How Does the Ca²⁺-paradox Injury Induce Contracture in the Heart?—A Combined Study of the Intracellular Ca²⁺ Dynamics and Cell Structures in Perfused Rat Hearts—

Abstract

The calcium (Ca²⁺)-paradox injury of the heart, induced by restoration of extracellular Ca²⁺ after its short-term depletion, is known to provoke cardiomyocyte contracture. However, undetermined is how the Ca²⁺-paradox provokes such a distinctive presentation of myocytes in the heart. To address this, we imaged sequential intracellular Ca²⁺ dynamics and concomitant structures of the subepicardial ventricular myocytes in fluo3-loaded, Langendorff-perfused rat hearts produced by the Ca²⁺ paradox. Under rapid-scanning confocal microscopy, repletion of Ca²⁺ following its depletion produced high-frequency Ca²⁺ waves in individual myocytes with asynchronous localized contractions, resulting in contracture within 10 min. Such alterations of myocytes were attenuated by 5-mM NiCl₂, but not by verapamil, SEA0400, or combination of ryanodine and thapsigargin, indicating a contribution of non-specific transmembrane Ca²⁺ influx in the injury. However, saponin-induced membrane permeabilization of Ca²⁺ showed no apparent contracture despite the emergence of high-frequency Ca²⁺ waves, indicating an essential role of myocyte-myocyte and myocyte-extracellular matrix (ECM) mechanical connections in the Ca²⁺ paradox. In immunohistochemistry Ca²⁺ depletion produced separation of the intercalated disc that expresses cadherin and dissipation of β-dystroglycan located along the sarcolemma. Taken together, along with the trans-sarcolemmal Ca²⁺ influx, disruption of cell-cell and cell-ECM connections is essential for contracture in the Ca²⁺-paradox injury.

I.  Introduction

The Ca²⁺-paradox injury, which is induced by short-term perfusion of the heart with Ca²⁺-free solution (Ca²⁺ depletion) and subsequent reperfusion with Ca²⁺ (Ca²⁺ repletion), provokes irreversible intracellular Ca²⁺ ([Ca²⁺]_i) overload and progressive contracture of cardiomyocytes [7, 25, 32, 33]. Because of the simple experimental procedure, the Ca²⁺ paradox is regarded as a useful experimental model for studying functional, morphological, and biochemical bases of myocardial injury, in particular [Ca²⁺]_i overload that simulates ischemia-reperfusion injury [25]. Despite such simple procedures, the mechanism(s) underlying the development of [Ca²⁺]_i overload is controversial [4, 7–9, 13, 15, 25]; some studies suggested that an influx of Ca²⁺ through either L-type Ca²⁺ channels [13, 15] or the Na⁺-Ca²⁺ exchanger (NCX) in the cell membrane [8] is responsible for the Ca²⁺ paradox-induced [Ca²⁺]_i overload, while others indicated the role of non-selective Ca²⁺ influx due to disruption of the cell membrane [4, 9]. In addition, electron microscopic studies suggested that the Ca²⁺ depletion-induced separation of the glycocalyx from the sarcolemma is speculated to be the cause of [Ca²⁺]_i overload [3, 10]. These different results stem from a lack of information regarding a direct link between structural changes and the concomitant [Ca²⁺]_i of the myocytes, in particular, exact sequential changes in myocyte morphology and [Ca²⁺]_i dynamics: morphological studies on the Ca²⁺-paradox injury have hitherto been performed mostly on fixed preparations [4, 7–9, 13, 15, 25]. In addition to the mechanisms for [Ca²⁺]_i overload, it is also unsettled how the Ca²⁺-paradox injury provokes myocyte contracture, the most distinctive presentation of the myocyte injury. In particular, unknown is whether the [Ca²⁺]_i overload is the only culprit for the contracture. With the advent of live-cell imaging technology, especially in situ real-time confocal microscopy, both the [Ca²⁺]_i dynamics and structure of the individual myocytes can be simultaneously visualized in the working heart [14, 18, 30]. This imaging modality, if applied to the heart under the Ca²⁺-paradox injury, would enable us to address the mechanism(s) underlying development of myocyte contracture. The present study was undertaken to elucidate the mechanistic link between the development of [Ca²⁺]_i overload and the corresponding morphological changes in the Ca²⁺ paradox, and the related molecules responsible for the myocyte contracture in the heart.

