A versatile high-throughput assay based on 3D ring-shaped cardiac tissues generated from human induced pluripotent stem cell-derived cardiomyocytes.

We developed a 96-well plate assay which allows fast, reproducible, and high-throughput generation of 3D cardiac rings around a deformable optically transparent hydrogel (polyethylene glycol [PEG]) pillar of known stiffness. Human induced pluripotent stem cell-derived cardiomyocytes, mixed with normal human adult dermal fibroblasts in an optimized 3:1 ratio, self-organized to form ring-shaped cardiac constructs. Immunostaining showed that the fibroblasts form a basal layer in contact with the glass, stabilizing the muscular fiber above. Tissues started contracting around the pillar at D1 and their fractional shortening increased until D7, reaching a plateau at 25±1%, that was maintained up to 14 days. The average stress, calculated from the compaction of the central pillar during contractions, was 1.4±0.4 mN/mm2. The cardiac constructs recapitulated expected inotropic responses to calcium and various drugs (isoproterenol, verapamil) as well as the arrhythmogenic effects of dofetilide. This versatile high-throughput assay allows multiple in situ mechanical and structural readouts.

[Experts' consensus on mammalian cardiomyocyte regeneration].

Myocardial infarction (MI) leads to a massive loss of cardiomyocytes, resulting in pathological cardiac remodeling and heart failure. Promoting cardiomyocyte regeneration is crucial for repairing the damaged heart. It is acknowledged that regenerative cardiomyocyte derives from the existing cardiomyocytes. In recent years, advancements in this field have updated our understanding of cardiomyocyte regeneration in many aspects, including intrinsic cell source and microenvironmental characteristics, extrinsic factors, molecular biology mechanisms, and intervention strategies. Here, we report a consensus by an expert committee on the definition, characteristics, evaluation, research methods, regulatory mechanisms, and intervention measures related to mammalian cardiomyocyte regeneration. The aim is to clarify important unresolved issues in this field and to promote myocardial regeneration research and its clinical translation.

Loss of Cardiac PFKFB2 Drives Metabolic, Functional, and Electrophysiological Remodeling in the Heart.

BACKGROUND: Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) is a critical glycolytic regulator responsible for upregulation of glycolysis in response to insulin and adrenergic signaling. PFKFB2, the cardiac isoform of PFK-2, is degraded in the heart in the absence of insulin signaling, contributing to diabetes-induced cardiac metabolic inflexibility. However, previous studies have not examined how the loss of PFKFB2 affects global cardiac metabolism and function. METHODS AND RESULTS: To address this, we have generated a mouse model with a cardiomyocyte-specific knockout of PFKFB2 (cKO). Using 9-month-old cKO and control mice, we characterized the impacts of PFKFB2 on cardiac metabolism, function, and electrophysiology. cKO mice have a shortened life span of 9 months. Metabolically, cKO mice are characterized by increased glycolytic enzyme abundance and pyruvate dehydrogenase activity, as well as decreased mitochondrial abundance and beta oxidation, suggesting a shift toward glucose metabolism. This was supported by a decrease in the ratio of palmitoyl carnitine to pyruvate-dependent mitochondrial respiration in cKO relative to control animals. Metabolomic, proteomic, and Western blot data support the activation of ancillary glucose metabolism, including pentose phosphate and hexosamine biosynthesis pathways. Physiologically, cKO animals exhibited impaired systolic function and left ventricular dilation, represented by reduced fractional shortening and increased left ventricular internal diameter, respectively. This was accompanied by electrophysiological alterations including increased QT interval and other metrics of delayed ventricular conduction. CONCLUSIONS: Loss of PFKFB2 results in metabolic remodeling marked by cardiac ancillary pathway activation. This could delineate an underpinning of pathologic changes to mechanical and electrical function in the heart.

Structural maturation of myofilaments in engineered 3D cardiac microtissues characterized using small angle x-ray scattering.

