Nature Biotechnology
22, 1282 - 1289 (2004)
Published online: 26 September 2004; | doi:10.1038/nbt1014
Electromechanical integration of cardiomyocytes derived from human embryonic stem cellsIzhak Kehat1, 3, Leonid Khimovich1, Oren Caspi1, Amira Gepstein1, Rona Shofti1, Gil Arbel1, Irit Huber1, Jonathan Satin5, Joseph Itskovitz-Eldor4
& Lior Gepstein1, 2, 31 The Sohnis Family Research Laboratory for the Regeneration of Functional Myocardium, Department of Biophysics and Physiology, the Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, P.O. Box 9649, Haifa, Israel. 2 The Rappaport Family Institute for Research in the Medical Sciences. 3 Department of Cardiology, Rambam Medical Center, Haifa, 31096 Israel. 4 Department of Obstetrics and Gynecology, Rambam Medical Center, Haifa, 31096 Israel. 5 Department of Physiology, University of Kentucky College of Medicine, Lexington, Kentucky, 40536-0298, USA.
Correspondence should be addressed to Lior Gepstein mdlior@tx.technion.ac.ilCell therapy is emerging as a promising strategy for myocardial repair. This approach is hampered, however, by the lack of sources for human cardiac tissue and by the absence of direct evidence for functional integration of donor cells into host tissues. Here we investigate whether cells derived from human embryonic stem (hES) cells can restore myocardial electromechanical properties. Cardiomyocyte cell grafts were generated from hES cells in vitro using the embryoid body differentiating system. This tissue formed structural and electromechanical connections with cultured rat cardiomyocytes. In vivo integration was shown in a large-animal model of slow heart rate. The transplanted hES cell−derived cardiomyocytes paced the hearts of swine with complete atrioventricular block, as assessed by detailed three-dimensional electrophysiological mapping and histopathological examination. These results demonstrate the potential of hES-cell cardiomyocytes to act as a rate-responsive biological pacemaker and for future myocardial regeneration strategies.Because the regenerative capacity of adult heart tissue is limited, any substantial cell loss or dysfunction, such as occurs during myocardial infarction, is mostly irreversible1 and may lead to progressive heart failure, a leading cause of morbidity and mortality2. Similarly, tissue loss or dysfunction at critical sites in the cardiac electrical conduction system may result in inefficient rhythm initiation or impulse conduction, requiring the implantation of a permanent electronic pacemaker3.
Transplantation of excitable myogenic cells within the dysfunctional zone is a possible therapeutic approach to restoring cardiac electromechanical functions. Although several cell types have been proposed4,
5,
6,
7,
8,
9,
10,
11,
12,
13, the inherent structural, electrophysiological and contractile properties of cardiomyocytes strongly suggest that they may be the ideal donor cell type. However, clinical application of this strategy is hampered by the paucity of cell sources for human cardiomyocytes and by the limited evidence of direct functional integration between host and donor cells14.
Human ES cells represent a promising source of donor cardiomyocytes. These unique cell lines, isolated from human blastocysts15,
16, can be propagated in the undifferentiated state in culture and coaxed to differentiate into derivatives of all three germ layers17. Recently, a reproducible cardiomyocyte differentiating system was established by culturing hES cells as three-dimensional differentiating cell aggregates termed embryoid bodies18,
19,
20,
21. Cells isolated from spontaneously beating areas of the cultures displayed structural, molecular and functional properties of early-stage cardiomyocytes18,
19,
20,
21. More recently, we have demonstrated that this differentiating system generates in vitro a functional cardiomyocyte syncytium with spontaneous pacemaker activity and action-potential propagation22.
Here we explore the utility of this unique tissue in cell therapy procedures aimed at restoring myocardial electromechanical functions. We show that excitable cardiac tissue generated from hES cells integrates structurally and functionally in vitro over the long term with rat cardiomyocyte cultures. Human ES cell−derived cardiomyocytes were also tested in a large animal model of complete atrioventricular block.
The cardiac conduction system consists of specialized cells that generate and conduct the electrical impulse in the heart. If this specialized conduction system is damaged at the atrioventricular junction, complete block of the electrical propagation between the atria and the ventricles ensues. This results in slow heart rate and circulatory compromise, currently one of the major indications for treatment with a permanent electronic pacemaker.
We found that hES cell−derived cardiomyocytes successfully pace the ventricle in swine with complete heart block. This result shows that the transplanted cells survive, function, and integrate with host cells following in vivo grafting and also provides proof-of-concept evidence for the ability of these cells to function as a biological alternative to the electronic pacemaker.
