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Functional human atrial myocytes derived from foetal atrial tissue can be massively expanded via a conditional cell-immortalization method, and used in in vitro models of atrial fibrillation.The lack of a scalable and robust source of well-differentiated human atrial myocytes constrains the development of in vitro models of atrial fibrillation (AF). Here we show that fully functional atrial myocytes can be generated and expanded one-quadrillion-fold via a conditional cell-immortalization method relying on lentiviral vectors and the doxycycline-controlled expression of a recombinant viral oncogene in human foetal atrial myocytes, and that the immortalized cells can be used to generate in vitro models of AF. The method generated 15 monoclonal cell lines with molecular, cellular and electrophysiological properties resembling those of primary atrial myocytes. Multicellular in vitro models of AF generated using the immortalized atrial myocytes displayed fibrillatory activity (with activation frequencies of 6-8 Hz, consistent with the clinical manifestation of AF), which could be terminated by the administration of clinically approved antiarrhythmic drugs. The conditional cell-immortalization method could be used to generate functional cell lines from other human parenchymal cells, for the development of in vitro models of human disease.

Aim: Channelrhodopsins (ChRs) are a large family of light-gated ion channels with distinct properties, which is of great importance in the selection of a ChR variant for a given application. However, data to guide such selection for cardiac optogenetic applications are lacking. Therefore, we investigated the functioning of different ChR variants in normal and pathological hypertrophic cardiomyocytes subjected to various illumination protocols.Methods and Results: Isolated neonatal rat ventricular cardiomyocytes (NRVMs) were transduced with lentiviral vectors to express one of the following ChR variants: H134R, CatCh, ReaChR, or GtACR1. NRVMs were treated with phenylephrine (PE) to induce pathological hypertrophy (PE group) or left untreated [control (CTL) group]. In these groups, ChR currents displayed unique and significantly different properties for each ChR variant on activation by a single 1-s light pulse (1 mW/mm2: 470, 565, or 617 nm). The concomitant membrane potential (Vm) responses also showed a ChR variant-specific profile, with GtACR1 causing a slight increase in average Vm during illumination (Vplateau: −38 mV) as compared with a Vplateau > −20 mV for the other ChR variants. On repetitive activation at increasing frequencies (10-ms pulses at 1–10 Hz for 30 s), peak currents, which are important for cardiac pacing, decreased with increasing activation frequencies by 17–78% (p < 0.05), while plateau currents, which are critical for arrhythmia termination, decreased by 10–75% (p < 0.05), both in a variant-specific manner. In contrast, the corresponding Vplateau remained largely stable. Importantly, current properties and Vm responses were not statistically different between the PE and CTL groups, irrespective of the variant used (p > 0.05).Conclusion: Our data show that ChR variants function equally well in cell culture models of healthy and pathologically hypertrophic myocardium but show strong, variant-specific use-dependence. This use-dependent nature of ChR function should be taken into account during the design of cardiac optogenetic studies and the interpretation of the experimental findings thereof.

Funding Acknowledgements Type of funding sources: Public hospital(s). Main funding source(s): LUMC Background Current in vitro models of atrial fibrillation have limited translational potential due to a lack of relevant human physiology or the inability to reach the high activation frequencies present in human atrial fibrillation. Absence of relevant models is the result of a general deficit of readily available and standardized sources of well-differentiated human atrial cardiomyocytes. Therefore, we aimed to immortalize native human atrial cardiomyocytes to produce natural and standardized lines of these cells. Methods Human fetal atrial cardiomyocytes were transduced with a lentiviral vector directing myocyte-specific and doxycycline-inducible expression of simian virus 40 large T antigen. Addition of doxycycline to the culture medium pushed cardiomyocytes towards a highly proliferative phenotype (proliferation up to 10^12 cells). These cells were labelled hiAMs (human immortalised Atrial Myocytes). After differentiation upon doxycycline removal, hiAM cells were characterized using various molecular, biological and electrophysiological assays. Results Following cardiomyogenic differentiation, hiAMs no longer expressed the proliferation marker Ki67, revealed striated α-actinin and troponin T staining patterns and displayed synchronous contractions. Optical voltage mapping of hiAM monolayers revealed excitable cells showing homogeneous spreading of action potentials at 22.5 ± 3.1 cm/s with a mean APD80 of 139 ± 22 ms. Addition of flecainide (10 µM) to hiAM monolayers decreased the conduction velocity by 35% and increased the APD80 by 107%. Dofetilide (10 nM) addition had no effect on the conduction velocity, but did increase the APD80 by 81%. Due to their scalability, monolayers of hiAMs as big as 10 cm2 showing homogenous action potential propagation could easily be created. Following high-frequency electrical pacing, rotors could be induced with an average activation frequency of 7.5 ± 0.9 Hz. Infusion of flecainide during arrhythmic activity resulted in termination of the rotor in 18 of 24 attempts (75%), whereas addition of 0.1% DMSO (vehicle control) did not result in termination in any of the attempts. Dofetilide infusion did not result in termination. However, it did lower the average activation frequency to 2.1 ± 0.7 Hz. Conclusion We have generated first-of-a-kind lines of human atrial cardiomyocytes, allowing massive cell expansion under proliferation conditions and robust formation of cross-striated, contractile and excitable cardiomyocytes after differentiation. These characteristics allow, for the first time, the modelling, at a large-scale, of human atrial arrhythmias with frequencies similar to human atrial fibrillation. With the generation of hiAMs, a user-friendly, clinically-relevant and much-anticipated human atrial research model has been produced. Abstract Figure. hiAM AF Model

Background Current in vitro models of atrial fibrillation have limited translational potential due to a lack of relevant human physiology or the inability to reach the high activation frequencies present in human atrial fibrillation. Absence of relevant models is the result of a general deficit of readily available and standardized sources of well-differentiated human atrial cardiomyocytes. Therefore, we aimed to immortalize native human atrial cardiomyocytes to produce natural and standardized lines of these cells. Methods Human fetal atrial cardiomyocytes were 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 highly proliferative phenotype (proliferation up to 1012 cells). These cells were labelled hiAMs (human immortalised Atrial Myocytes). After differentiation upon doxycycline removal, hiAM cells were characterized using various molecular, biological and electrophysiological assays. Results Following cardiomyogenic differentiation, hiAMs no longer expressed the proliferation marker Ki67, revealed striated α-actinin and troponin T staining patterns and displayed synchronous contractions. Optical voltage mapping of hiAM monolayers revealed excitable cells showing homogeneous spreading of action potentials at 22.5±3.1 cm/s with a mean APD80 of 139±22 ms. Addition of flecainide (10 μM) to hiAM monolayers decreased the conduction velocity by 35% and increased the APD80 by 107%. Dofetilide (10 nM) addition had no effect on the conduction velocity, but did increase the APD80 by 81%. Due to their scalability, monolayers of hiAMs as big as 10 cm2 showing homogenous action potential propagation could easily be created. Following high-frequency electrical pacing, rotors could be induced with an average activation frequency of 7.5±0.9 Hz. Infusion of flecainide during arrhythmic activity resulted in termination of the rotor in 18 of 24 attempts (75%), whereas addition of 0.1% DMSO (vehicle control) did not result in termination in any of the attempts. Dofetilide infusion did not result in termination. However, it did lower the average activation frequency to 2.1±0.7 Hz. Conclusion We have generated first-of-a-kind lines of human atrial cardiomyocytes, allowing massive cell expansion under proliferation conditions and robust formation of cross-striated, contractile and excitable cardiomyocytes after differentiation. These characteristics allow, for the first time, the modelling, at a large-scale, of human atrial arrhythmias with frequencies similar to human atrial fibrillation. With the generation of hiAMs, a user-friendly, clinically-relevant and much-anticipated human atrial research model has been produced. Large-scale AF model using hiAMs Funding Acknowledgement Type of funding source: None

