Journal of Applied Physiology
Vol. 82, No. 1, pp. 134-143, January 1997
EXERCISE AND MUSCLE

Short- and long-term effects of testosterone on diaphragm in castrated and normal male rats

David J. Prezant^1,2, Manoj L. Karwa¹, Helen H. Kim¹, Diane Maggiore¹, Virginia Chung¹, and David E. Valentine¹

¹ Department of Medicine, Pulmonary Division, and ² Department of Physiology and Biophysics, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York 10467

ABSTRACT

INTRODUCTION

METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Prezant, David J., Manoj L. Karwa, Helen H. Kim, Diane Maggiore, Virginia Chung, and David E. Valentine. Short- and long-term effects of testosterone on diaphragm in castrated and normal male rats. J. Appl. Physiol. 82(1): 134-143, 1997.The effects of short- and long-term testosterone absence or treatment on the diaphragm were studied in castrated and sexually normal male rats. Compared with control rats (untreated normal males), testosterone absence or treatment did not significantly affect costal weight. In untreated castrated males, there were significant decreases in specific forces, type II fiber cross-sectional area, and myosin heavy chain (MHC) isoform 2B after 2.5 wk. In castrated males that received testosterone, there were significant increases in specific forces, type II total fiber proportional area, and relative expression of all adult diaphragm fast MHC isoforms (MHC-2_all) after 2.5 wk. In normal males that received testosterone, the only significant finding was an increase in MHC-2B after 2.5 wk. Across all groups, there was close correlation between increases in maximum tetanic forces and MHC-2_all. Changes in diaphragm function and composition were closely related to changes in serum testosterone levels at 2.5 wk. The lack of significant change in diaphragm function at 10 wk occurred despite changes in serum testosterone levels and diaphragm composition similar to those at 2.5 wk. These findings support our hypothesis that the effects of testosterone are dependent on basal circulating androgen levels and study duration.

myosin heavy chain isoforms; anabolic steroids and sexual status

INTRODUCTION

DESPITE conflicting scientific data, with most human and animal skeletal muscle studies showing only minor changes in muscle mass, protein synthesis, and strength (19, 24), many remain convinced that anabolic steroid use can produce enhanced muscle bulk and strength. If this common belief can be proven true for diaphragm muscle, then it might be possible to exploit the anabolic potential of male sex hormones to improve diaphragm strength in patients with respiratory muscle weakness.

Our hypothesis is that the results of testosterone treatment are dependent on both basal circulating androgen levels and study duration. Preexperimental hormonal status may exert an important influence with greater effects expected in females and castrated males due to lower basal circulating testosterone levels (4). We have previously shown that testosterone treatment in female rats produced significant increases in body weight, costal diaphragm weight, and costal diaphragm specific forces along with significant decreases in costal diaphragm fatigue resistance indexes (FRIs) (23). In contrast, testosterone produced no significant effects in male rats. Second, treatment duration may be significant with greater effects after short-term treatment compared with long-term treatment. Human studies suggest that shorter treatment durations are more effective at improving limb muscle strength (6, 17, 25), and in female rats, we have shown that shorter treatment duration (2.5 vs. 10 wk) had a similar superior effect for diaphragm muscle (23).

It can be argued that differences in nonandrogen hormonal status (estrogen and progesterone) make direct comparisons between males and females potentially misleading. The present study excludes these confounding variables by comparing effects of testosterone in castrated male rats with sexually normal males. Serum testosterone levels are measured in all groups. Diaphragm function is assessed in vitro, fiber types are analyzed by myofibrillar adenosine triphosphatase staining, and, for the first time, the effect of testosterone on myosin heavy chain (MHC) isoforms is analyzed by gel electrophoresis.

METHODS

Animal model. Male Wistar Kyoto rats (Taconic Farms, Germantown, NY) were purchased either sexually normal or surgically castrated. At the time of surgery, the rats weighed 170-180 g (~42-49 days old). One week later, entry weight (180-220 g) was attained and the animals were divided into three experimental groups: 1) untreated castrated males, 2) castrated males that received testosterone, or 3) sexually normal males that received testosterone. Each experimental group had its own control group, which consisted of untreated sexually normal males (age and initial weight matched). Rats treated with testosterone propionate (Eli Lilly, Indianapolis, IN) received a daily dose (5 mg; 5 times/wk) by intramuscular hindlimb injection for 2.5 or 10 wk. This dose was selected to correspond with previous reports in the literature (15, 19, 23). Sexually normal males did not have sham surgery, but all groups that did not receive testosterone were injected daily with saline. All rats had free access to food (Rodent Laboratory Chow 50001, Purina Mills, St. Louis, MO) and water and were individually caged on a 12:12-h light-dark cycle. Adequacy of gonadectomy was determined by gross palpation and by serum testosterone levels at the time the animals were killed. Animals from each experimental and control group were randomly assigned to each analysis described below so that all analytical methods could be performed on the central mid-section of the costal diaphragm.

