AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 290: H1493-H1502, 2006. First published November 11, 2005; doi:10.1152/ajpheart.00669.2005
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Synchronous progression of calcium transient-dependent beating and sarcomere destruction in apoptotic adult cardiomyocytes

Rumi Maruyama,1 Genzou Takemura,1 Noritsugu Tohse,2 Tomoko Ohkusa,3 Yasuhiro Ikeda,3 Kunihiko Tsuchiya,1 Shinya Minatoguchi,1 Masunori Matsuzaki,3 Takako Fujiwara,4 and Hisayoshi Fujiwara1

1Second Department of Internal Medicine, Gifu University School of Medicine, Gifu; 2Department of Cellular Physiology and Signal Transduction, Sapporo Medical University School of Medicine, Sapporo; 3Second Department of Internal Medicine, Yamaguchi University School of Medicine, Ube; and 4Department of Food Science, Kyoto Women's University, Kyoto, Japan

Submitted 21 June 2005 ; accepted in final form 9 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During early apoptosis, adult cardiomyocytes show unusual beating, suggesting possible participation of abnormal Ca2+ transients in initiation of apoptotic processes in this cell type. Simultaneously with the beating, these cells show dynamic structural alteration resulting from cytoskeletal disintegration that is quite rapid. Because of the specialized structure and extensive cytoskeleton of cardiomyocytes, we hypothesized that its degradation in so short a time would require a particularly efficient mechanism. To better understand this mechanism, we used serial video microscopy to observe beta-adrenergic stimulation-induced apoptosis in isolated adult rat cardiomyocytes while simultaneously recording intracellular Ca2+ concentration and cell length. Trains of Ca2+ transients and corresponding rhythmic contractions and relaxations (beating) were observed in apoptotic cells. Frequencies of Ca2+ transients and beating gradually increased with time and were accompanied by cellular shrinkage. As the cells shrank, amplitudes of Ca2+ transients declined and diastolic intracellular Ca2+ concentration increased until the transients were lost. Beating and progression of apoptosis were significantly inhibited by antagonists against the L-type Ca2+ channel (nifedipine), ryanodine receptor (ryanodine), inositol 1,4,5-trisphosphate receptor (heparin), sarco(endo)plasmic Ca2+-ATPase (thapsigargin), and Na+/Ca2+ exchanger (KB-R7943). Electron-microscopic examination of beating cardiomyocytes revealed progressive breakdown of Z disks. Immunohistochemical analysis and Western blot confirmed that disappearance of Z disk constituent proteins ({alpha}-actinin, desmin, and tropomyosin) preceded degradation of other cytoskeletal proteins. It thus appears that, in adult cardiomyocyte apoptosis, Ca2+ transients mediate apoptotic beating and efficient sarcomere destruction initiated by Z disk breakdown.

apoptosis; calcium regulation; cardiac myocytes; sarcomere; Z disk

SEVERAL REPORTS have suggested that apoptosis among cardiomyocytes plays an important role in the progression of cardiovascular disease (1, 7, 11). This idea remains controversial, however, because the ultrastructural changes characteristic of apoptosis have only rarely been seen in cardiomyocytes from diseased hearts (8, 16, 34). However, cardiomyocyte apoptosis can be induced in vitro by a variety of stimuli. For instance, we and others previously described Fas-induced, hypoxia-induced, and beta-adrenergic pathway-stimulated apoptosis in cultured adult cardiomyocytes (5, 15, 17, 22).

Adult cardiomyocytes are terminally differentiated cells that are highly specialized in terms of structure and function. They express contractile proteins and a particularly well-developed cytoskeleton that are tightly packed into the cytoplasm and are sensitive to the Ca2+ load, mediating continual contraction and relaxation in vivo. In an earlier study, we used serial video and electron microscopy to show several unique features of apoptosis in adult cardiomyocytes, including the rhythmic contraction and relaxation (beating) that precede cellular deformation, accelerate as the cell shrinks, and are accompanied by the loss of myofibrillar striations (22). Cardiomyocyte beating is directly regulated by intracellular Ca2+ transients (26, 35), whereas elevation of the intracellular Ca2+ concentration ([Ca2+]i) is an important factor in the initiation of apoptosis in a variety of cells (27, 32). However, the kinetics of the changes in [Ca2+]i have not been recorded during the progression of cardiomyocyte apoptosis. In addition, [Ca2+]i is tightly regulated by multiple regulatory proteins in the sarcoplasmic reticulum (SR) and plasma membranes of cardiomyocytes, but the participation of these proteins in the progression of cardiomyocyte apoptosis is also not well understood.

