Functional Properties of Human Embryonic Stem CellCDerived Cardiomyocytes: Intra - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:11 |只看该作者 |倒序浏览 |打印

作者：Katya Dolnikova, Mark Shilkruta,b, Naama Zeevi-Levina, Sharon Gerecht-Nira,c, Michal Amita,c,d, Asaf Danona, Joseph Itskovitz-Eldora,c,d, Ofer Binaha作者单位：a Rappaport Family Institute for Research in Medical Sciences, Rappaport Faculty of Medicine, Technion, Haifa, Israel;

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      【摘要】9 ?* t) g# p! a3 @6 H: w, E; q; {7 _0 L
   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 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.
      【关键词】 Human embryonic stem cell-derived cardiomyocytes Excitation-contraction coupling i transients Contractions Sarcoplasmic reticulum
   INTRODUCTION

Cardiovascular diseases, including congestive heart failure, are the most frequent cause of death in the industrialized world .6 \/ g1 |8 H5 m1 j: ^: F
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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 .$ ?& I, G% S' ?, z& M: K

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., ß-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 , 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.* ^1 H" o4 h+ D( c8 V: ?

MATERIALS AND METHODS6 t( I: o( {% f+ |& y8 f$ O

Human Embryonic Stem Cells: Culture and Differentiation
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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 ß-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 Myocytes3 I# a5 i; p: r& A% A8 |8 H2 y) h: z

Ventricular myocytes from adult ICR mice were obtained by an enzymatic dissociation procedure as previously described .

Measurement of i Transients and EB Contractions# V) c9 Q2 o( m5 y" ]. U

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.
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Measurements of Protein Expression% _$ }+ m9 k" w, u" Q: Q) k" H
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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¨C913; ABR Affinity BioReagents, Golden, CO, http://www.bioreagents.com; 1:2,500 dilution), anti-SERCA2 (MA3¨C910, ABR Affinity BioReagents; 1:2,500), anti-phospholamban (MA3¨C922, ABR Affinity BioReagents; 1:500), anti-Na /Ca2  exchanger (NCX) (MA3-926, ABR Affinity BioReagents; 1:1,000), and anti-sarcomeric--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.6 N" ?* V' a0 y( M* P

Postrest Potentiation
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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.: |# H5 x4 I3 _) V- o, [+ M

Chemicals

Unless otherwise indicated, chemicals were purchased from Sigma-Aldrich Israel. Ryanodine and thapsigargin were purchased from Alomone (Jerusalem, Israel, http://www.alomone.com).$ I6 D. E, h& D' z3 t; j  ^

Statistical Analysis6 U, v. S$ y$ R. V. @% V

Results were expressed as mean ¡À standard error of mean. Means of two populations were compared using Student¡¯s t-test for unpaired observations. A value of p 5 ^" o! d: n4 e( \5 x% i
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RESULTS

Basic Functional Properties of hESC-CMs" P4 Y2 v% p5 r( e

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 i transient is associated with a corresponding contraction, which represents the normal functionality of the E-C coupling machinery.& h% j8 a+ w9 w6 d7 S  P( K1 g
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Figure 1. Excitation-contraction coupling in a spontaneously contracting 47-day-old EB from clone H9.2. (A): A typical EB containing a spontaneously contracting area (circled by the dotted ellipsoid). (B): Simultaneously recorded traces of i transient and the contraction. Abbreviations: EB, embryoid body; RSys, maximal (systolic) ratio; RDias, minimal (diastolic) ratio.7 u* K& k" [0 L, v4 t% G) v
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Force-Frequency Relations& V* [5 q7 ^! |' c8 B* j

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 i transient amplitude.! y) a7 I- _! @1 Z

Figure 2. Force-frequency relations in human embryonic stem cell-derived cardiomyocytes from clones H9.2 and I3. To generate the force-frequency relations, contracting embryoid bodies (EBs) were stimulated by means of electric field stimulation at 0.5, 1, 1.5, 2, and 2.5 Hz. (A, B): Representative recordings of i transient amplitude. (F): The contraction parameters (see G for details) dL/dtContrac, dL/dtRelax, and LAmpl. n = 5 EBs. The results are expressed as percent change from the values at 0.5 Hz.