II.  Materials and Methods

Preparation of the heart for Ca²⁺ imaging

The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was approved by the Institutional Committee for Animal Research (Approval reference No. M21-18). Adult male Wistar rats (9 weeks old) weighing 250–300 g were used (n=42). The procedure for in situ [Ca²⁺]_i imaging of the heart was essentially identical to those described previously [14, 18]. Briefly, Langendorff-perfused hearts with HEPES-buffered Tyrode’s solution containing (in mM): NaCl 145, KCl 5.4, MgCl₂ 1, CaCl₂ 1, HEPES 10, and glucose 10 (pH=7.4 by NaOH) were loaded with fluo3/AM (22 μM; Dojin, Japan) at 19–21°C for 45 min. Following 15 min the hearts were perfused with Tyrode’s solution containing probenecid (Nacalai Tesque, 1 mM) at 37°C for de-esterification of acetoxymethyl esters of fluo3/AM, and were subsequently used for experiments.

In situ real-time confocal microscopy

The confocal scanning system was principally analogous to that described by Hama et al. [14]. It consisted of a fixed stage upright microscope (BX50WI, Olympus), a multi-pinhole type confocal scanning device with a microlens array (CSU21, Yokogawa), an image intensifier (Videoscope, VS4-1845), an ICCD camera (310-TURBO, Roper), and a computer with an image capture board (IC-PCI, Imaging Technology). An Argon-Krypton laser beam focused with a water immersion objective lens (Olympus LUMPlan FL20X, numerical aperture=0.5) was applied to the subepicardial surface of the heart that was placed onto a custom-made perfusion chamber on the stage. The fluo3-fluorescence signals (8 bits, 512×480 pixels, 0.885 μm/pixel), scanned at 30 frames/sec on horizontal (X-Y) planes, were digitized with the ICCD camera, and were stored on a hard disc for later analysis.

Experimental procedures for the Ca²⁺ paradox

Before the Ca²⁺ paradox procedure the heart was perfused in a Langendorff manner at a constant rate of 10 mL/min with Ca²⁺-containing (1 mM) Tyrode’s solution under oxygenation with 100% O₂ at 25°C showing a regular sinus rhythm at 56±7 bpm (n=6). Stabilization of the heart with the Ca²⁺-containing solution was followed by perfusion with Ca²⁺-free Tyrode’s for 10 min at 37°C (Ca²⁺ depletion) and for another 5 min at 25°C. Thereafter, the Ca²⁺ paradox was created by reperfusion with Ca²⁺-containing (1 mM) solution (Ca²⁺ repletion). Upon repletion of Ca²⁺ the heart became pale due to the dissipation of the myoglobin (Fig. 1). Confocal visualization of [Ca²⁺]_i dynamics was made on the subepicardial myocardium at 25–26°C. In some hearts the cell membrane was stained with RH-237 (10 μM, Life Technologies) for visualization of the cell structure. Nickel chloride, verapamil, ryanodine, and thapsigargin were from Sigma, 2,3-butanedione monoxime (BDM), from Nacalai Tesque, and saponin, from Wako Pure Chemicals. SEA0400 was provided by Taisho Pharmaceutical Co., Ltd. [23].

Fig. 1. 

Schematic representation of procedure for Ca²⁺-paradox experiment and gross images of the heart before Ca²⁺ depletion (left) and after Ca²⁺ repletion (right).

Immunohistochemistry

The heart was fixed by perfusion with 2% paraformaldehyde for 30 min at room temperature for histochemical analysis. The fixed left ventricular free walls were cut into small pieces. Antibodies to β-dystroglycan (anti-mouse, monoclonal, Novo Castra) or to pan cadherin (anti-mouse, monoclonal, Abcam), diluted by 1:500, were applied to the specimen overnight at 4°C after 10-min treatment with 0.1% Triton X-100 and following 30-min treatment with 5% skim milk, and subsequently Alexa 488 goat anti-mouse IgG secondary antibody (Life Technologies) was applied for 2 hours at room temperature. Subepicardial myocytes of the left ventricle were imaged with a confocal laser scanning microscope (FV1000, Olympus) with a PlanApoN objective lens (60×, numerical aperture=1.42).

