Since cardiac transplantation is limited by the small availability of donor organs, regeneration of the diseased myocardium by cell transplantation is an attractive therapeutic modality. To determine the compatibility of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) (7 to 55 days old) with the myocardium, we investigated their functional properties regarding intracellular Ca2+ handling and the role of the sarcoplasmic reticulum in the contraction. The functional properties of hESC-CMs were investigated by recording simultaneously [Ca2+]i transients and contractions. Additionally, we performed Western blot analysis of the Ca2+-handling proteins SERCA2, calsequestrin, phospholamban, and Na+/Ca2+ exchanger (NCX). Our major findings are, first, that hESC-CMs displayed temporally related [Ca2+]i transients and contractions, negative force-frequency relations, and lack of post-rest potentiation. Second, ryanodine, thapsigargin, and caffeine did not affect the [Ca2+]i transient and contraction, indicating that at this developmental stage, contraction depends on transsarcolemmal Ca2+ influx rather than on sarcoplasmic reticulum Ca2+ release. Third, in agreement with the notion that a voltage-dependent Ca2+ current is present in hESC-CMs and contributes to the mechanical function, verapamil completely blocked contraction. Fourth, whereas hESC-CMs expressed SERCA2 and NCX at levels comparable to those of the adult porcine myocardium, calsequestrin and phospholamban were not expressed. Our study shows for the first time that functional properties related to intracellular Ca2+ handling of hESC-CMs differ markedly from the adult myocardium, probably due to immature sarcoplasmic reticulum capacity.
Cardiovascular diseases, including congestive heart failure, are the most frequent cause of death in the industrialized world . Although the current pharmacotherapy for congestive heart failure (which includes angiotensin-converting enzyme inhibitors and β-blockers) improves clinical outcomes, these treatment modalities as well as various interventional and surgical therapeutic methods are limited in their efficacy because of their inability to repair or replace damaged myocardium. Given the high morbidity and mortality rates associated with congestive heart failure, shortage of donor hearts for transplantation, complications resulting from immunosuppression, and long-term failure of transplanted organs , novel therapeutic means for improving cardiac function and preventing heart failure are in critical demand. Regeneration or repair of the damaged myocardium can be accomplished by the use of cell therapy, which is the transplantation of healthy, functional, and propagating cells to restore the viability or function of deficient tissues . In this regard, recent studies have shown that improvement of myocardial function can be achieved in experimental animal models of heart failure and infarction by transplanting stem cell–derived cardiomyocytes into the compromised myocardium .
Stem cells are fundamentally characterized by prolonged self-renewal and long-term potential to form differentiated cell types. Human embryonic stem cells (hESCs), which were derived from human preimplantation embryos at the blastocyst stage , were demonstrated to fulfill all of the criteria defining embryonic stem cells: immortality, capability to proliferate indefinitely in culture while maintaining the undifferentiated phenotype, and capacity to form derivatives of all three germ layers . Unlike other types of stem cells, embryonic stem cells can be easily induced to differentiate into spontaneously contracting cardiac-like cells, although the efficacy of this process is low . It was recently demonstrated that these contracting cells consist of functional syncytium with a spontaneous pacemaker activity and consequent action potential propagation . Further, transmembrane action potentials were recorded from these cells, and the mechanisms underlying action potential generation were investigated .
To improve the prospects of cardiac cell transplantation, it is widely realized that the functional properties as well as the hormonal and pharmacological responsiveness of hESC-derived cardiomyocytes (hESC-CMs) should be thoroughly investigated. Since it is desired that the transplanted cells fully integrate within the diseased myocardium, contribute to its contractile performance, and respond appropriately to various stimuli (e.g., β-adrenergic stimulation), it is important to decipher their compatibility with the host myocardium.
Whereas in recent studies the electrophysiological properties of hESC-CMs have been investigated in considerable depth and action potential properties and major ion currents were described [8 –10], much less is known about the downstream events of the excitation-contraction (E-C) coupling. Therefore, the major aim of this study was to investigate the functional properties of hESC-CMs regarding intracellular Ca2+ handling and the role of the sarcoplasmic reticulum (SR) in the contraction process. Our major findings show that the E-C coupling machinery of hESC-CMs is different from that of the mature myocardium, mainly due to a dysfunctional SR Ca2+ release capacity and a total dependency on extra-cellular Ca2+ for contraction. Given the attractive application of hESC-CMs in cell transplantation, this potential source for cell therapy should be further developed to attain functional compatibility with the adult myocardium.
