Biological pacemaking: In our lifetime?

15TH ANNUAL GORDON K. MOE LECTURE

Biological pacemaking: In our lifetime? Michael R. Rosen, MD From the Departments of Pharmacology and Pediatrics, Center for Molecular Therapeutics, College of Physicians and Surgeons of Columbia University, New York, New York.

Introduction The title of the Moe lecture is not selected by the speaker. It is assigned by committee, and this is a loaded title. Will we see biological pacemaking in the lifetime of a 60-something lecturer or a 20-something student in the audience? The answer should be “yes.” What else could the answer be in this age of unbridled vistas in medical biology? Doubters should consider the incredible advances made by electrophysiologists since World War II, from microelectrode1 to patch clamp to single channel2; from ECG3 to intracardiac recording4,5 to whole-heart mapping6,7; from catecholamines, digitalis, quinidine, and procainamide as antiarrhythmic mainstays8 to electronic pacemakers, ablation, and cardioversion-defibrillation.9,10 Now, new technologies in the molecular and genetic arenas have been provided to us. Can we and will we progress as rapidly, if not more rapidly, than before? Short of war or other major catastrophe, little can stop us. Yet the affirmative expectation is contingent on the desire to see progress happen— desire on the part of the investigators and on the part of the funders. In celebrating this year’s remembrance of Gordon Moe and his qualities as thinker, mentor, and communicator, it is only appropriate to try to see the biological pacemaker as he might have: as a challenge to understanding an issue to the point that one might make something good (in this case the electronic pacemaker) even better. Let us agree at the outset that little is wrong with the electronic pacemaker. It is life-saving, reliable, and generally affordable and implantable, at least in an affluent society. Will the electronic pacemaker continue to improve? I have little doubt it will. However, would a completely physiologic alternative that requires no instrumentation other than that for the initial insertion be a preferable alternative? For me, the answer is “yes.” Looking at it another way, accepting the electronic pacemaker as the sine qua non for sinus node disease or AV

The studies referred to were supported by USPHS-NHLBI Grants HL-28958, GM-55263, DK-60037, and HL-20558, and by the Guidant Corporation. Address reprint requests and correspondence: Dr. Michael R. Rosen, Department of Pharmacology, College of Physicians and Surgeons of Columbia University, 630 West 168 Street, PH 7West-321, New York, New York 10032. E-mail address: [email protected].

block would be like our predecessors having accepted the great passenger vessels as the standards for transoceanic travel. The ships were so good that there was no need for the airplane, or was there?

What is the biological pacemaker? Of course, the biological pacemaker is the sinoatrial (SA) node, a structure composed of specialized cardiocytes richly innervated by autonomic nerves. As shown in Figure 1, a family of currents contribute to phase 4 depolarization and to the action potential in the SA node, but the key to initiating the process is the pacemaker current If.11–33 Critical to the function of the responsible channel is that the inward Na current carried by the channel activates upon hyperpolarization, hence the designation “funny current,” or If. Figure 1 also shows a cartoon of the ␣-subunit of the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel, which carries If. There are four isoforms of the gene, labeled HCN1 to HCN4. The predominant isoform in the SA node is HCN4 and in ventricle is HCN2. HCN3 is not found in heart. The cyclic-AMP binding site on the HCN channel permits catecholamines to modulate activation, and it is largely this property that regulates the exquisite autonomic responsiveness of the pacemaker mechanism.

Avenues for creating biological pacemakers At least three avenues to creating a biological pacemaker can be explored. The avenue taken depends in part on whether the goal is repairing or creating an SA node in the atria of individuals who have a sick sinus node and intact AV nodal function; creating a demand pacemaker function in the ventricle of individuals with complete heart block and atrial fibrillation; or creating an AV node in individuals with normal sinus node function and AV nodal disease, thereby providing a biological therapy that restores normal pacemaking. The avenues available are to manipulate autonomic control; to manipulate ion channel number, structure, and/or function in order to create a nidus of pacemaker cells; or to create the SA or AV node

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Manipulating ion channel number, structure, and/ or function to create a nidus of pacemaker cells

