Protons Trigger Mitochondrial Flashes

Abstract

Emerging evidence indicates that mitochondrial flashes (mitoflashes) are highly conserved elemental mitochondrial signaling events. However, which signal controls their ignition and how they are integrated with other mitochondrial signals and functions remain elusive. In this study, we aimed to further delineate the signal components of the mitoflash and determine the mitoflash trigger mechanism. Using multiple biosensors and chemical probes as well as label-free autofluorescence, we found that the mitoflash reflects chemical and electrical excitation at the single-organelle level, comprising bursting superoxide production, oxidative redox shift, and matrix alkalinization as well as transient membrane depolarization. Both electroneutral H(+)/K(+) or H(+)/Na(+) antiport and matrix proton uncaging elicited immediate and robust mitoflash responses over a broad dynamic range in cardiomyocytes and HeLa cells. However, charge-uncompensated proton transport, which depolarizes mitochondria, caused the opposite effect, and steady matrix acidification mildly inhibited mitoflashes. Based on a numerical simulation, we estimated a mean proton lifetime of 1.42 ns and diffusion distance of 2.06 nm in the matrix. We conclude that nanodomain protons act as a novel, to our knowledge, trigger of mitoflashes in energized mitochondria. This finding suggests that mitoflash genesis is functionally and mechanistically integrated with mitochondrial energy metabolism.

Figure 1

Multifaceted signals underlying mitoflashes. (A–H) Mitoflashes in adult cardiomyocytes were measured by multiple reporters, including mt-cpYFP (A, F, and H), SNARF-1 (A), pHTomato (B), mitoSOX (C), DCF (D), grx1-roGFP2 (E), and TMRM (E and H), as well as autofluorescence of NADH (F) and FAD (G). Increases in SNARF-1 and pHTomato fluorescence indicate alkalinization of the mitochondrial matrix. MitoSOX was used for the detection of superoxide production, and DCF was used to report changes in the total ROS level. The DCF signal was processed by subtracting the baseline increase. An increase in grx1-roGFP2 fluorescence reflects the shift of the mitochondrial redox potential toward oxidation, and a decrease in the TMRM signal reflects mitochondrial depolarization. NADH autofluorescence (420–470 nm) was measured at 720 nm two-photon excitation. FAD autofluorescence was measured between 500 and 650 nm at 488 nm excitation. An increase of FAD autofluorescence indicates a decrease of FADH₂ content. All mitoflashes shown occurred spontaneously. To see this figure in color, go online.

Figure 2

Mitoflashes induced by electroneutral ionophores. (A) Nigericin (Nig) concentration-dependently activated cpYFP-flashes in adult cardiomyocytes. n = 9–22 cells per group. ^∗∗p < 0.01 versus control. (B) Averaged traces of cpYFP-flashes aligned by onset. Note that nigericin did not alter the properties of individual mitoflashes. n = 21 and n = 52 events from 10 cells for control and nigericin (Nig, 50–100 nM) groups, respectively. The mitoflashes in the control group occurred spontaneously. (C) Time course of the mitoflash response to nigericin in three representative cells. Individual mitoflash events are denoted by vertical ticks, and 50 nM nigericin was added at 100 s as indicated. (D and E) Nigericin (50 nM) stimulated mitoflashes reported with FAD (D) and DCF (E). n = 8–25 cells. ^∗∗p < 0.01 versus control. (F) Concentration-dependent effect of monensin (Mon) on cpYFP-flash activity. n = 11–14 cells. ^∗∗p < 0.01 versus control. (G) Concentration-dependent effects of nigericin on NADH, FAD, ΔΨ_m, and ATP in cardiomyocytes. Note that nigericin at 1–100 nM did not elicit discernible effects, but at 1 μM it caused a marked depletion of NADH, FADH₂, and ATP. The NADH level was measured by detecting the autofluorescence (420–470 nm) at 720 nm two-photon excitation. The FADH₂ level was indexed by monitoring FAD autofluorescence with excitation at 488 nm and emission at 500–650 nm. ΔΨ_m was measured using TMRM and the ATP content was measured with Mg²⁺-Green. An increase in Mg²⁺-Green fluorescence indicates a decrease in ATP content. n = 12–44 cells per trace. To see this figure in color, go online.

