GLUCOCORTICOID-INDUCED ALTERATIONS IN THE RATE OF DIAPHRAGMATIC FATIGUE

Pharmacological Research, Vol. 42, No. 1, 2000 doi:10.1006rphrs.1999.0658, available online at http:rrwww.idealibrary.com on

GLUCOCORTICOID-INDUCED ALTERATIONS IN THE RATE OF DIAPHRAGMATIC FATIGUE LOUISE FLETCHER, SCOTT K. POWERSU , JEFF S. COOMBES, HAYDAR DEMIREL, HEATHER VINCENT, STEPHEN L. DODD and JAMES MCLAUGHLIN Department of Exercise and Sport Sciences and Physiology, Center for Exercise Science, Uni¨ ersity of Florida, Gaines¨ ille, FL 32611, USA Accepted 16 December 1999

These experiments tested the hypothesis that in ¨ itro diaphragmatic fatigue resistance is enhanced in animals treated with glucocorticoids. Female Sprague]Dawley rats Ž4 months old. were randomly assigned to a control Ž N s 12. or glucocorticoid treatment group Ž N s 12.. Treatment animals were injected daily for 8 days with prednisolone Ž5 mg kgy1 .; control animals were injected with the same volume of the vehicle. Twenty-four hours after the last injection, the following in ¨ itro diaphragmatic contractile properties were examined in costal diaphragm strips: maximal twitch ŽPt . half time to peak tension Ž1r2 TPT., half relaxation time Ž1r2 RT., force-frequency relationship, and the rate of fatigue development. Diaphragmatic fatigue was assessed by monitoring the decrease in force production Žmeasured as percent of initial force. over a 60-min contractile period. The in ¨ itro fatigue protocol incorporated a supramaximal stimulus delivered at 30 Hz every 2 s with a train duration of 250 ms Žduty cycle 12.5%.. Citrate synthase ŽCS., superoxide dismutase ŽSOD., and water content of the costal diaphragm were also determined. Glucocorticoid administration induced an 18.9% Ž P- 0.05. decrease in animal body weight when compared to the control. Similar weight losses also occurred in the diaphragm with a decrease Ž P- 0.05. in mass of 16.5% compared to the control. Furthermore, prednisolone treatment resulted in a significant reduction in the cross-sectional area ŽCSA. of type IIb fibres with no change in the CSA area of type I and IIa fibres. 1r2 TPT and 1r2 RT were significantly prolonged Ž P- 0.05. in the glucocorticoid treated rats whereas the force-frequency curve was unaltered Ž P) 0.05.. Fatigue resistance was greater in the glucocorticoid group Ž P- 0.05.; the relative force production differed between groups at the end of 1 min of contractions and remained different throughout the 60-min fatigue protocol. Citrate synthase, SOD, and water content were not different between groups. These experiments support the hypothesis that costal diaphragm strips of glucocorticoid-treated rats possess a greater resistance to fatigue. We postulate that this fatigue resistance is due to glucocorticoid-induced changes muscle fibre type composition. Q 2000 Academic Press

KEY WORDS: glucocorticoid, fatigue, costal diaphragm, half time to peak tension, half relaxation time, force-frequency relationship, citrate synthase, superoxide dismutase.

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Corresponding author.