Figure 1 A: Representation of SA node action potential (control: solid lines) and some of the ion channels contributing to it. If is activated upon hyperpolarization and provides inward current during phase 4. T-type and L-type Ca currents are initiated toward the end of phase 4. The latter also contributes the major current to the action potential upstroke. Delayed rectifier current IK is responsible for repolarization. The acceleratory effects of norepinephrine (NE) are shown as broken lines. Note the prominent increase in phase 4, reflecting the actions of NE on If. B: Pacemaker channel. There are six transmembrane spanning domains. When the channel is in the open position, Na is the major ion transmitted. Cyclic AMP binding sites are present near the aminoterminus. ␤1-Adrenergic (〉1-AR) and M2-muscarinic receptors, which provide NE and acetylcholine (ACh) binding sites, respectively, are shown. Via G-protein coupling, these receptors regulate adenylyl cyclase (AC) activity, which in turn regulates intracellular cAMP levels, determining the availability of the second messenger for binding and channel modulation. (From reference 13, with permission.)

“from scratch.” In this paper, my focus is on creating an SA node.

Either an increase in inward currents (e.g., the pacemaker current If) or a decrease in outward potassium currents might convert a cell that normally does not manifest automaticity or is not a primary cardiac pacemaker cell into a pacemaker. One such outward current is the inward rectifier IK1, which is encoded by the Kir gene family, with Kir2.1 the major gene of interest. IK1 is minimally present in sinus node and is not represented in Figure 1. However, IK1 is a large current in ventricular myocardial cells, where it clamps resting membrane potential at a highly negative voltage. Any intervention that decreases IK1 (e.g., by deleting a member of the Kir family) is expected to increase pacemaker rate, as would any intervention that increases If (e.g., by overexpressing a member of the HCN gene family). Several approaches currently are being explored, including use of naked plasmids or viral vectors to deliver the genetic construct of interest to a selected region of the heart and use of cells to deliver pacemaker constructs, whether the cells naturally incorporate them (e.g., embryonic stem cells) or can be loaded with them (e.g., mesenchymal stem cells). Moreover, one can work with pore-forming ␣-subunits of the “wild-type” channel (altering channel number), with their modifying (␤) subunits (altering channel kinetics and conceivably number of channels inserted in the cell membrane), and/or with mutant genes (altering channel structure).

Recreating the SA node from scratch There is the possibility of introducing progenitor cells or stem cells into selected regions of the heart, with the intention that the progenitor or stem cells develop into cells having the characteristic physiologic function desired. This type of approach has been attempted to repair failed myocardium in the interest of creating healthy heart muscle and to fashion sinus node-like structures.

Manipulating autonomic control With regard to autonomic control, the assumption is made that there is a substrate in atrium (that is, sinus node cells and a full complement of ion channels) whose rate might be up-regulated by increasing ␤-adrenergic input, decreasing muscarinic input, or both. Any portion of the autonomic receptor– effector pathway is a potential target here. However, if the SA substrate in which the receptor– effector systems are to be genetically regulated is flawed in any way, then altering receptor– effector transduction pathways not only might be therapeutically ineffective but might be arrhythmogenic. Moreover, altering receptor– effector pathways outside the sinus node could further complicate rhythm control.

What characteristics should be embodied in a biological pacemaker? In a previous publication,14 we considered what might be the optimal characteristics of a biological pacemaker. These characteristics included the need to 1. create a stable physiologic rhythm for the lifetime of the individual 2. require no battery or electrode and no replacement 3. compete effectively in direct comparison with electronic pacemakers 4. confer no risk of inflammation/infection 5. confer no risk of neoplasia

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6. adapt to changes in physical activity and/or emotion with appropriate and rapid changes in heart rate 7. propagate through an optimal pathway of activation to maximize efficiency of contraction and cardiac output 8. have limited and preferably no arrhythmic potential 9. represent cure, not palliation. These considerations are not trivial. However, given the excellent successes of electronic pacemakers, anything less than the characteristics listed would not warrant the development of a biological alternative. In the remainder of this presentation, I provide more detail on gene therapy approaches to biological pacemaking and on cell therapy. I then summarize what has been done and what needs be done if the therapy is to become routine in humans “in our lifetime.” In so doing, I hope the reader will appreciate not only how far we have come but also how far we still need to go.

