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1 Division of
Pulmonary/Critical Care Medicine, The aim of this study was to evaluate the
potential mechanisms underlying the improved contractility of the
diaphragm (Dia) in adult intact male hamsters after nandrolone (Nan)
administration, given subcutaneously over 4 wk via a controlled-release
capsule (initial dose: 4.5 mg · kg
anabolic-androgenic steroids; respiratory muscles; muscle specific
force; muscle velocity of shortening; muscle fiber size; calcium
activated-myosin adenosinetriphosphatase; myosin heavy chains
RECENT STUDIES in both animals (5, 22, 23, 25) and in
humans (4, 26) have demonstrated a distinct impact of anabolic steroids
on respiratory and/or limb muscle structure and
function. In preliminary studies from our laboratory, we
reported improved isometric (10, 36) and isotonic (36) contractile function of the male hamster diaphragm after prolonged administration of anabolic steroids. There is, however, a paucity of literature in
which comprehensive studies on diaphragm contractility have been
evaluated after the use of anabolic steroids. Prezant and co-workers
(23) reported an increase in diaphragm specific force [i.e.,
force per unit cross-sectional area (CSA)] in female rats treated
with testosterone propionate for 2.5 wk. However, no effects on
diaphragm specific forces were reported by either Prezant et al. (22,
23) or Bisschop and colleagues (5) after the administration of anabolic
steroids to intact (i.e., noncastrated) male rats. With regard to limb
muscles, increased specific force of the tibialis anterior muscle was
noted in nandrolone (Nan)-treated rabbits by Salmons (25). Review of
the literature reveals no study in which velocity of shortening of the
diaphragm has been reported in animals receiving anabolic steroids.
The aim of this study was to evaluate the potential mechanisms
underlying the improved contractility of the diaphragm in intact male
hamsters after the prolonged administration of Nan, a synthetic anabolic-androgenic steroid agent. We thus evaluated, in addition to
isometric and isotonic diaphragm contractile function, the relative
contribution of diaphragm fiber types to total area of the costal
diaphragm, the relative interstitial space between muscle fibers, the
proportions of myosin heavy chain (MHC) isoforms, and a quantitative
analysis of Ca2+-activated myosin
ATPase activity within individual muscle fibers.
Animal groups.
Adult male Golden Syrian hamsters with an initial body weight of ~95
g were studied. The animals were divided into two groups: 1) control (Ctl;
n = 8) and
2) Nan treated
(n = 8). In our initial experimental
design, we planned to include an additional pair-weight control group,
because our prior experiments with testosterone resulted in significant
body weight loss in those animals (10). This is a major
factor to control for in the analysis of specific diaphragm fiber size.
As it became evident, however, that the Nan group did not lose weight,
and thus dietary manipulation was unnecessary in the
"pair-weight" controls, this group was dropped. All animals were
housed in individual cages with the ambient temperature in the vivarium
maintained at 22°C and the light cycle fixed at 12 h on and 12 h
off. Water and food (Purina rodent chow: 56% carbohydrate, 23%
protein, 4.5% fat, 6% fiber, and 10.5% ash minerals) were provided
ad libitum to all animals.
Anabolic-androgenic steroid administration.
Nan in pure powder form (Steraloids, Wilton, NH) was administered by
using a controlled-release Silastic capsule implanted subcutaneously
while the animals were anesthetized (100 mg/kg ketamine and 10 mg/kg
xylazine ip). In Ctl animals, a blank Silastic capsule was implanted.
By using this system (32), the major factor accounting for the delivery
rate of the steroid is the inner surface area of the tube. Because
circumference is constant, tube length can thus be modified to adjust
serum levels of the steroid. The size of the capsule (and the dose
equivalent of Nan) was determined from prior experiments by using
testosterone, in which serum levels six to seven times normal were
achieved (10). (Currently, no quantitative assay for serum Nan is
available for the hamster.) In our experience, when the Silastic
capsules were primed with testosterone, steady-state serum levels were
achieved by 48 h. Although the release of steroids from the Silastic
capsule is constant, increments in body weight will alter the relative dosages administered when normalized for body weight. Thus the initial
dose of Nan was 4.5 mg · kg In vitro isometric contractile properties.
