Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation. 2006;113:1832-1841
Published online before print April 10, 2006, doi: 10.1161/CIRCULATIONAHA.105.593038
CLINICAL PERSPECTIVE
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow HTML Page - index.htslp
Right arrow All Versions of this Article:
113/15/1832    most recent
CIRCULATIONAHA.105.593038v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chang, M. G.
Right arrow Articles by Abraham, M. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chang, M. G.
Right arrow Articles by Abraham, M. R.
Right arrowPubmed/NCBI databases
Medline Plus Health Information
*Arrhythmia
Related Collections
Right arrow Arrythmias-basic studies
Right arrow Arrhythmias, clinical electrophysiology, drugs

(Circulation. 2006;113:1832-1841.)
© 2006 American Heart Association, Inc.

Arrhythmia/Electrophysiology

Proarrhythmic Potential of Mesenchymal Stem Cell Transplantation Revealed in an In Vitro Coculture Model

Marvin G. Chang, BS; Leslie Tung, PhD; Rajesh B. Sekar, MS; Connie Y. Chang, BS; Josh Cysyk, MS; Peihong Dong, BS; Eduardo Marbán, MD, PhD; M. Roselle Abraham, MD

From the Department of Biomedical Engineering (L.T., R.B.S., J.C.) and Division of Cardiology (M.G.C., C.Y.C., P.D., E.M., M.R.A.), The Johns Hopkins University, Baltimore, Md.

Correspondence to M. Roselle Abraham, MD, Department of Cardiology, Johns Hopkins Hospital, 720 Rutland Ave, Ross Building, Room 871, Baltimore, MD 21205. E-mail mabraha3{at}jhmi.edu

Received October 7, 2005; revision received January 3, 2006; accepted February 2, 2006.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Background— Mesenchymal stem cells (MSCs) are bone marrow stromal cells that are in phase 1 clinical studies of cellular cardiomyoplasty. However, the electrophysiological effects of MSC transplantation have not been studied. Although improvement of ventricular function would represent a positive outcome of MSC transplantation, focal application of stem cells has the potential downside of creating inhomogeneities that may predispose the heart to reentrant arrhythmias. In the present study we use an MSC and neonatal rat ventricular myocyte (NRVM) coculture system to investigate potential proarrhythmic consequences of MSC transplantation into the heart.

Methods and Results— Human MSCs were cocultured with NRVMs in ratios of 1:99, 1:9, and 1:4 and optically mapped. We found that conduction velocity was decreased in cocultures compared with controls, but action potential duration (APD80) was not affected. Reentrant arrhythmias were induced in 86% of cocultures containing 10% and 20% MSCs (n=36) but not in controls (n=7) or cocultures containing only 1% MSCs (n=4). Immunostaining, Western blot, and dye transfer revealed the presence of functional gap junctions involving MSCs.

Conclusions— Our results suggest that mixtures of MSCs and NRVMs can produce an arrhythmogenic substrate. The mechanism of reentry is probably increased tissue heterogeneity resulting from electric coupling of inexcitable MSCs with myocytes.


Key Words: arrhythmia • conduction • electrophysiology • tachyarrhythmias


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Heart failure affects an estimated 4.9 million people in the United States, with 550 000 new cases reported annually.1 Despite major improvements in medical therapy, a significant proportion of patients remain symptomatic. Recent encouraging results from cellular therapy in animal models2,3 have greatly increased the enthusiasm for clinical trials in patients suffering from heart failure. Several cell types, including bone marrow mononuclear cells,4–6 have been used for transplantation in preclinical and clinical studies,7 the most recent involving mesenchymal stem cells (MSCs). MSCs are bone marrow stromal cells that have the potential of differentiating toward a number of cellular phenotypes.8

Advantages of cellular therapy based on MSCs include their rapid expansion in culture without discernible chromosomal abnormalities or loss of telomerase activity; in addition, MSCs lack B7 costimulatory molecules CD80 and CD86 and do not stimulate an allogeneic immune cell proliferation.9 Therefore, MSC therapy offers the tantalizing opportunity for "off-the-shelf" allogeneic stem cell therapy soon after a patient presents with myocardial infarction or heart failure.

Clinical Perspective p 1841

Like most undifferentiated cells, MSCs have a resting potential of –30 to –40 mV.10 MSCs possess delayed rectifier channels, and a small fraction expresses the L-type calcium channel. However, MSCs do not express sodium or inwardly rectifying potassium channels.10 Furthermore, despite expression of some cardiac muscle–specific proteins, complete cardiac transdifferentiation of MSCs to cardiomyocytes has not been confirmed.11 Significantly, the gap junction protein Cx43 is expressed in MSCs and forms functional gap junctions between MSCs as well as between MSCs and myocytes12; this attribute has been used to generate pacemaker-like areas of focal ventricular automaticity in animal models.13

On the basis of evidence that MSCs migrate to sites of tissue injury, allogeneic MSCs are in phase 1 clinical trials in the United States, where postmyocardial infarction patients are being enrolled for intravenous MSC therapy. Numerous animal studies have demonstrated the beneficial effects of MSC transplantation on cardiac hemodynamics11,14–19; the arrhythmic and electrophysiological consequences have not been systematically studied, however. The only study addressing possible proarrhythmic consequences of MSC therapy was performed by Pak et al,20 who found an increase in heterogeneously distributed sympathetic nerve sprouting in pigs after MSC transplantation. Amado and coworkers14 recently reported no significant increase in sudden death in pigs receiving intramyocardial injections of MSCs compared with placebo. However, they did not perform ECG monitoring or electrophysiological studies and were unable to accurately quantify MSC engraftment in these animals. Phase 1 studies of human skeletal myoblast transplantation have highlighted the importance of thorough assessment of arrhythmia risk before human trials.21,22

In the present study we used a reductionist in vitro coculture approach that allows a controlled study of the arrhythmogenic potential of MSCs. We hypothesized that inexcitable MSCs would act as microscopic current sinks, constitute abnormal electric loads, and predispose to conduction block and reentrant arrhythmias when electrically coupled with myocytes. The main advantage of an in vitro model of MSC transplantation is that it permits the evaluation of pure MSC–myocyte interactions without the confounding proarrhythmic effects of heart failure or sympathetic hyperinnervation20 seen in animal models.23 It also lends itself to manipulation of stem cell number and location and may provide important insights into the electrophysiological effects of MSC transplantation.

