Journal: Scientific Reports

Article Title: High throughput screening of mitochondrial bioenergetics in human differentiated myotubes identifies novel enhancers of muscle performance in aged mice

doi: 10.1038/s41598-018-27614-8

Figure Lengend Snippet: Multi-dimensional screen for mitochondrial activators. ( a ) Screening strategy: Primary screening of Sanofi’s pure natural compound library was conducted in differentiated immortalized human myotubes by measuring total ATP content after compound treatment in low glucose medium. 240 primary hits were further examined in a dual ATP assay with and without the oxidative phosphorylation inhibitor oligomycin to assess compound effects on mitochondrial and glycolytic ATP content. Finally, mitochondrial effects of active compounds were verified by direct measurement of cellular respiration in differentiated primary human myotubes. ( b ) Volcano plot illustrating nominal significance versus change of mean relative ATP content for all 7949 compounds screened. Standard deviation of data was 7.5% leading to the identification of 240 primary hits (shown in green box) which increased ATP by ≥2 SD. ( c ) Dot plot of mean ATP in % of control for all tested compounds. Selected active and toxic compounds are highlighted in green and red respectively. ( d ) Schematic representation of determination of mitochondrial, glycolytic and total ATP content from experimental data. For each compound cellular ATP levels were measured in untreated and oligomycin-treated myotubes (n ≥ 4); the difference was considered as mitochondrial ATP. ( e ) Venn diagram illustrating compound sets with combinatorial effects on mitochondrial, total and glycolytic ATP. Number of compounds in each category is given. The green circle marks the selected hit set defined by significantly increasing total ATP or decreasing glycolytic ATP and by increasing mitochondrial ATP level more than 7.5%. ( f ) Schematic overview of cellular respiration profiling using Seahorse XFe96 analyzer. Primary differentiated myotubes were treated 48 h with compounds or DMSO and cellular respiration was analyzed in real-time. Different components of mitochondrial respiration were isolated by treating myotubes with oligomycin, FCCP and antimycin/rotenone. Compounds that increased one or several components of mitochondrial respiration significantly were considered as hits. ( g ) Venn diagram highlighting the distribution of hits with differential effects on basal respiration, spare capacity, maximal respiration or ATP production (n ≥ 8, 1–3 independent experiments). OCR: oxygen consumption rate.

Article Snippet: Figure 1 Multi-dimensional screen for mitochondrial activators. ( a ) Screening strategy: Primary screening of Sanofi’s pure natural compound library was conducted in differentiated immortalized human myotubes by measuring total ATP content after compound treatment in low glucose medium.

Techniques: Drug discovery, ATP Assay, Phospho-proteomics, Standard Deviation, Control, Isolation