Defining and Identifying Satellite Cell-opathies within Muscular Dystrophies and Myopathies

Muscular dystrophies and congenital myopathies arise from specific genetic mutations causing skeletal muscle weakness that reduces quality of life. Muscle health relies on resident muscle stem cells called satellite cells, which enable life-course muscle growth, maintenance, repair and regeneration. Such tuned plasticity gradually diminishes in muscle diseases, suggesting compromised satellite cell function. A central issue however, is whether the pathogenic mutation perturbs satellite cell function directly and/or indirectly via an increasingly hostile microenvironment as disease progresses. Here, we explore the effects on satellite cell function of pathogenic mutations in genes (myopathogenes) that associate with muscle disorders, to evaluate muscle pathological hallmarks that define dysfunctional satellite cells. We deploy transcriptomic analysis and comparison between muscular dystrophies and myopathies to determine the contribution of satellite cell dysfunction using literature, expression dynamics of myopathogenes and correlation with expression of the satellite cell marker PAX7. Our multimodal approach extends current pathological classifications to define Satellite Cell-opathies: muscle disorders in which satellite cell dysfunction contributes to pathology. Primary Satellite Cell-opathies are conditions where mutations in a myopathogene directly affect satellite cell function, such as in Progressive Congenital Myopathy with Scoliosis (MYOSCO) and Carey-Fineman-Ziter Syndrome (CFZS). Primary satellite cell-opathies are generally characterised as being congenital with general hypotonia, and specific involvement of respiratory, trunk and facial muscles, although serum CK levels are usually within the normal range. Secondary Satellite Cell-opathies have mutations in myopathogene that affect both satellite cells and muscle fibres. Such classification aids diagnosis and predicting probable disease course, as well as informing on treatment and therapeutic development

Different Deformation Mechanisms Leading to Auxetic Behavior Exhibited by Missing Rib Square Grid Structures

This is the author accepted manuscript. The final version is available from Wiley via the DOI in this recordAn extensive investigation is carried out on the missing rib square grid structure using finite element simulations. This showed that, in the cases considered in this study, the type of deformation is primarily dependent on the ratio of the thickness of different ribs with the structure behaving like an anti-tetrachiral at particular ratios. The analysis also showed that the ability of the structure to deform predominantly through the bending of the ligament allows it to achieve much more negative values of the Poisson's ratio than previously reported. Confirmation of the numerical results is obtained through experimental methods involving the 3D printing of representative structures which are subsequently subjected to compression.European Regional Development Fun

Satellite cells derived from the mdx murine model of Duchenne muscular dystrophy retain their muscle regenerative capacity

Duchenne muscular dystrophy is an inherited disorder that is characterized by progressive skeletal muscle weakness and wasting, with a failure of muscle maintenance/repair mediated by satellite cells (muscle stem cells). The function of skeletal muscle stem cells resident in dystrophic muscle may be perturbed by being in an increasing pathological environment, coupled with constant demands for repairing muscle. To investigate the contribution of satellite cell exhaustion to this process, we tested the functionality of satellite cells isolated from the mdx mouse model of Duchenne muscular dystrophy. We found that satellite cells derived from young mdx mice contributed efficiently to muscle regeneration within our in vivo mouse model. To then test the effects of long-term residence in a dystrophic environment, satellite cells were isolated from aged mdx muscle. Surprisingly, they were as functional as those derived from young or aged wild type donors. Removing satellite cells from a dystrophic milieu reveals that their regenerative capacity remains both intact and similar to satellite cells derived from healthy muscle, indicating that the host environment is critical for mediating efficient satellite cell function

Integrated Functions of Pax3 and Pax7 in the Regulation of Proliferation, Cell Size and Myogenic Differentiation

