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The recent advancements in stem cell biology, molecular and cell biology, and tissue engineering have paved the way to the development of a new biomedical discipline: regenerative medicine. The heart represents an attractive candidate for this emerging discipline since these emerging technologies could be used to potentially treat a variety of myocardial disorders. Here we describe our efforts in using stem cell and cell therapy strategies to restore the myocardial electromechanical properties. Specifically, our research has focused on the potential role of human embryonic stem cells (hESC) for myocardial regeneration (for the treatment of heart failure) and on using genetically engineered cell grafts to modify the myocardial electrophysiological properties (for the treatment of cardiac arrhythmias). The recently described hESC lines are unique pluripotent cell lines that can be propagated in the undifferentiated state in culture and coaxed to differentiate into cell derivatives of all three germ layers, including cardiomyocytes. The current article describes this unique cardiomyocyte differentiating system and details the molecular, ultrastructural, and functional properties of the generated hESC-derived cardiomyocytes (hESC-CMs). The ability of the hESC-CMs to integrate structurally and functionally with host cardiomyocytes in both in vitro and in vivo studies will be described as well as their ability to restore the myocardial electromechanical function in animal models of diseased hearts. We will next present detailed in vitro, in vivo, and computer simulation studies performed in our laboratory testing the hypothesis that cell grafts, engineered to express specific ion channels, can be used to modify the myocardial electrophysiological properties of cardiac tissue. The potential and drawbacks of this novel approach for the treatment of both tachyarrhythmias (using cell grafts expressing potassium channels) and bradyarrhythmias (using hESC coaxed to differentiate into pacemaking cells or conducting tissue) will be described.

Excitation-contraction (EC) coupling is fundamental to the function of cardiac myocytes (CMs). In mature myocytes plasma membrane (PM) L-type Ca 2+ channels function in close juxtaposition to ryanodine receptors (RyR) on the sarcoplasmic reticulum (SR) membrane. Action potentials (APs) cause the opening of PM L-type Ca 2+ channels, which in turn provide trigger Ca 2+ for a larger RyR-mediated SR Ca 2+ release. In contrast, developing myocytes have a less well-developed SR. This incomplete development is observed in early stage and mid-maturation stages of murine embryonic stem cell-derived cardiac myocytes (ESC-CMs). Despite the absence of a well-developed t-tubule system, murine ESC-CMs use internal Ca 2+ stores for EC coupling. Direct measures of Ca 2+ handling, including pharmacological studies and investigation of genetically modified mouse ESC-CMs, established an important contribution of RyR-mediated internal Ca 2+ store to cell function. Similarly, early-stage human ESC-CMs use internal Ca 2+ store and partially share Ca 2+ handling characteristics with murine ESC-CMs. For example, elementary Ca 2+ release events are present in both murine and human ESC-CMs, and it is likely that Ca 2+ handling contributes to automatic rhythm generation in these cells. However, in human ESC-CMs, a unique voltage-gated Na + channel window current is critical for spontaneous, rhythmic depolarization. The advent of the murine and human ES cardiomyocyte differentiating systems has provided initial insights into the early steps of development of excitability and electromechanical coupling in the mammalian heart, including patterns of gene expression, myofibrillogenesis, ion channel development and function, and Ca 2+ handling. Here we discuss the information gained from these models to describe the nexus of voltage-gated channel currents and Ca 2+ handling on rhythmic activity.

Gene therapy, cell therapy, and tissue engineering are emerging as novel experimental therapeutic paradigms for a variety of cardiovascular disorders. In the current report we will review the possible implications of these emerging technologies in the field of cardiac electrophysiology. Initially, the possible role of myocardial gene and cell therapies in creating a biological alternative to electronic pacemakers for the treatment of bradyarrhythmias will be discussed. This will be followed by a description of the possible applications of using similar strategies for the treatment of common tachyarrhythmias. Finally, the electrophysiological implications of cardiac stem cell therapy for heart failure, as well as the possible in vitro applications of stem cell technology for electrophysiological studies and drug screening, will be discussed. While these emerging strategies provide a paradigm shift from conventional treatment modalities, this field is still at its infancy and several obstacles, discussed in this review, should be overcome before any clinical breakthroughs can be expected.

