Photo-expansion microscopy enables super-resolution imaging of cells embedded in 3D hydrogels.

Hydrogels are extensively used as tunable, biomimetic three-dimensional cell culture matrices, but optically deep, high-resolution images are often difficult to obtain, limiting nanoscale quantification of cell-matrix interactions and outside-in signalling. Here we present photopolymerized hydrogels for expansion microscopy that enable optical clearance and tunable ×4.6-6.7 homogeneous expansion of not only monolayer cell cultures and tissue sections, but cells embedded within hydrogels. The photopolymerized hydrogels for expansion microscopy formulation relies on a rapid photoinitiated thiol/acrylate mixed-mode polymerization that is not inhibited by oxygen and decouples monomer diffusion from polymerization, which is particularly beneficial when expanding cells embedded within hydrogels. Using this technology, we visualize human mesenchymal stem cells and their interactions with nascently deposited proteins at <120 nm resolution when cultured in proteolytically degradable synthetic polyethylene glycol hydrogels. Results support the notion that focal adhesion maturation requires cellular fibronectin deposition; nuclear deformation precedes cellular spreading; and human mesenchymal stem cells display cell-surface metalloproteinases for matrix remodelling.

TDP-43 and RNA form amyloid-like myo-granules in regenerating muscle.

A dominant histopathological feature in neuromuscular diseases, including amyotrophic lateral sclerosis and inclusion body myopathy, is cytoplasmic aggregation of the RNA-binding protein TDP-43. Although rare mutations in TARDBP-the gene that encodes TDP-43-that lead to protein misfolding often cause protein aggregation, most patients do not have any mutations in TARDBP. Therefore, aggregates of wild-type TDP-43 arise in most patients by an unknown mechanism. Here we show that TDP-43 is an essential protein for normal skeletal muscle formation that unexpectedly forms cytoplasmic, amyloid-like oligomeric assemblies, which we call myo-granules, during regeneration of skeletal muscle in mice and humans. Myo-granules bind to mRNAs that encode sarcomeric proteins and are cleared as myofibres mature. Although myo-granules occur during normal skeletal-muscle regeneration, myo-granules can seed TDP-43 amyloid fibrils in vitro and are increased in a mouse model of inclusion body myopathy. Therefore, increased assembly or decreased clearance of functionally normal myo-granules could be the source of cytoplasmic TDP-43 aggregates that commonly occur in neuromuscular disease.

Conference report on contractures in musculoskeletal and neurological conditions.

Limb contractures are debilitating complications associated with various muscle and nervous system disorders. This report summarizes presentations at a conference at the Shirley Ryan AbilityLab in Chicago, Illinois, on April 19-20, 2018, involving researchers and physicians from diverse disciplines who convened to discuss current clinical and preclinical understanding of contractures in Duchenne muscular dystrophy, stroke, cerebral palsy, and other conditions. Presenters described changes in muscle architecture, activation, extracellular matrix, satellite cells, and muscle fiber sarcomeric structure that accompany or predispose muscles to contracture. Participants identified ongoing and future research directions that may lead to understanding of the intersecting factors that trigger contractures. These include additional studies of changes in muscle, tendon, joint, and neuronal tissues during contracture development with imaging, molecular, and physiologic approaches. Participants identified the requirement for improved biomarkers and outcome measures to identify patients likely to develop contractures and to accurately measure efficacy of treatments currently available and under development.

Is Growth Differentiation Factor 11 a Realistic Therapeutic for Aging-Dependent Muscle Defects?

This "Controversies in Cardiovascular Research" article evaluates the evidence for and against the hypothesis that the circulating blood level of growth differentiation factor 11 (GDF11) decreases in old age and that restoring normal GDF11 levels in old animals rejuvenates their skeletal muscle and reverses pathological cardiac hypertrophy and cardiac dysfunction. Studies supporting the original GDF11 hypothesis in skeletal and cardiac muscle have not been validated by several independent groups. These new studies have either found no effects of restoring normal GDF11 levels on cardiac structure and function or have shown that increasing GDF11 or its closely related family member growth differentiation factor 8 actually impairs skeletal muscle repair in old animals. One possible explanation for what seems to be mutually exclusive findings is that the original reagent used to measure GDF11 levels also detected many other molecules so that age-dependent changes in GDF11 are still not well known. The more important issue is whether increasing blood [GDF11] repairs old skeletal muscle and reverses age-related cardiac pathologies. There are substantial new and existing data showing that GDF8/11 can exacerbate rather than rejuvenate skeletal muscle injury in old animals. There is also new evidence disputing the idea that there is pathological hypertrophy in old C57bl6 mice and that GDF11 therapy can reverse cardiac pathologies. Finally, high [GDF11] causes reductions in body and heart weight in both young and old animals, suggestive of a cachexia effect. Our conclusion is that elevating blood levels of GDF11 in the aged might cause more harm than good.