Results
Functional integration in hybrid cultures
The spontaneously contracting areas identified in some of the differentiating embryoid bodies comprised mainly small cells that stained positively for cardiac-specific markers (Fig. 1a). The myocytes were arranged in an isotropic pattern and were connected electrically through gap junctions. Interestingly, the hES cell−derived cardiomyocytes expressed both connexin-43 (Cx43) and Cx45, which in many cases colocalized to the same gap junctions (Fig. 1b), a phenomenon common to embryonic cardiomyocytes23.
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The beating embryoid bodies displayed stable and continuous activity for several weeks in culture. Whole-cell patch-clamp recordings from isolated beating hES cardiomyocytes demonstrated uniform embryonic-like action-potential morphology and confirmed the presence of an inherent pacemaking activity in these cells (Fig. 1c). Detailed electrophysiological investigation revealed that the biophysical basis for this spontaneous automaticity is a high-input resistance (generated by a low Kir current density) coupled with a relatively high sodium channel density24.
We next assessed the ability of the hES cardiomyocytes to integrate in vitro with primary cultures of neonatal rat ventricular myocytes. The contracting areas within the embryoid bodies were dissected and added to the cardiomyocyte cultures (Fig. 2a). Within 24 h after grafting we could already detect microscopically, in all 22 cocultures studied, synchronous mechanical activity (Supplementary Movie online).
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To further characterize the functional interactions within the cocultures, we mapped their electrical activity with a high-resolution microelectrode array (MEA) mapping technique22,
25 (Fig. 2a). By recording extracellular potentials simultaneously from 60 electrodes, we were able to generate high-resolution activation maps that characterize impulse initiation and conduction within the cocultures. A typical MEA map generated during spontaneous rhythm is shown in Fig. 2b. In this case, electrical activation was initiated within the rat tissue (red) and then propagated to the rest of the coculture. Electrograms recorded simultaneously from the human and rat tissues (Fig. 2b) demonstrated tight temporal coupling continuously for up to 21 d, the longest period studied.
We next carried out pacing studies in which either the rat (Fig. 2c) or human (Fig. 2d) tissues were stimulated through one of the MEA electrodes. Synchronous activity was maintained in the cocultures during both conditions. To assess electromechanical coupling between the two tissues, we correlated the mechanical contractions in the hES cardiomyocytes, as detected by a photodiode, with the electrical activity. As can be seen in Fig. 3a, the mechanical contractions of the embryoid bodies were time-locked with the electrical activity in both human and rat tissues.
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The degree of electrical coupling in the cocultures was assessed during long-term recordings and after adrenergic stimulation and partial gap-junction uncoupling. A histogram depicting the ratio between the activation cycle-lengths in the human and rat tissues is shown in Fig. 3b. The narrow peak at a ratio of 1 represents equal cycle lengths and confirms that electrical activity in the embryoid bodies was synchronous with that of the rat tissue. Isoproterenol (10  M) caused a significant increase in the spontaneous beating rate (from 1.5  0.6 to 1.9 0.8 Hz, P < 0.05), but electrical coupling between the two cell types was not hindered (Fig. 3c). Similarly, mild gap-junction uncoupling using 1-heptanol (0.5 mM) did not alter this tight coupling in the majority (five of the eight) of the cocultures (Fig. 3d), whereas in three cocultures occasional episodes of 2:1 conduction block were noted (Fig. 3e). Higher doses of heptanol (5 mM, causing total gap-junction uncoupling) totally abolished conduction in the hybrid cultures, indicating the significance of the generated gap junctions in electromechanical synchronization.
For electromechanical coupling to occur, specific structural interactions must develop at the interface between donor and host cells. Immunofluorescent staining for Cx43 (the major gap-junction protein) in conjunction with confocal microscopy demonstrated positive Cx43 immunostaining within the contracting embryoid bodies (Fig. 1b), between the neonatal rat ventricular myocytes, and at the border between the human and rat cardiomyocytes in the coculture experiments (Fig. 3f).