Understanding the structural and functional development of human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) is essential to engineering cardiac tissue that enables pharmaceutical testing, modeling diseases, and designing therapies. Here we use a method not commonly applied to biological materials, small angle x-ray scattering, to characterize the structural development of hiPSC-CMs within three-dimensional engineered tissues during their preliminary stages of maturation. An x-ray scattering experimental method enables the reliable characterization of the cardiomyocyte myofilament spacing with maturation time. The myofilament lattice spacing monotonically decreases as the tissue matures from its initial post-seeding state over the span of 10 days. Visualization of the spacing at a grid of positions in the tissue provides an approach to characterizing the maturation and organization of cardiomyocyte myofilaments and has the potential to help elucidate mechanisms of pathophysiology, and disease progression, thereby stimulating new biological hypotheses in stem cell engineering.

Acute Adenoviral Infection Elicits an Arrhythmogenic Substrate Prior to Myocarditis.

BACKGROUND: Viral cardiac infection represents a significant clinical challenge encompassing several etiological agents, disease stages, complex presentation, and a resulting lack of mechanistic understanding. Myocarditis is a major cause of sudden cardiac death in young adults, where current knowledge in the field is dominated by later disease phases and pathological immune responses. However, little is known regarding how infection can acutely induce an arrhythmogenic substrate before significant immune responses. Adenovirus is a leading cause of myocarditis, but due to species specificity, models of infection are lacking, and it is not understood how adenoviral infection may underlie sudden cardiac arrest. Mouse adenovirus type-3 was previously reported as cardiotropic, yet it has not been utilized to understand the mechanisms of cardiac infection and pathology. METHODS: We have developed mouse adenovirus type-3 infection as a model to investigate acute cardiac infection and molecular alterations to the infected heart before an appreciable immune response or gross cardiomyopathy. RESULTS: Optical mapping of infected hearts exposes decreases in conduction velocity concomitant with increased Cx43Ser368 phosphorylation, a residue known to regulate gap junction function. Hearts from animals harboring a phospho-null mutation at Cx43Ser368 are protected against mouse adenovirus type-3-induced conduction velocity slowing. Additional to gap junction alterations, patch clamping of mouse adenovirus type-3-infected adult mouse ventricular cardiomyocytes reveals prolonged action potential duration as a result of decreased IK1 and IKs current density. Turning to human systems, we find human adenovirus type-5 increases phosphorylation of Cx43Ser368 and disrupts synchrony in human induced pluripotent stem cell-derived cardiomyocytes, indicating common mechanisms with our mouse whole heart and adult cardiomyocyte data. CONCLUSIONS: Together, these findings demonstrate that adenoviral infection creates an arrhythmogenic substrate through direct targeting of gap junction and ion channel function in the heart. Such alterations are known to precipitate arrhythmias and likely contribute to sudden cardiac death in acutely infected patients.

Novel high-dense microelectrode array based multimodal bioelectronic monitoring system for cardiac arrhythmia re-entry analysis.

In recent decades, significant progress has been made in the treatment of heart diseases, particularly in the field of personalized medicine. Despite the development of genetic tests, phenotyping and risk stratification are performed based on clinical findings and invasive in vivo techniques, such as stimulation conduction mapping techniques and programmed ventricular pacing. Consequently, label-free non-invasive in vitro functional analysis systems are urgently needed for more accurate and effective in vitro risk stratification, model-based therapy planning, and clinical safety profile evaluation of drugs. To overcome these limitations, a novel multilayer high-density microelectrode array (HD-MEA), with an optimized configuration of 512 sensing and 4 pacing electrodes on a sensor area of 100 mm2, was developed for the bioelectronic detection of re-entry arrhythmia patterns. Together with a co-developed front-end, we monitored label-free and in parallel cardiac electrophysiology based on field potential monitoring and mechanical contraction using impedance spectroscopy at the same microelectrode. In proof of principle experiments, human induced pluripotent stem cell (hiPS)-derived cardiomyocytes were cultured on HD-MEAs and used to demonstrate the sensitive quantification of contraction strength modulation by cardioactive drugs such as blebbistatin (IC50 = 4.2 µM), omecamtiv and levosimendan. Strikingly, arrhythmia-typical rotor patterns (re-entry) can be induced by optimized electrical stimulation sequences and detected with high spatial resolution. Therefore, we provide a novel cardiac re-entry analysis system as a promising reference point for diagnostic approaches based on in vitro assays using patient-specific hiPS-derived cardiomyocytes.

Dissecting the roles of calcium cycling and its coupling with voltage in the genesis of early afterdepolarizations in cardiac myocyte models.

