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During cardiac development and morphogenesis, cardiac progenitor cells differentiate into cardiomyocytes that expand in number and size to generate the fully formed heart. Much is known about the factors that regulate initial differentiation of cardiomyocytes, and there is ongoing research to identify how these fetal and immature cardiomyocytes develop into fully functioning, mature cells. Accumulating evidence indicates that maturation limits proliferation and conversely proliferation occurs rarely in cardiomyocytes of the adult myocardium. We term this oppositional interplay the proliferation-maturation dichotomy. Here we review the factors that are involved in this interplay and discuss how a better understanding of the proliferation-maturation dichotomy could advance the utility of human induced pluripotent stem cell–derived cardiomyocytes for modeling in 3-dimensional engineered cardiac tissues to obtain truly adult-level function.

To extend the preservation of donor hearts beyond the current 4-6 h, this paper explores heart cryopreservation by vitrification-cryogenic storage in a glass-like state. While organ vitrification is made possible by using cryoprotective agents (CPA) that inhibit ice during cooling, failure occurs during convective rewarming due to slow and non-uniform rewarming which causes ice crystallization and/or cracking. Here an alternative, "nanowarming", which uses silica-coated iron oxide nanoparticles (sIONPs) perfusion loaded through the vasculature is explored, that allows a radiofrequency coil to rewarm the organ quickly and uniformly to avoid convective failures. Nanowarming has been applied to cells and tissues, and a proof of principle study suggests it is possible in the heart, but proper physical and biological characterization especially in organs is still lacking. Here, using a rat heart model, controlled machine perfusion loading and unloading of CPA and sIONPs, cooling to a vitrified state, and fast and uniform nanowarming without crystallization or cracking is demonstrated. Further, nanowarmed hearts maintain histologic appearance and endothelial integrity superior to convective rewarming and indistinguishable from CPA load/unload control hearts while showing some promising organ-level (electrical) functional activity. This work demonstrates physically successful heart vitrification and nanowarming and that biological outcomes can be expected to improve by reducing or eliminating CPA toxicity during loading and unloading.

As a precursor for cardiac arrhythmias such as atrial and ventricular fibrillations, which could cause sudden cardiac death (SCD), cardiac alternans is essentially an unstable heart rhythm with alternating long and short action potential durations (APD) of cardiac myocytes that usually occurs under fast pacing conditions. In this paper, the constant TR control method based on global pseudo-electrocardiogram (ECG) is studied and compared with the local constant diastolic interval (DI) control method using a 2-dimensional (2-D) cellular automata model (CAM), aiming at preventing or eliminating cardiac alternans before arrhythmias. The results show that both the constant TR and constant DI control methods are effective in stabling the alternans to a smaller basic cycle length (BCL). Also, the efficacy of the two control approaches depends on the “decrease step” Δ in the downsweep protocol, and a smaller Δ could significantly improve their performance. Besides, in general, constant TR control is superior to constant DI control in alternans prevention when a relatively large Δ is adopted.

Background: Rigorous study of ventricular fibrillation (VF) is not feasible in humans. The spatiotemporal characteristics of prolonged VF remain undefined, limiting our understanding of this lethal rhythm. Methods: VF was mapped in 4 canines. The endocardial and epicardial left ventricle (LV) and right ventricle (RV) were sequentially mapped at 0 to 15, 15 to 30, 30 to 45, and 45 to 60 minutes post-induction. Ten consecutive beats were used to determine the average cycle length and regularity index of ventricular and His-Purkinje system signals in discrete regions during each time interval. Results: Average VF time was 58±12 minutes. The shortest ventricular cycle length was present in the RV apical region (70±10 msec) at 0 to 15 minutes and at 15 to 30 minutes (89±31 msec) and LV apical region at 45 to 60 minutes (242±163 msec). The His-Purkinje system cycle length was the shortest at the RV outflow tract (75±3 msec) at 0 to 15 minutes, RV inflow and free wall (89±12 msec) at 15 to 30 minutes, LV apical region (83±14 msec) at 30 to 45 minutes, and inferior and inferolateral LV (145±23 msec) at 45 to 60 minutes. Regularity index was the highest in the RV inflow and free wall (78%) at 0 to 15 minutes, RV apical region (86%) at 15 to 30 minutes, LV septum and epicardial anterior RV (80%) at 30 to 45 minutes, and anterior and anterolateral LV (75%) at 45 to 60 minutes. Conclusions: These data suggest significant regional changes in electrical activity throughout VF in canines. A transition of fastest electrical activity from RV to LV apical regions across VF was observed. These novel insights of the temporal and spatial changes of VF merit further evaluation in diseased hearts to assess for reproducibility of findings.