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Q 2000 Academic Press

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INTRODUCTION

Glucocorticoids are frequently used to treat a variety of diseases including asthma, chronic obstructive pulmonary disease, chronic allergic rhinitis, systemic lupus, sarcoidosis, and pulmonary fibrosis w1x. Patients taking these drugs often experience weakness as a result of steroid induced myopathy and atrophy of skeletal muscles w2]4x. Indeed, it has long been known that glucocorticoids cause atrophy of locomotor muscles; however, only recently has it been shown that the diaphragm is also susceptible to steroid-induced atrophy w5]9x. A number of animal studies have examined the endurance capacity of the diaphragm in ¨ itro after glucocorticoid administration w5, 8, 10]15x. Unfortunately, the results of these studies provide conflicting results. A possible explanation for the controversy is the wide variation in the fatigue protocols employed. Differences between studies include the duration of the fatigue protocol, the stimulation frequency and duty cycle employed, and the type of contraction Žisotonic or isometric.. When studying diaphragmatic fatigue in ¨ itro it is important to induce a pattern of fatigue similar to that of the diaphragm in ¨ i¨ o. A gradual fatigue is more likely to develop in ¨ i¨ o than rapid fatigue w16, 17x. Most of the aforementioned reports used stimulation paradigms that induced rapid fatigue of the diaphragm which greatly exceed the rate of fatigue predicted in physiological conditions. In addition, numerous studies used isotonic contractions; these protocols are energetically demanding and also induce rapid fatigue of the diaphragm w10, 18x. Therefore, the previous investigations studying the effects of glucocorticoids on diaphragmatic fatigue may have limited physiological relevance. Additional research using contractile protocols that induce a pattern of fatigue similar to that of the diaphragm in ¨ i¨ o is warranted. This forms the basis for the current experiments. Hence, the purpose of this study was to determine whether glucocorticoid administration alters the fatigue resistance of the costal diaphragm. We postulated that glucocorticoid administration would enhance the fatigue resistance of the costal diaphragm. Our rationale for this postulate is that glucocorticoid administration promotes atrophy of the fast fatiguing type IIb and IIdrx fibres and has no effect on the slow fatiguing type I and type IIa fibres w5, 7, 13, 14x. Therefore, compared to control animals, diaphragms of glucocorticoid-treated rats would contain a higher percentage of fatigue resistant fibres. Thus, it is possible that fatigue resistance would be enhanced in the glucocorticoid-treated diaphragm strips. To test this hypothesis, diaphragmatic contractile performance was examined in ¨ itro using a

stimulation protocol designed to mimic the pattern of in ¨ i¨ o diaphragmatic fatigue.

METHODS

Animals Twenty-four female 4-month-old Sprague]Dawley rats were fed rat chow and water ad libitum and maintained on a 12-h lightrdark photo-period. Rats were randomly assigned to one of two groups Ž N s 12 per group.. The glucocorticoid-treated animals were injected subcutaneously with prednisolone Ž5 mg kgy1 dayy1 . suspended in an isotonic vehicle Ž1% aqueous carboxymethylcellulose in saline. for 8 days. The control group were sham-injected daily with the same volume of the vehicle. All injections were performed at approximately the same time of the day in both groups. Animal mass was recorded daily and glucocorticoid doses were adjusted to changes in body mass.

Muscle preparation for measurement of in vitro contractile properties At the completion of the 8-day treatment protocol, rats were injected with 90 mg kgy1 of sodium pentobarbital intraperitoneally. After reaching a surgical plane of anaesthesia the entire diaphragm was removed and placed immediately into a dissecting chamber containing Krebs]Hensleit solution aerated with a 95% O 2r5% CO 2 gas. A muscle strip including the tendinous attachments at the central tendon and rib cage Ždimensions s; 2 = 0.5 cm. was dissected from the ventral costal region. The diaphragm strip was then suspended vertically between two light weight Plexiglass clamps and connected to a transducer ŽGrass, model FT10. in a jacketed tissue bath. The bath contained Krebs] Hensleit solution with 12 m M D-tubocurarine to produce complete neuromuscular blockade. The jacketed tissue bath was aerated with gas Ž95% O 2r5% CO 2 ., osmolality was ; 290 mosmol, and pH was maintained at 7.4. The temperature of the bath was maintained at 24 " 0.58C, with the exception of the force frequency and the fatigue protocol which was maintained at 37 " 0.58C.

Determination of optimal length]tension relationship After 15 min of equilibration in the bath Žtemperature s 24 " 0.58C., the diaphragm strip was stimulated Žmodified Grass Instruments S48 stimulator. along its entire length with platinum wire electrodes to determine optimum contractile length Ž Lo . at which maximal tetanic tension was obtained. Determination of Lo was accomplished by systematically adjusting the length of the muscle using a micrometer while evoking isometric tetanic

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contractions. Muscle contractions were produced with a train of supramaximal Ž; 120 V. impulses Ž330 ms. each of which was 2 ms in duration delivered at 100 Hz. Thereafter, all contractile properties were measured at Lo . Force was monitored by a force transducer ŽGrass, model FT10.. The Grass transducer output was amplified and differentiated by operational amplifiers and underwent ArD conversion for analysis with a computer based data acquisition system ŽGW Instruments-Series II..

Isometric twitch contractions Peak isometric twitch tension was determined from a series of single pulses Ž2-ms duration.. Half relaxation time Ž1r2 RT. and half time to peak tension Ž1r2 TPT. were determined following a maximal isometric twitch by using computer generated algorithms.

Peak isometric tetanic contractions An isometric tetanic contraction was produced using a supra-maximal Ž; 120 V. stimulus train of 100 Hz, 2-ms pulse width, and 330-ms duration. Peak isometric tetanic tension was determined from three stimulations with a 2-min recovery between measurements.

