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J Physiol Volume 565, Number 3, 993-1005, June 15, 2005 DOI: 10.1113/jphysiol.2004.080663
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Age is no barrier to muscle structural, biochemical and angiogenic adaptations to training up to 24 months in female rats

H. B Rossiter1, R. A Howlett1, H. H Holcombe1, P. L Entin2, H. E Wagner1 and P. D Wagner1

1 Department of Medicine, Division of Physiology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0623, USA
2 Department of Exercise Science and Athletic Training, Northern Arizona University, Flagstaff, AZ 86011-5095, USA


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Ageing is associated with reduced transport and utilization of O2, diminishing exercise tolerance. Reductions may occur in cardiac output (delivery), and skeletal muscle oxidative capacity (utilization). To determine the reversibility of the declines in the muscular determinants of these limitations, skeletal muscle morphological, angiogenic and biochemical responses to acute exercise and endurance training were investigated in female Fischer 344 rats (n = 42; seven groups of six rats) aged 6 (Y) and 24 (O) months compared with resting untrained controls (YC, OC). Treadmill training lasted 8 weeks (10 deg incline, 1 h per day, 5 days per week). Two groups ran at maximum tolerated speeds (YTR, OTR), while an additional Y group (YTM) trained at OTR speed. There was no effect of age on vascular endothelial growth factor gene expression in gastrocnemius muscles after acute exercise. Similarly, age did not impair the effects of training, with increases (P < 0.05; ±S.E.M.) occurring in all of the following: 1 h exercise running speed (YTR 92 ± 4% versus OTR 140 ± 25%); citrate synthase (YTR 37 ± 8% versus OTR 97 ± 33%) and ß-hydroxyacyl-CoA-dehydrogenase (YTR 31 ± 7%, versus OTR 72 ± 24%) activities; and capillary-to-fibre ratio (YTR 5.2 ± 0.2% versus OTR 8.1 ± 0.2%). However, YTM muscle was unchanged in each measure compared with YC. In conclusion, these muscular responses to training were (1) not reduced by ageing, but (2) dependent on relative and not absolute work rate, since, at the same speed, OTR rats showed greater changes than YTM. Therefore, increases in exercise tolerance and muscle adaptations are not impaired in female rats up to 24 months of age, and require a smaller absolute exercise stimulus (than young) to be manifest.

(Received 7 December 2004; accepted after revision 18 April 2005; first published online 21 April 2005)
Corresponding author H. B Rossiter: School of Sport and Exercise Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS1 9JT, UK. Email: h.b.rossiter{at}leeds.ac.uk


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Sustained muscular exercise depends on the effective integration of O2 transport to, and O2 utilization by, skeletal-muscle mitochondria. However, during advancing age, the systems that determine this ability are often reported as being increasingly limited. In humans, ageing is associated with reductions in maximal cardiac output (e.g. Ehsani et al. 1991), muscle blood flow (e.g. Wahren et al. 1974), muscle mitochondrial capacity (e.g. Irrcher et al. 2003), and function – in some, but not consistently all, respiratory chain reactions (Proctor et al. 1995; Rasmussen et al. 2003). This not only diminishes the maximal O2 consumption (VO2max; Dehn & Bruce, 1972; Ogawa et al. 1992; Fitzgerald et al. 1997; Neder et al. 2001), but also the threshold parameters of aerobic performance, such as critical power (CP; Neder et al. 2000) and the lactate threshold (LT; Neder et al. 2001). However, it is well established that aerobic exercise training can induce positive adaptations in both cardiovascular and skeletal muscle systems (Saltin & Rowell, 1980; Ogawa et al. 1992; Hoppeler & Fluck, 2003), and hence has the potential to slow these age-related declines (Proctor et al. 1995; Terjung et al. 2002).

Longitudinal data suggest that age-related declines in the parameters of aerobic function are associated with a considerable impairment of peripheral muscle function, and particularly in maximal oxygen extraction (McGuire et al. 2001). These findings are reflected in a rat model of ageing, where Hepple et al. (2003) have shown reductions in muscle mass and mitochondrial oxidative capacity. The decreased ability of aged skeletal muscle to utilize O2 therefore occurs in concert with reductions (compared with young muscle) in oxidative enzyme activities, such as citrate synthase (CS; Cartee & Farrar, 1987; Klitgaard et al. 1989; Powers et al. 1992) and mitochondrial density, although many of these effects were not manifest in fast-twitch fibre properties (Walters et al. 1990). Exercise training, however, has been shown to equally benefit young and old rats in terms of maximal O2 consumption (in both male and female Fischer 344 rats aged between 3 and 24 months; Mazzeo et al. 1984; Cartee & Farrar, 1987).

The issues surrounding the ‘muscular component’ of O2 delivery and muscle capillarity (capillary-to-fibre (C/F) ratio or capillary-to-fibre-area ratio), however, are more complex and show varying effects with age (Mitchell et al. 1991; Coggan et al. 1992b; Suzuki et al. 1997). As muscle capillarity is one of the key steps in potentially limiting VO2max (Wagner, 1996), the ability to upregulate angiogenesis has the potential to be a key element of the endurance-training response. However, the interaction between training and ageing has shown conflicting reports in terms of alterations in C/F ratio (Mitchell et al. 1991; Yang et al. 1994; Suzuki et al. 1997). Furthermore, angiogenic gene activation (such as vascular endothelial growth factor, VEGF) has not been addressed in both ageing and training.