The methods for determining isometric contractile properties of the
diaphragm in vitro have been previously described (19, 27). Briefly, in
anesthetized animals (6 mg/100 g body wt pentobarbital sodium), the
entire diaphragm was rapidly excised. A narrow (3-4 mm wide) strip
of diaphragm was dissected from the right midcostal region while fiber
attachments to the ribs and central tendon were maintained intact. The
muscle strip was vertically mounted in a tissue bath containing
Krebs-Henseleit solution, which was maintained at a temperature of
26°C and constantly aerated with 95%
O2-5%
CO2. The costal margin clamp was
attached to a calibrated force transducer (model FT10, Grass, Quincy,
MA) and the central tendon clamp to a micromanipulator (Kopf, Topanga,
CA). The diaphragm strip was directly stimulated, by using 2-ms
monophasic impulses at supramaximal intensity (model S88 stimulator,
Grass). d-Tubocurare (12 µM) was
added to the tissue bath to block neuromuscular transmission. Muscle
length was adjusted until maximum isometric twitch force responses were
obtained. Isometric contractile properties were determined at this
optimal length
(Lo), which was
subsequently measured by using a digital caliper accurate to 1 µm (Mitutoyo).
ABSTRACT
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INTRODUCTION
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DISCUSSION
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1 · day1;
with weight gain, final dose: 2.7 mg · kg1 · day1).
Control (Ctl) animals received blank capsules. Isometric contractile properties of the Dia were determined in vitro after 4 wk. The maximum
velocity of unloaded shortening
(Vo) was
determined in vitro by means of the slack test. Dia fibers were
classified histochemically on the basis of myofibrillar ATPase staining
and fiber cross-sectional area (CSA), and the relative interstitial
space was quantitated. Ca2+-activated myosin ATPase
activity was determined by quantitative histochemistry in individual
diaphragm fibers. Myosin heavy chain (MHC) isoforms were identified
electrophoretically, and their proportions were determined by using
scanning densitometry. Peak twitch and tetanic forces, as well as
Vo, were
significantly greater in Nan animals compared with Ctl. The proportion
of type IIa Dia fibers was significantly increased in Nan animals. Nan
increased the CSA of all fiber types (26-47%), whereas the
relative interstitial space decreased. The relative contribution of
fiber types to total costal Dia area was preserved between the groups.
Proportions of MHC isoforms were similar between the groups. There was
a tendency for increased expression of
MHC2B with Nan.
Ca2+-activated myosin ATPase
activity was increased 35-39% in all fiber types in Nan animals.
We conclude that, after Nan administration, the increase in Dia
specific force results from the relatively greater Dia CSA occupied by
hypertrophied muscle fibers, whereas the increased ATPase activity
promotes a higher rate of cross-bridge turnover and thus increased
Vo. We speculate
that Nan in supraphysiological doses have the potential to offset or
ameliorate conditions associated with enhanced proteolysis and
disordered protein turnover.
INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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METHODS
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DISCUSSION
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1 · day1.
With progressive body weight gain over the 4-wk experimental period,
however, the dose of Nan delivered at the conclusion of treatment was
therefore relatively decreased to 2.7 mg · kg1 · day1.