Gene therapy Manipulating autonomic control Two reports by Edelberg et al15,16 provide an important transition between classical pharmacology and the gene therapy era. It has been known since Otto Loewi’s 3 AM Easter Sunday experiment in 1921 that autonomic modulation of atrial rate occurs via chemical transmission.17,18 Since Raymond Ahlquist’s 1948 discovery that a ␤-adrenergic action of catecholamines can increase sinus rate,19 and James Black’s 1964 development of propranolol20 that reducing the number of available ␤-adrenergic receptors competitively and effectively diminishes the effect of catecholamines to increase heart rate. The first transition to genetic control involved the ability to overexpress21 or knock out22 ␤-adrenergic receptors in experimental animals in the 1990s. From those insights, it was a logical, but nonetheless a major step, to attempt to regulate pacemaker function biologically through control of receptor number and/or receptor– effector pathways. The initial experiments by Edelberg et al15,16 provided the first proof of concept publication aimed at creating a biological pacemaker in situ. In their second study, Edelberg et al16 incorporated the gene encoding the ␤2-adrenergic receptor into a naked plasmid and injected it into the atrial muscle of swine. The result was increased ␤-adrenergic input to atrial fibers in vivo. The transfection was transient, as expected with this approach. Swine atria injected with the plasmid showed a 24-hour increase in atrial rate on day 2 after injection (Figure 2).

Manipulating ion channel number, structure, and/ or function to create a nidus of pacemaker cells The first proof of concept experiment of an alteration of ion channel expression causing an increased pacemaker rate

Figure 2 A: Surface ECGs from pigs injected with control constructs and those encoding the ␤2-adrenergic receptor (〉2-AR). Note the faster heart rate of the latter. B: Average percent change heart rates with control constructs and those encoding ␤2 receptor. On day 2 after administration, the atrial rate change of animals that received the ␤2-adrenergic receptor is significantly greater than that of controls. This effect is short-lived. (Modified from reference 16, with permission.)

since in vivo was reported by Miake et al.23 The strategy was to reduce repolarizing current. The process was not achieved by creating a nidus of pacemaker activity but by increasing pacemaker activity in a region(s) of unspecified dimension in the ventricle. Specifically, Miake et al replaced three amino acid residues in the pore of Kir2.1 to create a dominant negative construct. This action presumably formed multimeric, nonfunctional channels with endogenously expressed wild-type Kir2.1 and/or 2.2. They packaged the construct and green fluorescent protein (GFP) in a replication-deficient adenoviral vector and injected it into the left ventricular cavity of guinea pigs. Three to four days later, an approximately 80% reduction of IK1 density in individual ventricular myocytes and the emergence of ventricular ectopic rhythms on ECG (Figure 3) were observed. An initially theoretical objection that this approach might cause excess prolongation of repolarization by effectively removing a repolarizing current during the action potential plateau period was demonstrated to occur by the same team of authors in a follow-up publication.24 The other issue was the identity of the inward pacemaker current in the setting of reduced IK1. The Na/Ca exchanger has been hypothesized to be a likely current carrier,25 but preliminary experimental data favor several inward currents.26 Proof of concept that the If pacemaker current is overexpressed in myocytes, leading to increases in pacemaker

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421 the construct, and cells isolated from that region manifested If at least 100-fold greater than native current.31 In the ventricle, escape rates were approximately 25% faster than the idioventricular rates of control or sham-injected (GFP without HCN2) animals during vagal stimulation.31 Isolated left bundle branch fibers from the region of injection showed a significantly faster automatic rate at sites of HCN2 injection than at the same sites from control or sham-injected animals.31 Finally, current recordings showed approximately 100-fold overexpression of If in the HCN2-injected animals, and immunohistochemistry demonstrated increased fluorescence to an anti-HCN antibody.

Figure 3 Dominant negative strategy for gene therapy to create a biological pacemaker. A: Control guinea pig ventricular action potential. B: Phase 4 depolarization and regular automatic rhythm occurring in a myocyte from an animal that received the dominant negative construct and in which IK1 was suppressed. C: ECG from a control guinea pig. D: ECG from a transfected animal. Ventricular (V) and atrial (A) beats are clearly indicated. Dissociation between atrial and ventricular rhythms is seen. (From reference 23, with permission.)

