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2-agonist treatment
Departments of 1 Exercise and Sport Sciences, 2 Pharmacology, and 3 Physiology, University of Florida, Gainesville, Florida 32611
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Aging is associated with a decrease
in diaphragmatic maximal tetanic force production (Po) in
senescent rats. Treatment with the 
2-agonist
clenbuterol (CB) has been shown to increase skeletal muscle mass and
Po in weak locomotor skeletal muscles from dystrophic rodents. It is unknown whether CB can increase diaphragmatic mass and
Po in senescent rats. Therefore, we tested the hypothesis that CB treatment will increase specific Po (i.e., force
per cross-sectional area) and mass in the diaphragm of old rats. Young
(5 mo) and old (23 mo) male Fischer 344 rats were randomly assigned to
one of the following groups (n = 10/group):
1) young CB treated; 2) young control;
3) old CB treated; and 4) old control. Animals were injected daily with either CB (2 mg/kg) or saline for 28 days. CB
increased (P < 0.05) the mass of the costal diaphragm in both young and old animals. CB treatment increased
diaphragmatic-specific Po in old animals (~15%;
P < 0.05) but did not alter (P > 0.05) diaphragmatic-specific Po in young animals.
Biochemical analysis indicated that the improved maximal specific
Po in the diaphragm of CB-treated old animals was not due
to increased myofibrillar protein concentration. Analysis of the myosin
heavy chain (MHC) content of the costal diaphragm revealed a CB-induced
increase (P < 0.05) in type IIb MHC and a decrease in
type I, IIa, and IIx MHC in both young and old animals. These data
support the hypothesis that CB treatment can restore the age-associated
decline in both diaphragmatic-specific Po and muscle mass.
sarcopenia; clenbuterol; myosin heavy chain
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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THE DIAPHRAGM IS THE MOST important inspiratory muscle in mammals and plays a critical role in the maintenance of adequate alveolar ventilation. Unfortunately, diaphragmatic force-generating capacity has been shown to decrease with age (3, 10, 15, 28, 36). This age-related reduction in diaphragmatic maximal tetanic force production (Po) could negatively impact ventilatory muscle performance in the elderly. Therefore, developing a countermeasure to restore senescent diaphragm contractile function is important.
Chronic treatment with 2-adrenergic receptor agonists,
such as clenbuterol (CB), has been shown to promote a slow-to-fast shift in skeletal muscle fiber type in young and adult animals. These
drugs have also been shown to increase locomotor muscle mass and total
muscle force production in both adult and old animals (5,
7-9, 12, 16, 19, 22, 25, 27, 37, 38, 40, 41). Recent
studies reveal that CB has little or no effect on the Po of
locomotor skeletal muscles in healthy adult animals (19).
Although the influence of CB to restore muscle force production and
power output in distrophic muscles has been investigated, the impact of
CB on distrophic skeletal muscle remains controversial. Specifically, a
recent study indicates that CB has no effect (20) on
dystrophic muscle, whereas another investigation has reported that the
drug is effective in restoring specific Po (force per cross-sectional area) in compromised muscles of dystrophic animals (16). Furthermore, a current study has also indicated that
CB treatment is effective in improving diaphragmatic function in diaphragms from emphysematous animals (37). To date, the
effect of treatment with 2-adrenergic receptor agonists
on the senescent diaphragm is unknown.
Because aging is associated with a decrease in diaphragmatic-specific Po, we wanted to investigate the effects of chronic CB treatment on contractile function of the senescent diaphragm. We tested the hypothesis that chronic CB treatment would increase muscle mass and reverse the deficit in specific Po in the senescent rat diaphragm. Furthermore, we theorized that the CB-induced increase in maximal diaphragmatic-specific Po would be associated with both an increase in myofibrillar protein concentration and a slow-to-fast shift in the myosin heavy chain (MHC) pool in the senescent diaphragm.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals and Experimental Protocol
Male pathogen-free Fischer 344 (F-344) rats, obtained from the National Institute on Aging colony (Harlem Sprague Dawley, Indianapolis, IN), were chosen for the experiment because of their short life span and the extensive data available on this strain in age-related research. The University of Florida Institutional Animal Care and Use Committee approved the use of animals in this study.Both young adult (5-mo-old) and old (23-mo-old) rats (age at time of
death) were studied. Animals in both age groups were randomly divided
into the following experimental groups (n = 10/group): 1) young adult control (YC); 2) young adult
treated with CB (YCB); 3) old control (OC); and
4) old treated with CB (OCB). The animals were individually
housed and fed food and water ad libitum. Animals were injected daily
(intraperitoneal) for 28 consecutive days, with either CB (2 mg · kg
1 · day1) or an
equivalent volume of saline. Animal weights were measured and recorded
daily. To reduce the possibility of acute inotropic effects of CB on
the diaphragm, contractile measurements were obtained 48 h after
the last treatment.