Force]frequency relationship The force frequency relationship was determined using the tetanic tension stimulation parameters described above. The temperature of the bath was increased to 378C" 0.5. Maximal tetanic specific force ŽN cmy2 . was measured at 15, 30, 40, 50, 80, 120, 160, and 200 Hz. Two minutes of recovery separated each contraction.

Rate of fatigue In the context of these experiments we have defined fatigue as the rate of decline in diaphragmatic force production. This was measured at 37 " 0.58C by monitoring the decrease in isometric force production over a 60-min contractile protocol. The costal diaphragm strip was stimulated by unfused tetanic contractions using a supramaximal stimulus train of 30 Hz every 2 s with a train duration of 250 ms. The ratio of the period of muscle contraction to rest Žduty cycle. was 12.5%. At the completion of all contractile measurements, Lo was measured using calipers with the strip still suspended between the two plexiglass clamps. The total muscle cross-sectional area at right angles to the long axis was calculated by the following formula w19x: total muscle cross-sectional area Žcm2 . s wet mass Žg.rŽfibre length = 1.06..

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liquid nitrogen and stored at y808C until assay. Biochemical assays were conducted to determine muscle water content and citrate synthase ŽCS. and superoxide dismutase ŽSOD. enzyme activity. CS and SOD were analysed as markers of oxidative and antioxidant capacities, respectively.

Total muscle water content Water content was measured to determine any glucocorticoid-induced alterations in percent water and dry mass in the diaphragm. Total water content in the costal diaphragm was determined by using a freeze drying technique incorporating a vacuum pump with a negative pressure of ;y1 mmHg. The frozen samples were placed in a vacuum chamber and dried before measuring the dry mass. Freeze drying was terminated when the same weight was recorded three times in succession during a 6-h interval. Muscle water content was calculated as the difference between the wet weight and the dry weight of the same sample.

Tissue homogenization and determination of enzyme acti¨ ity The minced muscle sample Ž100 mg. was added to 2 ml of cold 100 mM phosphate buffer in a 15-ml glass homogenization tube. The homogenization process consisted of eight passes of the glass pestle through the homogenate using a low speed Ž; 50 rpm. motorized homogenizer ŽEberbach ConTorque, Ann Arbor, MI.. At the completion of eight passes an additional 8 ml of cold 100 mM phosphate buffer was then added and two additional passes of the pestle through the homogenate were performed. Homogenates were then centrifuged Ž48C; 400 g = 10 min. to remove the insoluble connective tissue from the homogenate. The supernatant was then removed and assayed for CS and SOD activity. These enzymes were chosen as representative markers of diaphragmatic oxidative and antioxidant capacities, respectively. Citrate synthase activity was analysed using a modified version of the technique described by Srere w20x. Superoxide dismutase activity was determined using the technique based on epinephrine auto oxidation described by Sun and Zigman w21x. All enzyme assays were performed at 258C in duplicate unless a variability of greater than 5% existed. If this was the case, subsequent assays were performed until two trials differed by 5% or less. The intra-assay coefficients of variation with the technicians for CS and SOD were approximately 2% and 3%, respectively.

Muscle fibre typing and morphometry Muscle preparation for biochemical assays After removal of fat and tendon, the costal diaphragm was blotted dry and weighed. The diaphragm was divided into sections and frozen in

To determine the effects of glucocorticoids on diaphragm muscle fibre types and morphometry, fibres were classified using myosin adenosine triphosphatase ŽATPase. histochemistry and the nomencla-

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ture of Brooke and Kaiser w22x. Fibre cross-sectional area ŽCSA. was then determined using computerized image analysis. A brief overview of this process follows. Six control and six glucocorticoid diaphragm strips were placed on aluminum foil Žresting, unstressed length. and rapidly frozen in liquid nitrogen. Serial cross-sections of each muscle were then cut at 12 m m in a cryostat maintained at y208C. Muscle fibres were classified as type I, IIa, or IIb based on their staining for myofibrillar ATPase after alkaline ŽpHs 10.3. and acid ŽpHs 4.6 and 4.3. preincubation using a modification of the procedure described by Brooke and Kaiser w22x. Briefly, the duration of the acid and alkaline preincubation was 7 and 20 min, respectively. Following preincubation, the tissue sections were rinsed in an 18 mM CaCl 2 Ž100 mM tris, pHs 7.8. solution and then placed in another incubation medium containing 4.5 mM ATP Ž100 mM Sigma 221 buffer, pHs 9.4.. The proportions of fibre types were determined from a sample of 200]300 fibres across the entire section of each muscle. After completion of myofibrillar ATPase stains, the different fibre types were identified and labelled on a photomicrograph. The CSA of each fibre was determined from non-dehydrated sections using computerized planimetry with the system being calibrated by a stage micrometer immediately prior to measurement. Note that this technique of fibre typing is not capable of separating type IIb and IIdrx fibres. Therefore, fibres classified as type IIb using the Brooke and Kaiser nomenclature include muscle fibres that express both IIb and IIdrx myosin heavy chains.

