TY - JOUR
AU - Gepstein, Lior
AB - Abstract The objective of the current study was to characterize calcium handling in developing human embryonic stem cell-derived cardiomyocytes (hESC-CMs). To this end, real-time polymerase chain reaction (PCR), immunocytochemistry, whole-cell voltage-clamp, and simultaneous patch-clamp/laser scanning confocal calcium imaging and surface membrane labeling with di-8-aminonaphthylethenylpridinium were used. Immunostaining studies in the hESC-CMs demonstrated the presence of the sarcoplasmic reticulum (SR) calcium release channels, ryanodine receptor-2, and inositol-1,4,5-trisphosphate (IP3) receptors. Store calcium function was manifested as action-potential-induced calcium transients. Time-to-target plots showed that these action-potential-initiated calcium transients traverse the width of the cell via a propagated wave of intracellular store calcium release. The hESC-CMs also exhibited local calcium events (“sparks”) that were localized to the surface membrane. The presence of caffeine-sensitive intracellular calcium stores was manifested following application of focal, temporally limited puffs of caffeine in three different age groups: early-stage (with the initiation of beating), intermediate-stage (10 days post-beating [dpb]), and late-stage (30–40 dpb) hESC-CMs. Calcium store load gradually increased during in vitro maturation. Similarly, ryanodine application decreased the amplitude of the spontaneous calcium transients. Interestingly, the expression and function of an IP3-releasable calcium pool was also demonstrated in the hESC-CMs in experiments using caged-IP3 photolysis and antagonist application (2 μM 2-Aminoethoxydiphenyl borate). In summary, our study establishes the presence of a functional SR calcium store in early-stage hESC-CMs and shows a unique pattern of calcium handling in these cells. This study also stresses the importance of the functional characterization of hESC-CMs both for developmental studies and for the development of future myocardial cell replacement strategies. Disclosure of potential conflicts of interest is found at the end of this article. Human embryonic stem cells, Calcium transients, Myogenesis, Fluorescence microscope, Embryoid body Introduction Human embryonic stem cells (hESCs) are pluripotent stem cell lines that by definition have the potential to give rise to any cell type [1]. The establishment of an in vitro cardiomyocyte differentiating system from these unique cells [2–5] raises the possibility that they may ultimately be used in the emerging field of cardiovascular regenerative medicine [6]. The suitability of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) for such a task depends, in part, on their contractile characteristics, which in turn are determined by their pattern of excitability, calcium handling, and excitation-contraction coupling. The initial reports describing the differentiation of hESC-CMs showed the development of spontaneous beating areas within embryoid bodies (EBs) [2–5]. Ultrastructural analysis showed that hESC-CMs develop in vitro from spherical cells to elongated cells with a more organized ultrastructural sarcomeric pattern [7]. Detailed electrophysiological analysis revealed that hESC-CMs express many of the same ion channels and ionic currents as mature cells [2–5, 8, 9]. hESC-CMs express a large sodium current density, and the action-potential (AP) duration is shortened by L-type calcium channel blockade, as in mature cardiac myocytes (CMs). At the multicellular level, hESC-CMs display APs with a variety of cardiac-like morphologies. The coupling between excitation and contraction in adult cardiomyocytes is by a calcium-induced calcium release (CICR) mechanism. In mature cardiac cells APs trigger the opening of L-type calcium channels, which in turn provide the trigger calcium for sarcoplasmic reticulum (SR) calcium release. Although calcium handling is fundamental to heart cell function, it has not been well studied in hESC-CMs. In fact, the existence of an SR calcium store in hESC-CMs, until now, has been thought to be of limited functional importance because no caffeine-releasable calcium store was observed [10]. In distinction, early-stage mouse ESC-CMs use ryanodine receptor (RyR)-sensitive stores, and they exhibit rhythmic internal calcium transients independently of AP-triggered CICR [11]. Later developmental stages of mouse ESC-CMs require ryanodine receptor-2 (RyR2) for spontaneous beating [12], and more recent studies confirm the presence of a functional SR in mouse ESC-CMs by showing spontaneous local calcium events and elevated caffeine-releasable calcium as a function of time of in vitro differentiation [13]. Moreover, the mouse RyR2 knockout model fails to develop a normal heart, highlighting the central importance of a functional SR in maturation. Although the role of RyR in adult cardiomyocytes is well established, the role of inositol-1,4,5-trisphosphate (IP3), an activator of a specific class of SR calcium release channels, known as inositol-1,4,5-trisphosphate receptors (IP3Rs), in the adult heart is still debated. Prior studies demonstrate evidence pointing to an important role of IP3-dependent signaling during cardiac development [14, 15]. Interestingly, in a recent study in mouse ESC-CMs, IP3Rs were also suggested to play a physiological role in calcium handling in developing cardiomyocytes [16], as well as in their pacemaking function [17]. The potential presence and functionality of a similar IP3R pathway in human cardiac tissue, however, has not been studied. In the current study, we tested the hypothesis that hESC-CMs contain functional SR calcium stores and that SR calcium is used similarly to mature adult CMs that lack a highly organized transverse tubules system, such as atrial myocytes. We start by showing that the principal features of hESC-CMs calcium handling include action-potential-initiated calcium (AP-Ca) transients, which initiate at the cell periphery and propagate to the center with a delay, and local calcium events (“sparks”-like), which initiate near the sarcolemma (SL). In addition, we show that RyR-mediated SR calcium stores are required for both AP-Ca transients and local calcium events. By testing age-dependent changes in caffeine-triggered calcium transients (in three hESC-CMs maturation stages) we show that RyR-mediated SR calcium stores are present and functional from the initiation of spontaneous beating activity and increase in load during in vitro maturation. Finally, we present evidence showing the expression and functional role of the IP3Rs in hESC-CMs. Materials and Methods Propagation of the hESC Lines and In Vitro Cardiomyocyte Differentiation Pluripotent hESCs of the H9.2 clone were grown in the undifferentiated state on top of mouse embryonic fibroblast feeder layer as previously described [2, 18]. To induce differentiation, hESCs were dispersed to small clamps using collagenase IV (1 mg/ml; Life Technologies, Rockville, MD, http://www.lifetech.com) and were then cultured in suspension for 7 days, where they formed EBs. The EBs were plated on gelatin-coated culture dishes (“date of plating”) and observed microscopically for the appearance of spontaneous contractions (“date of beating”). The spontaneously contracting EBs were mechanically isolated, dispersed into cells with 1 mg/ml collagenase B (Roche, Mannheim, Germany, http://www.roche.com), and adhered onto fibronectin-coated glass coverslips for study. Electrophysiology Coverslips containing the hESC-CMs were mounted in a chamber of an upright BX51WI Olympus microscope (Olympus, Tokyo, http://www.olympus-global.com) and superfused at a rate of 1 ml/minute and at a temperature of 37°C with physiological salt solution (PSS) containing the following (in mM): NaCl, 140; KCl, 5.4; CaCl2, 1.8; MgCl2, 1; HEPES, 10; glucose, 10; at pH 7.4 via 1 mM NaOH. Spontaneously beating single and monolayered clusters of hESC-CMs APs were measured using the whole-cell configuration of the patch-clamp technique. Patch pipette solution contained the following (in mM): potassium gluconate, 135; HEPES, 10; KCl, 10; phosphocreatinine, Na2, 10; Mg, ATP, 4; GTP-Na, 0.3; 0.005 Oregon