Assessing the function of mitochondria in cytosolic context in human skeletal muscle: Adopting high-resolution respirometry to homogenate of needle biopsy tissue samples
2015
Abstract
Using skeletal muscle homogenates for respirometry has many advantages, but the main challenge is avoiding the damage to outer mitochondrial membrane (OMM) and complex I. By optimising the amount of muscle and careful titration of substrates and inhibitors we developed a new protocol and compared it to isolated mitochondria.
We found acceptable damage to OMM (similar to 10-15% increment of oxygen flux after addition of cytochrome c) and to complex I (similar to 70% of electron flux). Homogenate retained similar to 90% of phosphorylation capacity of isolated mitochondria.
The use of fresh homogenate was crucial as mitochondrial function declined rapidly after 2-3 h of cold storage.