Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells

Abstract

Analysis of mitochondrial function is central to the study of intracellular energy metabolism, mechanisms of cell death and pathophysiology of a variety of human diseases, including myopathies, neurodegenerative diseases and cancer. However, important properties of mitochondria differ in vivo and in vitro. Here, we describe a protocol for the analysis of functional mitochondria in situ, without the isolation of organelles, in selectively permeabilized cells or muscle fibers using digitonin or saponin. A specially designed substrate/inhibitor titration approach allows the step-by-step analysis of several mitochondrial complexes. This protocol allows the detailed characterization of functional mitochondria in their normal intracellular position and assembly, preserving essential interactions with other organelles. As only a small amount of tissue is required for analysis, the protocol can be used in diagnostic settings in clinical studies. The permeabilization procedure and specific titration analysis can be completed in 2 h.

Are substrate use during exercise and mitochondrial respiratory capacity decreased in arm and leg muscle in type 2 diabetes?

“…Those parts were stored at -80°C until further analysis. The last part was put in BIOPS (see Kuznetsov et al [22] for content) and immediately analysed for mitochondrial function.…”

“…Respirometric protocol A detailed description for measuring mitochondrial respiration has been described elsewhere [22]. Minor changes were made and the temperature in the respirometer (Oxygraph-2k; Oroboros, Innsbruck, Austria) during the measurement was 37°C (physiological temperature).…”

Can Peripheral Blood Mononuclear Cells Be Used as a Proxy for Mitochondrial Dysfunction in Vital Organs During Hemorrhagic Shock and Resuscitation?

“…Given the inability to isolate an adequate amount of mitochondria from PBMCs in this model, PBMC oxygen consumption was measured using high-resolution respirometry (Oxygraph-2k Oroboros Instruments, Innsbruck, Austria) (17). Freshly isolated non-permeabilized PBMC (10 6 cells/mL) were suspended in the 2 mL glass chamber (37 C°) in Hank’s Balanced Salt Solution (HBSS) containing 5.5 mM glucose, 1 mM pyruvate and 10 mM HEPES (pH 7.4).…”

“…A standard substrate/inhibitor titration protocol was used for functional analysis of mitochondrial respiratory chain complexes (17). Individual complex activity directly impacts overall mitochondrial function and directly correlates with basal and maximal respiration.…”

“…Antimycin A (5 μM) was then added to inhibit complex III (CIII) in order to measure non-mitochondrial respiration; followed by addition of the artificial substrates for complex IV, TMPD (0.5 mM) and ascorbate (2 mM). In order to ensure that the respiratory capacity of complex IV (CIV) was not limited by cytochrome c depletion, respiration was measured after the addition cytochrome c (10 μM) (17). This protocol was completed within 60 to 70 min.…”