Cellular cardiomyoplasty—cardiomyocytes, skeletal myoblasts, or stem cells for regenerating myocardium and treatment of heart failure?

Time for primary review 23 days. In newts, excision of up to 50% of the ventricular myocardium results in spontaneous regeneration and rebuilding of the heart, a process that involves de-differentiation of the remaining cardiomyocytes, de-novo synthesis of DNA followed by proliferation and mitotic cell division of both cardiomyocytes and connective tissue, and subsequent replacing of the lost tissue [1–3]. In contrast, the mammalian heart loses its capability of a quantitatively sufficient proliferative response to various injuries soon after birth [4–6]. Interestingly, recent studies reported a certain degree of cardiomyocyte regeneration in various pathologic conditions, such as heart failure [7], orthotopic heart transplantation [8,9], or myocardial infarction [10,11], and even surmised the existence of resident cardiac stem cells [12], or extracardiac cardiomyocyte progenitor cells [13] in the mammalian heart. However, these findings are still controversial [14]. In some of these studies, the presence of the Y-chromosome in hearts from female donors after gender-incompatible heart transplantation was used as evidence for population of the hearts by extracardiac progenitors. However, recent in vitro studies reported the occurrence of spontaneous cell fusions of putative stem cells, such as bone-marrow [15] or neural stem cells [16], with embryonic stem cells when cultured together, which challenges the concept of transdifferention and requires additional investigations in this field. Nevertheless, the mechanisms of re-activated or preserved cardiomyocyte proliferation and regeneration in humans (for review see [17]) are obviously not sufficient to prevent extensive scar formation and the relentless disease progression of heart failure after myocardial infarction or loss of contractile function with pressure or volume overload [18,19]. Despite progress in pharmacological treatment of the syndrome of heart failure [20], mortality and curtailment of quality of life in advanced stages of congestive heart failure are significant [21], and the question remains, whether additional approaches can be developed for repairing human myocardial tissue. Taking the newt as a model of regenerating irreversibly damaged myocardium, the features of de-differentiation, proliferation, and re-differentiation, might be the basis for developing strategies for repairing and rebuilding the failing human heart: Presumably, the most desirable future goal would be the identification of the cellular and molecular mechanisms that prevent the adult mammalian cardiomyocyte from re-entering the cell cycle after loss of contractile tissue leading to therapeutic strategies that force surviving cardiomyocytes to de-differentiate, proliferate, and re-differentiate after myocardial damage [5,22]. However, another innovative approach, probably within nearer reach, is the exogenous delivery of less differentiated cells to the damaged heart, that retain to a certain degree their capability of proliferation, and subsequently may colonise the scar and differentiate into cells with contractile properties. This review will try to characterise the results of recent attempts to rebuild injured myocardial tissue by transplanting cells of various origin and stage of differentiation, such as fetal and neonatal cardiomyocytes, skeletal myoblasts, and embryonic and mesenchymal stem cells. Advantages, disadvantages, and major obstacles of using cells of various levels of differentiation are discussed. In an attempt to initiate a new discussion on the future direction of cellular cardiomyoplasty, we endeavour to delineate the value of some new techniques and approaches that are looming at the apparent (sometimes hazy) horizon for resolving problems and improving results in cellular cardiomyoplasty. Transplanting fetal or neonatal myocytes can be regarded as the primary model of cellular cardiomyoplasty. Being of myocardial origin, these cells differentiate towards an adult cardiomyocyte phenotype, when remaining in an appropriate environment, but still retain a certain capability of proliferation [4,23]. Less differentiated cells, delivered to scarred myocardial tissue, always must undergo differentiation into a contractile phenotype to serve as a substitute for injured myocardial tissue. The first pioneer studies reported survival of fetal or neonatal heart tissue over the first weeks, when transplanted into a non-physiological environment, such as the anterior chamber of the eye, the pinna of the ear, or the subcutaneous tissue of the rat hind limb [24–27]. Soon after, the formation of stable grafts over 2 months in normal hearts was demonstrated after injection of a suspension of cultured fetal cardiomyocytes in mice [28], and fetal or neonatal cardiomyocytes in rat hearts [29]. A recent quantitative analysis, using detection of the Sry gene of the Y-chromosome after injection of male neonatal cardiomyocytes into female normal rat hearts, suggested a survival rate of 15% of the initially injected number of cells after 12 weeks [30]. Similarly, in damaged myocardial tissue, such as scars created by cryoinjury [29], temporary [31], or permanent coronary