Acta Pharmacologica Sinica

Full-length article

Acta Pharmacologica Sinica 2006 July; 27 (7): 901-910

Developmental regulation of intracellular calcium transients during cardiomyocyte differentiation of mouse embryonic stem cells¹

Ji-dong Fu, Hui-mei Yu, Rong Wang, Ji Liang, Huang-tian Yang²

Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

¹Project supported in part by grants of the National Natural Science Foundation of China(No 30270656), the National Natural Science Foundation of China and The Hong Kong Research Grants Council(NSFC-RGC,30518003) and Programs (03DJ14019) from Science and Technology Committee of Shanghai Municipality.
²Correspondence to Dr Huang-tian YANG.
Phn/Fax 86-21-6385-2593.
E-mail htyang@sibs.ac.cn
Received 2006-04-15
Accepted 2006-05-08

Abstract

Aim: To investigate the developmental regulation of intracellular Ca²⁺ transients, an essential event in excitation-contraction coupling, during cardiomyocyte differentiation. Methods: Using the embryonic stem (ES) cell in vitro differentiation system and pharmacological intervention, we investigated the molecular and functional regulation of Ca²⁺ handling proteins on the Ca²⁺ transients at early, intermediate and later differentiation stages of ES cell-derived cardiomyocytes (ESCM). Results: Nifedipine, a selective antagonist of L-type Ca²⁺ channels, totally blocked Ca²⁺ transients even in the condition of field-electric stimulation in ESCM at three differentiation stages. The Ca²⁺ transients of ESCM were also inhibited by both ryanodine [an inhibitor of ryanodine receptors (RyRs)] and 2-aminoethoxydipheylborate [2-APB, an inhibitor of inositol-1,4,5-trisphosphate receptors (IP₃Rs)]. The inhibitory effect of ryanodine increased with the time of differentiation, while the effect of 2-APB decreased with the differentiation. Thapsigargin, an inhibitor of SR Ca²⁺-pump ATPase, inhibited Ca²⁺ transients equally at three differentiation stages that matched the expression profile. Na⁺ free solution, which inhibits Na⁺-Ca²⁺ exchanger (NCX) to extrude Ca²⁺ from cytosol, did not affect the amplitude of Ca²⁺ transients of ESCM until the latter differentiation stage, but it significantly enhanced the basal Ca²⁺concentration. Conclusion: The Ca²⁺ transients in ESCM depend on both the sarcolemmal Ca²⁺ entry via L-type Ca²⁺ channels and the SR Ca²⁺ release from RyRs and IP₃Rs even at the early differentiation stage; but NCX seems not to regulate the peak of Ca²⁺ transients until the latter differentiation stage.

Key words

Ca²⁺ transients; cardiac differentiation; embryonic stem cells; Ca²⁺ handing proteins