Generation of an in vivo biological pacemaker
To assess graft survival and functional integration in vivo, we measured the ability of hES cardiomyocytes to pace the heart of pigs with complete heart block. Complete block was induced by ablating the His bundle (the major electrical conduction pathway between the atria and the ventricles) with an electrophysiological ablation catheter. Complete atrioventricular block was immediately identified by the appearance of the typical dissociation between atrial (p waves) and ventricular (QRS deflections) activities (Fig. 4a). To prevent an extremely slow heart rate in the animals immediately following the creation of atrioventricular block, we also implanted an electronic pacemaker and positioned its electrode at the right ventricular apex.
| | Figure 4. ECG recordings in one of the animals with complete atrioventricular block. | | |  | (a−c) Typical ECG recordings (leads I, II and III) after the creation of complete atrioventricular block. During follow-up, episodes of the junctional escape rhythm (a), ventricular paced rhythm (b) and the new ventricular ectopic rhythm (c) were identified. (d,e) Long-term ECG recordings using an implantable loop-recorder. In each animal we could observe three different morphologies that correlate with the three different ECG patterns described in Figure 4a−c. Note in this example the presence of a stable and sustained ectopic rhythm after cell transplantation (d). Episodes of the sustained ectopic activity were interspersed with periods of the junctional escape rhythm (e, bottom) because of their similar rates. The first rhythm (e, top) was correlated, during electroanatomical mapping, with the new ventricular ectopic rhythm (electrical activation originating from the transplantation site in the posterolateral wall), whereas the second ECG pattern (e, bottom) was correlated with the junctional escape rhythm.
 Full Figure and legend (65K) |
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Electrocardiogram (ECG) recordings after complete atrioventricular block demonstrated the presence of a typical junctional escape rhythm (Fig. 4a). The source of this slow escape rhythm, which is characteristic of atrioventricular block, are cardiac cells with inherent pacemaking properties located distal to the site of conduction block. This pacemaking function is usually dormant during normal cardiac function but may become active when the ventricular beating rate slows substantially, as occurs during complete heart block. The ECG recordings also showed intermittent episodes of ventricular pacing (Fig. 4b) resulting from the programmed activity of the implanted electronic pacemaker when the spontaneous heart rate fell below a predetermined threshold.
After creation of atrioventricular block, we injected spontaneously contracting clusters of hES cardiomyocytes (40−150 beating embryoid bodies) into the posterolateral region of the left ventricle and marked the epicardial injection site with a suture. We also performed control injections of medium or nonmyocyte hES cell−derivatives into a completely different location in the anterior wall of each animal. A few days after cell transplantation, we could detect episodes of a new ventricular ectopic rhythm (Fig. 4c) that had a substantially different morphology (negative axis in leads I, II and III) compared with the junctional (Fig. 4a) or paced (Fig. 4b) rhythms.
The new ectopic rhythm was detected in 11 out of the 13 animals studied. In five animals this ectopic activity was limited to isolated beats or short runs of activity. In the remaining six animals, the ectopic activity was manifested by the presence of a regular, sustained and hemodynamically stable rhythm (Fig. 4d). The average rate of this new rhythm was 59 11 beats/min, which was similar to the junctional escape rhythm (61 6 beats/min), explaining the competition between the two rhythms observed in all animals (Fig. 4e). Interestingly, this new rhythm was sensitive to adrenergic stimulation, with the rate increasing to 94 18 beats/min after administration of isoproterenol (10−20 g/min, P < 0.05).
We next subjected the animals to an electrophysiological mapping procedure 1−3 weeks after cell transplantation. Mapping was done with a nonfluoroscopic mapping technique that uses special locatable catheters to generate detailed three-dimensional electroanatomical maps of the heart26,
27. We initially mapped the junctional escape rhythm (Fig. 5a,b; left panels). Not surprisingly, electrical activation was initiated at the superior septum (red), with the posterolateral wall being activated last (blue-purple). The average left ventricle activation time was 50 6 ms. In contrast, the new ventricular ectopic rhythm was characterized by a shift in the earliest electrical activation to the area of cell transplantation at the posterolateral wall (red area in Fig. 5a,b; right panels). Activation then propagated to the rest of the ventricle, with the septum being activated last. Total activation time of this new rhythm was 65 12 ms. In contrast, we did not note any ectopic activity from control sites (in which medium or noncardiomyocytes were injected) in the anterior wall, nor did we note any substantial ectopic activity in three control animals in which nonmyocyte cells were grafted.
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To verify that the origin of the new ventricular ectopic activity was the site of cell grafting, we navigated the catheter to the earliest activation site (arrow in Fig. 5c) and did a focal ablation at a nearby location (arrowhead). After harvesting the hearts, we noted an excellent spatial correlation between the electrophysiological and pathological findings (n = 8). Thus, the distances between the locations of earliest activation and ablation in the maps (19 5 mm) highly correlated (r2 = 0.93) with the measured distances between the ablation and injection sites (20 5 mm) in pathology (Fig. 5c, right). To verify the reproducibility of the source of the new ectopic activity, we repeated the mapping procedure in some animals. The acquired maps (Fig. 5d,e) were highly reproducible, and focal ablations delivered during each of these mapping procedures on opposite sides of the earliest activation were later identified with excellent spatial correlation in pathology (Fig. 5f).
