Mineralocorticoid Receptor Signaling Contributes to Normal Muscle Repair After Acute Injury

Abstract

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.

Keywords: conditional knockout mouse; mineralocorticoid receptor; mineralocorticoid receptor antagonist; muscle injury; myofiber; spironolactone.

Figure 1

CYP11B2 is present in acutely injured skeletal muscle. (A) The presence of CYP11B2 aldosterone synthase at 1, 4, and 7 days after acute muscle injury in tibialis anterior of wild-type mice was evaluated with immunohistochemistry (brown) and appeared to be present at the highest level at 4 days post-injury (n = 3 Cre− mice per time point). Scale bar = 50 μm. (B) CYP11B2 (red) co-localized (arrows) with CD11b⁺ immune cells (green) at 4 days post-injury (n = 4 Cre−) similar to that observed in chronic muscle injury (Chadwick et al., 2016). DAPI was used to stain nuclei (blue) in the merged image. The co-localization of CYP11B2 and CD11b⁺ immune cells can be better observed in the zoomed images (bottom panels) for each channel from the area represented by the white box in the top panels. Scale bars = 25 μm.

Figure 2

Evaluation of MRcko excision after acute muscle injury. (A) PCR was run to detect the level of MR excision by presence of the MR null allele in TAs from mice at 4 (n = 5 MRcko barium chloride injection, n = 1 MRcko PBS injection, n = 1 Cre− barium chloride injection) and 7 (n = 5 MRcko barium chloride injection, n = 1 MRcko PBS injection, n = 1 Cre− barium chloride injection) (two technical replicates) days post-injury. (B) The level of MR null allele at 4 and 7 days post-injury was quantified and normalized to the level of MR null allele from PBS injection at 7 days post-injury. There was restoration of MR excision at 7 days post-injury (p = 0.0002). Means are shown by lines for each group in the dot plot. BaCl₂, barium chloride injection; PBS, sterile phosphate buffered saline injection; TA, tibialis anterior; data were analyzed using a Student’s t-test; ^***p ≤ 0.001 compared to MRcko barium chloride injection at 4 days post-injury.

Figure 3

Increased percentages of degenerating myofibers in both MRcko and MR antagonist-treated wild-type mice. (A) Representative confocal images of IgG and laminin staining of 4 days post-injury tibialis anterior (TA) sections. Scale bar = 100 μm. (B) Representative TA sections at 4, 7, and 14 days post-injury stained with hematoxylin and eosin. Scale bar = 100 μm. (C) Dot plots showing number of IgG positive fibers at 4 days post-injury for MRcko and Cre− littermates (n = 15 Cre−, and 13 MRcko) and spironolactone treated and untreated wild-type littermate mice (n = 16 untreated, and 16 spironolactone). Means are shown by lines for each group in the dot plot. (D) Dot plots showing percent of myofibers with centralized nuclei in MRcko mice and control littermates at 4 (n = 15 Cre−, and 13 MRcko), 7 (n = 12 Cre−, and 14 MRcko), and 14 (n = 11 Cre−, and 13 MRcko) days post-injury and spironolactone-treated mice at 4 (n = 9 untreated, and 8 spironolactone), 7 (n = 8 untreated, and 10 spironolactone), and 14 (n = 12 untreated, and 13 spironolactone) days post-injury. Means are shown by lines for each group. H & E, hematoxylin and eosin; data were analyzed using a Student’s t-test; ^*p ≤ 0.05.

