Pulsed glucocorticoids enhance dystrophic muscle performance through epigenetic-metabolic reprogramming

Abstract

In humans, chronic glucocorticoid use is associated with side effects like muscle wasting, obesity, and metabolic syndrome. Intermittent steroid dosing has been proposed in Duchenne Muscular Dystrophy patients to mitigate the side effects seen with daily steroid intake. We evaluated biomarkers from Duchenne Muscular Dystrophy patients, finding that, compared with chronic daily steroid use, weekend steroid use was associated with reduced serum insulin, free fatty acids, and branched chain amino acids, as well as reduction in fat mass despite having similar BMIs. We reasoned that intermittent prednisone administration in dystrophic mice would alter muscle epigenomic signatures, and we identified the coordinated action of the glucocorticoid receptor, KLF15 and MEF2C as mediators of a gene expression program driving metabolic reprogramming and enhanced nutrient utilization. Muscle lacking Klf15 failed to respond to intermittent steroids. Furthermore, coadministration of the histone acetyltransferase inhibitor anacardic acid with steroids in mdx mice eliminated steroid-specific epigenetic marks and abrogated the steroid response. Together, these findings indicate that intermittent, repeated exposure to glucocorticoids promotes performance in dystrophic muscle through an epigenetic program that enhances nutrient utilization.

Trial registration: ClinicalTrials.gov NCT03319030.

Keywords: Metabolism; Muscle; Muscle Biology; Neuromuscular disease.

Conflict of interest statement

Conflict of interest: NU has filed a patent on intermittent glucocorticoid use based on this data (provisional number 62/876,238). SMH is an executive and shareholder of Amgen and holds equity stake in Tenaya Therapeutics. ZJ is an employee of Amgen as a member of the Amgen postdoctoral program.

Figures

Figure 1. Duchenne Muscular Dystrophy (DMD) patients treated with weekend glucocorticoid steroids have reduced markers of obesity and insulin resistance compared with those receiving daily glucocorticoids.

(A) Age, treatment duration, and BMI did not differ between daily (1–2.5 mg/kg/dose × 7 days/week) and high-dose weekend (1–4 mg/kg/dose × 2 consecutive days/week) cohorts. (B) Weekend dosing correlated with normalization of bone density score (DEXA scans at L1–L4 vertebrae) and less suppression of endogenous cortisol levels. (C) Compared with daily steroid treated patients, weekend steroid dosing correlated with decreased fat mass gain and increased lean mass preservation (TBLH, total body less head; DEXA scans). (D) Weekend steroid dosing, as compared with daily steroid dosing, associated with reduced serum insulin, glucose, branched chain amino acids (BCAA), and free fatty acids, consistent with reduced metabolic stress. Histograms depict single values and mean ± SEM; n = 12 patients/cohort; *P < 0.05 vs daily; Welch’s unpaired t test (2-tailed).

Figure 2. Epigenomic programs in steroid-treated dystrophic mouse muscles.

(A) PCA of H3K27ac profiles derived from quadriceps myofibers. Weekly vs. daily prednisone regimens cluster from each other and from vehicle-treated controls. (B) Motif enrichment analysis highlights GRE, KRE, and MEF2 as acetylation-enriched motifs after weekly prednisone, while FOXO3 motifs were enriched after daily prednisone. (C) Concordant genes enriched for both H3K27ac and expression included Klf15 and Mef2C after weekly prednisone, while daily prednisone promoted acetylation and expression of Foxo3 and other atrophy genes. (D) Representative H3K27ac markings across gene loci of interest demonstrated divergent acetylation enrichment with respect to weekly or daily prednisone (signal is tags/bp normalized to 1e7 reads; red arrows, gain; blue arrow, loss of H3K27ac signal). (E) Pathway analysis showed that pulsatile weekly prednisone increased transcription of genes regulating glucose, fatty acid, and BCAA metabolism. H3K27ac ChIP-seq showed GR enrichment after both weekly and daily steroids, but it showed increased enrichment of KRE and MEF2 sites only after weekly prednisone. ChIP-qPCR confirmed enriched occupancy for GR, KLF15, and MEF2C on the same sites (n = 3 mice/group for K27ac ChIP-seq, n = 5 mice/group for RNA-seq; q value, Benjamini-Hochberg test).

Figure 3. Pulsatile weekly prednisone acts through a GR-, KLF15-, and MEF2C-mediated transcriptional program and requires epigenetic remodeling.

(A) Genetic loss of Klf15 disrupts the transcriptional and metabolic changes induced by weekly prednisone in muscle, including Mef2C upregulation. (B) Luciferase reporter plasmids were electroporated into mdx myofibers, carrying regulatory regions from Mef2c, Pfkm, Ech1, and Bckdha loci. Prednisone pulse and Klf15 overexpression had additive effects in increasing GRE-KRE activation. Prednisone pulse, Klf15, and Mef2C overexpression had additive effects on MEF2 activation. Changes were lost after specific deletion of target sites (Δ). Histograms depict single values and mean ± SEM; n = 3 mice/group (A), n = 8 muscles/group (B). *P < 0.05 vs. own vehicle control, $P < 0.05 vs single-factor treatment, 1-way ANOVA with Tukey’s multiple comparison.

