Synergistic triggering of superoxide flashes by mitochondrial Ca2+ uniport and basal reactive oxygen species elevation

Abstract

Mitochondrial superoxide flashes reflect a quantal, bursting mode of reactive oxygen species (ROS) production that arises from stochastic, transient opening of the mitochondrial permeability transition pore (mPTP) in many types of cells and in living animals. However, the regulatory mechanisms and the exact nature of the flash-coupled mPTP remain poorly understood. Here we demonstrate a profound synergistic effect between mitochondrial Ca(2+) uniport and elevated basal ROS production in triggering superoxide flashes in intact cells. Hyperosmotic stress potently augmented the flash activity while simultaneously elevating mitochondrial Ca(2+) and ROS. Blocking mitochondrial Ca(2+) transport by knockdown of MICU1 or MCU, newly identified components of the mitochondrial Ca(2+) uniporter, or scavenging mitochondrial basal ROS markedly diminished the flash response. More importantly, whereas elevating Ca(2+) or ROS production alone was inefficacious in triggering the flashes, concurrent physiological Ca(2+) and ROS elevation served as the most powerful flash activator, increasing the flash incidence by an order of magnitude. Functionally, superoxide flashes in response to hyperosmotic stress participated in the activation of JNK and p38. Thus, physiological levels of mitochondrial Ca(2+) and ROS synergistically regulate stochastic mPTP opening and quantal ROS production in intact cells, marking the flash as a coincidence detector of mitochondrial Ca(2+) and ROS signals.

FIGURE 1.

Hyperactive superoxide flashes induced by hyperosmotic stress. A, representative deviation maps (upper row) and surface plots (lower row) of superoxide flashes registered in HeLa cells expressing mt-cpYFP. The data were extracted from time lapse image stacks (100 frames, 1 s/frame) in basal, high glucose (270 mm d-glucose in Tyrode's solution), and high mannitol (270 mm d-mannitol in Tyrode's solution). Scale bars, 10 μm. B, representative diaries of superoxide flash incidence. Each vertical tick denotes a flash event. C, time course of the flash response to high glucose or mannitol. The data are expressed as the means ± S.E. (n ≥ 15 cells/group). **, p < 0.01 high glucose or mannitol versus basal; ##, p < 0.01 high mannitol versus high glucose. D, time courses of averaged superoxide flashes in basal (gray), high glucose (red), and high mannitol (green). The data are expressed as the means ± S.E. (n = 15–46 flashes for each trace).

FIGURE 2.

Mitochondrial Ca²⁺ uniport is required for hyperosmotic stimulation of superoxide flashes. A, cytosolic Ca²⁺ (measured with Fluo-4) responses to high glucose and high mannitol in the presence or absence of extracellular Ca²⁺ (n = 50–90 cells for each trace). The error bars are omitted for clarity. Arrowheads mark the time of hyperosmotic stimulation. 0Ca²⁺: 5 mm EGTA in 0 Ca²⁺ Tyrode's solution; a.u., arbitrary units. B, removal of extracellular Ca²⁺ decreased hyperosmosis-associated flash activity. The data are expressed as the means ± S.E. (n ≥ 16 cells/group). **, p < 0.01 versus normal Ca²⁺; ##, p < 0.01 versus high glucose. C, effect of MICU1 or MCU knockdown on mitochondrial Ca²⁺ (measured with Rhod-2) responses to high glucose (left panel) and high mannitol (right panel). NC, siRNA negative control; MICU1 siRNA-a and MICU1 siRNA-b, two siRNAs for knocking down MICU1; MCU siRNA-a and MCU siRNA-b, two siRNAs for knocking down MCU; F₀, basal Rhod-2 fluorescence; F/F₀, normalized fluorescence signal. Arrowheads indicate the time of hyperosmotic stimulation. D, statistics of Rhod-2 amplitude as shown in C. The data are expressed as the means ± S.E. (n ≥ 50 cells/group). **, p < 0.01 versus negative control; ##, p < 0.01 versus high glucose. E, effect of MICU1 or MCU knockdown on superoxide flash activity induced by hyperosmotic stress. The data are expressed as the means ± S.E. (n ≥ 20 cells/group). **, p < 0.01 versus negative control; ##, p < 0.01 versus high glucose.