The mechanisms underlying the positive/negative force-frequency relations have been thoroughly investigated in a variety of animal species i.
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Figure 3. The effects of different bath Ca2  concentrations on the i transient and the contraction parameters. n = 3 EBs. The results are expressed as percent change from control at 2 mmol/l. *p

Postrest Potentiation8 }2 n8 P/ X1 Y5 a- G4 e
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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.3 `  t: P, \  U" E( N1 Y

Figure 4. Testing postrest potentiation in adult mouse ventricular myocytes and in human embryonic stem cell-derived cardiomyocytes (hESC-CMs) (see the Materials and Methods section for details). (A): Representative contraction tracings recorded from an adult mouse ventricular myocyte and from a contracting EB, depicting the control contraction recorded at 1 Hz and the first postrest contractions after rest periods of 5, 10, and 60 seconds. Note the prominent postrest potentiation in the mouse ventricular myocyte and its absence in hESC-CMs. (B): Average percent change in postrest contraction amplitude/pretest contraction amplitude versus the rest length in adult mouse ventricular myocytes (n = 3) and in contracting EBs (n = 3).- H; G$ J- [9 l9 z7 G! P

Does SR Ca2  Release Contribute to Contraction in hESC-CMs?
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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 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.8 N# M% \1 f# v+ q6 f, ^+ w% h# \
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Figure 5. The effects of thapsigargin and ryanodine on i transient and contraction parameters of hESC-CMs. Results are expressed as percent change from control. Abbreviations: hESC-CMs, human embryonic stem cell-derived cardiomyocytes; RSys, maximal (systolic) ratio; RDias, minimal (diastolic) ratio; RAmp = RSys ¨C RDias; dL/dtContrac, maximal rate of contraction; dL/dtRelax, maximal rate of relaxation; LAmp, the difference between minimal and maximal cursor position., ]) ?* o$ J+ f% ~# ^

Figure 6. The effects of caffeine and verapamil on the contractile machinery. (A): Representative i transient and contraction parameters. The results are expressed as percent change from control. *p # o$ k, d% Z7 |+ ]$ ]. P3 w

Intracellular Ca2 -Handling Machinery in hESC-CMs. d9 J2 k: u6 C, n1 c
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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  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.6 K4 {4 R! j7 }0 p
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Figure 7. Protein expression in human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and adult porcine ventricular myocardium. (A): Representative Western blots of the SR proteins SERCA2, calsequestrin (CSQ), phospholamban (PLB), and -actinin in porcine myocardium and hESC-CMs. These findings were repeated in three different experiments, each analyzing six to eight carefully excised spontaneously contracting areas of embryoid bodies. (B): Representative Western blots of Na /Ca2  exchanger (NCX) in porcine myocardium and hESC-CMs. The antibody directed against NCX identifies two bands, 120 and 70 kDa. These dual-molecular-weight bands have been reported in the literature to be associated with NCX . These findings were repeated in two different experiments, each analyzing six to eight carefully excised spontaneously contracting areas of EBs.

DISCUSSION% A2 M0 w+ [7 Y4 \

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.
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Functional Properties of hESC-CMs' Q% I% U3 B& d& \" R2 U3 p

At the outset of this section, it should be stated that our measurements of 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.
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Whereas hESC-CMs demonstrate the mature-like temporal relations between the 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  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.
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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,L in hESC-CMs. Studies in experimental animals and human cardiac specimens have shown that ICa,L is augmented by increasing the rate of stimulation¡ªa 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,L by elevated i is an important contributor to the negative force-frequency relations in hESC-CMs.$ A3 ~8 y0 m$ y) s/ Q' Q1 g8 e- I

Contribution of SR-Ca2  Release to the Contraction  B) t4 j. U) X; ^0 N: b5 R

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 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 i, was absolutely ineffective.5 Y0 m5 B, P, W6 _" T' G* y/ z0 }
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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.  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 ¨C800 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' p: [) h- V/ @+ l& @5 c+ H

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 i transient or contraction. Deciding whether this option is valid will await studies directly measuring SR Ca2  content.! }# F, D% o( ^9 ^' ?1 b
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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 . 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.
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Comparison with Animal Models of Myocardial Development% @- V( J1 g0 p# M* @

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  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.4 B( t3 w2 f, ]6 |8 s) P  c4 K
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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 i handling machinery is yet to be deciphered.2 |2 ~3 m/ N1 ~. y6 k; N
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Study Limitations: The Heterogeneity of the Contracting EBs  c; X' z6 x( A, G' s# H: p7 X6 E- [

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.  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.8 f: a9 N7 M9 g* k5 O
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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.' j8 [7 C+ {+ n1 i- }& Y, M6 T  m' n

ACKNOWLEDGMENTS: V9 I. h' c- l+ T) n
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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.) l4 |3 x# T* P! }* X/ ~4 R

