Secretoglobin 3A1 in activated muscle satellite cells contributes to myosin heavy chain IIX and IIB fiber differentiation

Figures

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  Fig. 1. Establishment of Scgb3a1 knockout mice. (A) Schematic illustration of the vector used to generate Scgb3a1 knock-out mice. (B) Southern blotting results. The 5’ and 3’ probe was used to detect bands after digestion with PacI/AflII and EcoRV, respectively. The probe positions and the size of the band detected by each probe are shown in A. (C) (left) PCR genotyping. Wild-type (WT: +/+), 200 bp; knockout (KO: -/-), 163 bp. Std, molecular weight standard. Lower band, 200 bp; upper band, 300 bp. (right) SCGB3A1 protein expression by immunohistochemistry and western blotting using lung sections and tissues, respectively. (D) Immunostaining of LAMININ B1 in 4-week-old Scgb3a1+/+ and Scgb3a1−/− TA muscles. Bar = 20 μm. (n = 3, representative image is shown). (E and F) Details of muscle fiber size distribution between Scgb3a1+/+ and Scgb3a1−/− mice (n = 4 mice, each 100 fibers). (G) Immunostaining of MyHC for E17.5 hindlimb cross section of Scgb3a1+/+ and Scgb3a1−/−. right Fig. are magnified images of the white rectangle regions in the left images. T, tibia; F, fibula. Bar = 20 μm. (n = 3, representative image is shown). (H) Analysis of MyHC positive area excluding interstitial area within…

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  Fig. 2. Muscle injury by injecting BaCl2 causes regeneration defects in Scgb3a1−/−. (A) Schematic illustration of BaCl2 injection and sample collection. BaCl2 injection was carried out at 3–4 months old. (B) Representative immunostaining images of MyHC-IIX and LAMININ B1 in TA muscle of Scgb3a1+/+ or Scgb3a1−/− after one week and one month of BaCl2 injection. Nuclei were counterstained with DAPI. Bar = 50 μm. (n = 3) (C) IIX (+) fiber size distribution between Scgb3a1+/+ and Scgb3a1−/− TA muscle after 1 month of BaCl2 injection (n = 3 mice, each 100 fibers). (D) Number of IIX fibers distribution after 1 month of BaCl2 injection shown in (C). (E) Representative immunostaining images of MyHC-IIB and LAMININ B1 in TA muscle of Scgb3a1+/+ or Scgb3a1−/− after one week and one month of BaCl2 injection. Nuclei were counterstained with DAPI. Bar = 50 μm. (n = 3) (F) IIB (+) fiber size distribution between Scgb3a1+/+ or Scgb3a1−/− TA muscle after 1 month of BaCl2 injection (n = 3 mice, each 100 fibers). (G) Number of IIB fibers distribution after 1 month of BaCl2 injection shown in (F). (H) Western blotting analysis for SCGB3A1 using whole cell extracts obtained from intact contra-lateral (CLA)…

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  Fig. 3. Pax7CreERT2; Scgb3a1f/fmice show reduced regeneration of BaCl2-induced injured muscle. (A) Schematic illustration of Pax7CreERT2;Scgb3a1f/f line. (B) Scheme for tamoxifen injection, BaCl2 injection and harvest of mice. Tamoxifen injection (Tam) was carried out at 4 weeks of age. (C) Western blotting analysis for the confirmation of Tam effects on SCGB3A1 deletion in BaCl2-injected TA muscles. (D,E) Representative immunostaining images of MyHC-IIX and DYSTROPHIN (D) or MyHC-IIB and DYSTROPHIN (E) in TA muscles of Pax7CreERT2;Scgb3a1f/fwith (Tam) and without tamoxifen (Oil), obtained after 1 month of BaCl2 injection. Nuclei were counterstained with DAPI. Bar=50 µm. (n=3) (F) Comparison of MyHC-IIX (+) and IIB (+) muscle fibers diameter in mice with (Tam) and without tamoxifen (Oil), carried out after 1 month of BaCl2 injection. n=3 mice. 100 myofibers per mouse. CLA, contra lateral TA, ** p<0.01 by Student’s t-test. ns, not significant

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  Fig. 4. SCGB3A1(+) cells reside in embryonic somites and contribute to muscle fiber type specification during regeneration. (A) Immunofluorescent staining of SCGB3A1(GFP antibody) and MyHC (anti-MyHC antibody, clone MF20) of mouse E10.5 somite tissue. Arrows indicate the overlapped staining of MyHC and GFP in myotome (m). Nuclei were counterstained with DAPI. nt, neural tube. Bar = 100 μm. (B) Immunofluorescent staining of SCGB3A1(GFP) and PAX7 of mouse E10.5 somite tissue. Arrowheads indicate the overlapped staining of PAX7 and GFP in dermomyotome (dm). Nuclei were counterstained with DAPI. nt, neural tube. Bar = 100 μm. (C) Schematic illustration of Scgb3a1CreERT2;mT/mG line. (D) Scheme for tamoxifen injection, BaCl2 injection and harvest of mice. Tamoxifen injection was carried out at 4 weeks of age. (E) Double immunofluorescent staining of tdTomato (red) and GFP, MyHC-IIX, IIB, and IIA (green) in mouse TA muscle after three weeks of BaCl2 injection. Nuclei were counterstained with DAPI. Each number depicts same muscle fiber position in serial sections. Bar = 50 μm

