QUANTITATIVE AND QUALITATIVE ADAPTATIONS OF MUSCLE FIBERS TO GLUCOCORTICOIDS MARCO ALESSANDRO MINETTO, MD, PhD,1 RIZWAN QAISAR, MD, PhD,2 VALENTINA AGONI, MS,2 GIOVANNA MOTTA, MD,1 EMANUELA LONGA, MD,2 DANILO MIOTTI, MD,3 MARIA ANTONIETTA PELLEGRINO, MS, PhD,2 and ROBERTO BOTTINELLI, MD, PhD2 1

Division of , Endocrinology, Diabetology and Metabolism, Department of Medical Sciences, University of Turin, Corso Dogliotti 14, 10126, Turin, Italy 2 Department of Molecular Medicine, University of Pavia, Pavia, Italy 3 Fondazione Salvatore Maugeri, Scientific Institute of Pavia, Pavia, Italy Accepted 7 January 2015 ABSTRACT: Introduction: The aim of this study was to understand the effects of short-term glucocorticoid administration in healthy subjects. Methods: Five healthy men received dexamethasone (8 mg/day) for 7 days. Vastus lateralis muscle biopsy and knee extension torque measurement were performed before and after administration. A large number of individual muscle fibers were dissected from the biopsy samples (pre-administration: n 5 165, post-administration: n 5 177). Results: Maximal knee extension torque increased after administration (13%), whereas both type 1 and type 2A fibers had decreased cross-sectional area (type 1: 11%, type 2A: 17%), myosin loss (type 1: 18%, type 2A: 32%), and loss of specific force (type 1: 24%, type 2A: 33%), which were preferential for fast fibers. Conclusion: Short-term dexamethasone administration in healthy subjects elicits quantitative and qualitative adaptations of muscle fibers that precede (and may predict) the clinical appearance of myopathy in glucocorticoidtreated subjects. Muscle Nerve 52: 631–639, 2015

Steroid myopathy is a well-known side effect of endogenous or exogenous glucocorticoid excess and results from reduced protein synthesis, increased protein breakdown, and impaired mitochondrial function.1,2 It remains to be elucidated which of these mechanisms is the primary cause. Further, a suppressive effect of glucocorticoids on sarcolemmal excitability has been reported in humans after short-term glucocorticoid administration,3,4 whereas a variable effect was observed in animals.5–8 Possible explanations for this effect include Abbreviations: BAP, brightness-area product; CK, creatine kinase; CSA, cross-sectional area; EGTA, ethylene-glycol tetraacetic acid; MHC, myosin heavy chain; MuRF-1, muscle RING-finger protein-1; P0/CSA, specific force; PGC-1a, peroxisome proliferator-activated receptor-gamma coactivator-1a; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; V0, unloaded maximal shortening velocity Additional Supporting Information may be found in the online version of this article. Key words: cross-sectional area; maximal shortening velocity; myosin; specific force; steroid myopathy The first 2 authors (M.A.M. and R.Q.) contributed equally to this work. This study was supported by grants from the Fondazione CARIPLO of Milan, Italy (2010.0764 to R.B.), the Ministry of Health (RF-2011-02350228 to R.B.), and the University of Turin (to M.A.M.). Correspondence to: M.A. Minetto; e-mail: [email protected] C 2015 Wiley Periodicals, Inc. V

Published online 16 January 2015 in Wiley Online Library (wileyonlinelibrary. com). DOI 10.1002/mus.24572