Regenerative medicine is a new biomedicine discipline that takes advantage of the recent advancements in the fields of stem cell biology, molecular biology, and tissue engineering to derive tissue substitutes, in an attempt to replace or modify the function of diseased organs. The heart represents an attractive candidate for these emerging technologies since adult cardiac tissue has limited regenerative capacity. Consequentially, myocardial cell replacement therapy has emerged as a novel therapeutic paradigm for restoration of the myocardial electromechanical function. This innovative strategy has been significantly hampered, however, by the paucity of cell sources for human cardiomyocytes. The recent establishment of the human embryonic stem cell (hESC) lines may provide a possible solution for this cell-sourcing problem. These unique pluripotent cell lines can be propagated in the undifferentiated state in culture and coaxed to differentiate into cell derivatives of all three germ layers, including cardiomyocytes. This review will describe the hESC system, their differentiation into cardiomyocytes, and the structural and functional characterization of these cardiac lineage derivatives. The potential applications of this unique differentiating system in several research areas will be discussed with special emphasis on the steps required to fully harness their unique potential in the emerging field of cardiovascular regenerative medicine.

The adult human heart has limited regenerative capacity and, therefore, functional restoration of the damaged heart presents a great challenge. Despite the progress achieved in the pharmacological and surgical treatment of degenerative myocardial diseases, they are still considered a major cause of morbidity and mortality in the western world. Repopulation of the damaged heart with cardiomyocytes represents a novel conceptual therapeutic paradigm but is hampered by the lack of sources for human cardiomyocytes. The recent derivation of pluripotent human embryonic stem cell lines may provide a solution for this cell sourcing problem. This review will focus on the derivation of the hESC lines, their mechanism of self-renewal, and their differentiation to cardiomyocytes. The possible signals and cues involved in the commitment and early differentiation of cardiomyocytes in this model will be discussed as well as the molecular, structural and electrophysiologic characteristics of the generated hESC-derived cardiomyocytes. Finally, the hurdles and challenges toward fully harnessing the potential clinical applications of these unique cells will be described.

Cell therapy is emerging as a promising strategy for myocardial repair. This approach is hampered, however, by the lack of sources for human cardiac tissue and by the absence of direct evidence for functional integration of donor cells into host tissues. Here we investigate whether cells derived from human embryonic stem (hES) cells can restore myocardial electromechanical properties. Cardiomyocyte cell grafts were generated from hES cells in vitro using the embryoid body differentiating system. This tissue formed structural and electromechanical connections with cultured rat cardiomyocytes. In vivo integration was shown in a large-animal model of slow heart rate. The transplanted hES cell-derived cardiomyocytes paced the hearts of swine with complete atrioventricular block, as assessed by detailed three-dimensional electrophysiological mapping and histopathological examination. These results demonstrate the potential of hES-cell cardiomyocytes to act as a rate-responsive biological pacemaker and for future myocardial regeneration strategies.

cardiac function depends on the appropriate timing and synchronization of the mechanical contraction in various regions of the heart as well as on achieving the appropriate heart rate. These properties are ensured through the hierarchical organization and electrical specialization of the cardiac