Muscle Preparation
Animals were anesthetized by an intraperitoneal injection of pentobarbital sodium (90 mg/kg). After the surgical plane of anesthesia was reached, the entire diaphragm was excised and placed in a dissecting chamber of Krebs-Henseleit solution equilibrated with 95% O2-5% CO2 gas. A muscle strip from each diaphragm was dissected from the left ventral costal diaphragm region, with part of the rib and central tendon attached. The remaining costal and crural diaphragm portions were carefully trimmed of fat and tendinous attachments, blotted dry, and weighed. Segments of the costal diaphragm were then rapidly frozen in liquid nitrogen and stored at
80°C until biochemical analyses were performed.
To determine the effects of CB on locomotor muscle mass, several locomotor muscles were removed and weighed from all experimental groups. Specifically, the soleus, tibialis anterior, plantaris, digitorum longus, and gastrocnemius muscles were dissected and weighed.
The diaphragm strips used for the in vitro contractile measurements were vertically suspended between two lightweight, Plexiglas clamps in a jacketed tissue bath containing Krebs-Henseleit solution. The bath was equilibrated with 95% O2-5% CO2 gas and maintained at 37°C, pH 7.4, and osmolality of ~290 mosmol/kgH2O.
Measurements of Diaphragmatic Contractile Properties
The in vitro contractile measurements began with determination of the optimal length (Lo) of the diaphragm strip for isometric tension development. After a 15-min equilibration in the bath, the muscle strip was connected to an isotonic force transducer (model 300B, Cambridge Instruments, Aurora) and was stimulated along its entire length with platinum wire electrodes. We have previously demonstrated that this method of stimulation results in optimal muscle stimulation compared with direct stimulation using stainless steel electrodes (10). Lo was determined systematically from a series of tetanic contractions at a frequency of 100 Hz and train duration of 1,000 ms using a modified Grass Instruments S48 stimulator. After Lo was found, all subsequent contractile properties were measured at Lo.Peak isometric tetanic tension. Maximal tetanic tension was determined by an isometric contraction induced by supramaximal stimulation (~150% of maximum) with a train duration of 1,000 ms at 150 Hz. Peak tetanic tension was determined from three stimulations separated by a recovery interval of 3 min each.
Force-frequency relationship. Maximal isometric tetanic tension was determined at varying frequencies of stimulation. Stimulation frequency was progressively increased from 10, 20, 30, 40, 60, 80, 100, to 150 Hz (500-ms train duration); each contraction was interspersed with 2 min of recovery.
Fatigue tolerance. Before the fatigue protocol, a maximal tetanic contraction was performed to assess the integrity of the muscle strip. Any diaphragm strip that exhibited >15% reduction in maximal Po, in reference to the first maximum isometric force measurement obtained after Lo was established, was eliminated from this protocol. To test fatigue resistance, the strip was stimulated submaximally at 2-s intervals (30 Hz; 250-ms train duration) for 20 min. Tolerance to fatigue was assessed by the percentage of initial force generated by the strip compared with the tension developed at the end of the 20-min protocol.