Background Ventricular tachyarrhytmias (VTs) are common among patients suffering from cardiac remodeling and cause significant morbidity and mortality. Current research and treatment options for such VTs are suboptimal, hence new strategies are urgently needed. Optogenetics offers efficacious means to control cardiac rhythm, including shock-free VT termination. However, this has not been demonstrated in diseased hearts in vivo, while clinical translation would not only require such demonstration, but also an in-depth understanding of cellular responses. Purpose To assess the optogenetic response at the cardiac cell, tissue, and whole heart level in terms of rhtyhm control under pathological conditions by an integrative experimental platform including in vitro and in vivo models of cardiac disease. Methods Remodeling was induced in neonatal rat ventricular cardiomyocytes (NRVMs) by phenylephrine (PE) exposure. Pathological conditions leading to ventricular remodeling were mimicked by transverse aortic constriction (TAC) surgery in adult rats. The light-activated ion channel ReaChR was ectopically expressed in NRVMs and in hearts of TAC and sham animals by viral vector-based gene delivery. Results Electrical and structural remodeling was evidenced by elongated action potential durations (p0.05). Illumination (1 s) caused a sudden shift in membrane potential leading to a plateau at −7.3 mV for PE-treated and −18.9 mV for CTL cells (p>0.05). Hearts explanted from TAC animals showed increased average heart weight to body weight ratio, ventricular fibrosis and expression of hypertrophy markers (ANP, aSkMA, p0.05) in VMs from TAC and sham animals, which currents led to comparable shifts in membrane potential (65.3 mV for TAC and 63.9 mV for CTL). In line with this, illumination caused marked depolarization in tissue preparations (from −77.6 to −16.4 mV) in TAC animals as assessed by conventional sharp electrode measurements. Importantly, as anticipated, electrically-induced VT episodes could be terminated in open chest experiments in TAC animals (n=6; 76.3% of cases) by epicardial illumination in vivo. Conclusions Key operational parameters of the optogenetic response remained unaffected in models of cardiac disease, which allowed efficacious optogenetic VT termination in the diseased rat heart exhibiting structural and electrical remodeling. These findings corroborate the translational potential of shock-free therapy of cardiac arrhythmia by optogenetics. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): This work was supported by personal funding from the Netherlands Organization for Scientific Research (NWO, Vidi grant 1714336 to D.A.P.). D.A.P. is also a recipient of the European Research Council (ERC), Starting grant (716509). Additional support was provided by the Netherlands Heart Institute (ICIN grant 230.148-04 to A.A.F.d.V.).

Background Diseased atria are characterised by functional and structural heterogeneities (e.g. dense fibrotic regions), which add to abnormal impulse generation and propagation, like ectopy and block. These heterogeneities are thought to play a role in the origin of complex fractionated atrial electrograms (CFAEs) under sinus rhythm (SR) in atrial fibrillation (AF) patients, but also in the onset and perpetuation (e.g. reentry) of this disorder. The underlying mechanisms, however, remain incompletely understood. Purpose To test the hypothesis that dense local fibrotic regions could create an electrically isolated conduction pathway in which reentry can be established via ectopy and block to become “trapped” (giving rise to CFAEs under SR), only to be “released” under dynamic changes at a connecting isthmus (causing acute focal arrhythmia (FA)). Methods The geometrical properties of such an electrically isolated pathway, under which reentry could be trapped and released, were explored in vitro using optogenetics by creating conduction blocks of any shape by means of light-gated depolarizing ion channels (CatCh) and patterned illumination. Insight from these studies was used for complementary computational investigation in virtual human atria to assess clinical translation and to provide deeper mechanistic insight. Results Optical mapping studies, in monolayers of CatCh-activated neonatal rat atrial cardiomyocytes, revealed that reentry could indeed be established and trapped by creating an electrically isolated pathway with a connecting isthmus causing source-sink mismatch. This proves that a tachyarrhythmia can exist locally with SR prevailing in the bulk of the monolayer. Next, it was confirmed under which conditions reentry could escape this pathway by widening of the isthmus (i.e. overcoming the source-sink mismatch), thereby converting this local dormant arrhythmic source into an active driver with global impact (i.e. acute monolayer-wide FA). This novel phenomenon was shown in circuits Conclusion This study reveals that “trapped reentry”, a previously undesignated phenomenon, can explain the origin of two designated ones: the observation of CFAEs under SR and acute onset of FA. Further exploration of the concept of “trapped reentry” may not only expand our understanding of AF initiation and perpetuation, but also termination, including ablation strategies by site-directed targeting. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): This study was funded by the European Research Council (Starting grant 716509) to D.A. Pijnappels

Rationale: Genome-wide association studies have identified a large number of common variants (single-nucleotide polymorphisms) associated with atrial fibrillation (AF). These variants are located mainly in noncoding regions of the genome and likely include variants that modulate the function of transcriptional regulatory elements (REs) such as enhancers. However, the actual REs modulated by variants and the target genes of such REs remain to be identified. Thus, the biological mechanisms by which genetic variation promotes AF has thus far remained largely unexplored. Objective: To identify REs in genome-wide association study loci that are influenced by AF-associated variants. Methods and Results: We screened 2.45 Mbp of human genomic DNA containing 12 strongly AF-associated loci for RE activity using self-transcribing active regulatory region sequencing and a recently generated monoclonal line of conditionally immortalized rat atrial myocytes. We identified 444 potential REs, 55 of which contain AF-associated variants ( P −8 ). Subsequently, using an adaptation of the self-transcribing active regulatory region sequencing approach, we identified 24 variant REs with allele-specific regulatory activity. By mining available chromatin conformation data, the possible target genes of these REs were mapped. To define the physiological function and target genes of such REs, we deleted the orthologue of an RE containing noncoding variants in the Hcn4 (potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4) locus of the mouse genome. Mice heterozygous for the RE deletion showed bradycardia, sinus node dysfunction, and selective loss of Hcn4 expression. Conclusions: We have identified REs at multiple genetic loci for AF and found that loss of an RE at the HCN4 locus results in sinus node dysfunction and reduced gene expression. Our approach can be broadly applied to facilitate the identification of human disease-relevant REs and target genes at cardiovascular genome-wide association studies loci.

Homeostatic regulation protects organisms against hazardous physiological changes. However, such regulation is limited in certain organs and associated biological processes. For example, the heart fails to self-restore its normal electrical activity once disturbed, as with sustained arrhythmias. Here we present proof-of-concept of a biological self-restoring system that allows automatic detection and correction of such abnormal excitation rhythms. For the heart, its realization involves the integration of ion channels with newly designed gating properties into cardiomyocytes. This allows cardiac tissue to i) discriminate between normal rhythm and arrhythmia based on frequency-dependent gating and ii) generate an ionic current for termination of the detected arrhythmia. We show in silico, that for both human atrial and ventricular arrhythmias, activation of these channels leads to rapid and repeated restoration of normal excitation rhythm. Experimental validation is provided by injecting the designed channel current for arrhythmia termination in human atrial myocytes using dynamic clamp. © Majumder et al. This study was supported by The European Research Council (ERC Starting grant 716509) to DAP. Additional support was provided by the Netherlands Organisation for Scientific Research (NWO Vidi grant 91714336) to DAP, and by Ammodo (to DAP and AAFdV). The line of conditionally immortalised human atrial myocytes used in this study was made with financial support of the research programme More Knowledge with Fewer Animals (MKMD) with project number 114022503 (to AAFdV), which is (partly) financed by the Netherlands Organisation for Health Research and Development (ZonMw) and the Dutch Society for the Replacement of Animal Testing (dsRAT), and of the Leiden Regenerative Medicine Platform Holding (LRMPH project 8212/41235 to AAFdeV). We would like to thank Marie-José Goumans and Tessa van Herwaarden for their involvement in human tissue handling, Prof. Gunnar Seemann for helping with the atrial modeling, and Prof. Leon Glass and Alexander S Teplenin for useful discussions.

