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Cardiovascular Research 2003 58(3):535-548; doi:10.1016/S0008-6363(03)00255-4
© 2003 by European Society of Cardiology
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Copyright © 2003, European Society of Cardiology

Fetal and postnatal development of Ca2+ transients and Ca2+ sparks in rat cardiomyocytes

Sumihiko Sekia,b,*, Masato Nagashimaa, Yoichi Yamadaa, Masaaki Tsutsuuraa, Takeshi Kobayashia, Akiyoshi Namikib and Noritsugu Tohsea

aDepartment of Cellular Physiology and Signal Transduction, Sapporo Medical University School of Medicine, Sapporo 060, Japan
bDepartment of Anesthesiology, Sapporo Medical University School of Medicine, Sapporo 060, Japan

sseki{at}kanazawa-med.ac.jp

* Corresponding author. Department of Anesthesiology, Kanazawa Medical University School of Medicine, Daigaku 1-1, Uchinada, Ishikawa, 920-0025 Japan. Tel.: +81-76-286-2211x3133; fax: +81-76-286-3475.

Received 2 July 2002; accepted 23 January 2003


   Abstract
Top
 Abstract
1 Introduction
 2 Method
 3 Results
 4 Discussion
 References
 
Objective: The aim of this study was to characterize the spatio-temporal dynamics of [Ca2+]i in rat heart in the fetal and neonatal periods. Methods: Using confocal scanning laser microscopy and the Ca2+ indicator fluo-3, we investigated Ca2+ transients and Ca2+ sparks in single ventricular myocytes freshly isolated from rat fetuses and neonates. T-tubules were labeled with a membrane-selective dye (di-8-ANEPPS). Spatial association of dihydropyridine receptors (DHPR) and ryanodine receptors (RyR) was also examined by double-labeling immunofluorescence. Results: Ca2+ transients in the fetal myocytes were characterized by slower upstroke and decay of [Ca2+]i compared to those in adult myocytes. The magnitude of fetal Ca2+ transients was decreased after application of ryanodine (1 µM) or thapsigargin (1 µM). However, Ca2+ sparks were rarely detected in the fetal myocytes. Frequent ignition of Ca2+ sparks was established in the 6–9-day neonatal period, and was predominantly observed in the subsarcolemmal region. The developmental change in Ca2+ sparks coincided with development of the t-tubule network. The immunofluorescence study revealed colocalization of DHPR and RyR in the postnatal period, which was, however, not observed in the fetal period. In the adult myocytes, Ca2+ sparks disappeared after disruption of t-tubules by glycerol incubation (840 mM). Conclusions: The sarcoplasmic reticulum (SR) of rat ventricular myocytes already functions early in the fetal period. However, ignition of Ca2+ sparks depends on postnatal t-tubule formation and resultant colocalization of DHPR and RyR.

KEYWORDS Calcium (cellular); Developmental biology; E–C coupling; Embryology; SR (function)


   1 Introduction
Top
 Abstract
 1 Introduction
2 Method
 3 Results
 4 Discussion
 References
 
In the early fetal period, the heart is tubular in shape. The heart tube starts to contract essentially as it is forming [1]. In mature ventricular myocytes, contraction is known to depend on the release of Ca2+ from sarcoplasmic reticulum (SR) through ryanodine receptor channels (RyR) [2], while in the fetal heart SR is scarce [3] and isolated SR vesicles have lower volume, lower density, and a decreased capability to load Ca2+ compared to those isolated from mature heart [4–6]. It has been reported that ryanodine, a RyR inhibitor, has little effect on fetal and neonatal cell contraction [3,7–9]. Therefore, it has been thought that contraction of fetal cells depends largely on trans-sarcolemmal Ca2+ influx rather than Ca2+ release from SR. However, recent studies have reported the expression of RyR-2 [10] and SR Ca2+-ATPase (SERCA2a) [11,12] in the early fetal stage. Thus, it is as of yet impossible to rule out functional roles of these SR proteins in fetal cells. On the other hand, in early-stage embryonic stem cell-derived cardiomyocytes, spontaneous [Ca2+]i oscillation is independent on Ca2+ influx [13]. The mechanism(s) of excitation–contraction (E–C) coupling during the fetal period have not yet been clarified.

In regard to E–C coupling during development, it is necessary to investigate subcellular [Ca2+]i dynamics in single cardiomyocytes from fetuses. Confocal Ca2+ imaging not only allows the visualization of Ca2+ transients, but also that of Ca2+ sparks, which involve localized Ca2+ release from clusters of RyR [14]. Ignition of Ca2+ sparks in the ventricular myocytes is already initiated in the neonatal period [15]. However, no information is available on Ca2+ sparks in the fetal period.

Using confocal scanning laser microscopy, we show, for the first time, Ca2+ transients and Ca2+ sparks in single ventricular myocytes freshly isolated from rat fetuses. We also perform t-tubule imaging and fluorescent immunolabeling of L-type Ca2+ channels and RyRs in order to characterize the development of E–C coupling from the fetal period to the adult stage. The present study reveals the developmental processes of establishment of E–C coupling in cardiomyocytes.


