Journal of Applied Physiology
Vol. 82, No. 1, pp. 125-133, January 1997
EXERCISE AND MUSCLE

Gender-specific effects of dexamethasone treatment on rat diaphragm structure and function

D. J. Prezant^1,2, M. L. Karwa¹, B. Richner¹, D. Maggiore¹, E. I. Gentry¹, and J. Cahill¹

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, D. J., M. L. Karwa, B. Richner, D. Maggiore, E. I. Gentry, and J. Cahill. Gender-specific effects of dexamethasone treatment on rat diaphragm structure and function. J. Appl. Physiol. 82(1): 125-133, 1997.The effects of long-term dexamethasone treatment on diaphragm muscle were studied in female and male rats. Compared with pair-fed control animals, dexamethasone treatment did not significantly affect estrous cycling or peak serum estradiol levels; however, testosterone levels were significantly increased in females and decreased in males. Dexamethasone significantly reduced body and costal diaphragm weights, but to a lesser extent in females than in males. Reductions in diaphragm weight were proportional to reductions in body weight. In females and males, dexamethasone treatment significantly decreased diaphragm fiber (types I and II) cross-sectional area and the relative expression of myosin heavy chain isoform 2B. With the exception of type I fiber atrophy, these changes occurred to a lesser extent in females. Dexamethasone did not significantly affect specific forces. Dexamethasone significantly increased twitch one-half relaxation time and fatigue resistance indexes in males but not in females. In conclusion, the effects of long-term dexamethasone treatment were gender specific, with significantly fewer effects in females, and changes in serum testosterone levels were associated with these findings.

respiratory muscles; gender differences

INTRODUCTION

CORTICOSTEROIDS are used clinically for the treatment of inflammatory diseases, both respiratory and systemic. Beneficial effects are unquestioned, but corticosteroids are not without numerous side effects, including skeletal limb muscle atrophy (8). Although muscle atrophy predominantly affects the least active limb muscles (20, 26), respiratory muscle dysfunction has been reported in humans (8).

Animal studies have shown that corticosteroids, especially when fluorinated, affect diaphragm mass, structure, and function. In adult male rats, cortisone (21), triamcinolone (5, 6, 31), prednisolone (20), and dexamethasone (28, 30) produced varying degrees of diaphragm atrophy, whereas specific forces [normalized for cross-sectional area (CSA)] were preserved (21), depressed (6), or varied (preserved or depressed, depending on age) (30). Fatigue resistance was increased (5, 6, 21, 32), decreased (19), unchanged (5, 11, 21, 28, 30), or unmeasured (31).

No study has evaluated the effect of corticosteroids on myosin phenotype expression. Furthermore, almost all studies have included only male animals given short-term treatment (4 wk), and none has compared males to females in the same laboratory or examined the effect of corticosteroids on other hormones (estradiol, testosterone, and thyroid hormones). In female rats, prednisolone produced costal diaphragm fiber (type IIb) atrophy, but we were unable to find any reference to the effect of corticosteroids on diaphragm function. Compared with male animals, females have slower growth rates, are more easily subject to stress-induced alterations in sex hormones, have higher basal corticosteroid levels with a more stress-responsive pituitary-adrenal axis, and may have more rapid steroid metabolism due to higher hydroxylating activity (9, 18). Adrenal-gonadal interactions may also occur that affect not only serum estrogen but also serum androgens and thyroid hormones (1, 27). In fact, we have shown that the effects of testosterone on diaphragm function are gender dependent with increased specific forces and decreased fatigue resistance in female, but not male, rats (25).