The role mechanical forces play in shaping the structure and function of the heart is critical to understanding heart formation and the etiology of disease but is challenging to study in patients. Engineered heart tissues (EHTs) incorporating human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have the potential to provide insight into these adaptive and maladaptive changes in the heart. However, most EHT systems are unable to model both preload (stretch during chamber filling) and afterload (pressure the heart must work against to eject blood). Here, we have developed a new dynamic EHT (dyn-EHT) model that enables us to tune preload and have unconstrained fractional shortening of >10%. To do this, 3D EHTs are integrated with an elastic polydimethylsiloxane (PDMS) strip that provides mechanical pre- and afterload to the tissue in addition to enabling contractile force measurements based on strip bending. Our results demonstrate in wild-type EHTs that dynamic loading is beneficial based on the magnitude of the forces, leading to improved alignment, conduction velocity, and contractility. For disease modeling, we use hiPSC–derived cardiomyocytes from a patient with arrhythmogenic cardiomyopathy (ACM) due to mutations in desmoplakin. We demonstrate that manifestation of this desmosome-linked disease state requires the dyn-EHT conditioning and that it cannot be induced using 2D or standard 3D EHT approaches. Thus, dynamic loading strategy is necessary to provoke a disease phenotype (diastolic lengthening, reduction of desmosome counts, and reduced contractility), which are akin to primary endpoints of clinical disease, such as chamber thinning and reduced cardiac output.Single Sentence SummaryDevelopment of a dynamic mechanical loading platform to improve contractile function of engineered heart tissues and study cardiac disease progression.

Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice with a large socioeconomic impact due to its associated morbidity, mortality, reduction in quality of life and health care costs. Currently, antiarrhythmic drug therapy is the first line of treatment for most symptomatic AF patients, despite its limited efficacy, the risk of inducing potentially life-threating ventricular tachyarrhythmias as well as other side effects. Alternative, in-hospital treatment modalities consisting of electrical cardioversion and invasive catheter ablation improve patients' symptoms, but often have to be repeated and are still associated with serious complications and only suitable for specific subgroups of AF patients. The development and progression of AF generally results from the interplay of multiple disease pathways and is accompanied by structural and functional (e.g., electrical) tissue remodeling. Rational development of novel treatment modalities for AF, with its many different etiologies, requires a comprehensive insight into the complex pathophysiological mechanisms. Monolayers of atrial cells represent a simplified surrogate of atrial tissue well-suited to investigate atrial arrhythmia mechanisms, since they can easily be used in a standardized, systematic and controllable manner to study the role of specific pathways and processes in the genesis, perpetuation and termination of atrial arrhythmias. In this review, we provide an overview of the currently available two- and three-dimensional multicellular in vitro systems for investigating the initiation, maintenance and termination of atrial arrhythmias and AF. This encompasses cultures of primary (animal-derived) atrial cardiomyocytes (CMs), pluripotent stem cell-derived atrial-like CMs and (conditionally) immortalized atrial CMs. The strengths and weaknesses of each of these model systems for studying atrial arrhythmias will be discussed as well as their implications for future studies.

During normal lifespan, the mammalian heart undergoes limited renewal of cardiomyocytes. While the exact mechanism for this renewal remains unclear, two possibilities have been proposed: differentiated myocyte replication and progenitor/immature cell differentiation. This study aimed to characterize a population of cardiomyocyte precursors in the neonatal heart and to determine their requirement for cardiac development. By tracking the expression of an embryonic Nkx2.5 cardiac enhancer, we identified cardiomyoblasts capable of differentiation into striated cardiomyocytes in vitro. Genome-wide expression profile of neonatal Nkx2.5+ cardiomyoblasts showed the absence of sarcomeric gene and the presence of cardiac transcription factors. To determine the lineage contribution of the Nkx2.5+ cardiomyoblasts, we generated a doxycycline suppressible Cre transgenic mouse under the regulation of the Nkx2.5 enhancer and showed that neonatal Nkx2.5+ cardiomyoblasts mature into cardiomyocytes in vivo. Ablation of neonatal cardiomyoblasts resulted in ventricular hypertrophy and dilation, supporting a functional requirement of the Nkx2.5+ cardiomyoblasts. This study provides direct lineage tracing evidence that a cardiomyoblast population contributes to cardiogenesis in the neonatal heart. The cell population identified here may serve as a promising therapeutic for pediatric cardiac regeneration.

Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice with a large socioeconomic impact due to its associated morbidity, mortality, reduction in quality of life and health care costs. Currently, antiarrhythmic drug therapy is the first line of treatment for most symptomatic AF patients, despite its limited efficacy, the risk of inducing potentially life-threating ventricular tachyarrhythmias as well as other side effects. Alternative, in-hospital treatment modalities consisting of electrical cardioversion and invasive catheter ablation improve patients' symptoms, but often have to be repeated and are still associated with serious complications and only suitable for specific subgroups of AF patients. The development and progression of AF generally results from the interplay of multiple disease pathways and is accompanied by structural and functional (e.g., electrical) tissue remodeling. Rational development of novel treatment modalities for AF, with its many different etiologies, requires a comprehensive insight into the complex pathophysiological mechanisms. Monolayers of atrial cells represent a simplified surrogate of atrial tissue well-suited to investigate atrial arrhythmia mechanisms, since they can easily be used in a standardized, systematic and controllable manner to study the role of specific pathways and processes in the genesis, perpetuation and termination of atrial arrhythmias. In this review, we provide an overview of the currently available two- and three-dimensional multicellular in vitro systems for investigating the initiation, maintenance and termination of atrial arrhythmias and AF. This encompasses cultures of primary (animal-derived) atrial cardiomyocytes (CMs), pluripotent stem cell-derived atrial-like CMs and (conditionally) immortalized atrial CMs. The strengths and weaknesses of each of these model systems for studying atrial arrhythmias will be discussed as well as their implications for future studies.

Atrial fibrillation (AF) is the most common clinical tachyarrhythmia with a strong tendency to progress in time. AF progression is driven by derailment of protein homeostasis, which ultimately causes contractile dysfunction of the atria. Here we report that tachypacing-induced functional loss of atrial cardiomyocytes is precipitated by excessive poly(ADP)-ribose polymerase 1 (PARP1) activation in response to oxidative DNA damage. PARP1-mediated synthesis of ADP-ribose chains in turn depletes nicotinamide adenine dinucleotide (NAD+), induces further DNA damage and contractile dysfunction. Accordingly, NAD+ replenishment or PARP1 depletion precludes functional loss. Moreover, inhibition of PARP1 protects against tachypacing-induced NAD+ depletion, oxidative stress, DNA damage and contractile dysfunction in atrial cardiomyocytes and Drosophila. Consistently, cardiomyocytes of persistent AF patients show significant DNA damage, which correlates with PARP1 activity. The findings uncover a mechanism by which tachypacing impairs cardiomyocyte function and implicates PARP1 as a possible therapeutic target that may preserve cardiomyocyte function in clinical AF., Atrial fibrillation (AF) is accompanied by a detrimental loss of functional cardiomyocytes. Here, Zhang et al. show that AF-induced cardiomyocyte dysfunction is a consequence of DNA damage-mediated PARP1 activation, which leads to depletion of NAD+ and further oxidative stress and DNA damage, and identify PARP1 inhibition as a potential therapeutic strategy in the treatment of AF.