rate, was provided by our group.27–30 We elected to overexpress If for two reasons: (1) If is the primary pacemaker current, and (2) If activates upon hyperpolarization and turns off at depolarized potentials. As a result, If has no effect to prolong action potential duration. Initial experiments demonstrated the feasibility of this approach in vitro (Figure 4). In rat ventricular myocytes in culture, If could be overexpressed by administration of the HCN2 gene using a plasmid as the vector.27 Importantly, the gene induced expression of a channel that retained its potential for autonomic regulation, that is, when HCN2 was administered to ventricular myocytes using an adenoviral vector, cAMP regulation of the current was clearly demonstrated,28 as expected for wild-type If. Another set of experiments demonstrated the effects of the ␤-subunit MiRP1, administered via a plasmid, in increasing pacemaker current density and kinetics in Xenopus oocytes via interaction with native HCN2.29 When HCN2 was overexpressed in neonatal myocytes in culture using an adenoviral vector, the spontaneous rate increased significantly, strongly suggesting that this approach leads to an increased pacemaker rate in situ.28 Having succeeded at the in vitro level, the next step was to attempt proof of concept in vivo, via injections of HCN2 into left atrial myocardium30 or the left bundle branch system31 of intact dogs. The vector was the replication deficient adenovirus. A second adenovirus expressing GFP was included in the injection mixture to permit visualization of adenovirus-transfected cells. Animals were maintained in sinus rhythm with intact AV nodal conduction. Sinoatrial slowing and/or AV nodal block were achieved in terminal experiments in which vagal stimulation was used to test for the emergence of ectopic rhythms. In both sets of experiments, the approach was successful (Figure 5). In the atria, the ectopic rhythms were mapped to the site of injection of

If gene therapy is to create biological pacemakers, what obstacles must be overcome? As shown earlier, gene therapy facilitates fabrication of biological pacemakers in experiments of up to 2 weeks’ duration. Research with HCN has seen more testing than the other gene therapy approaches to date. On balance at this time, I believe the HCN-based approach is the most attractive. Because it overexpresses members of the primary pacemaker gene family, it is not encumbered by excess prolongation of repolarization, and it incorporates autonomic modulation within the gene construct. The problems

Figure 4 A: Representative tracings from newborn (NB) rat ventricular myocytes transfected with HCN genes. Left: Native If in newborn myocyte. Center: Transfection with HCN2 gene. Right: Transfection with HCN4 gene. Note the markedly larger currents in center and right panels compared with the left panel. B: Effect of MiRP1 transfection in increasing current in Xenopus oocytes transfected with HCN2. Left: HCN2 alone. Center: HCN2 plus ␤-subunit minK. Right: HCN2 plus ␤-subunit MiRP1. Note the markedly larger current in the right panel. (Modified from references 28 and 29, with permission.)

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Heart Rhythm, Vol 2, No 4, April 2005 incidence of infection and inflammation arise with the use of any viral strategy. Moreover, a very real risk of neoplasia is associated with some viruses, including those expected to result in genomic incorporation of a pacemaker construct. Hence, the results of gene therapy to date are tantalizing, but the issues to be resolved are of major importance.

Cell therapy Figure 5 A, B: ECG recordings of leads I, II, and III from two different dogs. A: Animal injected with an adenoviral vector carrying green fluorescent protein (GFP) alone. B: Animal received the same vector incorporating GFP plus the HCN2 gene. The animals were anesthetized 6 days after injection and the vagi isolated and stimulated (arrows). Interval between traces is 22 seconds in panel A and 5 seconds in panel B. The animal that received GFP developed an idioventricular rhythm having a slower rate than the animal that received GFP plus HCN2. C: Posterior divisions of left bundle branch (construct injection site) were isolated from five control animals that had not been injected, six animals that received GFP, and five animals that received GFP plus HCN2 (each bar represents one animal). Note the rates generated by the bundle branches from the animals receiving HCN2 were significantly greater than the rates of the other two groups. D: If in a Purkinje myocyte isolated from the left bundle branch of a control animal (left) and one receiving HCN2 (right). The current is markedly greater in the latter (note the vertical gains of 5 pA/pF on the left and 50 pA/pF on the right). (Modified from references 14 and 31, with permission.)

I see with the dominant negative Kir strategy include the difficulty in translating this approach to a clinically convenient strategy, the lack of definition of the source of inward, pacemaker current in the setting of reduced outward current, the uncertainties regarding autonomic control, and the excess prolongation of repolarization with the possibility of increased dispersion of repolarization.23–26 The problem I see with the ␤2-adrenergic receptor up-regulation strategy15,16 is that it nonspecifically increases the responsiveness of any fibers transfected to catecholamine. As such, concerns regarding variability of response, and with this of arrhythmogenesis, are very real. It is too early to say that one, and only one, approach is the proper solution. For all strategies, we require more information comparing the range of pacemaker rates attainable with and without autonomic modulation, data regarding duration of efficacy, and direct comparisons of efficacy and safety with those of electronic pacemakers. Use of replication-deficient adenoviruses or naked plasmids represents an episomal strategy, such that the genetic material is not incorporated into the genome and the expression of biological pacemaking is impermanent. We need to achieve permanence; after all, the battery life of an electronic pacemaker is more than 5 years, and gene therapy must do better if it is to be competitive. In addition, questions of the

