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Loss of myocardial tissue remains a leading cause of disease and death, as the adult heart has insufficient regenerative potential. The (pre-)clinical effects of inducing cardiac regeneration by cardiac cell therapy have been disappointing. This lack of success may result from the fact that it remains largely unclear how the receiving pathological microenvironment affects this process of cardiomyogenic differentiation of implanted cells, and thereby may (negatively) influence the therapeutic outcome. However, the tools to address this lacuna in a proper manner are lacking, as this requires tightly controllable and quantitative models of cardiomyogenic differentiation. Therefore, we have recently generated lines of conditionally immortalized human CMCs (ciCMCs) through doxycycline-dependent expression of SV40 large T antigen after genetic modification and subsequent clonal expansion. In these cells, proliferation and differentiation can be tightly controlled, allowing cardiomyogenic differentiation to be i) induced on cue, ii) precisely monitored and quantified, and iii) manipulated. The aim of this study is to improve our understanding of cardiomyogenic differentiation of guest (transplanted) cells in the context of the host (receiving) microenvironment. To create pathological microenvironments and study the effects on cardiac differentiation, co-culture experiments with human ciCMCs and cardiac fibroblasts were conducted in different ratios (10%, 30%, 60% and 90% ciCMCs). Cardiomyogenic differentiation was determined by immunostaining for cardiac specific markers and electrophysiological analysis by optical voltage mapping. After 12 days of co-culture with cardiac fibroblasts, the amount of ciCMCs that expressed the sarcomeric protein cardiac troponin T was significantly and increasingly reduced (P Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Leiden University Fund

Background Preclinical cardiac research greatly depends on animal-derived cellular models, thereby hampering clinical translation. While upcoming human pluripotent stem cell technology seems to decrease this gap between bench and bedside, its complex/multi-step protocol to produce cardiac muscle cells, its required expertise, and its trouble to produce large numbers of phenotypically homogeneous cardiomyocytes so far has limited broad application. Purpose We aimed to conditionally immortalize native human atrial cardiomyocytes to produce natural and standardized lines of these cells by gaining full control over their proliferation and differentiation. Methods Human fetal atria (gestational age 18 weeks) were dissociated and transduced with a lentiviral vector directing myocyte-specific and doxycycline-inducible expression of simian virus 40 large T antigen (here defined as immortogenetics). Addition of doxycycline to the culture medium pushed cardiomyocytes towards a proliferative phenotype. In total, 125 proliferating monoclones were isolated, expanded and screened for their cardiomyogenic differentiation capacity upon doxycycline removal. Selected clones were characterised using various molecular biological and electrophysiological assays. Results Upon doxycycline removal (i.e. under differentiation conditions), cells spontaneously reacquired a cardiomyocyte-like appearance as judged by phase-contrast microscopy and were observed contracting. Simultaneously, these cells stopped proliferating, which was accompanied by a drop in large T level, loss of Ki67 expression and the development of sarcomeres with striated α-actinin and troponin T staining patterns. These cells were tagged conditionally immortalized human atrial cardiomyocytes (hereinafter called hiAMs). Optical voltage mapping of hiAM monolayers revealed excitable cells showing homogeneous spreading of action potentials at 22,5±3,1 cm/s following 1-Hz point stimulation, with a mean APD80 of 139±22 ms. Monolayers of hiAMs could easily be created as big as 10cm2 while continuing to display homogenous conduction throughout the culture. Single-cell patch clamp recordings of a hiAM clone in current-clamp mode confirmed excitability with a resting membrane potential of −62,2±4,3 mV, peak potential of 39,4±3,9 mV and APD80 of 339±9 ms. Excitable monolayer of hiAMs Conclusion We have generated first-of-a-kind lines of natural human atrial cardiomyocytes through immortogenetics, allowing massive cell expansion under proliferation conditions and robust formation of cross-striated, contractile and excitable cardiomyocytes after differentiation. Thereby, a user-friendly, clinically-relevant and much-anticipated research model has been produced, which application could range from multi-scale electrophysiological studies and drug response studies to disease modelling and myocardial regeneration.