We found that cocultures containing ≥10% MSCs exhibited decreased conduction velocity and were predisposed to reentrant arrhythmias, which are the most common cause of post–myocardial infarction arrhythmias.24


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Cell Culture
Human MSCs were obtained from Cambrex (Walkersville, Md) and grown in MSC basal growth medium (MSCBM, Clonetics Corp, Bio Whittaker, Inc, San Diego, Calif). MSCs were positive for CD105, CD166, CD29, and CD44 and negative for CD14, CD34, and CD45. The cells were seeded at 5000 to 6000 cells/cm2 and subcultured when they attained a density of 90%. Cells were frozen in liquid nitrogen at their third passage and were amplified up to 10 population doublings.

Immunostaining and Western Blot
MSCs were grown to confluence; immunostaining and Western blot were performed as previously described.25

Dye Transfer
Cell-to-cell coupling via gap junctions was assessed by dye transfer of calcein between human MSCs and neonatal rat ventricular myocytes (NRVMs). Details are included in the online-only Data Supplement.

Cardiac Myocytes
NRVMs were dissociated from ventricles of 2-day-old neonatal Sprague-Dawley rats (Harlan, Indianapolis, Ind) as previously described.25,26

Cocultures
MSCs and NRVMs were mixed and plated in ratios of 1:4 (20% MSCs), 1:9 (10% MSCs), and 1:99 (1% MSCs). The cell mixtures were cocultured for 9 to 11 days as isotropic monolayers on 21-mm plastic coverslips coated with human fibronectin (25 µg/mL, BD Biosciences, Becton, Dickinson and Co, San Jose, Calif) and then used for optical mapping (NRVMs mature to form rod-shaped, striated cells after 6 days in culture). On day 2 after cell plating, serum was reduced to 2%. For imaging of cocultures, MSCs were labeled with CMFDA green (10 µmol/L, Molecular Probes, Carlsbad, Calif) to enable cell tracking (n=12).

Recent reports by Amado et al14 of improved functional effects after focal intramyocardial injection of MSCs prompted us to perform a second set of experiments to simulate the in vivo situation in which MSCs would exist as islands mixed with myocytes (n=7). For this purpose, we preplated MSCs using poly-dimethyl-siloxane stencils and then added NRVMs 4 hours later, generating islands (6-mm diameter) of 20% MSC-NRVM mixtures surrounded by NRVMs. Optical mapping was performed after 9 days in culture.

Optical Mapping
Electric activity was monitored by optical mapping as previously described.25 Unipolar point stimulation via a platinum electrode was applied just above the monolayer with platinum field electrodes at the sides of the chamber acting as the return line.

Experimental Protocol
A 1-second recording was initially made to check for spontaneous activity. Thirty-five–beat drive trains of 10-ms monophasic pulses (1.5x diastolic threshold) were subsequently used for stimulation throughout the experiment. Stimulation was begun at 1 Hz and increased progressively by 1 Hz until 1:1 capture was no longer observed or reentry was initiated. Reentry was considered initiated if it lasted for >5 rotations and was considered sustained if it lasted for >5 minutes.

Data Analysis
Data was analyzed in MATLAB (The MathWorks Inc, Natick, Mass) with the use of custom-written scripts. Details are included in the online-only Data Supplement.

Statistics
Data are expressed as mean±SD. Differences between means were tested with the paired t test with the use of the statistical software GraphPad Prism (Graphpad Software, Inc, San Diego, Calif). Unpaired t tests were used to compare action potential duration (APD80) and conduction velocity between cocultures and controls, and paired t tests were used in all other cases. A probability value of <0.05 was considered statistically significant.

The authors had full access to the data and take full responsibility for its integrity. All authors have read and agree to the manuscript as written.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
MSCs Express Cx43 and Are Electrically Coupled With Myocytes
Structural characterization was performed before the electrophysiological phenotype of the cocultures was studied. Transmitted light (Figure 1A) and fluorescence microscopy (Figure 1B) revealed that MSCs are randomly distributed in culture and that large numbers of MSCs existed in a plane below the NRVMs with some interspersed in between the myocytes. This is perhaps due to the fact that MSCs adhere to the coverslips within 3 to 4 hours, whereas NRVMs take up to 24 hours to fully adhere.


Figure 1
View larger version (74K):
[in this window]
[in a new window]
  		Figure 1. A and B, Imaging of 1:4 MSC:NRVM cocultures on day 10 after plating. A, Transmitted light image of a 1:4 MSC:NRVM coculture (containing 2x105 MSCs and 8x105 NRVMs at initial plating) shows a confluent monolayer. B, Fluorescent image of CMFDA-labeled MSCs overlaying the transmitted light image seen in A shows random distribution of MSCs; MSCs attach, spread out, and continue to retain the label even 9 days after coculture.

An important characteristic of MSCs is their ability to form gap junctions between themselves and with neighboring cardiomyocytes.12 Figure 2A and 2B confirm Cx43 expression in MSC cultures by immunostaining and Western blot, respectively. We found high levels of Cx43 expression in MSCs, but, in contrast to adult myocytes in which Cx43 exists mainly as plaques, Cx43 was predominantly expressed in a punctate fashion throughout the plasma membrane. We confirmed the presence of functional gap junctions between MSCs and NRVMs by demonstrating calcein but not Di-I (membrane dye; Invitrogen) transfer from MSCs to NRVMs (Figure 2C and 2D).


