Van Balkom, Roland H. H., Wen-Zhi Zhan, Y. S. Prakash, P. N. Richard Dekhuijzen, and Gary C. Sieck. Corticosteroid effects on isotonic contractile properties of rat diaphragm muscle.J. Appl. Physiol. 83(4): 1062–1067, 1997.—The effects of corticosteroids (CS) on diaphragm muscle (Diam) fiber morphology and contractile properties were evaluated in three groups of rats: controls (Ctl), surgical sham and weight-matched controls (Sham), and CS-treated (6 mg ⋅ kg−1 ⋅ day−1prednisolone at 2.5 ml/h for 3 wk). In the CS-treated Diam, there was a selective atrophy of type IIx and IIb fibers, compared with a generalized atrophy of all fibers in the Sham group. Maximum isometric force was reduced by 20% in the CS group compared with both Ctl and Sham. Maximum shortening velocity in the CS Diamwas slowed by ∼20% compared with Ctl and Sham. Peak power output of the CS Diam was only 60% of Ctl and 70% of Sham. Endurance to repeated isotonic contractions improved in the CS-treated Diam compared with Ctl. We conclude that the atrophy of type IIx and IIb fibers in the Diam can only partially account for the CS-induced changes in isotonic contractile properties. Other factors such as reduced myofibrillar density or altered cross-bridge cycling kinetics are also likely to contribute to the effects of CS treatment.
- skeletal muscle
- fiber type
- shortening velocity
corticosteroid (CS) treatment is common in the clinical setting, despite a variety of contraindications, including skeletal muscle myopathy. Recently, considerable attention has focused on the possibility that CS treatment impairs diaphragm muscle (Diam) function in patients with chronic obstructive pulmonary disease (1). In these patients, CS treatment appears to contribute to Diam weakness, further reducing their functional reserve capacity. To date, animal studies have examined only the effects of CS treatment on isometric properties of the Diam. However, an examination of only the isometric properties of the Diam may not reveal the true impact of CS treatment. The force-velocity relationship is an essential characteristic of Diam contractile properties, and, to date, there is very little information concerning the effects of CS treatment on the ability of the Diam to shorten. This may explain the equivocal results of animal studies reporting either no effect of CS treatment on maximum isometric specific force (Po; force normalized for muscle cross-sectional area) of the Diam (2, 3, 10, 13, 22) or only a small reduction in specific force (20).
As in other skeletal muscles, the maximum shortening velocity (V max) of Diam fibers displays a strong association with myosin heavy chain (MHC) isoform composition (8, 18). In the Diam, type IIx and IIb fibers, expressing the MHC2X and MHC2B isoforms, respectively (16,19), have a fasterV max than type I and IIa fibers, expressing the MHCslow and MHC2A isoforms, respectively. An effect of CS treatment on the force-velocity relationship of the Diam is suggested by the selective atrophy of type IIx and/or IIb fibers (2, 3, 12, 14, 20, 22). Accordingly, we hypothesize that, in the Diam, CS treatment is associated with a slowing ofV max.
Fiber type differences inV max also correspond to differences in power output, with type IIx and IIb fibers generating greater power than type I and IIa fibers (18, 19). If CS treatment selectively affects the size of type IIx and IIb fibers, then the power output of the Diamshould be reduced. The increased power output of type IIx and IIb Diam fibers is also associated with greater energetic demands, compared with type I and IIa fibers (18). Thus a reduction in the relative contribution of type IIx and IIb fibers to total Diam mass should result in an overall reduction in energy requirements. If muscle fatigue is related to an imbalance between energy supply and energy demand, the effects of CS treatment may be reflected by an improvement in fatigue resistance (rate of force decline) or endurance (duration of sustained power output). Indeed, previous studies have reported an improvement in isometric fatigue resistance of the rat Diam after CS treatment (13, 22). However, since energy requirements increase with power output (4, 18), the effects of CS treatment on improving endurance should be even more pronounced during repetitive isotonic shortening.