Extract

Note: Please read the complete full text with Figures and Tables at
Introduction
Intracellular Ca²⁺ signaling regulates a wide variety of cellular functions and organ development^[1_4]. Intracellular Ca²⁺ transients, the cyclic variations in the concentration of cytosolic Ca²⁺ ([Ca²⁺]_i), play a crucial role in the contraction and relaxation of cardiomyocytes. The Ca²⁺ transients are the result of a spatio-temporal balance between cytosolic Ca²⁺ elevation and Ca²⁺ re-uptake by sarcoplasmic reticulum (SR) or cell extrusion. It arises via Ca²⁺-induced Ca²⁺ release (CICR) mechanism in adult cardiomyocytes, where a relatively small Ca²⁺ influx through sarcolemmal L-type Ca²⁺ channels triggers greater amounts of SR Ca²⁺ release from type-2 ryanodine receptor (RyR2). This is the base of cardiac excitation-contraction (E-C) coupling^[5,6]. Upon the recycling of a majority of cytosolic Ca²⁺ back to the SR by Ca²⁺-pump ATPase (SERCA2) and a small portion of cytosolic Ca²⁺ out of the sarcolemma by Na⁺-Ca²⁺ exchanger (NCX), a decrease of [Ca²⁺]_i occurs, leading to myocardial relaxation. Thus, SR plays a central role in the regulation of the contractile force of adult cardiac myocytes by modulating the amplitude and the rise or decay velocity of the Ca²⁺ transients. However, because of the known difficulties in obtaining cardiomyo-cytes from the very early mammalian embryos (eg, before d 12 to 13 of gestation in mice), there is only limited knowledge on the developmental aspects and the regulation of Ca²⁺ transients.
The heart is the first organ that becomes functional in the vertebrate embryo. On approximately embryonic day (E) 7.25 in mice, the precardiac mesoderm forms a primitive tubular heart that starts beating at E8^[7]. The heart is continuously remodeled until the four-chambered organ is formed, and maintains its physiologic pumping function in response to increasing circulatory demands^[7]. The ensuing development of E-C coupling is fundamental to the embryonic cardiac function during embryogenesis. In the embryonic heart the mRNA and protein abundance of the main Ca²⁺ handing proteins, such as RyR2, SERCA2, phospholamban (PLB), and NCX1, however, is different from those in neonatal and adult hearts^[8,9], suggesting that the regulation of Ca²⁺ transients in embryonic cardiomyocytes may be different from that in adult cardiac myocytes.
The embryonic stem (ES) cell-derived cardiomyocytes (ESCM) represent specialized cell types of the heart, such as atrial-like, ventricular-like, sinus nodal-like, and Purkinje-like cells^[10]. Published ultrastructural^[11], molecular biological^[12] and electrophysiological^[10,13] studies have demonstrated that within the ES cell-formed embryoid bodies (EB), the various stages of cardiomyocytes closely recapitulate the developmental pattern of murine early cardiogenesis. Therefore, the ES cell in vitro differentiation system can be used to investigate early cardiogenesis^{[10_12, 14_16]}. ESCM are also one possible source of transplantable cells. It is a therapeutic prerequisite to investigate the regulation of Ca²⁺ transients, one of the critical functional properties of potential replacement cells. Recently, we observed that RyR2-mediated SR Ca²⁺ release directly contributed to the spontaneous and b-adrenergic receptor-stimulated Ca²⁺ transients and contraction of ESCM even at very immature stages of development^[17]. However, the importance of sarcolemmal Ca²⁺ handing proteins, such as L-type Ca²⁺ channels and NCX, and SR Ca²⁺ release channels inositol triphosphate receptors (IP₃Rs) on Ca²⁺ transients of ESCM have not yet been fully clarified.
Therefore, in the present study, we investigated the developmental regulation of the main Ca²⁺ handling proteins on the Ca²⁺ transients in ESCM during cardiogenesis. Our results demonstrate that both sarcolemmal Ca²⁺ entry and SR Ca²⁺ release contribute to the Ca²⁺ transients even at the early differentiation stage, while NCX plays more crucial roles in maintaining normal basic Ca²⁺ concentration during whole ESCM differentiation and only regulates peak Ca²⁺ transients at the latter differentiation stage.

