Figure 4. Mutation of Pitx2/3 in adult muscle satellite cells leads to impairment of muscle regeneration by deregulation of their redox state.
(A) Double mutant Pitx2flox/flox:Pitx3flox/-:Pax7Cre-ERT2/+ and control Pitx2flox/+:Pitx3+/-:Pax7Cre- ERT2/+ adult mice were obtained by 4-hydroxytamoxifen (4-OHT) intra-peritoneal (IP) injection on 5 consecutive days (D) and subjected to muscle injury by cardiotoxin injection into the Tibialis Anterior (TA) muscle. (B) Three weeks (D21) post injury (PI), injured and contralateral uninjured TA muscles were dissected. Muscle cryo-sections were analysed by hematoxylin-eosin (HE) and senescence-associated−β-galactosidase (SA-βgal) staining. Experiments were performed with n = 4 animals for each condition and representative images are shown, PI (Post Injury). Scale bars, 100 μm. (C) Double mutant Pitx2flox/flox:Pitx3flox/-:Pax7Cre-ERT2/+:mdx and control Pitx2flox/+:Pitx3+/-: Pax7Cre-ERT2/+:mdx adult mice obtained as in (A) were maintained during 10 weeks under standard life conditions. From 1 week before the start of the experiment, half of the double mutant Pitx2flox/flox:Pitx3flox/-:Pax7Cre-ERT2/+:mdx mice were treated with N-Acetyl-Cysteine (NAC) in the drinking water. (D) After 10 weeks, all mice were sacrificed and diaphragm muscles were dissected and analysed by SA-βgal staining. The number of SA-βgal positive cells per field was counted in diaphragm sections of control and double mutant animals. (D) Experiments were performed with n ≥ 5 animals for each condition and representative images are shown (B). Error bars represent the mean ± s.d, with *p<0.05, ***p<0.001, ****p<0.001. Please see Figure 4—figure supplement 1 for additional data.
Figure 4—figure supplement 1. Deletion of Pitx2/3 in adult muscle satellite cells of mdx mice leads to aggravation of the dystrophic phenotype.
