A new immuno-, dystrophin-deficient model, the NSG-mdx(4Cv) mouse, provides evidence for functional improvement following allogeneic satellite cell transplantation.

Transplantation of a myogenic cell population into an immunodeficient recipient is an excellent way of assessing the in vivo muscle-generating capacity of that cell population. To facilitate both allogeneic and xenogeneic transplantations of muscle-forming cells in mice, we have developed a novel immunodeficient muscular dystrophy model, the NSG-mdx(4Cv) mouse. The IL2Rg mutation, which is linked to the Dmd gene on the X chromosome, simultaneously depletes NK cells and suppresses thymic lymphomas, issues that limit the utility of the SCID/mdx model. The NSG-mdx(4Cv) mouse presents a muscular dystrophy of similar severity to the conventional mdx mouse. We show that this animal supports robust engraftment of both pig and dog muscle mononuclear cells. The question of whether satellite cells prospectively isolated by flow cytometry can confer a functional benefit upon transplantation has been controversial. Using allogeneic Pax7-ZsGreen donors and NSG-mdx(4Cv) recipients, we demonstrate definitively that as few as 900 FACS-isolated satellite cells can provide functional regeneration in vivo, in the form of an increased mean maximal force-generation capacity in cell-transplanted muscles, compared to a sham-injected control group. These studies highlight the potency of satellite cells to improve muscle function and the utility of the NSG-mdx(4Cv) model for studies on muscle regeneration and Duchenne muscular dystrophy therapy.

Effects of ovarian hormones and estrogen receptor α on physical activity and skeletal muscle fatigue in female mice.

Menopause is associated with declines in physical activity and skeletal muscle strength. Physical activity is also reduced in rodents after ovariectomy (OVX) and whole-body estrogen receptor α (ERα) knockout. However, it is unclear if the effects are estradiol (E2) specific. Thus, the overall purpose of this study was to investigate the effects of the ovarian hormones, E2 and progesterone (P4), and skeletal muscle ERα (skmERα) on physical activity and skeletal muscle contractility in female mice. METHODS: Study 1: Forty female C57Bl/6J mice were given free access to running wheels for 2 weeks to assess baseline running and randomized into 4 treatment groups: OVX, OVX + E2, OVX + P4, OVX + E2 + P4. All mice underwent OVX, returned to wheels for 2 weeks, received hormone pellet implants and returned to running wheels for 6 weeks, after which soleus muscle contractility testing was completed. Study 2: Thirty-two skeletal muscle specific ERα knock-out (skmERαKO) mice and wildtype (WT) littermates were randomized into 4 groups: skmERαKO-Run, skmERαWT-Run, skmERαKO-Sed, and skmERαWT-Sed. Run mice were given free access to wheels for 20 wk and sedentary (Sed) mice maintained normal cage activities. At the end point, muscle contractility was tested. RESULTS: Study 1: OVX + E2 + P4 group ran greater distances than both the OVX and OVX + P4 groups (p ≤ 0.009). After fatiguing contractions, soleus muscles of the OVX + E2 + P4 group maintained greater submaximal force than those of other groups (p = 0.023). Immediately after the fatiguing contractions, OVX + E2 + P4 muscles had greater maximal force production than the OVX + E2 group (p = 0.027). Study 2: There were no differences in running distance between skmERαWT and skmERαKO mice (p = 0.240). Soleus muscles of skmERαKO mice were more fatigable (p < 0.001) and did not recover force as well as skmERαWT mice (p < 0.001). In vivo isometric, concentric and eccentric torque was decreased in skmERαKO mice compared to skmERαWT mice (p ≤ 0.029). CONCLUSIONS: Combined treatment of E2 + P4 in OVX mice restored physical activity, predominantly driven by E2, and protected soleus muscles against fatigue. Muscle of skmERαKO mice was weak regardless of physical activity. Although 20 wk of wheel running partially prevented force loss during fatigue in skmERαKO mice, force production during recovery remained low, indicating that estradiol functions through ERα in skeletal muscle.