Muscle Effects of Glucocorticoids

alterations of the sarcolemmal permeability and atrophy of muscle fibers. However, no previous studies have documented early atrophy of muscle fibers in subjects who receive short-term glucocorticoid administration, although it is generally accepted that preferential atrophy of type 2 fibers occurs as a long-term side effect of glucocorticoid excess.1,2 Therefore, we hypothesized that early adaptations in muscle mass may occur and precede the clinical appearance of myopathy in glucocorticoid-treated subjects. This would be relevant to the understanding of steroid myopathy and would highlight the importance of minimizing treatment duration to prevent undesirable side effects in both long- and short-term glucocorticoid use. As in other pathological states characterized by muscle atrophy, such as disuse atrophy and agerelated sarcopenia, changes in muscle mass (i.e., quantitative adaptations) are associated with qualitative adaptations, namely alterations in contractile properties and myosin concentration within individual muscle fibers.9–11 Therefore, we hypothesized that such qualitative adaptations would also occur after short-term glucocorticoid administration. The aim of this study was to determine whether short-term glucocorticoid administration would elicit ergogenic effects (documented in previous studies by improvements in maximal voluntary contraction force)3,4,12 as well as quantitative and qualitative adaptations of individual muscle fibers of healthy subjects. In addition, we also aimed to determine whether these quantitative and qualitative adaptations are paralleled by adaptations of master controllers of the balance between muscle protein breakdown and muscle protein synthesis (muscle-specific E3 ubiquitin ligases and myostatin) and energy homeostasis (peroxisome proliferator-activated receptor-gamma coactivator-1a). METHODS

Five men [age (mean 6 SD) 32.6 6 6.0 years, body mass 69.9 6 11.0 kg, height 178.0 6 7.7 cm, body mass index 21.0 6 2.0 kg/m2] volunteered to participate in the study. Subjects were free from neuromuscular or skeletal impairments. They were asked to Subjects.

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FIGURE 1. Outline of the sequence of experimental procedures. MVC, maximal voluntary contraction.

refrain from performing strenuous physical activity for 24 h before each experimental session. They received a detailed explanation of the study and gave written informed consent before participation. The study was performed in accordance with the Declaration of Helsinki and was approved by the ethics committee of the University of Pavia. Muscle samples were taken from the dominant vastus lateralis muscle (halfway along the line from the anterosuperior iliac spine to the superolateral border of the patella) by needle biopsy under local anesthesia, using the Bergstrom technique. Within 28 days of the initial muscle biopsy, subjects participated in 2 experimental sessions during which knee extension torque measurement and blood and saliva sampling were performed before and after (within 24 hours before administration and within 24 hours after the end of administration) 7 days of oral dexamethasone (8 mg once daily). Dexamethasone was specifically selected, because steroid myopathy has been reported to be associated with the use of fluorinated steroids.1,2 The dose of 8 mg/day was selected because we found significant effects on muscle force and excitability4 and corticospinal excitability12 in previous studies in normal-weight healthy subjects. Compliance with administration was assessed through assay of salivary cortisol levels (expected to be reduced after administration). For each subject, the 2 experimental sessions (pre- and post-administration) were performed at the same time of day. The pre-administration experimental session was preceded by a session during which subjects were familiarized with the knee extension torque measurements. The postadministration session also included repetition of the vastus lateralis biopsy (Fig. 1). Procedures.

Fasting saliva and blood samples were obtained on the day of each experiment at 8:00 A.M. (before and after the administration). Salivary cortisol levels were measured by an electrochemiluminescence immunoassay (Elecsys; Roche Diagnostics, Basel, Switzerland). The analytical sensitivity of the method was 0.5 nmol/L, and mean Laboratory Assays.

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inter- and intra-assay coefficients of variation were below 10% and 6%, respectively. Serum creatine kinase (CK) activity was measured using the standardized reverse reaction (creatine phosphate and adenosine triphosphate) and activation by N-acetylcysteine. The analytical sensitivity of the method was 3 U/L, and inter- and intra-assay coefficients of variation were 10% between the first and last activations. A total of 181 fibers were dissected from the preadministration biopsy samples (36.2 6 7.4 per subMuscle Effects of Glucocorticoids