For these reasons, we hypothesized that the effect of dexamethasone treatment on diaphragm function and structure is gender specific. To understand potential mechanisms and influences responsible for gender-specific differences, the estrous cycle was mapped by daily examination of vaginal cytology throughout this study and by obtaining serum estradiol, testosterone, thyroxine, and 3,3,5-triiodothyronine (T₃) levels. Male as well as female rats were studied because differences in multiple study variables (species, age, steroid type, dose, treatment duration, nutrition, and stimulation protocols) made comparisons with previously reported data from male rats (5, 6, 21, 28, 30, 31) unacceptable. We measured the effects of long-term (10-wk) dexamethasone treatment on costal diaphragm fiber types, contractile characteristics, and fatigue resistance in female and male rats. We also, for the first time, report the effects of corticosteroid treatment on diaphragm myosin heavy chain (MHC) isoforms.

METHODS

Animal model. Female (55-72 days old) and male (49-60 days old) Wistar VAF+ rats (Charles River Farms, Kingston, NC) weighing 180-220 g received daily (0.4 mg; 5 times/wk) dexamethasone sodium phosphate (Lyphomed, Rosemont, IL) intramuscular hindlimb injections for 10 wk. This dose was selected to correspond with previous animal studies in the literature (16, 28, 30). Because body weight for all experimental groups remained essentially unchanged throughout the study, the dose adjusted for body weight (2 mg/kg) was also equivalent. Control animals were injected daily with saline and pair-fed (Rodent Laboratory Chow Powder 5001, Purina Mills, St. Louis, MO) a volume amount equal to the prior day's food intake of their experimental counterpart. Specially designed feeding jars were used to avoid spillage. Despite pair feeding, control rats gained more weight than experimental rats. All rats had free access to water and were individually caged at normal ambient temperature on a 12:12-h light-dark cycle.

The experimental protocols described below proceeded in two phases. In phase 1, 44 female and 40 male rats were divided equally and randomly into pair-fed control and dexamethasone-treated groups. At the time the animals were killed, nearly one-half of each group was used for the in vitro contractility/fatigue protocol and the remaining animals were used for fiber-type analysis. In phase 2, 20 female and 14 male rats were divided equally and randomly into pair-fed control and dexamethasone-treated groups. These animals were used for determination of estrous cycling by daily vaginal cytologic examination smears for measurement of serum estradiol and testosterone levels and for diaphragm muscle MHC isoform analysis. Estrous cycling and serum hormone levels were also compared with an additional group of similar rats fed ad libitum. For all protocols, rats were anesthetized with ether, samples were obtained, and then the animals were killed by pneumothorax and/or decapitation. The actual numbers for each group varied slightly due to occasional technical problems (see Tables 1-5 and Figs. 1, 2, 3).

Table 1. Morphometric data after long-term (10 wk) treatment Dexamethasone Control Female rats n 22 22 Initial body wt, g 204 ± 5  205 ± 2  Final body wt, g 193 ± 4* 259 ± 5  Costal diaphragm wt, mg 378 ± 13* 476 ± 13  Costal diaphragm/body wt 2.1 ± 0.1  1.9 ± 0.1  Costal thickness, mm 0.536 ± 0.028  0.549 ± 0.025  Male rats n 20 20 Initial body wt, g 202 ± 3  200 ± 3  Final body wt, g 221 ± 7* 344 ± 5  Costal diaphragm wt, mg 363 ± 21* 575 ± 28  Costal diaphragm/body wt 1.8 ± 0.1  1.7 ± 0.1  Costal thickness, mm 0.574 ± 0.021* 0.653 ± 0.032 Values are means ± SE; n = no. of rats ( phase 1). For costal diaphragm thickness, muscle strips are from 11 dexamethasone-treated females, 11 pair-fed females, 11 dexamethasone-treated males, and 11 pair-fed males. * Significantly different compared with control values by analysis of variance (ANOVA), P < 0.05.