   2 Method
Top
 Abstract
 1 Introduction
 2 Method
3 Results
 4 Discussion
 References
 
This 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).

2.1 Cell preparation
Freshly isolated single cells were prepared from ventricles of 12- and 18-day fetal (full term is ~21 days), neonatal (1–18 days old) and adult (6–10 weeks old) Wistar rats as previously described [16,17].

2.2 Confocal imaging of Ca2+ transients and Ca2+ sparks
Ventricular myocytes were incubated with fluo-3 AM (10 µM; Molecular Probes) and pluronic F-127 (0.45%; Molecular Probes) for 15–30 min at room temperature. Cells were subsequently washed in modified Tyrode solution to allow de-esterification. The composition of the modified Tyrode solution was as follows (in mmol/l): 137 NaCl, 4 KCl, 1 MgCl2, 1.2 NaH2PO4, 1.8 CaCl2, 10 glucose, 10 Hepes. pH was adjusted to 7.4 with NaOH.

Confocal fluorescence imaging was performed with a confocal scanning laser microscope system (Fluoview, Olympus, Tokyo, Japan) coupled to an inverted microscope (IX70, Olympus) with a x40 objective (UPlanApo, Olympus, numerical aperture 0.11–0.23) or x60 oil immersion lens (UPlanFl, Olympus, numerical aperture 0.17), excitation at a wavelength of 488 nm, and emission at >515 nm. The line scan mode was used for quantitative analysis of Ca2+ transients and Ca2+ sparks. The pixel size used in the present study was 0.1–0.3 µm. Line-scan rate was 1.76 ms per 256-pixel line or 2.05 ms per 512-pixel line.

Confocal imaging of Ca2+ transients was performed with the myocytes in modified Tyrode solution (34±1°C). The myocytes, except spontaneously beating myocytes of 12-day fetuses, were electrically stimulated through parallel platinum field electrodes (0.5 Hz, 1–2 ms in duration). The resulting images were analyzed using Fluoview software (Olympus). Before analysis, line-scan image data were low-pass-filtered (3x3 pixels). Fluorescence signals (F) were normalized by dividing them by the basal cell fluorescence after dye loading (F0).

Confocal images of Ca2+ sparks were obtained without electrical field stimulation. In preliminary experiments with normal modified Tyrode solution, spontaneous Ca2+ sparks were unobservable in fetal myocytes. Therefore, the Ca2+ concentration in the solution was increased to 5 mM and 1 µM BayK8644 was added [18]. Ca2+ sparks were selected visually from the line scan images and accepted according to the following criteria: change in F/F0≥0.3, full duration ≥10 ms, and full width ≥1 µm. For precise estimation of the spatial and temporal distributions of Ca2+ sparks, the data were fitted to a Gaussian distribution (IGOR Pro, WaveMetrics, OR, USA) to calculate full-width at half-maximal fluorescence intensity (FWHM) and full-time at half-maximal fluorescence intensity (FTHM) [19].

2.3 Estimation of intracellular Ca2+ concentration
Intracellular calcium concentration was estimated according to the following equation [20]:

Formula
where R is normalized fluorescence (F/F0) and Kd is the dissociation constant of the Ca2+-fluo-3 complex (400 nM) [21].

In order to determine diastolic [Ca2+]i in fetal ventricular cells, we performed a preliminary experiment using a fluorescence emission ratio method. The device used was composed of an inverted fluorescence microscope (TE 300, Nikon, Tokyo, Japan) with a x40 objective (Plan Fluor, Nikon, numerical aperture 0.11–0.23) and a calcium imaging system (Argus 50 HiSCA, Hamamatsu Photonics, Hamamatsu, Japan). Adult and 12-day fetal ventricular myocytes were incubated with fura2 acetoxymethyl ester (10 µM; Molecular Probes) and pluronic F-127 for 10–20 min followed by superfusion with modified Tyrode solution. The emission ratio was measured by alternately stimulating the myocytes with 340 and 380 nm light. The intensity of emitted fluorescence was recorded at 510 nm. Calculation of [Ca2+]i was performed using the following equation [22]:

Formula
where Kd is the dissociation constant of fura-2 (224 nM) and R is the ratio of fura-2 fluorescence intensity at 510 nm elicited by excitation at 340 and 380 nm. Rmin and Rmax were determined using Ca2+-free (1 mM EGTA) and saturated solutions, respectively, in the presence of 4-bromo-A 23187 (10 µM). F0/F1 is the ratio of the 380-nm excitation intensity at zero [Ca2+]i to that at saturated [Ca2+]i level.