FIGURE 2. Electrophoretic separation (SDS-PAGE) of myosin heavy chain (MHC) isoforms. A typical electrophoretic separation of MHC isoforms in 5 single muscle fibers: slow fibers (containing MHC-1) in lanes 3 and 5; fast 2A fibers in lanes 1 and 6; fast 2A/2X fibers (containing both MHC-2A and MHC-2X) in lane 2; and mixed human muscle sample (containing all MHC isoforms) in lane 4.

ject), and 165 (33.0 6 8.5 per subject, range 18-39) were accepted after discarding 9% of the fibers. A total of 194 fibers were dissected from the postadministration biopsy samples (38.8 6 3.7 per subject), and 177 (35.4 6 4.3 per subject, range 33–43) were accepted after discarding 9% of the fibers. Fiber Typing and Myosin Isoform Identification. After the contractile measurements, each fiber segment was immersed in a small tube containing 20 ml of Laemmli solution and stored at 220 C until assayed. The MHC isoform composition of the individual muscle fiber segments was determined by polyacrylamide gel electrophoresis on 6% polyacrylamide slab gels, after denaturation in sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDSPAGE) using a procedure described previously.14,19 In the MHC region, 3 major bands corresponding to the 3 adult MHC isoforms (MHC-1, MHC-2A, and MHC-2X) were separated. As shown in the representative examples in Figure 2, in relation to the presence of 1 or 2 bands in the MHC region, fibers were classified as type 1, 2A, and 2X (pure fiber types) or 1–2A and 2A–2X (hybrid fiber types). The same electrophoretic protocol followed by densitometric analysis of MHC bands was used to determine the MHC isoform composition of whole muscle samples, as described previously.19 Myosin Quantification in Single Fibers. Quantification of myosin concentration was performed using an approach described previously.11 Briefly, myosin concentration was assessed using quantitative gel electrophoresis by determining the brightness-area product (BAP) of the MHC band of each fiber and calculating myosin concentration from BAP using a standard curve obtained in each gel (12% linear polyacrylamide gels) by loading known amounts of a myosin standard. Quantitative Real-Time Reverse Transcript-Polymerase

The relative mRNA levels of 2 E3 ubiquitin ligases [muscle RING finger 1 (MuRF-1) and muscle atrophy F-box (MAFbx), also called atrogin-1]20 that are major regulators of ubiquitinmediated protein degradation, as well as of myostatin Chain Reaction.

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and PGC-1a, were determined before and after dexamethasone administration using quantitative realtime reverse transcript-polymerase chain reaction (RT-PCR). Briefly, total RNA, from the frozen biopsy portion, was extracted using RNA isolation (Promega SV Total kit). The concentration of RNA was evaluated (NanoDrop; Thermo Scientific), and 300 ng RNA was reverse-transcribed with reverse transcriptase (SuperScript III; Invitrogen) to obtain cDNA. The cDNA was analyzed by RT-PCR (SYBR Green PCR kit; Applied Biosystems), and the data were normalized to cyclophilin expression. Oligonucleotide primers used for RT-PCR are listed in Table S1 in the Supplementary Material (available online). Statistics. Because the Shapiro–Wilk test for normal distribution of the data failed, non-parametric tests were used. The Wilcoxon signed rank sum test was used for comparing knee extension torque measurements, cortisol levels, CK, myoglobin, MHC isoform distribution, and mRNA levels before and after dexamethasone administration. The Fisher exact test was used for comparison between proportions, and the unpaired t-test with the Welch correction21 was adopted for comparing single-fiber CSA values, myosin concentration, and contractile properties between different fiber types (type 1 fibers vs. type 2 fibers pre-administration) and between different biopsy samples (pre- vs. post-administration). Spearman rank correlation analysis was used to test for linear correlations between the pre-/post-administration changes in individual fiber properties. Data are expressed as mean 6 standard deviation (SD). The threshold for statistical significance was set at P 5 0.05. Statistical tests were performed with Statistica 6 software (StatSoft, Inc., Tulsa, Oklahoma). RESULTS Laboratory Assays and Knee Extension Torque. All subjects completed the experiments, and compliance with administration was confirmed on the basis of a significant reduction in post-intervention salivary cortisol levels (before: 17.0 6 4.5 nmol/L, after: 2.2 6 1.8 nmol/L; P 5 0.04). Serum CK decreased significantly (P 5 0.04) after dexamethasone administration (before: 169.6 6 99.7 U/L, after: 70.2 6 62.2 U/L). Plasma myoglobin showed a non-significant (P 5 0.7) reduction after dexamethasone administration (before: 39.1 6 34.4 ng/ml, after: 29.1 6 8.3 ng/ml). Maximal torque of the dominant knee extensors increased significantly (P 5 0.04) after dexamethasone administration (before: 122.8 6 25.8 Nm, after: 138.0 6 26.0 Nm; mean relative increase: 12.8 6 4.3%). Fiber Type Distribution. Table 1 lists the percent distribution of pure and hybrid fiber types in the 634