Human embryonic stem cells are capable of unlimited proliferation in culture in the undifferentiated state and under the proper conditions can differentiate into different cell types including spontaneously beating cardiac myocytes (Kehat et al. 2001, 2002). Spontaneous beating or automaticity is not normally exhibited by mature atrial or ventricular myocytes. In contrast, embryonic heart cells display spontaneous activity (DeHaan & Gottlieb, 1968). The main requirement for automaticity is the presence of inward current at diastolic potentials. Although two recent reports of human embryonic stem cell-derived cardiac myocytes (hES-CMs) document action potentials (He et al. 2003; Mummery et al. 2003), there are no studies of ionic currents in hES-CMs. Moreover, the more thoroughly studied mouse ES-derived CMs exhibit AP morphologies that broadly reflect either atrial-like, ventricular-like, or nodal-like parameters (Hescheler et al. 1999; Sachinidis et al. 2003) that differ markedly from their human counterparts. The cardiac Na+ channel (termed NaV1.5) is expressed in relatively high density on surface membrane of mature heart cells in atria and ventricle. Despite the high NaV1.5 expression these cell types are not normally automatic because a high density of inward rectifier K+ channels (Kir) clamps the membrane potential to a value near the K+ reversal potential. At such hyperpolarized potentials the NaV1.5 channel's open probability approaches 0. In quiescent, mature heart cells, the initiating depolarization originates from neighbouring cells via gap junctions. Depolarization of membrane potential (Vm) activates NaV1.5 rapidly, driving the rapid AP upstroke, and within a few milliseconds of sustained depolarization NaV1.5 inactivates. Thus, NaV1.5 serves the role of generating a pathway for a rapid influx of depolarizing current. The maximum diastolic potential (MDP) is a key control point for NaV1.5, if MDP is relatively depolarized then NaV1.5 will be largely inactivated and unable to contribute to the AP upstroke. For this reason, there is a correlation between Na+ channel current density (not simply channel density), and the maximum upstroke velocity of the cellular AP (dV/dtmax). Moreover, in the developing heart there is a an increase in dV/dtmax from < 20 to 100–150 V s−1 that is concomitant with the onset of TTX sensitivity (McDonald et al. 1973), an increase in Na+ current density (Fujii et al. 1988), and a negative shift in the MDP (McDonald et al. 1973; DeHaan, 1980; Sperelakis, 1984). Given the exciting potential use of hES-CMs as replacement tissue in diseased heart (Gepstein, 2002; Kehat & Gepstein, 2003), and as an in vitro model for the study of early human cardiac development it is important to characterize their functional properties. We assessed the electrical properties of these cells in spontaneously beating embryoid bodies (EBs) using a multielectrode array mapping technique and detailed patch-clamp recordings and pharmacologically dissected the critical pathways in these structures. Our results provide the first description of the ionic currents in hES-CMs and show that the basis for spontaneous electrical activity in these cells is the absence of Kir conductance, a phenomenon that provides the substrate for a relatively large voltage-gated Na+ current to drive activity.

Terminally differentiated adult cardiomyocytes have limited regenerative capacity and therefore any significant cell loss may result in the development of progressive heart failure. Cell replacement therapy is a promising new approach for myocardial repair but has been limited by the paucity of cell sources for functional human cardiomyocytes. The recent establishment of the human pluripotent embryonic stem (ES) cell lines may present a novel solution for this cell-sourcing problem. The ES lines were derived from human blastocysts and were shown to be capable of continuous undifferentiated proliferation, in vitro, while retaining the capability to form derivatives of all three germ layers. More recently, a reproducible cardiomyocyte differentiation system was established using these unique cells. The current review describes the derivation and properties of human ES cells and the characteristics of the cardiomyocytes derived using this unique differentiating system. The possible applications in several research and clinical areas are discussed as well as the steps required to fully harness the potential of this new technology in the fields of myocardial cell replacement and tissue engineering.

Publisher Summary Cell transplantation is emerging as a novel strategy for myocardial regeneration, but it has been hampered by the lack of a source for human cardiomyocytes. Similarly, the lack of a human cardiomyocyte cell line has significantly limited a variety of experimental procedures. The recent advances in human embryonic stem cells (ES) research suggests a possible solution for this cell sourcing problem because these unique cells can be propagated in mass in vitro and coaxed to differentiate into the desired lineage. One of the most fascinating and important aspects of human ES cell lines is their ability to differentiate in vitro to advanced derivatives of all three germ layers. A prerequisite for this differentiation is the cultivation of the ES cells into three-dimensional cell aggregates. These aggregates, termed embryoid bodies (EBs) contain differentiating tissue from all three germ layers. Using the EB differentiation system cardiomyocytes can be reproducibly generated from the human ES cells. This chapter describes the methodologies associated with human ES cell cultivation and differentiation to cardiomyocytes, as well as the techniques used for studying the structural and functional properties of these unique cells.

The goal of the present report was to establish a new in vitro model for the study of impulse propagation in human cardiac tissue. By using the human embryonic stem cell differentiating system, spontaneously contracting areas were generated in three-dimensional differentiating cell aggregates (embryoid bodies). Morphological analysis revealed an isotropic tissue of early-stage cardiac phenotype. Gap junctions, assessed by immunostaining of connexin43 and connexin45, were distributed along the cell borders. High-resolution activation maps demonstrated the presence of a functional syncytium with stable focal activation and conduction properties. Conduction was significantly slower in narrow bands of contracting tissue compared with broad cardiomyocyte regions. Establishment of this unique in vitro human model may be used for the assessment of long-term structure-function relationships, for pharmacological studies, for tissue engineering, and may permit the study of genetically modified cardiomyocytes.

