Blood borne macrophages are essential for the triggering of muscle regeneration following muscle transplant
Introduction
Efficient muscle regeneration requires the recruitment of satellite cells [1]. With the possibility of expanding a population of satellite cells in vitro, able to fuse and form myotubes, came the idea that their transplantation might constitute a strategy for rebuilding a new muscle after an injury. However, this has not succeeded in providing a solution for treatment of inherited myopathies and a greater understanding of the mechanism of fibre formation during in vivo muscle repair is clearly required [2]. Indeed, in addition to satellite cell recruitment, the local environment also plays an important role 3, 4, 5, 6. For example, integration of grafted satellite cells by the host muscle is controlled by multiple factors exerting both inhibitory and enhancer activities 5, 7and whose origins are still open to debate. Furthermore, the neuromuscular system in fact comprises a considerable number of different cell types other than the muscle fibre itself and its surrounding satellite cells, namely, the motoneuron with its terminal Schwann cell, fibroblasts and resident macrophages. In addition, the abundant vascularisation of this tissue allows the arrival of blood borne cells, when required in the case of a lesion, for example, to such an extent that they have been proposed as cellular vectors for targeting therapeutic agents [8]. To fully understand the process of regeneration, the role of these blood borne cells must therefore be taken into consideration as well as that of those classically associated with the muscle tissue, and if we are finally to advance in this domain, it is essential to identify clearly not only the protagonists involved, but also the key events triggering and controlling the regeneration process in vivo.
We have previously developed a model of post-graft skeletal muscle regeneration 9, 10. This model is based on the use, as a muscle donor, of a transgenic mouse carrying the lacZ reporter gene under the control of a truncated human desmin promoter [11]. This truncated promoter is active during embryonic development and in the adult, fortuitously, uniquely during muscle regeneration 9, 12. Transgene activity allows the visualisation of replicating and differentiating myoblasts as well as myotubes 9, 11, 12. In this mouse, muscle regeneration, indicated by activation of the reporter gene, is evident 24–36 h after lesion. However, when muscle explants from this transgenic mouse are grafted into the muscle of a recipient non-transgenic mouse, regeneration occurs only after a delay of 3–4 days [9]. Because the transplant comprises destroyed muscle fibres transected at both ends, this, in fact, amounts to a lesion removed and disconnected from its original environment. The delay in activation of the regeneration program in the transplant could perhaps be explained by arguing that it corresponds to the time necessary to acquire an efficient vascularisation [13]. If this were indeed the case, the regeneration, rather than being governed by intrinsic muscle signals, would be triggered by blood borne factors.
Basically, a muscle lesion, whether accidental or as the result of a genetic defect, triggers a complex response which can be broken down into three phases. First, the lesion triggers a local inflammatory reaction. Second, each site of damage is subsequently infiltrated by blood borne cells. In exercise induced muscle injury, the predominant inflammatory cell type is the macrophage [14]. However, when acute muscle fibre necrosis is caused by a chemical agent such as bupivacaine, the predominant inflammatory cells are polymorphonuclear cells and macrophages, with, in addition, a small number of T and B lymphocytes [15]. This invasion of cells from the blood to the tissue begins immediately and reaches a maximum between 24 and 48 h 15, 16. Third, if the tissue plasticity allows, neighbouring muscle satellite cells, initially quiescent, are activated, proliferate, differentiate and then fuse giving a new replacement muscle fibre 10, 17. Although the first role of the macrophage is to phagocytose the debris at the site of damage, in vitro studies suggest that, in addition to their scavenger role, they could be implicated in muscle fibre regeneration by exerting mitogenic [18]and chemotactic effects on muscle precursor cells 19, 20.
The present study was undertaken to investigate whether the macrophages are simply recruited in the course of the necrosis-regeneration process, or whether they play a key role, `catalysing' the chain of events leading to regeneration. To this end, the relationship of this process to the homing of blood borne macrophages, was studied in vivo using the model of post-graft skeletal muscle regeneration previously described 9, 10. Muscle fragments from this transgenic mouse were implanted into non-transgenic host mice and the time course of activation of muscle regeneration was followed after having boosted or inhibited blood borne macrophage recruitment in the host mouse. We reveal here that homing of blood borne macrophages to the implant is essential for the triggering of muscle regeneration.
Section snippets
Animals
Adult transgenic pHuDes-nls-LacZ mice carrying the lacZ reporter gene under the control of a truncated human desmin promoter (1 kb cis-regulatory sequences), were used as muscle graft donors. In the muscle cell, the appearance of a blue nuclear product (as the β-galactosidase is targeted to the nucleus) in the presence of the X-gal substrate (Sigma) for the enzyme was indicative of early events of skeletal muscle regeneration 9, 10.
Transplantation surgery
After sacrifice by cervical dislocation, pectoralis abdominalis
Spacio-temporal relationship between activation of the skeletal muscle regeneration and monocyte-macrophage infiltration into transplants
Muscle fragments from the donor transgenic mouse were implanted into the tibialis anterior of recipient mice and subsequently examined at several time points (2, 3, 4 and 7 days) post-implantation, for the presence of β-galactosidase activity and monocyte-macrophage infiltration into the transplant. The transgene being under the control of a desmin promoter, its expression is an early marker of skeletal muscle regeneration labelling replicative, differentiated satellite cells as well as
Discussion
Although the molecular events involved in myotube formation in vitro are becoming increasingly well documented, a certain confusion surrounds the mechanisms of muscle repair in vivo, something which has doubtless contributed to the failure of muscle precursor grafts to provide a solution for treating inherited myopathies. Indeed, the term `muscle repair' encompasses a wide range of mechanisms from the simple reformation, or sealing, of damaged fibres, to their replacement, when irreparably
Acknowledgements
This work was supported by the University of Nantes, the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, and the Association Française contre les Myopathies.
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