An evaluation of leukaemia inhibitory factor as a potential therapeutic agent in the treatment of muscle disease

Abstract

The exogenous delivery of growth factors and cytokines is a potential therapeutic strategy to alleviate the degenerative effects of primary inherited myopathies such as Duchenne muscular dystrophy. The mdx mouse diaphragm is a model for examining the progressive degeneration of dystrophic muscle. We have delivered leukaemia inhibitory factor to the mdx diaphragm using slow release alginate gels. Previous studies have reported an improvement in the histology of mdx diaphragms after delivery of leukaemia inhibitory factor in a similar manner, but little attention has been paid to the mechanism by which leukaemia inhibitory factor acts. We have used autoradiography to examine cell proliferation, Evans Blue Dye to examine myofibre damage, and morphometric analysis to examine histology in leukaemia-inhibitory-factor-treated diaphragms and compared them with untreated mdx and normal C57Bl10/ScSn diaphragms. Autoradiography showed that although myoblast proliferation was significantly increased in leukaemia inhibitory factor-treated mdx diaphragms, leukaemia inhibitory factor did not reduce myofibre damage and no histological improvement was observed. The data presented here, while demonstrating a role for leukaemia inhibitory factor in myoblast proliferation, do not support a strong and consistent benefit of leukaemia inhibitory factor on dystrophic muscle in vivo as a means of alleviating the effects of chronic dystrophic muscle degeneration.

Introduction

Skeletal muscle has an excellent ability to regenerate and, in response to trauma, satellite cells (widely called myoblasts) become activated, proliferate, differentiate, and eventually fuse into myotubes that mature into myofibres [1]. Strategies to enhance regeneration are of clinical relevance in muscle disease such as Duchenne's muscular dystrophy (DMD) and after severe injury or transplantation of muscle tissue. Under certain circumstances, increased proliferation of myoblasts led to improved muscle regeneration [2], [3]. To this end, it is of interest to test in vivo the influence of growth factors that are known mitogens for myoblasts. One such growth factor is leukaemia inhibitory factor (LIF).

There is a strong evidence for a role for LIF in myogenesis and data from Austin and colleagues suggest a potentially very important role for LIF in clinical treatment of myopathies and DMD. In vivo, LIF is upregulated in diseased and injured muscle [4], [5] and the continual perfusion of LIF from osmotic pumps into undamaged skeletal muscle of the dystrophic mdx mouse, or into crush injured skeletal muscle, enhances regeneration in terms of the number and the size of regenerated myofibres [4], [6], [7]. In tissue culture, LIF accelerates proliferation of both mouse and human myoblasts [8], [9] and induces the formation of larger myotubes [10], although LIF has no effect on fibroblast cultures. Therefore, the in vivo action of LIF is probably related to specific myoblast proliferation with no effect on resident fibroblasts. The exogenous administration of basic fibroblast growth factor (bFGF) also increased replication of satellite and the proportion of myofibres showing evidence of regeneration (i.e. centralized nuclei) in the dystrophic mdx mouse [11]. The growth factors such as insulin-like growth factor-1 (IGF-1), bFGF and to a lesser extent nerve growth factor (NGF) also improve the histology of skeletal muscle regenerating after laceration injury [12]. In contrast with LIF and bFGF, other growth factors which stimulate myoblast proliferation in vitro, such as hepatocyte growth factor [13], interleukin 6 and transforming growth factor α [7] are either ineffective or detrimental in vivo when applied to injured muscle [14].

The exact mechanism by which LIF enhances skeletal muscle regeneration is not clear. The present study specifically tests the effect of LIF on (i) myoblast proliferation, (ii) myofibre size, and (iii) myofibre resistance to damage. To test whether the reported increase in the number and size of myotubes in vivo is indeed a result of increased myoblast numbers, autoradiography [15] is used to quantify the effect of LIF on myoblast proliferation in the regenerating diaphragm of dystrophic mdx mice.

The mdx mouse is an animal model for the lethal X-linked myopathy DMD where dystrophic muscles undergo repeated cycles of endogenous breakdown and regeneration [16]. In humans with DMD, muscle regeneration eventually fails and death results, whereas in the mdx mouse, muscle regeneration is generally successful and skeletal muscle function is preserved [17]. After this, significant degenerative changes are observed in the limb muscles of mdx mice only after eccentric exercise [18], [19]. However, in contrast to unexercised limb muscles, the diaphragm muscle of mdx mice does exhibit severe and long-term degeneration that begins before 3 months of age and progresses over the life of the mouse [20]. The diaphragm muscle more closely resembles the pathology of human DMD and therefore provides a suitable model for testing the efficacy of therapeutic substances as potential treatments for DMD. Previous studies have examined the effect of LIF on the histology and strength of mdx diaphragm muscles and reported a slowdown in the process of degeneration, as diaphragms continuously exposed to LIF contain more normal (peripherally nucleated) myofibres, larger regenerated fibres and less adipose tissue [21]. The amount of non-muscle (connective and adipose tissues) was significantly reduced and the maximum force producing the capacity of isolated diaphragm muscle strips was higher in LIF-treated mice [21].

LIF upregulates laminin expression in vitro [22] and it has been proposed that the beneficial effects of LIF on dystrophic muscle may (at least in part) be due to a strengthening of the myofibre, so that they are more resistant to damage and consequent necrosis (Austin personnel communication). A loss of myofibre integrity is observed in α7 integrin knockout mice [23] and myofibre integrity is enhanced after deflazacort administration [24]. Thus, agents that upregulate basement membrane components may be beneficial in ameliorating dystrophic progression. The present study is a direct extension of the previous work by Austin et al. [21]. It specifically tests the extent of myofibre damage in LIF treated-mdx muscles by using Evans Blue Dye (EBD) as a marker for myofibre integrity [25], [26], [27], [28].

The sustained delivery of LIF in vivo was achieved by using alginate rods containing LIF [21], [29] attached to the underside of the diaphragm of mdx mice. After 3 months, these LIF-treated dystrophic muscles were analysed autoradiographically and with EBD, to specifically address the impact of LIF on myoblast proliferation and myofibre integrity in vivo.