Deletion of periostin reduces muscular dystrophy and fibrosis in mice by modulating the transforming growth factor-β pathway

Abstract

The muscular dystrophies are broadly classified as muscle wasting diseases with myofiber dropout due to cellular necrosis, inflammation, alterations in extracellular matrix composition, and fatty cell replacement. These events transpire and progress despite ongoing myofiber regeneration from endogenous satellite cells. The degeneration/regeneration response to muscle injury/disease is modulated by the proinflammatory cytokine transforming growth factor-β (TGF-β), which can also profoundly influence extracellular matrix composition through increased secretion of profibrotic proteins, such as the matricellular protein periostin. Here we show that up-regulation and secretion of periostin is pathological and enhances disease in the δ-sarcoglycan null (Sgcd(-/-)) mouse model of muscular dystrophy (MD). Indeed, MD mice lacking the Postn gene showed dramatic improvement in skeletal muscle structure and function. Mechanistically, Postn gene deletion altered TGF-β signaling so that it now enhanced tissue regeneration with reduced levels of fibrosis. Systemic antagonism of TGF-β with a neutralizing monoclonal antibody mitigated the beneficial effects of Postn deletion in vivo. These data suggest that periostin functions as a disease determinant in MD by promoting/allowing the pathological effects of TGF-β, suggesting that inhibition of periostin could represent a unique treatment approach.

Fig. 1.

Evaluation of periostin expression in Sgcd^−/− mice. (A) Immunohistochemistry showing periostin (green) localization in muscle biopsy specimens from normal patients and patients with Duchenne MD (DMD). (Original magnification: 600×; scale bar: 50 μm.) (B) Periostin levels, detected by ELISA, in the serum of 6-wk old WT (Wt) and Sgcd^−/− mice. *P < 0.05. n = 4 mice each. (C) Western blot for periostin in Wt and Sgcd^−/− mice from the diaphragm at 6 wk and 6 mo of age. GAPDH was used as a loading control. (D) Immunohistochemistry showing increased periostin (green) in areas of fibrosis in the diaphragm (diaph.), gastrocnemius (gastroc.), and quadriceps (quad.) of Wt and Sgcd^−/− mice at 6 wk and 6 mo of age. Membranes are stained in red with wheat germ agglutinin conjugated to TRITC, and nuclei are stained with DAPI (blue). (Original magnification: 400×; scale bar: 100 μm.) (E) Immunohistochemistry showing ZsGreen staining (green) of the gastrocnemius in Sgcd^−/− and Sgcd^−/−Postn-ZsGreen TG mice. The ZsGreen-positive cells express periostin. Wheat germ agglutinin was conjugated to TRITC (red) and To-Pro nuclei (blue). Error bars represent SEM.

Fig. 2.

Genetic deletion of periostin diminishes the pathology in Sgcd^−/− muscle. (A and B) Muscle weights normalized to tibia length from the quadriceps, gastrocnemius, and diaphragm in the indicated groups of mice at 6 wk and 6 mo of age. (C) Quantification of the percent of fibers containing central nuclei in the gastrocnemius from 6-wk-old and 6-mo-old mice. (D) Fiber areas from the gastrocnemius of the indicated mice separated into ranges. Error bars represent SEM.*P < 0.05 vs. Wt; ^#P < 0.05 vs. Sgcd^−/−. The numbers of mice or muscles analyzed are shown in the bars.

Fig. 3.

Analysis of the ECM in skeletal muscle. (A) Representative Masson’s trichrome-stained diaphragm, gastrocnemius, and quadriceps from WT, Postn^−/−, Sgcd^−/−, and Sgcd^−/−Postn^−/− mice at 6 mo of age. (Original magnification: 100×.) (B and C) Quantification of fibrotic area by Masson’s trichrome staining at 6 wk and 6 mo of age. The number of muscles analyzed is shown in the bars. (D) Measurement of hydroxyproline content in the quadriceps from WT, Sgcd^−/−, and Sgcd^−/−Postn^−/− mice expressed as micrograms of hydroxyproline per milligram of tissue. (E) Western blot analysis for MMP9 in the indicated groups of mice from the gastrocnemius at 6 wk of age. GAPDH was used as a loading control. (F) MMP9-specific activity assay from the gastrocnemius of the indicated mice (6 wk of age). n = 3 or more for each group. (G) Real-time PCR from quadriceps muscle of the indicated mRNA in Sgcd^−/− vs. Sgcd^−/−Postn^−/− mice. mRNA levels were normalized to 18s RNA. Error bars represent SEM. *P < 0.05 vs. WT; ^#P < 0.05 vs. Sgcd^−/−. The number of mice analyzed is shown in or above the bars.

