Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006 Nov;116(11):2945-54.
doi: 10.1172/JCI28721.

Targeted ablation of IKK2 improves skeletal muscle strength, maintains mass, and promotes regeneration

Foteini Mourkioti  1 , Paschalis Kratsios, Tom Luedde, Yao-Hua Song, Patrick Delafontaine, Raffaella Adami, Valeria Parente, Roberto Bottinelli, Manolis Pasparakis, Nadia Rosenthal
Affiliations

Targeted ablation of IKK2 improves skeletal muscle strength, maintains mass, and promotes regeneration

Foteini Mourkioti et al. J Clin Invest. 2006 Nov.

Erratum in

  • J Clin Invest. 2007 Jan;117(1):277

Abstract

NF-kappaB is a major pleiotropic transcription factor modulating immune, inflammatory, cell survival, and proliferative responses, yet the relevance of NF-kappaB signaling in muscle physiology and disease is less well documented. Here we show that muscle-restricted NF-kappaB inhibition in mice, through targeted deletion of the activating kinase inhibitor of NF-kappaB kinase 2 (IKK2), shifted muscle fiber distribution and improved muscle force. In response to denervation, IKK2 depletion protected against atrophy, maintaining fiber type, size, and strength, increasing protein synthesis, and decreasing protein degradation. IKK2-depleted mice with a muscle-specific transgene expressing a local Igf-1 isoform (mIgf-1) showed enhanced protection against muscle atrophy. In response to muscle damage, IKK2 depletion facilitated skeletal muscle regeneration through enhanced satellite cell activation and reduced fibrosis. Our results establish IKK2/NF-kappaB signaling as an important modulator of muscle homeostasis and suggest a combined role for IKK inhibitors and growth factors in the therapy of muscle diseases.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Comparison of NF-κB activation in Ikk2f/f and Ikk2mko muscles.
(A) Deletion of the floxed alleles only in Ikk2mko skeletal muscles as shown by Southern blot analysis on genomic DNA isolated from different tissues. (B) Decreased expression of IKK2 in different Ikk2mko muscles, shown by immunoblotting of total muscle extracts. The other subunits, IKK1 and NEMO, remain intact. (C) Unchanged levels of IKK proteins in other tissues. H, heart; G, gastrocnemius; T, TA; D, diaphragm; S, soleus; B, brain; K, kidney; Sp, spleen; C, control from brain-specific IKK2 deletion. (D) IP in 300 μg of whole-muscle extracts from Ikk2f/f and Ikk2mko mice was performed with a polyclonal antibody against NEMO, followed by Western blot (WB) analysis with a monoclonal antibody against IKK1 or IKK2 as indicated. MEF, mouse embryonic fibroblasts. (E) Nuclear translocation of NF-κB was demonstrated by EMSA. (F) Evaluation of IKK activity. Total protein (300 μg) from normal or challenged (injured or denervated) muscles was IP with a NEMO antibody and subjected to a kinase assay using a truncated glutathione S-transferase–IκBa (aa residues 1–54) protein as substrate. DN, denervated; NDN, nondenervated.
Figure 2
Figure 2. IKK2 muscle depletion increases number of intermediate fibers.
(A and B) Increased number of intermediate fibers in Ikk2mko soleus with similar number of slow fibers. (C) Mean percentage increase in Ikk2mko soleus total CSA. (D–F) The number of intermediate fibers in the Ikk2mko EDL was modestly but not significantly increased while the number of other fiber types remained unaltered. (G) Mean percentage of total CSA of the EDL was similar between control and Ikk2mko animals. **Mean values differ significantly from those of control (P < 0.002).
Figure 3
Figure 3. IKK2 muscle deletion improves muscle strength.
(A) Increased specific twitch force of Ikk2mko EDL in comparison with control EDL from littermates. (B) Lower maximum specific tetanic force of EDL in control (n = 15) versus Ikk2mko animals (n = 11). (C) Time to exhaustion in treadmill test; control mice (n = 5) displayed a significantly lower performance than Ikk2mko mice (n = 5). *Mean values differ significantly from those of control (P < 0.05).
Figure 4
Figure 4. IKK2 muscle depletion maintains skeletal muscle mass and strength after denervation-induced atrophy.
(A and B) Four-month-old mice 28 days after denervation procedure. Left arrow points to denervated leg (skin removed from the hind limbs). (C–F) Representative pictures are shown from cross sections taken from gastrocnemius (28 days after denervation) of both legs and stained with H&E. Note the greater reduction of the muscle size of control compared with Ikk2mko atrophied gastrocnemius. (G) Quantitation of muscle mass in Ikk2mko muscles (solid line) compared with those of control littermates (dashed line), at 7, 14, and 28 days after denervation. Note greater protection in Ikk2mko soleus. All values represent the weight of denervated muscle after normalization compared with the weight of sham-operated muscle (n = 5 mice per time point) expressed as percentage. (H) Maximum EDL tetanic specific force of denervated control (n = 4) and denervated Ikk2mko (n = 4) muscle. *Mean value differs significantly from that of control (P < 0.05).
Figure 5
