Intermittent Glucocorticoid Dosing Improves Muscle Repair and Function in Mice with Limb-Girdle Muscular Dystrophy

Abstract

The muscular dystrophies are genetically diverse. Shared pathological features among muscular dystrophies include breakdown, or loss of muscle, and accompanying fibrotic replacement. Novel strategies are needed to enhance muscle repair and function and to slow this pathological remodeling. Glucocorticoid steroids, like prednisone, are known to delay loss of ambulation in patients with Duchenne muscular dystrophy but are accompanied by prominent adverse effects. However, less is known about the effects of steroid administration in other types of muscular dystrophies, including limb-girdle muscular dystrophies (LGMDs). LGMD 2B is caused by loss of dysferlin, a membrane repair protein, and LGMD 2C is caused by loss of the dystrophin-associated protein, γ-sarcoglycan. Herein, we assessed the efficacy of steroid dosing on sarcolemmal repair, muscle function, histopathology, and the regenerative capacity of primary muscle cells. We found that in murine models of LGMD 2B and 2C, daily prednisone dosing reduced muscle damage and fibroinflammatory infiltration. However, daily prednisone dosing also correlated with increased muscle adipogenesis and atrophic remodeling. Conversely, intermittent dosing of prednisone, provided once weekly, enhanced muscle repair and did not induce atrophy or adipogenesis, and was associated with improved muscle function. These data indicate that dosing frequency of glucocorticoid steroids affects muscle remodeling in non-Duchenne muscular dystrophies, suggesting a positive outcome associated with intermittent steroid dosing in LGMD 2B and 2C muscle.

Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Prednisone-accelerated sarcolemmal repair and repair cap formation in Dysf-null myofibers. A: A single dose of glucocorticoid steroids, either prednisone or deflazacort, decreased the extent of sarcolemmal injury in live myofibers, as quantitated by FM4-64 accumulation over time and injury area at analysis end point (arrows). Top left panels: Time-dependent real-time image stacks of injury site (10-μm-wide area). Top right panels: Z-stack rendering of FM4-64 accumulation at injury site at 300 seconds after injury. Bottom panels: Quantitative data are plotted. B: The single steroid dose associated with faster onset of the sarcolemmal repair cap, as monitored in real-time by green fluorescent protein (GFP)–labeled annexin A1 (ANXA1-GFP) accumulation at injury site (arrows). Top panels: Time-dependent image stacks (10-μm-wide area). Bottom panel: Quantitation of time (after injury) to cap onset is shown. Data are expressed as means ± SEM (marked line plots) or Tukey distribution (box plots). n = 5 mice (50 myofibers) per group (3 males and 2 females per group). ∗P < 0.05 versus vehicle (one-way analysis of variance test with Bonferroni multiple comparison); †P < 0.05 versus vehicle (two-way analysis of variance test with Bonferroni multiple comparison). Scale bar = 5 μm (A, right top panels). AU, arbitrary unit.

Figure 2

Weekly and daily prednisone regimens enhance sarcolemmal repair in Dysf-null myofibers. A:Dysf-null mice were treated for 4 weeks with either weekly or daily prednisone. Both prednisone regimens resulted in improved sarcolemmal repair after laser-induced injury, as shown by diminished accumulation of FM4-64 over time (10-μm-wide area) and decreased sarcolemmal injury area at end point (arrows). B: Both steroid regimens correlated with faster onset of the annexin A1 (ANXA1) repair cap, as monitored through ANXA1–green fluorescent protein (GFP) accumulation at injury site over time (10-μm-wide area). Data are expressed as means ± SEM (marked line plots) or Tukey distribution (box plots). n = 5 mice (50 myofibers) per group (3 males and 2 females in vehicle and daily groups and 2 males and 3 females in weekly group). ∗P < 0.05 versus vehicle (one-way analysis of variance test with Bonferroni multiple comparison); †P < 0.05 versus vehicle (two-way analysis of variance test with Bonferroni multiple comparison). Scale bar = 5 μm (A). AU, arbitrary unit.

Figure 3

Weekly and daily steroid regimens reduce muscle damage and fibrosis in Dysf-null mice. A: The same cohort of Dysf-null mice was assessed after 4 weeks of either weekly or daily prednisone. Both steroid regimens comparably decreased serum creatine kinase (CK) levels after a 4-week treatment, as compared to vehicle treatment. B: Immunostaining of quadriceps muscles revealed a decrease in infiltrating macrophages (F4/80+ cells; arrowheads and representative insets) in all steroid-treated mice, as compared to vehicle-treated animals. C: Fibrosis was comparably decreased in both prednisone regimens in hind limb skeletal muscles. Left panels: Masson trichrome staining of gastrocnemius muscle sections (arrows, fibrotic accumulation). Right panel: Quantitation of hydroxyproline content in quadriceps muscle tissue. Data are expressed as means ± SEM and single mouse values (histograms) or Tukey distribution (box plots). n = 5 mice per group (3 males and 2 females in vehicle and daily groups and 2 males and 3 females in weekly group). ∗P < 0.05 versus vehicle (one-way analysis of variance test with Bonferroni multiple comparison). Scale bars: 100 μm (B); 200 μm (C, left panels). HOP, hydroxyproline; WGA, wheat germ agglutinin.