Hormonal status. The experimental protocol consisted of 20 untreated castrated male rats, 20 castrated male rats that received testosterone, 20 untreated sexually normal male rats, and 20 normal male rats that received testosterone. One-half of the animals in each group were killed at 2.5 wk, and the remaining animals were killed at 10 wk. The actual numbers for each group (see Table 2) vary slightly due to occasional technical problems. At the time the animals were killed, serum was obtained, stored at 80°C, and then processed by solid-phase ¹²⁵I radioimmunoassay for total (free plus protein bound) testosterone levels (Diagnostics Products, Los Angeles, CA). For each assay, experimental and control samples were measured at the same time in random fashion. Additional samples were obtained in untreated ad libitum-fed male Wistar rats of similar age and weight as noted at the times of surgical castration (42-49 days old; 180 g; n = 4) and at entry into the study (50-60 days old; 205 g; n = 4) to determine the level of sexual development. All samples were run in duplicate and in conjunction with known standards to confirm reproducibility and standardization. The interassay coefficient of variation for testosterone at 111 ng/dl was 8.1%.

Table 2. Serum testosterone levels Castrated Male Rats Normal Male Rats Untreated Testosterone treated Untreated Testosterone treated 2.5 wk n 8 7 10 6 0.86 ± 0.01* >1,600* 557.9 ± 133.9  >1,600* 10 wk n 9 8 10 7 1.91 ± 0.01* >1,600* 726.6 ± 106.3  >1,600* Values are means ± SE of serum testosterone levels in ng/dl; n = no. of observations. * Significantly different compared with untreated normal male rats, P < 0.05.

In vitro contractile characteristics and fatigue protocol. The experimental protocol consisted of 18 untreated castrated male rats, 18 castrated male rats that received testosterone, 54 untreated sexually normal male rats, and 18 normal male rats that received testosterone. Nearly one-half of the animals in each group were killed (decapitation under light ether anesthesia) at 2.5 wk and the remaining were killed at 10 wk. The actual numbers for each group (see Tables 1 and 3) differ slightly due to occasional technical problems. The in vitro diaphragm costal muscle strip preparation used in this study has been previously described (22, 23). The diaphragm and adjacent rib sections were removed en bloc in <5 min and placed in a dissection tray filled with modified Krebs-Ringer bicarbonate (KRB) solution (in mM: 11.5 glucose, 138 NaCl, 5.9 KCl, 1.4 CaCl₂, 0.9 MgSO₄, 1.2 NaH₂PO₄, and 25 NaHCO₃; pH 7.42) and aerated continuously with 95% O₂-5% CO₂. Each diaphragm was divided along its central tendon into two hemicostal diaphragms. A central rectangular costal muscle strip (0.5 ± 0.2 cm wide) was dissected to facilitate normalization of force for cross-sectional area (CSA). The hemidiaphragms were suspended in water-jacketed organ baths filled with KRB and insulin (30 U, regular Humulin/100-ml KRB) and continuously aerated with 95% O₂-5% CO₂. Temperature was maintained at 37°C by circulating water through the external jacket of the organ bath by use of a thermostatically controlled water pump. The rib margin of each hemidiaphragm was anchored to the base of the organ bath, and the central tendon of the costal muscle strip was sutured to an isometric force transducer (model FTO3, Grass Instruments, Quincy, MA). Resting (precontraction) muscle length and tension could be altered by raising or lowering the force transducer.

Table 1. Morphometric data Castrated Male Rats Normal Male Rats Untreated Testosterone treated Untreated Testosterone treated 2.5 wk n  9  9 27 8 Initial body wt, g 190 ± 4  205 ± 1  210 ± 6  210 ± 2  Final body wt, g 274 ± 4  273 ± 5  298 ± 5  286 ± 4  Costal diaphragm   wt, mg 483 ± 24  415 ± 59  453 ± 15  502 ± 15  Diaphragm/body wt 1.8 ± 0.1  1.5 ± 0.1  1.5 ± 0.1  1.6 ± 0.1  10 wk n 10 11 30 8 Initial body wt, g 191 ± 3  207 ± 2  202 ± 3  203 ± 6  Final body wt, g 441 ± 11  416 ± 6* 460 ± 15  411 ± 7* Costal diaphragm   wt, mg 687 ± 26  687 ± 36  717 ± 22  707 ± 22  Diaphragm/body wt 1.6 ± 0.1  1.6 ± 0.1  1.5 ± 0.1  1.7 ± 0.1 Values are means ± SE; n = no. of rats. * Significantly different compared with untreated normal male rats, P < 0.05.

Table 3. Diaphragm contractile characteristics Castrated Male Rats Normal Male Rats Untreated Testosterone treated Untreated Testosterone treated 2.5 wk n 15 18 45 15 Lo, cm 2.1 ± 0.1  1.8 ± 0.05  1.8 ± 0.1  2.1 ± 0.1  CT, ms 14.7 ± 1.3* 22.5 ± 1.6  20.5 ± 1.1  19.7 ± 1.2  RT1/2, ms 35.7 ± 2.9  30.9 ± 1.4  34.7 ± 1.2  35.7 ± 2.3  Pt, N/cm2 3.33 ± 0.47* 6.15 ± 0.47* 4.56 ± 0.28  4.85 ± 0.31  Po, N/cm2 16.89 ± 1.19  20.76 ± 1.25* 17.72 ± 0.61  21.14 ± 1.56  10 wk n 19 17 48 15 Lo, cm 2.4 ± 0.05  2.2 ± 0.1  2.2 ± 0.1  2.2 ± 0.1  CT, ms 23.9 ± 1.8  18.1 ± 1.0  19.2 ± 1.2  22.3 ± 1.6  RT1/2, ms 37.1 ± 2.7  31.4 ± 1.5* 35.6 ± 1.2  27.7 ± 1.5* Pt, N/cm2 3.58 ± 0.20  4.72 ± 0.42  4.69 ± 0.25  5.22 ± 0.56  Po, N/cm2 16.91 ± 0.96  19.96 ± 1.73  18.60 ± 0.91  19.56 ± 1.69 Values are means ± SE; n = no. of hemidiaphragms. Lo, optimal muscle length; CT, time to peak force; RT1/2, one-half relaxation time; Pt, peak twitch force; Po, maximum tetanic force (100 Hz at 37°C). Significantly different at P < 0.05 compared with: * untreated normal male rats; untreated castrated male rats.

