The Notch signaling pathway in skeletal muscle health and disease.

The Notch signaling pathway is a key regulator of skeletal muscle development and regeneration. Over the past decade, the discoveries of three new muscle disease genes have added a new dimension to the relationship between the Notch signaling pathway and skeletal muscle: MEGF10, POGLUT1, and JAG2. We review the clinical syndromes associated with pathogenic variants in each of these genes, known molecular and cellular functions of their protein products with a particular focus on the Notch signaling pathway, and potential novel therapeutic targets that may emerge from further investigations of these diseases. The phenotypes associated with two of these genes, POGLUT1 and JAG2, clearly fall within the realm of muscular dystrophy, whereas the third, MEGF10, is associated with a congenital myopathy/muscular dystrophy overlap syndrome classically known as early-onset myopathy, areflexia, respiratory distress, and dysphagia. JAG2 is a canonical Notch ligand, POGLUT1 glycosylates the extracellular domain of Notch receptors, and MEGF10 interacts with the intracellular domain of NOTCH1. Additional genes and their encoded proteins relevant to muscle function and disease with links to the Notch signaling pathway include TRIM32, ATP2A1 (SERCA1), JAG1, PAX7, and NOTCH2NLC. There is enormous potential to identify convergent mechanisms of skeletal muscle disease and new therapeutic targets through further investigations of the Notch signaling pathway in the context of skeletal muscle development, maintenance, and disease.

Inhibition of FLT1 ameliorates muscular dystrophy phenotype by increased vasculature in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disease in which the dystrophin coding for a membrane stabilizing protein is mutated. Recently, the vasculature has also shown to be perturbed in DMD and DMD model mdx mice. Recent DMD transcriptomics revealed the defects were correlated to a vascular endothelial growth factor (VEGF) signaling pathway. To reveal the relationship between DMD and VEGF signaling, mdx mice were crossed with constitutive (CAGCreERTM:Flt1LoxP/LoxP) and endothelial cell-specific conditional gene knockout mice (Cdh5CreERT2:Flt1LoxP/LoxP) for Flt1 (VEGFR1) which is a decoy receptor for VEGF. Here, we showed that while constitutive deletion of Flt1 is detrimental to the skeletal muscle function, endothelial cell-specific Flt1 deletion resulted in increased vascular density, increased satellite cell number and improvement in the DMD-associated phenotype in the mdx mice. These decreases in pathology, including improved muscle histology and function, were recapitulated in mdx mice given anti-FLT1 peptides or monoclonal antibodies, which blocked VEGF-FLT1 binding. The histological and functional improvement of dystrophic muscle by FLT1 blockade provides a novel pharmacological strategy for the potential treatment of DMD.

[Massive Hemorrhage from the Delayed Diaphragmatic Injury due to the Rib Fracture after the Blunt Chest Trauma:Report of a Case].

We report a case of delayed diaphragmatic injury caused by lower rib fractures. A 56-year-old male was referred to our hospital due to the fractures of right lower ribs. Chest X-ray revealed pneumothorax, and the patient was hospitalized for follow-up. On the sixth day after the injury, the patient suddenly complained of chest pain and respiratory distress, and then shock status developed. Chest computed tomography (CT) revealed massive pleural effusion. An emergency operation was performed. The injury of the diaphragm was found. Fracture of the lower rib can cause diaphragmatic injury leading to massive hemorrhage.

Inhibition of microRNA-92a increases blood vessels and satellite cells in skeletal muscle but does not improve duchenne muscular dystrophy-related phenotype in mdx mice.