Materials and Methods
Human Embryonic Stem Cells: Culture and Differentiation
hESCs from clones H9.2 and I3 were grown on mouse embryonic fibroblast feeders (MEFs) in 80% knockout Dulbecco's modified Eagle's medium, 20% knockout serum replacement, 4 ng/ml basic fibroblast growth factor, 1 mmol/l glutamine, 0.1 mmol/l β-mercaptoethanol, and 1% nonessential amino acid stock (all from Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com). To induce embryoid body (EB) formation, hESCs were detached using 1 mg/ml type IV collagenase (Gibco-BRL) and transferred to Petri dishes to allow their aggregation. Resultant EBs were grown in 80% knockout Dulbecco's modified Eagle's medium, 20% fetal bovine serum defined (HyClone, Logan, UT, http://www.hyclone.com), 1 mmol/l glutamine, and 1% nonessential amino acid stock. The EBs were then cultured in suspension for 7 days and plated on gelatin-coated (0.1%; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) 24-well plates. Daily microscopic observations were conducted to detect the first spontaneous contractions. The contracting areas were then carefully dissected out by microscalpel and transferred to gelatin-coated 30-mm glass slides suitable for fluorescent measurements (Fisher Scientific International, Hampton, NH, http://www.fisherscientific.com). In the present study, we investigated 7- to 55-day-old EBs.
Adult Mouse Ventricular Myocytes
Ventricular myocytes from adult ICR mice were obtained by an enzymatic dissociation procedure as previously described .
Measurement of [Ca2+]i Transients and EB Contractions
[Ca2+]i transients and EB contractions were measured by means of Fura-2 fluorescence and video edge detector, respectively, routinely used in our laboratory . Briefly, spontaneously contracting areas with a diameter range of 0.5 to 1 mm were mechanically dissected out of the entire EB and adhered onto 30-mm-diameter glass slides. Subsequently, the contracting EBs were loaded for 25 minutes at room temperature with Fura 2-AM (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) at a final concentration of 5 μM. Fura 2-stained EBs were transferred to a chamber mounted on the stage of an inverted microscope and perfused with Tyrode's solution containing (in mmol/l) NaCl 140, KCl 5.4, MgCl2 1, sodium pyruvate 2, CaCl2 1, HEPES 10, glucose 10 (pH 7.4 with NaOH) at a rate of 1.5 to 2 ml/min at 37°C. EBs were stimulated at different rates by means of platinum wires embedded in the walls of the superfusion chamber. Fura-2 fluorescence was monitored (at a sampling rate of 100 Hz) by alternately illuminating the preparation with 340- and 380-nm light while measuring the emission at 510 nm (Photon Technologies Instruments, Princeton, NJ). The emitted fluorescence was detected by means of the Felix software (Photon Technology International, Birmingham, NJ, http://www.pti-nj.com), and the raw data were stored for offline analysis as the 340- and 380-nm counts and as the ratio R = F340/F380. To characterize [Ca2+]i transients, the maximal (systolic) ratio and minimal (diastolic) ratio were measured in 10 successive transients and averaged. To simultaneously record contractions and [Ca2+]i transients, the EB was illuminated with red light, and a dichroic mirror (630-nm cutoff) in the emission path deflected the EB image to a video optical system (Crescent Electronics), which tracked the motion of the edges during contractions. The motion signal was obtained at a rate of 60 Hz, and the digitized signal was stored along with the fluorescence data. Because contracting EBs have an irregular 3D structure, and unlike adult ventricular myocytes do not shorten along a single axis of contraction, the output of the video edge detector, which is expressed in arbitrary units, is not linearly indicative of the force of contraction. To characterize the contraction amplitude, the differences between minimal and maximal video cursor position (LAmp) were measured in 10 successive transients and averaged. Additionally, the maximal rate of contraction (dL/dtContrac) and the maximal rate of relaxation (dL/dtRelax) were calculated.