Cardiomyogenic differentiation of stem cells can be accomplished by coculture with cardiomyocytes (CMCs). To facilitate their identification, stem cells are often labeled through viral transduction with a fluorescent protein. A second marker to distinguish stem cell-derived CMCs from native CMCs is rarely used. This study aimed to investigate the occurrence of secondary transduction of unlabeled neonatal rat (nr) CMCs after coculture with human cells that had been transduced 0, 7, or 14 days earlier with a vesicular stomatitis virus (VSV) G protein-pseudotyped lentiviral vector (LV) encoding enhanced green fluorescent protein (GFP). To reduce secondary LV transfer, GFP-labeled cells were incubated with non-heat-inactivated human serum (NHI) or with VSV-neutralizing rabbit serum (αVSV). Heat-inactivated human serum and normal rabbit serum were used as controls. Immunostaining showed substantial GFP gene transfer to nrCMCs in cocultures started at the day of transduction indicated by the presence of GFP-positive/human lamin A/C-negative nrCMCs. The extent of secondary transduction was significantly reduced in cocultures initiated 7 days after GFP transduction, while it was completely abolished when human cells were added to nrCMCs 14 days post-transduction. Both NHI and αVSV significantly reduced the occurrence of secondary transduction compared to their controls. However, under all circumstances, GFP-labeled human cells had to be passaged for 14 days prior to coculture initiation to prevent any horizontal GFP gene transfer to the nrCMCs. This study emphasizes that differentiation experiments involving the use of viral vector-marked donor cells should be interpreted with caution and describes measures to reduce/prevent secondary transduction. STEM CELLS 2012;30:2830–2834

Signalling between the various cell types in the heart has been investigated for decades. However, relatively little is known about the interplay between the cardiac fibroblasts and myofibroblasts, which help to maintain myocardial tissue structure and function, and resident cardiac or extracardiac stem cells involved in tissue homeostasis and repair. Much of our knowledge about these interactions is derived from experimental animal models, especially those of myocardial infarction and stem cell transplantation. However, it still remains incompletely understood how stem cell therapy could modulate cardiac fibrosis in a beneficial manner and, how on the other hand, fibrotic processes in the heart may affect the therapeutic potential of stem cell therapy. A detailed and mechanistic insight into these matters would expedite the therapeutic optimization of cardiac cell therapy for the fibrotic heart and may even provide a basis for future biological therapies aiming for a reversal of cardiac fibrosis. Therefore, the main focus of this review is to discuss interactions between myofibroblasts and stem cells, especially in the adult and diseased, fibrotic myocardium, and emphasize those aspects that require more investigation using dedicated models and tools.

Background— After intramyocardial injection, mesenchymal stem cells (MSCs) may engraft and influence host myocardium. However, engraftment rate and pattern of distribution are difficult to control in vivo, hampering assessment of potential adverse effects. In this study, the role of the engraftment patterns of MSCs on arrhythmicity in controllable in vitro models is investigated. Methods and Results— Cocultures of 4×10 5 neonatal rat cardiomyocytes and 7% or 28% adult human MSCs (hMSCs) in diffuse or clustered distribution patterns were prepared. Electrophysiological effects were studied by optical mapping and patch-clamping. In diffuse cocultures, hMSCs dose-dependently decreased neonatal rat cardiomyocyte excitability, slowed conduction, and prolonged action potential duration until 90% repolarization (APD 90 ). Triggered activity (14% versus 0% in controls) and increased inducibility of re-entry (53% versus 6% in controls) were observed in 28% hMSC cocultures. MSC clusters increased APD 90 , slowed conduction locally, and increased re-entry inducibility (23%), without increasing triggered activity. Pharmacological heterocellular electric uncoupling increased excitability and conduction velocity to 133% in 28% hMSC cocultures, but did not alter APD 90 . Transwell experiments showed that hMSCs dose-dependently increased APD 90 , APD dispersion, inducibility of re-entry and affected specific ion channel protein levels, whereas excitability was unaltered. Incubation with hMSC–derived exosomes did not increase APD in neonatal rat cardiomyocyte cultures. Conclusions— Adult hMSCs affect arrhythmicity of neonatal rat cardiomyocyte cultures by heterocellular coupling leading to depolarization–induced conduction slowing and by direct release of paracrine factors that negatively affect repolarization rate. The extent of these detrimental effects depends on the number and distribution pattern of hMSCs. These results suggest that caution should be urged against potential adverse effects of myocardial hMSC engraftment.