Statistical analysis Comparisons between groups for the dependent variables were made by independent t-tests and repeated measures analysis of variance. When appropriate, contrasts were used to analyse where significant differences occurred. When contrasts were applied, data were corrected for multiple comparisons according to Bonferroni’s procedure. Significance was established at P- 0.05.

Fig. 1. Loss of body mass during 8 days of glucocorticoid administration. Weights shown are 24 h after each injection of prednisolone. Values are means " SEM. U indicates group differences Ž P - 0.05..

RESULTS Glucocorticoid administration for 8 days induced an 18.9% Ž P- 0.05. decrease in body weight when compared to the control ŽFig. 1.. The costal diaphragm of rats treated with prednisolone also experienced a significant weight loss Ž P- 0.05.. The mean costal diaphragm mass of the treatment group Ž420 " 7.7 mg. was 16.5% lower than that of the control group Ž502.8" 7.5 mg.. The optimal length of the costal strip did not differ Ž P) 0.05. between groups, however, strip weight was less Ž P- 0.05., and CSA smaller Ž P- 0.05., in glucocorticoid treated costal diaphragm strips. These results are reported in Table I. The contractile properties of the costal diaphragm in ¨ itro strip are reported in Table II. Maximal tetanic force ŽN cmy2 . did not differ between groups Ž P) 0.05., however, maximal twitch force ŽN cmy2 . was higher in the glucocorticoid treated group Ž P0.05.. Both 1r2 TPT and 1r2 RT were significantly

Table I Morphometric characteristics of control and glucocorticoid-treated rats, and properties of costal diaphragm

Initial body mass Žg. Final body mass Žg. Costal diaphragm Žmg. Lo Žcm. CSA Žcm2 . Strip weight Žmg.

Control (n s 12)

Glucocorticoid (n s 12)

P-¨ alue

280.3" 3.8 276 " 3 502.8" 7.5 2.16" 0.05 0.01088" 0.0008 24.8" 1.8

281.1" 4.9 223.8" 3.4 420 " 7.7 2.18" 0.04 0.00823" 0.0004 18.9" 0.8

NS - 0.05 - 0.05 NS - 0.05 - 0.05

Notes. Values are means " SEM. NS, non-significant Ž P) 0.05.; Lo , optimal length of in ¨ itro costal strip; muscle weight s wet weight; strip weight s in ¨ itro costal strip weight; CSAs in ¨ itro costal strip cross-sectional area.

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Fig. 2. Force-frequency relationship of costal diaphragm strips from control and glucocorticoid-treated animals. Values are means " SEM.

prolonged Ž P- 0.05. in the glucocorticoid-treated group. No differences Ž P) 0.05. in the force frequency curve existed between groups ŽFig. 2.. Development of fatigue Žmeasured as percent of initial force. occurred at a faster rate Ž P- 0.05. in the control diaphragms compared to glucocorticoid treated diaphragms ŽFig. 3.. Indeed, glucocorticoidtreated costal diaphragm strips demonstrated a greater resistance to fatigue by the end of the first minute and prevailed for the remainder of the 60-min protocol. Table III contains SOD and CS activities of the in ¨ itro costal diaphragm strip. Note that both CS and SOD activity did not differ Ž P) 0.05. between groups. Glucocorticoid treatment did not alter Ž P) 0.05. the percentage of water or dry mass in the costal diaphragm. Percent dry mass of the control and glucocorticoid treated groups were 31.3" 0.3 and 30.7" 0.3, respectively. Table IV contains the CSA of costal diaphragm fibres in both control and glucocorticoid-treated animals. Glucocorticoid treatment did not alter fibre number in any of the three fibre classifications Ždata not shown.. However, prednisolone treatment resulted in a significant reduction in the CSA of type IIb fibres with no change in the CSA area of type I

Fig. 3. Sixty minutes in ¨ itro fatigue protocol using costal diaphragm strips from control and glucocorticoid-treated animals. Values are means " SEM. v indicates group differences Ž P- 0.05..

