Fig 6. Conditional RGS12 deficiency leads to normal skeletal muscle development.

(A) Confirmation of Cre-dependent loss of the Rgs12 gene using a conditional knockout (‘floxed allele’) strategy. Rgs12^fl/fl mouse-derived primary myoblasts were cultured in vitro and infected with adeno-associated virus (AAV) expressing green fluorescent protein (GFP only) or Cre recombinase (GFP+Cre). Evidence of Cre recombinase-mediated excision of the Rgs12 gene (and hence loss of RGS12 expression) was obtained by immunoblotting whole cell lysate from AAV-infected myoblast cultures with indicated anti-RGS12 monoclonal antibodies (each generated by the Siderovski lab and deposited in the Developmental Studies Hybridoma Bank [Iowa City, IA]). (B) (i) Western blot confirmation of RGS12 expression in the tibialis anterior (TA) muscle of wild-type mice, Pax7::Cre-dependent loss of RGS12 in the TA of Rgs12^fl/fl mice, and expression of RGS12 in the cortex (brain) of wild-type mice using two independent monoclonal RGS12 antibodies. (ii, iii) Representative scanning micrographs of TA muscles from wild-type and Rgs12^fl/fl; Pax7::Cre mice labeled with wheat-germ agglutinin (WGA; green) and nuclear DNA (blue). (C) Relative frequency distribution of the cross-sectional area (CSA) of muscle fibers as measured by EVOS FL Auto software based on WGA staining (>800 fibers/mouse). No differences in fiber CSA were found between genotypes. Inset: The mean CSA of muscle fibers was not different in Rgs12^fl/fl; Pax7::Cre mice (n = 5) and wild-type Pax7::Cre mice (n = 5) as assessed by a Student’s t-test (ns, not significant; p > 0.05). Data are presented as mean ± SEM. (D) Tension-frequency display of electrically stimulated isometric plantar flexor contractions at the indicated frequencies. Mean ± SEM tension are displayed of Rgs12^fl/fl; Pax7::Cre and wild-type littermate control mice (n = 5/group). A 2-way ANOVA indicated no difference in the tension of wild-type and Rgs12-deficient mice at the indicated frequencies.