Gene expression effects of glucocorticoid and mineralocorticoid receptor agonists and antagonists on normal human skeletal muscle

Abstract

Mineralocorticoid and glucocorticoid receptors are closely related steroid hormone receptors that regulate gene expression through many of the same hormone response elements. However, their transcriptional activities and effects in skeletal muscles are largely unknown. We recently identified mineralocorticoid receptors (MR) in skeletal muscles after finding that combined treatment with the angiotensin-converting enzyme inhibitor lisinopril and MR antagonist spironolactone was therapeutic in Duchenne muscular dystrophy mouse models. The glucocorticoid receptor (GR) agonist prednisolone is the current standard-of-care treatment for Duchenne muscular dystrophy because it prolongs ambulation, likely due to its anti-inflammatory effects. However, data on whether glucocorticoids have a beneficial or detrimental direct effect on skeletal muscle are controversial. Here, we begin to define the gene expression profiles in normal differentiated human skeletal muscle myotubes treated with MR and GR agonists and antagonists. The MR agonist aldosterone and GR agonist prednisolone had highly overlapping gene expression profiles, supporting the notion that prednisolone acts as both a GR and MR agonist that may have detrimental effects on skeletal muscles. Co-incubations with aldosterone plus either nonspecific or selective MR antagonists, spironolactone or eplerenone, resulted in similar numbers of gene expression changes, suggesting that both drugs can block MR activation to a similar extent. Eplerenone treatment alone decreased a number of important muscle-specific genes. This information may be used to develop biomarkers to monitor clinical efficacy of MR antagonists or GR agonists in muscular dystrophy, develop a temporally coordinated treatment with both drugs, or identify novel therapeutics with more specific downstream targets.

Keywords: Duchenne muscular dystrophy; aldosterone; eplerenone; glucocorticoid receptor; mifepristone; mineralocorticoid receptor; prednisolone; spironolactone.

Fig. 1.

Mineralocorticoid receptor (MR) protein levels are maintained in aldosterone-treated human myotubes. Normal human myotubes were treated with 100 nM aldosterone from 0 to 48 h. Representative Western comparing MR protein levels from equivalent amounts (35 µg) of cell lysates shown. Western blots used a combination of MR-specific monoclonal antibodies MR1-18 1D5 and MRN 2B7 (20) (full-length MR predicted molecular weight ~107 kDa) or a GAPDH antibody (loading control; predicted molecular weight ~36 kDa).

Fig. 2.

Gene expression changes resulting from high-dose aldosterone treatment were conserved with prednisolone treatment. A: treatment with the endogenous MR agonist aldosterone resulted in 200 gene expression changes. Functional clustering of these genes revealed: 13 apoptotic, 30 immune or defense response, 14 transcriptional regulators, 16 ion binding, 13 transmembrane, 10 cell adhesion, 12 extracellular matrix (ECM) or cytoskeletal binding, 6 alternative splicing, 25 vasculature or muscle structure development, 19 oxidative stress responsive, 15 regulators of cell differentiation, 2 GTPases, and 25 genes with unknown or specific functions. B: prednisolone treatment resulted in 483 gene expression changes and functional clustering of these genes revealed: 34 apoptotic, 41 immune or defense response, 36 transcriptional regulators, 47 ion binding, 47 transmembrane, 29 cell adhesion, 31 ECM or cytoskeletal binding, 19 alternative splicing, 69 vasculature or muscle structure development, 23 oxidative stress responsive, 30 cell cycle, 5 oxidoreductases, 10 GTPase, and 62 genes with unknown or specific functions. C: treatment with aldosterone increased expression of 111 genes and decreased expression of 89 genes compared with untreated normal human myotubes. Of these gene changes, 189 (95%) were conserved in prednisolone-treated vs. untreated human myotubes. Treatment with standard-of-care prednisolone increased expression of 252 genes and decreased 231 genes. About 40% of these changes (189 genes) were conserved with aldosterone-treated myotubes; the remaining 294 genes are specific to prednisolone treatment.

Fig. 3.

Treatment with eplerenone or mifepristone alone caused gene expression changes in normal human myotubes. A: treatment with the selective MR antagonist eplerenone resulted in 42 gene expression changes and functional clustering of these genes revealed: 3 apoptotic, 5 immune or defense response, 3 transcriptional regulators, 4 ion binding, 3 cell adhesion, 3 alternative splicing, 12 muscle contraction, and 9 genes with unknown or specific functions. B: treatment with the GR antagonist mifepristone resulted in 27 gene expression changes and functional clustering of these genes revealed: 1 apoptotic, 4 immune or defense response, 9 regulators of transcription, 2 ion binding, 2 alternative splicing, 2 developmental, and 7 genes with unknown or specific functions.

Fig. 4.

Gene expression changes with low-dose aldosterone compared with aldosterone plus antagonists in normal human myotubes. A: co-incubation with aldosterone plus eplerenone increased 8 genes and decreased 85 genes (93 total) compared with myotubes treated with aldosterone alone. Functional clustering revealed: 4 apoptotic, 7 transcriptional regulators, 5 ion binding, 7 transmembrane, 6 cell adhesion, 11 cytoskeletal protein binding, 4 muscle contraction, 27 cell cycle, and 22 genes with unknown or specific functions. B: co-incubation with aldosterone plus spironolactone increased 24 genes and decreased 90 genes (114 total) compared with myotubes treated with aldosterone alone. Functional clustering revealed: 9 apoptotic, 8 immune or defense response, 4 transcriptional regulators, 9 ion binding, 6 transmembrane, 13 cell adhesion, 8 structural constituents of muscle, 30 tissue development, and 27 genes with unknown or specific functions. C: co-incubation with aldosterone plus mifepristone increased 251 genes and decreased 370 genes (621 total) compared with myotubes treated with aldosterone alone. Functional clustering revealed: 33 apoptotic, 38 immune or defense response, 62 transcriptional regulators, 63 ion binding, 45 transmembrane, 18 cell adhesion, 63 ECM or cytoskeletal protein binding, 30 alternative splicing, 106 vasculature or muscle structure development, 25 oxidative stress responsive, 54 cell cycle, 7 oxidoreductase, 9 GTPase, and 68 genes with unknown or specific functions.

Fig. 5.

Real-time PCR revealed PRG4 transcripts are decreased with spironolactone treatment and upregulated in dystrophic muscle. A: PRG4 is significantly decreased (P = 0.0004) in human myotube samples co-incubated with 100 nM aldosterone plus 10 µM spironolactone compared with myotubes treated with 100 nM aldosterone only. B: Prg4 is increased in gastrocnemius muscles from mdx (dystrophin-deficient), het (utrn^+/−, mdx) and dko (double knockout, utrn^−/−; mdx) mice relative to C57 (wild type). Prg4 expression was significantly increased (P = 0.0175) in mdx gastrocnemius muscle compared with wild type. Each bar represents the mean of 3 technical triplicates ± SE. Three independent biological replicates are shown for each genotype or treatment group. Human myotube samples were normalized to the mean of A-1 and mouse gastrocnemius samples were normalized to the mean of C57-2. GAPDH was used as the normalization control for human myotube samples and β-actin was used as the normalization control for mouse gastrocnemius samples. A, aldosterone; S, spironolactone. ***P < 0.001 and *P < 0.05.