Figure 5. IKK2 muscle depletion protects fiber size and maintains fiber type.
(A and B) Representative images of H&E-stained atrophied muscle 28 days after denervation. Note the absence of angular atrophic fibers and the nuclear accumulation in Ikk2mko denervated muscles (C) Histogram (frequency distribution of fiber CSA) shows the maintenance of fiber size in Ikk2mko gastrocnemius (white bars) compared with the control atrophied muscles (black bars). (D and E) Representative images of NADH staining that mark slow fibers with dark blue (red arrows) and intermediate fibers with light blue (purple arrows). (F) Fiber-type distribution in denervated soleus muscles: 37.5% decrease in slow fibers in control muscles, which become intermediate. Note the maintenance of fiber-type distribution in Ikk2mko denervated soleus.
Figure 6
Figure 6. Immunoblot analysis of protein extracts from control (Ikk2f/f) and Ikk2mko denervated and nondenervated muscles stained for anti–p-Akt Ser472/473, anti–p-GSK3α/β Ser21/9, anti–p-mTOR Ser 2448, and anti–p-p70S6K Thr421/Ser424.
Anti-tubulin served as a loading control.
Figure 7
Figure 7. Combined IGF-1 supplementation and IKK2 inhibition enhances protection against muscle atrophy.
(A) Comparison of gastrocnemius weights from control (black line), Ikk2mko (dashed line), mIgf-1 (light blue line), and mIgf-1/Ikk2f/f (dark blue line) mice at 7, 14, and 28 days after denervation. Values represent denervated muscle weights after normalization to sham-operated muscle weight (n = 5 mice per time point). (B–I) Enhanced maintenance of muscle-fiber size in gastrocnemius muscle from mIgf-1 and mIgf-1/Ikk2f/f denervated muscle. Magnification, ×100 (B–E); ×200 (F–I). (J) Sustenance in the gastrocnemius fiber CSA in Ikk2mko, mIgf-1, and mIgf-1/Ikk2f/f atrophied muscle. (K) MuRF1 and (L) atrogin-1/MAFbx mRNA levels in gastrocnemius muscles of control (n = 4), Ikk2mko (n = 4), mIgf-1 (n = 3), and mIgf-1/Ikk2f/f (n = 4) muscles as determined by quantitative real-time RT-PCR. Denervated values were normalized to nondenervated values. **Mean values differ significantly from those of control (P < 0.002).
Figure 8
Figure 8. IKK2 depletion promotes muscle regeneration.
(A–F) Trichrome staining of injured TA muscle sections from Ikk2f/f and Ikk2mko mice. (A and B) Extensive necrosis in both genotypes 2 days after CTX injection. Areas with infiltrated cells are marked with white arrows. (C and D) Recovery of the injured muscles in regenerating myofibers containing centralized nuclei (arrowheads) 5 days after injury. (E and F) Improved Ikk2mko fiber morphology after 10 days. Intercellular space is marked with asterisks. (G) Increased CSA in the Ikk2mko regenerated fibers. (H) Regenerating myocytes containing centralized nuclei close to injured area in the Ikk2mko injured muscles (n = 5).
Figure 10
Figure 10. Muscle-specific IKK2 depletion limits the inflammatory response.
(A and B) Anti-MHCII antibody was used to identify infiltrating cells from 3 independent injured mice (10 days after injury). (C) The graph represents the percentage of anti-MHCII cells at all time points as a percentage of the total DAPI nuclei. Note that at day 10, the number of infiltrating cells was significantly decreased in the Ikk2mko injured muscles. **Mean value differs significantly from that of control (P < 0.002).
Figure 9
Figure 9. IKK2 muscle deletion increases multinucleate regenerating fibers.
(A and B) Anti-LacZ immunostaining (red) of regenerating muscles (day 10) from control and Ikk2mko mice. (C) Negative control using only the secondary antibody (2nd) (no staining in red) in the Ikk2mko regenerating muscle. (D) Anti-LacZ immunostaining (red) of noninjured Ikk2mko muscle. (A–D, lower panels) DAPI-counterstained (blue staining) sections merged with green fluorescence (no staining). (E–F) Enlargement of regenerating areas. Note the increased desmin-nLacZ–positive cells in the regenerating area of Ikk2mko sections. Examples of fibers with double- or triple-LacZ–positive nuclei are indicated with arrowheads. (G) Graph showing nLacZ-positive nuclei counted from 3 independent injured mice, presented as a percentage of total DAPI nuclei. *Mean value differs significantly from that of control (P < 0.05). Magnification, ×100 (A–D); ×200 (E and F).
See this image and copyright information in PMC

Comment in

References

    1. Nowak K.J., Davies K.E. Duchenne muscular dystrophy and dystrophin: pathogenesis and opportunities for treatment. EMBO Rep. 2004;5:872–876. - PMC - PubMed
    1. Strassburg S., Springer J., Anker S.D. Muscle wasting in cardiac cachexia. Int. J. Biochem. Cell Biol. 2005;37:1938–1947. - PubMed
    1. Filippatos G.S., Anker S.D., Kremastinos D.T. Pathophysiology of peripheral muscle wasting in cardiac cachexia. Curr. Opin. Clin. Nutr. Metab. Care. 2005;8:249–254. - PubMed
    1. Tisdale M.J. Cachexia in cancer patients. Nat. Rev. Cancer. 2002;2:862–871. - PubMed
    1. Dalakas M.C. Inflammatory disorders of muscle: progress in polymyositis, dermatomyositis and inclusion body myositis. Curr. Opin. Neurol. 2004;17:561–567. - PubMed

Publication types

MeSH terms