Myeloid cells are capable of synthesizing aldosterone to exacerbate damage in muscular dystrophy

Abstract

FDA-approved mineralocorticoid receptor (MR) antagonists are used to treat heart failure. We have recently demonstrated efficacy of MR antagonists for skeletal muscles in addition to heart in Duchenne muscular dystrophy mouse models and that mineralocorticoid receptors are present and functional in skeletal muscles. The goal of this study was to elucidate the underlying mechanisms of MR antagonist efficacy on dystrophic skeletal muscles. We demonstrate for the first time that infiltrating myeloid cells clustered in damaged areas of dystrophic skeletal muscles have the capacity to produce the natural ligand of MR, aldosterone, which in excess is known to exacerbate tissue damage. Aldosterone synthase protein levels are increased in leukocytes isolated from dystrophic muscles compared with controls and local aldosterone levels in dystrophic skeletal muscles are increased, despite normal circulating levels. All genes encoding enzymes in the pathway for aldosterone synthesis are expressed in muscle-derived leukocytes. 11β-HSD2, the enzyme that inactivates glucocorticoids to increase MR selectivity for aldosterone, is also increased in dystrophic muscle tissues. These results, together with the demonstrated preclinical efficacy of antagonists, suggest MR activation is in excess of physiological need and likely contributes to the pathology of muscular dystrophy. This study provides new mechanistic insight into the known contribution of myeloid cells to muscular dystrophy pathology. This first report of myeloid cells having the capacity to produce aldosterone may have implications for a wide variety of acute injuries and chronic diseases with inflammation where MR antagonists may be therapeutic.

© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Figures

Figure 1.

11β-HSD2 is present in both human and mouse cultured myotubes and is increased in dystrophic skeletal muscle tissues. (A) 11β-HSD2 (44 kDa) was detected by western blots of human primary skeletal muscle (HSMM), C57/BL10 (C57), and mdx (MDX) myotubes differentiated for 5 days using equivalent amounts (35 µg) of cell lysates. Kidney is shown as a positive control tissue. GAPDH (36 kDa) shows equivalent loading of samples between C57 and MDX. (B) Representative western blot of quadriceps muscles from 3 biological replicates shows 11β-HSD2 protein levels from equivalent amounts (50 µg) of protein homogenates from: C57BL/10 wild-type control mice (C57), dystrophin-deficient mdx mice (MDX), dystrophin-deficient; utrophin haplo-insufficient mice (HET), and dystrophin/utrophin-deficient double knockout mice (DKO). Alpha-sarcomeric actin antibody (42 kDa) was used as a loading control.

Figure 2.

Detection of RT-PCR products verified the presence of enzymes required for aldosterone synthesis in muscle-derived leukocytes and dystrophic skeletal muscle tissue. RT-PCR products of transcripts encoding enzymes in the aldosterone synthesis pathway: StAR, CYP11A1, 3β-HSD2, CYP21, CYP11B1, CYP11B2, 11β-HSD1 and 11β-HSD2 from RNA isolated from DKO diaphragm muscle tissue (DKO Dia), differentiated myotubes from mdx primary myogenic cultures (MDX MT), and muscle-derived leukocytes isolated from C57 mice (C57 MdL). Only CYP11A1, StAR, 11β-HSD1 and 11β-HSD2 were detected in cultured mouse myotubes. CYP11B1 protein was detected in dystrophic skeletal muscle tissue but transcript levels were too low to verify expression. RNA isolated from an adrenal gland was used as a positive control for each PCR reaction. RT-PCR products were purified and sequenced. Sequences (not shown) were aligned to NCBI BLAST assembled genomes for confirmation of nucleic acid identity.

Figure 3.

CYP11B1 and CYP11B2 protein are present in normal healthy skeletal muscle tissues. Representative western blots of (A) CYP11B1 and (B) CYP11B2 protein levels from equivalent amounts of protein homogenates from C57 mouse heart, diaphragm, quadriceps, tibialis anterior, gastrocnemius, extensor digitorum longus, and soleus muscles. 50 µg of total protein was loaded for all lanes and standardization of loading was verified using an anti-GAPDH antibody (36 kDa). Adrenal glands were included as positive controls and standardization of loading was verified using an anti-GAPDH antibody. Skeletal muscles were abbreviated as follows: Dia= diaphragm, Quad= quadriceps, TA= Tibialis Anterior, GA= gastrocnemius, EDL= Extensor digitorum longus and Sol= soleus.

