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1 Department of Applied Physiology and Kinesiology, University of Florida, Gainesville, FL, USA 2 Laboratory of Biomechanics and Physiology, Yamaguchi University, Yamaguchi, Japan
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(Received 3 May 2007;
accepted after revision 27 June 2007; first published online 13 July 2007)
Corresponding author S. K. Powers: Department of Applied Physiology and Kinesiology, University of Florida, Gainesville, FL 32611, USA. Email: spowers{at}hhp.ufl.edu
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Introduction |
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The function of the UPP has been shown to decline as a result of ageing in a variety of cell types (Shang et al. 1997; Hayashi & Goto, 1998; Keller et al. 2000; Bulteau et al. 2002; Louie et al. 2002; Zeng et al. 2005). More specifically, age-related declines in the gene expression of key UPP components and diminished function of the proteasome have been reported in locomotor skeletal muscle with senescence (Radak et al. 2002b; Husom et al. 2004; Ferrington et al. 2005; Selsby et al. 2005). In contrast, Bardag-Gorce et al. (1999) report a parallel decline in proteasome content and activity during ageing and thus unaltered proteasome function. The physiological significance of the proteasome function remains an active area of research, and several investigators have suggested that diminished proteasome function is detrimental to cells because of the decreased ability to degrade damaged proteins (Grune et al. 2001, 2003). Thus, accumulation of damaged proteins in cells may contribute to the formation of large, cross-linked aggregates, which can threaten cell function and viability.
The diaphragm is the most important inspiratory muscle in mammals and is essential for normal ventilation (Poole et al. 1997) and, therefore, maintenance of diaphragm function is critical to overall health during senescence. To date, the function of the UPP in the aged diaphragm has not yet been described, and this forms the rationale for the present study. The objectives of this experiment were twofold: (1) to investigate the effects of ageing on the gene expression, protein abundance and ubiquitin-conjugating enzyme activity of key components of the UPP; and (2) to determine the impact of ageing on the three best-characterized activities of the 20S proteasome: chymotrypsin-like (CT-L), trypsin-like (T-L) and peptidylglutamyl peptide hydrolysing (PGPH) activities.
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Methods |
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These experiments were approved by the University of Florida Institutional Animal Care and Use Committee. Eight young adult (6 months) and eight senescent (24–26 months) male Fisher 344 rats were obtained from the National Institute on Ageing (Bethesda, MD, USA). The experimental animals were housed in pairs, maintained on a 12 h–12 h light–dark cycle, and were provided with food and water ad libitum. Animals were anaesthetized by an intraperitoneal injection of pentobarbitone sodium (100 mg kg–1). After reaching a surgical plane of anaesthesia, sections of the costal diaphragm were quickly excised, frozen in liquid nitrogen and stored at –80°C. The animals were euthenized by removal of the heart. One section was immediately frozen in Tissue-tek imbedding medium (Sakura Finetek, Torrance, CA, USA) at an unstressed length and stored at –80°C for subsequent histochemical analysis. Data investigating the effects of ageing on postural skeletal muscle collected from these animals has previously been published (Deruisseau et al. 2005b).
Isolation of total RNA, reverse transcription and quantitative real-time polymerase chain reaction (PCR)
Detailed methods for RNA isolation, reverse transcription and cDNA quantification employed by our laboratory have been previously described (Deruisseau et al. 2005b). Primers and probes for muscle atrophy F-box/Atrogin-1 (MAFbx) and muscle RING finger-1 (MuRF1) were obtained from Applied Biosystems (ABI; Assays-on-Demand). Sequences used by the manufacturer in the design of primers and probes from this service are proprietary and are therefore not reported. Gene expression was calculated using the relative standard curve method as described in the ABI User Bulletin no. 2. The mRNA input obtained from standard curves was normalized to hypoxanthine phosphoribosyltransferase (HPRT), since this gene does not demonstrate altered expression in the diaphragm in response to ageing (Deruisseau et al. 2006).
Western blot analysis
A section of the mid-costal diaphragm was homogenized and assayed for quantitative determination of the cytosolic protein levels of E214k, proteasome activator 28 (PA28), 20S 
-subunit 7 (C8) and ubiquitin–protein conjugates. Cytosolic proteins were separated via polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes followed by Ponceau-S staining and visual inspection for equal protein loading/transfer. The membranes were then incubated with a primary antibody directed against E214k, PA28 -subunit, C8 or anti-ubiquitin (Boston Biochem, Cambridge, MA, USA). This step was followed by incubation with a horseradish peroxidase–antibody conjugate directed against the primary antibody. The membranes were then treated with chemiluminescent reagents and exposed to film. Images of these films were captured and analysed using the 440CF Kodak Imaging System (Kodak, New Haven, CT, USA).
20S proteasome activity
The in vitro CT-L, T-L and PGPH activities of the 20S proteasome were measured fluorimetrically in cytosolic fractions by following the release of free 7-amido-4-methylcoumarin (AMC) from synthetic substrates (BioMol International LP, Plymouth Meeting, PA, USA) by a modification of the method of Stein et al. (1996) and as described by Betters et al. (2004).