All hemidiaphragms were equilibrated for 20 min in the presence of 6-µM D-tubocurarine. Optimal muscle length (L_o) for peak twitch force was established individually for each hemidiaphragm. All subsequent measurements were made at L_o. With a dual-channel stimulator (model S88, Grass Instruments; factory modified to provide 350- to 375-mA stimulation current/channel), direct stimulation was delivered via two platinum needle electrodes (subdermal needle electrode, Grass Instruments) implanted into the midportion of the costal muscle strip. Force transducer output was amplified and recorded by a polygraph (model 79D, Grass Instruments). For each muscle strip studied, twitch stimulation voltage was increased in 10-V increments until an increase in force was no longer achieved. To ensure supramaximal voltage throughout the protocol, stimulation voltage was thereafter delivered at 130% of maximal.

Control and experimental hemidiaphragms received the same in vitro stimulation protocol. Baseline single twitch (2-ms impulse duration at supramaximal voltage) and tetanic forces (400-ms trains of 2-ms impulses delivered at 2-min intervals at 10, 20, 60, and 100 Hz) were measured in duplicate to ensure reproducibility. At 37°C, a force plateau is reached after 400-ms stimulation and maximum tetanic force (P_o) occurs at 100 Hz. Time to peak contraction and one-half relaxation times were measured for single twitches.

Fatigue was then induced by using a 10-min stimulation program consisting of 30 trains/min of five impulses each at 5 Hz. Immediately after the fatigue run, single twitch and tetanic force measurements were repeated at 30-s intervals to construct a force-frequency curve for comparison with baseline. The 100-Hz measurement was then repeated 1 min later and confirmed that significant recovery had not occurred. FRI was defined as the force at the conclusion of the fatigue test divided by baseline force. FRI at 5 Hz was measured from the original stimulation tracing and therefore is also not influenced by recovery.

At the conclusion of each experiment, L_o of each strip was measured in the bath by using a vertical-ruled guide strip. The costal diaphragm was then removed, trimmed of all nonmuscular tissues, blotted dry, and weighed. All forces were normalized for size with the assumption that the shape of the muscle strip is roughly that of a rectangular solid and muscle density is 1.06 mg/mm³. In such case, dividing mass (volume) by L_o yields CSA. After dividing by CSA, force can be expressed in newtons per square centimeter.

Fiber type analysis. The experimental protocol consisted of 21 untreated castrated male rats, 14 castrated males that received testosterone, 40 untreated normal males, and 18 normal males that received testosterone. Nearly one-half of the animals in each group were killed at 2.5 wk, and the remaining were killed at 10 wk. The actual numbers for each group (see Table 4) vary slightly due to occasional technical problems. The procedure is as follows: a central region from the costal diaphragm was immediately removed and pinned at a length of 2.0 cm (~L_o), covered in a thin layer of OCT embedding medium (Miles Diagnostic Division, Elkhart, IN), quick frozen in isopentane (Fisher Scientific, Fairlawn, NJ) cooled by liquid nitrogen, and stored at 80°C. Serial cross sections of muscle fibers were cut at 10-µm thickness by using a cryostat (model 840, A/O Reichart, Leica Instruments, Deerfield, IL) kept at 20°C. Based on their staining reactions for myosin adenosine triphosphatase, after alkaline (pH = 9.0) preincubation, muscle fibers were classified as either type I or type II (23). Fiber type proportions and CSA values were determined from a sample of 150-250 fibers in the midcostal region by using nondehydrated sections, digitized by a computerized image processing system (model 920 Quantimet, Cambridge Instruments, Cambridge, UK). For each specimen, we calculated type II total fiber proportional area as 100 × (type II fiber number × type II fiber CSA)/ [(type I fiber number × type I fiber CSA) + (type II fiber number × type II fiber CSA)]. Midcostal diaphragm muscle thickness, equivalent to the height of the muscle section (measured at ×10 magnification), and fiber CSAs (measured at ×20 magnification) were determined from the number of pixels within the outlined fiber borders based on a calibrated pixel area of 0.676 µm² (×20 magnification).

Table 4. Fiber type analysis Castrated Male Rats Normal Male Rats Untreated Testosterone treated Untreated Testosterone treated 2.5 wk n  9 7 18 9 Total fiber no. 211 ± 11  207 ± 14  205 ± 8  173 ± 11  Fiber proportions   Type I 0.27 ± 0.01  0.23 ± 0.01* 0.29 ± 0.01  0.29 ± 0.01    Type II 0.73 ± 0.01  0.77 ± 0.01* 0.71 ± 0.01  0.71 ± 0.01  Fiber area, µm2   Type I 1,048 ± 40  1,199 ± 62  1,362 ± 72  1,283 ± 45    Type II 1,478 ± 67* 1,747 ± 57 1,999 ± 74  1,759 ± 74    Type II TPA 80 ± 1  83 ± 1* 79 ± 1  77 ± 1  10 wk n 12 7 21 9 Total fiber no. 204 ± 10  189 ± 14  181 ± 8  169 ± 12  Fiber proportions   Type I 0.31 ± 0.01  0.24 ± 0.02* 0.31 ± 0.01  0.35 ± 0.02    Type II 0.69 ± 0.01  0.76 ± 0.02* 0.69 ± 0.01  0.65 ± 0.02  Fiber area, µm2   Type I 1,823 ± 109  1,916 ± 126  1,569 ± 67  1,356 ± 101    Type II 2,833 ± 181  3,441 ± 170* 2,703 ± 125  2,342 ± 108    Type II TPA 78 ± 1  85 ± 1* 81 ± 1  81 ± 1 Values are means ± SE; n = no. of observations. Type II total fiber proportional area (TPA) = 100 × [type II fiber no. × type II fiber cross-sectional area (CSA)]/[(type II fiber no. × type II fiber CSA) + (type I fiber no. × type I fiber CSA)]. Significantly different at P < 0.05 compared with: * untreated normal male rats; untreated castrated male rats.