Healthy cardiomyocytes are electrically coupled at the intercalated discs by gap junctions. In infarcted hearts, adverse gap-junctional remodeling occurs in the border zone, where cardiomyocytes are chemically and electrically influenced by myofibroblasts. The physical movement of these contacts remains unquantified. Using scanning ion conductance microscopy, we show that intercellular contacts between cardiomyocytes and myofibroblasts are highly dynamic, mainly owing to the edge dynamics (lamellipodia) of the myofibroblasts. Decreasing the amount of functional connexin-43 (Cx43) at the membrane through Cx43 silencing, suppression of Cx43 trafficking, or hypoxia-induced Cx43 internalization attenuates heterocellular contact dynamism. However, we found decreased dynamism and stabilized membrane contacts when cellular coupling was strengthened using 4-phenylbutyrate (4PB). Fluorescent-dye transfer between cells showed that the extent of functional coupling between the 2 cell types correlated with contact dynamism. Intercellular calcein transfer from myofibroblasts to cardiomyocytes is reduced after myofibroblast-specific Cx43 down-regulation. Conversely, 4PB-treated myofibroblasts increased their functional coupling to cardiomyocytes. Consistent with lamellipodia-mediated contacts, latrunculin-B decreases dynamism, lowers physical communication between heterocellular pairs, and reduces Cx43 intensity in contact regions. Our data show that heterocellular cardiomyocyte–myofibroblast contacts exhibit high dynamism. Therefore, Cx43 is a potential target for prevention of aberrant cardiomyocyte coupling and myofibroblast proliferation in the infarct border zone.—Schultz, F., Swiatlowska, P., Alvarez-Laviada, A., Sanchez-Alonso, J. L., Song, Q., de Vries, A. A. F., Pijnappels, D. A., Ongstad, E., Braga, V. M. M., Entcheva, E., Gourdie, R. G., Miragoli, M., Gorelik, J. Cardiomyocyte–myofibroblast contact dynamism is modulated by connexin-43.

Because of suboptimal therapeutic strategies, restoration of sinus rhythm in symptomatic atrial fibrillation (AF) often requires in-hospital delivery of high-voltage shocks, thereby precluding ambulatory AF termination. Continuous, rapid restoration of sinus rhythm is desired given the recurring and progressive nature of AF. Here, we present an automated hybrid bioelectronic system for shock-free termination of AF that enables the heart to act as an electric current generator for autogenous restoration of sinus rhythm. We show that local, right atrial delivery of adenoassociated virus vectors encoding a light-gated depolarizing ion channel results in efficient and spatially confined transgene expression. Activation of an implanted intrathoracic light-emitting diode device allows for termination of AF by illuminating part of the atria. Combining this newly obtained antiarrhythmic effector function of the heart with the arrhythmia detector function of a machine-based cardiac rhythm monitor in the closed chest of adult rats allowed automated and rapid arrhythmia detection and termination in a safe, effective, repetitive, yet shock-free manner. These findings hold translational potential for the development of shock-free antiarrhythmic device therapy for ambulatory treatment of AF.

AimsThe generation of homogeneous cardiomyocyte populations from fresh tissue or stem cells is laborious and costly. A potential solution to this problem would be to establish lines of immortalized cardiomyocytes. However, as proliferation and (terminal) differentiation of cardiomyocytes are mutually exclusive processes, their permanent immortalization causes loss of electrical and mechanical functions. We therefore aimed at developing conditionally immortalized atrial myocyte (iAM) lines allowing toggling between proliferative and contractile phenotypes by a single-component change in culture medium composition.Methods and resultsFreshly isolated neonatal rat atrial cardiomyocytes (AMs) were transduced with a lentiviral vector conferring doxycycline (dox)-controlled expression of simian virus 40 large T antigen. Under proliferative conditions (i.e. in the presence of dox), the resulting cells lost most cardiomyocyte traits and doubled every 38 h. Under differentiation conditions (i.e. in the absence of dox), the cells stopped dividing and spontaneously reacquired a phenotype very similar to that of primary AMs (pAMs) in gene expression profile, sarcomeric organization, contractile behaviour, electrical properties, and response to ion channel-modulating compounds (as assessed by patch-clamp and optical voltage mapping). Moreover, differentiated iAMs had much narrower action potentials and propagated them at >10-fold higher speeds than the widely used murine atrial HL-1 cells. High-frequency electrical stimulation of confluent monolayers of differentiated iAMs resulted in re-entrant conduction resembling atrial fibrillation, which could be prevented by tertiapin treatment, just like in monolayers of pAMs.ConclusionThrough controlled expansion and differentiation of AMs, large numbers of functional cardiomyocytes were generated with properties superior to the differentiated progeny of existing cardiomyocyte lines. iAMs provide an attractive new model system for studying cardiomyocyte proliferation, differentiation, metabolism, and (electro)physiology as well as to investigate cardiac diseases and drug responses, without using animals.

Propagation of non-linear waves is key to the functioning of diverse biological systems. Such waves can organize into spirals, rotating around a core, whose properties determine the overall wave dynamics. Theoretically, manipulation of a spiral wave core should lead to full spatiotemporal control over its dynamics. However, this theory lacks supportive evidence (even at a conceptual level), making it thus a long-standing hypothesis. Here, we propose a new phenomenological concept that involves artificially dragging spiral waves by their cores, to prove the aforementioned hypothesis in silico, with subsequent in vitro validation in optogenetically modified monolayers of rat atrial cardiomyocytes. We thereby connect previously established, but unrelated concepts of spiral wave attraction, anchoring and unpinning to demonstrate that core manipulation, through controlled displacement of heterogeneities in excitable media, allows forced movement of spiral waves along pre-defined trajectories. Consequently, we impose real-time spatiotemporal control over spiral wave dynamics in a biological system., eLife digest From a spinning galaxy to a swarm of honeybees, rotating spirals are widespread in nature. Even within the muscles of the heart, waves of electrical activity sometimes rotate spirally, leading to irregular heart rhythms or arrhythmia – a condition that can be fatal. Irrespective of where they occur, spiral waves organize around a center or core with different biophysical properties compared to the rest of the medium. The properties of the core determine the overall dynamics of the spiral. This means that, theoretically, it should be possibly to completely control a spiral wave just by manipulating its core. Now, Majumder, Feola et al. have tested this long-standing hypothesis using a combination of computer modeling and experiments with single layers of rat heart cells grown in a laboratory. First, the heart cells were genetically modified so that their electrical properties could be altered with light; in other words, the cells were put under optical control. Next, by using of a narrow beam of light, Majumder, Feola et al. precisely controlled the electrical properties of a small number of cells, which then attracted and supported a rotating spiral wave by acting as its new core. Moving the light beam allowed the core of the spiral wave to be shifted too, meaning the spiral wave could now be steered along any desired path in the cell layer. Majumder, Feola et al. hope that these underlying principles may one day provide the basis of new treatments for irregular heartbeats that are more effective and less damaging to the heart than existing options. Yet first, more work is needed to translate these findings from single layers of cells to actual hearts.

Aims Electroanatomical voltage mapping (EAVM) is an important diagnostic tool for fibrosis identification and risk stratification in non-ischaemic cardiomyopathy (NICM); currently, distinct cut-offs are applied. We aimed to evaluate the performance of EAVM to detect fibrosis by integration with whole heart histology and to identify the fibrosis pattern in NICM patients with ventricular tachycardias (VTs). Methods and results Eight patients with NICM and VT underwent EAVM prior to death or heart transplantation. EAVM data was projected onto slices of the entire heart. Pattern, architecture, and amount of fibrosis were assessed in transmural biopsies corresponding to EAVM sites. Fibrosis pattern in NICM biopsies (n = 507) was highly variable and not limited to mid-wall/sub-epicardium. Fibrosis architecture was rarely compact, but typically patchy and/or diffuse. In NICM, biopsies without abnormal fibrosis unipolar voltage (UV) and bipolar voltage (BV) showed a linear association with wall thickness (WT). The amount of viable myocardium showed a linear association with both UV and BV. Accordingly, any cut-off to delineate fibrosis performed poorly. An equation was generated calculating the amount of fibrosis at any location, given WT and UV or BV. Conclusion Considering the linear relationships between WT, amount of fibrosis and both UV and BV, the search for any distinct voltage cut-off to identify fibrosis in NICM is futile. The amount of fibrosis can be calculated, if WT and voltages are known. Fibrosis pattern and architecture are different from ischaemic cardiomyopathy and findings on ischaemic substrates may not be applicable to NICM.

