Assessing the function of mitochondria in cytosolic context in human skeletal muscle: Adopting high-resolution respirometry to homogenate of needle biopsy tissue samples

• High-resolution respirometry in homogenates of human skeletal muscle has been compared to isolated mitochondria.
• Homogenisation technique is simple (sample preparation takes only ~ 10 min) and results are reproducible.
• Both outer mitochondrial membrane and complex I are well preserved during homogenization.
• Homogenates do not tolerate cold storage. Respirometry has to be performed immediately after homogenization.
• There is no difference in results from samples obtained by Bergstrom needle as compared to open surgical biopsy.

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 (~ 10–15% increment of oxygen flux after addition of cytochrome c) and to complex I (~ 70% of electron flux). Homogenate retained ~ 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.