A high–throughput digital script for multiplexed immunofluorescent analysis and quantification of sarcolemmal and sarcomeric proteins in muscular dystrophies

Scaglioni, Dominic; Ellis, Matthew; Catapano, Francesco; Torelli, Silvia; Chambers, D.; Feng, Lucy; Sewry, Caroline A.; Morgan, Jennifer E.; Muntoni, Francesco; Phadke, Rahul

doi:10.1186/s40478-020-00918-5

Cited by 9 publications

(23 citation statements)

References 46 publications

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“…By comparison, DMD generally exhibits high serum creatine kinase levels with severe pathology in many muscles ( 54 ). This is accompanied by a robust regenerative response initially in DMD: for example, the proportion of regenerating fibres expressing developmental MyHC isoforms varied from 38 to 47% in quadriceps biopsies from four DMD patients aged 4–13 years ( 55 ), 24–33% in muscle biopsies from five DMD patients aged 4.3–8.2 years ( 20 ) and a mean of 32% in muscle biopsies from three DMD patients aged 3.3–6.8 years ( 56 ). However, DMD clinical onset is within the first few years of life and so these are also growing muscles.…”

Section: Discussionmentioning

confidence: 99%

Skeletal muscle regeneration in facioscapulohumeral muscular dystrophy is correlated with pathological severity

Banerji

Henderson

Tawil

et al. 2020

Human Molecular Genetics

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Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant myopathy, characterised by slowly progressive skeletal muscle weakness and wasting. While a regenerative response is often provoked in many muscular dystrophies, little is known about whether a regenerative response is regularly elicited in FSHD muscle. For comparison, we also examined the similarly slowly progressing Myotonic Dystrophy type 2 (DM2). To investigate regeneration at the transcriptomic level, we first used the 200 human gene Hallmark Myogenesis list. This Myogenesis biomarker was elevated in FSHD and control healthy myotubes compared to their myoblast counterparts, so is higher in myogenic differentiation. The Myogenesis biomarker was also elevated in muscle biopsies from most independent FSHD, DM2 or Duchenne muscular dystrophy (DMD) studies compared to control biopsies, and on meta-analysis for each condition. The Myogenesis biomarker was also a robust binary discriminator of FSHD, DM2 and DMD from controls. We also analysed muscle regeneration at the protein level by immunolabelling muscle biopsies for Developmental Myosin Heavy Chain. Such immunolabelling revealed one or more regenerating myofibres in 76% of FSHD muscle biopsies from quadriceps and 91% from tibialis anterior. The mean proportion of regenerating myofibres per quadriceps biopsy was 0.48%, significantly less that the 1.72% in the tibialis anterior. All DM2 muscle biopsies contained regenerating myofibres, with a mean of 1.24% per biopsy. Muscle regeneration in FSHD was correlated with the pathological hallmarks of fibre size variation, central nucleation, fibrosis and necrosis/regeneration/inflammation. In summary, the regenerative response in FSHD muscle biopsies correlates with the severity of pathology.

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Section: Discussionmentioning

confidence: 99%

Skeletal muscle regeneration in facioscapulohumeral muscular dystrophy is correlated with pathological severity

Banerji

Henderson

Tawil

et al. 2020

Human Molecular Genetics

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“…We then assessed the fluorescence intensity of α-sarcoglycan and β-dystroglycan in the post-treatment biopsies by comparing the intensity of these DAPs in discrete regions of the sarcolemma that were classified as either dystrophin positive or dystrophin negative. The classification was performed utilising the same method published in our previous manuscript [ 40 ]. In all samples, regions of the sarcolemma that were dystrophin positive had greater α-sarcoglycan (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The analysis was implemented using Definiens Developer XD (Munich), version 2.7.0. A detailed review of the image analysis platform has previously been published [ 40 ].…”

Section: Methodsmentioning

confidence: 99%

“…In the present analysis, we quantified the colocalised expression of dystrophin and key DAPC proteins in clinical trial samples following 48 weeks of therapeutic intervention with golodirsen. Furthermore, using our recently published unbiased high-throughput digital script [ 40 ], we determined for the first time if the levels of dystrophin produced was sufficient to reduce the amount of degeneration in the muscle of the treated patients, an essential assessment of the molecular functionality of the induced dystrophin [ 9 , 31 , 34 ].…”

Section: Introductionmentioning

confidence: 99%

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The administration of antisense oligonucleotide golodirsen reduces pathological regeneration in patients with Duchenne muscular dystrophy

