Electrophysiological Coupling of Transplanted Cardiomyocytes
See related article, pages 484–492
The recent advances in stem cell biology and tissue engineering have paved the way to the development of a new field in biomedicine, regenerative medicine. This approach seeks to circumvent the shortage of organs for transplantation, by combining the above-mentioned technologies in an attempt to replace diseased or absent tissue. The heart represents an attractive candidate for these emerging technologies, and myocardial cell replacement therapy and tissue engineering present exciting new possibilities for assisting the failing myocardium.1,2
Despite the enormous enthusiasm associated with cardiovascular regenerative medicine, several obstacles need to be overcome before such strategies can become a clinical reality. Among the major targets that must be achieved are the needs to find an efficient cell source, which will provide the large number of cardiomyocytes required, overcoming the immune rejection barrier of nonautologous cell sources, and finding means for directing stem cells to differentiate to the cardiomyocyte lineage. Yet, concentration on these “major” unanswered questions may lead one to believe that all other issues with cell therapy have been solved. A closer inspection, however, reveals that this is not the case and many questions still remain open. Even if we could identify the ideal stem cell source, could direct the differentiation of these cells efficiently to cardiomyocytes, and prevent immune rejection we still do not know how many cells should be transplanted, what is the best timing and sites for cell transplantation, and most importantly whether the engrafted cells would survive and integrate appropriately with the existing cell-network of host cardiac tissue.
Numerous reports suggest that myocyte transplantation can improve cardiac performance in animal models of myocardial infarction.1,2 However, it is not entirely clear whether this functional improvement is attributable to direct contribution to contractility by the transplanted myocytes or by other indirect mechanisms such as attenuation of the remodeling process, amplification of an endogenous repair process by cardiac resident progenitor cells, or induction of angiogenesis. Because hundred millions of cardiomyocytes are lost during a myocardial infarction that result in heart failure, any significant systolic improvement would require the generation of a large working graft. Such a cell-graft would have to integrate both structurally and functionally with host tissue and participate in the synchronous contraction of the entire heart.
Several studies have documented the presence of gap junctions between host and grafted cells.3,4 Yet even the presence of such gap junctions, although necessary for integration, is by no means sufficient and does not guarantee appropriate functional integration. For such integration to occur, currents generated in one cell passing through gap junctions must be sufficient to depolarize neighboring cells.
The ability (or the lack of it) of the graft to functionally integrate with host myocardium is a key mechanistic question in cell therapy. This key question is addressed in the paper by Halbach and colleagues in this issue of Circulation Research.5 Murine fetal cardiomyocytes, derived from transgenic animals expressing eGFP, were transplanted into the necrotic area and to adjacent healthy myocardium in the mouse cryoinjury model. Using a novel ex vivo heart slice preparation6 and direct microelectrode recordings from the grafted cells, the authors show that 82% of transplanted fetal cardiac myocytes surrounded by viable host tissue were electrically integrated with host myocardium. In contrast, transplanted cardiomyocytes surrounded by cryoinjured tissue, although showing spontaneous electrical and contractile activity, were not integrated with host tissue.
This important study joins the very few published articles that focus on the electrophysiological integration of transplanted cells. In vitro studies showed that cocultured cells were able to form a single functional syncitium.7 Using 2-photon microscopy, Rubart and colleagues were able to document electrical integration (by way of intracellular calcium imaging) between transplanted fetal cardiomyocytes and host cells in the intact heart.4 A similar study by the same group noted the absence of such integration when skeletal myoblasts were used for transplantation.8 Kehat and colleagues were able to document stable long-term electrical integration of human embryonic stem cell derived cardiomyocytes in live animals using electroanatomical mapping.7 The former studies were hampered, however, by the limited ability to assess the efficacy of coupling and the latter was hampered by the low-spatial resolution of the mapping technique. Furthermore, both of these studies did not assess integration in the setting of diseased myocardium.
The study of Halbach and colleagues5 is in agreement with the aforementioned studies, claiming that transplanted cardiomyocyte can integrate with the host heart. In this respect, this study compliments previous work by demonstrating, unambiguously, cell integration, directly, at a single-cell resolution in the intact heart. This integration was highly efficacious, as several of the grafted cells were able to follow host myocardium in a 1:1 conduction at frequencies of up to 10 Hz.
The electrical integration of the transplanted cardiomyocytes required embedment within host cardiac tissue. Histological analysis showed that these functionally coupled cells generated gap junctions with host cardiomyocytes directly or through nonmyocytes that bridged the gap with nearby cardiomyocytes. Although it is normally assumed that cell-to-cell coupling by gap junctions is confined to the cardiomyocyte compartment, recent studies demonstrated that nonmyocytes (such as fibroblasts) can also couple with cardiomyocytes.9 Myocyte-to-nonmyocyte coupling causes local current circuits during propagation and may produce an electrotonic “action-potential” in the nonmyocyte cell. Consequentially, the nonmyocyte may act as a sink and contribute to conduction failure between host and graft cells. On the other hand, relatively short spans of nonmyocytes may potentially serve as a bridge and enable electrical integration despite a lack of direct physical contact between transplanted and host cardiomyocytes.9
In many cell therapy studies the grafted cells are usually injected in the center of the infarcted region. Halbach and colleagues5 showed that cells that were surrounded completely by the cryoinjured tissue did not integrate at all. Apparently, the thick scar tissue surrounding these cells acted functionally as an insulator and prevented coupling between the grafted and host tissue. The lack of integration in the cryoinfarct model should alert those in the field and prompt further study to validate this issue in more closer to real-life models such as LAD ligation. Nevertheless, if the lack of ability to integrate inside an injured tissue will be found to be generally true, then we will have reevaluate our current strategies of cell therapy and design methods to improve the ability of the grafted cells to engage in close-contact with host tissue.
