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Circulation. 2005;112:e82-e83
doi: 10.1161/CIRCULATIONAHA.104.534214
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(Circulation. 2005;112:e82-e83.)
© 2005 American Heart Association, Inc.

Correspondence

Letter Regarding the Article by Xue et al, "Functional Integration of Electrically Active Cardiac Derivatives From Genetically Engineered Human Embryonic Stem Cells With Quiescent Recipient Ventricular Cardiomyocytes"

Richard B. Robinson, PhD; Michael R. Rosen, MD

College of Physicians & Surgeons of Columbia University, Department of Pharmacology, Center for Molecular Therapeutics, New York, NY, mrr1{at}columbia.edu

Peter R. Brink, PhD; Ira S. Cohen, MD, PhD

Department of Physiology and Biophysics, Institute of Molecular Cardiology, Stony Brook University, Stony Brook, NY

To the Editor:

The article by Xue et al1 on human embryonic stem cell-derived pacemakers illustrates that embryonic stem cells differentiated into spontaneously beating cardiocytes may function as biological pacemakers and mentions a potential limitation: The intrinsic pacemaker rate was slower than desirable. They suggest that incorporating HCN pacemaker channel genes might achieve more desirable rates, an idea consistent with our published results using HCN2 in gene- and adult human mesenchymal stem cell (hMSC)-based therapies.2–4

However, certain of the comments by Xue et al misinterpret our own work on HCN-loaded hMSCs. They state that "...these modified, undifferentiated, human mesenchymal stem cells are incapable of pacing quiescent cells because the former are neither electrically active nor genuine cardiac cells" (p 19). This statement suggests a misunderstanding of the rationale and underlying biophysics of the hMSC experiments. In fact, generation of pacemaker activity does not require delivery of an "excitable differentiated cardiac cell," but only that the delivered cell (1) carry sufficient pacemaker current and (2) make gap junctions; thus, the hMSC-myocyte pair should behave as a pacemaker unit entirely equivalent to a single heart cell with substantial if. We clearly demonstrated both functions for our genetically engineered hMSCs.4,5

We further take issue with the statement by Xue et al that biological pacemakers must pace quiescent cells. Extrapolating this to human disease implies that biological pacemakers should initiate activity in non-beating hearts. Yet, this is not what an electronic or a biological pacemaker is expected to do clinically. Rather, both types of pacemaker should initiate activity in hearts beating perilously slowly, putting patients at risk of devastating arrhythmia, syncope, or death. Therefore, although our HCN-load hMSC biological pacemaker can pace quiescent cells, there is much to be said conceptually for a biological pacemaker that increases dangerously slow heart rates.

Xue et al also criticize our earliest in situ gene therapy publication.2 They imply a deficiency in this study derived from our use of vagal suppression to eliminate sinoatrial pacemaker function. Given that in the absence of atrioventricular block a physiologically adequate rate generated by gene therapy might be overdriven by the sinoatrial node, the requirement for vagal suppression is not relevant to examination of therapeutic potential. Moreover, in subsequent studies, we showed that during vagal stimulation, an HCN2-based biological pacemaker inserted locally into a bundle branch is functional,3 and that in chronic atrioventricular block and bundle branch insertion of HCN2, physiologically relevant rates are achieved.6

In closing, we welcome criticism and discourse but believe it essential that this be based on an accurate interpretation of the subject matter under discussion.


*    References
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 *References
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  1. Xue T, Cho HC, Akar FG, Tsang S-Y, Jones SP. Marban E, Tomaselli GF, Li RA. Functional integration of electrically active cardiac derivatives from genetically engineered human embryonic stem cells with quiescent recipient ventricular cardiomyocytes. Circulation. 2005; 111: 11–20.[Abstract/Free Full Text]
  2. Qu J, Plotnikov AN, Danilo P, Shlapakova I, Cohen IS, Robinson RB, Rosen MR. Expression and function of a biological pacemaker in canine heart. Circulation. 2003; 107: 1106–1109.[Abstract/Free Full Text]
  3. Plotnikov AN, Sosunov EA, Qu J, Shlapakova IN, Anyukhovsky EP, Liu L, Janse MJ, Brink PR, Cohen IS, Robinson RB, Danilo P Jr, Rosen MR. Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates. Circulation. 2004; 109: 506–512.[Abstract/Free Full Text]
  4. Potapova I, Plotnikov A, Lu Z, Danilo P, Valiunas V, Qu J, Doronin S, Zuckerman J, Shlapakova I, 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]
  5. 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; 555.3: 617–626.
  6. Plotnikov AN, Shlapakova IN, Kryukova Y, Danilo P Jr, Cohen IS, Robinson RB, Rosen MR. Biological pacemaker expresses wide range of physiologic rates and compares favorably with electrical pacemaker: a preliminary study in dogs. Circulation. 2004; 110 (suppl III): III-291.
 

Response

Tian Xue, PhD; Ronald A. Li, PhD

Department of Medicine, Johns Hopkins University, Baltimore, Md, ronaldli{at}jhmi.edu

We agree that Rosen et al have elegantly provided extensive proof-of-concept data that HCN2-transduced human mesenchymal stem cells (hMSCs), although otherwise quiescent by themselves, could facilitate pacing. Our approaches and designs for studying and engineering pacemaker activity, however, are different: We differentiate pluripotent human embryonic stem cells (hESCs)1 into an ex vivo sinoatrial (SA) node-like structure, which is capable of generating electrical rhythms on its own accord, for in vivo transplantation and other experiments.2 Unlike native nodal pacemaker cells, transduced hMSCs are indeed neither electrically-active nor genuine cardiac cells. The electrophysiological properties of hMSCs are also somewhat heterogeneous,3 a common property that undifferentiated mouse and hESCs also appear to share.4 It is uncertain over time whether hMSCs after transplantation will further differentiate3 into cardiac or even non-cardiac cells with electrical properties different from those of pre-transplanted cells.

