Specific effects of OP2113 on ROS/H₂O₂ production at different sites in isolated rat heart mitochondria.

The rates of ROS/H2O2 production were measured under conditions specifically designed by MD brand's group for the identification of the different mitochondrial ROS production sites [21, 22, 24, 38](see Methods section for details). The effects of increasing concentrations of OP2113 (from 2.5 to 80 μM) were tested for each condition of ROS/H2O2 production. Data are based on 3 to 5 independent experiments, each performed in duplicate or triplicate. *P versus control group.</p

Main sites of oxygen radical production by isolated mitochondria.

This scheme gives background information regarding the potential sites for ROS production, using different mitochondrial-targeted drugs in a protocol specifically designed for a study of the effect of a compound on mitochondrial ROS/H2O2 production. When mitochondria are energized by a combination of complex I (malate-glutamate) and complex II (succinate) substrates and in the absence of specific inhibitors of the complexes, ROS production is considered as mainly derived from reverse electron transport (RET) at site IQ (A). Of note, ROS produced by complex I, either at site IQ (quinone site) or at site If (flavin site), are delivered to the inner- (matrix-) side of the inner mitochondrial membrane. In the presence of rotenone, a specific inhibitor of complex I which blocks RET, ROS production is thought to occur predominantly at site IIIQO, possibly with residual production at site If (B) [24]. If complex III is inhibited as it is the case in the presence of antimycin a, the reduced to oxidized quinone ratio increases due to complex II activity and triggers an increase in ROS production, essentially at site IIIQO (C). Finally, myxothiazol (inhibitor of complex III site IIIQO) is supposed to block complex III ROS production, and the remaining production is usually ascribed to the flavin site of complex I for which there is no known inhibitor (D)[25]. However, due to matrix antioxidant machinery, the possibility that some ROS/H2O2 produced in the matrix may escape to the measurement has been suggested from experiments carried out with submitochondrial particles [37]. A typical recording of ROS production kinetics by mitochondria during the designed inhibitor sequence is presented in E.</p

OP2113 improves the recovery of the contractile performance of Langendorff-perfused rat heart during the reperfusion phase following ischemia.

This figure shows the time course of the rate-pressure product of two groups of isolated langendorff-perfused rat hearts (n = 6 in each group) during a protocol of ischemia-reperfusion. Rate Pressure Product (RPP), the product of the left ventricular developed pressure (mmHg/beat) by heart rate (beat/min) is used as an index of contractile performance and is expressed as % of baseline value measured at the end of the stabilization period (control 28932±2467 mmHg/min, OP2113 31653±4611 mmHg/min). Each heart was allowed to stabilize during 10 min (green background) before perfusion of vehicle (control group, black trace) or 10 μM OP2113 (+ OP2113 group, grey trace) during 10 more minutes (orange background). Hearts were then submitted to 30 min zero-flow ischemia (blue background) before 120 min of reperfusion in the absence of vehicle or OP2113 (purple background). For more details on the perfusion protocol see also the Methods section and S3 File. Data are expressed as the mean ± SEM for 6 independent experiments. The thickness of the line represents the error bars.</p

Scheme presenting the specific effects of OP2113 on ROS production by complex I.

Scheme presenting the specific effects of OP2113 on ROS production by complex I.</p

The lack of an effect of OP2113 on mitochondrial oxidative phosphorylation.

Panel A: This diagram illustrates a classical respiration assay and presents O2 concentration (solid lines) and O2 consumption slope (dotted lines) of rat heart mitochondria after a short incubation in the presence of the vehicle (blue lines) or 80 μM OP2113 (red lines). Respiratory substrates (glutamate + malate in this assay) were added, triggering the onset of oxygen consumption (substrate state, blue background) and then phosphorylation was promoted by the addition of 1 mM ADP in order to obtain the maximal oxidative phosphorylation rate (State 3, red background) [4, 35]. Finally, atractyloside (ATR), which inhibits the ADP/ATP translocator, was added to yield the mitochondrial leak rate (green background) under non phosphorylating conditions (State 4). Panels B to D: The three energetic states were studied in the presence of increasing concentrations of OP2113 (5, 20, and 80 μM), and with mitochondria oxidizing different respiratory substrate combinations: glutamate + malate (B), which feeds electrons to complex I; succinate in the presence of rotenone (C), supplying electrons to complex II; and glutamate + malate + succinate (D) feeding electrons to both complexes I & II. Results are based on 3 independent experiments. No significant differences in mitochondrial respiration rates were noted after the addition of OP2113.</p

Effect of OP2113 on infarct size.