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  Fig. 5. Regeneration defects after 2x BaCl2 injection affects the inverted grip test in Scgb3a1−/− mice. (A, B) Inverted grip test (IGT) results of Scgb3a1+/+ and Scgb3a1−/− mice. Duration time (in second) was normalized to each body weight (BW: gram). Test was carried out (A) using 2-month (n; +/+=3, -/-=3), 3-month (n; +/+=4, -/-=5), and 5-month (n; +/+=5, -/-=5)-old mice. (B) BaCl2 injection was given at 2.5 and 3.5 months of age, the test was carried out at 4.5 months of age, and the mice euthanized at 5 months of age (n; Scgb3a1+/+=5, Scgb3a1−/−=7). (C) Representative immunostaining images of MyHC-IIB and DYSTROPHIN in TA muscles after 2x BaCl2 injection. Arrow, representative muscle hypertrophy; arrowhead, reduced size MyHC-IIB-faintly positive or negative muscle fiber with central nucleus. Nuclei were counterstained with DAPI. Bar = 50 μm. (D) Average diameter size of each MyHC-IIB and IIX positive muscle fibers after 2x BaCl2 injection. n = 3 mice. 100 myofibers per mouse. *p < 0.05, *** p < 0.001 by Student’s t-test

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  Fig. 6. SCGB3A1 immunostaining co-localizes with PAX7 and NOTCH3 after BaCl2 injection. (A, B) Immunostaining of EDL muscle with (B) or without (A) BaCl2 injection carried out at 4 weeks of age. Muscles were collected 1 week after BaCl2 injection. Individually separated single muscle fibers were stained with anti-PAX7 and anti-SCGB3A1 antibodies. Arrowheads indicate PAX7(+)/SCGB3A1(-) satellite cells under control conditions (A). Arrows indicate PAX7(+)/SCGB3A1(+) and red arrowhead indicates PAX7(+)/SCGB3A1(-) cells after BaCl2 injection (B). (C) Immunostaining for PAX7 (green), NOTCH3 (purple) and SCGB3A1 (red) expression under BaCl2 injection. For all immunostaining, nuclei were counter-stained with DAPI. Bar = 30 μm. All data were representative images of three independent staining. (D) qPCR analysis and quantification of Notch3, HeyL, Hey1, and Hes1 mRNAs with (S) or without (C) 10 ng/ml recombinant SCGB3A1. n = 3, in triplicate per sample. (E) Notch3 expression in established cloned C2C12 cells overexpressing Scgb3a1. Both variant 1 and variant 2 sequences of Scgb3a1 (S1-variant 1 and S1-variant 2, respectively, see Materials and Methods) increased Notch3 mRNA in C2C12. (F) Notch3…

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  Fig. 7. Aged Scgb3a1−/− show reduced muscle mass and grip strength. (A) Comparison of body composition (lean mass and fat mass %) of Scgb3a1+/+ and Scgb3a1−/− mice at various ages were compared. (B) Inverted grip test (IGT) results of Scgb3a1+/+ and Scgb3a1−/− mice carried out at 22-month of age. A dot indicates a mouse. *p < 0.05, ***p < 0.001 by Student’s t-test. (C) Typical images of 22-month-old Scgb3a1+/+ and Scgb3a1−/− mice TA muscle (depicted with dotted blue line). Red arrowheads indicate thinner TA muscle of Scgb3a1−/− mice. (bottom) H&E staining of same specimen. Asterisk indicates the biggest muscle fiber in size in Scgb3a1+/+ TA muscle, while these big muscle fibers are rarely seen in Scgb3a1−/− tissue. Bar = 90 μm. (D) Total TA muscle CSA (µm2) and its distribution (n = 4 mice, each 100 fiber per mouse). (E) Immunostaining of MyHC-IIX and IIB of 22-month-old TA muscle. Bar = 170 μm. (F and G) Total IIX (F) or IIB(G) (+) fiber area (µm2) and detailed size distribution of each MyHC fibers both in Scgb3a1+/+ and Scgb3a1−/−. N = 3 mice, each 100 fibers per mouse. ***p < 0.001 by Student’s t-test. ns, not significant. (H) Representative immunostaining of PAX7 of 22-month-old EDL…