Mineralocorticoid receptors are present in skeletal muscle and represent a potential therapeutic target

Abstract

Early treatment with heart failure drugs lisinopril and spironolactone improves skeletal muscle pathology in Duchenne muscular dystrophy (DMD) mouse models. The angiotensin converting enzyme inhibitor lisinopril and mineralocorticoid receptor (MR) antagonist spironolactone indirectly and directly target MR. The presence and function of MR in skeletal muscle have not been explored. MR mRNA and protein are present in all tested skeletal muscles from both wild-type mice and DMD mouse models. MR expression is cell autonomous in both undifferentiated myoblasts and differentiated myotubes from mouse and human skeletal muscle cultures. To test for MR function in skeletal muscle, global gene expression analysis was conducted on human myotubes treated with MR agonist (aldosterone; EC50 1.3 nM) or antagonist (spironolactone; IC50 1.6 nM), and 53 gene expression differences were identified. Five differences were conserved in quadriceps muscles from dystrophic mice treated with spironolactone plus lisinopril (IC50 0.1 nM) compared with untreated controls. Genes down-regulated more than 2-fold by MR antagonism included FOS, ANKRD1, and GADD45B, with known roles in skeletal muscle, in addition to NPR3 and SERPINA3, bona fide targets of MR in other tissues. MR is a novel drug target in skeletal muscle and use of clinically safe antagonists may be beneficial for muscle diseases.

Keywords: aldosterone; gene expression microarray; muscular dystrophy; spironolactone; steroid hormone receptors.

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Figures

Figure 1.

MR is expressed in mouse skeletal muscle tissues and both mouse and human cultured myoblasts (MB) and myotubes (MT). Western blots using combination of MR-specific monoclonal antibodies MR1-18 1D5 and MRN 2B7 (31) show protein at predicted molecular weight for full-length MR of ∼107 kDa in all normal mouse (C57BL/10) skeletal muscles analyzed, and in heart (A). Second lower molecular weight isoform is also present in all tissues. Two MR protein bands are also present in both mouse (B) and human (C) myoblasts, but full-length MR becomes predominant in differentiated mouse myotubes. MR protein expression was detected using equivalent amounts (35 µg) of C57BL/10 protein homogenates, and C2C12 and human primary muscle (HSMM) cell lysates. Myotubes were differentiated for either 5 (HSMM) or 7 d (C2C12). Standardization of loading was verified using α-sarcomeric actin antibody; predicted molecular weight, ∼42 kDa. Differentiated myotubes contain higher levels of actin than undifferentiated myoblasts per microgram of total protein. Dia, diaphragm; Quad, quadriceps; TA, tibialis anterior; GA, gastrocnemius; Sol, soleus; EDL, extensor digitorum longus.

Figure 2.

Sequencing analysis verified presence of MR in skeletal muscle. A) Linearized structure of full-length MR gene (mouse and human MR genes are comparable) and human splice variant NR3C2 with exon 5 deletion. Translation is initiated in exon 2; exons 3, 4, and small portion of exon 5 encode DNA-binding domain; and exons 5–9 encode ligand-binding domain. Region spanned by human and mouse MR primers used for PCR and subsequent sequencing analysis are depicted by arrows. B) RT-PCR amplifying human MR from human myotubes yielded 2 bands corresponding to full-length MR (upper band) and NR3C2 transcript variant 2 (lower band) with deletion of exon 5 and truncation of DNA and ligand binding domains. C) Only 1 band corresponding to full-length MR was detected from RT-PCR of mouse MR using mouse quadriceps muscle; primers spanning exon 5 did not detect any splice variants with exon 5 deletion.

Figure 3.

MR protein levels are maintained in dystrophic muscle and agonist vs. antagonist treated human cells. A) Representative Western blots of quadriceps muscles from 3 biologic replicates are shown comparing MR protein levels from equivalent amounts of protein homogenates from C57BL/10 wild-type (C57) mice, dystrophin-deficient mdx (MDX) mice, het (HET) mice, and dystrophin/utrophin-deficient double knockout (DKO) mice. B) MR was also detected by Western blots of human primary muscle (HSMM) myotubes differentiated for 5 d (MT) and then treated for 5 d with 10 µM of MR antagonist spironolactone (S-10), MR agonist aldosterone (A-10), or ethanol vehicle untreated control (UC). For (A) and (B), 35 µg of total protein was used for each sample. Western blots used combination of MR-specific monoclonal antibodies MR1-18 1D5 or MR1-18 6G1 and MRN 2B7 (31) (full-length MR predicted molecular weight, ∼107 kDa) or α-sarcomeric actin antibody (loading control; predicted molecular weight, ∼42 kDa). C) MR localization is similar after treatment with MR antagonist and agonist. MR was detected by immunofluorescence staining (green) on human primary (HSMM) myotubes differentiated for 5 d then treated for 48 h with 10 µM of MR antagonist spironolactone or MR agonist aldosterone. Staining of subset of nuclei was present in all 3 groups. MR staining is similar with longer 5 d treatments (data not shown). Scale bars, 50 µm.

Figure 4.

Real-time RT-PCR revealed Ankrd1 mRNA levels are up-regulated in dystrophic muscle. Quadriceps muscles from MDX (dystrophin deficient) and HET (utrn+/−; mdx) mice relative to C57 (wild-type). Each bar represents mean of 3 technical triplicates ± se. Three biologic replicates are shown for each genotype; all samples are normalized to mean of C57-3. 18S was used as normalization control for all samples.

Figure 5.

ANKRD1 protein levels are conserved between normal and dystrophic mouse muscles but altered by treatment with MR agonists and antagonists in human cultured myotubes. A) Representative samples (quadriceps muscle) are shown comparing level of Ankrd1 protein (∼43 kDa) expression on equivalent amounts (35 µg) of protein homogenates from: C57BL/10 wild-type (C57) mice, dystrophin-deficient mdx (MDX) mice, het (HET) mice, and dystrophin/utrophin-deficient double knockout (DKO) mice (n = 3). These Western blots were run in conjunction with MR blots from Fig. 3 using same actin loading control. B) Human primary muscle (HSMM) myotubes (MT) were differentiated for 5 d and then treated for 5 d with 10 µM of MR agonist aldosterone (A-10), MR antagonist spironolactone (S-10), 2 µM of MR antagonist eplerenone (E-2), or ethanol vehicle untreated control (UC); 35 µg of total protein from cell lysates were used for each sample. Standardization of loading was verified using α-sarcomeric actin antibody; predicted molecular weight was ∼42 kDa. C) ANKRD1 staining is altered by treatment with MR antagonist. Ankrd1 was detected by immunofluorescence staining (red) on human primary (HSMM) myotubes differentiated for 5 d and then treated for ∼5 d with 10 µM of MR agonist aldosterone, MR antagonist spironolactone, or 2 µM of more selective MR antagonist eplerenone. Scale bars, 50 µm.