and IIa fibres. Furthermore, glucocorticoid treatment resulted in a significant decrease Ž P- 0.05. in the percent of the diaphragm made up by IIb fibres; there was a corresponding increase Ž P- 0.05. in the percentage of IIa fibres. DISCUSSION

O¨ er¨ iew of major findings While many investigators have studied the effects of glucocorticoids on the fatigue rate of the costal diaphragm, to our knowledge none have used a long-duration fatigue protocol designed to induce a pattern of fatigue similar to that of the diaphragm in ¨ i¨ o. This study, using a 60-min fatigue protocol, demonstrates that the fatigue resistance of the rat costal diaphragm is enhanced after 8 days of prednisolone treatment Ž5 mg kgy1 .. Furthermore, our data indicate that glucocorticoid treatment results in a selective atrophy of diaphragmatic type IIb fibres.

Glucocorticoids and diaphragmatic atrophy Glucocorticoid administration for 8 days induced a significant loss Ž19%. in body mass; this decrease

Table II In vitro contractile properties of costal diaphragm strips from control and glucocorticoid-treated rats

Maximal tetanic Po ŽN cmy2 . Maximal twitch Pt ŽN cmy2 . 1r2 TPT Žms. 1r2 RT Žms.

Control (n s 12)

Glucocorticoid (n s 12)

P-¨ alue

24.5" 0.53 8.49" 0.24 65.42" 1.16 50.02" 2.35

24.8" 0.56 9.35" 0.33 71.47" 2.06 64.06" 2.94

NS - 0.05 - 0.05 - 0.05

Notes. Values are means " SEM; NS, non-significant Ž P) 0.05.; Po , isometric tetanic specific force; Pt , isometric twitch specific force; 1r2 TPT, half time to peak tension; 1r2 RT, half relaxation time.

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Table III SOD and CS activities of control and glucocorticoidtreated costal diaphragm strips

SOD CS

Control (n s 12)

Glucocorticoid (n s 12)

P-¨ alue

4052.2" 156.6 62.5" 2.4

4227.2" 139.3 68.6" 2.2

NS NS

Values are means " SEM; NS, non-significant Ž P) 0.05.; SOD, superoxide dismutase activity Žunits miny1 100 mg proteiny1 .; CS, citrate synthase activity Ž m m miny1 100 mg proteiny1 ..

is within the range reported in previous studies w5, 6, 8, 9, 11, 12, 23x. The decrease in muscle mass of the costal diaphragm paralleled the loss in body mass. The present study ŽTable IV. and previous reports indicate that the glucocorticoid-induced decrease in diaphragmatic mass is due to the atrophy of both type IIb and IIdrx muscle fibres w5, 7, 13, 14, 23, 24x.

Glucocorticoids and diaphragmatic fatigue The principle finding of this study is that glucocorticoid treatment enhances fatigue resistance of the costal diaphragm. This observation is supported by Wilcox et al. w14x and Dekhuijzen et al. w5x but differs from other studies that reported no change in fatigue resistance w5, 8, 10]12x, or a decrease in fatigue resistance w8, 13, 15x. These conflicting results are possibly due to the many differences between the experimental fatigue protocols. Although the present study cannot provide a definite explanation for the enhanced fatigue resistance, a possible explanation is as follows. Again, the current data and the work of others clearly demonstrates that glucocorticoid administration causes atrophy of the fast fatiguing type IIb and IIdrx fibres with no effect on the slow fatigue resistant type I and type IIa fibres w5, 7, 13, 14, 24x. Therefore, compared to control animals, diaphragm strips of prednisolone-treated rats have a higher percentage of the total CSA that is composed of fatigue resis-

Table IV (a) Muscle fibre cross-sectional area (CSA) in the costal diaphragm. (b) Fibre type composition in costal diaphragm computed as % fibres by CSA Group

Type I

Type IIa

Type IIb

2

(a) Fibre CSA (m m ) Control 1087 " 69 1102 " 71 Glucocorticoid 1144 " 56 1221 " 66

2688 " 128 2084 " 71U

(b) Diaphragm composition, % fibre by area Control 23.7" 2.6 30.7" 2.5 42.6" 3.3 Glucocorticoid 27.3" 2.3 38.0" 2.1U 34.7" 2.1U U

Significantly different Ž P - 0.05. from the control. Note. The fibres classified as type IIb also include type IIdrx fibres. Values are means " SEM.

tant fibres Ži.e. type I and IIa. ŽTable IV.. Furthermore, the differences in the rate of fatigue development between control and glucocorticoid treated diaphragms is consistent with the notion that the fatigue resistance between the two experimental groups is due to fibre type differences ŽFig. 3.. Although diaphragm fatigue resistance is improved following prednisolone treatment, it seems likely that the maximal Žabsolute. force production of the diaphragm is reduced due to atrophy of the type IIb and IIdrx fibres. Hence, it seems likely that the total work performed by the diaphragm during the 60-min fatigue protocol would be lower in the glucocorticoid-treated animals compared to the control. The physiological significance of this event is unclear and warrants further investigation using in ¨ i¨ o models.

