Proliferin-1 Ameliorates Cardiotoxin-Related Skeletal Muscle Repair in Mice

Figures

• 

  Figure 1. Expressions of PLF-1 in the gastrocnemius muscles at the indicated timepoints after cardiotoxin (CTX) injection. (a) Photos of a gastrocnemius mass and representative microscopy images of H&E staining of the noninjured and injured muscles of mice on days 3 and 14 postinjury. Grip strength was calculated in both groups. (b) The changes of workload in the vertical direction were evaluated in both experimental groups as described in Materials and Methods. (c) Quantitative real-time PCR data show the levels of PLF-1 on days 0, 1, 3, 7, and 14 postinjury. (d) Real-time PCR data showed the PLF-1 gene expression in C2C12 myoblasts, fibroblasts, and endothelial cells inducted by CTX at 10 μM. Results are mean ± SE (n = 6–7). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. the corresponding day 0 by one-way ANOVA followed by Tukey's post hoc tests.

• 

  Figure 2. Recombinant proliferin-1 (rPLF-1) ameliorated skeletal muscle dysfunction and remodeling on day 14 after cardiotoxin (CTX) injection. (a) Grip strength was calculated in both groups. (b) The changes of workload in the vertical direction were evaluated in both experimental groups as described in Materials and Methods. (c-e) Quantitative data showing the cross-sectional area of the myofiber size and interstitial fibrosis (9 × 104 μm2). Results are mean ± SE (n = 6–7). ∗p < 0.05 and ∗∗p < 0.01 by two-way repeated-measures ANOVA and Tukey's post hoc tests or Student's t-test.

• 

  Figure 3. rPLF-1 ameliorated inflammation in response to CTX injury. (a) ELISA data show the levels of plasma TNF-α and IL-1β in both experimental groups on day 3 after CTX injection. (b) Quantitative real-time data show the levels of Pax7, MyoD, and Cyclin B1 mRNA expressions in the gastrocnemius muscles of both groups of mice. Results are mean ± SE (n = 6–7). ∗p < 0.05 and ∗∗p < 0.01 by Student's t-test or one-way ANOVA followed by Tukey's post hoc tests.

• 

  Figure 4. rPLF-1 alleviated the expressions of desmin and laminin proteins in the gastrocnemius muscles at the indicated days after CTX injection. (a, b) Double immunofluorescence was performed with the mouse mAb against desmin (red) and the rabbit polyclonal antibody (pAb) against laminin-5 (green). Representative images and quantitative data of the desmin protein expression in the gastrocnemius muscle of both experimental groups. (c) Double immunofluorescence was performed with goat pAb against integrin-α7 (red) and rabbit mAb against CD34 (green). Representative images and quantitative data show the numbers of CD34+/integrin-α7+ muscle stem cells (MuSCs). (d) Immunostaining was performed with mouse mAb against mouse monoclonal proliferating cell nuclear antigen (PCNA). Representative images and quantitative data show PCNA-positive cells. Red arrowheads: CD34+/integrin-α7+ cells or PCNA+ cells. Data are mean ± SE (n = 6–7). ∗p < 0.05 and ∗∗p < 0.01 vs. controls by Student's t-test.

• 

  Figure 5. rPLF-1 stimulated bone marrow (BM) MuSC production and mobilization in response to CTX injury. (a, b) Representative dot plots and quantitative data for the numbers of CD34+/integrin-α7+ MuSCs in BM and peripheral blood (PB). (c) After the isolation of BM-derived integrin-α7+ stem cells with magnetic beads, the cells were cultured on cover glasses for 24 hr and then subjected to double immunofluorescence with goat pAb against integrin-α7 (red) and mouse mAb against Ki67 (green). Representative double fluorescence images and quantitative data show the numbers of proliferating cells (×200 magnification). Results are mean ± SE (n = 7–8). ∗∗p < 0.01 vs. corresponding control groups by Student's t-test.

• 

  Figure 6. rPLF-1 increased the levels of proliferation-related signal proteins in the gastrocnemius muscle in response to CTX injury. Representative immunoblots and combined quantitative data show increased levels of p-Akt, p-mTOR, p-GSK3α/β, p-Erk1/2, p-p38MAKP, and IL-10 in the muscles of rPLF-1 mice. Results are mean ± SE (n = 3). ∗p < 0.05 and ∗∗p < 0.01 vs. corresponding control groups by one-way ANOVA followed by Tukey's post hoc tests.