Total glutathione
Diaphragmatic levels of total glutathione were measured by using a commercially avialable kit according to the manufacturer's instructions (Cayman Chemical, Ann Arbor, MI, USA).
Myofibre cross-sectional area
Serial sections from frozen diaphragm samples were cut at 10 µm using a cryotome (Shandon Inc., Pittsburgh, PA, USA). Sections for cross-sectional area analysis were stained using primary antibodies against type I, IIa and IIb/IIx myosin heavy chain as previously described (McClung et al. 2007). Subsequently, images were obtained at x10 magnification, and approximately 250 myofibres per sample were analysed to determine fibre cross-sectional area (in µm2) using Scion Image software (Scion Technologies, Frederick, MD, USA) by a blinded investigator. The decision to study 
250 myofibres per diaphragm sample was based on preliminary studies indicating that analysis of more than 250 myofibres per muscle section did not significantly alter the value of mean cross-sectional area of the fibre or the standard deviation.
Statistical analysis
Comparisons between groups for each dependent variable measured were made by one-way ANOVA. Significance was established at P < 0.05. Data are reported as means ± S.E.M.
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Results |
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We determined the effects of ageing on diaphragmatic mRNA expression of MAFbx and MuRF1 because these ubiquitin ligases are unique to skeletal muscle and both have been shown to play a key role in the development of skeletal muscle atrophy (Bodine et al. 2001). Ageing did not result in changes in the diaphragmatic mRNA levels of either MAFbx (young = 0.76 ± 0.11 and old = 0.47 ± 0.10 normalized input values) or MuRF1 (young = 37.56 ± 7.14 and old = 24.87 ± 9.22 normalized input values).
Diaphragmatic protein levels of UPP components
To further investigate the impact of ageing on diaphragmatic proteasome function, we analysed key elements of the ubiquitin conjugation cascade (i.e. E214k) and 20S proteasome (i.e. C8 and PA28 regulatory complex) in the diaphragm. Figure 1A illustrates representative Western blots of E214k, PA28 and C8 from young and old diaphragms. Figure 1B illustrates densiometric analysis of E214k, PA28 and C8 protein levels from Western blots; results are expressed as arbitrary optical density units. Note that no changes were detected in the protein levels of any of the three proteins between young and old diaphragms.
Diaphragmatic protein levels of ubiquitin–protein conjugates
A representative Western blot illustrating the degree of ubiquitin–protein conjugate accumulation is shown in Fig. 2A. Western blots were quantified using densitometry in which the analysis of ubiquitin–protein conjugates included all bands (i.e. the entire lane) for each sample. As illustrated in Fig. 2B, the total level of ubiquitin–protein conjugates was not different between the young and old animals.
Proteasome activity is maintained in the senescent diaphragm
To determine whether ageing influenced proteasome function in the diaphragm, we examined the three best-characterized activities of the 20S proteasome: CT-L, T-L and PGPH activities (Fig. 3). No changes in 20S activity for any of these three substrates were detected between the diaphragms of young and old animals.
Total glutathione content
Glutathione is the major non-protein thiol in cells and is considered to be the most important non-enzymatic antioxidant in the cell. Since it is well established that cellular oxidative stress results in lower levels of glutathione in tissue, we measured diaphragmatic levels of total glutathione as a marker of oxidative stress. Young and old diaphragms had similar total glutathione levels (1.16 ± 0.04 and 1.12 ± 0.05 mmol (g wet weight)–1, respectively), indicating that the ageing diaphragm is able to maintain normal cellular redox status.
Myofibre cross-sectional area
Figure 4A shows representative images of diaphragmatic fibres obtained from young and old animals. Interestingly, ageing resulted in an increase (P < 0.05) in cross-sectional area of type IIa fibres (Fig. 4B). Furthermore, compared with young adult animals, there was a trend towards a decrease (P = 0.052) in the percentage of the number of type IIa fibres in the senescent diaphragms (Fig. 4C).
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Discussion |
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This experiment provides new insight into the effects of ageing on UPP expression and function in the mammalian diaphragm. We have previously demonstrated that rat soleus muscle exhibits an age-related downregulation of selective mRNA levels of the ubiquitin conjugation cascade (Deruisseau et al. 2005b). Notably, our present results show that diaphragmatic gene expression, protein levels of several key proteasome components, content of endogenous ubiquitin–protein conjugates and 20S proteasome activity are not altered in the senescent diaphragm. Hence, it appears that the UPP pathway in old age is regulated differently between skeletal muscles with markedly different activation patterns. A discussion of these and other related findings follows.
