Long-term administration of the TNF blocking drug Remicade (cV1q) to mdx mice reduces skeletal and cardiac muscle fibrosis, but negatively impacts cardiac function

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Abstract

Duchenne muscular dystrophy (DMD) is a degenerative skeletal muscle disease caused by mutations in the gene encoding dystrophin (DYS). Tumor necrosis factor (TNF) has been implicated in the pathogenesis since short-term treatment of mdx mice with TNF blocking drugs proved beneficial; however, it is not clear whether long-term treatment will also improve long-term outcomes of fibrosis and cardiac health. In this investigation, short and long-term dosing studies were carried out using the TNF blocking drug Remicade and a variety of outcome measures were assessed. Here we show no demonstrable benefit to muscle strength or morphology with 10 mg/kg or 20 mg/kg Remicade; however, 3 mg/kg produced positive strength benefits. Remicade treatment correlated with reductions in myostatin mRNA in the heart, and concomitant reductions in cardiac and skeletal fibrosis. Surprisingly, although Remicade treated mdx hearts were less fibrotic, reductions in LV mass and ejection fraction were also observed, and these changes coincided with reductions in AKT phosphorylation on threonine 308. Thus, TNF blockade benefits mdx skeletal muscle strength and fibrosis, but negatively impacts AKT activation, leading to deleterious changes to dystrophic heart function. These studies uncover a previously unknown relationship between TNF blockade and alteration of muscle growth signaling pathways.

Introduction

Muscular dystrophies are genetically inherited disorders that cause progressive, clinical muscle weakness. In Duchenne muscular dystrophy (DMD), mutations in dystrophin lead to a destabilized cell membrane followed by muscle degeneration, inflammation and regeneration. While in acute muscle injury, inflammatory cells serve an important role in phagocytosis of debris and release of growth factors that facilitate repair; the chronic inflammatory environment that results from repeated degeneration/regeneration cycles in dystrophic muscle leads to development of fibrosis, which is highly detrimental to muscle function and satellite cell mediated repair. Thus, dystrophic muscle comprises a highly dynamic environment consisting of pro-necrotic, pro-regenerative and pro-fibrotic factors that can positively or negatively modulate the outcome of the disease.

The mdx mouse is the genetic homologue of DMD because it possesses a mutation in the dystrophin gene, lacks dystrophin protein and its muscles undergo mild degeneration, inflammation and regeneration in a process that approximates human DMD. While human DMD muscles experience significant fibrosis, most muscles of the mouse lack significant connective tissue deposition, due to efficient repair by murine satellite cells; however, the mdx diaphragm fibroses to a significant degree and is often studied as a model of progressive degeneration and fibrosis in DMD. Dissecting the role of inflammation in mdx dystrophy is complicated by the dynamic and interconnected nature of the muscle infiltrate and the robust regenerative response [1]. Mouse studies that have assessed immune interventions have mainly examined short term outcomes and failed to examine the final phenotypic end products of muscle fibrosis and cardiotoxicity [1], [2], [3]. Since cardiomyopathy occurs in all patients with DMD, it is critical that any drugs considered for clinical trials are assessed in long-term studies to evaluate the effects of these agents on the heart.

TNF is elevated in both human [4] and mouse [5] dystrophinopathies and is a cytokine secreted by a broad variety of cells including macrophages, T cells, mast cells and fibroblasts. TNF exerts pleiotropic effects on its target tissues, depending on the local concentration and the presence of either type I or type II TNF receptors. While generally considered a pro-inflammatory cytokine, there are instances where blockade of TNF leads to a worsened disease phenotype, such as in the case of TNF blockade in multiple sclerosis (MS). Prior to clinical trials, studies had demonstrated that TNF was elevated in the EAE mouse model of MS and in humans with MS, and most showed a positive response to TNF blockade; however some divergent reports had also emerged [6], [7], [8], [9]. In spite of the conflicting mouse data, clinical trials commenced in patients who were administered Lenercept, a recombinant TNF receptor (p55) immunoglobulin fusion protein [10], but unfortunately, the trials had to be suspended due to increased severity of symptoms. A similar set of circumstances led to failed trials of TNF blockade in patients with sepsis [11]. Thus, it is essential that studies proposing to block inflammatory mediators proceed with caution and examine long-term outcomes, due to the dynamic and interconnected nature of the inflammatory network and the potential danger of skewing the immune response towards a negative course.