Early afterdepolarizations (EADs) are abnormal depolarizations during the plateau phase of the action potential, which are known to be associated with lethal arrhythmias in the heart. There are two major hypotheses for EAD genesis based on experimental observations, i.e., the voltage (Vm)-driven and intracellular calcium (Ca)-driven mechanisms. In ventricular myocytes, Ca and Vm are bidirectionally coupled, which can affect each other's dynamics and result in new dynamics, however, the roles of Ca cycling and its coupling with Vm in the genesis of EADs have not been well understood. In this study, we use an action potential model that is capable of independent Vm and Ca oscillations to investigate the roles of Vm and Ca coupling in EAD genesis. Four different mechanisms of EADs are identified, which are either driven by Vm oscillations or Ca oscillations alone, or oscillations caused by their interactions. We also use 5 other ventricular action potential models to assess these EAD mechanisms and show that EADs in these models are mainly Vm-driven. These mechanistic insights from our simulations provide a theoretical base for understanding experimentally observed EADs and EAD-related arrhythmogenesis.

Experimentally-guided in silico design of engineered heart tissues to improve cardiac electrical function after myocardial infarction.

Engineered heart tissues (EHTs) built from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) showed promising results for cardiac function restoration following myocardial infarction. Nevertheless, human iPSC-CMs have longer action potential and lower cell-to-cell coupling than adult-like CMs. These immature electrophysiological properties favor arrhythmias due to the generation of electrophysiological gradients when hiPSC-CMs are injected in the cardiac tissue. Culturing hiPSC-CMs on three-dimensional (3D) scaffolds can promote their maturation and influence their alignment. However, it is still uncertain how on-scaffold culturing influences the overall electrophysiology of the in vitro and implanted EHTs, as it requires expensive and time consuming experimentation. Here, we computationally investigated the impact of the scaffold design on the EHT electrical depolarization and repolarization before and after engraftment on infarcted tissue. We first acquired and processed electrical recordings from in vitro EHTs, which we used to calibrate the modeling and simulation of in silico EHTs to replicate experimental outcomes. Next, we built in silico EHT models for a range of scaffold pore sizes, shapes (square, rectangular, auxetic, hexagonal) and thicknesses. In this setup, we found that scaffolds made of small (0.2 mm2), elongated (30° half-angle) hexagons led to faster EHT activation and better mimicked the cardiac anisotropy. The scaffold thickness had a marginal role on the not engrafted EHT electrophysiology. Moreover, EHT engraftment on infarcted tissue showed that the EHT conductivity should be at least 5% of that in healthy tissue for bidirectional EHT-myocardium electrical propagation. For conductivities above such threshold, the scaffold made of small elongated hexagons led to the lowest activation time (AT) in the coupled EHT-myocardium. If the EHT conductivity was further increased and the hiPSC-CMs were uniformly oriented parallel to the epicardial cells, the total AT and the repolarization time gradient decreased substantially, thus minimizing the likelihood for arrhythmias after EHT transplantation.

Interpretation of field and LEAP potentials recorded from cardiomyocyte monolayers.

Multielectrode arrays (MEAs) are the method of choice for electrophysiological characterization of cardiomyocyte monolayers. The field potentials recorded using an MEA are like extracellular electrograms recorded from the myocardium using conventional electrodes. Nevertheless, different criteria are used to interpret field potentials and extracellular electrograms, which hamper correct interpretation and translation to the patient. To validate the criteria for interpretation of field potentials, we used neonatal rat cardiomyocytes to generate monolayers. We recorded field potentials using an MEA and simultaneously recorded action potentials using sharp microelectrodes. In parallel, we recreated our experimental setting in silico and performed simulations. We show that the amplitude of the local RS complex of a field potential correlated with conduction velocity in silico but not in vitro. The peak time of the T wave in field potentials exhibited a strong correlation with APD90 while the steepest upslope correlated well with APD50. However, this relationship only holds when the T wave displayed a biphasic pattern. Next, we simulated local extracellular action potentials (LEAPs). The shape of the LEAP differed markedly from the shape of the local action potential, but the final duration of the LEAP coincided with APD90. Criteria for interpretation of extracellular electrograms should be applied to field potentials. This will provide a strong basis for the analysis of heterogeneity in conduction velocity and repolarization in cultured monolayers of cardiomyocytes. Finally, a LEAP is not a recording of the local action potential but is generated by intracellular current provided by neighboring cardiomyocytes and is superior to field potential duration in estimating APD90.NEW & NOTEWORTHY We present a physiological basis for the interpretation of multielectrode array-derived, extracellular, electrical signals.