Figure 2
View larger version (59K):
[in this window]
[in a new window]
  		Figure 2. Gap junction expression and coupling of MSCs with myocytes. A, Fluorescent images of Cx43 expression in MSCs in which nuclei were labeled with DAPI. B, Western blot analysis of Cx43 expression in MSC cultures. C and D, Fluorescent microscopic images of dye transfer from Di-I– and calcein-labeled MSCs to unlabeled NRVMs. Arrows indicate calcein-labeled NRVMs.

Electrophysiological Characterization Using Optical Mapping
In the first set of experiments, we characterized cocultures containing MSCs and NRVMs that were coplated in several ratios. We found that the conduction velocity (Figure 3C) was decreased (P=0.003, t test) by an average of 33% in 20% MSC/NRVM cocultures (14.7±2.8 cm/s; n=7) compared with controls (21.8±3.0 cm/s; n=7). The voltage maps for both the NRVM controls (Figure 3A) and 20% MSC/NRVM cocultures (Figure 3B) illustrate the differences in conduction velocity observed. Additionally, we found that, with the exception of 1-Hz pacing (P=0.007), APD80 as a function of the pacing interval (APD restitution) was not significantly changed in 20% MSC/NRVM cocultures (P=0.244) compared with controls (Figure 3D and 3E).


Figure 3
View larger version (37K):
[in this window]
[in a new window]
  		Figure 3. A and B, Impulse propagation. Voltage maps and optical action potentials during propagation (during 2-Hz pacing) of an impulse in an NRVM-only control monolayer (A) and a representative 1:4 MSC:NRVM coculture (B). The propagation wavefront is delayed in the coculture compared with control. Color bar corresponds to the normalized voltage level, with blue being resting state and red the peak of the action potential. Tracings are representative optical action potentials measured in monolayers. Bar graphs display conduction velocity (CV) (C) and APD80 (D) in NRVM-only controls (n=7) and 1:4 MSC:NRVM cocultures (n=7). Conduction velocity is significantly decreased (P=0.003, t test) but APD80 is not significantly different in cocultures compared with controls. With the exception of 1-Hz pacing frequency, the APD plotted as a function of CL, or APD restitution (E), is not significantly different (P=0.244) in 20% MSC cocultures compared with controls.

Reentrant Arrhythmias Are Easily Inducible in Mixtures of MSCs and Cardiomyocytes
Reentrant arrhythmias (spiral waves) were induced by rapid pacing between 5 to 7 Hz and were preceded by wavebreaks. This is seen in Figure 4A, which shows 6-Hz pacing in which wave propagation is fairly uniform in the second frame; with continued pacing, wavebreaks appear in the third frame and precede reentry initiation. Reentrant arrhythmias were induced in 86% of the 10% and 20% MSC cocultures that were mapped (n=36) but in none of the 1% MSC cocultures or NRVM-only controls. Reentries that sustained for >30 seconds and 5 minutes were observed in 83% and 64%, respectively, of the cocultures that were mapped.


Figure 4
View larger version (65K):
[in this window]
[in a new window]
  		Figure 4. Initiation of reentry and different reentry patterns. A, Voltage maps (during 1 stimulus) during pacing at 6 Hz in a representative 1:4 MSC:NRVM coculture illustrates wavebreaks in the third frame and reentry initiation in the sixth frame. B, Voltage maps during reentry in 4 different 1:4 MSC:NRVM cocultures showing a single spiral (B), figure-of-eight reentry (C), dual-arm reentry (D), and complex reentry (E). (The color bars in the figures are the same as in Figure 3.)

Single spiral waves similar to those seen in Figure 4B occurred most frequently; however, a variety of other reentry morphologies were also observed. Of the reentries that were sustained (n=23), 61% were initially single spirals (Figure 4B), 17% figure-of-eight spirals (Figure 4C), 13% dual-arm reentries (Figure 4D), and 9% complex reentries (Figure 4E). Single spiral wave reentry consists of a single spiral and usually manifests as monomorphic ventricular tachycardia in the clinical setting. Figure-of-eight reentry consists of 2 counterrotating spirals, and dual-arm reentry consists of 2 individual spirals that are coupled. Finally, complex reentry exhibits many spirals that may collide with one another, forming significant wavebreaks, and this morphology most resembles ventricular fibrillation. In all experiments in which reentries started out as dual-arm, complex, or figure-of-eight, the morphology eventually simplified to single spiral waves when reentry was sustained.

Cycle Length Adaptation in Sustained Reentry
Sustained reentry cycle length (CL) varied from 106 to 196 ms (n=20), with the average being 150±28 ms. Interestingly, the reentries accelerated (Figure 5A) by 17% from a mean of 185±53 ms within 30 seconds of initiation to 146±34 ms after reentry had been sustained for 5 minutes (n=9; P<0.005). To shed light on the possible mechanisms underlying reentry CL adaptation, we measured wavelength, APD80 (Figure 5B), wavefront conduction velocity (Figure 5C), wavefront curvature (Figure 5D), and spiral tip pathlength (Figure 5E) during reentry. We found that reentry conduction velocity was increased (10.5±2.9 versus 12.9±2.8 cm/s; P<0.005), whereas APD80 (103±16 versus 90±15 ms; P<0.001), curvature (0.33±0.02 versus 0.28±0.04; P=0.08), and pathlength (6.7±2.4 versus 5.1±2.5 mm; P<0.1) decreased without a significant change in reentry wavelength (7.87±0.37 versus 7.93±0.27 mm; P>0.4). Interestingly, the majority of the tip trajectories revealed initial hypocycloidal phenotypes that evolved into irregular phenotypes (Figure 5G) during sustained reentry.