Bionic-engineered tissues have been proposed for testing the performance of cardiovascular medical devices and predicting clinical outcomes ex vivo. Progress has been made in the development of compliant electronics that are capable of monitoring treatment parameters and being coupled to engineered tissues; however, the scale of most engineered tissues is too small to accommodate the size of clinical-grade medical devices. Here, we show substantial progress toward bionic tissues for evaluating cardiac ablation tools by generating a centimeter-scale human cardiac disk and coupling it to a hydrogel-based soft-pressure sensor. The cardiac tissue with contiguous electromechanical function was made possible by our recently established method to 3D bioprint human pluripotent stem cells in an extracellular matrix-based bioink that allows for in situ cell expansion prior to cardiac differentiation. The pressure sensor described here utilized electrical impedance tomography to enable the real-time spatiotemporal mapping of pressure distribution. A cryoablation tip catheter was applied to the composite bionic tissues with varied pressure. We found a close correlation between the cell response to ablation and the applied pressure. Under some conditions, cardiomyocytes could survive in the ablated region with more rounded morphology compared to the unablated controls, and connectivity was disrupted. This is the first known functional characterization of living human cardiomyocytes following an ablation procedure that suggests several mechanisms by which arrhythmia might redevelop following an ablation. Thus, bionic-engineered testbeds of this type can be indicators of tissue health and function and provide unique insight into human cell responses to ablative interventions.

During cardiac arrhythmias, dynamical patterns of electrical activation form and evolve, which are of interest to understand and cure heart rhythm disorders. The analysis of these patterns is commonly performed by calculating the local activation phase and searching for phase singularities (PSs), i.e. points around which all phases are present. Here we propose an alternative framework, which focuses on phase defect lines (PDLs) and surfaces (PDSs) as more general mechanisms, which include PSs as a specific case. The proposed framework enables two conceptual unifications: between the local activation time and phase description, and between conduction block lines and the central regions of linear-core rotors. A simple PDL detection method is proposed and applied to data from simulations and optical mapping experiments. Our analysis of ventricular tachycardia in rabbit hearts $(n=6)$ shows that nearly all detected PSs were found on PDLs, but the PDLs had a significantly longer lifespan than the detected PSs. Since the proposed framework revisits basic building blocks of cardiac activation patterns, it can become a useful tool for further theory development and experimental analysis., Comment: second version, modified according to reviewer comments

Cardiac alternans, an alternation in the action potential duration (APD), is suggested to be a precursor to lethal tachyarrhythmias such as ventricular tachycardia or ventricular fibrillation, which in turn can lead to sudden cardiac death. Linear methods for detection of alternans onset and suppression have shown limited success, predominantly due to presence of cardiac memory wherein APD prediction at any instant purportedly requires the knowledge of multiple previous APDs and diastolic intervals (DIs) to successfully characterize the present cardiac state. Taking into account the non-linear relationship between the APDs and preceding DIs, the transition from steady state to alternans has been captured successfully using non-linear analytical methods including bifurcation theory, principal component analysis and chaos control techniques. This chapter presents an overview of non-linear analytical approaches utilized for predicting the onset of alternans that hold potential to translate into preclinical modalities capable of alternans detection and control, thereby preventing fatal tachyarrhythmias and transition to sudden cardiac death. We present a preclinical evaluation using high resolution optical mapping of two novel techniques based on bifurcation analysis and eigenvalue decomposition to predict the onset of alternans. Furthermore, we correlate the spatial evolution of alternans with bifurcation parameters and dominant eigenvalues, providing a potential marker for alternans estimation.