Impairment of angiogenic responses (particularly VEGF) in aged animals has been shown in many tissues (Rivard et al. 1999, 2000; Swift et al. 1999; Kang et al. 2001; Wang et al. 2004), although Jozsi et al. (2000) did not detect such impairments in skeletal muscle of senescent humans. Therefore, if angiogenesis is impaired in ageing, it should be reflected in an attenuation of angiogenic gene activation following exercise (Hang et al. 1995; Breen et al. 1996; Olfert et al. 2001) and training (Gavin & Wagner, 2001; Amaral et al. 2001). Furthermore, as angiogenic gene activation has been shown to be dependent on exercise intensity (Breen et al. 1996), we were particularly interested in how exercise intensity (similar to Coggan et al. 1992a; in humans) modulated the training responses across age groups.

We were therefore interested in how angiogenic gene activation related to training-induced improvements in C/F ratio, and also how O2 utilization (skeletal muscle oxidative enzyme activity) and performance would be improved under conditions of age-related exercise limitations. We hypothesized that older rats (24 months old, female Fischer 344) would express a greater training response at lower absolute work rates than young (6 months old) rats, but that training at the same intensity (the maximum tolerated) would provide similar adaptations in youth and during advancing age.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Animals and group assignments

The study was conducted in accordance with the US National Institutes of Health guidelines, and approved by The University of California San Diego Animal Subjects and Animal Care Programs. Six-month-old (‘young’, Y; n = 24) and 24-month-old (‘old’, O; n = 18) female Fischer 344 rats (manifesting 100 and 65% survival rates, respectively; Turturro et al. 1999) were habituated to treadmill exercise, where rats were allowed to rest and walk on a four-lane, motor-driven, rodent treadmill (CL-4; Omnitech, Columbus, OH, USA) for 10 min per day for 3 days at 8 m min–1 (10 deg incline), increasing by 2 m min–1 on each consecutive day. Seven days following habituation, Y and O rats were randomly assigned to one of seven groups (n = 6 per group). Groups were: resting control (YC, OC), acute exercise (YA, OA), and endurance exercise training (YTR, OTR, and YTM; where subscript ‘T’ denotes ‘trained’, ‘R’ ‘relative’, and ‘M’ ‘matched’; see the section on Exercise training). This allowed comparisons of the following: (1) effect of ageing on structural, biochemical and functional adaptations to training at the maximum tolerated intensity (groups YC, OC versus YTR, OTR); (2) effect of intensity on training adaptations across age groups (YTR versus OTR versus YTM); (3) effects of age on training responses to acute exercise (groups YC, OC versus YA, OA), with particular reference to angiogenic mRNA and protein expression.

Exercise and training protocols

Acute exercise.  Acute running exercise was performed on the same treadmill ergometer (10 deg incline) as the familiarization. Running speeds were chosen to target fatigue in approximately 1 h, and performed at the maximum tolerable speed in both Y and O age groups.

Exercise training.  Endurance exercise training was prescribed at either the same relative intensity (YTR, OTR, i.e. the maximum tolerated, resulting in a higher absolute speed in young rats) or, for young rats only, at a speed matched to OTR (YTM; the same absolute speed, but a lower relative intensity than that tolerated by old rats). This allowed discrimination between the responses to absolute training speed or relative (maximal) intensity with ageing. Training consisted of 1 h per day treadmill running (10 deg incline), 5 days per week for 8 weeks. To identify the maximum tolerable intensity (relative; YTR, OTR) treadmill speeds were incremented by 1–2 m min–1 for the final 10 min of each training session. If the animal performed well, the running speed in the following session began at the level achieved on the previous session, and so on, in order to maintain the highest achievable running speeds on each day. YTM rats were run along side their matched (OTR) training partners.

During all treadmill exercise, rats were motivated by a shock grid and air jets at the rear of the treadmill and by treats (banana and raisins) following training sessions.

Muscle isolation

Rats were anaesthetized with pentobarbital sodium (Nembutal; 60 mg kg–1, I.P.) and the entire gasctrocnemius muscles of both legs were isolated, removed and weighed. Each muscle was transversely divided at the widest point of the muscle belly, weighed and either (alternating between the right an left gastrocnemius within each group) flash frozen in liquid N2 (for RNA and protein isolation, and enzymatic analysis) or a section of ~5 mm was cork mounted and fixed in liquid N2-cooled isopentane (for histological analysis), each being stored at –80°C thereafter. The procedure was conducted after at least 24 h recovery following the final bout of exercise training, except in the acutely exercised groups (OA, YA), where the muscles were frozen or fixed within 15 min of the end of exercise.

VEGF mRNA and protein analyses

Total cellular RNA was isolated from one portion (randomized between legs and including both heads) of the gastrocnemius muscle by the method of Chomczynski & Sacchi (1987). RNA solutes were quantified by absorbance at 260 nm, and integrity was checked from electrophoresis on a 1% agarose gel stained with ethidium bromide and examination of the 18S and 28S rRNA under ultraviolet light. Fractionated RNA was transferred by Northern blot to a Zeta probe membrane (Bio-Rad, Hercules, CA, USA), cross-linked by ultraviolet irradiation for 1 min, and probed with oligolabelled [{alpha}-32P]dCTP cDNA probes. The blot was probed for angiogenesis-related mRNAs using the following probes: rat VEGF, a 900 bp cDNA PstI/SmaI insert cloned into pBluescript II KS1 vector; rat VEGF-R1 (Flt-1), a 600 bp cDNA EcoRI/HindIII insert of pUC119; rat VEGF-R2 (Flk-1 or KDR), a 1.2 kb cDNA EcoRI insert of pUC18; rat transforming growth factor ß1 (TGFß1), a 985 bp cDNA HindIII/XbaI insert cloned into pBluescript II KS1 vector; and basic fibroblast growth factor (bFGF), a 1 kb XhoI fragment of human bFGF cDNA. Prehybridization and hybridizations were performed in 50% formamide, 10x saline sodium citrate (SSC; 20x SSC is 0.3 M sodium chloride, 0.3 M sodium citrate), 5% Denhardt's solution (100x Denhardt's is 2% Ficoll and 2% polyvinyl pyrrolidone, 2% bovine serum albumin factor V), 50 mM sodium phosphate (pH 7.0), 1% sodium dodecyl sulphate (SDS), and 250 mg ml–1 sonicated salmon sperm DNA, at 42°C. Blots were washed with 2% SSC and 0.1% SDS at room temperature, and 0.1% SSC and 0.1% SDS at 65°C (VEGF), 60°C (bFGF) or 50°C (TGFß1, VEGF-R1, and VEGF-R2). Blots were exposed to BIOMAX MR X-ray film (Eastman Kodak, New Haven, CT, USA) using a Cronex Lighting Plus screen at –80°C. Autoradiographs were quantified via a densitometry (Gel-Pro Analyser; Media Cybernetics, Silver Spring, MD, USA) and normalized to ribosomal 28S RNA levels.

