Decreased EBD uptake and increased annexin A1 up-regulation in the diaphragm of male <i>mdx</i> mice after injection with corticosterone or PD2 female <i>mdx</i> mice.

<p>EBD-positive area per total diaphragm section area was calculated after injecting male (A and B) and female (C) <i>mdx</i> mice with vehicle, corticosterone, estradiol, or progesterone for 2 days. Vehicle, n = 8 (male) and n = 4 (female); corticosterone, n = 9 (male) and n = 5 (female); estradiol, n = 9 (male) and n = 5 (female); progesterone, n = 9 (male) and n = 5 (female). Bar (200 μm). (D) mRNA levels of <i>Anxa1</i> in vehicle or corticosterone-treated mice, or virgin or PD2 female mice were quantified with real-time qPCR. Vehicle, n = 4 (male) and n = 6 (female); corticosterone, n = 6 (male) and n = 6 (female); n = 7 (virgin) and n = 6 (PD2). <i>GAPDH</i> was used for internal control.</p

<i>Annexin A1</i> (<i>Anxa1</i>) is up-regulated after treatment with corticosterone.

<p>(A, B) Female <i>mdx</i> myotubes were incubated for 20 hours with vehicle, corticosterone or corticosterone and mifepristone. After 1 hour of incubation with 100% medium or 50% hypo-osmotic shock medium, relative dead cells were calculated as Trypan blue-positive cells (red arrows) per total cells (n = 3). Bar (200 μm). (C) mRNA levels of <i>Inta7</i>, <i>MG53</i>, <i>Anxa1</i>, and <i>Serca1</i> in vehicle or corticosterone-treated wild-type or <i>mdx</i> myotubes were quantified with real-time qPCR (n = 3). <i>β-Actin</i> was used for internal control. (D) Female <i>mdx</i> myotubes were incubated for 20 hours with vehicle or corticosterone, and stained for MHC (green) and annexin A1 (red). DAPI (blue) denotes all nuclei. Bar (50 μm).</p

Pregnancy-Induced Amelioration of Muscular Dystrophy Phenotype in <i>mdx</i> Mice via Muscle Membrane Stabilization Effect of Glucocorticoid

<div><p>Duchenne muscular dystrophy (DMD), the most common and severe type of dystrophinopathy, is an X-linked recessive genetic disease caused by the absence of dystrophin, which leads to fragility and vulnerability of the sarcolemma to mechanical stretching with increased membrane permeability. Currently, glucocorticoids such as prednisolone are the only medication available for DMD. However, molecular pathways responsible for this effect are still unclear. In addition, it remains unclear whether sex-related factors, including pregnancy and the postpartum period, affect the phenotype of dystrophinopathy. Here, we report the amelioration of muscle membrane permeability in the diaphragm muscle of pregnant and postpartum, but not in nulliparous, <i>mdx</i> mice, an animal model for DMD, during the physiological surge of corticosterone, the most abundant glucocorticoid in rodents. Cultures of single muscle fibers and myotubes isolated from <i>mdx</i> mouse diaphragm demonstrate resistance to hypo-osmotic shock when treated with corticosterone but not with estradiol or progesterone. This corticosterone-mediated resistance was diminished by an antagonist of corticosterone, indicating that the glucocorticoid-glucocorticoid receptor axis plays a role in this membrane stabilization effect on muscle. Moreover, subcutaneous injection of corticosterone into <i>mdx</i> mice showed decreased membrane permeability. This is the first report to demonstrate that pregnancy-related resistance to muscle fiber damage in <i>mdx</i> mice due to the membrane stabilization effect of corticosterone. We also propose that this membrane stabilization effect is exerted through annexin A1 up-regulation as the molecular mechanisms of glucocorticoid effects on DMD muscle. Furthermore, single muscle fiber culture studies provide a sensitive chemical screening platform for muscular dystrophies.</p></div

Muscle fiber characterizations in the diaphragm from virgin, pregnant and postpartum <i>mdx</i> mice.

<p>(A) Diaphragm from 2.5-month-old virgin and pregnant female <i>mdx</i> mice with EBD injection. Gestation day 7.5 (GD7.5) and GD14.5 mean 7 and 14 days after the recognition of vaginal plug in mated female mice, respectively. Postpartum day 2 (PD2), PD7 and PD14 mean 2, 7 and 14 days after parturition, respectively. Bar (2 mm) (B) Diaphragm sections examined by fluorescence microscopy. Bars (200 μm). (C) The EBD uptake at GD14.5 (n = 3), PD2 (n = 4), PD7 (n = 3) and the virgin female <i>mdx</i> mice (n = 5). (D) Fiber size (μm) distribution in virgin (n = 4) and PD2 <i>mdx</i> mice (n = 4). (E) Histogram of number of fibers with centrally located nuclei (CLN) in virgin (n = 9), PD2 (n = 7) and PD7 (n = 5) <i>mdx</i> mice.</p

Over-expression of annexin A1 can protect muscle fiber death in hypo-osmotic shock.

<p>(A) Female <i>mdx</i> myoblasts were infected with empty retroviral vector (pMX), annexin A1 retroviral vector (pMX-Anxa1), control lentiviral shRNA vector or annexin A1 lentiviral shRNA vector. After myotube formation, cells were stained for MHC (green) and annexin A1 (red). DAPI (blue) denotes all nuclei. Bar (50 μm). Relative mRNA level of <i>annexin A1</i> in <i>mdx</i> myotubes was also quantified with real-time qPCR after viral infection (n = 3). <i>β-Actin</i> was used for internal control. (B) Myotubes were incubated with 100% medium or 50% hypo-osmotic shock medium for 1 hour, and stained with Trypan blue (red arrows). Relative dead cells were calculated as Trypan blue-positive cells per total cells (n = 3). Bar (200 μm).</p