Myogenic Lineage Progression Research Articles

3D organization of enhancers in MuSCs.

Skeletal muscle stem cells (MuSCs), also known as satellite cells, are essential for muscle growth and injury induced regeneration. In healthy adult muscle, MuSCs remain in a quiescent state located in a specialized niche beneath the basal lamina. Upon injury, these dormant MuSCs can quickly activate to re-enter the cell cycle and differentiate into new myofibers, while a subset undergoes self-renewal and returns to quiescence to restore the stem cell pool. The myogenic lineage progression is intricately controlled by complex intrinsic and extrinsic cues and coupled with dynamic transcriptional programs. In transcriptional regulation, enhancers are key regulatory elements controlling spatiotemporal gene expression through physical contacting promoters of target genes. The three-dimensional (3D) chromatin architecture is known to orchestrate the establishment of proper enhancer-promoterinteractions throughout development and aging. However, studies dissecting the 3D organization of enhancers in MuSCs are just emerging. Here, we provide an overview of the general properties of enhancers and newly developed methods for assessing their activity. In particular, we summarize recent discoveries regarding the 3D rewiring of enhancers during MuSC specification, lineage progression as well as aging.

Inhibitory SMAD6 interferes with BMP-dependent generation of muscle progenitor cells and perturbs proximodistal pattern of murine limb muscles.

The mechanism of pattern formation during limb muscle development remains poorly understood. The canonical view holds that naïve limb muscle progenitor cells (MPCs) invade a pre-established pattern of muscle connective tissue, thereby forming individual muscles. Here, we show that early murine embryonic limb MPCs highly accumulate pSMAD1/5/9, demonstrating active signaling of bone morphogenetic proteins (BMP) in these cells. Overexpression of inhibitory human SMAD6 (huSMAD6) in limb MPCs abrogated BMP signaling, impaired their migration and proliferation, and accelerated myogenic lineage progression. Fewer primary myofibers developed, causing an aberrant proximodistal muscle pattern. Patterning was not disturbed when huSMAD6 was overexpressed in differentiated muscle, implying that the proximodistal muscle pattern depends on BMP-mediated expansion of MPCs before their differentiation. We show that limb MPCs differentially express Hox genes, and Hox-expressing MPCs displayed active BMP signaling. huSMAD6 overexpression caused loss of HOXA11 in early limb MPCs. In conclusion, our data show that BMP signaling controls expansion of embryonic limb MPCs as a prerequisite for establishing the proximodistal muscle pattern, a process that involves expression of Hox genes.

Effect of Mechanical Loading of Senescent Myoblasts on Their Myogenic Lineage Progression and Survival

During aging, muscle cell apoptosis increases and myogenesis gradually declines. The impaired myogenic and survival potential of the aged skeletal muscle can be ameliorated by its mechanical loading. However, the molecular responses of aged muscle cells to mechanical loading remain unclear. This study examined the effect of mechanical loading of aged, proliferating, and differentiated myoblasts on the gene expression and signaling responses associated with their myogenic lineage progression and survival. Control and aged C2C12 cells were cultured on elastic membranes and underwent passive stretching for 12 h at a low frequency (0.25 Hz) and different elongations, varying the strain on days 0 and 10 of myoblast differentiation. Activation of ERK1/2 and Akt, and the expression of focal adhesion kinase (FAK) and key myogenic regulatory factors (MRFs), MyoD and Myogenin, were determined by immunoblotting of the cell lysates derived from stretched and non-stretched myoblasts. Changes in the expression levels of the MRFs, muscle growth, atrophy, and pro-apoptotic factors in response to mechanical loading of the aged and control cells were quantified by real-time qRT-PCR. Mechanical stretching applied on myoblasts resulted in the upregulation of FAK both in proliferating (day 0) and differentiated (day 10) cells, as well as in increased phosphorylation of ERK1/2 in both control and aged cells. Moreover, Akt activation and the expression of early differentiation factor MyoD increased significantly after stretching only in the control myoblasts, while the late differentiation factor Myogenin was upregulated in both the control and aged myoblasts. At the transcriptional level, mechanical loading of the proliferating myoblasts led to an increased expression of IGF-1 isoforms and MRFs, and to downregulation of muscle atrophy factors mainly in control cells, as well as in the upregulation of pro-apoptotic factors both in control and aged cells. In differentiated cells, mechanical loading resulted in an increased expression of the IGF-1Ea isoform and Myogenin, and in the downregulation of atrophy and pro-apoptotic factors in both the control and aged cells. This study revealed a diminished beneficial effect of mechanical loading on the myogenic and survival ability of the senescent muscle cells compared with the controls, with a low strain (2%) loading being most effective in upregulating myogenic/anabolic factors and downregulating atrophy and pro-apoptotic genes mainly in the aged myotubes.