Another portion of the frozen muscle sample was homogenized in 1x PBS, 0.1% SDS with a complete protease inhibitor tablet (Roche, Indianapolis, IN, USA). Total protein was measured by the bicinchoninic acid method (BCA protein assay kit; Bio-Rad Laboratories, Hercules, CA, USA). VEGF protein was measured from 25 µg of total protein, and was analysed in duplicate. A commercial VEGF ELISA kit was used according to the manufacturer's instructions (MMV00; R & D Systems, Minneapolis, MN, USA) to detect the 121 and165 kDa isoforms of VEGF protein. VEGF levels were obtained by use of a microplate reader at 450 nm, and corrected by readings at 540 nm.

Capillarity, fibre typing and morphometry

Transverse 8 µm serial sections were cut from the isopentane-fixed muscle portions using a cryotome (Cryostat) at –26°C, and mounted on slides for histochemical capillary and fibre-type analyses.

Capillaries were stained using a combined alkaline phosphatase (AP) and dipeptidylpeptidase (DPP) reaction to identify all capillaries (Lojda, 1979; Grim & Carlson, 1990). Sections were soaked in a cooled 1:1 mixture of acetone and chloroform for 5 min and then air-dried. Slides were incubated for 60 min at 37°C in a 0.1 M phosphate-buffered solution of 0.08% gly-pro 4-methoxy-ß-naphthylamide and 0.034% fast blue (pH 7.2). Slides were then rinsed in phosphate buffer, and transferred to 0.04% naphthol ASMX phosphate and 0.21% variance blue in 0.1 M Tris buffer (pH 9.2) for 2 h at 37°C.

A modified assay of Ogilvie & Feeback (1990) was used to delineate between the myosin-ATPase of type I and type II muscle fibres. Sections were preincubated (8 min) in a solution containing 0.49% potassium acetate and 0.26% calcium chloride (pH 4.4) and rinsed in 0.1 M TRIS buffer (pH 7.8). Sections were then incubated for 30 min at 37°C in 0.4% glycine, 0.42% calcium chloride, 0.38% sodium chloride, 0.19% sodium hydroxide and 0.15% ATP (pH 9.4). Slides were rinsed in 1% calcium chloride and stained in 0.1% toluidine blue for 1 min, rinsed in distilled H2O, dehydrated in ethanol, and cleared in Hemo-De. All the sections were mounted with Permount.

The entire medial gastrocnemius cross-section was digitally imaged (30–40 images, each image 1.15 mm by 0.86 mm), and morphometric measurements of the entire set of images were made for each muscle using MATLAB 5.3, with images displayed on a computer screen at x200 magnification. Prior to capillary and fibre counts the ‘mixed’ (defined here as the mixed type I and type II muscle-fibre region; see Fig. 4 below) and ‘predominantly type II’ fibre regions of the gastrocnemius cross-sections were visually identified by an experimenter blinded to the experimental condition. Counts were recorded for each of the two muscle regions, and also summed for whole-muscle morphometry. The reason for this was that capillary counts were found to be very different in the two regions. ‘Edge’ effects were minimized by counting all capillaries and fibres of the entire cross-section. Less than 1% of capillaries lay along the demarcation zone, and these few were assigned equally to the two regions – no capillaries were double counted. Also, the mean fibre-area, as well as those for the mixed and predominantly type II regions of the muscle alone, were measured from the digital images. From these data, the mean C/F ratio, and mean capillary density, were calculated for the entire muscle, as well as separately for the mixed and predominantly type II portions.



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  		Figure 4.  Cross-section of a medial gastrocnemius muscle from an untrained young (6-month-old) Fischer 344 rat stained for fibre type
Staining protocol is modified from Ogilvie & Feeback (1990) for metachromatic staining of type I and II myosin-ATPases; type I fibres stain blue. All fibres and capillaries from the whole muscle section were counted (see Methods, Capillarity, fibre typing and morphometry). The muscle was visually separated into the ‘predominantly type II’ and ‘mixed’ (mixed, type I and II) fibre regions. Fibre and capillary counts were recorded separately for these two muscle regions and also summed for whole-muscle morphometry.

 
Muscle enzyme analysis

A muscle portion (including both left and right heads of the gastrconemius) was ground to a fine powder under liquid N2. CS and ß-hydroxyacyl-CoA dehydrogenase (ß-HAD) activities were measured in whole-muscle homogenates, prepared using 6–10 mg of pulverized wet tissue. Samples were homogenized in 80–90 vols (w/v) of buffer (175 mM KCl, 2 mM EDTA; pH 7.4) with a Polytron mixer for 35–45 s, and subjected to four cycles of freeze–thaw in liquid N2. The thawed samples were then centrifuged at 5800 g for 1 min to isolate particulate matter. Enzyme activities were determined spectrophotometrically (Beckman, model 64) at 20°C from the supernatant, as per Srere (1969) for CS, and Bergmeyer (1974) for ß-HAD.

