Subcellular metabolic transients and mitochondrial redox waves in heart cells

Abstract

Precise matching of energy supply with demand requires delicately balanced control of the enzymes involved in substrate metabolism. In response to a change in substrate supply, the nonlinear properties of metabolic control may induce complex dynamic behavior. Using confocal imaging of flavoprotein redox potential and mitochondrial membrane potential, we show that substrate deprivation leads to subcellular heterogeneity of mitochondrial energization in intact cells. The complex spatiotemporal patterns of redox and matrix potential included local metabolic transients, cell-wide coordinated redox transitions, and propagated metabolic waves both within and between coupled cells. Loss of metabolic synchrony during mild metabolic stress reveals that intra- and intercellular control of mitochondrial function involves diffusible cytoplasmic messengers.

Figure 1

Mitochondrial redox in a cardiomyocyte. (A, a–c) Confocal images of endogenous flavoprotein fluorescence in the control (10 mM glucose-containing modified Tyrode’s; A, a), fully reduced (4 mM cyanide; A, b), and fully oxidized (200 μM 2,4-dinitrophenol; A, c) states. Each image was the average of 10 frames taken during the time segments shown in B (labeled a, b, c). Pixel brightness increases with oxidation of the mitochondrial flavoproteins. The spacing of the sarcomeric z-lines (transverse dark bands in A, c) is ≈1.8 μm, giving an internal scale of the images. (B) The time course of mean cell autofluorescence intensity change (arbitrary units) under fully reduced (cyanide) or oxidized (2,4-dinitrophenol) conditions. Image frames in the series were collected ≈10 sec apart. (C) Cross-correlation plot for simultaneously collected flavoprotein and TMRE fluorescence images (images not shown, but similar to Fig. 5A, a) demonstrating localization of the redox signal to the mitochondrial matrix.

Figure 2

Oscillations in mitochondrial redox potential in zero-glucose medium. (A, a–d) Confocal images of flavoprotein redox oscillations (lower cell) or a transition from reduced to oxidized (upper cell). Each image was the average of 10 taken during the time segments shown in B (labeled a, b, c, d). A pseudocolor palette graded from light blue (reduced) to red (oxidized) was applied. Note the local oxidation of a mitochondrial cluster at the right end of the upper myocyte in A, c. (B) The time course of mean cell fluorescence change (arbitrary units) during mitochondrial redox oscillation (lower plot corresponds to the lower cell in images shown in A) or a maintained transition of redox (upper plot corresponds to upper cell in A). The decline in fluorescence maxima late in the series is due to photobleaching. Frames were collected ≈6 sec apart.

Figure 3

Focal oxidation of mitochondrial flavoproteins. (A, a–c) The bright regions evident in A, b and A, c indicate highly localized oxidation of independent clusters of mitochondria. Each image is an average of 16 frames collected during the time segments shown in B (labeled a, b, c). (B) The time course of flavoprotein redox change in different subcellular regions. Values are the mean fluorescence intensities across the width of the cell at levels I, II, and III, as denoted on image. Regions I and III displayed oxidation of mitochondrial clusters, whereas the intervening region (II) remained reduced throughout the experiment. Image frames were collected ≈6 sec apart.

Figure 4

Propagated intra- and intercellular mitochondrial redox wave. (A, a–e) A sequential series of confocal images of flavoprotein fluorescence in a pair of cardiomyocytes coupled by an intercalated disc. A wave of oxidation propagated from the end of the first myocyte (A, b) to the cell–cell junction (A, c) and crossed over to the second myocyte (A, d and e). Single image frames were processed with a 3 × 3 median filter, and a pseudocolor palette graded from light blue (reduced) to red (oxidized) was applied. The time interval between images was (in seconds) 8.7 (A, a to b), 8.7 (A, b to c), 13 (A, c to d), 22.9 (A, d to e), 51.1 (A, d to e). (B) Three-dimensional plots of flavoprotein fluorescence intensity. Each image shown in A was sectioned into a 64 × 64 grid, and pixel intensities within each section were averaged and plotted in three dimensions to illustrate redox gradients within and between myocytes in the cell pair.

Figure 5

Simultaneous imaging of mitochondrial redox and matrix electrical potential during metabolic oscillations. (A, a–c) Two-color confocal images during reduced (A, a and c) and oxidized (A, b) phases of metabolic oscillation. Averages of six frames corresponding to time points labeled a, b, c in B are shown, with the green component of each image indicating flavoprotein redox state and the red component indicating TMRE fluorescence. The marked redistribution of TMRE and spatial heterogeneity of mitochondrial membrane potential evident in A, b returned to the basal distribution (A, a) during a subsequent reduction (A, c). The islands of polarized mitochondria visible in A, b had increased TMRE fluorescence, as a result of TMRE redistributing from depolarized mitochondria to the cytoplasm. (B) Heterogeneity of TMRE fluorescence during mitochondrial oxidation was quantitatively assessed by dispersion analysis (dispersion was calculated as 0.05–0.95 interfractile range from histograms of each TMRE image in the time series). The correlation of TMRE dispersion (upper plot) and the flavoprotein redox state (lower plot) demonstrates the association of redox transients with heterogeneous mitochondrial depolarizations.