Regulation of skeletal muscle stem cells by fibroblast growth factors.

Fibroblast growth factors (FGFs) are essential for self-renewal of skeletal muscle stem cells (satellite cells) and required for maintenance and repair of skeletal muscle. Satellite cells express high levels of FGF receptors 1 and 4, low levels of FGF receptor 3, and little or no detectable FGF receptor 2. Of the multiple FGFs that influence satellite cell function in culture, FGF2 and FGF6 are the only members that regulate satellite cell function in vivo by activating ERK MAPK, p38α/ß MAPKs, PI3 kinase, PLCγ and STATs. Regulation of FGF signaling is complex in satellite cells, requiring Syndecan-4, a heparan sulfate proteoglycan, as well as ß1-integrin and fibronectin. During aging, reduced responsiveness to FGF diminishes satellite cell self-renewal, leading to impaired skeletal muscle regeneration and depletion of satellite cells. Mislocalization of ß1-integrin, reductions in fibronectin, and alterations in heparan sulfate content all contribute to reduced FGF responsiveness in satellite cells. How these cell surface proteins regulate satellite cell self-renewal is incompletely understood. Here we summarize the current knowledge, highlighting the role(s) for FGF signaling in skeletal muscle regeneration, satellite cell behavior, and age-induced muscle wasting. Developmental Dynamics 246:359-367, 2017. © 2017 Wiley Periodicals, Inc.

NEDD4 Regulates PAX7 Levels Promoting Activation of the Differentiation Program in Skeletal Muscle Precursors.

The transcription factor Pax7 regulates skeletal muscle stem cell (satellite cells) specification and maintenance through various mechanisms, including repressing the activity of the muscle regulatory factor MyoD. Hence, Pax7-to-MyoD protein ratios can determine maintenance of the committed-undifferentiated state or activation of the differentiation program. Pax7 expression decreases sharply in differentiating myoblasts but is maintained in cells (re)acquiring quiescence, yet the mechanisms regulating Pax7 levels based on differentiation status are not well understood. Here we show that Pax7 levels are directly regulated by the ubiquitin-ligase Nedd4. Our results indicate that Nedd4 is expressed in quiescent and activated satellite cells, that Nedd4 and Pax7 physically interact during early muscle differentiation-correlating with Pax7 ubiquitination and decline-and that Nedd4 loss of function prevented this effect. Furthermore, even transient nuclear accumulation of Nedd4 induced a drop in Pax7 levels and precocious muscle differentiation. Consequently, we propose that Nedd4 functions as a novel Pax7 regulator, which activity is temporally and spatially controlled to modulate the Pax7 protein levels and therefore satellite cell fate.

Activation of Notch signaling during ex vivo expansion maintains donor muscle cell engraftment.

Transplantation of myogenic stem cells possesses great potential for long-term repair of dystrophic muscle. However, a single donor muscle biopsy is unlikely to provide enough cells to effectively transplant the muscle mass of a patient affected by muscular dystrophy. Expansion of cells ex vivo using traditional culture techniques significantly reduces engraftment potential. We hypothesized that activation of Notch signaling during ex vivo expansion would maintain donor cell engraftment potential. In this study, we expanded freshly isolated canine muscle-derived cells on tissue culture plates coated with Delta-1(ext) -IgG to activate Notch signaling or with human IgG as a control. A model of canine-to-murine xenotransplantation was used to quantitatively compare canine muscle cell engraftment and determine whether engrafted donor cells could function as satellite cells in vivo. We show that Delta-1(ext) -IgG inhibited differentiation of canine muscle-derived cells and increased the level of genes normally expressed in myogenic precursors. Moreover, cells expanded on Delta-1(ext) -IgG resulted in a significant increase in the number of donor-derived fibers, as compared to cells expanded on human IgG, reaching engraftment levels similar to freshly isolated cells. Importantly, cells expanded on Delta-1(ext) -IgG engrafted to the recipient satellite cell niche and contributed to further regeneration. A similar strategy of expanding human muscle-derived cells on Notch ligand might facilitate engraftment and muscle regeneration for patients affected with muscular dystrophy.

Growth of limb muscle is dependent on skeletal-derived Indian hedgehog.