Both embryonic and adult mesenchymal stem cells have been used in attempts to fabricate biological pacemakers. Both are stem cells, but the commonality ends there. With embryonic stem cells, which are pluripotent and have the potential to differentiate into any cell type in the body, the general strategy is to direct the cells down a lineage that will incorporate pacemaker properties in its own right, couple to adjacent myocytes, and be integrated as a new sinus node cell.32 With adult mesenchymal stem cells, which are multipotent and are expected to differentiate only further along mesenchymally derived lineages, the strategy is to use the cells as platforms to carry genes of interest to regions of the heart where the cells would need to couple with adjacent myocytes.14 Both cell types require gap junctional coupling if they are to integrate effectively into the myocardial syncytium. This is not a requisite for gene therapy, which avails itself of the existing gap junctional connections among transfected and nontransfected myocytes. The embryonic stem cell is considered more a tool for tissue engineering (although it can be used as a platform for genes as well), whereas the mesenchymal stem cell is considered more a tool for delivering traditional gene therapy (although some data from studies of adult rat bone marrow suggest differentiation along cardiogenic cell lines may be possible33).

Embryonic stem cells Embryonic stem cells have almost a quarter-century of research behind them, since the initial reports of their study in the mouse.34,35 Human cell lines are far more recent, having been reported only within the past 6 years.36 As pluripotent cells, embryonic stem cells differentiate into neuronal,37 pancreatic islet,38, hematopoietic progenitor,39 endothelial,40 and heart41 cells. Not only do they have the potential to differentiate along many lineages, but as a cardiac lineage they have the property of performing as a syncytium, no doubt facilitated by the presence of the gap junctional proteins connexin43 (Cx43) and connexin45 (Cx45).42 Human embryonic stem cells have been studied with regard to their cellular electrophysiologic properties. Whether in culture43 or as embryoid bodies,44 human embryonic stem cells generate action potentials having a range of characteristics, from those of pacemaker cells (low membrane potential, phase 4 depolarization, low upstroke velocities) to those of relatively mature myocardium (high membrane potential, no phase 4 depolarization, rapid upstroke

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velocities). Hescheler et al45 studied ion channel properties of murine embryoid bodies. They described L-type Ca currents in precursor cells and voltage-dependent Na channels and inwardly and outwardly rectifying K channels in more mature cells. Embryoid bodies demonstrate stable pacemaker activity, but the source of this activity in terms of the responsible ionic current is not known.42,46 Hence, human embryonic stem cells provide a rich source of material for regenerating myocardium and initiating electrical activity in heart. The possibility that their use requires immunosuppressive treatment remains an issue. Kehat et al47 recently reported the use of cardiomyocyte cell grafts derived from human embryonic stem cells to create a biological pacemaker. The cells were implanted into the ventricles of swine with complete heart block, and mapping experiments revealed that pacemaker activity originated at the implantation site.

Adult human mesenchymal stem cells To the best of our knowledge, ours is the only group to have reported data using adult human mesenchymal stem cells as a platform for gene therapy.48 In contemplating this approach, we saw two attractive aspects to the cells. First, the cells were readily available, and there were no objections to, or constraints on, their use for research purposes. Second, there is a literature suggesting that these cells are immunoprivileged,49 that is, they might not elicit the immune response that complicates homologous or heterologous transplantation. We viewed this as a major benefit to our work, which contemplated implanting human mesenchymal stem cells into canine hearts. In addition, we believed that if the work advanced to the point of clinical trial, this property might facilitate the use of banked rather than autologous cells as a source for human administration. A summary of the strategy for isolating human mesenchymal stem cells, loading them with HCN genes, and transferring them to the heart is shown in Figure 6.The first experimental issue was the gene complement of human mesenchymal stem cells. Of particular interest was the presence/absence of pacemaker genes or other genes that control the ion channels contributing to action potentials and to gap junctions. A preliminary gene chip analysis suggested that human mesenchymal stem cells do not encode message for HCN2 or HCN4. They have some message for KCNQ1 (the gene encoding the ␣-subunit for IKs), and they have abundant message for the gap junctional protein Cx43. Heubach et al50 reported data indicating essentially the same result. Moreover, Heubach et al50 provided biophysical data indicating that human mesenchymal stem cells express a Caactivated K current, a clofilium-sensitive outward current, and occasionally an L-type Ca current. Our main focus in early experiments was on the connexins because the coupling of stem cells to myocytes was critical to any planned use of human mesenchymal stem cells as a platform for gene therapy. Initial experiments performed in vitro demonstrated coupling of human mes-