In electrically stimulated (0.5 Hz) adult myocytes, diastolic [Ca2+]i was 151±4 nmol/l (n = 7). In 12-day fetal myocytes which beat spontaneously, diastolic [Ca2+]i was 102±1 nmol/l (n = 6). Therefore, we used values of 150 and 100 nmol/l for [Ca2+]0 to calibrate fluo-3 signals acquired from adult and fetal myocytes, respectively.

2.4 Imaging of Ca2+ transients in hearts from fetuses earlier than fetal day 12
The heart of fetuses earlier than fetal day 12 is too small to subject to single-cell preparations. Therefore, whole bodies of the 9- and 10-day fetuses were loaded with fluo-3 AM and changes in fluorescence were observed. First, to estimate the presence of spontaneous Ca2+ transients, a cooled CCD camera system (I-PentaMAX, Princeton Instruments, Trenton, NJ) with imaging software (WinView, Princeton Instruments) mounted on the microscope (IX70, Olympus) was used to capture the fluorescent images with excitation at 480 nm and emission at >510 nm at approximately 50 frames/s. Then, the 10-day fetuses, which exhibited Ca2+ transients, were observed using the confocal imaging system as described above.

2.5 Confocal imaging of t-tubules
For confocal imaging of t-tubules, the cells were incubated with the membrane-selective fluorescent dye di-8-ANEPPS (5 µM; Molecular Probes) and pluronic F-127 for 5–10 min. After washing with normal modified Tyrode solution, the residual dye on the surface of the membrane was excited with the 488-nm line of an argon laser and emitted fluorescence >510 nm was recorded.

2.6 Indirect immunofluorescent labeling of DHPR and RyR
Double labeling was performed with mouse monoclonal anti-RyR-2 antibodies (Affinity Bioreagents, Golden, CO, USA; 1 mg/ml) [23] and rabbit polyclonal antibodies to the {alpha}1c subunit of L-type Ca2+ channels (Alomone Laboratories, Jerusalem, Israel; 0.3 mg/ml) [24]. Freshly isolated myocytes were adhered to Laminin-coated coverslips, and initially fixed with 3% buffered paraformaldehyde in phosphate-buffered saline (PBS) for 10 min. After washing with PBS, the cells were permeabilized with Triton X-100 (0.1%) for 10 min. The cells were then incubated overnight with primary antibodies in antibody buffer solution (2% goat serum, 1% BSA, 0.05% Triton X-100, and 3 mM NaN3 in PBS). The primary antibody dilution was 1:80 for anti-{alpha}1c, and 1:100 for anti-RyR-2. Excess primary antibodies were washed twice with antibody wash solution {0.05% Triton X-100 in SSC (150.7 mM NaCl and 17.5 mM sodium citrate)} [25] and once with PBS. The cells were then incubated with fluorescein (FITC)-conjugated goat anti-mouse IgG (H+L) (binding to anti-RyR-2 antibodies: Jackson Immuno Research; 1.5 mg/ml) and Texas Red-conjugated anti-rabbit IgG (H+L) (binding to anti-{alpha}1c antibodies: Molecular Probes; 2 mg/ml) secondary antibodies, diluted to 1:100, for 2 h. The cells were then washed twice with the antibody wash solution and once with PBS. Finally, they were mounted on a slide glass using Gel/Mount (Biomeda). To determine nonspecific binding, control staining experiments with secondary antibody in the absence of primary antibody were also performed. Samples were examined by confocal scanning laser microscopy using a Zeiss LSM 510 equipped with an ArKr laser and a HeNe laser. We used the ‘Multi Track’ scanning mode to avoid cross-talk of fluorescences.

2.7 Disruption of t-tubules in adult myocytes
In order to prove that Ca2+ sparks are directly related to t-tubule formation, Ca2+ sparks were investigated after disruption of t-tubules in adult myocytes. Cells were incubated with control solution (modified Tyrode solution) or glycerol solution (modified Tyrode solution containing 840 mM of glycerol) for 1 h before being examined for sparks. Disruption of t-tubules was verified by loss of di-8-ANEPPS fluorescence in the cell interior.

2.8 Statistical analysis
Values are expressed as mean±S.E.M. Statistical significance of differences in means was evaluated by paired t-test or ANOVA followed by Fisher's test. Differences were considered significant when P values were less than 0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Method
 3 Results
4 Discussion
 References
 
3.1 Characteristics of fetal Ca2+ transients
Because the ventricular portion becomes easily distinguishable from the atrial portion of the heart in rat at fetal day 12, we used ventricular myocytes from 12-day fetus as the most primitive ventricular cells in the present study. The 12-day fetal ventricular myocytes were consistently round (Fig. 1A) and exhibited spontaneous Ca2+ transients without electrical stimulation. Fetal Ca2+ transients were characterized by slower upstroke and decay of [Ca2+]i in comparison with those in adult ventricular myocytes evoked by electrical stimulation (Fig. 1B,C). In the adult myocytes, fluorescence increased simultaneously across the entire width of the cell, whereas in the fetal myocytes, the increase in [Ca2+]i at the centre exhibited slower rise and decay than that in the subsarcolemmal region (Fig. 1C). These findings are summarized in Table 1. The 18-day fetal Ca2+ transients evoked by electrical stimulation exhibited a rate of rise and time constant of decay similar to those of the 12-day fetal Ca2+ transients (Table 1).