MHC isoforms. The experimental protocol consisted of 16 untreated castrated male rats, 16 castrated males that received testosterone, 16 untreated normal males, and 16 normal males that received testosterone. Nearly one-half of the animals in each group were killed at 2.5 wk, and the remaining were killed at 10 wk. The actual numbers for each group (Table 5) vary slightly due to occasional technical problems. Costal diaphragm sections were removed and immediately minced and extracted for 40 min in 4 vol of buffer I (300 mM NaCl, 100 mM NaH₂PO₄, 50 mM Na₂HPO₄, 1 mM MgCl₂, 10 mM Na₄P₂O₇, 10 mM EDTA, and 0.1% phenylmethylsulfonyl fluoride) at pH 6.5 at 0°C. All subsequent steps were performed at 0°C. Extracts were centrifuged at 13,000 g for 30 min, and the supernatant was recovered and diluted in 9 vol of 1 mM EDTA, 0.1% phenylmethylsulfonyl fluoride, and 0.1% -mercaptoethanol (vol/vol). The diluted extracts were stored overnight at 0°C to allow precipitation of myosin filaments. The filament containing solution was centrifuged at 13,000 g for 30 min. The supernatant was discarded, and the remaining pellet was resuspended in buffer II [62.5 mM tris(hydroxymethyl)aminomethane base (pH 6.80), 2% (wt/vol) sodium dodecyl sulfate (SDS), and 30% glycerol]. The concentration of protein was determined with the Pierce micro-bicinchoninic acid protein assay kit (Pierce, Rockford, IL). The samples were then resuspended in a loading buffer [62.5 mM tris(hydroxymethyl)aminomethane base (pH 6.50), 2% (wt/vol) SDS, 30% glycerol, 5% (vol/vol) -mercaptoethanol, and 0.001% (wt/vol) bromophenol blue] to give a final protein concentration of 0.2 mg/ml. Samples were stored at 80°C.

Table 5. MHC phenotype analysis Castrated Male Rats Normal Male Rats Untreated Testosterone treated Untreated Testosterone treated 2.5 wk n 8 8 7 7 MHC-1 16 ± 1  12 ± 1 13 ± 1  14 ± 0  MHC-2A 39 ± 1  37 ± 1  40 ± 1  32 ± 1* MHC-2X 41 ± 1  47 ± 1* 41 ± 1  40 ± 2  MHC-2B 3 ± 0* 3 ± 1  6 ± 1  14 ± 3* MHC-2all 84 ± 1  88 ± 1 87 ± 1  86 ± 1  10 wk n 8 8 8 8 MHC-1 17 ± 1* 12 ± 1 13 ± 1  12 ± 1  MHC-2A 38 ± 1* 35 ± 1  32 ± 1  35 ± 2  MHC-2X 42 ± 1* 44 ± 1* 48 ± 2  44 ± 2  MHC-2B 3 ± 0* 9 ± 2 7 ± 1  9 ± 2  MHC-2all 83 ± 1* 88 ± 1 87 ± 1  88 ± 1 Values are means ± SE of percent of total myosin; n = no. of observations. MHC, myosin heavy chain; MHC-2all, relative expression of all adult diaphragm fast MHC isoforms [(MHC-2A + MHC-2X + MHC-2B)/(MHC-2A + MHC-2X + MHC-2B + MHC-1)]. Significantly different at P < 0.05 compared with: * untreated normal male rats; untreated castrated male rats.

MHC isoforms were separated by SDS-polyacrylamide gel electrophoresis by using a modification of the method of Talmadge and Roy (29). Separating gels measured 5.3 × 10.2 × 0.75 cm, and stacking gels measured 2 × 10.2 × 0.75 cm. Both gels were prepared from a 30% (wt/vol) stock solution of acrylamide: N, N-bis-methylene acrylamide (BIS) (50:1). Final total concentrations of acrylamide and BIS were 8 and 4% for separating and stacking gels, respectively. The concentration of monomer due to BIS was 2%. Two microliters of myosin extract (300-400 ng) were loaded on the gel, and electrophoresis was performed on a Bio-Rad Mini Protean II dual slab system (Bio-Rad, Hercules, CA) with separate upper and lower running buffers. The gels were run at a constant 70 V (1,000/500 power supply; Bio-Rad) at 4°C for 25.5 h. Separating gels were silver stained (Silver Stain Plus, Bio-Rad) and dried. With this electrophoretic technique, adult costal diaphragm samples separate MHC isoforms according to their mobility: MHC-1 (slow/) > MHC-2B > MHC-2X > MHC-2A (14, 29). The relative compositions of the different MHC isoforms were determined by laser densitometry (model 300E, Molecular Dynamics, Sunnyvale, CA) by using integration software (version 3.3, Image Quant, Molecular Dynamics) and normalizing the average density of each band for the total peak densities for all isoforms combined. The relative expression of all adult diaphragm fast MHC isoforms (MHC-2_all) was calculated as (MHC-2A + MHC-2X + MHC-2B)/ (MHC-2A + MHC-2X + MHC-2B + MHC-1).