In vitro maximum velocity of unloaded shortening (Vo). The protocol to determine the Vo was similar to that originally described for its measurement by using the slack test by Edman (12). All experiments were performed on fresh costal diaphragm strips at 21°C. For these studies, a computer-controlled ergometer (model 300B, Cambridge Technology, Watertown, MA) linked to a Keithley MetraByte/Asyst (Taunton, MA) DAS-1602 I/O interface board with customized data-acquisition and signal-analysis software (ISR's MUSCLE, Integrated Scientific Resources, Santa Monica, CA), designed to control muscle force and length as well as pulse train, was used. Data sampling was at 2 kHz. The muscle strip was maximally stimulated for 600 ms at 75 pps and allowed to reach its isometric plateau. Then, changes in length steps of known distances (8-15% of Lo) were rapidly made, with a resultant drop in the force to zero. The duration of unloaded shortening for each quick release is a measure of the time interval between the release and the beginning of force redevelopment. The stimuli and length steps were presented with 1-min intervals between each train. The slope of the line describing this distance-time relationship defines the muscle Vo.
Histochemical procedures: diaphragm fiber type proportions and CSA.
After completion of the physiological studies, the muscle segments
(midcostal region), adjacent separate strips, and the remaining portion
of the costal diaphragm were weighed, and the fresh adjacent strips
were stretched to
Lo (as determined
for the stimulated strip), mounted on cork, and then rapidly frozen in
isopentane (which had been cooled to its melting point by liquid
nitrogen). Serial cross sections of the unstimulated diaphragm segments
were cut at 10-µm thickness by using a cryostat (model 2800E,
Reichert-Jung) kept at 
20°C.
Histochemical procedures: diaphragm fiber Ca2+-activated myosin ATPase activity. Our detailed methodological procedures have been previously published (7). These methods ensure specificity for Ca2+-activated myosin ATPase by excluding the contributions of mitochondrial and sarcoplasmic reticulum ATPases as well as the role of other divalent cations (7). Briefly, tissue serial sections (6-µm thickness) were reacted for Ca2+-activated myosin ATPase activity with or without ATP (substrate). With hydrolysis of ATP, Pi ions are liberated and reacted with a lead ammonium citrate-acetate complex to form a precipitate of lead phosphate in the tissue. The latter is converted to a brown-colored lead sulfide precipitate by a reaction with sodium sulfide (peak absorbance wavelength of 550 nm). The Beer-Lambert equation was used to measure the concentration of lead sulfide (molar extinction coefficient = 1,450 mol/cm for lead sulfide). On the basis of our prior studies (7), the linearity of the reaction was verified up to 9 min. We chose a single end point of 4 min to determine the maximum velocity (Vmax) of Ca2+-activated myosin ATPase reaction in duplicate sections by using varying ATP concentrations in the incubation media (0.0, 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mM ATP). Alternate sections were incubated without substrate, and density was subtracted from paired sections containing substrate. The Vmax and the apparent Michaelis-Menten rate constant (kapp) of the ATPase reaction were derived by a Lineweaver-Burke transformation of the data relating velocity of the ATPase reaction (optical density measured at 570 nm/min) and ATP concentration. The slope of the relationship is equal to kapp/Vmax, whereas the y-intercept is equal to 1/Vmax. The Vmax of the ATPase activity was expressed as millimoles of Pi per liter of tissue per minute, on the basis of the stoichiometric relationships of the Pi-sulfide exchange in the reaction. At least 200 diaphragm fibers (of all types) were sampled for the determination of the ATPase activity.
Electrophoretic identification of MHC isoforms. For the myofibril extraction (28), 10-mg muscle samples were homogenized by hand on ice in 20 vol of cold buffer containing 250 mM sucrose, 100 mM KCl, 20 mM Tris, and 5 mM EDTA, pH, 6.8. The homogenate was centrifuged at 1,000 g at 4°C for 10 min. The supernatant was discarded, and the pellet was resuspended in 20 vol of a washing buffer containing 175 mM KCl, 2 mM EDTA, 20 mM Tris, and 0.5% Triton X-100, pH at 6.8. After centrifugation, the final pellet was resuspended in 10 vol of a buffer containing 150 mM KCl and 20 mM Tris, pH at 7.0. Myofibril content was determined by using a micro-bicinchoninic acid protein assay kit (Pierce) and quantified by using an ELISA reader at 550 nm. Samples of purified myofibrils were diluted 1:8 in a denaturing sample buffer where the final concentration was 1.5% dithiotreitol, 2% SDS, 10% glycerol, 0.012% bromophenol blue, and 80 mM Tris (pH 6.8). Final myofibrillar protein concentration was ~0.125 µg/µl. Proteins were denatured in the same buffer by boiling samples for 2 min before loading.