Ageing and cellular proteasome function
The function of the UPP declines as a result of ageing in a variety of cell types, including the liver (Hayashi & Goto, 1998), lens (Shang et al. 1997), retina (Louie et al. 2002), heart (Bulteau et al. 2002) and brain (Zeng et al. 2005). Furthermore, age-related deficits in the mRNA expression of key UPP components (Deruisseau et al. 2005b; Ferrington et al. 2005) and diminished function of the proteasome have recently been reported in locomotor skeletal muscle with senescence (Radak et al. 2002b; Husom et al. 2004; Ferrington et al. 2005; Selsby et al. 2005). In contrast, a parallel decline in proteasome content and activity has been reported in the ageing gastrocnemius, implying that proteasome function is unaltered (Bardag-Gorce et al. 1999). The physiological significance of the activity and function of the proteasome in ageing skeletal muscle remains an active area of research. Progressive accumulation of biological waste (i.e. damaged proteins) is evident in aged cells, and it inevitably interferes with cellular functions, resulting in decreased adaptability and death of postmitotic cells (Terman, 2006). Age-related accumulation of damaged proteins may result from either an increase in the amount of damaged proteins or a decline in the degradation of damaged proteins. Davies and collaborators suggest that a combination of both processes contributes to the accumulation of damaged proteins with age (Grune et al. 2001). Furthermore, accumulated cross-linked proteins and declining proteasome activity during senescence are highly correlated (Grune et al. 2001). Thus, if damaged proteins are not removed rapidly, they tend to accumulate in cells and may contribute to the formation of large, cross-linked aggregates, which can threaten cell function and viability. It has been reported that antioxidant enzyme activity is elevated in ageing diaphragm (Powers et al. 1992) and that protein carbonyl derivatives are similar between young and old diaphragms (Criswell et al. 2003). Furthermore, total glutathione level, which is sensitive to changes in cellular redox status, is not altered in the ageing diaphragm. Therefore, it appears that cellular redox status is maintained in the diaphragm throughout the lifespan.
Maintenance of proteasome function in the senescent diaphragm
No age-related differences in diaphragmatic levels of MAFbx and MuRF1 mRNA were detected between young and old animals. However, it is possible that mRNA abundance is not directly representative of changes in protein content owing to translational regulation of protein expression. Our data also reveal that ageing is not associated with alterations in protein levels of diaphragmatic components of the ubiquitin conjugation cascade (i.e. E214k) or the 20S proteasome (i.e. C8 and PA28). In contrast to these findings in the aged diaphragm, we have previously demonstrated an age-related downregulation of mRNA levels of select components of the ubiquitin conjugation cascade (i.e. E214k and MuRF1) in rat soleus muscle (Deruisseau et al. 2005b). Investigators have reported a diminished function of the proteasome in locomotor skeletal muscle with senescence (Radak et al. 2002b; Husom et al. 2004; Ferrington et al. 2005; Selsby et al. 2005), while others report no changes in proteasome activity with ageing (Radak et al. 2002a; Clavel et al. 2006). These divergent results may be due to differences in tissue processing (i.e. muscle extracts versus purified proteasome), age and species/strain of the animals used in the study or the assay conditions used to measure activity.
Several factors may contribute to age-related differences in proteasome function between locomotor (i.e. gastrocnemious) and postural (i.e. soleus) skeletal muscles and diaphragm. For example, unlike locomotor muscle, the diaphragm remains chronically active throughout the lifespan. In addition, during ageing the soleus muscle maintains its activity and may exhibit a relative overload (i.e. due to increased body weight), resulting in a selective fibre hypertrophy (Hepple et al. 2004). Thus, the variation in activity patterns may at least explain the differences observed in different skeletal muscles. Furthermore, during periods of disuse (i.e. denervation, immobilization and gravitational unloading) the ubiquitin-conjugating activity increases markedly (Bodine et al. 2001; Jagoe & Goldberg, 2001; Reid, 2005; Powers et al. 2007). In addition, unloading the diaphragm via mechanical ventilation results in increased diaphragmatic ubiquitin–protein conjugates, chymotrypsin-like activity and trypsin-like activity (Betters et al. 2004; DeRuisseau et al. 2005a).
Interestingly, markers of the UPP activity are also altered by the increased muscle contractile activity that occurs during exercise (Reid, 2005), but the effect of repetitive, life-long activity on the UPP in locomotor skeletal muscle remains unknown. Response of the UPP to a change in muscle activity, however, appears to vary according to intensity and duration of the intervention. For example, strenuous exercise stimulates skeletal muscle adaptation, which requires selective degradation of existing proteins, a process primarily regulated by the UPP. To meet this challenge, activity of the proteasome pathway is increased (Glickman & Ciechanover, 2002; Reid, 2005). Although one study suggests that repetitive exercise has a training effect that tends to inhibit the UPP (Willoughby et al. 2000), others have reported that prolonged exercise training enhances the proteasome function of postural skeletal muscle compared with levels in untrained animals (Radak et al. 1999).
Summary and conclusions
This is the first study to examine the ageing response of the UPP in the mammalian diaphragm specifically. Of particular importance is the observation of maintained proteasome function in the diaphragm of senescent animals compared with diaphragm of young adult animals. Further experiments are required to clearly define the factor(s) involved in the differential regulation of proteasome function between the diaphragm and locomotor muscles of senescent animals. A more thorough understanding of the molecular mechanisms regulating skeletal muscle mass, including the homeostatic balance between protein degradation and synthesis, could lead to the enhancement of therapeutic strategies aimed at improving morbidity and mortality outcomes in clinically relevant situations.
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Footnotes |
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References |
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