David
Lloyd
*a,
Sonia
Cortassa
b,
Brian
O'Rourke
b and
Miguel A.
Aon
b
aSchool of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AT Wales, UK. E-mail: lloydd@cardiff.ac.uk; Fax: +44(0) 2920 874305; Tel: +44(0)2920 842649
bJohns Hopkins University, School of Medicine, 1059 Ross Bldg., 702 Rutland Av., Baltimore, MD 21205, USA
First published on 5th December 2011
The coherent and robust, yet sensitively adaptable, nature of organisms is an astonishing phenomenon that involves massive parallel processing and concerted network performance at the molecular level. Unravelling the dynamic complexities of the living state underlines the essential operation of ultradian oscillations, rhythms and clocks for the establishment and maintenance of functional order simultaneously on fast and slower timescales. Non-invasive monitoring of respiration, mitochondrial inner membrane potentials, and redox states (especially those of NAD(P)H, flavin, and the monochlorobimane complex of glutathione), even after more than 50 years research, continue to provide both new insights and biomedical applications. Experiments with yeast and in cardiac cells reveal astonishing parallels and similarities in their dynamic biochemical organization.
Insight, innovation, integrationInsight is lacking about the overall integration, coordination and coherence of living systems, especially with respect to temporal organisation. Here we have developed a method whereby yeast or cardiomyocyte single cells are observed by two photon laser scanning fluorescence microscopy that enables mitochondrial membrane potential, NAD(P)H, and glutathione redox states, as well as reactive O2 species to be monitored simultaneously in identical fields. Concerted oscillatory performance (period range sec to min) was observed in both systems, emphasizing the central role of redox balance. Membrane inlet mass spectrometry on self-synchronised yeasts also indicated coordinated and self-similar multiple time scales for respiratory oscillations (period range min to h). These features are central for spatiotemporal synchrony and network coherence. |
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Fig. 1 The temporal structure of biological organization as characterized by the relaxation times of biological processes. The different time scales exhibited by living systems are shown by the typical relaxation times (after perturbation) in decades on a logarithmic scale. This way of representation accounts for the broad temporal scale spanned by the occurrence of biological processes, i.e. from picoseconds [10−12 s] to many years. The biological processes indicated represent many of the main molecular and cellular functional properties as well as those related to conformational changes in molecules (e.g. following ligand interactions), metabolism, energy transduction, solute transport, action potentials in neurons and cardiomyocytes, macromolecule polymerization and cell growth and division. (Reproduced from Lloyd and Rossi (2008) in Ultradian Rhythms from Molecules to Mind). |
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Fig. 2 Periodic phenomena across temporal scales. The period of cyclic processes in the ultradian (<24 h), circadian (24 h) and infradian (>24 h) time domains are shown in a logarithmic scale. Several clocks (temperature-compensated biological mechanisms) can be found in the ultradian temporal domain, in addition to the ubiquitous circadian clock. A prominent ultradian clock is the circahoralian (about 1 h period) that is indicated as occurring in many lower eukaryotes and is always of shorter duration that the cell division cycle, as shown in the figure. (Reproduced from Lloyd and Rossi (2008) in Ultradian Rhythms from Molecules to Mind). |
Fig. 3 indicates common features not only between widely-separated taxa, but also across widely-based time domains.14–16 The scheme shows that generation of rhythms entails the cycling of cytoplasmic and mitochondrial proteins between their oxidized and reduced states, mainly driven by ROS and NAD(P)H and reflected in the redox potentials of the thiol pools.15,17,18Mitochondria are the main source of ROS produced by the respiratory chain; oxidative stress results from an imbalance between ROS production and ROS scavenging. The glutathione redox potential, and the absolute concentrations of reduced (GSH) and oxidised (GSSG) glutathione, modulate glutathione reductase (GR) and transhydrogenase (THD). Although in yeast it has not been confirmed that THD plays a part, thioredoxin and glutaredoxin play a crucial role. In turn, the redox status in the mitochondrial matrix represented by NADH and NADPH pools,19 which are interconverted by THD activity, drives GSH regeneration.15 Mitochondrial GSH is also replenished via cytoplasmic import of reducing power through carriers and metabolic shuttle systems.20–22 In cardiomyocytes, mitochondrial oscillations are triggered when a threshold level of ROS is attained12,13 which happens when the glutathione redox potential oxidizes to GSH:
GSSG ratios between 150
:
1 to 100
:
1. A critical ratio of GSH-GSSG of 50
:
1 elicits the opening of the permeability transition pore, cell hyper-contraction, and death.15,18 In yeast, numerous processes (magenta) have proven to be oscillatory and we propose that ensembles of oscillators are coupled via this primordial mechanism.5,14 In yeast, these processes may be modulated by illumination, temperature changes or chemical perturbation. Perturbation analysis of the yeast ultradian system utilising NO+ donors19 confirms the central role of this redox system; the numbers on Fig. 3 represent the site of perturbation. The assay, biological significance, physiology and pathophysiology of mitochondrial glutathione have been reviewed.20–22 The pivotal roles of the GSH/GSSG redox couple in the generation of oscillatory respiration in yeast has been confirmed.23
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Fig. 3 Redox cycling of intracellular thiols at the core of yeast and heart rhythmicity. The scheme shows that generation of rhythms entails the cycling of cytoplasmic and mitochondrial proteins between their oxidized and reduced states mainly driven by ROS and the redox potential of the thiols pool. Mitochondria are the main source of ROS produced by the respiratory chain; oxidative stress results from an imbalance between ROS production and ROS scavenging. The glutathione and thioredoxin redox potentials, and the absolute concentrations of reduced (GSH) and oxidized (GSSG) glutathione, modulate ROS emission in mitochondria. Mitochondrial GSH and thioredoxin (Trx2) regeneration in the matrix are essential for keeping the ROS balance. In yeast, numerous processes (magenta I–III) have proven to be oscillatory and we propose that ensembles of oscillators are coupled via this primordial mechanism. In heart, the redox cycling involves at least glutathione, Trx2, and NAD(P)H couples in the mitochondrial matrix, as modulators of ROS generation. Perturbation analysis of the yeast ultradian system utilising NO+ donors, 5-nitro-2-furaldehyde(I), D,L-butathionine (S,R)-sulphoximine(II) or protonophores(III), confirms the central role of this redox system. The numbers on the figure represent the site of perturbation. (Reproduced from Aon MA, Roussel MR, Cortassa S, B. O'Rourke, Murray DB, et al. (2008) The Scale-Free Dynamics of Eukaryotic Cells. PLoS ONE 3(11): e3624. doi: 10.1371/journal.pone.0003624). |
The history of the concept and evidence for the pivotal role of the cycling of low molecular weight thiols and protein thiols during ultradian cell-division cycle, and circadian rhythmicity goes back to the 1930's. Key advances were made with the pioneering research by Rapkine24 employing experiments on sea urchin eggs. Subsequent theoretical treatment and mathematical modelling led to an unified basic understanding of the fundamental significance of oscillations in living systems.
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Fig. 4 Mitochondria in yeast and cardiomyocytes. (a,b) Confocal microscopy of a Saccharomyces cerevisiaecell from an aerobically-grown culture loaded in (a) with tetramethyl rhodamine ethyl ester (TMRE). (b) autofluorescence (NAD(P)H) during perfusion of untreated tethered organisms with 5mM glucose in aerated Na phosphate-buffered saline. (c,d) Two photon laser scanning fluorescence microscopy of a freshly isolated cardiomyocyte loaded with (c) TMRE or after (d) autofluorescence. Notice the reticulate or lattice organization of the mitochondrial network in yeast and heart, respectively. |
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Fig. 5 ROS production, and ΔΨm, NADH, and GSH status as a function of GSH![]() ![]() |
A single focused laser flash produced similarly rapid processes within both heart cells and a population of yeast organisms.12,29 A fast localized oxidation of nicotinamide nucleotides and depolarisation of ΔΨm was followed by a rapid migration of both processes as a wave spreading throughout the mitochondrial network in cardiomyocyte and across the epicardium,12,13,18,30,31 and also between mitochondria within and between yeast cells.29 Depletion of cellular GSH and accumulation of ROS closely follow the trigger of the laser flash: all the observed changes follow oscillatory kinetics in the minute domain.