Life‐threatening ventricular arrhythmias and sudden cardiac death are often preceded by cardiac alternans, a beat‐to‐beat oscillation in the T‐wave morphology or duration. However, given the spatiotemporal and structural complexity of the human heart, designing algorithms to effectively suppress alternans and prevent fatal rhythms is challenging. Recently, an antiarrhythmic constant diastolic interval pacing protocol was proposed and shown to be effective in suppressing alternans in 0‐, 1‐, and 2‐dimensional in silico studies as well as in ex vivo whole heart experiments. Herein, we provide a systematic review of the electrophysiological conditions and mechanisms that enable constant diastolic interval pacing to be an effective antiarrhythmic pacing strategy. We also demonstrate a successful translation of the constant diastolic interval pacing protocol into an ECG‐based real‐time control system capable of modulating beat‐to‐beat cardiac electrical activity and preventing alternans. Furthermore, we present evidence of the clinical utility of real‐time alternans suppression in reducing arrhythmia susceptibility in vivo. We provide a comprehensive overview of this promising pacing technique, which can potentially be translated into a clinically viable device that could radically improve the quality of life of patients experiencing abnormal cardiac rhythms.

Ischemic cardiomyopathy is associated with an increased risk of sudden death, activation of the unfolded protein response (UPR), and reductions in multiple cardiac ion channels. When activated, the protein kinase-like ER kinase (PERK) branch of the UPR reduces protein translation and abundance. We hypothesized that PERK inhibition could prevent ion channel downregulation and reduce arrhythmic risk after myocardial infarct (MI). MI induced by coronary artery ligation resulted in mice exhibited reduced ion channel levels, ventricular tachycardia (VT), and prolonged corrected intervals between the Q and T waves of the ECGs (QTc). Protein levels of major cardiac ion channels were decreased. MI cardiomyocytes showed significantly prolonged action potential duration and decreased maximum upstroke velocity. Cardiac-specific PERK knockout (PERKKO) reduced electrical remodeling in response to MI with shortened QTc intervals, less VT episodes, and higher survival rates (P

Cardiac alternans, beat-to-beat alternations in action potential duration, is a precursor to fatal arrhythmias such as ventricular fibrillation. Previous research has shown that voltage driven alternans can be suppressed by application of a constant diastolic interval (DI) pacing protocol. However, the effect of constant-DI pacing on cardiac cell dynamics and its interaction with the intracellular calcium cycle remains to be determined. Therefore, we aimed to examine the effects of constant-DI pacing on the dynamical behavior of a single-cell numerical model of cardiac action potential and the influence of voltage-calcium (V-Ca) coupling on it. Single cell dynamics were analyzed in the vicinity of the bifurcation point using a hybrid pacing protocol, a combination of constant-basic cycle length (BCL) and constant-DI pacing. We demonstrated that in a small region beneath the bifurcation point, constant-DI pacing caused the cardiac cell to remain alternans-free after switching to the constant-BCL pacing, thus introducing a region of bistability (RB). The size of the RB increased with stronger V-Ca coupling and was diminished with weaker V-Ca coupling. Overall, our findings demonstrate that the application of constant-DI pacing on cardiac cells with strong V-Ca coupling may induce permanent changes to cardiac cell dynamics increasing the utility of constant-DI pacing.

As a leading cause of death in 325,000 adults per year in the United States, a significant proportion of sudden cardiac arrest (SCA) result from arrhythmias. To better understand the onset of arrhythmias and its potential treatment with more rapid and effective control approaches, a two-dimensional 50 × 50 cellular automata (CA) model is used in this study to illustrate the propagation of electrical waves across its tissue, and a constant diastolic interval (DI) control mechanism is adopted to help stabilize and prevent cardiac arrhythmias. Simulations of various scenarios including normal conduction and spiral waves in the presence of scar, normal conduction and alternans under control conditions are shown. The results validate that the CA model and constant DI control method are very efficient and effective in the study of dynamics and control of cardiac arrhythmias.

