Thesis - Aurélien Brun

Context - Skeletal muscle integrity is vital for metabolic health and physical resilience. It is compromised by aging and disease, leading to sarcopenia (muscle mass and function loss) and myosteatosis (aberrant lipid accumulation). While both are major defects, their precise mechanistic interplay remains poorly understood. Sarcopenia stems from protein imbalance, whereas myosteatosis, characterized by intra- and extramyocellular fat depositions linked to fibroadipogenic progenitor (FAP) expansion, is hypothesized to culminate in mitochondrial exhaustion and myofiber atrophy. Critically, both conditions impact myogenic progenitor regenerative capacity. Given their frequent co-occurrence in patients, understanding if they co-evolve or trigger one another is crucial. This thesis investigates cellular mechanisms of lipid-induced muscle stress and assesses the therapeutic potential of long-chain polyunsaturated fatty acids (LCPUFAs) to counteract muscle atrophy and myosteatosis. We hypothesized that specific LCPUFAs could modulate muscle protein balance and improve muscle cell differentiation under metabolic stress.
Method - Our approach combined reductionist cell models with a translational primary cell model derived from muscle biopsies from a human cohort. We developed a robust C2C12 myotube lipotoxicity model using palmitic acid (500µM), precisely characterizing the timeline of lipid accumulation, protein dynamics (e.g., 50% reduction in insulin-mediated protein anabolism), oxidative stress, and mitochondrial network disaggregation. Subsequent co-incubation with 50µM LCPUFAs revealed their rescue potential. Following promising C2C12 results, we transitioned to human primary myotubes, conducting live confocal microscopy, insulin-induced protein synthesis kinetics, and transcriptomic analyses. To extend these findings, we established the first primary human muscle cell biobank from 47 gastrointestinal cancer patients for whom muscle mass and whether muscle is lost or gained at the time of biopsy was known. Systematic screening of myogenic progenitor fusion index matched with muscle characteristics was determined. This platform enabled the evaluation of the effect of LCPUFA in human plasma-derived media, mimicking physiological fatty acid levels.
Results - Key findings demonstrated that in both C2C12 and human myotube lipotoxicity models, docosahexaenoic acid (DHA) and arachidonic acid (ARA) robustly reversed metabolic defects, lipid accumulation, anabolic resistance, and mitochondrial disaggregation, supported by transcriptomic evidence. eicosapentaenoic acid (EPA) and α-linolenic acid (ALA) showed only temporary attenuation. Furthermore, systematic screening in human-derived myogenic stem cell pools revealed that physiological doses of ALA and EPA significantly promoted fusion index (+20%), highlighting a distinct role in muscle regeneration. Methodologically, this work also led to the development of TRUEFAD, a bioimagery package for automated morphometry in muscle cell culture and cross-sections.
Conclusion - This research provides compelling in vitro evidence, from C2C12 to human patient-derived primary cells, that specific LCPUFAs can mitigate core aspects of muscle pathology relevant to atrophy and myosteatosis. Our findings suggest LCPUFAs can restore muscle protein balance under lipotoxic stress and enhance muscle regeneration through improved differentiation, positioning them as a viable nutritional or therapeutic strategy against age-related and chronic disease-induced muscle wasting. The observed complexity in PUFA response warrants further investigation. Importantly, the establishment of a human myogenic progenitor biobank and the development of high-throughput bioimagery tools significantly enhance future myology research capacity.