Modeling Ischemia/Reperfusion Injury in Rat Heart Isolated Mitochondria
Authors: Sonju Anna A. , Salzman Michele M. , Pille Johannes A. , Hees Josephine E. , Lotze Felicia P. , Cleveland William J. , Balzer Claudius , Riess Matthias L. 
Published: June 2021

Introduction

Severe cellular injuries arise from both the prolonged ischemia during cardiac arrest (CA), as well as during the early phase of reperfusion when cardiopulmonary resuscitation (CPR) is provided. These cellular injuries can include decreased intracellular pH, altered calcium influx, and increased reactive oxygen species (ROS), which in turn can affect mitochondrial function. Our lab has shown that several post‐conditioning strategies delivered at the start of reperfusion attenuate cellular and mitochondrial damage, however it is unclear whether these post‐conditioning strategies directly act on both the cardiac cells and mitochondria. Therefore, the aim of this project is to develop a rat heart isolated mitochondria model in which we can replicate the increase in ROS (e.g., hydrogen peroxide [H2O2]) that occurs in the cellular cytoplasm during ischemia, affecting both cellular and mitochondrial function (e.g., ATP synthesis, oxygen consumption). Development of this model will allow the investigation of whether our post‐conditioning strategies work directly on mitochondria.

Methods

Sprague‐Dawley rats (adult, male) were euthanized, and their hearts removed. Mitochondria were isolated by differential centrifugation, then kept on ice to slow their bioenergetic activity prior to experimental use. To replicate exposure to ROS during ischemia, mitochondria underwent treatment with or without H2O2 (100μM, 200μM, or 500μM) for 10 minutes at room temperature (~23°C) followed by dilution (½) with experimental buffer to simulate reperfusion. Oxygen consumption was recorded using closed cell respirometry and an oxygen meter. Electron transport chain Complex I was examined using the substrates glutamate/malate (G/M), and Complex II was examined using the substrate succinate plus the Complex I inhibitor rotenone (S/R). Statistics: Data expressed as mean ± standard error of the mean (SEM); Unpaired two‐tailed Student's t‐test, p<0.05.

Results

Respiratory control index (RCI) values (n=4 rat hearts; 2 replicates per treatment per experiment) for Complex I were not significantly different between Control vs 100μM, 200μM, and 500μM H2O2. Similar results occurred for Complex II (Control vs 100μM, 200μM, and 500μM H2O2).

Conclusion

Our study shows that exposure of rat heart isolated mitochondria to concentrations of 100μM, 200μM and 500μM H2O2 for 10 minutes does not appear to have a significant impact on mitochondrial oxygen consumption. Further studies will test higher concentrations of H2O2 and/or longer exposure periods to determine whether these changes will mimic the injuries that occur during CA and the early phase of CPR in these isolated mitochondria.

Support or Funding Information

This work was supported by institutional funds, NIH grant 5R01 HL123227, and a Merit Review Award (I01 BX003482) from the U.S. Department of Veterans Affairs Biomedical Laboratory R&D Service.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.