Targeted ablation of the cellular inhibitor of apoptosis 1 (cIAP1) attenuates denervation-induced skeletal muscle atrophy

Figures

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  Fig. 1. cIAP1 expression is induced in denervation-induced muscle atrophy. A small (5–10 mm) piece of the right sciatic nerve was removed to induce denervation of the entire right hind limb of 6-week-old female C57BL/6 mice. The contralateral leg served as an internal control. Mice were sacrificed 14 days post denervation. a Muscle mass of tibialis anterior (TA), soleus (Sol) and extensor digitorum longus (EDL) from non-denervated (black bars) or denervated (white bars) hind limbs. b Average fiber size of muscle fibers from non-denervated or denervated TA, Sol, and EDL muscles. c Representative Western blots of Atrogin-1, MuRF1, MyHC, cIAP1, and GAPDH protein expression in Gas muscle from non-denervated (N) and denervated (D) hind limbs. Quantification of d Atrogin-1, e MuRF1, f MyHC, and g cIAP1 protein expression normalized with total protein and relative to non-denervated (N) muscle. Data is the mean ± SEM, n = 4, *p < 0.05, ***p < 0.001

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  Fig. 2. cIAP1 is sufficient to induce muscle atrophy in vitro. a C2C12 myoblasts were allowed to differentiate for 4 days to produce myotubes. Myotubes were infected with adenovirus expressing GFP (Ad-GFP) or cIAP1 (Ad-cIAP1) for 24 h at an MOI of 400. Myotubes were stained with MyHC (pink) and GFP (green), and nuclei were counterstained with DAPI (blue). Representative images are shown. Scale bar =50um. b Myotube diameter of MyHC+/GFP+ myotubes stained as in a (n = 5). c Fusion index (number of nuclei/myotubes) of MyHC+/GFP+ myotubes stained as in a (n = 5). d Representative western blots of cIAP1, Atrogin-1, MuRF1, and GAPDH expression in cells cultured as described in a. e Quantification of cIAP1, Atrogin-1, and MuRF-1 protein expression normalized with total protein and relative to Ad-GFP infected C2C12 myotubes (n = 5). f Primary myoblasts were isolated from C57BL/6 mice and allowed to differentiate for 48 h to produce myotubes. Myotubes were infected with adenovirus expressing GFP (Ad-GFP) or cIAP1 (Ad-cIAP1) for 24 h at an MOI of 400. Myotubes were stained with MyHC (pink) and GFP (green) and nuclei were counterstained with DAPI (blue). Representative images are shown. Scale bar =…

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  Fig. 3. Genetic ablation of cIAP1 attenuates denervation-induced atrophy. A small (5–10 mm) piece of the right sciatic nerve was removed to induce denervation of the entire right hind limb of 6-week-old C57BL/6 mice (blue bars) or sex- and age-matched cIAP1-null mice (orange bars). The contralateral leg served as an internal control. Mice were sacrificed 14 days post-denervation. a Loss of muscle mass as a percentage of non-denervated control leg mass of tibialis anterior (TA), gastrocnemius (Gas), soleus (Sol), and extensor digitorum longus (EDL) (n = 4). b Representative images of TA muscle sections stained with H&E from mice described above. Scale bar = 20um. c Loss in TA, Sol, and EDL fiber size as a percentage of non-denervated control leg cross-sectional area (n = 4). d Representative western blots of cIAP-1, Atrogin-1, MuRF1, and GAPDH protein expression in Gas muscle from non-denervated (NDN) and denervated (DEN) hind limbs of C57BL/6 and cIAP1-null mice. Quantification of e cIAP1, f Atrogin-1, and g MuRF-1 protein expression normalized with total protein and relative to wild-type non-denervated (WT NDN) muscle (n = 3). Data is the mean ± SEM, *p < 0.05, **p < 0.01, and ***p <…

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  Fig. 4. Genetic ablation of cIAP2 does not attenuate denervation-induced atrophy. A small (5–10 mm) piece of the right sciatic nerve was removed to induce denervation of the entire right hind limb of 6-week-old C57BL/6 mice (blue bars) or sex- and age-matched cIAP2-null mice (yellow bars). The contralateral leg served as an internal control. Mice were sacrificed 14 days post-denervation. a Representative Western blots of cIAP1 and cIAP2 protein expression of the heart and spleen of C57BL/6, cIAP1-null, and cIAP2-null mice. b Representative images of TA muscle sections stained with H&E from mice described above. Scale bar = 20 um. c Loss in TA fiber size as a percentage of non-denervated cross-sectional area (n = 3). d Representative Western blots of cIAP1, MuRF1, and GAPDH protein expression in gastrocnemius muscle from non-denervated (NDN) and denervated (DEN) hind limbs of C57BL/6 and cIAP2-null mice. Data is the mean ± SEM, n.s. not significant

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  Fig. 5. cIAP1 induces atrophy through upregulation of classical NF-κB signaling. a Representative Western blots of phospho-p65, p65, and GAPDH expression in gastrocnemius muscle from non-denervated (NDN) and denervated (DEN) hind limbs of C57BL/6 and cIAP1-null mice. b Quantification of phospho-p65 expression normalized with total protein and relative to wild-type non-denervated (WT NDN) muscle (n = 3). c Representative Western blots of phospho-p65, p65, and GAPDH expression in C2C12 myotubes infected with adenovirus expressing GFP (Ad-GFP) or human cIAP1 (Ad-cIAP1) for 24 h. d Quantification of phospho-p65 expression normalized with total protein and relative to Ad-GFP infected myotubes (n = 5). e Nascent (DM day 2) C2C12 myotubes were transfected with non-targeting (NT) siRNA or siRNA targeting murine IKKβ, allowed to form mature myotubes (DM day 4) and then infected with Ad-GFP or Ad-cIAP1 for 24 h. Myotubes were stained with MyHC (pink) and nuclei were counterstained with DAPI (blue). Representative images are shown. Scale bar = 50 um. f Myotube diameter of myotubes stained as in e (n = 5). g Representative Western blots of cIAP1, IKKβ, Atrogin-1, MuRF1, and GAPDH expression in cells…

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  Fig. 6. Pharmacological ablation of cIAP1 with LCL161 attenuates muscle atrophy. a Primary myoblasts were isolated from C57BL/6 mice and allowed to differentiate for 48 h to produce myotubes. Myotubes were treated with 10 ng/ml TNFα or 50 um dexamethasone (Dex) in the absence or presence of 500 nM LCL161. Untreated (UT) myotubes served as controls. Myotubes were collected for protein 24 h after treatment. Representative Western blots of cIAP1 and GAPDH expression. b Myotubes cultured as in a were stained with MyHC (green) and nuclei were counterstained with DAPI (blue). Representative images are shown. Scale bar = 50 um. Myotube diameter of MyHC+ myotubes in c UT vehicle and LCL161-treated cultures, d TNFα-treated cultures, and e Dex-treated cultures (n = 4). f C57BL/6 mice were treated with vehicle or LCL161 (75 mg/kg) at 4, 7, and 11 days after denervation of the right hind limb. Mice were sacrificed 14 days post-denervation. Representative Western blots of cIAP1 and GAPDH expression in the gas muscle. g Representative images of EDL muscle sections stained with H&E from mice described in f. Scale bar = 20 um. h Loss in TA, Sol, and EDL fiber size as a percentage of non-denervated…