Figure 1
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  		Fig. 1 Representative Ca2+ transients obtained from an adult ventricular myocyte and a 12-day fetal ventricular myocyte. (A) x–y scan images in resting state from a 12-day fetal cell (left) and an adult cell (right). (B) The line scan images illustrate a spontaneous Ca2+ transient in a 12-day fetal cell (top) and a field-stimulation evoked-Ca2+ transient in an adult cell (lower). Arrows indicate the cell centre (red) and the subsarcolemmal region (blue). (C) Changes in fluorescence at the centre (red) and in the subsarcolemmal region (blue) of a 12-day fetal cell (top) and an adult cell (bottom) were calculated as [Ca2+]i and plotted as a function of time. (D) Effects of caffeine (10 mM) on Ca2+ transients of 12-day fetal ventricular myocytes. The time course of line scan images is shown in the top panel. Change in fluorescence was calculated as [Ca2+]i and plotted as a function of time (bottom). Representative data are shown from experiments in 12-day fetal ventricular myocytes (n = 3).

 

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  		Table 1 Characteristics of Ca2+ transients in fetal and adult ventricular myocytes

 
We investigated the effects of Co2+ (2 mM CoCl2) or nifedipine (100 nM) on the spontaneous Ca2+ transients in 12-day fetal myocytes. After exposure to either Co2+ (n = 3) or nifedipine (n = 3), the fetal Ca2+ transients disappeared completely (data not shown). These findings indicate the importance of Ca2+ influx through L-type Ca2+ channels for 12-day fetal Ca2+ transients.

3.2 Contribution of SR function to fetal Ca2+ transients
To test for the existence of functional Ca2+ stores, 12-day fetal ventricular myocytes (n = 3) were superfused with caffeine (10 mM), which is capable of emptying Ca2+ stores. Diastolic [Ca2+]i increased uniformly across the cell immediately, and reached 2.5-fold the control level of [Ca2+]i at 20 s after the application of caffeine (Fig. 1D). Ca2+ stores (i.e., SR) thus appeared to exist in the 12-day fetal myocytes. The magnitude of Ca2+ transients gradually declined 20 s after the application due to depletion of SR Ca2+ content.

To assess the contribution of Ca2+ stores to fetal Ca2+ transients, we compared Ca2+ transients in the absence and presence of ryanodine or thapsigargin. In accordance with a previous report [26], we confirmed that ryanodine (1 µM) decreased the magnitude and rate of rise of Ca2+ transients in adult myocytes (Table 2). In 12-day fetal myocytes, the magnitude of Ca2+ transients was also decreased by ryanodine (Fig. 2A,B), accompanied by a significant decrease in the maximum rate of rise (Table 2). Representative records of the Ca2+ transient (Fig. 2C) illustrated that their magnitudes were decreased by ryanodine in both the subsarcolemmal region and the centre of the cell. After exposure to 1 µM thapsigargin, the magnitude of fetal Ca2+ transients was also decreased (Table 3, Fig. 2D–F), and the time constant of decay was significantly prolonged in both regions (Table 3). As shown in Fig. 2D, diastolic [Ca2+]i transiently increased after thapsigargin application, probably due to inhibition of SR Ca2+ uptake by thapsigargin. Similar results were observed in the adult ventricular myocytes (Table 3) and were consistent with those of a previous study [26]. These results indicate that the main source of fetal Ca2+ transients is Ca2+ released from SR through RyR.


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  		Table 2 Effects of ryanodine (1 µM) on Ca2+ transients in fetal and adult ventricular myocytes

 

Figure 2
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  		Fig. 2 Effects of ryanodine and thapsigargin on Ca2+ transients recorded from 12-day fetal ventricular myocytes. (A) Time course of change in [Ca2+]i after application of 1 µM ryanodine recorded from a 12-day fetal cell. (B) Line scan images of a Ca2+ transient in a 12-day fetal cell under control conditions (top) and after exposure to 1 µM ryanodine (bottom). Arrows indicate the cell centre (red) and the subsarcolemmal region (blue). (C) Changes in [Ca2+]i before and after the application of 1 µM ryanodine in the subsarcolemmal region (left) and at the centre (right) of a 12-day fetal cell. (D) Time course of change in [Ca2+]i after application of 1 µM thapsigargin recorded from a 12-day fetal cell. (E) Line scan images of a Ca2+ transient in a 12-day fetal cell under control conditions (top) and after exposure to 1 µM thapsigargin (bottom). Arrows indicate the cell centre (red) and the subsarcolemmal region (blue). (F) Changes in [Ca2+]i before and after application of 1 µM thapsigargin in the subsarcolemmal region (left) and at the centre (right) of a 12-day fetal cell.