Figure 4

Weekly and daily prednisone regimens induce divergent effects on Dysf-null muscle function. A: Unlike weekly dosing, daily prednisone dosing reduced relative body mass over time. B: Weekly and daily prednisone regimens produced opposite effects on muscle performance, as measured by run-to-exhaustion treadmill and grip strength tests. Weekly dosing improved performance, whereas daily dosing decreased performance. C: Left panel: Weekly prednisone associated with increased maximum tetanic force of whole tibialis anterior muscle, as compared to vehicle-treated animals. Right panel: The increase in force remained stable for 30 consecutive isometric contractions. Daily prednisone dosing induced opposite effects. D: Weekly prednisone dosing induced hypertrophic remodeling, whereas daily prednisone dosing induced atrophic remodeling, of skeletal muscle, as quantitated by cross-sectional area (CSA) of gastrocnemius myofibers. E: Whole-body plethysmography showed that weekly prednisone treatment induced improved respiratory capacity, increasing minute volume and decreasing average inspiration time. Daily prednisone dosing yielded the opposite effects. F: Myofiber CSA and transverse thickness of diaphragm muscles followed trends observed in hind limb muscles (ie, increased after weekly prednisone and decreased after daily prednisone, as compared to vehicle-treated control animals). Data are expressed as means ± SEM (marked line plots and histograms), single mouse values (histograms), or Tukey distribution (box plots). n = 5 mice per group (3 males and 2 females in vehicle and daily groups and 2 males and 3 females in weekly group). ∗P < 0.05 versus vehicle (one-way analysis of variance test with Bonferroni multiple comparison); †P < 0.05 versus vehicle (two-way analysis of variance test with Bonferroni multiple comparison). AU, arbitrary unit.

Figure 5

Weekly prednisone dosing promotes myogenic capacity, whereas daily dosing promotes adipogenesis, in Dysf-null mice. A: Real-time quantitative PCR of quadriceps muscle tissue showed transcriptional trends correlating with divergent ergogenic effects of weekly versus daily prednisone in Dysf-null muscle, including divergent effects on the myogenic factor Mef2C. B: Immunostaining of differentiated primary myoblasts (CD56+/CD140a− fraction, passage 0) showed that, after weekly prednisone, myoblasts increased fusion efficiency in multinucleated myotubes. Daily prednisone induced the opposite effects. C: The adipogenic factor Pparg was up-regulated in muscle after daily prednisone (left panels), and the extent of fatty infiltrates in quadriceps muscle was dramatically increased after daily prednisone dosing (right panel), as shown by Oil Red O staining. D: Daily prednisone dosing induced a dramatic increase in spontaneous adipocyte production by proliferating primary fibroadipogenic progenitors (FAPs; CD56−/CD140a+ fraction, passage 0), as demonstrated by Oil Red O staining of lipid-storing cells (left panels show representative images; right panel, quantitationData are expressed as means ± SEM, single mouse values (histograms), or Tukey distribution (box plots). n = 5 mice per group (3 males and 2 females in vehicle and daily groups and 2 males and 3 females in weekly group). ∗P < 0.05 versus vehicle (one-way analysis of variance test with Bonferroni multiple comparison). Scale bars: 100 μm (B and D); 200 μm (C). AU, arbitrary unit; MyHC, myosin heavy chain.

Figure 6

Prednisone treatment of Sgcg-null mice enhances sarcolemmal repair and repair cap formation after injury. A: Laser injury was performed on Sgcg-null myofibers after 4 weeks of either weekly or daily prednisone treatment. Both weekly and daily prednisone reduced the extent of sarcolemmal damage after laser-induced injury, as shown by decreased FM4-64 accumulation over time (10-μm-wide area) and at end point (arrows). B: Both weekly and daily prednisone regimens correlated with faster onset (arrows) of accumulation of annexin A1–green fluorescent protein (ANXA1-GFP) in repair caps on the site of sarcolemmal injury (10-μm-wide area). Data are epressed as means ± SEM (marked line plots) or Tukey distribution (box plots). n = 5 mice (50 myofibers) per group (3 males and 2 females per group). ∗P < 0.05 versus vehicle (one-way analysis of variance test with Bonferroni multiple comparison); †P < 0.05 versus vehicle (two-way analysis of variance test with Bonferroni multiple comparison). Scale bar, 5 μm (A). AU, arbitrary unit.

