Morphological and functional analyses of skeletal muscles from an immunodeficient animal model of limb-girdle muscular dystrophy type 2E

Abstract

Introduction: Limb-girdle muscular dystrophy type 2E (LGMD2E) is caused by mutations in the β-sarcoglycan gene, which is expressed in skeletal, cardiac, and smooth muscles. β-Sarcoglycan-deficient (Sgcb-null) mice develop severe muscular dystrophy and cardiomyopathy with focal areas of necrosis.

Methods: In this study we performed morphological (histological and cellular characterization) and functional (isometric tetanic force and fatigue) analyses in dystrophic mice. Comparison studies were carried out in 1-month-old (clinical onset of the disease) and 7-month-old control mice (C57Bl/6J, Rag2/γc-null) and immunocompetent and immunodeficient dystrophic mice (Sgcb-null and Sgcb/Rag2/γc-null, respectively).

Results: We found that the lack of an immunological system resulted in an increase of calcification in striated muscles without impairing extensor digitorum longus muscle performance. Sgcb/Rag2/γc-null muscles showed a significant reduction of alkaline phosphate-positive mesoangioblasts.

Discussion: The immunological system counteracts skeletal muscle degeneration in the murine model of LGMD2E. Muscle Nerve, 2018.

Keywords: EDL; immunodeficient dystrophic mice; mesoangioblasts; smooth muscle; β-sarcoglycan.

Figure 1

Histological analysis of gastrocnemius (GCN), diaphragm (DIA), and cardiac muscles from C57Bl/6J, Rag2/γc‐null mice, IC‐Sgcb‐null, and ID‐Sgcb‐null mice. Paraffin sections were obtained from 7‐month‐old mice and stained with hematoxylin and eosin (H&E) (A) and Masson trichrome (B). Fibrosis, fiber degeneration, and central nucleation of fibers are present only in dystrophic samples (asterisks and arrows, respectively). Similarly, large areas of necrosis and mild fibrosis are seen in dystrophic cardiac muscles. Bar = 50 μm. Muscle calcifications (arrows) can be observed directly in intact diaphragms from dystrophic mice (C). Fibrosis quantification (mm²) of gastrocnemius (GCN), diaphragm (DIA), and cardiac muscles is presented as mean ± standard deviation in (D). Calcification in GCN muscles, DIA, and cardiac muscles is reported as mean ± standard deviation in (E). One‐way ANOVA test (n = 4): *P < 0.05; **P < 0.01; ***P < 0.001. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 2

Inflammatory infiltration in skeletal muscle and cardiac muscles from C57Bl/6J, Rag2/γc null mice, IC‐Sgcb null, and ID‐Sgcb null mice. Muscle sections were obtained from 7‐month‐old mice. Distribution of type M1 and M2 macrophages is shown in (A). F4/80Ab was used to identify M1 macrophages (red). CD206‐Ab was used to detect M2 macrophages (green). Nuclei were counterstained with Hoecht stain (blue). Bar = 50 μm. M1 macrophage quantification (cells/mm²) in the hindlimb is shown as mean ± standard deviation in (B). M2 macrophage quantification (cells/mm²) in the heart is shown as mean ± standard deviation in (C). Ratio of M2/M1 in hindlimb and heart in shown as mean ± standard deviation in (D). One‐way ANOVA test (n = 4): ***P < 0.001. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 3

Immunofluorescence staining for fiber types from skeletal muscle sections of 7‐month‐old C57Bl/6J, Rag2/γc‐null, IC‐Sgcb‐null, and ID‐Sgcb‐null mice. Antibodies used for this immunostaining indicate fiber types 1 (A) and 2A (B). Bar = 50 μm. Fiber cross‐sectional area (CSA) was measured and the distribution is shown as mean ± standard deviation for both slow and glycolytic type A fibers. Several sections from 3 mice for each cohort were evaluated. One‐way ANOVA test: compared with IC‐Sgcb ($) and compared with ID‐Sgcb (*) (n = 3): **,^$$ P < 0.01; ***P < 0.001; ****,^$$$$ P < 0.0001. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 4

Functional muscle analysis from C57Bl/6J, Rag2/γc‐null mice, IC‐Sgcb‐null and ID‐Sgcb‐null mice. Isometric force measured during a fused tetanus stimulation at 180 Hz is reported in (A) as maximum force, and normalization by muscle cross‐sectional is reported in (B) as specific force. Another specific force, calculated with stimulation at 200 Hz, is reported as a bar chart in (C). The time course of force decline during 40 s of fatiguing stimulation is shown in (D) as fatigue. Data are presented as mean from 1‐month‐old mice (panels at left) and 7‐month‐old mice (panels at right) (n = 5). One‐way ANOVA test: C57Bl/6J vs. IC‐Sgcb‐null (‡); Rag2/γc null vs. ID‐Sgcb null (§); IC‐Sgcb null vs. ID‐Sgcb null (*) (n = 4); ^‡‡,§§,**P < 0.01; ^{‡‡‡,§§§},***P < 0.001. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 5

Pericyte quantification from C57Bl/6J, Rag2/γc‐null mice, IC‐Sgcb‐null and ID‐Sgcb‐null mice. Anti‐alkaline phosphatase antibodies (red fluorophore) reveal pericytes in red in GCN sections. Nuclei are counterstained in blue with Hoecht stain (A). Bar = 25 µm. Quantification of AP⁺ cells is reported in (B). AP enzymatic staining was performed on a mix population obtained by primary culture of biopsies from hindlimb muscle and heart of 1‐month‐old (C) (bar = 25 µm) and 7‐month‐old (E) mice. Bar = 10 µm. Quantification of the AP⁺ cells in hindlimbs and hearts from 1‐month‐old (D) and 7‐month‐old (F) mice is shown as mean ± SEM. One‐way ANOVA test (n = 5): *P < 0.05; **P < 0.01; ***P < 0.001. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 6

Smooth muscle differentiation of interstitial cells and pericytes from C57Bl/6J, Rag2/γc‐null, IC‐Sgcb‐null, and ID‐Sgcb‐null mice. Quantification of α‐SMA⁺, calponin⁺, αSMA/calponin double‐positive and double‐negative cells from 1‐month‐old (A) and 7‐month‐old (B) mice is shown. Immunofluorescence analysis for α‐SMA (red) and calponin (green) and quantification of positive cells derived from cardiac and hindlimb pericytes of 1‐month‐old mice is shown in (C). One‐way ANOVA test (n = 5): *P < 0.05; **P < 0.01; ***P < 0.001; t‐test: *P < 0.05; **P < 0.01. [Color figure can be viewed at http://wileyonlinelibrary.com]