 

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  		Table 3 Effect of thapsigargin (1 µM) on Ca2+ transients in fetal and adult ventricular myocytes

 
3.3 Ca2+ transients in fetal heart in the very early period
The above findings indicate that SR of rat ventricular myocytes is already functional as early as fetal day 12. Subsequently, we investigated cardiac Ca2+ transients of fluo-3-loaded fetuses earlier than fetal day 12. Using a cooled CCD camera system, spontaneously evoked Ca2+ transients were detected in the cardiac area of the 10-day fetus. In most fetuses at this age, the heart is tubular in shape and the ventricular portion is roughly distinguishable from the atrial portion (Fig. 3A). The heart in some of them was not yet tubular in shape, as shown in Fig. 3B. Nevertheless, significant Ca2+ transients were observed in the cardiac area (Fig. 3C). The Ca2+ transients were initiated inferior to this area and propagated toward posteriorly, as shown in Fig. 3C. In 9-day old fetuses, the heart has not differentiated morphologically and no significant changes in fluorescence were detected (results not shown).


Figure 3
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  		Fig. 3 Ca2+ transients recorded from whole heart of a 10-day fetus. (A) Confocal x–y scan image from whole heart of a 10-day fetus. In the heart of this fetus, the ventricular portion was roughly distinguishable from the atrial portion (A, atrium; V, ventricle; Bc, bulbus cordis). The white vertical line on the image indicates the position of subsequent line scanning. Scale bar: 50 µm. (B) Fluorescence image obtained by cooled CCD camera system from whole body of a 10-day fetus. The heart area is a projection seen at left. The heart in this fetus had not yet become tubular in shape. Scale bar: 0.5 mm. (C) The changes in [Ca2+]i obtained from the pacemaker area (red circle in panel B) and the follower area (blue circle in panel B) of the 10-day fetal heart are illustrated as red and blue lines, respectively. (D) The changes in [Ca2+]i observed on line scanning at the position shown in panel A are plotted for control conditions (top) and after exposure to 1 µM ryanodine (bottom). Because of the small size of 10-day fetal cardiac myocytes, averaged fluorescence intensity across the entire width of the cells was analyzed. (E) Percentage changes in peak [Ca2+]i and rate of rise of Ca2+ transients after exposure to ryanodine in 10-day fetal, 12-day fetal and adult ventricular myocytes. The averaged fluorescence intensity across the entire width of the cells was used for calculation of [Ca2+]i. The values for the 10-day fetal (f10d) cells were obtained from whole-heart preparations, and the values for 12-day fetal (f12d) and adult cells were obtained from single-cell preparations. *P<0.05 versus f12d; {dagger}P<0.05 versus adult.

 
We then positioned the scan line close to the edge of the heart wall in the 10-day fetuses (Fig. 3A) to perform confocal line scanning. As shown in the representative traces (Fig. 3D), maximum systolic [Ca2+]i was significantly decreased after 1 µM ryanodine application (164±2 nM) compared to control (205±9 nM) in 14 hearts of 10-day fetuses. This was accompanied by a significant decrease in the maximum rate of rise (4.9±0.4 F/F0 per second in control, 1.7±0.3 F/F0 per second in the presence of ryanodine). However, percentage changes in peak [Ca2+]i and the maximum rate of rise were significantly smaller than those observed in the 12-day fetal or adult ventricular myocytes (Fig. 3E). These results indicate that SR function plays a role in Ca2+ transients by rat fetal day 10 at the latest and develops rapidly by rat fetal day 12.

3.4 Ca2+ sparks in fetal ventricular cells
Since our results showed that fetal Ca2+ transients depended on Ca2+ release from SR, we investigated Ca2+ sparks, an elemental form of Ca2+ release [14,27], in fetal ventricular myocytes. Only two Ca2+ sparks (shown in Fig. 4B,C) were detected in 20 myocytes from 12-day fetuses examined with 5 mM Ca2+ and 1 µM BayK8644. Therefore, we extended our investigation to the postnatal period. Ca2+ sparks were only minimally detected in the myocytes from newborn and 3-day old cells. Ignition of Ca2+ sparks constantly appeared in 6-day-old cells, and was observed predominantly in proximity to the sarcolemmal membrane (Fig. 4B). Frequent ignitions of Ca2+ sparks in the cell centre were observed in neonatal myocytes older than 12 days (Fig. 4B). The Ca2+ spark frequency (µm–1 s–1) in each period is summarized in Fig. 4D. The developmental change in percentage of sparks located in the subsarcolemmal region, which was defined as the area within 2 µm from the edge of the entire cell image, is shown in Fig. 4E. We emphasize that the sparks initially appear in close proximity to the surface membrane and become prominent throughout the cell during subsequent development.


