Abstract
BackgroundThe 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.MethodsIndividual 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.ResultsWe 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0030-1) contains supplementary material, which is available to authorized users.
Highlights
The stiffness of the myogenic stem cell microenvironment markedly influences the ability to regenerate tissue
We observed a dramatic increase in X-gal staining in collapsed myofibers, which was associated with Myf5+ myogenic progenitor cells (MPCs), after 6 days (Figure 1A)
Unlike intact myofibers, collapsed myofibers displayed a marked increase in cellular budding on their periphery after 6 days in culture, which is a hallmark of MPC proliferation
Summary
The stiffness of the myogenic stem cell microenvironment markedly influences the ability to regenerate tissue. Satellite cells are sequestered between the myofiber plasma membrane and the basal lamina [2], and their function is tightly regulated by numerous biochemical and cellular signals in their microenvironment or niche. These consist of subjacent myofiber anchoring; the extracellular matrix (ECM) that sequesters various growth factors; the neighboring cells from the stroma, including immune cells; and the environment of the circulatory system (for a recent review, see [3]). First considered as a structural tissue that maintains the integrity of muscle fibers during contraction, it is well established that the ECM plays a major role in the function of myogenic stem cell and muscle regeneration [11,12,13]. Excessive accumulation of ECM (fibrosis) and conditions in which muscle passive stiffness is heightened, such as during myopathy and aging, can impair satellite cell function and muscle regeneration [14,15,16,17]
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