• 

  Figure 7. PLF-1 blocking accelerated skeletal muscle dysfunction and remodeling on day 14 after CTX injection. (a) Grip strength was calculated in both groups. (b) The changes of workload in the vertical direction were evaluated in both groups as described in Materials and Methods. (c-e) Quantitative data showing the cross-sectional area of muscle fiber size and interstitial fibrosis (9 × 104 μm2). Results are mean ± SE (n = 6–7). ∗p < 0.05 and ∗∗p < 0.01 by two-way repeated-measures ANOVA and Tukey's post hoc tests or Student's t-test.

• 

  Figure 8. PLF-1 blocking accelerated the inflammation in response to CTX injury. (a) ELISA data show the levels of plasma TNF-α and IL-1β in both experimental groups on day 3 after CTX injection. (b) Quantitative real-time data show the levels of Pax7, MyoD, and Cyclin B1 mRNA expressions in the gastrocnemius muscles of both groups of mice. Results are mean ± SE (n = 6–8). ∗p < 0.05 and ∗∗p < 0.01 by Student's t-test or one-way ANOVA followed by Tukey's post hoc tests.

• 

  Figure 9. PLF-1 blocking accelerated the impaired desmin and laminin expressions in the gastrocnemius muscles postinjury. (a, b) Double immunofluorescence was performed with the mouse monoclonal antibody (mAb) against desmin (red) and the rabbit polyclonal antibody (pAb) against laminin-5 (green). Representative images and quantitative data show the contents of desmin proteins in the gastrocnemius of both experimental groups. (c) Double immunofluorescence was performed with goat pAb against integrin-α7 (red) and rabbit mAb against CD34 (green). Representative images and quantitative data show the numbers of CD34+/integrin-α7+ muscle stem cells (MuSCs) in the gastrocnemius muscles of both experimental groups. (d) Immunostaining was performed with mouse mAb against proliferating cell nuclear antigen (PCNA). Representative images and quantitative data show PCNA-positive cells in the gastrocnemius muscles of both groups. Red arrowheads: CD34+/integrin-α7+ cells or PCNA+. Data are mean ± SE (n = 7–8). ∗p < 0.05 and ∗∗p < 0.01 vs. controls by Student's t-test.

• 

  Figure 10. PLF-1 depletion reduced the numbers of MuSCs in bone marrow (BM) and peripheral blood (PB) in response to CTX injury. (a, b) Representative dot plots and quantitative data for the numbers of CD34+/integrin-α7+ MuSCs in BM and PB of both experimental groups. (c) After the isolation of BM-derived integrin-α7+ stem cells with magnetic beads, the cells were cultured on cover glasses for 24 hr and then subjected to double immunofluorescence with goat pAb against integrin-α7 (red) and mouse mAb against Ki67 (green). Representative double images and quantitative data show the numbers of proliferating cells (×200 magnification). Results are mean ± SE (n = 6–8). ∗∗p < 0.01 vs. corresponding control groups by Student's t-test.

• 

  Figure 11. PLF-1 depletion accelerated the impaired proliferation signaling activation in the gastrocnemius muscle in response to CTX injury. Representative immunoblots and combined quantitative data show decreased levels of p-Akt, p-mTOR, p-GSK3α/β, p-Erk1/2, p-p38MAPK, and IL-10 in the muscles of nPLF-1-treated mice. Results are mean ± SE (n = 3). ∗p < 0.05 and ∗∗p < 0.01 vs. corresponding control groups by one-way ANOVA followed by Tukey's post hoc tests.

• 

  Figure 12. rPLF-1 promotes muscle cell proliferation. (a) C2C12 myoblasts were treated with rPLF-1 (100 nM) for 24 hr for proliferation. (b) Representative immunoblots and quantitative data show the dose-dependent levels of p-Akt, p-mTOR, p-p38MAPK, and p-Erk1/2 proteins in the cells. Results are mean ± SE (n = 3–6). ∗p < 0.05 and ∗∗p < 0.01 by Student's t-test or one-way ANOVA followed by Tukey's post hoc tests.