Figure 4
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  		Fig. 4 Developmental changes in ignition of Ca2+ sparks. (A) x–y scan images in resting state recorded from a 12-day fetal ventricular myocyte (f12d), a 6-day neonatal ventricular myocyte (n6d), a 12-day neonatal ventricular myocyte (n12d) and an adult ventricular myocyte in modified Tyrode solution containing 5 mM Ca2+ and 1 µM BayK8644. The red arrow indicates a peripheral Ca2+ spark in a 6-day neonatal cell, and the white arrows indicate Ca2+ sparks in the cell centre of a 12-day neonatal cell and an adult cell. Scale bar: 10 µm. (B) Line scan images recorded from the cells shown in (A). (C) A fluorescence at the lines indicated by the arrows in (B) are plotted as a function of time. The only two Ca2+ sparks detected from the 20 fetal myocytes examined are shown at the top. (D) Ca2+ spark frequency (events/µm/s) in ventricular myocytes from 12- and 18-day fetuses (f12–18; two events from 20 cells), 6–9-day neonates (n6–9; 112 events from 23 cells), 12–14-day neonates (n12–14; 66 events from 12 cells) and adults (2946 events from 20 cells), respectively. (*P<0.05 versus adult. (E) Developmental changes in percentage of Ca2+ sparks in the subsarcolemmal region in ventricular myocytes in each developmental period. The subsarcolemmal region was defined as the area within 2 µm from the edge of the entire cell image. *P<0.05 versus adult. (F) Developmental changes in characteristics of Ca2+ sparks. Mean values of peak F/F0 (normalized fluorescence), FWHM (full-width at half-maximal fluorescence intensity) and FTHM (full-time at half-maximal fluorescence intensity) in each developmental period are summarized. *P<0.05 versus adult.

 
Developmental changes in the characteristics of Ca2+ sparks are summarized in Fig. 4F. There were no significant differences in FWHM among the periods. On the other hand, the mean values of peak F/F0 were significantly smaller and the mean values of FTHM significantly longer in the neonatal than in the adult cells.

3.5 Developmental changes in t-tubule images
Since the location of Ca2+ sparks has been reported to coincide with that of t-tubules in mature ventricular rat myocytes [27], we performed t-tubule labeling with di-8-ANEPPS, as shown in Fig. 5. The regularly spaced and uniform staining in adult myocytes was indicative of a well-developed t-tubule network. In contrast, in the fetal myocytes staining was apparent only in the surface membrane area, indicating lack of t-tubules. t-Tubule images were observed first at neonatal days 6–9 only in the subsarcolemmal region as short regions of staining, but not in the cell interior. In the myocytes from 12- to 14-day neonatal rats, t-tubules extended to the cell interior although staining was still irregular.


Figure 5
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  		Fig. 5 Confocal imaging of t-tubules from membrane-selective fluorescent dye di-8-ANEPPS-loaded ventricular myocytes. The t-tubule images were obtained with x–y scan mode from a 12-day fetal myocyte (f12d), an 18-day fetal myocyte (f18d), a 7-day neonatal myocyte (n7d), a 14-day neonatal myocyte (n14d), an 18-day neonatal myocyte (n18d) and an adult myocyte. Scale bars: f12d, 5 µm; f18d, 10 µm; n7d, 10 µm; n14d, 5 µm; n18d, 10 µm; adult, 20 µm.

 
3.6 Developmental changes in colocalization of DHPR and RyR
The confocal images in Fig. 6 illustrate the subcellular location of the {alpha}1c subunit of L-type Ca2+ channels (DHPR; dihydropyridine receptors) and RyR-2 proteins at four stages of rat cardiomyocytes development. In 12-day fetal myocytes, uniform RyR-2 (FITC, green) labeling was observed. The DHPR labeling (Texas Red) inside the cells probably reflects {alpha}1c subunits being delivered to the subsarcolemma. In 7-day neonatal myocytes, colocalization of DHPR and RyR-2 (yellow) was predominant in the periphery of cells. In 14-day neonatal myocytes, discrete spots associated with RyR-2 were observed inside of cells and were spaced at regular intervals, although t-tubule staining was irregular at that time (see Fig. 6). DHPR labeling was still predominant in the periphery, but could be seen in the cell interior. Colocalization with RyR thus appeared in the cell interior. Therefore, there was a temporal correlation among three events (i.e., development of the t-tubule network, colocalization of DHPR and RyR-2, and frequent observation of Ca2+ sparks) in the cell interior. In 18-day neonatal myocytes, the distributions of both DHPR and RyR were regularly spaced and by this age the t-tubule network had almost completely developed (see Fig. 6).


Figure 6
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  		Fig. 6 Confocal imaging of Texas Red labeling with antibodies against the {alpha}1c subunit of L-type Ca2+ channel (left panels, red), FITC labeling with antibodies against the RyR2 (middle panels, green), and the overlay images indicating colocalization of the two receptors (right panels, yellow) in ventricular myocytes isolated from a 12-day fetus (f12d), 7-day neonate (n7d), 14-day neonate (n14d) and 18-day neonate (n18d). Scale bars: f12d, 5 µm; n7d, 10 µm; n14d, 5 µm; n18d, 10 µm.