The cardiomyocyte origins of diastolic dysfunction: cellular components of myocardial "stiffness".

The impaired ability of the heart to relax and stretch to accommodate venous return is generally understood to represent a state of "diastolic dysfunction" and often described using the all-purpose noun "stiffness." Despite the now common qualitative usage of this term in fields of cardiac patho/physiology, the specific quantitative concept of stiffness as a molecular and biophysical entity with real practical interpretation in healthy and diseased hearts is sometimes obscure. The focus of this review is to characterize the concept of cardiomyocyte stiffness and to develop interpretation of "stiffness" attributes at the cellular and molecular levels. Here, we consider "stiffness"-related terminology interpretation and make links between cardiomyocyte stiffness and aspects of functional and structural cardiac performance. We discuss cross bridge-derived stiffness sources, considering the contributions of diastolic myofilament activation and impaired relaxation. This includes commentary relating to the role of cardiomyocyte Ca2+ flux and Ca2+ levels in diastole, the troponin-tropomyosin complex role as a Ca2+ effector in diastole, the myosin ADP dissociation rate as a modulator of cross bridge attachment and regulation of cross-bridge attachment by myosin binding protein C. We also discuss non-cross bridge-derived stiffness sources, including the titin sarcomeric spring protein, microtubule and intermediate filaments, and cytoskeletal extracellular matrix interactions. As the prevalence of conditions involving diastolic heart failure has escalated, a more sophisticated understanding of the molecular, cellular, and tissue determinants of cardiomyocyte stiffness offers potential to develop imaging and molecular intervention tools.

Fast creation of data-driven low-order predictive cardiac tissue excitation models from recorded activation patterns.

Excitable systems give rise to important phenomena such as heat waves, epidemics and cardiac arrhythmias. Understanding, forecasting and controlling such systems requires reliable mathematical representations. For cardiac tissue, computational models are commonly generated in a reaction-diffusion framework based on detailed measurements of ionic currents in dedicated single-cell experiments. Here, we show that recorded movies at the tissue-level of stochastic pacing in a single variable are sufficient to generate a mathematical model. Via exponentially weighed moving averages, we create additional state variables, and use simple polynomial regression in the augmented state space to quantify excitation wave dynamics. A spatial gradient-sensing term replaces the classical diffusion as it is more robust to noise. Our pipeline for model creation is demonstrated for an in-silico model and optical voltage mapping recordings of cultured human atrial myocytes and only takes a few minutes. Our findings have the potential for widespread generation, use and on-the-fly refinement of personalised computer models for non-linear phenomena in biology and medicine, such as predictive cardiac digital twins.

Unlocking cardiac motion: assessing software and machine learning for single-cell and cardioid kinematic insights.

The heart coordinates its functional parameters for optimal beat-to-beat mechanical activity. Reliable detection and quantification of these parameters still represent a hot topic in cardiovascular research. Nowadays, computer vision allows the development of open-source algorithms to measure cellular kinematics. However, the analysis software can vary based on analyzed specimens. In this study, we compared different software performances in in-silico model, in-vitro mouse adult ventricular cardiomyocytes and cardioids. We acquired in-vitro high-resolution videos during suprathreshold stimulation at 0.5-1-2 Hz, adapting the protocol for the cardioids. Moreover, we exposed the samples to inotropic and depolarizing substances. We analyzed in-silico and in-vitro videos by (i) MUSCLEMOTION, the gold standard among open-source software; (ii) CONTRACTIONWAVE, a recently developed tracking software; and (iii) ViKiE, an in-house customized video kinematic evaluation software. We enriched the study with three machine-learning algorithms to test the robustness of the motion-tracking approaches. Our results revealed that all software produced comparable estimations of cardiac mechanical parameters. For instance, in cardioids, beat duration measurements at 0.5 Hz were 1053.58 ms (MUSCLEMOTION), 1043.59 ms (CONTRACTIONWAVE), and 937.11 ms (ViKiE). ViKiE exhibited higher sensitivity in exposed samples due to its localized kinematic analysis, while MUSCLEMOTION and CONTRACTIONWAVE offered temporal correlation, combining global assessment with time-efficient analysis. Finally, machine learning reveals greater accuracy when trained with MUSCLEMOTION dataset in comparison with the other software (accuracy > 83%). In conclusion, our findings provide valuable insights for the accurate selection and integration of software tools into the kinematic analysis pipeline, tailored to the experimental protocol.

