Fig 1.
Rgs12 expression is high in mouse embryonic skeletal muscle and active satellite cells, upregulated early during muscle repair, and downregulated upon differentiation of the myoblastic C2C12 cell line.
(A) Relative expression (mean ± SEM of Fragments Per Kilobase of transcript per Million mapped reads [FPKM]) of Rgs12, Rgs14, and Pax7 gene transcripts within indicated ages of mouse gastrocnemius muscle samples was obtained via RNA-Seq using an Illumina HiSeq 2000. Based on Gene Expression Omnibus dataset GSE108402 [50]. Inset, detection of RGS12 protein expression via immunoblotting of (a) 100 μg of whole gastrocnemius muscle lysate from a 3-month-old C57BL/6J mouse and (b) 50 μg of lysate from myoblasts isolated from cardiotoxin-injected tibialis anterior (TA) muscle. (B) Normalized expression levels (mean ± SEM) of Rgs12, Rgs14, and Pax7 gene transcripts within indicated ages of flow-sorted, Pax7+ mouse skeletal muscle satellite cells were obtained via an Affymetrix Mouse Gene 1.0 ST microarray, based on Gene Expression Omnibus dataset GSE47401; published in [51]. (C) Relative expression of Rgs12 and eMHC in TA muscle following CTX-induced muscle damage. RNA was extracted from muscle at the indicated time points and quantified using qRT-PCR, with Gapdh abundance as an internal control. *, p < 0.01 Rgs12 level compared to time zero (one-way ANOVA with Dunnett’s test); #, p < 0.0001 eMHC level compared to time zero (one-way ANOVA with Dunnett’s test). Inset, TA muscle was injected with 0.1 ml of 10 μM cardiotoxin (CTX) diluted in PBS; contralateral, PBS-injected TA muscle was used as a control. After four days, the muscles were harvested and used for RGS12 protein expression analysis by immunoblotting (with GAPDH protein levels interrogated in parallel as a loading control). (D) C2C12 cell line cultures (4 x 105 cells/well) were maintained in growth medium (DMEM containing 10% fetal bovine serum [FBS]) for two days. Differentiation was induced by replacing growth medium with differentiation medium (DMEM containing 2% horse serum [HS] instead of FBS). Total RNA and protein lysates were separately isolated from cell cultures at the indicated time points (hours) after the switch to differentiation medium. Rgs12 mRNA and RGS12 protein levels were determined by qRT-PCR and immunoblotting, respectively. GAPDH mRNA and protein levels were used as internal controls for each experiment. **, p < 0.01; ***, p < 0.001 versus level observed at time zero (one-way ANOVA with Dunnett’s test).
Fig 2.
RGS12 expression is observed in cytosolic puncta within the myoblastic C2C12 cell line, but localized in the nucleus in the poorly differentiating human RD cell line.
(A) Immunoblotting (IB) of cell lysates from all three cell types indicates RGS12 protein expression, as detected using a rabbit anti-RGS12 polyclonal antibody previously described [7]; β-tubulin protein levels were also examined by immunoblotting in parallel as a loading control. (B) Cultures of the poorly differentiating, human rhabdomysarcoma RD cell line and the more-easily differentiated, mouse C2C12 cell line were separately fixed and stained with DAPI; RGS12 protein was detected by indirect immunofluorescence using UNC60-26.2.1. Panels (i-vi) represent: (i, iv) anti-RGS12 antibody detection with Alexa-fluor-594 secondary antibody; (ii, v) DAPI nuclear stain; and, (iii, vi) overlay of both images. (C) Endogenous RGS12 localizes to early (APPL1-positive) endosomes within C2C12 cells. C2C12 cell line cultures (4 x 105 cells/well) were maintained in growth medium (DMEM containing 10% fetal bovine serum [FBS]) for two days. Cells were then fixed in paraformaldehyde, permeabilized, and stained with primary mouse monoclonal antibody UNC60-26.2.1 and secondary Alexa-fluor-594 anti-mouse antibody, alone or in combination with primary anti-APPL1 or -Rab9 rabbit polyclonal antibodies followed by Alexa-fluor-488 secondary anti-rabbit antibody. Overlays in panels (vii-ix) are absent the DAPI nuclear stain (blue) to highlight lack of overlap between RGS12 and Rab9 signals. (D) RGS12 N-terminus binds to select phosphatidylinositides in a lipid dot-blot protein overlay assay. A “PIP Strip” nitrocellulose membrane pre-spotted with the indicated phospholipid species was probed with 20 μg/mL of GST alone, recombinant GST-RGS12 protein (amino-acids 9–406 spanning PDZ and PTB domains; “WT”), or GST-RGS12(aa 9–406) protein with alanine substitutions to four arginines in the PTB domain (Arg-255, -260, -262, and -308; “4R→A”) previously predicted by electrostatic contouring [61] to be involved in phospholipid binding. After extensive washing, the binding of protein to phospholipid spots was detected by chemiluminescence using anti-GST mouse monoclonal antibody and anti-mouse-horseradish peroxidase conjugated secondary antibody. Lipid abbreviations: LPA, lysophosphatidic acid; LPC, lysophosphocholine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine-1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine.