Statistical analysis. Values are means ± SE. All outcome indicators were continuous, and distributions were examined to determine deviations from normality. Hypothesis tests for the following outcome indicators were considered independently: initial body weight, final body weight, serum testosterone levels, diaphragm mass, L_o, forces, FRIs, fiber type proportions, fiber type CSAs, type II total fiber proportional area, and the relative expression of individual MHC isoforms. Differences between experimental and control groups due to factors in the statistical model were assessed for significance by using analysis of variance (ANOVA) (Statgraphics software, version 6.1, 1993, Statistical Graphics, Rockville, MD). ANOVA factors included treatment, study duration, and sexual status, with corresponding interaction terms. For contractility (force and FRI) measurements, stimulation frequency and animal were additional factors included for ANOVA, and Duncan's multiple-range test was used for contractile measurement differences. Contractility (force and FRI) measurements were analyzed for the entire force-frequency relationship, for low-frequency (twitch, 10, 20 Hz) stimulation or for high-frequency (60, 100 Hz) stimulation, by using ANOVA after the assessment that assumptions of equal variances were not violated. For the ratio FRI, absolute rather than logarithmic ratios were used because the former followed a more normal distribution. For serum hormone levels, a logarithmic transformation was used to meet assumptions of equal variances. Type III sums of squares were used for hypothesis testing of this unbalanced design. The design was unbalanced due to differences in numbers of animals between groups (see legends of Tables 1-5 and Figs. 1, 2, 3, 4). For two- or three-way interactions between factors identified by ANOVA as significant, comparisons between groups were performed by using Student's two-tailed unpaired t-tests, and incorporating a Bonferroni multiple-comparison procedure. All pairwise comparisons were listed, and, before further analysis, those comparisons considered to be relevant were identified, the number of which was used to derive the adjusted type I error for the Bonferroni procedure. Linear regression, after logarithmic transformation, was used to study the following relationships: type II total fiber proportional area vs. MHC-2_all; P_o vs. type II total fiber proportional area, and P_o vs. MHC-2_all. In all cases, statistical significance was defined by using an overall type I error of 0.05 and is reported as P < 0.05, even though P values varied from 0.0001 to 0.05. No significant differences were found among control animals (untreated sexually normal males); therefore, throughout this paper, although analyzed separately for statistical purposes, control animals were grouped together for presentation only (see Figs. 1, 2, 3, 4 and Tables 1-5).

RESULTS

Animal and diaphragm weights. All animals started at similar weights (Table 1). Numbers for each group are provided in legends of Tables 1-5 and Figs. 1, 2, 3, 4. Final body weight was affected by study duration (ANOVA interaction between treatment and duration). Testosterone absence (castration) or treatment (castrated or sexually normal males that received testosterone) did not significantly affect final body weight after 2.5 wk but did significantly affect final body weight after 10 wk (ANOVA interaction between treatment and sexual status). Compared with 10-wk control animals (untreated sexually normal males), final body weight was significantly decreased in castrated males that received testosterone and sexually normal males that received testosterone. The decrease in final body weight was not significantly different between the two treatment groups. For costal diaphragm weight, all preplanned comparisons between control and experimental groups at 2.5 or 10 wk were not significant. Compared with control animals, neither testosterone absence nor treatment significantly affected the ratio of costal diaphragm to body weight, regardless of study duration.

Hormonal status. To determine whether rats were sexually mature at the onset of this study, serum testosterone levels were measured in untreated ad libitum-fed male Wistar rats of similar age and weight as noted at the time of surgical castration (42-49 days old; 180 g; n = 4) and at entry into the study (50-60 days old; 205 g; n = 4). Compared with reported norms for male Wistar rats (13), serum testosterone levels measured at the time of surgical castration (102 ± 23 ng/dl) were typical of prepubescent development, whereas serum testosterone levels measured at the time of entry into the study (315.0 ± 108.9 ng/dl) were typical of postpubescent development (>120 ng/dl; Ref. 13). To determine the effects of castration and/or treatment on male rats, serum testosterone levels were also obtained at the time the animals were killed (2.5 and 10 wk). Compared with control animals, serum testosterone levels were significantly decreased in untreated castrated males and significantly increased in castrated males that received testosterone and normal males that received testosterone, regardless of study duration. In fact, at both 2.5 and 10 wk, castration without treatment reduced testosterone levels to nearly zero, whereas testosterone treatment in normal or castrated males increased levels to >1,600 ng/dl (Table 2).

Diaphragm contractile characteristics. Diaphragm twitch contraction kinetics (Table 3) were affected by study duration with a significant three-way interaction found among duration, treatment, and sexual status for contraction time and for one-half relaxation time. Compared with 2.5-wk control animals, costal diaphragm peak contraction time was significantly shortened in untreated castrated males but was not significantly affected by testosterone treatment in castrated or sexually normal males. Costal diaphragm one-half relaxation time was not significantly affected by testosterone absence or treatment. Compared with 10-wk control animals, costal diaphragm peak contraction time was not significantly affected by testosterone absence or treatment and diaphragm one-half relaxation time was significantly decreased in castrated males that received testosterone and in sexually normal males that received testosterone.

