Mitochondrial flash as a novel biomarker of mitochondrial respiration in the heart

Abstract

Mitochondrial respiration through electron transport chain (ETC) activity generates ATP and reactive oxygen species in eukaryotic cells. The modulation of mitochondrial respiration in vivo or under physiological conditions remains elusive largely due to the lack of appropriate approach to monitor ETC activity in a real-time manner. Here, we show that ETC-coupled mitochondrial flash is a novel biomarker for monitoring mitochondrial respiration under pathophysiological conditions in cultured adult cardiac myocyte and perfused beating heart. Through real-time confocal imaging, we follow the frequency of a transient bursting fluorescent signal, named mitochondrial flash, from individual mitochondria within intact cells expressing a mitochondrial matrix-targeted probe, mt-cpYFP (mitochondrial-circularly permuted yellow fluorescent protein). This mt-cpYFP recorded mitochondrial flash has been shown to be composed of a major superoxide signal with a minor alkalization signal within the mitochondrial matrix. Through manipulating physiological substrates for mitochondrial respiration, we find a close coupling between flash frequency and the ETC electron flow, as measured by oxygen consumption rate in cardiac myocyte. Stimulating electron flow under physiological conditions increases flash frequency. On the other hand, partially block or slowdown electron flow by inhibiting the F0F1 ATPase, which represents a pathological condition, transiently increases then decreases flash frequency. Limiting electron entrance at complex I by knocking out Ndufs4, an assembling subunit of complex I, suppresses mitochondrial flash activity. These results suggest that mitochondrial electron flow can be monitored by real-time imaging of mitochondrial flash. The mitochondrial flash frequency could be used as a novel biomarker for mitochondrial respiration under physiological and pathological conditions.

Keywords: biomarker; electron transport chain; mitochondrial flash; mitochondrial respiration; real-time confocal imaging.

Fig. 1.

Physiological substrates modulated mitochondrial flash in the perfused heart. A: experimental protocol for manipulating physiological substrates in the perfused mouse heart. B: representative images of the myocardium of mt-cpYFP (mitochondrial-circularly permuted yellow fluorescent protein) hearts perfused with mixed substrate (left) or glucose-only substrate (right) showing a representative mitochondrial flash at its peak (boxed areas and the inset enlarged images). C: traces showing fluorescence changes of the two flashes highlighted in B. D: summarized data showing mitochondrial flash frequency during substrate manipulations in the perfused mt-cpYFP transgenic (TG) mouse hearts. TMRM, tetramethylrhodamine, methyl ester; ΔF/F₀, amplitude, where F₀ refers to basal fluorescence intensity. Values are means ± SE; N = 30–70 cells from 3–4 mice for each group. #P < 0.01 and †P < 0.001 vs. control (with substrate). *P < 0.05 vs. no substrate.

Fig. 2.

Electron transport chain (ETC) activity underlay substrate-induced mitochondrial flashes in intact adult cardiac myocyte. A: representative traces of typical mitochondrial flashes in cultured adult rat cardiac myocytes supplemented with various substrates [5 mM glucose (Glu), 0.5 mM palmitate (Pal), or 5 mM Glu plus 0.5 mM Pal]. B: stimulation of mitochondrial flash frequency by physiological substrates and the subsequent inhibition by rotenone (+Rot, 0.5 μM). C: galactose (Gal; 10 mM) had no effect on mitochondrial flash frequency, while pyruvate (Pyr; 10 mM) increased flash frequency. N = 11–22 cells from 3–4 rats. †P < 0.001 and #P < 0.01 vs. no substrate (No sub). D: manipulation of Glu-supported mitochondrial flash frequency by cyclosporine A (CsA, 1 μM), Tiron (1 mM), or adenovirus-mediated SOD2 overexpression (SOD2). N = 17–26 cells from 3–4 rats. #P < 0.01 vs. No sub. †P < 0.001 vs. Glu. E: unitary properties of mitochondrial flash in cultured rat myocytes. T_pk, time to peak; T₅₀, time from peak to 50% decay. N = 143–323 flashes from 11–46 cells in 3–7 rats. #P < 0.01 and †P < 0.001 vs. No sub. Values are means ± SE.

Fig. 3.

