Abstract
The primary molecular endpoint for many Duchenne muscular dystrophy (DMD) clinical trials is the induction, or increase in production, of dystrophin protein in striated muscle. For accurate endpoint analysis, it is essential to have reliable, robust and objective quantification methodologies capable of detecting subtle changes in dystrophin expression. In this work, we present further development and optimisation of an automated, digital, high-throughput script for quantitative analysis of multiplexed immunofluorescent (IF) whole slide images (WSI) of dystrophin, dystrophin associated proteins (DAPs) and regenerating myofibres (fetal/developmental myosin-positive) in transverse sections of DMD, Becker muscular dystrophy (BMD) and control skeletal muscle biopsies. The script enables extensive automated assessment of myofibre morphometrics, protein quantification by fluorescence intensity and sarcolemmal circumference coverage, colocalisation data for dystrophin and DAPs and regeneration at the single myofibre and whole section level. Analysis revealed significant variation in dystrophin intensity, percentage coverage and amounts of DAPs between differing DMD and BMD samples. Accurate identification of dystrophin via a novel background subtraction method allowed differential assessment of DAP fluorescence intensity within dystrophin positive compared to dystrophin negative sarcolemma regions. This enabled surrogate quantification of molecular functionality of dystrophin in the assembly of the DAP complex. Overall, the digital script is capable of multiparametric and unbiased analysis of markers of myofibre regeneration and dystrophin in relation to key DAPs and enabled better characterisation of the heterogeneity in dystrophin expression patterns seen in BMD and DMD alongside the surrogate assessment of molecular functionality of dystrophin. Both these aspects will be of significant relevance to ongoing and future DMD and other muscular dystrophies clinical trials to help benchmark therapeutic efficacy.
Highlights
There is a growing need for reliable, replicable, automated, high-throughput, digital image analysis techniques that can be utilised for the pathological assessment of diagnostic and clinical trial samples
There has been a rapid expansion in the number of Duchenne muscular dystrophy (DMD) clinical trials [49]
This is achieved via interaction of dystrophin with the subsarcolemmal actin network, and many other proteins at the sarcolemma, such as αsarcoglycan, β-dystroglycan and nNOS, that together form the dystrophin associated protein complex (DAPC) [19]
Summary
There is a growing need for reliable, replicable, automated, high-throughput, digital image analysis techniques that can be utilised for the pathological assessment of diagnostic and clinical trial samples. There is a growing interest in the use of automated digital scripts for the analysis of pathological end-points and/or outcome measures in clinical trial samples [35]. Dystrophin is essential for stabilisation of muscle fibres during contraction by linking the extracellular matrix and myofibre cytoskeleton. This is achieved via interaction of dystrophin with the subsarcolemmal actin network, and many other proteins at the sarcolemma, such as αsarcoglycan, β-dystroglycan and nNOS, that together form the dystrophin associated protein complex (DAPC) [19]. Regenerating myofibres re-express developmental and fetal myosin heavy chains – isoforms that are normally highly expressed in embryonic and fetal skeletal muscles [42]
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