Synchronized Whole Cell Oscillations in Mitochondrial Metabolism Triggered by a Local Release of Reactive Oxygen Species in Cardiac Myocytes
2003; Elsevier BV; Volume: 278; Issue: 45 Linguagem: Inglês
10.1074/jbc.m302673200
ISSN1083-351X
AutoresMiguel A. Aon, Sonia Cortassa, Eduardo Marbán, Brian O’Rourke,
Tópico(s)ATP Synthase and ATPases Research
ResumoReactive oxygen species (ROS) and/or Ca2+ overload can trigger depolarization of mitochondrial inner membrane potential (ΔΨm) and cell injury. Little is known about how loss of ΔΨm in a small number of mitochondria might influence the overall function of the cell. Here we employ the narrow focal excitation volume of the two-photon microscope to examine the effect of local mitochondrial depolarization in guinea pig ventricular myocytes. Remarkably, a single local laser flash triggered synchronized and self-sustained oscillations in ΔΨm, NADH, and ROS after a delay of ∼40s, in more than 70% of the mitochondrial population. Oscillations were initiated only after a specific threshold level of mitochondrially produced ROS was exceeded, and did not involve the classical permeability transition pore or intracellular Ca2+ overload. The synchronized transitions were abolished by several respiratory inhibitors or a superoxide dismutase mimetic. Anion channel inhibitors potentiated matrix ROS accumulation in the flashed region, but blocked propagation to the rest of the myocyte, suggesting that an inner membrane, superoxide-permeable, anion channel opens in response to free radicals. The transitions in mitochondrial energetics were tightly coupled to activation of sarcolemmal K ATP currents, causing oscillations in action potential duration, and thus might contribute to catastrophic arrhythmias during ischemia-reperfusion injury. Reactive oxygen species (ROS) and/or Ca2+ overload can trigger depolarization of mitochondrial inner membrane potential (ΔΨm) and cell injury. Little is known about how loss of ΔΨm in a small number of mitochondria might influence the overall function of the cell. Here we employ the narrow focal excitation volume of the two-photon microscope to examine the effect of local mitochondrial depolarization in guinea pig ventricular myocytes. Remarkably, a single local laser flash triggered synchronized and self-sustained oscillations in ΔΨm, NADH, and ROS after a delay of ∼40s, in more than 70% of the mitochondrial population. Oscillations were initiated only after a specific threshold level of mitochondrially produced ROS was exceeded, and did not involve the classical permeability transition pore or intracellular Ca2+ overload. The synchronized transitions were abolished by several respiratory inhibitors or a superoxide dismutase mimetic. Anion channel inhibitors potentiated matrix ROS accumulation in the flashed region, but blocked propagation to the rest of the myocyte, suggesting that an inner membrane, superoxide-permeable, anion channel opens in response to free radicals. The transitions in mitochondrial energetics were tightly coupled to activation of sarcolemmal K ATP currents, causing oscillations in action potential duration, and thus might contribute to catastrophic arrhythmias during ischemia-reperfusion injury. Mitochondria play a multifunctional role as key arbiters of cell life and death. In addition to oxidative phosphorylation, mitochondria are involved in thermogenesis, free radical production, and intracellular Ca2+ homeostasis. Impairment of mitochondrial function during and after ischemia causes rapid energy depletion, contractile failure, and loss of cellular integrity, which may lead to necrotic or apoptotic cell death (1Crompton M. Virji S. Doyle V. Johnson N. Ward J.M. Biochem. Soc. Symp. 1999; 66: 167-179Crossref PubMed Scopus (186) Google Scholar, 2Duchen M.R. J. Physiol. 