In the present study, we evaluated the effects of CS treatment on the isotonic contractile and endurance properties of the rat Diam. We hypothesized that CS treatment induces a selective atrophy of type IIx and IIb Diam fibers and that, as a result, there is a slowing ofV max, a decrease in power output, and an improvement in isotonic endurance.
Male Sprague-Dawley rats (initial body weights 315 ± 5 g) were divided into three groups: 1) untreated controls (Ctl; n = 8);2) surgical sham and weight-matched controls (Sham; n = 8); and3) CS-treated (CS;n = 8). All animals were housed in separate cages under a 12:12-h light-dark cycle, fed with Purina rat chow, and provided with water ad libitum. Animals in the Ctl and CS groups were provided food ad libitum, whereas rats in the Sham group were food restricted to match their weight growth curve with that of the CS group. Body weights were monitored daily in all groups.
All procedures used in this study were approved by the Institutional Animal Care and Use Committee of the Mayo Clinic and were in strict accordance with the American Physiological Society animal care guidelines. Surgical procedures were performed under aseptic conditions. The recovery of animals from surgery was carefully monitored.
CS treatment. Animals were anesthetized by the administration of ketamine (60 mg/kg im) and xylazine (2.5 mg/kg im), and a miniosmotic pump (Alzet 2M4) was implanted sucutaneously in the neck. In the CS group, the miniosmotic pump contained a 37.5 mg/ml aqueous suspension of prednisolone sodium succinate (Upjohn), whereas in the Sham group the pump contained a sterile physiological saline solution. Based on a flow rate of 2.5 μl/h for the osmotic pump, a dose of 6 mg/kg prednisolone was provided continuously for a 3-wk period. Measurements of the remaining amount of solution in the pump at the end of the 3-wk treatment period were used to estimate total drug delivery. At the terminal experiment, blood samples were obtained to measure prednisolone, 3,3′,5-triiodo-l-thyronine (T3), and thyroxine (T4).
Fiber type composition and morphology. After the 3-wk treatment period, the rats were anesthetized with pentobarbital sodium (70 mg/kg), and the right Diam was rapidly excised. Muscle segments were dissected from the midcostal region, and the resting excised length of the strip was measured by using digital calipers. The muscle strips were then stretched to 1.5 times this excised length [an approximation for optimal fiber length (L o) (15)], pinned on cork, and rapidly frozen in melting isopentane cooled to its melting point by liquid nitrogen.
Transverse sections of muscle fibers were cut at 6 μm by using a cryostat (Reichert Jung 2000E) kept at −20°C. The muscle sections were then reacted with antibodies to different MHC isoforms:1) mouse anti-MHCslow immunoglobulin (Ig) G (Novocastra) for identification of type I fibers by positive immunoreactivity; 2) mouse anti-MHC2A IgG (7) for identification of type IIa fibers by positive immunoreactivity;3) mouse anti-MHCall-2X IgG (16) for identification of type IIx fibers by negative immunoreactivity; and4) mouse anti-MHC2B IgM (16) for identification of type IIb fibers by positive immunoreactivity. After a 2- to 3-h incubation with the primary antibody, the sections were washed in 0.1 M phosphate buffer and incubated further in Cy3-conjugated donkey anti-mouse IgG or IgM.
The fluorescently stained sections were visualized by using an Olympus BH-2 microscope. Images of the stained muscle sections were digitized into a 1,024 × 1,024 array of picture elements (pixels) by using a charge-coupled diode camera attached to a calibrated image-processing system (19). With the use of a ×20 microscope objective, each pixel had a projected area of 0.15 μm2. The cross-sectional area of individual muscle fibers was determined from the number of pixels within the delineated boundary of the fiber. To determine fiber type proportions, ∼500 muscle fibers were sampled from each Diam. Cross-sectional areas were measured for at least 25 fibers of each type within a given muscle. The relative contribution of each fiber type to the total area of the muscle segment (an estimate of total mass whenL o was similar) was calculated based on the proportion and average cross-sectional area of each fiber type.
