Equine atypical myopathy: towards a targeted therapy | Myopathie atypique équine : vers une thérapie ciblée

Equine atypical myopathy (AM) is an environmental intoxication linked to the ingestion of seeds and seedlings of the sycamore maple (Acer pseudoplatanus) in Europe, while the box elder (Acer negundo) is implicated in the United States. The toxins primary involved in this condition are hypoglycin A (HGA) and methylenecyclopropylglycine (MCPrG). The poisoning is characterized by a disturbance of the energy metabolism inducing degeneration and necrosis of predominantly postural and respiratory skeletal muscles, as well as the myocardium. Clinical signs resemble those of fatal acute rhabdomyolysis in the majority of cases. Poisoning mainly occurs in equids grazing in pastures, and clinical repercussions have also been described in camels, gnus and Père David’s deer. The intoxication, which has increased in prevalence since the turn of the millennium, is influenced by various factors, including climate change and the invasion of forest ecosystems by the sycamore maple. These factors have the potential to worsen the prevalence of the disease in the future. A decade ago, the cause of this disease was elucidated. However, without targeted therapy, the outcome of this intoxication remains largely fatal to this day. The present thesis is thus situated in a context focused on the development of tools necessary for exploring therapeutic perspectives. It employs innovative techniques through the lens of fundamental research. The goal is to deepen our understanding of the pathophysiological mechanisms triggered by sycamore maple intoxication. In a first study, we undertook an extensive label-free proteomic investigation using tandem mass spectrometry (LC-MS/MS), subsequently validated by Western blots. The primary objective of this study was to gain a deeper understanding of the mechanisms underlying toxin-induced damage. This understanding could potentially provide new approaches for therapeutic interventions. Furthermore, by leveraging proteomic-based technologies, we were able to identify potential diagnostic biomarkers, assess pathogenicity, and improve the interpretation of functional pathways involved in this intoxication. This initial study also helped refine the selection of cell lines subsequently used in the experimental section dedicated to cell culture as well as guide the choice of therapeutic molecule categories to target as a priority. Our second and third studies were conducted with the aim of developing an in vitro model of equine atypical myopathy using equine skeletal myoblasts. To validate this model, a variety of tools were employed, including high-resolution respirometry, as well as fluorescence and luminescence-based cytotoxicity and viability analyses. These analytical procedures significantly contributed to a deepening 8 of our understanding of the molecular and physiological mechanisms of equine atypical myopathy. The analyses sought to elucidate changes in various parameters when cells were exposed to both the toxin and its unconjugated toxic metabolite, methylenecyclopropylacetic acid. The results of this second study led to considering the use of immortalized cell lines to ensure the reproducibility of the in vitro model. Therefore, we used two immortalized cell lines, namely human-origin liver cancer cells (HepG2) and murine-origin immortalized myoblast cells (C2C12). This study aimed to replicate the cytotoxicity and viability tests initially conducted. The primary objective of this third study was to validate a model that could be used for high-throughput screening of therapeutic molecules.