Fig 1.
Laminopathy-like phenotypes in muscle-selective Lmna-KO mice.
(A) Kyphotic-like phenotype in 3-mo LmnaHSA-cko mice. Shown are representative images of mice with the indicated genotype. The control mice are littermates, Lmnaf/f. (B) Age-dependent decrease of body weight in LmnaHSA-cko mice. *P < 0.05, **P < 0.01. (C) Representative μCT images of thoracic spines of 6-mo Lmnaf/f and LmnaHSA-cko mice. (D and G) Representative images of HE-stained skin sections from Lmnaf/f and LmnaHSA-cko mice at of 3 mo (D) and 6 mo (G). Scale bar, 200 μm. (E,F,H,I) Quantification of adipocyte size and subcutaneous fat thickness in Lmnaf/f and LmnaHSA-cko mice at 3 mo (E,F) and 6 mo (H,I). The values shown are means ± SD (n = 4 animals per genotype). **P < 0.01, ***P < 0.001. (J) Representative images of spine muscle fibers and bone by HE staining analysis. Scale bar, 200 μm. (K,L) Quantification analyses of data in (J). The percentage of muscle fibers (K) and the spine trabecular BV/TV (L) were presented. The values shown are means ± SD (n = 4 animals per genotype). *P < 0.05, **P < 0.01. The underlying data for this figure can be found in S1 Data. μCT, microcomputer tomographic; BV/TV, bone volume over total volume; cko, conditional knockout; HSA, human alpha-skeletal actin; KO, knockout; Lmna, lamin A/C gene; Lmnaf/f, floxed Lmna mice; LmnaHSA-cko, skeletal muscle–specific Lmna-cko mice; mo, months old; P, postnatal day.
Fig 2.
Decreases in muscle size, muscle force, and TB mass in 3-mo LmnaHSA-cko mice.
(A) Representative images of gastrocnemius cross sections. Scale bar, 20 μm. (B,C) Quantification analyses of cross-section area and central nuclei distribution. The values shown are means ± SD (n = 5 animals per genotype). **P < 0.01, ***P < 0.001. (D) Representative twitch curves and tetanic curves at stimulation frequencies 50 and 150 Hz by muscle stimulation in LmnaHSA-cko mice. (E,F) Quantification analyses of twitch force and tetanic force. The values shown are means ± SD (n = 4 animals per genotype). *P < 0.05, **P < 0.01, ***P < 0.001. (G) Representative μCT 3D images of femurs from 3-mo Lmnaf/f and LmnaHSA-cko littermates. (H) Quantification analyses of TB BV/TV, Tb.N, TB.Th, Tb.Sp, and CB BV/TV by direct model of μCT analysis. Data are shown as means ± SD (n = 4 male mice of each genotype, examined blindly). **P < 0.01, ***P < 0.001, significant difference by two-way ANOVA. The underlying data for this figure can be found in S1 Data. μCT, microcomputer tomographic; BV/TV, bone volume over total volume; CB, cortical bone; cko, conditional knockout; HSA, human alpha-skeletal actin; KO, knockout; Lmna, lamin A/C gene; Lmnaf/f, floxed Lmna mice; LmnaHSA-cko, skeletal muscle–specific Lmna-cko mice; mo, months old; TB, trabecular bone; Tb.N, TB number; Tb.Sp, TB space; TB.Th, TB thickness.
Fig 3.
No change in bone formation, but an increase in bone resorption, in LmnaHSA-cko mice.
