Jean Gautron

GROWTH FACTORS IN SKELETAL MUSCLE REGENERATION

Adult skeletal muscles are able to regenerate after injury. This process is due to the activation of quiescent muscle precursor cells, also called satellite cells, which proliferate and differentiate to form new myotubes. In this regeneration process, several growth factors which come from the muscle and/or from the motor nerve and inflammatory cells have been shown to play key roles. However, most of our knowledge comes from in vitro studies, where, during myogenesis, proliferation of satellite cells is regulated by FGFs, TGF beta s, PDGF, IGF-I and II, while differentiation appears to be promoted mainly by IGFs. During regeneration in vivo, most of these factors have been shown to operate and interact. Other factors also appear to condition the regeneration process, such as LIF, which acts predominantly as a proliferative factor; and HARP/PTN/HB-GAM and other neurotrophic factors, which may be necessary for the formation of new neuromuscular junctions. TGF beta has a major influence on the reorganisation of the extracellular matrix. This review presents a critical summary of the known effects of growth factors on skeletal muscle regeneration.

Differential myogenicity of satellite cells isolated from extensor digitorum longus (EDL) and soleus rat muscles revealed in vitro

Abstract Following muscle damage, fast- and slow-contracting fibers regenerate, owing to the activation of their satellite cells. In rats, crush-induced regeneration of extensor digitorum longus (EDL, a fast muscle) and soleus (a slow muscle) present different characteristics, suggesting that intrinsic differences exist among their satellite cells. An in vitro comparative study of the proliferation and differentiation capacities of satellite cells isolated from these muscles is presented there. We observed several differences between soleus and EDL satellite cell cultures plated at high density on gelatin-coated dishes. Soleus satellite cells proliferated more actively and fused into myotubes less efficiently than EDL cells. The rate of muscular creatine kinase enzyme appeared slightly lower in soleus than in EDL cultures at day 11 after plating, when many myotubes were formed, although the levels of muscular creatine kinase mRNA were similar in both cultures. In addition, soleus cultures expressed higher levels of MyoD and myogenin mRNA and of MyoD protein than EDL satellite cell cultures at day 12. A clonal analysis was also carried out on both cell populations in order to determine if distinct lineage features could be detected among satellite cells derived from EDL and soleus muscles. When plated on gelatin at clonal density, cells from both muscles yielded clones within 2 weeks, which stemmed from 3–15 mitotic cycles and were classified into three classes according to their sizes. Myotubes resulting from spontaneous fusion of cells from the progeny of one single cell were seen regardless of the clone size in the standard culture medium we used. The proportion of clones showing myotubes in each class depended on the muscle origin of the cells and was greater in EDL- than in soleus-cell cultures. In addition, soleus cells were shown to improve their differentiation capacity upon changes in the culture condition. Indeed, the proportions of clones showing myotubes, or of cells fusing into myotubes in clones, were increased by treatments with a myotube-conditioned medium, with phorbol ester, and by growth on extra-cellular matrix components (Matrigel). These results, showing differences among satellite cells from fast and slow muscles, might be of importance to muscle repair after trauma and in pathological situations.

A substituted dextran enhances muscle fiber survival and regeneration in ischemic and denervated rat EDL muscle

Ischemia and denervation of EDL muscle of adult rat induce a large central zone of degeneration surrounded by a thin zone of peripheral surviving muscle fibers. Muscle regeneration is a complex phenomenon in which many agents interact, such as growth factors and heparan sulfate components of the extracellular matrix. We have shown that synthetic polymers, called RGTA (as regenerating agents), which imitate the heparan sulfates, are able to stimulate tissue repair when applied at the site of injury. In crushed muscles, RGTA were found to accelerate both regeneration and reinnervation. In vitro, RGTA act as protectors and potentiators of various heparin binding growth factors (HBGF). It was postulated that in vivo their tissue repair properties were due in part to an increase of bioavailability of endogenously released HBGF. In the present work, we show that ischemic and denervated EDL muscle treated by a unique injection of RGTA differs from the control after 1 wk in several aspects: 1) the epimysial postinflammatory reaction is inhibited and the area of fibrotic tissue among fibers is reduced; 2) the peripheral zone, as measured by the number of intact muscle fibers, was increased by more than twofold; and 3) In the central zone, RGTA enhances the regeneration of the muscle fibers as well as muscle revascularization. These results suggest that RGTA both protects muscle fibers from degeneration and preserves the differentiated state of the surviving fibers. For the first time it is demonstrated that a functionalized polymeric compound can prevent some of the damage resulting from muscle ischemia. RGTA may therefore open a new therapeutic approach for muscle fibrosis and other postischemic muscle pathologies.—Desgranges, P., Barbaud, C., Caruelle, J.‐P., Barritaoult, D., Gautron, J. A substituted dextran enhances muscle fiber survival and regeneration in ischemic and denervated rat EDL muscle. FASEB J. 13, 761–766 (1999)