The determination of the MHC composition was performed by using a SDS-PAGE separation technique (29). The separating gels were composed of 30% glycerol, 8% acrylamide:bis (50:1), 0.2 M Tris (pH 8.8), 0.1 M glycine, and 0.4% SDS. The stacking gels were composed of 30% glycerol, 4% acrylamide:bis (50:1), 70 mM Tris (pH 6.7), 4 mM EDTA, and 0.4% SDS. Polymerization of the gels was achieved with 0.1% ammonium persulfate and 0.05% TEMED. Each well was loaded with 0.7 to 0.9 µg of protein extract. Electrophoresis was performed by using a Bio-Rad Mini-Protean II system with a power supply for a duration of 25 h at constant 80 V with running buffers kept at 4-7°C by placing the gel unit in a box containing ice and/or cold packs. The upper buffer was composed of 0.1 M Tris, 150 mM glycine, and 0.1% SDS. The lower buffer contained 50 mM Tris, 75 mM glycine, and 0.05% SDS. The separating gels were stained with silver nitrate (Bio-Rad Silver Stain Plus kit). Dried stained gels with duplicate samples were scanned twice by using an Ultra-Violet Products Image Store 5000 System, and densitometric measurements were performed with its Gel Documentation and Software System. After background subtraction, the relative contribution of each band within a gel was determined by the ratio of the total gray level within the area of a specific band to that of the cumulative gray level of all the bands present in a sample. The specificity of each band has been demonstrated based on immunoblotting identification after electrophoretic transfer (14). This SDS-PAGE method allows the clear separation of MHC isoforms from denatured myofibrils of skeletal muscle. In adult hamster muscle, the fastest migrating band corresponds to MHC1 (
/slow), followed in order by
MHC2B,
MHC2X, and
MHC2A. This migration pattern is
identical to that demonstrated in skeletal muscle of other rodent
species (e.g., rat, mouse, guinea pig). The densitometric analysis of
each migrated band corresponding to the identified MHC isoforms was
performed in duplicate on two samples for each muscle, and the average
relative content of each MHC isoform was estimated.
Statistical analysis. Statistical analysis was performed by using a one-way ANOVA with the experimental factor being the administration of Nan. In comparing the force-frequency curves, ANOVA with repeated measures was used to compare differences in independent groups. An alpha level of 0.05 was used to compare differences in independent groups and to determine overall significance. All data are presented as means ± SD.
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Body and muscle weights.
The initial mean body weight of the animals was 94 ± 3 g. During
the experimental period, both the Nan and Ctl groups exhibited significant body weight gain. The final body weights and percent increment in body weights were similar between the groups, with the
mean for both groups being 68.4 ± 10.1% (Fig.
1A).
The weights of the costal diaphragm, medial gastrocnemius, extensor
digitorum longus, and soleus muscles were also similar between the
groups (Fig. 1B).
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In vitro diaphragm contractile properties.
The Lo of the
diaphragm was similar between Ctl and Nan animals (Table
1). Assessment of twitch characteristics
indicated significant prolongation of CT in Nan animals compared with
the Ctl group (P < 0.05), whereas
RT1/2 was similar between the
groups (Table 1). Pt and
Po were both significantly
increased in the Nan group compared with Ctl animals
(P < 0.05; Table
1). Figure 2
depicts the force-frequency relationships of the costal diaphragm expressed in absolute values (Fig.
2A) and normalized to
Po (Fig. 2B). Consistently greater specific
forces were observed in the force-frequency curve of the Nan group
across all frequencies from 5 to 100 pps
(P < 0.05), whereas normalized
forces were comparable between the groups.