Although cardiac derivatives from hESCs, genetically-modified or not, are genuine heart cells,5 we likewise do not yet know how long and to what extent electrically active hESC-derived cardiomyocytes will continue to retain their "nodal-like" phenotype once transplanted into the in vivo ventricular or atrial environment. As Rosen et al correctly pointed out, the induced rate observed in our reported experiment was slow. This could be attributed to the above reason. Alternatively, the graft size, transplantation site, purity of nodal cells in the graft, time course for maturation, etc, are other contributing factors that also need to be considered. Our naïve working hypothesis, which remains to be experimentally tested, is that ex vivo overexpression of particular HCN channels can perhaps somehow compensate for the reduced efficacy after transplantation by (1) boosting the basal firing frequency in vitro6 and/or (2) increasing the percentage of nodal (or nodal-like) cardiomyocytes. This strategy of HCN manipulations, however, is somewhat complicated by the complex molecular identity of native If (as the 4 isoforms, whose expression levels differ depending on the cell type and species, can coassemble with one another7) and the presence of such ionic components as IK1 that can influence the ultimate functional effects of If per se.8 Protein engineering may be useful for custom-tailoring their biophysical properties for achieving specific outcomes.9,10

To unleash and study the induced pacing activity of our transplanted human cells, we needed to surgically remove the atria and cryoablate the atrioventricular node because the induced rate by human cells is overridden (by the intrinsically faster native guinea pig SA and atrioventricular nodes).2 Rosen et al chose to perform vagal stimulation in dogs. Although not explicitly stated, we hinted that future in vivo experiments of this kind can perhaps be facilitated by developing a novel animal model that enables the efficacy of cell transplantation or gene transfer approaches be more specifically investigated, free from undesirable effects due to extensive surgical manipulations, escape rhythms, etc.

Given that the native SA node is such a complex structure consisting of a gradient of nodal pacemaker cells with a range of phenotypic properties, etc,11 it is likely that all of the above-mentioned factors will need to be carefully taken into consideration for future experiments designed to improve our understanding of the basis of pacing, and to bioengineer a surrogate SA node.


*    References 
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 up arrowReferences
 *References 
 

  1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. 1998; 282: 1145–1147.[Abstract/Free Full Text]
  2. Xue T, Cho HC, Akar FG, Tsang SY, Jones SP, Marban E, Tomaselli GF, Li RA. Functional integration of electrically active cardiac derivatives from genetically engineered human embryonic stem cells with quiescent recipient ventricular cardiomyocytes: insights into the development of cell-based pacemakers. Circulation. 2005; 111: 11–20.[Abstract/Free Full Text]
  3. Boheler KR. Functional markers and the "homogeneity" of human mesenchymal stem cells. J Physiol. 2004; 554: 592.[Free Full Text]
  4. Wang K, Xue T, Tsang SY, Huizen RV, Wong J, Lai KW, Ye Z, Cheng L, Au K, Zhang J, Li GR, Lau CP, Tse HF, Li RA. Electrophysiological properties of pluripotent human and mouse embryonic stem cells. Stem Cell. In press.
  5. Moore JC, van Laake LW, Braam SR, Xue T, Tsang SY, Ward D, Passier R, Tertoolen LL, Li RA, Mummery CL. Human embryonic stem cells: genetic manipulation on the way to cardiac cell therapies. Reprod Toxicol. 2005; 20: 377–391.[CrossRef][Medline] [Order article via Infotrieve]
  6. Xue T, Chan CW, Henrikson CA, Sang DP, Marban E, Li RA. Lentivirus-mediated genetic modifications of human embryonic stem cells and their cardiac derivatives. Circulation. 2003; 108 (suppl IV): IV-33.
  7. Xue T, Marban E, Li RA. Dominant-negative suppression of HCN1- and HCN2-encoded pacemaker currents by an engineered HCN1 construct: insights into structure-function relationships and multimerization. Circ Res. 2002; 90: 1267–1273.[Abstract/Free Full Text]
  8. Azene EM, Xue T, Marbán E, Tomaselli GF, Li RA. Non-equilibrium behavior of HCN-channels: insights into the role of HCN-channels in native and engineered pacemakers. Cardiovasc Res. 2005; 67: 263–273.[Abstract/Free Full Text]
  9. Tsang SY, Lesso H, Li RA. Dissecting the structural and functional roles of the S3-S4 linker of pacemaker (hyperpolarization-activated cyclic nucleotide-modulated) channels by systematic length alterations. J Biol Chem. 2004; 279: 43752–43759.[Abstract/Free Full Text]
  10. HCho, Xue T, Li RA. Induced automaticity in ventricular cardiomyocytes by overexpression of an engineered HCN1 construct. Biophys J. 2004; 86: 293a.
  11. Boyett MR, Honjo H, Kodama I. The sinoatrial node, a heterogeneous pacemaker structure. Cardiovasc Res. 2000; 47: 658–687.[Abstract/Free Full Text]




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