Research into fundamental principles and the testing of therapeutic hypotheses for treatment of human disease is commonly performed on mouse models of human diseases. Although this is often the only ...practicable approach, it carries a number of caveats arising from differences between the two species. This review focuses on the example of skeletal muscle disease, in particular muscular dystrophy, to identify some of the principal classes of obstacles to translation of data from mouse to humans. Of these, the difference in scale is one of the most commonly ignored, and is of particular interest because it has quite major repercussions for evaluation of some classes of intervention and of outcome criteria, while having comparatively little bearing on others. Likewise, inter‐species differences and similarities in cell and molecular biological mechanisms underlying development, growth and response to pathological processes should be considered on an individual basis. An awareness of such distinctions is crucial if we are to avoid misjudging the likely applicability to humans of results obtained on mouse models.
The mdx mouse is heavily used as a model of Duchenne muscular dystrophy. In order to make the best use of it, we need to understand in what ways it is similar and in what ways it is different. Most important are the differences in size and in the mode of growth.
Skeletal muscle tissue provides mechanical force for locomotion of all vertebrate animals. It is prone to damage from acute physical trauma and physiological stress. To cope with this, it possesses a ...tremendous capacity for rapid and effective repair that is widely held to be accomplished by the satellite cells lying between the muscle fiber plasmalemma and the basement membrane. Cell transplantation and lineage-tracing studies have demonstrated that Pax7-expressing (Pax7(+)) satellite cells can repair damaged muscle tissue repeatedly after several bouts of acute injury. These findings provided evidence that Pax7(+) cells are muscle stem cells. However, stem cells from a variety of other origins are also reported to contribute to myofibers upon engraftment into muscles, questioning whether satellite cells are the only stem cell source for muscle regeneration. Here, we have engineered genetic ablation of Pax7(+) cells to test whether there is any significant contribution to muscle regeneration after acute injury from cells other than this source. We find that such elimination of Pax7(+) cells completely blocks regenerative myogenesis either following injury to the tibialis anterior (TA) muscle or after transplantation of extensor digitorum longus (EDL) muscles into nude mice. As Pax7 is specifically expressed in satellite cells, we conclude that they are essential for acute injury-induced muscle regeneration. It remains to be established whether there is any significant role for stem cells of other origins. The implications of our results for muscle stem cell-based therapy are discussed.
Muscle loss due to fibrotic or adipogenic replacement of myofibers is common in muscle diseases and muscle-resident fibro/adipogenic precursors (FAPs) are implicated in this process. While ...FAP-mediated muscle fibrosis is widely studied in muscle diseases, the role of FAPs in adipogenic muscle loss is not well understood. Adipogenic muscle loss is a feature of limb girdle muscular dystrophy 2B (LGMD2B) - a disease caused by mutations in dysferlin. Here we show that FAPs cause the adipogenic loss of dysferlin deficient muscle. Progressive accumulation of Annexin A2 (AnxA2) in the myofiber matrix causes FAP differentiation into adipocytes. Lack of AnxA2 prevents FAP adipogenesis, protecting against adipogenic loss of dysferlinopathic muscle while exogenous AnxA2 enhances muscle loss. Pharmacological inhibition of FAP adipogenesis arrests adipogenic replacement and degeneration of dysferlin-deficient muscle. These results demonstrate the pathogenic role of FAPs in LGMD2B and establish these cells as therapeutic targets to ameliorate muscle loss in patients.
Satellite cells are situated beneath the basal lamina that surrounds each myofiber and function as myogenic precursors for muscle growth and repair. The source of satellite cell renewal is ...controversial and has been suggested to be a separate circulating or interstitial stem cell population. Here, we transplant single intact myofibers into radiation-ablated muscles and demonstrate that satellite cells are self-sufficient as a source of regeneration. As few as seven satellite cells associated with one transplanted myofiber can generate over 100 new myofibers containing thousands of myonuclei. Moreover, the transplanted satellite cells vigorously self-renew, expanding in number and repopulating the host muscle with new satellite cells. Following experimental injury, these cells proliferate extensively and regenerate large compact clusters of myofibers. Thus, within a normally stable tissue, the satellite cell exhibits archetypal stem cell properties and is competent to form the basal origin of adult muscle regeneration.
We sought to determine the mechanisms underlying failure of muscle regeneration that is observed in dystrophic muscle through hypothesis generation using muscle profiling data (human dystrophy and ...murine regeneration). We found that transforming growth factor β-centered networks strongly associated with pathological fibrosis and failed regeneration were also induced during normal regeneration but at distinct time points. We hypothesized that asynchronously regenerating microenvironments are an underlying driver of fibrosis and failed regeneration. We validated this hypothesis using an experimental model of focal asynchronous bouts of muscle regeneration in wild-type (WT) mice. A chronic inflammatory state and reduced mitochondrial oxidative capacity are observed in bouts separated by 4 d, whereas a chronic profibrotic state was seen in bouts separated by 10 d. Treatment of asynchronously remodeling WT muscle with either prednisone or VBP15 mitigated the molecular phenotype. Our asynchronous regeneration model for pathological fibrosis and muscle wasting in the muscular dystrophies is likely generalizable to tissue failure in chronic inflammatory states in other regenerative tissues.