Apoptosis is known to be an energy-requiring process causing dramatic structural alterations within a relatively short period of time (31). Video recording of various apoptotic cell types in culture has revealed apoptosis to be a dynamic process that is complete within hours and is characterized by several common features: cellular shrinkage and rounding, budding or blebbing, and apoptotic body formation (3, 23, 24). These structural changes are accompanied by disassembly of the cytoskeletal and nuclear scaffold as a result of proteolysis catalyzed by caspase family proteases (25, 36). Moreover, we previously showed that apoptosis in cardiomyocytes, despite their highly specialized structure and extensive cytoskeleton, proceeds at a rate comparable to that of other cell types (22). We therefore hypothesized that cardiomyocytes express a particularly robust mechanism for executing apoptosis, enabling efficient degradation of their stout structure.

We sought to test our hypothesis to obtain a fuller understanding of the pathophysiology of adult cardiomyocyte apoptosis by first examining the changes in [Ca2+]i during apoptosis in adult cardiomyocytes and to investigate the roles of the L-type Ca2+ channel, ryanodine receptor, inositol 1,4,5-trisphosphate receptor (IP3R), sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), and Na+/Ca2+ exchanger (NCX) in the process. In addition, we carried out ultrastructural and immunohistochemical analyses of the cytoskeleton of beating cardiomyocytes with the aim of determining the mechanism underlying the efficient collapse of apoptotic cardiomyocytes. Our findings suggest that adult cardiomyocyte apoptosis is exquisitely regulated by Ca2+ transients, which are responsible for the apoptotic beating and sarcomere destruction initiated by Z disk breakdown.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cardiomyocyte culture and treatments. All animals received humane care in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85-23, Revised 1985]. This study was approved by our Institutional Animal Research Committee. Cardiomyocytes were isolated from the ventricles of adult male Sprague-Dawley rats (200–250 g body wt) as previously described (22, 29). The cells were plated on laminin-coated dishes or in glass slide chambers at a concentration of 5.0 x 104 cells/ml and incubated in MEM with 1.8 mM Ca2+ and 50 µg/ml gentamicin in 95% air-5% CO2 before experimentation.

Cardiomyocyte apoptosis was induced by incubation of the cells with the beta-adrenergic agonist isoproterenol (Iso, 10–5 mol/l; Sigma, St. Louis, MO) (5, 6, 38, 39). Initially, apoptosis and the associated beating were characterized in this model using light and electron microscopes, as well as serial video microscopy. Thereafter, we continually monitored changes in [Ca2+]i in relation to long-axial cell shortening and evaluated the contributions of the L-type Ca2+ channel, ryanodine receptor, IP3R, SERCA, and NCX to the apoptotic process by testing the effects of the selective inhibitors nifedipine, ryanodine, heparin, thapsigargin (Sigma), and KB-R7943 (Tocris Cookson, Bristol, UK). Finally, electron and fluorescence microscopy were applied to characterize the destruction of the sarcomeres, and their proteins were determined in the beating cardiomyocytes during the early stage of apoptosis. For comparison, an oncosis model was prepared by addition of 0.01% Triton X-100 (Sigma) to samples of cardiomyocytes as previously described (22).

Cell morphology and membrane permeability. Cells were morphologically classified into two groups according to their length-to-width ratio: rod-shaped cells with a cell length-to-width ratio >3 and non-rod-shaped cells with a cell length-to-width ratio <3 (19). The latter included square and round cells, which sometimes also showed budding or blebbing and cellular fragmentation. To evaluate membrane permeability, the cells were exposed to 0.1% trypan blue for 5 min, and the numbers of stained and unstained cells in the dishes were counted (18). Six independent experiments were carried out with each group.