(A) Serum Ocn levels in 3-mo Lmnaf/f and LmnaHSA-cko mice. The serum Ocn levels were measured by ELISAs. The values shown are means ± SD from four males per genotype. (B) Representative images of histologic sections showing calcein labeling of endocortical bone in femur mid-diaphysis of LmnaHSA-cko and Lmnaf/f mice. (C-E) Ec.MAR (C), MS/BS (D), and Ec.BFR (E) are presented. The values shown are means ± SD from four males per genotype. (F) ELISA analysis of serum PYD levels. The values presented are means ± SD (n = 3). *P < 0.05, significant difference. (G) Colorimetric analysis of serum calcium levels. The values presented are means ± SD (n = 3). **P < 0.01, significant difference. (H) TRAP staining analysis of femur sections from 1-mo and 3-mo Lmnaf/f and LmnaHSA-cko mice. Scale bar, 100 μm. (I) Quantification analysis as means ± SD (n = 5 femur samples for each group). *P < 0.05, ***P < 0.001, significant difference. (J) TRAP staining analysis of cultured OCs derived from 3-mo Lmnaf/f and LmnaHSA-cko mice. Cells were treated with 100 ng/ml RANKL for 7 days. (K) Quantitative data of TRAP+ MNCs (more than three nuclei) per randomly selected visual field. Scale bar, 100 μm. Data shown are means ± SD from five different cultures. **P < 0.01. The underlying data for this figure can be found in S1 Data. BFR, bone formation rate; BS, bone surface; cko, conditional knockout; Ec.BFR, endocortical bone formation rate; Ec.MAR, endocortical mineral apposition rate; HSA, human alpha-skeletal actin; Lmna, lamin A/C gene; Lmnaf/f, floxed Lmna mice; LmnaHSA-cko, skeletal muscle–specific Lmna-cko mice; MAR, mineral apposition rate; MNC, multinucleated cell; mo, months old; MS, mineral surface; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; OC, osteoclast; Ocn, osteocalcin; PYD, pyridinoline; RANKL, receptor activator of NF-κB ligand; TRAP, tartrate-resistant acid phosphatase.
Fig 4.
Increased osteoclast differentiation in coculture system in the CM of Lmna-KO myotubes.
(A) The experimental treatment strategy. WT BMMs were treated with CMs of Ctrl or Lmna-KO myotubes. (B) The representative images of TRAP staining. Scale bar, 100 μm. (C) The quantitative data of TRAP+ MNCs. (D) Experimental strategy. WT OBs and WT BMMs were treated with CM of Ctrl or Lmna-KO myotubes for 10 days. (E) Representative images of the cultures treated with CM for 10 days. Scale bar, 100 μm. (F) The quantitative analyses of TRAP+ MNCs per field. The values shown are means ± SD from five different cultures. ***P < 0.001, significant difference from the Ctrl. The underlying data for this figure can be found in S1 Data. BMM, bone marrow macrophage/monocyte; CM, conditioned medium; Ctrl, control; KO, knockout; Lmna, lamin A/C gene; M-CSF, macrophage colony-stimulating factor; MNC, multinucleated cell; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; OB, osteoblast; RANKL, receptor activator of NF-κB ligand; TRAP, tartrate-resistant acid phosphatase WT, wild-type.
Fig 5.
Increased expression levels of IL-6 in Lmna-KO myotubes and in muscles and serum samples of LmnaHSA-cko mice.
(A) Proteome profile of Mouse Cytokine Array of CM of Ctrl and Lmna-KO myotubes. (B) Volcano plot comparing Log2 (fold change). Up-regulated proteins were marked in red, and down-regulated proteins were indicated in blue (P < 0.05). (C) Real-time PCR analysis of gene expression in muscles of 1-mo Lmnaf/f and LmnaHSA-cko mice. *P < 0.05, **P < 0.01, significant difference. (D) Western blot analysis of IL-6 expression in muscles of 1-mo HSA-Cre, Lmnaf/f, and LmnaHSA-cko mice. GAPDH was used as the loading Ctrl. (E) ELISA analysis of serum IL-6 level in 1-mo and 3-mo HSA-Cre, Lmnaf/f, and LmnaHSA-cko mice. *P < 0.05, **P < 0.01, ***P < 0.001, significant difference. The underlying data for this figure can be found in S1 Data. BMP, bone morphogenetic protein; cko, conditional knockout; CM, conditioned medium; Cre, cyclization recombination enzyme; Ctrl, control; GAPDH, glyceraldehyde-3-phosphate hydrogenase; HSA, human alpha-skeletal actin; IL, interleukin; KO, knockout; Lmna, lamin A/C gene; Lmnaf/f, floxed Lmna mice; LmnaHSA-cko, skeletal muscle–specific Lmna-cko mice; M-CSF, macrophage colony-stimulating factor; MIG, monokine induced by gamma interferon; mo, months old; RANTES, regulated on activation, normal T cell expressed and secreted; TGF, transforming growth factor; TNF, tumor necrosis factor.
Fig 6.
Diminished osteoclastogenesis in OBs/OCs coculture system in the presence of IL-6 blocking Ab.
