Suppressing PDGFRβ Signaling Enhances Myocyte Fusion to Promote Skeletal Muscle Regeneration

Figure 1.. Pdgfrβ is expressed and activated in stimulated muscle progenitor cells (A) Schematic of the experimental approach. Pax7tdTomato male mice were administered tamoxifen (TMX) at postnatal day 60 (P60) to induce Cre-mediated recombination. Following TMX administration, mice were either uninjured or subjected to a single intramuscular injection of 1.2% BaCl2 to induce injury in the TA muscle. The TA muscles were harvested, and muscle cells were isolated. Fluorescence-activated cell sorting (FACS) was then employed to separate Pax7tdTomato-positive and -negative cells. (B) Quantitative PCR (qPCR) analysis of Pax7 and Pdgfrβ mRNA expression in quiescent Pax7tdTomato-positive and -negative cells isolated from the mice described in (A), providing insights into gene expression patterns in muscle progenitor cells (n = 4 biologically independent mice/group). (C) Comparative mRNA expression analysis of Pdgfrβ within Pax7tdTomato+ cells under quiescent and injured conditions, highlighting the dynamic upregulation of Pdgfrβ following muscle injury in the mice described in (A) (n = 3 biologically independent mice/group). (D) Representative flow cytometric histogram showing PDGFRβ expression on…

Figure 2.. Pdgfrβ activity regulates myotube development and muscle regeneration. (A) Schematic overview of the experimental procedure. Hindlimb muscles were collected from P30 male mice, and isolated muscle progenitor cells (MPCs) were cultured. Throughout differentiation, cells were treated with vehicle, PDGF-BB (25 ng/mL), or SU16f (1 μM) for five days and then myotube formation was assessed. (B) Representative images of myosin heavy chain (MyHC) showing myotube development in cultures as described in (A), providing visual evidence of the effects of PDGF-BB and SU16f on myotube formation. (C) Quantification of the fusion index of differentiated cultures described in (B), reflecting the efficiency of myoblast fusion into myotubes in the treated cultures (n = 3 biologically independent mice per group). (D) Quantification of myotube nuclei count and distribution from cultures described in (B) (n = 4 biologically independent mice per group). (E) Muscle progenitor cells were isolated from hindlimb muscles of MyoGtdTomato mice and cultured at low density. Cells were treated with vehicle, PDGF-BB (25 ng/mL), or SU16f (1 μM), and the differentiation index was determined by monitoring…

Figure 3.. Genetically altering Pdgfrβ expression changes muscle regeneration and myotube development. (A) Schematic of the experimental approach. ControlPax7, PdgfrβPax7KO, and PdgfrβPax7-D849V mice were administered TMX at postnatal day 60 (P60) to induce Cre-mediated recombination. TA muscles were subsequently injured with 1.2% BaCl2 and analyzed at seven d.p.i. (B) Representative images of injured TA muscle sections immunostained for eMyHC and laminin from mice described in (A), illustrating the regenerative response across different genotypes. (C) Quantification of mean cross-sectional area (CSA) of injured TA myofibers from sections described in (B) from mice described in (A), reflecting the differences in muscle regeneration (n = 3 biologically independent mice per group). (D) Quantification of myonuclear accretion within injured myofibers, providing insights into cellular fusion and muscle repair dynamics in the different experimental groups (n = 4 biologically independent mice per group). (E) Quantification of eMyHC immunostaining from TA muscle sections described in (B), indicating the level of ongoing regeneration post-injury (n = 3 biologically independent mice per group). (F)…