Figure 5
View larger version (24K):
[in this window]
[in a new window]
  		Figure 5. Reentry CL adaptation. Each panel shows data from individual experiments within 30 seconds of reentry initiation and after reentry had sustained for 5 minutes. A, Reentry CL decreased significantly in all reentries (n=7; P<0.005). Reentry APD80 (B) was significantly decreased (n=7; P<0.001), and reentry conduction velocity (CV) (C) was significantly increased (n=6; P<0.005). Wavefront curvature (D) was decreased (n=7; P=0.08), and spiral tip pathlength (E) was decreased in 6 of 7 experiments (n=7; P<0.1). F illustrates a "phase singularity" in which all phases converge and indicates the spiral wave tip. G illustrates a hypocycloidal trajectory (left) within 30 seconds of initiation that evolves into an irregular trajectory after reentry sustained for 5 minutes (right). The spacing is 2 mm apart, and the insets represent a x4 magnified version of the tip trajectory over 3 cycles.

Optical Mapping of MSC-NRVM Islands Surrounded by NRVMs
Because uniform mixtures of MSCs and NRVMs were arrhythmogenic, we investigated whether MSC-NRVM islands surrounded by myocytes could support reentry. Optical mapping of these cocultures (n=7) revealed delayed activation and repolarization of the MSC-NRVM region even at low pacing rates (Figure 6A), reentry initiation at high pacing rates, and pinning of the spirals to the MSC-NRVM islands (Figure 6B) in 6 of 7 cultures that were mapped. This suggests that MSCs have the potential of introducing microscopic and macroscopic heterogeneities that are proarrhythmic.


Figure 6
View larger version (60K):
[in this window]
[in a new window]
  		Figure 6. A and B, Voltage maps in an MSC:NRVM coculture consisting of a mixture of MSCs and NRVMs (20% MSCs) in the center surrounded by NRVMs. A, Delayed activation and recovery from activation (repolarization) during 2-Hz pacing is evident in the region of MSC coculture that is in the center (indicated by white circle in first panel). B, Reentry was initiated by 7-Hz pacing and was pinned to the central MSC:NRVM coculture region. (The color bar in the figures is the same as in Figure 3.) Arrows in both panels indicate direction of wavefront propagation.

Triggered Activity and Increased Automaticity Are Not Observed in MSC-NRVM Cocultures
Because abnormal automaticity and triggered activity can also induce arrhythmias, we examined the susceptibility of these cocultures to triggered activity. Early afterdepolarizations were not observed spontaneously or at low pacing rates; delayed afterdepolarizations were only seen with rapid pacing in the presence of 2.5 µmol/L BayK 8644, an L-type calcium channel agonist.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Our study characterizes for the first time the electrophysiological consequences of mixing MSCs and myocytes at a syncytial level. The main findings of this study are (1) that mixtures of MSCs and NRVMs containing ≥10% MSCs resulted in an arrhythmogenic substrate that is characterized by decreased conduction velocity and easily inducible sustained reentrant arrhythmias and (2) that the mechanism underlying arrhythmogenicity of the cocultures is probably coupling of cardiomyocytes with inexcitable MSCs.

This study points to possible undesirable consequences of MSC therapy and emphasizes the importance of quantifying MSC engraftment and performing ECG monitoring and detailed electrophysiological studies in large-animal models of intramyocardial MSC therapy. On the basis of our results, we would not expect a proarrhythmic effect of intravenous MSC therapy, which is currently in phase 1 trials. This is due to the fact that in mouse models of intravenous MSC therapy, the injected cells were monodispersed27 and tended to resemble our 1% MSC/NRVM cocultures.

Possible Mechanisms Underlying Decreased Conduction Velocity Observed in MSC-NRVM Cocultures
Conduction velocity was significantly depressed in MSC cocultures containing >10% MSCs despite the ability of MSCs to couple with their cardiomyocyte and MSC neighbors. These observations can be explained by the dependence of conduction velocity on both gap junction coupling and excitability.28–30 The observed decrease in conduction velocity in these MSC cocultures may be attributable to the inexcitable nature of the MSCs and their ability to act as current sinks. The coupled MSCs, which have a resting membrane potential of {approx}–40 mV,10 could partially depolarize neighboring myocytes, inactivate Na+ channels, and decrease conduction velocity.

Surprisingly, APD80 was not significantly changed in our cocultures compared with controls. MSCs secrete an important array of growth factors and cytokines including macrophage, granulocyte, and granulocyte-macrophage colony stimulating factors, stem cell factor 1, leukemia inhibitory factor, stromal cell–derived factor-1, Flt-3 ligand, and interleukin (IL)-1, -6, -7, -8, -11, -14, and -15.31,32 On the basis of our results with human skeletal myoblast and NRVM cocultures,25 we speculate that these paracrine factors secreted by MSCs may induce myocyte hypertrophy and consequently increase APD, which is balanced by increased electric load imposed by coupled MSCs, resulting in a net insignificant change in APD.

Factors Underlying Initiation and Maintenance of Reentrant Arrhythmias
Factors predisposing to reentrant arrhythmias include a heterogeneous distribution of MSCs and electric coupling of inexcitable MSCs with myocytes. Consequently, MSCs may induce Na+ channel inactivation in myocytes in a heterogeneous fashion. These effects would be exacerbated at high pacing rates because of incomplete recovery of Na+ channels and result in conduction block, wavebreak, and reentrant arrhythmias. We also observed lower conduction velocities at all pacing rates in MSC cocultures compared with NRVM-only (Figure 7A) controls, which could predispose to reentrant arrhythmias in the in vivo setting after intramyocardial injection and engraftment of large numbers of MSCs. Delayed activation of areas containing a mixture of MSCs and myocytes (as seen in Figure 4) would also delay the time of recovery of that region. For this reason, high heart rates or premature impulses may encounter refractory tissue in the MSC area, leading to localized block of impulse propagation and susceptibility to reentry.


