Methodological considerations for the determination of mitochondrial ADP sensitivity in skeletal muscle

Abstract

In skeletal muscle, mitochondria adapt to physiological (i.e. exercise, aging) and pathological scenarios (i.e. insulin resistance, muscle atrophy). Due to the kinetic regulation by adenylates within the oxidative phosphorylation system, a small increase in free ADP (_f[ADP]) within the cell results in a rapid compensatory increment in ATP production and oxygen consumption, conferring to mitochondria the unique ability to detect, and respond, to small changes in the energetic status of the cells. The advent of high-resolution oxygraphy potentiated the studies on mitochondrial bioenergetics where it is now possible to record mitochondrial O₂ consumption in real-time with high sensitivity in various tissues and substrate protocols. While most of the studies rely on saturating concentrations of substrates and ADP to test the maximal respiratory capacity of mitochondria, such approaches may not fully recapitulate physiological conditions by which these organelles are exposed within skeletal muscle cells. Over the years, we and others have employed a mitochondrial ADP sensitivity assay, where we determine mitochondrial bioenergetic responses to a wide range of ADP concentrations, scaling from the physiological levels found in resting skeletal muscle cells (mM) to saturating values (mM). Here, we reviewed this methodology by offering practical guidance and insights from experiments performed in our laboratory, as well as examples of the applicability of such a protocol.

Cite:

Brunetta HS, Holloway GP (2024) Methodological considerations for the determination of mitochondrial ADP sensitivity in skeletal muscle. Bioenerg Commun 2024.4. https://doi.org/10.26124/bec:2024-0004