We next validated the presence of the grafted cells at the site of earliest electrical activity. Histological sections from this area identified the transplanted cells, which were organized as cell clusters and were aligned in the appropriate juxtaposition with host cardiomyocytes (Fig. 6a−c). The grafted cells were identified and their human nature was established by positive immunostaining with anti-human mitochondrial antibodies (Fig. 6b−e). The cardiomyocyte phenotype of many (but not all) of the grafted cells was confirmed using anti-cardiac  -actinin antibodies (Fig. 6c−e). The morphology of the grafted hES cardiomyocytes (small cells with early-striated staining pattern, typical of embryonic-like cardiomyocytes) closely resembled their in vitro structure (Fig. 1a) and thus had not matured substantially after in vivo transplantation. In a minority of cases, we noted a more advanced maturation stage of the grafted cells (Fig. 6e). In contrast, we did not detect any transplanted cells in sections taken from remote myocardial areas.
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Discussion
Cell therapy is a promising therapeutic approach to myocardial repair28. In this report, we generated spontaneously excitable cardiomyocyte tissue from hES cells and showed that it integrates structurally, electrically and mechanically with rat cardiac cells in vitro. We then demonstrated that the transplanted hES cardiomyocytes survive, integrate and function in vivo by showing that they pace the ventricle in pigs with complete heart block.
Recent reports suggest that myocyte transplantation may improve cardiac function in animal models of myocardial infarction4,
12,
29,
30,
31,
32. However, it is not clear whether this functional improvement is due to direct contribution to contractility by the transplanted myocytes, attenuation of the remodeling process, amplification of an endogenous repair process or induction of angiogenesis. For example, although transplantation of skeletal myoblasts was shown to improve myocardial performance, gap junctions were not observed between graft and host tissues4,
33. Yet even the presence of such gap junctions between host and donor cardiomyocyte tissues, as observed in some studies8,
9, does not guarantee functional integration. For such integration to occur, currents generated in one cell passing through gap junctions must be sufficient to depolarize neighboring cells.
Our results demonstrate long-term electromechanical integration between host and donor tissues at several levels. Electromechanical coupling was initially demonstrated in vitro by the presence of positive Cx43 immunostaining at the interface between the hES and rat cardiomyocytes and by the appearance of synchronized electrical and mechanical activities in these cocultures. The high degree of coupling was evident by the lack of local conduction delay at the tissues' junction, by the continuous long-term coupling and by the persistent coupling during altered pacemaker position, adrenergic stimulation and partial (but not total) gap-junction uncoupling.
This study also provides evidence for the in vivo functional integration of donor cells by demonstrating the ability of hES-cell cardiomyocytes to pace the heart in swine with complete atrioventricular block. Electroanatomical mapping and subsequent pathological examination confirmed that the source of the new ventricular ectopic rhythm was the site of cell transplantation. Nevertheless, because it is not possible in these whole-organ experiments to map the electrical activity at the cellular level, we could not rule out that this new activity resulted from an indirect effect of the transplanted cells on neighboring host cardiomyocytes. Potential mechanisms for such an effect include the secretion of certain factors from the grafted cardiomyocytes or the generation of electronic currents between grafted and neighboring cardiomyocytes. Similarly, we could not rule out cell fusion as a possible mechanism of the observed results.
Disturbances in the pacemaker function or impulse propagation through the cardiac conduction system may result in severe bradycardia, circulatory failure and even death, and usually require the implantation of a permanent electronic pacemaker. Our proof-of-concept study suggests the use of excitable cell grafts as a biological alternative to implantable devices. We chose to transplant spontaneously excitable cardiomyocyte cell clusters rather than single cardiomyocytes because we hypothesized that because of sink-source mismatches, isolated donor cells connected simultaneously to a number of neighboring cells will not be able to depolarize these cells to threshold and capture the ventricle.
The ability of hES-cell cardiomyocytes to generate stable spontaneous pacemaking activity was demonstrated at several levels. Patch-clamp studies of isolated cells confirmed that they generate repetitive, spontaneous action-potentials and that this automaticity stems from a high-input resistance (low Kir channel density) coupled with a prominent sodium current and the presence of the hyperpolarization-activated current24. Continuous pacemaking activity and stable conduction properties were also demonstrated at the multicellular level in vitro during long-term recordings of isolated embryoid bodies22 and in the coculture experiments. Finally, studies in the complete atrioventricular block model also demonstrated the pacemaking capacity of the hES-cell cardiomyocytes in vivo.
