The functional role of Pax7-expressing satellite cells (SCs) in postnatal skeletal muscle development beyond weaning remains obscure. Therefore, the relevance of SCs during prepubertal growth, a period after weaning but prior to the onset of puberty, has not been examined. Here, we have characterized mouse skeletal muscle growth during prepuberty and found significant increases in myofiber cross-sectional area that correlated with SC-derived myonuclear number. Remarkably, genome-wide RNA-sequencing analysis established that post-weaning juvenile and early adolescent skeletal muscle have markedly different gene expression signatures. These distinctions are consistent with extensive skeletal muscle maturation during this essential, albeit brief, developmental phase. Indelible labeling of SCs with Pax7CreERT2/+; Rosa26nTnG/+ mice demonstrated SC-derived myonuclear contribution during prepuberty, with a substantial reduction at puberty onset. Prepubertal depletion of SCs in Pax7CreERT2/+; Rosa26DTA/+ mice reduced myofiber size and myonuclear number, and caused force generation deficits to a similar extent in both fast and slow-contracting muscles. Collectively, these data demonstrate SC-derived myonuclear accretion as a cellular mechanism that contributes to prepubertal hypertrophic skeletal muscle growth.
Identification of pro-regenerative approaches to improve tendon healing is critically important as the fibrotic healing response impairs physical function. In the present study we tested the hypothesis that S100a4 haploinsufficiency or inhibition of S100a4 signaling improves tendon function following acute injury and surgical repair in a murine model. We demonstrate that S100a4 drives fibrotic tendon healing primarily through a cell non-autonomous process, with S100a4 haploinsufficiency promoting regenerative tendon healing. Moreover, inhibition of S100a4 signaling via antagonism of its putative receptor, RAGE, also decreases scar formation. Mechanistically, S100a4 haploinsufficiency decreases myofibroblast and macrophage content at the site of injury, with both cell populations being key drivers of fibrotic progression. Moreover, S100a4-lineage cells become α-SMA+ myofibroblasts, via loss of S100a4 expression. Using a combination of genetic mouse models, small molecule inhibitors and in vitro studies we have defined S100a4 as a novel, promising therapeutic candidate to improve tendon function after acute injury.
Despite the requirement for Scleraxis-lineage (Scx Lin ) cells during tendon development, the function of Scx Lin cells during adult tendon repair, post-natal growth, and adult homeostasis have not been defined. Therefore, we crossed Scx-Cre and Diphtheria Toxin Receptor (DTR) mouse models to inducibly deplete Scx Lin cells prior to acute flexor tendon injury and repair (ScxLin DTR ) and hypothesized that ScxLin DTR mice would exhibit worse healing than wildtype littermates. Surprisingly, depletion of Scx Lin cells resulted in increased biomechanical properties without detriments to tendon gliding function at 28 days post-repair, indicative of regeneration. These improvements in biomechanical properties correspond to increased αSMA+ myofibroblasts in ScxLin DTR repairs, indicating that increased myofibroblast-mediated matrix remodeling may be responsible for accelerated recovery of tensile strength. Furthermore, we have defined the relative timing of Scx Lin cell localization relative to collagen deposition in bridging tissue of the repair and demonstrated a predominance of non-Scx Lin cells in the early bridging tissue, consistent with lack of bridging tissue disruption in ScxLin DTR repairs. Lastly, we utilized ScxLin DTR mice to assess effects of tendon cell depletion on post-natal tendon growth and adult tendon homeostasis. Collectively, these findings enhance our understanding of tendon cell localization, function, and fate during healing, growth, and homeostasis.3 Introduction:Despite the significant efforts toward improving tendon healing and regeneration, the specific cellular contributions during tendon healing have not been extensively characterized(1). While many studies have examined the potential of using various stem cell populations to promote healing, originating from both tendon intrinsic(2) and extrinsic sources(3), little focus has been directed toward defining the functions and therapeutic potential of tendon cells during tendon healing following an acute injury. Tendon cells are increasingly being recognized as a heterogenous population of cells where many, but not all, express the gene Scleraxis (Scx)(4-6).Understanding the localization and function of tendon cell subpopulations during healing could be instrumental in discovering why tendon heals using scar-mediated processes, resulting in poor patient outcomes, and could be used to develop pro-regenerative approaches to improve healing.Scx, a basic helix-loop-helix transcription factor, is currently the most well-characterized marker that the field possesses to study tendon (7). Scx has been utilized to examine tendon biology and development(8-10), healing and regeneration(4, 11-13), differentiation(14-17), and mechano-transduction (18,19). Functionally, Scx expression has previously been shown to drive matrix production and remodeling (11,20,21), epithelial-tomesenchymal transition(22), development of force-transmitting tendons(8), and effect focal adhesion morphology (19). Despite the effort to understand the functions of Scx as a transcr...
Flexor tendon injuries heal with excessive scar tissue that limits range of motion and increases incidence of re-rupture. The molecular mechanisms that govern tendon healing are not well defined. Both the canonical nuclear factor kappa B (NF-κB) and mitogen activated protein kinase (MAPK) pathways have been implicated in tendon healing. The gene NFKB1 (proteins p105/p50) is involved in both NF-κB and MAPK signaling cascades. In the present study, we tested the hypothesis that global NFKB1 deletion would increase activation of both NF-κB and MAPK through loss of signaling repressors, resulting in increased matrix deposition and altered biomechanical properties. As hypothesized, NFKB1 deletion increased activation of both NF-κB and MAPK signaling. While gliding function was not affected, NFKB1 deletion resulted in tendons that were significantly stiffer and trending towards increased strength by four weeks post-repair. NFKB1 deletion resulted in increased collagen deposition, increase macrophage recruitment, and increased presence of myofibroblasts. Furthermore, NFKB1 deletion increased expression of matrix-related genes ( Col1a1 , Col3a1 ), macrophage-associated genes ( Adgre1 , Ccl2 ), myofibroblast markers ( Acta2 ), and general inflammation ( Tnf ). Taken together, these data suggest that increased activation of NF-κB and MAPK via NFKB1 deletion enhance macrophage and myofibroblast content at the repair, driving increased collagen deposition and biomechanical properties.
Despite the requirement for Scleraxis-lineage (ScxLin) cells during tendon development, the function of ScxLin cells during adult tendon repair, post-natal growth, and adult homeostasis have not been defined. Therefore, we inducibly depleted ScxLin cells (ScxLinDTR) prior to tendon injury and repair surgery and hypothesized that ScxLinDTR mice would exhibit functionally deficient healing compared to wild-type littermates. Surprisingly, depletion of ScxLin cells resulted in increased biomechanical properties without impairments in gliding function at 28 days post-repair, indicative of regeneration. RNA sequencing of day 28 post-repair tendons highlighted differences in matrix-related genes, cell motility, cytoskeletal organization, and metabolism. We also utilized ScxLinDTR mice to define the effects on post-natal tendon growth and adult tendon homeostasis and discovered that adult ScxLin cell depletion resulted in altered tendon collagen fibril diameter, density, and dispersion. Collectively, these findings enhance our fundamental understanding of tendon cell localization, function, and fate during healing, growth, and homeostasis.
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