Figure 4.

CYP11B2, but not CYP11B1 protein levels are increased in dystrophic skeletal muscle tissues compared to wild-type. Representative western blots of (A) CYP11B2 protein levels from equivalent amounts of protein homogenates from C57 and HET mouse: quadriceps, tibialis anterior, heart, diaphragm, gastrocnemius, extensor digitorum longus and soleus muscles; (B) CYP11B1 protein levels from equivalent amounts of protein homogenates from C57 and HET mouse: quadriceps, tibialis anterior, heart, and diaphragm muscles from 2 biological replicates. 50 µg of total protein was loaded for all lanes and standardization of loading was verified using an anti-GAPDH antibody (36 kDa). Adrenal glands were included as positive controls.

Figure 5.

CYP11B2 is present at high levels in myeloid cells localized to areas of damage in dystrophic muscles. (A) CYP11B2 was detected in areas of damage within skeletal muscle tissue by immunofluorescence staining (red) of quadriceps muscle histological sections from C57 and dystrophic HET and DKO mice. Bar = 100 µm. (B) Immunofluorescence co-stain of CYP11B2 (red) and the immune cell marker CD11b (green) in quadriceps muscles from DKO mice revealed CYP11B2 co-localizes with a subset of CD11b-positive cells. The DNA dye DAPI is shown as a marker of nuclei (blue). Bar = 50 µm. (C) Western blot comparing CYP11B2 protein levels in myeloid cells isolated from hind limb musculature of C57 and MDX mice. CYP11B2 protein levels were lower compared to adrenal positive control and skeletal muscle tissue homogenates likely due to the series of enzymatic digestions and additional processing required to isolate leukocytes from skeletal muscles. Equivalent amounts (15 µg) of cell lysates were run with an adrenal gland sample included as a positive control. Standardization of loading was verified using an anti-GAPDH antibody. (D) CYP11B2 is present in a higher percentage of myeloid cells from MDX (35.7% ± 12.32%) muscle compared to C57 (10.90% ± 1.84%). CYP11B2 was detected by immunofluorescence staining (red) on C57 and MDX derived myeloid cells. Nuclei are stained with DAPI (blue). Top panel: confocal image without optical zoom, bar = 20 µm; and bottom panel: z-stack image of 0.22 μm optical slices, bar = 5 µm.

Figure 6.

Aldosterone levels are significantly higher in dystrophic quadriceps and diaphragm muscles compared to wild-type, despite comparable circulating levels. (A) Aldosterone was extracted from quadriceps (n =3) and diaphragm (n =2) muscles from C57/BL10 wild-type mice (C57) and dystrophin-deficient mdx mice (MDX); samples were run in triplicate and assay was repeated with biological replicates to confirm results. Aldosterone concentrations are represented as mean value ± SEM. Student t-test was used to determine differences between C57 and MDX groups; *= quadriceps (P = 0.02) and diaphragm (P = 0.05). (B) Baseline plasma aldosterone levels are not increased in dystrophic mice compared to wild-type. Plasma was collected from age matched C57 (n = 6) and MDX (n = 6) mice.

Figure 7.

Enzymes necessary for local aldosterone synthesis are present in muscle-derived leukocytes. Schematic diagram depicting the aldosterone synthesis pathway and enzyme required for each step. Presence of each enzyme was confirmed by sequencing of RT-PCR products. Yellow star = detected in muscle cultures (myotubes generated from dystrophin-deficient mdx mouse neonatal myoblasts), green star = detected in immune cells (leukocytes isolated from C57BL/10 wild-type mice), and red star = detected in whole muscle tissue (diaphragm muscle tissue from dystrophin/utrophin-deficient double knockout dko mouse).