Pax3 and Pax7 are paired-box transcription factors with roles in developmental and adult regenerative myogenesis. Pax3 and Pax7 are expressed by postnatal satellite cells or their progeny but are down regulated during myogenic differentiation. We now show that constitutive expression of Pax3 or Pax7 in either satellite cells or C2C12 myoblasts results in an increased proliferative rate and decreased cell size. Conversely, expression of dominant-negative constructs leads to slowing of cell division, a dramatic increase in cell size and altered morphology. Similarly to the effects of Pax7, retroviral expression of Pax3 increases levels of Myf5 mRNA and MyoD protein, but does not result in sustained inhibition of myogenic differentiation. However, expression of Pax3 or Pax7 dominant-negative constructs inhibits expression of Myf5, MyoD and myogenin, and prevents differentiation from proceeding. In fibroblasts, expression of Pax3 or Pax7, or dominant-negative inhibition of these factors, reproduce the effects on cell size, morphology and proliferation seen in myoblasts. Our results show that in muscle progenitor cells, Pax3 and Pax7 function to maintain expression of myogenic regulatory factors, and promote population expansion, but are also required for myogenic differentiation to proceed

Further Characterisation of the Molecular Signature of Quiescent and Activated Mouse Muscle Satellite Cells

Satellite cells are the resident stem cells of adult skeletal muscle. To date though, there is a paucity of native markers that can be used to easily identify quiescent satellite cells, with Pax7 probably being the best that is currently available. Here we have further characterized a number of recently described satellite cell markers, and also describe novel ones. Caveolin-1, integrin α7 and the calcitonin receptor proved reliable markers for quiescent satellite cells, being expressed by all satellite cells identified with Pax7. These three markers remained expressed as satellite cells were activated and underwent proliferation. The nuclear envelope proteins lamin A/C and emerin, mutations in which underlie Emery-Dreifuss muscular dystrophy, were also expressed in both quiescent and proliferating satellite cells. Conversely, Jagged-1, a Notch ligand, was not expressed in quiescent satellite cells but was induced upon activation. These findings further contribute to defining the molecular signature of muscle satellite cells

MicroRNA-125a and -b inhibit A20 and MAVS to promote inflammation and impair antiviral response in COPD

Influenza A virus (IAV) infections lead to severe inflammation in the airways. Patients with chronic obstructive pulmonary disease (COPD) characteristically have exaggerated airway inflammation and are more susceptible to infections with severe symptoms and increased mortality. The mechanisms that control inflammation during IAV infection and the mechanisms of immune dysregulation in COPD are unclear. We found that IAV infections lead to increased inflammatory and antiviral responses in primary bronchial epithelial cells (pBECs) from healthy nonsmoking and smoking subjects. In pBECs from COPD patients, infections resulted in exaggerated inflammatory but deficient antiviral responses. A20 is an important negative regulator of NF-κB-mediated inflammatory but not antiviral responses, and A20 expression was reduced in COPD. IAV infection increased the expression of miR-125a or -b, which directly reduced the expression of A20 and mitochondrial antiviral signaling (MAVS), and caused exaggerated inflammation and impaired antiviral responses. These events were replicated in vivo in a mouse model of experimental COPD. Thus, miR-125a or -b and A20 may be targeted therapeutically to inhibit excessive inflammatory responses and enhance antiviral immunity in IAV infections and in COPD

PAX7 target genes are globally repressed in facioscapulohumeral muscular dystrophy skeletal muscle

Facioscapulohumeral muscular dystrophy (FSHD) is a prevalent, incurable myopathy, linked to hypomethylation of D4Z4 repeats on chromosome 4q causing expression of the DUX4 transcription factor. However, DUX4 is difficult to detect in FSHD muscle biopsies and it is debatable how robust changes in DUX4 target gene expression are as an FSHD biomarker. PAX7 is a master regulator of myogenesis that rescues DUX4-mediated apoptosis. Here, we show that suppression of PAX7 target genes is a hallmark of FSHD, and that it is as major a signature of FSHD muscle as DUX4 target gene expression. This is shown using meta-analysis of over six FSHD muscle biopsy gene expression studies, and validated by RNA-sequencing on FSHD patient-derived myoblasts. DUX4 also inhibits PAX7 from activating its transcriptional target genes and vice versa. Furthermore, PAX7 target gene repression can explain oxidative stress sensitivity and epigenetic changes in FSHD. Thus, PAX7 target gene repression is a hallmark of FSHD that should be considered in the investigation of FSHD pathology and therapy