Fig 3.
RGS12 interacts with activated H-Ras and is downregulated within the C2C12 cell line during differentiation.
(A) Immunoblotting (IB) of cell lysates from three stable clones of the C2C12 cell line indicating their stable expression of a constitutively-active, GTPase-deficient (G12V) mutant of H-Ras protein (via detection of its N-terminal myc-epitope tag). (B) Co-immunoprecipitation of myc-tagged, activated H-Ras (G12V mutant) with endogenous RGS12 protein expressed in the C2HRas cell line clone 9. IP: immunoprecipitation; IB: immunoblotting. (C) To test the effect of stable expression of activated H-Ras on myoblast differentiation in vitro, multi-nucleated myotube content of indicated cell populations (either parental C2C12 cells [panels i, ii] or C2HRas clone #9 cells [panels iii, iv]) was measured by fixation and staining for sarcomere myosin (MHC; green) and nuclear DNA content (DAPI; pseudocolored red), either pre-differentiation (panels i, iii) or post-differentiation by culture for 5 days in low serum (2% horse serum; panels ii, iv). The mean fusion index for several C2C12 cell populations was 40% (± 2.5%; SEM), consistent with other reports [68, 69]. In contrast, the C2HRas cell line clone 9 was incapable of myotube formation (panel iv) and no fusion index could be calculated. (D) Co-immunoprecipitation of myc-tagged, activated H-Ras (G12V mutant) with ectopically co-expressed, HA-tagged RGS12 in C2C12 cells. IgH: immunoglobulin heavy-chain. (E) Endogenous levels of RGS12 protein are down-regulated during C2C12 differentiation into myotubes (i.e., 7 days of culture in low serum medium), whereas the same culture conditions did not lower RGS12 levels within the C2HRas cell line clone #9. (F) Mammalian RGS12 proteins encode a “destruction box” recognition motif, conforming to the consensus RxxLxxxx(D/N), where “x” is any amino-acid, that is found in most substrates of the Anaphase-Promoting Complex (APC). This destruction box sequence is completely conserved across human (h), mouse (m), and rat (r) RGS12 proteins (i.e., amino acids 402–410 of mouse RGS12: RAFLDGDAD); this consensus motif is preserved in zebrafish (z) and Drosophila (d) RGS12 orthologs, as well as in the APC targets Skp2 and Myf5.
Fig 4.
Ectopic RGS12 over- and under-expression affects myotube formation by the C2C12 cell line.
(A) RGS12-overexpressing and control C2C12 cell line cultures were each switched from growth medium (DMEM, 10% FBS) to differentiation medium (DMEM, 2% horse serum) and cultured for two days. Cells were then fixed with paraformaldehyde and stained with anti-MHC antibody (MF20) and Alexa-fluor-594 secondary antibody (red). Nuclei were visualized with DAPI staining (blue). (B) Differentiation to myotubes was quantitated by the fusion index [36, 42): namely, the percentage of nuclei present in fused, MHC-positive cells vs total nuclei in the field. Graphed below the fusion index is the quantitation of MHC-positive cells observed in each field examined. Statistical significance was tested by one-way ANOVA: **, p<0.01; ***, p<0.001. (C) Expression of epitope-tagged, full-length RGS12 was confirmed by immunoblotting (IB) with indicated anti-epitope tag antibodies. (D) Ectopic expression of Myc-epitope tagged, full-length RGS12 was verified by immunoblotting (IB) of whole cell lysates; equal total protein loading was verified by β-tubulin detection. (E) RGS12-overexpressing and mock transfected (empty vector) C2C12 cell line cultures in growth medium (DMEM, 10% FBS) were separately incubated for 60 minutes with 5-ethynyl-2’-deoxyuridine (EdU) to detect DNA synthesis by proliferating cells. Click-iT labeling with Alexa-fluor-488 identified cells with newly synthesized DNA. The percentage of EdU positive cells were counted using ImageJ software. N = 3 independent experiments. Statistical significance was established by Student’s t-test: ***, p<0.001. (F) C2C12 cell cultures were infected with lentiviridae encoding either a non-specific (NS) control shRNA or one of two different shRNAs targeting Rgs12 (#2, #5), and then selected in puromycin-containing growth medium (DMEM + 10% FBS) for two weeks. Cell cultures were then switched from growth medium to differentiation medium (DMEM + 2% HS) for five days; as a positive control for low-to-nil fusion index, the poorly-differentiating, human RD cell line was also cultured for five days in differentiation medium (rightmost panel). Cell cultures were immunolabeled with a primary antibody directed against sarcomere MHC (MF20) and Alexa-fluor-594 secondary antibody; nuclei were counterstained with DAPI. (G) Underneath each fluorescence micrograph is the plot of phospho-ERK content (quantified by densitometry and normalized to total ERK content) from parallel cultures harvested at indicated timepoints; immunoblot inset within graph presents representative data from nontransfected RD cells over five days of culture. (H) shRNA-mediated knockdown of RGS12 expression was confirmed by immunoblotting of whole cell lysates from indicated, shRNA-expressing C2C12 cell lines; β-actin protein levels were also examined by immunoblotting in parallel as a loading control. (I) Myotube formation within indicated C2C12 cell cultures was quantitated by calculation of the fusion index: nuclei from sarcomere MHC-positive, multi-nucleated cells and MHC-negative, non-fused cells were separately counted using ImageJ software and the fusion index calculated as the ratio of nuclei present in fused MHC-positive cells to the total number of nuclei in the field (expressed as a percentage). ***, p < 0.005 versus control shRNA-expressing C2C12 cell line; Student’s t-test.