Hormonal status. To assess cornification of cells, vaginal smears were obtained daily during the first and last 3 wk of the study in unanesthethized dexamethasone-treated, pair-fed, and ad libitum-fed female rats. Because the normal estrous cycle in rats is the repetitive regular passage from estrous to metestrous to diestrous to proestrous over a 4- to 5-day period, there was ample time for determining whether treatment affected cycling. Serum was obtained at the time the animals were killed (10 wk), stored at 80°C, and then processed by double-antibody ¹²⁵I radioimmunoassay for estradiol levels (Pantex, Santa Monica, CA) in female rats and by solid-phase ¹²⁵I radioimmunoassay for testosterone (Diagnostic Products, Los Angeles, CA), thyroxine (Pantex), and T₃ (Pantex) levels in males and females. Because estradiol levels peak during proestrous, all females were killed at proestrous as determined by vaginal cytologic examination. For each assay, experimental and control animals were measured at the same time in random fashion. Each sample was run in duplicate and in conjunction with standards to confirm reproducibility and standardization. The interassay coefficient of variation for estradiol at 119 pg/ml was 8.7%, testosterone at 111 ng/dl was 8.1%, thyroxine at 6.8 µg/dl was 3.9%, and T₃ at 137 ng/dl was 5.9%.

MHC isoforms. Costal diaphragm sections were removed and immediately minced, homogenized, 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, 0.1% phenylmethylsulfonyl fluoride; 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 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.

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%. A volume of 2 µl of myosin extract (300-400 ng) was loaded on the gel, and electrophoresis was performed on a Biorad Mini Protean II dual slab system (BioRad, Hercules, CA) with separate upper and lower running buffers. The gels were run at a constant 70 V (1,000/500 power supply; Biorad) at 4°C for 25.5 h. Separating gels were silver stained (Silver Stain Plus, Biorad) 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 (15, 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 Qaunt, 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).

Fiber-type analysis. In similarly prepared animals, the central costal region of the diaphragm was pinned at a length of 2.0 cm [which is approximately optimal muscle length (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 (24, 25). Fiber-type proportions and CSA values were determined from a sample of 150-250 fibers by using nondehydrated sections, digitized by a computerized image processing system (model 920, Quantimet, Cambridge Instruments, Cambridge, UK). Midcostal diaphragm muscle thickness, equivalent to the height of the muscle section (measured at ×10 magnification), and fiber CSA values (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).

In vitro contractility/fatigue protocol. The in vitro diaphragm costal muscle strip preparation used in this study has been previously described (25). 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 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.

All hemidiaphragms were equilibrated for 20 min in the presence of 6-µM D-tubocurarine to block indirect muscle stimulation (T. K. Aldrich, personal communication). 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 occurs at 100 Hz (34). Time to peak contraction and one-half relaxation times were measured for single twitches.

Fatigue was then induced with 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. The fatigue resistance index (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 was 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.

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 estradiol levels, serum testosterone levels, diaphragm mass, L_o, forces, FRI values, fiber-type proportions, fiber-type CSAs, 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 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 we assessed that assumptions of equal variances were not violated. For the FRI ratio, 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). 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, 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. In all cases, statistical significance was defined by using an overall type I error of 0.05 and is reported as P < 0.05, although actual P values varied from 0.0001 to 0.05.

RESULTS

Animal and diaphragm weights. For all groups, differences between initial weights were not significant. Numbers for each group are provided in the legends of Tables 1-5 and Figs. 1, 2, 3. Dexamethasone-treated rats were anorexic but showed no visual evidence of respiratory distress, altered activity levels, or pneumonia. After dexamethasone treatment, there were no significant differences between initial and final body weights regardless of gender (Table 1). In contrast, the significant weight gain found in control animals despite pair-feeding indicated that dexamethasone-treated rats were catabolic. Due to catabolism, final body weights were significantly different among all experimental and control groups. Due to greater weight increases in pair-fed control males compared with females, dexamethasone-induced weight impairment can be compared between genders by expressing dexamethasone final weights relative to pair-fed control final weights. There was a significant difference in dexamethasone-induced weight impairment (ANOVA interaction between treatment and gender) between females (74 ± 4% of pair-fed control) and males (64 ± 2% of pair-fed control), with less effect in females, after long-term dexamethasone treatment.