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Table 1. Distribution of fiber types identified on the basis of MHC isoform composition in the population of individual muscle fibers dissected from pre- and post-administration biopsy samples. Pre-administration Fiber type Type Type Type Type Type Total

1 1-2A 2A 2A-2X 2X

Post-administration

Number Percentage Number Percentage P-value 70 11 57 16 11 165

42.4 6.7 34.5 9.7 6.7 100.0

68 13 60 14 22 177

38.4 7.3 34.0 7.9 12.4 100.0

0.51 0.83 0.91 0.57 0.10 —

population of individual muscle fibers dissected from pre-administration (n 5 165) and postadministration (n 5 177) biopsy samples. The percentages of different fiber types did not change after treatment. Accordingly, no MHC isoform distribution change after dexamethasone administration was observed for the whole muscle samples (pre-administration: MHC-1 30.7 6 2.7%, MHC-2A 39.4 6 1.8%, MHC-2X 29.8 6 2.9%; post-administration: MHC-1 30.8 6 3.4%, MHC-2A 40.0 6 3.4%, MHC-2X 29.1 6 4.3%; range of P-values: 0.34–0.89). CSA and Myosin Concentration of Individual Muscle Fibers. Figure 3 shows the mean values of CSA and myosin concentration of type 1 and 2A fibers pre- and post-administration. No significant difference was observed preadministration between CSA values of type 1 and 2A fibers (type 1 fibers: 6357.7 6 2493.4 mm2, type 2A fibers: 6342.5 6 2445.2 mm2; P 5 0.97). Both fiber types went through atrophy after administration; however, there was fiber type variability in the sensitivity to dexamethasone-induced atrophy. In fact, the percent decrease in CSA was 11% and not statistically significant (P 5 0.06) for type 1 fibers and was 17% and statistically significant (P 5 0.008) for type 2A fibers. Myosin concentration pre-administration was significantly higher in type 2A compared with type 1 fibers (type 2A fibers: 324.0 6 156.1 mM/L, type 1 fibers: 251.5 6 136.2 mM/L; P 5 0.01). Both fiber types had significant loss of myosin after administration, with a greater loss for type 2A fibers. In fact, the percent decrease in myosin concentration was 18% for type 1 fibers (P 5 0.04) and 32% for type 2A fibers (P < 0.0001). Type 2X fibers were rare (7% and 12% of preand post-administration fibers, respectively; see Table 1) and showed no significant change in CSA and myosin concentration after dexamethasone administration (pre-administration: CSA 4928 6 998 mm2, myosin concentration: 264.3 6 62.2 mM/L; MUSCLE & NERVE

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FIGURE 3. Cross-sectional area (CSA) and myosin concentration [Myosin] of type 1 and type 2A fibers dissected from vastus lateralis biopsy samples before and after dexamethasone administration (8 mg/day for 7 days) in 5 subjects. The number of fibers per type is reported in Table 1. *Significantly different (P < 0.05) post- vs. pre-administration.

post-administration: CSA 5893 6 1877 mm2, myosin concentration: 342.8 6 207.9 mM/L; range of P-values: 0.12-0.41). Contractile