Estrogen Regulates the Satellite Cell Compartment in Females.

Skeletal muscle mass, strength, and regenerative capacity decline with age, with many measures showing a greater deterioration in females around the time estrogen levels decrease at menopause. Here, we show that estrogen deficiency severely compromises the maintenance of muscle stem cells (i.e., satellite cells) as well as impairs self-renewal and differentiation into muscle fibers. Mechanistically, by hormone replacement, use of a selective estrogen-receptor modulator (bazedoxifene), and conditional estrogen receptor knockout, we implicate 17ß-estradiol and satellite cell expression of estrogen receptor α and show that estrogen signaling through this receptor is necessary to prevent apoptosis of satellite cells. Early data from a biopsy study of women who transitioned from peri- to post-menopause are consistent with the loss of satellite cells coincident with the decline in estradiol in humans. Together, these results demonstrate an important role for estrogen in satellite cell maintenance and muscle regeneration in females.

Intravenous Administration of Stable-Labeled N-Acetylcysteine Demonstrates an Indirect Mechanism for Boosting Glutathione and Improving Redox Status.

There is an increasing interest in using N-acetylcysteine (NAC) as a treatment for neurodegenerative disorders to increase glutathione (GSH) levels and its redox status. The purpose of this study was to characterize the biosynthesis of NAC to GSH using a novel stable isotope-labeled technique, and investigate the pharmacodynamics of NAC in vivo. Female wild-type mice were given a single intravenous bolus dose of 150 mg kg(-1) stable-labeled NAC. Plasma, red blood cells (RBC), and brain tissues were collected at predesignated time points. Stable-labeled NAC and its metabolite GSH (both labeled and unlabeled forms) were quantified in blood and brain samples. Molar ratios of the reduced and oxidized forms of GSH (GSH divided by glutathione disulfide, redox ratio) were also determined. The elimination phase half-life of NAC was approximately 34 min. Both labeled and unlabeled GSH in RBC were found to increase; however, the area under the curve above baseline (AUCb0-280 ) of labeled GSH was only 1% of the unlabeled form. These data indicate that NAC is not a direct precursor of GSH. In addition, NAC has prolonged effects in brain even when the drug has been eliminated from systemic circulation.

Comparison of colloid, thyroid follicular epithelium, and thyroid hormone concentrations in healthy and severely sick dogs.

OBJECTIVES: To compare serum concentrations of total thyroxine (TT4), free thyroxine (fT4), and thyroid-stimulating hormone (TSH), as well as measures of thyroid follicular colloid and epithelium, between groups of healthy dogs and severely sick dogs. DESIGN: Cross-sectional study. ANIMALS: 61 healthy dogs and 66 severely sick dogs. PROCEDURE: Serum samples were obtained before euthanasia, and both thyroid lobes were removed immediately after euthanasia. Morphometric analyses were performed on each lobe, and serum TT4, fT4, and TSH concentrations were measured. RESULTS: In the sick group, serum TT4 and fT4 concentrations were less than reference range values in 39 (59%) and 21 (32%) dogs, respectively; only 5 (8%) dogs had high TSH concentrations. Mean serum TT4 and fT4 concentrations were significantly lower in the sick group, compared with the healthy group. In the healthy group, a significant negative correlation was found between volume percentage of colloid and TT4 or fT4 concentrations, and a significant positive correlation was found between volume percentage of follicular epithelium and TT4 or fT4 concentrations. A significant negative correlation was observed between volume percentages of colloid and follicular epithelium in both groups. CONCLUSIONS AND CLINICAL RELEVANCE: TT4 and fT4 concentrations are frequently less than reference range values in severely sick dogs. Therefore, thyroid status should not be evaluated during severe illness. The absence of any significant differences in mean volume percentages of follicular epithelium between healthy and severely sick dogs suggests that these 2 groups had similar potential for synthesizing and secreting thyroid hormones.