Assays for DNA fragmentation. In situ TdT-mediated dUTP nick end-labeling (TUNEL) assays were carried out 24 h after stimulation using a commercially available kit (Chemicon International, Temecula, CA) with cardiomyocytes fixed in 4% paraformaldehyde according to the supplier's instructions. Double-stranded DNA breaks were assessed by gel electrophoresis with genomic DNA isolated from the cardiomyocytes as previously described (22). Six independent experiments were carried out with each group.

Serial video microscopy. Twenty-four-hour serial video microscopy of cultured cardiomyocytes was carried out as described elsewhere (22). Normal rod-shaped adult cardiomyocytes (n = 166) were recorded beginning immediately after treatment with 10–5 mol/l Iso. In separate experiments, 146 normal rod-shaped cells were incubated with medium alone and similarly recorded for 24 h (control group), and 140 other normal rod-shaped cells were incubated with medium containing 0.01% Triton X-100 (oncosis model).

Under a video microscope, the apoptotic process in adult cardiomyocytes was designated as one of three types (22): long-axial shortening to a bonelike, clublike, or square shape followed by rounding and bud formation, formation of apoptotic bodies, and secondary necrosis (type A1); long-axial shortening similar to that described for type A1 followed by rounding, budding, and secondary necrosis, without formation of apoptotic bodies (type A2); and long-axial shortening followed immediately by secondary necrosis (type A3).

Scanning and transmission electron microscopy. Cells on dishes and in slide chambers (n = 3 each per group) were fixed with phosphate-buffered 2.5% glutaraldehyde (pH 7.4) for 4 h, postfixed with 1% osmium tetroxide for 1 h, and conventionally prepared for scanning (model S-450, Hitachi, Tokyo, Japan) and transmission (model H-800, Hitachi) electron microscopy.

Measurement of [Ca2+]i and cell length. After cardiomyocytes were loaded with fura-2 AM (Molecular Probes, Eugene, OR) as previously described (13), they were superfused with 1.8 mM Ca2+-containing Tyrode solution (pH 7.4) at 37°C. Fura 2 fluorescence ratios and cell shortening, which was detected by video edge detection, were simultaneously recorded using real-time power spectrum analysis software (IonWizard, Optix, Milton, MA). Because it is difficult to anticipate the timing of initiation of apoptosis and which cell would undergo apoptosis, we measured the fura 2 fluorescence ratio and cell length in many (n = 10–20) rod-shaped cells at the beginning of each experiment. When one of the cardiomyocytes was found to be beating during observation, continuous measurement was performed, with the focus on that cell. Collectively, [Ca2+]i and cell length could be measured in 12 apoptotic cardiomyocytes stimulated by Iso.

Immunohistochemistry and confocal microscopy. Cells plated on glass slides were fixed with 4% paraformaldehyde and conventionally processed for immunohistochemical analysis using primary antibodies raised against tropomyosin (clone TM311, Sigma), {alpha}-actinin (EA-53, Sigma), desmin (DE-U-10, Sigma), myosin (NOQ7.5.4D, Sigma), troponin T (H169, Santa Cruz Biotechnology, Santa Cruz, CA), or the active form of caspase-3 (Chemicon International). F-actin was stained with rhodamine-phalloidin (Molecular Probes). Hoechst 33342 (Wako, Tokyo, Japan) was used for nuclear staining. The immunostained preparations were observed under a confocal microscope (model LSM510, Zeiss). Three independent experiments were carried out with each group.

Western blotting. Cells were stimulated with Iso and then washed twice with PBS and lysed in Laemmli buffer. The amount of protein in each lysate was determined using a protein assay kit (Bio-Rad, Hercules, CA). The samples (4 µg) were heated in a boiling water bath for 5 min and subjected to electrophoresis on a 12.5% polyacrylamide-SDS slab gel. Proteins were transferred onto a polyvinylidene difluoride membrane after electrophoresis in 25 mmol/l Tris·HCl, 192 mmol/l glycine, and 0.1% SDS. The membrane was blocked with 10% nonfat dry milk overnight at 4°C and then incubated with the primary antibody against tropomyosin, {alpha}-actinin, desmin, myosin, or troponin T in PBS containing 0.05% (vol/vol) Tween 20 at room temperature for 60 min. The immunoreactive protein bands were then visualized using a peroxidase-conjugated secondary antibody, diaminobenzidine HCl, and enhanced chemiluminescence (ECL Plus, Amersham Biosciences, Buckinghamshire, UK). Three independent experiments were carried out with each group.

