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IUPS KEYNOTE LECTURE
Mitochondrial flashes: new insights into mitochondrial ROS signalling and beyond Tingting Hou, Xianhua Wang, Qi Ma and Heping Cheng
The Journal of Physiology
State Key Laboratory of Biomembrane and Membrane Biotechnology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China
Abstract Respiratory mitochondria undergo stochastic, intermittent bursts of superoxide production accompanied by transient depolarization of the mitochondrial membrane potential and reversible opening of the membrane permeability transition pore. These discrete events were named ‘superoxide flashes’ for the reactive oxygen species (ROS) signal involved, and ‘mitochondrial flashes’ (mitoflashes) for the entirety of the multifaceted and intertwined mitochondrial processes. In contrast to the flashless basal ROS production of ‘homeostatic ROS’ for redox regulation, bursting ROS production during mitoflashes may provide ‘signalling ROS’ at the organelle level, fulfilling distinctly different cell functions. Mounting evidence indicates that mitoflash frequency is richly regulated over a broad range, and represents a novel, universal, and ‘digital’ readout of mitochondrial functional status and of the mitochondrial stress response. An emerging view is that mitoflashes participate in vital processes including metabolism, cell differentiation, the stress response and ageing. These recent advances shed new light on the role of mitochondrial functional dynamics in health and disease. (Received 9 April 2014; accepted after revision 10 July 2014; first published online 18 July 2014) Corresponding author H. Cheng: State Key Laboratory of Biomembrane and Membrane Biotechnology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China. Email:
[email protected] Abbreviations CsA, cyclosporin A; CypD, cyclophilin D; cpYFP, circularly permuted yellow fluorescent protein; DCF, 2,7-dichlorodihydrofluorescein diacetate; ETC, electron transport chain; fMPT, putative reversible mitochondrial permeability transition underlying the mitoflash; MAPK, mitogen-activated protein kinase; MPT, mitochondrial permeability transition; mPTP, MPT pore; NPCs, neural progenitor cells; pMPT, permanent MPT; ROS, reactive oxygen species; SOD, superoxide dismutase; tMPT, transient MPT.
Introduction
The mitochondrion is arguably the most complex and intriguing cellular organelle in eukaryotic cells – not only for its unique symbiotic origin, retention of its
own genome, maternal inheritance and double-layered membranous structure, but also for its unparalleled functional diversity. More than a powerhouse central to cellular bioenergetics, it plays pivotal roles in calcium
Tingting Hou is a PhD candidate (expected to graduate in 2015) in biophysics at the Institute of Molecular Medicine, Peking University. Instructed by professor Heping (Peace) Cheng and Professor Xianhua Wang, she focuses on the regulatory mechanisms and molecular basis of mitochondrial flashes as well as on novel functions of mitochondrial proteins. Heping (Peace) Cheng has a PhD in physiology and biophysics and is a founder of the Institute of Molecular Medicine at Peking University. His early achievements include the co-discovery of ‘calcium sparks’. His current research focus is on the mechanism, regulation and biology of mitochondrial flashes as well as on methodology for flash study.