After completion of the contractile measurements, diaphragm muscle strips were subsequently weighed, and Lo was measured. Muscle cross-sectional area was determined using the method described by Metzger and Fitts (25), and this calculation was used to obtain an estimation of diaphragmatic-specific force production.Determination of Citrate Synthase Activity
To determine citrate synthase (CS) activity, muscle samples were homogenized in cold 100 mM phosphate buffer with 0.05% bovine serum albumin (1:20 wt/vol, pH 7.4). Homogenates were then centrifuged at 4°C for 10 min at 400 g. The resulting supernatant was decanted and assayed for enzyme activity and protein content. CS activity was measured at 25°C using the technique described by Srere (32), whereas protein content was determined by using techniques described by Watters (39).Myofibrillar Protein Isolation
Muscle samples for biochemical analysis were taken from the ventral costal sections of the diaphragm proximal to the strip used for contractile measurements. Myofibrillar protein was isolated by a modified version of the technique used by Solaro et al. (31). In brief, minced muscle portions were homogenized in 4 ml of sucrose buffer (250 mM sucrose, 100 mM KCl, 5 mM EDTA, and 20 mM Tris, pH 6.8). Total protein concentration (mg/g) was determined from the homogenate by using the biuret technique described by Watters (39). The remaining homogenate was centrifuged for 10 min at 2,500 g. The supernatant was discarded, and the pellet was suspended in a KCl buffer with Triton X-100 (175 mM KCl, 0.5% Triton X-100, and 20 mM Tris, pH 6.8) and then centrifuged for 10 min at 2,500 g. A pellet was again obtained, resuspended, and centrifuged using the aforementioned process (4). The biuret technique was performed to measure the insoluble protein portion from the third pellet. Insoluble protein concentration (mg/g) was used as an estimate of myofibrillar protein concentration (mg/g).MHC Concentration Measurements
MHC isoforms were separated electrophoretically according to the method described by Suigiura et al. (34). Briefly, MHCs were separated in 5-8% SDS-polyacrylamide gradient slab gel (7 cm × 9 cm × 1 mm). A 10-µl portion of denatured sample homogenate (~5 µg of myofibrillar protein), diluted with SDS sample buffer containing 30% (vol/vol) glycerol, 5% (vol/vol)-mercaptoethanol, 2.3% (wt/vol) SDS, 62.5 mM Tris · HCl (pH
6.8), and 0.05% (wt/vol) bromophenol blue, was applied to the gel.
Electrophoresis began at 50 V until the tracking dye completely entered
the separating gel; the voltage was then increased to 150 V for ~18 h
at 8°C. The gels were then stained with a solution containing 0.1%
Coomassie brilliant blue and destained by diffusion in a
methanol-glacial acetic acid solution. To identify MHC bands, standard
myosin and molecular weight markers (Sigma Chemical, St. Louis, MO)
along a sample of protein from a soleus muscle and extensor digitorium longus muscle were loaded to different cells throughout the gel. On the
basis of their migration pattern on SDS-PAGE, four different MHC bands,
corresponding to the ~205-kDa myosin standard, were identified as MHC
I, MHC IIb, MHC IIx, and MHC IIa. The relative proportions of MHC
isoforms were estimated densitometrically by a scanning technique using
a computerized image analysis system.
Total Muscle Water Content
Frozen costal diaphragm samples were analyzed for total water content using a freeze-drying technique. Samples were placed in a vacuum chamber with a pump generating a negative pressure of ~1 mmHg. The samples were dried in the chamber for 48 h, and weights were recorded for each. The samples were then placed in the chamber for another 4 h and reweighed to ensure that no change in weight had occurred, indicating that drying was successful. In all cases, muscle weights were unchanged after this additional 4-h drying period. Water content was determined by subtracting the original weight from the weight after the freeze-drying protocol and normalizing this mass to muscle wet weight.Data Analysis
Data were statistically analyzed using a 2 × 2 ANOVA (age × CB treatment). When appropriate, Tukey's test was used for a post hoc analysis where differences occurred. Significance for all data was established at P < 0.05.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Food Intake and Body Weight Changes
In response to CB treatment, both young and old animals displayed an initial period of anorexia, which paralleled a decrease in body mass. This was anticipated based on previous work that observed CB's influence on the2-receptors of the hypothalamus, causing suppression of appetite (2). In contrast, the
control animals exhibited no aberrant change in food intake or body
weight after the saline injections. Food intake returned to
preexperiment levels in the CB-treated animals by day 5 of
the injections; thereafter, animal body weights (both age groups) began
a paralleled increase. Note, at the completion of the CB treatment,
animal body weights were not significantly altered by CB treatment
(Fig. 1).