Modeling mutation-specific arrhythmogenic phenotypes in isogenic human iPSC-derived cardiac tissues.

Disease modeling using human induced pluripotent stem cells (hiPSCs) from patients with genetic disease is a powerful approach for dissecting pathophysiology and drug discovery. Nevertheless, isogenic controls are required to precisely compare phenotypic outcomes from presumed causative mutations rather than differences in genetic backgrounds. Moreover, 2D cellular models often fail to exhibit authentic disease phenotypes resulting in poor validation in vitro. Here we show that a combination of precision gene editing and bioengineered 3D tissue models can establish advanced isogenic hiPSC-derived cardiac disease models, overcoming these drawbacks. To model inherited cardiac arrhythmias we selected representative N588D and N588K missense mutations affecting the same codon in the hERG potassium channel gene KCNH2, which are reported to cause long (LQTS) and short (SQTS) QT syndromes, respectively. We generated compound heterozygous variants in normal hiPSCs, and differentiated cardiomyocytes (CMs) and mesenchymal cells (MCs) to form 3D cardiac tissue sheets (CTSs). In hiPSC-derived CM monolayers and 3D CTSs, electrophysiological analysis with multielectrode arrays showed prolonged and shortened repolarization, respectively, compared to the isogenic controls. When pharmacologically inhibiting the hERG channels, mutant 3D CTSs were differentially susceptible to arrhythmic events than the isogenic controls. Thus, this strategy offers advanced disease models that can reproduce clinically relevant phenotypes and provide solid validation of gene mutations in vitro.

Gene correction and overexpression of TNNI3 improve impaired relaxation in engineered heart tissue model of pediatric restrictive cardiomyopathy.

Research on cardiomyopathy models using engineered heart tissue (EHT) created from disease-specific induced pluripotent stem cells (iPSCs) is advancing rapidly. However, the study of restrictive cardiomyopathy (RCM), a rare and intractable cardiomyopathy, remains at the experimental stage because there is currently no established method to replicate the hallmark phenotype of RCM, particularly diastolic dysfunction, in vitro. In this study, we generated iPSCs from a patient with early childhood-onset RCM harboring the TNNI3 R170W mutation (R170W-iPSCs). The properties of R170W-iPSC-derived cardiomyocytes (CMs) and EHTs were evaluated and compared with an isogenic iPSC line in which the mutation was corrected. Our results indicated altered calcium kinetics in R170W-iPSC-CMs, including prolonged tau, and an increased ratio of relaxation force to contractile force in R170W-EHTs. These properties were reversed in the isogenic line, suggesting that our model recapitulates impaired relaxation of RCM, i.e., diastolic dysfunction in clinical practice. Furthermore, overexpression of wild-type TNNI3 in R170W-iPSC-CMs and -EHTs effectively rescued impaired relaxation. These results highlight the potential efficacy of EHT, a modality that can accurately recapitulate diastolic dysfunction in vitro, to elucidate the pathophysiology of RCM, as well as the possible benefits of gene therapies for patients with RCM.

Two decades of heart regeneration research: Cardiomyocyte proliferation and beyond.

Interest in vertebrate cardiac regeneration has exploded over the past two decades since the discovery that adult zebrafish are capable of complete heart regeneration, contrasting the limited regenerative potential typically observed in adult mammalian hearts. Undercovering the mechanisms that both support and limit cardiac regeneration across the animal kingdom may provide unique insights in how we may unlock this capacity in adult humans. In this review, we discuss key discoveries in the heart regeneration field over the last 20 years. Initially, seminal findings revealed that pre-existing cardiomyocytes are the major source of regenerated cardiac muscle, drawing interest into the intrinsic mechanisms regulating cardiomyocyte proliferation. Moreover, recent studies have identified the importance of intercellular interactions and physiological adaptations, which highlight the vast complexity of the cardiac regenerative process. Finally, we compare strategies that have been tested to increase the regenerative capacity of the adult mammalian heart. This article is categorized under: Cardiovascular Diseases > Stem Cells and Development.