Prelamin A mediates inflammation in dilated and HIV associated cardiomyopathies

Cardiomyopathies are complex heart muscle diseases that can be inherited or acquired. Dilated cardiomyopathy can result from mutations in LMNA, encoding the nuclear intermediate filament proteins lamin A/C. Some LMNA mutations lead to accumulation of the lamin A precursor, prelamin A, which is disease causing in a number of tissues yet its impact upon the heart is unknown. Here we discovered myocardial prelamin A accumulation occurred in a case of dilated cardiomyopathy and show that a novel mouse model of cardiac specific prelamin A accumulation exhibited a phenotype consistent with ‘inflammatory cardiomyopathy’ which we observed to be similar to HIV associated cardiomyopathy, an acquired disease state. Numerous HIV protease therapies are known to inhibit ZMPSTE24, the enzyme responsible for prelamin A processing, and we confirmed that accumulation of prelamin A occurred in HIV+ patient cardiac biopsies. These findings: (1) confirm a unifying pathological role for prelamin A common to genetic and acquired cardiomyopathies; (2) have implications for the management of HIV patients with cardiac disease suggesting protease inhibitors should be replaced with alternative therapies i.e. non-nucleoside reverse transcriptase inhibitors; and (3) suggest that targeting inflammation may be a useful treatment strategy for certain forms of inherited cardiomyopathy

Dynamic transcriptomic analysis reveals suppression of PGC1α/ERRα drives perturbed myogenesis in facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is a prevalent, incurable myopathy, linked to epigenetic de-repression of D4Z4 repeats on chromosome 4q, leading to ectopic DUX4 expression. FSHD patient myoblasts have defective myogenic differentiation, forming smaller myotubes with reduced myosin content. However, molecular mechanisms driving such disrupted myogenesis in FSHD are poorly understood. We performed high-throughput morphological analysis describing FSHD and control myogenesis, revealing altered myogenic differentiation results in hypotrophic myotubes. Employing polynomial models and an empirical Bayes approach, we established eight critical time-points during which human healthy and FSHD myogenesis differ. RNA-sequencing at these eight nodal time-points in triplicate, provided temporal depth for a multivariate regression analysis, allowing assessment of interaction between progression of differentiation and FSHD disease status. Importantly, the unique size and structure of our data permitted identification of many novel FSHD pathomechanisms undetectable by previous approaches. Selected for further analysis here, were pathways that control mitochondria: of interest considering known alterations in mitochondrial structure and function in FSHD muscle, and sensitivity of FSHD cells to oxidative stress. Notably, we identified suppression of mitochondrial biogenesis, in particular via PGC1α, the co-factor and activator of ERRα. PGC1α knock-down caused hypotrophic myotubes to form from healthy myoblasts. Known ERRα agonists and safe food supplements Biochanin A, Genistein or Daidzein, each rescued the hypotrophic FSHD myotube phenotype. Together our work describes transcriptomic changes in high resolution that occur during myogenesis in FSHD ex-vivo, identifying suppression of the PGC1α-ERRα axis leading to perturbed myogenic differentiation, which can effectively be rescued by readily-available food supplements

Three-Dimensional Human iPSC-Derived Artificial Skeletal Muscles Model Muscular Dystrophies and Enable Multilineage Tissue Engineering

Generating human skeletal muscle models is instrumental for investigating muscle pathology and therapy. Here, we report the generation of three-dimensional (3D) artificial skeletal muscle tissue from human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) from patients with Duchenne, limb-girdle, and congenital muscular dystrophies. 3D skeletal myogenic differentiation of pluripotent cells was induced within hydrogels under tension to provide myofiber alignment. Artificial muscles recapitulated characteristics of human skeletal muscle tissue and could be implanted into immunodeficient mice. Pathological cellular hallmarks of incurable forms of severe muscular dystrophy could be modeled with high fidelity using this 3D platform. Finally, we show generation of fully human iPSC-derived, complex, multilineage muscle models containing key isogenic cellular constituents of skeletal muscle, including vascular endothelial cells, pericytes, and motor neurons. These results lay the foundation for a human skeletal muscle organoid-like platform for disease modeling, regenerative medicine, and therapy development