Figure 7
View larger version (18K):
[in this window]
[in a new window]
  		Figure 7. A, Conduction velocity (CV) restitution. Rate dependence of conduction velocity was decreased in 1:4 MSC:NRVM cocultures compared with NRVM-only controls. B, Table shows that slope of APD restitution (rate dependence of APD80) at reentry rates was <0.5.

Because dynamic instabilities in the setting of steep APD restitution slopes (slopes >0.5, when CL is plotted against APD) can also result in wavebreak and reentry initiation,33 we measured APD restitution (CL dependence of APD) slopes at different reentry CLs (Figure 3E). We found that APD80 slopes were <0.5, ruling out dynamic instability as cause of wavebreaks (Figure 7B).

Functional, Not Anatomic, Reentry Is the Cause of Arrhythmias Observed in MSC-NRVM Cocultures
Anatomic reentry occurs because of rotation of the cardiac impulse around a fixed obstacle like scar, whereas functional reentry can occur in homogeneous tissue and takes the form of an Archimedean spiral that rotates around an unexcited but excitable core.34,35 Several observations suggest that reentries observed in our cocultures are functional. (1) Tip trajectories of the reentries reveal hypocycloidal or irregular patterns of tip meandering (Figure 8A) that have been described experimentally in ventricular and atrial tissue36 as well as in numerical models of functional reentry.37–39 (2) Spiral drift, in which the reentry would move from one distinct location to another, was frequently observed (Figure 8B). (3) The reentry wavefront could be closely fit to an Archimedean spiral (Figure 8C). (4) In addition, we did not find evidence of action potential activation in the core (at 1-mm resolution); the core region was partially depolarized during reentry with a "double-humped" morphology characteristic of such regions (Figure 8D) and excited when the reentry wave drifted.


Figure 8
View larger version (58K):
[in this window]
[in a new window]
  		Figure 8. Characteristics of functional reentry. Spiral tip trajectories were tracked by phase analysis and revealed a variety of phenotypes. A, Hypocycloid tip trajectory (inset, x3 magnification). B, Spiral drift (inset, x1.5 magnification). The trajectory for each CL of reentry was plotted in the following order: red, blue, green, cyan, and pink; the scale is 2 mm. C, Representative fit of Archimedean spiral (red tracing) to the spiral wavefront obtained experimentally (blue tracing) in MSC:NRVM cocultures. D, Representative optical action potential recordings from 253 channels during reentry show that the reentry core (indicated by the yellow circle) is partially depolarized, lacks fully activated action potentials, and has a characteristic doubled-humped potential during a single reentry cycle. The snapshot of the tableau (middle) is of normalized raw data in which the drift is due to photo bleaching of the dye. The panels below and above the tableau are nonnormalized detrended and filtered data of a representative channel within and far away from the core, respectively.

Mechanism of Reentry CL Adaptation
Previous work40,41 has revealed the importance of reentry wavelength and pathlength on the CL and stability of anatomic reentry. However, to date, there has been very little quantitative experimental analysis of CL adaptation in functional reentry. In our experiments, acceleration of reentry was associated with a decrease in reentry APD80, tip pathlength, and wavefront curvature and increase in reentry conduction velocity without any change in reentry wavelength.

Reentry rate is dependent on APD (tissue refractoriness), tissue excitability, wavefront curvature, source-load effects, and reentry core size (pathlength).42 Decreased APD may occur because of an increase in intracellular calcium resulting from high-frequency stimulation that occurs during reentry, enhancing calcium-dependent ICaL inactivation. Decreased APD would also be expected to shorten refractoriness that would decrease wavefront–waveback interactions, resulting in increased wavefront conduction velocity. Because calcium is a major contributor to transmembrane potential at sites of steep wavefront curvature,43 as seen in the core, the increased intracellular calcium, by increasing excitability in the region of the spiral tip, may also decrease core size.

Limitations
One potential limitation of this study is the use of a 2-dimensional experimental model to predict effects of cell therapy in the 3-dimensional heart. Because of differences in electric loading of cells in the 2-dimensional monolayer versus the 3-dimensional heart, the impact of a noncardiac cell is clearer and would be expected to be magnified in 2-dimensional compared with 3-dimensional tissue because there are fewer shunt pathways available for propagation to circumvent the noncardiac cell. Thus, on this basis, one might expect to find a higher incidence of arrhythmia in 2-dimensional than in 3-dimensinoal tissue. However, the difference in arrhythmia incidence would be a quantitative and not a qualitative difference and would not preclude our results showing an increased incidence of arrhythmias with increasing numbers of MSCs. Indeed, our results are to be expected on the basis of fundamental biophysical principles.

Bearing in mind this limitation, in a separate study we were able to reproduce features of clinical arrhythmias seen after skeletal myoblast transplantation25 that were not seen in small-animal models of myoblast transplantation. This underscores the importance of using both in vitro and in vivo approaches to arrhythmic risk assessment before clinical application of intramyocardial stem cell therapy.

Another limitation of our investigation involves the use of NRVMs for our cultures. NRVMs are electrophysiologically different from adult human cardiomyocytes. However, it is not possible at the present time to culture cells of other species that more closely resemble the human ventricular myocyte. Nevertheless, the NRVM has proven to be an effective model for the study of cardiac conduction45 and reentry.26,46,47

Conclusions
Our results indicate that in vitro, mixtures of MSCs and cardiomyocytes result in an arrhythmogenic substrate when the MSC concentration is ≥10%. Reentry, not triggered activity or increased automaticity, was observed to underlie arrhythmias seen in these cocultures.

