Figure 1. Simultaneous knockout of both Acvr1b and Tgfbr1 caused muscle hypertrophy.

(A) Scheme showing cross-breeding of HSA-Cre mouse line with conditional knockout mouse lines Acvr1b^fl/fl and Tgfbr1^fl/fl. LoxP sites are indicated by black arrows. A loxP-flanked neomycin (neo) cassette is inserted upstream of exon3 of Acvr1b genome. (B) Scheme demonstrating receptor knockout induced by tamoxifen (TMX) injection for consecutive 5 days. (C) Relative mRNA expression of Acvr1b and Tgfbr1 in TA muscles of experimental groups. (D) Histology stainings of TA muscles 35 days after first TMX injection. H&E staining and immunofluorescent staining of eMyHC (green) of TA showed regenerative regions containing eMyHC⁺ myofibres with central nuclei (DAPI, blue) in Acvr1b:Tgfbr1 CKO mice, wheat glucose agglutinin (WGA, red) was used to visualise cell membranes and ECM. Acvr1b:Tgfbr1 CKO mice showed lower staining intensity for SDH activity in low oxidative region of TA. MyHCs staining demonstrated type IIA (green), IIB (red), IIX (green) and I (red) myofibres in low and high oxidative regions of TA. Scale bars = 250 μm. (E) TA and EDL muscle mass and myofibre cross-sectional areas (CSAs) were increased in Acvr1b:Tgfbr1 CKO mice. (F) In TA, specifically CSA of type IIB myofibres was increased in Acvr1b:Tgfbr1 CKO animals, while in EDL CSA of all type II myofibres was increased. Myofibre types were stained in EDL. (G) Percentage of type IIB in low oxidative region of TA was reduced. No differences were observed in myofibre distribution in high oxidative region of TA or EDL. (H) SDH activity (absorbance units (∆A₆₆₀) per micrometer section thickness per second of incubation time (∆A₆₆₀∙μm^–1∙s^–1)) was decreased, while the integrated SDH activity, SDH activity multiplied by CSA (∆A₆₆₀∙μm∙s^–1), increased in low oxidative region of TA of Acvr1b:Tgfbr1 CKO animals. N = 5–8 mice. Results are presented as mean + SEM. *: p < 0.05. Significant difference between individual groups is indicated by lines with a *. Single * indicates significant difference compared to all other groups.