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Fig. 6 Multi-oscillatory behavior in a self-organized (synchronous) continuous culture of S. cerevisiae. Relative membrane-inlet mass spectrometry (MIMS) signals of the m/z = 32 component corresponding to dissolved O2versus time. Time is given in hours after the start of fermentor continuous operation. Periods of ∼13 h, ∼40 min and ∼4 min can be detected (see the sub-panel). The biological bases for all three oscillatory outputs of the yeast culture have been confirmed by exclusion of the possible influences of variations of aeration or stirring, pulsed medium addition, cycles of NaOH addition and pH variation, or cycles of temperature control. (Reproduced from Aon MA, Roussel MR, Cortassa S, O'Rourke B, Murray DB, et al. (2008) The Scale-Free Dynamics of Eukaryotic Cells. PLoS ONE 3(11): e3624. doi: 10.1371/journal.pone.0003624). |
The 40 min period, the synchronisation time-base for the coherent operation of all the parallel processing necessary, is the output of the ultradian clock, a temperature-compensated redox oscillator.40 A spike of H2S produced by sulphite reductase once per 40 min cycle, alongside acetaldehyde and probably other synchronizers, is essential for the auto-dynamic coordination of the yeast population.41 A strange attractor indicating chaotic behaviour is revealed in plots of the concentrations of the three gases (O2, CO2 and H2S).35,42 Deterministic chaos is characteristic of adaptable and robust systems showing very complex behaviour, predictable only in the short-term, but underpinned by a set of simple non-linear kinetic equations.43,44
However, close inspection of the apparently ‘noisy’ output indicates that the highly responsive mass spectrometric monitoring (t1/2 = 2 s) also reveals an oscillator with a 4 min period: this presumably corresponds to the ROS-driven mitochondrial oscillator observed in cardiomyocytes.45 In fact, the presence of the short period oscillations in yeast35 represents evidence of the common redox core shared with cardiomyocytes (Fig. 3). The ability of 4-chlorodiazepam, a mitochondrial benzodiazepine receptor antagonist, to block the oscillations in the minute time domain both in yeast and heart cells14 argues in favor of a common set of redox processes as the mechanistic base of the rhythmicity observed in both systems (Fig. 3), at least in the minute time scale. The long period (12 h) may correspond to a sub-harmonic of the circadian oscillator as the experiments were conducted under natural day-night conditions.
Periodic behaviour was also revealed in the mitochondrial network of cardiac muscle cells at the most fundamental level of their spatio-temporal energetic organization. The ROS-dependent mitochondrial oscillator described in cardiomyocytes exhibits at least two modes of function under physiological conditions or in response to metabolic and oxidative stress. Both modes depend upon network behaviour of mitochondria. In their normal physiological states these organelles behave as a highly integrated network of coupled oscillators (Fig. 7a) with a broad range of frequencies.46 ROS normally weakly couples mitochondria but becomes a strong coupling messenger when under oxidative stress;12,47 the mitochondrial network attains criticality when a threshold level is attained. Then a certain density of mitochondria forms a cluster that spans the whole volume of the cell.13 Under these conditions, the slightest perturbation triggers a cell-wide collapse of ΔΨm, visualized as a wave of depolarization travelling through the cell. There then follows whole-cell synchronized oscillations in NADH, ROS and GSH. This behaviour scales from the mitochondria to the cell by driving cell excitability and to the whole heart in which potentially catastrophic arrhythmias rapidly ensue.17,30,31,48–50 Next-amplitude plots reveal the sharp transition to the patho-physiological state in which an apparently noisy regime gives way to a slow oscillatory pathophysiology in which a high-amplitude period of about 100 s is dominant (Fig. 7b).
