Intermittent glucocorticoid steroid dosing enhances muscle repair without eliciting muscle atrophy

Abstract

Glucocorticoid steroids such as prednisone are prescribed for chronic muscle conditions such as Duchenne muscular dystrophy, where their use is associated with prolonged ambulation. The positive effects of chronic steroid treatment in muscular dystrophy are paradoxical because these steroids are also known to trigger muscle atrophy. Chronic steroid use usually involves once-daily dosing, although weekly dosing in children has been suggested for its reduced side effects on behavior. In this work, we tested steroid dosing in mice and found that a single pulse of glucocorticoid steroids improved sarcolemmal repair through increased expression of annexins A1 and A6, which mediate myofiber repair. This increased expression was dependent on glucocorticoid response elements upstream of annexins and was reinforced by the expression of forkhead box O1 (FOXO1). We compared weekly versus daily steroid treatment in mouse models of acute muscle injury and in muscular dystrophy and determined that both regimens provided comparable benefits in terms of annexin gene expression and muscle repair. However, daily dosing activated atrophic pathways, including F-box protein 32 (Fbxo32), which encodes atrogin-1. Conversely, weekly steroid treatment in mdx mice improved muscle function and histopathology and concomitantly induced the ergogenic transcription factor Krüppel-like factor 15 (Klf15) while decreasing Fbxo32. These findings suggest that intermittent, rather than daily, glucocorticoid steroid regimen promotes sarcolemmal repair and muscle recovery from injury while limiting atrophic remodeling.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Pulse dosing of GC steroids improves sarcolemmal repair.

Prednisone and deflazacort, both GC steroids, were given 1 day prior to injury. (A) Laser injury was applied to isolated muscle fibers in the presence of FM4-64, which marks sarcolemmal injury. A single dose of GC steroid reduced FM4-64 accumulation. Shown is Z-stack rendering of FM4-64 dye accumulation of laser-injured sarcolemmal sites at 300 seconds after injury. Quantitation shows that GC pulse associated with decreased dye accumulation over time as well as a decreased area of injury. (B) Imaging of annexin A6 (ANXA6) cap formation at the site of sarcolemmal injury. Pulse dosing of prednisone and deflazacort associated with smaller repair caps, consistent with reduced injury and enhanced repair. Shown is Z-stack rendering of GFP-tagged ANXA6 cap of laser-injured sarcolemmal sites at 300 seconds after injury. Quantitation of GC dosing demonstrated faster, smaller cap formation over time and faster recovery of GFP-tagged ANXA6 at injury site. FM4-64 and ANXA6-GFP pictures were acquired simultaneously. F/F0, average fluorescence ratio versus average fluorescence at time 0 of imaging series. n = 50 myofibers (5 mice)/group. *P < 0.05 vs. vehicle, 1-way ANOVA test with Bonferroni’s multiple comparison; #P < 0.05 vs. vehicle, 2-way ANOVA test with Bonferroni’s multiple comparison.

Figure 2. GC pulse upregulates Anxa1 and Anxa6 through increased GR binding of their GRE sites.

(A) Model of transactivation pathway linking GC action in muscle to regulation of Anxa1 and Anxa6 expression; dashed lines, interrogated interactions. (B) Anxa1 and Anxa6 were upregulated in the skeletal muscle (quadriceps) and primary myoblasts (isolated from tibialis anterior) of mice 24 hours after a single pulse of GC steroids. (C) Twenty-four hours after a single GC pulse in C2C12 cells, ChIP-qPCR analysis showed that GRE sites of Anxa1 and Anxa6 were significantly enriched in GR occupancy as compared with vehicle. (D) Diagram shows experimental design of electroporation into muscles and 24 hours after i.p. prednisone pulse in WT mice. (E) Histograms show that prednisone pulse upregulated luciferase activity when constructs contained the GRE sites (white bars). Conversely, GC-related upregulation was ablated after deletion of the GRE-binding site in the constructs (black bars). Klf15 GRE was monitored as positive control. Data are expressed as fold change to luminescence from vehicle-treated muscles electroporated with the same plasmids (dashed line). n = 5 mice/group (B); n = 4 assays or mice/group (C and D). *P < 0.05 vs. vehicle, 1-way ANOVA test with Bonferroni’s multiple comparison (B and C); *P < 0.05 vs. WT site construct, unpaired 2-tailed t test with Welch’s correction (D).

Figure 3. miR-383 contributes to GC-associated Anxa6 regulation.