Statistical analysis. Values are means ± SD. Statistical comparisons were made using analysis of variance followed by Newman-Keuls multiple comparisons test. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Morphology, membrane permeability, and the apoptotic process in adult cardiomyocytes. At 24 h after plating, rod-shaped cells accounted for 82 ± 3% of the cells attached to control plates; 95 ± 1% of these cells excluded trypan blue, indicating that their plasma membranes were intact (Fig. 1, A and B). Treatment with Iso markedly reduced the percentage of rod-shaped cells (36 ± 3%) but had only a small effect on the numbers of cells excluding trypan blue (90 ± 2%). Treatment with Triton X-100 reduced the percentages of rod-shaped (50 ± 4%) and dye-excluding cells (0 ± 0%), with a much more profound effect on trypan blue exclusion. Thus, in contrast to Iso-induced apoptosis, which almost always left the plasma membrane intact, Triton X-100-induced oncosis always disrupted the plasma membrane. This was confirmed by scanning electron-microscopic examination, which revealed that Iso-treated cardiomyocytes showed various deformities, including clublike, bonelike, square, and round shapes, as well as cells containing multiple lobules (apoptotic bodies), but they all presented a smooth surface with an intact plasma membrane (Fig. 1C). Iso images in Fig. 1C show a clublike cardiomyocyte (top) and apoptotic bodies formation (bottom). By contrast, cardiomyocytes treated with Triton X-100 were rod-shaped, square, or oval, and all had a rough surface, indicative of a disrupted plasma membrane (Fig. 1C).


Figure 1
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  		Fig. 1. Morphology of isoproterenol (Iso)-induced apoptosis in adult cardiomyocytes. A: light photomicrographs of control, Iso-stimulated, and Triton X-100-stimulated cardiomyocytes. B: relation between morphology and membrane permeability in cardiomyocytes. *P < 0.05 vs. baseline. #P < 0.05 vs. percentage of trypan blue-containing cells at the corresponding time. C: scanning electron micrographs of control, Iso-stimulated, and Triton X-100-stimulated cardiomyocytes. Iso images in C show a clublike cardiomyocyte (top) and apoptotic body formation (bottom). Scale bars, 10 µm.

 
We then used serial video microscopy to record the overall process leading to the structural and functional alterations seen in cardiomyocytes exposed to Iso or Triton X-100. The modes and incidences of cell death among the cells studied are summarized in Table 1. All 166 of the Iso-treated cells were rod-shaped, and none were beating at the start of the experiment; moreover, 51 of these cells (31%) showed no morphological changes and no beating after 24 h of exposure to Iso. On the other hand, the remaining 115 cells (69%), which were subsequently deemed apoptotic, began to beat after an average of 8.2 ± 4.2 h of stimulation (Fig. 2). At ~1 h after the onset of beating, long-axial shortening began, leading to a bonelike shape with rounded edges, a clublike shape with rounding of the edge on one side, or a square shape without deformity of the cellular edges. In 108 of the apoptotic cells (types A1 and A2), progression of the cellular shrinkage was accompanied by increases in the beat frequency (Fig. 2), which continued until, after an additional 3.7 ± 3.9 h, the cells were almost completely round and the beating had stopped (Fig. 2). After 0.2 ± 0.3 h, multiple buds were observed; 1.3 ± 0.7 h later, 22 cells (type A1; 13%) developed apoptotic bodies, whereas 86 cells (type A2; 52%) did not. In all these cases, the cell surfaces remained smooth. By contrast, in the seven other apoptotic cells (type A3; 4%), the cell surfaces of the club- and bone-shaped cells assumed the rough appearance characteristic of secondary postapoptotic necrosis 4.9 ± 1.2 h after the start of beating, at which time the beating ceased, with no budding or apoptotic body formation. There was no indication of oncosis in any of the 140 cells treated with Iso.