Figure 7

Comparable improvement in histopathological findings by weekly and daily prednisone in Sgcg-null mice. A: Weekly and daily prednisone similarly decreased serum creatine kinase (CK) levels after a 4-week treatment, as compared to vehicle treatment. B: Immunostaining of quadriceps muscles revealed a decrease in infiltrating macrophages (F4/80+ cells; arrowheads and representative insets) in all prednisone-treated mice, as compared to vehicle-treated animals. C: Fibrosis (arrows) was comparably decreased in hind limb skeletal muscles by both prednisone regimens. Left panels: Masson trichrome staining of gastrocnemius muscle sections. Right panel: Quantitation of hydroxyproline content in quadriceps muscle tissue. Data are expressed as means ± SEM, single mouse values (histograms), or Tukey distribution (box plots). n = 5 mice per group (3 males and 2 females per group). ∗P < 0.05 versus vehicle (one-way analysis of variance test with Bonferroni multiple comparison). Scale bars: 100 μm (B); 200 μm (C, left panels). HOP, hydroxyproline; WGA, wheat germ agglutinin.

Figure 8

Weekly prednisone improves muscle function in Sgcg-null mice, whereas daily prednisone induces atrophy. A: Unlike weekly dosing, daily prednisone reduced relative body weight over time. B: Weekly prednisone improved run to exhaustion and grip strength, whereas daily prednisone resulted in a reduction in both treadmill and grip strength performance, in Sgcg-null mice. C: Left panel: Weekly prednisone associated with increased tetanic force of whole tibialis anterior muscle, as compared to vehicle-treated animals. Right panel: The increase in force remained stable for 10 consecutive isometric contractions. Daily prednisone dosing induced opposite effects. D: Weekly prednisone induced hypertrophic remodeling, whereas daily dosing induced atrophic remodeling, of skeletal muscle, as quantitated by cross-sectional area (CSA) of gastrocnemius myofibers. E: Whole-body plethysmography showed that weekly prednisone treatment induced improved respiratory capacity, increasing minute volume and decreasing average inspiration time. Daily prednisone dosing induced opposite effects. F: Real-time quantitative PCR of quadriceps muscle tissue showed transcriptional trends correlating with divergent ergogenic effects of steroid regimens in muscle, including the ergogenic factor Klf15, which was up-regulated after weekly dosing, and the atrophic factors Fbxo32 and Trim63, which were up-regulated after daily dosing. Data are expressed as means ± SEM (marked line plots and histograms), single mouse values (histograms), or Tukey distribution (box plots). n = 5 mice per group (3 males and 2 females per group). ∗P < 0.05 versus vehicle (one-way analysis of variance test with Bonferroni multiple comparison); †P < 0.05 versus vehicle (two-way analysis of variance test with Bonferroni multiple comparison). AU, arbitrary unit.

Figure 9

Weekly versus daily prednisone induces divergent effects on myogenic and adipogenic capacities in Sgcg-null muscles. A: Real-time quantitative PCR on quadriceps muscle tissue showed up-regulation of the myogenic factor Mef2C after weekly prednisone, and its down-regulation after daily prednisone. B: Immunostaining of differentiated primary myoblasts (passage 0) showed that, after weekly prednisone, myoblasts increased fusion efficiency in multinucleated myotubes. Daily prednisone induced the opposite effects. C: The adipogenic factor Pparg was up-regulated in muscle after daily prednisone dosing. D: Daily prednisone dosing also associated with an increase in spontaneous adipocyte production by proliferating primary fibroadipogenic progenitors (FAPs), as demonstrated by Oil Red O staining of lipid-storing cells (left panels show representative images; right panel, quantitation). Data are expressed as means ± SEM and single mouse values (histograms) or Tukey distribution (box plots). n = 5 mice per group (3 males and 2 females per group). ∗P < 0.05 versus vehicle (one-way analysis of variance test with Bonferroni multiple comparison). Scale bar = 100 μm (B and D, left panels). AU, arbitrary unit.