Materials and methods
Cell culture, differentiation and isolation of beating cardiomyocytes R1 ES cell lines were cultivated and differentiated into spontaneously beating cardiomyocytes as described in a previous study^[16]. Undifferentiated ES cells were cultivated on mitomycin C-inactivated mouse feeder layers in the presence of leukemia inhibitory factor. The differentiation of ES cells into cardiac cells was initiated by a hanging drop technique to form embryoid bodies (EB). After 7 d in suspension, EB were plated onto gelatin-coated tissue culture dishes. Cardiomyocytes appeared in the form of spontaneously contracting cell clusters, and single cardiomyocytes were isolated at three distinct differentiation stages [early (EDS, 7+2_4 d); intermediate (IDS, 7+6_8 d), and late differentiation stages (LDS, 7+11_14 d)] by enzymatic dissociation with collagenase followed by plating on laminin/gelatin-coated glass coverslips^[16]. All cultivation medium and other substances for cell cultures were purchased from Gibco BRL (Grand Island, NY, USA).
Detection of gene transcripts ES cells, EB and adult mouse hearts were used to isolate total RNA^[16]. In brief, 0.5 mg total RNA from each tissue was converted to cDNA by using Superscript II reverse transcriptase (Life Tech, MD) and oligodT (T16, 500 ng) in a final volume of 20 µL, according to the manufacturer’s instructions, and 0.4 µL of this was used for each PCR reaction. Semi-quantitative reverse transcriptase polymerase chain reactions (RT-PCR) were carried out with Tth DNA polymerase (Promega, Madison, WI, USA)) and DNA amplifications were carried out according to the manufacturer’s instructions. Reactions were carried out in a Mastercycler gradient (Eppendorf, Hamburg, Germany) under the following conditions. PCR amplification involved 5 min at 95 ^oC followed by 30_35 cycles of 45 s at 95 ^oC, 45 s at the appropriate annealing temperature and 45 s at 72 ^oC for elongation ending with 5 min at 72 ^oC for final PCR product extension. DNA was visualized on a 1% agarose gel containing ethidium bromide. The primers of L-type Ca²⁺ channel (L-type channel, forward: 5’-GTTCCTGAAGGA-GGTGTGCTGGACG-3’, reverse: 5’-AAAGGCAG TTCCCA-TGCCGG-3’), cardiac Na⁺/Ca²⁺ exchanger (NCX1, forward: 5’-CAGCTTCCAAAACTGAAATCGA-3’, reverse: 5’-GTCCC-TCTCATCGACTTC CAAAA-3’), RyR2 (forward: 5’-GACGG-CAGAAGCCACTCACCTGCG-3’, reverse: 5’-CCTGCAGAG-AAACTGACAACTGG-3’), type 2 IP₃R (IP₃R2, forward: 5’-GGCTCGGTCAATGGCTTC-3’, reverse: 5’-CCCCTGTTTCG-CCTGCTT-3’), SERCA2a (forward: 5’-TGTGTGATGTGGA-GGAAATGTGTA-3’, reverse: 5’-TACAACTGAAGGCATG-CATTACAA-3’), and house-keeping gene b-tubulin (forward: 5’-GGAACATAGCCGTAAA-CTGC-3’, reverse: 5’-TCACTG-TGCCT GAACTTACC-3’) were used in RNA samples.
Measurement of Ca²⁺ transients Isolated ESCM were loaded with 5 µmol/L Indo-1AM and 0.45% pluronic F-127 (Molecular Probes, Eugene, Oregon, USA) for 10 min at room temperature^[17,18]. Loaded cells were washed with a solution containing 140 mmol/L NaCl, 5.4 mmol/L KCl, 1.8 mmol/L CaCl₂, 1.0 mmol/L MgCl₂, 5.0 mmol/L NaHCO₃, 10.0 mmol/L glucose and 10 mmol/L HEPES (pH 7.4 at 35 ºC). Fluorescence signals of Indo-1 were detected by a Fluorescence/Contractility System (IonOptix, Milton, MA, USA). Fluorescence signals were excited at 360±5 nm with an ultraviolet light source, and the emitted fluorescence was measured at 405 and 480 nm using two photomultipliers attached to an inverted microscope (Olympus, Tokyo, Japan). After subtraction of background fluorescence, the ratio of fluorescence (R) emitted at 405 and 480 nm was recorded^[19] and analyzed by IonWizard 4.4 software (IonOptix).
Sodium free solutions were produced by equimolar replacement of Na⁺ by Li⁺. 2-Aminoethoxydipheylborate (2-APB), thapsigargin (Calbiochem, Darmstadt, Germany), nifedipine and, ryanodine (Sigma, St Louis, MO, USA) were used in our experiments.
Statistics Data are expressed as mean±SEM. Statistical significance of differences in means was estimated by one- way ANOVA, by Student’s t-test or a paired t-test, where appropriate (StatSoft, Version 5.1, StatSoft, Tulsa, OK, USA). P<0.05 was considered significant.