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).
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. Rgs12fl/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 Rgs12fl/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 Rgs12fl/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 Rgs12fl/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 Rgs12fl/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.
Fig 7.
Loss of RGS12 leads to a transient impairment in muscle repair.
A, Representative cross-sections of tibialis anterior (TA) muscle from indicated mice, labeled 8 days after cardiotoxin (CTX) injection with wheat-germ agglutinin (WGA; green) and nuclear DNA (blue). B, Representative micrographs of H&E-stained TA muscle 8 days after CTX-induced injury. C, Relative frequency distribution of the cross-sectional area (CSA) of regenerating (i.e., centrally-nucleated) muscle fibers 8 days after CTX injection as measured by EVOS FL Auto software based on WGA staining (>800 fibers/mouse). Differences between Rgs12fl/fl; Pax7::Cre mice (n = 5) and wild-type Pax7::Cre mice (n = 6) were evaluated using a two-way ANOVA with Sidak’s post hoc test. ****, p<0.0001; *, p<0.05. Inset: The mean CSA of centrally nucleated fibers was shifted towards smaller area in Rgs12fl/fl; Pax7::Cre mice vs wild-type Pax7::Cre mice as assessed by a Student’s t-test; *, p<0.05. Data are presented as mean ± SEM. D, Representative cross-sections of TA muscle from indicated mice, labeled 24 days after CTX injection with WGA (green) and nuclear DNA (blue). E, CSA of regenerated myofibers in TA muscle of Rgs12fl/fl; Pax7::Cre mice (n = 5) and wild-type Pax7::Cre mice (n = 5) 24 days after CTX injury were measured by EVOS FL Auto software based on WGA staining. No differences in fiber CSA were found between genotypes. Inset: The mean CSA of centrally nucleated fibers was not different in Rgs12fl/fl; Pax7::Cre mice (n = 5) and wild-type Pax7::Cre mice (n = 5) as assessed by a Student’s t-test. Data are presented as mean ± SEM. F. The maximal tetanic isometric plantar flexor tension of Rgs12fl/fl; Pax7::Cre mice (n = 5) and wild-type Pax7::Cre mice (n = 5) was obtained in both CTX and PBS treated legs at 14 and 28 days following injection. 14 days post-injection Rgs12fl/fl; Pax7::Cre mice trended towards more impairment in the CTX injected leg relative to wild-type mice, as evaluated by a Student’s t-test (p = 0.06). By 28 days post-CTX injection, both genotypes had recovered to an equivalent level relative to their PBS-injected leg. Data are presented as mean ± SEM.
Fig 8.
Rgs12 mRNA is upregulated in skeletal muscles of Dmdmdx mice, but RGS12 loss does not appear to alter the muscle wasting phenotype of the Dmdmdx mouse model.
A, Representative micrographs of quadriceps femoris muscle from indicated mice, labeled with wheat-germ agglutinin (WGA; green) and nuclear DNA (blue). Loss of RGS12 in satellite cells does not alter skeletal muscle morphology of mdx mice. B, Rgs12 expression was elevated in the diaphragm (*, p<0.05) and quadriceps femoris (***, p<0.001) muscles of mdx mice as assessed by qRT-PCR analysis. Bar graph displays the mean ± SEM; differences between wild-type and mdx mice were shown to be statistically significant by Student’s t-test. C, The maximal tetanic isometric plantar flexor tension normalized to body weight of Rgs12fl/fl; Pax7::Cre mice (n = 5), wild-type Pax7::Cre mice (n = 5), Rgs12fl/fl; Pax7::Cre; mdx mice (n = 5), and wild-type Pax7::Cre; mdx mice (n = 5) obtained at 6, 12, and 24 weeks of age are displayed as mean ± SEM. A two-way ANOVA indicated mdx mice had impaired muscle function relative to Dmd wild-type mice at each age; however, Rgs12 deficiency did not alter muscle function in mdx or Dmd wild-type mice.