During embryogenesis, muscle and bone develop in close temporal and spatial proximity. We show that Indian Hedgehog, a bone-derived signaling molecule, participates in growth of skeletal muscle. In Ihh(-/-) embryos, skeletal muscle development appears abnormal at embryonic day 14.5 and at later ages through embryonic day 20.5, dramatic losses of hindlimb muscle occur. To further examine the role of Ihh in myogenesis, we manipulated Ihh expression in the developing chick hindlimb. Reduction of Ihh in chicken embryo hindlimbs reduced skeletal muscle mass similar to that seen in Ihh(-/-) mouse embryos. The reduction in muscle mass appears to be a direct effect of Ihh since ectopic expression of Ihh by RCAS retroviral infection of chicken embryo hindlimbs restores muscle mass. These effects are independent of bone length, and occur when Shh is not expressed, suggesting Ihh acts directly on fetal myoblasts to regulate secondary myogenesis. Loss of muscle mass in Ihh null mouse embryos is accompanied by a dramatic increase in myoblast apoptosis by a loss of p21 protein. Our data suggest that Ihh promotes fetal myoblast survival during their differentiation into secondary myofibers by maintaining p21 protein levels.

Reciprocal inhibition between Pax7 and muscle regulatory factors modulates myogenic cell fate determination.

Postnatal growth and regeneration of skeletal muscle requires a population of resident myogenic precursors named satellite cells. The transcription factor Pax7 is critical for satellite cell biogenesis and survival and has been also implicated in satellite cell self-renewal; however, the underlying molecular mechanisms remain unclear. Previously, we showed that Pax7 overexpression in adult primary myoblasts down-regulates MyoD and prevents myogenin induction, inhibiting myogenesis. We show that Pax7 prevents muscle differentiation independently of its transcriptional activity, affecting MyoD function. Conversely, myogenin directly affects Pax7 expression and may be critical for Pax7 down-regulation in differentiating cells. Our results provide evidence for a cross-inhibitory interaction between Pax7 and members of the muscle regulatory factor family. This could represent an additional mechanism for the control of satellite cell fate decisions resulting in proliferation, differentiation, and self-renewal, necessary for skeletal muscle maintenance and repair.

The regenerating skeletal muscle niche drives satellite cell return to quiescence.

Skeletal muscle stem cells, or satellite cells (SCs), are essential to regenerate and maintain muscle. Quiescent SCs reside in an asymmetric niche between the basal lamina and myofiber membrane. To repair muscle, SCs activate, proliferate, and differentiate, fusing to repair myofibers or reacquiring quiescence to replenish the SC niche. Little is known about when SCs reacquire quiescence during regeneration or the cellular processes that direct SC fate decisions. We find that most SCs reacquire quiescence 5-10 days after muscle injury, following differentiation and fusion of most cells to regenerate myofibers. Single-cell sequencing of myogenic cells in regenerating muscle identifies SCs reacquiring quiescence and reveals that noncell autonomous signaling networks influence SC fate decisions during regeneration. SC transplantation experiments confirm that the regenerating environment influences SC fate. We define a window for SC repopulation of the niche, emphasizing the temporal contribution of the regenerative muscle environment on SC fate.

RNA-binding proteins direct myogenic cell fate decisions.

RNA-binding proteins (RBPs), essential for skeletal muscle regeneration, cause muscle degeneration and neuromuscular disease when mutated. Why mutations in these ubiquitously expressed RBPs orchestrate complex tissue regeneration and direct cell fate decisions in skeletal muscle remains poorly understood. Single-cell RNA-sequencing of regenerating Mus musculus skeletal muscle reveals that RBP expression, including the expression of many neuromuscular disease-associated RBPs, is temporally regulated in skeletal muscle stem cells and correlates with specific stages of myogenic differentiation. By combining machine learning with RBP engagement scoring, we discovered that the neuromuscular disease-associated RBP Hnrnpa2b1 is a differentiation-specifying regulator of myogenesis that controls myogenic cell fate transitions during terminal differentiation in mice. The timing of RBP expression specifies cell fate transitions by providing post-transcriptional regulation of messenger RNAs that coordinate stem cell fate decisions during tissue regeneration.

Pax7-FKHR transcriptional activity is enhanced by transcriptionally repressed MyoD.