INTRODUCTION: The vasculature and blood flow in muscle are perturbed in Duchenne muscular dystrophy (DMD) and its mdx mouse model. MicroRNA-92a (miR-92a) is enriched in endothelial cells, especially during ischemic injury. METHODS: Because antagonizing miR-92a was shown to result in increased proliferation and migration of endothelial cells and recovery from ischemia, we assessed the effects of Antagomir-92a in vitro in muscle stem cell culture and in vivo in mdx mice. RESULTS: miR-92a was found to be highly expressed in muscle endothelial cells and satellite cells. Treatment with Antagomir-92a increased capillary density and tissue perfusion, which was accompanied by an increase in satellite cells. However, Antagomir-92a-treated mdx mice showed no histological improvement and had worse muscle function. Antagomir-92a suppressed myogenic differentiation in satellite cell culture. DISCUSSION: AntagomiR-92a improves the vasculature but not the muscle in mdx mice, possibly due to its side effects on satellite cell differentiation. Muscle Nerve 59:594-594, 2019.

Imaging analysis for muscle stem cells and regeneration.

Composed of a diverse variety of cells, the skeletal muscle is one of the body's tissues with the remarkable ability to regenerate after injury. One of the key players in the regeneration process is the muscle satellite cell (MuSC), a stem cell population for skeletal muscle, as it is the source of new myofibers. Maintaining MuSC quiescence during homeostasis involves complex interactions between MuSCs and other cells in their corresponding niche in adult skeletal muscle. After the injury, MuSCs are activated to enter the cell cycle for cell proliferation and differentiate into myotubes, followed by mature myofibers to regenerate muscle. Despite decades of research, the exact mechanisms underlying MuSC maintenance and activation remain elusive. Traditional methods of analyzing MuSCs, including cell cultures, animal models, and gene expression analyses, provide some insight into MuSC biology but lack the ability to replicate the 3-dimensional (3-D) in vivo muscle environment and capture dynamic processes comprehensively. Recent advancements in imaging technology, including confocal, intra-vital, and multi-photon microscopies, provide promising avenues for dynamic MuSC morphology and behavior to be observed and characterized. This chapter aims to review 3-D and live-imaging methods that have contributed to uncovering insights into MuSC behavior, morphology changes, interactions within the muscle niche, and internal signaling pathways during the quiescence to activation (Q-A) transition. Integrating advanced imaging modalities and computational tools provides a new avenue for studying complex biological processes in skeletal muscle regeneration and muscle degenerative diseases such as sarcopenia and Duchenne muscular dystrophy (DMD).

Endothelial cell signature in muscle stem cells validated by VEGFA-FLT1-AKT1 axis promoting survival of muscle stem cell.

Endothelial and skeletal muscle lineages arise from common embryonic progenitors. Despite their shared developmental origin, adult endothelial cells (ECs) and muscle stem cells (MuSCs; satellite cells) have been thought to possess distinct gene signatures and signaling pathways. Here, we shift this paradigm by uncovering how adult MuSC behavior is affected by the expression of a subset of EC transcripts. We used several computational analyses including single-cell RNA-seq (scRNA-seq) to show that MuSCs express low levels of canonical EC markers in mice. We demonstrate that MuSC survival is regulated by one such prototypic endothelial signaling pathway (VEGFA-FLT1). Using pharmacological and genetic gain- and loss-of-function studies, we identify the FLT1-AKT1 axis as the key effector underlying VEGFA-mediated regulation of MuSC survival. All together, our data support that the VEGFA-FLT1-AKT1 pathway promotes MuSC survival during muscle regeneration, and highlights how the minor expression of select transcripts is sufficient for affecting cell behavior.

Vascular therapy for Duchenne muscular dystrophy (DMD).