Cardiac alternans is a proarrhythmic state in which the action potential duration (APD) of cardiac myocytes alternate between long and short values and often occurs under conditions of rapid pacing of cardiac tissue. In the ventricles, alternans is especially dangerous due to the life-threatening risk of developing arrhythmias, such as ventricular fibrillation. Alternans can be formed in periodically paced tissue as a result of pacing itself. Recently, it has been demonstrated that this pacing-induced alternans can be prevented by performing constant diastolic interval (DI) pacing, in which DI is independent of APD. However, constant DI pacing is difficult to implement in experimental settings since it requires the real-time measurement of APD. A more practical way was proposed based on electrocardiograms (ECGs), which give an indirect measure of the global DI relaxation period through the TR interval assessment. Previously, we demonstrated that constant TR pacing prevented alternans formation in isolated Langendorff-perfused rabbit hearts. However, the efficacy of “local” constant DI pacing vs “global” constant TR pacing in preventing alternans formation has never been investigated. Thus, the purpose of this study was to implement an ECG-based constant TR pacing in a 1D numerical model of human ventricular tissue and to compare the dynamical behavior of cardiac tissue with that resulted from a constant DI pacing. The results showed that both constant TR and constant DI pacing prevented the onset of alternans until lower basic cycle length when compared to periodic pacing. For longer cable lengths, constant TR pacing was shown to exhibit greater control on alternans than constant DI pacing.

Atrial Fibrillation (AF) is most common sustained cardiac arrhythmia and a precursor to many fatal cardiac conditions. Catheter ablation, which is a minimally invasive treatment, is associated with limited success rates in patients with persistent AF. Rotors are believed to maintain AF and core of rotors are considered to be robust targets for ablation. Recently, multiscale entropy (MSE) was proposed to identify the core of rotors in ex-vivo rabbit hearts. However, MSE technique is sensitive to intrinsic parameters, such as scale factor and template dimension, that may lead to an imprecise estimation of entropy measures. The purpose of this research is optimize MSE approach to improve its accuracy and sensitivity in rotor core identification using simulated EGMs from human atrial model. Specifically, we have identified the optimal time scale factor (τopt) and optimal template dimension (Τopt) that are needed for efficient rotor core identification. The τopt was identified to be 10, using a convergence graph, and the Τopt (~20 ms) remained the same at different sampling rates, indicating that optimized MSE will be efficient in identifying core of the rotor irrespective of the signal acquisition system.

Over the last few years, the use of cardiac mapping for effective diagnosis and treatment of arrhythmias has increased significantly. In the clinical environment, electroanatomical mapping (EAM) is performed during the electrophysiological procedures using proprietary systems such as CARTO, EnSite Precision, RHYTHMIA, etc. These systems generate the 3D model of patient-specific atria with the electrical activity (i.e., intracardiac electrograms (iEGMs)) displayed on it, for further identification of the sources of arrhythmia and for guiding cardiac ablation therapy. Recently, several novel techniques were developed to perform iEGMs analysis to more accurately identify the arrhythmogenic sites. However, there is a difficulty in incorporating the results of iEGMs analysis back to EAM systems due to their proprietary constraints. This created a hurdle in the further development of novel techniques to help navigate patient-specific clinical ablation therapy. Thus, we developed an open source software, VIEgram1, that allows researchers to visualize the results of the various iEGMs analysis on a patient-specific 3D atria model. It eliminates the dependency of the academic environment on the proprietary EAM systems, thereby making the process of retrospective mapping extremely convenient and time efficient. Here, we demonstrate the features of VIEgram such as visual inspection of iEGMs, flexibility in implementing custom iEGMs analysis techniques and interpolation schemes, and spatial analysis.