Two-dimensional blue native/SDS gel electrophoresis

Immunoblotting for β-dystroglycan was performed by a combination of blue native poly-acrylamide gel electrophoresis (BN-PAGE) and sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) in a two-dimensional approach [5]. The excised rat heart, after washout of the blood with Tyrode’s solution, was subjected to the analysis. The whole cell lysates after SDS-PAGE, separated of protein complex via BN-PAGE, were compared before and during Ca²⁺ depletion.

Data analysis

Analysis of the in situ confocal real-time image was conducted on the digitized fluorescence intensity for fluo3 and RH-237 by Image J software (NIH). Quantitative analyses of Ca²⁺ waves were performed from X-t images, which were reconstructed by cumulatively layering a series of consecutive X-Y image frames scanned by a line along the longitudinal axis of the cells. The cell length and intercellular gap were measured from X-Y images stained with RH-237. In immunohistochemistry, intensity of the Alexa 488 fluorescence was analyzed by averaging the fluorescence intensity along the cell membrane at 10-μm length. The quantitative data (mean±SD) were statistically analyzed by ANOVA, and significance was defined as P<0.05.

III.  Results

Changes in [Ca²⁺]_i dynamics and cell structure by Ca²⁺ paradox

The [Ca²⁺]_i dynamics of cardiomyocytes were distinct among the three different conditions; i.e., before and during Ca²⁺ depletion, and after Ca²⁺ repletion (Figs. 2 and 3). Under control conditions (Fig. 2A) the myocytes exhibited spatially homogenous Ca²⁺ transients on excitation with no discernible rise of [Ca²⁺]_i during diastole as shown in the sequential X-Y (Fig. 2A-b) and X-t (cells 1–3 in Fig. 2A-c) images. The corresponding fluorescent image of the cell membranes stained with RH-237 indicated that the individual myocytes showed tight connections at the intercalated discs (Fig. 2A-a). In sharp contrast, depletion of extracellular Ca²⁺ diminished Ca²⁺ transients, and instead, Ca²⁺ waves emerged sporadically in individual cells (Fig. 2B). The waves occurred within the myocytes (Fig. 2B-a) at an incidence of 11.7±3.8/min/cell showing longitudinal propagation at a velocity (V_prop) of 77.5±13.5 μm/sec (n=33). Of note, myocytes appeared disconnected at intercalated discs having a remarkable intercellular gap (approx. 7 μm) during Ca²⁺ depletion (Fig. 2B-b). Upon repletion of Ca²⁺ high-frequency, ripple-like Ca²⁺ waves developed in individual myocytes (Fig. 3) within 2 min. Such high-frequency Ca²⁺ waves were asynchronous among the myocytes at a frequency of 123.3±21.0/min/cell with V_prop of 90.4±21.1 μm/sec (n=23), and were accompanied by localized, wave-like contractions as evident in fluctuations of the intercellular gaps (Fig. 3b). Moreover, intercellular gaps widened gradually and myocytes eventually showed contracture (Fig. 3c) after 10-min of Ca²⁺ repletion, where myocytes exhibited no apparent Ca²⁺ waves, but instead showed high-static fluo3-fluorescence intensity.

Fig. 2. 

The [Ca²⁺]_i dynamics of the subepicardial surface of the left ventricle in the Langendorff-perfused rat heart before (A) and during (B) Ca²⁺ depletion. A(a): An X-Y image stained with RH-237 and the corresponding schematic illustration of myocytes at the same region. A(b): Sequential X-Y images (1–11, displayed every 150 msec) of the [Ca²⁺]_i dynamics before Ca²⁺ depletion. The individual myocytes show homogenous Ca²⁺ transients (2 and 9) on excitation. A(c): An X-t image of the myocytes (cells 1–3) scanned along the arrow in A(a). The dotted lines denote the cell borders. B: The [Ca²⁺]_i dynamics during Ca²⁺ depletion. B(a): Sequential X-Y images (1–10) displayed every 210 msec. B(b): X-t image of the cells indicated by an arrow in B(a). The myocytes exhibit Ca²⁺ waves propagating sporadically along the longitudinal axis instead of Ca²⁺ transients. Note that the cells are separated at intercalated discs (indicated by two white arrowheads).