Results
Expressions of genes coding main Ca²⁺ handling proteins during cardiac differentiation of ES cells ES cells formed spontaneously contracting cardiomyocytes that were visible 1 d after EB plating during in vitro differentiation. The number of EB with spontaneous contracting cardio-myocytes increased significantly and reached maximum on d 5 after plating as was observed in a previous study^[17]. Concomitant with the differentiation of ESCM, transcripts of sarcolemmal L-type Ca²⁺ channels and NCX1 increased with the cardiac development; transcripts of SR Ca²⁺release-related proteins IP₃R2 was expressed very early and increased in early differentiation stages but not in the latter one. RyR2 was present prior to the occurrence of spontaneous beating activity and increased in abundance from early to late differentiation stages. SERCA2a was also present prior to initial contractions but had no obvious changes during ESCM differentiation (Figure 1). Therefore, the main Ca²⁺ handling proteins were already expressed, even at the early differentiation stage.
Contribution of L-type Ca²⁺ channels to Ca²⁺ transients during cardiomyocyte differentiation L-type Ca²⁺ channels are thought to be the main transporter for trans-sarcolemmal Ca²⁺ influx in adult cardiomyocytes^[20], and play an important role in E-C coupling in early embryonic cardiomyocytes^[21,22]. We therefore used an L-type Ca²⁺ channel selective anta-gonist, nifedipine, to identify the role of L-type Ca²⁺ channels in the regulation of Ca²⁺ transients during cardiomyocyte differentiation. When L-type Ca²⁺ channels were blocked by 3 µmol/L of nifedipine, spontaneous and field-electric stimulated Ca²⁺ transients were totally inhibited in ESCM at three differentiation stages. In order to evaluate the importance of L-type Ca²⁺ channels in the regulation of ESCM Ca²⁺ transients at different differentiation stages, we selected a lower concentration of nifedipine. Spontaneous Ca²⁺ transients in the ESD (n=17) and IDS (n=12) ESCM were completely inhibited by nifedipine at 1 µmol/L (Figure 2A), but were only partially inhibited in some of the LDS ESCM (8 out of 30 cells). Under field-electric stimulation, the Ca²⁺ transients in 24% EDS (n=17) and 8% IDS ESCM (n=12) were totally inhibited by nifedipine at 1 µmol/L and others were partially inhibited. But Ca²⁺ transients in all LDS ESCM examined were only partially inhibited (n=11, Figure 2A and 2B). We then analyzed the inhibitory degree of nifedipine on the cells showing partially inhibited Ca²⁺ transients. The inhibitory effect of nifedipine in EDS and IDS ESCM was obviously stronger than that in LDS cells (P <0.05, Figure 2C). These results indicate that the Ca²⁺ influx through the L-type Ca²⁺ channel is essential for Ca²⁺ transients of ESCM at the three differentiation stages, but it is more dominant in the EDS and IDS.
Contribution of SR to Ca²⁺ transients during cardiomyo-cyte differentiation The contraction in adult cardiomyocytes is highly dependent on the SR Ca²⁺release from RyR2. To identify whether RyR2 contributes to the upstroke of Ca²⁺ transients during cardiac differentiation, ESCM were treated with ryanodine (10 µmol/L, 30 min) to inhibit RyRs. Ryanodine significantly decreased the amplitude and reduced the upstroke velocity (dP/dT) of ESCM Ca²⁺ transients at the three differentiation IDS stages (Figure 3). The inhibitory effects of ryanodine were larger in the LDS ESCM than in the EDS and IDS cells (Figure 3B, 3C). These results indicate that the SR Ca²⁺ release from RyR2 is one of the Ca²⁺ sources of Ca²⁺ transients even in early differentiating ESCM, and the role of RyR2 tends to increase with the development.
Besides RyRs, there is an IP₃ sensitive Ca²⁺ release channel IP₃R2 on the SR membrane of adult cardiomyocytes. We then used 2-APB, an IP₃R inhibitor^[23], to investigate the role of IP₃R in the regulation of Ca²⁺ transients in the ESCM. 2-APB (20 µmol/L, 15 min) inhibited Ca²⁺ transients significantly by decreasing the amplitude and reducing the upstroke dP/dT of Ca²⁺ transients of ESCM from the EDS to LDS (Figure 4). The inhibitory effect of 2-APB was larger in EDS than in and LDS ESCM (Figure 4B, 4C). These data demonstrate that IP₃Rs also contribute to the upstroke of Ca²⁺ transients, but this effect decreases with the ESCM differen-tiation.
To maintain steady-state contraction of cardiomyocytes, the SR released Ca²⁺ should be sequestered by the SR Ca²⁺-pump ATPase. In our experiment thapsigargin, a Ca²⁺-pump ATPase inhibitor, was used to detect the role of SR Ca²⁺-pump ATPase in the regulation of Ca²⁺ transients during cardiac differentiation. Thapsigargin (0.5 µmol/L, 15 min) also significantly inhibited Ca²⁺ transients by decreasing the amplitude and the decay dP/dT of Ca²⁺ transients in differentiating ESCM, but unlike ryanodine and 2-APB, there was no obvious difference in the thapsigargin-induced changes between the EDS and LDS (Figure 5B, 5C). These results indicate that SR Ca²⁺-pump ATPase functions in the Ca²⁺ reuptake into the SR and contributes to the Ca²⁺ decay of Ca²⁺ transients at three differentiation stages examined.
Some studies suggest that 2-APB is not only an antagonist to inhibit IP₃-induced Ca²⁺ release, but is also an inhibitor of SR Ca²⁺ pump ATPase in non-excitable cells^[24]. To further confirm that the observed inhibitory effect of 2-APB is not related to its effect on SR Ca²⁺-pump ATPase function. The ESCM were treated with a combination of ryanodine with thapsigargin or 2-APB, respectively. Ryanodine-inhibited peak Ca²⁺ transients were reversed by thapsigargin, although the duration of Ca²⁺ transients was still significantly prolonged (Figure 6A). In contrast, ryanodine-inhibited Ca²⁺ transients were decreased further by 2-APB (Figure 6B). These observations confirm that the target of 2-APB is not the same as that of thapsigargin.
Contribution of NCX to Ca²⁺ transients during cardio-myocyte differentiation In the normal resting myocytes, the NCX functions in a "forward" (Na⁺ in/Ca²⁺ out) mode and serves as the main extrusion mechanism. It is clear that in adult cardiomyocytes NCX is critical in maintaining a low cytosolic [Ca²⁺]_i and that it extrudes the intracellular Ca²⁺ before the subsequent contraction^[5]. To evaluate the role of NCX in the regulation of the cardiac Ca²⁺ transients with differentiation, we used the Na⁺ free solution, which inhibits NCX to extrude Ca²⁺ from cytosol^[5]. From EDS to LDS, the basal [Ca²⁺]_i of ESCM increased dramatically when cells were treated with the Na⁺ free solution, indicating that NCX is already functional in maintaining the low cytosolic [Ca²⁺]_i even in early differentiating ESCM (Figure 7A). But Na⁺ free solution had no effect on the Ca²⁺ transients in EDS ESCM, while it completely blocked the spontaneous Ca²⁺ transients in the LDS cells (Figure 7A). To evaluate the relative role of NCX in the decay of Ca²⁺ transients, we analyzed the stimulated Ca²⁺ transients of ESCM using field-electric stimulation. The Na⁺ free solution did not inhibit the amplitude of Ca²⁺ transients of ESCM until the LDS (Figure 7A, 7B). Moreover, the decay dP/dT of Ca²⁺ transients were significantly increased in EDS cells but reduced in LDS ESCM. These results demonstrated that NCX already regulates basal [Ca²⁺]_i and the exclusion of cytosolic Ca²⁺during differentiation of ESCM, but it does not regulate the amplitude of Ca²⁺ transients until the LDS.

