2. Electrophysiologic Aspects of Cell Transplant Saeed Oraii MD, Cardiologist Interventional electrophysiologist Tehran Arrhythmia Clinic

3. Tehran Arrhythmia Center Statistics Cardiovascular disease affects approximately 58.8 million people in the United States. About 400,000 new cases of congestive heart failure occur each year. More than 2,600 deaths occur each day from cardiovascular disease -- 1 death every 33 seconds. Currently the most effective treatment strategies include lifestyle, medication and devices.

4. Tehran Arrhythmia Center Cell Transplant The adult heart was once thought of as a post-mitotic and terminally differentiated organ. This dogma is being challenged by recent findings that the adult heart contains cardiomyocytes that undergo proliferation. “new paradigm sees heart as a highly dynamic organ in which old, poorly functioning myocytes & vascular smooth muscle cells replaced by activation & commitment of resident Cardiac Stem Cells” Limited cardiac regeneration through either recruitment of stem cell populations from the bone marrow or through the activation of resident cardiac progenitor cells has been suggested.

5. Tehran Arrhythmia Center Regenerating Heart Old dogma for last 30 years: “the heart is a post-mitotic organ Incapable of regenerating parenchymal cells… Cardiomyocytes can undergo cellular hypertrophy, but cannot be replaced” “… you have so many beats of your heart, so use it wisely” In other words, the heart cells you have at birth is it!! When they are damaged, or die – that’s it!! Why the change? Observation of male cells in female hearts transplanted into a male (progeny of primitive cells in the heart, or migrated from elsewhere – bone) Anversa P, Kajstura J, Leri A, Bolli R. Life and death of cardiac stem cells: a paradigm shift in cardiac biology. Circulation. 2006 Mar 21;113(11):1451-63.

6. Tehran Arrhythmia Center New Paradigm Heart is viewed as a self-renewing organ (cell # controlled by stem cell compartment) Primitive cells may represent 2% of cells Clustered in atria, apex & throughout ventricular myocardium Entire cell population of heart re-populated every 4.5 years Old concept false - parenchymal cells do not live as long as the organism (approx. 80 years) Old concept false - parenchymal cells do not live as long as the organism (approx. 80 years) Stem cells within infarcted area also die Do not migrate from healthy myocardium to infarcted area to replace dead cells

7. Tehran Arrhythmia Center New Paradigm Intense myocyte formation in non-infarcted tissue, and acute & chronic heart failure 11 fold higher than normal physiological turnover 11 fold higher than normal physiological turnover Myocardial aging – telomere dysfunction, decrease pool of competent stem cells. View of aging & heart failure from perspective of stem cell disorder. “new paradigm sees heart as a highly dynamic organ in which old, poorly functioning myocytes & vascular smooth muscle cells replaced by activation & commitment of resident Cardiac Stem Cells”

8. Tehran Arrhythmia Center Implications for Cardiac Rehabilitation Awareness of trials for stem cell implantation Potential for arrhythmias Potential for arrhythmias Tracking other side effects Tracking other side effects Sense of hope for CR patients There is a healing capacity of the heart There is a healing capacity of the heart What is the effect of our interventions (exercise, dietary, stress management) on stem cell regeneration Exercise – when to start and how much from the perspective of stem cell health e.g. effects of ischemia on stem cell health e.g. effects of ischemia on stem cell health

9. Tehran Arrhythmia Center Arrhythmias in Heart Failure The failing heart is prone to ventricular tachyarrhythmias. Ventricular arrhythmias are common in patients with CHF. During 24- to 48-hour period, 5% to 10% of patients have runs of sustained (>30 seconds) VT and 40% to 80% have nonsustained VT. Of the annual mortality of up to 50% in severe heart failure, about half the deaths are sudden and presumably arrhythmic. Zalmen Blanck, MD Nicholas D. Georgakopoulos, MD Marcie Berger, MD et al. Electrical Therapy in Patients with Congestive Heart Failure. Current Problems in Cardiology Volume 27 Number 2 February 2002

10. Tehran Arrhythmia Center Mechanism of Arrhythmias Reentry due to fibrosis Myocardial ischemia Enhanced automaticity Dispersion of repolarization Electrolyte abnormalities

11. Tehran Arrhythmia Center Transmural Heterogeneity Normal cardiac cells exhibit significant transmural heterogeneity of action potential duration. A delicate balance has to be maintained to prevent arrhythmia. When abnormal shortening or prolongation of action potential duration in some, but not all, of the myocardial cells, disturbs this balance, the incidence of arrhythmia increases.