Background — Traditional pharmacological therapies aiming to modify the abnormal electrophysiological substrate underlying cardiac arrhythmias may be limited by their relatively low efficacy, global cardiac activity, and significant proarrhythmic effects. We suggest a new approach, in which transfected cellular grafts expressing various ionic channels may be used to manipulate the local electrophysiological properties of cardiac tissue. To examine the feasibility of this concept, we tested the hypothesis that transfected fibroblasts expressing the voltage-sensitive potassium channel Kv1.3 can modify the electrophysiological properties of cardiomyocytic cultures. Methods and Results — A high-resolution multielectrode mapping technique was used to assess the electrophysiological and structural properties of primary cultures of neonatal rat ventricular myocytes. The transfected fibroblasts, added to the cardiomyocytic cultures, caused a significant effect on the conduction properties of the hybrid cultures. These changes were manifested by significant reduction in extracellular signal amplitude and by the appearance of multiple local conduction blocks. The location of all conduction blocks correlated with the spatial distribution of the transfected fibroblasts assessed by vital staining. All electrophysiological changes were reversed after the application of Charybdotoxin, a specific Kv1.3 blocker. In contrast, conduction remained uniform in the control hybrid cultures when nontransfected fibroblasts were used. Conclusions — Transfected fibroblasts are able to electrically couple with cardiac myocytes, causing a significant local and reversible modification of the tissue’s electrophysiological properties. More broadly, this study suggests that transfected cellular grafts expressing various ionic channels may be used to modify cardiac excitability, providing a possible future novel cell therapy strategy.

ObjectivesWe tested the hypothesis that spatial association of low-amplitude intracardiac electrograms can identify the presence, location and extent of dysplastic regions in arrhythmogenic right ventricular dysplasia (ARVD).BackgroundArrhythmogenic right ventricular dysplasia is a right ventricular (RV) cardiomyopathy characterized pathologically by fibrofatty infiltration and clinically by a spectrum of arrhythmias, sudden cardiac death and RV failure. Diagnosis of ARVD still remains a clinical challenge.MethodsA three-dimensional electroanatomic mapping technique was used to map the RV of two groups of patients: 1) those with ARVD presenting with typical clinical, electrocardiographic and echocardiographic or magnetic resonance imaging (MRI) findings; and 2) those with structurally normal ventricles.ResultsThe dysfunctional RV area could be identified only in the first group and was characterized by the presence of discrete areas of abnormally low-amplitude electrograms. Hence, the normal voltage values observed in the control group (unipolar: 11.9 ± 0.3 mV; bipolar: 4.6 ± 0.2 mV [mean ± SEM]) and in the nonaffected zones in the ARVD group (unipolar: 10.4 ± 0.2 mV; bipolar: 4.6 ± 0.2 mV) were reduced significantly (p < 0.05) in the dysplastic areas (unipolar: 3.3 ± 0.1 mV; bipolar: 0.5 ± 0.1 mV). The pathologic process mainly involved the RV anterolateral free wall, apex and inflow and outflow tracts and ranged from patchy areas to uniform and extensive involvement. Concordance between electroanatomic findings and MRI or echocardiographic findings was noted in all patients.ConclusionsThe pathologic substrate in ARVD can be identified by spatial association of low-amplitude endocardial electrograms, reflecting replaced myocardial tissue. The ability to accurately identify the presence, location and extent of the pathologic substrate may have important diagnostic, prognostic and therapeutic implications.