Discussion
In this study, we used the ES cell in vitro differentiation system to focus on the developing regulation of Ca²⁺ transients in ESCM, which until now has not yet been fully clarified. We have demonstrated that: (i) the Ca²⁺ influx through L-type Ca²⁺ channels is essential for Ca²⁺ transients in ESCM at three differentiation stages, but it is more dominating in the earlier stage; (ii) Ca²⁺ release from IP₃Rs also contributes to the amplitude and upstroke of Ca²⁺ transients, but its contribution decreases with the cardiomyocyte differentiation. This temporal change is complementary to the development of RyR2 in the regulation of Ca²⁺ transients during cardiac differentiation; (iii) NCX already functions in EDS in the regulation of basal [Ca²⁺]_i, but it does not regulate the peak Ca²⁺ transients until the LDS.
Ca²⁺ influx in the E-C coupling of the developing cardiomyocytes In adult cardiac myocytes, a relatively small Ca²⁺ influx via voltage-activated L-type Ca²⁺channels triggers greater amounts of SR Ca²⁺ release from the RyR2 by the process of CICR, which leads to a rapid and high enough increase of [Ca²⁺]_i to initiate the interaction of contractile filaments and subsequent contractions^[5]. For cardiac contraction, an extracellular Ca²⁺-influx is required because the removal of Ca²⁺ from extracellular solution abolishes cardiac contraction. This phenomenon is also observed in ESCM during differentiation (data not shown). L-type Ca²⁺ channels are thought to be the main transporter for trans-sarcolemmal Ca²⁺ influx in adult cardiomyocytes^[20]. In the early stage (3 d) of development, the L-type Ca²⁺ channels occur in embryonic chick-heart cells, and the density of L-type Ca²⁺ current in 3-d cells was higher than in 17-d cells^[25]. In 9.5 d postcoitum (dpc) mouse heart^[21] and in early-stage ESCM^[22], b-adrenergic receptor stimulation already modulates L-type Ca²⁺ channel currents. In this study, nifedipine (>2 µmol/L) blocked the Ca²⁺ transients of ESCM during differentiation. Those results demonstrate that the Ca²⁺ influx via L-type Ca²⁺ channels is required for E-C coupling in early embryonic cardiomyocytes. The decreased inhibitory effects of nifedipine with differentiation, observed in our experiment, can be partially explained by the increased density of Ca²⁺ current in mouse and rat hearts during fetal development^[26,27], but it may also indicate that the contribution of the L-type Ca²⁺ channels in the regulation of Ca²⁺ transients decreases with the differentiation upon the development of RyR2.
Importance of SR Ca²⁺ release in the E-C coupling of the developing cardiomyocytes In fetal heart cells the SR is scarce when observed under electron microscopy^[28]. Isolated SR vesicles from the fetal heart have a lower volume, lower density, and a lower capability to load Ca²⁺ compared to those isolated from the mature heart^[29_31]. Ryanodine, a specific inhibitor of RyR, has little to no effect on Ca²⁺ transients in fetal cells^[8,28]. Therefore, it was proposed that the contraction of fetal cardiomyocyte is regulated predominantly by sarcolemmal Ca²⁺ influx rather than Ca²⁺release from SR. However, RyR2 knockout fetal cardiomyocytes^[32] and ESCM^[17,18] have slow, weak, and irregular Ca²⁺ transients, which demonstrates that RyR2-released Ca²⁺ is critical for Ca²⁺ homeostasis and normal Ca²⁺ transients in early developmental cardiomyocytes. The observation of the amplitude of Ca²⁺ transients in early differentiating ESCM being inhibited by either ryanodine or thapsigargin is consistent with our recent data^[17] and with the findings from the fetal cardiomyocytes^[33].
Not like the inhibitory effect of ryanodine or thapsigargin when used alone, ryanodine-inhibited Ca²⁺ transients in ESCM could be reversed by thapsigargin. The latter is in line with the observation that the Ca²⁺ transients of the fetal cardiomyocytes at 9.5 dpc was unaffected by the combined presence of both ryanodine and thapsigargin, while the Ca²⁺ transients in the adult stage are largely inhibited by this combination^[8]. Therefore, care should be taken in interpreting the results from using a combination of thapsigargin and ryanodine in these early cardiomyocytes, although we do not know why this combination can reverse the inhibitory effect of ryanodine or thapsigargin when used alone.
