Current Pharmaceutical Design - Volume 16, Issue 8, 2010

Skeletal musculature plays a crucial role in locomotor activity, postural behaviour and breathing, thus the maintenance of a working musculature is fundamental for animal survival: for this reason the mechanisms involved in muscular regeneration processes have been widely investigated. Skeletal muscle can be damaged not only by direct trauma (such as intensive physical activities or lacerations) but also by neurological dysfunction or innate genetic defects. In the last years several studies have been addressed to find out new therapeutic strategies to recover some of the pathological conditions associated with poor muscle regenerative capacity, such as in myogenic dystrophies. For a long time mammalian animal models have been considered the best model for studying muscle regeneration and for dissecting a human disease. However, during the last decade a lot of evidence has shown that the mechanisms controlling muscle development and regeneration have been highly conserved. This fact encouraged researcher to employ invertebrate models to identify and characterize the different population of cells involved in the regenerative process and the molecular pathways which finely orchestrate the activation of these set of cells during the repair process. These collected reviews contribute to the current knowledge about muscle biology and pathology and the potential of different therapeutic strategies (drug, gene, cell-based therapies) to promote muscle regeneration using both vertebrate and invertebrate models. The paper by Ciciliot and Schiaffino [1] contains different aspects about muscle regeneration. Firstly they review the three phases that are involved in regeneration of mammalian skeletal muscle and underline the role of nerve activity in this phenomenon. The occurrence of the regeneration process is then discussed in a chronic degenerative setting, i.e. muscular dystrophies, and in relation to traumatic injuries common in sport medicine. A final description of the age-dependent decline in muscle regeneration potential leads to a discussion of the molecular factors underlying muscle growth that could thus be recruited to boost regeneration and rescue muscle loss in aging muscle and muscular dystrophies. Formigli and collaborators [2] review the current knowledge about the use of skeletal myoblasts for cardiac regeneration. Although the use of these cells for a therapeutic purpose has always been controversial, the possibility to genetically engineer them to potentiate their paracrine attitude and their function versus cardiac regeneration appears now attracting and is raising great expectations. To this purpose, the Authors focus on key aspects underlying the interactions between skeletal myoblasts and the host cardiac tissues, with a particular attention towards the cell-derived factors that are involved in cardiac repair and regeneration. The high conservation of mechanisms that control muscle development and repair has led to the establishment of some invertebrate models of human muscular disorders. In this context, Daczewska et al. [3] compare the cellular and molecular events underlying muscle development and regeneration in normal and pathological conditions in Drosophila, a classical model system which is amenable to global genomic/transcriptomic approaches, genetic manipulation and high throughput chemical compound screening. The extensive similarities in the myogenic pathways between fruit fly and vertebrates provide a powerful platform for the identification of candidate genes and to test their potential to rescue mutant phenotypes. The paper by Garcia-Arrarás and Dolmatov [4] emphasizes the use of a less conventional animal model, echinoderms, for studies on muscle regeneration. These animals show amazing regenerative capabilities and, due to their close phylogenetic relation to vertebrates, can surely represent interesting model systems to determine cellular and molecular processes involved in muscle regeneration and to set up pharmacological studies for muscular diseases. In contrast to what previously thought, skeletal muscle satellite cells are not the only source of myogenic precursors in skeletal muscles. In their paper, Tamaki and colleagues [5] provide data about skeletal muscle-derived stem cells, with a particular attention to two stem cell populations previously identified in their lab, that are able to differentiate into myogenic-vasculogenic cells in the interstitial spaces of murine skeletal muscle. The Authors discuss not only the possible physiological role of these cell in vivo but also their contribution to muscular regeneration and use for the treatment of severely damaged muscle. The paper by Grimaldi et al. [6] emphasizes the use of an unusual invertebrate, the leech Hirudo medicinalis, as a new emerging model for studying endothelial and hematopoietic precursor cells involved in muscle post-natal growth and regeneration processes. Moreover, the Authors propose a new “in vivo cell sorting method” to isolate a specific population of hematopoietic/endothelial precursors cells which can differentiate in muscle. Muscular dystrophies are a heterogeneous group of diseases affecting both children and adults which lead to progressive loss of muscle strength and mass in patients. The review by Lamperti and Moggio [7] focuses on the clinical features and genetic classification of Congenital Muscular Dystrophies, dystrophinopathies and Limb Girdle Muscular Dystrophies. In addition a survey of three main strategies to develop a therapy (i.e. gene, cell and drug therapy) is presented. Among the strategies to treat Duchenne Muscular Dystrophy, exon skipping has emerged as one of the most promising and has recently undergone completion of Phase I clinical trials in humans. In their paper Wilton and Fletcher [8], starting from an overview of the history of splice intervention therapy, focus on the pre-clinical antisense oligomer splice-switching studies and the ongoing clinical trials to by-pass disease causing dystrophin mutations. They also discuss commercial/ethical issues and future perspectives related to this strategy. Type 1 diabetes is an autoimmune disorder characterized by absence of insulin. The absolute dependence of patients on exogenous insulin for survival has boost research towards the search for new therapies and among them the use of gene therapy is of wide interest. In their paper, Mann et al. [9] focus attention towards the muscle, a target tissue that is not only amenable to gene therapy technology, but its central role in whole body metabolism and glucose homeostasis makes it a good candidate for treatment of diabetes, through its modification in order to produce and secrete insulin into the blood and/or increase muscle glucose uptake.