The study of human cardiac tissue development is hampered by the lack of a suitable in vitro model. We describe the phenotypic properties of cardiomyocytes derived from human embryonic stem (ES) cells. Human ES cells were cultivated in suspension and plated to form aggregates termed embryoid bodies (EBs). Spontaneously contracting areas appeared in 8.1% of the EBs. Cells from the spontaneously contracting areas within EBs were stained positively with anti-cardiac myosin heavy chain, anti--alpha-actinin, anti-desmin, anti--cardiac troponin I (anti-cTnI), and anti-ANP antibodies. Electron microscopy revealed varying degrees of myofibrillar organization, consistent with early-stage cardiomyocytes. RT-PCR studies demonstrated the expression of several cardiac-specific genes and transcription factors. Extracellular electrograms were characterized by a sharp component lasting 30 +/- 25 milliseconds, followed by a slow component of 347 +/- 120 milliseconds. Intracellular Ca(2+) transients displayed a sharp rise lasting 130 +/- 27 milliseconds and a relaxation component lasting 200--300 milliseconds. Positive and negative chronotropic effects were induced by application of isoproterenol and carbamylcholine, respectively. In conclusion, the human ES cell--derived cardiomyocytes displayed structural and functional properties of early-stage cardiomyocytes. Establishment of this unique differentiation system may have significant impact on the study of early human cardiac differentiation, functional genomics, pharmacological testing, cell therapy, and tissue engineering.

Background and Objective The aim of the present study was to investigate the possibility that low-energy laser irradiation attenuates infarct size formation after induction of chronic myocardial infarction (MI) in small and large experimental animals. Study Design/Materials and Methods Laser irradiation was applied to the infarcted area of rats and dogs at various power densities (2.5 to 20 mW/cm2) after occlusion of the coronary artery. Results In infarcted laser-irradiated rats that received laser irradiation immediately and 3 days after MI at energy densities of 2.5, 6, and 20 mW/cm2, there was a 14%, 62% (significant; P

Background —Generation of long and continuous linear ablations is required in a growing number of atrial arrhythmias. However, deployment and assessment of these lesions may be difficult, and there are few data regarding their short- and long-term effects on atrial electrophysiology and pathology. Methods and Results —A nonfluoroscopic mapping and navigation technique was used to generate 3-dimensional (3D) electroanatomic maps of the right atrium in 8 pigs. The catheter was then used to deliver sequential radiofrequency (RF) applications (power output gradually increased until 80% reduction in the amplitude of the unipolar electrogram) to generate a continuous lesion between the superior and inferior venae cavae. The animals were remapped 4 weeks after ablation during septal pacing. Lesion continuity was confirmed in all cases by the following criteria: (1) activation maps indicating conduction block [significant disparities in activation times (52.0±16.0 ms) and opposite orientation of the activation wave front on opposing sides of the lesion], (2) evidence of double potentials (interspike time difference of 52.3±17.1 ms), and (3) low peak-to-peak amplitude of the bipolar electrograms (0.7±0.6 mV) along the lesion. At autopsy, all lesions were continuous and transmural, averaged 50.5±6.7 mm, and were characterized histologically by transmural fibrosis throughout the length of the lesion. Conclusions —Long linear atrial ablation, created by sequential RF applications (using unipolar amplitude attenuation as the end point for energy delivery), results in long-term continuous and transmural lesions. Lesion continuity is associated with evidence of conduction block in the 3D activation maps and the presence of double potentials and low electrogram amplitude along the lesion.

Background —A catheter-based left ventricular (LV) endocardial mapping procedure using electromagnetic field energy for positioning of the catheter tip was designed to acquire simultaneous measurements of endocardial voltage potentials and myocardial contractility. We investigated such a mapping system to distinguish between infarcted and normal myocardium in an animal infarction model and in patients with coronary artery disease. Methods and Results —Measurements of LV endocardial unipolar (UP) and bipolar (BP) voltages and local endocardial shortening were derived from dogs at baseline (n=12), at 24 hours (n=6), and at 3 weeks (n=6) after occlusion of the left anterior descending coronary artery. Also, 12 patients with prior myocardial infarction (MI) and 12 control patients underwent the LV endocardial mapping study for assessment of electromechanical function in infarcted versus healthy myocardial regions. In the canine model, a significant decrease in voltage potentials was noted in the MI zone at 24 hours (UP, 42.8±9.6 to 29.1±12.2 mV, P =0.007; BP, 11.6±2.3 to 4.9±1.2 mV, P P P P Conclusions —These preliminary data suggest that infarcted myocardium could be accurately diagnosed and distinguished from healthy myocardium by a reduction in both electrical voltage and mechanical activity. Such a diagnostic electromechanical mapping study might be clinically useful for accurate assessment of myocardial function and viability.

