Measurement of Mitochondrial Respiration ‐ Comparison of Two Respirometers: MitoCellMt200A versus O2k

Abstract

Background Mitochondrial function is a critical determinant of cardiomyocyte survival after ischemia/reperfusion (I/R) and thus an important target of cardioprotection. A reliable measurement of mitochondrial respiration is therefore essential for cardioprotection research. Methods Mitochondria were isolated by differential centrifugation from naïve rat hearts (n=8-14) or isolated buffer-perfused rat hearts subjected to 30 min global zero-flow ischemia and 10 min reperfusion (n=8-14). Mitochondrial respiration was measured in 100 µg/ml mitochondria at 37°C during magnetic stirring using two different Clark-type electrode respirometers, the MitoCellMt200A from Strathkelvin (Glasgow, UK) and the O2k from Oroboros (Innsbruck, AT). Two different respiration buffers (incubation buffer and MiR05 buffer) were used as recommended: in mmol/L; incubation buffer: 125.0 KCl, 10.0 3-(N-morpholino) propane sulfonoic acid, 2.0 MgCl2, 5.0 KH2PO4, and 0.2 ethylene glycol tetra-acetic acid; MiR05 buffer: 0.5 ethylene glycol tetra-acetic acid, 3.0 MgCl2, 60.0 lactobionic acid, 20.0 taurine, 10.0 KH2PO4, 20.0 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid, and 110.0 D-sucrose. Glutamate and malate (5.0 mmol/L, each) were added as complex I substrates. Complex I respiration was measured after adding adenosine diphosphate (ADP, 1.0 mmol/L). Complex IV respiration was stimulated by adding N,N,N′,N′-tetra-methyl-p-phenylene diamine (TMPD, 300 µmol/L) and ascorbate (3 mmol/L). Mitochondrial respiration was normalized to mitochondrial protein content. Results Baseline, ADP-stimulated complex I and complex IV respiration were higher in mitochondria isolated from naïve rat hearts than in those isolated from rat hearts, which had undergone global zero-flow ischemia and reperfusion - independently of the used respirometers and the used respiration buffers (see Table 1). Baseline respiration was l higher in the MitoCellMt200A respirometer - independently of the respiration buffer used (see Table 1). ADP-stimulated complex I respiration was comparable between the respirometers, whereas the MiR05 buffer resulted in higher respiration than the incubation buffer (see Table 1). Complex IV respiration, as an indicator for equal loading of the chambers with viable mitochondria, was comparable between the respirometers and the buffers but, as expected, reduced after ischemia/reperfusion (see Table 1). Conclusion Independently of the Clark-type electrode respirometers or respiration buffers used, we identify a reduction of mitochondrial respiration after myocardial I/R. However, the respiration buffer composition determines the magnitude of mitochondrial ADP-stimulated complex I respiration. A lower sensitivity of the MitoCellMt200A respirometer in comparison to the O2k high-resolution respirometer at low oxygen consumption may affect the accuracy of baseline respiration measurements.