Vo was normalized
for costal diaphragm
Lo and expressed
as Lo per second.
In Nan animals, the
Vo of the diaphragm was significantly greater than the Ctl group
(P < 0.05; Fig.
3).
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Diaphragm fiber type proportions and CSA.
Evaluation of fiber type proportions of the costal diaphragm revealed a
significant increase in the proportions of type IIa fibers in Nan
animals compared with Ctl (P < 0.05;
Fig. 4). The mean CSAs of costal diaphragm
fibers are depicted in Fig. 5. A significant increase in the CSA of types I, IIa, and IIx diaphragm fibers was observed in the Nan animals compared with Ctl animals (P < 0.01). The relative
contribution of specific fiber types to total costal diaphragm area
(computed from fiber proportions and CSA) remained unchanged in the Nan
animals compared with Ctl. Assessment of the relative proportions of
interstitial space and muscle area in the costal diaphragm revealed
that in Nan animals, compared with Ctl animals, cumulative muscle fiber
area increased and the relative interstitial space decreased
significantly (P < 0.05; Fig.
6).
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Ca2+-activated
myosin ATPase activity.
Ca2+-activated myosin ATPase
activity was significantly elevated in types I (increased 35%;
P = 0.02) and IIa (increased 39%; P < 0.01) diaphragm muscle fibers of
Nan animals compared with Ctl (Fig. 7).
Although Ca2+-activated myosin
ATPase was increased to a similar extent (~35%) in type IIx
fibers of Nan vs. Ctl animals, this was not quite significant
(P = 0.07; Fig. 7).
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MHC isoforms.
The proportions of MHC isoforms were similar between the groups (Fig.
8). However, the proportion of
MHC2B in Nan-treated hamsters was
increased ~42% compared with Ctl animals (Fig. 8). Although this did
not quite reach statistical significance
(P = 0.06), a distinct trend for
increased expression of the isoform with Nan administration was
evident.
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This study demonstrated significantly improved diaphragm contractile function, assessed isometrically (specific forces) and isotonically (Vo), after prolonged administration of Nan to intact male hamsters. Hypertrophy of all fiber types was evident, which resulted in a significant increase in cumulative muscle fiber area per unit area, together with a reduction in the interstitial space between muscle fibers. Despite a slight increase in the proportion of type IIa fibers in Nan animals, the relative contribution of fiber types to total costal diaphragm area remained similar between Nan and Ctl animals. The proportions of MHC isoforms were similar between groups, although a trend for increased expression of MHC2B was evident in the Nan group. The activity of Ca2+-activated myosin ATPase was increased by 35-39% in all diaphragm fiber types.
Critique of methods. In the present study, supraphysiological doses of Nan were administered. The rationale for the dosage regimen is as follows. It has recently become evident that supraphysiological doses of anabolic-androgenic steroids may upregulate protein expression in motoneurons (6) or muscles (4, 31) not traditionally viewed as androgen sensitive. Indeed, it has been suggested that 10 times normal physiological levels are required to increase muscle mass in normal men (2) or upregulate choline acetyltransferase mRNA in lumbar motoneurons of intact male rats (6). Athletes commonly administer doses 10-100 times normal therapeutic recommended ranges, and thus controversy over a large number of studies in humans or animals may in part reflect the dose of the agent administered (13, 35).