Figure 6 Experimental plan for human mesenchymal stem cellbased pacemaker. See text for discussion. (Modified after Dougherty M. Research on a biological pacemaker. In Vivo, Columbia University Medical Center 2004;3(11):6, with permission.)

enchymal stem cells to one another, to other cell lines, and to cardiac myocytes.51 The latter information was demonstrated physiologically via transfer of dye between cells and by passage of current between cells in a pair. The nature of the current traces was asymmetrical, suggesting that the connexins might not be forming homotypic channels (Figure 7). Immunostaining experiments showed that human mesenchymal stem cells have both Cx43 and Cx40. Given the presence of Cx43 only in ventricular myocytes, the junctional connections likely are heteromeric (Figure 7). The criticality of functional gap junctions in the cell pairs can be appreciated from the diagrams in Figure 8, which highlights the basis for conceiving of human mesenchymal stem cells as a platform therapy. In the normal sinus node (upper panel), cells have a complete complement of ion channel genes, including the pacemaker gene HCN4. The resultant current, If, activates upon hyperpolarization, initiating phase 4 depolarization and an action potential that propagates via low-resistance gap junctions to other cells. With platform therapy (lower panel), the human mesenchymal stem cell has been transfected with an HCN gene. However, the human mesenchymal stem cell does not have either a complete complement of channels to permit it to initiate an action potential or the means to hyperpolarize to a point where the overexpressed HCN channel will open. The human mesenchymal stem cell is in close proximity to a ventricular myocyte, which has a complete complement of channels to generate an action potential and actually has

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Figure 7 A: Immunohistochemical stains for connexin43 (left), connexin40 (center), and connexin45 (right) in human mesenchymal stem cells (hMSCs). Note the punctate staining that is positive for Cx43 and Cx40. There is no staining for Cx45. B, left: Human mesenchymal stem and canine ventricular myocyte in culture before (top) and after (bottom) impalement with electrodes. B, right: Voltage protocol (top) and current traces recorded (bottom). Note the asymmetrical nature of the currents. (Modified from reference 51, with permission.)

Heart Rhythm, Vol 2, No 4, April 2005 brane potential of the myocyte initiates HCN channel opening in the stem cell and the occurrence of a pacemaker potential. This action would generate current flow from stem cell to myocyte and depolarize the latter. When the myocyte reached threshold potential, it would initiate an action potential whose positive voltages would turn off the pacemaker current. The action potential also would propagate to other cells in the myocardial syncytium, as per usual. The final piece of the platform-building puzzle was transfecting the human mesenchymal stem cells with a pacemaker gene. Here, we did not need to rely on a viral vector; electroporation resulted in a transfection efficacy that now approaches 50%.48 At this point, we had a means of loading human mesenchymal stem cells with the appropriate gene. We could demonstrate that the human mesenchymal stem cell so loaded generated an If-like current (Figure 9) that responded to cesium and to autonomic modulation much like native If. The major difference from native If in sinus node was the fact that acetylcholine, in its

HCN2 and can generate If current. However, in the myocyte, If activates at a highly negative level of membrane potential (approximately ⫺140 mV),52 a level so negative that the cell never reaches this voltage and channel opening is not realized. We hypothesized that when myocyte and stem cell communicate via gap junctions, the high mem-

Figure 8 Rationale for human mesenchymal stem cell (hMSC)based pacemaker. Top: Sinoatrial node myocyte coupled to an atrial myocyte via gap junctions. The HCN gene in the membrane of the SA node cell is activated upon hyperpolarization, generating an action potential that propagates to the atrial myocyte. Bottom: Human mesenchymal stem cell in which HCN gene has been overexpressed is coupled via gap junction to a myocyte. The high membrane potential in the myocyte activates If, which in turn leads the cell to depolarize such that it can propagate an action potential further in the conducting system. See text for discussion. (Modified from reference 14, with permission.)