Measurements of Protein Expression
Western blot analysis was performed on hESC-CMs and left ventricular porcine myocardium lysates (the latter served as control) treated with phosphatase and protease inhibitors. Porcine lysates were prepared by three brief homogenizations of 1 to 1.5 g of freshly frozen trimmed myocardial tissue in ice-cold RIPA buffer with freshly added inhibitors. Homogenized tissue was centrifuged twice at 13,000 rpm for 20 minutes. For hESC-CMs lysate preparation, only spontaneously contracting EBs were used. To minimize contamination of cardiomyocyte lysate with proteins from cells of noncardiac origin, spontaneously contracting areas were carefully dissected from five to eight EBs, rinsed in phosphate-buffered saline, placed in ice-cold RIPA buffer with inhibitors, and transferred several times through a 21-gauge needle. The total cell lysate was obtained after 30 minutes of incubation on ice and a subsequent centrifugation at 13,000 rpm for 20 minutes. Protein concentration was determined by the Bradford assay. Two micrograms of total porcine protein and 50 μg of total hESC-CMs protein were loaded on 7.5% SDS-polyacrylamide gel, followed by electrophoretic transfer to nitrocellulose membranes. The following antibodies were used for the Western blot analysis: anti-calse-questrin (PA1–913; ABR Affinity BioReagents, Golden, CO, http://www.bioreagents.com; 1:2,500 dilution), anti-SERCA2 (MA3–910, ABR Affinity BioReagents; 1:2,500), anti-phospholamban (MA3–922, ABR Affinity BioReagents; 1:500), anti-Na+/Ca2+ exchanger (NCX) (MA3-926, ABR Affinity BioReagents; 1:1,000), and anti-sarcomeric-α-actinin (Sigma-Aldrich Israel, Rehovot, Israel, http://www.sigmaaldrich.com; 1:500). Immune complexes were detected using the enhanced chemiluminescence detection system (Biological Industries, Beit Ha'Emek, Israel, http://www.bioind.com) with a secondary antibody coupled to horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) followed by autoradiography.
To characterize postrest potentiation in hESC-CMs and in adult mouse ventricular myocytes, the regular stimulation protocol at 1 Hz was interrupted by pauses (each at a time) of varying lengths (5, 10, 30, and 60 seconds), followed by resumption of the regular stimulation protocol. Postrest potentiation is defined as the ratio between the first postrest contraction and the prerest contraction.
Unless otherwise indicated, chemicals were purchased from Sigma-Aldrich Israel. Ryanodine and thapsigargin were purchased from Alomone (Jerusalem, Israel, http://www.alomone.com).
Results were expressed as mean ± standard error of mean. Means of two populations were compared using Student's t-test for unpaired observations. A value of p < .05 was considered significantly different.
Basic Functional Properties of hESC-CMs
The functional properties of hESC-CMs regarding the intracellular Ca2+ handling and the role of the SR in the contraction process were investigated by recording simultaneously the [Ca2+]i transients and contractions from spontaneously contracting or from electrically stimulated EBs. Figure 1A depicts an EB in which the contracting area is circled by the dotted ellipsoid. Figures 1B and 1C depict in an EB contracting spontaneously at 0.2 Hz that each [Ca2+]i transient is associated with a corresponding contraction, which represents the normal functionality of the E-C coupling machinery.
A fundamental property of the adult human myocardium is the ability to increase the contraction force in response to increased rate of stimulation. The phenomenon, which is termed positive force-frequency relations, is utilized by the heart to increase cardiac output under exercise or stress conditions. To generate force-frequency relations, spontaneously contracting EBs were paced at 0.5, 1.0, 1.5, 2.0, and 2.5 Hz and the [Ca2+]i transients and contractions were measured at each frequency after steady state was attained. As depicted by a representative experiment (Figs. 2A, 2B), in contrast to the mature myocardium, in hESC-CMs, the increase in stimulation rate caused a reduction in the amplitude of both the [Ca2+]i transient and the contraction, thus presenting a negative force-frequency relation. To exclude the possibility that the negative force-frequency relations are clone-or age-specific, we repeated the experiment depicted in Figures 2A and 2B (clone H9.2, 55-day-old) in a 7-day-old EB from clone I3. As seen in Figures 2C and 2D, this young EB from clone I3 also depicted negative force-frequency relations. The proposed explanation for the elevated diastolic [Ca2+]i seen in Figures 2A and 2C is provided in the Discussion section. In summary, as seen in Figures 2E and 2F, increasing the stimulation rate decreased the maximal rate of shortening (dL/dtContract), maximal rate of relaxation (dL/dtRelax), and contraction amplitude (LAmp), most likely caused by the reduction in the [Ca2+]i transient amplitude.