In the present study, serum levels of Nan were not measured because techniques for the quantitative determination of serum Nan levels in rodents have not been well developed or validated (personal communication, Scientific Division of Organon, Oss, The Netherlands). The Silastic capsule used in the present study, if primed with testosterone instead of Nan, would produce serum testosterone levels ~6.7 times control values in intact male hamsters (10). [This assumes a similar rate of release and tissue uptake of both steroids from the capsule and similar pharmacokinetics (30, 33).] Because the anabolic-to-androgenic ratio of Nan is 2.5-3:1, compared with a ratio of 1:1 with testosterone, a dose- for-dose comparison would produce a relatively greater anabolic effect of nandrolone. Thus, although it is true that the dose of testosterone could be increased to produce equivalent anabolic effects, this would be at the cost of markedly increased androgenic effects. The relative binding affinities of different anabolic-androgenic steroid agents to the androgen receptor in skeletal muscle (rats, rabbits) have been compared, and comparisons revealed that Nan binds strongly to the androgen receptor (24) compared with other anabolic-androgenic steroid agents (e.g., stanozolol). Thus the anabolic-androgenic-steroid-specific agent used may also influence the results. Another possible confounder, may be the method of administration of the steroids. Continuous release of Nan by using a Silastic tube produces steady-state levels over time (32), whereas daily intermittent injections, as used by Prezant and colleagues (23), for example, may produce high peaks and prolonged troughs, during which time the levels of anabolic-androgenic steroids may fall below effective levels. Clinically, it is of interest to note that controlled-released transdermal (nonscrotal) testosterone patches have recently been made available with the intent of providing steady-state levels of the hormone (3). Physiologically, in the intact adult male subject, plasma testosterone levels tend to fluctuate around the mean (16). In the present study, adjunctive exercise was not introduced as an added factor, and animals were fed a balanced diet ad libitum only, with no attempt to augment protein intake above normal requirements.Isometric contractile properties: potential mechanisms. Nan-treated animals had improved specific force of the diaphragm (Pt and all tetanic forces). Similarly, preliminary data obtained from testosterone-treated male hamsters revealed augmentation of diaphragm specific force (10). Improved specific force of the tibialis anterior muscle was also noted in Nan-treated rabbits (25). Similarly, recent studies on testosterone-treated male rats showed increased specific force of the diaphragm (Sieck, unpublished observations). Prezant et al. (23), however, reported an increase in diaphragm specific force only in female rats treated with testosterone propionate after 2.5 wk, but not after 10 wk, of treatment. No effects on diaphragm specific forces were reported by Prezant et al. (22, 23) or Bisschop et al. (5) after anabolic-androgenic steroid administration to intact male rats.
Although there may be several mechanisms underlying the improved diaphragm specific forces in the present study, one likely possibility is an increase in myofibrillar density. The increase in cumulative muscle fiber area per unit area, together with a decrease in interstitial space and preserved Lo, supports this hypothesis. Thus an increased amount of contractile material per unit area could, in part, account for the improved specific forces observed. The relative contribution of diaphragm fiber types to total costal diaphragm area was similar between Nan and Ctl animals. This likely reflects hypertrophy of all diaphragm fibers in Nan animals and only a minor change in the proportions of type IIa diaphragm fibers. Thus the improved specific force of the diaphragm in Nan animals could not be explained by type II fibers contributing more to total costal area. [Fast muscle fibers exhibit specific forces 1.5-2 times that of slow fibers (11).] Similarly, the proportions of MHC isoforms were similar between Nan and Ctl hamsters. It is, therefore, unlikely that a major shift in the expression of MHCs accounted for the increased specific force. We cannot, however, exclude the possibility of coexpression of MHCs or the trend for increased expression of MHC2B as minor contributing factors. It should be emphasized, however, that there is controversy in the literature regarding physiological correlations between different MHC isoforms and force generation (e.g., 9, 11).Isotonic contractile properties: potential mechanisms. Nan significantly increased Vo compared with Ctl. We speculate that alteration of the ATP consumption rate of myosin in Nan-treated animals is a major factor affecting diaphragm Vo. The enhanced activity of the Ca2+-activated myosin ATPase in all diaphragm fiber observed in Nan animals suggests a greater rate of cross-bridge turnover, which would result in fiber shortening occurring at a greater rate. Altered expression of MHC was not observed in Nan animals and thus does not appear to account for the increased Vo in general. We cannot, however, exclude that coexpression of MHCs within single fibers (that may not be translated into differences in the intensity of the mATPase stains) or the distinct trend for increased MHC2B expression contributed to a small extent, although the physiological significance of the latter change to total MHC expression is debatable. Of interest, Prezant and co-workers (22) reported significant increases in MHC2B in intact male rats after 2.5 wk of testosterone administration but not after 10 wk. Whether alterations in the proportions and expression of myosin light chain (MLC) isoforms could influence velocity of shortening is speculative and has not been evaluated in the context of anabolic steroid administration. For example, it has been reported that for a given proportion of MHC, the greater the amount of MLC3f bound to MHC, the greater the reported velocity of contraction (8, 20). This may account for 4-16% of the increment, depending on the particular MHC isoform involved (8).