1999; 516: 1-17Crossref PubMed Scopus (531) Google Scholar, 3Murphy M.P. Biochim. Biophys. Acta. 2001; 1504: 1-11Crossref PubMed Scopus (52) Google Scholar). ROS 1The abbreviations used are: ROS, reactive oxygen species; DIDS, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid; CM-H2DCFDA, 5-(-6)-chloromethyl-2′,7′-dichlorohydrofluorescein diacetate; CM-H2DCFH, 5-(-6)-chloromethyl-2′,7′-dichlorohydrofluorescein; FRET, fluorescence energy transfer; BKA, bongkrekic acid; TMPyP, Mn(III)-tetrakis(1-methyl-4-pyridil)porphyrin pentachloride; TMRE, tetramethylrhodamine ethyl ester; PTP, permeability transition pore; CsA, cyclosporin A; IMAC, inner membrane anion channel; mBzR, mitochondrial benzodiazepine receptor.1The abbreviations used are: ROS, reactive oxygen species; DIDS, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid; CM-H2DCFDA, 5-(-6)-chloromethyl-2′,7′-dichlorohydrofluorescein diacetate; CM-H2DCFH, 5-(-6)-chloromethyl-2′,7′-dichlorohydrofluorescein; FRET, fluorescence energy transfer; BKA, bongkrekic acid; TMPyP, Mn(III)-tetrakis(1-methyl-4-pyridil)porphyrin pentachloride; TMRE, tetramethylrhodamine ethyl ester; PTP, permeability transition pore; CsA, cyclosporin A; IMAC, inner membrane anion channel; mBzR, mitochondrial benzodiazepine receptor. have been implicated in ischemic dysfunction; however, they play a dual role as determinants of cell survival, on the one hand contributing to Ca2+ overload and the induction of a mitochondrial permeability transition, but on the other acting as second messengers that protect cells against injury (4Sundaresan M. Yu Z.X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2305) Google Scholar, 5Baines C.P. Goto M. Downey J.M. J. Mol. Cell Cardiol. 1997; 29: 207-216Abstract Full Text PDF PubMed Scopus (418) Google Scholar, 6Vanden Hoek T.L. Becker L.B. Shao Z. Li C. Schumacker P.T. J. Biol. Chem. 1998; 273: 18092-18098Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar). Mitochondria are a major site of physiological ROS production in the cardiomyocyte, with ∼1-5% of the electrons flowing through the electron transport chain leaking into the production of ROS (7Kowaltowski A.J. Vercesi A.E. Free Radical Biol. Med. 1999; 26: 463-471Crossref PubMed Scopus (701) Google Scholar, 8Chance B. Sies H. Boveris A. Physiol. Rev. 1979; 59: 527-605Crossref PubMed Scopus (4797) Google Scholar). The negative effects of ROS on metabolism are evident in several studies showing rapid and spatiotemporally heterogeneous discharge of ΔΨm in response to oxidative stress (1Crompton M. Virji S. Doyle V. Johnson N. Ward J.M. Biochem. Soc. Symp. 1999; 66: 167-179Crossref PubMed Scopus (186) Google Scholar, 2Duchen M.R. J. Physiol. 1999; 516: 1-17Crossref PubMed Scopus (531) Google Scholar) and/or Ca2+ overload (9Ichas F. Jouaville L.S. Mazat J.P. Cell. 1997; 89: 1145-1153Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar), including protocols employing laser-induced photooxidation (10Hüser J. Blatter L.A. Biochem. J. 1999; 343: 311-317Crossref PubMed Scopus (232) Google Scholar, 11Siemens A. Walter R. Liaw L.H. Berns M.W. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 466-470Crossref PubMed Scopus (35) Google Scholar) and mitochondrial ROS-induced ROS release (12Zorov D.B. Filburn C.R. Klotz L.O. Zweier J.L. Sollott S.J. J. Exp. Med. 2000; 192: 1001-1014Crossref PubMed Scopus (1105) Google Scholar). In light of our previous work showing that substrate deprivation can initiate synchronized oscillations of mitochondrial redox and membrane potential (13O'Rourke B. Ramza B.M. Marbán E. Science. 1994; 265: 962-966Crossref PubMed Scopus (225) Google Scholar), and that a diffusible cytoplasmic messenger may be involved (14Romashko D.N. Marbán E. O'Rourke B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1618-1623Crossref PubMed Scopus (178) Google Scholar), the present study tests whether similar global self-organizing behavior can be triggered by a highly localized perturbation of a few mitochondria among the thousands packed within the cardiac myocyte. We demonstrate that ROS release and mitochondrial depolarization in less than 1% of the volume of the cell can trigger spatiotemporally synchronized oscillations in ΔΨm, ROS production, and mitochondrial redox potential throughout the entire volume of the cell. Close coupling of the metabolic responses to cardiac electrical excitability highlights the importance of this form of intraorganellar communication in determining whole cell function. Cardiomyocyte Isolation—All experiments were carried out at 37 °C on freshly isolated adult guinea pig ventricular myocytes prepared by enzymatic dispersion as previously described (13O'Rourke B. Ramza B.M. Marbán E. Science. 