Figure 9 If recorded from a native human mesenchymal stem cell (A) and IHCN2 from a human mesenchymal stem cell in which HCN2 has been overexpressed via electroporation (B). Note the essential absence of If in the former. C: Current-voltage relationship for IHCN2 (note tail currents in inset), which activates in a physiologically relevant voltage range. (From reference 48, with permission.)

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own right, did not shift activation negatively or diminish current. However, accentuated antagonism occurred in the presence of isoproterenol, a result consistent with the actions of acetylcholine in vivo and with the function of If in the Purkinje system. The suggestion that coupling between myocyte and human mesenchymal stem cell resulted in effective pacemaker function was demonstrated in vitro, on a coverslip on which a small node of human mesenchymal stem cells transfected with either HCN2 plus GFP or GFP alone was overlaid with neonatal rat myocytes. The former developed beating rates approximately twice the rates of the latter.48 The tissue culture experiments predicted what we found in intact animal experiments.48 Approximately one million human mesenchymal stem cells loaded with HCN2 plus GFP or GFP alone were injected transepicardially into the left ventricular free wall. Animals subsequently were subjected to the same vagal stimulation protocol we used in the gene therapy experiments.31 The result was expression of an idioventricular rhythm pace-mapped to the site of injection and at a rate of approximately 60 bpm (Figure 10), which was significantly faster than the rates generated by GFP-

Figure 10 Expression of human mesenchymal stem cell-based biological pacemaker function in canine heart. Top panels: Experiment in a dog that received human mesenchymal stem cells containing green fluorescent protein (GFP) plus HCN2 as a left ventricular transepicardial injection 3 days before the experiment. On this day, the animal was anesthetized and the vagi isolated. Vagal stimulation led to a 20-second period of cardiac arrest, after which the escape pacemaker function demonstrated in the center panel was seen. This activity persisted for 3 minutes, at which vagal stimulation was discontinued and a postvagal sinus tachycardia occurred. The origin of pacemaker activity was pacemapped to the site of injection (data not shown). Lower panels: Histologic sections of tissue removed from the injection site. Left: Hematoxylin and eosin stain showing basophilic cells abutting on normal myocardium. Right: Higher magnification of vimentin staining of interface between the two cell types. Note the brownstaining mesenchymal cells overlying the myocytes. These cells also were positive for the CD44 antigen, consistent with their human origin. (Modified from reference 48, with permission.)

425 injected hearts.48 Subsequent excision of the injected site revealed the presence of basophilic cells that stained positively for vimentin (consistent with mesenchymal origin) (Figure 10) and CD44 (a human antigen).48 Moreover, abundant gap junctions between human mesenchymal stem cell and human mesenchymal stem cell and between human mesenchymal stem cell and muscle were shown using immunostaining techniques for Cx43.48

If cell therapy is to work, what obstacles must be overcome? Human embryonic stem cell therapy and human mesenchymal stem cell therapy present some challenges in common and others unique to the individual cell type. In considering these challenges, let us start from the understanding that although both are stem cells, in using them we are working from opposite ends of a spectrum. With the human embryonic stem cell, the investigator has available cells with the potential to become any form of myocyte with every ion channel that characterizes myocytes. The investigator has the additional option to overexpress specific genes. In contrast, human mesenchymal stem cells are cells that may be able to develop along cardiogenic lines (although this awaits demonstration) but in themselves have only the property to couple with other cells and to be loaded with a gene or complement of genes selected by the investigator. As a result, the human embryonic stem cell can be viewed as an embryonic Rosetta stone, with every part in place whether or not one wants all the parts (and to which other parts can be added). The investigator needs to decipher the complex workings of the human embryonic stem cell, an exercise that is ongoing in several laboratories. By comparison, the human mesenchymal stem cell is more the skeleton of a Rosetta stone to which the investigator can add those parts he/she wishes. Among the limitations common to both types of stem cell are concerns regarding their maintenance as stem cells vs their differentiation into other cell types. If one is dealing with myocardial repair and desires an embryonic stem cell population to evolve into cardiocytes, then one must select for cardiogenic cells and “educate” them—provide the environment needed—to evolve in that direction. If one prefers that the cells maintain the properties of a sinus node, it is important that the cells not be permitted to differentiate into mature ventricular myocytes with no pacemaking function. For both human embryonic stem cells and human mesenchymal stem cells, it is important that the cells not evolve into unwanted cell lineages, as we would not want to see cartilage or lakes of hematopoietic cells in the heart. Similarly, issues regarding localization of cells vs migration elsewhere in the heart— or in the body—are critical. The migration of stem cells housing HCN2 to the bladder might be a scientific curiosity but also a social disaster. Additional limitations related to inflammation, infection, graft rejection, and neoplasia remain to be adequately ad-

