Muscular dystrophies (MDs) are heterogeneous diseases, characterized by primary wasting of skeletal muscle, which in severe cases, such as Duchenne Muscular Dystrophy (DMD), leads to wheelchair ...dependency, respiratory failure, and premature death. Research is ongoing to develop efficacious therapies, particularly for DMD. Most of the efforts, currently focusing on correcting or restoring the primary defect of MDs, are based on gene-addition, exon-skipping, stop codon read-through, and genome-editing. Although promising, most of them revealed several practical limitations. Shared knowledge in the field is that, in order to be really successful, any therapeutic approach has to rely on spared functional muscle tissue, restricting the number of patients eligible for clinical trials to the youngest and less compromised individuals. In line with this, many therapeutic strategies aim to preserve muscle tissue and function. This Review outlines the most interesting and recent studies addressing the secondary outcomes of DMD and how to better deliver the therapeutic agents. In the future, the effective treatment of DMD will likely require combinations of therapies addressing both the primary genetic defect and its consequences.
Inflammation and tissue regeneration follow tissue damage, but little is known about how these processes are coordinated. High Mobility Group Box 1 (HMGB1) is a nuclear protein that, when released on ...injury, triggers inflammation. We previously showed that HMGB1 with reduced cysteines is a chemoattractant, whereas a disulfide bond makes it a proinflammatory cytokine. Here we report that fully reduced HMGB1 orchestrates muscle and liver regeneration via CXCR4, whereas disulfide HMGB1 and its receptors TLR4/MD-2 and RAGE (receptor for advanced glycation end products) are not involved. Injection of HMGB1 accelerates tissue repair by acting on resident muscle stem cells, hepatocytes, and infiltrating cells. The nonoxidizable HMGB1 mutant 3S, in which serines replace cysteines, promotes muscle and liver regeneration more efficiently than the wild-type protein and without exacerbating inflammation by selectively interacting with CXCR4. Overall, our results show that the reduced form of HMGB1 coordinates tissue regeneration and suggest that 3S may be used to safely accelerate healing after injury in diverse clinical contexts.
Abstract
The transcription factor NF-Y promotes cell proliferation and its activity often declines during differentiation through the regulation of NF-YA, the DNA binding subunit of the complex. In ...stem cell compartments, the shorter NF-YA splice variant is abundantly expressed and sustains their expansion. Here, we report that satellite cells, the stem cell population of adult skeletal muscle necessary for its growth and regeneration, express uniquely the longer NF-YA isoform, majorly associated with cell differentiation. Through the generation of a conditional knock out mouse model that selectively deletes the NF-YA gene in satellite cells, we demonstrate that NF-YA expression is fundamental to preserve the pool of muscle stem cells and ensures robust regenerative response to muscle injury. In vivo and ex vivo, satellite cells that survive to NF-YA loss exit the quiescence and are rapidly committed to early differentiation, despite delayed in the progression towards later states. In vitro results demonstrate that NF-YA-depleted muscle stem cells accumulate DNA damage and cannot properly differentiate. These data highlight a new scenario in stem cell biology for NF-Y activity, which is required for efficient myogenic differentiation.
Nfix belongs to a family of four highly conserved proteins that act as transcriptional activators and/or repressors of cellular and viral genes. We previously showed a pivotal role for Nfix in ...regulating the transcriptional switch from embryonic to fetal myogenesis. Here, we show that Nfix directly represses the Myostatin promoter, thus controlling the proper timing of satellite cell differentiation and muscle regeneration. Nfix-null mice display delayed regeneration after injury, and this deficit is reversed upon in vivo Myostatin silencing. Conditional deletion of Nfix in satellite cells results in a similar delay in regeneration, confirming the functional requirement for Nfix in satellite cells. Moreover, mice lacking Nfix show reduced myofiber cross sectional area and a predominant slow twitching phenotype. These data define a role for Nfix in postnatal skeletal muscle and unveil a mechanism for Myostatin regulation, thus providing insights into the modulation of its complex signaling pathway.
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•Muscle differentiation is delayed in Nfix-null satellite cells in vitro•Mice lacking Nfix in satellite cells show delayed regeneration upon injury•Nfix directly regulates Myostatin expression during regeneration•In vivo silencing of Myostatin rescues the regeneration deficit in Nfix-null mice
Rossi et al. highlight a function for Nfix during postnatal skeletal muscle regeneration and a link with Myostatin. During regeneration, Nfix regulates Myostatin expression, thus controlling the temporal progression of the regeneration process. When Nfix is deficient, regeneration is markedly delayed.
Cells derived from blood vessels of human skeletal muscle can regenerate skeletal muscle, similarly to embryonic mesoangioblasts. However, adult cells do not express endothelial markers, but instead ...express markers of pericytes, such as NG2 proteoglycan and alkaline phosphatase (ALP), and can be prospectively isolated from freshly dissociated ALP(+) cells. Unlike canonical myogenic precursors (satellite cells), pericyte-derived cells express myogenic markers only in differentiated myotubes, which they form spontaneously with high efficiency. When transplanted into severe combined immune deficient-X-linked, mouse muscular dystrophy (scid-mdx) mice, pericyte-derived cells colonize host muscle and generate numerous fibres expressing human dystrophin. Similar cells isolated from Duchenne patients, and engineered to express human mini-dystrophin, also give rise to many dystrophin-positive fibres in vivo. These data show that myogenic precursors, distinct from satellite cells, are associated with microvascular walls in the human skeletal muscle, may represent a correlate of embryonic 'mesoangioblasts' present after birth and may be a promising candidate for future cell-therapy protocols in patients.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Due to the physiological properties of l-carnosine (l-1), supplementation of this dipeptide has both a nutritional ergogenic application and a therapeutic potential for the treatment of numerous ...diseases in which ischemic or oxidative stress are involved. Quantitation of carnosine and its analogs in biological matrices results to be crucial for these applications and HPLC-MS procedures with isotope-labeled internal standards are the state-of-the-art approach for this analytical need. The use of these standards allows to account for variations during the sample preparation process, between-sample matrix effects, and variations in instrument performance over analysis time. Although literature reports a number of studies involving carnosine, isotope-labeled derivatives of the dipeptide are not commercially available. In this work we present a fast, flexible, and convenient strategy for the synthesis of the 13C-labeled carnosine analogs and their application as internal standards for the quantitation of carnosine and anserine in a biological matrix.
