title: Assessing the function of mitochondria in cytosolic context in human skeletal muscle: adopting high-resolution respirometry to homogenate of needle biopsy tissue samples.
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 (~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 2-3h of cold storage.
authors.affiliation: Laboratory of Bioenergetics, 3rd Faculty of Medicine, Charles University in Prague, Czech Republic.
authors.forename: Jakub
authors.initials: J
authors.lastname: Ziak
authors.affiliation: Laboratory of Bioenergetics, 3rd Faculty of Medicine, Charles University in Prague, Czech Republic.
authors.forename: Adela
authors.initials: A
authors.lastname: Krajcova
authors.affiliation: Laboratory of Bioenergetics, 3rd Faculty of Medicine, Charles University in Prague, Czech Republic.
authors.forename: Katerina
authors.initials: K
authors.lastname: Jiroutkova
authors.affiliation: Department of Molecular Biology, 3rd Faculty of Medicine, Charles University in Prague, Czech Republic.
authors.forename: Vlasta
authors.initials: V
authors.lastname: Nemcova
authors.affiliation: Department of Orthopaedic Surgery, Kralovske Vinohrady University Hospital in Prague, Czech Republic.
authors.forename: Valer
authors.initials: V
authors.lastname: Dzupa
authors.affiliation: Laboratory of Bioenergetics, 3rd Faculty of Medicine, Charles University in Prague, Czech Republic. Electronic address: fduska@yahoo.com.
authors.forename: Frantisek
authors.initials: F
authors.lastname: Duska
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001707:Biopsy, Needle; D002457:Cell Extracts; D019069:Cell Respiration; D003584:Cytological Techniques / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D008928:Mitochondria / Q000502:physiology*; D028361:Mitochondrial Diseases / Q000175:diagnosis*; D018482:Muscle, Skeletal / Q000502:physiology*; D013048:Specimen Handling / Q000379:methods*
chemical_list: D002457:Cell Extracts
keywords: High resolution respirometry; Homogenate; Human skeletal muscle; Mitochondria; Needle biopsy