The mechanisms underlying the positive/negative force-frequency relations have been thoroughly investigated in a variety of animal species , and it is commonly thought that the positive relations result from augmented SR [Ca2+]i release at higher rates, which generates increased force. As a first step in deciphering the mechanisms responsible for the negative force-frequency relations in hESC-CMs, we tested the hypothesis that the contractile machinery is either not sensitive to or is attenuated by increased Ca2+. The hypothesis was tested by measuring [Ca2+]i transients and contractions in the presence of 2 (the regular bath concentration), 4, and 6 mmol/l [Ca2+]0. As seen in Figures 3A and 3B, elevating [Ca2+]0 caused a concentration-dependent increase in diastolic [Ca2+]i, systolic [Ca2+]i, and [Ca2+]i transient amplitude. Concomitantly, the contraction parameters dL/dtContrac, dL/dtRelax, and LAmp were also increased (Figs. 3C, 3D). Based on these findings, we concluded that the negative force-frequency relations in hESC-CMs do not result from the inability to respond to increased [Ca2+]i.
An important contractile feature related to a fully functional SR is postrest potentiation. This function is commonly tested by interrupting the regular stimulation (at 1 Hz) with a pause of varying lengths, followed by resumption of the regular stimulation protocol. In principle, due to a larger filling of the SR with Ca2+ during the rest, the postrest contraction is larger than the prerest contraction. Indeed, as seen by the representative tracings recorded from an adult mouse ventricular myocyte (Fig. 4A) and by the summary of three experiments (Fig. 4B), the first postrest contraction gradually increased as the rest period was lengthened. In contrast, in hESC-CMs, postrest potentiation of contraction was absent, supporting the concept of a nonfunctional SR concomitant with a major contribution of transsarcolemmal Ca2+ influx to contraction.
Does SR Ca2+ Release Contribute to Contraction in hESC-CMs?
A key aspect of the E-C coupling machinery is Ca2+-induced Ca2+ release (CICR), which provides (in most mature hearts) ∼70% of the entire Ca2+ ions utilized by the contractile machinery . Since the SR function is indispensable for the generation of positive force-frequency relations and studies have shown that SR Ca2+ release in the immature myocardium is not as robust as in the mature muscle, we tested the contribution of SR Ca2+ release to the contraction in hESC-CMs. First, we determined the effect of the SERCA inhibitor thapsigargin (100 nmol/l), which, by depleting SR Ca2+ stores, causes a strong negative inotropic effect . In contrast to the adult myocardium, thapsigargin did not affect the [Ca2+]i transient or the contraction (Figs. 5A, 5C, 5D). Second, we determined the effect of 10 μmol/l ryanodine, which at this blocking concentration causes a prominent negative inotropic effect . Again, in contrast to the mature heart, ryanodine did not affect the [Ca2+]i transient or the contraction parameters of hESC-CMs (Figs. 5B–5D). Similar results were obtained with a 7-day-old EB from clone I3 (data not shown). Finally, in accordance with the failure of both ryanodine and thapsigargin to affect hESC-CM contraction, exposure of contracting EBs to caffeine (10 mmol/l), which commonly causes an abrupt SR Ca2+ release in a variety of cardiac preparations , did not affect the [Ca2+]i transient or the contraction (Fig. 6). Collectively, these findings show for the first time that in hESC-CMs, compared with the mature myocardium, SR Ca2+ release does not contribute to the contraction. In addition to demonstrating indirectly (by the unresponsiveness of the contraction to ryanodine, thapsigargin, and caffeine) that transsarcolemmal Ca2+ influx is the major contributor to the contraction in hESC-CMs, we tested the effect of verapamil (a Ca2+ channel blocker) on the contraction. As depicted by a representative experiment (Fig. 6), shortly after its administration to the bath, verapamil completely blocked the contraction, supporting the role of the L-type calcium current (ICa,L) in hESC-CMs contraction. Similar findings were repeated in three different experiments.
