Gene-mediated restoration of normal myofiber elasticity in dystrophic muscles.
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ABSTRACT:
SUBMITTER: Puttini S
PROVIDER: S-EPMC2834969 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Autophagy is emerging as a key regulatory process during skeletal muscle development, regeneration and homeostasis, and deregulated autophagy has been implicated in muscular disorders and age-related muscle decline. We have monitored autophagy in muscles of mdx mice and human Duchenne muscular dystrophy (DMD) patients at different stages of disease. Our data show that autophagy is activated during the early, compensatory regenerative stages of DMD. A progressive reduction was observed during mdx disease progression, in coincidence with the functional exhaustion of satellite cell-mediated regeneration and accumulation of fibrosis. Moreover, pharmacological manipulation of autophagy can influence disease progression in mdx mice. Of note, studies performed in regenerating muscles of wild-type mice revealed an essential role of autophagy in the activation of satellite cells upon muscle injury. These results support the notion that regeneration-associated autophagy contributes to the early compensatory stage of DMD progression, and interventions that extend activation of autophagy might be beneficial in the treatment of DMD. Thus, autophagy could be a 'disease modifier' targeted by interventions aimed to promote regeneration and delay disease progression in DMD.
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Project description:Homogenates of hindleg muscle were obtained from control and dystrophic male hamsters, 30 and 190 days of age, and were used to prepare the postmicrosomal pH5-supernatant fraction. The activity of this fraction in the incorporation of [14C]phenylalanyl-tRNA into peptides was increased in the dystrophic-muscle preparations. No such increase was found in brain or liver preparations from dystrophic hamsters. The increased capacity for aminoacyl-tRNA binding that was observed in preparations from dystrophic animals is discussed.
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Project description:PurposeThe cytoskeleton of the extraocular muscles (EOMs) is significantly different from that of other muscles. We aimed to investigate the role of obscurin, a fundamental cytoskeletal protein, in the EOMs.MethodsThe distribution of obscurin in human and zebrafish EOMs was compared using immunohistochemistry. The two obscurin genes in zebrafish, obscna and obscnb, were knocked out using CRISPR/Cas9, and the EOMs were investigated using immunohistochemistry, qPCR, and in situ hybridization. The optokinetic reflex (OKR) in five-day-old larvae and adult obscna-/-;obscnb-/- and sibling control zebrafish was analyzed. Swimming distance was recorded at the same age.ResultsThe obscurin distribution pattern was similar in human and zebrafish EOMs. The proportion of slow and fast myofibers was reduced in obscna-/-;obscnb-/- zebrafish EOMs but not in trunk muscle, whereas the number of myofibers containing cardiac myosin myh7 was significantly increased in EOMs of obscurin double mutants. Loss of obscurin resulted in less OKRs in zebrafish larvae but not in adult zebrafish.ConclusionsObscurin expression is conserved in normal human and zebrafish EOMs. Loss of obscurin induces a myofiber type shift in the EOMs, with upregulation of cardiac myosin heavy chain, myh7, showing an adaptation strategy in EOMs. Our model will facilitate further studies in conditions related to obscurin.
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Project description:Progressive muscle degeneration followed by dilated cardiomyopathy is a hallmark of muscular dystrophy. Stem cell therapy is suggested to replace diseased myofibers by healthy myofibers, although so far, we are faced by low efficiencies of migration and engraftment of stem cells. Chemokines are signalling proteins guiding cell migration and have been shown to tightly regulate muscle tissue repair. We sought to determine which chemokines are expressed in dystrophic muscles undergoing tissue remodelling. Therefore, we analysed the expression of chemokines and chemokine receptors in skeletal and cardiac muscles from Sarcoglycan-? null, Sarcoglycan-? null and immunodeficient Sgc?-null mice. We found that several chemokines are dysregulated in dystrophic muscles. We further show that one of these, platelet-derived growth factor-B, promotes interstitial stem cell migration. This finding provides perspective to an approachable mechanism for improving stem cell homing towards dystrophic muscles.
