Real-Time 2-Photon Imaging of Mitochondrial Function in Perfused Rat Hearts Subjected to Ischemia/Reperfusion
Background— Mitochondria play pivotal roles in cell death; the loss of mitochondrial membrane potential (ΔΨm) is the earliest event that commits the cell to death. Here, we report novel real-time imaging of ΔΨm in individual cardiomyocytes within perfused rat hearts using 2-photon laser-scanning microscopy, which has unique advantages over conventional confocal microscopy: greater tissue penetration and lower tissue toxicity.
Methods and Results— The Langendorff-perfused rat heart was loaded with a fluorescent indicator of ΔΨm, tetramethylrhodamine ethyl ester. Tetramethylrhodamine ethyl ester was excited with an 810-nm line of a Ti:sapphire laser, and its fluorescence in the heart cells was successfully visualized up to ≈50 μm from the epicardial surface. Taking advantage of this system, we monitored the spatiotemporal changes of ΔΨm in response to ischemia/reperfusion at the subcellular level. No-flow ischemia caused progressive ΔΨm loss and a more prominent ΔΨm loss on reperfusion. During ischemia/reperfusion, cells maintained a constant ΔΨm for the cell-to-cell specific period of latency, followed by a rapid, complete, and irreversible ΔΨm loss, and this process did not affect the neighboring cells. Within a cell, ΔΨm loss was initiated in a particular area of mitochondria and rapidly propagated along the longitudinal axis. These spatiotemporal changes in ΔΨm resulted in marked cellular and subcellular heterogeneity of mitochondrial function. Ischemic preconditioning reduced the number of cells undergoing ΔΨm loss, whereas cyclosporin A partially inhibited ΔΨm loss in each cell.
Conclusions— Investigation of cellular responses in the natural environment will increase knowledge of ischemia/reperfusion injury and provide deeper insights into antiischemia/reperfusion therapy that targets mitochondria.
Received March 22, 2006; revision received June 26, 2006; accepted July 14, 2006.
Mitochondria are crucial regulators of life and death in a variety of cells1,2 and play pivotal roles in cardiomyocyte death in response to myocardial ischemia/reperfusion.3,4 The loss of mitochondrial membrane potential (ΔΨm) is the earliest event that commits the cell to death, and this process is potentially a prime target for therapeutic interventions.5,6 We and other investigators have studied the spatiotemporal changes in ΔΨm using confocal imaging of isolated cardiomyocytes in vitro that were exposed to a variety of stresses.7–12 Investigation of such time-dependent changes in mitochondrial function within single cardiomyocytes of an intact living heart would have tremendous advantages over the in vitro models, because the heart is working as a syncytium in which individual cardiomyocytes are anatomically and functionally connected. Moreover, the pathological stimulus (ischemia/reperfusion) applied to the intact heart model is more clinically relevant to ischemic heart disease in humans than those used in the in vitro models, such as oxidant stress,7–9 metabolic inhibition,10 hypoxia,11 glucose deprivation,12 or other insults. The visualization of mitochondrial function in the living heart has been a technically difficult task for conventional confocal imaging, however, because the heart tissue is highly light-scattering, and mitochondria are quite susceptible to photodamage.
Editorial p 1452
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Two-photon laser-scanning microscopy (TPLSM) has unique advantages over conventional single-photon confocal microscopy.13,14 First, the penetration depth of the excitation beam is increased. Second, because no out-of-focus fluorescence is generated, no pinhole is necessary during image acquisition, which results in increased fluorescence collection efficiency. Third, 2-photon excitation markedly reduces overall photobleaching and photodamage, which results in extended viability of biological specimens during long-term imaging. These properties are particularly advantageous in monitoring mitochondrial function in the living heart.
Taking advantage of these properties of TPLSM, we, for the first time, successfully visualized ΔΨm in ex vivo heart specimens and monitored the progressive changes of ΔΨm in response to ischemia/reperfusion. Direct imaging of these spatial and temporal changes in mitochondrial function would revolutionize our understanding of ischemia/reperfusion injury and provide deeper insights into the establishment of a novel cardioprotective therapy that targets mitochondria.
All procedures were performed in accordance with the Kyoto University animal experimentation committee, which conforms to the “Guide for the Care and Use of Laboratory Animals” published by the US National Institutes of Health.