Properties

of

Individual

Muscle

Figure 4 shows the mean values of P0/CSA and V0 of type 1 and 2A fibers pre- and postadministration. As expected, on the basis of the known differences in contractile properties between type 1 and 2A fibers,14 both P0/CSA and V0 pre-administration were significantly (P < 0.0001 and P 5 0.0001, respectively) lower in type 1 (P0/ CSA: 95.6 6 48.6 kN/m2, V0: 0.81 6 0.86 L/s) than in type 2A (P0/CSA: 152.0 6 53.3 kN/m2, V0: 1.42 6 0.83 L/s) fibers. Both fiber types went through significant loss of specific force after administration, with a greater loss for type 2A fibers. In fact, the percent decrease in P0/CSA was 24% for type 1 fibers (P 5 0.004) and 33% for type 2A fibers (P < 0.0001). Fibers.

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The 2 fiber types went through different changes in V0 after administration: type 1 fibers showed no significant (P 5 0.7) change in V0, whereas type 2A fibers did show a significant increase in V0 (33%, P 5 0.009). Type 2X fibers were rare (7% and 12% of preand post-administration fibers, respectively; see Table 1) and showed no significant change in P0/ CSA and V0 after dexamethasone administration (pre-administration: P0/CSA: 107.2 6 30.3 kN/m2, V0: 2.93 6 1.03 L/s; post-administration: P0/CSA: 140.4 6 90.6 kN/m2, V0: 3.46 6 1.51 L/s; range of P-values: 0.22–0.32). Correlations between Dexamethasone-Induced Changes in Individual Fiber Properties. No significant correlation (R 5 –0.04, P 5 0.91) was observed between the pre-/post-administration change in CSA and the pre-/post-administration change in myosin concentration (n 5 10, pooled data from the 5 MUSCLE & NERVE

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FIGURE 4. Specific force (P0/CSA) and unloaded maximal shortening velocity (V0) of type 1 and type 2A fibers dissected from vastus lateralis biopsy samples before and after dexamethasone administration (8 mg/day for 7 days) in 5 subjects. The number of fibers per type is reported in Table 1. *Significantly different (P < 0.05) post- vs. pre-administration.

subjects combining the average changes for type 1 and type 2A fibers). On the contrary, the pre-/post-administration change in myosin concentration was significantly and positively associated with the change in P0/CSA (n 5 10; R 5 0.71, P 5 0.02). A disproportionate loss of myosin with respect to CSA occurred and was correlated with the loss of specific force of muscle fibers. mRNA levels. MuRF-1 and atrogin-1 mRNA levels were unaffected by dexamethasone administration (P 5 0.89 and P 5 0.22, respectively). Myostatin and PGC-1a mRNA levels were unchanged (P 5 0.68 and P 5 0.50, respectively) in response to dexamethasone administration (Fig. 5). DISCUSSION

In this study we have characterized the quantitative and qualitative adaptations of muscle fibers of healthy subjects to short-term dexamethasone administration. Both fiber types investigated under636

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went atrophy, decreased specific force, and decreased myosin concentration, which was preferential for fast compared with slow fibers, whereas variable changes in unloaded shortening velocity were observed between different fibers. Glucocorticoids

Decrease

CSA

and

Myosin

Although muscle atrophy is a well-known side effect of long-term glucocorticoid excess that may result from (a combination of) reduced protein synthesis, increased protein breakdown, and impaired mitochondrial function, we have shown that short-term administration of glucocorticoids in doses well within the range used clinically22–24 could reduce fiber CSA and myosin concentration, with preferential reductions in fast compared with slow fibers. Preferential atrophy of fast fibers is a well-known histopathologic feature of steroid myopathy.1,2,13 The observation that myosin concentration was reduced after administration indicates that the Concentration of Muscle Fibers.

