RIPK3‐mediated cell death is involved in DUX4‐mediated toxicity in facioscapulohumeral dystrophy

Figures

• 

  Figure 1. DUX4 expression triggers cell death in iC2C12‐DUX4 myoblasts. ( A, B, D–F ) Expression of DUX4, Tm7sf4, Ripk1, Mlkl, or Ripk3, respectively (RT‐qPCR) 24 h after addition of different concentration of dox in iC2C12‐DUX4 myoblasts. (C) Cell viability assay [adenosine triphosphate (ATP) assay] at 24 h after dox induction at various concentrations. Data represent the mean ± standard deviation on three independent experiments; ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05 by one‐way analysis of variance with Dunnett's post hoc test.

• 

  Figure 2. Necroptosis participates in DUX4‐mediated cell death. (A) Cell viability on iC2C12‐DUX4 myoblasts at 48 h of induction without or with dox (200 ng/mL) and treatment with various concentrations of the pan‐caspase inhibitor Z‐VAD. (B) ATP assay on iC2C12‐DUX4 myoblasts at 48 h of induction with 100 or 200 ng/mL of dox and treatment with different molecules (Z: ZVAD 20 μM, N: necrostatin‐1 30 μM; G: GSK'872 2 μM; C: cyclosporin A 0.2 μM) alone or in combination. Data are presented as means ± standard deviation; *: P < 0.05 by one‐way analysis of variance with Dunnett's post hoc test.

• 

  Figure 3. DUX4 expression triggers cell death in iC2C12‐DUX4 myotubes. (A, B, D–F) Expression of DUX4, Tm7sf4, Ripk1, Mlkl, or Ripk3, respectively (RT‐qPCR) 24 h after addition of different concentration of dox in iC2C12‐DUX4 myotubes after 4 days of differentiation. (C) Cell viability assay [adenosine triphosphate (ATP) assay] at 24 h after dox induction at various concentrations. Data represent the mean ± standard deviation on three independent experiments; ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05 by one‐way analysis of variance with Dunnett's post hoc test.

• 

  Figure 4. Ripk3 inhibition decreased DUX4‐mediated cell death in iC2C12‐DUX4 myotubes. (A) iC2C12‐DUX4 viability in the presence of dox 1000 ng/mL and treated with different doses of Z‐VAD, necrostatin‐1, or GSK'872. (B) Cell viability in the presence of dox 1000 ng/mL with the different compounds alone or in combination. Z: Z‐VAD (20 μM), N: necrostatin‐1 (30 μM), N+: necrostatin‐1 (90 μM), G: GSK'872 (2 μM); G+: GSK'872 (4 μM). Data are presented as means ± standard deviation; ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05 by one‐way analysis of variance with Dunnett's post hoc test.

• 

  Figure 5. In vivo DUX4‐mediated toxicity leading to weight loss is triggered by Ripk3. (A) Variation of the total body weight gain (in percentage) from the beginning of treatment to animal death. (B) Total running time was measured on a 15° angled treadmill system. (C and D) Weights of the tibialis anterior (TA) and quadriceps (QUA) in the different models. N = 4–8 animals per group. Male (C) or female (D). Six‐week‐old animals were given a 2 day treatment of 10 mg/kg tamoxifen delivered intraperitoneally, and the mice were killed 5 days after the last injection. CD: DUX4−/+ animals; CDR: DUX4−/+Ripk3−/−. Data are presented as means ± standard deviation; ****: P < 0.0001; **: P < 0.01; *: P < 0.05 by one‐way analysis of variance with Dunnett's post hoc test. (E) The quadriceps muscles (female only) were sectioned and labelled with laminin, and the min Feret was calculated for each muscle fibre. (F and G) The min Feret average (F) and the ratio min Feret/Feret were calculated in the quadriceps of CD‐ and CDR‐Cre+ females. (H and I) Haematoxylin and eosin staining of CD‐Cre+ (H) and CDR‐Cre+ (I) muscle cross sections.

• 

  Figure 6. Expression of necroptotic and DUX4‐network genes in the CD mice. Ripk3, Ripk1, and Mlkl and Tm7sf4, Duxbl, and Wfdc3 were measured in the quadriceps. Six‐week‐old female mice were given a 2 day treatment of 10 mg/kg tamoxifen delivered intraperitoneally, and the mice were killed 5 days after the last injection. N = 2 to 5 muscles per group. CD: DUX4−/+ animals; CDR: DUX4−/+Ripk3−/−. Data are presented as means ± standard deviation; ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05 by one‐way analysis of variance with Tukey's post hoc test.

• 

  Figure 7. Ripk3 deficiency ameliorates muscle phenotype in mouse. Representative images of transversed sections of mouse quadriceps labelled with Laminin and CD68 (A) or Laminin and IgG uptake (B). Quantification of CD68‐ (C) or IgG‐uptake‐ (D) positive areas. Scale bar 200 μm. Data represent the mean ± standard error of the mean on three to five animals per group. CD: DUX4−/+ animals; CDR: DUX4−/+Ripk3−/−. The Student's T‐test was performed. *P < 0.05.