Intracellular Ca2+-Handling Machinery in hESC-CMs
To decipher the molecular basis for the different Ca2+-handling machinery in hESC-CMs, we tested the expression of four key proteins participating in intracellular Ca2+ handling: SERCA2, calsequestrin, phospholamban (PLB), and NCX. As shown by the representative Western blots (Fig. 7), SERCA2 (the myocardial SERCA isoform) and NCX are expressed in comparable levels both in hESC-CMs and in porcine left ventricle. Regarding SERCA expression in hESC-CMs, it should be noted that the SERCA2 gene family consists of two isoforms: SERCA2a, which is located in the SR membrane and expressed mostly in cardiomyocytes and slow-twitch skeletal muscle, and SERCA2b, which is considered a housekeeping gene and expressed in a variety of cells. Because there is no specific antibody to distinguish between the two isoforms, the Western blot presented in Figure 7 may represent both isoforms. In contrast to SERCA2, calsequestrin, a Ca2+-binding protein expressed in the SR lumen , is abundant in the adult porcine ventricle but is absent in hESC-CMs. Further, phospholamban, a small, reversibly phosphorylated SR-transmembrane regulatory protein of SERCA2  was also expressed in the porcine heart but was missing from hESC-CMs. The absence of phospholamban and calsequestrin in hESC-CMs is the likely cause for the dysfunctional SR, consequently contributing to the negative force-frequency relations.
The initiative for the present work was the realization that stem cells in general and hESC-CMs in particular hold great potential for treating intractable heart diseases. Because of the deficiency of effective means for treating myocardial ischemia and infarction, cell transplantation is proposed as a new therapeutic modality to regenerate or repair the diseased myocardium . Since it is desired that the transplanted cells fully integrate within the diseased myocardium, contribute to its contractile performance, and respond appropriately to various stimuli (e.g., changes in heart rate), it is important to decipher their adaptability with the host myocardium. Therefore, the objective of this work was to investigate functional properties of hESC-CMs regarding intracellular Ca2+ handling and the role of the SR in the contraction process.
Functional Properties of hESC-CMs
At the outset of this section, it should be stated that our measurements of [Ca2+]i transients and contractions were conducted from contracting clusters of cells, likely comprised of several types of cardiomyocytes (e.g., atrial and ventricular cells), thus lacking the ability to discriminate between the different types. Hence, the ensemble recordings represent the average behavior of the cluster rather than the individual functional properties, which may vary among the different cell types. This issue is further addressed in the Study Limitations section towards the end of the Discussion.
Whereas hESC-CMs demonstrate the mature-like temporal relations between the [Ca2+]i transient and the contraction, in contrast to the adult myocardium, hESC-CMs display negative force-frequency relations, which reflect the underlying E-C coupling and the [Ca2+]i handling machinery. Increased heart rate either during exercise or due to other stimuli (e.g., emotional stress) increases cardiac output by increasing the number of contractions per minute, as well as by its action on the E-C coupling . As depicted in Figure 2, in contrast to most mature myocardium (e.g., rabbit, guinea pig, human) [17, 22, 23], in hESC-CMs, both the [Ca2+]i transient and the contraction amplitudes were reduced in response to increased stimulation rate. As reviewed by Lederer and colleagues , the final outcome of the increased rate on contraction depends on the fine balance between Ca2+ influx, SR Ca2+ content, and release. Thus, in the case of positive force-frequency relations, increases in SR Ca2+ content and [Ca2+]i transient amplitude are expected, whereas in the case of negative force-frequency relations, the reverse situation is likely to occur. Thus, the occurrence of negative or positive force-frequency relations depends on whether the SR releases more Ca2+ than it acquires.
Understanding the mechanisms underlying the nature (positive or negative) of the force-frequency relations was enhanced by studies showing that whereas the healthy human myocardium displays positive force-frequency relations, in failing hearts these relations are either flattened or reversed [23, 25, 26]. For example, Pieske et al.  reported that whereas in the nonfailing human myocardium increased stimulation rate from 0.5 Hz to 2 Hz increased the isometric force and the [Ca2+]i transient signal, in a failing heart with dilated cardiomyopathy, the increased rate attenuated both the [Ca2+]i transient and contraction amplitude. Several studies focusing on the [Ca2+]i handling proteins addressed the molecular mechanisms underlying the altered force-frequency relations in the failing myocardium. These studies have shown that in the failing human heart, SR Ca2+ uptake was reduced whereas the Na+-Ca2+ exchange increased [28, 29]. These changes resulted from decreased SERCA protein expression, increased NCX protein expression, and altered function of ryanodine receptor. In this regard, Hasenfuss and colleagues [28, 30] examined ventricular preparations from normal and failing hearts and found a close positive correlation between the frequency-dependent changes in contraction and SERCA protein levels measured in myocardium from the same hearts; namely, the higher the SERCA expression, the stronger the positive inotropic effect of increased rate of stimulation.
