Research ArticleSarcopenia

An embryonic CaVβ1 isoform promotes muscle mass maintenance via GDF5 signaling in adult mouse

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Science Translational Medicine  06 Nov 2019:
Vol. 11, Issue 517, eaaw1131
DOI: 10.1126/scitranslmed.aaw1131
  • Fig. 1 Expression of an embryonic CaVβ1 isoform by alternative first exon splicing after muscle denervation.

    (A) RT-qPCR for Cacnb1 from mouse tibialis anterior muscles (TAs) innervated [day zero (D0)] or after the indicated number of days of denervation. Primers were designed for exons 2 and 3 of the Cacnb1 coding region. (B and C) Western blot (B) and quantification (C) of CaVβ1 bands in innervated or denervated TAs after the indicated number of days of denervation. Actin was the loading control. (D) CaVβ1 protein and Cacnb1 gene and transcript variants [adapted from (40)]. Cacnb1-A (NM_031173), Cacnb1-B (NM_145121), Cacnb1-C (NM_001159319), Cacnb1-D (NM_001159320), Cacnb1-E (NM_001282977), and Cacnb1-F (NM_001282978). Molecular weights (MW) are in brackets. (E) Cacnb1 gene showing two putative open reading frames (ATG1 and ATG2). Thin colored vertical lines indicate the start of transcription at ATG1 in innervated muscle and at ATG1 and ATG2 in denervated muscle. (F and G) RT-PCR (F) and quantification (G) of different Cacnb1 regions in innervated (Inn) or denervated (Den) TAs validating the RNAseq data. Primers were designed for different exons (Ex) of the predicted coding region of Cacnb1. Ribosomal phosphoprotein (PO) was the loading control. (H and I) RT-PCR (H) and quantification (I) of Cacnb1-E–specific region in exon 14 and the Cacnb1-D–specific region in exon 13 in Inn or Den TAs. PO was the loading control. (J) RT-PCR of the expression of Cacnb1 (Ex 5 to 8/9) in adult Inn and Den TAs and in the spinal cord (SC). PO was the loading control. (K) RT-PCR for the Cacnb1-E–specific region in exon 14 and the Cacnb1-D–specific region in exon 13 in embryonic and neonatal muscles [day 12.5 (E12.5) and 16 (E16) since fertilization; postnatal 0 (P0)], in adult TAs Inn or Den for 15 days. 3T3 cells were the negative control. PO was the loading control. (L) Western blot of CaVβ1 in TAs Inn or Den for 15 days and in embryonic muscle (E16; diluted 1:10) using AbCaVβ1 (central peptide). Actin was the loading control. (A, C, and I) Means ± SEM [(A), n = 6 mice per group; (C) and (I), n = 3 independent experiments]; *P < 0.05, ***P < 0.001 (ordinary one-way ANOVA, Dunnett’s test). (G) Means ± SEM (n = 3 independent experiments); ***P < 0.001 (ordinary two-way, Sidak’s test).

  • Fig. 2 CaVβ1E down-regulation and increased muscle atrophy by reduced GDF5 signaling after denervation.

    (A) RT-qPCR for Cacnb1-E (Ex2 and Ex3) in adult TAs Inn or Den for 15 days treated with AAV-Sh scrambled (Scra) or AAV-ShCaVβ1E (ShCaVβ1E). (B and C) Western blot (B) using AbCaVβ1 (central peptide) and quantification (C) of CaVβ1 expression in adult TAs Inn or Den treated with Scra or ShCaVβ1E. Actin was the loading control. (D and E) Inn or Den muscle/body weight ratio (D) and percentage of atrophy (E) after denervation of adult TAs treated with Scra or ShCaVβ1E. (F) Frequency distribution of fiber size of TAs Inn or Den treated with Scra or ShCaVβ1E. (G to I) RT-qPCR for (G) Gdf5, (H) Gdf8, and (I) Bmp7 in Inn or Den adult TAs treated with Scra or ShCaVβ1E. (J) Western blot (top) and quantification (bottom) of phosphorylated SMAD1/5 and SMAD5 in adult TAs Inn or Den treated with Scra or ShCaVβ1E. (K to M) Immunofluorescence images (K and L) and quantification (M) of TAs Inn or Den, treated with (K) Scra or (L) ShCaVβ1E, stained with AchR (magenta), SMAD4 (yellow), Cav3 (gray), DAPI (4′,6-diamidino-2-phenylindole, cyan). Scale bars, 10 μm. (N and O) RT-qPCR for (N) Id-1 and (O) Id-2 in adult TAs Inn or Den treated with Scra or ShCaVβ1E. (P) Firefly/Renilla ratio in 24- or 48-hour–differentiated C2C12 cells cotransfected with a Renilla and HSVTK-Gdf5 promoter-Luc3′ with either pCDNA3-Scrambled (Scra) or pCDNA3-ShCaVβ1E (ShCaVβ1E). (A, C, D, G to J, and M to O) Means ± SEM [n = 4 to 7 mice per group; (M) n = 3 cryosections quantified]; *P < 0.05, ** P < 0.01, and ***P < 0.001 (ordinary one-way ANOVA, Sidak’s test). (E) Means ± SEM (n = 6 mice per group); *P < 0.05 [independent samples t test (two tailed)]. (F and P) Means ± SEM [(F) n = 3 cryosections quantified per condition; (O) n = 3 independent experiments]; *P < 0.05, **P < 0.01, and ***P < 0.001 [ordinary two-way ANOVA, (F) Tukey’s test, (O) Sidak’s test].

