Inhibition of the IKK/NF-κB pathway by AAV gene transfer improves muscle regeneration in older mdx mice

Abstract

The IκB kinase (IKKα, β and the regulatory subunit IKKγ) complex regulates nuclear factor of κB (NF-κB) transcriptional activity, which is upregulated in many chronic inflammatory diseases. NF-κB signaling promotes inflammation and limits muscle regeneration in Duchenne muscular dystrophy (DMD), resulting in fibrotic and fatty tissue replacement of muscle that exacerbates the wasting process in dystrophic muscles. Here, we examined whether dominant-negative forms of IKKα (IKKα-dn) and IKKβ (IKKβ-dn) delivered by adeno-associated viral (AAV) vectors to the gastrocnemius (GAS) and tibialis anterior (TA) muscles of 1, 2 and 11-month-old mdx mice, a murine DMD model, block NF-κB activation and increase muscle regeneration. At 1 month post-treatment, the levels of nuclear NF-κB in locally treated muscle were decreased by gene transfer with either AAV-CMV-IKKα-dn or AAV-CMV-IKKβ-dn, but not by IKK wild-type controls (IKKα and β) or phosphate-buffered saline (PBS). Although treatment with AAV-IKKα-dn or AAV-IKKβ-dn vectors had no significant effect on muscle regeneration in young mdx mice treated at 1 and 2 months of age and collected 1 month later, treatment of old (11 months) mdx with AAV-CMV-IKKα-dn or AAV-CMV-IKKβ-dn significantly increased levels of muscle regeneration. In addition, there was a significant decrease in myofiber necrosis in the AAV-IKKα-dn- and AAV-IKKβ-dn-treated mdx muscle in both young and old mice. These results demonstrate that inhibition of IKKα or IKKβ in dystrophic muscle reduces the adverse effects of NF-κB signaling, resulting in a therapeutic effect. Moreover, these results clearly demonstrate the therapeutic benefits of inhibiting NF-κB activation by AAV gene transfer in dystrophic muscle to promote regeneration, particularly in older mdx mice, and block necrosis.

Figure 1. IKKs gene transfer efficiency and decreased nuclear NF-κB in treated muscle

(A) In vitro gene transfer of IKKα-(dn and wt) and IKKβ-(dn and wt) in AAV constructs. Studies were performed to confirm gene transfer efficiencies of AAV2-IKKα-dn, AAV2-IKKα-wt, AAV2-IKKβ-dn, and AAV2-IKKβ-wt in infected-293 cells 48 hours post-treatment. Western blot analysis by the use of anti-C-terminuses of IKKα and anti-antibodies shows high levels of IKKα-(dn and wt) as well as IKKβ-(dn and wt) expressions (MWs of 85 kD and 87 kD). (B) One month after intramuscular gene transfer with four different AAV2 vectors, the cryosections of GAS muscles treated at 2 months of age and TA muscles treated at 11 months of age were determined the efficiencies of gene transfer by Western blot analysis. The increased levels of IKKα-(dn and wt) and IKKβ-(dn and wt) were found in AAV2-IKKα-dn, AAV2-IKKα-wt, AAV2-IKKβ-dn, and AAV2-IKKβ-wt treated young and old mdxmuscles compared to PBS treated mdx muscle at same age. (C) The electrophoretic mobility shift assay (EMSA) demonstrates the level of nuclear NF-κB in the gastrocnemius (GAS) muscle of both 1 and 2 month-old young mdx mice and the tibialis anterior (TA) muscle of 11 month-old mdx mice treated by AAV2-IKKα-dn, AAV2-IKKα-wt, AAV2-IKKβ-dn, AAV2-IKKβ-wt, and PBS. A total of 45 μg of nuclear extract from each mouse was analyzed at 1 month post -treatment.

Figure 2. Myogenic regeneration in young and old mdx mice

Tissue sections were immunostained for embryonic myosin heavy chain (eMyHC) expression (red), to assay levels of muscle regeneration. The first panel from the left represents tissues from negative control mdx mice treated with PBS. The next two panels represent the tissues treated by AAV2-IKKα-dn and AAV2-IKKα-wt. Finally, the last two panels represent the tissues treated by AAV2-IKKβ-dn and AAV2-IKKβ-wt. (A) At 1 month post -treatment, gastrocnemius (GAS) muscles were isolated from AAV vector-treated young mdx mice at 1 and 2 months of age. (B) GAS and tibialis anterior (TA) muscle tissues of old mdx mice, treated at 11 months of age, were collected at 1 month post-treatment. All panels are shown at 100x magnification.

Figure 3. AAV-IKKs-dn inhibits muscle necrosis

Cryosections of muscle, from mice treated at 1, 2 and 11 months of age and sacrificed 1 month after gene transfer, were incubated with fluorescently-labeled mouse IgG. From left to right, many IgG positive necrotic (green) were found in GAS muscles treated by PBS (first panel), AAV2-IKKα-wt (third panel) and AAV2-IKKβ-wt (fifth panel) in both 1, 2 and 11 month-old groups. In contrast, the GAS muscle treated by AAV2-IKKα-dn (second panel) and AAV2-IKKβ-dn (fourth panel) showed only few necrotic myofibers. All panels are shown at 100x magnification.

Figure 3. AAV-IKKs-dn inhibits muscle necrosis

Cryosections of muscle, from mice treated at 1, 2 and 11 months of age and sacrificed 1 month after gene transfer, were incubated with fluorescently-labeled mouse IgG. From left to right, many IgG positive necrotic (green) were found in GAS muscles treated by PBS (first panel), AAV2-IKKα-wt (third panel) and AAV2-IKKβ-wt (fifth panel) in both 1, 2 and 11 month-old groups. In contrast, the GAS muscle treated by AAV2-IKKα-dn (second panel) and AAV2-IKKβ-dn (fourth panel) showed only few necrotic myofibers. All panels are shown at 100x magnification.