Hormonal status. To determine whether rats were sexually mature at the onset of this study, serum testosterone levels were measured in a similar group of untreated ad libitum-fed male Wistar rats (50-60 days old; 205 ± 3 g) and daily vaginal smears were examined in all female rats during the first week of the study. Baseline serum testosterone levels (315.0 ± 108.9 ng/dl; n = 4) were at adult values (>120 ng/dl; see Fig. 4 of Ref. 17). All female rats were observed to be in estrous (sexually mature) during week 1.

To determine the effect of pair feeding and dexamethasone on the estrous cycle, vaginal smears were examined daily during the first and last 3 wk of this 10-wk study (Table 2). The estrous cycle lasted 4-5 days for both control and treated groups. Compared with pair-fed control animals, dexamethasone treatment produced no significant difference in the number of rats experiencing normal estrous cycles.

Table 2. Hormonal status after long-term (10 wk) treatment Dexamethasone Treated Pair Fed Control Normal Ad Libitum Fed Female rats n 9  9  10  No. in normal estrous 8 (88%) 9 (90%) 10 (100%) Serum peak estradiol, pg/ml 25.9 ± 11.3 28.2 ± 7.3 39.9 ± 9.8  Serum testosterone, ng/dl 19.7 ± 6.5* 1.9 ± 0.9 9.9 ± 3.7  Serum thyroxine, µg/dl 4.9 ± 0.3* 2.9 ± 0.3 4.3 ± 0.6  Serum T3, ng/dl 87.6 ± 8.8* 38.2 ± 4.1 65.0 ± 3.9  Male rats n 7  6  10  Serum testosterone, ng/dl 535.9 ± 136.2* 788.0 ± 214.3  726.6 ± 106.3  Serum thyroxine, µg/dl 4.5 ± 0.3  4.7 ± 0.2  4.7 ± 0.4  Serum T3, ng/dl 89.0 ± 5.2* 36.6 ± 3.4  44.6 ± 7.5 Values are means ± SE; n = no. of rats. T3, 3,3,5-triiodothyronine. Significantly different (P < 0.05) by ANOVA compared with: * pair-fed control values; ad libitum-fed values.

After 10 wk, serum hormone levels were obtained at the time the animals were killed (Table 2). There was no significant difference in serum peak estradiol levels between groups. The effect of dexamethasone on serum testosterone levels was gender specific (ANOVA interaction between treatment and gender). Compared with control animals of the same gender, dexamethasone treatment increased serum testosterone levels in females and decreased serum testosterone levels in males. The effect of dexamethasone on serum thyroxine, but not on serum T₃, was gender specific (interaction between treatment and gender). Compared with pair-fed control animals, dexamethasone increased both serum thyroxine and T₃ levels in females but only increased serum T₃ levels in males (Table 2).

Diaphragm structure. The effects of long-term dexamethasone treatment on costal diaphragm weight and thickness in females and males are shown in Table 1. Compared with pair-fed control animals, long-term dexamethasone treatment significantly decreased costal diaphragm weight in females and males, but this decrease was significantly smaller in females (ANOVA interaction between treatment and gender). Diaphragm-to-body weight ratios were not significantly different compared with pair-fed control animals of the same gender. Costal diaphragm thickness was significantly decreased in males but not females after long-term dexamethasone treatment. Crural diaphragms were not obtained.

MHC phenotype expression. A representative gel electrophoresis sample is shown in Fig. 1. Based on differences in electrophoretic migration, four adult costal diaphragm MHC-isoforms were identified: MHC-1 (slow), MHC-2B, MHC-2X, and MHC-2A (15, 29). Although dexamethasone treatment significantly affected several MHC isoforms in both females and males, the most noticeable effect was on MHC-2B expression. The effect of dexamethasone treatment on the relative expression of individual MHC isoforms was not significantly different between females and males (ANOVA interaction between treatment and gender). Dexamethasone treatment produced substantial decreases in the relative expression of MHC-2B in females and males as well as significant but less impressive increases in the relative expression of MHC-2A in females and males (Table 3). Because these changes occurred to a somewhat lesser degree in dexamethasone-treated females (~40% decrease in MHC-2B) compared with males (~65% decrease in MHC-2B), a significant decrease in MHC-2_all isoform expression with a reciprocal increase in MHC-1 expression was found only in dexamethasone-treated males.