We thank Houston et al for their interest in our study. In their letter, they raise the question whether the rotors and accompanied spiral waves observed in our study represent microreentrant circuits anchored to lines of conduction block/slowing (ie, anatomical reentry), instead of reentrant activity around an unexcited, yet excitable core region (ie, functional reentry). Their comment is based on a movie published on their website, showing high-resolution mapping, in an HL-1 culture, of reentrant activity that seems anchored to microregions of conduction abnormalities. Although appraisal of these data is difficult without a detailed description of methods and results, we still would like to …

The generation of abnormal excitations in pathological regions of the heart is a main trigger for lethal cardiac arrhythmias. Such abnormal excitations, also called ectopic activity, often arise from areas with local tissue heterogeneity or damage accompanied by localized depolarization. Finding the conditions that lead to ectopy is important to understand the basic biophysical principles underlying arrhythmia initiation and might further refine clinical procedures. In this study, we are the first to address the question of how geometry of the abnormal region affects the onset of ectopy using a combination of experimental, in silico, and theoretical approaches. We paradoxically find that, for any studied geometry of the depolarized region in optogenetically modified monolayers of cardiac cells, primary ectopic excitation originates at areas of maximal curvature of the boundary, where the stimulating electrotonic currents are minimal. It contradicts the standard critical nucleation theory applied to nonlinear waves in reaction-diffusion systems, where a higher stimulus is expected to produce excitation more easily. Our in silico studies reveal that the nonconventional ectopic activity is caused by an oscillatory instability at the boundary of the damaged region, the occurrence of which depends on the curvature of that boundary. The onset of this instability is confirmed using the Schrödinger equation methodology proposed by Rinzel and Keener [SIAM J. Appl. Math. 43, 907 (1983)SMJMAP0036-139910.1137/0143058]. Overall, we show distinctively novel insight into how the geometry of a heterogeneous cardiac region determines ectopic activity, which can be used in the future to predict the conditions that can trigger cardiac arrhythmias.

Background: Recently, a new ablation strategy for atrial fibrillation has emerged, which involves the identification of rotors (ie, local drivers) followed by the localized targeting of their core region by ablation. However, this concept has been subject to debate because the mode of arrhythmia termination remains poorly understood, as dedicated models and research tools are lacking. We took a unique optogenetic approach to induce and locally target a rotor in atrial monolayers. Methods and Results: Neonatal rat atrial cardiomyocyte monolayers expressing a depolarizing light-gated ion channel (Ca 2+ -translocating channelrhodopsin) were subjected to patterned illumination to induce single, stable, and centralized rotors by optical S1-S2 cross-field stimulation. Next, the core region of these rotors was specifically and precisely targeted by light to induce local conduction blocks of circular or linear shapes. Conduction blocks crossing the core region, but not reaching any unexcitable boundary, did not lead to termination. Instead, electric waves started to propagate along the circumference of block, thereby maintaining reentrant activity, although of lower frequency. If, however, core-spanning lines of block reached at least 1 unexcitable boundary, reentrant activity was consistently terminated by wave collision. Lines of block away from the core region resulted merely in rotor destabilization (ie, drifting). Conclusions: Localized optogenetic targeting of rotors in atrial monolayers could lead to both stabilization and destabilization of reentrant activity. For termination, however, a line of block is required reaching from the core region to at least 1 unexcitable boundary. These findings may improve our understanding of the mechanisms involved in rotor-guided ablation.

Aims RHOA-ROCK signalling regulates cell migration, proliferation, differentiation, and transcription. RHOA is expressed in the developing cardiac conduction system in chicken and mice. In early development, the entire sinus venosus myocardium, including both the transient left-sided and the definitive sinoatrial node (SAN), has pacemaker potential. Later, pacemaker potential is restricted to the right-sided SAN. Disruption of RHOA expression in adult mice causes arrhythmias including bradycardia and atrial fibrillation, the mechanism of which is unknown but presumed to affect the SAN. The aim of this study is to assess the role of RHOA-ROCK signalling in SAN development in the chicken heart. Methods and results ROCK signalling was inhibited chemically in embryonic chicken hearts using Y-27632. This prolonged the immature state of the sinus venosus myocardium, evidenced by up-regulation of the transcription factor ISL1, wide distribution of pacemaker potential, and significantly reduced heart rate. Furthermore ROCK inhibition caused aberrant expression of typical SAN genes: ROCK1, ROCK2, SHOX2, TBX3, TBX5, ISL1, HCN4, CX40, CAV3.1, and NKX2.5 and left-right asymmetry genes: PITX2C and NODAL. Anatomical abnormalities in pulmonary vein development were also observed. Patch clamp electrophysiology confirmed the immature phenotype of the SAN cells and a residual left-sided sinus venosus myocardium pacemaker-like potential. Conclusions RHOA-ROCK signalling is involved in establishing the right-sided SAN as the definitive pacemaker of the heart and restricts typical pacemaker gene expression to the right side of the sinus venosus myocardium.

Aims Current treatments of ventricular arrhythmias rely on modulation of cardiac electrical function through drugs, ablation or electroshocks, which are all non-biological and rather unspecific, irreversible or traumatizing interventions. Optogenetics, however, is a novel, biological technique allowing electrical modulation in a specific, reversible and trauma-free manner using light-gated ion channels. The aim of our study was to investigate optogenetic termination of ventricular arrhythmias in the whole heart. Methods and results Systemic delivery of cardiotropic adeno-associated virus vectors, encoding the light-gated depolarizing ion channel red-activatable channelrhodopsin (ReaChR), resulted in global cardiomyocyte-restricted transgene expression in adult Wistar rat hearts allowing ReaChR-mediated depolarization and pacing. Next, ventricular tachyarrhythmias (VTs) were induced in the optogenetically modified hearts by burst pacing in a Langendorff setup, followed by programmed, local epicardial illumination. A single 470-nm light pulse (1000 ms, 2.97 mW/mm2) terminated 97% of monomorphic and 57% of polymorphic VTs vs. 0% without illumination, as assessed by electrocardiogram recordings. Optical mapping showed significant prolongation of voltage signals just before arrhythmia termination. Pharmacological action potential duration (APD) shortening almost fully inhibited light-induced arrhythmia termination indicating an important role for APD in this process. Conclusion Brief local epicardial illumination of the optogenetically modified adult rat heart allows contact- and shock-free termination of ventricular arrhythmias in an effective and repetitive manner after optogenetic modification. These findings could lay the basis for the development of fundamentally new and biological options for cardiac arrhythmia management.

