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Stem cell therapy appears to be a promising modality for myocardial repair of both hearts post myocardial infarction and those with other forms of structural cardiac disease (eg. congestive heart failure). In fact recent experimental and clinical work has suggested that stem cell therapy contributes to cardiac regeneration. Unfortunately at this time we contend that stem cell therapy is proarrhythmic. Accordingly, in this review we will approach this subject by restating this potential in the framework of traditional mechanisms of arrhythmias (automaticity and reentry). Lastly we will address recent clinical work with stem cells commenting on the proarrhythmic outcomes.
Before assessing the proarrhythmic potential of stem cells, we think it important to first address the nature of the players. After all these “players” are the cells selected for use in trials.
Obviously since this cell population has the capacity to develop into differentiated cardiac cells, they have been phenotyped in terms of ionic current makeup, intracellular Ca2+ handling, and connexin expression.1 ESCs have been shown to have at least fast sodium current, L type Ca2+ current, If, and IK12,3 and immature excitation-contraction (EC) coupling.4 Thus, implanting them into damaged myocardium you would be implanting areas of additional excitable cells that presumably would form gap junctions not only with fellow ESCs, but also with surviving myocytes of the damaged substrate. Evidence for resident cardiac stem cells 5,6 has surfaced and these excitable cells could show promise for use in stem cell therapy. For example, c-Kit+ cardiac derived cells isolated from a normal rat heart when delivered via the aortic root, seem to invade the infarcted myocardium and regenerate muscle to improve left ventricular (LV) function.7 Unfortunately, there was no mention of rhythm instability (or stability) of the injected hearts in this study. Some have suggested that Kit+ expressing cells are actually not heart cells but bone marrow cells out of place. Importantly human ESCs (hESCs) can be cultured as three dimensional differentiating cell aggregates called embryoid bodies (Figure 1).8
Perhaps one of the first classes of cell types used for replacement therapy was the autologous skeletal myoblast derived cell. While these cells are able to contract and show some excitability, the phenotype of EC coupling in these cells differs completely from that of normal cardiac cells.9 Importantly undifferentiated myoblasts can express connexins and form gap junctions. However, after time in the new substrate, the myoblast tends to lose this capacity.10 Thus myoblasts may survive where replacement tissue is needed but could then lack both mechanical synchronization and electrical integration forming islands of tissue.
Various types of bone marrow cells can differentiate into important stem cells. One population is the mesenchymal stem cell (MSC). They can be differentiated into neuronal type cells and have been implanted into the ischemic brain.11 Studies have described a complement of ionic currents in both the undifferentiated and differentiated MSC12;13 resulting in cells with resting potentials in the −30 to −40mV range. Other studies have described critical cardiac proteins such as troponin I and connexin 43 (Cx43).14 Whether MSCs fully differentiate into cardiac cell types remains controversial.15,16
If stem cells are implanted into myocardium for replacement or to become the pacemaker of the heart, they should functionally couple with remaining myocytes of the substrate to allow for a more homogeneous myocardium. As we know from numerous years of study of the pacemaker cells of the normal sinus node, if implanted cells have intrinsic abnormal electrophysiology and/or show spontaneous electrical activity, they then can become the source of electrical excitation. Cell electrophysiology studies of ESCs have shown slow upstroke action potentials and triggered activity (Figure 1).8,17-19 This inherent pacemaking activity is thought to be due to high input resistance and high sodium current density.3 In culture studies of both neonatal rat and hESCs, as the implanted cells, intrinsic pacemaker activity of the implanted cells cannot overcome normal rhythm since they were only located in a small area (eg. 200μm by 20μm).8 In contrast, larger denser areas of cell implantation can cause pacemaker potentials derived from the hESCs, a strategy often used for biopacemaker treatment of bradyarrhythmias.20-22 Interestingly, in a rather exhaustive study using mouse hearts post myocardial infarction (MI),23 there is no mention of enhanced spontaneous automatic rhythms after in vivo engraftment of ESCs, fibroblasts, or SkMs alone. Perhaps these events may have happened but were not reported. Only inducible rhythms were reported.
Experimental work with SkMs have reported that grafted myoblasts differentiate into peculiar hyperexcitable cells24 with EC coupling independent of the host cardiac cells. Experimental work with inexcitable MSCs has not led to increased automaticity of implanted cultures, but did alter conduction.25 However, if MSCs are made to express HCN channels and show pacemaker function, then good escape rhythms exist in the injected hearts. 21,22 hESCs, when transplanted in AV blocked animals, also show the potential for pacemaker activity. Interestingly, these experiments required only hundreds of hESCs for an effect.20 Thus, depending on your outlook, implanted cells can show enhanced automaticity and be arrhythmic, or can show enhanced automaticity and be antiarrhythmic.
a. Stem cells could lead to an increase in the area of conduction block in the damaged heart if and only if the stem cells DO NOT electrically couple to surviving myocytes. So is there evidence that stem cells electrically couple to nonmyocytes?