IP₃Rs, another Ca²⁺ release channel on the SR membrane, played an important role in the regulation of cellular proliferation and apoptosis, whereas RyR-released Ca²⁺ is required for muscle contraction^[34]. The IP₃R mRNA expression and IP₃-induced intracellular Ca²⁺ release are detected as early as 5.5 dpc in the mouse embryo, which is earlier than the time of expression of RyR2 mRNA^[35], which is also supported by our in vitro study. In adult guinea pig ventricular myocytes, the low concentrations of IP₃ (1_10 µmol/L) transiently increases isotonic contractions, which is in accordance with the receptor-initiated SR Ca²⁺ release^[36]. Recent studies showed that IP₃-dependent shuttle of free Ca²⁺ in and out of the SR is essential for a proper generation of pacemaker activity during early cardiomyogenesis and fetal life^[37]. From EDS to LDS, the Ca²⁺ transients of ESCM were inhibited significantly by 2-APB, but its inhibitory effect decreased with development. Although 2-APB is also an inhibitor of SR Ca²⁺-pump in non-excitable cells^[24], the different effects between 2-APB and thapsigargin we observed indicate that IP₃Rs have a critical role in regulation of Ca²⁺ transients in early developing cardiomyocytes. The complementary temporal changes in the molecular and function of RyR2 and IP₃Rs on the regulation of Ca²⁺ transients demonstrates a developmental mechanism of the Ca²⁺ release from SR.
Decay of [Ca²⁺]_i in the E-C coupling of the developing cardiomyocytes The decay of the Ca²⁺ transients occurs in adult cardiomyocytes because of a reuptake of Ca²⁺ into the SR through the Ca²⁺-pump ATPase and extrusion of Ca²⁺ from the myocytes by the NCX^[20]. The two fundamental principles for maintaining a steady-state contraction of cardiac physiological function are that^[5]: (i) a balance exits between the amount of Ca²⁺ entering the cells mainly via the L-type Ca²⁺ channels and the amount of Ca²⁺ extruded via the NCX; and (ii) the amount of Ca²⁺ released by the SR equals that sequestered by the SR Ca²⁺-pump ATPase. There is dynamic competition among NCX and SERCA2 during relaxation, and the SERCA2 and NCX contribute a variable amount toward [Ca²⁺]_i decline depending on species, development stages, and physiologic conditions.
The expression of SERCA2 increases at the time when beating cardiomyocytes appear and maintains a stable level during ESCM differentiation. This is consistent with the observation that the inhibitory effect of thapsigargin does not appear significantly different during ESCM differentia-tion. The NCX expression level increases upon the appearance of cardiomyocytes as observed here and its level in early developed cells is twice that in adult myocytes^[8,9,38,39]. This is supported by the experiment with the Na⁺ free solution showing that NCX is functional to maintain the low cytosolic [Ca²⁺]_i even in the early differentiating ESCM. But the Na⁺ free solution did not affect the peak Ca²⁺ transients until the LDS. This is consistent with the observation that KB-R 7943, an inhibitor of the reverse mode of NCX, has no effect on the Ca²⁺ transients in the 9.5 dpc fetal cardiomyocytes^[8]. Thus, the sarcolemmal Ca²⁺-ATPase may play a major role in maintaining the balance between the amount of Ca²⁺ entering the cells and the amount of Ca²⁺ extruded at the early developmental period.
As discussed above, the dynamic changes in the regulation of Ca²⁺ transients occur during the cardiac development, and the sources of Ca²⁺ for producing contraction is altered during development. The failing heart has an altered program of gene expression with embryonic characteristics^{[40, 41]}. Moreover, transplantation of exogenous cells into injured myocardium, such as fetal cardiomyocytes^[42], bone marrow cells^[43] and ESCM^[44], has emerged for regeneration of damaged myocardium and for improvement of cardiac function in post-infarcted hearts in recent years. Therefore, it is significant to further investigate the establishment of the E-C coupling and the regulation of Ca²⁺ homeostasis during development, which is important for a better understanding of normal development aspects and abnormalities in cardiac diseases.