Previous studies have shown that both Th1 (pro-inflammatory) and Th2 (anti-inflammatory, pro-regenerative, pro-fibrotic) type inflammatory cytokines are elevated in mdx muscles including the Th1 cytokines TNF, interferon gamma (IFNγ) and interleukin 6 (IL6) and the Th2 cytokines interleukin 10 (IL10) and transforming growth factor β (TGFβ) [5]. In the muscular dystrophy literature, much focus has been placed on TNF, due to the availability of TNF blocking drugs and their success in reducing the severity of several inflammatory diseases such as inflammatory bowel disease and rheumatoid arthritis. Several FDA approved, TNF blocking drugs are available including Enbrel® (etanercept), Remicade® (infliximab) and Humira® (adalimumab) and are in widespread use for a variety of diseases. Early investigations of TNF blockade in the mdx mouse were somewhat conflicting, but primarily supported TNF blockade as beneficial for the phenotype. While ablation of TNF in the mdx mouse was not advantageous in short-term studies [12], long term investigation of pulmonary function showed promise [13]. Subsequent studies examining the efficacy of TNF blocking drugs Remicade (cV1q) [2], [14], [15] or Enbrel [3], [16] on early features of mdx dystrophy also reported reduced necrosis and improved exercise fatigue, but also demonstrated that TNF blockade interfered with muscle repair [14]. The latter finding is consistent with the documented role of TNF in myogenesis and post-natal muscle repair [17]. TNF has been shown to play both positive and negative roles on muscle mass and myogenesis. On the one hand, high levels of TNF can induce cachexia [18], [19], [20], but on the other hand, TNF can promote muscle regeneration [17]. Since regenerated dystrophin deficient fibers are more stable than mature dystrophin deficient fibers, they significantly slow the course of the dystrophic process; thus, the effect of any pharmacological intervention for Duchenne on muscle repair is important to evaluate.

In this investigation, we examined the effect of TNF blockade on muscular dystrophy pathogenesis with a particular focus on the long-term outcomes of fibrosis and cardiomyopathy. Furthermore, we employed both traditional methods of quantitative histology and biochemistry in parallel with non-invasive methods, such as functional strength testing and imaging, to obtain a robust assessment of the phenotype. We show that higher dosing of Remicade (10 and 20 mg/kg) does not confer benefit on either muscle strength or fibrosis, but that long-term administration of low dose Remicade is beneficial for both muscle strength and fibrosis. However, improvements in skeletal muscle strength coincide with reduced cardiac function and alterations in cardiac AKT signaling. These studies call into question the potential utility of TNF blocking drugs for treatment of DMD, and suggest that commencement of trials proceed with great caution and attention to cardiotoxic side effects.

Section snippets

Animals

mdx (C57BL/10ScSn-mdx/J) breeder mice were obtained from the Jackson Laboratories and housed and bred in the UCLA vivarium in compliance with the regulations of the Department of Laboratory and Animal Medicine. All experimental protocols and use of animals were conducted in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals and approved by the UCLA Institutional Animal Care and Use Committee.

Injection regimen

Four different treatment regimens, that varied by the age of

Short-term treatment of either young or old mdx mice with 10 mg/kg Remicade (cV1q) does not result in obvious histological or strength benefits

Short-term treatment with Remicade (10 mg/kg or 20 mg/kg administered once weekly) was previously shown to improve strength and reduce necrosis of mdx muscles [2], [3]. We sought to replicate those earlier results, prior to carrying out long-term treatment studies with this drug. Injections of 10 mg/kg Remicade were initiated in male mdx mice at either 2 weeks or 30 weeks of age and mice were dosed for 6 weeks total (2–8 weeks vs 28–36 weeks of age). Mice were exercised on a treadmill for the last 3 

Discussion

Duchenne muscular dystrophy results from mutations in the gene encoding dystrophin (DYS), a cytoskeletal protein that resides on the inner surface of the sarcolemmal membrane. Dystrophin links cytoskeletal actin to the dystrophin glycoprotein complex (DGC) [28], [29]. In the absence of dystrophin, the entire DGC complex is lost and plasma membrane integrity is compromised [30]. While muscle regeneration by satellite cells can help counter the degeneration, it does not completely compensate for

Acknowledgements

This work was supported by funding from the National Institute of Arthritis, Musculoskeletal and Skin Diseases for a Wellstone Cooperative Muscular Dystrophy Center (U54AR052646-Sweeney) and a P30 Muscular Dystrophy Core Center (P30AR057230-01-Spencer). Funding was also provided by Parent Project Muscular Dystrophy (Spencer) and the Muscular Dystrophy Association (Spencer). Mouse specific Remicade (infliximab) was generously supplied by Centocor. Mouse specific Enbrel (etanercept) was

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    Current address: Whittier College, Department of Biology, Whittier, CA 90608, USA.

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