Another important issue that may contribute to the ultimate success of cell transplantation is the degree of structural and functional maturation of the grafted cells. For the grafted cells to contribute optimally to ventricular systolic contraction they should possess an adult working ventricular myocyte phenotype. On the other hand, previous studies have documented an inverse relationship between the degree of maturation of the transplanted cells and their long-term engraftment and survival rates.10 One of the most interesting finding in the study of Halbach and colleagues5 is that the well-integrated transplanted fetal cardiomyocytes displayed plasticity and underwent significant changes to assume electrophysiological properties of more mature murine cardiomyocytes. The persistence of this effect, even in the presence of gap junction uncouplers, suggests that this resulted from altered expression of ionic channels and not from electrotonic interactions. In contrast, the transplanted cells that remained isolated within the cryoinjured area continued to display an “immature” action-potential phenotype (lower maximal upstroke velocity and longer action-potential duration).
Unfortunately the authors did not attempt to try to identify the mechanisms underlying this interesting maturation process in the well-coupled cells. It seems, nevertheless, that this process requires a niche provided by host cardiac tissue, whose elements require direct contact with the immature grafted cells.
An important, but frequently overlooked, obstacle to the development of successful cell therapy strategies is the possibility of induction of malignant ventricular arrhythmias. In the initial clinical skeletal myoblast trials, for example, a disturbingly high incidence of ventricular arrhythmias was noted. One possible reason for this arrhythmogenesis may be the lack of formation of gap junctions in the myotubes that may completely uncouple these cells from surrounding ventricular myocytes, resulting in the formation of anatomical obstacles, increasing tissue inhomogenities, slowing conduction, and increasing the likelihood of the formation of reentrant arrhythmias.11
Although early-stage cardiomyocytes can form gap junctions and functionally integrate with host tissue, as shown elegantly in this article,5 they too in theory may have the potential for arrhythmogenesis through a number of mechanisms.12 Cell transplantation may theoretically increase the likelihood of reentrant arrhythmias by generating slow conducting viable channels within the border-zone of the infarct (the anatomical substrate responsible for postmyocardial infarction ventricular tachycardia in patients). On the other hand, this same strategy may become antiarrhythmic if cardiomyocyte transplantation can effectively regenerate the infarct and improve conduction in this area.
Cell therapy may also contribute to the generation of local heterogeneities in tissue architecture or cellular electrical properties, a property that may contribute to the formation of unidirectional conduction block, a key ingredient in the reentrant circuit. Even if optimal coupling between host and grafted myocytes could be achieved, the immature engrafted cells may differ in their electrophysiological properties from those of host cells. Simulations and experimental studies have shown that such gradients can affect spiral waves and wave splitting in ventricular fibrillation. In this respect it is encouraging to learn that well-integrated transplanted fetal cardiomyocytes displayed plasticity and underwent significant changes to assume electrophysiological properties of more mature murine cardiomyocytes thereby reducing local heterogeneity.5
Besides conduction blocks at the host-transplant junction that were observed in a number of preparations during rapid pacing, Halbach and colleagues5 also noted the presence of early after-depolarizations in a few of the transplanted cells as well as spontaneous automaticity that was confined to the nonintegrated cells. Encouragingly, none of these findings (that may predispose to arrhythmogenesis through reentrant, triggered activity, and abnormal automaticity mechanisms respectively) resulted in the generation of arrhythmias in the transplanted animals. Nevertheless, these findings call for further studies that should focus on studying the effects of cell transplantation on the electrophysiological substrate in more clinically-related animal models and using state-of-the-art electrophysiological methodologies.
Finally, whereas the issues discussed in this editorial regarding the electrophysiological aspects of cardiomyocyte cell transplantation may have seemed only academic a few years ago, because of the lack of sources for human cardiomyocytes, the exciting developments in the fields of cardiac tissue–derived13,14 and embryonic stem cell biology15 has made cardiac regenerative therapy using human cardiomyocytes a more realistic prospect. Consequentially, we believe that to generate a more rational approach to cell therapy strategies it is imperative that in addition to the “traditional” cell therapy studies, analyzing the effects of cell transplantation on histology and global cardiac function, significant efforts should also be made in the future on improving our understanding of the overall balance and interactions between host and donor myocytes. Yet, even the thousand mile journey must begin where you stand, and the study of Halbach and colleagues5 makes our current standing better understood.
Sources of Funding
Israel Science Foundation (#520/01) and the Grand Family Research Grant.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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