The possible use of hES-cell cardiomyocytes for biological pacemaking is further strengthened by some of their unique properties. Besides the capability to screen the phenotypic properties of the cells ex vivo, they could be genetically engineered to enhance their function. Moreover, the observation that hES-cell cardiomyocytes possess functional adrenergic and cholinergic receptors that respond with appropriate chronotropic changes to specific agonists both in vitro18,
22 and in vivo suggests that a biological pacemaker could function in a physiological, rate-responsive manner.
The clinical application of such an approach, however, would require continuous fail-safe and long-term function of the grafted pacemaking cells, verification of which was beyond the scope of this study. We noted sustained ectopic activity in only half the animals, and these episodes were interspersed with episodes of junctional escape rhythm. These rhythm changes were probably due to similarities in the rates and therefore competition between the two rhythms, but we could not rule out pacing failure of the transplanted cells. It is also possible that early-stage embryonic myocytes would eventually mature into working ventricular myocytes and lose their propensity for spontaneous pacemaking. In this respect, one might be able to combine innovative gene therapy approaches34,
35,
36,
37 to biological pacing with a cell therapy strategy to create cell grafts with well-characterized, long-term pacemaking properties.
Human ES-cell cardiomyocytes may have advantages over other cell candidates for cardiac repair, such as the availability of potentially unlimited numbers of cardiomyocytes, the possibility of generating different cardiomyocyte cell types, the relative ease of genetic manipulation of these cells and their inherent structural and functional cardiomyocyte properties19. Nevertheless, several obstacles must be overcome before this strategy can reach the clinic, including the generation of large quantities of pure cardiomyocyte cultures, the prevention of immune rejection and the demonstration that the grafts survive, function and improve myocardial performance in diseased hearts. A major concern is the possible development of ES cell−related tumors such as teratomas, which were not observed in the current study. To minimize this risk, cells grafts must be free of undifferentiated ES cells.
In summary, this report provides evidence that transplanted hES-cell cardiomyocytes can integrate in vitro and in vivo with host cardiac tissue. These results suggest the potential utility of these cells to serve as a biological pacemaker and for cardiac regenerative medicine in general.
Methods
hES cell culturing and generation of embryoid bodies.
Human undifferentiated ES cells of the clone H9.238 were grown on a mitotically inactivated (mitomycin C) mouse embryonic fibroblast feeder layer (MEF) as previously described18,
38. The culture medium consisted of 20% FBS (HyClone), 80% knockout DMEM supplemented with 1 mM L-glutamine, 0.1 mM mercaptoethanol and 1% nonessential amino acids (all from Life Technologies). To induce differentiation, hES cells were dispersed to small clamps (3−20 cells) using collagenase IV (1 mg/ml, Life Technologies). The cells were then transferred to plastic Petri dishes at a cell density of about 5  106 cells in a 58-mm dish, where they were cultured in suspension for 7−10 d. During this stage the cells aggregated to form embryoid bodies, which were then plated on 0.1% gelatin-coated culture dishes and observed for the appearance of spontaneous contractions. Intact contracting areas within the embryoid bodies were then mechanically dissected using a pulled glass micropipette for use in the different experiments.
Patch-clamp studies.
For single-cell action-potential analysis, the whole-cell configuration of the patch-clamp technique was used. Cells were isolated from beating embryoid bodies by 1-h digestion at 37 °C with collagenase B (1 mg/ml, Roche Molecular Biochemical). After dissociation, cells were replated for 1−3 d on gelatin-coated glass coverslips. The patch pipette solution consisted of (in mM): 120 KCl, 1 MgCl2, 3 Mg-ATP, 10 HEPES, 10 EGTA, pH 7.3. The bath recording solution consisted of (in mM): 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, pH 7.4. Upon seal formation and after patch-break, analog capacitance compensation was used. Series resistance compensation was used up to 80%. Axopatch 200B, Digidata1322 and pClamp8 (Axon) were used for data amplification, acquisition and analysis.
Generation of primary neonatal rat ventricular myocyte cultures.
Primary monolayer cultures of neonatal rat (Sprague-Dawley) ventricular myocytes were prepared as previously described25. Briefly, after excision, the ventricles were minced in Dulbecco's phosphate buffered saline (Biological industries) and later treated with RDB (IIBR). After centrifugation, the dispersed cells were suspended in culture medium (Ham's F10), 5% fetal calf serum, 5% horse serum, 100 U/ml penicillin, 100 mg/ml streptomycin (all from Biological industries), 1 mM CaCl2 and 50 mg/100 ml bromodeoxyuridine (Sigma). Dispersed cells were then cultured on gelatin-coated (0.1%) microelectrode culture plates or on glass coverslips at a density of 1.5 106 cells/ml.