Despite the difficulty in extrapolating in vitro results to clinical cardiology, these findings of easily inducible sustained reentrant arrhythmias motivate a careful assessment of arrhythmogenic potential by performing electrophysiological studies in large-animal models of MSC transplantation before proceeding to intramyocardial MSC therapy in patients. Our results also suggest that intramyocardial MSC injections restricted to areas of scar and intravenous MSC therapy (which results in few MSCs surrounded by myocytes) would probably be safe from an arrhythmia standpoint. Finally, these findings indicate that if current engraftment rates are increased by genetic modification44 or other means to improve therapeutic outcome, the resulting tissue substrate may then be prone to arrhythmias.


*    Acknowledgments
 
This study was supported by the Donald W. Reynolds Foundation, a Scientist Development Grant of the American Heart Association (Dr Abraham), and National Institutes of Health grant HL66239 (Dr Tung). Dr Marbán holds the Michel Mirowski, MD, Professorship of Cardiology. We thank Dr Flavio Fenton, Dr Alan Garfinkel, Dr Zhilin Qu, and Dr Mark Pittenger for helpful discussions and Roland Emokpae, Jr, for technical assistance.

Disclosures

None.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Hunt SA, Baker DW, Chin MH, Cinquegrani MP, Feldman AM, Francis GS, Ganiats TG, Goldstein S, Gregoratos G, Jessup ML, Noble RJ, Packer M, Silver MA, Stevenson LW, Gibbons RJ, Antman EM, Alpert JS, Faxon DP, Fuster V, Jacobs AK, Hiratzka LF, Russell RO, Smith SC Jr. ACC/AHA guidelines for the evaluation and management of chronic heart failure in the adult: executive summary: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee to Revise the 1995 Guidelines for the Evaluation and Management of Heart Failure): developed in collaboration with the International Society for Heart and Lung Transplantation; endorsed by the Heart Failure Society of America. Circulation. 2001; 104: 2996–3007.[Free Full Text]
  2. Silva GV, Litovsky S, Assad JA, Sousa AL, Martin BJ, Vela D, Coulter SC, Lin J, Ober J, Vaughn WK, Branco RV, Oliveira EM, He R, Geng YJ, Willerson JT, Perin EC. Mesenchymal stem cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a canine chronic ischemia model. Circulation. 2005; 111: 150–156.[Abstract/Free Full Text]
  3. Dai W, Hale SL, Martin BJ, Kuang JQ, Dow JS, Wold LE, Kloner RA. Allogeneic mesenchymal stem cell transplantation in postinfarcted rat myocardium: short- and long-term effects. Circulation. 2005; 112: 214–223.[Abstract/Free Full Text]
  4. Wollert KC, Meyer GP, Lotz J, Ringes-Lichtenberg S, Lippolt P, Breidenbach C, Fichtner S, Korte T, Hornig B, Messinger D, Arseniev L, Hertenstein B, Ganser A, Drexler H. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet. 2004; 364: 141–148.[CrossRef][Medline] [Order article via Infotrieve]
  5. Strauer BE, Brehm M, Zeus T, Kostering M, Hernandez A, Sorg RV, Kogler G, Wernet P. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation. 2002; 106: 1913–1918.[Abstract/Free Full Text]
  6. Schachinger V, Zeiher AM. Stem cells and cardiovascular and renal disease: today and tomorrow. J Am Soc Nephrol. 2005; 16 (suppl 1): S2–S6.[Abstract/Free Full Text]
  7. Laflamme MA, Murry CE. Regenerating the heart. Nat Biotechnol. 2005; 23: 845–856.[CrossRef][Medline] [Order article via Infotrieve]
  8. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science. 1999; 284: 143–147.[Abstract/Free Full Text]
  9. Tse WT, Pendleton JD, Beyer WM, Egalka MC, Guinan EC. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation. 2003; 75: 389–397.[CrossRef][Medline] [Order article via Infotrieve]
  10. Heubach JF, Graf EM, Leutheuser J, Bock M, Balana B, Zahanich I, Christ T, Boxberger S, Wettwer E, Ravens U. Electrophysiological properties of human mesenchymal stem cells. J Physiol. 2004; 554: 659–672.[Abstract/Free Full Text]
  11. Toma C, Pittenger MF, Cahill KS, Byrne BJ, Kessler PD. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation. 2002; 105: 93–98.[Abstract/Free Full Text]
  12. Valiunas V, Doronin S, Valiuniene L, Potapova I, Zuckerman J, Walcott B, Robinson RB, Rosen MR, Brink PR, Cohen IS. Human mesenchymal stem cells make cardiac connexins and form functional gap junctions. J Physiol. 2004; 555: 617–626.[Abstract/Free Full Text]
  13. Potapova I, Plotnikov A, Lu Z, Danilo P Jr, Valiunas V, Qu J, Doronin S, Zuckerman J, Shlapakova IN, Gao J, Pan Z, Herron AJ, Robinson RB, Brink PR, Rosen MR, Cohen IS. Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers. Circ Res. 2004; 94: 952–959.[Abstract/Free Full Text]
  14. Amado LC, Saliaris AP, Schuleri KH, St John M, Xie JS, Cattaneo S, Durand DJ, Fitton T, Kuang JQ, Stewart G, Lehrke S, Baumgartner WW, Martin BJ, Heldman AW, Hare JM. Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells after myocardial infarction. Proc Natl Acad Sci U S A. 2005; 102: 11474–11479.[Abstract/Free Full Text]
  15. Shake JG, Gruber PJ, Baumgartner WA, Senechal G, Meyers J, Redmond JM, Pittenger MF, Martin BJ. Mesenchymal stem cell implantation in a swine myocardial infarct model: engraftment and functional effects. Ann Thorac Surg. 2002; 73: 1919–1925;discussion 1926.
  16. Katritsis DG, Sotiropoulou PA, Karvouni E, Karabinos I, Korovesis S, Perez SA, Voridis EM, Papamichail M. Transcoronary transplantation of autologous mesenchymal stem cells and endothelial progenitors into infarcted human myocardium. Catheter Cardiovasc Interv. 2005; 65: 321–329.[CrossRef][Medline] [Order article via Infotrieve]
  17. Chen SL, Fang WW, Qian J, Ye F, Liu YH, Shan SJ, Zhang JJ, Lin S, Liao LM, Zhao RC. Improvement of cardiac function after transplantation of autologous bone marrow mesenchymal stem cells in patients with acute myocardial infarction. Chin Med J (Engl). 2004; 117: 1443–1448.[Medline] [Order article via Infotrieve]
  18. Nagaya N, Fujii T, Iwase T, Ohgushi H, Itoh T, Uematsu M, Yamagishi M, Mori H, Kangawa K, Kitamura S. Intravenous administration of mesenchymal stem cells improves cardiac function in rats with acute myocardial infarction through angiogenesis and myogenesis. Am J Physiol. 2004; 287: H2670–H2676.
  19. Chen SL, Fang WW, Ye F, Liu YH, Qian J, Shan SJ, Zhang JJ, Chunhua RZ, Liao LM, Lin S, Sun JP. Effect on left ventricular function of intracoronary transplantation of autologous bone marrow mesenchymal stem cell in patients with acute myocardial infarction. Am J Cardiol. 2004; 94: 92–95.[CrossRef][Medline] [Order article via Infotrieve]
