatory proteins was examined in the EDL and gastrocnemius muscles. Beclin-1, autophagyrelated gene 7 (ATG7), and lysosome-associated membrane protein were found to be lower in the EDL and gastrocnemius muscles of old mice compared to those in the young mice, then Beclin-1, ATG7, and musclespecific RING finger protein-1 upregulated after regular exercise. Moreover, the muscle weight/body weight was significantly increased only in the gastrocnemius muscle of the old trained mice. These data suggest that autophagy regulatory events are attenuated in old skeletal muscle. However, this effect is upregulated when animals are subjected to exercise training. Keywords Aging . Skeletal muscle . Exercise training . Autophagy . MuRF-1
Introduction Aging is characterized by a gradual loss of muscle proteins (i.e., sarcopenia). The size of stable postmitotic
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tissues, such as skeletal and cardiac muscles, is regulated by protein turnover, and skeletal muscle is influenced by a balance between protein synthesis and degradation and the turnover of contractile proteins. Thus, sarcopenia should occur due to an imbalance between the rates of protein synthesis and degradation. Protein degradation in skeletal muscle cells is essentially mediated by the activity of two highly conserved pathways: the autophagic lysosomal pathway and the ubiquitin–proteasome pathway. In the first pathway, portions of the cytoplasm and cell organelles are sequestered into vacuoles, called autophagosomes. The autophagosomes then fuse with lysosomes, where the content of the vacuoles are digested by lysosomal hydrolases [16, 26, 28]. In the second pathway, target proteins are conjugated to multiple ubiquitin moieties, and ubiquitin-tagged proteins are degraded within the proteasome complex [8]. The autophagic process can be divided into the following distinct steps: signaling and induction, autophagosome nucleation, membrane expansion and vesicle completion, and autophagosome targeting, docking, and fusion with the lysosome. The initial step in autophagy is vesicle induction, which results in the formation of autophagosomes. Inhibition of mTOR leads to dephosphorylation of autophagy-related gene 13 (ATG13) and the association of ATG13 with a protein complex containing ATG1 kinase and ATG17 [7]. Vesicle nucleation depends on the formation of a multiprotein complex involving the interaction of Beclin-1 with the class III phosphatidyl-inositol 3kinase and vacuolar protein sorting 34 [3]. Subsequent vesicle elongation involves ATG7, which is an ubiquitin E1-like enzyme homolog. This pathway also requires the conjugation of phosphatidylethanolamine to microtubule-associated protein 1b light chain 3 (LC3) by the conversion of the soluble form of LC3 (LC3-I) into the autophagic vesicle-associated form (LC3-II), which is required for membrane expansion. LC3-II is used as a marker of autophagy because its lipidation and specific recruitment to autophagosomes produce a shift from a diffuse to a punctuate staining pattern of the protein and an increase in its electrophoretic mobility on gels when compared with LC3-I [2, 7, 13]. Autophagosomes undergo maturation by fusion with lysosomes to create autolysosomes, and the lysosomeassociated membrane protein 2a (LAMP2a) is believed to play a role in this process [13]. Fusion of the autophagosome with a lysosome results in the exposure of the autophagosome content to hydrolase enzymes [7].
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Recent studies have shown that the impaired autophagy seen in ATG7 null muscles is characterized by muscle atrophy, weakness, and features of myofiber degeneration. Thus, autophagy was found to be essential for myofiber maintenance and for the clearance of damaged proteins and altered organelles [15]. In addition, muscle-specific RING finger protein-1 (MuRF-1) was also shown to be involved in the regulation of myofibril replacement and turnover [18]. However, the role of MuRF-1 during autophagy regulation in response to exercise is largely unknown. Autophagy, which is activated when skeletal muscle is under nutritional stress (such as metabolic stress) plays a role in the catabolic condition and in the degradation of macromolecules and organelles [7]. Catabolic pathways are accelerated during exercise to supply energy and substrates to the muscle for continuation of contractions. It has been well established that the rates of amino acid (relatively small) and glucose oxidation are increased during endurance exercise, and increased energy consumption is likely required to induce autophagy. Recently, autophagy was shown to be required for myofiber maintenance and for the clearance of damaged proteins and altered organelles [15, 16, 26– 28]. This basal autophagy plays a primary role in and is very important for intracellular quality control through the constitutive turnover of cytoplasmic components [21]. In addition, manipulation of autophagy genes has demonstrated that there is a tight connection between autophagy and life span and aging [20, 29, 31, 33]. However, autophagy has been shown to be attenuated with age because autophagy plays a pivotal role in cellular quality control and in the maintenance of homeostasis [4, 19, 23–25, 33–35]. This age-related attenuation of autophagy diminishes the efficiency of protein degradation and the clearance of damaged organelles [4, 23, 24, 35]. A decrease in proteolytic activity has been considered responsible, at least in part, for the accumulation of damaged cellular components in almost all tissues of aging organisms [23, 24, 30, 35]. A recent study showed that mild caloric restriction can increase the expression of Beclin-1, ATG7, and LC3-II, and no additive effect of life-long voluntary wheel running was detected when combined with caloric restriction (8 %) in 24-month-old rats compared to 6-month-old rats [34]. However, a limitation of this previous study was that an exercise-only group was not included, making it difficult to differentiate between the effects of exercise and of caloric restriction.
Autophagic response to regular exercise training
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Table 1 Body weight, EDL weight, relative EDL weight (g/BW), gastrocnemius weight (g), and relative gastrocnemius weight in young (4 months), old (12 months), control, and exercise-trained ICR mice Young
Old
Control (n=5)
Trained (n=6)
Control (n=7)
Trained (n=6)
Body weight (g)
44.103±1.81
43.035±0.79
52.904±2.71a
47.484±2.01b
EDL weight (g)
0.243±0.021
0.274±0.034
0.221±0.021
0.227±0.0413 a
Relative EDL weight
0.551±0.043
0.637±0.063
0.409±0.044
Gastrocnemius weight (g)
0.181±0.012
0.214±0.021
0.194±0.013
0.238±0.017b
Relative gastrocnemius weight
0.442±0.052
0.529±0.075
0.367±0.020
0.502±0.022b
0.479±0.039
Data are presented as mean±SEM; same indices represent significant difference at p