Atrial fibrillation (AF) is the most frequent form of arrhythmia occurring in the industrialized world. Because of its complex nature, each identified form of AF requires specialized treatment. Thus, an in-depth understanding of the bases of these arrhythmias is essential for therapeutic development. A variety of experimental studies aimed at understanding the mechanisms of AF are performed using primary cultures of neonatal rat atrial cardiomyocytes (NRAMs). Previously, we have shown that the distinct advantage of NRAM cultures is that they allow standardized, systematic, robust re-entry induction in the presence of a constitutively-active acetylcholine-mediated K+ current (IKACh-c). Experimental studies dedicated to mechanistic explorations of AF, using these cultures, often use computer models for detailed electrophysiological investigations. However, currently, no mathematical model for NRAMs is available. Therefore, in the present study we propose the first model for the action potential (AP) of a NRAM with constitutively-active acetylcholine-mediated K+ current (IKACh-c). The descriptions of the ionic currents were based on patch-clamp data obtained from neonatal rats. Our monolayer model closely mimics the action potential duration (APD) restitution and conduction velocity (CV) restitution curves presented in our previous in vitro studies. In addition, the model reproduces the experimentally observed dynamics of spiral wave rotation, in the absence and in the presence of drug interventions, and in the presence of localized myofibroblast heterogeneities., Author Summary A fundamentally important element in cardiac in silico research is a model for the cardiac cell. It provides a link between measurable characteristics at the subcellular level and biological processes at the whole cell level, thereby allowing the researcher to study mechanisms of cardiac arrhythmias from a molecular cell biological perspective. Such studies are of vast importance for the advancement of understanding of living systems from cells to patient populations. This paper is a joint in silico-experimental study in which we propose the first model for the action potential of an NRAM. To develop this model, we fitted patch-clamp data from recent literature, while additionally performing specific measurements of IKACh-c in NRAMs. IKACh-c is an important factor in atrial arrhythmogenesis and a promising target for pharmacological AF-management. The model reproduces in vitro results such as standard characteristics of AP morphology, restitution, and spiral wave dynamics in monolayers, with effects of a subsequent drug-intervention and in the presence of localized myofibroblast heterogeneities. Thus it can be used as a tool to provide computational support to a variety of systematic experimental studies that investigate the mechanisms underlying atrial fibrillation (AF) in NRAM cultures.

Aims Anatomical re-entry is an important mechanism of ventricular tachycardia, characterized by circular electrical propagation in a fixed pathway. It's current investigative and therapeutic approaches are non-biological, rather unspecific (drugs), traumatizing (electrical shocks), or irreversible (ablation). Optogenetics is a new biological technique that allows reversible modulation of electrical function with unmatched spatiotemporal precision using light-gated ion channels. We therefore investigated optogenetic manipulation of anatomical re-entry in ventricular cardiac tissue. Methods and results Transverse, 150-μm-thick ventricular slices, obtained from neonatal rat hearts, were genetically modified with lentiviral vectors encoding Ca2+-translocating channelrhodopsin (CatCh), a light-gated depolarizing ion channel, or enhanced yellow fluorescent protein (eYFP) as control. Stable anatomical re-entry was induced in both experimental groups. Activation of CatCh was precisely controlled by 470-nm patterned illumination, while the effects on anatomical re-entry were studied by optical voltage mapping. Regional illumination in the pathway of anatomical re-entry resulted in termination of arrhythmic activity only in CatCh-expressing slices by establishing a local and reversible, depolarization-induced conduction block in the illuminated area. Systematic adjustment of the size of the light-exposed area in the re-entrant pathway revealed that re-entry could be terminated by either wave collision or extinction, depending on the depth (transmurality) of illumination. In silico studies implicated source-sink mismatches at the site of subtransmural conduction block as an important factor in re-entry termination. Conclusions Anatomical re-entry in ventricular tissue can be manipulated by optogenetic induction of a local and reversible conduction block in the re-entrant pathway, allowing effective re-entry termination. These results provide distinctively new mechanistic insight into re-entry termination and a novel perspective for cardiac arrhythmia management.

Background— Ventricular arrhythmias as a result of unintentional blockade of the K v 11.1 (hERG [human ether-à-go-go–related gene]) channel are a major safety concern in drug development. In past years, several highly prescribed drugs have been withdrawn for their ability to cause such proarrhythmia. Here, we investigated whether the proarrhythmic risk of existing drugs could be reduced by K v 11.1 allosteric modulators. Methods and Results— Using [ 3 H]dofetilide-binding assays with membranes of human K v 11.1-expressing human embryonic kidney 293 cells, 2 existing compounds (VU0405601 and ML-T531) and a newly synthesized compound (LUF7244) were found to be negative allosteric modulators of dofetilide binding to the K v 11.1 channel, with LUF7244 showing the strongest effect at 10 μmol/L. The K v 11.1 affinities of typical blockers (ie, dofetilide, astemizole, sertindole, and cisapride) were significantly decreased by LUF7244. Treatment of confluent neonatal rat ventricular myocyte (NRVM) monolayers with astemizole or sertindole caused heterogeneous prolongation of action potential duration and a high incidence of early afterdepolarizations on 1-Hz electric point stimulation, occasionally leading to unstable, self-terminating tachyarrhythmias. Pretreatment of NRVMs with LUF7244 prevented these proarrhythmic effects. NRVM monolayers treated with LUF7244 alone displayed electrophysiological properties indistinguishable from those of untreated NRVM cultures. Prolonged exposure of NRVMs to LUF7244 or LUF7244 plus astemizole did not affect their viability, excitability, and contractility as assessed by molecular, immunological, and electrophysiological assays. Conclusions— Allosteric modulation of the K v 11.1 channel efficiently suppresses drug-induced ventricular arrhythmias in vitro by preventing potentially arrhythmogenic changes in action potential characteristics, raising the possibility to resume the clinical use of unintended K v 11.1 blockers via pharmacological combination therapy.

Fibrosis and altered gap junctional coupling are key features of ventricular remodelling and are associated with abnormal electrical impulse generation and propagation. Such abnormalities predispose to reentrant electrical activity in the heart. In the absence of tissue heterogeneity, high-frequency impulse generation can also induce dynamic electrical instabilities leading to reentrant arrhythmias. However, because of the complexity and stochastic nature of such arrhythmias, the combined effects of tissue heterogeneity and dynamical instabilities in these arrhythmias have not been explored in detail. Here, arrhythmogenesis was studied using in vitro and in silico monolayer models of neonatal rat ventricular tissue with 30% randomly distributed cardiac myofibroblasts and systematically lowered intercellular coupling achieved in vitro through graded knockdown of connexin43 expression. Arrhythmia incidence and complexity increased with decreasing intercellular coupling efficiency. This coincided with the onset of a specialized type of spatially discordant action potential duration alternans characterized by island-like areas of opposite alternans phase, which positively correlated with the degree of connexinx43 knockdown and arrhythmia complexity. At higher myofibroblast densities, more of these islands were formed and reentrant arrhythmias were more easily induced. This is the first study exploring the combinatorial effects of myocardial fibrosis and dynamic electrical instabilities on reentrant arrhythmia initiation and complexity.

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

Cardiac development is characterized by a complex interplay of chemical, mechanical, and electrical forces, which together contribute to the proper formation of the heart muscle. In adult myocardium, cardiomyocytes are elongated, well-coupled by gap junctions, and organized in spatially well-defined muscle fibers. This specific tissue architecture affects electromechanical activation and global cardiac function. Since the adult heart has only limited capacity for repair after injury, a significant loss of myocardial tissue often leads to impaired cardiac function. Recent efforts to transplant autologous cells to counteract this cardiomyocyte loss have resulted in marginal functional improvement and no evidence of myocyte regeneration. In order to achieve durable therapeutic efficiency, the transplanted cells will need to not only be cardiomyogenic, but also functionally integrate with host myocardial tissue and thereby contribute to both structural and functional restoration.