Langendorff-Perfused Rat Heart
Adult Sprague-Dawley rats (weighing 250 to ≈300 g, all females; Shimizu Laboratory Supplies, Kyoto, Japan) were used for all studies. The heart was excised, the ascending aorta was cannulated with a customized needle, and hearts were perfused in the Langendorff mode. Perfusion was performed at a constant mean perfusion pressure with oxygenated (95% O2) Tyrode’s solution containing (in mmol/L) 134 NaCl, 4 KCl, 1.2 MgSO4, 1.2 NaH2PO4, 10 HEPES, 11 d-glucose, and 2 CaCl2 (pH 7.4, adjusted with 1 mol/L NaOH). After loading of fluorescent indicators as indicated below, the heart was perfused further by a solution containing 10 mmol/L 2,3-butanedione monoxime (BDM) for 15 minutes to eliminate contraction-induced movement of the heart and was placed in a circular glass-bottomed dish (35-mm diameter) for 2-photon imaging. To optimize the focal plane, the heart was gently pressed against the coverslip (170-μm thickness) at the bottom of the chamber.
Loading of Fluorescent Dyes
After an initial perfusion period of ≈10 minutes, the buffer was switched to Tyrode’s solution containing 100 nmol/L tetramethylrhodamine ethyl ester (TMRE; Molecular Probes; Eugene, Ore), which is an indicator of ΔΨm, and this was followed by a 20-minute washout with dye-free solution.
Cytolysis was monitored with 20 μmol/L 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM; Molecular Probes). After a 30-minute loading period, the heart was perfused with dye-free Tyrode’s solution for 20 minutes to allow for deesterification of the dyes by endogenous esterases. In this case, the heart was further loaded with TMRE, followed by washout. The detection of cell death was also performed by staining with propidium iodide (PI; Molecular Probes). In this experiment, the heart was preloaded with TMRE and BCECF-AM and subjected to 30 minutes of ischemia followed by 30 minutes of reperfusion. At the end of the experimental period, the heart was further loaded with PI by perfusing it with Tyrode’s solution containing PI 1.5 mmol/L for 20 minutes.
The perfusate was equilibrated with 95% O2 (pH 7.4). The temperature was maintained at 37°C with a solution heater and a platform heater (Warner Instruments; Hamden, Conn) installed on the microscope stage. After a stabilization period (≈10 minutes), the hearts were subjected to either ischemia (60 minutes; myocardial ischemia [MI]) or ischemia/reperfusion (30 minutes/30 minutes; MI/reperfusion [MI/R]). Ischemia was achieved by clamping the perfusion line. Reperfusion was achieved by releasing the clamp. In the cyclosporin A (CsA)–treated group, the hearts were treated with CsA 0.2 or 1.0 μmol/L dissolved in the perfusate for 30 minutes immediately before BDM treatment and MI/R. In the ischemic preconditioning (IPC) group, 3 cycles of 5 minutes of ischemia and 5 minutes of reperfusion were followed by BDM treatment and MI/R.
Two-Photon Laser-Scanning Microscopy
Images were recorded with a Zeiss LSM510 laser scanning microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany) modified for TPLSM. Illumination for 2-photon excitation was provided by a mode-locked Ti:sapphire laser (Spectra-Physics; Irvine, Calif); the excitation wavelength was 810 nm. Hearts were imaged through a Zeiss 63× 1.40 numerical aperture oil-immersion objective with a working distance of 180 μm (Carl Zeiss Microimaging, Inc). Emitted light was collected by 2 photomultiplier tubes fitted with band-pass filters for 500 to 550 nm (for BCECF-AM) and 565 to 615 nm (for TMRE and PI), respectively. For full-frame-mode analyses (512×512 pixels), hearts were scanned on horizontal (x, y) planes, and the resulting images were digitized at an 8-bit resolution and stored directly on a hard disk. The images are representative ones from at least 3 independent experiments, and we have confirmed the reproducibility of the responses.
Postacquisition analysis was performed with an image-analysis software program (ImageJ, National Institutes of Health; available at http://rsb.info.nih.gov/ij/). From the image sequences, we quantitatively assessed the kinetics of ΔΨm loss in individual cells. Regions of interest were drawn over a part of an individual cell, and fluorescence signals within these regions were collected over time. ΔΨm was monitored by mean TMRE brightness within the regions. Three-dimensional image processing was performed with a software program (Imaris; Bitplane Inc; St Paul, Minn).
Quantitative data are presented as mean±SEM. Comparisons were performed with either the unpaired Student t test or 1-way ANOVA with Bonferroni procedure as the post hoc test. A level of P<0.05 was accepted as statistically significant.
The authors had full access to the data and take full responsibility for its integrity. All authors have read and agree to the manuscript as written.