1994; 265: 962-966Crossref PubMed Scopus (225) Google Scholar). After isolation, cells were stored in Dulbecco's modified Eagle's medium (10-013, Mediatech, Inc., Herndon, VA) in laminin-coated Petri dishes in a 5% CO2 incubator at 37 °C and used within 6-8 h of isolation. Experimental recordings started after exchange of the Dulbecco's modified Eagle's medium with an experimental solution containing 140 mm NaCl, 5 mm KCl, 1 mm MgCl2, 10 mm HEPES, 1 mm CaCl2, pH 7.5 (adjusted with NaOH), supplemented with 10 mm glucose. The dish containing the cardiomyocytes was equilibrated at 37 °C with unrestricted access to atmospheric oxygen on the stage of a Nikon E600FN upright microscope. Fluorescent Probes for Two-photon Laser Scanning Microscopy—The cationic potentiometric fluorescent dye tetramethylrhodamine ethyl ester (TMRE) was used to monitor changes in ΔΨm. The large potential gradient across the inner mitochondrial membrane results in the accumulation of TMRE within the matrix compartment according to its Nernst potential (15Loew L.M. Tuft R.A. Carrington W. Fay F.S. Biophys. J. 1993; 65: 2396-2407Abstract Full Text PDF PubMed Scopus (192) Google Scholar). ROS production was monitored with the ROS-sensitive fluorescent probe 5-(-6)-chloromethyl-2′,7′-dichlorohydrofluorescein diacetate (CM-H2DCFDA). The acetate group of CM-H2DCFDA is hydrolyzed by esterases when it enters the cell and is trapped inside as the nonfluorescent 5-(-6)-chloromethyl-2′,7′-dichlorohydrofluorescein (CMH2DCFH). CM-H2DCFH was chosen because, unlike underivatized dichlorofluorescein (H2DCFDA), it is well retained in cells (16Xie Z. Kometiani P. Liu J. Li J. Shapiro J.I. Askari A. J. Biol. Chem. 1999; 274: 19323-19328Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar) and, in our case, in the mitochondrial matrix. Similar results demonstrating matrix localization of ROS production were also obtained with the carboxyl derivative, 5-(-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (Cbx-DCF). Oxidation of CM-H2DCFH by ROS, particularly by hydrogen peroxide (H2O2) and hydroxyl radical (17Vanden Hoek T.L. Li C. Shao Z. Schumacker P.T. Becker L.B. J. Mol. Cell Cardiol. 1997; 29: 2571-2583Abstract Full Text PDF PubMed Scopus (332) Google Scholar), yields the fluorescent product CM-DCF, and in an indirect manner, measures mitochondrially produced O2˙- that has dismutated to H2O2 through the action of mitochondrial manganese-dependent superoxide dismutase (8Chance B. Sies H. Boveris A. Physiol. Rev. 1979; 59: 527-605Crossref PubMed Scopus (4797) Google Scholar, 18Turrens J.F. Alexandre A. Lehninger A.L. Arch. Biochem. Biophys. 1985; 237: 408-414Crossref PubMed Scopus (1064) Google Scholar). To image the distribution of ΔΨm and ROS production simultaneously, 100 nm TMRE and 2-6 μm CM-H2DCFDA were added to the external solution and allowed to equilibrate for at least 20 min. Under these conditions, CM-DCF fluorescence was largely localized to the mitochondrial matrix space. The influence of partial fluorescence energy transfer (FRET) (19Stryer L. Annu. Rev. Biochem. 1978; 47: 819-846Crossref PubMed Scopus (1956) Google Scholar) between CM-DCF and TMRE is described in the Supplemental Materials. Retention of calcein in the mitochondrial matrix was tested by loading myocytes for 20 min with 2 μm calcein-AM at room temperature. After dye loading the cells were resuspended in the experimental solution used for imaging. Intracellular esterase action then resulted in loading of both the cytoplasmic and mitochondrial compartments of the cell. Calcein-AM desterification was allowed to proceed for at least 1 h before imaging. To reduce the contribution of the cytoplasmic component to the fluorescence images, myocytes were patch-clamped with fluorophore-free pipette solution, which permitted diffusion of the cytoplasmic calcein into the large pipette volume. Image Acquisition and Analysis—Images were recorded using a two-photon laser scanning microscope (Bio-Rad MRC-1024MP) with excitation at 740 nm (Tsunami Ti:Sa laser, Spectra Physics). Because of the overlap in the cross-sections for two-photon excitation of the three fluorophores of interest (20Xu C. Zipfel W. Shear J.B. Williams R.M. Webb W.