Several studies have indicated an interaction between the renin-angiotensin (ANG II) system and endothelin (ET) in the regulation of vascular tone. Previously, we have shown that both ET and ANG II exert a vasoconstrictor influence on the coronary resistance vessels of awake normal swine. Here, we investigated whether the interaction between ANG II and ET exists in the control of coronary resistance vessel tone at rest and during exercise using single and combined blockade of angiotensin type 1 (AT1) and ETA/ETB receptors. Since both circulating ANG II and ET levels are increased after myocardial infarction (MI), we investigated if the interaction between these systems is altered after MI. In awake healthy swine, coronary vasodilation in response to ETA/ETB receptor blockade in the presence of AT1 blockade was similar to vasodilation produced by ETA/ETB blockade under control conditions. In awake swine with a 2- to 3-wk-old MI, coronary vasodilator responses to individual AT1 and ETA/ETB receptor blockade were virtually abolished, despite similar coronary arteriolar AT1 and ETA receptor expression compared with normal swine. Unexpectedly, in the presence of AT1 blockade (which had no effect on circulating ET levels), ETA/ETB receptor blockade elicited a coronary vasodilator response. These findings suggest that in normal healthy swine the two vasoconstrictor systems contribute to coronary resistance vessel control in a linear additive manner, i.e., with negligible cross-talk. In contrast, in the remodeled myocardium, cross-talk between ANG II and ET emerges, resulting in nonlinear redundant control of coronary resistance vessel tone.

Cardiovascular disease is the leading cause of death globally, and stem cell therapy remains one of the most promising strategies for regeneration or repair of the damaged heart. We report that human placenta-derived multipotent cells (hPDMCs) can modulate cardiac injury in small and large animal models of myocardial ischemia (MI) and elucidate the mechanisms involved. We found that hPDMCs can undergo in vitro cardiomyogenic differentiation when cocultured with mouse neonatal cardiomyocytes. Moreover, hPDMCs exert strong proangiogenic responses in vitro toward human endothelial cells mediated by secretion of hepatocyte growth factor, growth-regulated oncogene-α, and interleukin-8. To test the in vivo relevance of these results, small and large animal models of acute MI were induced in mice and minipigs, respectively, by permanent left anterior descending (LAD) artery ligation, followed by hPDMC or culture medium-only implantation with follow-up for up to 8 weeks. Transplantation of hPDMCs into mouse heart post-acute MI induction improved left ventricular function, with significantly enhanced vascularity in the cell-treated group. Furthermore, in minipigs post-acute MI induction, hPDMC transplantation significantly improved myocardial contractility compared to the control group ( p=0.016) at 8 weeks postinjury. In addition, tissue analysis confirmed that hPDMC transplantation induced increased vascularity, cardiomyogenic differentiation, and antiapoptotic effects. Our findings offer evidence that hPDMCs can modulate cardiac injury in both small and large animal models, possibly through proangiogenesis, cardiomyogenesis, and suppression of cardiomyocyte apoptosis. Our study offers mechanistic insights and preclinical evidence on using hPDMCs as a therapeutic strategy to treat severe cardiovascular diseases.

Electrical cardioversion (ECV), a mainstay in atrial fibrillation (AF) treatment, is unsuccessful in up to 10–20% of patients. An important aspect of the remodeling process caused by AF is the constitutive activition of the atrium-specific acetylcholine-dependent potassium current (IK,ACh → IK,ACh-c), which is associated with ECV failure. This study investigated the role of IK,ACh-c in ECV failure and setting the atrial defibrillation threshold (aDFT) in optically mapped neonatal rat cardiomyocyte monolayers. AF was induced by burst pacing followed by application of biphasic shocks of 25–100 V to determine aDFT. Blocking IK,ACh-c by tertiapin significantly decreased DFT, which correlated with a significant increase in wavelength during reentry. Genetic knockdown experiments, using lentiviral vectors encoding a Kcnj5-specific shRNA to modulate IK,ACh-c, yielded similar results. Mechanistically, failed ECV was attributed to incomplete phase singularity (PS) removal or reemergence of PSs (i.e. re-initiation) through unidirectional propagation of shock-induced action potentials. Re-initiation occurred at significantly higher voltages than incomplete PS-removal and was inhibited by IK,ACh-c blockade. Whole-heart mapping confirmed our findings showing a 60% increase in ECV success rate after IK,ACh-c blockade. This study provides new mechanistic insight into failing ECV of AF and identifies IK,ACh-c as possible atrium-specific target to increase ECV effectiveness, while decreasing its harmfulness.

Aims Fibrosis increases arrhythmogenicity in myocardial tissue by causing structural and functional disruptions in the cardiac syncytium. Forced fusion of fibroblastic cells with adjacent cardiomyocytes may theoretically resolve these disruptions. Therefore, the electrophysiological effects of such electrical and structural integration of fibroblastic cells into a cardiac syncytium were studied. Methods and results Human ventricular scar cells (hVSCs) were transduced with lentiviral vectors encoding enhanced green fluorescent protein alone (eGFP-hVSCs) or together with the fusogenic vesicular stomatitis virus G protein (VSV-G/eGFP- hVSCs) and subsequently co-cultured (1:4 ratio) with neonatal rat ventricular cardiomyocytes (NRVMs) in confluent monolayers yielding eGFP- and VSV-G/eGFP-co-cultures, respectively. Cellular fusion was induced by brief exposure to pH = 6.0 medium. Optical mapping experiments showed eGFP-co-cultures to be highly arrhythmogenic [43.3% early afterdepolarization (EAD) incidence vs. 7.7% in control NRVM cultures, P , 0.0001], with heterogeneous prolongation of action potential (AP) duration (APD). Fused VSV-G/eGFP-co-cultures displayed markedly lower EAD incidence (4.6%, P , 0.001) than unfused co-cultures, associated with decreases in APD, APD dispersion, and decay time of cytosolic Ca2+ waves. Heterokaryons strongly expressed connexin43 (Cx43). Also, maximum diastolic potential in co-cultures was more negative after fusion, while heterokaryons exhibited diverse mixed NRVM/hVSC whole-cell current profiles, but consistently showed increased outward Kv currents compared with NRVMs or hVSCs. Inhibition of Kv channels by tetraethylammonium chloride abrogated the anti-Arrhythmic effects of fusion in VSV-G/ eGFP-co-cultures raising EAD incidence from 7.9 to 34.2% (P , 0.001). Conclusion Forced fusion of cultured hVSCs with NRVMs yields electrically functional heterokaryons and reduces arrhythmogenicity by preventing EADs, which is, at least partly, attributable to increased repolarization force.

Aims Atrial fibrillation (AF) is the most common cardiac arrhythmia and often involves reentrant electrical activation (e.g. spiral waves). Drug therapy for AF can have serious side effects including proarrhythmia, while electrical shock therapy is associated with discomfort and tissue damage. Hypothetically, forced expression and subsequent activation of light-gated cation channels in cardiomyocytes might deliver a depolarizing force sufficient for defibrillation, thereby circumventing the aforementioned drawbacks. We therefore investigated the feasibility of light-induced spiral wave termination through cardiac optogenetics. Methods and results Neonatal rat atrial cardiomyocyte monolayers were transduced with lentiviral vectors encoding light-activated Ca2+-translocating channelrhodopsin (CatCh; LV.CatCh∼eYFP↑) or eYFP (LV.eYFP↑) as control, and burst-paced to induce spiral waves rotating around functional cores. Effects of CatCh activation on reentry were investigated by optical and multi-electrode array (MEA) mapping. Western blot analyses and immunocytology confirmed transgene expression. Brief blue light pulses (10 ms/470 nm) triggered action potentials only in LV.CatCh∼eYFP↑-transduced cultures, confirming functional CatCh-mediated current. Prolonged light pulses (500 ms) resulted in reentry termination in 100% of LV.CatCh∼eYFP↑-transduced cultures ( n = 31) vs. 0% of LV.eYFP↑-transduced cultures ( n = 11). Here, CatCh activation caused uniform depolarization, thereby decreasing overall excitability (MEA peak-to-peak amplitude decreased 251.3 ± 217.1 vs. 9.2 ± 9.5 μV in controls). Consequently, functional coresize increased and phase singularities (PSs) drifted, leading to reentry termination by PS–PS or PS–boundary collisions. Conclusion This study shows that spiral waves in atrial cardiomyocyte monolayers can be terminated effectively by a light-induced depolarizing current, produced by the arrhythmogenic substrate itself, upon optogenetic engineering. These results provide proof-of-concept for shockless defibrillation.

