Development of an in vitro platform to predict and screen combinations of endogenous metabolic modulators capable of influencing muscle physiology

INTRODUCTION: Dysregulated metabolism contributes to muscle wasting which results in decreased quality of life and morbidity and mortality in aging and disease. We previously published that AXA2678, a composition of Endogenous Metabolic Modulators (EMMs) anchored by amino acids (AA), promotes maintenance of muscle mass, prevents muscle fat accumulation, decreases pro-inflammatory cytokines and promotes myogenic myokines in healthy subjects undergoing single limb immobilization (Holloway et al, 2019). To date, no culture system has existed to systematically evaluate muscle metabolism and physiology in vitro. Therefore, we developed a primary human myotube model that enables prediction and screening of EMM compositions and their impact on muscle physiology. Objectives: Primary human myoblasts were differentiated for 5 days using Myoscreen™ (Young et al, 2018). Established myotubes were cultured for 2-4 days in AA concentrations reflecting plasma levels of healthy humans. Metabolic dysfunction was modeled by culturing cells in high glucose, TNFα (10ng/mL), Free Fatty Acids (400uM, 2:1 oleate:palmitate), and AA concentrations reported in frail and sarcopenic individuals. Myotube area, fusion index, protein synthesis (PS), and lipid accumulation were assessed via fluorescence microscopy. Extracellular and intracellular metabolite profiling was performed via LC-MS.

RESULTS: We demonstrated that metabolic activity of this model system was consistent with human skeletal muscle physiology. Primary human myotube cultures consumed branched chain AAs (BCAAs, eg. Leucine, Valine, and Isoleucine) and generated non-essential AAs (NEAAs, eg. Alanine and Glutamine). Changes in extracellular and intracellular metabolite profiles were measured and informed identification of AAs important for influencing anabolism. Altering concentrations and ratios of specific AAs, alone and in combinations, differentially impacted myotube growth and PS. AA compositions that promoted PS also increased BCAA consumption and NEAA production, highlighting the link between AA metabolism and anabolic activity. Simulating metabolic dysfunction in the culture (inflammation, glucose intolerance, and myosteatosis) inhibited myotube anabolism and altered AA metabolism. Additionally, EMM combinations were found to impact myotube physiology in these altered culture conditions.

CONCLUSION: We were able to predict EMM compositions that differentially impacted myotube physiology using a primary cell platform that models human skeletal muscle metabolism. This platform is adaptable to pathophysiological conditions to model the impact of AA metabolism on disease.