occlusion [32,33], stable transplants were described after intramyocardial injection of fetal or neonatal cardiomyocytes or fragments of fetal ventricular tissue, but not after injection of adult cardiomyocytes [34], that showed signs of coagulation necrosis already 1 day after transplantation [29]. The notable exception to the growing body of evidence supporting survival in the damaged heart, reported in a porcine model of chronic myocardial infarction by Watanabe et al. [35] remains unexplained: In this study, fetal as well as neonatal cells only survived in normal myocardium, but surprisingly not in the infarct. Viability of engrafted cells in infarcted myocardium now has been demonstrated for up to 6–7 months after transplantation of fetal and neonatal cardiomyocytes [34,32] with a survival rate of 60% of the initial number of injected neonatal cells in a permanent coronary occlusion model in the rat after 6 months [32]. Reasons for greater survival in the infarct model than the non-infarcted model from Müller-Ehmsen's [28] work may be related to less washout of cells from an infarct with compromised blood supply than from viable myocardium with an intact vasculature. Also, a lack of contractile force within the scar may have led to better cell survival by less trauma, or less pushing of the cells out of the region of initial injection. The proliferative activity of transplanted cells may be essential for colonising the scar tissue after transplantation. In the investigations by Reinecke et al. neonatal grafts were positive for PCNA (proliferating cell nuclear antigen) from 3 days to 2 weeks after transplantation with a maximum at day six after transplantation [29]. Labelling with tritiated thymidine in Soonpaa's mice transplantation study, demonstrated a decline from 29% 3H-thymidine-positive cells at gestational day 15 to 0.6% 19 days after transplantation [28]. After injection of neonatal cardiomyocytes at three sites into the ischemic risk zone, Scorsin et al. described the cells as remaining grouped around the injection site [36]. Even 5–7 months after transplanting fetal cardiomyocytes, Connold et al. reported that the majority of cells was detected around the previous injection site [34] with a limited degree of migration. Function of the heart depends crucially on its microanatomic structure as an electrical and mechanical syncytium. Therefore, integration into the host and differentiation toward the adult phenotype are critical for the transplant to successfully contribute to cardiac function. In the fundamental investigations by Soonpaa et al., transplanting fetal cardiomyocytes into normal mice hearts led to a close juxtaposition of graft and host cells with circumferential alignment of graft cells parallel to the host myocardium, and importantly intercalated disks between host and graft cells, as demonstrated by electron microscopy [28]. After transplantation of neonatal cells in cryoinjured hearts, graft cells demonstrated an increasingly polar alignment of N-cadherin, an adherens junction protein, and connexin-43 ([29], also [37]). Albeit close spatial approximation between host and graft cells early after transplantation in the study by Reinecke et al., most grafts were separated from the host by scar tissue by 8 weeks after transplantation with only few contact sites and gap junctions between host and graft, visible by confocal microscopy. This tendency of separation between host and graft was even more pronounced in the long-term follow-up by Müller-Ehmsen et al. [32], who described the graft as ‘confluent clusters of myocardial cells in the scar’ 6 months after transplantation, and importantly did not find any gap junctions between graft and host, but frequently between grafted cells. Transplanted immature cardiomyocytes, characterized by their small cell size, high nuclear to cytoplasm ratio and loose myofibrillar organization early after transplantation [36], showed an increase in graft-cell diameters up to 8 weeks post-transplantation and myofibrillae, forming complete sarcomeres, junctional complexes and abundant mitochondria [28,29], indicating a certain differentiation and switch to hypertrophic growth patterns. Fetal cardiomyocytes in culture appeared spherical with disorganised myofilaments and sarcomeric structures, while 4 weeks after transplantation the graft had developed sarcomeres, desmosomes and fasciae adherentes [38]. However, 65 days after engrafting of fetal myocardial tissue in Leor's experiments, the transplant was still positive for α-actin on immunostaining, the actin-isoform typical of fetal and smooth muscle cells [31,33], indicating incomplete differentiation at the level of protein expression. Summarizing these basic findings, one has to state that complete regeneration of the damaged heart by transplantation, i.e. replacement of the scar by contractile tissue, has not yet been accomplished. In longer follow-up studies, the graft appeared mainly surrounded by collagen and scar tissue, meaning incomplete mechanical and electrical integration, although the principle development of intercalated disks between host and graft cell was convincingly demonstrated in normal hosts. To date, no study has been able to show synchronous beating of the graft within the infarcted myocardium in vivo. Thus, does