Figure 4. Pulsatile weekly prednisone requires epigenetic remodeling.

(A) Coadministration of anacardic acid, a histone acetyl-transferase inhibitor, blunted gain of H3K27ac marking in identified GRE, KRE, and MEF2 sites in muscle observed after weekly prednisone. (B) Coadministration of prednisone and anacardic acid blunted gains in oxygen consumption rates with glucose, palmitate, or valine. (C) Coadministration of prednisone and anacardic acid blunted gains in muscle force (Tibialis anterior). Histograms depict single values and mean ± SEM; n = 3 mice/group (C). *P < 0.05 vs. vehicle, 1-way ANOVA with Tukey’s multiple comparison.

Figure 5. Pulsatile glucocorticoids enhanced nutrient utilization for mitochondrial respiration in dystrophic muscle.

(A) PCA of 171 metabolites showed treatment-specific clustering of muscle tissues. (B) Heatmaps of metabolite levels showed that pulsatile prednisone activated consumption of glucose, BCAA, and glutamine, increasing ATP and phosphocreatine levels. (C) Muscle respirometry showed that weekly prednisone led to higher basal oxygen consumption in the presence of either glucose, palmitate, or valine. (D) Weekly prednisone increased BCKDHA levels and reduced its phosphorylation in muscle. (E) Weekly prednisone increased glucose uptake in muscle, as shown by 2-NBDG uptake in live dystrophic myofibers. (F) Weekly prednisone increased whole-body insulin sensitivity, while daily regimen induced insulin resistance. Curves depict mean ± SEM; histograms depict single values and mean ± SEM; box plots, Tukey distribution; n = 3 mice/group (A and B), n = 6 mice/group (C–F). *P < 0.05 vs. vehicle, 2-way ANOVA with Tukey’s multiple comparison (C), 1-way ANOVA with Tukey’s multiple comparison (D and E). #P < 0.05 vs. vehicle, 2-way ANOVA.

Figure 6. Isotope tracing in ex vivo contracting muscle confirms higher nutrient flux to the TCA cycle after pulsatile weekly prednisone treatment.

(A) 13C incorporation into muscle metabolites was analyzed through mass resolution in an ex vivo contraction system. (B) Using either glucose, palmitate, or valine as source of 13C, weekly prednisone–treated muscles showed higher 13C incorporation than vehicle-treated muscles. In the same mass spectrometry analysis, ATP and phosphocreatine levels increased in prednisone-treated muscle. Histograms depict mean ± SEM. (stacked histograms), or single values and mean ± SEM; n = 3 mice/group. #P < 0.05 vs. vehicle, 2-way ANOVA; *P < 0.05 vs. vehicle, Welch’s t test (2-tailed).

Figure 7. Long-term pulsatile prednisone improves insulin sensitivity, energy production, and muscle function in mdx mice.

(A) Multimodal imaging in live mice showed that weekly prednisone associated with a nonsignificant trend in fat mass reduction, no loss of bone mineral density, and significant changes in FDG uptake, upregulated in muscle and downregulated in fat (MRI-controled volumetric quantitation). (B) Treatment correlated with improved strength, respiratory function, and cardiac function over time, as compared with vehicle-treated mdx animals. (C) At endpoint, treated mice showed improved morbidity and increased VO2 and energy expenditure during the nocturnal activity phase. Treatment increased muscle ATP and force (Tibialis anterior), did not induce hyperglycemia, and reduced circulating levels of BCAA and free fatty acids. Curves, mean ± SEM; histograms depict single values and mean ± SEM; box plots, Tukey distribution; n = 3 mice/group (A), n = 10 mice/group (B and C). *P < 0.05 vs vehicle, Welch’s t test (2-tailed); #P < 0.05 vs vehicle, 2-way ANOVA.

Figure 8. Pulsatile prednisone enhanced muscle metabolism and function in a distinct genetic model of muscular dystrophy, Dysf-null mice, a genetic model of Limb Girdle Muscular Dystrophy type 2B.

Mice lacking dysferlin (Dysf) serve as a model of Limb Girdle Muscular Dystrophy type 2B, and loss of dysferlin induces muscular dystrophy by impairing muscle repair. (A) Weekly prednisone exposure mitigated loss of grip strength over time seen in this model, and treatment increased muscle force at study endpoint (Tibialis anterior). (B and C) Weekly prednisone–treated mice had improved respiratory and cardiac muscle function over time, as compared with vehicle-treated controls. (D) ATP levels were increased in both muscle and heart tissues at endpoint. Curves, mean ± SEM; histograms depict single values and mean ± SEM; n = 10 mice/group. *P < 0.05 vs vehicle, Welch’s t-test (two-tailed); #P < 0.05 vs vehicle, 2-way ANOVA.