Similar efficacy from specific and non-specific mineralocorticoid receptor antagonist treatment of muscular dystrophy mice

Abstract

Background: Combined treatment with an angiotensin-converting enzyme inhibitor and a mineralocorticoid receptor (MR) antagonist improved cardiac and skeletal muscle function and pathology in a mouse model of Duchenne muscular dystrophy. MR is present in limb and respiratory skeletal muscles and functions as a steroid hormone receptor.

Objective: The goals of the current study were to compare the efficacy of the specific MR antagonist eplerenone with the non-specific MR antagonist spironolactone, both in combination with the angiotensin-converting enzyme inhibitor lisinopril.

Methods: Three groups of n=18 dystrophin-deficient, utrophin-haploinsufficient male mice were given chow containing: lisinopril plus spironolactone, lisinopril plus eplerenone, or no drug, from four to 20 weeks-of-age. Eighteen C57BL/10 male mice were used as wild-type controls. In vivo measurements included cardiac magnetic resonance imaging, conscious electrocardiography, and grip strength. From each mouse in the study, diaphragm, extensor digitorum longus, and cardiac papillary muscle force was measured ex vivo, followed by histological quantification of muscle damage in heart, diaphragm, quadriceps, and abdominal muscles. MR protein levels were also verified in treated muscles.

Results: Treatment with specific and non-specific MR antagonists did not result in any adverse effects to dystrophic skeletal muscles or heart. Both treatments resulted in similar functional and pathological improvements across a wide array of parameters. MR protein levels were not reduced by treatment.

Conclusions: These data suggest that spironolactone and eplerenone show similar effects in dystrophic mice and support the clinical development of MR antagonists for treating skeletal muscles in Duchenne muscular dystrophy.

Keywords: Duchenne muscular dystrophy; eplerenone; lisinopril; mineralocorticoid receptors; spironolactone.

Conflict of interest statement

The authors declare no conflict of interest relevant to this study.

Figures

Fig. 1. Myofiber damage in quadriceps, heart, diaphragm and abdominal muscles

Representative images of immunofluorescence stain for serum IgG on quadriceps, heart, diaphragm and abdominal sections. Sections from treated mice groups (EL and LS) show less ongoing damage than untreated het mice (LS heart compared to het, P = 0.0059; LS and EL diaphragm sections compared to het, P = 0.0003 and P = 0.0117 respectively, see also Table 1). C57BL/10 (C57) wild-type control mice had no visible muscle damage. Bar = 200 μm.

Fig. 2. EDL force measurements

(A) EDL specific force shows a larger average force generation in the LS and EL treated groups compared with the untreated het group. C: C57BL/10 wild-type control mice, U: untreated het mice, and LS: lisinopril / spironolactone treated mice (n = 18 per group); and EL: lisinopril / eplerenone treated mice (n = 17). (B) EDL forces measured during 10 eccentric contractions (ecc1-10) and during a final contraction (ecc 11) after a rest period recovery to allow recovery from fatigue, show lower force generation in the untreated het group compared to C57 control mice. Force for LS and EL treated groups had values closer to wild-type values. Force are represented mean ± SEM.

Fig. 3. MR protein levels are maintained in quadriceps muscles from het mice after 16 weeks of LS or EL treatment

Representative western blots of quadriceps muscles from 3 biological replicates are shown comparing MR protein levels from equivalent amounts (50 μg) of protein homogenates from: C57BL/10 wild-type mice (C57), dystrophin-deficient; utrophin haplo-insufficient mice (Het), lisinopril plus spironolactone treated Het mice (LS) and eplerenone plus lisinopril treated HET mice (EL). Western blots used a combination of MR-specific monoclonal antibodies MR1-18 1D5 & MRN 2B7 (full length MR predicted molecular weight ~107kDa, arrow) or a GAPDH antibody (loading control; predicted molecular weight ~36kDa). MR blots detected an additional lower molecular weight (~90 kDa) doublet, which are known degradation products resulting from snap freezing samples prior to processing [22].