Most in vitro experimental work has been done using neonatal myocytes and implanted stem cells. These cells couple differently than the typical adult cell surviving in a host myocardium. In fact, hESC cells did show positive staining for Cx43 but no functional electrical coupling (note here; it was presumed based on “normal mechanical contractions”).8 Furthermore, when bone marrow derived cells were efficiently grafted into the ischemic region of the adult heart, they were located in clusters within the infarct scar or border zone, but showed no electronically evoked Ca2+ transients.16 Staining for gap junction proteins was absent in these studies.
The efficacy and arrhythmia occurrence of stem cell therapy depends on the cell number as well as the cells' delivery route. An intramyocardial route tends to cause cell clusters embedded in nonmyocardium10,24 leading to heterogeneity in conduction and perhaps conduction block. The intracoronary route could provide more homogeneous delivery, but hopefully cells will aggregate in sufficient quantity at the correct anatomical locale. In fact, in rat hearts post MI,26 intramyocardial BMC injections, while improving cardiac function, increased the risk of ventricular premature complexes (VPCs) for 28 days post injection. When the intracoronary route was used in these studies, VPC occurrence was markedly decreased. Importantly these animal studies were done in the ABSENCE of antiarrhythmic drugs, which is often not the case for patients in clinical trials (see below).
b. Stem cells could promote slowed conduction between substrate myocytes. What is the nature of propagation between “normal” myocardium and stem cells if they are coupled? Is there INa and gap conductance?
Some implanted cells have intrinsic INa function (eg. ESCs2,3) and when implanted could provide reasonable fast sodium dependent conduction between host myocardium and stem cell areas. On the other hand, if propagation is only Ca2+ dependent (so called slow response conduction27) or purely electronic, it may be that the implanted stem cells would provide areas of slowed conduction, setting the stage for reentry.
While experimental work has shown a temporal increase in conduction velocity (CV) over a combined culture of human MSCs (hMSCs) and neonatal host cells,28 the actual values of CV measured are quite slow, ranging from 4 to 17 cm/sec. Furthermore, there was still a 4 fold difference in CV between the graft and host sites even at the longest time post culture (14 days). Presumably cells under these conditions have reduced and differing resting potentials (MSCs −40mV vs Host −67mV), suggesting that this preparation is potentially arrhythmogenic.
Direct calculation of conduction paths and velocities of excitatory waves over integrated hMSCs with rat cell cultures again suggest that hMSCs, which show Cx43 positive staining, do indeed provide conduction between two channels of neonatal cells, therefore conduction block is relieved. However, propagation is extremely slow (0.9 cm/s), perhaps electronic29 and the hMSCs in the conducting channel show reduced resting potentials and action potential amplitude. As above, the implanted stem cells seem to provide areas of slowed conduction setting the stage for reentry.
When others have transplanted SkMs into adult canine myocardium with and without MI and then mapped conduction30, they also found clear conduction slowing particularly in the epicardium of the SkM transplanted wedge sections.
c. Is refractoriness or action potential duration (APD) dispersion promoted with stem cell replacement?
While there has been emphasis of the repair of conduction between the disparate areas of host myocardium by the implanted stem cells, there has been little appraisal of the changes in refractoriness or APD dispersion of substrate with the grafted cells on board. Experimental work has suggested APDs differ considerably between host and graft cells28,29 and other important work has shown that the increase in tissue heterogeneities of host/graft (MSCs) cell cultures do not align with altered APD restitution curves but with reduced conduction velocity, and easily inducible spiral wave reentry (Figure 2).25 In these MSC/neonatal cocultures, MSCs expressed Cx43 and were coupled to the host cells. However in cocultures containing >10% MSCs, transplanted cells became areas of inexcitable sinks and delayed activation and repolarization, which led to a proarrhythmogenic substrate.
Cell-based therapy for cardiac regeneration has been evaluated in the clinic in three distinct clinical scenarios: (1) recent acute myocardial infarction, (2) chronic myocardial ischemia in no-option revascularization patients and (3) chronic infarct-related heart failure. Cell types that have been transplanted in these clinical settings include skeletal myoblasts (SkMs) and bone marrow-derived stem cells (BMCs) (mononuclear stem cells, hematopoietic stem cells, mesenchymal stem cells (MSCs), endothelial progenitor cells and circulating progenitor cells). Two cell delivery methods have been used: intracoronary and intramyocardial injections (transendocardial during cardiac catheterization and transepicardial during open-chest surgery). The amount of injected cells also varies among studies. Thus, a wide range of clinical situations, cell preparations, routes and doses employed make it difficult to totally interpret and compare the results from human trials. However, in this next section we will evaluate the consequences of stem cell transplantation and arrhythmia occurrence.