FIGURE 3.

Increasing mitochondrial Ca²⁺ alone is insufficient to augment superoxide flash activity. A and B, cytosolic and mitochondrial Ca²⁺ transients stimulated by 100 μm histamine (A) or 2 μm ionomycin (B) (n = 70–100 cells for each trace). The error bars are omitted for clarity. C, histamine or ionomycin treatment was unable to alter superoxide flash activity. The data are expressed as the means ± S.E. (n ≥ 10 cells/group). NS, no significant difference.

FIGURE 4.

Mitochondrial ROS elevation augments superoxide flash activity. A, hyperosmotic stress-induced mitochondrial ROS production (measured with mitoSOX) in the absence and presence of the mitochondria-targeted ROS scavengers, mitoTEMPO (500 nm) or SS31 (100 μm). Arrowhead indicates the time of hyperosmotic stimulation (n ≥ 50 cells for each trace). B, statistics. The mitochondrial ROS level was indexed by the slope of the mitoSOX signal. Basal, before hyperosmotic stimulation; hyperosmotic stress, high glucose or high mannitol stimulation; mitoTEMPO, pretreatment with 500 nm mitoTEMPO; SS31, pretreatment with 100 μm SS31. The data are expressed as the means ± S.E. (n ≥ 30 cells/group). ##, p < 0.01 versus basal; **, p < 0.01 SS31 or mitoTEMPO treatment versus untreated group. C, effect of mitochondrial ROS scavengers on superoxide flash activity induced by hyperosmotic stress. The data are expressed as the means ± S.E. (n = 11–66 cells/group). **, p < 0.01 SS31 or mitoTEMPO treatment versus untreated group. D, menadione treatment increased mitochondrial basal ROS production. An arrowhead indicates the time of menadione addition. Dashed lines represent linear fitting of the averaged mitoSOX traces before (gray) and after addition of 25 μm (red) or 200 μm menadione (green) (n = 11–34 cells). E, statistics of D. The data are the means ± S.E. (n = 11–34 cells). **, p < 0.01 versus basal group. F, menadione treatment increased superoxide flash frequency in a dose-dependent manner. The data are expressed as the means ± S.E. (n = 12–43 cells). **, p < 0.01 versus group without menadione treatment. NS, no significant difference.

FIGURE 5.

Synergistic effect of mitochondrial Ca²⁺ uniport and ROS elevation on superoxide flash activity. A, synergistic triggering of superoxide flashes by subthreshold menadione (25 μm) combined with ionomycin (2 μm). This effect was blocked by extracellular Ca²⁺ removal (EGTA 5 mm) and by treatment with SS31 (100 μm) or cyclosporine A (CsA, 2 μm). The data are expressed as the means ± S.E. (n = 10–40 cells/group). **, p < 0.01 versus control; ##, p < 0.01 versus menadione-treated group. B and C, bar graphs showing time-dependent superoxide flash activity induced by ionomycin (2 μm, B) or histamine (100 μm, C) in combination with 25 μm menadione. The red trace overlay reflects mitochondrial Ca²⁺ measured by Rhod-2. A dashed line marks the basal mitochondrial Ca²⁺ level before ionomycin or histamine stimulation. The data are expressed as the means ± S.E. (n = 10–60 cells/group). **, p < 0.01 versus control.

FIGURE 6.

Role of superoxide flashes in hyperosmotic stress-induced activation of JNK and p38. HeLa cells were pretreated with or without cyclosporine A (CsA, 2 μm, 0.5 h) or mitoTEMPO (500 nm, 0.5 h) and then challenged with high glucose for different time (0, 0.5, and 1 h). Cell lysates were subjected to immunoblotting with anti-phospho-JNK (p-JNK), total JNK (t-JNK), phospho-p38 (p-p38), and total p38 (t-p38) antibodies. A, representative Western blot results. B, statistics. The ratios of p-JNK to t-JNK and phospho-JNK to total p38 were normalized with that for 0.5-h high glucose challenge alone. The data are expressed as the means ± S.E. (n = 3 for JNK and n = 4 for p38). *, p < 0.05 versus control.