A role for Regulator of G protein Signaling-12 (RGS12) in the balance between myoblast proliferation and differentiation
Fig 5
Myotube formation by primary myoblasts isolated from RGS12-null mice is disrupted, but restored upon RGS12 re-expression.
(A) Confirmation of RGS12 loss in primary myoblasts from Rgs12Δ5-8/Δ5–8 mice via immunoblotting of myoblast lysates with indicated antibodies. Skeletal muscle myoblasts were isolated from tibialis anterior (TA) muscles of 2- to 3-week old homozygous Rgs12Δ5-8/Δ5–8 mice, heterozygous Rgs12+/Δ5–8 mice, or wild-type littermate controls, and cultured in F10 Ham’s medium with 20% FBS and 2.5 ng/mL bFGF prior to lysis. (B) Cultures of wild-type myoblasts or Rgs12Δ5-8/Δ5–8 myoblasts were each switched to differentiation medium (DMEM, 2% horse serum) when at 80% confluence and cultured for an additional 2 days. Cell cultures were then fixed, permeabilized, and stained with anti-MHC (MF20; red). Nuclei were visualized with DAPI (blue). (C) Myoblast cultures identical to those of panel B were cultured in differentiation medium for 2 days and lysed (“post” = post-differentiation over 2 days); parallel cultures were treated as in panel A before lysis (“pre” = pre-differentiation). Resultant lysates were immunoblotted with antibodies against indicated proteins (β-actin as a loading control). Below the representative immunoblots is a bar-graph of quantitated protein levels normalized as percentage change in expression between pre- and post-differentiation samples (*, p < 0.05; Student’s t-test). (D) Separate Rgs12Δ5-8/Δ5–8 myoblast cultures were transiently transfected using liposomes with an expression plasmid encoding full-length, HA-tagged RGS12 prior to being switched into differentiation medium (DMEM, 2% horse serum) when at 80% confluence and cultured for an additional 2 days. Cell cultures were then fixed, permeabilized, and stained with anti-MHC (MF20; red). Nuclei were visualized with DAPI (blue). Inset: Ectopic expression of HA-RGS12 in Rgs12Δ5-8/Δ5–8 myoblasts was confirmed by immunoblotting with anti-RGS12 antibody. (E) Differentiation of myoblasts to myotubes ex vivo, from micrographs as depicted in panels B and D, was quantitated by the fusion index; statistical significance was tested by one-way ANOVA: ***, p<0.0001, RGS12-null myoblasts exhibited a significantly lower fusion index compared to wild-type, Rgs12+/+ myoblasts; ###, p<0.0001, Rgs12Δ5-8/Δ5–8 myoblasts expressing HA-RGS12 showed statistically significant recovery of fusion index as compared to mock-transfected Rgs12Δ5-8/Δ5–8 myoblasts. (There was no statistically significant difference between the fusion index of wild-type myoblasts and Rgs12Δ5-8/Δ5–8 myoblasts re-expressing RGS12).