The specific (normalized for muscle strip CSA) force-frequency relationship of the costal diaphragm was affected by study duration with a significant three-way interaction found among duration, treatment, and sexual status when the force-frequency relationship was analyzed in its entirety or separately over twitch, low-frequency (10 and 20 Hz), or high-frequency (60 and 100 Hz) range. After 2.5 wk (Table 3; Fig. 1A), the effects of testosterone on the specific force relationship of the costal diaphragm showed a significant interaction between treatment and sexual status when the force-frequency relationship was analyzed in its entirety or separately over twitch, low-frequency (10 and 20 Hz), or high-frequency (60 and 100 Hz) range. In untreated castrated males (2.5 wk), costal diaphragm- specific forces were decreased at twitch and low-frequency stimulation compared with control animals. In castrated males that received testosterone (2.5 wk), costal diaphragm-specific forces were increased at twitch and low- and high-frequency stimulation compared with control animals and were also increased at twitch and low- and high-frequency stimulation compared with untreated castrated males. In sexually normal males that received testosterone (2.5 wk), diaphragm-specific forces were not significantly affected. Compared with 10-wk control animals (Table 3; Fig. 1B), diaphragm-specific forces were not significantly affected by testosterone absence or treatment, regardless of whether the analysis included the entire force-frequency relationship or was performed separately for forces at low- or high-frequency stimulation.

Diaphragm fatigue resistance. Figure 2, A and B, shows the effect of testosterone on costal diaphragm FRI values after 2.5 and 10 wk. All preplanned comparisons between control and experimental groups were found to show no significant effects of testosterone absence or treatment on diaphragm fatigue resistance, regardless of study duration.

Fiber type analysis. Effects of testosterone on types I and II costal diaphragm fiber types are detailed in Table 4. For fiber type proportions, the interaction between testosterone treatment and sexual status was significant, whereas interactions with study duration were not significant. Compared with control animals, costal diaphragm fiber type proportions were not significantly affected in untreated castrated males (2.5 or 10 wk), were significantly affected (type I decreased; type II increased) in castrated males that received testosterone after 2.5 and 10 wk, and were not significantly affected in normal males that received testosterone (2.5 or 10 wk).

Type I costal diaphragm fiber CSA was not significantly affected by testosterone absence or treatment, regardless of study duration. Type II costal diaphragm fiber CSA was affected by study duration with a significant three-way interaction found among duration, treatment, and sexual status. After 2.5 wk, the interaction between testosterone treatment and sexual status was significant (Table 4). Type II fiber CSA was significantly decreased in untreated castrated males compared with control animals and significantly increased in castrated males that received testosterone compared with untreated castrated males. After 10 wk, the interaction between treatment and sexual status was also significant. Type II fiber CSA was not significantly affected in untreated castrated males compared with control animals but was significantly increased in castrated males that received testosterone compared with control animals or to untreated castrated males. In sexually normal males that received testosterone, type II fiber CSA was not significantly affected regardless of study duration.

To further analyze the effects of testosterone absence or treatment on costal fiber types, we calculated type II total fiber proportional area for each specimen (Table 4). This calculation is independent of potential fixation errors that may occur due to variability in fixation length (2.0 cm) relative to L_o (1.8-2.4 cm). Type II total fiber proportional area also accounts for the fact that testosterone treatment in castrated males affected both fiber type proportions and CSAs. For type II total fiber proportional area, the interaction between treatment and sexual status was significant, whereas interactions with study duration were not. Type II total fiber proportional area was decreased in untreated castrated males after 2.5 and 10 wk compared with castrated males that received testosterone and increased in castrated males that received testosterone after 2.5 and 10 wk compared with control animals.

MHC phenotype expression. Representative gel electrophoresis samples at 2.5 and 10 wk are shown in Fig. 3, A and B. Based on differences in electrophoretic migration, four MHC-isoforms were identified: MHC-1 (slow), MHC-2B, MHC-2X, and MHC-2A (19, 35). Although testosterone absence or treatment affected several MHC isoforms after 2.5 or 10 wk, the most noticeable and consistent effect was on MHC-2B expression. Costal diaphragm MHC-isoform expression was affected by study duration with a significant three-way interaction found among duration, treatment, and sexual status. After 2.5 wk (Table 5; Fig. 3A), the effects of testosterone on costal diaphragm MHC isoform composition showed a significant interaction between treatment and sexual status. In untreated castrated males (2.5 wk), MHC-2B was decreased to nearly one-half the average control value. In castrated males that received testosterone (2.5 wk), the decrease in MHC-2B was no longer significant compared with control animals. In untreated castrated males, the decrease in MHC-2_all was not significant compared with control animals but was significant compared with castrated males that received testosterone. In sexually normal males that received testosterone (2.5 wk), MHC-2B was increased to nearly twice the average control value; however, as MHC-2A was decreased, there was no significant effect on MHC-2_all isoforms compared with control animals.

After 10 wk (Table 5; Fig. 3B), the effects of testosterone on costal diaphragm MHC isoform composition also showed a significant interaction between treatment and sexual status. In untreated castrated males (10 wk), MHC-2B was decreased to nearly one-third of average control values. In castrated males that received testosterone (10 wk), MHC-2B was no longer significantly decreased compared with control animals and was increased compared with untreated castrated males. The overall effect was a decrease in MHC-2_all isoforms and a reciprocal increase in MHC-1 when untreated castrated males were compared with control animals or with castrated males that received testosterone. In sexually normal males that received testosterone (10 wk), there was no significant effect on MHC isoforms.