Table 3. Costal diaphragm MHC phenotype analysis after long-term (10 wk) treatment Dexamethasone Control Female rats n 8 8 MHC-1 19 ± 1  17 ± 1  MHC-2A 32 ± 1* 28 ± 1  MHC-2X 44 ± 1  44 ± 1  MHC-2B 6 ± 1* 10 ± 1  MHC-2all 81 ± 1  83 ± 1  Male rats n 7 6 MHC-1 20 ± 1* 15 ± 1  MHC-2A 32 ± 1* 27 ± 1  MHC-2X 43 ± 2  44 ± 1  MHC-2B 5 ± 1* 14 ± 2  MHC-2all 80 ± 1* 85 ± 1 Values are means ± SE of percentage of total myosin; n = no. of observations. MHC, myosin heavy chain; MHC-2all, (MHC-2A + MHC-2X + MHC-2B)/(MHC-2A + MHC-2X + MHC-2B + MHC-1). * Significantly different compared with control values by ANOVA, P < 0.05.

Fiber-type analysis. In female and male rats, histochemical analysis revealed no significant difference in the proportions of type I or type II fibers to the total number of fibers after dexamethasone treatment (Table 4). Long-term dexamethasone treatment did produce significant atrophy of types I and II costal diaphragm fibers for both females and males. The effect of dexamethasone treatment on type I costal diaphragm fiber CSA values was not significantly affected by gender (ANOVA interaction between treatment and gender); significant atrophy occurred in both females and males. For descriptive purposes only, mean ratios (experimental-to-control) show that dexamethasone decreased the CSA values of type I costal fibers by 14 and 16% in long-term-treated females and males, respectively. The effect of dexamethasone treatment on type II costal diaphragm fiber CSAs was significantly affected by gender (ANOVA interaction between treatment and gender) due to less atrophy in females compared with males. For descriptive purposes only, mean ratios show that dexamethasone decreased the CSA values of type II costal fibers by 24% in females and 36% in males.

Table 4. Costal diaphragm fiber type analysis after long-term (10 wk) treatment Dexamethasone Control Female rats n 11 11 Fiber ratio   Type I 0.29 ± 0.01  0.32 ± 0.01    Type II 0.71 ± 0.01  0.68 ± 0.01  Fiber area, µm2   Type I 1,195 ± 51* 1,392 ± 62    Type II 1,540 ± 79* 2,033 ± 89  Male rats n 11 11 Fiber ratio   Type I 0.29 ± 0.01  0.31 ± 0.02    Type II 0.71 ± 0.01  0.69 ± 0.02  Fiber area, µm2   Type I 1,230 ± 65* 1,469 ± 62    Type II 1,655 ± 81* 2,584 ± 149 Values are means ± SE; n = no. of observations. * Significantly different compared with control values by ANOVA, P < 0.05.

Diaphragm contractility. Diaphragm twitch contraction kinetics and contractility data are shown in Table 5. Costal diaphragm twitch peak contraction times were not significantly affected by dexamethasone treatment in females or males. Diaphragm twitch one-half relaxation times were not significantly affected by dexamethasone treatment in females but were significantly increased by dexamethasone treatment in males.

