Figure 3: Deletion of Ebf3 impairs muscle relaxation and expression of Atp2a1.

(A,B) Analysis of the ultrastructure of muscle fibres of the diaphragm by electron microscopy of wild-type (a) and Ebf3-deficient (b) newborn mice. Representative electron microscopy pictures of sarcomeres showing myofilaments at 10.000 × (bar, 2.5 nm) and 25.200 × magnification (bar, 1 nm); b shows hypercontracted myofibres. (C) Determination of sarcomere length in the diaphragm of Ebf3^+/+, Ebf3^+/− and Ebf3^−/− newborn mice. n=3; error bars=s.d.; *P<0.05. (D,E) Single twitch stimulation (50 V, 1 ms) of diaphragm muscle from newborn mice of the indicated genotypes. (D) Measurement of single twitch normalized to the cross-sectional area, representing muscle force in response to the stimulation. (E) Total duration of muscle contraction after single twitch stimulation. n=5 Ebf3^+/+, n=7 Ebf3^+/−, n=4 Ebf3^−/−; error bars=s.d.; *P<0.05, **P<0.01, ***P<0.001. (F) Ebf3-β-gal-expressing cells were isolated from the diaphragm of E18.5 Ebf3^+/− embryos by flow cytometry and analysed for the expression of the indicated genes by qPCR. Expression in Ebf3^+/− cells was set to 1 and relative expression in Ebf3^−/− cells is shown. n=3, *P<0.05, **P<0.01. (G) Cells of the diaphragm of E18.5 animals were sorted as positive or negative for the expression of Ebf3-β-gal and analysed by quantitative PCR for the expression of Ebf3 and Atp2a1. Unsorted cells of wild-type diaphragm were used as control and set to 1; n=3, *P<0.05, **P<0.01. (H) Representative western blot analysis of Serca1 expression in skeletal muscle (SM) and diaphragm (D) of E18.5 embryos of the indicated genotypes; n=3. In all PCR experiments, values are normalized to wild-type levels, error bars represent s.d., and P-values were calculated in comparison to the wild-type control.