Fast and slow rat muscles degenerate and regenerate differently after whole crush injury.

SummaryThe whole-crush injured rat skeletal muscle was used as a model to explore the regenerating potentialities of fast and slow muscles. Laminin was chosen to follow changes in basal lamina and desmin to visualize new muscular elements; they were revealed by immunofluorescence on cryostat sections of either fast (extensor digitorum longus) or slow (soleus) regenerating muscle. Soleus myolysis was rapid, extensive and heterogeneous. Basal laminae were nearly destroyed. In contrast, extensor digitorum longus maintained its basal lamina framework during myolysis. Soleus reconstruction began early, following the pattern of remaining basal laminae as closely as possible, but regeneration stagnated from day 16 and the regenerated muscle was fibrotic. In extensor digitorum longus, reconstruction progressed slower than in soleus, but regularly from the periphery toward the centre of the muscle. The regenerated extensor digitorum longus showed a quasi-normal structure from day 16. At the end of the process, the elimination of old basal lamina was completed in extensor digitorum longus, but was not achieved in soleus. We propose that the old basal lamina should help the initiation of reconstruction. This new model also underlines the importance of the turnover of basal laminae in muscular regeneration, and will be useful to understand the background of the different regenerative response of both muscles.

Proliferating satellite cells express acidic fibroblast growth factor duringin vitro myogenesis

Recent in vitro studies have indicated that the proliferation of satellite cells, which are involved in muscular regeneration in vivo, is stimulated by exogenous addition of fibroblast growth factor (FGF). We present evidence that satellite cell cultures produce acidic, but not basic FGF. Acidic or basic FGF content was measured by enzyme immunoassay on cellular extracts after partial purification by heparin-Sepharose chromatography. During maximal cell proliferation, the level of acidic fibroblast growth factor (aFGF) was increased over fivefold from the values obtained before plating. aFGF content drastically dropped at the postmitotic stage to almost the threshold of detection, and remained weak as differentiation was completed. The immunolocalization of aFGF using highly purified anti-aFGF antibodies confirmed these results and indicated that aFGF was cytoplasma- or membrane-associated. Our work suggests that an endogenous production of aFGF by satellite cells may trigger cell proliferation by an intra- or autocrine mechanism, and therefore play an important role in muscular regeneration.

Immunolocalization of Fibronectin and other Macromolecules of the Intercellular Matrix in the Striated Muscle Fiber of the Adult Rat

The distribution pattern of fibronectin, laminin and type IV collagen in the striated muscle fiber of adult rat was studied using immunofluorescence staining and electron microscopy. The results indicate that fibronectin as well as laminin and type IV collagen precisely delineate each muscle fiber. Fibronectin is present on the sarcolemma extending from the cell membrane to the intercellular collagen fibers beyond the basal lamina lucida externa. This suggests a role for fibronectin in making contact between the cell membrane and the intercellular matrix.