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Muscle Mass
CB resulted in significant increases in the muscle mass of the rat hindlimb muscles in both the young and old animals (P < 0.01; Table 1). These differences existed both when absolute muscle mass was compared and by expressing muscle mass as a percentage of body weight. The mass of the costal diaphragm was significantly increased after CB treatment in both the young and old animals (P < 0.01; Table 1). This increase in muscle mass without an increase in body mass in CB-treated animals was likely due to the combination of fat loss and increased lean mass associated with chronic CB treatment (6, 7, 27, 29, 32).
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Finally, note that, compared with young animals, aging did not result in significant atrophy in the diaphragm and five of the six locomotor muscles studied in these experiments (Table 1). A potential explanation for this observation is that, at the time of death, our old animals were 23 mo old. Hence, it seems likely that this age may precede the point on the aging continuum for the F-344 rat at which skeletal muscles experience significant sarcopenia.
Contractile Measurements
Lo ranged from 21 to 23 mm in all diaphragm strips, and the mean Lo did not differ across the four experimental groups. Compared with YC animals, both submaximal (i.e., 100 Hz) and maximal diaphragmatic Po were significantly reduced in the OC animals (Figs. 2 and 3). Treatment with CB restored both submaximal and maximum diaphragmatic Po in the old animals to values comparable to those of the YC animals. It is noteworthy that CB did not alter (P > 0.05) maximal diaphragmatic-specific Po in the young animals (Fig. 2).
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With the use of percentage of initial force as the basis for
comparison, there was a marked reduction in diaphragmatic fatigue resistance in the OCB animals compared with all other groups in the
study (P < 0.05; Fig.
4). In contrast, CB did not significantly alter diaphragmatic fatigue resistance in the young animals.
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Diaphragmatic CS Activity
Compared with young animals, diaphragmatic CS activity was significantly lower (P < 0.05) in old animals. CB treatment decreased CS activity in the young animals and further decreased the activity in the already aerobically inferior senescent diaphragm (P < 0.01; Fig. 5).
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Muscle Protein and Water Content
No differences existed (P > 0.05) among experimental groups in percentage of total muscle water content (data not shown). This suggests that the increases in muscle mass are likely due to an increase in total muscle protein and not water. This observation was consistent with protein concentration measurements, which revealed no differences among groups in insoluble or soluble protein concentrations (mg/g) (P > 0.05; Fig. 6). Therefore, compared with control, CB treatment resulted in an increase (P < 0.05) in total diaphragmatic protein content in both young and old animals (P < 0.05) (Fig. 6).
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Diaphragmatic MHC Analysis
Figure 7 illustrates the separation of MHC isoforms using gel electrophoresis. Electrophoretic measurements of diaphragm MHC concentration indicated that CB treatment resulted in a slow-to-fast shift in MHC expression in both young and old animals (Table 2). Specifically, note that CB treatment resulted in large increases in type IIb MHC accretion in the costal diaphragm (Table 2 and Fig. 7).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Overview of Major Findings
This is the first experiment to examine the effects of the2-agonist CB on the contractile properties, muscle mass,
and biochemistry of the senescent diaphragm. Our results indicate that,
after 28 days of CB treatment, diaphragm weakness associated with aging (i.e., specific Po decrement) was restored to values
consistent with young adult animals. The increased diaphragmatic
force-generating capacity was accompanied by an increase in diaphragm
mass and total protein content. Furthermore, CB treatment resulted in
an increase in the relative content of type IIb MHC in the diaphragm. Finally, CB treatment in the old animals resulted in a decreased resistance to diaphragmatic fatigue. A brief discussion of these major
points follows.