Green 488 BAPTA, 1 (Molecular Probes, Eugene, OR, http://probes.invitrogen.com); at pH 7.4 via 1 mM KOH. Confocal Calcium Imaging Cells were loaded with fluo-4 (Molecular Probes) to follow intracellular calcium transients and with Di-8-ANEPPS (Molecular Probes) to delineate surface membrane [19]. A final concentration of 5 μM fluo-4 AM was used in the presence of Pluronic F-127 (Molecular Probes) at a dilution of 2:1, respectively. Di-8-ANEPPS (10 μM; Molecular Probes) was then applied for 5 minutes at 37°C. Intracellular calcium transients were recorded with a confocal imaging system (Fluoview; Olympus) mounted on an upright BX51WI Olympus microscope equipped with a ×60 water objective [20]. The temporal resolution of the line scan was 512 Hz. Line scans were set at mid-cell z-section depth and oriented to avoid the cell nuclei. Data were analyzed using custom routines written in IDL (ITT Visual Information Solutions, Boulder, CO, http://www.ittvis.com). Local calcium events were identified with an automated local calcium event detection algorithm, and their dimensions were analyzed for full width at half-maximum and kinetic parameters. We used the “time-to-target” plot [19] to determine whether a propagating wave of calcium release underlies the AP-triggered whole-cell calcium transient. Data were fitted offline with Origin software (OriginLab, Northampton, MA, http://www.originlab.com). Caged-IP3 Loading A modified saponin-permeabilization protocol [21] was used to load the hESC-CMs with the caged-IP3 compound. Initially, the temperature of the cells was gradually brought down by incubation with chilled phosphate-buffered saline (PBS) in an ice bath. At this stage, the cells were placed in a permeabilization buffer (20 mmol/l HEPES, pH 7.4, 10 mmol/l EGTA, 140 mmol/l KCl, 5 mmol/l oxalic acid dipotassium salt 5 mmol/l sodium azide) containing saponin (50 μg/ml; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) and caged IP3 (20 μM; Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and incubated for 10 minutes. ATP was added just before adding the permeabilization buffer to the cells (30 μl of 200 mmol/l ATP per milliliter of permeabilization buffer). Following repeated washouts with chilled PBS, the cells were slowly brought back to 37°C and then loaded with fluo-4 for calcium imaging as described above. Caged-IP3 Photolysis The active form of IP3 was liberated by photolysis induced with localized UV illumination (351 nm). IP3 uncaging was performed in a spatially restricted area in the cytoplasm. A possible source of error in the use of caged compounds may be the triggering response of the flash. Therefore UV illumination intensity was tested prior to each experiment and was decreased to a level where artifact triggering response was not observed. Real-Time PCR Real-time PCR was performed in 96-well optical plates in triplicate using an ABI 7700 Sequence Detector (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). Samples (0.2 ng of cDNA) were prepared using the TaqMan PCR core reagent kit (Applied Biosystems). Cycle conditions (50 cycles) were 15 seconds at 95°C and 1 minute at 60°C. Primer sequences were as follows: RyR2, forward primer, 5′-agaacttacacacgcgacctg-3′, reverse primer, 5′-catctctaaccggaccatactgc-3′ (accession number X98330); Cav1.2, forward primer: 5′-gagaacagcaagtttgactttgacaa-3′, reverse primer, 5′-cgaaggtggagacggtgaa-3′ (accession number NM_000719); CaVβ2, forward primer, 5′-gacagacgccttatagctcctcaa-3′, reverse primer, 5′-cggtcctcctccagagatacat-3′ (accession number AF285239); NaV1.5, forward primer, 5′catcttcacaggcgagtgtattg-3′, reverse primer, 5′-gatattccagctgttggtgaagtagta-3′ (accession number AF482988). In all cases a single amplicon of the appropriate size was detected using gel electrophoresis. Results are expressed normalized to cyclophilin A according to the 2−ΔCt method as in our previous work [8]. For IP3R2 amplification, Applied Biosystems master mix Hs00181916 was used. Immunocytochemistry Single cells and small monolayered clusters were fixed using 4% paraformaldehyde and permeabilized with 0.05% Triton. Cells were then blocked with 10% normal goat serum and 1% bovine serum albumin and incubated overnight at 4°C with primary antibodies for sarcomeric α-actinin (Sigma-Aldrich) at 1:200 and ryanodine receptor (Chemicon, Temecula, CA, http://www.chemicon.com) at 1:1,000. Immunostaining for calreticulin and IP3Rs was performed under light fixation (2% PFA, 30 minutes) using the calreticulin (1:50; Millipore, Billerica, MA, http://www.millipore.com) antibody and monoclonal pan-IP3R (1:10; Millipore) antibody, which reacts with the C-terminal cytoplasmic domain of IP3R types 1, 2, and 3. The secondary antibodies were Cy3-conjugated donkey anti-mouse IgG antibody at 1:200 and Cy2-conjugated goat anti-rabbit IgG antibody at 1:300 for 1 hour (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) at room temperature. Nuclei were counterstained with 4,6-diamidino-2-phenylindole at a dilution of 1:500. Control experiments included testing staining specificity by omitting the primary antibodies. Confocal microscopy was performed using a Nikon Eclipse E600 microscope (Nikon, Tokyo, http://www.nikon.com) and the Bio-Rad Radiance 2000 scanning system (Bio-Rad, Hercules, CA, http://www.bio-rad.com). Results Expression of Calcium-Handling Molecules To focus on whether mature calcium-handling molecular components are present in hESC-CMs, we first used real-time reverse transcription (RT)-PCR to evaluate the presence and relative expression of RyR2, CaV1.2, and CaVβ2 transcripts in <40 days post-beating (dpb) hESC-CMs in relation to those in mature human ventricle (Fig. 1A). The mRNA level for the cardiac voltage-dependent calcium channel, CaV1.2, had an abundance similar to that of mature tissue. The mRNA level for CaVβ2, a critical regulatory subunit, was 20-fold lower, and RyR2 was ∼1,000-fold less abundant in the hESC-CMs compared with adult heart. Figure 1. Open in new tabDownload slide Evaluation of the presence and relative expression of calcium-handling proteins in developing hESC-CMs. (A): Real-time PCR of CaV1.2 (cardiac L-type calcium channel), CaVβ2 (cardiac L-type channel auxiliary subunit), and RYR2 mRNA in hESC-CMs. Mean ± SEM of calculated log molecules present in adult human ventricle are normalized to that in hESC-CMs. (B, C): RYR2 localization in hESC-CMs. Confocal micrograph of a representative isolated cell (B) and small cluster (C) colabeled with antibodies for RYR2 (middle panels) and sarcomeric α-actinin (top panels). Merged images are presented in bottom panels. (D–F): Transmission electron microscopy images of the hESC-CMs. (D): Relatively low-power view of a cell with sarcomere in a slightly oblique longitudinal orientation. (E): Expanded view of the dotted box from (D) showing nSR. The t is in an axial-like orientation. The sets of arrows are placed approximately perpendicular to the longitudinal plane of myofilaments to illustrate the somewhat disorganized orientation. (F): Expanded view of the solid box in (D) showing t that is juxtaposed between z and m. Abbreviations: jSR, junctional sarcoplasmic reticulum; L, lipid droplet; m, mitochondria; n, nucleus; nSR, network sarcoplasmic reticulum; RYR2, ryanodine receptor-2; t, t-tubule; z, z-disc. Figure 1. Open in new tabDownload slide Evaluation of the presence and relative expression of calcium-handling proteins in developing hESC-CMs. (A): Real-time PCR of CaV1.2 (cardiac L-type calcium channel), CaVβ2 (cardiac L-type channel auxiliary subunit), and RYR2 mRNA in hESC-CMs. Mean ± SEM of calculated log molecules present in adult human ventricle are normalized to that in hESC-CMs. (B, C): RYR2 localization in hESC-CMs. Confocal micrograph of a representative isolated cell (B) and small cluster (C) colabeled with antibodies for RYR2 (middle panels) and sarcomeric α-actinin (top panels). Merged images are presented in bottom panels. (D–F): Transmission electron microscopy images of the hESC-CMs. (D): Relatively low-power view of a cell with sarcomere in a slightly oblique longitudinal orientation. (E): Expanded view of the dotted box from (D) showing nSR. The t is in an axial-like