Background While abnormalities of activation and repolarization play an important role in arrhythmogenesis, little information is available on the interaction between their spatial dispersions in the heart. This study examined the effects of activation spread on the spatial distribution of the repolarization properties during different depolarization patterns. Methods and Results Left ventricular (LV) endocardial activation and repolarization patterns were mapped in 13 healthy pigs. LV local activation, repolarization, and activation-recovery interval (ARI) times were determined from the intracardiac unipolar electrograms, color-coded, and superimposed on a three-dimensional anatomic map of the ventricle generated with a nonfluoroscopic mapping system. ARI values correlated with the duration of monophasic activation potential recorded from onset of activation to time of 90% repolarization ( r =.97, P r 2 =.76±.03) and paced ( r 2 =.77±.02) rhythms. The longest ARIs were located at the sites of earliest activation and shortest at the latest activation areas, with gradual shortening between them. Conclusions The spatial distribution of repolarization is dependent on the activation pattern. Repolarization dispersion in the healthy swine heart is relatively small as the result of tight coupling of the action potential duration to the activation process, assigning longer ARIs to sites activated earlier. This coupling reduces global and regional dispersion of repolarization and may serve as an important antiarrhythmic mechanism present in normal myocardium.

Background Clinical cardiac volumetric measurement techniques are essential for assessing cardiac performance but produce significant inaccuracies in extrapolation of the volume of a three-dimensional (3D) object from two-dimensional images and lack the ability to associate cardiac electrical and mechanical activities. In this study, we tested the accuracy of cardiac volumetric measurements using a new catheter-based system. Methods and Results The system uses magnetic technology to accurately locate a special catheter at a frequency of 125 Hz and is currently used in the field of electrophysiology, in which activation maps are superimposed on the 3D geometry of the cardiac chamber. The mapping procedure is based on sequentially acquiring the location of the tip and local electrogram while in contact with the endocardium. The 3D geometry of the chamber is reconstructed in real time, and its volume could be calculated at every time step (8 ms). The volumetric measurements of the system were found to be highly accurate for simple phantoms (mean±SEM deviation, 2.3±1.1%), left ventricular casts (9.6±1.3%), and a dynamic test jig. In addition, left ventricular volumes of 12 swine were measured. Intraobserver and interobserver variabilities were found to be minimal (ejection fraction, 6.5±1.9% and 7.1±2.0%; stroke volume, 4.5±1.0% and 11.3±2.4%). Comparison with the thermodilution method for measuring stroke volume showed an average deviation of 8.1±2.2%. Typical pressure-volume loops were also obtained. Conclusions The new mapping image provides, for the first time, simultaneous information regarding cardiac mechanics, hemodynamics, and electrical properties. Furthermore, all this information is achieved without the use of fluoroscopy, contrast medium, or complicated image processing.

Background Ablation therapy for certain arrhythmias requires the formation of complex lesions based on electrical and anatomic mapping. We tested the accuracy and reproducibility of a nonfluoroscopic mapping and navigation (NFM) system to guide delivery of radiofrequency (RF) energy in the right atrium (RA) of swine. Methods and Results The NFM system uses an ultralow magnetic field to measure the real-time three-dimensional (3D) location of the tip of the locatable catheter. While in stable contact with the endocardium, between 30 and 40 consecutive tip locations were sampled and used for the 3D reconstruction of the RA geometry. The location of the catheter tip was presented in real time, superimposed over the RA geometry. We selected a point on the 3D reconstruction and delivered RF energy to that site via the tip of the locatable catheter. The catheter was then completely withdrawn and renavigated twice to the same point, at which RF energy was delivered again. At autopsy, the distance between the centers of the three ablation points (mean±SEM) was 2.3±0.5 mm (n=27). Similarly, we used the NFM system to guide the generation of linear lesions. The measured length of the linear lesions on the NFM 3D view was close to the actual lesion length measured at autopsy (correlation coefficient, .96; P =.002; n=6). Furthermore, the location, shape, and continuity of the linear lesions corresponded to the autopsy findings. Conclusions We conclude that the NFM system can guide the application of RF energy without the use of fluoroscopy in a highly accurate and reproducible manner.

A novel method for three dimensional (3D) electromechanical mapping of the heart is presented. The new method is based on utilizing special magnetically locatable catheters connected to a mapping and navigation system. The 3D electromechanical map of the chamber is reconstructed by sampling the location of the catheter tip throughout the cardiac cycle at a plurality of endocardial sites together with their local electrograms. The ability to spatially combine electrical and mechanical information may provide a useful tool for both research and clinical cardiology.