Effect of Mechanical Loading of Aged Myotubes on their Myogenic Lineage Progression and Apoptosis

Effect of Mechanical Loading of Aged Myotubes on their Myogenic Lineage Progression and Apoptosis

The Stat3-Fam3a axis promotes muscle stem cell myogenic lineage progression by inducing mitochondrial respiration

Metabolic reprogramming is an active regulator of stem cell fate choices, and successful stem cell differentiation in different compartments requires the induction of oxidative phosphorylation. However, the mechanisms that promote mitochondrial respiration during stem cell differentiation are poorly understood. Here we demonstrate that Stat3 promotes muscle stem cell myogenic lineage progression by stimulating mitochondrial respiration in mice. We identify Fam3a, a cytokine-like protein, as a major Stat3 downstream effector in muscle stem cells. We demonstrate that Fam3a is required for muscle stem cell commitment and skeletal muscle development. We show that myogenic cells secrete Fam3a, and exposure of Stat3-ablated muscle stem cells to recombinant Fam3a in vitro and in vivo rescues their defects in mitochondrial respiration and myogenic commitment. Together, these findings indicate that Fam3a is a Stat3-regulated secreted factor that promotes muscle stem cell oxidative metabolism and differentiation, and suggests that Fam3a is a potential tool to modulate cell fate choices.

Effect of the In Vitro Mechanical Loading of Myotubes on their Myogenic Lineage Progression

Effect of the In Vitro Mechanical Loading of Myotubes on their Myogenic Lineage Progression

Fas-Associated Protein with Death Domain Regulates Notch Signaling during Muscle Regeneration

Notch signaling plays critical roles during myogenesis by promoting the proliferation and inhibiting the differentiation of myogenic progenitors. However, the mechanism of the temporal regulation of Notch signaling during the myogenic lineage progression remains elusive. In the present study, we show that a constitutively phosphoryl-mimicking mutation of Fas-associated death domain (FADD-D) enhances Notch-1 signaling and compromises Wnt signaling in both cultured myoblasts and regenerating muscles, which results in inhibited myogenic differentiation and muscle regeneration. Inhibition of Notch signaling recovers the regeneration ability in injured FADD-D muscles through rescuing Wnt signaling. Furthermore, we found that protein kinase Cα mediates FADD-D-induced Notch-1 signaling by stabilizing Notch-1. Collectively, these data identify a novel mechanism for the temporal regulation of Notch signaling during myogenic lineage progression and muscle regeneration.

Wnt/β-catenin controls follistatin signalling to regulate satellite cell myogenic potential.

BackgroundAdult skeletal muscle regeneration is a highly orchestrated process involving the activation and proliferation of satellite cells, an adult skeletal muscle stem cell. Activated satellite cells generate a transient amplifying progenitor pool of myoblasts that commit to differentiation and fuse into multinucleated myotubes. During regeneration, canonical Wnt signalling is activated and has been implicated in regulating myogenic lineage progression and terminal differentiation.MethodsHere, we have undertaken a gene expression analysis of committed satellite cell-derived myoblasts to examine their ability to respond to canonical Wnt/β-catenin signalling.ResultsWe found that activation of canonical Wnt signalling induces follistatin expression in myoblasts and promotes myoblast fusion in a follistatin-dependent manner. In growth conditions, canonical Wnt/β-catenin signalling prime myoblasts for myogenic differentiation by stimulating myogenin and follistatin expression. We further found that myogenin binds elements in the follistatin promoter and thus acts downstream of myogenin during differentiation. Finally, ectopic activation of canonical Wnt signalling in vivo promoted premature differentiation during muscle regeneration following acute injury.ConclusionsTogether, these data reveal a novel mechanism by which myogenin mediates the canonical Wnt/β-catenin-dependent activation of follistatin and induction of the myogenic differentiation process.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0038-6) contains supplementary material, which is available to authorized users.

