NEDD4-1 deficiency impairs satellite cell function during skeletal muscle regeneration

Figures

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  Fig. 1. NEDD4-1 expression during skeletal muscle regeneration. A Nedd4-1 expression during skeletal muscle regeneration at different days post injury (dpi) with BaCl2 (1.2% w/v). Upper panels show H&E staining of TA cryo-sections. Degenerating myofibers together with abundant mononuclear cells were observed at 1–3 dpi, while muscle cytoarchitecture was progressively recovered by 30 dpi. Scale bar = 100 μm. Lower panels show representative immunofluorescence of TA cryo-sections, marked with anti-NEDD4-1 (green), LAMININ (red), and DAPI for nuclear staining (blue). Regenerating myofibers exhibit centrally located nuclei. NEDD4-1 expression peaked at 3–7 dpi and decreased by 30 dpi at levels comparable to uninjured muscle. Arrowheads indicate interstitial cells; arrows indicate Nedd4(+) cells closely associated with myofiber’s basal lamina. Scale bar = 20 μm. B Double immunofluorescence for NEDD4-1 and MYOD (left panel) and NEDD4-1 and MYOGENIN (right panel) at 3 and 5 dpi. Arrowheads indicate non-myogenic NEDD4-1(+) cells; arrows indicate myogenic cells co-expressing MYOD or MYOGENIN and NEDD4-1. DAPI was used for nuclear staining (blue). Scale bar = 20 μm

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  Fig. 2. Nedd4-1 kinetics expression in activated and differentiated SCs. A Skeletal muscle fibers or SCs were isolated from wild-type c57/bl6 adult mice and maintained in proliferation (PM) or differentiation (DM) culture conditions. At the indicated time points, Nedd4-1 expression was evaluated by IF, qPCR, or Western blot (WB). B Isolated skeletal muscle fibers were maintained in proliferation conditions, and NEDD4-1 expression in associated SCs was analyzed by IF (green) at the indicated time points. PAX7 expression was used as SCs marker (red). Nuclei were marked by DAPI staining (blue). Scale bar = 10 μm. C Isolated SCs were maintained in PM for 2 days (2d) or DM for 7 days (7d), and Nedd4-1 mRNA relative levels were measured by qPCR. Pax7, MyoD, and Myogenin mRNA levels were measured to monitor myogenic progression. Freshly isolated (Fi) SCs were used to determine mRNA basal levels. 18S RNA was used as housekeeping for normalization. Results were expressed as the average RQ ± SD; n = 3. *P-value < 0.05; **P-value < 0.01, ***, P-value < 0.001. D Western blot shows the expression of NEDD4-1 in SCs cultures maintained in PM and DM for 2 and 7 days, respectively. For quantitative…

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  Fig. 3. Effect of Nedd4-1 deletion in SCs during early muscle regeneration. A Pax7CreERT2/+; Nedd4-1f/f mice were injected with tamoxifen (TMX) or vehicle (Veh) for five days (see “Materials and methods” section). 48 h after the final dose, myofibers were isolated for further analysis. B Myofibers were isolated from mice treated as in A and maintained in proliferation culture conditions for 24 h. NEDD4-1 expression was analyzed by IF. PAX7 and SYNDECAN-4 (Sdc-4) were used as independent SC markers. Quantification shows average percentage of NEDD4-1(+)/PAX7(+)/SYNDECAN-4(+) cells upon treatment with vehicle (SC-N4wt) or TMX (SC-N4KO); n = 3, ***P-value < 0.001. Scale bar: 10 μm. C Pax7CreERT2/+; Nedd4-1f/f mice were injected with tamoxifen (TMX) or vehicle (Veh) for five days. 48 h after the final dose, TA muscles were injured by BaCl2 intramuscular injection (see “Materials and methods” section). Injured and contra-lateral uninjured TAs were isolated at 9 dpi for further analysis. D Representative image of frozen injured TAs obtained from SC-N4wt and SC-N4KO animals, respectively. Quantification shows normalized TA weight of contra-lateral (uninj.) and injured (inj.) muscles isolated…

