Superoxide flashes in mouse skeletal muscle are produced by discrete arrays of active mitochondria operating coherently

Abstract

Reactive Oxygen Species (ROS) constitute important intracellular signaling molecules. Mitochondria are admitted sources of ROS, especially of superoxide anions through the electron transport chain. Here the mitochondria-targeted ratiometric pericam (RPmt) was used as a superoxide biosensor, by appropriate choice of the excitation wavelength. RPmt was transfected in vivo into mouse muscles. Confocal imaging of isolated muscle fibers reveals spontaneous flashes of RPmt fluorescence. Flashes correspond to increases in superoxide production, as shown by simultaneous recordings of the fluorescence from MitoSox, a mitochondrial superoxide probe. Flashes occur in all subcellular populations of mitochondria. Spatial analysis of the flashes pattern over time revealed that arrays of mitochondria work as well-defined superoxide-production-units. Increase of superoxide production at the muscle fiber level involves recruitment of supplemental units with no increase in per-unit production. Altogether, these results demonstrate that superoxide flashes in muscle fibers correspond to physiological signals linked to mitochondrial metabolism. They also suggest that superoxide, or one of its derivatives, modulates its own production at the mitochondrial level.

Figure 1. Targeting of RPmt to mitochondria.

A, B Confocal images (x,y) of fluorescence of a skeletal muscle fiber transfected with RPmt (B) and loaded with TMRM (A). Images are averaged 4 times in line mode. C Overlay, showing the colocalisation of the two fluorescence patterns. SM: subsarcolemmal mitochondria; IM: intermyofibrillar mitochondria. D 3D reconstruction of a 4 µm-thick slice of the mitochondrial network. Images are averaged 2 times in line mode. Depth scale is shown in the right lower corner. CN: thin column; CK: thick column; TM: transversal mitochondria. Both white scale bars on the picture correspond to 5 µm.

Figure 2. Spectral properties of the spontaneous bursts of fluorescence.

A Left, confocal image (x,y) of fluorescence of a fiber transfected with RPmt. RPmt was excited at 488 nm. Right, time course of changes in normalized RPmt fluorescence within the Regions Of Interest (ROI) 1, 2 and 3. B Left, confocal image (x,y) of fluorescence of a skeletal muscle fiber transfected with RPmt. Right, time course of changes in normalized fluorescence of RPmt, stimulated either at 488 nm or at 458 nm, within the ROI. C This trace, recorded in a different cell, shows the time course of changes in normalised fluorescence of RPmt, stimulated at 477 nm, which is close to the isoemissive point for calcium. D Excitation spectrum of RPmt recorded in transfected skeletal muscle fibers. Excitation wavelength increases by 10 nm steps from 360 to 490 nm. Emission was measured at 520–560 nm. Fibers were incubated in tyrode with 100 µM EGTA AM (0 Ca²⁺) or 20 µM ionomycine (Ca²⁺).

Figure 3. Spontaneous bursts of fluorescence reflect increases of superoxide levels in mitochondria.

A Confocal images of a fiber transfected with RPmt (excitation wavelength 491 nm and 405 nm) and loaded with Rhod-2. Traces represent the time course of changes in normalised RPmt fluorescence (green: 491 nm excitation; blue: 405 nm) or Rhod-2 fluorescence (red) in the ROI. B Confocal images of a fiber transfected with RPmt and loaded with MitoSox. Traces represent the time course of change in normalized RPmt (green) or MitoSox (red) fluorescence in the ROI. Results were reproduced on 6 cells. C Total area of flashing mitochondria per 1000 µm² cell per 100 s (left) and average amplitude of flashes (right) in 7 cells under control conditions and after 20 min of treatment with 15 mM tiron (*: p<0.05 for tiron vs control, paired Student's t test). D Average amplitude of flashes for each of the 7 cells (C.1–C.7) included in C, under control condition and after 20 min of treatment with 15 mM tiron.

Figure 4. Superoxide flashes are not induced by photostimulation.

A Total area of flashing mitochondria per 1000 µm² cell per 100 s (left), average amplitude of flashes (middle) and number of flashes per active units (right) in 7 cells during two successive time series of 3 min (CN 1 and CN 2). Paired Student's t tests have been performed. B Total area of flashing mitochondria per 1000 µm² cell per 100 s (left, n = 8), and number of flashes per active units (right, n = 5) during 3 successive time series of 100 s, with increasing laser intensity. Friedman ANOVA has been performed. Note: Cri in the detection macro was set to 1.5 in this series of experiments, as the signal over noise ratio was greatly reduced for the data obtained with 30% laser transmission. This reduced the resolution of the detection algorithm, and false positives had to be removed manually.

Figure 5. Subcellular distribution of superoxide flashes.

A Left, confocal image (x,y) of fluorescence of a fiber transfected with RPmt. Middle, binary mask of all flashes detected in the 3 min time series. Right, overlay showing in blue the flashes arising in subsarcolemmal mitochondria and in yellow the flashes in intermyofibrillar mitochondria. B Left, percentage of flashing mitochondria among subsarcolemmal or intermyofibrillar mitochondria during a 3 min time series (n = 6, *: p<0.05 for SM vs IM, paired Wilcoxon Signed Rank test). SM: subsarcolemmal mitochondria; IM: intermyofibrillar mitochondria. Right, percentage of flashing mitochondria among subsarcolemmal or intermyofibrillar mitochondria during a 3 min time series for each of the 6 cells included in the left panel.

