Myofiber-specific inhibition of TGFβ signaling protects skeletal muscle from injury and dystrophic disease in mice

Abstract

Muscular dystrophy (MD) is a disease characterized by skeletal muscle necrosis and the progressive accumulation of fibrotic tissue. While transforming growth factor (TGF)-β has emerged as central effector of MD and fibrotic disease, the cell types in diseased muscle that underlie TGFβ-dependent pathology have not been segregated. Here, we generated transgenic mice with myofiber-specific inhibition of TGFβ signaling owing to expression of a TGFβ type II receptor dominant-negative (dnTGFβRII) truncation mutant. Expression of dnTGFβRII in myofibers mitigated the dystrophic phenotype observed in δ-sarcoglycan-null (Sgcd(-/-)) mice through a mechanism involving reduced myofiber membrane fragility. The dnTGFβRII transgene also reduced muscle injury and improved muscle regeneration after cardiotoxin injury, as well as increased satellite cell numbers and activity. An unbiased global expression analysis revealed a number of potential mechanisms for dnTGFβRII-mediated protection, one of which was induction of the antioxidant protein metallothionein (Mt). Indeed, TGFβ directly inhibited Mt gene expression in vitro, the dnTGFβRII transgene conferred protection against reactive oxygen species accumulation in dystrophic muscle and treatment with Mt mimetics protected skeletal muscle upon injury in vivo and improved the membrane stability of dystrophic myofibers. Hence, our results show that the myofibers are central mediators of the deleterious effects associated with TGFβ signaling in MD.

Figure 1.

Generation of skeletal muscle-specific TG mice expressing a dominant-negative TGFβ-receptor. (A) Schematic representation of the transgene construct. pA is the abbreviation for polyadenylation sequence. (B) Western blot analysis for dnTGFβRII protein expression in quadriceps isolated from WT and two different TG mouse lines at 2 months of age. (C) Western blot analysis for dnTGFβRII protein expression in different muscle groups isolated from WT and TG mice at 2 months of age. Quad, quadriceps; Gast, gastrocnemius; TA, tibialis anterior; Diap, diaphragm. (D) H&E and Masson's trichrome-stained histological sections of quadriceps from 6-month-old mice of the indicated genotypes. Original magnification is ×200. (E) Percentage of myofibers from histological sections of the indicated muscles with centrally located nuclei from WT and TG mice at 6 months of age. (F) Measurement of grip strength in the indicated genotypes of mice at 2 and 6 months of age. *P < 0.05 versus WT controls. The number of mice used is shown in each of the bars in each panel. (G) Immunocytochemistry for phospho-SMAD3 (top row) in WT or TG myofibers isolated from the EDL and left untreated or stimulated with TGFβ for 20 min. The bottom row shows bright field images of the same fibers and nuclei in blue.

Figure 2.

Myofiber-specific inhibition of TGFβ signaling reduces MD histopathology. (A) Representative immunohistochemical images for phospho-SMAD3 in quadriceps (green, top panels) sections from 2-month-old Sgcd^−/− mice versus Sgcd^−/− mice crossed with the dnTGFβRII transgene (Sgcd^−/− TG). The lower panels show blue staining of nuclei (DAPI) and red staining of the myofiber outlines with WGA-TRITC. Original magnification is ×400. (B) Quantification of phospho-SMAD3-positive fibers and interstitial cells from histological sections stained as shown in A, from the indicated genotypes. (C) Representative H&E-stained histological sections from quadriceps of 2-month-old mice of the indicated genotypes. Original magnification is ×200. (D) MW/TL from the indicated muscle groups of mice at 2 months of age. (E) Percentage of myofibers with centrally located nuclei from mice at 2 months of age from the indicated muscles in Sgcd^−/− versus Sgcd^−/− TG mice. (F) Measurement of hydroxyproline content for fibrosis from the indicated muscle groups of mice at 2 months of age of the indicated genotypes. *P < 0.05 versus WT controls; ^#P < 0.05 versus Sgcd^−/− mice. The number of mice used is shown in the bars within each panel.

Figure 3.