Fig. 3. 

The [Ca²⁺]_i dynamics of the subepicardial surface of the left ventricle in the Langendorff-perfused rat heart after Ca²⁺ repletion. (a): The X-Y fluo3-fluorescence image recorded 2 min after Ca²⁺ repletion and the corresponding outline drawing are shown on the right panel. (b): Two X-t images of myocytes showing high-frequency Ca²⁺ waves with asynchronous localized contractions are shown. Note that the intercalated disc is remarkably detached (indicated by “gap”). (c): Shown are X-Y images (left, fluo3 fluorescence; middle, RH-237) of the myocytes acquired 10 min after Ca²⁺ repletion and the corresponding outline drawing on the right panel. Cells are remarkably detached from the intercalated discs showing contracture.

Role of transmembrane Ca²⁺ influx in the Ca²⁺ paradox-induced contracture

Sequential changes in the intercellular gap and myocyte length revealed that the gap gradually widened with concomitant shortening of the cells having a sigmoid time course of changes in cell length with a half shortening time of 3.3±0.6 min (Fig. 4). We also examined the time courses of changes in the myocyte length and intercellular gap under pharmacological inhibition of various pathways for the [Ca²⁺]_i rise. In the presence of SEA0400 (3 μM), a selective inhibitor of the NCX, the Ca²⁺ paradox-induced emergence of Ca²⁺ waves and contracture were not attenuated: the time course of changes in cell length and the properties of Ca²⁺ waves were identical to those found in the absence of this agent (Fig. 5). Blockade of L-type Ca²⁺ channels by verapamil (20 μM) also resulted in progressive contracture with concomitant Ca²⁺ waves with the incidence and V_prop equivalent to those in the absence of the drug, although the latent period for cell shortening was prolonged for about 2.5 min (Fig. 5). Diminution of Ca²⁺ release from the sarcoplasmic reticulum (SR) by combined application of ryanodine (10 μM) and thapsigargin (0.5 μM) also failed to prevent the contracture: time courses of the changes in cell length were almost identical to those in the absence of these inhibitors (Fig. 5A). In contrast, nickel chloride (5 mM) was effective in preventing the progressive contracture and [Ca²⁺]_i overload, where myocytes exhibited neither Ca²⁺ waves, wave-like contractions, nor widening of the intercalated discs (Fig. 5).

Fig. 4. 

Sequential changes in cell length and intercellular gap during Ca²⁺ repletion. Time zero denotes the start of Ca²⁺ repletion.

Fig. 5. 

A: Pharmacological effects on the sequential changes in cell length (upper) and intercellular gap (lower) during Ca²⁺ repletion. B: The bar graphs showing frequency (upper) and propagation velocity, V_prop, (lower) of Ca²⁺ waves during Ca²⁺ repletion under various pharmacological interventions. The acronym ‘cont’ denotes control, “vera”, verapamil at 20 μM, “SEA”, SEA0400 at 3 μM, “rya”, ryanodine at 10 μM and thapsigargin at 0.5 μM, and “Ni”, nickel chloride at 5 mM.

Role of [Ca²⁺]_i overload and mechanical motion in the development of contracture

When myocytes were rendered [Ca²⁺]_i overload by membrane permeabilization with saponin (0.4%, 0.1 mM-Ca²⁺-containing Tyrode’s solution), they showed high-frequency Ca²⁺ waves (Fig. 6A) at 120.8±8.5/min/cell showing rapid V_prop of 117.7±11.0 μm/sec (n=33, 3 hearts) and asynchronous wave-like contractions; the former values were similar to, and the latter, significantly higher than, those observed in Ca²⁺-paradox injury (Fig. 6B-a, b). However, the saponin treatment failed to mimic the Ca²⁺-paradox injury; no widening of the intercellular gap or myocyte contracture occurred (Fig. 6B-c). Thus, irreversible [Ca²⁺]_i overload by trans-membrane Ca²⁺ influx alone did not lead to development of the contracture.

Fig. 6. 