Alveolar rhabdomyosarcoma (ARMS) are characterized by the expression of chimeric transcription factors Pax3-FKHR and Pax7-FKHR, due to chromosomal translocations fusing PAX3 or PAX7 with the FKHR gene. Although ARMS exhibits a muscle lineage phenotype, the cells evade terminal differentiation despite expressing the potent myogenic transcriptional regulator MyoD. Here we show that while Pax7-FKHR inhibits MyoD-dependent transcription, MyoD enhances Pax7-FKHR activity in myogenic cell cultures. Importantly, this effect is not recapitulated by close related transcription factor myogenin and involves specific MyoD functional domains, distinct from those required for Pax7 to regulate MyoD during muscle formation. Together, these results suggest that although repressed as a myogenic regulatory factor, MyoD can play an active role in ARMS by augmenting Pax7-FKHR function.

MAP kinase phosphatase-1 deficiency impairs skeletal muscle regeneration and exacerbates muscular dystrophy.

In skeletal muscle, the mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) is a critical negative regulator of the MAPKs. Since the MAPKs have been reported to be both positive and negative for myogenesis, the physiological role of MKP-1 in skeletal muscle repair and regeneration has remained unclear. Here, we show that MKP-1 plays an essential role in adult regenerative myogenesis. In a cardiotoxin-induced muscle injury model, lack of MKP-1 impaired muscle regeneration. In mdx mice, MKP-1 deficiency reduced body weight, muscle mass, and muscle fiber cross-sectional area. In addition, MKP-1-deficient muscles exhibit exacerbated myopathy accompanied by increased inflammation. Lack of MKP-1 compromised myoblast proliferation and induced precocious differentiation, phenotypes that were rescued by pharmacological inhibition of p38alpha/beta MAPK. MKP-1 coordinates both myoblast proliferation and differentiation. Mechanistically, MyoD bound to the MKP-1 promoter and activated MKP-1 expression in proliferating myoblasts. Later, during myogenesis, MyoD uncoupled from the MKP-1 promoter leading to the down-regulation of MKP-1 and facilitation of promyogenic p38alpha/beta MAPK signaling. Hence, MKP-1 plays a critical role in muscle stem cells and in the immune response to coordinate muscle repair and regeneration.

Post-Transcriptional Regulation in Skeletal Muscle Development, Repair, and Disease.

Skeletal muscle formation is a complex process that requires tight spatiotemporal control of key myogenic factors. Emerging evidence suggests that RNA processing is crucial for the regulation of these factors, and that multiple post-transcriptional regulatory pathways work dependently and independently of one another to enable precise control of transcripts throughout muscle development and repair. Moreover, disruption of these pathways is implicated in neuromuscular disease, and the recent development of RNA-mediated therapies shows enormous promise in the treatment of these disorders. We discuss the overlapping post-transcriptional regulatory pathways that mediate muscle development, how these pathways are disrupted in neuromuscular disorders, and advances in RNA-mediated therapies that present a novel approach to the treatment of these diseases.

Myoblast mechanotransduction and myotube morphology is dependent on BAG3 regulation of YAP and TAZ.

Skeletal muscle tissue is mechanically dynamic with changes in stiffness influencing function, maintenance, and regeneration. We modeled skeletal muscle mechanical changes in culture with dynamically stiffening hydrogels demonstrating that the chaperone protein BAG3 transduces matrix stiffness by redistributing YAP and TAZ subcellular localization in muscle progenitor cells. BAG3 depletion increases cytoplasmic retention of YAP and TAZ, desensitizing myoblasts to changes in hydrogel elastic moduli. Upon differentiation, muscle progenitors depleted of BAG3 formed enlarged, round myotubes lacking the typical cylindrical morphology. The aberrant morphology is dependent on YAP/TAZ signaling, which was sequestered in the cytoplasm in BAG3-depleted myotubes but predominately nuclear in cylindrical myotubes of control cells. Control progenitor cells induced to differentiate on soft (E' = 4 and 12 kPa) hydrogels formed circular myotubes similar to those observed in BAG3-depleted cells. Inhibition of the Hippo pathway partially restored myotube morphologies, permitting nuclear translocation of YAP and TAZ in BAG3-depleted myogenic progenitors. Thus, BAG3 is a critical mediator of dynamic stiffness changes in muscle tissue, coupling mechanical alterations to intracellular signals and inducing changes in gene expression that influence muscle progenitor cell morphology and differentiation.

Injury-mediated stiffening persistently activates muscle stem cells through YAP and TAZ mechanotransduction.