References
1 Clapham DE. Calcium signaling. Cell 1995; 80: 259_68.
2 Webb SE, Miller AL. Calcium signaling during embryonic development. Nat Rev Mol Cell Biol 2003; 4: 539_51.
3 Puceat M, Jaconi M. Ca(2+) signaling in cardiogenesis. Cell Calcium 2005; 38: 383-9
4 Evenas J, Malmendal A, Forsen S. Calcium. Curr Opin Chem Biol 1998; 2: 293_302.
5 Barry WH, Bridge JH. Intracellular calcium homeostasis in cardiac myocytes. Circulation 1993; 87: 1806_15.
6 Bers DM. Cardiac excitation-contraction coupling. Nature 2002; 415: 198_205.
7 Brand T. Heart development: molecular insights into cardiac specification and early morphogenesis. Dev Biol 2003; 258: 1_19.
8 Liu W, Yasui K, Opthof T, Ishiki R, Lee JK, Kamiya K, et al. Developmental changes of Ca(2+) handling in mouse ventricular cells from early embryo to adulthood. Life Sci 2002; 71: 1279_92.
9 Reed TD, Babu GJ, Ji Y, Zilberman A, Ver Heyen M, Wuytack F, et al. The expression of SR calcium transport ATPase and the Na(+)/Ca(2+)exchanger are antithetically regulated during mouse cardiac development and in hypo/hyperthyroidism. J Mol Cell Cardiol 2000; 32: 453_64.
10 Hescheler J, Fleischmann BK, Lentini S, Maltsev VA, Rohwedel J, Wobus AM, et al. Embryonic stem cells: a model to study structural and functional properties in cardiomyogenesis. Cardiovasc Res 1997; 36: 149_62.
11 Westfall MV, Pasyk KA, Yule DI, Samuelson LC, Metzger JM. Ultrastructure and cell-cell coupling of cardiac myocytes differentiating in embryonic stem cell cultures. Cell Motil Cytoskeleton 1997; 36: 43_54.
12 Boheler KR, Czyz J, Tweedie D, Yang HT, Anisimov SV, Wobus AM. Differentiation of pluripotent embryonic stem cells into cardiomyocytes. Circ Res 2002; 91: 189_201.
13 Banach K, Halbach MD, Hu P, Hescheler J, Egert U. Development of electrical activity in cardiac myocyte aggregates derived from mouse embryonic stem cells. Am J Physiol Heart Circ Physiol 2003; 284: H2114_23.
14 Doevendans PA, Kubalak SW, An RH, Becker DK, Chien KR, Kass RS. Differentiation of cardiomyocytes in floating embryoid bodies is comparable to fetal cardiomyocytes. J Mol Cell Cardiol 2000; 32: 839_51.
15 Fijnvandraat AC, van Ginneken AC, Schumacher CA, Boheler KR, Lekanne Deprez RH, Christoffels VM, et al. Cardiomyocytes purified from differentiated embryonic stem cells exhibit characteristics of early chamber myocardium. J Mol Cell Cardiol 2003; 35: 1461_72.
16 Wobus AM, Guan K, Yang HT, Boheler KR. Embryonic stem cells as a model to study cardiac, skeletal muscle, and vascular smooth muscle cell differentiation. Methods Mol Biol 2002; 185: 127_56.
17 Fu JD, Li J, Tweedie D, Yu HM, Chen L, Wang R, et al. Crucial role of the sarcoplasmic reticulum in the developmental regulation of Ca²⁺ transients and contraction in cardiomyocytes derived from embryonic stem cells. FASEB J 2006; 20: 181_3.
18 Yang HT, Tweedie D, Wang S, Guia A, Vinogradova T, Bogdanov K, et al. The ryanodine receptor modulates the spontaneous beating rate of cardiomyocytes during development. Proc Natl Acad Sci USA 2002; 99: 9225_30.
19 Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440_50.
20 Tibbits GF, Xu L, Sedarat F. Ontogeny of excitation-contraction coupling in the mammalian heart. Comp Biochem Physiol A Mol Integr Physiol 2002; 132: 691_8.
21 Liu W, Yasui K, Arai A, Kamiya K, Cheng J, Kodama I, et al. beta-adrenergic modulation of L-type Ca²⁺-channel currents in early-stage embryonic mouse heart. Am J Physiol 1999; 276: H608_13.
22 Maltsev VA, Ji GJ, Wobus AM, Fleischmann BK, Hescheler J. Establishment of beta-adrenergic modulation of L-type Ca²⁺ current in the early stages of cardiomyocyte development. Circ Res 1999; 84: 136_45.