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FIGURE 5. Vastus lateralis relative mRNA levels of muscle RING-finger protein-1 (MuRF-1), atrogin-1, myostatin, and peroxisome proliferator-activated receptor-gamma coactivator (PGC)21a before and after dexamethasone administration (8 mg/day for 7 days) in 5 subjects.

decrease in myosin content overcame the decrease in CSA; that is, a disproportionate loss of myosin with respect to CSA occurred. This has been observed previously in young healthy subjects after immobilization11 and in atrophic fibers of elderly subjects.9 Because the intervention duration was short and all subjects continued their normal daily activities during the administration, it is unlikely that the decrease in CSA and the myosin loss were secondary to muscle disuse. The post-intervention reduction in circulating muscle proteins points toward the anti-anabolic effect of glucocorticoids as the most plausible explanation for the decrease in CSA and myosin loss. In fact, post-treatment levels of CK and myoglobin were reduced compared with baseline, consistent with previous observations.4,13 The smaller decrease in myoglobin compared with CK could reflect preferential impairment of muscle synthesis in fast fibers, which have high levels of CK expression, whereas myoglobin is preferentially expressed in slow fibers.25 Despite the aforementioned findings, myostatin expression, a well-known negative regulator of muscle mass that inhibits muscle cell proliferation and protein synthesis26,27 and plays a role in Muscle Effects of Glucocorticoids

mediating glucocorticoid-induced muscle atrophy,28,29 was not increased after short-term administration of dexamethasone, in line with a previous study.30 The lack of changes in mRNA expression in any of the other target genes could indicate that no changes in muscle protein breakdown and mitochondrial oxidative metabolism (as indirectly inferred by the assessment of PGC-1a, which is a transcriptional coactivator that induces and coordinates gene expression stimulating mitochondrial oxidative metabolism in skeletal muscle cells)31,32 occurred after short-term glucocorticoid administration. However, because the time course of glucocorticoid-induced changes in expression of myostatin, atrogenes, and transcriptional factors such as PGC-1a has not been established in humans, it may be hypothesized that a muscle biopsy performed after 7 days of glucocorticoid administration failed to detect earlier changes in mRNA levels. It cannot be excluded that glucocorticoid-induced upregulation of target genes occurred early after the administration onset, died away by the end of treatment (i.e., 1 week), but was still responsible for the imbalance between protein synthesis and breakdown that MUSCLE & NERVE

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resulted in the observed myosin loss and decrease in fiber CSA. Glucocorticoid Effects on Muscle Force. Short-term administration of dexamethasone showed 2 divergent effects on muscle force. It improved maximal voluntary isometric torque of the knee extensors in vivo and decreased the specific force of individual muscle fibers in vitro. It has been observed recently that a single intravenous dose of hydrocortisone in healthy subjects increased the amplitude of motor evoked potentials detected from the first dorsal interosseous in response to transcranial magnetic stimulation.33 Moreover, it has also been observed that short-term administration increased the maximal force of elbow flexors and knee extensors,4 increased the maximal force of ankle dorsiflexors,12 and decreased the threshold of motor evoked potentials detected from the tibialis anterior.12 Although no direct recordings of central nervous system activity were obtained in this study, it may be hypothesized that the observed increase of voluntary isometric torque of knee extensors can be related to glucocorticoid actions on cortical and/or corticospinal excitability. Moreover, an alteration in calcium handling is another factor that may affect the maximal force of muscle fibers in vivo. In fact, a change in calcium handling would result in a modification in cross-bridge cycling kinetics. Previous studies of the effects of glucocorticoids on calcium handling demonstrated improved sarcoplasmic reticulum function in both skeletal muscle34,35 and myocardium.36,37 The force generation capacity per CSA (specific force) of individual muscle fibers depends on the number of actin-myosin interactions (crossbridges) per unit of CSA and on the force developed by a single interaction.38 The most plausible mechanism underlying the observed reduction in specific force of type 1 and 2A fibers is myosin loss. A further mechanism that could account for the reduction in specific force is impairment of excitation-contraction coupling. However, as chemically skinned muscle fibers lack excitationcontraction coupling, the latter cannot be involved in specific force loss, and myosin loss remains the most likely mechanism. A significant positive correlation was found between pre-/post-administration changes in myosin concentration and specific force. Glucocorticoid

Effects

on

Unloaded

Shortening

Unloaded shortening velocity is an index of cross-bridge kinetics and is largely determined by MHC composition.10,39 However, it was observed previously that V0 of individual muscle fibers can change with no change in Velocity of Muscle Fibers.