This review is based on an IUPS Keynote Lecture delivered at IUPS 2013, the XXXVII International Congress of Physiological Sciences, Birmingham, UK, 24 July 2013. C 2014 The Authors. The Journal of Physiology C 2014 The Physiological Society
DOI: 10.1113/jphysiol.2014.275735
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signalling, redox homeostasis and cell fate regulation (Green, 1998; Duchen, 2000; Droge, 2002; Nunnari & Suomalainen, 2012). Since Jensen reported that the respiratory chain produced reactive oxygen species (ROS) in 1966 (Jensen, 1966) followed by the pioneering work of Chance and colleagues on the mitochondrial production of hydrogen peroxide (H2 O2 ) (Loschen et al. 1971; Boveris & Chance, 1973; Chance et al. 1979), a huge literature has been developed on the sources and consequences of mitochondrial ROS production (see Sies, 2014 for a review). Normally, the flux of electrons from substrates flows through various redox centres in the electron transport chain (ETC), and is ultimately terminated to water in a four-electron reduction of molecular oxygen, catalysed by cytochrome c oxidase. However, a small fraction of ‘leak’ electrons participate in a single-electron, incomplete reduction of O2 to produce the free radical superoxide anion (O2 •− ). Superoxide is subsequently converted to H2 O2 through spontaneous or superoxide dismutase (SOD)-catalysed dismutation and then to oxygen and water by antioxidant enzymes such as catalase, thioredoxin and glutathione peroxidases (Droge, 2002). The production of ROS by mitochondria can lead to oxidative damage that underlies many pathologies including malignant diseases, diabetes mellitus, atherosclerosis, ischaemia–reperfusion injury, chronic inflammatory processes and neurodegenerative diseases (Halliwell, 2001; Droge, 2002; Newsholme et al. 2007). A paradigm-shifting concept in recent years, however, is that mitochondrial ROS also contribute to retrograde redox signalling from the organelle to the cytosol and nucleus (Droge, 2002; Balaban et al. 2005; Wallace, 2012). In so doing, mitochondrial ROS play signalling roles in a variety of pathways in differentiation and organogenesis (Owusu-Ansah & Banerjee, 2009), cell fate regulation (Maryanovich & Gross, 2013) and the stress response (Adler et al. 1999). In this brief review, we focus on the surprising recent discovery of a novel mode of ROS generation or ‘superoxide flash’ (Wang et al. 2008), also known as ‘mitochondrial flash’ (mitoflash) (Shen et al. 2014), in respiratory mitochondria. In particular, we present evidence that mitoflashes are universal and multifaceted, and highlight features that distinguish them from basal mitochondrial ROS production and regulation. We also synthesize the data on possible signalling roles of mitoflashes in the context of metabolism, cell differentiation, stress response, disease and ageing. Universality of mitoflashes
By serendipity, we found that the fluorescent moiety of the Ca2+ indicator pericam (Nagai et al. 2001), a circularly permuted yellow fluorescent protein (cpYFP), is a novel biosensor of O2 •− , the primal ROS generated by the mitochondrial ETC (Wang et al. 2008). By targeting cpYFP
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to the mitochondrial matrix using the signal peptide of cytochrome c oxidase subunit IV (COX IV), we discovered superoxide flashes – sudden, quantal, 10 s bursts of superoxide production – in single mitochondria in intact cells (Wang et al. 2008). More than a free radical-producing event, a superoxide flash is always accompanied by transient depolarization of the mitochondrial membrane potential (m), but not vice versa (Wang et al. 2008; Li et al. 2012). There is also a transient, reversible increase of mitochondrial permeability at the onset of a superoxide flash, evidenced by a concomitant, irreversible loss of matrix-loaded indicators such as Rhod-2 (mol. mass 752 Da) (Wang et al. 2008) or its Ca2+ -insensitive analogue (mol. mass 980 Da) (Wang et al. 2012) (Fig. 1). In the following discussion, we use ‘mitoflash’ when referring to this complex mitochondrial phenomenon in toto, and ‘superoxide flash’ when specifically referring to its free radical-producing component. Since their discovery, mitoflashes have been detected in many types of cells and tissues, including cardiomyocytes, skeletal muscle myotubes and fibres, neurons, glial cells, fibroblasts, chondrocytes, and