  • Fig. 3 Muscle mass loss during aging and alteration of the CaVβ1E/GDF5 axis.

    (A) Muscle/body weight ratio of innervated adult TAs from 12-, 52-, and 78-week-old mice. (B and C) RT-qPCR for (B) Cacnb1-E (Ex2 and Ex3) and (C) Cacnb1-D (Ex13) in innervated TAs from 12-, 52-, and 78-week-old mice. (D to F) RT-qPCR for (D) Cacnb1 (Ex2 and Ex3), (E) Cacnb1-D (Ex13), and (F) Gdf5 in TAs Inn or Den for 15 days from 12-, 52-, and 78-week-old mice. (G to I) Western blot (G) using CaVβ1 (central peptide) and quantification (H and I) of CaVβ1E and CaVβ1D in Inn or Den TAs from 12-, 52-, and 78-week-old mice. Actin was the loading control. (J and K) Western blot (J) and quantification (K) of phosphorylated SMAD1/5 and SMAD5 in Inn or Den TAs from 12-, 52-, and 78-week-old mice. (L and M) RT-qPCR for (L) Id-1 and (M) Id-2 in Inn or Den TAs from 12-, 52-, and 78-week-old mice. (A to C) Means ± SEM (12, n = 8 mice per group; 52, n = 9 mice per group; and 78, n = 10 mice per group); *P < 0.05 (ordinary one-way ANOVA, Dunnett’s test). (D to F, L, and M) Means ± SEM (n = 6/7); *P < 0.05, ** P < 0.01, and ***P < 0.001 (ordinary one-way ANOVA, Sidak’s test). (H, I, and K) Means ± SEM (n = 3; Western blots used for quantification are showed in raw data); *P < 0.05, ** P < 0.01, and ***P < 0.001 (ordinary one-way ANOVA, Sidak’s test).

  • Fig. 4 NMJ-associated genes and MyHC expression and effect of exercise on CaVβ1E expression in aged muscle.

    (A to D) RT-qPCR for (A) Chrna1, (B) Chrne, (C) Chrng, and (D) Musk in innervated TAs from 12-, 52-, and 78-week-old mice. (E to H) RT-qPCR for (E) MyHC-I, (F) MyHC-IIA, (G) MyHC-IIX, and (H) MyHC-IIB in innervated TAs from 12- and 78-week-old mice. (I and J) Immunofluorescence images of TAs from (I ) 12- and (J) 78-week-old mice stained with MyHC-IIA (cyan), MyHC-IIB (magenta), and CaVβ1E (gray). MyHC-IIX are black (unstained). Scale bars, 25 μm. (K) Fluorescence intensity of CaVβ1E staining in MyHC-IIA, MyHC-IIB, and MyHC-IIX positive fibers in TA muscles from 12- and 78-week-old mice. a.u., arbitrary unit. ( L and M) RT-qPCR for (L) Cacnb1-E and (M) Cacnb1-D in innervated TAs from 12- and 78-week-old mice in resting or exercised conditions. (N and O) RT-qPCR for (N) MyHCII-A and (O) MyHCII-X in innervated TAs from 78-week-old mice in resting or exercised conditions. (A to D, and K to M) Means ± SEM [(A to D, L, and M), n = 6/8; (K) MyHC-IIA, n = 24; MyHC-IIX, n = 65; and MyHC-IIB, n = 41]; *P < 0.05, **P < 0.01, and ***P < 0.001 [ordinary one-way ANOVA, (A to D) Dunnett’s test, (K) Tukey’s test, (L and M) Sidak’s test). (E to H, N, and O) Means ± SEM (n = 6); *P < 0.05, **P < 0.01, and ***P < 0.001 [independent samples t test (two tailed)].