12. Tehran Arrhythmia Center Cell Regenerative Therapies Many of the cell therapy strategies under investigation will themselves be applied in a patchy or regional distribution, which may or may not be targeted to the areas of greatest contractile dysfunction. One important question that this raises is whether this approach will suppress an arrhythmic tendency, by restoring greater uniformity of healthy tissue architecture and function, or whether it will further add to the heterogeneity, thereby enhancing any arrhythmic tendency.

13. Tehran Arrhythmia Center EXAMPLES OF NATURALLY OCCURRING STEM CELLS ADAPTED FROM WWW.NIH.GOV/NEWS/STEMCELL/FIG2.GIF TOTIPOTENT CELL (ZYGOTE) PLURIPOTENT STEM CELLS (EMBRYONIC STEM CELLS) OTHER COMMITTED STEM CELLS (Myoblast, ETC.) WHITE BLOOD CELLS PLATELETS RED BLOOD CELLS SPECIALIZED CELLS: BLOOD STEM CELLS “ADULT” STEM CELLS

14. Tehran Arrhythmia Center Which Cell Type? Tissue-specific stem cells: Germ-line

15. Tehran Arrhythmia Center Skeletal Myoblasts Skeletal myoblasts are one of the earliest cell types that have been tested as a regenerative agent for structural heart disease. Myoblasts are derived from skeletal muscle satellite cells that normally lie quiescent under the basal membrane of skeletal muscle fibers. They are harvested by muscle biopsy, expanded in culture, and then injected into the heart of the same patient.

16. Tehran Arrhythmia Center Skeletal Myoblasts From a clinical perspective, they are attractive for the following reasons: They are easily obtained by muscle biopsy They are autologous, circumventing any histocompatibility concerns They have a high proliferative potential in vitro They have commitment to a well-differentiated myogenic lineage They have high resistance to ischemia, which is an advantage given the hypovascular nature of post-infarct scars

17. Tehran Arrhythmia Center First Cell Transplants Autologous myoblast transplantation was first performed by Taylor et al in rabbit hearts following cryoinjury in 1996. Skeletal myoblasts were the first cell type tested in humans for cellular cardiomyoplasty by Menasche’s group. Menasche P, Hagege AA, Vilquin JT, et al. Autologous skeletal myoblast transplantation for severe postinfarction left ventricular dysfunction. J Am Coll Cardiol 2003;41:1078–83.

18. Tehran Arrhythmia Center Gap Junctions Although transplantation of skeletal myoblasts was demonstrated to improve myocardial performance in animal models, gap junctions and functional coupling were not observed between grafted and host tissues. Yet even the presence of such gap junctions between host and donor cardiomyocyte tissues, as observed in some studies, does not guarantee functional integration.

19. Tehran Arrhythmia Center Arrhythmogenic Risk The second important electrophysiological consideration relates to the possible arrhythmogenic risk of these procedures. In the skeletal myoblast trials, a disturbingly high incidence of ventricular arrhythmias was noted in the initial stages of clinical follow-up. 10 out of the first 22 patients undergoing skeletal myoblast transplantation experienced ventricular arrhythmias.

20. Tehran Arrhythmia Center Ventricular Arrhythmias One of the five patients had sustained episodes of ventricular tachycardia and required implantable cardioverter-defibrillator placement. The investigators also describe a subsequent unpublished experience of two sudden deaths and three serious ventricular arrhythmias in eight additional patients. These data seem to correspond to the Menasche et al. experience in which 4 of 10 patients required ICD implantation for ventricular arrhythmias after open chest autologous myoblast transplantation. Smits PC, van Geuns R-J, Poldermans D, et al. 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;42:2063–9. Menasche P, Hagege AA, Scorsin M, et al. Myoblast transplantation for heart failure. Lancet 2001;357:279–80.