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  		Table 1. Mode and incidences of cell death among cultured adult cardiomyocytes after incubation for 24 h under control conditions or in the presence of Iso or Triton X-100

 

Figure 2
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  		Fig. 2. Real-time serial recording of apoptotic beating in adult cardiomyocytes. Two representative examples are shown in separate movie files (supplemental video files may be found at http://ajpheart.physiology.org/cgi/content/full/00669.2005/DC1). Focus on cardiomyocytes circled in A and B.

 
Of the 146 cells incubated with medium alone (control), 120 (82%) were unchanged, 9 (6%) underwent oncosis, and the remaining 17 (12%) showed apoptosis similar to that seen in the Iso-treated group: 14 cells (10%) were type A2, and 3 (2%) were type A3. In the Triton X-100-treated oncosis model, the cells showed rigorous contracture after the cell surface became very rough. Within 12 h after application of Triton X-100, all the cells were fluffy and square or oval. None of the cells beat during the course of these changes.

Relation between [Ca2+]i and beating during adult cardiomyocyte apoptosis. Before induction of apoptosis, normal rod-shaped cardiomyocytes loaded with fura-2 AM showed no spontaneous Ca2+ transients or shortening (Fig. 3A). When electrically stimulated (40 V) at a frequency of 1 Hz, all the cells showed Ca2+ transients and corresponding long-axial shortening (Fig. 3B).


Figure 3
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  		Fig. 3. Simultaneous recording of intracellular Ca2+ concentration ([Ca2+]i) and cell length in adult cardiomyocytes. A: untreated quiescent cells. B: cell shortening (top) and Ca2+ transients (bottom) induced by electrical stimulation. C: cell length (top) and [Ca2+]i (bottom) recorded from an apoptotic cardiomyocyte that was beating and shrinking. D: enlargement of C1–C8.

 
Thereafter, by simultaneously recording [Ca2+]i and cell length in 12 cardiomyocytes exposed to Iso, we found that these cells initially exhibit rapid and intense changes in [Ca2+]i, i.e., Ca2+ transients, that are accompanied by corresponding contractions and relaxations (beating; Fig. 3, C and D). With time, the frequency of the Ca2+ transients and the beating increased, and the cells became shorter, even during the periods of relaxation. There was also a gradual increase in the diastolic [Ca2+]i as the cells shortened, as well as a gradual decline in cell length (Fig. 3, C and D). Although beating was almost invisible in round cells, Ca2+ transients were still apparent (Fig. 3D), indicating excitation-contraction dissociation in the apoptotic cells at the late stage.

Because [Ca2+]i is tightly regulated by the cooperative actions of various Ca2+ regulatory proteins in the plasma and SR membranes in cardiomyocytes, we next investigated the contributions of these proteins during apoptosis. We found that blockade of L-type Ca2+ channels with nifedipine or SR Ca2+ release channels with ryanodine significantly and dose dependently (10–9–10–6 mol/l) inhibited Iso-induced cardiomyocyte shortening, as did blockade of IP3R with 10–500 µg/ml heparin (Fig. 4A). Interestingly, thapsigargin (a SERCA antagonist) and KB-R7943 (an NCX inhibitor) also inhibited Iso-induced shortening in cardiomyocytes. TUNEL positivity was confined not in rod-shaped, but in already deformed, cardiomyocytes, mostly in round cardiomyocytes at the late stage of apoptosis. All these antagonists significantly reduced the incidence of TUNEL-positive cardiomyocytes and inhibited DNA ladder formation (Fig. 4, B and C). Pretreatment with 10–6 mol/l nifedipine completely blocked the occurrence of Ca2+ transients and beating in all the cells (n = 24) during 24 h of Iso stimulation (Fig. 4D), as did all the other antagonists (n = 12–20 each; data not shown).