Duchenne muscular dystrophy (DMD) is a progressive disease characterized by the wasting of the muscles that eventually lead to difficulty moving and, ultimately, premature death from heart and respiratory complications. DMD deficiency is caused by mutations in the gene encoding dystrophin, which prevents skeletal muscle, cardiac muscle, and other cells from producing the functional protein. Located on the cytoplasmic face of the plasma membrane of muscle fibers, dystrophin serves as a component of the dystrophin glycoprotein complex (DGC), mechanically reinforces the sarcolemma, and stabilizes the DGC, preventing it from contraction-mediated muscle degradation. In DMD muscle, dystrophin deficiency leads to progressive fibrosis, myofiber damage, chronic inflammation, and dysfunction of the mitochondria and muscle stem cells. Currently, DMD is incurable, and treatment involves the administration of glucocorticoids in order to delay disease progression. In the presence of developmental delay, proximal weakness, and elevated serum creatine kinase levels, a definitive diagnosis can usually be made after an extensive review of the patient's history and physical examination, as well as confirmation through muscle biopsy or genetic testing. Current standards of care include the use of corticosteroids to prolong ambulation and delay the onset of secondary complications, including respiratory muscle and cardiac functions. However, different studies have been carried out to show the relationship between vascular density and impaired angiogenesis in the pathogenesis of DMD. Several recent studies on DMD management are vascular targeted and focused on ischemia as a culprit for the pathogenesis of DMD. This review critically discusses approaches-such as modulation of nitric oxide (NO) or vascular endothelial growth factor (VEGF)-related pathways-to attenuate the dystrophic phenotype and enhance angiogenesis.

Tissue Clearing and Confocal Microscopic Imaging for Skeletal Muscle.

Skeletal muscle is a highly ordered tissue composed of a complex network of a diverse variety of cells. The dynamic spatial and temporal interaction between these cells during homeostasis and during times of injury gives the skeletal muscle its regenerative capacity. To properly understand the process of regeneration, a three-dimensional (3-D) imaging process must be conducted. With the advancement of imaging and computing technology, it has become powerful to analyze spatial data from confocal microscope images. In order to prepare whole tissue skeletal muscle samples for confocal imaging, the muscle must be subjected to tissue clearing. With the use of an ideal optical clearing protocol - one that minimizes light scattering via refractive index mismatching - a more accurate 3-D image of the muscle can be produced as it does not involve the physical sectioning of the muscle. While there have been several protocols relating to the study of 3-D biology in whole tissue, these protocols have primarily been focused on the nervous system. In this chapter, we present a new method for skeletal muscle tissue clearing. In addition, this protocol aims to outline the specific parameters required for taking 3-D images of immunofluorescence-stained skeletal muscle samples using a confocal microscope.

Three-Dimensional Imaging Analysis for Skeletal Muscle.

Skeletal muscle is a highly ordered tissue composed of a complex network of a diverse variety of cells. The dynamic spatial and temporal interaction between these cells during homeostasis and during times of injury gives the skeletal muscle its regenerative capacity. In order to properly understand the process of regeneration, a three-dimensional (3-D) imaging process must be conducted. While there have been several protocols studying 3-D imaging, it has primarily been focused on the nervous system. This protocol aims to outline the workflow for rendering a 3-D image of the skeletal muscle using spatial data from confocal microscope images. This protocol uses the ImageJ, Ilastik, and Imaris software for 3-D rendering and computational image analysis as both are relatively easy to use and have powerful segmentation capabilities.

Flt-1 haploinsufficiency ameliorates muscular dystrophy phenotype by developmentally increased vasculature in mdx mice.

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disease caused by mutations in the gene coding for the protein dystrophin. Recent work demonstrates that dystrophin is also found in the vasculature and its absence results in vascular deficiency and abnormal blood flow. This induces a state of ischemia further aggravating the muscular dystrophy pathogenesis. For an effective form of therapy of DMD, both the muscle and the vasculature need to be addressed. To reveal the developmental relationship between muscular dystrophy and vasculature, mdx mice, an animal model for DMD, were crossed with Flt-1 gene knockout mice to create a model with increased vasculature. Flt-1 is a decoy receptor for vascular endothelial growth factor, and therefore both homozygous (Flt-1(-/-)) and heterozygous (Flt-1(+/-)) Flt-1 gene knockout mice display increased endothelial cell proliferation and vascular density during embryogenesis. Here, we show that Flt-1(+/-) and mdx:Flt-1(+/-) adult mice also display a developmentally increased vascular density in skeletal muscle compared with the wild-type and mdx mice, respectively. The mdx:Flt-1(+/-) mice show improved muscle histology compared with the mdx mice with decreased fibrosis, calcification and membrane permeability. Functionally, the mdx:Flt-1(+/-) mice have an increase in muscle blood flow and force production, compared with the mdx mice. Consequently, the mdx:utrophin(-/-):Flt-1(+/-) mice display improved muscle histology and significantly higher survival rates compared with the mdx:utrophin(-/-) mice, which show more severe muscle phenotypes than the mdx mice. These data suggest that increasing the vasculature in DMD may ameliorate the histological and functional phenotypes associated with this disease.