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Project description:PurposeTo investigate changes in myofiber composition in the global layer (GL) and orbital layer (OL) of extraocular muscles (EOMs) from terminal amyotrophic lateral sclerosis (ALS) donors.MethodsMedial recti muscles collected postmortem from spinal-onset ALS, bulbar-onset ALS, and healthy control donors were processed for immunofluorescence with antibodies against myosin heavy chain (MyHC) IIa, MyHCI, MyHCeom, laminin, neurofilaments, synaptophysin, acetylcholine receptor γ-subunit, and α-bungarotoxin.ResultsThe proportion of myofibers containing MyHCIIa was significantly smaller and MyHCeom was significantly larger in the GL of spinal-onset ALS and bulbar-onset ALS donors compared to control donors. Changes in the GL were more prominent in the bulbar-onset ALS donors, with a significantly larger proportion of myofibers containing MyHCeom being present compared to spinal-onset ALS donors. There were no significant differences in the myofiber composition in the OL. In the spinal-onset ALS donors, the proportions of myofibers containing MyHCIIa in the GL and MyHCeom in the OL were significantly correlated with the disease duration. Neurofilament and synaptophysin were present at motor endplates of myofibers containing MyHCeom in ALS donors.ConclusionsThe EOMs of terminal ALS donors displayed changes in the fast-type myofiber composition in the GL, with a more pronounced alteration in bulbar-onset ALS donors. Our results align with the worse prognosis and subclinical changes in eye movement function previously observed in bulbar-onset ALS patients and suggest that the myofibers in the OL might be more resistant to the pathological process in ALS.
| S-EPMC10207955 | biostudies-literature
Project description:Gene therapy approaches for DMD using recombinant adeno-associated viral (rAAV) vectors to deliver miniaturized (or micro) dystrophin genes to striated muscles have shown significant progress. However, concerns remain about the potential for immune responses against dystrophin in some patients. Utrophin, a developmental paralogue of dystrophin, may provide a viable treatment option. Here we examine the functional capacity of an rAAV-mediated microutrophin (?Utrn) therapy in the mdx4cv mouse model of DMD. We found that rAAV-?Utrn led to improvement in dystrophic histopathology & mostly restored the architecture of the neuromuscular and myotendinous junctions. Physiological studies of tibialis anterior muscles indicated peak force maintenance, with partial improvement of specific force. A fundamental question for ?Utrn therapeutics is not only can it replace critical functions of dystrophin, but whether full-length utrophin impacts the therapeutic efficacy of the smaller, highly expressed ?Utrn. As such, we found that ?Utrn significantly reduced the spacing of the costameric lattice relative to full-length utrophin. Further, immunostaining suggested the improvement in dystrophic pathophysiology was largely influenced by favored correction of fast 2b fibers. However, unlike ?Utrn, ?dystrophin (?Dys) expression did not show this fiber type preference. Interestingly, ?Utrn was better able to protect 2a and 2d fibers in mdx:utrn-/- mice than in mdx4cv mice where the endogenous full-length utrophin was most prevalent. Altogether, these data are consistent with the role of steric hindrance between full-length utrophin & ?Utrn within the sarcolemma. Understanding the stoichiometry of this effect may be important for predicting clinical efficacy.