Elevating intracellular action potential recording in cardiomyocytes: A precision-enhanced and biosafe single-pulse electroporation system.

Action potentials play a pivotal role in diverse cardiovascular physiological mechanisms. A comprehensive understanding of these intricate mechanisms necessitates a high-fidelity intracellular electrophysiological investigative approach. The amalgamation of micro-/nano-electrode arrays and electroporation confers substantial advantages in terms of high-resolution intracellular recording capabilities. Nonetheless, electroporation systems typically lack precise control, and commonly employed electroporation modes, involving tailored sequences, may escalate cellular damage and perturbation of normal physiological functions due to the multiple or higher-intensity electrical pulses. In this study, we developed an innovative electrophysiological biosensing system customized to facilitate precise single-pulse electroporation. This advancement serves to achieve optimal and uninterrupted intracellular action potential recording within cardiomyocytes. The refinement of the single-pulse electroporation technique is realized through the integration of the electroporation and assessment biosensing system, thereby ensuring a consistent and reliable means of achieving stable intracellular access. Our investigation has unveiled that the optimized single-pulse electroporation technique not only maintains robust biosafety standards but also enables the continuous capture of intracellular electrophysiological signals across an expansive three-day period. The universality of this biosensing system, adaptable to various micro/nano devices, furnishes real-time analysis and feedback concerning electroporation efficacy, guaranteeing the sustained, secure, and high-fidelity acquisition of intracellular data, thereby propelling the field of cardiovascular electrophysiological research.

Animal models to study cardiac regeneration.

Permanent fibrosis and chronic deterioration of heart function in patients after myocardial infarction present a major health-care burden worldwide. In contrast to the restricted potential for cellular and functional regeneration of the adult mammalian heart, a robust capacity for cardiac regeneration is seen during the neonatal period in mammals as well as in the adults of many fish and amphibian species. However, we lack a complete understanding as to why cardiac regeneration takes place more efficiently in some species than in others. The capacity of the heart to regenerate after injury is controlled by a complex network of cellular and molecular mechanisms that form a regulatory landscape, either permitting or restricting regeneration. In this Review, we provide an overview of the diverse array of vertebrates that have been studied for their cardiac regenerative potential and discuss differential heart regeneration outcomes in closely related species. Additionally, we summarize current knowledge about the core mechanisms that regulate cardiac regeneration across vertebrate species.

Optimizing the Cell-Nanostructure Interface: Nanoconcave/Nanoconvex Device for Intracellular Recording of Cardiomyocytes.

Nanostructures are powerful components for the development of high-performance nanodevices. Revealing and understanding the cell-nanostructure interface are essential for improving and guiding nanodevice design for investigations of cell physiology. For intracellular electrophysiological detection, the cell-nanostructure interface significantly affects the quality of recorded intracellular action potentials and the application of nanodevices in cardiology research and pharmacological screening. Most of the current investigations of biointerfaces focus on nanovertical structures, and few involve nanoconcave structures. Here, we design both nanoconvex and nanoconcave devices to perform intracellular electrophysiological recordings. The amplitude, signal-to-noise ratio, duration, and repeatability of the recorded intracellular electrophysiological signals provide a multifaceted characterization of the cell-nanostructure interface. We demonstrate that devices based on both convex and concave nanostructures can create tight coupling, which facilitates high-quality and stable intracellular recordings and paves the way for precise electrophysiological study.

Cardiac structure discontinuities revealed by ex-vivo microstructural characterization. A focus on the basal inferoseptal left ventricle region.