23 Maruyama T, Kanaji T, Nakade S, Kanno T, Mikoshiba K. 2APB, 2-aminoethoxydiphenyl borate, a membrane-penetrable modulator of Ins(1,4,5)P3-induced Ca²⁺ release. J Biochem (Tokyo) 1997; 122: 498_505.
24 Bootman MD, Collins TJ, Mackenzie L, Roderick HL, Berridge MJ, Peppiatt CM. 2-aminoethoxydiphenyl borate (2-APB) is a reliable blocker of store-operated Ca²⁺ entry but an inconsistent inhibitor of InsP3-induced Ca²⁺ release. FASEB J 2002; 16: 1145_50.
25 Tohse N, Meszaros J, Sperelakis N. Developmental changes in long-opening behavior of L-type Ca²⁺ channels in embryonic chick heart cells. Circ Res 1992; 71: 376_84.
26 Davies MP, An RH, Doevendans P, Kubalak S, Chien KR, Kass RS. Developmental changes in ionic channel activity in the embryonic murine heart. Circ Res 1996; 78: 15_25.
27 Masuda H, Sumii K, Sperelakis N. Long openings of calcium channels in fetal rat ventricular cardiomyocytes. Pflugers Arch 1995; 429: 595_7.
28 Nakanishi T, Seguchi M, Takao A. Development of the myocardial contractile system. Experientia 1988; 44: 936_44.
29 Mahony L, Jones LR. Developmental changes in cardiac sarcoplasmic reticulum in sheep. J Biol Chem 1986; 261: 15257_65.
30 Pegg W, Michalak M. Differentiation of sarcoplasmic reticulum during cardiac myogenesis. Am J Physiol 1987; 252: H22_31.
31 Olivetti G, Anversa P, Loud AV. Morphometric study of early postnatal development in the left and right ventricular myocardium of the rat. II. Tissue composition, capillary growth, and sarcoplasmic alterations. Circ Res 1980; 46: 503_12.
32 Takeshima H, Komazaki S, Hirose K, Nishi M, Noda T, Iino M. Embryonic lethality and abnormal cardiac myocytes in mice lacking ryanodine receptor type 2. EMBO J 1998; 17: 3309_16.
33 Seki S, Nagashima M, Yamada Y, Tsutsuura M, Kobayashi T, Namiki A, et al. Fetal and postnatal development of Ca²⁺ transients and Ca²⁺ sparks in rat cardiomyocytes. Cardiovasc Res 2003; 58: 535_48.
34 Marks AR. Intracellular calcium-release channels: regulators of cell life and death. Am J Physiol 1997; 272: H597_605.
35 Rosemblit N, Moschella MC, Ondriasa E, Gutstein DE, Ondrias K, Marks AR. Intracellular calcium release channel expression during embryogenesis. Dev Biol 1999; 206: 163_77.
36 Saeki T, Shen JB, Pappano AJ. Inositol-1,4,5-trisphosphate increases contractions but not L-type calcium current in guinea pig ventricular myocytes. Cardiovasc Res 1999; 41: 620_8.
37 Mery A, Aimond F, Menard C, Mikoshiba K, Michalak M, Puceat M. Initiation of embryonic cardiac pacemaker activity by inositol 1,4,5-trisphosphate-dependent calcium signaling. Mol Biol Cell 2005; 16: 2414_23.
38 Koban MU, Moorman AF, Holtz J, Yacoub MH, Boheler KR. Expressional analysis of the cardiac Na-Ca exchanger in rat development and senescence. Cardiovasc Res 1998; 37: 405_23.
39 Qu Y, Ghatpande A, el Sherif N, Boutjdir M. Gene expression of Na⁺/Ca²⁺ exchanger during development in human heart. Cardiovasc Res 2000; 45: 866_73.
40 Ghatpande S, Goswami S, Mascareno E, Siddiqui MA. Signal transduction and transcriptional adaptation in embryonic heart development and during myocardial hypertrophy. Mol Cell Biochem 1999; 196: 93_7.
41 Marks AR. Ryanodine receptors/calcium release channels in heart failure and sudden cardiac death. J Mol Cell Cardiol 2001; 33: 615_24.
42 Soonpaa MH, Koh GY, Klug MG, Field LJ. Formation of nascent intercalated disks between grafted fetal cardiomyocytes and host myocardium. Science 1994; 264: 98_101.
43 Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001; 410: 701_5.
44 Min JY, Yang Y, Converso KL, Liu L, Huang Q, Morgan JP, et al. Transplantation of embryonic stem cells improves cardiac function in postinfarcted rats. J Appl Physiol 2002; 92: 288_96.