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Fig. 7 Physiological and pathophysiological behavior of the network of coupled mitochondrial oscillators in cardiac cells. (a) Temporally correlated behavior of the mitochondrial network in cardiac cells. The statistical analysis of the TMRM signal showed that the mitochondrial network of the heart cell functions as a highly correlated network of oscillators. Shown in left panel are the results obtained by Relative Dispersional Analysis (RDA) as a log-log plot of the CV (=SD/mean) of the fluorescence distribution obtained at increasing values of the aggregation parameter, m. This gives a fractal dimension, Df, close to 1.0, either for myocytes showing large (“pathophysiological”) oscillations in ΔΨm (top right panel A) or those under “physiological” conditions (bottom right panel). A completely random process gives Df ∼ 1.5. (b) Before the mitochondrial network reaches criticality, the ΔΨm oscillates at high frequencies and small amplitudes. After criticality, the network behavior evolves into “pathophysiological” behavior characterized by low-frequency, high amplitude oscillations. |
Both yeast and cardiomyocyte function on several temporal scales simultaneously; their multi-oscillatory behaviour became evident when investigated by power spectral analysis. Relative dispersional analysis reveals that both exhibit self-similar (fractal) features characteristic of scale-free systems.14,51 These systems have a common feature in that they show a long-term memory in a time series and on all scales share statistically similar characteristics in their frequency-amplitude relationships. Thus these fluctuations are not random excursions but correlated oscillations. Power spectral analysis further confirms these features: the inverse power relationship between amplitude and frequency shows that β (the spectral exponent) has values of 1.8 for cardiomyocytes ΔΨm and 1.95 (i.e. close to that for “colored noise” expected for a chaotic system,52,53 for a similar analysis of yeast respiratory oscillations. Were these time series randomly noisy (“white noise”), β would be 0. These relationships hold over three orders of magnitude (12 h–4 min for yeast and 100 s–220 ms for cardiac muscle cells).14,51,54
The dynamic balance between ROS production and ROS scavenging is one of the underlying basic mechanisms at the origin of oscillations in the redox core (Fig. 3), according to mathematical modelling of the mitochondrial oscillator. Basic network motifs participating in the regulation of this dynamic balance are essentially positive and negative feedbacks. More specifically, the positive feedback is given by mitochondrial respiration and the percentage of the respiratory flux diverted to the free radical superoxide anion, O2˙−; when the latter attains a certain threshold in the matrix facilitates the opening of an inner membrane anion channel (IMAC) which depolarizes the ΔΨm. In order to regenerate the ΔΨm, a negative feedback driven by Cu, Zn SOD on O2˙− closes the IMAC allowing the repolarization of the inner membrane. The computational model of the mitochondrial oscillator is able to produce oscillatory periods ranging from msec to several hours.
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Fig. 8 Effect on cell viability and ΔΨm (TMRM) and GSH (GSB) pool of a pro-reductive or a pro-oxidative shift in redox status. (a, b) Cardiomyocytes in the absence (control) or in the presence of 2 mM dithiothreitol (DTT) or 1 mM diallyldisulphide (DADS) were preincubated for 2 h and examined by two-photon microscopy. The pro-oxidative shift in the intracellular thiol pool significantly decreases both the levels of intracellular GSH and ΔΨm with respect to the control (a); in contrast, both variables are increased in the presence of DTT (b). Eighty five percent of cardiomyocytes became non-viable in the presence of DADS (c, left panel) whereas DTT protected them to even slightly, but significantly, higher (85%) values (c, right panel) than the control (80%; c, mid panel) (n = 400; 2 experiments). |
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Fig. 9 Kinetics of cell death induction in Candida albicans by a short treatment with DADS. (a) Culture-grown C. albicanscells were exposed to 0.5 mM DADS for 30 min at room temperature, while untreated cells remained as control. Cell death was monitored after DADS removal by sampling at the times specified, in which the cells were washed and subjected to protoplasting. Cell death by apoptosis and necrosis was monitored with Alexa Fluor 488 annexin V and propidium iodide on coverslips coated with polylysine, in a thermostated chamber at 30 °C, on the stage of a Nikon E600FN upright microscope. (b) Images of green and red fluorescence were recorded by two photon microscopy. Representative snapshots of the kinetics of appearance of necrotic (red) or apoptotic (green) cells are shown. Key to symbols: A, apoptotic; N, necrotic; V, viable; DADS, diallyl disulphide. (Reproduced from Lemar, Aon, Cortassa, O'Rourke, Muller and Lloyd (2007) Yeast 24, 695–706). |
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Fig. 10 DADS-triggered oxidative stress, ΔΨm depolarization, and cell death. Freshly isolated guinea pig cardiomyocytes were loaded with TMRM and the ROS probe CM-H2DCFDA at 37 °C and imaged by two-photon laser scanning fluorescence microscopy in a perfusion chamber. After exposure to the thiol-oxidizing agent DADS (1 mM), the oxidation of the ROS probe steadily increased after oxidation of the NAD(P)H pool triggering ΔΨm depolarization and sudden cell death. |
The apoptotic/necrotic effects of DADS on C.utilis (Fig. 9) can be quantified by the propidium iodide/annexin-fluorescein antibody procedure whereby the permeabilization of plasma membranes is indicated by red fluorescence and the externalization of serine-phospholipid gives a green fluorescence. These processes are measured by the changing distributions of populations of the dying yeasts (Fig. 9a).