21. Tehran Arrhythmia Center Potential Hazards of Skeletal Myoblasts Itsik Ben-Dor, Shmuel Fuchs and Ran Kornowski. Cell Transplantation Protocols for Ischemic Myocardial Syndrome J. Am. Coll. Cardiol. 2006;48;1519-1526

22. Tehran Arrhythmia Center Time Course The time course of these events appear to show a peak at 11–30 days post cell transplantation. There is also a hint from the pooled findings that there may be an early period following cell transplantation, possibly extending for the first 20–30 days, during which there is enhanced arrhythmic tendency.

23. Tehran Arrhythmia Center Why? In the case of skeletal myoblasts, the generated myotubes have completely different physiological properties than host myocytes (extremely short APD). Moreover, due to their lack of gap junctions, these myotubes are completely uncoupled to the surrounding ventricular myocytes and may therefore act as anatomical obstacles, increasing tissue inhomogeneity, slowing conduction, and increasing the likelihood for the formation of reentrant arrhythmias.

24. Tehran Arrhythmia Center Cell Therapy Proarrhythmia Proarrhythmia after stem cell therapy might be attributed to one or more of the following reasons: Heterogeneity of action potentials between the native and the transplanted stem cells Intrinsic arrhythmic potential of injected cells Increased nerve sprouting induced by stem cell injection Local injury or edema induced by intramyocardial injection

25. Tehran Arrhythmia Center Slow Conduction Zones This hypothesis was recently demonstrated experimentally. Co-culturing of human skeletal myoblasts with primary rat cardiomyocyte cultures resulted in the formation of slow conduction zones that led to the generation of spiral (reentrant) wave in this in vitro model. Interestingly, genetic modification of the myoblasts to express the major gap junction protein, Cx43, improved conduction and decreased the tendency for arrhythmias

26. Tehran Arrhythmia Center Different Cell Types The nature of the injected cell may have the most impact on arrhythmogenesis after transplantation. Myoblasts and stem cells differ in their inherent electrophysiologic properties and in their ability to couple electromechanically both among themselves and with host cardiomyocytes. Limited clinical data available thus far suggest that arrhythmias are more likely to occur after myoblast than after stem cell transplantation. Finally, limited clinical experience suggests that proarrhythmic effects of cell therapy may be transient. Nonetheless, because the occurrence of cardiac arrhythmia is highly unpredictable, long-term follow-up studies of cell transplant recipients would seem to be essential for understanding the natural course of myoblast and stem cell induced arrhythmogenesis.

27. Tehran Arrhythmia Center Potential Antiarrhythmic Effect Although cell grafting could theoretically increase the potential for arrhythmias, the opposite may also occur. Cardiomyocyte transplantation in the infarct border zone may facilitate the emergence of new reentrant ventricular arrhythmias by generating slow conduction channels in this area. The same strategy, on the other hand, may also be utilized as a novel antiarrhythmic strategy. Thus, if cardiomyocyte transplantation will result in efficient regeneration of the infarct, existing slow conduction pathways within the scar may be eliminated, reducing the arrhythmogenic risk in these patients.

28. Tehran Arrhythmia Center Border or Center Soliman et reported more frequent and polymorphic premature ventricular contractions, couplets, triplets, longer post-PAC pauses, and bradycardiac death following injection of myoblasts in the infarct border zone compared with central scar injection in a rabbit model. One might expect that myoblast injection into the border zone, but not scar, may be proarrhythmic. Because functional improvement is independent of electrical integration of the injected myoblasts, injection of myoblasts into regions of scar may improve hemodynamics via a paracrine mechanism without the potential proarrhythmic consequences.

29. Tehran Arrhythmia Center Cell Specificities This mechanism may be specific for transplanted myoblasts, because embryonic stem cells have been reported to differentiate into a spontaneously contracting functional syncytium with gap junctions distributed along the cell borders. Kehat I, Gepstein A, Spira A, Itskovitz-Eldor J, Gepstein L. Highresolution electrophysiological assessment of human embryonic stem cell-derived cardiomyocytes: a novel in vitro model for the study of conduction. Circ Res 2002;91:659–61.