Since the absence of a functional SR in hESC-CMs negates its direct role in the negative force-frequency relations, the following options can be considered. First, increased stimulation rate steeply decreases action potential duration in hESC-CMs. Numerous studies have shown that action potential duration (APD) is inversely related to the rate of stimulation, which in the absence of a concomitant increase in ICa,L and augmented SR Ca2+ release may decrease the force of contraction. If indeed in hESC-CMs, APD versus stimulation rate relations are much steeper than in the adult myocardium, such relations may contribute to the negative force-frequency relations. Second, increased stimulation rate decreases ICa,Lin hESC-CMs. Studies in experimental animals and human cardiac specimens have shown that ICa,L is augmented by increasing the rate of stimulation—a mechanism contributing to the positive force-frequency relations. If in hESC-CMs ICa,L density is inversely dependent on the stimulation rate, then increasing the rate may cause negative force-frequency relations. The third possibility is inactivation of ICa,Lby elevated [Ca2+]i. An interesting phenomenon observed in the presence of both higher stimulation rate and [Ca2+]0 was increased diastolic [Ca2+]i on the order of 10%–15% (Figs. 2, 3), which was accompanied by a corresponding elevation in the resting length. We propose that this phenomenon results from the inability of cardiomyocytes to maintain sufficiently low diastolic [Ca2+]i when the regular [Ca2+]i homeostasis is perturbed. We further propose that this may be caused by ineffective [Ca2+]i buffering capacity due to nonfunctional SR and/or from inefficient NCX and/or membrane Ca2+-ATPase activity. Thus, based on these findings, we suggest that the well-established inactivation of ICa,L by elevated [Ca2+]i is an important contributor to the negative force-frequency relations in hESC-CMs.
Contribution of SR-Ca2+ Release to the Contraction
The observation that hESC-CMs display negative force-frequency relations is not only important from the cardiac developmental aspect but also suggests to us that in hESC-CMs the [Ca2+]i handling machinery differs from that of the mature myocardium. Having found that hESC-CMs can readily increase both [Ca2+]i transient and contraction amplitude in response to elevating [Ca2+]0 from 2 to 4 and 6 mmol/l (Fig. 3), we directed our attention to the SR function.
Although our data do not directly address the issue of Ca2+ influx as the major source of Ca2+ for contraction, based on the following findings, we concluded that the CICR machinery is nonfunctional in hESC-CMs. First, in contrast to adult mouse ventricular myocytes (as well as other ventricular preparations), hESC-CMs do not exhibit postrest potentiation (Fig. 4). Second, ryanodine (a RyR blocker), which causes a negative inotropic effect in a variety of cardiac preparations, did not affect the [Ca2+]i transient or the contraction. Third, thapsigargin, a SERCA inhibitor and a negative inotropic agent, did not affect the [Ca2+]i transient and contraction. Fourth, caffeine, which rapidly increases [Ca2+]i, was absolutely ineffective.
The notion that hESC-CMs express functional ICa,L which contributes directly to the contraction is supported by the following findings. First, as shown in Figure 6, verapamil blocked the contraction. Theoretically, the inhibitory effect of verapamil on contraction may have been caused indirectly, due to blocking of the action potential (as would occur with a slow response). That this is not the case is indicated by the following findings by Satin et al.  showing that in 20- to 35-day-old hESC-CMs (clone H9.2, the same clone used in our study), the action potentials are fast responses (insensitive to verapamil): the action potentials were sensitive to TTX and nifedipine did not inhibit the spontaneous action potentials. Thus, because fast responses (as opposed to slow responses) are blocked by TTX and not by Ca2+ channel blocker, the marked attenuation of the contraction by verapamil did not result from a direct effect on the action potential generation but from a blocking effect of verapamil on ICa,L. Second, Mummery et al.  showed that hESC-CMs express the α catalytic subunit of the cardiac-specific ICa,L. Third, Satin et al.  have recently shown that nifedipine (a Ca2+ channel blocker) markedly shortened the action potential recorded from isolated hESC-CMs. Finally, and most importantly, Dr. Timothy Kamp (personal communication, Gordon Conference on Cardiac Arrhythmia Mechanisms, Santa Ynez, CA, February 2005) recently recorded from hESC-CMs Ca2+ currents featuring cardiac-like behavior with peak current amplitude of −800 pA at a membrane potential of 0 mV. Thus, collectively, these data clearly demonstrate that voltage-dependent Ca2+ current is present and functional in hESC-CMs.