Table 5. Costal diaphragm contractile characteristics after long-term (10 wk) treatment Dexamethasone Control Female rats n 21 21 Lo, cm 2.1 ± 0.1  2.0 ± 0.1  CT, ms 21.0 ± 2.0  20.0 ± 1.0  RT1/2, ms 45.0 ± 1.0  42.0 ± 2.0  Pt, N/cm2 4.05 ± 0.28  4.52 ± 0.34  Po, N/cm2 18.21 ± 0.73  19.60 ± 1.08  Male rats n 17 17 Lo, cm 1.9 ± 0.1  2.0 ± 0.2  CT, ms 32.0 ± 1.0  28.0 ± 1.0  RT1/2, ms 44.0 ± 2.0* 38.0 ± 1.0  Pt, N/cm2 3.86 ± 0.26  4.06 ± 0.42  Po, N/cm2 17.57 ± 0.88  18.43 ± 1.19 Values are means ± SE; n = no. of hemidiaphragms. Lo, optimal muscle length; CT, time to peak contraction; RT1/2, half-relaxation time; Pt, peak twitch force; Po, peak tetanic force (100 Hz at 37°C). * Significantly different compared with control values by ANOVA, P < 0.05.

The effect of dexamethasone on diaphragm-specific forces is shown in Table 5 and Fig. 2. The interaction between treatment and gender was not significant regardless of whether the force-frequency relationship was analyzed over its entirety or over the low-frequency (twitch, 10, 20 Hz) or high-frequency (60, 100 Hz) range. There was no significant effect on the specific force-frequency relationship of the costal diaphragm after long-term dexamethasone treatment in females (Fig. 2A) or males (Fig. 2B).

Diaphragm fatigue resistance. The effect of dexamethasone on costal diaphragm FRI values was gender specific (ANOVA interaction between treatment and gender) when the analysis included all stimulation frequencies or at high-frequency stimulation, but it was not gender specific at low-frequency stimulation. Compared with pair-fed control animals of the same gender, significant effects on costal diaphragm FRI values after long-term dexamethasone treatment were observed in males (Fig. 3B) but not females (Fig. 3A).

DISCUSSION

This study was the first to evaluate the effects of corticosteroid treatment on skeletal muscle structure and function in both female and male animals. Both genders received the same dose of dexamethasone (2.0 mg ^· kg^{1 ·} day¹ for 10 wk). Compared with pair-fed control animals, dexamethasone significantly decreased body weight, costal diaphragm weight, fiber (types I and II) CSAs, and MHC-2B expression and significantly increased MHC-2A expression, twitch one-half relaxation times, and FRI values. Consistent with our hypothesis, these effects occurred to a lesser extent in females.

Because obvious differences exist in growth rate and hormonal milieu, direct comparisons between females and males must be interpreted cautiously. It is nevertheless important to know the effect of corticosteroids in females and to consider our findings in males for contrast and perspective. The same differences that make comparison between genders problematic may afford relative protection to females from dexamethasone-induced diaphragm muscle atrophy. First, slower growth rates in females may reduce or prevent dexamethasone-induced catabolism. Because female growth rates are slower, any study comparing female and male animals must choose between matching initial weight or age. By choosing the former, it was necessary to use females older than their male counterparts; this is a potential drawback of the study. In male rats, youth increases the response of the diaphragm to corticosteroids, but this effect occurs only in neonates (30), which are far younger than those used in our study. Furthermore, we do not believe that age-related differences in sexual maturity were introduced into the study design because vaginal smears and serum testosterone levels (Table 2) demonstrate that both the female and male rats were sexually mature from the onset of this study (7, 17).

Second, stress and undernutrition inhibit female reproduction and induce anestrous (18). Immature ovarian follicles decrease estradiol and increase progesterone. Increased progesterone has been associated with increased in vivo respiratory muscle endurance (2). In our study, stress and undernutrition associated with pair feeding did reduce the number of rats undergoing normal estrous cycles only compared with rats fed ad libitum. No differences in ovarian status (estrous cycling or serum peak estradiol levels) were observed when dexamethasone-treated females were compared with pair-fed control animals. This emphasizes the importance of using pair-fed rather than ad libitum-fed control animals when corticosteroids are studied in females. If control animals had been ad libitum fed, estrous cycling would have been greatly reduced in the dexamethasone experimental group compared with control animals. If control animals had been "pair weighted" (severe dietary restriction), estrous cycling would have been reduced in the pair-weighted control animals compared with the dexamethasone group. Furthermore, severe undernutrition, as undoubtedly occurs with pair-weighted control animals, may increase serum cortisol levels (13), thereby further confounding the analysis.

