CLINICAL PERSPECTIVE

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(Circulation. 2006;114:1497-1503.)
© 2006 American Heart Association, Inc.

Imaging

Real-Time 2-Photon Imaging of Mitochondrial Function in Perfused Rat Hearts Subjected to Ischemia/Reperfusion

Madoka Matsumoto-Ida, MD; Masaharu Akao, MD, PhD; Toshihiro Takeda, MD; Masashi Kato, MD; Toru Kita, MD, PhD

From the Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan.

Correspondence to Masaharu Akao, MD, PhD, Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. E-mail akao{at}kuhp.kyoto-u.ac.jp

Received March 22, 2006; revision received June 26, 2006; accepted July 14, 2006.

	Abstract

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Background— Mitochondria play pivotal roles in cell death; the loss of mitochondrial membrane potential (_m) is the earliest event that commits the cell to death. Here, we report novel real-time imaging of _m 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 _m, 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 _m in response to ischemia/reperfusion at the subcellular level. No-flow ischemia caused progressive _m loss and a more prominent _m loss on reperfusion. During ischemia/reperfusion, cells maintained a constant _m for the cell-to-cell specific period of latency, followed by a rapid, complete, and irreversible _m loss, and this process did not affect the neighboring cells. Within a cell, _m loss was initiated in a particular area of mitochondria and rapidly propagated along the longitudinal axis. These spatiotemporal changes in _m resulted in marked cellular and subcellular heterogeneity of mitochondrial function. Ischemic preconditioning reduced the number of cells undergoing _m loss, whereas cyclosporin A partially inhibited _m 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.

Key Words: imaging • ischemia • reperfusion • mitochondria

	Introduction

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Mitochondria are crucial regulators of life and death in a variety of cells^1,2 and play pivotal roles in cardiomyocyte death in response to myocardial ischemia/reperfusion.^3,4 The loss of mitochondrial membrane potential (_m) 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 _m 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

Clinical Perspective p 1503

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 _m in ex vivo heart specimens and monitored the progressive changes of _m 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.

	Methods

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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 _m, 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.

Ischemia/Reperfusion Protocol
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 63x 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 (512x512 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.

Image Analysis
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 _m 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. _m 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).

Statistical Analysis
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.

	Results

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We visualized mitochondria in the Langendorff-perfused rat heart loaded with TMRE, a fluorescent indicator of _m (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).

View larger version (76K): [in this window] [in a new window]   Figure 1. Two dimensional and 3-dimensional imaging of mitochondria in perfused rat hearts. A, Schematic drawing of the perfused heart and optical slices. The images were obtained every 0.5 µm x100 slices. B, Representative optical slices of TMRE-loaded heart obtained at various depths. Numbers indicate depth (in micrometers) from the epicardial surface. Scale bar=20 µm. C, Zoomed image (x3) showing individual mitochondria. Scale bar=10 µm. D, Three-dimensional processing images of a single cardiomyocyte.

Using this imaging system, we monitored _m 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 _m for >60 minutes (Figure 2A, control). No-flow MI (60 minutes of ischemia) caused progressive _m 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 _m loss during the ischemic period and a more prominent _m loss on reperfusion (Figure 2A, MI/R; online Data Supplement, Movie IV). The _m 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 _m loss; instead, there was a period of latency that varied from cell to cell, after which _m started to decrease. Once it began, this rapid loss of _m was complete within 5 minutes, and it was irreversible. Figure 2C shows the time courses of representative cells undergoing the collapse of _m, monitored at 1-minute intervals. The kinetics of _m 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 _m 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 _m loss were comparable between the ischemic and reperfusion periods, which suggests a common underlying mechanism. Figure 2E shows that the duration of _m loss did not vary with latency in any given cell: _m dissipation occurred within 5 minutes, regardless of the delay with which it began.

View larger version (81K): [in this window] [in a new window]   Figure 2. Progressive loss of m during ischemia/reperfusion. A, Time-lapse images taken every 5 minutes. Control indicates control perfusion for 60 minutes. Data are from a single experiment, representative of at least 3 independent experiments. Scale bar=50 µm. B, Time course of TMRE fluorescence in each individual cell, monitored at 5-minute intervals. C, Time course of TMRE fluorescence in representative cells undergoing m loss, monitored at 1-minute intervals. Solid lines indicate ischemic period; dashed lines, reperfusion period. "Ischemic period" represents MI and the first 30 minutes of MI/R. A.U. indicates arbitrary units. D, Summarized data of the duration required for m loss in the ischemic period (Isc) and the reperfusion period (Rep); n=6, from 3 independent scans. E, Duration required for m loss vs latency period of m loss. Black circles indicate ischemic period; gray circles, reperfusion period; and line, regression line, indicating no correlation between duration and latency of m loss.