Manipulation of ETC electron flow in permeabilized adult rat cardiac myocyte. A: experimental protocol for manipulating substrate in permeabilized rat cardiac myocytes. B: oxygen consumption rate (OCR) measured in permeabilized rat myocytes with substrates for complex I (Pyr/Mal/ADP: 10 mM Pyr, 5 mM malate, and 2 mM ADP), complex II (Succ/ADP: 10 mM succinate and 2 mM ADP), or complex IV (TMPD/Asc/ADP: 0.5 mM N,N,N′,N′ tetramethyl p-phenylenediamine, 2 mM ascorbate, and 2 mM ADP) and subsequent addition of ETC inhibitors [0.5 μM Rot, 5 μM antimycin A (AA)]. N = 5–10 experiments from 3–4 rats. C: summarized results showing increased MitoSOX Red signals (slope of 405-nm or 514-nm excitation) in permeabilized rat myocytes during substrate stimulation. N = 9–25 cells from 3–4 rats. D: correlation between OCR (B) and MitoSOX signal (C) in permeabilized rat myocytes. ○, 405-nm excitation of MitoSOX vs. OCR; ▲: 514-nm excitation of MitoSOX vs. OCR. Values are means ± SE. †P < 0.001 vs. No sub. #P < 0.01 vs. with substrate.

Fig. 4.

ETC electron flow supported mitochondrial flash generation in permeabilized adult rat cardiac myocyte. A, left: representative images of a permeabilized rat cardiac myocyte showing flash events (highlighted in white boxes) during the 100-s scan in the absence (No sub) or presence of complex I substrates (Pyr/Mal/ADP). Scale bar = 10 μm. Right: representative traces showing a typical flash before or after the complex I substrate. B: flash frequency supported by Pyr/Mal/ADP, Succ/ADP, or TMPD/Asc/ADP and subsequent inhibition by ETC inhibitors: Rot (0.5 μM), AA (5 μM), FCCP (1 μM) or NaCN (1 mM). N = 11–58 cells from 3–8 rats. C: unitary properties of mitochondrial flash in permeabilized rat myocytes with or without substrates. N = 194–524 flashes in 16–57 cells from 6–8 rats. D: correlation between OCR and mitochondrial flash frequency in permeabilized rat myocytes. The data points are from Figs. 3B and 4B. Values are means ± SE. †P < 0.001 vs. No sub.

Fig. 5.

Slowdown electron flow transiently increased mitochondrial flashes in intact rat cardiac myocyte. A: time-dependent change of mitochondrial flash frequency in intact rat myocytes after oligomycin A (OA; 5 μM) treatment. N = 12–38 cells from 3–8 rats. B: OA (5 μM)-induced flashes were sensitive to ETC blockers. The inhibitors used were Rot (0.5 μM), AA (5 μM), and FCCP (1 μM). Top shows representative traces of flashes before and after OA treatment. C: OA (5 μM) or Pyr (10 mM) added separately or sequentially increased flash frequency in intact rat myocytes. N = 10–17 cells from 2–3 rats. #P < 0.01 and †P < 0.001 vs. No sub. D: OA (5 μM) had no effect on Pyr/Mal/ADP-induced flashes in permeabilized rat myocytes. N = 12–38 cells from 3–9 rats. †P < 0.001 vs. No sub. E: OA-induced flashes in intact rat myocytes were blocked by CsA (1 μM). N = 15–41 cells from 3–8 rats. †P < 0.001 vs. control. F: adenovirus-mediated overexpression of human SOD2 but not SOD1 suppressed basal and OA (5 μM)-induced flashes. N = 15–32 cells from 3–5 rats. #P < 0.01 and †P < 0.001 vs. −OA. G: representative traces showing OA (5 μM) accelerated the increase rate of MitoSOX Red signal in intact rat myocytes. Similar effects were observed in 12–25 cells from 3 rats. Values are means ± SE.

Fig. 6.

Complex I deficiency suppressed mitochondrial flashes in mouse cardiac myocyte. A: representative images of a freshly isolated and intact cardiac myocyte from heart-specific Ndufs4 knockout and mt-cpYFP TG mouse (cKO). White boxes indicate the onset of a mitochondrial flash. B: representative traces showing the flash highlighted in A. C: frequency of mitochondrial flash in freshly isolated myocytes from wild-type (WT) or cKO mice in physiological solutions containing 5 mM Glu as substrate. N = 32–40 cells from 5 mice. D: complex I substrate (Pyr 10 mM)-induced flashes in WT and cKO myocytes. N = 17–31 cells from 3–5 mice. E: unitary properties of flashes in freshly isolated myocytes from WT or cKO mice. N = 66–159 flashes in 32–40 cells from 5 mice. Values are means ± SE. *P < 0.05 vs. WT. †P < 0.001 vs. No sub.

Fig. 7.

Model of the coupling between ETC electron flow and mitochondrial flash. Respiration substrate initiates electron flow to support mitochondrial respiration and flash generation. The amount of electrons transported along ETC is a determining factor for the respiration-flash coupling. The electron flow leads to superoxide production and matrix alkalization, which are components of mitochondrial flash. IMM, inner mitochondrial membrane; Qo, quinol-oxidizing center; Qi, quinone-reducing center; CytoC, cytochrome C.