orientation. The sets of arrows are placed approximately perpendicular to the longitudinal plane of myofilaments to illustrate the somewhat disorganized orientation. (F): Expanded view of the solid box in (D) showing t that is juxtaposed between z and m. Abbreviations: jSR, junctional sarcoplasmic reticulum; L, lipid droplet; m, mitochondria; n, nucleus; nSR, network sarcoplasmic reticulum; RYR2, ryanodine receptor-2; t, t-tubule; z, z-disc. We next evaluated RyR2 protein expression in hESC-CMs by immunocytochemistry, probing for both RyR2 and sarcomeric α-actinin in dispersed single cells (Fig. 1B) and small clusters (Fig. 1C). As also shown previously [7], the hESC-CMs showed a relatively disorganized sarcomeric pattern, typical of immature early-stage CMs (Figs. 1B, 1C, top panel). Costaining for RyR2 showed its expression throughout the cytosol (with some colocalization with the myofilaments) with intense staining in the perinuclear region (Figs. 1B, 1C, middle and bottom panels). This subcellular localization is similar to that previously reported in the mouse ESC-CMs [13]. We next continued to study the ultrastructural properties of the hESC-CMs. As is evident from the electron micrographs in Figure 1D–1F, hESC-CMs (at 30–40 dpb) display an early-stage ultrastructural phenotype. Note the presence of parallel nascent Z-bands confining the myofibrils to an early-stage sarcomeric pattern. These sarcomeric structures, although they showed clearly defined Z-bands, did not display well-defined M-lines, A-bands, or I-bands. More importantly, these electron micrographs (Fig. 1D–1F) also demonstrate the presence of a developing SR system. Note the presence of structures consistent with network SR located adjacent to the myofilaments, as well as junctional SR (Fig. 1E, 1F). These higher magnification images also reveal structures consistent with a transverse (t)-tubular system. Nevertheless, whereas t-tubules juxtaposed to z-discs and mitochondria could be identified, their abundance, distribution, and maturation pattern did not reach the level seen in mature cardiomyocytes [22]. Two Types of Calcium Transients Are Apparent During Calcium Imaging Simultaneous AP recordings and calcium imaging in the hESC-CM (Fig. 2) reveal two distinct types of calcium transients: an AP-driven whole-cell calcium transient (AP-Ca transients; Fig. 2B, gray arrow) and local calcium transients (Fig. 2B, white arrows). The AP-Ca transient is a relatively high-amplitude event that traverses the entire width of the cell (Fig. 2B, gray arrows) and follows membrane depolarization (Fig. 2C). In contrast, the spark-like local calcium events are confined to the cell periphery (Fig. 2B, white arrows) and are not temporally related to the AP (Fig. 2C). Figure 2D shows an expanded view of local calcium events visible during diastole. Note that the frequency of the local calcium events is much greater than the frequency for AP-Ca transients (n = 5). Figure 2. Open in new tabDownload slide Action-potential (AP)-initiated and local calcium transients. (A): Two-dimensional confocal image of fluo-4-loaded human embryonic stem cell-derived cardiomyocyte. Whole-cell patch pipette position is indicated by the arrow. The diagonal line is the line scan used in (B). (B): Line scan presenting a distance-time plot of changes in intracellular calcium. AP-initiated calcium and local calcium transients are highlighted by the gray and white arrows, respectively. (C): Simultaneous calcium imaging (top) and whole-cell recordings of AP (bottom). (D): Expanded aspect ratio (amplitude/time) for fluo-4 signal (left) and AP recordings (right). Abbreviations: F/Fo, fluorescence (F) normalized to baseline fluorescence (Fo); s, seconds. Figure 2. Open in new tabDownload slide Action-potential (AP)-initiated and local calcium transients. (A): Two-dimensional confocal image of fluo-4-loaded human embryonic stem cell-derived cardiomyocyte. Whole-cell patch pipette position is indicated by the arrow. The diagonal line is the line scan used in (B). (B): Line scan presenting a distance-time plot of changes in intracellular calcium. AP-initiated calcium and local calcium transients are highlighted by the gray and white arrows, respectively. (C): Simultaneous calcium imaging (top) and whole-cell recordings of AP (bottom). (D): Expanded aspect ratio (amplitude/time) for fluo-4 signal (left) and AP recordings (right). Abbreviations: F/Fo, fluorescence (F) normalized to baseline fluorescence (Fo); s, seconds. The whole-cell configuration perturbs the native intracellular environment and can create artifact leak currents, resulting in non-native calcium handling and pacemaker activity. We therefore performed a series of experiments to investigate calcium handling in intact, spontaneously beating cell clusters. This preserves native cytosolic conditions. Figure 3A shows calcium transients (fluo-4 fluorescence) for a representative hESC-CM cluster displayed in Figure 3B. Cells were loaded with fluo-4 to measure calcium and Di-8-ANEPPS to delineate cell surface membrane [19]. As with cells recorded in the whole-cell configuration, two types of calcium transients were recorded: AP-Ca transients and local calcium events. Average amplitude recordings are shown along with the line-scan plot. Two features are readily apparent. First, AP-Ca transients are rhythmic and traverse the width of the cell. Second, local calcium events are confined to the SL. Figure 3. Open in new tabDownload slide Action-potential-initiated calcium (AP-Ca) traverses the cell via a propagated wave. (A): A 23-s line scan of the cell shown in (B). The tracings correspond to regions indicated by arrows in the line scan. The pseudocolored segment to the right of the line scan shows Di-8-ANEPPS staining. (B): A photograph of a cell loaded with fluo-4. The yellow X mark is for reference of plasma membrane. (C): AP-Ca transients from the central region (red) and upper (black) and lower (green) surface membranes for a single beat are superimposed. (D): Time-to-target plot for AP-Ca transients. Abbreviations: F/Fo, fluorescence (F) normalized to baseline fluorescence (Fo); s, seconds. Figure 3. Open in new tabDownload slide Action-potential-initiated calcium (AP-Ca) traverses the cell via a propagated wave. (A): A 23-s line scan of the cell shown in (B). The tracings correspond to regions indicated by arrows in the line scan. The pseudocolored segment to the right of the line scan shows Di-8-ANEPPS staining. (B): A photograph of a cell loaded with fluo-4. The yellow X mark is for reference of plasma membrane. (C): AP-Ca transients from the central region (red) and upper (black) and lower (green) surface membranes for a single beat are superimposed. (D): Time-to-target plot for AP-Ca transients. Abbreviations: F/Fo, fluorescence (F) normalized to baseline fluorescence (Fo); s, seconds. AP-Ca transients originate from the SL and spread to the center with a delay. This is illustrated in Figure 3C where the central region (red tracing), lower plasma membrane (PM) (Fig. 3A, 3B, green tracing recorded from the site marked by the yellow X), and upper PM (black tracing) calcium transients are superimposed. The spread of calcium from periphery to center during a whole-cell calcium transient could theoretically occur solely by diffusion or by a propagating wave of regenerative store calcium release (calcium-wave). To distinguish between these two possibilities we analyzed the shape of the time-to-target plot (Fig. 3D). The time-to-target plot [19] is a map displaying the time it takes for fluo-4 fluorescence to attain a fixed (target) value as a function of the distance from the cell periphery where initiation takes place. If calcium spreads via a propagated wave, then we expect a linear relationship between the time it takes to reach a given target level of calcium and the distance from the cell edge. In contrast, diffusion would result in a time-to-target plot with a concave-up configuration, as calcium spread slows as it diffuses in three dimensions from its point of origin. The time-to-target plot (Fig. 3D) of the hESC-CM AP-Ca transient shows an initial flat region spanning ∼1.8 μm, displaying instantaneous