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myosin isoform content; that is, it can increase after muscle disuse9,40 and decrease in aging.9,39,41 It has been suggested that the increase in V0 after disuse is related to a disproportionate loss of thin filaments with respect to thick filaments,10,42,43 whereas its decrease in aging is thought to depend on impairment of myosin motor function9,44 as a possible result of myosin glycosylation.45 The pre-/post-administration increase of V0 we observed in type 2A fibers could have originated at the sarcomere or muscle fiber level. A disproportionate loss of thin filaments with respect to thick filaments, which is a possible mechanism underlying the increase in V0 after disuse,10,42,43 is unlikely in response to glucocorticoid administration, because glucocorticoid excess triggers a preferential loss of myosin associated with relative preservation of thin, actin-containing filaments.38,46 Another mechanism should account for the increase in V0 observed in type 2A fibers. Only a small fraction (1%–5%) of cross-bridges that would be firmly attached during isometric contractions occupy strong-binding states during maximum unloaded shortening,47 whereas the majority of cross-bridges cycle through weak-binding configurations. Dexamethasone administration may recruit weak-binding states in individual fibers; that is, it can be hypothesized that cross-bridge cycling increases (even though force production decreases) after dexamethasone administration. The demonstration of accelerated contraction kinetics during unloaded shortening is in line with recent results obtained in an acute septic myopathy model48 and suggests a direct action of glucocorticoids at the cross-bridge kinetics level, which seems preferential for fast muscle fibers. Limitations and Clinical Implications. There are several limitations to this study. First, we focused entirely on a small number of healthy subjects. Second, only 1 muscle was investigated through biopsy sampling, and only 1 biopsy was performed after glucocorticoid administration. Nonetheless, we have provided a clear demonstration that shortterm administration of glucocorticoids in healthy subjects, in doses well within the range used clinically,22–24 has positive ergogenic effects, as it increased maximal torque during voluntary contractions. These positive effects are in line with previous observations.4,12 However, individual muscle fibers had atrophy, decreased specific force, and decreased myosin concentration. These findings are relevant to increasing our understanding of steroid myopathy. The glucocorticoid-induced imbalance between protein synthesis and breakdown that results in the observed quantitative and qualitative adaptations of muscle fibers develops, MUSCLE & NERVE