In Figure 3, a representative cell undergoing _m loss is shown. These 1-minute interval images suggest that _m 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 _m loss in a single cell with higher spatiotemporal resolution, time-lapse images were taken every 10 seconds (Figure 4A; online Data Supplement, Movie V). _m 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 _m 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

View larger version (55K): [in this window] [in a new window]   Figure 3. Intercellular propagation of m loss. A, Time-lapse images were taken every 1 minute. Representative scan of the MI/R group (30 minutes of ischemia followed by 30 minutes of reperfusion). Scale bar=20 µm. B, Middle, Time-line image of TMRE fluorescence by analysis of a white line drawn in the left panel; right, time course of TMRE fluorescence in each individual cell numbered in the left panel. A.U. indicates arbitrary units.

View larger version (62K): [in this window] [in a new window]   Figure 4. Intracellular propagation of m loss. A, Time-lapse images were taken every 10 seconds. Representative scan of the MI group (60 minutes of ischemia). Scale bar=10 µm. B, Middle, Time-line image of TMRE fluorescence by analysis of a white dashed line drawn in the left panel. White arrow indicates propagation of m loss starting from a particular area of mitochondria; right, time-area montage image of TMRE fluorescence by analysis of a white open box drawn in the left panel. White arrowhead indicates the single mitochondrion where propagation of m loss appeared to start.

We confirmed that cells that underwent _m loss eventually experienced cytolysis, as evidenced by the fluorescent indicators BCECF-AM and PI. As shown in Figure 5, a cell with _m loss was PI-positive (left; white arrows) and showed leakage of BCECF (middle), indicating cytolysis. Meanwhile, a cell with intact _m was PI-negative (left; white arrowhead) and showed no leakage of BCECF.

View larger version (84K): [in this window] [in a new window]   Figure 5. m loss followed by cytolysis. Images were taken at the end of 30 minutes of ischemia followed by 30 minutes of reperfusion. Red channel shows TMRE and PI (left); green channel shows BCECF (middle); and merged image (right).

A key mechanism of _m 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 _m 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 _m, but the cells appeared to lose _m 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 _m 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 _m loss (Figure 6A, IPC; Movie VIII), whereas it did not change the duration of _m loss in unprotected cells. Moreover, _m 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 _m-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 _m loss in each individual cell (Figure 6B). The duration of _m 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 _m loss, whereas CsA did not (Figure 6E). The wave velocity of _m loss propagation in the IPC group was 1.9 µm/s, which was comparable to that of the MI/R (IPC absent) group.

View larger version (78K): [in this window] [in a new window]   Figure 6. Effects of CsA and IPC. A, Time-lapse images taken every 5 minutes. CsA indicates infusion of 0.2 or 1.0 µmol/L CsA, followed by 30 minutes of ischemia and 30 minutes of reperfusion. IPC indicates 3 cycles of 5 minutes of ischemia and 5 minutes of reperfusion, followed by 30 minutes of ischemia and 30 minutes of reperfusion. Data are from a single experiment, representative of at least 3 independent experiments. Scale bar=50 µm. B, Time course of TMRE fluorescence in each individual cell, monitored at 5-minute intervals. C, Mean fluorescence intensity at the end of the experimental period (60 minutes) from 10 cells randomly and prospectively selected in each group. *P<0.05 vs MI/R. D, Duration required for m loss in each group. *P<0.05 vs MI/R. E, Latency period of m loss in each group. *P<0.05 vs MI/R. A.U. indicates arbitrary units.

	Discussion

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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 _m Loss
During ischemia/reperfusion, cells maintained a constant _m for the cell-to-cell specific period of latency, followed by a rapid, complete, and irreversible depolarization of _m. Once initiated, the duration of _m depolarization was constant and did not depend on the length of the latency. The kinetics of _m loss appeared almost identical between ischemia and reperfusion, which suggests a common mechanism. Considering the similar kinetics of _m 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 _m 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 _m loss.

Cell Death During Ischemia and Reperfusion
There was progressive _m loss during the ischemic period and a more prominent _m 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, _m loss was initiated sporadically, and it did not affect neighboring cells, which suggests closure of the gap junction before _m loss. At the subcellular level, _m 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 _m 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 _m loss was similarly observed in the absence of BDM (data not shown), which indicates that sporadic _m 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 _m loss but also delayed the onset of _m loss (Figure 6E), whereas it changed neither the duration of _m loss (Figure 6D) nor the intracellular wave propagation velocity of _m loss in unprotected cells. IPC fully preserved the _m level in protected cells. CsA, in contrast, did not change the latency period (Figure 6E) but did slow the process of _m 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 _m 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 _m loss; IPC may affect the latency period but not the depolarization period, and CsA may blunt the depolarization process but not affect the latency.

Study Limitations
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.

Clinical Implications
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.

	Acknowledgments

 
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.

Disclosures

None.

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CLINICAL PERSPECTIVE

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.

	Footnotes

 
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.106.628834/DC1.

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