Gap junctional coupling is important for functional integration of transplanted cells with host myocardium. However, the role of gap junctions in cardiomyogenic differentiation of transplanted cells has not been directly investigated. The objective of this work is to study the role of connexin43 (Cx43) in cardiomyogenic differentiation of human mesenchymal stem cells (hMSCs). Knockdown of Cx43 gene expression (Cx43↓) was established in naturally Cx43-rich fetal amniotic membrane (AM) hMSCs, while Cx43 was overexpressed (Cx43↑) in inherently Cx43-poor adult adipose tissue (AT) hMSCs. The hMSCs were exposed to cardiomyogenic stimuli by coincubation with neonatal rat ventricular cardiomyocytes (nrCMCs) for 10 days. Differentiation was assessed by immunostaining and whole-cell current clamping. To establish whether the effects of Cx43 knockdown could be rescued, Cx45 was overexpressed in Cx43↓ fetal AM hMSCs. Ten days after coincubation, not a single Cx43↓ fetal AM hMSC, control adult AT MSC, or Cx43↑ adult AT mesenchymal stem cell (MSC) expressed α-actinin, while control fetal AM hMSCs did (2.2% ± 0.4%, n = 5,000). Moreover, functional cardiomyogenic differentiation, based on action potential recordings, occurred only in control fetal AM hMSCs. Of interest, Cx45 overexpression in Cx43↓ fetal AM hMSCs restored their ability to undergo cardiomyogenesis (1.6% ± 0.4%, n = 2,500) in coculture with nrCMCs. Gap junctional coupling is required for differentiation of fetal AM hMSCs into functional CMCs after coincubation with nrCMCs. Heterocellular gap junctional coupling thus plays an important role in the transfer of cardiomyogenic signals from nrCMCs to fetal hMSCs but is not sufficient to induce cardiomyogenic differentiation in adult AT hMSCs. Disclosure of potential conflicts of interest is found at the end of this article.

Aims Cardiac fibrosis is associated with increased incidence of cardiac arrhythmias, but the underlying proarrhythmic mechanisms remain incompletely understood and antiarrhythmic therapies are still suboptimal. This study tests the hypothesis that myofibroblast (MFB) proliferation leads to tachyarrhythmias by altering the excitability of cardiomyocytes (CMCs) and that inhibition of MFB proliferation would thus lower the incidence of such arrhythmias. Methods and results Endogenous MFBs in neonatal rat CMC cultures proliferated freely or under control of different dosages of antiproliferative agents (mitomycin-C and paclitaxel). At Days 4 and 9, arrhythmogeneity of these cultures was studied by optical and multi-electrode mapping. Cultures were also studied for protein expression and electrophysiological properties. MFB proliferation slowed conduction from 15.3 ± 3.5 cm/s (Day 4) to 8.8 ± 0.3 cm/s (Day 9) ( n = 75, P < 0.01), whereas MFB numbers increased to 37.4 ± 1.7 and 62.0 ± 2%. At Day 9, 81.3% of these cultures showed sustained spontaneous reentrant arrhythmias. However, only 2.6% of mitomycin-C-treated cultures ( n = 76, P < 0.0001) showed tachyarrhythmias, and ectopic activity was decreased. Arrhythmia incidence was drug–dose dependent and strongly related to MFB proliferation. Paclitaxel treatment yielded similar results. CMCs were functionally coupled to MFBs and more depolarized in cultures with ongoing MFB proliferation in which only L-type Ca2+-channel blockade terminated 100% of reentrant arrhythmias, in contrast to Na+ blockade (36%, n = 12). Conclusion Proliferation of MFBs in myocardial cultures gives rise to spontaneous, sustained reentrant tachyarrhythmias. Antiproliferative treatment of such cultures prevents the occurrence of arrhythmias by limiting MFB-induced depolarization, conduction slowing, and ectopic activity. This study could provide a rationale for a new treatment option for cardiac arrhythmias.