Effects of membrane permeabilization on Ca²⁺ waves and myocyte shortening. A: Two representative X-t images of [Ca²⁺]_i dynamics after application of saponin. Saponin (0.4% in 0.1 mM Ca²⁺-containing Tyrode’s solution) produced high-frequency, rapidly propagating Ca²⁺ waves in individual myocytes. B: Frequency (a) and V_prop (b) of Ca²⁺ waves during saponin perfusion compared with those induced by Ca²⁺ paradox. (c) Time course of the myocyte dimensions, cell length and intercellular gap, before and during perfusion with saponin at 5 and 10 min. *, P<0.01.

In the presence of the mechanical uncoupler BDM [21] at 30 mM, no myocyte contracture occurred after Ca²⁺ repletion, where most myocytes exhibited quiescent [Ca²⁺]_i without contraction, and Ca²⁺ waves or excitation-evoked Ca²⁺ transients were barely observed. However, washing out of BDM during Ca²⁺ repletion resulted in emergence of high-frequency Ca²⁺ waves with asynchronous local contractions of individual myocytes and eventual contracture (3 hearts, data not shown). Thus, mechanical contraction per se is indispensable for contracture.

Immunohistochemistry for cadherin and β-dystroglycan

Contraction of the myocytes is mediated by the costameres, which physically connect the myofilaments to the extracellular matrix (ECM) via dystroglycan complex [6, 16, 20]. To test whether or not Ca²⁺ depletion alters β-dystroglycan, we conducted immunohistochemistry for this protein. In intact hearts β-dystroglycan was expressed continuously along the cell membranes, whereas depletion of Ca²⁺ remarkably reduced the distribution of this protein (Fig. 7A-a). Quantitatively the fluorescence intensity for β-dystroglycan was significantly reduced by Ca²⁺ depletion (Fig. 7A-b). Furthermore, two-dimensional gel immunoblotting for β-dystroglycan revealed that multiple protein complexes were identified during control, whereas the largest component of the complex was significantly diminished by Ca²⁺ depletion (Fig. 7A-c). In addition, immunohistochemistry revealed that separation of intercalated discs produced by Ca²⁺ depletion was associated with detachment of cadherin (Fig. 7B). Thus, Ca²⁺ depletion appeared to diminish β-dystroglycan along the sarcolemmal membrane, in addition to the separation of cadherin at the intercalated disc.

Fig. 7. 

A: (a) Immunohistochemical images of β-dystroglycan before (control) and during Ca²⁺ depletion. β-dystroglycan was distributed continuously along cell membranes, whereas its distribution was reduced after 10-min depletion of Ca²⁺. (b): Comparison of fluorescence intensity for β-dystroglycan under control conditions (cont) and during Ca²⁺ depletion (dep). *, P<0.01. (c): Two-dimensional blue native/SDS gel electrophoresis for β-dystroglycan before and during Ca²⁺ depletion. B: Immunohistochemistry of cadherin of the heart before and during Ca²⁺ depletion.

IV.  Discussion

The present study addressed the mechanisms for myocyte contracture produced by Ca²⁺-paradox injury in the heart. On restoration of extracellular Ca²⁺ following short-term Ca²⁺ depletion in the Langendorff-perfused rat heart, individual cardiomyocytes exhibited asynchronous, high-frequency Ca²⁺ waves. Ca²⁺ waves of this type would correspond to the “agonal” waves we previously identified in the rat heart [18, 30], a presentation of irreversible [Ca²⁺]_i overload leading to cell death. Such irreversible [Ca²⁺]_i dynamics accompanied the progressive contracture of the myocytes with a sigmoid time course of the cell-length shortening during the Ca²⁺ repletion. This sequence of events was nearly superimposable on that under blockade of either NCX or SR Ca²⁺ release, indicating no major role of these Ca²⁺ handling proteins in the contracture. A slight retardation of the development of contracture by verapamil (~2.5 min) may indicate a minor, if any, contribution of the voltage-gated L-type Ca²⁺ channels to this phenomenon. The Ca²⁺ paradox-induced contracture was found to be mediated by transmembrane influx of Ca²⁺ via Ni²⁺-sensitive pathway(s) other than the L-type Ca²⁺ channels or NCX, e.g., Ca²⁺-permeable non-specific cation channels [24], although no definitive pathway was eventually specified in this study. A relatively recent study in isolated mouse ventricular myocytes suggested that transient receptor potential (TRP) channels mediate the influx of Ca²⁺ under the Ca²⁺-paradox injury [19]. Considering that the TRP channels are activated by various environmental changes, in particular, mechanical stretch [22], the myocyte contraction per se may contribute to the development of [Ca²⁺]_i overload via this channel. In practice, we observed that mechanical arrest by BDM precluded the generation of the high-frequency Ca²⁺ waves, whereas its washout resulted in generation of the agonal waves and hypercontracture. This indicates that certain mechano-sensitive pathway(s) may provide progressive, synergistic augmentation of [Ca²⁺]_i overload through the transmembrane influx of Ca²⁺. It remains to be determined whether or not the TRP channels are the culprit for the irreversible [Ca²⁺]_i overload in the multicellular myocardium.