  20. Pak HN, Qayyum M, Kim DT, Hamabe A, Miyauchi Y, Lill MC, Frantzen M, Takizawa K, Chen LS, Fishbein MC, Sharifi BG, Chen PS, Makkar R. Mesenchymal stem cell injection induces cardiac nerve sprouting and increased tenascin expression in a swine model of myocardial infarction. J Cardiovasc Electrophysiol. 2003; 14: 841–848.[CrossRef][Medline] [Order article via Infotrieve]
  21. Menasche P, Hagege AA, Vilquin JT, Desnos M, Abergel E, Pouzet B, Bel A, Sarateanu S, Scorsin M, Schwartz K, Bruneval P, Benbunan M, Marolleau JP, Duboc D. Autologous skeletal myoblast transplantation for severe postinfarction left ventricular dysfunction. J Am Coll Cardiol. 2003; 41: 1078–1083.[Abstract/Free Full Text]
  22. Smits PC, van Geuns RJ, Poldermans D, Bountioukos M, Onderwater EE, Lee CH, Maat AP, Serruys PW. Catheter-based intramyocardial injection of autologous skeletal myoblasts as a primary treatment of ischemic heart failure: clinical experience with six-month follow-up. J Am Coll Cardiol. 2003; 42: 2063–2069.[Abstract/Free Full Text]
  23. Tomaselli GF, Beuckelmann DJ, Calkins HG, Berger RD, Kessler PD, Lawrence JH, Kass D, Feldman AM, Marban E. Sudden cardiac death in heart failure: the role of abnormal repolarization. Circulation. 1994; 90: 2534–2539.[Abstract/Free Full Text]
  24. Janse MJ, Wit AL. Electrophysiological mechanisms of ventricular arrhythmias resulting from myocardial ischemia and infarction. Physiol Rev. 1989; 69: 1049–1169.[Free Full Text]
  25. Abraham MR, Henrikson CA, Tung L, Chang MG, Aon M, Xue T, Li RA, O’Rourke B, Marban E. Antiarrhythmic engineering of skeletal myoblasts for cardiac transplantation. Circ Res. 2005; 97: 159–167.[Abstract/Free Full Text]
  26. Iravanian S, Nabutovsky Y, Kong CR, Saha S, Bursac N, Tung L. Functional reentry in cultured monolayers of neonatal rat cardiac cells. Am J Physiol. 2003; 285: H449–H456.
  27. Kraitchman DL, Tatsumi M, Gilson WD, Ishimori T, Kedziorek D, Walczak P, Segars WP, Chen HH, Fritzges D, Izbudak I, Young RG, Marcelino M, Pittenger MF, Solaiyappan M, Boston RC, Tsui BM, Wahl RL, Bulte JW. Dynamic imaging of allogeneic mesenchymal stem cells trafficking to myocardial infarction. Circulation. 2005; 112: 1451–1461.[Abstract/Free Full Text]
  28. Shaw RM, Rudy Y. Ionic mechanisms of propagation in cardiac tissue: roles of the sodium and L-type calcium currents during reduced excitability and decreased gap junction coupling. Circ Res. 1997; 81: 727–741.[Abstract/Free Full Text]
  29. Kleber AG, Rudy Y. Basic mechanisms of cardiac impulse propagation and associated arrhythmias. Physiol Rev. 2004; 84: 431–488.[Abstract/Free Full Text]
  30. Rohr S, Kucera JP, Kleber AG. Slow conduction in cardiac tissue, I: effects of a reduction of excitability versus a reduction of electrical coupling on microconduction. Circ Res. 1998; 83: 781–794.[Abstract/Free Full Text]
  31. Haynesworth SE, Baber MA, Caplan AI. Cytokine expression by human marrow-derived mesenchymal progenitor cells in vitro: effects of dexamethasone and IL-1 alpha. J Cell Physiol. 1996; 166: 585–592.[CrossRef][Medline] [Order article via Infotrieve]
  32. Majumdar MK, Thiede MA, Mosca JD, Moorman M, Gerson SL. Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells. J Cell Physiol. 1998; 176: 57–66.[CrossRef][Medline] [Order article via Infotrieve]
  33. Qu Z, Garfinkel A, Chen PS, Weiss JN. Mechanisms of discordant alternans and induction of reentry in simulated cardiac tissue. Circulation. 2000; 102: 1664–1670.[Abstract/Free Full Text]
  34. Pertsov AM, Davidenko JM, Salomonsz R, Baxter WT, Jalife J. Spiral waves of excitation underlie reentrant activity in isolated cardiac muscle. Circ Res. 1993; 72: 631–650.[Abstract/Free Full Text]
  35. Athill CA, Ikeda T, Kim YH, Wu TJ, Fishbein MC, Karagueuzian HS, Chen PS. Transmembrane potential properties at the core of functional reentrant wave fronts in isolated canine right atria. Circulation. 1998; 98: 1556–1567.[Abstract/Free Full Text]
  36. Kim DT, Kwan Y, Lee JJ, Ikeda T, Uchida T, Kamjoo K, Kim YH, Ong JJ, Athill CA, Wu TJ, Czer L, Karagueuzian HS, Chen PS. Patterns of spiral tip motion in cardiac tissues. Chaos. 1998; 8: 137–148.[CrossRef][Medline] [Order article via Infotrieve]
  37. Winfree AT. Varieties of spiral wave behavior: an experimentalist’s approach to the theory of excitable media. Chaos. 1991; 1: 303–334.[CrossRef][Medline] [Order article via Infotrieve]
  38. Zykov VS, Steinbock O, Muller SC. External forcing of spiral waves. Chaos. 1994; 4: 509–518.[Medline] [Order article via Infotrieve]
  39. Fenton FH, Cherry EM, Hastings HM, Evans SJ. Multiple mechanisms of spiral wave breakup in a model of cardiac electrical activity. Chaos. 2002; 12: 852–892.[CrossRef][Medline] [Order article via Infotrieve]
  40. Girouard SD, Rosenbaum DS. Role of wavelength adaptation in the initiation, maintenance, and pharmacologic suppression of reentry. J Cardiovasc Electrophysiol. 2001; 12: 697–707.[CrossRef][Medline] [Order article via Infotrieve]
  41. Mines GR. On dynamic equilibrium in the heart. J Physiol (Lond). 1913; 46: 350–383.
  42. Fast VG, Kleber AG. Role of wavefront curvature in propagation of cardiac impulse. Cardiovasc Res. 1997; 33: 258–271.[Abstract/Free Full Text]
  43. Pastore JM, Girouard SD, Laurita KR, Akar FG, Rosenbaum DS. Mechanism linking T-wave alternans to the genesis of cardiac fibrillation. Circulation. 1999; 99: 1385–1394.[Abstract/Free Full Text]
  44. Tang YL, Tang Y, Zhang YC, Qian K, Shen L, Phillips MI. Improved graft mesenchymal stem cell survival in ischemic heart with a hypoxia-regulated heme oxygenase-1 vector. J Am Coll Cardiol. 2005; 46: 1339–1350.[Abstract/Free Full Text]
  45. Rohr S, Kucera JP, Fast VG, Kleber AG. Paradoxical improvement of impulse conduction in cardiac tissue by partial cellular uncoupling. Science. 1997; 275: 841–844.[Abstract/Free Full Text]
  46. Entcheva E, Lu SN, Troppman RH, Sharma V, Tung L. Contact fluorescence imaging of reentry in monolayers of cultured neonatal rat ventricular myocytes. J Cardiovasc Electrophysiol. 2000; 11: 665–676.[Medline] [Order article via Infotrieve]
  47. Bursac N, Aguel F, Tung L. Multiarm spirals in a two-dimensional cardiac substrate. Proc Natl Acad Sci U S A. 2004; 101: 15530–15534.[Abstract/Free Full Text]
 

