Quantification of Mitochondrial Oxidative Phosphorylation in Skeletal Muscle

Mitochondrial oxygen consumption stimulated by ADP and inorganic phosphate is often measured as an index of ATP synthesis derived from oxidative phosphorylation. This measurement is typically performed by monitoring the rate at which oxygen disappears from a solution containing isolated mitochondria or either intact or permeabilized cells and tissue. Termed "respirometry," this method has become increasingly popular in numerous research disciplines focused on cellular metabolism, including the broader field of skeletal muscle physiology. Here, we describe how respirometry is performed in permeabilized skeletal muscle fiber bundles. This technique involves mechanically separating skeletal muscle fibers followed by exposure to a cell membrane-permeabilizing agent (e.g., saponin). This technique is thought to preserve the mitochondrial network, morphology, and interactions with surrounding intracellular structures. The method can be performed on small tissue samples and permits flexibility in designing substrate titration protocols to target specific mitochondrial pathways.