Supplemental Figure S1

Prednisone treatment associated with up-regulation of annexin gene expression in Dysf-null muscles. A: Weekly and daily prednisone treatment induced a comparable increase in Anxa1 and Anxa6 expression levels in quadriceps muscles of Dysf-null mice. B: Steroid uptake was confirmed by down-regulation of Gzmb and Ifng, markers of immune cell activation, in primary splenocytes of steroid-treated mice. Data are expressed as means ± SEM and single mouse values (histograms). n = 5 mice (50 myofibers) per group (3 males and 2 females in vehicle and daily groups and 2 males and 3 females in weekly group). ∗P < 0.05 versus vehicle (one-way analysis of variance test with Bonferroni multiple comparison). AU, arbitrary unit.

Supplemental Figure S2

Weekly and daily prednisone-induced divergent functional effects on Dysf-null muscle. A: Daily prednisone dosing reduced grip strength normalized to body mass, suggesting a significant impact on intrinsic muscle weakness. B: Specific force and relative fatigue of tibialis anterior muscles did not significantly change across prednisone regimen types. C: Times of tibialis anterior muscle contraction and relaxation were reduced after weekly prednisone, and increased after daily prednisone. D: Weekly prednisone correlated with expansion of type 2B fast, glycolytic fibers and reduction of type 2A fast, oxidative fibers, as quantitated by immunostaining of quadriceps muscle sections. Daily prednisone had the opposite effects. Type 1 myofibers (blue bars) were found in negligible amounts in all conditions. E: Prednisone dosing schemes associated with the opposite effects on average expiration time, as quantitated through whole-body plethysmography, paralleling the effects on inspiration time. Data are expressed as means ± SEM (histograms). n = 5 mice per group (3 males and 2 females in vehicle and daily groups and 2 males and 3 females in weekly group). ∗P < 0.05 versus vehicle (one-way analysis of variance test with Bonferroni multiple comparison); †P < 0.05 versus vehicle (two-way analysis of variance test with Bonferroni multiple comparison). AU, arbitrary unit.

Supplemental Figure S3

Daily prednisone dosing produced adipocyte hypertrophy. Left panels: Representative images of ventral fat pad histology. Right panel: Quantitation of adipocyte cross-sectional area (CSA), showing significantly increased CSA values after daily prednisone dosing. Data are expressed as box plots (Tukey distribution). n = 5 mice per group (3 males and 2 females in vehicle and daily groups and 2 males and 3 females in weekly group). ∗P < 0.05 versus vehicle (one-way analysis of variance test with Bonferroni multiple comparison).

Supplemental Figure S4

Prednisone increased annexin gene expression and improved diaphragm histopathology in Sgcg-null muscles. A: Weekly and daily steroid regimens induced comparable increases in Anxa1 and Anxa6 expression levels in quadriceps muscles of Sgcg-null mice. B: Steroid uptake was confirmed by down-regulation of Gzmb and Ifng, markers of immune cell activation, in splenocytes of steroid-treated mice. C: Fibrosis of dystrophic diaphragm muscle was comparably reduced after both steroid regimens, as shown by Masson trichrome staining. Data are expressed as means ± SEM and single mouse values (histograms). n = 5 mice per group (3 males and 2 females in vehicle and daily groups and 2 males and 3 females in weekly group). ∗P < 0.05 versus vehicle (one-way analysis of variance test with Bonferroni multiple comparison). Scale bar = 100 μm (C). AU, arbitrary unit.

Supplemental Figure S5

Weekly prednisone induced hypertrophy, and daily prednisone induced atrophy, of Sgcg-null muscles. A: Weekly prednisone injection increased the grip strength, as normalized to body weight, as compared to vehicle treatment. Daily prednisone dosing had the opposite effects. B: Weekly prednisone correlated with expansion of type 2B fast, glycolytic and reduction of type 2A fast, oxidative myofibers, as quantitated by immunostaining of quadriceps muscle sections. Daily dosing had the opposite effects. C: Histological analysis of gastrocnemius muscle sections showed hypertrophic remodeling of myofibers after weekly prednisone dosing, and atrophic remodeling after the daily regimen. D: Weekly steroid regimen decreased expiration time, whereas daily dosing correlated with increased time, as quantitated through whole-body plethysmography. E and F: Divergent effects on muscle remodeling were evidenced in diaphragm muscles, as shown by myofiber cross-sectional area (CSA) values, transverse thickness, and representative histology images. G: Daily steroid dosing correlated with adipocyte hypertrophy, as quantitated through histology analysis of ventral fat pad sections. Data are expressed as means ± SEM, single mouse values (histograms), or box plots (Tukey distribution). n = 5 mice per group (3 males and 2 females per group). ∗P < 0.05 versus vehicle (one-way analysis of variance test with Bonferroni multiple comparison); †P < 0.05 versus vehicle (two-way analysis of variance test with Bonferroni multiple comparison). Scale bars = 100 μm (C and F). H&E, hematoxylin and eosin.