A number of features make myoblasts an attractive cell type for cardiac cell transplantation. They can be obtained in sufficient quantity directly from the patient and are resistant to ischemia, making them possible to survive in the low capillary environment of the infarcted myocardium.10 SkMs can differentiate into myotubes in vivo, but do not integrate with surviving cardiomyocytes. In addition, there is lack of evidence supporting their effectiveness in improving cardiac function.31
Several small trials investigating the safety and feasibility of myoblast transplantation in patients with ischemic cardiomyopathy have been published (Table 1).32-41 It is known that patients with left ventricular dysfunction and heart failure after a myocardial infarction have a favorable substrate for ventricular arrhythmias. However, these initial experiences suggested a proarrhythmic effect of SkM cell therapy. In the first phase I clinical trial with skeletal myoblasts, Menasche et al. reported sustained monomorphic ventricular tachycardia (VT) in 4 of 10 patients (one of them syncopal) early after the operation (11 to 22 days) that was not related to myocardial ischemia.32 The four patients had had an implanted cardioverter-defibrillator (ICD) implanted after the VT episode. At follow-up visits, two of these patients still experienced ventricular arrhythmias, despite antiarrhythmic drug therapy with beta-blockers and amiodarone. Indeed, due to the major concern of the potential arrhythmogenic effect of the new therapy, amiodarone was prophylactically instituted in the last three patients included in this study. Despite this, at a median follow-up 52 months after transplantation, there were 14 appropriate shocks for 3 arrhythmic storms in 3 patients.33 Shortly after these initial alarming data, Smits et al. reported episodes of sustained VT in one of five patients after transendocardial injection of SkMs for the treatment of ischemic heart failure.34 Subsequently, these same investigators have described an unpublished experience of two sudden cardiac deaths and three serious ventricular arrhythmias in eight additional patients.
In other studies, arrhythmias after myoblast transplantation have been reported. Siminiak et al. gave prophylactic amiodarone to prevent ventricular arrhythmias to the last 8 patients included in their study of epicardial SkMs transplantation during CABG, after the first 2 had ventricular tachycardias in the early postoperative period.39 Two other studies also included prophylactic amiodarone as a standard pretransplantation therapy.35,40 In fact, in a different study by Siminiak et al., the only patient not receiving amiodarone developed episodes of ventricular tachycardia and experienced two interventions from its ICD at day 8 post procedure.40 This strongly suggests that the proarrhythmic effect of SkM transplantation might be prevented by amiodarone, even though it is not known how amiodarone may exert this effect.
To date, the only placebo-controlled randomized study evaluating the efficacy of SkMs has been the MAGIC II trial (Myoblast Autologous Grafting in Ischemic Cardiomyopathy). Here the efficacy of this cell-based therapy in patients with a history of myocardial infarction, left ventricular dysfunction and indication for coronary surgery was evaluated.31 Investigators tested two different doses of transepicardial injected SkMs versus placebo during CABG. An ICD and antiarrhythmic therapy were used in all patients. Even though the patients included in the trial are among the highest risk for ventricular arrhythmias, it seems that the investigators had significant concerns about the safety of the new procedure since they not only implanted an ICD, but they also recommended antiarrhythmic drugs. This trial failed to detect an incremental improvement in regional or global left ventricular function over that provided by CABG alone. However, at the 6 month follow-up, the number of ventricular arrhythmias was 2 times greater in patients of the treated groups. Notably these investigators called attention to the proarrhythmic risk of myoblast transplantation (Table 2). It is important to mention that the MAGIC II trial was the first large study providing exhaustive rhythm monitoring to the entire population.
In sum, small trials using SkMs have shown the treatment to be proarrhythmic.
The effects of adult bone marrow-derived progenitor cells have been investigated in patients with recent acute myocardial infarction after successful primary percutaneous coronary intervention. Other studies have also been performed in chronic MI and heart failure patients. Intracoronary, transendocardial and transepicardial administration routes have been used. Initially, the several small and non-randomized clinical trials which evaluated both safety and feasibility of BMCs in these situations, reported no obvious evidence of arrhythmic risk associated with the procedure or during follow-up care.42-49 Perin et al. reported one sudden cardiac death in a patient 14 weeks after transendocardial autologous BMC transplantation for chronic severe heart failure.50 When direct intramyocardial percutaneous delivery of autologous bone marrow cells in 10 patients with refractory myocardial angina was used, one patient experienced acute heart failure 7 days after the procedure due to acute atrial fibrillation.48 In another study, 4 of 12 patients developed transient atrial fibrillation after transepicardial injection of BMCs during coronary artery bypass grafting.49 Interestingly, no other arrhythmic episodes were described in these phase I safety studies. It is important to mention that arrhythmia monitoring was not continuous in any of these studies except during the periprocedural time. Only occasional 24-hour Holter ECGs and clinical evaluations were carried out and the follow-up period was no more than a few months.