The skeletal muscle microenvironment transiently remodels and stiffens after exercise and injury, as muscle ages, and in myopathic muscle; however, how these changes in stiffness affect resident muscle stem cells (MuSCs) remains understudied. Following muscle injury, muscle stiffness remained elevated after morphological regeneration was complete, accompanied by activated and proliferative MuSCs. To isolate the role of stiffness on MuSC behavior and determine the underlying mechanotransduction pathways, we cultured MuSCs on strain-promoted azide-alkyne cycloaddition hydrogels capable of in situ stiffening by secondary photocrosslinking of excess cyclooctynes. Using pre- to post-injury stiffness hydrogels, we found that elevated stiffness enhances migration and MuSC proliferation by localizing yes-associated protein 1 (YAP) and WW domain-containing transcription regulator 1 (WWTR1; TAZ) to the nucleus. Ablating YAP and TAZ in vivo promotes MuSC quiescence in postinjury muscle and prevents myofiber hypertrophy, demonstrating that persistent exposure to elevated stiffness activates mechanotransduction signaling maintaining activated and proliferating MuSCs.

The p38alpha/beta MAPK functions as a molecular switch to activate the quiescent satellite cell.

Somatic stem cells cycle slowly or remain quiescent until required for tissue repair and maintenance. Upon muscle injury, stem cells that lie between the muscle fiber and basal lamina (satellite cells) are activated, proliferate, and eventually differentiate to repair the damaged muscle. Satellite cells in healthy muscle are quiescent, do not express MyoD family transcription factors or cell cycle regulatory genes and are insulated from the surrounding environment. Here, we report that the p38alpha/beta family of mitogen-activated protein kinases (MAPKs) reversibly regulates the quiescent state of the skeletal muscle satellite cell. Inhibition of p38alpha/beta MAPKs (a) promotes exit from the cell cycle, (b) prevents differentiation, and (c) insulates the cell from most external stimuli allowing the satellite cell to maintain a quiescent state. Activation of satellite cells and p38alpha/beta MAPKs occurs concomitantly, providing further support that these MAPKs function as a molecular switch for satellite cell activation.

Muscling in on the Awesome Proliferative Power of the Terrible Teratoma.

In this issue of Cell Stem Cell, Chan et al. (2018) report that in vivo differentiation of pluripotent stem cells in induced teratomas produces functional embryonic-like muscle stem cells. These purified muscle stem cells engraft with high efficiency and regenerate serially injured muscle.

Muscle stem cell dysfunction impairs muscle regeneration in a mouse model of Down syndrome.

Down syndrome, caused by trisomy 21, is characterized by a variety of medical conditions including intellectual impairments, cardiovascular defects, blood cell disorders and pre-mature aging phenotypes. Several somatic stem cell populations are dysfunctional in Down syndrome and their deficiencies may contribute to multiple Down syndrome phenotypes. Down syndrome is associated with muscle weakness but skeletal muscle stem cells or satellite cells in Down syndrome have not been investigated. We find that a failure in satellite cell expansion impairs muscle regeneration in the Ts65Dn mouse model of Down syndrome. Ts65Dn satellite cells accumulate DNA damage and over express Usp16, a histone de-ubiquitinating enzyme that regulates the DNA damage response. Impairment of satellite cell function, which further declines as Ts65Dn mice age, underscores stem cell deficiencies as an important contributor to Down syndrome pathologies.

Striated muscle gene therapy for the treatment of lipoprotein lipase deficiency.

Excessive circulating triglycerides due to reduction or loss of lipoprotein lipase activity contribute to hypertriglyceridemia and increased risk for pancreatitis. The only gene therapy treatment for lipoprotein lipase deficiency decreases pancreatitis but minimally reduces hypertriglyceridemia. Synthesized in multiple tissues including striated muscle and adipose tissue, lipoprotein lipase is trafficked to blood vessel endothelial cells where it is anchored at the plasma membrane and hydrolyzes triglycerides into free fatty acids. We conditionally knocked out lipoprotein lipase in differentiated striated muscle tissue lowering striated muscle lipoprotein lipase activity causing hypertriglyceridemia. We then crossed lipoprotein lipase striated muscle knockout mice with mice possessing a conditional avian retroviral receptor gene and injected mice with either a human lipoprotein lipase retrovirus or an mCherry control retrovirus. Post-heparin plasma lipoprotein lipase activity increased for three weeks following human lipoprotein lipase retroviral infection compared to mCherry infected mice. Human lipoprotein lipase infected mice had significantly lower blood triglycerides compared to mCherry controls and were comparable to wild-type blood triglyceride levels. Thus, targeted delivery of human lipoprotein lipase into striated muscle tissue identifies a potential therapeutic target for lipoprotein lipase deficiency.