Artificial intelligence workflow quantifying muscle features on Hematoxylin-Eosin stained sections reveals dystrophic phenotype amelioration upon treatment.

Cell segmentation is a key step for a wide variety of biological investigations, especially in the context of muscle science. Currently, automated methods still struggle to perform skeletal muscle fiber quantification on Hematoxylin-Eosin (HE) stained histopathological whole slide images due to low contrast. On the other hand, the Deep Learning algorithm Cellpose offers new perspectives considering its increasing adoption for segmentation of a wide range of cells. Combining two open-source tools, Cellpose and QuPath, we developed MyoSOTHES, an automated Myofibers Segmentation wOrkflow Tuned for HE Staining. MyoSOTHES enables solving segmentation inconsistencies encountered by default Cellpose model in presence of large range size cells and provides information related to muscle Feret's diameter distribution and Centrally Nucleated Fibers, thus depicting muscle health and treatment effects. MyoSOTHES achieves high quality segmentation compared to baseline workflow with a detection F1-score increasing from 0.801 to 0.919 and a Root Mean Square Error (RMSE) on diameter improved by 31%. MyoSOTHES was validated on an animal study featuring gene transfer in [Formula: see text]-Sarcoglycanopathy, for which dose-response effect is visible and conclusions drawn are consistent with those previously published. MyoSOTHES thus paves the way for wide quantification of HE stained muscle sections and retrospective analysis of HE labeled slices used in laboratories for decades.

Muscle regeneration affects Adeno Associated Virus 1 mediated transgene transcription.

Duchenne muscular dystrophy is a severe neuromuscular disease causing a progressive muscle wasting due to mutations in the DMD gene that lead to the absence of dystrophin protein. Adeno-associated virus (AAV)-based therapies aiming to restore dystrophin in muscles, by either exon skipping or microdystrophin expression, are very promising. However, the absence of dystrophin induces cellular perturbations that hinder AAV therapy efficiency. We focused here on the impact of the necrosis-regeneration process leading to nuclear centralization in myofiber, a common feature of human myopathies, on AAV transduction efficiency. We generated centronucleated myofibers by cardiotoxin injection in wild-type muscles prior to AAV injection. Intramuscular injections of AAV1 vectors show that transgene expression was drastically reduced in regenerated muscles, even when the AAV injection occurred 10 months post-regeneration. We show also that AAV genomes were not lost from cardiotoxin regenerated muscle and were properly localised in the myofiber nuclei but were less transcribed leading to muscle transduction defect. A similar defect was observed in muscles of the DMD mouse model mdx. Therefore, the regeneration process per se could participate to the AAV-mediated transduction defect observed in dystrophic muscles which may limit AAV-based therapies.