30. Tehran Arrhythmia Center Arrhythmias With Other Cell Types Itsik Ben-Dor, Shmuel Fuchs and Ran Kornowski. Cell Transplantation Protocols for Ischemic Myocardial Syndrome J. Am. Coll. Cardiol. 2006;48;1519-1526

31. Tehran Arrhythmia Center And we draw conclusions: what do we learn by examining the evidence?

32. Tehran Arrhythmia Center Factors Affecting Risk Current data suggest that the risk of arrhythmia occurring after myocardial cell transplantation may be increased by several factors: The type of cell injected The local myocardial milieu and electrical properties of the recipient tissue The presence of global and regional left ventricular function; The ex vivo cell expansion technique; and The timing of the transplantation relative to the ischemic or infarction events

33. Tehran Arrhythmia Center We Need More Basic Research on Stem Cells to Define Antiarrhythmic or Proarrhythmic Potentials ?

34. Tehran Arrhythmia Center Conclusion One implication of this clinical observation may be a requirement for a prophylactic ICD implantation in all patients participating in these trials. Another implication is the need for a clinical electrophysiologist to be actively involved in the designing and execution of these trials. This latter would allow better patient selection and better understanding of the nature, prevention, and treatment of the arrhythmia episodes. 1997: Dolly, a Cloned Mammal

35. Tehran Arrhythmia Center Cell Therapy for Cardiac Arrhythmias Cardiac arrhythmias represent one of the most common causes of worldwide morbidity and mortality and result in a major burden on the health care systems. The possible applications of these emerging technologies is establishing novel antiarrhythmic therapeutic paradigms.

36. Tehran Arrhythmia Center Cell Therapy for Cardiac Arrhythmias LIOR YANKELSON and LIOR GEPSTEIN. From Gene Therapy and Stem Cells to Clinical Electrophysiology. PACE 2006; 29:996–1005

37. Tehran Arrhythmia Center Implanted Pacemakers Implanted pacemakers have become a highly effective and safe treatment modality. Nevertheless, these devices are not without limitations. The need for a surgical procedure with its associated small but existing risks The need for a surgical procedure with its associated small but existing risks The requirement of repeated procedures for battery replacement The requirement of repeated procedures for battery replacement The inability to adjust heart rate and the resulting electrical activation sequence in the same effectiveness as the native pacemaker and cardiac conduction system. The inability to adjust heart rate and the resulting electrical activation sequence in the same effectiveness as the native pacemaker and cardiac conduction system.

38. Tehran Arrhythmia Center Biological Pacemakers In recent years, a number of novel gene and cell therapy approaches have emerged as experimental platforms for the creation of biological pacemakers.

39. Tehran Arrhythmia Center Avenues for Creating Biological Pacemakers To manipulate autonomic control; To manipulate ion channel number, structure, and/or function in order to create a nidus of pacemaker cells; or or To create the SA or AV node “from scratch.”

40. Tehran Arrhythmia Center Cell Therapy Both embryonic and adult mesenchymal stem cells have been used in attempts to fabricate biological pacemakers. With embryonic stem cells (pluripotent), 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. With adult mesenchymal stem cells (multipotent), 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.

41. Tehran Arrhythmia Center Embryonic Stem Cells 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.

42. Tehran Arrhythmia Center Human Embryonic Stem Cell Lines LIOR YANKELSON and LIOR GEPSTEIN. From Gene Therapy and Stem Cells to Clinical Electrophysiology. PACE 2006; 29:996–1005

43. Tehran Arrhythmia Center Mesenchymal Stem Cells Hyperpolarization-activated, Cyclic Nucleotide-gated (HCN) channel (I f current) Michael R. Rosen, MD. Biological pacemaking: In our lifetime? Heart Rhythm, Vol 2, No 4, April 2005

44. Tehran Arrhythmia Center Mesenchymal Stem Cells Michael R. Rosen, MD. Biological pacemaking: In our lifetime? Heart Rhythm, Vol 2, No 4, April 2005

45. Tehran Arrhythmia Center Limitations In the case of the engineered Mesenchymal Stem Cells it is not clear whether the transfected cells will eventually discontinue expressing the channel or whether these multipotent cells may differentiate into unwanted cell types such as bone and cartilage. The genetically engineered cells only partially recapitulate the phenotype of the SA nodal cells. In the case of the hESC-derived cells, it is not clear whether these early stage cardiomyocytes, that display some pacemaker like properties, will eventually mature into adult ventricular-like cells and lose their capabilities for spontaneous automaticity.

46. Tehran Arrhythmia Center Challenge: Knowledge Explosion “We are drowning in information but starved for knowledge.”—Naisbitt, ‘82