 
3.7 Disappearance of Ca2+ sparks after disruption of t-tubules
As shown in Fig. 7A, we performed t-tubule labeling with di-8-ANEPPS in adult ventricular myocytes incubated with or without glycerol (840 mM) for 1 h. In the glycerol-treated cells, disappearance of the regularly spaced staining of the cell interior indicated disruption of the t-tubule network (Fig. 7A, right). Line-scan images from fluo-3-loaded cells confirmed that caffeine elicited release of Ca2+ from SR stores after glycerol treatment (Fig. 7B). However, Ca2+ sparks were not detected in 20 glycerol-treated cells examined with 5 mM Ca2+ and 1 µM BayK8644 (Fig. 7C).


Figure 7
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  		Fig. 7 Effects of disruption of t-tubules with glycerol treatment on Ca2+ sparks in adult ventricular myocytes. (A) Confocal imaging of t-tubules from di-8-ANEPPS-loaded ventricular myocytes incubated with control solution (left) and glycerol solution (right). (B) Effect of caffeine (10 mM) on [Ca2+]i in glycerol-treated myocytes without stimulation. The time course of line scan images is shown in the top panel. Change in fluorescence was calculated as [Ca2+]i and plotted as a function of time (bottom). (C) Line-scan images of fluo-3-loaded control myocytes (left) and glycerol treated myocytes (right), with the time course of Ca2+ sparks indicated below, determined over the line indicated by the arrows.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Method
 3 Results
 4 Discussion
References
 
This is the first study to reveal the spatio-temporal dynamics of [Ca2+]i in single ventricular myocytes freshly isolated from rat fetuses. The major findings of the present study can be summarized as follows. The heart tube in the early fetal period already exhibited Ca2+ transients. The Ca2+ transients of the most primitive ventricular cells are characterized by markedly slower rise and decay than those of mature myocytes. The fetal Ca2+ transients depend not only on Ca2+ influx through L-type Ca2+ channels but also on Ca2+ release through RyR of SR. However, ignition of Ca2+ sparks is only minimally observed in the fetal period. Ignition of Ca2+ sparks requires postnatal t-tubule formation and resultant colocalization of L-type Ca2+ channels and ryanodine receptors.

4.1 Role of SR function in fetal Ca2+ transients
Previous studies have reported that ryanodine has little effect on the contraction of fetal and neonatal ventricular cells [3,7–9,28]. However, we observed significant inhibition of fetal Ca2+ transients by ryanodine and thapsigargin. The inhibitory effect of ryanodine was more prominent on 12-day fetal Ca2+ transients than on 10-day fetal ones. This finding suggests that SR function contributes to Ca2+ transients by the time the heart tube forms, and then develops rapidly by the time the ventricular portion becomes distinguishable from the atrial portion of the heart.

Although we clearly demonstrated the possibility of involvement of SR function in fetal Ca2+ transients, two studies that investigated the Ca2+ transients in immature heart cells obtained results conflicting with ours. First, Takeshima et al. [29] showed that Ca2+ transients in the early fetal period were still present in the hearts of RyR-2 knockout mice and ryanodine-treated wild-type mice. However, the amplitude of Ca2+ transients was slightly decreased with SR inhibition. Therefore, it is impossible to rule out the possibility that SR function may develop dramatically just after the fetal period in mouse myocytes. Since the RyR-2 knockout mice died early in the fetal period, this possibility could not be tested completely. Second, Haddock et al. [15] reported that the Ca2+ transients in cardiomyocytes from newborn rabbits were affected by thapsigargin application neither at cell centre nor in the periphery. Since the rabbit ventricle has a higher threshold of [Ca2+] for calcium-induced calcium release (CICR) than the rat ventricle [2], developmental changes in Ca2+ signaling in rabbit heart may differ from those in rat heart.

4.2 Mechanism of propagation of Ca2+ to the cell interior
In cells which lack t-tubules, such as Purkinje cells [30,31] and atrial cells [32], Ca2+ transients are initiated by Ca2+ influx to the peripheral area. However, the mechanism of subsequent propagation of Ca2+ to the interior is still unclear. For example, in rabbit Purkinje cells [31], decrease in Ca2+ diffusion from peripheral SR could explain the depressant effect of ryanodine on central Ca2+ transients. On the other hand, in canine Purkinje cells [30] and cat atrial cells [32], Ca2+ could propagate via the CICR mechanism toward the centre of the cell. It is still unknown whether Ca2+ release from peripheral SR can subsequently induce further Ca2+ release from stores in more central regions in rat fetal ventricular myocytes. However, the finding of caffeine-induced elevation in [Ca2+]i across the cell indicated that functional SR exists in the cell interior. Our immunolabeling findings also indicated that RyR are present in the interior of fetal myocytes. Furthermore, the RyR-2 gene is detectable in rat cardiomyocytes in the early fetal stage [10]. Therefore, the possibility of Ca2+ propagation via the CICR mechanism cannot be excluded. In any case, our findings indicate that the rat fetal Ca2+ transient requires SR Ca2+ release, at least in the subsarcolemmal region.