Correlations between diaphragm function and composition. Relationships between the relative expression of MHC-2_all isoforms, type II total fiber proportional area, and P_o for mean data are shown in Fig. 4, A-C. There was a significant correlation between type II total fiber proportional area and MHC-2_all isoforms (Fig. 4A; r = 0.70), although the former yielded slightly lower measurements. There was a significant correlation between increasing P_o and the increase in MHC-2_all isoforms (Fig. 4B; r = 0.71). Although there was a trend, the correlation between increasing P_o and type II total fiber proportional area was not significant (Fig. 4C; r = 0.63; P < 0.10).

DISCUSSION

Controversy exists as to whether anabolic steroid treatment produces hypertrophy and whether androgen-deficient states (female animals or castrated males) are more responsive to treatment. In humans (6, 17, 25) and in sexually normal male laboratory animals (2, 7, 11, 14, 18, 28), variable effects have been found. In androgen-deficient states, the effects of androgen treatment on limb muscle structure and function are difficult to interpret because of small numbers of studies, varying treatment durations, conflicting results, and the fact that differences in circulating androgen levels have always been assumed rather than measured. In female rats, androgen treatment increased (7), decreased (7), or did not affect (7, 15) limb muscle function. Castration, in male rats, prevented (19) or did not affect (10, 20, 21) compensatory limb muscle hypertrophy, and androgen treatment after castration restored (19), augmented (20), or did not affect (21) compensatory hypertrophy. In cardiac muscle, testosterone in large part prevented the negative effects on cardiac function and contractile protein profiles observed in gonadectomized male and female rats (26).

For respiratory muscles, we previously showed that short-term (2.5 wk) but not long-term (10 wk) testosterone treatment significantly increased diaphragm weight and specific forces along with significantly decreasing fatigue resistance in sexually normal female rats but not in sexually normal males (23). However, comparisons between males and females may be misleading because of differences in growth rates, sexual maturity, and other circulating hormones (estradiol and progesterone). The potential confounding impact of such differences is eliminated by studying testosterone treatment in castrated male rats in which, like females, there are lower circulating testosterone levels (Table 2 and Ref. 4) and higher skeletal muscle cytosolic androgen receptor concentrations (3, 18, 24) than found in sexually normal males; however, unlike females, growth rates and nonandrogen hormonal status are more similar to those found in sexually normal males (Table 1 and Ref. 13).

In castrated and sexually normal males, we found that short-term (2.5 wk) testosterone absence or treatment did not significantly affect body or diaphragm weight. After 2.5 wk, specific forces and diaphragm type II fiber CSA were decreased in castrated males that did not receive testosterone and increased in castrated males that received testosterone but were not significantly affected in sexually normal males that received testosterone. Rather than a dose response to testosterone, these short-term results show that the near absence of testosterone in castrated rats can affect diaphragm structure/function and that testosterone treatment can produce significant augmentation, but only when basal levels are nearly zero. These findings support our hypothesis that results of testosterone treatment are dependent on the basal circulating androgen level. In contrast, testosterone absence or treatment affects MHC phenotype expression in all short-term experimental groups, even sexually normal males, and in this aspect suggests a dose response. Specifically, after 2.5 wk, MHC-2B expression was relatively decreased in castrated males that did not receive testosterone, MHC-2_all expression was relatively increased in castrated males that received testosterone, and MHC-2B expression was relatively increased in sexually normal males that received testosterone.

After long-term (10 wk) treatment, body weight was decreased in castrated males that received testosterone and in sexually normal males that received testosterone. This has been previously noted (14, 26) and is likely due to androgen-induced increases in fat metabolism (14). Diaphragm weight was not significantly affected. After 10 wk, the relative expression of MHC-2B, MHC-2X, and MHC-2_all was decreased in untreated castrated males. After 10 wk, the relative expression of MHC-2B, MHC-2X, and MHC-2_all was increased, type II CSA and type II total fiber proportional area was increased, and one-half relaxation twitch time was reduced in castrated males that received testosterone. Diaphragm-specific forces were not significantly affected in castrated rats with or without long-term testosterone treatment. With the exception of decreased one-half relaxation twitch time, there were no significant effects on diaphragm structure or function in sexually normal males that received long-term testosterone treatment. Again, these findings support our hypothesis that the results of testosterone treatment are dependent on the basal circulating androgen level. The near absence of testosterone has an effect, and testosterone treatment has the opposite effect but only in castrated males where basal levels are nearly zero.

Across all groups (2.5 and 10 wk), we observed a significant correlation between type II total fiber proportional area and MHC-2_all isoforms (Fig. 4A), thereby demonstrating that both methodologies (e.g., histochemistry and gel electrophoresis) yield similar results, although histochemical results were slightly lower. Type II fiber hypertrophy, after anabolic steroid treatment, has been reported in limb muscles (7, 18). Our study is the first to report effects of androgen manipulation on MHC phenotype expression. The major effect of testosterone absence or treatment was on MHC-2B expression, and overall effects on MHC-2_all isoforms generally reflect this change. Because immunohistochemical stains were not performed in this or other studies, we cannot state whether this correlation reflects homogeneous MHC isoform shifts within individual fibers or heterogenous MHC isoform shifts within the total population of type II fibers. The fact that type II fiber total proportional area changed without measurable changes in diaphragm weight also suggests a change in nonmyocyte compartments (e.g., interstitium, adipocytes, vascular). Direct measurement of these compartments was not obtained.