MyoD gene suppression by Oct4 is required for reprogramming in myoblasts to produce induced pluripotent stem cells.

Expression of the four transcription factors, that is, Oct4, Sox2, cMyc, and Klf4 has been shown to generate induced pluripotent stem cells (iPSCs) from many types of specialized differentiated somatic cells. It remains unclear, however, whether fully committed skeletal muscle progenitor cells (myoblasts) have the potency to undergo reprogramming to develop iPSCs in line with previously reported cases. To test this, we have isolated genetically marked myoblasts derived from satellite cell of adult mouse muscles using the Cre-loxP system (Pax7-CreER:R26R and Myf5-Cre:R26R). On infection with retroviral vectors expressing the four factors, these myoblasts gave rise to myogenic lineage tracer lacZ-positive embryonic stem cell (ESC)-like colonies. These cells expressed ESC-specific genes and were competent to differentiate into all three germ layers and germ cells, indicating the successful generation of myoblast-derived iPSCs. Continuous expression of the MyoD gene, a master transcription factor for skeletal muscle specification, inhibited this reprogramming process in myoblasts. In contrast, reprogramming myoblasts isolated from mice lacking the MyoD gene led to an increase in reprogramming efficiency. Our data also indicated that Oct4 acts as a transcriptional suppressor of MyoD gene expression through its interaction with the upstream enhancer region. Taken together, these results indicate that suppression of MyoD gene expression by Oct4 is required for the initial reprogramming step in the development of iPSCs from myoblasts. This data suggests that the skeletal muscle system provides a well-defined differentiation model to further elaborate on the effects of iPSC reprogramming in somatic cells.

Rhabdomyosarcomagenesis-Novel pathway found.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Recent work presented in this issue of Cancer Cell by Fleischmann et al. on gene knockout mice revealed a remarkable molecular pathway for rhabdomyosarcomagenesis, in which Trp53/Fos double mutant mice developed RMS of the facial and orbital regions with high penetrance. This finding may provide novel molecular mechanisms for Rhabdomyosarcomagenesis and therapeutic implications for RMS patients.

Immunofluorescence analysis of myogenic differentiation.

Skeletal muscle is a highly regenerative tissue that can efficiently recover from various damages caused by injuries and excessive exercises. In adult muscle, stem cells termed satellite cells are mitotically quiescent but activated upon muscle damages to enter the cell cycle as myogenic precursor cells or myoblasts. After several rounds of cell cycles, they exist the cycle and fuse to each other to form multinucleated myotubes, and eventually mature to become contractile myofibers. Satellite cells can be readily isolated from mouse skeletal muscle with enzymatic digestion and magnetic separation with antibodies against specific surface markers. C2C12 cells are an immortalized mouse myoblast cell line that is commercially available and more readily expandable than primary myoblasts. Both primary myoblasts and C2C12 cells have been extensively used as useful in vitro models for myogenic differentiation. Proper examination of this process requires monitoring specific protein expression in subcellular compartments, which can be accomplished through immunofluorescence staining. This chapter describes the workflow for the isolation of satellite cells from mouse skeletal muscle and subsequent immunofluorescence staining to assess the proliferation and differentiation of primary myoblasts and C2C12 cells.

Implications of notch signaling in duchenne muscular dystrophy.