many types of cancer cells (Wang et al. 2008; Pouvreau, 2010; Fang et al. 2011; Ma et al. 2011; Wei et al. 2011; Hou et al. 2012; Cao et al. 2013; Hou et al. 2013; Wei-LaPierre et al. 2013) (Fig. 2). The experimental systems used range from isolated single mitochondria (Wei-LaPierre et al. 2013; Zhang et al. 2013) and intact cells to ex vivo beating hearts (Wang et al. 2008) and even live animals (Fang et al. 2011; Shen et al. 2014) (Fig. 2). That mitoflashes occur in isolated single mitochondria attests that the mechanism of their formation is intrinsic to this organelle; on the other hand, the manifestation of mitoflashes in vivo underscores the physiological relevance of this dynamic mitochondrial activity. Remarkably, mitoflashes are evolutionarily conserved – events of nearly identical properties have been found in different cell types and experimental systems across species from Caenorhabditis elegans (Shen et al. 2014), to zebrafish (M Zhang & J Xiong, unpublished observations), to humans (Wang et al. 2008; Ma et al. 2011; Hou et al. 2013). In worms and mammals alike, the generation of mitoflashes requires the integrity of the ETC and mitochondrial respiratory function (Wang et al. 2008; Shen et al. 2014). Moreover, the intersection of mitoflashes with the key mitochondrial function – respiration – strongly suggests that they represent a conserved and fundamental activity of this ancient organelle, and are quintessential to mitochondrial ROS signalling and other functions (see below). Nature of mitoflashes
It has been increasingly appreciated that the mitoflash is a complex phenomenon comprising multifaceted and intertwined processes: superoxide flashes are not C 2014 The Authors. The Journal of Physiology C 2014 The Physiological Society
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Mitochondrial flashes
only coupled with transient depolarization of m, but are also concurrent with a rapid depletion of the electron donors NADH and FADH2 (Fig. 1). In some cases, mitoflashes are associated with conspicuous, reversible mitochondrial swelling that may masquerade as mitochondrial ‘contraction’ (Ma et al. 2011; Breckwoldt et al. 2014) and, in filamentous mitochondria, give rise to the transient beads-on-thread shape during a mitoflash (Ma et al. 2011). Among all these dynamic activities, bursting ROS signals constitute one but not the sole message that mitoflashes could convey. Given that cpYFP is also a pH sensor with a pKa of 8.5 (Wang et al. 2008; Schwarzl¨ander et al. 2011), Schwarzl¨ander et al. proposed that a similar mitochondrial phenomenon found in Arabidopsis reflects a transient matrix alkalization rather than a superoxide burst (Schwarzl¨ander et al. 2011). Specifically, they suggested that spontaneous membrane depolarization accelerates proton pumping by the ETC and results in an increase in matrix pH, or ‘pH flash’ as termed by Schwarzl¨ander et al. This controversy on the origin of mitoflashes has been examined in recent reviews by ourselves and others (Quatresous et al. 2012; Schwarzl¨ander et al. 2012b; Wang et al. 2014); however, it is not yet fully resolved. Because mitochondrial superoxide metabolism and pH regulation are inseparably interlinked, this fact renders most evidence ambivalent and inconclusive for either hypothesis. For instance, both hypotheses would be compatible with the requirement of an intact ETC (Wang et al. 2008; Schwarzl¨ander et al. 2011; Shen et al. 2014), dependence
on oxygen (Wang et al. 2008; Huang et al. 2011), activation by ROS (Huang et al. 2011; Hou et al. 2013; Shen et al. 2014), inhibition by antioxidants (Wang et al. 2008; Schwarzl¨ander et al. 2011), and the coupling with transient depolarization of m (Wang et al. 2008; Schwarzl¨ander et al. 2012a). The evidence in favour of the pH hypothesis includes the finding that SypHer, a mutant of Hyper, with its two cysteine residues critical to H2 O2 sensing changed to serine, which is insensitive to H2 O2 but sensitive to pH with a pKa of 8.7 (Poburko et al. 2011), detects similar flashes in astrocyte (Azarias & Chatton, 2011) and HeLa mitochondria (Santo-Domingo et al. 2013). However, caution has been voiced for this interpretation because the possibility that SypHer might also sense superoxide has not yet been excluded (Quatresous et al. 2012). While the pH hypothesis predicts a mirror relationship between m and the cpYFP signal, which is supported by data from Arabidopsis, experimental data from mammalian cells have shown that this is not always the case: oscillatory m depolarizations are often uncoupled from or, when coupled, outlast the cpYFP flashes (Wang et al. 2008; Li et al. 2012; Wei-LaPierre et al. 2013). Furthermore, nigericin, a H+ /K+ antiporter, used in the micromolar range to clamp matrix pH without altering m, inhibits mitoflashes, and this observation was interpreted as strong evidence to bolster the pH hypothesis of mitoflash origin (Schwarzl¨ander et al. 2012a). However, a counter-argument is that nigericin at high doses also perturbs mitochondrial proton