In isolated cardiomyocytes perfusion with 1 mM diamide, a powerful glutathione oxidant,15,55 leads to an oscillatory mode that presages oxidation of the autofluorescent nicotinamide nucleotides and collapse of ΔΨm during progressive ROS accumulation.15 By 20–30 min to 2 h cell death was evident (Fig. 5, 8–10). These effects can be clearly demonstrated by poising the redox state of the GSH pool in a series of successively increased oxidation states (Fig. 5). In the perfused guinea pig heart, 30 min exposure to this thiol oxidant produces similar results and the whole organ progresses to an arrhythmic state17 (Fig. 11).
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Fig. 11 Imaging of ΔΨm (top) and GSB (bottom) in intact guinea pig hearts using 2-photon microscopy after exposure to diamide. Hearts were perfused under normoxic conditions in the presence of 200 μM diamide. Mitochondria remained polarized until approximately 20 min of exposure, at which point Ψm, began to heterogeneously depolarize (top row), and GSB (the adduct marker of GSH, bottom row) became markedly oxidized in parallel with the onset of arrhythmias. Bar is equal to 100 μm. (Modified from Brown, Aon, Frasier, Sloan, Maloney, Anderson, O'Rourke (2010) J. Mol. Cell. Cardiol. 48, 673–679). |
Applications of redox state monitoring, pioneered by Britton Chance, have been perfected over 50 years,56 culminating in the in vivo fluorimetric assessment of the effects of cyclosporine A on mitochondrial function during protection against infarction in myocardial ischemia and reperfusion.57
Minimally invasive continuous monitoring of mitochondrial functions in yeasts (by membrane inlet mass spectrometry), and in cardiomyocytes by two-photon microscopy, reveal that these two evolutionary-distant systems exhibit strikingly similar oscillatory features. The evidence suggests conservation of an indispensable core metabolic function in the form of intrinsically rhythmic organization involving redox balance. Continuous oscillatory states are observable on multiple timescales, an obligatory requirement for the synchronization and coordination of reactions, events and processes of the coherently-functioning cellular network. Beyond the necessary boundary conditions for adaptable and robust performance, various stress factors (physical, chemical) perturb oscillatory behaviour to signal and initiate the pathways to cell death. Notwithstanding, whether the intrinsic oscillatory dynamics emerging from the redox core exert timekeeping functions that synchronize the spatio-temporal occurrence of processes happening in, e.g. tissue, organ, is still an open theoretical and experimental question. These processes include metabolic-, information-, and signalling-network dynamics which in turn regulate and control essential functions involved in cell growth, division, and differentiation, within tissues, organs, and organisms. This represents a wide research avenue into the future.
Footnote |
† Dedication: This contribution is dedicated to the memory of Britton Chance, who passed away on November 18th 2010, a mentor and life-long friend of DL, whose many achievements can be viewed at: http://www.med.upenn.edu/biocbiop/chance/reflections.html |
This journal is © The Royal Society of Chemistry 2012 |