This review focuses upon the implications of the Notch signaling pathway in muscular dystrophies, particularly Duchenne muscular dystrophy (DMD): a pervasive and catastrophic condition concerned with skeletal muscle degeneration. Prior work has defined the pathogenesis of DMD, and several therapeutic approaches have been undertaken in order to regenerate skeletal muscle tissue and ameliorate the phenotype. There is presently no cure for DMD, but a promising avenue for novel therapies is inducing muscle regeneration via satellite cells (muscle stem cells). One specific target using this approach is the Notch signaling pathway. The canonical Notch signaling pathway has been well-characterized and it ultimately governs cell fate decision, cell proliferation, and induction of differentiation. Additionally, inhibition of the Notch signaling pathway has been directly implicated in the deficits seen with muscular dystrophies. Here, we explore the connection between the Notch signaling pathway and DMD, as well as how Notch signaling may be targeted to improve the muscle degeneration seen in muscular dystrophies.

Fine-Tuning of Piezo1 Expression and Activity Ensures Efficient Myoblast Fusion during Skeletal Myogenesis.

Mechanical stimuli, such as stretch and resistance training, are essential in regulating the growth and functioning of skeletal muscles. However, the molecular mechanisms involved in sensing mechanical stress during muscle formation remain unclear. Here, we investigated the role of the mechanosensitive ion channel Piezo1 during myogenic progression of both fast and slow muscle satellite cells. We found that Piezo1 level increases during myogenic differentiation and direct manipulation of Piezo1 in muscle stem cells alters the myogenic progression. Indeed, Piezo1 knockdown suppresses myoblast fusion, leading to smaller myotubes. Such an event is accompanied by significant downregulation of the fusogenic protein Myomaker. In parallel, while Piezo1 knockdown also lowers Ca2+ influx in response to stretch, Piezo1 activation increases Ca2+ influx in response to stretch and enhances myoblasts fusion. These findings may help understand molecular defects present in some muscle diseases. Our study shows that Piezo1 is essential for terminal muscle differentiation acting on myoblast fusion, suggesting that Piezo1 deregulation may have implications in muscle aging and degenerative diseases, including muscular dystrophies and neuromuscular disorders.

The endothelial Dll4-muscular Notch2 axis regulates skeletal muscle mass.

Adult skeletal muscle is a highly plastic tissue that readily reduces or gains its mass in response to mechanical and metabolic stimulation; however, the upstream mechanisms that control muscle mass remain unclear. Notch signalling is highly conserved, and regulates many cellular events, including proliferation and differentiation of various types of tissue stem cell via cell-cell contact. Here we reveal that multinucleated myofibres express Notch2, which plays a crucial role in disuse- or diabetes-induced muscle atrophy. Mechanistically, in both atrophic conditions, the microvascular endothelium upregulates and releases the Notch ligand, Dll4, which then activates muscular Notch2 without direct cell-cell contact. Inhibition of the Dll4-Notch2 axis substantively prevents these muscle atrophy and promotes mechanical overloading-induced muscle hypertrophy in mice. Our results illuminate a tissue-specific function of the endothelium in controlling tissue plasticity and highlight the endothelial Dll4-muscular Notch2 axis as a central upstream mechanism that regulates catabolic signals from mechanical and metabolic stimulation, providing a therapeutic target for muscle-wasting diseases.

Per1/Per2-Igf2 axis-mediated circadian regulation of myogenic differentiation.

Circadian rhythms regulate cell proliferation and differentiation, but circadian control of tissue regeneration remains elusive at the molecular level. Here, we show that proper myoblast differentiation and muscle regeneration are regulated by the circadian master regulators Per1 and Per2. Depletion of Per1 or Per2 suppressed myoblast differentiation in vitro and muscle regeneration in vivo, demonstrating their nonredundant functions. Both Per1 and Per2 were required for the activation of Igf2, an autocrine promoter of myoblast differentiation, accompanied by Per-dependent recruitment of RNA polymerase II, dynamic histone modifications at the Igf2 promoter and enhancer, and the promoter-enhancer interaction. This circadian epigenetic priming created a preferred time window for initiating myoblast differentiation. Consistently, muscle regeneration was faster if initiated at night, when Per1, Per2, and Igf2 were highly expressed compared with morning. This study reveals the circadian timing as a significant factor for effective muscle cell differentiation and regeneration.