BACKGROUND: While the microstructure of the left ventricle (LV) has been largely described, only a few studies investigated the right ventricular insertion point (RVIP). It was accepted that the aggregate cardiomyocytes organization was much more complex due to the intersection of the ventricular cavities but a precise structural characterization in the human heart was lacking even if clinical phenotypes related to right ventricular wall stress or arrhythmia were observed in this region. METHODS: MRI-derived anatomical imaging (150 µm3) and diffusion tensor imaging (600 µm3) were performed in large mammalian whole hearts (human: N = 5, sheep: N = 5). Fractional anisotropy, aggregate cardiomyocytes orientations and tractography were compared within both species. Aggregate cardiomyocytes orientation on one ex-vivo sheep whole heart was then computed using structure tensor imaging (STI) from 21 µm isotropic acquisition acquired with micro computed tomography (MicroCT) imaging. Macroscopic and histological examination were performed. Lastly, experimental cardiomyocytes orientation distribution was then compared to the usual rule-based model using electrophysiological (EP) modeling. Electrical activity was modeled with the monodomain formulation. RESULTS: The RVIP at the level of the inferior ventricular septum presented a unique arrangement of aggregate cardiomyocytes. An abrupt, mid-myocardial change in cardiomyocytes orientation was observed, delimiting a triangle-shaped region, present in both sheep and human hearts. FA's histogram distribution (mean ± std: 0.29 ± 0.06) of the identified region as well as the main dimension (22.2 mm ± 5.6 mm) was found homogeneous across samples and species. Averaged volume is 0.34 cm3 ± 0.15 cm3. Both local activation time (LAT) and morphology of pseudo-ECGs were strongly impacted with delayed LAT and change in peak-to-peak amplitude in the simulated wedge model. CONCLUSION: The study was the first to describe the 3D cardiomyocytes architecture of the basal inferoseptal left ventricle region in human hearts and identify the presence of a well-organized aggregate cardiomyocytes arrangement and cardiac structural discontinuities. The results might offer a better appreciation of clinical phenotypes like RVIP-late gadolinium enhancement or uncommon idiopathic ventricular arrhythmias (VA) originating from this region.

The benign nature and rare occurrence of cardiac myxoma as a possible consequence of the limited cardiac proliferative/ regenerative potential: a systematic review.

BACKGROUND: Cardiac Myxoma is a primary tumor of heart. Its origins, rarity of the occurrence of primary cardiac tumors and how it may be related to limited cardiac regenerative potential, are not yet entirely known. This study investigates the key cardiac genes/ transcription factors (TFs) and signaling pathways to understand these important questions. METHODS: Databases including PubMed, MEDLINE, and Google Scholar were searched for published articles without any date restrictions, involving cardiac myxoma, cardiac genes/TFs/signaling pathways and their roles in cardiogenesis, proliferation, differentiation, key interactions and tumorigenesis, with focus on cardiomyocytes. RESULTS: The cardiac genetic landscape is governed by a very tight control between proliferation and differentiation-related genes/TFs/pathways. Cardiac myxoma originates possibly as a consequence of dysregulations in the gene expression of differentiation regulators including Tbx5, GATA4, HAND1/2, MYOCD, HOPX, BMPs. Such dysregulations switch the expression of cardiomyocytes into progenitor-like state in cardiac myxoma development by dysregulating Isl1, Baf60 complex, Wnt, FGF, Notch, Mef2c and others. The Nkx2-5 and MSX2 contribute predominantly to both proliferation and differentiation of Cardiac Progenitor Cells (CPCs), may possibly serve roles based on the microenvironment and the direction of cell circuitry in cardiac tumorigenesis. The Nkx2-5 in cardiac myxoma may serve to limit progression of tumorigenesis as it has massive control over the proliferation of CPCs. The cardiac cell type-specific genetic programming plays governing role in controlling the tumorigenesis and regenerative potential. CONCLUSION: The cardiomyocytes have very limited proliferative and regenerative potential. They survive for long periods of time and tightly maintain the gene expression of differentiation genes such as Tbx5, GATA4 that interact with tumor suppressors (TS) and exert TS like effect. The total effect such gene expression exerts is responsible for the rare occurrence and benign nature of primary cardiac tumors. This prevents the progression of tumorigenesis. But this also limits the regenerative and proliferative potential of cardiomyocytes. Cardiac Myxoma develops as a consequence of dysregulations in these key genes which revert the cells towards progenitor-like state, hallmark of CM. The CM development in carney complex also signifies the role of TS in cardiac cells.