The key finding of this study is that the massive trans-sarcolemmal influx of Ca²⁺, presented as the high-frequency Ca²⁺ waves, was not sufficient for the development of contracture, because saponin-induced Ca²⁺ permeabilization of the membrane barely produced myocyte separation or contracture despite the severe [Ca²⁺]_i overload that was equivalent to that emerged during the Ca²⁺-paradox injury. Two major alterations were identified under Ca²⁺ depletion: separation of the myocytes at intercalated discs and dissipation of the β-dystroglycan complex along the lateral membrane surface of the myocytes. The former change would stem from impairment of the Ca²⁺-dependent intercellular connection at the intercalated disc via N-cadherin [17] as confirmed by its separation in immunohistochemistry. Such cell-end to cell-end separation of the myocytes, however, would not fulfill the requirement for the hypercontracture, because myocytes are tightly connected to the lateral sides with the ECM, i.e., the scaffold of the myocardium [6, 16, 20]. We assume the deletion of β-dystroglycan by Ca²⁺ depletion is responsible for the progressive contracture in the Ca²⁺-paradox injury because this membrane-spanning protein forms a complex that connects with the actin-binding protein dystrophin in the cells [6, 16, 20, 26], and provides trans-sarcolemmal linkage between the actin filaments and the ECM at the costamere. Thus, in addition to the separation of the cell-cell connection via N-cadherin, weakening of the cell-ECM connection by diminution of β-dystroglycan, may lead to the contracture. Although it may be practically difficult to address the role of β-dystroglycan in the development of contracture, an instantaneous, whole-cell ablation of this target protein, by using chromophore-assisted laser inactivation (CALI) [28, 29], may provide a direct evidence for this assumption.

At present it is unknown how Ca²⁺ depletion leads to dissipation of β-dystroglycan. In this regard, Rentschler et al. [26] identified Ca²⁺-binding EF-hand regions in dystrophin that are essential for binding with β-dystroglycan. This may indicate that Ca²⁺ depletion promotes detachment of β-dystroglycan from dystrophin by impairment of these regulatory sites, and thereby, diminishes β-dystroglycan located along the cell membrane. It is also undetermined whether and how the depletion of β-dystroglycan contributes to irreversible Ca²⁺ influx in the myocytes. In this respect, dissipation of the dystroglycan-dystrophin complex in the skeletal muscle of the muscular dystrophy (mdx) mice reportedly promotes Ca²⁺ influx via Ca²⁺ leakage [1, 2] due possibly to disruption of the membrane integrity and subsequent activation of protease(s), e.g., calpain, and thereby, augment permeability of the cell membrane to Ca²⁺. In practice, mediation of calpain was suggested in the Ca²⁺-paradox injury of the heart [11, 31]. It should also be noteworthy that mdx skeletal muscles showed increased expression of TRP channels [12].

To our knowledge the present observations are the first to propose a possible contribution of cell-ECM disconnection by dissipation of β-dystroglycan and the resultant [Ca²⁺]_i overload in the development of myocyte contracture under the Ca²⁺-paradox injury. Our experimental design, however, may not directly reflect the real pathological conditions in the heart. In this regard, as Rodriguez et al. [27] demonstrated, several membrane-spanning proteins including β-dystroglycan are predominantly diminished in ischemic myocardium. Thus, during ischemia-reperfusion injury, alterations of β-dystroglycan may contribute to impairment of the structure and mechanical functions of the myocardium. Our present results provide important insights into the mechanism for myocardial injury that leads to contracture.

V.  Disclosures

None.

VI.  Acknowledgment

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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