Myofiber-specific inhibition of TGFβ signaling improves muscle performance and protects the sarcolemmal membrane in MD. (A) Time to fatigue in seconds with forced downhill treadmill running in the indicated groups of mice at 2 and 6 months of age. (B) Quantification of CK levels in the blood of the indicated groups of mice at 2 and 6 months of age. (C and D) Representative immunofluorescence images and quantitation of EBD (red) uptake in histological sections from quadriceps of 2-month-old mice subjected to running. Membranes are stained with wheat germ agglutinin (WGA-FITC, green). Original magnification is ×400. (E and F) Representative images and quantitation of FM1-43 dye entry (green fluorescence) in isolated FDB myofibers from the indicated genotypes of mice before and after laser-induced injury. Eight or more fibers were analyzed from each genotype. *P < 0.05 versus WT controls; ^#P < 0.05 versus Sgcd^−/− mice. The number of mice used is shown in the bars of each panel.

Figure 4.

Myofiber-specific inhibition of TGFβ signaling protects skeletal muscle upon CTX injury. (A) Representative H&E-stained histological sections from CTX injury to the TA muscle in WT or TG mice 5 and 14 days afterward. Original magnification is ×200. (B) TA muscle weights (MW) normalized to body weight (BW) from the indicated genotypes of mice with vehicle control or CTX injection. (C and D) Histological sections and quantification of eMHC-positive area (green) within the greater injury area (dark red) of the TA muscle 5 days after CTX injury. The white outline shows the regenerating area whereas the yellow outline is the entire area of injury. Original magnification is ×40. *P < 0.05 versus WT control; ^#P < 0.05 versus CTX-injured WT mice. The number of mice used is shown in the bars of each panel.

Figure 5.

Altered metallothionein mRNA expression underlies TGFβ effects on myofibers. (A–C) Real-time PCR from quadriceps muscle of WT and TG mice for metallothionein 1, 2, 3 (Mt1, Mt2, Mt3) mRNA expression normalized to Rpl7 control mRNA. *P < 0.05 versus WT. (D–I) Real-time PCR from C2C12-derived myotubes with vehicle control (con) or treated with recombinant TGFβ (TGF), recombinant interleukin-6 (IL6) or zinc (Zn) for Mt1, Mt2 and Mt3 mRNA expression normalized to Rpl7 control mRNA. *P < 0.05 versus control; ^#P < 0.05 versus Zn or IL6 treatment. The number of mice or biological replicates used is shown in each of the graphs.

Figure 6.

Signals involved in TGFβ-mediated inhibition of Mt expression. (A–C) Real-time PCR for Mt1, 2, 3 mRNA expression from C2C12 cells with no treatment (control) or treated with recombinant TGFβ (TGF) or zinc (Zn) with Adβgal control or AdSMAD6/7, Ad-dnJNK1/2, Ad-dnMEK1, Ad-dnMKK3/6 adenoviral infection. *P < 0.05 versus Adβgal. Data were normalized to Rpl7 control mRNA. (D and E) Representative images and quantitation of the ROS marker DHE (red) from cultured C2C12 myotubes treated with vehicle (control) or recombinant TGFβ. The same numbers of cells are present in each image. Original magnification is ×200.

Figure 7.

Treatment with Mt mimetics protects skeletal muscle upon injury. (A and B) Representative histological images and quantitation of the ROS marker DHE (red) from quadriceps sections of the indicated genotypes of mice. Original magnification is ×200. (C) Schematic of the protocol used for CTX injury to the TA along with EMTIN B treatment times. (D) Representative H&E-stained histological sections from the TA injured with CTX and treated with vehicle or EMTIN B, 5 days post-injury. Original magnification is ×200. (E) MW/BW from the indicated mouse treatment groups. (F and G) Representative immunohistochemical images and quantitation for eMHC expression (green) from CTX-injured TAs treated with vehicle or EMTIN B. (H and I) Time course and representative images of FM1-43 dye entry into EDL myofibers from Sgcd^−/− mice with or without EMTIM B-enriched antioxidant mix (AO) following laser-induced injury. Eight or more fibers were analyzed each. *P < 0.05 versus WT or vehicle control; ^#P < 0.05 versus Sgcd^−/− or CTX-injured vehicle-treated mice. The number of mice used is shown in each of the graphs.