Deep Bioenergetic Phenotyping for Healthy Human Aging

Mitochondrial dysfunction(definition) is a hallmark of aging and driver of pathologies such as frailty, diabetes, and neurodegeneration. Due to the varied nature of these disease states, a diagnostic tool capable of assessing mitochondrial bioenergetics paired with a scalable platform for testing drug interventions will improve the success rate of mitochondrial-focused therapeutics. To this end, the objectives of this study were to: (1) improve two major mitochondrial bioenergetic assays—respirometry and mitochondrial membrane potential (ΔψM)—in order to standardize surveying of mitochondrial health in human cohort studies; and (2) leverage scalable assay technology for the design of high-throughput drug screenings to reveal mitochondrial modulators. We carried out deep bioenergetic profiling—a comprehensive functional assessment of substrate utilization, electron transfer, and energy production in mitochondria—in a plate-based respirometry assay using isolated mitochondria from human skeletal muscle biopsies (SkM) and analyzed fluorescent microscopy data from human peripheral blood mononuclear cells (PBMCs). Each assay was validated before use in clinical studies. We examined the correlations between exercise, mitochondrial respiration, and clinical parameters in elderly individuals by comparing the “gold standard”—a high-level benchmark—found in competition-trained endurance athletes with their age-equivalent, untrained counterparts. We also quantified bioenergetic differences in subpopulations of PBMCs between old and young donors using immunocytochemistry (ICC), precluding the need for flow-based cell sorting. Our work utilized automation-enabled robotics in the development of two novel assays which improve the depth of bioenergetic phenotyping in clinical settings for both skeletal muscle and PBMCs.