  • Fig. 5 CaVβ1E and GDF5 overexpression in aged muscle.

    (A, C, and D) RT-qPCR for (A) Cacnb1-E (Ex2 and Ex3), (C) Cacnb1-D (Ex13), and (D) Gdf5 in innervated TAs from 92-week-old mice treated with Scra or AAV-CaVβ1E (CaVβ1E). (A) Minimum top axis = 15. (B) Immunofluorescence images of innervated TAs from 92-week-old mice treated with Scra or CaVβ1E stained with Cav3 (magenta), CaVβ1E (yellow), and DAPI (cyan). Scale bars, 10 μm. (E and F) Western blot (E) and quantification (F) of phosphorylated SMAD1/5 and SMAD5 in innervated TAs from 92-week-old mice treated with Scra or CaVβ1E. (G) Immunofluorescence images of innervated TAs from 92-week-old mice treated with Scra or CaVβ1E stained with SMAD4 (yellow), Cav3 (magenta), and DAPI (cyan). Scale bar, 10 μm. (H and I) RT-qPCR for (H) Id-1 and (I) Id-2 in innervated TAs from 92-week-old mice treated with Scra or CaVβ1E. (J) Muscle/body weight ratio of innervated TAs from 12- and 92-week-old mice treated with Scra or from 92-week-old mice treated with CaVβ1E. (K) Specific force generated (gr/mg: gram per milligram) by innervated TAs from 12- and 92-week-old mice treated with Scra or from 92-week-old mice treated with CaVβ1E. (A, C, D, H, and I) Means ± SEM (Scra, n = 8; CaVβ1E, n = 8); *P < 0.05, **P < 0.01, and ***P < 0.001 [independent samples t test (two tailed)]. (F) Means ± SEM (n = 2 to 3); *P < 0.05 [independent samples t test (two tailed)]. (J and K) Means ± SEM [(J) Scra 12 weeks, n = 12; Scra 92 weeks, n = 11; CaVβ1E 92 weeks, n = 25; GDF5 92 weeks, n = 11; (K) Scra 12 weeks, n = 7; Scra 92 weeks, n = 15; CaVβ1E 92 weeks, n = 19; GDF5 92 weeks, n = 6]; *P < 0.05, ** P < 0.01, and ***P < 0.001 (ordinary one-way ANOVA, Sidak’s test).

  • Fig. 6 Expression of CaVβ1E in human muscle: Conserved compensatory mechanism?

    (A) Representation of human CaVβ1 protein and hCACNB1 gene and transcript variants [adapted from (40)]. hCACNB1-A (NM_199247), hCACNB1-B (NM_000723), hCACNB1-C (NM_199248), predicted hCACNB1-E (XM_006722072.2). MW are in brackets. (B) RT-PCR for different isoforms of hCACNB1 in human quadriceps (Q) and fascia lata (FL1 and FL2) muscle biopsies from three healthy adults. RNA from one human SC biopsy was the positive control for hCACNB1-B. Primers were designed for different exons (Ex) of the coding region of hCACNB1. Human ribosomal phosphoprotein (hPO) is the loading control. (C) RT-PCR for hCACNB1-E in human Q, FL1, and FL2 muscle biopsies from three healthy adults. RNA from SC biopsy was the positive control for the expression of exon 14 of hCACNB1-B. Primers were designed for different exons of the coding region of hCACNB1. hPO was the loading control. (D) Western blot using CaVβ1 (central peptide) in human FL1 and FL2 muscle biopsies from the same adult healthy participants as in (B) and (C). Actin was the loading control. (E) Hematoxylin/eosin (H/E) and immunofluorescence (IF) images of human FL1 and FL2 muscle used in (B), (C), and (D) stained with hCaVβ1E (yellow), Cav3 (magenta), and DAPI (cyan). Scale bars, 50 μm (H/E) and 10 μm (IF). (F and G) Distribution of (F) lean mass percentage and (G) power in human quadriceps biopsies from healthy young and aged volunteers (Table 1). (H and I) Distribution of (H) hCACNB1-E or (I) hCACNB1-A expression in human quadriceps biopsies from healthy young and aged volunteers (Table 1). (J) Linear regression between hCACNB1-E expression and lean mass percentage in human quadriceps biopsies from healthy young and aged volunteers (Table 1). (K) Distribution of hGDF5 (pink triangles, left y axis) and hCACNB1-E (blue circles, right y axis) expression in human quadriceps biopsies from healthy aged volunteers (Table 1) having increasing lean mass percentage. Dotted black line indicates the average of lean mass percentage of the young group. (F to I) Means ± SEM (young, n = 8; aged, n = 17); **P < 0.01, ***P < 0.001 [independent samples t test (two-tailed)].