Figure 6. Shape and size of intermyofibrillar flashes.

A Left, confocal image (x,y) of the fluorescence of a fiber transfected with RPmt. Middle, binary mask of all flashes detected in the 3 min time series. Right, overlay, showing in blue the flashes involving probably a single mitochondrion, in orange the flashes involving a pair of longitudinal mitochondria, and in green the flashes involving larger clusters of longitudinal and transversal mitochondria. The array of mitochondria involved in the same flash is called an active unit. B Imaging of an active unit involving a cluster of mitochondria, at 1 s intervals. The image is shown in pseudo-colored scale. C Histogram of distribution of active units sizes with x-axis ranging from 0 to 4.8 µm². The histogram corresponds to 490 flashes recorded on 7 cells. Binning interval was 0.4 µm². Point shaped units have an area of approximately 0.088–0.8 µm², and column shaped units of 0.8–1.6 µm². Unit constituted of a pair of longitudinal mitochondria measure roughly 1.6–2.4 µm². Larger size-units (up to 18 µm²) are clusters of transversal and longitudinal mitochondria.

Figure 7. Superoxide flashes are linked to metabolism and involve the electron transport chain.

A Total area of flashing mitochondria per 1000 µm² cell per 100 s (left, n = 12 cells), average amplitude of flashes (middle, n = 10) and number of flashes per active unit (right, n = 10 cells) in cells incubated in a tyrode solution devoid of metabolites, and after application of a tyrode solution containing 10 mM glucose and 5 mM pyruvate. (*: p<0.05 for “No metabolites” vs “Glucose + Pyruvate”, paired Student's t test) B Histogram of distribution of active units sizes in 9 cells incubated in a tyrode solution devoid of metabolites, and after application of a tyrode solution containing 10 mM glucose and 5 mM pyruvate. X-axis ranges from 0 to 5 µm, as there are very few counts beyond this value. Binning interval is 0.4 µm². C Total area of flashing mitochondria per 1000 µm² cell per 100 s (left), average amplitude of flashes (middle) and number of flashes per active unit (right) in 5 cells under control conditions, and after a treatment with 2.5 µM antimycin A. (*: p<0.05 for control vs antimycin A, paired Wilcoxon Signed Rank test).

Figure 8. Superoxide flashes induce mitochondrial depolarizations, independently from the activity of the permeability transition pore.

A Left, confocal images (x,y) of fluorescence of a skeletal muscle fiber transfected with RPmt and loaded with TMRM. Right, time course of changes in normalised RPmt (green) or TMRM (red) fluorescence in ROI. B Enlarged views showing imaging of RPmt and TMRM fluorescence at 1 s interval. The images are shown in pseudo-coloured scale. Note the spatial and temporal colocalization of the increase in RPmt and decrease in TMRM fluorescence. C Total area of mitochondria presenting a flash or a depolarization per 1000 µm² cell per 100 s, in 5 cells under control conditions and after 20 min of treatment with 15 mM tiron (*: p<0.05 for control vs tiron, paired Wilcoxon Signed Rank test). D Number of depolarizations concomitant to superoxide flashes (left), and depolarization amplitude (right) in 6 cells under control conditions and after a 20 min treatment with 5 µM cyclosporin A (cyclo A). Paired Wilcoxon Signed Rank tests have been performed.

Figure 9. Superoxide scavenger decreases the number of active units and extends the flashes rising phase.

A Number of flashes per active unit in 7 cells under control conditions and after 20 min of treatment with 15 mM tiron. B Histogram of distribution of active units sizes in 7 cells under control conditions and after 20 min of treatment with 15 mM tiron. C Time to peak (s) of the flashes in 7 cells under control conditions and after 20 min of treatment with 15 mM tiron (*: p<0.05 for control vs tiron, paired Student's t test).

Figure 10. Superoxide flashes are not induced by openings of the permeability transition pore.

A Total area of flashing mitochondria per 1000 µm² cell per 100 s (left), average amplitude of flashes (middle) and number of flashes per active unit (right) in 5 cells under control conditions and after a 20 min treatment with 5 µM cyclosporin A. Paired Wilcoxon Signed Rank tests have been performed. B Total area of flashing mitochondria per 1000 µm² cell per 100 s (left), average amplitude of flashes (middle) and number of flashes per active unit (right) in 8 cells under control conditions and after a 20 min treatment with 50 µM atractyloside. Paired Student's t tests have been performed.

Figure 11. Anion channel inhibitor does not affect superoxide flashes properties.

Total area of flashing mitochondria per 1000 µm² cell per 100 s (left), average amplitude of flashes (middle) and number of flashes per active unit (right) in 8 cells under control conditions and after treatment with 40 µM 4′-chlorodiazepam (4-ChlDZP). Paired Student's t tests have been performed.