calcium spread near the border of the cell. This is followed by a segment of a linear region with a mean velocity of 0.25 ± 0.09 μm/millisecond (n = 14), indicating that hESC-CMs use propagated calcium waves. Hence, the linearity of the time-to-target curve indicates that the subsequent spread of calcium into the cell is due to regenerative store calcium release. In hESC-CMs, local calcium events (sparks) occur during all phases of the beat cycle (Figs. 2D, 4A). Local calcium events are present during the diastolic intervals, superimposed on the AP-Ca transient upstroke, and possibly also embedded in the AP-Ca transient (Fig. 4A, arrows). We limited quantification of local calcium events to the diastolic interval to minimize superimposition of AP-Ca transients (Fig. 4B–4E). The local calcium event dimension distribution is bimodal (Fig. 4B). Amplitude and rise time distributions (Fig. 4C, 4D) are indistinguishable, but the wider transients have a significantly longer duration (Fig. 4E). Figure 4. Open in new tabDownload slide Local calcium transients. (A): The calcium imaging tracing shows the presence of high-frequency, relatively small-amplitude transients (local calcium events, vertical arrows) that occur throughout the contraction/relaxation cycle (systolic events are indicated by dashed line) and are superimposed on the high-amplitude action-potential-initiated calcium transients. (B): Local calcium event dimensions are bimodally distributed. (B–E): Low FWHM events are represented by open bars and high FWHM events by stippled bars. (C, D): Local calcium event amplitude (C) and rise time (D) are indistinguishable between low- and high-FWHM populations. (E): Decay time of higher-FWHM events is significantly longer than that of the lower-FWHM population (p = .0002). Abbreviations: F/Fo, fluorescence (F) normalized to baseline fluorescence (Fo); FWHM, full width at half-maximum; s, seconds. Figure 4. Open in new tabDownload slide Local calcium transients. (A): The calcium imaging tracing shows the presence of high-frequency, relatively small-amplitude transients (local calcium events, vertical arrows) that occur throughout the contraction/relaxation cycle (systolic events are indicated by dashed line) and are superimposed on the high-amplitude action-potential-initiated calcium transients. (B): Local calcium event dimensions are bimodally distributed. (B–E): Low FWHM events are represented by open bars and high FWHM events by stippled bars. (C, D): Local calcium event amplitude (C) and rise time (D) are indistinguishable between low- and high-FWHM populations. (E): Decay time of higher-FWHM events is significantly longer than that of the lower-FWHM population (p = .0002). Abbreviations: F/Fo, fluorescence (F) normalized to baseline fluorescence (Fo); FWHM, full width at half-maximum; s, seconds. hESC-CMs Express Caffeine- and Ryanodine-Sensitive Calcium Stores We next determined whether hESC-CMs use mature calcium handling components in the form of caffeine- and ryanodine-sensitive calcium stores. To measure caffeine mobilization of store calcium and its effect on intracellular calcium transients, a caffeine puff (10 mM, <2 seconds) was applied to 30–40-dpb hESC-CMs by pressure ejection through a pipette located approximately 100 μm away from the cells. Caffeine rapidly released calcium from its intracellular stores, increasing cytosolic calcium and reversibly inhibiting the spontaneous calcium transients (Fig. 5A). This was followed by the reappearance of the AP-Ca transients, with a progressive increase in their amplitude as calcium gradually loaded the SR stores. Finally, to confirm that the actual pressure injected puff was not mechanically stimulating the cell surface and causing the apparent changes in calcium transients, we also conducted control PSS puff experiments. These control puffs did not elicit any apparent effect on the intracellular calcium transients (data not shown). Figure 5. Open in new tabDownload slide Caffeine-releasable calcium stores. (A): A line scan of a representative spontaneously beating 30–40 dpb human embryonic stem cell-derived cardiomyocyte (hESC-CM) (confocal image displayed in the upper left panel). Shown is the calcium imaging tracing during the response to rapid 10 mM caffeine application. Note the initial local bolus release of calcium initiating a calcium wave propagation, which is followed by the disappearance of calcium transients and finally by the reappearance of these transients, which gradually increase in amplitude. (B): Two-dpb hESC-CMs showing similar caffeine responsiveness. (C): Caffeine-releasable calcium increases progressively during in vitro maturation. The caffeine transient amplitude was normalized to preceding mean AP-Ca transients amplitude. One-way analysis of variance with Tukey post hoc tests: *, p = .005 for 10 dpb, and #, p < .001 for 30–40 dpb, compared with 2 dpb. n = 6, 6, and 10 for 2, 10, and 30–40 dpb, respectively. Box and whisker plot shows 25%–75% level (box); vertical lines display the range. (D): Response of the local calcium events to a brief application of 10 mM caffeine. Abbreviations: dpb, days post-beating; F/Fo, fluorescence (F) normalized to baseline fluorescence (Fo); s, seconds. Figure 5. Open in new tabDownload slide Caffeine-releasable calcium stores. (A): A line scan of a representative spontaneously beating 30–40 dpb human embryonic stem cell-derived cardiomyocyte (hESC-CM) (confocal image displayed in the upper left panel). Shown is the calcium imaging tracing during the response to rapid 10 mM caffeine application. Note the initial local bolus release of calcium initiating a calcium wave propagation, which is followed by the disappearance of calcium transients and finally by the reappearance of these transients, which gradually increase in amplitude. (B): Two-dpb hESC-CMs showing similar caffeine responsiveness. (C): Caffeine-releasable calcium increases progressively during in vitro maturation. The caffeine transient amplitude was normalized to preceding mean AP-Ca transients amplitude. One-way analysis of variance with Tukey post hoc tests: *, p = .005 for 10 dpb, and #, p < .001 for 30–40 dpb, compared with 2 dpb. n = 6, 6, and 10 for 2, 10, and 30–40 dpb, respectively. Box and whisker plot shows 25%–75% level (box); vertical lines display the range. (D): Response of the local calcium events to a brief application of 10 mM caffeine. Abbreviations: dpb, days post-beating; F/Fo, fluorescence (F) normalized to baseline fluorescence (Fo); s, seconds. We next continued to evaluate the presence of caffeine-sensitive stores in hESC-CMs of earlier developmental stages. To this end, three different age groups were studied: with the initiation of spontaneous beating activity (2 dpb; early stage), at 10 dpb (intermediate stage), and at 30–40 dpb (late stage). Interestingly, even at the earliest stage of differentiation (2 dpb), the hESC-CMs were already responsive to a puff of 10 mM caffeine (n = 7) (Fig. 5B). To provide additional information regarding developmental changes in the intracellular calcium stores, we also assessed potential differences in the amplitude of the caffeine-induced calcium transient (normalized and expressed as the relative increase over the normal AP-induced calcium transient) between the three developmental groups. As can be noted in Figure 5C, a statistically significant increase in this value was noted during hESC-CM maturation (p = .005 for 10 dpb and p < .001 for 30–40 dpb compared with 2 dpb), suggesting an increase in the calcium-store content over time. Spontaneously beating hESC-CMs clusters with a relatively slow interbeat interval of ∼9 seconds unmasked the rhythmic nature of local calcium events and allowed us to test whether local calcium events (sparks) are also sensitive to caffeine. Rapid caffeine application elicited a large release of calcium, followed by reversible quiescence of local calcium events (Fig. 5D). In summary, caffeine, which acts by reversibly depleting stored calcium, prevented both local calcium events and AP-Ca transients. Finally, to determine whether store calcium uses RyR-mediated release, we also tested the effect of ryanodine, a specific