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10763-10768Crossref PubMed Scopus (1071) Google Scholar) (NADH, CM-DCF, and TMRE), this wavelength permitted recording of redox potential, ROS production, and ΔΨm simultaneously. The red emission of TMRE was collected at 605 ± 25 nm and the green emission of CM-DCF was recorded at 525 ± 25 nm. NADH emission was collected as the total fluorescence <490 nm. At 3.5-s intervals, 512 × 512 pixel 8-bit grayscale images of the three emission channels were collected simultaneously and stored. The total illumination time was 3.0 s/image, unless otherwise specified. Average power from the Ti:Sa laser was 1000 milliwatts and the pulse bandwidth was ∼12 nm, corresponding to <60 fs pulse duration at 80 MHz repetition rate. This excitation was attenuated by the optical system and by a combination of neutral density filters such that the average intensity at the focal plane was <10 milliwatts. Light-induced mitochondrial depolarization was applied in a small cytoplasmic volume by zooming the laser beam in on a 20 × 20-pixel (8.7 × 8.7 μm square, <1 μm focal depth) region of the cell. Because the total scan duration was the same as that used for full frame imaging, the laser dwell time in a given cell volume during a flash was ∼655 times normal. This caused the local generation of ROS because of direct interaction with molecular O2 to promote triplet state excitation of local fluorophores (21Tsien R.Y. Waggoner A. Pawley J.B. Fluorophores for Confocal Microscopy. Handbook of Biological Confocal Microscopy. Plenum Press, New York1995: 267-279Google Scholar, 22Hüser J. Rechenmacher C.E. Blatter L.A. Biophys. J. 1998; 74: 2129-2137Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Local photon-induced ROS elaboration, reinforced by the restricted irreversible depolarization of ΔΨm, allowed us to perturb a small region of the myocyte to look for propagating effects. In the absence of the local perturbation, with few exceptions (see "Results"), myocyte behavior was stable over the duration of the experiments. Image Analysis—Images were analyzed offline using ImageJ software (Wayne Rasband, National Institutes of Health) 2rsb.info.nih.gov/ij. with customized plugins (B. O'R.). For visualization of the spatio-temporal responses of TMRE and CM-DCF presented in Figs. 1, 2, 4, 6, and 7, a 2 to 3-pixel wide line was drawn along the length of the myocyte (as shown in Fig. 1) and the average fluorescence profile along the line was determined for the entire time series of two-dimensional images for a given experiment. A new image was then created, showing the line fluorescence as a function of time (time-line image).Fig. 2Quantitative characterization of cell-wide mitochondrial oscillations. A, the asterisk on top of the TMRE time-line image shown in panel B of Fig. 1 points out the cycle of ΔΨm depolarization and repolarization expanded as an image montage in panel A. The frame interval in panel A is 7 s. B, quantitative analysis of the fraction of polarized mitochondria from two-dimensional TMRE images as a % of the total cell area (see "Experimental Procedures"). Arrow indicates the timing of the flash. The trace labeled Control refers to a cell not exposed to a flash in the same microscopic field. C, simultaneous recordings of the temporal evolution of fluorescence intensity (a.u., arbitrary intensity units) for TMRE, CM-DCF, and the endogenous NADH signals in the flashed region before and after the laser flash (arrow). D, time course of average whole cell fluorescence of TMRE and NADH, and E, CM-DCF and the derivative of CM-DCF signals (dF/dt, purple). The precise phase relationship between all signals can be clearly appreciated from the vertical reference line drawn. The plots were from a different cell than that shown in panel A.