Dexamethasone produced significant effects on serum testosterone levels, with an increase in females and a decrease in males. This has been reported after short-term dexamethasone treatment in bulls (1) and male rats (27). It remains unknown whether this occurs in humans. In females, in which androgen receptors are increased (4), an increase in serum testosterone might protect against corticosteroid-induced muscle atrophy. In males, a decrease in serum testosterone might accentuate or be partially responsible for corticosteroid-induced muscle atrophy. We have previously shown that testosterone absence (castrated males) decreases diaphragm contractility (23); testosterone treatment, albeit at substantially higher serum levels, increases diaphragm weight and contractility in normal female (25) and castrated male (23) rats; and our preliminary data in male rats (22), as well as recently published data in male rabbits (10), indicate that combined treatment (testosterone and dexamethasone) prevents the effects of corticosteroids on diaphragm structure and function.

Dexamethasone may also affect thyroid status. In humans, short-term dexamethasone treatment does not produce clinical hypothyroidism but does modestly reduce T₃ and thyroid hormone-binding globulin levels, increases reverse T₃, and variably affects thyroxine levels (3). In rats, the effect of corticosteroid treatment on thyroid hormone levels has not been reported. Compared with control animals, we found that dexamethasone treatment increased serum T₃ levels in both female and male rats and increased serum thyroxine levels in females (Table 2). We do not believe that alterations in thyroid hormones were a contributing cause for our results because, despite these relative changes, thyroxine and T₃ levels remained within normal limits (3) after dexamethasone treatment in both female and male rats. Furthermore, the effect of dexamethasone treatment on MHC-isoform expression (Table 3) was far more diverse than that reported for hypo- or hyperthyroidism. Hypothyroidism affects diaphragm muscle MHC-isoform expression by downregulating MHC-2B to undetectable levels without significantly affecting MHC-slow or MHC-2A, and hyperthyroidism upregulates MHC-2B expression (14).

Compared with pair-fed control animals, dexamethasone significantly decreased body weight, costal diaphragm weight, and costal fiber (types I and II) CSA. With the exception of type I fiber atrophy, effects were gender specific, with less weight reduction and type II fiber atrophy in treated females. Prior studies, with one exception (20), measured diaphragm fiber atrophy after short-term treatment (6 wk) in males but not females. Cortisone (3 wk) produced atrophy of all diaphragm fiber types (I, IIa, and IIb) in male rabbits (12). Dexamethasone (3 wk) produced type II diaphragm fiber atrophy in male hamsters (19). Triamcinolone (3-4 wk) produced types IIa and IIb diaphragm fiber atrophy in male hamsters (32) and type IIb diaphragm fiber atrophy in male rats (5, 6). Prednisolone produced type IIb diaphragm fiber atrophy in female rats (20). Potential reasons of why type II fibers are more susceptible to corticosteroid catabolism are 1) the number of cortisol specific and nonspecific receptors vary according to fiber type (16); 2) type I fibers are partially protected by their greater contractile activity (16); and 3) undernutrition, only partially controlled for by pair feeding, has been shown to produce type II, greater than type I, fiber atrophy (5, 24, 26).

The effect of corticosteroid treatment on diaphragm muscle MHC isoform expression has not been reported. At the start of this study, rat diaphragm MHC-isoform expression was known to have reached stable adult levels (15). Dexamethasone treatment significantly decreased relative expression of MHC-2B and increased relative expression of MHC-2A in both females and males (Table 3). This pattern is a reversal of the normal maturational change found in male rat diaphragms from postnatal day 14 to adult (15). We noted a significant decrease in MHC-2_all isoform expression with a reciprocal increase in MHC-1 expression only in dexamethasone-treated males because the MHC-2B isoform change occurred to a lesser extent in females. Because immunohistochemical fiber stains were not performed, we cannot state whether this correlation reflects homogeneous MHC isoform shifts within individual fibers or heterogenous MHC isoform shifts within the total population of fibers.

