The decline in function of dystrophin-deficient skeletal muscle is due to progressive muscle necrosis and the ultimate failure of muscle regeneration. Prior studies demonstrated that NF-κB signaling is central to the processes of inflammation, necrosis and regeneration in dystrophin-deficient muscle (Acharyya et al., 2007
). Therefore, we expanded on a prior study demonstrating the potential of the Antp-NBD peptide to ameliorate the pathology of dystrophin-deficient limb muscle (Acharyya et al., 2007
). In this study we demonstrated that systemic delivery of the NBD peptide fused to multiple different PTDs was effective in decreasing necrosis and increasing regeneration of dystrophin-deficient muscle. The therapeutic effect was observed in diaphragm muscle and was confirmed in limb muscle of the mdx
mouse model of DMD. The observed effect on diaphragm muscle histology and our demonstration of improved ex vivo
physiological function, as measured by specific force and response to repetitive lengthening activations, is significant because of the more severe histological phenotype in the mdx
mouse diaphragm, which provides the best histological model of the changes observed in human DMD muscle (Stedman et al., 1991
The rationale for delivering PTD-NBD peptides for the treatment of DMD is based on increased levels of activated NF-κB and cytokine production in dystrophic muscle (Acharyya et al., 2007
; Dogra et al., 2008
; Hnia et al., 2007
; Kumar and Boriek, 2003
; Messina et al., 2004
; Messina et al., 2006
; Whitehead et al., 2008
). Furthermore, heterozygous deletion of the NF-κB p65 subunit on the mdx
genetic background demonstrated improved muscle pathology compared to age matched mdx
mice (Acharyya et al., 2007
). In this study, we experimentally demonstrated that multiple PTD-NBD peptides interrupt NF-κB activation, presumably by the disruption of the IKK complex. This disruption is predicted to modulate downstream events in the NF-κB signaling pathway, leading to decreased expression of NF-κB dependent pro-inflammatory genes and decreasing NF-κB-mediated repression of genes whose expression is required for muscle regeneration.
Analysis of NF-κB activation by EMSA in normal C57BL/10 and mdx
mouse muscle confirmed previous studies (Acharyya et al., 2007
; Kumar and Boriek, 2003
; Messina et al., 2004
; Messina et al., 2006
). Interestingly, both the diaphragm and hind limb skeletal muscle of 4-week-old C57BL/10 mice revealed increased levels of activated NF-κB, presumably due to the role of NF-κB in key developmental cellular processes. However, by 9 and 12 weeks of age very little NF-κB activation was detected in normal mouse muscle, while the levels of NF-κB activation increased in mdx
Treatment of mdx
mice with PTD-NBD peptides, either with a single injection or over a period of 4 or 7 weeks, effectively decreased NF-κB activation in hind limb skeletal muscle and diaphragm, as detected by EMSA, highlighting a critical role that NF-κB plays in dystrophic muscle. From previous studies in other disease models, including neonatal hypoxia-ischemia (Nijboer et al., 2008
), collagen induced arthritis (Jimi et al., 2004
), and carrageenan-induced paw edema (Di et al., 2005
), treatment with PTD-NBD peptide therapy was shown to decrease NF-κB activation, as determined by EMSA, in tissue extracts from brain, ankle joints, and mouse paws, respectively.
In the prior study that demonstrated use of a single PTD-NBD peptide and its effect on hind limb skeletal muscle, 200µg of Antp-NBD peptide was administered by intraperitoneal injection to 23 day old mdx
mice every other day for 27 days. Improved muscle histopathology and increased force output was observed in the wild type Antp-NBD peptide treated mdx
mice compared to those receiving mutant peptide (Acharyya et al., 2007
). The present study builds on this prior data to demonstrate that 8K and TAT are also effective PTDs for the systemic delivery of NBD peptide to skeletal muscle. All 3 PTDs were effective and no single PTD stood out as consistently superior to any other when viewed over 2 muscle types (limb and diaphragm) and 2 lengths of treatment (4 and 7 weeks).
Some observed differences across the experiments reported here provide further insights into the treatment of dystrophic muscle with NBD peptide-mediated inhibition of NF-κB activation. In the tibialis anterior muscle of mice treated for 4 weeks, there was improvement in dystrophic histopathology, decreased necrotic fibers and increased levels of muscle fiber regeneration, compared to age-matched untreated mdx
mice. Following 7 weeks of treatment, we observed continued protection of the tibialis anterior muscle from necrosis with each of the PTD-NBD peptides, but there was a lower effect at this time point. The temporal pattern of degeneration and regeneration in mdx
skeletal muscle is characterized by an increase in the degree of pathological changes of limb muscle degeneration and regeneration between 4 and 10 weeks of age in mdx
mice (Grounds et al., 2008
). By 12 weeks of age, progressive degeneration and regeneration in mdx
limb muscle continues at a lower level. Consistent with this, we did not observe an increase in activated NF-κB levels by EMSA in hind limb muscle between 9 and 12 weeks of age.
