Cooling Uncouples Differentially ROS Production from Respiration and Ca2+ Homeostasis Dynamic in Brain and Heart Mitochondria

Abstract

Hypothermia provides an effective neuro and cardio-protection in clinical settings implying ischemia/reperfusion injury (I/R). At the onset of reperfusion, succinate-induced reactive oxygen species (ROS) production, impaired oxidative phosphorylation (OXPHOS), and decreased Ca²⁺ retention capacity (CRC) concur to mitochondrial damages. We explored the effects of temperature from 6 to 37 °C on OXPHOS, ROS production, and CRC, using isolated mitochondria from mouse brain and heart. Oxygen consumption and ROS production was gradually inhibited when cooling from 37 to 6 °C in brain mitochondria (BM) and heart mitochondria (HM). The decrease in ROS production was gradual in BM but steeper between 31 and 20 °C in HM. In respiring mitochondria, the gradual activation of complex II, in addition of complex I, dramatically enhanced ROS production at all temperatures without modifying respiration, likely because of ubiquinone over-reduction. Finally, CRC values were linearly increased by cooling in both BM and HM. In BM, the Ca²⁺ uptake rate by the mitochondrial calcium uniporter (MCU) decreased by 2.7-fold between 25 and 37 °C, but decreased by 5.7-fold between 25 and 37 °C in HM. In conclusion, mild cold (25-37 °C) exerts differential inhibitory effects by preventing ROS production, by reverse electron transfer (RET) in BM, and by reducing MCU-mediated Ca²⁺ uptake rate in BM and HM.

Keywords: Ca2+ retention capacity; MCU; brain mitochondria; cold; heart mitochondria; ischemia/reperfusion; mitochondrial functions; mitochondrial respiration; reactive oxygen species; reverse electron transport.

Figure 1

The dependance of mitochondrial oxygen consumption on the temperature. (A) Mitochondrial oxygen consumption in brain mitochondria, in nmol O₂/min/mg protein, according to temperature (6, 10, 15, 20, 25, 31, and 37 °C). For each temperature, each experiment was performed on the same sample in the same oxygraph cuvette: GluMP was first added, followed by rotenone and succinate. n = 4 to 7 for each temperature. Data are expressed as mean ± IC95%. (B) as A for heart mitochondria. (C,D) Arrhenius plots of data from (A,B) panels, respectively, expressed as % of maximum. Mean ± IC95%. Arrows indicate characteristic inflexion point in the activation energy.

Figure 2

The dependance of mitochondrial ROS production rate on the temperature. (A) ROS production rate in brain mitochondria, in arbitrary fluorescence unit/sec, according to temperature (6, 10, 15, 20, 25, 31, and 37 °C). For each temperature, each experiment was performed on the same sample in the same cuvette: GluMP (respectively, succinate) was first added, followed by rotenone. n = 4 to 7 for each temperature. Data are expressed as mean ± IC95%. (B) as A for heart mitochondria. (C,D) Arrhenius plots of data from (A,B) panels, respectively, expressed as % of maximum. Mean ± IC95%. Arrows indicate characteristic inflexion point in the activation energy.

Figure 3

The dependance of mitochondrial ROS production rate on increasing succinate concentrations. (A) ROS production rate in brain mitochondria, in arbitrary fluorescence unit/s (F.I.s⁻¹), according to increasing succinate concentrations at different temperature (15, 20, 25, 31, and 37 °C). For each temperature, each experiment was performed on the same sample in the same cuvette: GluMP was first added, followed by increased amount of succinate. n = 3 for each temperature. Data are expressed as mean ± SEM. Dotted lines represent logistic regression IC95%. (B) as A for heart mitochondria. (C). Succinate concentration EC50 for maximal ROS production rate at each temperature, extracted from the logistic regression shown in the panels (A,B). Data are expressed as mean ± SEM. (D). Histogram represents Succ-induced burst in ROS production. The values were calculated by divided the ratio of ROS production rate calculated in presence of Succ (w) over its value in absence of Succ (wo). Data are expressed as mean ± IC95%; n = 3 for brain mitochondria and =4 for heart mitochondria. (E). Representative time trace of O₂ consumption in mitochondria fed with 5 mM GluMP. Addition of unlimited ADP induced complex V-induced ETC activity. Gradual supplementation of either 1 m Succ or 1 mM GluMP did not significantly modified the respiration rate. (F). Representative time trace of O₂ consumption in mitochondria fed with 5 mM Succ. Addition of unlimited ADP was not sufficient to induce significantly complex V-induced ETC activity. Gradual supplementation of either 0.1 mM or 1 mM GluMP significantly increased respiration until it reached maximal activity as in panel E. Absence of the initial 5 mM Succ (red line) did not prevent respiration induction when 0.1 mM GluMP was added.

Figure 4

The dependance of Ca²⁺ retention capacity on the temperature. Example of Ca²⁺ retention capacity (CRC) traces for brain (A) and heart (B) at different temperatures. (C,D). CRC in nmol of Ca²⁺ calculated by the calibration curve method at different temperature (15, 20, 25, 31, and 37 °C). n = 3 for each temperature. Data are expressed as median ± 95%CI. (E). CRC value normalized by the estimated content of live mitochondria in each brain and heart mitochondria samples.

Figure 5

The dependance of Ca²⁺ uptake rate and steady-state [Ca²⁺] concentration in mitochondria on the temperature. (A), [Ca²⁺] over time at each temperature presented as the Calcium Green 5N fluorescence decay after the second pulse of Ca²⁺ of each CRC assay. Values were normalized by the peak value at origin of each curve. Experiments conducted with or without NCLX inhibitor (CGP). Dotted lines represent logistic regression IC95%. (B), as A for heart mitochondria. (C,D), biplots represent interplay between [Ca²⁺] at equilibrium and Ca²⁺ uptake rate in mitochondria of brain and heart, respectively. Data were extracted from Figure 5A,B. Data are expressed as mean ± SEM (E), Correlation between steady-state [Ca²⁺] concentration and CRC values in BM and HM. Ψm estimation was figured out in either state 2 respiration (F) or state 3 respiration (G) in both brain and heart mitochondria at different temperatures as reported in the M and M Section.