MHC isoform composition. The techniques for determination of MHC isoform composition of the rat Diam have been previously described (8, 19). Briefly, myosin was extracted from scissor-minced Diam tissue, the extracts were centrifuged, and supernatants were recovered. After overnight storage to allow precipitation of myosin filaments, the solution was centrifuged, and the pellet was dissolved in a sample buffer, boiled, and then stored frozen. Different MHC isoforms were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The identity of specific MHC bands in silver-stained gels had been previously determined by using immunoblotting techniques (9, 19). The relative composition of the different MHC isoforms was determined by densitometry, normalizing the average density of each band for the total peak densities for all the isoforms combined.
Contractile and endurance properties. Muscle strips (∼3 mm wide) were dissected from the midcostal region, with fiber insertions at the costal margin and central tendon left intact. The muscle strip was mounted vertically in a glass tissue chamber containing oxygenated mammalian Ringer solution of the following composition (mM): 135 Na+, 5 K+, 2 Ca2+, 1 Mg2+, 121 Cl−, 25 , 11 glucose, 0.3 glutamic acid, 0.4 glutamate, andN, N-bis(2-hydroxyethyl)-2-aminoethane-sulfonic acid buffer (pH = 7.4). A 0.0008% solution ofd-tubocurarine chloride was added to prevent neuromuscular transmission. The solution was oxygenated with 95% O2-5% CO2 and maintained at 26°C. The origin of the muscle bundle along the costal margin was attached to a metal clamp mounted in series with a micromanipulator at the base of the tissue chamber. The central tendon was glued to a thin, stiff plastic rod that was firmly fixed to the lever arm of a dual-mode length-force servo-control system (Cambridge Technologies, model 300B).
The muscle was stimulated directly by using platinum plate electrodes placed in close apposition on either side of the muscle. Rectangular current pulses (0.5-ms duration) were generated by using a Grass S88 stimulator and amplified by a current amplifier (Mayo Foundation, Section of Engineering). The stimulus intensity producing the maximum twitch force response was determined, and the stimulus intensity was set at ∼125% of this value for the remainder of the experiment (∼220 mA). Muscle preload was adjusted by using the micromanipulator until L o for maximal twitch force was achieved.
The Cambridge system was controlled by using custom-built software (LabView), implemented on an IBM 486 personal computer. Length and force were independently controlled, allowing the Cambridge system to operate either in isometric or isotonic modes, respectively. Length and force outputs were digitized by using a data-acquisition board (National Instruments) at a sampling frequency of 1 kHz.
Peak isometric twitch force (Pt) and Po (600-ms duration train) were measured. The force-velocity relationship of the Diam was then determined. While the muscle was maximally stimulated at 75 Hz for 330 ms, afterloads were clamped at values ranging from 3 to 100% of Po. A shorter stimulus duration was used to accommodate the limited range of lever movement of the Cambridge system during muscle shortening. At least 1 min intervened between each load level. The velocity of shortening at each load clamp was calculated as the change in muscle length (normalized forL o) during a 50-ms period. To eliminate the dynamics of connective and other noncontractile tissue in the muscle, the time window for this measurement was set to begin at 25 ms after the first detectable change in length. V maxwas calculated by fitting the force-velocity curve by using the modified Hill equation and extrapolating the fitted curve to zero-load (21).
Power output during isotonic contraction was calculated as the product of force and velocity, and the load clamp level yielding maximum power was determined. The load clamp was set to this value, and endurance was assessed during repetitive isotonic shortening induced by stimulating the muscle at 75 Hz in 330-ms duration trains repeated every second. The time at which power output declined to zero (no detectable muscle shortening) was defined as endurance time.
After the experiment, the muscle was weighed, and cross-sectional area was estimated based on the following formula: muscle weight (g)/[L o(cm) ⋅ 1.056 (g/cm3)]. Forces were then normalized for cross-sectional area of the muscle segments.