In contrast in diaphragm muscle, we demonstrated that NF-κB activation levels increase between 9 and 12 weeks of age. In diaphragm, we observed a trend towards improved histopathology after 4 weeks of PTD-NBD peptide delivery and this trend continued following 7 weeks of PTD-NBD peptide treatment. Diaphragm pathology in mdx
mice more closely models the dystrophic pathology of human DMD muscle with progressive degeneration, attempted regeneration that ultimately fails and replacement with connective tissue (Lynch et al., 1997
; Niebroj-Dobosz et al., 1997
; Stedman et al., 1991
). Since we observed increasing levels of NF-κB activation in diaphragm muscle tissue between 9 and 12 weeks of age, the ongoing benefit seen over time in mdx
diaphragm with continued treatment by PTD-NBD peptide in our study is encouraging for its ultimate therapeutic utility.
The improvements observed in the functional properties of the costal diaphragm reflected in specific force production and the response to repetitive lengthening activations demonstrates a functional correlate to the improvements observed in histopathology. We demonstrate that mice treated with either the 8K- or TAT-NBD peptide show significantly higher specific force production compared to saline treated mdx mice. Additionally, both of the peptide treated mdx mice groups had greater sustained force production following repetitive lengthening activations compared to saline treated mdx mice after each individual lengthening activation. However, mdx mice treated with 8K-NBD peptide achieved the greatest functional correction, as measured by repetitive lengthening activations; 8K-NBD peptide treated mdx mice were significantly different from saline treated mdx mice, and not significantly different from the C57BL/10 control mice group. Provision of a functional benefit to respiration, which is a vital physiological process, by systemic administration of PTD-NBD peptide to a murine model of dystrophin deficiency suggests a clinically significant treatment effect in this preclinical model of DMD.
Our findings strongly support an in vivo
mechanism of NBD peptide therapy in muscle. The beneficial effect on necrosis and regeneration in dystrophic muscle reflects the effect of decreased NF-κB activation on downstream targets of the NF-κB pathway. We hypothesize that a lower level of NF-κB activation decreases the inflammatory microenvironment in dystrophic skeletal muscle and this in turn plays a role in the decreased necrosis and increased regeneration observed in PTD-NBD peptide treated mdx
mouse muscle. Reactive oxygen species (ROS) are increased in pre-necrotic dystrophic muscles (Disatnik et al., 1998
), and have been shown to activate the NF-κB pathway (Kumar and Boriek, 2003
; Whitehead et al., 2006
). A previous study utilizing the anti-oxidant N
-acetylcysteine in mdx
mice resulted in significant reduction in ROS in muscle cells in conjunction with increased force output following stretch-induced injury and decreased centralized nuclei in hind limb muscle (Whitehead et al., 2008
). The increased levels of ROS in dystrophic muscle coupled with the ability of ROS to activate NF-κB, in addition to knowledge that NF-κB regulates pro-inflammatory cytokines, such as TNF-α and IL-1β, which are also elevated in dystrophic tissue, presents a complex picture of activation and feedback loops that contribute to the inflammatory response in muscular dystrophy (Kumar and Boriek, 2003
). Future studies will need to comprehensively examine these relationships to determine how PTD-NBD fusion peptide treatment affects this balance.
Administration of PTD-NBD peptides by intraperitoneal injection is a relatively non-invasive means of therapy and offers the advantage of treating a disease of widespread muscle tissue by systemic delivery. However, it is difficult to determine the biodistribution of PTD-NBD peptides in treated mice and whether there are effects on other non-target organs. Evaluation of serum creatine kinase levels from mdx mice treated with PTD-NBD peptide for 4 weeks did not show different results from untreated mdx controls (data not shown). The demonstration that NF-κB activation is decreased in muscle of treated mice indicates that the NBD peptide is active in muscle tissue following intraperitoneal delivery. Dosage studies will be required to determine the optimal treatment effect and the potential effects on non-target tissues.
In this study, the use of PTD-NBD peptide therapy for treatment of dystrophic muscle of mdx mice supports continued research toward its use for DMD patients. While the primary cause of DMD is the lack of a functional dystrophin protein to maintain structural and signaling links from the internal cytoskeleton to the extracellular matrix of muscle cells, therapies that limit inflammation associated with dystrophy and promote muscle regeneration could serve a critical therapeutic purpose. Highly effective treatment of DMD has been elusive to date due to the extent of affected muscle tissue, eventual exhaustion of muscle satellite cells and persistence of inflammation. PTD-NBD peptide therapy has potential both as a primary treatment for DMD and as an adjunct to dystrophin gene transfer.
- Systemic PTD-NBD therapy of mdx mice improves diaphragmatic muscle function.
- NBD peptide effectively blocks NF-κB activation in dystrophic mdx mice in vivo.
- NBD peptide treatment improves histopathology in dystrophic mdx mice.
- Both hindlimb and diaphragm muscles show improvement with NBD peptide therapy.
- Inhibition of NF-κB signaling pathway has therapeutic potential for dystrophic muscle.