STAT3 signaling controls satellite cell expansion and skeletal muscle repair

The progressive loss of muscle regenerative capacity with age or disease results in part from a decline in the number and function of satellite cells, the direct cellular contributors to muscle repair. However, little is known about the molecular effectors underlying satellite cell impairment and depletion. Elevated levels of inflammatory cytokines, including interleukin-6 (IL-6), are associated with both age-related and muscle-wasting conditions. The levels of STAT3, a downstream effector of IL-6, are also elevated with muscle wasting, and STAT3 has been implicated in the regulation of self-renewal and stem cell fate in several tissues. Here we show that IL-6-activated Stat3 signaling regulates satellite cell behavior, promoting myogenic lineage progression through myogenic differentiation 1 (Myod1) regulation. Conditional ablation of Stat3 in Pax7-expressing satellite cells resulted in their increased expansion during regeneration, but compromised myogenic differentiation prevented the contribution of these cells to regenerating myofibers. In contrast, transient Stat3 inhibition promoted satellite cell expansion and enhanced tissue repair in both aged and dystrophic muscle. The effects of STAT3 inhibition on cell fate and proliferation were conserved in human myoblasts. The results of this study indicate that pharmacological manipulation of STAT3 activity can be used to counteract the functional exhaustion of satellite cells in pathological conditions, thereby maintaining the endogenous regenerative response and ameliorating muscle-wasting diseases.

Expanding Donor Muscle‐Derived Cells for Transplantation

Studies in mice showed tremendous promise for the eventual clinical utility of myoblast transplantation to treat human muscular dystrophies. Initial attempts to translate the murine studies to humans, however, were not successful, due in part to limited engraftability of expanded donor myoblasts. Conventionally, muscle cells have been cultured on collagen-coated tissue culture-treated polystyrene. However, this promotes lineage progression and differentiation of cells, which limits engraftment potential. This unit describes the isolation of canine muscle-derived cells, ex vivo expansion of cells on plates coated with a modified Notch ligand, and the xenotransplant method used to evaluate engraftment potential. Activation of Notch signaling in freshly isolated canine muscle-derived cells with Delta-1(ext)-IgG inhibits myogenic differentiation, and maintains cells earlier in myogenic lineage progression. Delta-1(ext)-IgG-expanded cells engraft into the regenerating muscle of NOD/SCID mice more effectively than control cells expanded on human IgG, as evidenced by a significant increase in the number of muscle fibers expressing canine dystrophin in recipient murine muscle. Therefore, this protocol provides the basis for further developing culture conditions for ex vivo expansion of donor muscle cells for transplant.

Wnt7a–Fzd7 signalling directly activates the Akt/mTOR anabolic growth pathway in skeletal muscle

Wnt7a signals through its receptor Fzd7 to activate the planar-cell-polarity pathway and drive the symmetric expansion of satellite stem cells resulting in enhanced repair of skeletal muscle. In differentiated myofibres, we observed that Wnt7a binding to Fzd7 directly activates the Akt/mTOR growth pathway thereby inducing myofibre hypertrophy. Notably, the Fzd7 receptor complex was associated with Gαs and PI3kinase and these components were required for Wnt7a to activate the Akt/mTOR growth pathway in myotubes. Wnt7a/Fzd7 activation of this pathway was completely independent of IGF-receptor activation. Together, these experiments demonstrate that Wnt7a/Fzd7 activates distinct pathways at different developmental stages during myogenic lineage progression, and together identify a novel non-canonical anabolic signalling pathway for Wnt7a and its receptor Fzd7 in skeletal muscle.