Figure 4
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  		Fig. 4. Effect of Ca2+ regulatory protein inhibitors on Iso-induced apoptosis. A: dose-dependent effects of inhibitors on cell viability assessed as percent rod-shaped cells. *P < 0.05 vs. Iso alone. {dagger}P < 0.05 vs. the same group at the different concentration. B: effect of inhibitor on incidence of TdT-mediated dUTP nick-end labeling (TUNEL)-positive Iso-treated cardiomyocytes. KB-R, KB-R7943. *P < 0.05 vs. Iso alone. C: DNA ladder formation in Iso-stimulated cardiomyocytes and its inhibition by nifedipine. D: effect of nifedipine on cell length (top) and [Ca2+]i (bottom) in Iso-stimulated cardiomyocytes. D1 and D2 are enlarged at right.

 
Sarcomere destruction in adult cardiomyocytes during apoptosis. Electron-microscopic examination revealed that, in contrast to normal cardiomyocytes, which show regular striations (Fig. 5A), rounded apoptotic cardiomyocytes, as well as apoptotic cardiomyocytes forming apoptotic bodies, show disarrangement of their myofibrils into whirlpool-like structures with the loss of striations (Fig. 5, C1–C4). This feature was completely distinct from the supercontracted myofibrils seen in necrotic cardiomyocytes (Fig. 5B). The earliest sign of apoptosis-related structural change was deformation of the cell edges with slight long-axial shortening, resulting in the aforementioned bonelike, clublike, and square shapes. We therefore focused on these cells to investigate early-stage apoptosis. We found that these cells exhibited sarcomere destruction at their edges but that the sarcomeres at their centers remained intact (Fig. 5, D1–D3). The most conspicuous finding was the breakdown of the Z disks at the border between the intact and disrupted sarcomeres, which were being broken into pieces.


Figure 5
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  		Fig. 5. Transmission electron micrographs of adult cardiomyocytes. A: normal rod-shaped cardiomyocyte with intact striations. B: oncotic cardiomyocyte treated with Triton X-100. C1 and C2: round apoptotic cardiomyocyte during late-stage apoptosis. C3 and C4: apoptotic cardiomyocyte forming apoptotic bodies during late-stage apoptosis. D1–D3: bone-shaped cardiomyocyte during early-stage apoptosis. At cell edges, Z disks have been broken into pieces, and sarcomeres have been completely lost; near the center of the cell, sarcomeres remain intact. N, nucleus; Mt, mitochondria. Scale bars, 1 µm.

 
We then investigated which sarcomeric proteins were the targets of the apoptotic proteolysis. In the apoptotic cardiomyocytes, the immunoreactivity of several Z disk constituents, including {alpha}-actinin, desmin, and tropomyosin, disappeared from regions in which the Z disks appeared to have been broken down, i.e., at the edges of the cells during early-stage apoptosis and throughout the entire cell during late-stage apoptosis (Fig. 6A). Other cytoskeletal proteins, including actin, myosin and troponin T, were preserved until the late stage (Fig. 6). By contrast, no cytoskeletal immunoreactivity was lost from cells treated with Triton X-100 (Fig. 6A). Western blotting confirmed the immunohistochemical findings, i.e., earlier reduction of Z disk constituent proteins ({alpha}-actinin and tropomyosin) than of the other protein (myosin; Fig. 7A).


Figure 6
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  		Fig. 6. Confocal photomicrographs of adult cardiomyocytes. A: an intact cell, an early-stage apoptotic cell with a bone-like shape, a late-stage apoptotic cell with a round shape, and an oncotic cell. Green, {alpha}-actinin; red, troponin T; blue, Hoechst 33342. Scale bars, 10 µm. *, Deficit of immunoreactive {alpha}-actinin or troponin T. B: incidences of cytoskeletal protein defects in cardiomyocytes during early- and late-stage apoptosis. Z disk constituents ({alpha}-actinin, desmin, and tropomyosin) disappeared during the early stage, but the other proteins (actin, myosin, and troponin T) were preserved until a later stage.

 

Figure 7
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  		Fig. 7. A: Western blot of {alpha}-actinin, tropomyosin, and myosin in adult cardiomyocytes before and 6 and 24 h after Iso stimulation (a) and densitometric analysis of Western blot results (b). {alpha}-Actinin and tropomyosin (Z disk constituent proteins), but not myosin, were significantly reduced as early as 6 h after Iso stimulation. *P < 0.05. B: immunohistochemical preparations for the active form of caspase-3 in a normal rod-shaped, a bone-shaped, and a round cell. Scale bars, 10 µm.