With great interest, we read the commentary of Anderson et al1 on the recent publication of Chong et al2 about the large-scale use of human embryonic stem cell–derived cardiomyocytes to regenerate infarcted, nonhuman primate myocardium. In their commentary, they focus on the development of ventricular tachyarrhythmias after injection of large numbers of human embryonic stem cell–derived cardiomyocytes, as reported by Chong et al.2 We like to extent the discussion about the origin of these arrhythmias. In our opinion, a crucial aspect of cell injection in infarcted myocardium remains unnoticed in both the article of Chong et al2 and the commentary of Anderson et al,1 being the anisotropic nature of myocardial tissue requiring proper spatial integration (ie, alignment) of the implanted cells.3, …

A thorough understanding of the developmental signals that direct pluripotent stem cells (PSCs) toward a cardiac fate is essential for translational applications in disease modeling and therapy. We screened a panel of 44 cytokines/signaling molecules for their ability to enhance Nkx2.5+ cardiac progenitor cell (CPC) formation during in vitro embryonic stem cell (ESC) differentiation. Treatment of murine ESCs with insulin or insulin-like growth factors (IGF1/2) during early differentiation increased mesodermal cell proliferation and, consequently, CPC formation. Furthermore, we show that downstream mediators of IGF signaling (e.g., phospho-Akt and mTOR) are required for this effect. These data support a novel role for IGF family ligands to expand the developing mesoderm and promote cardiac differentiation. Insulin or IGF treatment could provide an effective strategy to increase the PSC-based generation of CPCs and cardiomyocytes for applications in regenerative medicine. Stem Cells 2014;32:1493–1502

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— Atrial fibrillation is the most common cardiac arrhythmia. Ventricular proarrhythmia hinders pharmacological atrial fibrillation treatment. Modulation of atrium-specific Kir3.x channels, which generate a constitutively active current ( I K,ACh-c ) after atrial remodeling, might circumvent this problem. However, it is unknown whether and how I K,ACh-c contributes to atrial fibrillation induction, dynamics, and termination. Therefore, we investigated the effects of I K,ACh-c blockade and Kir3.x downregulation on atrial fibrillation. Methods and Results— Neonatal rat atrial cardiomyocyte cultures and intact atria were burst paced to induce reentry. To study the effects of Kir3.x on action potential characteristics and propagation patterns, cultures were treated with tertiapin or transduced with lentiviral vectors encoding Kcnj3 - or Kcnj5 -specific shRNAs. Kir3.1 and Kir3.4 were expressed in atrial but not in ventricular cardiomyocyte cultures. Tertiapin prolonged action potential duration (APD; 54.7±24.0 to 128.8±16.9 milliseconds; P I K,ACh-c . Furthermore, tertiapin decreased rotor frequency (14.4±7.4 to 6.6±2.0 Hz; P P Kcnj3 or Kcnj5 gave similar results. Blockade of I K,ACh-c prevented/terminated reentry by prolonging APD and changing APD and conduction velocity restitution slopes, thereby altering the probability of APD alternans and rotor destabilization. Whole-heart mapping experiments confirmed key findings (eg, >50% reduction in atrial fibrillation inducibility after I K,ACh-c blockade). Conclusions— Atrium-specific Kir3.x controls the induction, dynamics, and termination of fibrillation by modulating APD and APD/conduction velocity restitution slopes in atrial tissue with I K,ACh-c . This study provides new molecular and mechanistic insights into atrial tachyarrhythmias and identifies Kir3.x as a promising atrium-specific target for antiarrhythmic strategies.

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.

AIMS Sustained ventricular fibrillation (VF) is maintained by multiple stable rotors. Destabilization of sustained VF could be beneficial by affecting VF complexity (defined by the number of rotors). However, underlying mechanisms affecting VF stability are poorly understood. Therefore, the aim of this study was to correlate changes in arrhythmia complexity with changes in specific electrophysiological parameters, allowing a search for novel factors and underlying mechanisms affecting stability of sustained VF. METHODS AND RESULTS Neonatal rat ventricular cardiomyocyte monolayers and Langendorff-perfused adult rat hearts were exposed to increasing dosages of the gap junctional uncoupler 2-aminoethoxydiphenyl borate (2-APB) to induce arrhythmias. Ion channel blockers/openers were added to study effects on VF stability. Electrophysiological parameters were assessed by optical mapping and patch-clamp techniques. Arrhythmia complexity in cardiomyocyte cultures increased with increasing dosages of 2-APB (n > 38), leading to sustained VF: 0.0 ± 0.1 phase singularities/cm(2) in controls vs. 0.0 ± 0.1, 1.0 ± 0.9, 3.3 ± 3.2, 11.0 ± 10.1, and 54.3 ± 21.7 in 5, 10, 15, 20, and 25 µmol/L 2-APB, respectively. Arrhythmia complexity inversely correlated with wavelength. Lengthening of wavelength during fibrillation could only be induced by agents (BaCl(2)/BayK8644) increasing the action potential duration (APD) at maximal activation frequencies (minimal APD); 123 ± 32%/117 ± 24% of control. Minimal APD prolongation led to transient VF destabilization, shown by critical wavefront collision leading to rotor termination, followed by significant decreases in VF complexity and activation frequency (52%/37%). These key findings were reproduced ex vivo in rat hearts (n = 6 per group). CONCLUSION These results show that stability of sustained fibrillation is regulated by minimal APD. Minimal APD prolongation leads to transient destabilization of fibrillation, ultimately decreasing VF complexity, thereby providing novel insights into anti-fibrillatory mechanisms.

Aims Cardiac hypertrophy and fibrosis are associated with potentially lethal arrhythmias. As these substrates often occur simultaneously in one patient, distinguishing between pro-arrhythmic mechanisms is difficult. This hampers understanding of underlying pro-arrhythmic mechanisms and optimal treatment. This study investigates and compares arrhythmogeneity and underlying pro-arrhythmic mechanisms of either cardiac hypertrophy or fibrosis in in vitro models. Methods and results Fibrosis was mimicked by free myofibroblast (MFB) proliferation in neonatal rat ventricular monolayers. Cultures with inhibited MFB proliferation were used as control or exposed to phenylephrine to induce hypertrophy. At Day 9, cultures were studied with patch-clamp and optical-mapping techniques and assessed for protein expression. In hypertrophic ( n = 111) and fibrotic cultures ( n = 107), conduction and repolarization were slowed. Triggered activity was commonly found in these substrates and led to high incidences of spontaneous re-entrant arrhythmias [67.5% hypertrophic, 78.5% fibrotic vs. 2.9% in controls ( n = 102)] or focal arrhythmias (39.1, 51.7 vs. 8.8%, respectively). Kv4.3 and Cx43 protein expression levels were decreased in hypertrophy but unaffected in fibrosis. Depolarization of cardiomyocytes (CMCs) was only found in fibrotic cultures (−48 ± 7 vs. −66 ± 7 mV in control, P < 0.001). L-type calcium-channel blockade prevented arrhythmias in hypertrophy, but caused conduction block in fibrosis. Targeting heterocellular coupling by low doses of gap-junction uncouplers prevented arrhythmias by accelerating repolarization only in fibrotic cultures. Conclusion Cultured hypertrophic or fibrotic myocardial tissues generated similar focal and re-entrant arrhythmias. These models revealed electrical remodelling of CMCs as a pro-arrhythmic mechanism of hypertrophy and MFB-induced depolarization of CMCs as a pro-arrhythmic mechanism of fibrosis. These findings provide novel mechanistic insight into substrate-specific arrhythmicity.