We visualized mitochondria in the Langendorff-perfused rat heart loaded with TMRE, a fluorescent indicator of ΔΨm (Figure 1). Tightly focused images of TMRE fluorescence could be obtained at various depths up to ≈50 μm (≈3 to 4 layers of myocardial cells) below the epicardial surface of the heart (Figure 1B; online-only Data Supplement, Movie I). The temporal resolution was sufficient to observe each single mitochondrion (Figure 1C). The image clearly demonstrates that cardiomyocytes have considerable numbers of mitochondria aligned in an orderly fashion along the myofilaments. Three-dimensional image reconstruction was made possible by stacking the optical slices with 3-dimensional processing software (Figure 1D; online Data Supplement, Movie II).
Using this imaging system, we monitored ΔΨm changes in perfused hearts subjected to ischemia/reperfusion. Time-lapse images were taken every 5 minutes (Figure 2A). Control perfusion with oxygenated Tyrode’s solution maintained ΔΨm for >60 minutes (Figure 2A, control). No-flow MI (60 minutes of ischemia) caused progressive ΔΨm loss (Figure 2A, MI; online Data Supplement, Movie III). In the MI/R group (30 minutes of ischemia followed by 30 minutes of reperfusion), there was progressive ΔΨm loss during the ischemic period and a more prominent ΔΨm loss on reperfusion (Figure 2A, MI/R; online Data Supplement, Movie IV). The ΔΨm loss was sporadically initiated over the observation area (arrows in Figure 2A, MI and MI/R). Figure 2B shows the time courses of fluorescence obtained from representative cells. In both the MI and MI/R groups, ischemia/reperfusion did not immediately result in ΔΨm loss; instead, there was a period of latency that varied from cell to cell, after which ΔΨm started to decrease. Once it began, this rapid loss of ΔΨm was complete within 5 minutes, and it was irreversible. Figure 2C shows the time courses of representative cells undergoing the collapse of ΔΨm, monitored at 1-minute intervals. The kinetics of ΔΨm loss during the ischemic period (solid lines) were identical to those during the reperfusion period (dashed lines). Here, “ischemic period” represents MI and the first 30 minutes of MI/R. Summarized data of duration of ΔΨm loss in each cell are shown in Figure 2D; “duration” was defined as the time required for 50% loss of TMRE fluorescence in this analysis. The durations of ΔΨm loss were comparable between the ischemic and reperfusion periods, which suggests a common underlying mechanism. Figure 2E shows that the duration of ΔΨm loss did not vary with latency in any given cell: ΔΨm dissipation occurred within 5 minutes, regardless of the delay with which it began.
In Figure 3, a representative cell undergoing ΔΨm loss is shown. These 1-minute interval images suggest that ΔΨm loss was proceeding almost homogeneously throughout the cell, but it never affected the neighboring cells (Figure 3A). This was confirmed by a quantitative assessment (Figure 3B; line scan shown in the middle, and fluorescence time course of individual cells shown on the right). To further investigate the time-dependent changes of ΔΨm loss in a single cell with higher spatiotemporal resolution, time-lapse images were taken every 10 seconds (Figure 4A; online Data Supplement, Movie V). ΔΨm loss was initiated in a particular area of mitochondria (Figure 4B, right, white arrowhead), and it rapidly propagated along the longitudinal axis within the cell (Figure 4B, middle panel, white arrows). The calculated velocity of the ΔΨm loss propagation was 2.2 μm/s, which was comparable to a previously reported propagation rate of the mitochondrial apoptotic signal in H9c2 cells.15
We confirmed that cells that underwent ΔΨm loss eventually experienced cytolysis, as evidenced by the fluorescent indicators BCECF-AM and PI. As shown in Figure 5, a cell with ΔΨm loss was PI-positive (left; white arrows) and showed leakage of BCECF (middle), indicating cytolysis. Meanwhile, a cell with intact ΔΨm was PI-negative (left; white arrowhead) and showed no leakage of BCECF.