 
Next, to check the topographic relation between caspase-3 and sarcomeric protein degradation, we used immunohistochemistry to determine the active form of caspase-3 in Iso-stimulated cardiomyocytes at various stages of apoptosis. Activated caspase-3 was absent in normal rod-shaped cardiomyocytes but was observed at the edges of bone- or club-shaped cardiomyocytes (early stage of apoptosis) and throughout round cardiomyocytes (late stage; Fig. 7B).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Apoptosis is a morphological term indicating a particular mode of cell death that is most reliably identified through electron microscopy (21, 37). Communal et al. (5) were the first to report that beta-adrenergic stimulation could induce apoptosis in adult cardiomyocytes, a finding that was confirmed by the present study at the ultrastructural level using scanning and transmission electron microscopy. Moreover, we were able to use serial video microscopy to document the entire apoptotic process culminating at a stage in which the cardiomyocytes had shrunk and showed smooth plasma membranes and apoptotic bodies. Thus, with respect to cardiomyocyte ultrastructure and dynamics, apoptosis induced by Iso is strikingly similar to that induced by Fas (22). Adult cardiomyocytes began to beat just before initiation of apoptotic morphological changes and continued to beat until they had shrunk maximally. This beating is known to be regulated directly by [Ca2+]i, and in the present study we were able to record the Ca2+ transients that were responsible for the beating. Felzen et al. (10) described Fas-induced alterations in the electrophysiological properties of cardiomyocytes, which included reduced resting potential, reduced action potential amplitude, increased action potential duration, elevated diastolic [Ca2+]i, and increased arrhythmogenic activity. They did not describe the dynamics of the morphological and structural changes associated with the electrophysiological and [Ca2+]i alterations. We believe that the present study is the first in which the changes in [Ca2+]i and the beating of adult cardiomyocytes committed to apoptosis were simultaneously recorded. One of the confounders associated with Iso is that, by itself, it induces enhanced Ca2+ cycling. However, such a specific change in [Ca2+]i accompanying beating may be a common phenomenon in apoptotic cardiomyocytes, because similar beating is observed in the Fas-stimulated apoptotic process in adult cardiomyocytes (22).

It has been shown that an increase in [Ca2+]i is essential for initiation of apoptosis in a variety of cell types (27, 32) and that blockade of L-type Ca2+ channels can inhibit apoptosis in cardiomyocytes (30, 33). beta-Adrenergic agonists such as Iso elicit increases in [Ca2+]i by accelerating Ca2+ influx though L-type Ca2+ channels via activation of protein kinase A (PKA) (27). Activated PKA can also induce inositol trisphosphate synthesis by phospholipase C, which elicits Ca2+ release from the SR via the IP3R, the expression of which may be upregulated during apoptosis (10, 20). However, in a more recent report, Zhu et al. (39) showed that Iso-induced cardiomyocyte apoptosis is mediated by PKA-independent activation of the Ca2+ channel and subsequent activation of Ca2+/calmodulin-dependent protein kinase II. Our finding that heparin inhibited apoptosis is consistent with earlier reports (10, 20). Ryanodine inhibition of Iso-induced cardiomyocyte apoptosis is consistent with the idea that reagents acting to reduce [Ca2+]i, whether by interfering with Ca2+ influx through the plasma membrane or Ca2+ release from the SR, will inhibit apoptosis. More surprisingly, thapsigargin and KB-R7943 also inhibited Iso-induced apoptosis, even though both of these reagents would be expected to increase [Ca2+]i by interfering with SR Ca2+ uptake through SERCA and by interfering with Ca2+ efflux through NCX on the plasma membrane, respectively. Our finding that thapsigargin inhibited Iso-induced cardiomyocyte apoptosis confirms that of Zhu et al., who did not speculate on this phenomenon. We suggest that the ability to sequester Ca2+ is also necessary for apoptosis, which is consistent with the idea that Ca2+ transients, and not just a steady rise in [Ca2+]i, are necessary for initiation of cardiomyocyte apoptosis, which would account for the beating observed in the present study.

