TY - JOUR
T1 - Increased microenvironment stiffness in damaged myofibers promotes myogenic progenitor cell proliferation
AU - Trensz, Frédéric
AU - Lucien, Fabrice
AU - Couture, Vanessa
AU - Söllrald, Thomas
AU - Drouin, Geneviève
AU - Rouleau, André Jean
AU - Grandbois, Michel
AU - Lacraz, Gregory
AU - Grenier, Guillaume
N1 - Funding Information:
We are grateful to Dr. Anthony Scimè for critically reading the manuscript, Dr. Michael Rudnicki for providing the Myf5-nLacZ mice, Gene Bourgeau for proofreading the manuscript, Marc-Antoine Lauzon for the statistical analyses, and Jennifer Downey for editing the figures. This work was supported by grants from the Canadian Institutes of Health Research (CIHR), the Canada Foundation for Innovation (CFI), and the Fonds de Recherche du Québec - Santé (FRQS). FT received a scholarship from FORMSAV and the Fondation pour la Recherche et l’Enseignement en Orthopédie de Sherbrooke (FREOS). GL received a post-doctoral scholarship from CIHR. GG received a New Investigator Award from FRQS.
Publisher Copyright:
© 2015 Trensz et al.
PY - 2015/2/17
Y1 - 2015/2/17
N2 - Background: The stiffness of the myogenic stem cell microenvironment markedly influences the ability to regenerate tissue. We studied the effect of damaged myofibers on myogenic progenitor cell (MPC) proliferation and determined whether the structural integrity of the microenvironment contributes to phenotypic changes. Methods: Individual myofibers were isolated and cultured for 6 days. During this period, the cytoskeleton of myofibers and transcription factors regulating MPC differentiation were characterized by immunostaining. Atomic Force Microscopy (AFM) was performed to measure stiffness of cultured myofibers. Healthy and damaged myofibers, and their associated MPCs, were studied in skeletal muscle from dystrophic and tenotomy mouse models. MPCs were cultured on stiffness-tunable substrates, and their phenotypes were assessed by immunostaining of myogenic transcription factors. Results: We showed that individual myofibers tend to shrink or collapse when cultured ex vivo starting from day 1 and that this is associated with a marked increase in the number of proliferative MPCs (Pax7+MyoD+). The myofibers collapsed due to a loss of viability as shown by Evans blue dye uptake and the disorganization of their cytoskeletons. Interestingly, collapsed myofibers in mdx skeletal muscles were similar to damaged myofibers in that they lose their viability, have a disorganized cytoskeleton (actin and α-actinin), and display local MPC (MyoD+) proliferation at their periphery. In a tenotomy model that causes loss of muscle tension, the cytoskeletal disorganization of myofibers also correlated with the activation/proliferation of MPCs. A deeper analysis of collapsed myofibers revealed that they produce trophic factors that influence MPC proliferation. In addition, collapsed myofibers expressed several genes related to the basal lamina. Immunostaining revealed the presence of fibronectin in the basal lamina and the cytoplasm of damaged myofibers. Lastly, using atomic force microscopy (AFM), we showed that collapsed myofibers exhibit greater stiffness than intact myofibers. Growing MPCs on a 2-kPa polyacrylamide-based substrate, exempt of additional microenvironmental cues, recapitulated proliferation and reduced spontaneous differentiation compared to growth on a 0.5-kPa substrate. Conclusions: Our results support the notion that collapsed or damaged myofibers increase the structural stiffness of the satellite cell microenvironment, which in addition to other cues such as trophic factors and changes in extracellular matrix composition, promotes the proliferation and maintenance of MPCs, required for myofiber repair.
AB - Background: The stiffness of the myogenic stem cell microenvironment markedly influences the ability to regenerate tissue. We studied the effect of damaged myofibers on myogenic progenitor cell (MPC) proliferation and determined whether the structural integrity of the microenvironment contributes to phenotypic changes. Methods: Individual myofibers were isolated and cultured for 6 days. During this period, the cytoskeleton of myofibers and transcription factors regulating MPC differentiation were characterized by immunostaining. Atomic Force Microscopy (AFM) was performed to measure stiffness of cultured myofibers. Healthy and damaged myofibers, and their associated MPCs, were studied in skeletal muscle from dystrophic and tenotomy mouse models. MPCs were cultured on stiffness-tunable substrates, and their phenotypes were assessed by immunostaining of myogenic transcription factors. Results: We showed that individual myofibers tend to shrink or collapse when cultured ex vivo starting from day 1 and that this is associated with a marked increase in the number of proliferative MPCs (Pax7+MyoD+). The myofibers collapsed due to a loss of viability as shown by Evans blue dye uptake and the disorganization of their cytoskeletons. Interestingly, collapsed myofibers in mdx skeletal muscles were similar to damaged myofibers in that they lose their viability, have a disorganized cytoskeleton (actin and α-actinin), and display local MPC (MyoD+) proliferation at their periphery. In a tenotomy model that causes loss of muscle tension, the cytoskeletal disorganization of myofibers also correlated with the activation/proliferation of MPCs. A deeper analysis of collapsed myofibers revealed that they produce trophic factors that influence MPC proliferation. In addition, collapsed myofibers expressed several genes related to the basal lamina. Immunostaining revealed the presence of fibronectin in the basal lamina and the cytoplasm of damaged myofibers. Lastly, using atomic force microscopy (AFM), we showed that collapsed myofibers exhibit greater stiffness than intact myofibers. Growing MPCs on a 2-kPa polyacrylamide-based substrate, exempt of additional microenvironmental cues, recapitulated proliferation and reduced spontaneous differentiation compared to growth on a 0.5-kPa substrate. Conclusions: Our results support the notion that collapsed or damaged myofibers increase the structural stiffness of the satellite cell microenvironment, which in addition to other cues such as trophic factors and changes in extracellular matrix composition, promotes the proliferation and maintenance of MPCs, required for myofiber repair.
KW - Microenvironment
KW - Myofiber
KW - Myogenic progenitor cells
KW - Stiffness
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U2 - 10.1186/s13395-015-0030-1
DO - 10.1186/s13395-015-0030-1
M3 - Article
AN - SCOPUS:84924603008
SN - 2044-5040
VL - 5
JO - Skeletal Muscle
JF - Skeletal Muscle
IS - 1
M1 - 5
ER -