Aging, CB, and Diaphragmatic-specific Po
The mechanism responsible for the age-related decrease in skeletal muscle-specific Po continues to be investigated. Theoretically, an age-related decrease in specific force production of individual muscle fibers could be due to one of several factors, including impaired excitation-contraction coupling, reduced force production per myosin cross bridge, a reduction in myofibrillar protein concentration, and/or a reduced number of myosin cross bridges in the strong binding state (10, 11, 18, 24, 30). Of these potential mechanisms, experiments measuring force production in skinned single-muscle fibers from both young and old animals indicate that impairment in excitation-contraction coupling is not responsible for age-related skeletal muscle weakness (35). Furthermore, to date, there are no published reports indicating that age-associated muscle weakness is due to reduced force production per myosin cross bridge. In contrast, evidence exists that both a reduction in myofibrillar protein concentration and a reduced number of myosin cross bridges in the strong binding state are contributory factors to age-related changes in muscle force production. For example, previous experiments have shown a loss of myofibrillar protein and an increase in intermyofibrillar spaces in very old animals (1, 10). Also, a recent experiment using electron paramagnetic resonance reveals age-related structural changes in rat muscle fibers, leading to a reduced number of myosin cross bridges in the strong binding state (18). These experiments provide a molecular explanation for the age-related decline in muscle force production in muscles in which myofibrillar protein concentration is not reduced.In regard to the role that these specific mechanisms play in
age-related muscle weakness, Thompson and Brown (35) have
suggested that age-associated muscle contractile dysfunction is time
dependent and may be due to different mechanisms, depending on the age
of the animal. Therefore, it is possible that one or both of the aforementioned factors could be responsible for losses in
Po in a time-dependent manner. For example, at the onset of
senescence, a reduced number of myosin cross bridges in the strong
binding state may be the primary factor contributing to the attenuation in force production, and, as the animal ages, other mechanisms (i.e.,
reduction in myofibrillar protein concentration) may be present to
further reduce skeletal muscle-specific Po. Regardless of
the mechanism responsible for the age-related decrease in
diaphragmatic-specific Po, the observation that treatment
with CB eliminated the age-related diaphragmatic force deficit
indicates that the abnormality responsible for the contractile
dysfunction was corrected by treatment with this
2-agonist.
In the present experiments, we hypothesized that CB treatment would improve diaphragmatic-specific Po in old animals. This postulate was based on reports indicating that this drug is effective in restoring Po in compromised muscles from dystrophic or emphysematous animals (16, 37). Our results clearly support this hypothesis. We also postulated that, if CB improved diaphragmatic-specific Po in old animals, the increase in diaphragmatic force production would be accompanied by an increase in myofibrillar protein concentration. This hypothesis evolved from previous work in our laboratory that indicated that the reduction in diaphragmatic-specific Po in very old animals (26-mo-old F-344 rats) is due, at least in part, to an age-associated decrease in myofibrillar protein concentration (10). However, in the present study with 23-mo-old rats, muscle water content and myofibrillar protein concentration were unaltered by age or CB treatment. These results do not support our hypothesis that CB will promote an increase in myofibrillar protein concentration and indicate that a reduction in myofibrillar protein concentration was not responsible for differences in diaphragmatic-specific Po between young and old animals. An obvious difference between the present investigation and our laboratory's previous study is the age differences of the rats. The present study investigated 23-mo-old F-344 rats, whereas our previous study investigated very old F-344 animals (i.e., 26 mo old), which would be considered old-age survivors. It seems possible that an age-related threshold exists, whereby myofibrillar protein concentration is not reduced until a critical level of senescence is reached. This notion is supported by previous research (1).
Whereas it is clear that CB treatment elevated maximal diaphragmatic-specific Po at high-stimulation frequencies (i.e., 150 Hz) in the old animals, the physiological significance of this observation is unknown. Indeed, because skeletal muscles are rarely maximally activated in vivo, it is unclear whether a CB-induced increase in maximal specific Po would translate into improved muscle function in senescent animals. Additional experiments using in vivo methodologies to investigate diaphragmatic function will be required to determine whether CB treatment translates into improved ventilatory function in senescent animals.
CB-induced Alterations in Diaphragmatic Fiber Type
It is known that chronic CB treatment results in an increase in the number of fast muscle fibers in locomotor skeletal muscle (9). It contrast, to date, there are no published reports regarding the effects of CB on the myosin phenotype in the diaphragm. Therefore, we determined the effects of CB treatment on diaphragmatic MHC content via gel electrophoresis. Our results revealed a significant decrease in the relative content of type I, IIa, and IIx MHC and a large relative increase in type IIb MHC in the diaphragm of both young and old animals (Table 2).There are at least two mechanisms that could explain the increased expression of type IIb MHC in the diaphragm of CB-treated animals. First, CB could activate satellite cells to differentiate into new muscle fibers that express type IIb MHC. A second possibility is that CB promoted a slow-to-fast transition of MHC isoforms (e.g., I > IIa > IIx > IIb MHC) within existing muscle fibers. Although the present study cannot determine which of these two mechanisms is responsible for the slow-to-fast fiber shift, work by Maltin et al. (23) indicates that CB does not increase the number of total muscle fibers. Furthermore, Maltin and Delday (22) report that CB does result in satellite cell division or differentiation in the soleus muscles of CB-treated rats. Collectively, these experiments suggest that the CB-induced changes in skeletal muscle fiber type occur by a fiber-type transition rather than the formation of new muscle fibers. Other investigators have reached similar conclusions (9).
