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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.
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