A key mechanism of ΔΨm loss is by the opening of the mitochondrial permeability transition pore (MPTP), a nonspecific pore that opens at the contact site between outer and inner mitochondrial membranes.5 We have confirmed in an isolated cardiomyocyte model that ΔΨm loss is mediated by the MPTP opening.7 Accordingly, we tested the effect of an MPTP blocker, CsA,16 in this system. CsA (0.2 or 1.0 μmol/L) blocked the rapid dissipation of ΔΨm, but the cells appeared to lose ΔΨm with slower kinetics (Figure 6A, CsA 0.2 and CsA 1.0; online Data Supplement Movies VI and VII). The time course of fluorescence obtained from 10 representative cells (Figure 6B) indicates prolonged duration of ΔΨm loss and smaller cellular heterogeneity in the CsA-treated group. Finally, we tested the effect of IPC. IPC is an endogenous mechanism whereby brief periods of ischemia and reperfusion paradoxically protect the myocardium against the damaging effects of subsequent prolonged ischemia.17 IPC decreased the number of cells undergoing ΔΨm loss (Figure 6A, IPC; Movie VIII), whereas it did not change the duration of ΔΨm loss in unprotected cells. Moreover, ΔΨm level was fully polarized in the protected cells (Figure 6B). Figure 6C shows the average of TMRE fluorescence intensity at the end of the experimental period (60 minutes) from 10 randomly and prospectively selected cells in each group. The ΔΨm-preserving effect of CsA 0.2 and IPC was remarkable, but that of CsA 1.0 was less and was modest. Although CsA 0.2 and IPC achieved a similar level of protection (Figure 6C), the mechanism of action was suggested to be distinct, as evidenced by the differential kinetics of ΔΨm loss in each individual cell (Figure 6B). The duration of ΔΨm loss was much longer in the CsA 0.2 group than in the MI/R and IPC groups (Figure 6D). Moreover, IPC significantly delayed the onset of ΔΨm loss, whereas CsA did not (Figure 6E). The wave velocity of ΔΨm loss propagation in the IPC group was 1.9 μm/s, which was comparable to that of the MI/R (IPC absent) group.
Visualization of Living Mitochondria in the Perfused Heart With TPLSM
This is the first demonstration of living mitochondria in perfused hearts and their responses to ischemia/reperfusion. The importance of the detection of cell death processes at the subcellular level in the heart in situ has attracted attention,18,19 especially when compared with conventional studies, which depend on rough end points such as myocardial enzyme release, recovery of left ventricular pressure, or macroscopic measurement of infarct size. TPLSM is ideal for bioimaging of the intact heart and has been used in pioneering studies by Rubart et al.20,21
Real-Time Monitoring of Mitochondrial Function During Ischemia/Reperfusion
Taking advantage of this novel imaging system, we monitored real-time mitochondrial function under MI/R at the single cardiomyocyte level, or single mitochondrion level. Our observations provide important insights into the understanding of ischemia/reperfusion injury.
Kinetics and Mechanisms of ΔΨm Loss
During ischemia/reperfusion, cells maintained a constant ΔΨm for the cell-to-cell specific period of latency, followed by a rapid, complete, and irreversible depolarization of ΔΨm. Once initiated, the duration of ΔΨm depolarization was constant and did not depend on the length of the latency. The kinetics of ΔΨm loss appeared almost identical between ischemia and reperfusion, which suggests a common mechanism. Considering the similar kinetics of ΔΨm loss demonstrated in our previous study with isolated cardiomyocytes,7 the opening of the MPTP is most likely involved in the mitochondrial collapse during ischemia as well as reperfusion. CsA, a potent MPTP blocker, showed partial protective effects against ischemia/reperfusion–induced ΔΨm loss. Although a potent MPTP inhibitor in isolated mitochondria, the effect of CsA is inconsistent in intact cells.22 In addition, CsA is not completely specific and inhibits calcineurin, which also plays an important role in modulating mitochondrial death signals.23 Further studies are required to elucidate the mechanisms of ΔΨm loss.
Cell Death During Ischemia and Reperfusion
There was progressive ΔΨm loss during the ischemic period and a more prominent ΔΨm loss on reperfusion. Obviously, permanent ischemia eventually kills all the cells, and reperfusion is necessary to salvage them. However, during reperfusion, the heart undergoes further damage due in large part to the generation of reactive oxygen species.24 Many drugs have been shown to reduce infarct size when administered before ischemia, but it was recently reported that cytochrome P450 inhibitors administered at reperfusion reduced infarct size.25 Our observations in the present study support the possibility that therapies directed at inhibiting mitochondrial dysfunction and resultant cell death could be successful even if applied during resumption of coronary blood flow.
Cellular and Subcellular Heterogeneity of Mitochondrial Function
Interestingly, ΔΨm loss was initiated sporadically, and it did not affect neighboring cells, which suggests closure of the gap junction before ΔΨm loss. At the subcellular level, ΔΨm loss was initiated in a particular area of mitochondria and rapidly propagated along the longitudinal axis within the cell. These cellular and subcellular dispersions of the ΔΨm level after ischemia/reperfusion can lead to spatiotemporal heterogeneity of excitability in the heart. Recently, Akar and colleagues26 demonstrated that this heterogeneity may underlie the genesis of potentially lethal cardiac arrhythmias. In the present study, the hearts were perfused with buffer containing 10 mmol/L BDM to halt contraction. BDM reportedly has a cardioprotective effect.27 The closure of the gap junction mediated by BDM28 may potentially underlie its cardioprotective property,29 but we confirmed that sporadic ΔΨm loss was similarly observed in the absence of BDM (data not shown), which indicates that sporadic ΔΨm loss and the resultant mitochondrial heterogeneity were not an effect of BDM. This was further confirmed by the similar sequence of events observed under perfusion with cytochalasin D (50 μmol/L), which uncouples excitation/contraction.
