Mineralocorticoid Receptor Signaling Contributes to Normal Muscle Repair After Acute Injury.

Acute skeletal muscle injury is followed by a temporal response of immune cells, fibroblasts, and muscle progenitor cells within the muscle microenvironment to restore function. These same cell types are repeatedly activated in muscular dystrophy from chronic muscle injury, but eventually, the regenerative portion of the cycle is disrupted and fibrosis replaces degenerated muscle fibers. Mineralocorticoid receptor (MR) antagonist drugs have been demonstrated to increase skeletal muscle function, decrease fibrosis, and directly improve membrane integrity in muscular dystrophy mice, and therefore are being tested clinically. Conditional knockout of MR from muscle fibers in muscular dystrophy mice also improves skeletal muscle function and decreases fibrosis. The mechanism of efficacy likely results from blocking MR signaling by its endogenous agonist aldosterone, being produced at high local levels in regions of muscle damage by infiltrating myeloid cells. Since chronic and acute injuries share the same cellular processes to regenerate muscle, and MR antagonists are clinically used for a wide variety of conditions, it is crucial to define the role of MR signaling in normal muscle repair after injury. In this study, we performed acute injuries using barium chloride injections into tibialis anterior muscles both in myofiber MR conditional knockout mice on a wild-type background (MRcko) and in MR antagonist-treated wild-type mice. Steps of the muscle regeneration response were analyzed at 1, 4, 7, or 14 days after injury. Presence of the aldosterone synthase enzyme was also assessed during the injury repair process. We show for the first time aldosterone synthase localization in infiltrating immune cells of normal skeletal muscle after acute injury. MRcko mice had an increased muscle area infiltrated by aldosterone synthase positive myeloid cells compared to control injured animals. Both MRcko and MR antagonist treatment stabilized damaged myofibers and increased collagen infiltration or compaction at 4 days post-injury. MR antagonist treatment also led to reduced myofiber size at 7 and 14 days post-injury. These data support that MR signaling contributes to the normal muscle repair process following acute injury. MR antagonist treatment delays muscle fiber growth, so temporary discontinuation of these drugs after a severe muscle injury could be considered.

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

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. 11ß-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.

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

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.

The Angiotensin Converting Enzyme Inhibitor Lisinopril Improves Muscle Histopathology but not Contractile Function in a Mouse Model of Duchenne Muscular Dystrophy.

BACKGROUND: Angiotensin converting enzyme inhibitors (ACEi) are the current standard of care treatment for cardiac dysfunction in Duchenne muscular dystrophy patients. We previously showed treatment with an ACEi plus mineralocorticoid receptor (MR) antagonist improves limb and respiratory skeletal muscles, in addition to cardiac muscles, in a dystrophic mouse model at 20 weeks-of-age. OBJECTIVE: To determine whether previously observed preclinical benefits of an ACEi plus MR antagonist on dystrophic skeletal muscles can be reproduced by increasing ACEi dosage alone. We also compared functional and histological outcome measures at 10 and 20 weeks-of-age. METHODS: Dystrophin deficient utrophin haplo-insufficient (utrn+/-; mdx) "het" mice were treated with 10, 20, or 50 mg/kg × day of the ACEi lisinopril from 4 to 10 weeks-of-age via water bottles and compared with C57BL/10 wild-type control mice and untreated hets. Data from 10 week-old het mice were also compared to data collected from an untreated het group at 20 weeks-old. In vivo cardiac and grip strength measurements, in vitro diaphragm and extensor digitorum longus muscle force measurements, and histopathological analyses were performed. One-way ANOVA followed by Dunnett post hoc comparison was used to determine significance. RESULTS: ACEi treatment reduced skeletal muscle damage but had no significant effect on muscle force. Body weight, heart rate, grip strength and blood pressure were unaffected by treatment. Limb muscle histopathology was more informative at 10 than 20 weeks-of-age. CONCLUSIONS: These results suggest increased ACEi dosage alone cannot improve all dystrophic parameters. Further optimization of MR antagonists in 20 week-old mice is warranted.

Claudin-5 levels are reduced from multiple cell types in human failing hearts and are associated with mislocalization of ephrin-B1.

Claudin-5 is transcriptionally downregulated resulting in dramatically reduced protein levels in human heart failure. Studies in mice have demonstrated that reduced claudin-5 levels occur prior to cardiac damage and far before reduced whole heart function. Therefore, claudin-5 may be a useful early therapeutic target for human heart failure. However, the cell types in which claudin-5 is localized in human heart and from which claudin-5 is reduced in heart failure is not known. The recent identification of claudin-5's interaction with ephrin-B1 in mouse hearts has also not been investigated in non-failing or failing human hearts. In this study we collected human left ventricular mid-myocardium histological samples from 7 non-failing hearts and 16 end-stage failing hearts. Immunoblots demonstrate severe reductions of claudin-5 protein in 14 of 16 failing hearts compared to non-failing controls. Claudin-5 was observed to localize to cardiomyocytes, endothelial cells, and a subset of fibroblasts in non-failing human heart sections. In isolated cardiomyocytes, the transmembrane claudin-5 protein localized in longitudinal striations in lateral membranes. In failing heart, both cardiomyocyte and endothelial claudin-5 localization was severely reduced, but claudin-5 remained in fibroblasts. Absence of claudin-5 staining also correlated with the reduction of the endothelial cell marker CD31. Ephrin-B1 localization, but not protein levels, was altered in failing hearts supporting that claudin-5 is required for ephrin-B1 localization. These data support that loss of claudin-5 in cardiomyocytes and endothelial cells is prevalent in human heart failure. Investigating claudin-5/ephrin-B1 protein complexes and gene regulation may lead to novel therapies.