Mammalian skeletal muscles can regenerate following injury and this response is mediated by a specific type of stem cell, the satellite cell. We review here the three main phases of muscle regeneration, including i) the initial inflammatory response and the dual role of macrophages as both scavengers involved in the phagocytosis of necrotic debris and promoters of myogenic differentiation, ii) the activation and differentiation of satellite cells and iii) the growth and remodeling of the regenerated muscle tissue. Nerve activity is required to support the growth of regenerated myofibers and the specification of muscle fiber types, in particular the activation of the slow gene program. We discuss the regeneration process in two different settings. Chronic degenerative diseases, such as muscular dystrophies, are characterized by repeated cycles of segmental necrosis and regeneration involving scattered myofibers. In these conditions the regenerative capacity of satellite cells becomes exhausted with time and fibrosis prevails. Acute traumatic injuries, such as strain injuries common in sport medicine, cause the rupture of large myofiber bundles leading to muscle regeneration and formation of scar tissue and new myotendinous junctions at the level of the rupture. Mechanical loading is essential for muscle regeneration, therefore, following initial immobilization to avoid the risk of reruptures, early remobilization is required to induce correct growth and orientation of regenerated myofibers. Finally, we discuss the causes of age-dependent decline in muscle regeneration potential and the possibility of boosting regeneration in aging muscle and in muscular dystrophies.

Until recently, skeletal myoblasts (SkMBs) have been the most widely used cells in basic research and clinical trials of cellbased therapy for cardiac repair and regeneration. Although SkMB engraftment into the postinfarcted heart has been consistently found to improve cardiac contractile function, the underlying therapeutic mechanisms remain still a matter of controversy and debate. This is basically because SkMBs do not attain a cardiac-like phenotype once homed into the diseased heart nor they form a contractile tissue functionally coupled with the surrounding viable myocardium. This issue of concern has generated the idea that the cardiotropic action of SkMBs may depend on the release of paracrine factors. However, the paracrine hypothesis still remains ill-defined, particularly concerning the identification of the whole spectrum of cell-derived soluble factors and details on their cardiac effects. In this context, the possibility to genetically engineering SkMBs to potentate their paracrine attitudes appears particularly attractive and is actually raising great expectation. Aim of the present review is not to cover all the aspects of cell-based therapy with SkMBs, as this has been the object of previous exhaustive reviews in this field. Rather, we focused on novel aspects underlying the interactions between SkMBs and the host cardiac tissues which may be relevant for directing the future basic and applied research on SkMB transplantation for postischemic cardiac dysfunction.