Figure 4.. Genetically altering Pdgfrβ activity in myocytes modifies muscle regeneration. (A) Schematic overview of the experimental setup: TA muscles from ControlMyoG, PdgfrβMyoGKO, and PdgfrβMyoG-D849V mice were intramuscularly injected with 1.2% BaCl2. Muscle tissues were harvested and analyzed at seven days post-injury (d.p.i.). (B) Representative immunofluorescence images illustrating eMyHC and laminin staining in injured TA muscle sections. The images provide a visual context for the extent of muscle regeneration across the different genotypes. (C) Quantitative assessment of myofiber cross-sectional area (CSA) in the injured regions of TA muscles from sections and images described in (B) (n = 4 biologically independent mice per group). (D) Quantification of myonuclear accretion within injured myofibers, a metric of cellular fusion and muscle repair (n = 3 biologically independent mice per group). (E) Quantification of eMyHC-positive fibers within the injured TA muscle, providing insights into the ongoing myogenic processes in the different experimental groups (n = 3 biologically independent mice per group). (F) Muscle progenitor cells isolated from the hindlimb muscles of ControlMyoG,…

Figure 5.. Pdgfrβ signaling cooperates with Tgfβ signaling to control myocyte morphology. (A) Schematic of the in vitro cell mixing experimental procedure. Muscle progenitor cells were isolated from the hindlimb muscle groups of from ControlMyoG, PdgfrβMyoG-D849V and wild-type mice. ControlMyoG and PdgfrβMyoG-D849V cells were plated grown and differentiated whereas PdgfrβPax7-D849V were co-cultured with wild-type. Myotube and chimeric myotube development was assessed by MyoGtdTomato fluorescence. (B) Representative images of myotube development as assessed by MyoGtdTomato fluorescence from muscle progenitors isolated in (A). (C) Quantification of the fusion index from myotube cultures described in (B), providing a measure of muscle cell fusion in response cell mixing (n = 3 biologically independent mice per group). (D) Quantification of nuclei within myotubes from images described in (B) and cultures described in (A) (n = 3 biologically independent mice per group). (E, F) Heatmap of gene expression profiles comparing hindlimb muscles from ControlMyoG, PdgfrβMyoGKO, and PdgfrβMyoG-D849V P30 male mice. Gene transcript analysis revealed changes in TGFβ signaling (E) and focal adhesion (F)…

Figure 6.. STAT1 mediates PDGFRβ signaling to control myocyte fusion and myofiber regeneration. (A) Schematic of the in vitro experimental procedure. Muscle progenitor cells were isolated from the hindlimb muscle groups from ControlMyoG and PdgfrβMyoG-D849V male mice. Cells were grown and differentiated in the presence of either vehicle (1% DMSO) or fludarabine (1 μM). Myotube formation was assessed after five days of treatment. (B) Representative images of myotube development as assessed by MyoGtdTomato fluorescence from muscle progenitors isolated in (A). (C) Quantification of the fusion index from myotube cultures described in (B), providing a measure of muscle cell fusion in response to STAT1 inhibition (n = 3 biologically independent mice per group). (D) Quantification of nuclei within myotubes from cultures described in (B) (n = 3 biologically independent mice per group). (E) Muscle progenitor cells were isolated from the hindlimb muscle groups from ControlPax7 and PdgfrβPax7-D849V male mice. Cells were grown and differentiated for one day in the presence of vehicle (1% DMSO) or fludarabine (1 μM). Cells were stained with F-actin to visualize cell size and actin orientation. (F)…

Figure 7.. Blocking Pdgfrβ signaling in human muscle cells boosts myotube development. (A) Schematic of the experimental approach. Muscle progenitor cells were FACS-isolated from human quadricep muscles and cultured. These cells were treated with vehicle, PDGF-BB (25 ng/mL), or SU16f (1 μM) throughout the myogenesis process. Myotube development was assessed to evaluate the effects of these treatments. (B) Representative images of myotube development as assessed by MyHC immunostaining from human muscle cell cultures described in (A). (C) Quantification of the fusion index from cultures and images described in (A and B) offering insights into human muscle cell fusion under different treatment conditions (n = 5 biologically independent samples per group). (D) Quantification of nuclei and distribution within myotubes from images described in (B), reflecting myonuclear accretion in response to targeting PDGFRβ (n = 5 biologically independent samples per group). (E) Quantification of myotube length from cultures described in (A) and (B), providing a measure of myotube development under the influence of PDGF-BB and SU16f (n = 5 biologically independent samples per group). (F) Quantification of…