Alignment of cardiomyocytes (CMCs) contributes to the anisotropic (direction-related) tissue structure of the heart, thereby facilitating efficient electrical and mechanical activation of the ventricles. This study aimed to investigate the effects of forced alignment of stem cells during cardiomyogenic differentiation on their functional integration with CMC cultures. Labeled neonatal rat (nr) mesenchymal stem cells (nrMSCs) were allowed to differentiate into functional heart muscle cells in different cell-alignment patterns during 10 days of coculture with nrCMCs. Development of functional cellular properties was assessed by measuring impulse transmission across these stem cells between 2 adjacent nrCMC fields, cultured onto microelectrode arrays and previously separated by a laser-dissected channel (230±10 μm) for nrMSC transplantation. Coatings in these channels were microabraded in a direction (1) parallel or (2) perpendicular to the channel or were (3) left unabraded to establish different cell patterns. Application of cells onto microabraded coatings resulted in anisotropic cell alignment within the channel. Application on unabraded coatings resulted in isotropic (random) alignment. After coculture, conduction across seeded nrMSCs occurred from day 1 (perpendicular and isotropic) or day 6 (parallel) onward. Conduction velocity across nrMSCs at day 10 was highest in the perpendicular (11±0.9 cm/sec; n=12), intermediate in the isotropic (7.1±1 cm/sec; n=11) and lowest in the parallel configuration (4.9±1 cm/sec; n=11) ( P

Aims Ventricular tachyarrhythmias (VTs) are common in the pathologically remodelled heart. These arrhythmias can be lethal, necessitating acute treatment like electrical cardioversion to restore normal rhythm. Recently, it has been proposed that cardioversion may also be realized via optically controlled generation of bioelectricity by the arrhythmic heart itself through optogenetics and therefore without the need of traumatizing high-voltage shocks. However, crucial mechanistic and translational aspects of this strategy have remained largely unaddressed. Therefore, we investigated optogenetic termination of VTs (i) in the pathologically remodelled heart using an (ii) implantable multi-LED device for (iii) in vivo closed-chest, local illumination. Methods and results In order to mimic a clinically relevant sequence of events, transverse aortic constriction (TAC) was applied to adult male Wistar rats before optogenetic modification. This modification took place 3 weeks later by intravenous delivery of adeno-associated virus vectors encoding red-activatable channelrhodopsin or Citrine for control experiments. At 8–10 weeks after TAC, VTs were induced ex vivo and in vivo, followed by programmed local illumination of the ventricular apex by a custom-made implanted multi-LED device. This resulted in effective and repetitive VT termination in the remodelled adult rat heart after optogenetic modification, leading to sustained restoration of sinus rhythm in the intact animal. Mechanistically, studies on the single cell and tissue level revealed collectively that, despite the cardiac remodelling, there were no significant differences in bioelectricity generation and subsequent transmembrane voltage responses between diseased and control animals, thereby providing insight into the observed robustness of optogenetic VT termination. Conclusion Our results show that implant-based optical cardioversion of VTs is feasible in the pathologically remodelled heart in vivo after local optogenetic targeting because of preserved optical control over bioelectricity generation. These findings add novel mechanistic and translational insight into optical ventricular cardioversion.