Electrophysiological and mechanical assessment of the hybrid cultures.
Once a well-synchronized activity was established in the neonatal rat cardiomyocyte cultures, spontaneously contracting areas within the human embryoid bodies were mechanically dissected and added to the cultures. The electrophysiological properties of the hybrid cultures were examined using a microelectrode array (MEA) data acquisition system (Multi Channel Systems)22,
25. The MEA plates consist of a matrix of 60 titanium nitride-gold contact (30 m) electrodes with an interelectrode distance of 100 or 200 m allowing simultaneous recording of the extracellular potentials at a sampling rate of 10 kHz. All recordings were made at 37 °C and a pH of 7.4.
Local activation time (LAT) at each electrode was determined by the timing of the maximal negative intrinsic deflection (dV/dtmin). This information was then used for the generation of color-coded activation maps by interpolating the LAT values between the electrodes using MATLAB standard two-dimensional plotting function (MATLAB 5.3.0, The MathWorks). Mechanical contractions were detected through a microscope (Axiovert 135, Zeiss) using a photodiode (UV100BG, EG&G).
Immunohistochemistry.
In the in vitro studies isolated embryoid bodies or the cocultures were fixed in 4% paraformaldehyde. In the in vivo studies, the hearts were harvested, frozen in liquid nitrogen and cryosectioned. Immunostaining was done using anti-human mitochondria antibodies, anti-Cx43, anti-cardiac troponin I (all from Chemicon) and anti- sarcomeric - actinin antibodies (Sigma). Secondary antibodies were FITC-conjugated anti-rabbit IgG and Cy3-conjugated anti-mouse IgG (Chemicon) or using the Zenon Labeling Kit (Molecular Probes). Nuclei were counterstained by ToPro3 (Molecular Probes). Confocal microscopy was done using a Nikon Eclipse E600 microscope and Bio-Rad Radiance 2000 scanning system.
Establishment of the swine complete atrioventricular block model and cell transplantation.
The study involved 13 study pigs (30−50 kg) and 3 controls. All animal experimental protocols were approved by the Animal Use and Care Committee of the Technion Faculty of Medicine. Anesthesia was maintained after intubation and mechanical ventilation with isoflurane 1%. Vascular access was obtained using cutdown of the jugular veins and carotid arteries. A 7F electrophysiological catheter was introduced to the right atrium through the jugular vein. The catheter was then navigated to the His bundle position (the major electrical conduction pathway, connecting the atria with the ventricles). Complete atrioventricular block was then created by ablating this bundle using radio-frequency energy (using a 500-kHz RF generator; RFG-3C, Radionics in a temperature control mode, 60 °C). After the generation of complete atrioventricular block, we implanted a single chamber pacemaker (ELA Medical) and positioned its electrode at the right ventricular apex to allow ventricular pacing if the junctional escape rate was <50 beats/min.
Cell transplantation.
Through a left thoracotomy, we injected the hES cardiomyocytes (40−150 contracting embryoid bodies) at a site in the posterolateral wall of the left ventricle. A suture was used to mark the exact locations where injections were made. In each animal, control injections using either medium or nonmyocyte ES derivatives were made at a different site in the anterior wall. Similar grafting experiments using nonmyocyte cell transplantation were done in the posterolateral wall in three control animals. After the procedure, the animals were treated by daily injections of cyclosporin A (10 mg/kg) and methylprednisolone (2 mg/kg) to prevent immune rejection. Body-surface electrocardiographic recordings were made daily to characterize the rate and configuration of the escape rhythm. In three animals we also implanted subcutaneously an implantable loop recorder (Reveal Plus, Medtronic) that allows continuous recording of body-surface ECGs.
Electroanatomical mapping.
A nonfluoroscopic, catheter-based, electroanatomical mapping technique (Carto, Biosense-Webster) was used to assess the electrophysiological activation patterns of the different ventricular rhythms. This system, described in detail elsewhere26,
27, uses magnetic technology to accurately detect the location of a special locatable catheter while it is deployed within the heart. By sampling the location of the catheter together with the local electrogram recorded from its tip at a plurality of endocardial sites, detailed three-dimensional electroanatomical maps of the cardiac chambers can be generated. The LAT at each sampled site was determined as the time interval between a fiducial point on the body-surface ECG and the steepest negative intrinsic deflection from the unipolar recordings. The LATs were color coded (red being earliest activation site and purple the latest) and superimposed on the three-dimensional geometry of the map.