Ca2+-Handling Proteins in hESC-CMs
The likely main cause for the adult-dissimilar E-C coupling in hESC-CMs is altered expression and/or function of SR Ca2+ handling proteins and its regulatory counterparts. The first step in the [Ca2+]i handling process is RyR-mediated CICR. In this regard, although Mummery et al.  have shown that hESC-CMs express RyR mRNA, they did not determine whether the RyR was functional. Indeed, that the RyR receptor is not functional (although expressed) is suggested by our finding that caffeine, which binds to RyR and releases large amounts of Ca2+, was ineffective (Fig. 6). In view of the expression of RyR in hESC-CMs, another explanation for the lack of effect of caffeine is empty SR Ca2+ stores. Lederer and colleagues  recently emphasized the critical role of SR luminal Ca2+ in regulating the RyR and cardiac Ca2+ signaling. According to their working model, the decrease in luminal Ca2+ underlies RyR closure and the termination of the Ca2+ spark. To the best of our knowledge, the fundamental process that initially fills the SR with Ca2+ is unknown. What is known, however, is that the SR Ca2+ content is determined by SERCA, which ensures SR refilling after Ca2+ influx due to CICR. With regards to SERCA2 expression/function in hESC-CMs, our work shows that, on the one hand, SERCA2 is expressed in levels comparable to those in the porcine heart, but, on the other hand, thapsigargin, a SERCA inhibitor that commonly causes a negative inotropic effect, was ineffective. These findings can be accounted for by one or more of the following possibilities. First, SERCA is not functional, despite its expression. Here, the underlying mechanism can be within the protein itself, in its cellular localization, and/or due to its interaction with phospholamban. Phospholamban, which is not expressed in contracting EBs (Fig. 7), is an endogenous inhibitor of SERCA, and its phosphorylation/dephosphorylation balance determines the extent of SERCA inhibition . A recent study has shown that phospholamban expression closely correlates with SERCA2 levels, and disruption of a single copy of the murine SERCA2 gene resulted, apart from decreased SR-Ca2+ load and deranged cardiomyocyte contraction and Ca2+ transients, in a ∼50% decrease in phospholamban levels . Our finding that contracting EBs do not express phospholamban but express SERCA2 may imply also that at this developmental stage, SERCA2 is not incorporated in the SR and therefore is not functional. Second, SERCA is functional, but because the SR is practically empty (since calsequestrin is absent; Fig. 7), thapsigargin does not affect the [Ca2+]i transient or contraction. Deciding whether this option is valid will await studies directly measuring SR Ca2+ content.
Our finding showing that calsequestrin is not expressed in hESC-CMs may indicate indirectly that the SR Ca2+ stores are either empty or inaccessible. Calsequestrin is the major intra-SR Ca2+ binding protein and is localized at the junctional face membrane in the SR [18, 32]. Calsequestrin has a large number of acidic amino acids that enable it to coordinately bind 40 to 50 Ca2+ ions , and it allows the Ca2+ required for contraction to be stored in the SR lumen at concentrations of up to 20 mmol/l. In addition to its Ca2+-binding capacity, calsequestrin is an important luminal regulator of RyR, thus making this molecule a major player in Ca2+ homeostasis that extends beyond its ability to modulate intracellular Ca2+ . Hence, our key finding that hESC-CMs do not express calsequestrin at all (compared with the mature myocardium) may provide a plausible explanation for the immaturity of the E-C coupling in hESC-CMs.