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  Fig. 4. Effect of Nedd4-1 deletion in SCs during late muscle regeneration. A Pax7CreERT2/+; Nedd4-1f/f mice were injected with tamoxifen (TMX) or vehicle (Veh) for five days. 48 h after the final dose, TA muscles were injured by BaCl2 intramuscular injection (see materials and methods). Injured and contra-lateral uninjured TAs were isolated at 30 dpi for further analysis. B Comparison of freshly isolated contra-lateral (uninjured) and injured (30 dpi) TA muscle treated as in A with vehicle (SC-N4wt) or tamoxifen (SC-N4KO) prior to muscle injury. C H&E staining of cryosections from TA muscles obtained as in A at × 4 (left panels) and × 40 (right panels) magnification. Scale bars = 500 μm and 40 μm, respectively. D Differences in average whole TA cross-sectional area (CSA) between contra-lateral (uninj.) and injured (30 dpi) obtained from SC-N4wt and SC-N4KO animals; n = 3, *P < 0.05. E Quantification shows individual fiber CSA for regenerating (centrally located nuclei) myofibers. Uninjured contralateral myofiber CSA are shown as internal control. mean values are indicated for each condition. n = 3, ***P < 0.001

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  Fig. 5. Nedd4-1 ablation results in decreased expansion and differentiation of myogenic progenitors in vivo. A SC-N4wt and SC-N4KO TA muscles were obtained at 5, 7, and 15 days post-injury (dpi), cryosectioned, and analyzed by IF for PAX7 expression (red; arrowheads). Nuclei were stained with DAPI (blue). Scale bar = 30 μm. B Number of myogenic progenitors (PAX7 +) per 100 nuclei from cryosections obtained as in A; n = 3; **P-value < 0.01. C Number of myogenic progenitors (PAX7+) per section area (mm2) from cryosections obtained as in A; n = 3; **P-value < 0.01. D SC-N4wt and SC-N4KO TA muscles obtained as in A were cryosectioned and analyzed by IF for MYOGENIN (MGN) expression (magenta, arrowheads). Nuclei were stained with DAPI (blue). Scale bar = 20 μm. E Number of myogenic progenitors (MGN+) per 100 nuclei from cryosections obtained as in D; n = 3, *P-value < 0.05. F Number of myogenic progenitors (MGN+) per section area (mm2) from cryosections obtained as in D; n = 3, **P-value < 0.01

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  Fig. 6. Nedd4-1 ablation results in decreased expansion and differentiation of myogenic progenitors in vitro. A Representative images of primary myoblasts colonies by phase contrast microscopy from SC-N4wt and SC-N4KO mice, maintained in proliferation conditions for 48 h. Quantification shows the average number of cells per colony (n = 3, **P < 0.01). Scale bar = 100 μm. B Primary myoblasts isolated from SC-N4wt and SC-N4KO mice and maintained in proliferation conditions were labeled with EdU for 6 h prior to fixation. EdU (magenta) incorporation and PAX7 (green) or MYOD (red) expression were analyzed by IF. Quantification shows the average number of myogenic EdU(+) cells, normalized by the total number of cells, in each condition; n = 3, *P < 0.05. Scale bar = 100 μm. C Single myofiber-associated SCs were transfected upon isolation with control (siScramble) or Nedd4-1 targeting siRNA (siNedd4-1) for 24 h prior to fixation and IF. Transfection was monitored by co-transfection with a FAM-labeled non-targeting siRNA (FAM; green). SYNDECAN-4 (Sdc-4; blue), NEDD4-1 (red, upper panel), and MYOD (red, lower panel) were detected by IF. Quantification shows the percentage of…

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  Fig. 7. Effect of Nedd4-1 ablation in SC upon repeated injury and regeneration. A Schematic of TMX and BaCl2 treatment to induce consecutive injury in TA muscles. Samples were collected 30 days after the second injury (30 dpi; 52 days) for further analysis. B H&E staining of contra-lateral (uninjured) and injured (30 dpi) TAs from SC-N4wt and SC-N4KO mice. Scale bar = 200 μm. C Quantification shows the average TA weights ± SEM (normalized by total body weight) from SC-N4wt (white) and SC-N4KO (black bars) at 30 dpi. D Quantification shows the average whole TA CSA ± SEM from SC-N4wt (white) and SC-N4KO (black bars) at 30 dpi. For C and D, n = 3, ***P < 0.001. E Quantification shows individual fiber CSA for regenerating (centrally located nuclei) myofibers. Uninjured contralateral myofiber CSA are shown as internal control. mean values are indicated for each condition. n = 3, ***P < 0.001. F TA cryosections from SC-N4wtmT/mG and SC-N4KOmT/mG Cre-reporter mice after 30 days of BaCl2-induced injury (30 dpi). mGFP(+) myofibers show contribution of recombined SCs to muscle regeneration. Representative of 3 independent experiments. Scale bar = 200 μm. G Differences in regenerating diameter from…