Glucocorticoids and twitch contractile properties Many studies investigating the effect of glucocorticoids on diaphragmatic contractile properties have not reported a change in either 1r2 TPT or 1r2 RT w5, 8, 9, 11]13x. In the present study, however, both 1r2 TPT and 1r2 RT were prolonged in the costal diaphragm of rats treated with prednisolone. These results are in agreement with the work of Wilcox et al. w14x and Dekhuijzen et al. w5x. The mechanism to explain this prolongation in the active state of the diaphragm has not been determined, although a number of possibilities exist. Due to the selective atrophy of type IIb and IIdrx fibres, the diaphragm of glucocorticoid-treated animals contains a relatively higher percentage of type I and type IIa fibres as computed by CSA ŽTable IV.. Because slow Žtype I. fibres contract and relax more slowly than fast twitch fibres, a higher proportion of slow fibres in the diaphragm may result in slower contraction and relaxation times. Additional explanations for the prolongation of 1r2 TPT and 1r2 RT are a change in myosin ATPase activity toward a slower isoform, changes in calcium flux in the sarcoplasmic reticulum, and changes in the elastic components of the diaphragm. In this regard, Shoji et al. w25x demonstrated that calcium-dependent ATPase activity and calcium uptake by the sarcoplasmic reticulum were reduced in human lateral vastus and rabbit quadriceps treated with glucocorticoids. If these changes also occur in the rat costal diaphragm, it is feasible that these alterations may alter contraction and relaxation times.

Critique of experimental model The rat was chosen as the experimental model because the degree of glucocorticoid-induced atrophy and the fibre type composition in the rat costal diaphragm is similar to that reported in humans w26, 27x. Four-month-old rats were used because, by this age, body weight has reached a plateau so growth

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would not be a confounding factor and the extent of the glucocorticoid-induced atrophy could be accurately determined. Prednisolone was chosen as the experimental drug because it is prototypical of non-fluorinated glucocorticoids used to treat human inflammatory diseases w1x. Using a scaling formula for inter-species scaling of equivalent drug doses, a 5 mg kgy1 in the rat is equated to ; 0.9 mg kgy1 in humans w28x. Therefore, the dose used in this study is similar to doses Ž0.5]1 mg kgy1 dayy1 . used to treat inflammatory diseases in humans w1x. The in ¨ itro fatigue protocol employed in this study was designed to induce a pattern of fatigue similar to that of the diaphragm in ¨ i¨ o. The temperature of the bath was maintained at 378C to mimic physiological temperature. The rationale for using a low duty cycle of 12.5% was based on findings demonstrating that increases in the duty cycle above 12.5% accelerate muscular fatigue w18, 29x. We choose 30 Hz as the stimulation frequency in our fatigue protocol because frequencies of 10]30 Hz represent the physiologic range of intrinsic neural firing rates w30, 31x. An isometric protocol was chosen because studies have shown that in ¨ itro isotonic fatigue protocols induce rapid fatigue of the diaphragm w10, 18x. Therefore, since a slow rate of fatigue would be more likely to develop in humans being treated with glucocorticoids than rapid fatigue w16, 17x, an isometric fatigue protocol was chosen over an isotonic fatigue protocol.

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3. 4. 5.

6. 7.

8. 9. 10. 11. 12.

13.

CONCLUSIONS This study demonstrates that the fatigue resistance of the rat costal diaphragm is enhanced after 8 days of prednisolone treatment. We postulate that the physiological mechanism to explain these results could be due to the selective atrophy of type IIb and IIdrx fibres. The glucocorticoid-induced selective atrophy would cause an increase in the percentage of total CSA that is composed of the slow and fatigue resistant type I and type IIa fibres and may contribute to the enhanced fatigue resistance illustrated in this study.

ACKNOWLEDGEMENTS This work was supported by a grant from the American Lung Association-Florida ŽSKP..

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