Of further interest is the possibility that the CB-induced changes in myosin phenotype could impact the contractile performance of the diaphragm. Although conflicting reports in the literature exist, a recent well-designed, single-fiber experiment lends convincing evidence that faster fibers (i.e., types IIb and IIx) in the rat diaphragm possess a greater specific Po than do slower fibers (i.e., types I and IIa) (14). Again, our results reveal that CB treatment results in a significant shift toward a greater percentage of the IIb isoform at the expense of IIx, IIa, and type I MHC fibers in diaphragms of both young and old animals. Nonetheless, despite the increased number of IIb fibers in both young and old animals, it seems unlikely that the shift in the MHC isoform played an important role in the CB-induced improvement of diaphragmatic Po in the old animals. If the slow-to-fast change in diaphragmatic fiber type were primarily responsible for the increase in diaphragmatic-specific Po in the old animals, it would be predicted that a similar increase in diaphragmatic-specific Po would have occurred in the YCB animals. However, this was not the case. Therefore, it seems unlikely that the observed change in myosin phenotype was the dominant factor contributing to the CB-mediated improvement in diaphragmatic Po in the old animals.
CB and Diaphragmatic Fatigue
CB treatment in old animals decreased diaphragmatic fatigue tolerance expressed as a percentage of initial force production. Consistent with previous findings, CB treatment in the present experiments did not alter diaphragmatic endurance in young adult animals (37). The mechanism(s) responsible for the differential impact of CB on diaphragmatic fatigue in young and old animals is unclear. In this regard, although the difference in diaphragmatic oxidative capacity between OCB and YCB animals is noteworthy (Fig. 5), it is not a decisive explanation for the reduced fatigue resistance observed in the OCB animals. The role of oxidative capacity as a potential mechanism is complicated by the lack of differences in fatigue resistance between the YCB and YC animals (i.e., groups that differed in CS activity). Furthermore, CB-induced alterations in fiber type did not substantially contribute to differences in fatigue performance in these experiments, because treatment with CB resulted in similar slow-to-fast shifts in diaphragmatic MHC phenotype in both young and old animals (Table 2).Regardless of the mechanism to explain the age-related difference in
diaphragmatic response to chronic CB treatment, the decreased tolerance
to fatigue in the OCB animals may serve as a contraindication when
2-agonists are considered as a potential treatment for
senescent muscle weakness. However, the possibility exists that, after
CB treatment in old animals, the senescent diaphragm with increased muscle mass and specific Po may perform some ventilatory
tasks better than a senescent diaphragm suffering from impaired
Po. Additional research is needed to evaluate the effects
of chronic CB treatment on diaphragmatic performance in vivo.
Conclusions and Recommendations for Further Study
Our results reveal that, despite sustained contractile activity throughout the normal life span, the senescent diaphragm is subject to a reduction in maximal force-generating capacity. Importantly, these experiments demonstrate that chronic treatment with a2-agonist can restore diaphragmatic-specific
Po in old animals to a level that is comparable to that of
young adult animals. The molecular mechanism to explain the improved
diaphragmatic-specific Po is unclear but may be related to
changes in myosin structure that result in an increased number of
myosin cross bridges in the strong binding state. This is a testable
hypothesis and warrants further research.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute on Aging Grant R03 AG-14779 (to S. K. Powers).
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. K. Powers, PO Box 118206, Center for Exercise Science, Univ. of Florida, Gainesville, FL 32611 (E-mail: spowers{at}hhp.ufl.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/japplphysiol.00576.2001
Received 6 June 2001; accepted in final form 2 November 2001.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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