inhibitor of RyR. Three micromolar ryanodine had no effect, but 10 μM ryanodine acted slowly (>5 minutes) to progressively decrease the amplitude of calcium release (Fig. 6A, 6B; p = .004; n = 10). Figure 6. Open in new tabDownload slide Response of the calcium transients to bulk flow of Ry. (A): Average amplitude recordings of calcium transients from a small intact cluster under control conditions (top row, left), after 1 minute of 10 μM Ry application (middle), and after 6 minutes of continual Ry exposure (bottom row, left). Line scans are shown for control conditions (top row, right) and 6 minutes of continual Ry exposure (bottom row, right), with the arrow indicating position for the calcium transient plots shown to the left. (B): Pooled data of the difference in amplitude between <2-minute or >5-minute Ry and control. Abbreviations: F/Fo, fluorescence (F) normalized to baseline fluorescence (Fo); Ry, ryanodine; s, seconds. Figure 6. Open in new tabDownload slide Response of the calcium transients to bulk flow of Ry. (A): Average amplitude recordings of calcium transients from a small intact cluster under control conditions (top row, left), after 1 minute of 10 μM Ry application (middle), and after 6 minutes of continual Ry exposure (bottom row, left). Line scans are shown for control conditions (top row, right) and 6 minutes of continual Ry exposure (bottom row, right), with the arrow indicating position for the calcium transient plots shown to the left. (B): Pooled data of the difference in amplitude between <2-minute or >5-minute Ry and control. Abbreviations: F/Fo, fluorescence (F) normalized to baseline fluorescence (Fo); Ry, ryanodine; s, seconds. IP3-Mediated Calcium Release in hESC-CMs It was recently demonstrated that mouse ESC-CMs require IP3R-mediated calcium release for spontaneous activity [16, 17]. We therefore tested for IP3R expression, localization, and function in 30–40 dpb hESC-CMs. Quantitative RT-PCR showed IP3R2 expression, appearing with cardiomyocyte differentiation and then gradually declining with maturation (Fig. 7A). Immunostaining of these hESC-CMs revealed positive IP3R and calreticulin staining with a subcellular distribution that was similar to that reported in neonatal rat cardiomyocytes [23] and mouse ESC-CMs [17] (Fig. 7B). This was manifested by the colocalization of the IP3R and the much denser calreticulin immunosignal around the nucleus. Figure 7. Open in new tabDownload slide IP3R localization and function in human embryonic stem cell-derived cardiomyocytes (hESC-CMs). (A): Quantitative reverse transcription-PCR shows appearance of IP3R2 mRNA with beating and then a progressive decline of mRNA with maturation. (B): Confocal micrograph of a representative isolated cell costained with antibodies for pan-IP3R (middle panel) and calreticulin (left panel). Merged images are presented in right panel. The nucleus is highlighted in blue by 4,6-diamidino-2-phenylindole staining. (C): A line scan displaying localized inositol-1,4,5-trisphosphate (IP3)-induced calcium release (upper left) by photolysis of 20 μM caged IP3 (indicated by arrow). The tracing below corresponds to the region indicated by the arrow in the line scan. On the right is shown a region expanded on time base illustrating a higher magnification of the IP3-dependent signals. (D): A line scan tracing presenting IP3-induced calcium release in the presence of 20 μM TTX. (E): An example of a cell in which application of 2-APB (2 μM) completely and reversibly blocked all calcium transients. (F): An example of a cell in which application of 2-APB decreased action-potential-initiated calcium transients. (G): Summary of the reduction in calcium transient amplitudes following application of 2-APB in the hESC-CMs tested (*, p = .0006, paired t test). Abbreviations: dpb, days post-beating; F/Fo, fluorescence (F) normalized to baseline fluorescence (Fo); IP3R, inositol-1,4,5-trisphosphate receptor; s, seconds. Figure 7. Open in new tabDownload slide IP3R localization and function in human embryonic stem cell-derived cardiomyocytes (hESC-CMs). (A): Quantitative reverse transcription-PCR shows appearance of IP3R2 mRNA with beating and then a progressive decline of mRNA with maturation. (B): Confocal micrograph of a representative isolated cell costained with antibodies for pan-IP3R (middle panel) and calreticulin (left panel). Merged images are presented in right panel. The nucleus is highlighted in blue by 4,6-diamidino-2-phenylindole staining. (C): A line scan displaying localized inositol-1,4,5-trisphosphate (IP3)-induced calcium release (upper left) by photolysis of 20 μM caged IP3 (indicated by arrow). The tracing below corresponds to the region indicated by the arrow in the line scan. On the right is shown a region expanded on time base illustrating a higher magnification of the IP3-dependent signals. (D): A line scan tracing presenting IP3-induced calcium release in the presence of 20 μM TTX. (E): An example of a cell in which application of 2-APB (2 μM) completely and reversibly blocked all calcium transients. (F): An example of a cell in which application of 2-APB decreased action-potential-initiated calcium transients. (G): Summary of the reduction in calcium transient amplitudes following application of 2-APB in the hESC-CMs tested (*, p = .0006, paired t test). Abbreviations: dpb, days post-beating; F/Fo, fluorescence (F) normalized to baseline fluorescence (Fo); IP3R, inositol-1,4,5-trisphosphate receptor; s, seconds. To evaluate the potential functional role of IP3R in modulating calcium handling, we studied the effects of both IP3R blockade and activation. Initially, to test whether IP3R are indeed functional, caged-IP3 photolysis was used. We chose to work with this compound, despite the complex experimental protocol, to allow rapid increase of IP3 concentration in close vicinity of the SR. Since caged IP3 is not cell-permeant, we used a modified saponin-permeabilization protocol [21] to allow intracellular loading of the compound. The method is reported to allow permeabilization with minimal effects on cell viability or physiology. Following intracellular loading, we used UV flash to release the caged IP3 in a spatially defined manner (along the line-scan). In four of seven clusters tested, uncaging the caged IP3 (20 μM) resulted in a localized calcium release (Fig. 7C). Because of the small dimensions of the caged IP3 induced calcium release, it was sometimes difficult to distinguish the small IP3-dependent calcium signals from the large propagating AP-induced calcium transients. We therefore performed additional experiments in which TTX (20 μM) was used to inhibit AP-induced calcium-transients. This allowed the observation of a localized IP3-induced calcium release with minimal distortion (Fig. 7D). We next assessed the effect of IP3R blockade on hESC-CMs calcium handling. To this end, hESC-CMs were exposed to a low concentration of 2-APB (2 μM), a well-known membrane-permeant IP3 antagonist. 