the graft contribute to systolic force development, a process that requires adequate electrical and mechanical synchronisation? Four weeks after transplantation of fetal cardiomyocytes in cryoinjured hearts, the graft beat spontaneously when excised en bloc, as reported by Sakai et al. [39]; and the graft obtained after injection of fetal and neonatal cells in the subcutaneous tissue by Li et al., demonstrated a local electrogram resembling an ‘idioventricular rhythm’ [27], showing the principally preserved contractile capacity after engraftment. A substantial number of investigations demonstrated improved left ventricular function after cellular cardiomyoplasty: Four weeks after transplantation of fetal cardiomyocytes in cryoinured hearts, Li et al. demonstrated higher developed left-ventricular pressures in the transplanted group in an ex vivo Langendorff-preparation [40]. In vivo assessment of left ventricular function by transthoracic echocardiography in a model of reperfused myocardial infarction in rats revealed higher ejection fractions, as assessed from two-dimensional images, and cardiac output, calculated from Doppler profile in the pulmonary outflow tract 1 month after transplantation in a study by Scorsin et al. [41]. A long-term follow-up over 6 months after transplantation in infarcted rat hearts, created by permanent coronary occlusion, similarly showed higher developed pressures in a Langendorff-preparation, along with smaller end-diastolic left ventricular diameters and increased fractional shortening, measured by transthoracic echocardiography [42]. Are the observed improvements in left ventricular performance really due to an active synchronous by host cell or mechanical the grafts beat to excised or does cellular the process after myocardial infarction by the and of the infarcted myocardium by and the scar with subsequent of systolic months after intramyocardial injection of neonatal cardiomyocytes, left ventricular as assessed by by Müller-Ehmsen et al. [32], was improved in of higher ejection fractions, less in the infarcted zone, along with of the scar on the separation of graft and host tissue by connective tissue. Similarly, of fetal cardiomyocytes in study, using a permanent coronary occlusion model in showed that transplantation scar and left ventricular over days after transplantation, at on the process as the of improved cardiac performance A recent study, in which fetal cardiomyocytes were injected 4 weeks after permanent coronary that cellular did not already developed left ventricular indicating that the on cardiac on the the cells are transplanted also However, has to be that the of transplanted cells is Sakai et al. the results after transplantation of fetal cardiomyocytes, smooth muscle cells and and convincingly demonstrated a higher degree of of ex vivo of left ventricular function in the cardiomyocyte indicating that and contractile of the transplanted cells are not In their permanent occlusion et al. reported a tendency toward of the scar after transplantation, did not Four weeks after fetal cell transplantation, Li et al. a higher number of and in the transplanted tissue and a recent study demonstrated a increase of with of myocardial blood after cardiomyocyte transplantation into scars created by cryoinjury might that cardiomyocyte transplantation some of its by scar and infarct or in by growth with on the the a recent study in infarcted mice hearts demonstrated pronounced of and higher force development in muscle after transplantation, along with intact cellular electrical of the transplanted cells The that a of the graft to systolic force development might be the of improved left ventricular function. This study, which was the first to an of survival in the transplanted but the follow-up was and most in the group within the first studies assessed the of cellular in of heart in which the separation of the graft by scar tissue might be less The and survival was demonstrated in mice and hearts by et al. et al. evidence for at survival of transplanted cardiomyocytes in for 4 vivo in a demonstrated higher developed pressures in the transplanted group Interestingly, Scorsin et al. demonstrated improved left ventricular function in heart failure in mice 1 month after fetal as assessed in vivo by transthoracic although the transplanted cells for one out of 12 did not over the of 1 month In transplantation of immature cardiomyocytes in infarcted myocardium on cardiac function between fetal and neonatal in long-term studies, the might be due to on the process than replacing contractile tissue with beating graft tissue. 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heart the myocardium after in requires de-differentiation, proliferation and of and the mechanisms and for the loss of this capacity in close the mammalian heart might be the basis for cardiac in the these basic features of cellular to be a leading to strategies for the damaged heart: of immature cardiomyocytes mainly a model for can be by approaches of transplantation. of cells at various stages of differentiation, but with the capability of into a contractile phenotype might be such as skeletal or embryonic or mesenchymal stem cells. The on the of heart the therapeutic approach, and techniques of to results after transplantation to be