So far, few of the randomized clinical BMC therapy studies suggests either no or a small benefit in patients with ischemic heart disease.51,52 From the published data of these randomized placebo-controlled trials, there does not seem to be an enhanced risk of clinical arrhythmias related to this type of cell transplantation, but again, the method of evaluating the arrhythmic risk is generally not exhaustive (Table 3).53-67 Only Wollert et al. tested arrhythmia inducibility with programmed ventricular stimulation 6 months after intracoronary BMC in 30 cell treated and 30 control patients.66 Most groups never report any specific rhythm monitoring during follow-up after transplantation thus arrhythmia occurrence is unknown.56, 59-63 On the other hand, most of the patients included in these few clinical trials were taking β blocker agents, as they are indicated for ischemic heart disease. Treatment with β blockers might mask a potential proarrhythmic effect of the transplanted cells in humans.
Two other non-randomized studies have specifically evaluated the electrophysiological and arrhythmogenic effects of transplantation of autologous bone marrow-derived progenitor cells.68;69 The study by Beeres et al. was carried out in 20 patients with drug-refractory angina and myocardial ischemia. Immediately before intramyocardial BMC injection, 3-dimensional electroanatomical LV mapping was performed to evaluate the local bipolar electrogram characteristics of the myocardial region with ischemia in which BMCs were to be injected. Three months later, mapping was repeated in the same area and electrograms showed no prolongation, no decrease in amplitude or increase in fragmentation suggesting conduction was not affected.68 Twenty-four hour Holter monitoring was performed at baseline and 3 and 6 months later. The total number of ventricular premature beats remained unchanged. However, this was a non-randomized study, without a control group and with no programmed ventricular stimulation protocol to evaluate the inducibility of ventricular tachycardia. Also, the authors state that the measurement of electrogram duration by the electrophysiological mapping is influenced by the direction of the wavefront in relation to the bipole, which could limit the interpretation of the results. In the second study, Katritsis et al. followed patients with a history of myocardial infarction and ICD for ventricular arrhythmias in whom intracoronary transplantation of MSCs and endothelial progenitor cells was performed.69 Before stem cell transplantation, clinical non-sustained ventricular tachycardia and inducible monomorphic ventricular tachycardia or ventricular flutter were demonstrated in all 5 patients. At 16-36 months follow-up, the interrogation of the ICD failed to detect sustained or non-sustained ventricular arrhythmias in any patient and a repeated electrophysiological study induced sustained ventricular arrhythmias in only two patients. This was a small and non-randomized study and should be regarded as a preliminary experience and not as proof of an antiarrhythmic potential of this type of stem cell.
The exact mechanism of “electrical” action following BMC transplantation is unknown. With intracoronary administration, fewer than 5% of cells are retained in the infarcted myocardium. If the cells do not remain in the areas of interest, neither important long lasting effects nor arrhythmic potential might be expected. In fact, a lack of sustained long-term beneficial effects of BMC has recently been reported.55,65,67
Further clinical experience and more exhaustive studies are necessary before reaching valid conclusions regarding the electrical safety of BMCs or MSCs.
SkMs and BMCs might have some beneficial indirect effects on the myocardium (paracrine mechanisms, potential to induce angiogenesis),37 but they do not differentiate into cardiomyocytes. Thus, they may not have clinical significant long-term favorable effects over the heart pump function.31,55,65,67 Other sources of potential regenerative cells like ESCs and endogenous cardiac stem cells have not yet been tested in humans. Embryonic stem cells are the prototypical stem cells. However, there are several difficulties in using hESC. First, these cells are allogeneic, and immunosuppressive therapy might be needed. Second, they have the potential to form teratomas when injected in vivo, an issue that will be probably solved with technical advances to lead their differentiation only into cardiomyocytes.70 Finally, the use of hESC is still surrounded by ethical problems.
In conclusion, given both the experimental and clinical data available so far we content that stem cell therapy is arrhythmogenic. Experimental studies have provided some electrical basis for such in that stem cells can show intrinsic pacemaker function and provide for areas of slowed conduction. These latter changes in the substrate could set the stage for arrhythmias. Clinical studies so far are not exhaustive in their rhythm monitoring and usually have some type of antiarrhythmic therapy accompanying the treatment. This is a wise idea since stem cell therapy can be proarrhythmic.
Sources of Funding: Supported by grant HL58860 from the National Heart Lung and Blood Institute Bethesda, Maryland; Dr. Macia is supported by Medtronic fellowship.