Strategies Toward Cardioprotection
This imaging system can be used to assess potential therapeutic agents for ameliorating myocardial infarction. Indeed, the feasibility of the system was confirmed by the remarkable cardioprotective effect of IPC and CsA. IPC not only decreased the number of cells undergoing ΔΨm loss but also delayed the onset of ΔΨm loss (Figure 6E), whereas it changed neither the duration of ΔΨm loss (Figure 6D) nor the intracellular wave propagation velocity of ΔΨm loss in unprotected cells. IPC fully preserved the ΔΨm level in protected cells. CsA, in contrast, did not change the latency period (Figure 6E) but did slow the process of ΔΨm loss (Figure 6D), and all the cells were partially depolarized. Therefore, cellular heterogeneity was much greater in the IPC-treated group than in the CsA-treated group. The slower ΔΨm loss observed in the CsA group may be mediated by a partial (or low-conductance) opening of the MPTP or by another mechanism that comes into play under the inhibition of the MPTP, but further studies are needed to confirm this. Collectively, CsA and IPC may exert a cardioprotective effect with distinct mechanisms of action, as evidenced by the differential kinetics of ΔΨm loss; IPC may affect the latency period but not the depolarization period, and CsA may blunt the depolarization process but not affect the latency.
Although this technique can be performed in whole tissue, the heart is not contracting at the time of observation, and hence, the mitochondrial response excludes the energetic demands of contractile function. Also, although TPLSM has allowed increased depth assessment, there is still room for improvement to assess the transmural mitochondrial response to ischemic and reperfusion stress.
In the present study, we have developed a real-time imaging system to monitor mitochondrial function in the perfused heart. Because mitochondria play important roles in cell death pathways, these organelles are potentially prime targets for therapeutic intervention.6,30,31 The factors regulating the maintenance or disruption of mitochondrial function can be investigated in detail with this system. Mechanistic dissection of the sequence of events will pinpoint the therapeutic targets against ischemia/reperfusion injury. The effects of various candidate drugs can be tested with this system. Thus, this system will provide valuable information for a more integrated understanding of ischemia/reperfusion injury, and furthermore, it will provide deeper insights into the establishment of antiischemia/reperfusion therapy that targets mitochondria.
We thank Dr Steven P. Jones (University of Louisville) for his helpful comments and critical reading of the manuscript.
Sources of Funding
This work was supported by research grants (grants-in-aid in scientific research, leading project for biosimulation) from the Ministry of Education, Culture, Science, and Technology of Japan; a Japan Heart Foundation/Novartis grant for research award on molecular and cellular cardiology; and a research grant from Mitsubishi Pharma Research Foundation.
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Mitochondria are crucial regulators of life and death in a variety of cells and play pivotal roles in cardiomyocyte death in response to myocardial ischemia/reperfusion. In the present study, for the first time, we successfully visualized mitochondrial function in living heart specimens and monitored the progressive changes of mitochondrial function in response to ischemia/reperfusion using 2-photon laser-scanning microscopy. Monitoring the spatiotemporal changes in mitochondrial function within single cardiomyocytes of an intact living heart would have tremendous advantages over the conventional in vitro models, because the heart is working as a syncytium in which individual cardiomyocytes are anatomically and functionally connected. Moreover, the pathological stimulus (ischemia/reperfusion) applied to the intact heart model is clinically relevant to ischemic heart disease in humans. Cardiac myocytes can undergo mitochondrial collapse during ischemia and can do so more prominently during reperfusion, which emphasizes the importance of reperfusion injury in the clinical setting. Ischemia/reperfusion results in marked cellular and subcellular heterogeneity in mitochondrial function, perhaps leading to electrical instability and arrhythmogenesis. Cardioprotective interventions (pharmacological or nonpharmacological) are now being applied in clinical medicine to reduce infarct size and improve the clinical status of patients with acute myocardial infarction. Thus, investigation of cellular responses in the natural environment will increase knowledge of ischemia/reperfusion injury and provide deeper insights into antiischemia/reperfusion therapy that targets mitochondria.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.106.628834/DC1.