Statistical analyses

All data are presented as means ± S.E.M. Measurements from the acutely exercised rats were compared with control animals using ANOVA (with Scheffe's post-hoc test). To test for differences between training status and age as well as their interaction, measurements from exercise-trained and untrained rats were subjected to a two-way ANOVA (with Scheffe's post-hoc test). Statistical analyses were made using Statview software (version 5.0.1; SAS Institute, USA) and significance was accepted at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
YC rats had a significantly lower mean body mass compared with OC rats (YC 197 ± 2 g, OC 258 ± 6 g; P < 0.05), a difference which was also evident between YA and OA (YA 203 ± 2 g, OA 272 ± 4 g; P < 0.05), and which was preserved even following exercise training (YTR 209 ± 5 g, OTR 260 ± 12 g, YTM 223 ± 9 g; P < 0.05); however, there were no significant differences within the Y and O rat groups. The salient comparisons are presented according to the three main aims set out in Methods.

Effect of ageing on adaptations to training at the maximum tolerated intensity

Running speed.  The absolute and relative improvements in running speed achieved during endurance exercise training are shown in Fig. 1A and B (OTR and YTM, n = 5; YTR, n = 6). Because one OTR rat was injured on day 20, and did not sufficiently recover to continue, this rat and its YTM training partner were omitted from further analysis. OTR and matched YTM rats began at a lower absolute speed in the first training week (11.1 ± 0.6 m min–1) than YTR (16.2 ± 0.3 m min–1), but showed a greater improvement relative to pre-training speed, such that the two groups were not statistically different by the end of week 6 (OTR reaching 24 ± 2 m min–1 versus YTR 29 ± 1 m min–1 at 8 weeks). It is of note that the running speeds achieved by the most responsive OTR rats (30 m min–1; n = 2) were similar to those in the best YTR rats (31 m min–1; n = 4). Also, whereas the YTR showed little improvement in 1 h running speed after ~5 weeks of training, OTR continued to increase for the entire 8 week training duration (Fig. 1A and B) – a relative difference which became significant after 6 weeks of training (P < 0.05; Fig. 1B).



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  		Figure 1.  End-exercise treadmill running speed for 8 weeks of training in young and old rats
End-exercise treadmill running speeds (at 10 deg incline) for young ({circ}; 6 months; YTR) and old (•; 24 months; OTR) rats trained for 1 h per day, 5 days per week for 8 weeks. A, absolute running speed; B, running speed relative to pre-training baseline. YTR rats ran at identical, matched, speeds to OTR (•), and are therefore not shown.

 
Mitochondrial enzyme adaptations.  Resting CS activity was significantly (P < 0.05) greater prior to training in YC (17.5 ± 0.7 mmol kg–1 min–1) compared with OC (14.0 ± 1.5 mmol kg–1 min–1), with no difference (P > 0.05) in ß-HAD. The difference in resting CS activity was more than overcome by training, however, as both OTR (25.1 ± 1.6 mmol kg–1 min–1) and YTR (23.5 ± 1.1 mmol kg–1 min–1) manifest similar (P > 0.05) values post-training. Similar fold-increases were observed in ß-HAD activity (YC 3.2 ± 0.1, YTR 4.1 ± 0.2 mmol kg–1 min–1 versus OC 2.9 ± 0.2, OTR 4.7 ± 0.4 mmol kg–1 min–1; Fig. 2A). CS and ß–HAD activities were significantly correlated and independent of age or state of training (Fig. 2B; R2 = 0.89; P < 0.05), and were also correlated with maximum 1 h running speed (R2 = 0.79; P = 0.1; data not shown).



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  		Figure 2.  Mitochondrial enzyme adaptations to 8 weeks exercise training in gastronemius muscle of young and old rats
A, citrate synthase (CS) and ß-hydroxyacyl-CoA-dehydrogenase (ß-HAD) activities in young (6 months; open bars) and old (24 month; filled bars) control and exercise-trained rat gastrocnemius muscle. Old, untrained control rats (OC) had a significantly lower CS activity than their young control (YC) counterparts. However, this difference was overcome by maximal exercise training (YTR, OTR) such that both ß-HAD and CS activities were significantly (P < 0.05) greater post-training, but not different between ages. B, CS and ß-HAD activities were significantly correlated (R2 = 0.89, P < 0.05), with increases from young and old control rats (YC, OC) with maximal exercise training (YTR, OTR) being independent of age.

 
Structural adaptations.  In the absence of training (YC and OC), there were no significant differences in absolute overall gastrocnemius mass or cross-sectional area, total number of fibres or capillaries, fibre-type composition or C/F ratio, between the ages (P > 0.05; Table 1). However, gastrocnemius mass as a percentage of body mass was lower in OC than YC (OC 0.36 ± 0.01%, YC 0.47 ± 0.01%; P < 0.05). Following maximal training, the percentage of type I fibres remained unchanged and similar across age groups (Table 1). Both total gastrocnemius and medial gastrocnemius muscle mass, however, increased (P < 0.05), but total muscle cross-sectional area was slightly decreased (although this was not significant). Similarly, total capillary number tended to increase (n.s.). The net result of these small changes was that overall C/F ratio increased significantly (P < 0.05) by ~5% in YTR and ~8% in OTR with maximal training (Fig. 3A; Table 1). When these changes were separated according to mixed-fibre-type region and predominantly-type-II-fibre region (Fig. 4), it was noted that the predominant improvement (P < 0.05) was in the mixed-fibre-type region, with similar increases in both YTR and OTR groups (Table 1). An interaction between age and training approached significance (ANOVA, P = 0.08) for C/F ratio in the predominantly-type-II-fibre region; however, it was the OTR rats that showed a greater increase (~11%), with no change in YTR (~1% reduction). Interestingly, C/F ratio in the whole muscle increased in concert with CS activity following maximal training (R2 = 0.75; P = 0.1), the slope of the relationship being independent of age (Fig. 3B).