Hearts were pre-treated (OP2113) or not (Control) with OP2113 before 30 min of ischemia followed by 120 min of reperfusion (see Fig 5). Left panel, grey dots represent individual experiments and black dots mean ± SEM (see Methods, n = 6 independent experiments for each condition). Right panel, typical pictures obtained in the control and OP2113 groups following TTC staining. The tissues appearing in red are living tissues stained with TTC, whereas damaged tissue appears white.</p

Effect of OP2113 on ROS/H₂O₂ production by isolated rat heart mitochondria.

The rate of ROS/H2O2 production by isolated rat heart mitochondria respiring on glutamate + malate + succinate was measured under the different conditions described in Fig 2 in the presence of increasing concentrations of OP2113 (5 to 80 μM). In the absence of specific inhibitors of the complexes (see Fig 2A), ROS/H2O2 production is maximal and is mainly derived from reverse electron transport at site IQ (see comments in Fig 2). Following blockade of reverse electron transport by addition of rotenone (1.5 μM) (Fig 2B), ROS/H2O2 production is reduced and occurs essentially at site IIIQO. The subsequent addition of Antimycin a (2 μM) (Fig 2C), which blocks the transfer of electrons to oxygen, increases this ROS/H2O2 production. Finally, myxothiazol (0.2 μM) blocks ROS/H2O2 production at site IIIQO (Fig 2D). Data are based on 4 independent experiments, each performed in duplicate. *P versus control group.</p

Experimental determination of oxidation rate, phosphorylation rate and ADP/ATP concentrations.

<p>Our experimental set-up was composed of an oxygraph, a spectrophotometer and a luminometer. An optic fiber, connected to the spectrophotometer, was inserted in the oxygraphic vessel (picture on the top-left hand corner). Mitochondrial oxidation rate was determined using the Clark electrode of the oxygraph. Phosphorylation rate was assessed, with the help of the optic fiber, by the continuous monitoring of NADPH production in the oxygraphic vessel. Samplings were performed at the onset and the end of the recording to assess both ADP and ATP concentrations using a bioluminescence-based assay with the help of a luminometer (picture on the top-right hand corner). For clarity, all parameters that were measured during each experiment are highlighted by colored circles. HK: hexokinase, G6PDH: glucose 6 phosphate dehydrogenase, G6P: glucose 6 phosphate, OM: outer membrane, IM: inner membrane.</p

Changes in the P/O ratio as a function of ADP concentration in liver and muscle mitochondria.

<p>P/O ratio was determined by calculating the phosphorylation to oxidation rates ratio. Data for liver (n = 4) and muscle (n = 5) are presented as mean ± SD. Differences were tested using an unpaired bilateral student's t-test. ** p<0.01 between liver and muscle.</p

Typical recording of oxidation rate, phosphorylation rate and ADP/ATP concentrations.

<p>Oxidation and phosphorylation rates were recorded simultaneously in liver and muscle mitochondria oxidizing glutamate+malate+succinate as substrates. Mitochondrial protein concentration in the oxygraphic vessel was 25 µg.ml⁻¹. Steady states of oxygen consumption and phosphorylation rates were obtained using the coupled enzymatic system composed of Glucose (5 mM) - Hexokinase (2.5 U.ml⁻¹, Sigma-Aldrich, H4502) - Glucose-6-phosphotate dehydrogenase (2.5 U.ml⁻¹, Sigma-Aldrich, G6378) - NADP⁺ (1.6 mM). Dashed arrows correspond to the sampling of measurement medium taken from the oxygraphic vessel during each recording for determination of ADP and ATP concentrations.</p