Figure 1. Mitoflash is a complex phenomenon comprising multifaceted and intertwined processes A cpYFP flash is associated with a transient loss of mitochondrial membrane potential (indexed by tetramethyl rhodamine methyl ester (TMRM)) (A), mitoSOX signal (B), DCF signal (modified from Zhang et al. 2013) (C), transient depletion of NADH (D) and FADH2 (reflected by FAD+ autofluorescence) (E), and an MPT (evidenced by irreversible loss of Rhod-2 analogue) (modified from Wang et al. 2012) (F). NADH autofluorescence was measured by 720 nm two-photon excitation. FAD+ autofluorescence was measured between 500 and 650 nm at 488 nm excitation. Scale bar: 10 s for x-axis and 0.2F/F0 for y-axis in A, D, E and F; 20 s for x-axis and 1 arbitrary unit for y-axis in B and C. C 2014 The Authors. The Journal of Physiology C 2014 The Physiological Society
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gradients that might be essential to superoxide production, and nigericin at nanomolar concentrations markedly stimulates, rather than inhibits, mitoflashes (Wei-LaPierre et al. 2013). The observation that mitoflash events comprise a burst of superoxide production (superoxide flash) has been independently corroborated by several groups using multiple approaches. The synthetic ROS indicators mitoSOX (Pouvreau, 2010; Wei-LaPierre et al. 2013; Zhang et al. 2013) and 2,7-dichlorodihydrofluorescein diacetate (DCF) (Zhang et al. 2013), both pH insensitive in the milieu of intact mammalian cells at extracellular pH ranging from 6.0 to 9.5, faithfully report mitoflashes in both single-cell and single-mitochondrion systems. When used individually to avoid possible fluorescence resonance energy transfer (FRET) effects and spectral cross-contamination, these pH-insensitive ROS sensors confirmed flash events of nearly identical frequencies (Zhang et al. 2013). Azarias and Chatton demonstrated concurrent mitoSOX-reported bursts of superoxide during the SypHer-reported pH flashes (Azarias &
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Chatton, 2011). More recently, Breckwoldt et al. reported reversible redox changes lasting 200 s arising from brief oxidative bursts and coincident with spontaneous ‘contractions’ of axonal mitochondria in transgenic mice with neuron-specific expression of the redox sensor Grx1-roGFP (Breckwoldt et al. 2014). However, mitoSOX and DCF are not universally accepted as reliable ROS indicators and, so far, the structural information and biochemistry of how cpYFP senses superoxide also remain a mystery. A third possibility remains open: the mitoflash could be sphinx-like; the superoxide flash and the pH flash are just two facets of this complex phenomenon (Wang et al. 2014). In this regard, quantitative appraisal of the respective contributions of pH (measured with SNARF-1) and superoxide have revealed superoxide as the predominant signal with a minor pH (0.08 units) contribution for mitoflashes in the heart and skeletal muscles (Wei-LaPierre et al. 2013). It should be informative to implement similar quantitative analyses in Arabidopsis, because it is conceivable that the relative contributions of superoxide and pH to mitoflash
Figure 2. Mitoflashes represent a universal and conserved mitochondrial activity Mitoflashes are found across species in different tissues, cells and experimental systems. Their unitary properties are highly comparable. MTS: mitochondrial targeting signal peptide of cytochrome c oxidase subunit IV (COX IV) (for transgenic mouse and zebrafish) or succinate dehydrogenase (ubiquinone) iron-sulfur subunit (SDHB-1) (for transgenic C. elegans). C 2014 The Authors. The Journal of Physiology C 2014 The Physiological Society
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events vary in a species-, cell type-, and context-dependent manner.