4.3 Role of Ca2+ influx through L-type Ca2+ channels in fetal Ca2+ transients
In early-stage embryonic stem cell-derived cardiomyocytes, spontaneous Ca2+ oscillation is not generated by transmembrane ion currents, but by intracellularly stored Ca2+ [13]. However, in 12-day fetal ventricular myocytes, spontaneous Ca2+ transients disappeared completely after nifedipine application in the present study. Therefore, Ca2+ influx through the L-type Ca2+ channels may be required for fetal Ca2+ transients. In fact, L-type Ca2+ current has already been observed in the early fetal period [33]. The spontaneous beating of 12-day cells may be due to the spontaneous firing of action potentials that we observed previously [16]. This pacemaker potential had disappeared in the 18-day fetal ventricular myocytes [16]. In addition, the 18-day fetal ventricular myocytes were quiescent during measurement of Ca2+ fluorescence.

It is still controversial whether the Ca2+ influx through L-type Ca2+ channels triggers Ca2+ release from SR in fetal myocytes. A short distance between the sarcolemmal Ca2+ channels and SR Ca2+ release channels is essential for efficient Ca2+ influx to release Ca2+ from SR. Löhn et al. [19] reported that neonatal rat cardiomyocytes lack the t-tubule system but that in them infoldings of the surface membrane (i.e., caveolae) are abundant. The short distance between the caveolae and SR (20–50 nm) could be responsible for the proximity of the Ca2+ sparks in neonatal myocytes. Takeshima et al. [34] observed two types of junctional membrane complexes with gap sizes of ~12 and ~30 nm in the cardiomyocytes of mice at fetal day 9.5. Because the junctional gap in mature cardiomyocytes of mice is always ~12 nm, fetal cardiomyocytes might also possess functional peripheral couplings, as do adult cells. This supports the present finding that Ca2+ influx was capable of triggering Ca2+ release from peripheral coupling SR in rat fetal myocytes.

4.4 Lack of Ca2+ sparks in fetal myocytes
Although the number of RyR underlying the Ca2+ sparks is disputed, the Ca2+ sparks detectable by confocal imaging are postulated to arise from a cluster of multiple RyRs [35]. In the present study, the width of the Ca2+ sparks observed in neonatal myocytes did not differ from that in adult myocytes. This finding suggests that a number of RyR had already clustered before t-tubule formation or may cluster rapidly as soon as t-tubule formation has occurred. We speculate that subsequent participation of additional RyR in clusters and increase in Ca2+-ATPase activity after birth [11] may be responsible for the postnatal increase in amplitude and decrease in duration of Ca2+ sparks, respectively. On the other hand, the observed changes in characteristics of postnatal Ca2+ sparks imply that the amount of Ca2+ released was not altered. It thus seems likely that cooperation between RyRs in clusters was improved during the postnatal period.

Close correlation was found among the development of Ca2+ sparks, t-tubule formation and colocalization of DHPR and RyR. This suggests that t-tubule formation may lead to colocalization of DHPR and RyR, resulting in ignition of Ca2+ sparks. Therefore, we used a hyperosmolar solution containing glycerol to disrupt the t-tubules of adult myocytes, and the finding of disappearance of Ca2+ sparks after this disruption strongly supports this hypothesis. Recently, Takeshima et al. [34] identified important protein of the junctional membrane complex, termed junctophilin-2. It is essential for Ca2+ signaling between sarcolemmal Ca2+ channels and RyR of SR, since cardiomyocytes from junctophilin-2 knockout fetal mice exhibited deficiency of morphological peripheral coupling. Although junctophilin-2 appears already to play an important role in functional peripheral coupling in fetuses, we detected almost no significant Ca2+ sparks in fetal myocytes even at the periphery of cells. Lipp and Niggli [36] proposed a fundamental Ca2+ release event from a limited number of RyR (possibly one), i.e., a Ca2+ quark, which is not detectable with presently available method of confocal microscopy. Therefore, individual RyR may have not yet clustered and may exist diffusely in the cytoplasm of rat fetal cardiomyocytes.

In summary, our findings show that the release of Ca2+ from SR contributes to Ca2+ transients in fetal cardiomyocytes more prominently than has been believed previously. Although SR already functions in the fetal period, ignition of Ca2+ sparks requires postnatal development of t-tubules and colocalization of DHPR and RyR, which complete spatially uniform Ca2+ transients.

Time for primary review 38 days


   References
Top
 Abstract
 1 Introduction
 2 Method
 3 Results
 4 Discussion
 References
 

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