| S-EPMC7682874 | biostudies-literature
Project description:Ectopic calcification (EC) of myofibers is a pathological feature of muscle damage in Duchenne muscular dystrophy (DMD). Mineralisation of muscle tissue occurs concomitantly with macrophage infiltration, suggesting a link between ectopic mineral deposition and inflammation. One potential link is the P2X7 purinoceptor, a key trigger of inflammation, which is expressed on macrophages but also up-regulated in dystrophic muscle cells. To investigate the role of P2X7 in dystrophic calcification, we utilised the Dmd mdx-βgeo dystrophin-null mouse model of DMD crossed with a global P2X7 knockout (P2rx7 -/- ) or with our novel P2X7 knockin-knockout mouse (P2x7 KiKo ), which expresses P2X7 in macrophages but not muscle cells. Total loss of P2X7 increased EC, indicating that P2X7 overexpression is a protective mechanism against dystrophic mineralisation. Given that muscle-specific P2X7 ablation did not affect dystrophic EC, this underlined the role of P2X7 receptor expression on the inflammatory cells. Serum phosphate reflected dystrophic calcification, with the highest serum phosphate levels found in genotypes with the most ectopic mineral. To further investigate the underlying mechanisms, we measured phosphate release from cells in vitro, and found that dystrophic myoblasts released less phosphate than non-dystrophic cells. Treatment with P2X7 antagonists increased phosphate release from both dystrophic and control myoblasts indicating that muscle cells are a potential source of secreted phosphate while macrophages protect against ectopic mineralisation. Treatment of cells with high phosphate media engendered mineral deposition, which was decreased in the presence of the P2X7 agonist BzATP, particularly in cultures of dystrophic cells, further supporting a protective role for P2X7 against ectopic mineralisation in dystrophic muscle.
| S-EPMC9333129 | biostudies-literature
Project description:?-Dystroglycanopathy (?-DGP) is a group of muscular dystrophy characterized by abnormal glycosylation of ?-dystroglycan (?-DG), including Fukuyama congenital muscular dystrophy (FCMD), muscle-eye-brain disease, Walker-Warburg syndrome, and congenital muscular dystrophy type 1D (MDC1D), etc. LARGE, the causative gene for MDC1D, encodes a glycosyltransferase to form [-3Xyl-?1,3GlcA?1-] polymer in the terminal end of the post-phosphoryl moiety, which is essential for ?-DG function. It has been proposed that LARGE possesses the great potential to rescue glycosylation defects in ?-DGPs regardless of causative genes. However, the in vivo therapeutic benefit of using LARGE activity is controversial. To explore the conditions needed for successful LARGE gene therapy, here we used Large-deficient and fukutin-deficient mouse models for MDC1D and FCMD, respectively. Myofibre-selective LARGE expression via systemic adeno-associated viral gene transfer ameliorated dystrophic pathology of Large-deficient mice even when intervention occurred after disease manifestation. However, the same strategy failed to ameliorate the dystrophic phenotype of fukutin-conditional knockout mice. Furthermore, forced expression of Large in fukutin-deficient embryonic stem cells also failed to recover ?-DG glycosylation, however coexpression with fukutin strongly enhanced ?-DG glycosylation. Together, our data demonstrated that fukutin is required for LARGE-dependent rescue of ?-DG glycosylation, and thus suggesting new directions for LARGE-utilizing therapy targeted to myofibres.
| S-EPMC4321163 | biostudies-literature
Project description:INTRODUCTION: Several evidences show that muscles have an endocrine function. Glucocorticoid, estrogen, progesterone, and testosterone receptors have already been found in normal skeletal muscles, but not in dystrophic muscles. METHODS: The gene expression of hormone receptors was compared between dystrophic and healthy muscles in mdx and C57BL6 mice strains. RESULTS: The mdx mice showed a significant increase in the steroid receptors mRNA when compared to the C57BL6 mice: levels of androgen(s) receptors in the heart, estrogen receptors alpha in the EDL, and estrogen receptors beta in the quadriceps were increased. In addition, significant lowered levels of some other hormone receptors were found: corticosteroid receptors in the EDL and estrogen receptors alpha in the quadriceps. CONCLUSION: Dystrophic muscles bear significant differences in the expression of hormone receptors when compared to the C57BL6 mice strain. The importance of such differences is yet to be better understood.
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Project description:Skeletal muscles are composed of different myofiber types characterized by the expression of myosin heavy chain isoforms, which can be affected by physical activity, aging, and pathological conditions. Here, we present a step-by-step high-throughput semi-automated approach for performing myofiber type quantification of entire human or mouse muscle tissue sections, including immunofluorescence staining, image acquisition, processing, and quantification. For complete details on the use and execution of this protocol, please refer to Abbassi-Daloii et al. (2022).1.
| S-EPMC9898062 | biostudies-literature