Experimental oxygen concentration influences rates of mitochondrial hydrogen peroxide release from cardiac and skeletal muscle preparations

Mitochondria utilize the majority of oxygen (O2) consumed by aerobic organisms as the final electron acceptor for oxidative phosphorylation (OXPHOS) but also to generate reactive oxygen species (mtROS) that participate in cell signaling, physiological hormesis, and disease pathogenesis. Simultaneous monitoring of mtROS production and oxygen consumption ( Jo2) from tissue mitochondrial preparations is an attractive investigative approach, but it introduces dynamic changes in media O2 concentration ([O2]) that can confound experimental results and interpretation. We utilized high-resolution fluorespirometry to evaluate Jo2 and hydrogen peroxide release ( Jh2o2) from isolated mitochondria (Mt), permeabilized fibers (Pf), and tissue homogenates (Hm) prepared from murine heart and skeletal muscle across a range of experimental [O2]s typically encountered during respirometry protocols (400–50 µM). Results demonstrate notable variations in Jh2o2 across tissues and sample preparations during nonphosphorylating (LEAK) and OXPHOS-linked respiration states at 250 µM [O2] but a linear decline in Jh2o2 of 5–15% per 50-µM decrease in chamber [O2] in all samples. Jo2 was generally stable in Mt and Hm across [O2]s above 50 µM but tended to decline below 250 µM in Pf, leading to wide variations in assayed rates of Jh2o2/O2 across chamber [O2]s and sample preparations. Development of chemical background fluorescence from the H2O2 probe (Amplex Red) was also O2 sensitive, emphasizing relevant calibration considerations. This study highlights the importance of monitoring and reporting the chamber [O2] at which Jo2 and Jh2o2 are recorded during fluorespirometry experiments and provides a basis for selecting sample preparations for studies addressing the role of mtROS in physiology and disease.