VEGFR-1/Flt-1 inhibition increases angiogenesis and improves muscle function in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy is characterized by structural degeneration of muscle, which is exacerbated by localized functional ischemia due to loss of nitric oxide synthase-induced vasodilation. Treatment strategies aimed at increasing vascular perfusion have been proposed. Toward this end, we have developed monoclonal antibodies (mAbs) that bind to the vascular endothelial growth factor (VEGF) receptor VEGFR-1 (Flt-1) and its soluble splice variant isoform (sFlt-1) leading to increased levels of free VEGF and proangiogenic signaling. The lead chimeric mAb, 21B3, had high affinity and specificity for both human and mouse sFlt-1 and inhibited VEGF binding to sFlt-1 in a competitive manner. Proof-of-concept studies in the mdx mouse model of Duchenne muscular dystrophy showed that intravenous administration of 21B3 led to elevated VEGF levels, increased vascularization and blood flow to muscles, and decreased fibrosis after 6-12 weeks of treatment. Greater muscle strength was also observed after 4 weeks of treatment. A humanized form of the mAb, 27H6, was engineered and demonstrated a comparable pharmacologic effect. Overall, administration of anti-Flt-1 mAbs in mdx mice inhibited the VEGF:Flt-1 interaction, promoted angiogenesis, and improved muscle function. These studies suggest a potential therapeutic benefit of Flt-1 inhibition for patients with Duchenne muscular dystrophy.

Post-mitotic role of nucleostemin as a promoter of skeletal muscle cell differentiation.

Nucleostemin (NS) is a nucleolar protein abundantly expressed in a variety of proliferating cells and undifferentiated cells. Its known functions include cell cycle regulation and the control of pre-rRNA processing. It also has been proposed that NS has an additional role in undifferentiated cells due to its downregulation during stem cell differentiation and its upregulation during tissue regeneration. Here, however, we demonstrate that skeletal muscle cell differentiation has a unique expression profile of NS in that it is continuously expressed during differentiation. NS was expressed at similar levels in non-proliferating muscle stem cells (satellite cells), rapidly proliferating precursor cells (myoblasts) and post-mitotic terminally differentiated cells (myotubes and myofibers). The sustained expression of NS during terminal differentiation is necessary to support increased protein synthesis during this process. Downregulation of NS inhibited differentiation of myoblasts to myotubes, accompanied by striking downregulation of key myogenic transcription factors, such as myogenin and MyoD. In contrast, upregulation of NS inhibited proliferation and promoted muscle differentiation in a p53-dependent manner. Our findings provide evidence that NS has an unexpected role in post-mitotic terminal differentiation. Importantly, these findings also indicate that, contrary to suggestions in the literature, the expression of NS cannot always be used as a reliable indicator for undifferentiated cells or proliferating cells.

MyoD induces myogenic differentiation through cooperation of its NH2- and COOH-terminal regions.

MyoD and Myf5 are basic helix-loop-helix transcription factors that play key but redundant roles in specifying myogenic progenitors during embryogenesis. However, there are functional differences between the two transcription factors that impact myoblast proliferation and differentiation. Target gene activation could be one such difference. We have used microarray and polymerase chain reaction approaches to measure the induction of muscle gene expression by MyoD and Myf5 in an in vitro model. In proliferating cells, MyoD and Myf5 function very similarly to activate the expression of likely growth phase target genes such as L-myc, m-cadherin, Mcpt8, Runx1, Spp1, Six1, IGFBP5, and Chrnbeta1. MyoD, however, is strikingly more effective than Myf5 at inducing differentiation-phase target genes. This distinction between MyoD and Myf5 results from a novel and unanticipated cooperation between the MyoD NH2- and COOH-terminal regions. Together, these results support the notion that Myf5 functions toward myoblast proliferation, whereas MyoD prepares myoblasts for efficient differentiation.