Figure 4

MR antagonism reduces myofiber size after acute muscle injury. (A) Bar graphs showing the average size of centrally nucleated tibialis anterior myofibers at 4 (n = 15 Cre−, and 13 MRcko) (n = 9 untreated, and 8 spironolactone), 7 (n = 12 Cre−, and 14 MRcko) (n = 8 untreated, and 10 spironolactone), and 14 (n = 11 Cre−, and 13 MRcko) (n = 12 untreated, and 13 spironolactone) days post-injury in MRcko versus Cre− mouse littermates (left panel) and MR antagonist-treated and untreated wild-type littermate mice (right panel). The data are presented as mean ± SEM. ^**p ≤ 0.01. (B) The distribution of centrally nucleated myofiber percentages was broken down by size in line graphs into small myofibers 75–600 μm² and larger myofibers 600–3,600 μm². The centrally nucleated myofiber size was analyzed at 4 (n = 15 Cre−, and 13 MRcko) (n = 9 untreated, and 8 spironolactone), 7 (n = 12 Cre−, and 14 MRcko) (n = 8 untreated, and 10 spironolactone), and 14 (n = 11 Cre−, and 13 MRcko) (n = 12 untreated, and 13 spironolactone) days post-injury. For size analysis, the following numbers of centrally nucleated myofibers were analyzed for each group and days post-injury: 2,653 ± 105 Cre− and 2,584 ± 161 MRcko at 4 days post-injury, 2,810 ± 166 Cre− and 2,818 ± 181 MRcko at 7 days post-injury, 3,103 ± 157 Cre− and 3,173 ± 193 MRcko at 14 days post-injury, 2,466 ± 167 untreated and 2,697 ± 167 spironolactone treated at 4 days post-injury, 2,465 ± 165 untreated and 2,629 ± 130 spironolactone treated at 7 days post-injury, and 2,730 ± 159 untreated and 3,019 ± 157 spironolactone treated at 14 days post-injury. (C) Bar graph showing representative myogenic factor and myofiber growth genes assessed in spironolactone treated (n = 5) and untreated (n = 5) littermates at 7 days post-injury. Beta-actin levels are used as a normalization control for each sample and fold-changes are normalized to the same untreated control. Mstn, myostatin; Igf1, insulin-like growth factor 1; Myog, myogenin; Myod1, myoblast determination protein 1. The data are presented as mean ± SEM. All data were analyzed using a Student’s t-test, ^*p ≤ 0.05 and ^**p ≤ 0.01 for the bins of fiber-sizes shown by the bracket.

Figure 5

MRcko mice have increased CYP11B2 infiltration at 4 days post-injury. (A) Acutely injured tibialis anterior (TA) muscles of spironolactone treated and untreated mice at 4 days after acute injury were stained for CD11b to visualize myeloid immune cells (green), vimentin to visualize fibroblasts (red), and with DAPI to visualize nuclei (blue in merged image) and were imaged with confocal microscopy. Zoomed out images are shown to depict the areas of muscle infiltrated by immune cells (green dots) or fibroblasts (red dots) quantified in B. Scale bar = 100 μm. (B) Dot plots of spironolactone treated and untreated mouse (n = 16 spironolactone, and 16 untreated) TA muscle percent area at 4 days post-injury of immune cells and fibroblast infiltration together or percentage of myeloid immune cells infiltration alone. Means are shown by lines for each group in the dot plots. (C) CYP11B2 immunohistochemistry was analyzed at 4 (n = 6 Cre−, and 6 MRcko) (n = 5 untreated, and 6 spironolactone) days post-injury. Scale bar = 100 μm. (D) Dot plots of CYP11B2 percent infiltration at 4 days post-injury. Means are shown by lines for each group in the dot plots. All data was analyzed using a Student’s t-test. ^*p ≤ 0.05.

Figure 6

MRcko and MR antagonist treatment results in increased detrimental collagen infiltration. (A) Representative images of tibialis anterior muscle after picrosirius red staining under brightfield to quantify total collagen and polarized light to quantify collagen packaging. Loosely packaged collagen appears green and tightly packaged collagen appears red under polarized light. Scale bar = 40 μm (B) Dot plots of percent muscle area containing collagen infiltration and percent of loosely and tightly packaged collagen at 4 days after acute muscle injury for MRcko mice (n = 7 Cre−, and 8 MRcko) and spironolactone-treated mice (n = 8 untreated, 8 spironolactone) Means are shown by lines for each group in the dot plots. (C) Bar graph showing four representative collagen genes involved in fibrosis assessed in spironolactone treated (n = 5) and untreated (n = 5) littermates. Beta-actin levels were used as a normalization control. Col1, collagen 1 alpha 1; Col3, collagen 3 alpha 1; Col8, collagen 8 alpha 1; Col11, collagen 11, alpha 1. Data are presented as mean ± SEM. All data were analyzed using a Student’s t-test, ^*p ≤ 0.05 and ^**p ≤ 0.01.