Statistical analysis. Data were compared by using a one-way analysis of variance followed by Duncan’s multiple-range test. Repeated-measures analysis of variance was used for analysis of force-frequency, force-velocity, and force-power relationships, as well as for the analysis of the decline in maximum power output during the isotonic fatigue test. Statistical significance was tested at the 0.05 level. All data were expressed as means ± SE.
Efficacy of CS treatment. After 3 wk of treatment, there was very little residual solution (<5% of total volume) remaining in the miniosmotic pumps. Prednisolone levels measured in blood serum of Ctl and Sham animals were below detectable levels (<0.5 μg/dl). In contrast, the serum prednisolone level measured in the CS-treated animals at the time of the terminal experiment was 4.9 ± 1 μg/dl. Serum T3 and T4 levels were not significantly different across the three experimental groups (Ctl: T3 46 ± 3 ng/dl, T4 4.0 ± 0.2 μg/dl; Sham: T3 48 ± 6 ng/dl, T4 4.2 ± 0.4 μg/dl; and CS: T3 47 ± 4 ng/dl, T4 3.9 ± 0.4 μg/dl).
Body weights. Over the 3-wk experimental period, body weights of Ctl animals increased by 26% (315 ± 7 g initial and 397 ± 9 g final body weights). In the CS and Sham animals, body weight gain was significantly reduced compared with Ctl (P < 0.05), increasing by only 6 and 4%, respectively (CS: 319 ± 5 g initial and 338 ± 9 g final body weights; Sham: 313 ± 7 g initial and 327 ± 9 g final body weights).
Fiber type composition and morphology. In all three experimental groups, fiber types could be readily classified by immunoreactivity for the different MHC antibodies. The incidence of coexpression of MHC isoforms appeared to be very low (<1%) in all three groups. However, it was not possible to detect coexpression of MHC2X and MHC2B isoforms by immunohistochemistry, and it is likely that such coexpression was more frequent (19). There were no differences across groups in the proportions of different fiber types (Table1).
In the CS-treated animals, cross-sectional areas of type IIx and IIb Diam fibers were significantly smaller than those of type IIx and IIb fibers in Ctl (P < 0.05; Table 1). In contrast, cross-sectional areas of type I and IIa fibers in the CS Diam were comparable to similar fiber types in Ctl animals. In the Sham Diam, there was a generalized atrophy of all fiber types compared with Ctl (P < 0.05; Table 1). Type I fibers in the Sham Diam were also smaller than type I fibers in the CS Diam(P < 0.05; Table 1). Cross-sectional areas of type IIa, IIx, and IIb fibers in the CS Diam were comparable to similar fiber types in Sham animals.
In the CS Diam, the relative contribution of type I fibers to total Diam cross-sectional area increased (P < 0.05; Table 1). Otherwise, there were no differences across groups in the relative contribution of different fiber types to total Diam cross-sectional area. However, the combined contribution of type IIx and IIb fiber areas was ∼60% of total Diamcross-sectional area in Ctl and Sham animals but only ∼48% in the CS group (Table 1).
MHC isoform composition. On the basis of electrophoretic separation, the relative expression of the MHC2B isoform decreased in the CS-treated Diam(P < 0.05; Table2). The MHC isoform composition of Ctl and Sham Diam was comparable (Table2).
Contractile and endurance properties.After 3 wk of CS treatment, Pt and Po of the Diam were reduced compared with both Ctl and Sham groups (P < 0.05, Table 3). Pt and Po were not different between Ctl and Sham animals. Compared with Ctl and Sham groups, the force-velocity relationships of the CS Diam were shifted to the left (P < 0.05; Fig.1 A). The V max of the CS Diam was significantly slower than that of both Ctl and Sham Diam(P < 0.05, Fig.1 B). In all Diam, peak power output occurred at ∼33% of Po and at 33% ofV max (Fig.2). Peak power output of the CS Diam was significantly lower than that of both Ctl and Sham groups (Fig. 2; P < 0.05). The peak power output of the Sham Diam was also slightly lower than that of Ctl animals (Fig. 2;P < 0.05).