Rationale: One goal of cardiac tissue engineering is the generation of a living, human pump in vitro that could replace animal models and eventually serve as an in vivo therapeutic. Models that replicate the geometrically complex structure of the heart, harboring chambers and large vessels with soft biomaterials, can be achieved using 3-dimensional bioprinting. Yet, inclusion of contiguous, living muscle to support pump function has not been achieved. This is largely due to the challenge of attaining high densities of cardiomyocytes—a notoriously nonproliferative cell type. An alternative strategy is to print with human induced pluripotent stem cells, which can proliferate to high densities and fill tissue spaces, and subsequently differentiate them into cardiomyocytes in situ. Objective: To develop a bioink capable of promoting human induced pluripotent stem cell proliferation and cardiomyocyte differentiation to 3-dimensionally print electromechanically functional, chambered organoids composed of contiguous cardiac muscle. Methods and Results: We optimized a photo-crosslinkable formulation of native ECM (extracellular matrix) proteins and used this bioink to 3-dimensionally print human induced pluripotent stem cell–laden structures with 2 chambers and a vessel inlet and outlet. After human induced pluripotent stem cells proliferated to a sufficient density, we differentiated the cells within the structure and demonstrated function of the resultant human chambered muscle pump. Human chambered muscle pumps demonstrated macroscale beating and continuous action potential propagation with responsiveness to drugs and pacing. The connected chambers allowed for perfusion and enabled replication of pressure/volume relationships fundamental to the study of heart function and remodeling with health and disease. Conclusions: This advance represents a critical step toward generating macroscale tissues, akin to aggregate-based organoids, but with the critical advantage of harboring geometric structures essential to the pump function of cardiac muscle. Looking forward, human chambered organoids of this type might also serve as a test bed for cardiac medical devices and eventually lead to therapeutic tissue grafting.

Paroxysmal atrial fibrillation (Paro. AF) is challenging to identify at the right moment. This disease is often undiagnosed using currently existing methods. Nonlinear analysis is gaining importance due to its capability to provide more insight into complex heart dynamics. The aim of this study is to use several recently developed nonlinear techniques to discriminate persistent AF (Pers. AF) from normal sinus rhythm (NSR), and more importantly, Paro. AF from NSR, using short-term single-lead electrocardiogram (ECG) signals. Specifically, we adapted and modified the time-delayed embedding method to minimize incorrect embedding parameter selection and further support to reconstruct proper phase plots of NSR and AF heart dynamics, from MIT-BIH databases. We also examine information-based methods, such as multiscale entropy (MSE) and kurtosis (Kt) for the same purposes. Our results demonstrate that embedding parameter time delay (&tau, ), as well as MSE and Kt values can be successfully used to discriminate between Pers. AF and NSR. Moreover, we demonstrate that &tau, and Kt can successfully discriminate Paro. AF from NSR. Our results suggest that nonlinear time-delayed embedding method and information-based methods provide robust discriminating features to distinguish both Pers. AF and Paro. AF from NSR, thus offering effective treatment before suffering chaotic Pers. AF.

Goal: Clinical studies identifying rotors and confirming these sites for ablation in treating cardiac arrhythmias have had inconsistent results with the currently available analysis techniques. The aim of this study is to evaluate four new signal analysis approaches—multiscale frequency (MSF), Shannon entropy (SE), Kurtosis (Kt), and multiscale entropy (MSE)—in their ability to identify the pivot point of rotors. Methods: Optical mapping movies of ventricular tachycardia were used to evaluate the performance and robustness of SE, Kt, MSF, and MSE techniques with respect to several clinical limitations: decreased time duration, reduced spatial resolution, and the presence of meandering rotors. To quantitatively assess the robustness of the four techniques, results were compared to the “true” rotor(s) identified using optical mapping-based phase maps. Results: The results demonstrate that MSF, Kt, and MSE accurately identified both stationary and meandering rotors. In addition, these techniques remained accurate under simulated clinical limitations: shortened electrogram duration and decreased spatial resolution. Artifacts mildly affected the performance of MSF, Kt, and MSE, but strongly impacted the performance of SE. Conclusion: These results motivate further validation using intracardiac electrograms to see if these approaches can map rotors in a clinical setting and whether they apply to more complex arrhythmias including atrial or ventricular fibrillation. Significance: New techniques providing more accurate rotor localization could improve characterization of arrhythmias and, in turn, offer a means to accurately evaluate whether rotor ablation is a viable and effective treatment for chaotic cardiac arrhythmias.