Scaglioni

Catapano

Ellis

et al. 2021

acta neuropathol commun

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During the last decade, multiple clinical trials for Duchenne muscular dystrophy (DMD) have focused on the induction of dystrophin expression using different strategies. Many of these trials have reported a clear increase in dystrophin protein following treatment. However, the low levels of the induced dystrophin protein have raised questions on its functionality. In our present study, using an unbiased, high-throughput digital image analysis platform, we assessed markers of regeneration and levels of dystrophin associated protein via immunofluorescent analysis of whole muscle sections in 25 DMD boys who received 48-weeks treatment with exon 53 skipping morpholino antisense oligonucleotide (PMO) golodirsen. We demonstrate that the de novo dystrophin induced by exon skipping with PMO golodirsen is capable of conferring a histological benefit in treated patients with an increase in dystrophin associated proteins at the dystrophin positive regions of the sarcolemma in post-treatment biopsies. Although 48 weeks treatment with golodirsen did not result in a significant change in the levels of fetal/developmental myosins for the entire cohort, there was a significant negative correlation between the amount of dystrophin and levels of regeneration observed in different biopsy samples. Our results provide, for the first time, evidence of functionality of induced dystrophin following successful therapeutic intervention in the human.

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“…Restoration of the dystrophin-associated complex (DAPC) is a readout of the functionality of the restored dystrophin in myofibres [ 36 , 37 , 38 ]. We have previously investigated the restoration of the DAPC on donor-derived myofibres in mdx nude mice muscle that had been transplanted with mini-dystrophin (ΔR3R13 and ΔH2R23)-expressing muscle cells.…”

Section: Discussionmentioning

confidence: 99%

Effects of Mini-Dystrophin on Dystrophin-Deficient, Human Skeletal Muscle-Derived Cells

Meng

Counsell

Morgan

2020

IJMS

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Background: We are developing a novel therapy for Duchenne muscular dystrophy (DMD), involving the transplantation of autologous, skeletal muscle-derived stem cells that have been genetically corrected to express dystrophin. Dystrophin is normally expressed in activated satellite cells and in differentiated muscle fibres. However, in past preclinical validation studies, dystrophin transgenes have generally been driven by constitutive promoters that would be active at every stage of the myogenic differentiation process, including in proliferating muscle stem cells. It is not known whether artificial dystrophin expression would affect the properties of these cells. Aims: Our aims are to determine if mini-dystrophin expression affects the proliferation or myogenic differentiation of DMD skeletal muscle-derived cells. Methods: Skeletal muscle-derived cells from a DMD patient were transduced with lentivirus coding for mini-dystrophins (R3–R13 spectrin-like repeats (ΔR3R13) or hinge2 to spectrin-like repeats R23 (ΔH2R23)) with EGFP (enhanced green fluorescence protein) fused to the C-terminus, driven by a constitutive promoter, spleen focus-forming virus (SFFV). Transduced cells were purified on the basis of GFP expression. Their proliferation and myogenic differentiation were quantified by ethynyl deoxyuridine (EdU) incorporation and fusion index. Furthermore, dystrophin small interfering ribonucleic acids (siRNAs) were transfected to the cells to reverse the effects of the mini-dystrophin. Finally, a phospho-mitogen-activated protein kinase (MAPK) array assay was performed to investigate signalling pathway changes caused by dystrophin expression. Results: Cell proliferation was not affected in cells transduced with ΔR3R13, but was significantly increased in cells transduced with ΔH2R23. The fusion index of myotubes derived from both ΔR3R13- and ΔH2R23 -expressing cells was significantly compromised in comparison to myotubes derived from non-transduced cells. Dystrophin siRNA transfection restored the differentiation of ΔH2R23-expressing cells. The Erk1/2- signalling pathway is altered in cells transduced with mini-dystrophin constructs. Conclusions: Ectopic expression of dystrophin in cultured human skeletal muscle-derived cells may affect their proliferation and differentiation capacity. Caution should be taken when considering genetic correction of autologous stem cells to express dystrophin driven by a constitutive promoter.

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A high–throughput digital script for multiplexed immunofluorescent analysis and quantification of sarcolemmal and sarcomeric proteins in muscular dystrophies

Cited by 9 publications

References 46 publications

Skeletal muscle regeneration in facioscapulohumeral muscular dystrophy is correlated with pathological severity

Skeletal muscle regeneration in facioscapulohumeral muscular dystrophy is correlated with pathological severity

The administration of antisense oligonucleotide golodirsen reduces pathological regeneration in patients with Duchenne muscular dystrophy

Effects of Mini-Dystrophin on Dystrophin-Deficient, Human Skeletal Muscle-Derived Cells

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