Influence of a dextran derivative on myosin heavy chain expression during rat skeletal muscle regeneration

We recently described dextran derivatives (RGTA) which stimulate tissue repair in several in vivo models. One of them, RGTA11, has been shown to accelerate crush-induced regeneration and reinnervation of rat EDL and Soleus muscles. In this study we wanted to know if RGTA11 alters the pattern of myosin heavy chain expression during regeneration. In both EDL and Soleus muscles, RGTA11, injected at the moment of the crush, was found to accelerate the shift from neonatal to adult myosin heavy chain isoforms within 2 weeks. The proportion of slow fibers increased considerably, especially in the Soleus where RGTA11 induced a precocious and permanent expression of slow myosin isoform, thus confirming that a more efficient innervation had occurred in the presence of RGTA11. These results illustrate the interesting potential pharmacological use of such dextran derivatives in neuromuscular disease.

Protein kinase C activity and phorbol ester binding to rat myogenic cells during growth and differentiation.

Phorbol esters have been reported to induce opposite responses in fetal myoblasts and in satellite cells isolated from adult skeletal muscles. We examined the possibility that different levels of protein kinase C (PKC) activity and different phorbol ester binding characteristics account for these responses. For this purpose, the subcellular distributions of PKC were compared in primary cultures of myogenic cells from fetal and adult rat muscles and in the L6 cell line. Cells were used at the proliferative stage or after differentiation into myotubes. Binding of phorbol dibutyrate (PDBu) was assayed. In all three cell types, the levels of PKC specific activity were comparable at the proliferating and the differentiated stages, and partial translocation of PKC activity from the membrane to the cytosolic compartment was observed after differentiation into myotubes. PDBu binding, which had a Kd of 6 to 13 nM in proliferative cells, rose to between 30 and 52 nM in myotubes. Simultaneously, a small increase was observed in the total number of PDBu binding sites. These results suggest that the role of PKC might change with the stage of differentiation. They also imply that the difference described by others between the sensitivity to phorbol esters of fetal myoblasts and satellite cells is not connected with the phorbol ester receptor (i.e., PKC), but might be caused by events subsequent to PKC activation.

Biosynthesis of laminin and fibronectin by rat satellite cells during myogenesis in vitro

The biosynthesis of fibronectin and laminin was studied in satellite cells cultured from adult rat muscles before (day 4) and after fusion and formation of myotubes (day 14) using (35S) methionine as a tracer. The kinetics of incorporation into immunoprecipitable fibronectin and laminin were recorded at intervals from 1.5 to 24 hours of incubation with the tracer from the cells, the pericellular matrix and the culture medium. The rate of synthesis of fibronectin and laminin expressed as dpm/microgram DNA were constant from the mononucleated cell to the myotube state. Both glycoproteins were detected in the cells and in the pericellular matrix. When the results were expressed as the percentage of incorporation into total protein, major changes could be observed in the early phase of the kinetic studies in the cells and the pericellular matrix. Both showed an increase from the mononucleated myoblast to myotube, suggesting that an increasing fraction of total protein biosynthesis is directed towards these two extracellular matrix glycoproteins. At the same time, there was a decrease in the secretion into the medium of freshly synthesized radiolabeled fibronectin and laminin. Our results confirm the synthesis of varying ratios of both extracellular matrix macromolecules by undifferentiated mononucleated myogenic cells as well as myotubes.

Activity, localization and molecular form of acetylcholinesterase in the male accessory glands of metamorphosing Tenebrio molitor L.

Abstract Despite their mesodermal origin, male accessory glands of the reproductive system of Tenebrio molitor exhibited marked acetylcholinesterase (AChE) activity throughout metamorphosis (i.e. from the newly ecdysed pupa to the mature adult stages). This activity was true AChE activity, since it was inhibited by high concentrations of substrate and by 10 −5 M BW284C51 dibromide, but not by iso-OMPA. The AChE activity was exclusively localized in the muscular coat of the glands and not in the epithelial cells which secreted the spermatophore proteins. The enzyme extracted from the glands with 1% Triton X-100 and 1 M NaCl sedimented as a single peak in a sucrose density gradient, with a sedimentation coefficient of 5.4 S . This single AChE sedimentation peak was not altered after treatment with DTT or β-mercaptoethanol. AChE activity per gland increased throughout metamorphosis. Activity per mg of wet weight of glands rose at the mid pupal stage. It decreased at adult ecdysis to the level of the newly edysed pupa. AChE activity per mg of protein exhibited a peak in the young pupa.