With repetitive shortening contractions, maximum power output of the Diam rapidly declined in all three groups (Fig. 3;P < 0.05). After 60 s of repetitive contractions, Diam power output was comparable in all three groups (Fig. 3). However, given the differences in the initial peak power output of each group, the rate of decline in power was slower in the CS Diam compared with both Sham and Ctl animals (Fig. 3; P < 0.05). Endurance time of the CS Diam was 120 ± 6 s compared with 96 ± 4 s for Ctl (P < 0.05) and 108 ± 7 s for Sham (Fig. 3).
The results of the present study support our hypotheses that CS treatment induces a selective atrophy of type IIx and IIb fibers in the rat Diam, which is associated with a slowing ofV max, a reduction in power output, and an improvement in isotonic endurance. However, the CS-induced changes in Diamisotonic properties were disproportionately greater than the changes in type IIx and IIb fiber morphology and MHC isoform expression. Therefore, we conclude that, in addition to the selective atrophy of type IIx and IIb fibers, CS treatment exerts an influence on cross-bridge cycling kinetics.
Across the 3-wk period, the normal increase in body weight observed in Ctl rats was blunted by CS treatment. The final body weight of the CS-treated animals was ∼15% lower than that of Ctl rats. Because alterations in nutritional status alone can affect morphology and function of the rat Diam (2, 11,17), interpretation of the direct effects of CS treatment is confounded. However, the morphological and contractile adaptations of the Diam in the Sham group, where body weight was matched to that of the CS group by food restriction, were generally dissimilar to those observed in the CS-treated animals. These results suggest that the effects of CS treatment on Diam structural and functional properties cannot be solely attributed to a nonselective catabolic effect.
The CS-induced selective atrophy of type IIx and IIb Diam fibers observed in the present study is in general agreement with several previous studies (2,3, 12, 14, 20, 22). However, these previous studies did not classify fiber types based on expression of different MHC isoform, nor did they distinguish between type IIx and IIb fibers. Standard histochemical classification of fiber types based on the pH lability of myofibrillar adenosinetriphosphatase (ATPase) staining, as used in these studies, cannot distinguish type IIx fibers, which are abundant in the rat Diam (9, 19). In addition, the MHC2B isoform is often coexpressed with the MHC2X isoform. Therefore, it is not surprising that type IIx and IIb fibers displayed a similar pattern of atrophy in response to CS treatment. In the Sham Diam, there was a generalized atrophy of all fiber types, which has also been previously observed (2,10, 11, 17, 20, 22). When combined, type IIx and IIb fibers comprised ∼60% of both Ctl and Sham Diambut only ∼48% of the CS Diam. In the CS-treated Diam, there was a reduction in the relative expression of the MHC2B isoform, whereas no changes in MHC isoform expression were observed in the Sham group. When combined, MHC2X and MHC2B isoforms comprised ∼40% of the CS Diam compared with ∼49% of Ctl and ∼46% of Sham Diam. The relatively modest change in MHC isoform composition of the CS Diam was consistent with the normal T3 and T4 levels of these animals. Clearly, the CS-induced morphological adaptations of type IIx and IIb Diam fibers and the alterations in MHC isoform expression were not as pronounced as the changes in isotonic contractile properties.
Three weeks of CS treatment resulted in a 20% reduction in Po compared with both Ctl and Sham groups. These results are in agreement with the previous report of van Balkom et al. (20) but contrast with several other studies in which no effect of CS treatment on DiamPo was observed (2, 3, 10, 13,22). The reasons for these discrepant results are unclear but may relate to the type, dose, and duration of CS treatment used. It is unlikely that the reduction in specific force of the CS-treated Diam observed in the present study was attributable only to the selective atrophy of type IIx and IIb fibers or the reduction in MHC2Bisoform expression. A reduction in specific force could also arise from a number of alternative mechanisms, including a decrease in myofibrillar density and/or changes in cross-bridge cycling kinetics. Lieu and colleagues (12) reported that CS treatment is associated with a reduction of myofibrillar and sarcoplasmic protein concentration in the rat Diam, albeit not as pronounced as in the plantaris muscle. Such alterations in myofibrillar and sarcoplasmic protein concentration could reflect a decrease in the number of available cross bridges and/or changes in calcium handling.
