Decreased proliferation kinetics of mouse myoblasts overexpressing FRG1

Abstract

Although recent publications have linked the molecular events driving facioscapulohumeral muscular dystrophy (FSHD) to expression of the double homeobox transcription factor DUX4, overexpression of FRG1 has been proposed as one alternative causal agent as mice overexpressing FRG1 present with muscular dystrophy. Here, we characterize proliferative defects in two independent myoblast lines overexpressing FRG1. Myoblasts isolated from thigh muscle of FRG1 transgenic mice, an affected dystrophic muscle, exhibit delayed proliferation as measured by decreased clone size, whereas myoblasts isolated from the unaffected diaphragm muscle proliferated normally. To confirm the observation that overexpression of FRG1 could impair myoblast proliferation, we examined C2C12 myoblasts with inducible overexpression of FRG1, finding increased doubling time and G1-phase cells in mass culture after induction of FRG1 and decreased levels of pRb phosphorylation. We propose that depressed myoblast proliferation may contribute to the pathology of mice overexpressing FRG1 and may play a part in FSHD.

Figure 1. FRG1 expression and dystrophic phenotype of mice.

A) Muscles and tissue from either H-FRG1 ^TG transgenic mice (FRG1) or wild-type littermate control (C57BL/6) were collected from 10-week old mice. 300 µg of total protein from tissue lysates isolated from quadriceps muscle (Q), gastrocnemius muscle (G), diaphragm muscle (D), whole heart (H), lung tissue (Lu), liver tissue (Li) and brain tissue (B) were probed with α-FRG1 antibody after SDS-PAGE. B) Diaphragm and quadriceps muscle from 13-week old H-FRG1 ^TG mouse (FRG1) or wild-type littermate control (C57BL/6) stained with hemotoxylin/eosin and viewed under 100× magnification. Arrows note location of centrally located nuclei present in FRG1 cross-section. Scale bar notes 50 µm.

Figure 2. Clonal analysis of mouse-derived myoblasts.

A) Myoblasts isolated from diaphragm (D) or thigh (T) of 18-week old H-FRG1 ^TG mice (FRG1) or wild-type littermate controls (WT) were cultured and plated at low density. Cells were fixed at regular time intervals and stained for myosin heavy chain. Total number of nuclei per clone was counted and a representative graph of data from 96-hours post-plating is shown (n = 100). B) Myoblasts isolated from diaphragm (D) or thigh (T) of 4-week old H-FRG1 ^TG mice (FRG1) or wild-type littermate controls (WT) were scored for proliferation as above at 72-hours post-plating (50<n<80).

Figure 3. Expression of FRG1 in iC2C12-FRG1 myoblasts.

A) Western blot of lysates from iC2C12-FRG1 myoblasts before induction with doxycycline or after induction for 24 hours with concentrations ranging from 250 ng/mL to 1000 ng/mL using α-FLAG antibody. β-actin loading control shown below. B) Localization of FRG1 by immunofluorescence in iC2C12-FRG1either uninduced or induced with 500 ng/mL doxycycline for 24 hours. DAPI stain is represented in the blue channel and α-FRG1 antibody staining is represented in the green channel.

Figure 4. Characterization of proliferation defect in iC2C12-FRG1 myoblasts.

A) Mass culture doubling times calculated from hemocytometer counts of iC2C12-FRG1 myoblasts with or without induction of expression by doxycycline over 120 hours. Below are the cell cycle profiles of uninduced iC2C12-FRG1 myoblasts as well as after 24 hours and 72 hours in the presence of doxycycline. Cell cycle profiles were calculated from DNA content analysis by flow cytometry on proliferating myoblasts that were fixed and DAPI stained. *denotes statistical significance p<0.05, **p<0.005. B) Cell cycle profiles of synchronized iC2C12-FRG1 myoblasts over 24 hours in the absence or presence of doxycycline showing fraction of cells in G1, S or G2-phase. Comparison of data is plotted in a line graph fashion showing G1 in green, S in blue and G2 in red to demonstrate the generalized lag exhibited by iC2C12-FRG1 myoblasts expressing FRG1. Uninduced iC2C12-FRG1 myoblasts are graphed with a dashed line, while iC2C12-FRG1 myoblasts with doxycycline-induced FRG1 expression are graphed with a solid line. n>20,000 cells analyzed for each time point.

Figure 5. Phosphorylation of pRb is perturbed by FRG1 expression.

Western blot analysis for pRb, pRb Ser807/811 and FRG1 in iC2C12-FRG1 cells grown in the presence or absence of doxycycline. β-actin loading control shown below.