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Effects of inhibitors of ROS production or scavengers on cell-wide mitochondrial oscillations. Conditions are as described in the legend to Fig. 1. A, scheme of the possible pathway of ROS generation in the electron transport chain (adapted from Ref. 60Hockenbery D.M. Oltvai Z.N. Yin X.M. Milliman C.L. Korsmeyer S.J. Cell. 1993; 75: 241-251Abstract Full Text PDF PubMed Scopus (3289) Google Scholar). The protonmotive Q-cycle of cytochrome bc1 (complex III) is also shown explicitly, along with the sites of action of its two inhibitors, myxothiazol (Myx) and antimycin (Ant). In this branched pathway of electron transfer, the ubisemiquinone anion (UQ·) can be generated from ubiquinol (UQH2) at the periplasmic (UQp·) or matrix (UQm·) faces, respectively, of the mitochondrial membrane. High ROS production in Ant is because of accumulation of the precursors of O2˙-, UQp·, and UQm·. UQp· anion reduces the b 566 and b 562 hemes, as indicated by arrows through the center of the Q-cycle, while reduction of the FeS(III) center is required for the oxidation of UQH2 to UQp· (25Raha S. McEachern G.E. Myint A.T. Robinson B.H. Free Radical Biol. Med. 2000; 29: 170-180Crossref PubMed Scopus (194) Google Scholar, 41Nicholls D.G. Ferguson S.J. Bioenergetics 2. Academic Press, San Diego1992Google Scholar). B and C, time-line images of TMRE for typical experiments of cells acutely exposed to rotenone (B, 15 μm) or antimycin A (C, 6 μm), respectively. Thick lines above panels indicate the presence of inhibitor. D, TMRE traces of myocytes showing cell-wide mitochondrial oscillations after a laser flash and, subsequently, being subjected to an acute addition of rotenone (15 μm, Rot) (see also B), myxothiazol (5 μm, Myx), NaCN (5 mm, CN -), or bongkrekic acid (25 μm, BKA) while the image acquisition was briefly paused. After imaging resumed (within ∼3 min of inhibitor addition), the oscillations were abolished. The thick line in each panel indicates the presence of inhibitor. E, the effect of the different inhibitors on ROS production as measured by the normalized CM-DCF signal per unit time. Antimycin (Ant) and oligomycin (Olig) concentrations were 6 μm and 10 μg/ml, whereas the other inhibitor concentrations were as described in D. ROS production following a flash was quantified for cells preincubated in the presence of each inhibitor. Under these conditions, the cells did not exhibit oscillations after the laser flash. Each bar of the normalized CM-DCF signal corresponds to n = 6 cells, obtained from two independent cell preparations. The n value exceeds that required according to retrospective power calculations based on the p value and the actual differences between the control and treatment means. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control. F, the superoxide dismutase mimetic TMPyP was acutely added to myocytes at a final concentration of 250 or 500 μm. Prolonged incubation (1 h) with the scavenger completely suppressed the synchronized oscillations (n = 10, three experiments). The control corresponds to cells exhibiting cell-wide mitochondrial oscillations before addition of the scavenger. The time-dependent effect of TMPyP to suppress oscillations correlated with a significant decrease of ROS production to basal levels (not shown).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Blockage of mitochondrial inner membrane anion channels abolishes oscillations. Conditions are as described in the legend to Fig. 1. Either acute addition (not shown) or preincubation with PK11195 (panel A; 50 μm) or DIDS (panel C; 100 μm) abolished whole cell mitochondrial oscillations triggered by a flash, but did not prevent local depolarization of ΔΨm and ROS production in the flashed region, as shown in representative time-line images of TMRE (n = 4) or CM-DCF (n = 4) fluorescence. The time course plots of TMRE (red) and CM-DCF (green) demonstrate that matrix ROS still increases markedly during mitochondrial depolarization of the flashed region in the presence of PK11195 (B) or DIDS (D). E, shows close-up views of ΔΨm and CM-DCF distributions and their corresponding surface plots in the flashed and neighboring regions in the presence of the inhibitors. Fluorescence of CM-DCF remained localized within the mitochondria of the flashed region without affecting neighboring areas. PK11195 or DIDS had no apparent effect on unflashed controls (not shown). Brackets point out the flashed region. Prior to each experiment carried out in the presence of inhibitors, myocytes from the same cell isolation showed flash-triggered oscillations in ΔΨm (not shown). F, the acute effect of 4′-chlorodiazepam (32 μm, left panel) on mitochondrial ΔΨm is shown. Preincubation with 64 μm 4′-chlorodiazepam completely inhibited the flash-induced ΔΨm depolarizations (n = 10). Preincubation with the agonist FGIN-I-27 (92 μm, right panel) provoked permanent mitochondrial ΔΨm depolarization in 80% of the flashed cells (n = 10). 