Skeletal muscle growth and regeneration are attributed to satellite cells - muscle stem cells resident beneath the basal lamina that surrounds each myofibre. Quiescent satellite cells express the ...transcription factor Pax7 and when activated, coexpress Pax7 with MyoD. Most then proliferate, downregulate Pax7 and differentiate. By contrast, others maintain Pax7 but lose MyoD and return to a state resembling quiescence. Here we show that Pax7 is able to drive transcription in quiescent and activated satellite cells, and continues to do so in those cells that subsequently cease proliferation and withdraw from immediate differentiation. We found that constitutive expression of Pax7 in satellite-cell-derived myoblasts did not affect MyoD expression or proliferation. Although maintained expression of Pax7 delayed the onset of myogenin expression it did not prevent, and was compatible with, myogenic differentiation. Constitutive Pax7 expression in a Pax7-null C2C12 subclone increased the proportion of cells expressing MyoD, showing that Pax7 can act genetically upstream of MyoD. However these Pax7-null cells were unable to differentiate into normal myotubes in the presence of Pax7. Therefore Pax7 may be involved in maintaining proliferation and preventing precocious differentiation, but does not promote quiescence.
While spinal muscular atrophy (SMA) is characterized by motor neuron degeneration, it is unclear whether and how much survival motor neuron (SMN) protein deficiency in muscle contributes to the ...pathophysiology of the disease. There is increasing evidence from patients and SMA model organisms that SMN deficiency causes intrinsic muscle defects. Here we investigated the role of SMN in muscle development using muscle cell lines and primary myoblasts. Formation of multinucleate myotubes by SMN-deficient muscle cells is inhibited at a stage preceding plasma membrane fusion. We found increased expression and reduced induction of key muscle development factors, such as MyoD and myogenin, with differentiation of SMN-deficient cells. In addition, SMN-deficient muscle cells had impaired cell migration and altered organization of focal adhesions and the actin cytoskeleton. Partially restoring SMN inhibited the premature expression of muscle differentiation markers, corrected the cytoskeletal abnormalities and improved myoblast fusion. These findings are consistent with a role for SMN in myotube formation through effects on muscle differentiation and cell motility.
The amount and distribution of dystrophin protein in myofibers and muscle is highly variable in Becker muscular dystrophy and in exon-skipping trials for Duchenne muscular dystrophy. Here, we ...investigate a molecular basis for this variability. In muscle from Becker patients sharing the same exon 45–47 in-frame deletion, dystrophin levels negatively correlate with microRNAs predicted to target dystrophin. Seven microRNAs inhibit dystrophin expression in vitro, and three are validated in vivo (miR-146b/miR-374a/miR-31). microRNAs are expressed in dystrophic myofibers and increase with age and disease severity. In exon-skipping-treated mdx mice, microRNAs are significantly higher in muscles with low dystrophin rescue. TNF-α increases microRNA levels in vitro whereas NFκB inhibition blocks this in vitro and in vivo. Collectively, these data show that microRNAs contribute to variable dystrophin levels in muscular dystrophy. Our findings suggest a model where chronic inflammation in distinct microenvironments induces pathological microRNAs, initiating a self-sustaining feedback loop that exacerbates disease progression.
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•miRNAs in muscle microenvironments cause variable dystrophin in muscular dystrophy•miRNAs are elevated in dystrophic myofibers and increase with disease severity•Inflammatory cytokines induce miRNAs, and anti-inflammatories block their expression•miRNAs provide a precision medicine target in dystrophy and exon skipping
Fiorillo et al. find that miRNAs in muscle promote variable dystrophin levels in muscular dystrophies. Dystrophin-targeting miRNAs reduce dystrophin and increase with disease severity. Innate inflammatory pathways induce miRNAs, whereas NFκB inhibition dampens induction. These events initiate a self-sustaining feedback loop, exacerbating disease progression. Thus, miRNA inhibition in dystrophic muscle could provide therapeutic targets.
Exon skipping is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD), employing morpholino antisense oligonucleotides (PMO-AO) to exclude disruptive exons from the mutant DMD ...transcript and elicit production of truncated dystrophin protein. Clinical trials for PMO show variable and sporadic dystrophin rescue. Here, we show that robust PMO uptake and efficient production of dystrophin following PMO administration coincide with areas of myofiber regeneration and inflammation. PMO localization is sustained in inflammatory foci where it enters macrophages, actively differentiating myoblasts and newly forming myotubes. We conclude that efficient PMO delivery into muscle requires two concomitant events: first, accumulation and retention of PMO within inflammatory foci associated with dystrophic lesions, and second, fusion of PMO-loaded myoblasts into repairing myofibers. Identification of these factors accounts for the variability in clinical trials and suggests strategies to improve this therapeutic approach to DMD.Exon skipping is a strategy for the treatment of Duchenne muscular dystrophy, but has variable efficacy. Here, the authors show that dystrophin restoration occurs preferentially in areas of myofiber regeneration, where antisense oligonucleotides are stored in macrophages and delivered to myoblasts and newly formed myotubes.
Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the
gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino ...oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the
reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig
gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy.
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