Despite costal diaphragm atrophy, dexamethasone did not significantly affect costal diaphragm-specific forces (Fig. 2, A and B). Specific forces were not significantly affected in all studies, including ours, where corticosteroids produced equivalent reductions in diaphragm and body weights (5, 12, 19, 28, 30, 32). Decreased diaphragm-specific forces have only been observed in studies where dexamethasone or triamcinolone reduced diaphragm mass out of proportion to body mass (6, 28, 31).

Fatigue resistance was significantly increased in males (Fig. 3B) but not females (Fig. 3A) after long-term dexamethasone treatment. Prior studies, again only in males, have shown diaphragm fatigue resistance to be unchanged (11, 21) or increased (21) after cortisone, increased after triamcinolone (5, 6, 32), and unchanged (28, 30) or decreased (19) after dexamethasone treatment. Disparate results might be explained by the degree of muscle atrophy and by differences in stimulation paradigms. Our study used a low-frequency (5-Hz) fatigue paradigm, whereas others used higher stimulation frequencies (20 Hz). In cortisone-treated rats, diaphragm fatigue resistance was increased or unchanged, depending on the stimulation duty cycle of the fatigue paradigm (21). In another model, acute starvation (26), diaphragm fatigue resistance was increased or unchanged, depending on the stimulation frequency of the fatigue paradigm (5 vs. 100 Hz).

Increased in vitro fatigue resistance in atrophic skeletal muscle could result from 1) an increase in substrate/oxygen supply due to a decrease in muscle thickness; 2) a shift in fiber types due to a relative increase in type I (fatigue resistant) fiber area; or 3) an increase in the oxidative capacity of all fibers, occurring independent of changes in fiber size (19). Diaphragm thickness increases with aging and decreases (in males) after long-term dexamethasone treatment (Table 1), but not to the extent where improved oxygen diffusion could explain dexamethasone-induced increases in fatigue resistance. Rather, the increase in fatigue resistance in males after long-term dexamethasone treatment may be explained by the relative increase in both MHC-1 isoform (more energy efficient) expression and the greater atrophy of type II (fatigue sensitive) than type I (fatigue resistant) fibers. This is also consistent with our finding that diaphragm twitch one-half relaxation time was prolonged in males after long-term dexamethasone treatment. Increased fatigue resistance was not observed in females after dexamethasone treatment because the magnitude of the change in MHC isoform expression and costal fiber atrophy was far less in females than in males.

In conclusion, dexamethasone significantly decreased body weight, costal diaphragm weight, and costal fiber (types I and II) CSA and the relative expression of the MHC-2B isoform while significantly increasing the relative expression of MHC-2A, twitch one-half relaxation times, and FRI values. These effects occurred to a lesser extent in females than in males. Serum testosterone levels, which increased in dexamethasone-treated females and decreased in dexamethasone-treated males, may be partially responsible for these gender-specific effects. Despite corticosteroid-induced diaphragm muscle atrophy, preservation of specific force and an increase in fatigue resistance allow for maintenance of ventilation under normal load conditions. However, these adaptations may be of limited value during periods of increased respiratory work when absolute force, decreased due to atrophy, may be of more functional relevance.

ACKNOWLEDGEMENTS

The authors appreciate Drs. T. K. Aldrich, M. H. Williams, J. Scheuer, B. Wittenberg, and J. Wittenberg for advice and support. We also thank Dr. K. Freeman for statistical advice; Dr. L. Brown for access to and advice in using the Quantimet imaging system; Dr. E. Bloch for help with rat vaginal cytologic analysis; and Dr. B. Thyssen for help with serum estradiol and testosterone measurements. 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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