4-ChlDZP, 4′-chlorodiazepam.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 7Effects of mitochondrial oscillation on the electrical excitability of the cardiomyocyte. A, action potentials (upper panel) evoked by brief current injections were recorded in current-clamp mode during whole cell patch clamp while simultaneously imaging ΔΨm with TMRE (lower panel). During a synchronized cell-wide depolarization-repolarization cycle, the action potential shortened in synchrony with fast mitochondrial depolarization, and the cell became unexcitable in the fully depolarized state (remaining upward spikes are from the stimulus only). Recovery of ΔΨm coincided with restoration of the action potential. B, correlation between the action potential duration at 90% repolarization and ΔΨm. C, voltage-clamp ramps demonstrate that the current-voltage relationship of the oscillatory membrane current fits the profile of the sarcolemmal K ATP current (green trace, mitochondria polarized; red trace, mitochondria depolarized). D, correlation between sarcolemmal current measured at 0 mV (from voltage ramps) and ΔΨm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To quantitatively determine whether mitochondrial depolarizations were spatially synchronized in the presence of glucose or inhibitors (Fig. 1), we applied grid analysis to the two-dimensional images. A binary mask of the cell TMRE fluorescence was made and the cell area, excluding nuclei, was divided into small squares approximately the size of individual mitochondria (∼2 × 2 μm). The average fluorescence within each grid object was measured and histograms were made of the distribution of fluorescence in polarized and depolarized mitochondria, giving two major peaks of fluorescence intensity. A cutoff value halfway between these peaks was then used to determine whether a given grid object was classified as "polarized" and the fraction of polarized mitochondria with respect to the total number of objects at time 0 was calculated for the image series. The initial value of ∼80% in most experiments reflects an underestimation of the total number of polarized mitochondria because of overlap between the histogram distributions, causing some objects in the polarized population to fall below the cutoff. Cardiomyocyte Electrophysiological Studies—Isolated ventricular myocytes were whole cell patch-clamped using borosilicate glass pipettes (1-4 megaohm tip resistance) and action potentials (current-clamp mode) or membrane currents (voltage-clamp mode) were recorded by means of an Axopatch 200A amplifier coupled to a Digidata 1200A interface (Axon Instruments, Union City, CA) using custom acquisition and analysis software. Myocytes were superfused with (in millimole/liter) NaCl 140, KCl 5, MgCl2 1, CaCl2 2, glucose 10, and HEPES 10 (pH 7.4 with NaOH). Intracellular solutions contained (in millimole/liter) potassium glutamate 130, KCl 9, NaCl 10, MgCl2 0.5, MgATP 5, EGTA 1, and HEPES 10 (pH 7.2 with KOH). Action potentials were evoked by brief (4 ms) current injections applied at 2-s intervals. Voltage ramps from -90 to +30 mV were applied in voltage-clamp mode over a pulse duration of 200 ms at a 2-s interval. Imaging was performed as described for the other experiments, except that a ×40 lens was used instead of a ×60, to provide additional clearance between the cell and the lens for pipette access. Statistical Analysis—Data were analyzed with the software Graph-Pad Prism (version 2; San Diego, CA). The statistical significance of the differences between treatments (respiratory inhibitors, comparison between flashed cells, and spontaneous ones) was evaluated with a