Clinical implications. The striking changes noted in diaphragm contractile function after the administration of Nan in the present study have a number of clinical implications. The enhanced specific force of the diaphragm together with increased contractile muscle mass suggest that the total diaphragm force generation would be greater. This would be expected to improve the functional force reserve of the diaphragm and endurance capacity by reducing the ratio of the force generated during breathing efforts and maximal force. This may be particularly important under conditions of increased respiratory loads requiring enhanced recruitment and/or frequency coding of motor units to augment force generation. It is interesting to note that in animal studies the provision of anabolic-androgenic steroids to male rats resulted in a 41% increment in endurance running time, as well as a 29% increase in maximal sprinting speed, compared with saline-treated controls (34). Although no specific muscle or muscle groups were studied, it is interesting to speculate that augmentation of limb muscle force and/or velocity (as was noted in the diaphragm in our studies) may account, at least in part, for those observations. The improvement in diaphragm contractility and muscle fiber hypertrophy observed in our studies with the use of Nan suggests that anabolic-androgenic steroids may be useful in conditions in which loss of muscle mass and/or alterations in contractility may curtail the reserve of the respiratory muscles.
A valid concern with the use of anabolic-androgenic steroids for clinical indications is the possibility of serious side effects. These include liver disorders, including peliosis hepatitis, decreased high-density-lipoprotein cholesterol and potential for increased risk of cardiovascular disorders, and potentiation of sleep apnea and behavioral disorders (1). In particular, the alkylated androgens appear to be linked to some of these serious reported side effects (e.g., liver disorders, reduced high-density-lipoprotein cholesterol; Ref. 1). However, recent studies evaluating the efficacy of high dose anabolic-androgenic steroids over prolonged periods for male contraception (21), in chronic obstructive pulmonary disease (18, 26), and in normal men (4, 31) reported no evidence of systemic toxicity. In summary, Nan administered to intact male hamsters resulted in improved diaphragm contractility (improved specific force, increased Vo) and hypertrophy of diaphragm muscle fibers (types I, IIa, IIx). We postulate that the increased activity of Ca2+- activated myosin ATPase within diaphragm fibers mediates, in part, the improved Vo noted in Nan-treated animals while the enhanced contractile mass of the diaphragm per unit area and/or increased myofibrillar density in part explains the improved specific forces noted in the Nan group. Our provocative results in normal animals with intact protein turnover and energy metabolism suggest that Nan may well be of value in conditions associated with disordered protein turnover and a concomitant reduction in diaphragm mass and/or contractility.| |
ACKNOWLEDGEMENTS |
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The authors gratefully acknowledge the superb assistance of Xiayou Da and Ling Tang in conducting these studies and the secretarial support of Debbie Craig.
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FOOTNOTES |
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This research was supported by National Heart, Lung, and Blood Institute Grant HL-47537 and California Tobacco-Related Disease Research Program Grant 6RT-0144.
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. §1734 solely to indicate this fact.
Address for reprint requests: M. I. Lewis, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Rm. 6732, Los Angeles, CA 90048 (E-mail: michael.lewis{at}cshs.org).
Received 24 July 1998; accepted in final form 27 October 1998.
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METHODS
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