(A) Model of pathway linking GC uptake in muscle to regulation of miR-383 and Anxa6 expression. Solid lines, reported interactions; dashed lines, interrogated interactions. (B) Nr0b1 and miR-383 were upregulated and downregulated, respectively, in GC–pulse dosed skeletal muscle (quadriceps) and primary myoblasts. (C) Electroporation directly into muscle with targeting oligonucleotides altered miR-383 and Anxa6 levels. (D) miR-383 mimic correlated with worse sarcolemmal injury, whereas anti–miR-383 correlated with smaller injury. (Left) Z-stack rendering of laser-injured sarcolemmal sites at 300 seconds after injury; (center) quantitation of FM4-64 dye accumulation at sarcolemmal site after laser injury; (right) end-point area of injury. (E) Luciferase assay to validate GC-dependent regulation of Anxa6-3′ UTR targeting by miR-383 in skeletal muscle. Diagram depicting experimental design after introducing 3′ UTR reporter constructs into muscle followed by ex vivo Fluc measurement. miR-383 reduced luminescence, while 3′ UTR without the miR-383–binding site (Δ miR-383) increased luminescence. n = 5 mice/group (B); n = 3 mice (30 myofibers)/group (C–E). *P < 0.05 vs. vehicle, 1-way ANOVA test with Bonferroni’s multiple comparison; #P < 0.05 vs. vehicle, 2-way ANOVA test with Bonferroni’s multiple comparison.

Figure 4. GC steroids decrease extent of acute muscle injury in WT muscle.

Acute muscle injury was induced with cardiotoxin (ctx) injection in the tibialis anterior muscles of normal mice. (A) Diagram depicting the treatments performed in parallel in WT mice. Colored arrows, GC steroid injection; gray arrows, vehicle injections. (B) GC steroid regimens comparably reduced the extent of injury 7 days after cardiotoxin injection. (Left) Representative H&E images of tibialis anterior muscles with prednisone treatments. The dotted lines outline the injury area, which includes necrosis, fibrosis, immune cell infiltrates, and centrally nucleated fibers. (Right) Injury extent quantitation (10 replicates). (C) GC regimens comparably decreased macrophage infiltration within the area of injury 7 days after cardiotoxin injection. Data are depicted as quantitation of F4-80+ cells/mm2 in gastrocnemius muscles by immunostaining. (D) GC steroid regimens comparably reduced serum CK at 24 hours after injury, with no significant changes after 7 days. (E) Fibrosis was comparably reduced in the presence of all GC regimens both 7 and 14 days after muscle injury. Data are depicted as quantitation of hydroxyproline content in gastrocnemius muscles. Gray, WT vehicle; light blue, WT prednisone predose; blue, WT prednisone weekly; dark blue, WT prednisone daily; light purple, WT deflazacort predose; purple, WT deflazacort weekly; dark purple, WT deflazacort daily. n = 6 mice/group. *P < 0.05 vs. vehicle, 1-way ANOVA test with Bonferroni’s multiple comparison; #P < 0.05 vs. vehicle, 2-way ANOVA test with Bonferroni’s multiple comparison.

Figure 5. Weekly GC steroid dosing enhanced muscle performance, while daily GC treatments promoted atrophy in normal muscles 14 days after injury.

(A) Weekly GC steroid dosing enhanced running distance after injury, while daily GC steroid dosing reduced performance. (B) Maximum tetanic force and contraction time were increased and accelerated, respectively, in tibialis anterior muscles of injured mice treated with weekly GC. Daily GC steroid dosing induced opposite effects. (C) CSA of myofibers remote from injury site was increased by weekly but not daily GC steroid dosing, while no significant change in CSA was seen within area of injury. (D) Representative blots and densitometric quantitation of p-Akt (Ser473) from duplicate blots run in parallel from tibialis anterior muscles 14 days after injury (3 replicates). (E) Fourteen days after injury, Fbxo32 gene expression was increased after daily GC dosing and reduced by weekly GC dosing. Klf15 was stimulated by weekly dosing and reduced after daily GC dosing. Daily GC steroid treatments induced opposite trends. Mef2a and Igf1 followed similar divergent trends. Gray, WT vehicle; light blue, WT prednisone predose; blue, WT prednisone weekly; dark blue, WT prednisone daily; light purple, WT deflazacort predose; purple, WT deflazacort weekly; dark purple, WT deflazacort daily. n = 6 mice/group. *P < 0.05 vs. vehicle, 1-way ANOVA test with Bonferroni’s multiple comparison.

Figure 6. Weekly and daily GC steroid regimens enhanced sarcolemmal repair in mdx mice.