t test (small samples, paired t test with two tail p values). Summary statistics, presented as mean ± S.E. (95% confidence interval), were determined for periods of oscillation and rates of depolarization and repolarization. The statistical analysis of the rates of ΔΨm depolarization or repolarization during mitochondrial oscillations was performed after nonlinear regression analysis performed with a Levenberg-Marquardt algorithm (Microcal Origin™, Northampton, MA). The normality of the data was tested with a Kolmogorov-Smirnov test (GraphPad Prism). Materials—TMRE, CM-H2DCFDA, carboxy-H2DCFDA, and calcein AM were purchased from Molecular Probes, Inc., and bongkrekic acid (BKA) and Mn(III)tetrakis(1-methyl-4-pyridil)porphyrin pentachloride (TMPyP) from Calbiochem. All other reagents were from Sigma. Stock solutions of cyclosporin A, rotenone, thapsigargin, oligomycin, antimycin A, DIDS, 4′-chlorodiazepam (Ro5-4864), FGIN-I-27, and PK11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide) were prepared in Me2SO and concentrated enough to avoid exceeding 0.1% Me2SO (v/v) in the final solution. The stock solution of bongkrekic acid was prepared in 2 n NH4OH as recommended by the manufacturer. All stock solutions were stored at -20 °C. Global Transitions of Mitochondrial Energetics Triggered by Local Perturbation of Mitochondrial Function—Under otherwise normal physiological conditions (normoxic cells in the presence of external substrates), we tested whether a highly localized metabolic perturbation could have widespread effects on the mitochondrial network of the myocyte. The thin optical section excited by the two-photon laser (23Denk W. Strickler J.H. Webb W.W. Science. 1990; 248: 73-76Crossref PubMed Scopus (8218) Google Scholar) was employed to perturb a small fraction (<0.3% of the total cell volume) of the mitochondrial population while monitoring the behavior of the remainder of the cell. After collecting 10-20 control images, an 8.7 × 8.7-μm region of the cell was excited in a single flash (Fig. 1A). This resulted in a rapid, but not instantaneous (exponential time constant 18.2 s) (see Fig. 2C), depolarization of ΔΨm in the flashed region (see white square in Fig. 1A). Thereafter, ΔΨm remained depolarized in the flashed area throughout the experiment (Fig. 1A). Unflashed control cells in the same field remained polarized throughout the experiment (Fig. 1, C and D). In contradistinction to the response in the flashed region, mitochondria throughout the rest of the cell were initially unaffected, but began to oscillate in a synchronized manner after a substantial delay. The spatiotemporal pattern of mitochondrial depolarization and repolarization can be readily appreciated from the time-line images created from the entire image sequence (Fig. 1B; the time-line image is not a "line scan" image, but is a two-dimensional representation of the full three-dimensional time series of the experiment, as described in under "Experimental Procedures") and by inspection of the image montage for a single oscillation (Fig. 2A, from the oscillation marked with an asterisk in Fig. 1B). Plots of whole cell fluorescence illustrate that synchronized and periodic transitions in ΔΨm and the NADH redox pool (Fig. 2D) were associated with bursts of mitochondrial ROS production (Fig. 2E), triggered by the initial depolarization and ROS accumulation in the flashed region (see white squares in Fig. 1A and flashed zone in B). Quantitative Analysis of Mitochondrial Oscillations—On average, 72 ± 2.8% of the total mitochondrial population depolarized during each cycle of oscillation (n = 9 cells; 7 experiments; see "Experimental Procedures" for grid analysis), whereas in control cells, mitochondria remained polarized throughout the experiment (Fig. 2B, see also Fig. 1, C and D). The probability of inducing cell-wide oscillations in response to a flash exceeded 80% (n = 65; 7 experiments), whereas in unflashed controls, oscillations were observed in less than 18% of the cells (n = 244 cells analyzed in 61 fields; 9 experiments). Furthermore, the few cells that oscillated in the absence of a flash did so only after a significantly longer delay (448 ± 64
Referência(s)