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  		Table 1. Gastrocnemius muscle characteristics of 6- and 24-month-old control and exercise-trained rats
 


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  		Figure 3.  Capillary/fibre (C/F) ratio adaptations to 8 weeks exercise training in gastrocnemius muscle of young and old rats
A, capillary/fibre (C/F) ratio in gastrocnemius muscle of young (6 months; open bars) and old (24 months; filled bars) control (YC and OC) and exercise-trained rats (YTR, OTR). Capillary and fibre counts were made from the entire cross-section of the medial gastrocnemius, and separated for analysis into ‘mixed’ (mixed type I and type II muscle fibres) and ‘predominantly type II’ muscle-fibre regions (cf. Fig. 4). Training induced significant (P < 0.05) increases in C/F ratio in the mixed muscle of both young and old rats, there being no difference across the age groups within the ‘control’ or ‘training’ conditions. OTR rats manifest the largest percentage increase (although not significant, P > 0.05) in the predominantly type II muscle C/F ratio. B, C/F ratio and CS activity were correlated with no apparent age-effect either pre-training or post-training. C/F ratio and CS post-training values were significantly greater than control in both young (6 months; {circ}) and old (24 month; •) rats; both increasing with a similar slope.

 
Resting angiogenic mRNAs and VEGF protein expression.  There were no differences (P > 0.05) between resting controls and resting trained rats in muscular angiogenic mRNAs for VEGF, VEGF-R1, and bFGF, or for VEGF protein concentrations. However, there was a significant effect of age in the resting expression of TGFß1 and VEGF-R2 mRNAs (similar to that seen in the acute exercise responses, see below). Both TGFß1 and VEGF-R2 mRNA were significantly greater (by 1.7 ± 0.4- and 2.1 ± 0.4-fold, respectively; P < 0.05) in OTR rats compared with YTR.

Effects of intensity on training adaptations across age groups

The biochemical and structural adaptations to training in YTR and OTR were not mirrored in young rats that trained at absolute speeds matched to OTR (YTM). These YTM rats did not show training adaptations in any of the variables measured other than running speed (Fig. 1A and B) and total mass of the gastrocnemius (Table 1). YTM rats showed no change (P > 0.05) in enzyme activities with training (CS 19.1 ± 0.9 mmol kg–1 min–1, ß-HAD 3.3 ± 0.2 mmol kg–1 min–1) compared with YC (CS 17.5 ± 0.7 mmol kg–1 min–1, ß-HAD 3.2 ± 0.1 mmol kg–1 min–1). Unlike either YTR or OTR rats, YTM rats also did not increase C/F ratio, remaining the same as YC (YC 1.35 ± 0.03, YTM 1.32 ± 0.06; P > 0.05). The YTM group was therefore included with control animals for further analyses.

Effects of age on responses to acute exercise

Running speed.  Untrained young (YA) rats achieved a higher absolute running speed during the acute run to fatigue than untrained old (OA) rats (19.7 ± 0.2 versus 14.0 ± 0.6 m min–1, respectively), with both YA and OA reaching fatigue at an average of 53 ± 3 min of the 1 h target.

Angiogenic mRNAs and VEGF protein expression.  While the resting expression of angiogenic mRNAs tended to be slightly higher in YC rats compared with OC, the values were not statistically different; therefore, mRNA expression normalized to control (resting) levels was compared between the age groups (this second normalization process aimed to alleviate any potential differences in 28S ribosomal RNA synthesis with age). Figure 5 shows that the exercise/rest ratio of VEGF, VEGF-R1 and bFGF mRNA expression was similar (P > 0.05) between YA and OA, with VEGF mRNA increasing ~2.5-fold following exercise in both age groups. However, TGFß1 showed an ~1.5-fold increase during exercise in YA compared with a tendency to decrease in OA; similarly, VEGF-R2 expression was greater in YA compared with OA. However, acute exercise resulted in a decrease of OA VEGF-R2 expression compared with both resting control (OC) and YA (P < 0.05; Fig. 5). The ~2.5-fold increase in VEGF mRNA expression following exercise did not translate into an increase in protein expression, at least by the time of muscle sampling (~15 min post-exercise), with no difference between groups (YC 0.034 ± 0.01, OC 0.032 ± 0.01, YA 0.033 ± 0.01 and OA 0.031 ± 0.01 pg µg–1; P > 0.05).



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  		Figure 5.  Angiogenic mRNA expression responses to 8 weeks exercise training in gastrocnemius muscle of young and old rats
Acute exercise-induced increases in angiogenic mRNA concentrations in rat gastrocnemius muscle. RNA densitometry was normalized to 28S and is expressed as a fold-increase from control (no exercise, resting) to exercise (1 h treadmill running). Exercise-induced fold-increases in vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and VEGF receptor-1 (VEGF-R1) were independent of age (6 months, open bars; 24 months, filled bars). However, exercise-induced increases were attenuated in old rats for transforming growth factor ß1 (TGFß1) and VEGF receptor-2 (VEGF-R2), although absolute densitometry units remained greater at rest and following exercise in old rats compared with young rats.