CLINICAL PERSPECTIVE

Is mesenchymal stem cell (MSC) transplantation potentially arrhythmogenic? In the present study, this question is addressed with optical mapping of an in vitro coculture model of MSC transplantation. Sustained reentrant arrhythmias, the in vitro equivalent of sustained monomorphic ventricular tachycardia, were easily induced by rapid pacing in cocultures containing at least 10% MSCs but not in cocultures containing 1% MSCs. These results indicate that MSCs may be proarrhythmic if large numbers of cells were to engraft at injection sites in the heart and suggest a need for arrhythmia risk assessment in large-animal models of MSC transplantation before clinical trials of intramyocardial MSC therapy.


*    Footnotes
 
The online-only Data Supplement can be found at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.105.593038/DC1.




This article has been cited by other articles:


Home page
 J Am Coll CardiolHome page
J.-Y. Hahn, H.-J. Cho, H.-J. Kang, T.-S. Kim, M.-H. Kim, J.-H. Chung, J.-W. Bae, B.-H. Oh, Y.-B. Park, and H.-S. Kim
Pre-treatment of mesenchymal stem cells with a combination of growth factors enhances gap junction formation, cytoprotective effect on cardiomyocytes, and therapeutic efficacy for myocardial infarction.
J. Am. Coll. Cardiol., March 4, 2008; 51(9): 933 - 943.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
R. Tao, C.-P. Lau, H.-F. Tse, and G.-R. Li
Functional ion channels in mouse bone marrow mesenchymal stem cells
Am J Physiol Cell Physiol, November 1, 2007; 293(5): C1561 - C1567.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
S. Fukushima, A. Varela-Carver, S. R. Coppen, K. Yamahara, L. E. Felkin, J. Lee, P. J.R. Barton, C. M.N. Terracciano, M. H. Yacoub, and K. Suzuki
Direct Intramyocardial But Not Intracoronary Injection of Bone Marrow Cells Induces Ventricular Arrhythmias in a Rat Chronic Ischemic Heart Failure Model
Circulation, May 1, 2007; 115(17): 2254 - 2261.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow HTML Page - index.htslp
Right arrow All Versions of this Article:
113/15/1832    most recent
CIRCULATIONAHA.105.593038v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chang, M. G.
Right arrow Articles by Abraham, M. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chang, M. G.
Right arrow Articles by Abraham, M. R.
Right arrowPubmed/NCBI databases
Medline Plus Health Information
*Arrhythmia
Related Collections
Right arrow Arrythmias-basic studies
Right arrow Arrhythmias, clinical electrophysiology, drugs

Circulation Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 2006 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.