One to three weeks after cell injection, animals were subjected to an additional electrophysiological study. Detailed electroanatomical mapping of the left ventricle was done during the appearance of the new ectopic ventricular rhythm. In some animals we also mapped the junctional escape rhythm. After establishment of the origin of the new ectopic rhythm (earliest activation site), we navigated the catheter to a nearby site (usually 2 cm away) and created a focal radio frequency ablation (temperature, 60 °C; output, 10−40 W) to allow for pathological correlation.
Note: Supplementary information is available on the Nature Biotechnology website.
Received 23 February 2004; Accepted 19 August 2004; Published online: 26 September 2004.
REFERENCES
- Pasumarthi, K.B. & Field, L.J. Cardiomyocyte cell cycle regulation. Circ. Res. 90, 1044−1054 (2002). | Article | PubMed | ISI | ChemPort |
- Cohn, J.N. et al. Report of the National Heart, Lung, and Blood Institute Special Emphasis Panel on Heart Failure Research. Circulation 95, 766−770 (1997). | PubMed | ISI | ChemPort |
- Kusumoto, F.M. & Goldschlager, N. Cardiac pacing. N. Engl. J. Med. 334, 89−97 (1996). | Article | PubMed | ISI | ChemPort |
- Taylor, D.A. et al. Regenerating functional myocardium: improved performance after skeletal myoblast transplantation. Nat. Med. 4, 929−933 (1998). | Article | PubMed | ISI | ChemPort |
- Murry, C.E., Wiseman, R.W., Schwartz, S.M. & Hauschka, S.D. Skeletal myoblast transplantation for repair of myocardial necrosis. J. Clin. Invest. 98, 2512−2523 (1996). | PubMed | ISI | ChemPort |
- Menasche, P. et al. Autologous skeletal myoblast transplantation for severe postinfarction left ventricular dysfunction. J. Am. Coll. Cardiol. 41, 1078−1083 (2003). | Article | PubMed | ISI |
- Muller-Ehmsen, J. et al. Rebuilding a damaged heart: long-term survival of transplanted neonatal rat cardiomyocytes after myocardial infarction and effect on cardiac function. Circulation 105, 1720−1726 (2002). | Article | PubMed | ISI |
- Reinecke, H., Zhang, M., Bartosek, T. & Murry, C.E. Survival, integration, and differentiation of cardiomyocyte grafts: a study in normal and injured rat hearts. Circulation 100, 193−202 (1999). | PubMed | ISI | ChemPort |
- Soonpaa, M.H., Koh, G.Y., Klug, M.G. & Field, L.J. Formation of nascent intercalated disks between grafted fetal cardiomyocytes and host myocardium. Science 264, 98−101 (1994). | PubMed | ISI | ChemPort |
- Leor, J., Patterson, M., Quinones, M.J., Kedes, L.H. & Kloner, R.A. Transplantation of fetal myocardial tissue into the infarcted myocardium of rat. A potential method for repair of infarcted myocardium? Circulation 94, II332−336 (1996). | PubMed | ChemPort |
- Toma, C., Pittenger, M.F., Cahill, K.S., Byrne, B.J. & Kessler, P.D. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation 105, 93−98 (2002). | Article | PubMed | ISI |
- Orlic, D. et al. Bone marrow cells regenerate infarcted myocardium. Nature 410, 701−705 (2001). | Article | PubMed | ISI | ChemPort |
- Klug, M.G., Soonpaa, M.H., Koh, G.Y. & Field, L.J. Genetically selected cardiomyocytes from differentiating embryonic stem cells form stable intracardiac grafts. J. Clin. Invest. 98, 216−224 (1996). | PubMed | ISI | ChemPort |
- Rubart, M. et al. Physiological coupling of donor and host cardiomyocytes after cellular transplantation. Circ. Res. 92, 1217−1224 (2003). | Article | PubMed | ISI | ChemPort |
- Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145−1147 (1998). | Article | PubMed | ISI | ChemPort |
- Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A. & Bongso, A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat. Biotechnol. 18, 399−404 (2000). | Article | PubMed | ISI | ChemPort |
- Itskovitz-Eldor, J. et al. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol. Med. 6, 88−95 (2000). | PubMed | ISI | ChemPort |
- Kehat, I. et al. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J. Clin. Invest. 108, 407−414 (2001). | Article | PubMed | ISI | ChemPort |
