Elsevier

Experimental Cell Research

Volume 283, Issue 2, 15 February 2003, Pages 196-205
Experimental Cell Research

Original article
Mitotic activity of rat muscle satellite cells in response to serum stimulation: relation with cellular metabolism

https://doi.org/10.1016/S0014-4827(02)00030-7Get rights and content

Abstract

Cellular and molecular adaptations of satellite cells isolated from rat hindlimb muscles (n = 10) were investigated in response to serum stimulation. Flow cytometry analysis of the amounts of DNA and RNA indicated that 97.7 ± 0.7% of satellite cells were in G0 at the end of the isolation procedure, whereas 93.2 ± 2.0% of cells were cycling after serum exposure. The length of cell division was 34.0 ± 2.8 h. Myoblast proliferation was asynchronous, suggesting the existence of heterogeneous cell populations in skeletal muscle. Myoblast proliferation was also accompanied by a significant increase in c-met expression, and major adaptations of energetic and proteolytic metabolisms, including an increase in the relative contribution of glycolytic metabolism for energy production, an increase in proteasome and matrix metalloproteinases 2 and 9 activities, and a decrease in plasminogen activator activities. Our data suggest that, along with molecular adaptations leading to cell cycle activation itself, adaptations in energetic and proteolytic metabolisms are crucial events involved in satellite cell activation and myoblast proliferation.

Introduction

Satellite cells are a population of myogenic precursor cells that reside between the plasmalemma and basal lamina of mature muscle fibers [1]. In adult skeletal muscle, satellite cells constitute the pool of myogenic precursor cells involved in muscle regeneration and the maintenance of muscle mass. Activation of satellite cells occurs in response to multiple stimuli, such as muscle damage, compensatory muscular hypertrophy, exercise, or hereditary muscular disorders (reviewed in [2]). The descendants of activated satellite cells, called myoblasts, proliferate by multiple rounds of cell division. Myoblasts can then fuse together and with preexisting muscle fibers to form differentiated myofibers [3], [4] and/or return to quiescence and contribute to the self-renewal of satellite cell population [4], [5], [6].

Several growth factors, such as hepatocyte growth factor (HGF), fibroblast growth factors (FGFs), insulin-like growth factors I and II, leukemia inhibitory factor, and platelet-derived growth factor, have been implicated in stimulating satellite cell proliferation (see [2] for review). The combinatorial action of these growth factors orchestrates profound changes in the gene expression profile of satellite cells, which ultimately leads to the formation of differentiated myofibers. There are evidences to suggest that myoblast proliferation can become “rate limiting” in certain situations including muscle senescence or muscular dystrophies [7], [8], [9]. In such circumstances, the proliferative capacity of satellite cells could be dramatically reduced as a consequence of the number of cell division already expended in vivo, as well as a consequence of a decrease in growth factor biodisponibility or a decrease in the responsiveness of satellite cells to mitogenic signals. In this context, a detailed knowledge of the proliferative characteristics of satellite cells would be of critical importance in understanding physiological and pathological events related to skeletal muscle biology.

Several growth factors are involved in stimulating satellite cell proliferation, but only HGF and FGF appear capable of affecting initial activation of satellite cells [10], [11], [12], [13]. HGF is a multifunctional cytokine expressed in skeletal muscle [14], which activates satellite cells through interaction with its transmembrane tyrosine kinase receptor c-met [15], [16]. Regulation of c-met expression must be therefore critical in controlling satellite cell activation and myoblast proliferation.

Among the molecular events regulating satellite cell activation and myoblast proliferation, adaptations in the expression of proteins involved in metabolic pathways must be considered. First, several reports suggest that ATP supply by glycolytic and mitochondrial oxidative metabolisms is necessary for the execution of regulatory and biosynthesis events that occur during myogenesis [17], [18], [19]. Second, cell cycle regulation is highly dependent on the proteasomal proteolysis of regulatory proteins such as cyclins ([20]; see [21] for review), suggesting that adaptations in proteasome activity could be important for the regulation of satellite cell activation and myoblast proliferation. Finally, proteolysis of extracellular matrix (ECM) proteins is necessary in vivo for the expansion of proliferating cells [22], [23] and the activation/liberation of growth factors sequestrated in the ECM [24].

Despite an increasing number of studies focusing on satellite cells, little information on energetic and proteolytic metabolism adaptations of rat muscle satellite cells upon serum stimulation are available. To determine the physiological relevance of the above factors in satellite cell biology, the present study provides a characterization of satellite cells and myoblasts with special reference to the relationship between mitotic activity and cellular metabolism.

Section snippets

Isolation of primary muscle cells

The animal protocol was approved by the Ministère de l’Agriculture et de la Forêt. Six-month-old Sprague–Dawley male rats (304 ± 10 g; n = 10) were euthanized with an intraperitoneal injection of Nesdonal. Gastrocnemius and quadriceps muscles were aseptically removed and carefully cleaned of blood vessels, fat, and connective tissues. Muscles were minced in PBS, weighed, and washed extensively in PBS. Primary muscle cells were isolated by pronase digestion ([25], [26], with some modifications).

Efficiency and quality of primary cultures

Before proceeding with the experiment to determine the mitotic activity of proliferating myoblasts, it was essential first to ensure the myogenicity of the cells isolated from gastrocnemius and quadriceps muscles. Under our conditions, cell yield averaged 4.8 × 105 ± 0.3 × 105 cells per gram of tissue (n = 10). Flow cytometry analysis of desmin expression on the overall cell population at the myoblast stage, indicated that we were able to routinely harvest 80 ± 5% (n = 10) of desmin-positive

Discussion

Satellite cells are a population of myogenic precursor cells that play major roles in muscle growth, muscle regeneration, and the regulation of muscle mass. In this study, we have evaluated the kinetic behavior of proliferating myoblasts and tentatively determined some of the biochemical events that could be essential for satellite cell activation and myoblast proliferation.

Isolation of myogenic precursor cells by pronase digestion is commonly used to release monucleated cells from skeletal

Acknowledgements

We thank Professor David A. Hood for the kind donation of L6E9 cells and Dr. Georges Stépien for the provision of C2C12 cells. We also thank Dr. Isabelle Tchou for her helpful assistance in immunolabeling experiments and Dr. Yann Bassaglia for his helpful comments in satellite cell extraction procedure. Thanks are also due to Léonard Féasson, Anne-Cécile Durieux, and Stéphanie Duguez for their helpful comments. A.E.B. is a recipient of a doctoral fellowship from the Ministère de l’Education

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