2-APB application completely eliminated (in a reversible manner) all calcium transients in 4 of 22 cells tested (Fig. 7E) and significantly decreased AP-Ca transient amplitude, by 30% ± 20% in 18 of 22 cells tested (p = .0006; paired t-test) (Fig. 7F, 7G). Discussion For an hESC-CM to become a bona fide working heart muscle cell that can be used in future cell replacement therapy strategies, it is required to possess functional contractile proteins, calcium handling, and excitability properties. In this work we focused on studying the calcium handling properties of developing hESC-CMs. Our major findings include the following: (a) mature molecular components CaV1.2, CaVβ2, RyR2, and IP3R are expressed; (b) there are two types of calcium transients, periphery-initiated local calcium events (sparks) and cell-wide AP-driven calcium transients; (c) caffeine- and ryanodine-sensitive calcium stores are required for both modes of calcium transients; (d) the AP-Ca transients traverse the width of the cell via a propagated wave of intracellular store calcium release; (e) caffeine-sensitive calcium stores are present and functional from the initiation of spontaneous beating, and SR calcium load increases during in vitro maturation; and (f) an IP3-releasable calcium pool is expressed and functional. We conclude that despite the relatively brief maturation period, <40-dpb hESC-CMs express functional intracellular calcium handling components, albeit in a spatially somewhat immature state. AP-Ca Transient Spread In mature mammalian ventricular cells, the highly organized t-tubule system serves as a pathway enabling uniform transmission of sarcolemmal depolarization and a nearly synchronous SR calcium release throughout the interior of the cell [24–26]. Consequently, line-scan images of fluo-4- and Di-8-ANEPPS-labeled adult rat ventricular cells show AP-triggered whole-cell calcium transients with onsets that appear to be aligned as straight horizontal bands, indicating near-simultaneous release of calcium throughout the cell [19]. Ultrastructural analysis of the hESC-CMs demonstrated that although some t-tubules can be identified in these cells, they do not display a highly developed and organized t-tubule system typical of adult ventricular myocytes. As a result, the AP-Ca transients in the hESC-CMs are characterized by an initial rise in intracellular calcium originating at the PM and then spreading to the center of the cell with a delay via a propagated wave of intracellular store calcium release. In this regard, these cells may resemble the observations made in guinea pig [27], cat [28] and rat [19] adult atrial cells. Local Calcium Events An interesting phenomenon noted in this study was the presence of local calcium events that were of lower amplitude and higher frequency than the AP-Ca transients. Spontaneous local calcium events (sparks) are common findings in several cardiomyocyte preparations and are usually observed in areas where RyRs are located in the immediate vicinity of the sarcolemmal membrane [19, 28–30]. Kirk et al. [19] showed that in adult rat atrial cells most local calcium events take place within 1 μm of either the sarcolemmal membrane or in close apposition to the interior transverse-axial tubular system. To determine the spatial association of the local calcium events identified in the hESC-CMs, we performed simultaneous fluo-4 (for calcium imaging) and Di-8ANEPPS (for membrane localization) imaging. The origination of the local calcium events was found to be confined to the cell periphery in a similar manner to that in adult cat atrial myocytes, rabbit Purkinje cells, and neonatal rat myocytes [28–30]. The local calcium events were not associated with calcium wave propagation throughout the cells. This phenomenon argues that release units in these cells are separated by critical distances [31]. Caffeine-Sensitive SR Calcium Stores In contrast to the adult heart, the contribution of SR calcium release to calcium handling in embryonic and fetal cardiomyocytes remains controversial. Several studies have reported the SR to be structurally and functionally underdeveloped in fetal cardiomyocytes, displaying a lower volume, resulting in a limited capacity to load calcium [32, 33]. In adult myocardium, the SR is the main intracellular calcium store responsible for the release and uptake of approximately 70%–90% of cytosolic calcium [34]. SR calcium release, via RyR2, plays a critical role in the regulation of the CICR mechanism and greatly contributes to EC coupling. We used caffeine as a tool to determine the presence of SR calcium in hESC-CMs. The mechanism of caffeine release of store calcium is via an increased sensitivity of the RyR2 to calcium [35], which increases RyR2 opening, manifesting in a single large calcium transient that decays within seconds. In hESC-CMs a local puff of 10 mM caffeine clearly elicits a local bolus release of calcium (Fig. 5), and this release then initiates calcium wave propagation, similar to that of mature rat ventricular myocytes [36]. The caffeine-induced bolus release results in SR calcium depletion, leading to the disappearance of both AP-Ca transients and local calcium events, suggesting an important role for SR calcium in the generation of both transients. This is followed by the reappearance and a gradual increase (Fig. 5A) in the amplitude of the AP-Ca transient as calcium gradually loads the SR stores. To further confirm that hESC-CMs calcium stores use RyR-mediated release, we tested the effect of ryanodine application. Ryanodine administration slowly and progressively decreased the amplitude of calcium release (Fig. 6A, 6B) in the hESC-CMs in a manner similar to that reported in mouse ESC-CMs (10 μM, 30 minutes) [37]. Increase in SR Calcium Load As a Function of In Vitro Maturation Another important finding of the current study is that RyR-mediated calcium stores are operational from the earliest stages of hESC-CM development (simultaneous with the initiation of spontaneous beating and prior to completion of the electrophysiological maturation process). Hence, we were able to demonstrate the presence of caffeine-induced calcium release in all age groups studied (2, 10, and 30–40 dpb). The relative amplitude of caffeine-induced calcium transients is considered a descriptive index of the level of SR calcium load [38]. It is well appreciated that caffeine is capable of triggering an additional amount of calcium release that is not normally released during an AP-initiated calcium transient. To provide additional information regarding developmental changes in the intracellular calcium stores, we assessed for potential differences in the amplitude of the caffeine-induced calcium transient (normalized and expressed as the relative increase over the normal averaged AP-induced calcium transient) among the three developmental groups. Our results suggest that caffeine-sensitive calcium stores increase in calcium load during in vitro maturation. Similar results were also obtained in the mouse-ESC model [16], where RyR-mediated calcium stores were present and functional from the onset of spontaneous activity and their contribution to the AP-initiated calcium transients increased with temporal cell maturation. In contradiction to our caffeine application results, a prior study by Dolnikov et al. [10] reported the lack of caffeine effect in hESC-CM EBs of a similar age range. We speculate that the difference in the acquired results may stem from the different experimental setups. For example, different perfusion techniques and experimental preparations were used. In the current study we found gravitational caffeine perfusion to be a inefficient method for rapid caffeine application. To overcome the technical limitation, a caffeine puff (10 mM, <2 seconds) was applied by pressure ejection through a pipette located approximately 100 μm away from the target cell, promising immediate and localized exposure of the target cell or cluster to the applied caffeine. Moreover, Dolnikov et al. studied whole portions of EBs and used nonconfocal fluorescent microscopy for calcium imaging [10]. The possibility exists that caffeine did not have sufficient access to cardiac myocytes in the interior of the EBs. In this work we studied single cells and small monolayered clusters (using laser-confocal calcium imaging) promising direct contact of most of the cells' surface with the applied caffeine. IP3R Expression and Function in hESC-CMs In electrically nonexcitable cells, IP3-dependent calcium release constitutes the fundamental pathway of intracellular calcium release [39]. Although a few studies have demonstrated the expression of IP3Rs (more in atrial cells and less so in ventricular myocytes) and IP3-dependent calcium release in adult cardiac tissue [40, 41], their functional importance in cardiac physiology has remained elusive and controversial [42–46]. Nevertheless, IP3-dependent calcium signaling seems to play an important role during cardiac development [14, 15]. Furthermore, IP3R are reported to be the first calcium release channels expressed in embryos [14, 43] and have been reported to contribute to spontaneous activity in mouse ESC-CMs [16, 17]. Méry et al. [17], for example, demonstrated that depolarization of early embryonic pacemaker cells is triggered by IP3-sensitive intracellular calcium stores in the ER. To evaluate the potential