The recent demonstration that, throughout evolution, many molecular mechanisms have been highly conserved is fundamental to the advancement of our knowledge on muscle development and regeneration. Research has provided new insights into genetic cascades governing early steps of embryonic myogenesis and the regeneration of adult muscle in normal and pathological conditions, thus revealing significant similarity of both processes. Here we provide a current view on genetic mechanisms underlying muscle regeneration with a special focus on regeneration processes that take place in diseased and aging human muscle. Through examples of Drosophila models of human muscular diseases, we discuss potential impact they might have on uncovering molecular bases and identifying new treatments of muscle disorders. Taking advantage of evolutionarily conserved aspects of muscle development and the relative ease by which molecular pathways can be uncovered and dissected in a simple animal model, the fruit fly, we provide a comprehensive analysis of muscle development in Drosophila. Importantly, identification of muscle stem cell like adult muscle precursors in Drosophila makes fruit fly an attractive model system for studying muscle stem cell biology and muscle regeneration. In support of this assumption, recent studies in our laboratory provide arguments that important insights into the biology of vertebrate muscle stem cells can be gained from genetic analysis in Drosophila.

Organisms of the phylum Echinodermata show some of the most impressive regenerative feats within the animal kingdom. Following injury or self-induced autotomy, species in this phylum can regenerate most tissues and organs, being the regeneration of the muscular systems one of the best studied. Even though echinoderms are closely related to chordates, they are little known in the biomedical field, and therefore their uses to study pharmacological effects on muscle formation and/or regeneration have been extremely limited. In order to rectify this lack of knowledge, we describe here the echinoderm muscular systems, particularly the somatic and visceral muscle components. In addition, we provide details of the processes that are known to take place during muscle regeneration, namely dedifferentiation, myogenesis and new muscle formation. Finally, we provide the available information on molecular and pharmacological studies that involve echinoderm muscle regeneration. We expect that by making this information accessible, researchers consider the use of echinoderms as model systems for pharmacological studies in muscle development and regeneration.

Stem cells other than satellite cells that can give rise to primary myoblasts, which are able to form additional new fibers postnatally, are present in the interstitial spaces of skeletal muscle. These cells are sorted into CD34+/45- (Sk-34) and CD34-/45- (Sk-DN) cell fractions, and they are wholly (>99%) negative for Pax7 at initial isolation. Colony-forming units of these cells typically include nonadherent type myogenic cells, while satellite cells are known to be adherent in cell culture. In addition, both Pax7- and Pax7+ cells are produced, depending on asymmetric cell division. A large number of myotubes are also formed in each colony, thus suggesting that putative Pax7+ satellite cells also present in each colony. Interestingly, interstitial myogenic cells show basal lamina formation at early stages of myogenesis in response to various types of stimulation in compensatory enlarged muscle, a property that satellite cells do not possess in the parent fiber basal lamina cylinder. Basal lamina formation and production of satellite cells are essential before muscle fiber establishment in vivo. It is therefore likely that myogenic cells in skeletal muscle can be divided into two populations: 1) basal laminaproducing myogenic cells; and 2) basal lamina-non-producing myogenic cells. The latter population may be Pax7+ satellite cells showing adherent capacity in cell culture, while the lamina-producing myogenic population derived from interstitial multipotent stem cells, which is predominant among Sk-34 and Sk-DN cells, plays a role in primary myoblast generation and shows non-adherent behavior in culture. Therefore, the physiological role of interstitial myogenic cells is as a source for new postnatal muscle fiber formation, and multinucleated muscle fibers (cells) are potentially formed clonally.