  • Table 1 Characteristics of human muscles.

    Lean mass (%) was assessed by dual-energy x-ray absorptiometry. Power is expressed by watt on kilograms (W/kg). Y, young participants, from 20 to 42 years old; A, aged participants, from 70 to 81 years old; M, male; F, female; ND, not determined.

    ParticipantGenderAge (years)Height (m)Weight (kg)Lean mass (%)Power (W/kg)
    Y1M20.91.8284.482.354.7
    Y2F24.51.6356.278.451.1
    Y3F26.11.5353.070.438.4
    Y4M26.41.7656.789.248.6
    Y5M27.11.8373.875.942.4
    Y6M37.0NDNDNDND
    Y7M38.0NDNDNDND
    Y8M42.0NDNDNDND
    A1F70.81.6870.258.922.3
    A2M70.91.5865.170.434.5
    A3M71.41.7690.764.529.7
    A4M71.41.6884.466.327
    A5F71.51.6153.367.621.2
    A6M71.71.6769.479.235.3
    A7M72.51.6774.370.434.7
    A8M72.91.7491.664.228.1
    A9M73.81.6776.071.736
    A10F74.21.5458.364.026.3
    A11M75.01.6980.068.329.4
    A12M76.41.5867.774.725.6
    A13M76.71.7685.567.425.8
    A14F77.81.5855.570.822.2
    A15M78.01.7580.368.522.6
    A16M79.61.6767.980.926.3
    A17F80.61.5956.371.229

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/517/eaaw1131/DC1

    Materials and Methods

    Fig. S1. Expression of embryonic CaVβ1 isoform.

    Fig. S2. CaVβ1 isoform localization on adult skeletal muscle.

    Fig. S3. Histological characterization and myogenin signaling in CaVβ1E knockdown muscles.

    Fig. S4. Expression of Cacnb1-E and Gdf5 in C2C12.

    Fig. S5. Effects of CaVβ1E, CaVβ1D, and GDF5 overexpression on AAV-ShCaVβ1E–treated muscles.

    Fig. S6. GDF5 overexpression in aged mice muscle.

    Fig. S7. CaVβ1E overexpression in young mice muscle.

    Fig. S8. GDF5 overexpression in young mice muscle.

    Fig. S9. Schematic representation of CaVβ1E/GDF axis in skeletal muscle.

    Table S1. Expression data.

    Table S2. The 100 top up-regulated genes in innervated and denervated samples.

    Table S3. List of human samples.

    Table S4. Primer list.

    Data file S1. List of 1022 differentially regulated alternative splicing events.

    Data file S2. Uncut Western blotting raw data.

    Data file S3. Individual participant data.

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Expression of embryonic CaVβ1 isoform.
    • Fig. S2. CaVβ1 isoform localization on adult skeletal muscle.
    • Fig. S3. Histological characterization and myogenin signaling in CaVβ1E knockdown muscles.
    • Fig. S4. Expression of Cacnb1-E and Gdf5 in C2C12.
    • Fig. S5. Effects of CaVβ1E, CaVβ1D, and GDF5 overexpression on AAV-ShCaVβ1E–treated muscles.
    • Fig. S6. GDF5 overexpression in aged mice muscle.
    • Fig. S7. CaVβ1E overexpression in young mice muscle.
    • Fig. S8. GDF5 overexpression in young mice muscle.
    • Fig. S9. Schematic representation of CaVβ1E/GDF axis in skeletal muscle.
    • Table S1. Expression data.
    • Table S2. The 100 top up-regulated genes in innervated and denervated samples.
    • Table S3. List of human samples.
    • Table S4. Primer list.
    • Legends for data files S1 to S3

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). List of 1022 differentially regulated alternative splicing events.
    • Data file S2 (.pdf format). Uncut Western blotting raw data.
    • Data file S3 (Microsoft Excel format). Individual participant data.

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