Fig. 4.

Loss of periostin improves in vivo muscle function. (A and B) Quantification of serum creatine kinase (CK) levels in the indicated groups of mice at 6 wk and 6 mo of age. (C and D) Time to fatigue in seconds with forced downhill treadmill running in the indicated groups of mice. Error bars represent SEM. *P < 0.05 vs. WT; ^#P < 0.05 vs. Sgcd^−/−. The number of mice analyzed is shown in or above the bars.

Fig. 5.

Loss of periostin improves skeletal muscle regeneration in dystrophic mice. (A) Quantification of the percent of embryonic myosin heavy chain (eMHC)-positive fibers and myogenin-positive nuclei in the gastrocnemius of Sgcd^−/− and Sgcd^−/−Postn^−/− mice at 6 wk of age. (B) Representative immunohistochemical images for the activated macrophage marker Mac-3 (green cells) in the gastrocnemius muscle of Sgcd^−/− and Sgcd^−/−Postn^−/− mice at 6 mo of age. Red staining was with WGA-TRITC, and blue denotes nuclei (TO-PRO). (Original magnification: 400×.) (C and D) Quantification of Mac-3–positive cells per field in the indicated groups of mice at 6 wk and 6 mo of age. (E) ELISA detection of TGF-β levels in the quadriceps of 6-wk old WT and Sgcd^−/− mice. (F) Immunohistochemistry of phosphorylated (p) Smad2/3 (green nuclei) in the quadriceps of Sgcd^−/− and Sgcd^−/−Postn^−/− mice at 6 wk of age. Membranes are stained red with WGA-TRITC, and nuclei are stained blue with DAPI. (Scale bar: 100 μm.) (G) Quantification of percentage of phospho-Smad2/3–positive nuclei in the diaphragm, gastrocnemius, and quadriceps of Sgcd^−/− and Sgcd^−/−Postn^−/− mice at 6 wk of age. *P < 0.05 vs. WT; ^#P < 0.05 vs. Sgcd^−/−. Error bars represent SEM. The numbers of mice or individual muscles examined are shown in or above the bars throughout the figure.

Fig. 6.

Periostin leads to altered regeneration through TGF-β signaling in MD and muscle injury. (A) Representative immunohistochemistry of phospho-Smad2/3 in the quadriceps showing the nearly complete loss of signal in Sgcd^−/−Postn^−/− mice injected with TGF-β neutralizing antibody. Original magnification: 600×; scale bar: 50 μm.) (B) Time to fatigue in seconds with forced downhill treadmill running in the indicated groups of mice injected with anti–TGF-β or vehicle. ^#P < 0.05, Sgcd^−/−Postn^−/− vehicle vs. anti-TGF-β. (C) Western blots for MyoD, myogenin, periostin, and Pax7 in the gastrocnemius of the indicated groups of mice, injected or not injected with TGF-β antibody. GAPDH was used as a loading control. (D) Immunohistochemistry showing increased periostin (green) staining after 7 d of freeze injury in the gastrocnemius muscle. Red staining was with WGA-TRITC, and blue denotes nuclei (DAPI). (Original magnification: 400×; scale bar: 100 μm.) (E) Quantification of eMHC-positive fibers in the injured area of WT and Postn^−/− mice after 7 d of injury. (F) Quantification of myogenin-positive nuclei in the injured area of WT and Postn^−/− mice after 7 d of injury. (G) Fiber areas from the peri-injury area in the gastrocnemius of WT and Postn^−/− mice, separated into ranges. *P < 0.05 vs. WT; ^#P < 0.05 vs. vehicle. Error bars represent SEM. The numbers of mice or individual muscles examined are shown in or above the bars throughout the figure.