(A) Experimental strategy. WT OBs and WT BMMs were treated with CM of myotubes from Ctrl, Lmna-KO, or Lmna-KO plus IL-6 Ab for 10 days. (B) Representative images of the cultures treated with CM for 10 days. Scale bar, 100 μm. (C) The quantitative analyses of TRAP+ MNCs per field. The values shown are means ± SD from three different cultures. *P < 0.05, **P < 0.01, significant difference. (D) Real-time PCR analysis of RANKL/OPG expression in OBs treated with CM of Ctrl, Lmna-KO, or Lmna-KO plus IL-6 Ab myotube CM. *P < 0.05, significant difference. The underlying data for this figure can be found in S1 Data. Ab, antibody; BMM, bone marrow macrophage/monocyte; CM, conditioned medium; Ctrl, control; IL, interleukin; KO, knockout; Lmna, lamin A/C gene; NF-κB, nuclear factor κB; MNC, multinucleated cell; OB, osteoblast; OC, osteoclast; OPG, osteoprotegerin; RANKL, receptor activator of NF-κB ligand; TRAP, tartrate-resistant acid phosphatase; WT, wild-type.
Fig 7.
Attenuated trabecular bone loss phenotype in LmnaHSA-cko mice by IL-6 KO.
(A) Representative μCT 3D images of femurs from 3-month-old Lmnaf/f, LmnaHSA-cko, IL-6–KO, and IL-6–KO; LmnaHSA-cko littermates. (B-F) Quantification analyses by direct model of μCT analysis. Three different male mice of each genotype per group were examined blindly. *P < 0.05, **P < 0.01, significant difference. (G) The serum osteocalcin levels were measured by ELISAs. The values shown are means ± SD from three males per genotype. (H) ELISA analysis of serum PYD levels. The values presented are means ± SD (n = 3). *P < 0.05, **P < 0.01, significant difference. The underlying data for this figure can be found in S1 Data. μCT, microcomputer tomographic; BV/TV, bone volume over total volume; CB, cortical bone; cko, conditional knockout; HSA, human alpha-skeletal actin; IL, interleukin; KO, knockout; Lmna, lamin A/C gene; Lmnaf/f, floxed Lmna mice; LmnaHSA-cko, skeletal muscle–specific Lmna-cko mice; PYD, pyridinoline; Tb.N, trabecular bone number; Tb.Sp, trabecular bone space; Tb.Th, trabecular bone thickness.
Fig 8.
Increased cellular senescence in Lmna-KO muscles and the requirement of p16INK4a for the increase of IL-6 expression in Lmna-KO muscle cells.
(A) SA-β-gal staining counterstained with eosin of gastrocnemius cross-section from 3-mo HSA-Cre, Lmnaf/f, and LmnaHSA-cko mice. Scale bar, 20 μm. (B) Quantification of SA-β-gal densities (mean ± SD; n = 3). *P < 0.05, significant difference. (C) Western blot analysis of indicated protein expression in muscles from mice with indicated genotypes (at 1 mo). GAPDH was used as a loading Ctrl. (D) Quantification analysis (mean ± SD; n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. (E) Real-time PCR analysis of p16INK4a and p19ARF expression in muscles from mice with indicated genotypes (at 1 mo) (mean ± SD; n = 3). **P < 0.01, significant difference. (F) Western blot analysis of p16INK4a, which was reduced in p16INK4a-KD muscle cells. (G) ELISA analysis of IL-6 level in CM of Ctrl, Lmna-KO, p16INK4a-KD, and Lmna-KO; p16INK4a-KD C2C12 muscle cells (mean ± SD; n = 3). **P < 0.01, significant difference. (H) Illustration of a working model. Lmna loss in skeletal muscles results in increased cellular senescence and p16INK4a, which increases the expression and secretion of SASP factors, including IL-6, thus up-regulating RANKL expression in OBs and promoting osteoclastogenesis and trabecular bone loss. The underlying data for this figure can be found in S1 Data. BubR1, Bub1-related kinase; cko, conditional knockout; CM, conditioned medium; Cre, cyclization recombination enzyme; Ctrl, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HSA, human alpha-skeletal actin; IκBα, inhibitor of nuclear factor kappa B; IL, interleukin; KD, knockdown; KO, knockout; Lmna, lamin A/C gene; Lmnaf/f, floxed Lmna mice; LmnaHSA-cko, skeletal muscle–specific Lmna-cko mice; mo, months old; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; OB, osteoblast; OC, osteoclast; OPG, osteoprotegerin; p19ARF, the alternate reading frame tumor-suppressor protein; RANKL, receptor activator of NF-κB ligand; SA-β-gal, senescence-associated beta-galactosidase; SASP, senescence-associated secretory phenotype.