We also observed the progressive breakdown of the Z disks during early cardiomyocyte apoptosis that was associated with the initial cellular deformation after exposure to Iso. The Z disks define the lateral boundaries of the sarcomeres. They are a dynamic and complex filamentous lattice with a unique structural symmetry and play a number of pivotal roles in striated muscle cells (12). In addition to serving as an elastic element, Z disks are involved in diverse aspects of sarcomere assembly and organization and in the generation and transmission of muscle force (2, 4, 12). In apoptotic cardiomyocytes, myofibrillar striations are effectively lost as the Z disks are broken down, resulting in sarcomere destruction. Such a key event during early-stage apoptosis could efficiently facilitate the subsequent catastrophic destruction of the tightly packed cytoskeleton of adult cardiomyocytes. For instance, tropomyosin is a constituent protein that provides the Z disk with structural stability and modulates the function of actin filaments (14, 28). Without tropomyosin, actin filaments are unable to maintain their filamentous structure and break down, leading to disintegration of the sarcomere. We found in the present study that tropomyosin, along with {alpha}-actinin and desmin, immunoreactivity disappeared with the breakdown of the Z disks, although other cytoskeletal proteins, including actin, myosin, and troponin T, were unaffected until a later stage of the apoptotic process. Also, caspase-3 was activated at the corresponding sites with the loss of immunoreactivity of those sarcomeric proteins. This strongly suggests that Z disk constituents are the initial targets of apoptotic proteolysis in adult cardiomyocytes. These systematic changes were entirely different from those seen in oncotic cardiomyocytes, in which myofibrils were supercontracted and exhibited contraction bands with preserved Z disks.

Ca2+ transients appear to be related to the dynamics of apoptosis ubiquitously observed among various cell types (3, 9, 23, 24). These dynamics would be especially apparent in the beating of cells with well-developed myofilament systems, e.g., adult cardiomyocytes, although they would also be reflected in the proteolysis and DNA fragmentation catalyzed by Ca2+-dependent protease and DNases. We observed that, during the synchronous propagation of Ca2+ transient-dependent beating and sarcomere destruction in apoptotic cardiomyocytes, the beating frequency increased as the cell shortened. This increase in beat frequency may be related not only to the cell length but also to the number of remaining intact sarcomeres able to contract and relax. It would be apparent that Ca2+ transients preceding apoptosis in cardiomyocytes are different in nature from those in the physiological situation, because in vivo cardiomyocytes beat continuously. In cardiomyocytes from neonatal rats, Iso-induced increases in [Ca2+]i reportedly activate the Ca2+-dependent phosphatase calcineurin, which promotes apoptosis by reducing Bcl-2 and releasing cytochrome c from mitochondria into the cytosol (33). This apoptosis was inhibited by nifedipine and calcineurin inhibitors; however, the specific underlying mechanisms remain substantially unknown. Similarly, the steps in adult cardiomyocyte apoptosis occurring downstream of the Ca2+ transients remain to be described.

In summary, we have recorded the changes in [Ca2+]i (Ca2+ transients) that are responsible for the beating observed during cardiomyocyte apoptosis and demonstrated the regulation of the apoptotic process by multiple Ca2+ regulatory proteins. We have also shown that the systematic destruction of sarcomeres is initiated by Z disk breakdown. Most of the findings are indeed descriptive but new. It thus appears that, in adult cardiomyocytes, apoptosis is exquisitely regulated by Ca2+ transients, which are responsible for apoptotic beating and efficient sarcomere destruction.


   ACKNOWLEDGMENTS
 
We thank Tomoko Nao, Tomo Matsumoto, and Yuji Hisamatsu (Yamaguchi University School of Medicine) for helpful discussion and Akiko Tsujimoto and Hatsue Ohshika (Gifu University School of Medicine) for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Fujiwara, Second Dept. of Internal Medicine, Gifu Univ. School of Medicine, 1-1 Yanagido, Gifu 501-1194, Japan (e-mail: gifuim-gif{at}umin.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 DISCUSSION
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