Assaying Mitochondrial Respiration as an Indicator of Cellular Metabolism and Fitness.

Mitochondrial respiration is an essential component of cellular metabolism. It is a process of energy conversion through enzymatically mediated reactions, the energy of taken-up substrates transformed to the ATP production. Seahorse equipment allows to measure oxygen consumption in living cells and estimate key parameters of mitochondrial respiration in real-time mode. Four key mitochondrial respiration parameters could be measured: basal respiration, ATP-production coupled respiration, maximal respiration, and proton leak. This approach demands the application of mitochondrial inhibitors-oligomycin to inhibit ATP synthase, FCCP-to uncouple the inner mitochondrial membrane and allow maximum electron flux through the electron transport chain, rotenone, and antimycin A to inhibit complexes I and III, respectively. This chapter describes two protocols of seahorse measurements performed on iPSC-derived cardiomyocytes and TAZ knock-out C2C12 cell line.