We focused our studies on the leech, Hirudo medicinalis. This invertebrate has a relative anatomical simplicity and is a reliable model for studying a variety of basic events, such as tissue repair, which has a striking similarity with vertebrate responses. Hirudo is also a good invertebrate model to test the actions of drugs and gene products, since the responses evoked by the different stimuli are clear and easily detectable due to their small size and anatomical simplicity. Here we review the use of this invertebrate model to investigate muscle regeneration and the role of hematopoietic stem cells in this process. Our recent data, summarized in this review, demonstrate that the injection of an appropriate combination of the matrigel biopolymer supplemented with Vascular Endothelial Growth Factor (VEGF) in the leech Hirudo medicinalis is a remarkably effective tool for isolating a specific population of hematopoietic/endothelial precursor cells, which in turn can differentiate in muscle cells. Thus leeches can be considered as a new emerging model for studying endothelial and hematopoietic precursors cells involved in muscle post-natal growth and regeneration processes.

Muscle degeneration and regeneration are two of the most evident pathological events characterizing muscular diseases and in particular muscular dystrophies. Muscular dystrophies are an heterogeneous group of hereditary diseases affecting both children and adults, and are characterized by muscle wasting and weakness. Until now at least 30 different genes have been associated with muscular dystrophies. They have been divided into several subgroups depending on the distribution of the muscle weakness. Thus, the histopathological markers of all these forms are dystrophic changes at the muscle biopsy characterized by fiber size variability, fibres necrosis, regeneration, inflammation and connective tissues deposition. As for now, no effective therapy is available for these diseases but new inside has now been expanded in regenerative therapy such as cell therapy and gene therapy. This review is focused on muscular dystrophies and new acknowledgments in regenerative therapy.

In little more than a decade, induced exon skipping as a therapy to treat Duchenne muscular dystrophy (DMD) has progressed from a concept tested in vitro, to pre-clinical evaluation in mouse and dog models, and recent completion of Phase I clinical trials in man. There is no longer any doubt that antisense oligomers can redirect dystrophin gene processing and by-pass protein truncating mutations after direct injection into muscle. Proof-of-concept has been demonstrated in human dystrophic muscle, with trials in Leiden and London showing that two different oligomer chemistries can restore the reading-frame in selected DMD patients by excising dystrophin exon 51. Systemic delivery of both oligomer types into DMD patients has commenced with promising results but it remains to be established if this therapy will have measurable clinical benefits. Targeted removal of exon 51 will only be directly applicable to about one in ten DMD individuals, and the immediate challenges include development of appropriate and effective delivery regimens, and extending spliceswitching therapies to other dystrophin gene lesions. The success of induced exon skipping has spawned a number of “fusion therapies”, including vector-mediated dystrophin exon skipping and ex vivo viral delivery of splice-switching antisense molecules into myogenic stem cells, followed by implantation, which may address long term oligomer delivery issues. This review summarizes the pivotal events leading to the completion of the first proof-of-concept trials and speculates on some of the scientific, ethical, regulatory and commercial challenges facing targeted exon skipping for the treatment of DMD.

Type 1 diabetes is characterised by the absence of circulating insulin due to the autoimmune destruction of ß-cells in the pancreas. Patients are traditionally treated with multiple daily injections of exogenous insulin analogues. However, although these therapies improve quality of life, they are associated with the risk of hypoglycemic episodes and do not prevent the development of debilitating secondary complications. For these reasons, there is increasing demand for new therapies and preventions. One approach is the use of viral or non-viral gene therapy to modify skeletal muscle to produce and secrete insulin into the circulation and/or to increase muscle glucose uptake. Skeletal muscle is a desirable target tissue for the treatment of diabetes not only for its central role in whole body metabolism and glucose homeostasis, but also for its accessibility and amenability to many potential gene therapy technologies. Here, we review the basic metabolic principles of skeletal muscle in the absorptive and post-absorptive states at rest and during exercise and discuss how these processes are affected in type 1 diabetes. Finally, current viral and non-viral strategies for modification of skeletal muscle and their application to the treatment of type 1 diabetes are also presented.