Figure 3

Effects of FCCP and steady matrix pH on mitoflash activity. (A) Inhibition of mitoflash activity by FCCP. n = 9–10 cells per group. ^∗∗p < 0.01 versus control. (B) FCCP reversed the nigericin (Nig)-induced mitoflash response. n = 6–8 cells. ^∗∗p < 0.01 versus control; ^##p < 0.01 versus Nig group. (C) Effects of FCCP on NADH, FADH₂ (inversely related to FAD autofluorescence), ΔΨ_m (measured with TMRM), and ATP (measured with Mg²⁺-Green). n = 13–23 cells for each trace. Note that membrane depolarization and NADH oxidation started at 10 nM, followed by FADH₂ oxidation at 50 nM and ATP depletion at 500 nM FCCP. (D) Mitochondrial pH changes in response to altered extracellular pH (pH_o) in adult cardiomyocytes. Mitochondrial matrix pH was measured with mt-EYFP, and a decrease of its fluorescence indicates acidification of the matrix. n = 7 cells per group. ^∗∗p < 0.01 versus pH_o 7.3 group. (E) Effect of matrix pH changes (as shown in D) on mitoflash activity. n = 17–25 cells per group. ^∗∗p < 0.01 versus pH_o 7.3. To see this figure in color, go online.

Figure 4

Proton uncaging triggers mitoflashes. Adult cardiomyocytes expressing mt-cpYFP were loaded with 1 mM NBA, and proton uncaging at 405 nm illumination was alternated with imaging acquisition at 488 nm excitation (see Materials and Methods). (A) Mitoflash response to proton uncaging at 3% laser power. Top: intracellular mapping of mitoflash events before (yellow boxes, 0–60 s recording) and during (red boxes, 60–120 s recording) photolysis. Scale bar, 10 μm. Bottom: time course of mitoflash incidence (marked by vertical ticks) in three representative cells. The top row corresponds to the cell shown above. (B) Laser-power dependence of proton uncaging-induced mitoflashes. Cells without NBA treatment were used as controls. n = 5–15 cells per group. ^∗∗p < 0.01 versus control. (C) Space-time confinement of mitoflashes responding to subcellular proton uncaging. Surface plots of mitoflashes registered in 10-s periods overlay the respective confocal images of the cell. A spike indicates a mitoflash event. The 405 nm laser (3% power) illumination was restricted to the central segment of the cell (red box) and applied between 50 and 100 s of the recording. Scale bar, 10 μm. (D) Statistics. n = 22 cells. ^∗∗p < 0.01 versus control region; ^##p < 0.01 versus photolysis. (E) Mitoflash response to short-duration NBA photolysis. The arrow denotes a 0.5-s 405-nm laser illumination at 3% intensity of full laser power (15 mW). Note that the increase in the rate of mitoflash occurrence was well confined within 2 s after proton uncaging. n = 62 cells. ^∗∗p < 0.01 versus baseline or postphotolysis levels. (F) Averaged traces of cpYFP-flashes aligned by onset. n = 21 events from 11 cells for the control group; n = 52 events from 15 cells for the NBA photolysis group (uncaging). (G) Proton uncaging triggers mitoflashes in permeabilized adult cardiomyocytes bathed with 20 mM HEPES. n = 5–7 cells per group. ^∗∗p < 0.01 versus control.

Figure 5

Simulation of a proton spike and schematic model for mitoflash genesis. (A) Matrix proton spike induced by proton uncaging. In this simulation, uncaging at time = 0 ns elicited a sudden drop in pH (from 8 to 4) or a surge of protons (upper inset) and a steady bulk matrix pH change of 0.02 unit (lower inset). The mean lifetime of the uncaged protons was 1.42 ns and the mean distance of diffusion was 2.06 nm. (B) A stochastic, flickering opening of the mPTP results in water and ionic fluxes to strain and depolarize the IMM. The mechanical strain causes dislocation of ETC molecules and disruption of the normal electron path, and thus greatly increases electron leakage to oxygen, resulting in a burst of superoxide formation. Meanwhile, depolarization accelerates electron transfer from the donor pool to ETC acceptors (e.g., from NADH to complex I, and from FADH₂ to complex II) and shifts the redox potential toward oxidation. Accelerated ETC activity, driven dually by depolarization and oxygen deprivation of ETC electrons, stimulates coupled proton pumping across the IMM, giving rise to matrix alkalinization. Among others, nanodomain protons at the IMM may directly bind to a putative proton-binding site and thereby trigger transient opening of the mPTP to initiate a cascade of changes in a mitoflash. To see this figure in color, go online.