Comparison with Animal Models of Myocardial Development
In agreement with our findings, several studies performed in mouse, rat, and sheep hearts indicate that myocardial contraction at early developmental stages is largely dependent on transsarcolemmal Ca2+ influx rather than on SR Ca2+ release [35–40]. Among other findings, this major conclusion was based on biochemical data as well as on studies demonstrating that the negative inotropic effects of ryanodine in the fetal and neonatal hearts are less pronounced than in adults. For example, Liu et al.  have recently studied the developmental changes of Ca2+ handing in mouse ventricular cells from early embryo to adulthood. Their main findings pertaining to the present study were that from early to late embryonic stage, mRNAs for RyR2, SERCA2, and phospholamban increased by 3- to 15-fold, whereas NCX was unchanged. After birth, there was a further increase in mRNA of RyR2, SERCA2, and phospholamban by 18- to 33-fold. The protein levels of RyR2, SERCA2, and phospholamban showed quantitatively parallel developmental changes. Based on these results, the authors concluded that “activator Ca2+ for contraction in the early embryonic stage depends almost entirely on ICa-L.” Although it is difficult to compare the developmental stages of the EBs studied here versus rat embryonic hearts, the recent findings by Moorman et al.  differ from ours. This group tested the effects of SR blockers on Ca2+ transients and determined SR function biochemically in different heart sections in day-13 rat embryos (E13). Their main findings were that in all cardiac tissue compartments, verapamil abolished the Ca2+ transients, and that functional SR was absent in the embryonic outflow tract but present in atria and ventricles. Whether this disparity results from species- or developmental-stage differences remains to be determined.
Since the mouse embryonic stem cell model has been extensively utilized to study cardiac development, it is important to address the apparent discrepancy between our findings and those of Hescheler and colleagues . Their major finding was that at an early developmental stage (day 9.5), the contractions were evoked by [Ca2+]i oscillations originating from intracellular stores rather than from transmembrane ion currents. Further (in agreement with our findings), they found that CICR is not required for the functioning of the early embryonic heart. However, whether these differences result from dissimilar developmental stages investigated in the two studies and/or from principle differences between human and mouse [Ca2+]i handling machinery is yet to be deciphered.
Study Limitations: The Heterogeneity of the Contracting EBs
The use of fine contracting clusters (surgically removed from contracting EBs) as a model for hESC-CMs implies that these areas probably include the three major types of myocytes: ventricular, atrial, and nodal-like cells. Although it is currently impossible to accurately determine the proportion of individual cardiomyocytes within a contracting area, several studies demonstrate the prevalent presence of ventricular-like myocytes among the cardiomyocyte population within the contracting EBs. In a recent study, He et al.  used the ES cell lines H1, H7, H9, and H14 and demonstrated by means of microelectrode recordings that of the 105 action potentials recorded in 20 contracting EBs, 26 were nodal-like, 19 atrial-like, and 60 (the majority, 57%) ventricular-like. Importantly, of the total action potential recorded, 75% were of the fast-response phenotype. Mummery et al.  have shown that in hESC-derived contracting areas, of the 33 action potential recordings, 28 impalements (85%) were characterized by a ventricular-like phenotype. Satin et al.  recorded action potentials from contracting cell clusters of the clone H9.2 and reported, “All of our AP recordings have a definitive plateau and they are similar to the ventricular-like action potentials reported by He et al.” Furthermore, using the Micro-Electrode-Array techniques and micro-electrode recordings, this group has shown that the spontaneous action potential generation and conduction were TTX-sensitive (and unaffected by nifedipine), indicating that the activation was of the fast-response phenotype. Collectively, these findings suggest that the majority of cardiomyocytes within the contracting areas are ventricular-like.
In summary, in the present study, we investigated the basic properties of the E-C coupling in hESC-CMs. The principal finding was that E-C coupling properties in hESC-CMs differ from mature myocardium by featuring negative force-frequency relations and because the Ca2+ used by the contractile machinery is provided by transsarcolemmal influx and not by SR Ca2+ release. Among other possibilities, these differences from the mature myocardium may be due to lack of calsequestrin and phospholamban expression in hESC-CMs.
K.D., M.S., and N.Z.-L. contributed equally to this study. This work was supported by the Ministry of Science and Technology, Israel, the Israel Science Foundation (a grant to O.B. and J.I.-E.), the Rappaport Family Institute for Research in the Medical Sciences, and by the Sylvia and Stanley Shirvan Chair in Cell and Tissue Regeneration Research (J.I.-F.). The superb technical assistance of Danit Ohayon, Irina Reiter, Hanna Segev, Bettina Fishman, Rita Shulman, and Anna Ziskind is gratefully acknowledged.
The authors indicate no potential conflicts of interest.