Mitochondrial Na⁺overload is caused by oxidative stress and leads to activation of the caspase 3‐dependent apoptotic machinery

Abstract

Oxidative stress is one of the major causes of cell death. Using time-lapse confocal recording of live cardiomyocytes, we showed that H2O2 (OH*) caused a marked increase in Na+ and Ca2+ levels in both the cytosol ([Na]cyt, [Ca]cyt) and mitochondria ([Na]m, [Ca]m). The H2O2-induced intracellular Na+ ([Na]i) overload contributed to the H2O2-induced [Ca]cyt/[Ca]m overload via activation of the reverse mode of the Na-Ca exchanger. When myocytes were treated for 40 min with 100 microM H2O2 in normal medium, then returned to H2O2-free medium, the percentage of apoptotic cells increased from 4% at 0 h to 55 and 85% at 4.5 and 16 h, respectively. H2O2-induced apoptosis was completely prevented by using Na-free, but not Ca-free, medium. When a Na+ ionophore cocktail in Ca-free medium was used instead of H2O2 to increase the [Na]i by more than 30 mM without any change in the [Ca]i, cytochrome c release and caspase 3-dependent apoptosis occurred, showing that [Na]i overload per se induced apoptosis. We also showed that the increase in the mitochondrial, but not the cytosolic, Na+ levels resulted in the opening of the permeation transition pore, followed by cytochrome c release. Our findings therefore suggest that H2O2-induced [Na]m overload is an important upstream signal for the apoptotic machinery, and the prevention of [Na]m overload thus represents a particularly attractive target for strategies aimed at preventing oxidative stress-induced cell death.

Non-Lethal Levels of Oxidative Stress in Response to Short-Term Intermittent Hypoxia Enhance Ca2+Handling in Neonatal Rat Cardiomyocytes

“…Thus, cardiomyocytes were treated with H 2 O 2 (10, 30, 50 and 100 µM) to mimic different degrees of ROS-induced cardiomyocyte death. H 2 O 2 -induced ROS generation, which significantly increases cell death in cardiomyocytes, was not noted at lower concentrations (approximately 10 µM) [20,32] but was observed at higher concentrations (>50 µM) [33,34]. Compared with the number of cardiomyocytes treated with 10 µM H 2 O 2 , the number of cardiomyocytes was not decreased by 4 days of IH exposure, which suggests that 4 days of IH induces non-lethal oxidative stress in cardiomyocytes.…”

Activation of the transient receptor potential M2 channel and poly(ADP-ribose) polymerase is involved in oxidative stress-induced cardiomyocyte death

“…However, other explanations cannot be ruled out. 4,11,33 apoptosome formation, and caspase 3-dependent nuclear condensation/ fragmentation. Intracellular ATP/NAD þ are also depleted, due to NAD þ re-synthesis from ATP and nicotineamide (NA), 13 resulting in membrane permeabilization and DNA fragmentation/condensation in a single myocyte One important finding is that TRPM2 opening, resulting in the Na þ /Ca 2 þ overload, is involved in the H 2 O 2 -activated apoptotic machinery.…”

“…34 Using confocal microscopy with a thin optical section (0.04 mm), it can be used to simultaneously report changes in the [Na þ ] cyt and [Na þ ] m , since only a small proportion of the probe is found in the cytosolic compartment, 34 the majority being trapped by the negative mitochondrial potential because of the positive charge of the probe. Since the nuclear membrane is no barrier to cytosolic ion movement, 35,36 averaging the signal over a small nuclear optical section (ER-free and mitochondria-free) or the mitochondria (identified by a mitochondrial marker, MTG, last frame in Figures 2a and c Figure S1) or after clamping the [Na þ ] i at 60 mM using Na-ionophore cocktail (5 mM gramicidin D, 40 mM monensin, and 100 mM strophanthidine) in Ca-free medium, 33 Significant chromatin condensation (arrows in Figure 2fi) and DNA fragmentation ( Figure 2fii, green for TUNEL ( þ ) staining) were seen after 4.5 h wash-out of H 2 O 2 or of Naionophore cocktail/Ca-free medium (Figure 2g). Approximately 18% inhibition of condensation was seen in Ca-free medium (Figure 2g), in which the [Ca 2 þ ] m overload should be totally abolished.…”

“…12 33 The Na-ionoph/Ca-free medium contained 5 mM gramicidin D, 40 mM monensin, and 100 mM strophanthidine in 60 mM NaCl-containing medium. Note that the Ca-free medium has little inhibitory effect on the…”

“…A Leica SP confocal laser-scanning imaging system equipped with a 63 Â nicotinamide (NA) 1.32 oil immersion objective was used. The following equations 33 were used in Figure 2 …”