 
Enzyme activities.  There was a significant increase in CS enzyme activity following acute exercise in OA compared with OC (19.2 ± 1.4 versus 14.0 ± 1.5 mmol kg–1 min–1, respectively); however, there were no other differences in enzymatic activities within or between ages following acute exercise.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
These data demonstrate that ageing is no barrier to adaptation in almost every variable measured (structural, angiogenic and biochemical) in response to either a single exercise bout or aerobic exercise training. The data demonstrated that, in female rats at 24 months of age, (a) training at the maximal tolerated intensity resulted in similar (or greater) improvements in exercise tolerance compared with young animals (Fig. 1A and B), (b) the gastrocnemius aerobic enzyme activity ‘deficit’ (compared with YC) was eliminated by maximal training (Fig. 2A and B), (c) training-induced increases in C/F ratio were the same as those seen in young rats (Fig. 3A and B), (d) structural or biochemical adaptations were clearly manifest, whereas young rats trained at the same absolute speeds showed no such training effect.

The only differences apparent between untrained rats aged 6 and 24 months were in the responses of TGFß1 and VEGF-R2 mRNA expression following ~1 h of treadmill running to exhaustion (Fig. 5). However, because both TGFß1 and VEGF-R2 mRNA values were twofold greater at rest in aged rats compared with controls (a difference that was maintained following 8 weeks training), the lack of an increase following acute exercise resulted in similar absolute values between the two groups.

These findings are in accordance with the suggestions of Walters et al. 1990) that some observed influences of ageing on skeletal muscle are not universal phenomena. Indeed, untrained control values for muscle weight, capillarity and fibre-type proportion were similar between YC and OC rats, suggesting few muscular differences (CS activity excepted) with age, up to at least 24 months. Similarly, following maximally tolerated aerobic exercise training, biochemical and structural adaptations in female 24-month-old Fischer 344 rats paralleled those of 6-month-old rats (Figs 2B and 3B), demonstrating that both availability and utilization indices were upregulated. These findings are discussed in reference to the three main aims of the study.

Effect of ageing on structural, biochemical and functional adaptations to training at the maximum tolerated intensity

Performance.  The aerobic exercise training protocol used in the present study was particularly effective in improving 1 h exercise speed, with >100% increases. Rather than training at a constant speed and measuring time to exhaustion (Lloyd et al. 2003; Steinberg et al. 2004; among others) we used an approach allowing relative intensity to remain approximately constant throughout training, increasing running speed every session as tolerated (more similar to Mazzeo et al. 1984; Olfert et al. 2001). While the average improvement in 1 h running speed was the same between age groups (~15 m min–1), improvements continued to occur in OTR rats for the entire training duration, whereas the YTR group peaked at a speed of ~30 m min–1 in the fifth week of training. This may be due, in part, to older rats only tolerating lower speeds in the sedentary state (beginning training at ~11 m min–1 compared with ~16 m min–1 for the 6-month-old rats) allowing a greater potential for improvement. Of particular note were the two OTR rats that achieved the same end-training running speeds as the YTR group. Indeed, these rats maintained 30 m min–1 for 1 h, out-performing two of the YTR group and their own YTM training partners (these rats being unable to maintain 30 m min–1 for the entire hour).

Mitochondrial enzymes.  Increases in oxidative enzyme activity with aerobic exercise training are well described (Beyer et al. 1984, for example). CS activity was significantly lower in aged control (untrained) rats compared with young. However, and in accordance with Beyer et al. (1984), the improvement in oxidative enzyme activities following training resulted in CS and ß-HAD activities in old rats that were not only greater than in YC rats, but exceeded (n.s.) that of YTR rats trained at the same intensity. It was not surprising that CS and ß-HAD activities were tightly correlated (R2 = 0.89), but it is of interest that this relationship was unaffected by both age and state of training (Fig. 2B). It is also of note that both young and old trained rats manifest greater CS and ß-HAD activities than rats bred for running tolerance (Howlett et al. 2003). This finding could add to the suggestion that the significant impediment to endurance exercise tolerance is a peripheral limitation of O2 utilization (McGuire et al. 2001; Hepple et al. 2003). It is clear that this limitation can be successfully attenuated via training, but it is of interest that a training programme as short as 8 weeks can provide a greater increase in muscle oxidative enzyme capacity (at any time up to 24 months of age) than several generations of selective breeding for endurance capacity (Howlett et al. 2003). The correlation between C/F ratio and CS (or ß-HAD) activity over the training period observed here (Fig. 3B) also supports the notion that capillary supply increases in proportion to O2 utilization potential (Hepple et al. 2000). In contrast to the findings of Hepple & Vogell, 2004), we found that C/F ratio and oxidative enzyme capacity were both increased by training independent of age up to 24 months. Whether this is due to strain, sex or other differences, remains to be determined. Indeed, while running performance is clearly dependent on a large number of interrelated variables (Wagner, 1996), both CS and C/F ratio were reasonably well correlated to end-training running performance in the present study (R2 = 0.79 and 0.61 for CS and C/F ratio, respectively). The slope of the relationship between C/F ratio and CS with training was similar for both the 6 and 24 month age groups. Twenty-four-month-old rats began from a lower CS value with the same C/F ratio as rats aged 6 months, suggesting that perhaps aerobic enzyme activity was lower than ‘expected’ for the potential O2 delivery defined by C/F ratio. It may be, therefore, that intramuscular processes associated with O2 utilization do indeed pose the most significant limitation to exercise tolerance in ageing (Hepple et al. 2000, 2003; McGuire et al. 2001). However, the ability to overcome this ‘deficit’ was not limited by ageing; the concomitant increase in C/F ratio is indicative that age (up to 24 months) did not provide a barrier to overcome both delivery and utilization measures.