Prolongation of the refractory period is one of the therapeutical strategies for the treatment of cardiac arrhythmias. The Kv1.3 channel have a long tail current causing it to be attractive as a potential modulator of cardiac effective refractory period (ERP) when overexpressed by cardiomyocytes or other cells electrically coupled with cardiomyocytes. It was previously demonstrated that Kv1.3 channel in which the histidine at position 401 was replaced by tryptophan (Kv1.3 H401W) introduced a rapid inactivation property to the channel. Preliminary analysis suggested that the effect of the mutation is caused by slowing down of the recovery rate from inactivation. In this channel recovery from inactivation involved passing through the open state. It was therefore expected that the mutant channel will have extremely long tail current. In this work the possibility of prolonging the refractory period of pig hearts at specific loci by transplantation of fibroblasts overexpressing GFP, the wild type (WT) Kv1.3 channel, and the Kv1.3 H401W was evaluated by computer simulation and in vivo experiments.

Background Optogenetics has proved to be a reliable method for precise control of excitable tissue both in vitro and in vivo. Much of the research on optogenetics has been devoted to neural cell excitation. Therefore, our aim was to investigate whether optogenetics could be used to control cardiac depolarization and hyperpolarization by using combined gene and cell therapy. Methods NIH3T3 fibroblasts and HEK293 cells were transfected with either the light-sensitive depolarizing Channelrhodopsin-2 (NIH-ChR2) ion channel or the hyperpolarizing light-sensitive Archaerhodopsin-3 (HEK-Arch) proton pump, respectively. A whole-cell patch-clamp study of the transfected cells was conducted, and the mean light-induced ion currents were calculated. Transfected cells were cocultured with either neonatal rat cardiomyocytes (NRCM) or human embryonic stem cell-derived cardiomyocytes (hESC-CMs), and plated on a 252-electrode multielectrode array (MEA). Conduction maps, tissue activation times, and beating frequencies were calculated before and after illumination (470 or 590 nm). Results A whole-cell patch-clamp study of NIH-ChR2 cells demonstrated inward cation currents dependent on the presence and intensity of a 470-nm monochromic light. NIH-ChR2 cells were able to induce precisely controlled rapid cardiomyocyte depolarization and cause cardiac cell resynchronization by shortening tissue activation time. HEK-Arch cells, in contrast, were able to completely silence electrical and mechanical activity of vigorous cardiac cells and to cause conduction blocks in a beating cardiomyocytes monolayer. Hyperpolarization was reversed following termination of light exposure. Conclusions Combined gene and cell therapy, using light-sensitive proteins, could be applied efficiently for controlling cardiomyocyte activation and inactivation. Effects were similarly demonstrated in both NRCM and hESC-CMs. Therefore, it is suggested that this novel method could be used for treatment of tachyarrhythmias and bradyarrhythmias.

We report a case of a 55-year-old woman with idiopathic ventricular fibrillation (VF) who suffered from recurrent implantable cardioverter-defibrillator shocks triggered by short coupled ventricular premature beat (VPB). This VPB was mapped and ablated from the myocardium of right ventricle close to the lateral tricuspid annulus. Learning objective: Triggering ventricular premature beat (VPB) in idiopathic ventricular fibrillation was reported to originate from the myocardium of right ventricular outflow tract or from Purkinje system. In this case, the origin of triggering VPB is the myocardium of right ventricle close to the lateral tricuspid annulus.>