role of IP3 pathway in affecting calcium handling in hESC-CMs, we first demonstrated the presence of the IP3R at both the mRNA and protein level. Immunostaining for IP3R and calreticulin resulted in intense staining around the vicinity of the nucleus as previously observed in neonatal rat cardiomyocytes and in mESC-CMs [17, 23]. Next, to test whether these IP3R are functional and can affect calcium handling we tested both an antagonist approach and an agonist approach. To block IP3R we used the cell-permeant compound 2-APB. We chose to study only a relatively low concentration (2 μM) of this compound because higher concentrations were found to be less specific and to affect several other cellular targets [47, 48]. Application of 2-APB had a significant blocking effect (either completely abolishing or significantly reducing the amplitude of the intracellular calcium transients). To evaluate the agonist effect on IP3R we used caged-IP3 photolysis. Despite the complex protocol associated with its intracellular loading (saponin permeabilization) and uncaging technique, we chose to work with caged IP3 to overcome potential diffusion delays and to allow rapid increase of IP3 concentrations in close vicinity of the SR. Uncaging of the caged IP3 resulted in the development of a spatially defined calcium transient. This elementary nonpropagating, spatially restricted nature of IP3R-dependent calcium release events was also previously reported in neonatal rat cardiomyocytes [23], oocytes [49], Hela cells [50], and vascular endothelial cells [51]. Conclusion In order for hESC-CMs to ultimately serve as cell source for cardiovascular regenerative medicine, successful functional and structural integration with the diseased host myocardium must take place. Such functional and structural compatibility requires hESC-CMs to possess cellular/molecular functional properties that resemble a mature cardiomyocyte phenotype. Previous ultrastructure studies show that hESC-CMs have intact contractile machinery in the form of myofibrillar strands [2, 7] and functional ion channels typical of CMs [8, 9]. In this study we show that hESC-CMs express functional RyR regulated intracellular calcium stores from the initiation of spontaneous beating activity and that these calcium stores increase in calcium content with in vitro maturation. We show the presence of both AP-Ca transients and local calcium events, both of which require the caffeine-sensitive, ryanodine-sensitive stores. In addition, we present evidence suggesting the presence and functionality of an IP3-releasable pool in <40-dpb hESC-CMs. The relatively immature spatial organization of these cells, however, leads to a unique phenotype. In this respect, the AP-Ca transient origination and spread and local calcium events are similar to those reported in adult atrial CMs models lacking an orderly t-tubule system [19]. Many obstacles still need to be overcome to realize the potential benefits of hESC-CMs for cell replacement therapy [6]. Nevertheless, the evidence gathered in recent years with regards to hESC-CM cell expandability; their ability to integrate functionally with host cardiac tissue [52]; and their cardiac-specific contractile apparatus [3], excitability [4], and calcium handling (as shown here) makes them an attractive candidate for this exciting field. However, additional studies are warranted to provide a better understanding of the properties of these cells both in vitro and in vivo. Acknowledgements We thank the Lady Davis Foundation (J.S.). This work was partially funded by the NIH (HL074091; to J.S.), Israel Science Foundation (520/01; L.G.), the ISF-Converging Technologies (1781/07; to L.G. and J.S.), and the Nancy & Stephen Grand Philanthropic Fund. We thank Drs. Edith Suss-Toby and Ofer Shenkar and the multidisciplinary laboratories (microscopy and imaging unit) for support. Disclosure of Potential Conflicts of Interest The authors indicate no potential conflicts of interest. References 1 Thomson JA , Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts . Science 1998 ; 282 : 1145 – 1147 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Kehat I , Kenyagin-Karsenti D, Snir M et al. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes . J Clin Invest 2001 ; 108 : 407 – 414 . Google Scholar Crossref Search ADS PubMed WorldCat 3 Mummery C , Ward-van Oostwaard D, Doevendans P et al. Differentiation of human embryonic stem cells to cardiomyocytes: Role of coculture with visceral endoderm-like cells . Circulation 2003 ; 107 : 2733 – 2740 . Google Scholar Crossref Search ADS PubMed WorldCat 4 He JQ , Ma Y, Lee Y et al. Human embryonic stem cells develop into multiple types of cardiac myocytes: Action potential characterization . Circ Res 2003 ; 93 : 32 – 39 . Google Scholar Crossref Search ADS PubMed WorldCat 5 Xu C , Police S, Rao N et al. Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells . Circ Res 2002 ; 91 : 501 – 508 . Google Scholar Crossref Search ADS PubMed WorldCat 6 Gepstein L . Derivation and potential applications of human embryonic stem cells . Circ Res 2002 ; 91 : 866 – 876 . Google Scholar Crossref Search ADS PubMed WorldCat 7 Snir M , Kehat I, Gepstein A et al. Assessment of the ultrastructural and proliferative properties of human embryonic stem cell-derived cardiomyocytes . Am J Physiol Heart Circ Physiol 2003 ; 285 : H2355 – H2363 . Google Scholar Crossref Search ADS PubMed WorldCat 8 Satin J , Kehat I, Caspi O et al. Mechanism of spontaneous excitability in human embryonic stem cell derived cardiomyocytes . J Physiol 2004 ; 559 : 479 – 496 . Google Scholar Crossref Search ADS PubMed WorldCat 9 Sartiani L , Bettiol E, Stillitano F et al. Developmental changes in cardiomyocytes differentiated from human embryonic stem cells: A molecular and electrophysiological approach . Stem Cells 2007 ; 25 : 1136 – 1144 . Google Scholar Crossref Search ADS PubMed WorldCat 10 Dolnikov K , Shilkrut M, Zeevi-Levin N et al. Functional properties of human embryonic stem cell-derived cardiomyocytes: Intracellular Ca2+ handling and the role of sarcoplasmic reticulum in the contraction . Stem Cells 2006 ; 24 : 236 – 245 . Google Scholar Crossref Search ADS PubMed WorldCat 11 Viatchenko-Karpinski S , Fleischmann BK, Liu Q et al. Intracellular Ca2+ oscillations drive spontaneous contractions in cardiomyocytes during early development . Proc Natl Acad Sci U S A 1999 ; 96 : 8259 – 8264 . Google Scholar Crossref Search ADS PubMed WorldCat 12 Yang H-T , Tweedie D, Wang S et al. The ryanodine receptor modulates the spontaneous beating rate of cardiomyocytes during development . Proc Natl Acad Sci U S A 2002 ; 99 : 9225 – 9230 . Google Scholar Crossref Search ADS PubMed WorldCat 13 Sauer H , Theben T, Hescheler J et al. Characteristics of calcium local Ca events in cardiomyocytes derived from embryonic stem cells . Am J Physiol Heart Circ Physiol 2001 ; 281 : H411 – H421 . Google Scholar Crossref Search ADS PubMed WorldCat 14 Rosemblit N , Moschella MC, Ondriasá E et al. Intracellular calcium release channel expression during embryogenesis . Dev Biol 1999 ; 206 : 163 – 177 . Google Scholar Crossref Search ADS PubMed WorldCat 15 Poindexter BJ , Smith JR, Buja LM et al. Calcium signaling mechanisms in dedifferentiated cardiac myocytes: Comparison with neonatal and adult cardiomyocytes . Cell Calcium 2001 ; 30 : 373 – 382 . Google Scholar Crossref Search ADS PubMed WorldCat 16 Kapur N , Banach K. Inositol-1,4,5-trisphosphate-mediated spontaneous activity in mouse embryonic stem cell-derived cardiomyocytes . J Physiol 2007 ; 581 : 1113 – 1127 . Google Scholar Crossref Search ADS PubMed WorldCat 17 Méry A , Aimond F, Ménard C et al. Initiation of embryonic cardiac pacemaker activity by inositol 1,4,5-trisphosphate-dependent calcium signaling . Mol Biol Cell 2005 ; 16 : 2414 – 2423 . Google Scholar Crossref Search ADS PubMed WorldCat 18 Kehat I , Amit M, Gepstein A et al. Development of cardiomyocytes from human ES cells . Methods Enzymol 2003 ; 365 : 461 – 474 . Google Scholar Crossref Search ADS PubMed WorldCat 19 Kirk MM , Izu LT, Chen-Izu Y et al. Role of the transverse-axial tubule system in generating calcium local Ca events and calcium