The force-velocity relationship of the Diam was altered by CS treatment such that V maxwas slowed by ∼20% and peak power output was reduced by 40% compared with Ctl animals. The slowing ofV max in the CS Diam is generally consistent with the selective atrophy of type IIx and IIb fibers and the reduction in MHC2B expression. However, the slowing of V maxinduced by CS treatment was substantially greater than that which would be predicted by the relatively modest reduction in MHC2B expression. Therefore, it is unlikely that the slowing ofV max in the CS-treated Diam was solely attributable to a selective atrophy of type IIx and IIb fibers and/or the reduction in MHC2B expression. In muscle fibers, V max is correlated with actomyosin ATPase activity (18) and cross-bridge cycling rate. Type I and IIa fibers have lower actomyosin ATPase activities than type IIx and IIb fibers (18, 19) and, as a result, a slower V max. It is possible that the slowing ofV max in the CS-treated Diam reflects a decrease in actomyosin ATPase activity of muscle fibers independent of MHC isoform expression.
In all groups, the Diam displayed very rapid fatigue during repetitive isotonic contractions at a load corresponding to peak power output. During shortening contractions, muscle fiber energy utilization increases (4, 18); thus the rapid fatigue may be related to an imbalance between energy utilization and energy production. CS-treated animals displayed a slower rate of power decrement during repetitive isotonic contractions compared with Ctl and Sham groups and prolonged endurance time compared with Ctl rats. These results are in general agreement with the improved fatigue resistance during repetitive isometric contractions noted in previous studies (13,22). However, the results of the present study are in contrast to the report of Ferguson and colleagues (5), who found that CS-treated rabbits displayed less endurance to an incremental inspiratory threshold load. However, Diamfatigue was not directly verified in this study, and respiratory failure, used to define endurance, could have resulted from a number of mechanisms other than Diamfatigue.
The results of the present study suggest that CS treatment reduces energy utilization during repetitive isotonic contractions and thus improves the balance between energy supply and energy demand. A reduction in energy utilization would result from the selective atrophy of type IIx and IIb fibers, which have higher actomyosin ATPase activities (18, 19). In addition, as suggested above, CS treatment may directly reduce actomyosin ATPase activity independent of MHC isoform expression. Other studies have also suggested that CS treatment impairs muscle energy utilization. For example, after CS treatment, there is an accumulation of glycogen (5) and a reduction in creatine kinase activity (6). There may also be an effect of CS treatment on energy production. For example, it has been reported that CS treatment reduces citrate synthase activity in the rat Diam (12, 20). However, no effect of CS treatment on succinate dehydrogenase activity was observed (10).
In conclusion, CS treatment causes a reduction in specific force, a slowing of V max, a decrease in power output, and an improvement in endurance during repetitive isotonic contractions. These contractile adaptations are generally consistent with the selective atrophy of type IIx and IIb fibers and the reduction in MHC2Bexpression that was observed in the CS Diam. However, the contractile adaptations are disproportionately greater than the morphological changes induced by CS treatment. Therefore, we conclude that the impairment of Diam function associated with CS treatment involves additional mechanisms including a reduction in myofibrillar density and/or a slowing of cross-bridge cycling kinetics.
The authors are grateful to Yun-Hua Fang for her assistance in this study.
Address for reprint requests: G. C. Sieck, Anesthesia Research, Mayo Clinic, Rochester, MN 55905 (E-mail:).
This work was supported by National Heart, Lung, and Blood Institute Grants HL-34817 and HL-37680 and by Grant 92.17 from the Dutch Asthma Foundation.
- Copyright © 1997 the American Physiological Society