Structural adaptations.  The C/F ratio values, established from counts of the entire gastrocnemius muscle, are similar to those previously reported (Suzuki et al. 1997; Tyml et al. 1999; Howlett et al. 2003). It has been suggested, however, that the size of the CF interface may be the paramount parameter limiting O2 flux to the mitochondria (Hoppeler et al. 1985; Hepple et al. 2000; Gayeski & Honig, 1986). In the present study, C/F ratio was significantly increased by a similar degree in young and old rats following intensity-matched training; this was similar to Coggan et al. (1992a) and Hepple et al. (1997) following training in older human subjects. However, the present data also manifest a tendency for the mean fibre area to be reduced in both groups (Table 1). Taken together our data suggest that size of CF interface would be increased (by a greater extent, on average, than C/F ratio alone) following exercise training in both young and old rats. Unlike both Mitchell et al. (1991) and Suzuki et al. (1997), the present study suggests that the potential to improve O2 transport from capillaries to mitochondria (by either measure) is age independent. Quite why these discrepancies were manifest is not entirely clear, but they may be related to the different muscles investigated. Mitchell et al. (1991) investigated the soleus and extensor digitorum longus muscles, and Suzuki et al. (1997) the soleus alone; thus, any differences in fibre populations of the investigated muscles may be of significance (Lloyd et al. 2003). For example, in humans, Proctor et al. (1995) have suggested that ageing appears to selectively decrease C/F ratio and oxidative enzyme activity in type II muscle fibres, but that this reduction is overcome in both fibre populations by endurance training.

Effect of intensity on training adaptations across age groups

A striking feature of the present study was the complete lack morphometric or biochemical training responses in the YTM group. While all rats improved their running speed over the 8 weeks of training, young rats running at the same absolute speeds as old rats did not elicit any of the physiological adaptations observed in the old group. Towards the end of the training protocol, this resulted in two of the YTM rats having difficulty in completing the 1 h training duration and being outperformed by OTR rats. These findings highlight the importance of the relative exercise intensity (and not absolute work rate) in both acute exercise and training (similar to Gute et al. 1994, 1996). For example, training-induced improvements in CS and ß-HAD activities, and C/F ratio, in maximally trained 24-month-old rats exceeded (P < 0.05) those of YTM, despite undergoing the same training protocol. A potential caveat could be that while the absolute work rate for treadmill exercise is unknown here, it is likely that OTR rats, being heavier (P < 0.05), exercised at a slightly greater work rate than their speed-matched YTM partners, which might partly explain the disparity between responses. However, absolute speeds in YTM did not exceed those tolerated by young acutely exercised rats until the sixth week of training, and, as such, were only exposed to an intensity that was known to achieve a training response for 2–3 weeks. The lack of structural and biochemical adaptations for this short duration therefore may not be surprising, although Amaral et al. (2001) demonstrated significant increases in capillarity as early as 3 days after training onset. These data highlight the importance of intensity compared with work rate in the successful induction of training responses.

The findings regarding training intensity are in accordance with the acute-exercise angiogenic responses to the different exercise intensities employed by Breen et al. (1996). These authors demonstrated that in young rats, 1 h of running at 15 m min–1 resulted in small (< twofold) increases in VEGF mRNA, while running at 20 m min–1 induced >threefold increases. When training was continued, Gute et al. (1994) showed that low-intensity exercise (similar to that used here) resulted in a greater capillarization in more oxidative fibre regions of muscle, compared with high-intensity training, where the biggest changes were observed in glycolytic fibres. This intensity dependence of angiogenic regulation can reasonably be extended to biochemical adaptations and exercise training. Interestingly, and consistent with the present study, Olfert et al. (2001) demonstrated that training young rats at 15–18 m min–1 in hypoxia induced a significant increase in capillarity, whereas the same speeds in normoxia produced no effect. While hypoxia itself might be an important stimulus for angiogenesis, via HIF-mediated VEGF expression, hypoxia could also reasonably be viewed as a manipulation of relative exercise intensity, because maximal aerobic capacity and the LT and CP thresholds are dependent (at least in part) on inspired partial pressure of O2. As such, normoxic low-intensity training in the study of Olfert et al. (2001), and the YTM group of the present study, could be seen to be analogous and suboptimal, with YTR and hypoxic training (Olfert et al. 2001) eliciting an effective training intensity to induce adaptations.

Effects of age on angiogenic mRNA and protein expression responses to acute exercise

To the extent that C/F ratio is an important determinant of O2 conductance in intensely active muscle, the potential for exercise training to increase O2 conductance in skeletal muscle is crucially dependent on the pathways of capillary growth regulation (Breen et al. 1996; Lloyd et al. 2003). Transcription of bFGF, TGFß1, VEGF, and its receptors VEGF-R1 (Flt-1) and VEGF-R2 (Flk-1 or KDR), in skeletal muscle, has been shown to be upregulated by acute exercise (Breen et al. 1996; Olfert et al. 2001; Lloyd et al. 2003). The degree of this rise in angiogenic mRNAs is dependent on the exercise mode; electrical stimulation or exercise in hypoxia generating a greater increase than normoxic exercise of similar work rate and/or duration. Nevertheless, the modest ~2.5-fold increase in VEGF responses we observed in the entire gasctrocnemius muscle were in accordance with previous reports for 1 h exercise (e.g. ~3.5-fold from Breen et al. 1996; or ~threefold from Olfert et al. 2001). Importantly, however, this response was not affected by age, the increase being identical between 6- and 24-month-old rats. These findings support the suggestions of Jozsi et al. (2000) that the VEGF mRNA response to acute exercise in humans is unaffected by age. This, in turn, suggests that skeletal muscle may express a different interaction of regulatory mechanisms than other tissues, where ageing was found to impair vascular regulation (smooth muscle, Rivard et al. 2000; kidney, Kang et al. 2001; and subcutaneous vessel growth, Wang et al. 2004). VEGF protein increases concomitant to that of VEGF mRNA expression following acute exercise, however, were not evident in the present study. Amaral et al. (2001) have demonstrated that muscular VEGF protein is elevated ~twofold on the third day of aerobic exercise training, and that the ensuing increases in capillarity are attenuated by inhibition of VEGF actions. The timing of muscle sampling after acute exercise may be crucial in interpreting these results, however. Post-exercise VEGF responses are dependent on both state of training and time of sampling (Kraus et al. 2004). Also, training-induced increases in resting VEGF levels may be complete by 10–15 days after training onset (Lloyd et al. 2003). Therefore, the timing of muscle tissue removal (either 15 min following acute exercise, or at rest following 8 weeks of training) may have resulted in an inability to detect increases in VEGF protein following mRNA upregulation. Of course, we cannot distinguish between these confounding factors. The present study and results from other models (such as peripheral arterial insufficiency; Yang et al. 1994) show that the angiogenic response to aerobic exercise training in ageing animals was successful, in that ~5–10% increases in C/F ratio were manifest in the different portions of the gastrocnemius.