There was a significant correlation between increasing P_o and the increase in MHC-2_all isoforms (Fig. 4B), but major changes in specific forces were not observed. Prior reports indicate that P_o is greater in limb muscle fibers that express MHC fast isoforms than in those that express the MHC-slow isoform (2). In diaphragm muscle, fibers (5) and motor units (27) histochemically classified as type II (fast twitch) generate greater specific force than fibers and motor units classified as type I (slow twitch). Recently, Johnson et al. (12) reported a strong correlation between P_o and MHC fast isoform transitions in the rat diaphragm during early postnatal development. However, other studies have not found a consistent relationship between P_o and histochemically characterized fibers (8, 9).

Shifts in MHC isoform expression may influence P_o production by affecting myofibrillar density, cross-bridge cycling kinetics, or the force generated per cross bridge (12). However, given modest changes in type II total fiber proportional area and MHC-2_all isoforms observed in our study, such alterations serve only as a partial explanation for androgen-induced changes in contractile characteristics. Furthermore, alterations in type II total fiber proportional area or MHC-2_all isoforms could not explain the diaphragm's entire functional response to testosterone. Changes in contractile kinetics and the lack of change in FRI values did not correlate with changes in type II total fiber proportional area or MHC-2_all isoforms. In females, we previously observed increased diaphragm-specific forces and decreased fatigue resistance without significant change in histochemical fiber types (23). For these reasons, we believe that additional cellular alterations, perhaps in concentrations of contractile or oxidative proteins, may be partially responsible for effects of testosterone on diaphragm function.

The effect of study duration on androgen therapy has not been directly evaluated by other investigators. Despite identical changes in serum testosterone levels and similar shifts in diaphragm composition and/or structure to those observed after 2.5 wk, specific forces were not significantly affected by testosterone absence or treatment after 10 wk. The loss of functional effects over time due to treatment duration and/or animal aging is similar to our findings in female rats in which short-term, but not long-term, testosterone treatment produced significant increases in diaphragm-specific forces (23). Human studies also suggest that shorter treatment durations are more effective at improving limb muscle strength (6, 17, 25). We can only speculate on the cause for this significant interaction between testosterone treatment and study duration. First, rats in long-term castrated groups had a longer time to recover from any unknown influence of surgery. However, surgery below the umbilicus has not been shown to influence diaphragm function, and it is unlikely that surgery that far removed from the diaphragm would influence MHC phenotype expression. Moreover, this would not explain our similar findings in short-term treated female rats where surgery was not part of the protocol (22). Second, feedback inhibition of synergistic hormones (growth hormone or thyroxine) might have dampened the response to long-term treatment. This appears unlikely because Scheuer et al. (26) used a similar dose of testosterone (3 mg/day for 10 wk) and found no significant effects on serum thyroxine, thyroid binding globulins, insulin, or plasma glucose levels in sexually normal or castrated male rats. Third and most important, aging rather than treatment duration may have led to differences between short- and long-term results. At the end point of the study, rats in long-term groups were 7.5 wk older than those in short-term groups. Younger rats, in the midst of a major growth phase, may show a greater response and/or dependence on testosterone than older long-term-treated rats. Castrated rats may show the greatest response and/or dependence on testosterone because they were prepubescent and without treatment would remain unexposed to the rise in serum testosterone levels normally induced by puberty. Older rats may be less responsive to testosterone due to downregulation of muscle cytosol androgen receptors from long-term treatment or perhaps from aging. Measurement of androgen receptors is beyond our investigative scope, but other studies have shown that testosterone treatment in castrated males can reduce increased androgen binding capacity to normal levels (24). For these reasons, differences between short- and long-term treatment results may be due to aging and/or treatment duration.

In conclusion, changes in diaphragm function were consistent with, but not entirely explained by, changes in diaphragm composition. After 2.5 wk, changes in diaphragm function and fiber type CSA were closely related to changes in serum testosterone levels in castrated males but not sexually normal males. Changes in diaphragm MHC isoform expression were related to changes in serum testosterone levels in both castrated and sexually normal males. After 10 wk, there were no significant changes in diaphragm function despite identical changes in serum testosterone levels and similar changes in diaphragm composition to those observed after 2.5 wk. These findings support our hypothesis that the results of testosterone treatment are dependent both on the basal circulating androgen level and on study duration. The latter may represent effects of aging and/or treatment duration.

ACKNOWLEDGEMENTS

The authors thank Drs. T. K. Aldrich, M. H. Williams, J. Scheuer, B. Wittenberg, and J. Wittenberg for advice and support. We thank Dr. K. Freeman for statistical guidance; Dr. L. Brown for providing the Quantimet computerized microscopic imaging system; and Dr. B. Thyssen for help with serum testosterone assays. We appreciate Drs. A. Malhotra, A. Andersen, V. Hatcher, G. Sieck, J. F. Watchko, and M. J. Daood; A. Nakusi and D. Elliot; and The Cancer Research Center at Albert Einstein College of Medicine for providing advice and guidance with MHC-isoform gel electrophoresis.

FOOTNOTES

Address for reprint requests: D. J. Prezant, Montefiore Medical Center, Pulmonary Division, Centennial 423, Bronx, NY 10467.

Received 5 June 1995; accepted in final form 6 September 1996.

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