- Gepstein, L. Derivation and potential applications of human embryonic stem cells. Circ. Res. 91, 866−876 (2002). | Article | PubMed | ISI | ChemPort |
- Xu, C., Police, S., Rao, N. & Carpenter, M.K. Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells. Circ. Res. 91, 501−508 (2002). | Article | PubMed | ISI | ChemPort |
- Mummery, C. et al. Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. Circulation 107, 2733−2740 (2003). | Article | PubMed | ISI | ChemPort |
- Kehat, I., Gepstein, A., Spira, A., Itskovitz-Eldor, J. & Gepstein, L. High-resolution electrophysiological assessment of human embryonic stem cell-derived cardiomyocytes: a novel in vitro model for the study of conduction. Circ. Res. 91, 659−661 (2002). | Article | PubMed | ISI | ChemPort |
- Alcolea, S. et al. Downregulation of connexin 45 gene products during mouse heart development. Circ. Res. 84, 1365−1379 (1999). | PubMed | ISI | ChemPort |
- Satin, J. et al. Mechanism of spontaneous excitability in human embryonic stem cell derived cardiomyocytes. J. Physiol. (2004). | PubMed |
- Feld, Y. et al. Electrophysiological modulation of cardiomyocytic tissue by transfected fibroblasts expressing potassium channels: a novel strategy to manipulate excitability. Circulation 105, 522−529 (2002). | Article | PubMed | ISI | ChemPort |
- Ben-Haim, S.A. et al. Nonfluoroscopic, in vivo navigation and mapping technology. Nat. Med. 2, 1393−1395 (1996). | Article | PubMed | ChemPort |
- Gepstein, L., Hayam, G. & Ben-Haim, S.A. A novel method for nonfluoroscopic catheter-based electroanatomical mapping of the heart. In vitro and in vivo accuracy results. Circulation 95, 1611−1622 (1997). | PubMed | ISI | ChemPort |
- Reinlib, L. & Field, L. Cell transplantation as future therapy for cardiovascular disease?: A workshop of the National Heart, Lung, and Blood Institute. Circulation 101, E182−187 (2000). | PubMed | ISI | ChemPort |
- Etzion, S. et al. Influence of embryonic cardiomyocyte transplantation on the progression of heart failure in a rat model of extensive myocardial infarction. J. Mol. Cell. Cardiol. 33, 1321−1330 (2001). | Article | PubMed | ISI | ChemPort |
- Li, R.K. et al. Natural history of fetal rat cardiomyocytes transplanted into adult rat myocardial scar tissue. Circulation 96, II 179−186, discussion 177−186 (1997). | ISI |
- Scorsin, M. et al. Does transplantation of cardiomyocytes improve function of infarcted myocardium? Circulation 96, II−188−193 (1997). | ISI |
- Menasche, P. et al. Myoblast transplantation for heart failure. Lancet 357, 279−280 (2001). | Article | PubMed | ISI | ChemPort |
- Reinecke, H., MacDonald, G.H., Hauschka, S.D. & Murry, C.E. Electromechanical coupling between skeletal and cardiac muscle. Implications for infarct repair. J. Cell. Biol. 149, 731−740 (2000). | Article | PubMed | ISI | ChemPort |
- Edelberg, J.M., Aird, W.C. & Rosenberg, R.D. Enhancement of murine cardiac chronotropy by the molecular transfer of the human beta2 adrenergic receptor cDNA. J. Clin. Invest. 101, 337−343 (1998). | PubMed | ISI | ChemPort |
- Miake, J., Marban, E. & Nuss, H.B. Biological pacemaker created by gene transfer. Nature 419, 132−133 (2002). | Article | PubMed | ISI | ChemPort |
- Qu, J. et al. Expression and function of a biological pacemaker in canine heart. Circulation 107, 1106−1109 (2003). | Article | PubMed | ISI |
- Potapova, I. et al. Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers. Circ. Res. 94, 952−959 (2004). | Article | PubMed | ISI | ChemPort |
- Amit, M. et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 227, 271−278 (2000). | Article | PubMed | ISI | ChemPort |
Acknowledgments
We thank Asaf Zaretzki and Edith Cohen for their valuable help in the animal studies. We thank Ofer Shenker and the interdisciplinary unit for their technical assistance. This research was supported in part by the Israel Science Foundation (grant no. 520/01), by the Israel Health Ministry, by the Johnson & Johnson Focused Research Grant and by the Nahum Guzik Research Fund.
Competing interests statement:
The authors declare that they have no competing financial interests. |