Steroids were given to mdx mice daily or weekly for 4 weeks. (A) (Left) Weekly and daily GC steroid regimens reduced FM4-64 dye accumulation and sarcolemmal injury in myofibers. Z-stack rendering of FM4-64 dye accumulation at laser-injured sarcolemmal sites at 300 seconds after injury. (Right) Quantitation of FM4-64 dye accumulation at sarcolemmal site after laser injury and end-point area of injury. (B) Fibrotic infiltration (blue scars; arrows) was reduced after all GC regimens, as evidenced by Masson’s trichrome staining of gastrocnemius muscle sections (10 replicates). (C) GC steroid regimens reduced hydroxyproline content in quadriceps muscles. (D) Macrophage infiltrates in skeletal muscles were reduced after all GC regimens. Chart shows quantitation of infiltrating F4-80+ cells, assessed by immunostaining on quadriceps muscle sections. (E) Diaphragm muscles followed trends similar to those of hind limb muscles, as fibrosis (blue scars; arrows) appeared comparably reduced after all GC regimens (10 replicates). n = 5 mice (50 myofibers)/group. *P < 0.05 vs. vehicle, 1-way ANOVA test with Bonferroni’s multiple comparison; #P < 0.05 vs. vehicle, 2-way ANOVA test with Bonferroni’s multiple comparison.

Figure 7. Daily GC dosing elicits atrophic mdx skeletal muscles, while weekly GC dosing does not.

(A) Daily, but not weekly, GC steroid dosing caused loss of body mass. (B) Weekly GC steroid dosing enhanced grip strength and run-to-exhaustion performance, while daily GC steroid-dosing treatments correlated with reduced grip strength and run performance. (C) Myofiber CSA was increased after weekly, but decreased after daily GC administration in gastrocnemius muscle sections. (D) Max tetanic force of tibialis anterior muscles was increased after weekly treatments and decreased after daily GC administration. (E) Fatigue analysis showed that tetanic force was increased over consecutive contraction bouts after weekly dosing, while daily dosing induced opposite trends. (F) Weekly GC regimen correlated with improved respiratory function, as assessed by WBP. Minute volume was increased and inspiration time was decreased in weekly GC dosing. Daily dosing reversed these beneficial trends. (G) Weekly GC treatments promoted increased CSA and diaphragm thickness, while daily CG dosing reduced CSA and diaphragm thickness. n = 5 mice/group. *P < 0.05 vs. mdx vehicle, 1-way ANOVA test with Bonferroni’s multiple comparison; #P < 0.05 vs. mdx vehicle, 2-way ANOVA test with Bonferroni’s multiple comparison.

Figure 8. Histone mark enrichment in GRE sites in gastrocnemius myofibers of treated mdx mice and GR occupancy.

(A) ChIP-qPCR for the repressive histone mark pH3k9me3 and the active histone mark H3k27ac (active histone mark) revealed that GREs of Anxa1 and Anxa6 were comparably enriched in permissive histone signature (higher H3k27ac, lower H3k9me3) after all GC treatments. However, the Klf15 GRE presented a divergent histone signature in response to weekly and daily regimens, consistent with its gene-expression trends. (B) After 4 weeks of GC treatment, ChIP-qPCR on the myofiber fraction from gastrocnemius muscles revealed that GRE sites were enriched in GR (NR3C1) binding in all steroid-treated muscles. GR occupancy on Klf15 was significantly higher after daily GC administration, as compared with weekly regimens, consistent with dose-dependent occupancy. n = 5 mice/group. *P < 0.05 vs. mdx vehicle or indicated sample, 1-way ANOVA test with Bonferroni’s multiple comparison.

Figure 9. Transcriptome profiling of prednisone-treated mdx muscles.

(A) RNA-seq was conducted on GC-treated mdx quadriceps muscle (n = 5 per condition). Principal component analysis discriminated gene-expression profiles according to mdx vehicle and weekly and daily GC dosing. (B) Unbiased hierarchical clustering based on differentially expressed genes also separated samples in regimen-specific groups. (C) Analysis of differentially expressed genes in paired comparisons for enriched GO terms. Highlighted are GO term groups of interest. Both daily and weekly GC steroids enriched for GO terms of membrane repair, growth regulation, immune response, and steroid metabolism. Weekly versus daily GC steroids only enriched for steroid metabolism. (D–F) Heat maps of annotated gene lists. Green denotes higher values, while red indicates lower values of gene mRNA quantitation, as compared with row mean value. n = 5 mice/group. (B–F) Adjusted P value < 0.05 for all genes and GO terms was reported using edgeR differential expression (treated vs. vehicle paired comparisons) and Panther GO enrichment tests (details in Supplemental Methods).