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•Quantitation of carnosine in biological matrices is essential for its applications.•Isotope-labeled internal standards allow accurate quantitation in mass spectroscopy.•A flexible and convenient synthesis of 13C-labeled d-carnosine and d-anserine is presented.•13C-labeled carnosine analogs are employed as internal standards in HPLC-MS analysis.•Carnosine and anserine are quantified in mouse muscle tissue.
Muscular dystrophies are severe disorders due to mutations in structural genes, and are characterized by skeletal muscle wasting, compromised patient mobility, and respiratory functions. Although ...previous works suggested enhancing regeneration and muscle mass as therapeutic strategies, these led to no long-term benefits in humans. Mice lacking the transcription factor Nfix have delayed regeneration and a shift toward an oxidative fiber type. Here, we show that ablating or silencing the transcription factor Nfix ameliorates pathology in several forms of muscular dystrophy. Silencing Nfix in postnatal dystrophic mice, when the first signs of the disease already occurred, rescues the pathology and, conversely, Nfix overexpression in dystrophic muscles increases regeneration and markedly exacerbates the pathology. We therefore offer a proof of principle for a novel therapeutic approach for muscular dystrophies based on delaying muscle regeneration.
Macrophages (MPs) are immune cells which are crucial for tissue repair. In skeletal muscle regeneration, pro-inflammatory cells first infiltrate to promote myogenic cell proliferation, then they ...switch into an anti-inflammatory phenotype to sustain myogenic cells differentiation and myofiber formation. This phenotypical switch is induced by dead cell phagocytosis. We previously demonstrated that the transcription factor Nfix, a member of the nuclear factor I (Nfi) family, plays a pivotal role during muscle development, regeneration and in the progression of muscular dystrophies. Here, we show that Nfix is mainly expressed by anti-inflammatory macrophages. Upon acute injury, mice deleted for Nfix in myeloid line displayed a significant defect in the process of muscle regeneration. Indeed, Nfix is involved in the macrophage phenotypical switch and macrophages lacking Nfix failed to adopt an anti-inflammatory phenotype and interact with myogenic cells. Moreover, we demonstrated that phagocytosis induced by the inhibition of the RhoA-ROCK1 pathway leads to Nfix expression and, consequently, to acquisition of the anti-inflammatory phenotype. Our study identified Nfix as a link between RhoA-ROCK1-dependent phagocytosis and the MP phenotypical switch, thus establishing a new role for Nfix in macrophage biology for the resolution of inflammation and tissue repair.
In contrast to conventional gene therapy vectors, human artificial chromosomes (HACs) are episomal vectors that can carry large regions of the genome containing regulatory elements. So far, HACs have ...not been used as vectors in gene therapy for treating genetic disorders. Here, we report the amelioration of the dystrophic phenotype in the mdx mouse model of Duchenne muscular dystrophy (DMD) using a combination of HAC-mediated gene replacement and transplantation with blood vessel-associated stem cells (mesoangioblasts). We first genetically corrected mesoangioblasts from dystrophic mdx mice with a HAC vector containing the entire (2.4 Mb) human dystrophin genetic locus. Genetically corrected mesoangioblasts engrafted robustly and gave rise to many dystrophin-positive muscle fibers and muscle satellite cells in dystrophic mice, leading to morphological and functional amelioration of the phenotype that lasted for up to 8 months after transplantation. Thus, HAC-mediated gene transfer shows efficacy in a preclinical model of DMD and offers potential for future clinical translation.
Insights into the evolution of non-model organisms are limited by the lack of reference genomes of high accuracy, completeness, and contiguity. Here, we present a chromosome-level, ...karyotype-validated reference genome and pangenome for the barn swallow (Hirundo rustica). We complement these resources with a reference-free multialignment of the reference genome with other bird genomes and with the most comprehensive catalog of genetic markers for the barn swallow. We identify potentially conserved and accelerated genes using the multialignment and estimate genome-wide linkage disequilibrium using the catalog. We use the pangenome to infer core and accessory genes and to detect variants using it as a reference. Overall, these resources will foster population genomics studies in the barn swallow, enable detection of candidate genes in comparative genomics studies, and help reduce bias toward a single reference genome.
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•Generation of a high-quality annotated reference genome and pangenome for barn swallow•Generation of comprehensive barn swallow genetic variants catalog•Multispecies alignment and variants catalog detected list of candidate genes•Pangenome improves read mapping and variant calling
Secomandi et al. present a chromosome-level genome and pangenome for the barn swallow. They generate a large catalog of worldwide genetic variants and identify genomic regions potentially under selection. They also compare the barn swallow genome with that of other bird species to detect conserved and accelerated genes.
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