DISCLOSURES0 u3 M. F8 B  q( _' u
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The authors indicate no potential conflicts of interest.; k* S4 z. E) ~( ]& ], i6 z. q1 f- _& N! C
      【参考文献】* {% a2 J4 u+ J* d# ?* k/ }

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Cohn JN, Bristow MR, Chien KR et al. Report of the National Heart, Lung, and Blood Institute Special Emphasis Panel on Heart Failure Research. Circulation 1997;95:766¨C770., z2 b; i# }% k# P$ B+ W9 Y7 B) `! O

Hunt SA. Current status of cardiac transplantation. JAMA 1998;280: 1692¨C1698.
; w1 b& P3 F# u' G" m% q7 D( l
Nir SG, David R, Zaruba M et al. Human embryonic stem cells for cardiovascular repair. Cardiovasc Res 2003;58:313¨C323.* ]8 s2 g1 M$ r# D
8 j8 D4 o8 Q" J8 L. n
Dowell JD, Rubart M, Pasumarthi KB et al. Myocyte and myogenic stem cell transplantation in the heart. Cardiovasc Res 2003;58:336¨C350.
3 }; G% B" n8 b: K2 n  z
Amit M, Itskovitz-Eldor J. Derivation and spontaneous differentiation of human embryonic stem cells. J Anat 2002;200:225¨C232.9 j# j- F( m/ `! }& N5 N

Amit M, Carpenter MK, Inokuma MS et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol 2000;227:271¨C278.
: `7 \. O8 E( r7 {) z$ X
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¨C414.# O5 }9 J, y0 X, M8 h1 _
& v! @5 v& G( [  O( z4 Y
Satin J, Kehat I, Caspi O et al. Mechanism of spontaneous excitability in human embryonic stem cell derived cardiomyocytes. J Physiol 2004;559: 479¨C496.
! X$ n- h* a% Y" G" j" S0 i$ Z, C( J$ G
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¨C39., G5 x) A5 y  W2 t
, @( s& U! F4 B
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¨C2740.: c1 a. r: ^5 e7 s, t( D1 k% `

Less H, Shilkrut M, Rubinstein I et al. Diseased ventricular myocytes form the pathogenic process of heart failure in murine autoimmune myocarditis. J Autoimmun 1999;12:209¨C220.

Felzen B, Shilkrut M, Less H et al. Fas (CD95/Apo-1)-mediated damage to ventricular myocytes induced by cytotoxic T lymphocytes from per-forin-deficient mice: A major role for inositol 1,4,5-trisphosphate. Circ Res 1998;82:438¨C450.

Alpert NR, Leavitt BJ, Ittleman FP et al. A mechanistic analysis of the force-frequency relation in non-failing and progressively failing human myocardium. Basic Res Cardiol 1998;93(suppl 1):23¨C32.5 d* W0 a) @+ q" t( N

Bers DM. Cardiac excitation-contraction coupling. Nature 2002;415: 198¨C205.
* T" r8 S( x+ g
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¨C382.

Borzak S, Murphy S, Marsh JD. Mechanisms of rate staircase in rat ventricular cells. Am J Physiol 1991;260:H884¨CH892.

Balaguru D, Haddock PS, Puglisi JL et al. Role of the sarcoplasmic reticulum in contraction and relaxation of immature rabbit ventricular myocytes. J Mol Cell Cardiol 1997;29:2747¨C2757.4 U8 X$ r9 `, m$ I* {5 e/ p1 `5 Z
. n0 l& A+ U* E, q9 \" ^7 V. z5 B6 g
Beard NA, Laver DR, Dulhunty AF. Calsequestrin and the calcium release channel of skeletal and cardiac muscle. Prog Biophys Mol Biol 2004;85:33¨C69.

Brittsan AG, Kranias EG. Phospholamban and cardiac contractile function. J Mol Cell Cardiol 2000;32:2131¨C2139.

Hassink RJ, Goumans MJ, Mummery CL et al. Human stem cells shape the future of cardiac regeneration research. Int J Cardiol 2004;95:S20¨CS22.
( r, ^/ ?3 k: e
Schillinger W, Lehnart SE, Prestle J et al. Influence of SR Ca2 -ATPase and Na -Ca2 -exchanger on the force-frequency relation. Basic Res Cardiol 1998;93(suppl 1):38¨C45.6 E* m9 _, s/ _" A) H

Bers DM. Sarcoplasmic reticulum Ca release in intact ventricular myocytes. Front Biosci 2002;7:d1697¨Cd1711.5 L1 Y9 l) S4 W! o2 {1 d. P

