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

Abstract

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.

Mitochondrial capacity and reactive oxygen species production during hypoxia and reoxygenation in the ocean quahog, Arctica islandica

“…Thus, the observed ROS efflux in A. islandica mitochondria in both the LEAK and OXPHOS states is probably due to Complex III and, possibly, Complex II, which is also a ROS-generating site in some species (Dröse, 2013;Grivennikova et al, 2017). Notably, the fraction of O 2 converted to H 2 O 2 in A. islandica mitochondria is relatively high (1.9-11.5%) compared with that in other species, such as the hypoxia-tolerant bivalve M. arenaria with 5% (Ouillon et al, 2021), Salmo salar with 0.5% (Gerber et al, 2021), the hypoxia-tolerant epaulette shark with 2.5% (Hickey et al, 2012) and terrestrial organisms such as mice with 3% (Li Puma et al, 2020). This indicates that low ROS efflux in A. islandica is a by-product of slow mitochondrial metabolism rather than exceptionally low electron leak.…”

Developing chicken cardiac muscle mitochondria are resistant to variations in incubation oxygen levels

“…While there are no differences in the ROS production under the mitochondria substrate and inhibitor conditions used in this study, it is still possible that ROS levels differ within the cardiac tissues in vivo under the in ovo incubation conditions ( Murphy, 2009 ). The ROS production measured from permeabilized fibers must be done under high oxygen conditions in the respirometer so as oxygen flux is not limited ( Li Puma et al, 2020 ) and thus may provide an upper estimation of ROS production capacity under the metabolic states studied. In the in vivo state, cardiac mitochondria will likely be in an OXPHOS state somewhere below the maximal OXPHOS state measured here and not a LEAK state, so ROS production would be lower ( Murphy, 2009 ).…”

“…Additionally, it is unclear what cellular antioxidant capacity the permeabilized cardiac fibers are capable of in our preparation. It is possible the ROS production levels are similar because of differential antioxidant capacities retained within the permeabilized cells and they were all measured at elevated oxygen levels in the respirometry chamber ( Li Puma et al, 2020 ).…”

Direct Cardiac Actions of Sodium-Glucose Cotransporter 2 Inhibition Improve Mitochondrial Function and Attenuate Oxidative Stress in Pressure Overload-Induced Heart Failure

“…A high-resolution respirometer (Oxygraphy2k, Oroboros Instruments GmbH) was equipped with a two-channel fluorescence optical module to simultaneously monitor oxygen concentration and H 2 O 2 production as previously described ( 16 , 17 ) with a data collection interval of 0.2 s. H 2 O 2 release was measured from the fluorescence of resorufin formed from Amplex UltraRed reacting with H 2 O 2 in the presence of horseradish peroxidase. Oxygen polarography was performed at 37°C in O2k-chambers.…”