Flash and flashless ROS production: similarities and differences
As the primary source of intracellular ROS, mitochondria contain a sophisticated, multilayered system for ROS metabolism and signalling. On the one hand, mitochondria contain at least 10 known sites that are capable of generating ROS, with 9 for superoxide anions (Andreyev et al. 2005; Starkov, 2008). The basal ROS production is believed to be a continuous and flashless process. On the other hand, mitochondria harbour numerous antioxidants and ROS-defence enzymes including MnSOD, catalase, glutathione, glutathione-S-transferase and phospholipid hydroperoxide glutathione peroxidase (for the removal of lipid peroxides) (Andreyev et al. 2005). The concerted actions of these molecular players help to set the steady-state ROS level and maintain redox homeostasis. In contrast, superoxide flashes are bursting superoxide-producing events that occur only intermittently and are spatially well confined. As a hallmark of ‘digital’ signals, the properties of individual superoxide flashes appear to be stereotypical. Depending on the tissue and cell type, metabolic rate, developmental stage, age and degree of oxidative stress, the frequency of superoxide flashes can vary by orders of magnitude, but there are only mild to moderate changes in the amplitude and duration of individual events. Mitoflash regulation and signalling occur predominantly in a frequency-modulated manner (Wang et al. 2014). Apart from the distinctive digital feature, superoxide flashes differ from basal ROS in the high levels of ROS attained, as evidenced by the steep rise of cpYFP, mitoSOX or DCF (Zhang et al. 2013), and Grx1-roGFP signals (Breckwoldt et al. 2014). Thus, whereas sustained elevation of global ROS is undoubtedly detrimental, brief ROS pulses may activate high-threshold ROS pathways locally, fulfilling signalling roles while limiting cellular and mitochondrial damage. Indeed, while the rate of superoxide flashes waxes and wanes, the global ROS level and redox balance are exquisitely safeguarded by sophisticated multi-layered systems. Recently, we have proposed that basal ROS production may fulfil housekeeping roles, such as maintaining redox balance (i.e. homeostatic ROS); against this homeostatic background, superoxide flashes as dynamic events are well poised to serve as physiological signalling units (i.e. signalling ROS) (Wang et al. 2012). In analogy to ‘calcium sparks’ for calcium signalling (Cheng & Lederer, 2008), mitoflashes with their spatiotemporally controlled high ROS signals may also build up hierarchical multi-scaled intracellular ROS C 2014 The Authors. The Journal of Physiology C 2014 The Physiological Society
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dynamics, conferring efficiency, specificity and diversity on ROS signalling (see below). It is also instructive to compare and contrast the similarities and differences in the regulation of basal ROS and superoxide flashes. Both depend on m (Starkov, 2008; Wang et al. 2008), and are sensitive to matrix pH (Murphy, 2009; Wei-LaPierre et al. 2013) and oxygen tension (Huang et al. 2011), as well as the concentration of substrate supplied (Starkov, 2008). In addition, no flashes have been detected in ρ0 143B cells, human osteosarcoma cells devoid of all mitochondrial (mt) DNA-encoded ETC components (Wang et al. 2008). In ρ− PC12 cells, rat PC12 phechromocytoma cells treated with ethidium bromide to partially deplete mitochondrial DNA, both the superoxide flash incidence and mtDNA decrease proportionally (Wang et al. 2008). More recently, it has been shown that C. elegans mutants with defective respiratory chain complexes, including the complex I mutants gas-1, nuo-6, the complex II mutant mev-1, the complex III mutants cyc-1, isp-1, and the complex V mutant atp-3, all exhibit extremely low mitoflash activity (Shen et al. 2014). Interestingly, the ETC inhibitors differentially regulate mitoflash and basal ROS production. For basal ROS, they can be either inhibitory or stimulatory depending on the sites of the ETC complex affected, the direction of electron flow, and the ‘side-ness’ of ROS emission (Starkov, 2008). In contrast, all the ETC inhibitors tested, including antimycin A, rotenone, myxothiazole and oligomycin, suppress or even abolish mitoflash production (Wang et al. 2008). As to the pH sensitivity, alkalization of the mitochondrial matrix accelerates basal ROS production and acidification suppresses it (Murphy, 2009), but the mitoflash incidence in cardiac cells shows little change over the physiological pH range and is even depressed at extreme pH values (X. Wang & H. Cheng, unpublished observations). Future investigation is warranted to establish the superoxide-generating sites and the mechanism of their synchronous activation as well as their prompt termination after a 10 s burst.
Mitochondrial permeability transition during mitoflashes
An important feature of mitoflashes is that their ignition is tightly coupled with another dynamic mitochondrial activity, the mitochondrial permeability transition (MPT). The MPT is defined as a sudden increase of inner mitochondrial membrane permeability to ions and small solutes (Haworth & Hunter, 1979; Hunter & Haworth, 1979a,b), and is thought to be mediated by the opening of a high-conductance channel, the MPT pore (mPTP) which is also known as the mitochondrial megachannel (Kinnally et al. 1989; Petronilli et al. 1989; Szabo & Zoratti, 1991; Szabo et al. 1992). Extensive functional
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Table 1. Properties of pMPT, tMPT and fMPT pMPT
tMPT
fMPT Up to 980 Da, permeable to Rhod-2 (Wang et al. 2008) and Rhod-2 analogue (Wang et al. 2012) Yes (reversible) Transient disruption Yes No Yes (except in skeletal muscle) Yes (except in skeletal muscle)
Molecular cutoff (permeability)