Faggiano P, Colucci WS. The force-frequency relation in normal and failing heart. Cardiologia 1996;41:1155¨C1164.2 _2 V; U# N! H% M$ P
' |; ?/ e* ?2 y3 ~/ c6 p
Guatimosim S, Dilly K, Santana LF et al. Local Ca2  signaling and EC coupling in heart: Ca2  sparks and the regulation of the/ A4 i: b1 y" t4 Z% C& F  Q

Gwathmey JK, Slawsky MT, Hajjar RJ et al. Role of intracellular calcium handling in force-interval relationships of human ventricular myocardium. J Clin Invest 1990;85:1599¨C1613./ e. S0 E% I' ?! L

Mulieri LA, Hasenfuss G, Leavitt B et al. Altered myocardial force-frequency relation in human heart failure. Circulation 1992;85:1743¨C1750.
" u( n+ b& L0 m: z. A% y
Pieske B, Kretschmann B, Meyer M et al. Alterations in intracellular calcium handling associated with the inverse force-frequency relation in human dilated cardiomyopathy. Circulation 1995;92:1169¨C1178.
9 {" K9 P) Q( O. S. Z5 I* m
Hasenfuss G, Reinecke H, Studer R et al. Relation between myocardial function and expression of sarcoplasmic reticulum Ca2 -ATPase in failing and nonfailing human myocardium. Circ Res 1994;75:434¨C442.
/ ~# z$ _7 h& |' F+ O/ f. Y! V2 o  q
Reinecke H, Studer R, Vetter R et al. Cardiac Na /Ca2  exchange activity in patients with end-stage heart failure. Cardiovasc Res 1996; 31:48¨C54.4 R2 L6 R; Y) D% j6 d
7 T8 I, y' u) M( j# Q0 R4 D
Kambayashi M, Miura T, Oh BH et al. Enhancement of the force-frequency effect on myocardial contractility by adrenergic stimulation in conscious dogs. Circulation 1992;86:572¨C580.# y: x) r3 C* ]7 e) q! A$ q3 b

Ji Y, Lalli MJ, Babu GJ et al. Disruption of a single copy of the SERCA2 gene results in altered Ca2  homeostasis and cardiomyocyte function. J Biol Chem 2000;275:38073¨C38080.' k; h% j2 \' @& O

Bers DM. Macromolecular complexes regulating cardiac ryanodine receptor function. J Mol Cell Cardiol 2004;37:417¨C429.9 |3 z3 j6 f( F; g( t0 }7 p

MacLennan DH, Wong PT. Isolation of a calcium-sequestering protein from sarcoplasmic reticulum. Proc Natl Acad Sci U S A 1971;68:1231¨C1235.
# Z, n4 ^( W5 R+ T/ B- o
Terentyev D, Viatchenko-Karpinski S, Gyorke I et al. Calsequestrin determines the functional size and stability of cardiac intracellular calcium stores: Mechanism for hereditary arrhythmia. Proc Natl Acad Sci U S A 2003;100:11759¨C11764.

Pegg W, Michalak M. Differentiation of sarcoplasmic reticulum during cardiac myogenesis. Am J Physiol 1987;252:H22¨CH31.3 k( `3 H: L: Q  L; t$ r
/ S0 z$ R" ], M* o, d7 c
Klitzner TS, Friedman WF. A diminished role for the sarcoplasmic reticulum in newborn myocardial contraction: Effects of ryanodine. Pediatr Res 1989;26:98¨C101.

Tanaka H, Shigenobu K. Effect of ryanodine on neonatal and adult rat heart: Developmental increase in sarcoplasmic reticulum function. J Mol Cell Cardiol 1989;21:1305¨C1313.* D, j$ ~  V  K2 W1 o/ _) B
! A6 Q1 W4 D+ i0 K! ^' A
Klitzner TS. Maturational changes in excitation-contraction coupling in mammaliam myocardium. J Am Coll Cardiol 1991;17:218¨C225.
) b, `; Q: b; @/ S9 ~2 B' X
Ramesh V, Kresch MJ, Katz AM et al. Characterization of Ca2 -release channels in fetal and adult rat hearts. Am J Physiol 1994;269:H778¨CH782. .

Liu W, Yasui K, Opthof T et al. Developmental changes of Ca2  handling in mouse ventricular cells from early embryo to adulthood. Life Sci 2002;71:1279¨C1292.

Moorman AFM, Schumacher CA, de Boer PAJ et al. Presence of functional sarcoplasmic reticulum in the developing heart and its confinement to chamber myocardium. Dev Biol 2000;223:279¨C290.  [6 u& [& R$ N: y% z" ]8 y* u

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¨C8264.

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