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even in healthy subjects, after only a few days of glucocorticoid administration. It is therefore recommended to minimize not only the dose but also the duration of administration to prevent (or minimize) undesirable side effects, even with shortterm administration. The authors thank Dr. F. Lanfranco (University of Turin, Italy) and L. Guidotti (University of Pavia, Italy) for their valuable assistance with experimental measures. REFERENCES 1. Minetto MA, Lanfranco F, Motta G, Allasia S, Arvat E, D’Antona G. Steroid myopathy: some unresolved issues. J Endocrinol Invest 2011; 34:370–375. 2. Schakman O, Kalista S, Barb e C, Loumaye A, Thissen JP. Glucocorticoid-induced skeletal muscle atrophy. Int J Biochem Cell Biol 2013;45:2163–2172. 3. van der Hoeven JH. Decline of muscle fiber conduction velocity during short-term high-dose methylprednisolone therapy. Muscle Nerve 1996;19:100–102. 4. Minetto MA, Botter A, Lanfranco F, Baldi M, Ghigo E, Arvat E. Muscle fiber conduction slowing and decreased levels of circulating muscle proteins after short-term dexamethasone administration in healthy subjects. J Clin Endocrinol Metab 2010;95:1663–1671. 5. Gruener R, Stern LZ. Corticosteroids. Effects on muscle membrane excitability. Arch Neurol 1972;26:181–185. 6. Dlouha H, Vyskocil F. The effect of cortisol on the excitability of the rat muscle fiber membrane and neuromuscular transmission. Physiol Bohemoslov 1979;28:485–494. 7. Ruff RL, Martyn D, Gordon AM. Glucocorticoid-induced atrophy is not due to impaired excitability of rat muscle. Am J Physiol 1982;243: E512–521. 8. Ruff RL, St€ uhmer W, Almers W. Effect of glucocorticoid treatment on the excitability of rat skeletal muscle. Pflugers Arch 1982;395:132– 137. 9. D’Antona G, Pellegrino MA, Adami R, Rossi R, Carlizzi CN, Canepari M, et al. The effect of ageing and immobilization on structure and function of human skeletal muscle fibers. J Physiol 2003;552:499–511. 10. Canepari M, Pellegrino MA, D’Antona G, Bottinelli R. Single muscle fiber properties in aging and disuse. Scand J Med Sci Sports 2010;20: 10–19. 11. Borina E, Pellegrino MA, D’Antona G, Bottinelli R. Myosin and actin content of human skeletal muscle fibers following 35 days bed rest. Scand J Med Sci Sports 2010;20:65–73. 12. Baudry S, Lanfranco F, Merletti R, Duchateau J, Minetto MA. Effects of short-term dexamethasone administration on corticospinal excitability. Med Sci Sports Exerc 2014;46:695–701. 13. Khaleeli AA, Edwards RH, Gohil K, McPhail G, Rennie MJ, Round J, et al. Corticosteroid myopathy: a clinical and pathological study. Clin Endocrinol (Oxf) 1983;18:155–166. 14. Bottinelli R, Canepari M, Pellegrino MA, Reggiani C. Force-velocity properties of human skeletal muscle fibers: myosin heavy chain isoform and temperature dependence. J Physiol 1996;495:573–586. 15. Bottinelli R, Betto R, Schiaffino S, Reggiani C. Unloaded shortening velocity and myosin heavy chain and alkali light chain isoform composition in rat skeletal muscle fibers. J Physiol 1994;478:341–349. 16. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–685. 17. Brenner B. Technique for stabilizing the striation pattern in maximally calcium-activated skinned rabbit psoas fibers. Biophys J 1983; 41:99–102. 18. Bottinelli R, Schiaffino S, Reggiani C. Force-velocity relations and myosin heavy chain isoform compositions of skinned fibers from rat skeletal muscle. J Physiol 1991;437:655–672. 19. Pellegrino MA, Canepari M, Rossi R, D’Antona G, Reggiani C, Bottinelli R. Orthologous myosin isoforms and scaling of shortening velocity with body size in mouse, rat, rabbit and human muscles. J Physiol 2003;546:677–689. 20. Bodine SC, Latres E, Baumhueter S, Lai VK, Nunez L, Clarke BA, et al. Identification of ubiquitin ligases required for skeletal muscle atrophy. Science 2001;294:1704–1708. 21. Welch BL. The generalisation of student’s problems when several different population variances are involved. Biometrika 1947;34:28–35. 22. Leppert W, Buss T. The role of corticosteroids in the treatment of pain in cancer patients. Curr Pain Headache Rep 2012;16:307–313. 23. Gaujoux-Viala C, Gossec L. When and for how long should glucocorticoids be used in rheumatoid arthritis? International guidelines and recommendations. Ann NY Acad Sci 2014;1318:32–40.

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MUSCLE & NERVE

October 2015

639

Quantitative and qualitative adaptations of muscle fibers to glucocorticoids.

The aim of this study was to understand the effects of short-term glucocorticoid administration in healthy subjects...
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