Background The short QT syndrome (SQTS) is an inherited arrhythmogenic syndrome characterized by abnormal ion channel function, life-threatening arrhythmias, and sudden cardiac death. Objectives The purpose of this study was to establish a patient-specific human-induced pluripotent stem cell (hiPSC) model of the SQTS, and to provide mechanistic insights into its pathophysiology and therapy. Methods Patient-specific hiPSCs were generated from a symptomatic SQTS patient carrying the N588K mutation in the KCNH2 gene, differentiated into cardiomyocytes, and compared with healthy and isogenic (established by CRISPR/Cas9-based mutation correction) control hiPSC-derived cardiomyocytes (hiPSC-CMs). Patch-clamp was used to evaluate action-potential (AP) and IKr current properties at the cellular level. Conduction and arrhythmogenesis were studied at the tissue level using confluent 2-dimensional hiPSC-derived cardiac cell sheets (hiPSC-CCSs) and optical mapping. Results Intracellular recordings demonstrated shortened action-potential duration (APD) and abbreviated refractory period in the SQTS-hiPSC-CMs. Similarly, voltage- and AP-clamp recordings revealed increased IKr current density due to attenuated inactivation, primarily in the AP plateau phase. Optical mapping of the SQTS-hiPSC-CCSs revealed shortened APD, impaired APD-rate adaptation, abbreviated wavelength of excitation, and increased inducibility of sustained spiral waves. Phase-mapping analysis revealed accelerated and stabilized rotors manifested by increased rotor rotation frequency, increased rotor curvature, decreased core meandering, and increased rotor complexity. Application of quinidine and disopyramide, but not sotalol, normalized APD and suppressed arrhythmia induction. Conclusions A novel hiPSC-based model of the SQTS was established at both the cellular and tissue levels. This model recapitulated the disease phenotype in the culture dish and provided important mechanistic insights into arrhythmia mechanisms in the SQTS and its treatment.

The clinical use of electromechanical mapping of the endocardium requires better understanding of the endocardial electrogram morphology. In this study, principal component analysis was used in order to identify the signal segments that change as a result of regional acute ischemia in a canine model. For each ischemic stage, (immediately, 5 hours and 3 days following ligation of the left anterior descending coronary artery), parameters were defined as linear combinations of regional average principal components and were compared to Echo scores. It was found that the major changes occur in the depolarization segment of the signal as well as in the ST segment.

Assessment of early ultrastructural development and cell-cycle regulation in human cardiac tissue is significantly hampered by the lack of a suitable in vitro model. Here we describe the possible utilization of human embryonic stem cell (ES) lines for investigation of these processes. With the use of the embryoid body (EB) differentiation system, human ES cell-derived cardiomyocytes at different developmental stages were isolated and their histomorphometric, ultrastructural, and proliferative properties were characterized. Histomorphometric analysis revealed an increase in cell length, area, and length-to-width ratio in late-stage EBs (>35 days) compared with early (10–21 days) and intermediate (21–35 days) stages. This was coupled with a progressive ultrastructural development from an irregular myofibrillar distribution to an organized sarcomeric pattern. Cardiomyocyte proliferation, assessed by double labeling with cardiac-specific antibodies and either [3H]thymidine incorporation or Ki-67 immunolabeling, demonstrated a gradual withdrawal from cell cycle. Hence, the percentage of positively stained nuclei in early-stage cardiomyocytes ([3H]thymidine: 60 ± 10%, Ki-67: 54 ± 23%) decreased to 36 ± 7% and 9 ± 16% in intermediate-stage EBs and to

Funding Acknowledgements Type of funding sources: None. Background The diagnosis of atrial fibrillation (AF) induced cardiomyopathy can be challenging. It relies on ruling out other causes of dilated cardiomyopathy, upon recovery of left ventricular ejection fraction (LVEF) following return to sinus rhythm (SR). Aim The aim of this study was to identify clinical and echocardiographic predictors for developing new dilated cardiomyopathy in patients with AF or atrial flutter (AFL). Methods This is a retrospective study conducted in a large tertiary care center. Patients that suffered deterioration of LVEF under 50% during AF demonstrated by pre-cardioversion trans-esophageal echocardiography (TEE) were compared to those with preserved LV function during AF. All patients had documented preserved LVEF at baseline (EF >50%) while in SR. Patients with a previous history of reduced LVEF during SR were excluded. Results From a total of 482 patients included in the final analysis, 80 (17%) patients had reduced LV function and 402 (83%) had preserved LV function during the pre-cardioversion TEE. Patients with reduced LVEF were more likely to be male and with a more rapid ventricular response during AF/AFL. A history of prosthetic valves was also identified as a risk factor for reduced LVEF. Patients with reduced LVEF also had higher incidence of TR and RV dysfunction. Conclusion In "real world" experience, male patients with rapid ventricular response during AF or AFL are more prone to LVEF reduction. Patients with prosthetic valves are also at risk for LVEF reduction during AF/AFL. Lastly, TR and RV dysfunction may indicate relatively long-standing AF with an associated reduction in LVEF.