transients in rat atrial myocytes . J Physiol 2003 ; 547 : 441 – 451 . Google Scholar Crossref Search ADS PubMed WorldCat 20 Polsky A , Mel BW, Schiller J. Computational subunits in thin dendrites of pyramidal cells . Nat Neurosci 2004 ; 7 : 621 – 627 . Google Scholar Crossref Search ADS PubMed WorldCat 21 Johnson JA , Gray MO, Karliner JS et al. An improved permeabilization protocol for the introduction of peptides into cardiac myocytes. Application to protein kinase C research . Circ Res 1996 ; 79 : 1086 – 1099 . Google Scholar Crossref Search ADS PubMed WorldCat 22 Lukyanenko V , Ziman A, Lukyanenko A et al. Functional groups of ryanodine receptors in rat ventricular cells . J Physiol 2007 ; 583 : 251 – 269 . Google Scholar Crossref Search ADS PubMed WorldCat 23 Jaconi M , Bony C, Richards SM. Inositol 1,4,5-trisphospate directs Ca(2+) flow between mitochondria and the endoplasmic/sarcoplasmic reticulum: A role in regulating cardiac autonomic Ca(2+) spiking . Mol Biol Cell 2000 ; 11 : 1845 – 1858 . Google Scholar Crossref Search ADS PubMed WorldCat 24 Cheng H , Cannell MB, Lederer WJ. Propagation of excitation-contraction coupling into ventricular myocytes . Pflugers Arch 1994 ; 428 : 415 – 417 . Google Scholar Crossref Search ADS PubMed WorldCat 25 Shacklock PS , Wier WG, Balke CW. Local Ca2+ transients (Ca2+ local Ca events) originate at transverse tubules in rat heart cells . J Physiol 1995 ; 487 : 601 – 608 . Google Scholar Crossref Search ADS PubMed WorldCat 26 Wier WG , Lopez-Lopez JR, Shacklock PS et al. Calcium signaling in cardiac muscle cells . Ciba Found Symp 1995 ; 188 : 146 – 160 ; discussion 160–164. Google Scholar PubMed OpenURL Placeholder Text WorldCat 27 Berlin JR . Spatiotemporal changes of Ca2+ during electrically evoked contractions in atrial and ventricular cells . Am J Physiol 1995 ; 269 : H1165 – H1170 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 28 Hüser J , Lipsius SL, Blatter LA. Calcium gradients during excitation-contraction coupling in cat atrial myocytes . J Physiol 1996 ; 494 : 641 – 651 . Google Scholar Crossref Search ADS PubMed WorldCat 29 Löhn M , Furstenau M, Sagach V et al. Ignition of calcium local Ca events in arterial and cardiac muscle through caveolae . Circ Res 2000 ; 87 : 1034 – 1039 . Google Scholar Crossref Search ADS PubMed WorldCat 30 Cordeiro JM , Spitzer KW, Giles WR et al. Location of the initiation site of calcium transients and local Ca events in rabbit heart Purkinje cells . J Physiol 2001 ; 531 : 301 – 314 . Google Scholar Crossref Search ADS PubMed WorldCat 31 Izu LT , Means SA, Shadid JN et al. Interplay of ryanodine receptor distribution and calcium dynamics . Biophys J 2006 ; 91 : 95 – 112 . Google Scholar Crossref Search ADS PubMed WorldCat 32 Pegg W , Michalak M. Differentiation of sarcoplasmic reticulum during cardiac myogenesis . Am J Physiol 1987 ; 252 : H22 – H31 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 33 Nakanishi T , Seguchi M, Takao A. Development of the myocardial contractile system . Experientia 1988 ; 44 : 936 – 944 . Google Scholar Crossref Search ADS PubMed WorldCat 34 Bers DM , Despa S. Cardiac myocytes Ca(2+) and Na(+) regulation in normal and failing hearts . J Pharmacol Sci 2006 ; 100 : 315 – 322 . Google Scholar Crossref Search ADS PubMed WorldCat 35 Rousseau E , Meissner G. Single cardiac sarcoplasmic reticulum Ca2+-release channel: Activation by caffeine . Am J Physiol 1989 ; 256 : H328 – H333 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 36 Trafford AW , Lipp P, O'Neill SC et al. Propagating calcium waves initiated by local caffeine application in rat ventricular myocytes . J Physiol 1995 ; 489 : 319 – 326 . Google Scholar Crossref Search ADS PubMed WorldCat 37 Fu JD , Li J, Tweedie D et al. Crucial role of the sarcoplasmic reticulum in the developmental regulation of Ca2+ transients, contraction in cardiomyocytes derived from embryonic stem cells . FASEB J 2006 ; 20 : 181 – 183 . Google Scholar Crossref Search ADS PubMed WorldCat 38 Weber AM , Herz R. The relationship between caffeine contracture of intact muscle and the effect of caffeine on isolated reticulum . J Gen Physiol 1968 ; 52 : 750 – 759 . Google Scholar Crossref Search ADS PubMed WorldCat 39 Berridge MJ . Elementary and global aspects of calcium signalling . J Physiol 1997 ; 499 : 291 – 306 . Google Scholar Crossref Search ADS PubMed WorldCat 40 Hirata M , Suematsu E, Hashimoto T et al. Release of Ca2+ from a non-mitochondrial store site in peritoneal macrophages treated with saponin by inositol 1,4,5-trisphosphate . Biochem J 1984 ; 223 : 229 – 236 . Google Scholar Crossref Search ADS PubMed WorldCat 41 Nosek TM , Williams MF, Zeigler ST et al. Inositol trisphosphate enhances calcium release in skinned cardiac and skeletal muscle . Am J Physiol 1986 ; 250 : C807 – C811 . Google Scholar Crossref Search ADS PubMed WorldCat 42 Gutstein DE , Marks AR. Role of inositol 1,4,5-trisphosphate receptors in regulating apoptotic signaling and heart failure . Heart Vessels 1997 , ( suppl 12 ): 53 – 57 . Google Scholar OpenURL Placeholder Text WorldCat 43 Moschella MC , Marks AR. Inositol 1,4,5-trisphosphate receptor expression in cardiac myocytes . J Cell Biol 1993 ; 120 : 1137 – 1146 . Google Scholar Crossref Search ADS PubMed WorldCat 44 Woodcock EA , Matkovich SJ, Binah O. Ins(1,4,5)P3 and cardiac dysfunction . Cardiovasc Res 1998 ; 40 : 251 – 256 . Google Scholar Crossref Search ADS PubMed WorldCat 45 Marks AR . Cardiac intracellular calcium release channels: Role in heart failure . Circ Res 2000 ; 87 : 8 – 11 . Google Scholar Crossref Search ADS PubMed WorldCat 46 Blatter LA , Kockskämper J, Sheehan KA et al. Local calcium gradients during excitation-contraction coupling and alternans in atrial myocytes . J Physiol 2003 ; 546 : 19 – 31 . Google Scholar Crossref Search ADS PubMed WorldCat 47 Bootman MD , Collins TJ, Mackenzie L et al. 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 – 1150 . Google Scholar Crossref Search ADS PubMed WorldCat 48 Peppiatt CM , Collins TJ, Mackenzie L et al. 2-Aminoethoxydiphenyl borate (2-APB) antagonises inositol 1,4,5-trisphosphate-induced calcium release, inhibits calcium pumps and has a use-dependent and slowly reversible action on store-operated calcium entry channels . Cell Calcium 2003 ; 34 : 97 – 108 . Google Scholar Crossref Search ADS PubMed WorldCat 49 Parker I , Yao Y. Ca2+ transients associated with openings of inositol trisphosphate-gated channels in Xenopus oocytes . J Physiol 1996 ; 491 : 663 – 668 . Google Scholar Crossref Search ADS PubMed WorldCat 50 Bootman M , Niggli E, Berridge M et al. Imaging the hierarchical Ca2+ signalling system in HeLa cells . J Physiol 1997 ; 499 : 307 – 314 . Google Scholar Crossref Search ADS PubMed WorldCat 51 Hüser J , Blatter LA. Elementary events of agonist-induced Ca2+ release in vascular endothelial cells . Am J Physiol 1997 ; 273 : C1775 – C1782 . Google Scholar Crossref Search ADS PubMed WorldCat 52 Kehat I , Khimovich L, Caspi O et al. Electromechanical integration of cardiomyocytes derived from human embryonic stem cells . Nat Biotechnol 2004 ; 22 : 1282 – 1289 . Google Scholar Crossref Search ADS PubMed WorldCat Author notes Author contributions: J. Satin and I.I.: joint first co-authors, conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; S.R. and E.A.S.: collection and/or assembly of data, data analysis and interpretation; L.I.: data analysis and interpretation; G.A.: provision of study material, collection and/or assembly of data; R.B. and C.W.B.: conception and design, final approval of manuscript; J. Schiller: conception and design, collection and/or assembly of data, data analysis and interpretation; L.G.: conception and design, financial support, data analysis and interpretation, manuscript writing, final approval of manuscript. Copyright © 2008 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Calcium Handling in Human Embryonic Stem Cell-Derived Cardiomyocytes
JF - Stem Cells
DO - 10.1634/stemcells.2007-0591
DA - 2008-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/calcium-handling-in-human-embryonic-stem-cell-derived-cardiomyocytes-uWY2W4lVGx
SP - 1961
EP - 1972
VL - 26
IS - 8
DP - DeepDyve
ER -