TGFß1 and VEGF-R2 were the only angiogenic variables measured to be affected by age. The TGFß1 response to exercise is known to be lower than that of VEGF (Breen et al. 1996). TGFß1 is an indirect angiogenic growth factor with a wide variety of effects on cell proliferation and regulation. The tyrosine kinase VEGF-R2 receptor is thought to play a central role in angiogenesis and vascular permeability (Gille et al. 2001), while VEGF-R1 may be a competitive regulator of VEGF action manifesting a higher affinity for the VEGF165 isoform than VEGF-R2 (Gille et al. 2001). While 24-month-old rats failed to increase TGFß1 and VEGF-R2 mRNA expression with acute exercise, this effect in 6-month-old rats was minor, with < twofold increase. Furthermore, the resting expression of both angiogenic mRNAs was greater in muscle in the OC group compared with the YC group. This, coupled with the similar increases in C/F ratio in both OTR and YTR groups, suggests that the age dependence of exercise-induced expression of TGFß1 and VEGF-R2 mRNAs was of little functional significance (for angiogenesis at least). Other molecules important in the stabilization of the capillary network, such as angiopoietin-1 and -2 and their receptor, were not measured here. While Lloyd et al. (2003) have highlighted the importance of this signalling pathway, an ischaemic exercise-training model may not be directly comparable to the present study in that exercise under femoral artery ligation would be expected to have a greater reliance on anaerobic metabolism in type II muscle fibres. Indeed, their data suggest a greater angiogenic response in the white gastrocnemius of trained rats compared with either the red gastrocnemius or soleus muscles. An endurance training protocol under ‘normal’ conditions has previously been demonstrated to result in greater increases in C/F ratio in the regions of muscle that were active during exercise (Gute et al. 1994, 1996), which would be expected to be predominantly oxidative muscle regions in the present study. While Lloyd et al. (2003) only report capillarity in the white gastrocnemius, the present study resulted in the greatest increases in capillarity in the mixed-fibre-type portion of the muscle. These discrepancies in relation to angiogenic gene activation and fibre-type-specific signalling pathways clearly require further study.

Limitations

Sex specificity.  We used only female Fischer 344 rats in the present study as they have been shown to better regulate body mass during endurance training than their male counterparts (Oscai et al. 1971; Mazzeo et al. 1984). However, we did not include males in the present study (unlike Coggan et al. 1992a, with human subjects), and we cannot therefore be certain that males would have behaved similarly. While circulating oestrogens have been implicated in vascular regulation (Schnaper et al. 1996), it is unlikely that there was a significant fall in circulating oestrogens by 24 months in our rats (although this was not measured). The possibility remains then, that angiogenic regulation may be different between the sexes during advancing age.

Age limitations.  The older rats in the present study were 24 months of age, which may not have been old enough to produce age-related declines in exercise capacity. However, ageing is known to result in declines in VO2max in rats by as early as 12 months (Mazzeo et al. 1984) – declines that are observed up to at least 35 months of age (e.g. Hepple et al. 2003; Olfert et al. 2004). In our study, it was clear that both maximal running speed and CS activity were lower in the untrained 24-month-old rats than in their younger counterparts. Thus, our untrained rats did show evidence of an age-related decline in exercise capacity. Linderman & Blough (2002) have suggested that because of lack of muscle atrophy, this strain of rat may not be ideal for studies of muscle ageing. However, here we found, in the absence of training, that gasctrocnemius mass as a percentage of body mass was ~23% lower in OC than in YC (P < 0.05), a difference consistent with muscle atrophy. Collectively, this evidence of age-related differences supports the use of this strain, sex and age for the present study.

Conclusion

In female Fischer 344 rats, muscular responses to endurance exercise training (1) were not reduced by ageing (up to 24 months), and (2) were dependent on relative and not absolute work rate. Trained 24-month-old female Fischer 344 rats increased both O2-delivery potential and O2-utilization potential, which not only exceeded those of their untrained young counterparts, but also were at least as great as for 6-month-old trained animals. The improvement in performance (running speed) was similar between the age groups, and while the old rats increased performance from a lower level, some achieved speeds equal to those of trained young rats. Also, OTR rats showed greater muscular structural and biochemical changes than YTM – young rats trained at the matched absolute speed as old. Age (up to at least 24 months) it seems is no barrier to increases in exercise tolerance and muscular structural, biochemical, angiogenic adaptations in female Fischer 344 rats, provided that the training intensity is sufficient.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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