426 dressed. Although the current data are encouraging, longterm studies related to these aspects of potential toxicity have not been performed. Data defining the duration of effectiveness and a direct comparison with electronic pacemakers also are needed. Finally, concerns remain regarding delivery systems. Whether administered via a needle/electrode combination operating through a catheter, a hollow-lumen steerable catheter, or a combination of both, the ability to place constructs in the proper region without inducing trauma remains a challenge. The design of systems in which biological and electronic pacemakers can be used to complement each other’s operation as hybrid therapy also is challenging.

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Conclusion Are we ready for biological pacemakers? Yes. Will we see phase I trials beginning in 3 to 5 years? This is not outside the realm of possibility, but only if literally everything goes right. As is always the case, each setback ensures delay. However, if we are given the combination of a long lifetime and enthusiastic and informed investigation, then I have no doubt that whether or not I personally am home to a biological pacemaker other than my sinus node by the time I die, others will have this technology available to them.

Acknowledgments What needs to be done in our lifetime? Much of what needs to be done I have addressed in the sections on “what obstacles must be overcome.” We must address the areas of concern for each gene and cell therapy approach, as summarized in Table 1. In the process, we likely will learn that some concerns are groundless while others require novel approaches to put them to rest. Beyond this, we need to learn a great deal more about gene and stem cell therapies from the point of view of therapeutic potential and toxicity. Will the gene ultimately used for biological pacemaking be one of the wild-type genes HCN1 to HCN4, or some combination? Or will the mutant genes that we and others are making have properties of activation, inactivation, and autonomic response that make them better suited to the creation of an artificial pacemaker? I strongly suspect that when the final gene(s) is(are) established, we will have identified mutants that are better suited to this purpose. It also is possible that those mutants best suited to stem cells will differ from those fit for gene therapy. In addition, will the therapy used involve alterations in several genes or just one gene?

Table 1 What is now needed to see a biologic pacemaker “In our lifetime”? For virus or stem cell ● Evidence that it is superior to the electronic pacemaker in terms of adaptability to the body’s physiology and duration of effectiveness ● Evidence regarding long-term incidence of inflammation, infection, rejection, neoplasia ● Evidence for/against long-term proarrhythmic potential ● Localization at site of implantation vs migration to other sites ● Other toxicity ● Optimization of delivery systems For stem cell (embryonic, mesenchymal) ● Evidence regarding persistence of the administered cell type vs differentiation into other cell types; in the latter event, evidence regarding persistence of pacemaker function (current and coupling)

Because this is a lecture, it can be given by only one person, and I am very proud and honored to have been selected as the Moe lecturer. However, this particular Moe lecture should not have been given by one person; rather, it should have been “sung” by a barbershop quartet consisting of Peter Brink, Ira Cohen, Richard Robinson, and me. The nature of lectures and the quality of our voices is such that we spared the audience the singing performance, but everything presented and everything in this paper is the result of the thought, leadership, and cooperation of all our laboratories. Thus, this paper should be considered as having been written by the four of us and is dedicated to those in our laboratories who have done much of the thinking and all of the work. I single out (alphabetically) Peter Danilo, Alexei Plotnikov, and Iryna Shlapakova in my laboratory; Virginijus Valiunas in Peter Brink’s laboratory; Sergei Doronin, Zhongju Lu, and Irina Potapova in Ira Cohen’s laboratory; and Yelena Kryukova and Jihong Qu in Richard Robinson’s laboratory. Outstanding administrative support was central to the progress of these projects, and I thank Eileen Franey, Ruth Troncoso, and Laureen Pagan. Finally, I acknowledge the central role played by NHLBI in bringing the work presented here to fruition. Support through HL-28958, a Program Project in its 22nd year incorporating the efforts of Drs. Cohen’s and Robinson’s laboratories, as well as my own, has been critical. The support and partnership of Guidant Corporation, although more recent, has become a mainstay in our plans and in our progress. We particularly acknowledge the efforts of Beverly Lorell, Bruce KenKnight, Steven Girouard, and August Watanabe and the early encouragement of Jay Warren.

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