Neutrophils facilitate the epicardial regenerative response after zebrafish heart injury.

Despite extensive studies on endogenous heart regeneration within the past 20 years, the players involved in initiating early regeneration events are far from clear. Here, we assessed the function of neutrophils, the first-responder cells to tissue damage, during zebrafish heart regeneration. We detected rapid neutrophil mobilization to the injury site after ventricular amputation, peaking at 1-day post-amputation (dpa) and resolving by 3 dpa. Further analyses indicated neutrophil mobilization coincides with peak epicardial cell proliferation, and recruited neutrophils associated with activated, expanding epicardial cells at 1 dpa. Neutrophil depletion inhibited myocardial regeneration and significantly reduced epicardial cell expansion, proliferation, and activation. To explore the molecular mechanism of neutrophils on the epicardial regenerative response, we performed scRNA-seq analysis of 1 dpa neutrophils and identified enrichment of the FGF and MAPK/ERK signaling pathways. Pharmacological inhibition of FGF signaling indicated its' requirement for epicardial expansion, while neutrophil depletion blocked MAPK/ERK signaling activation in epicardial cells. Ligand-receptor analysis indicated the EGF ligand, hbegfa, is released from neutrophils and synergizes with other FGF and MAPK/ERK factors for induction of epicardial regeneration. Altogether, our studies revealed that neutrophils quickly motivate epicardial cells, which later accumulate at the injury site and contribute to heart regeneration.

Histomorphological evaluation of acellularized bovine pericardium in breast implant coverage.

Bovine pericardium (BP) has been used as a biomaterial for several decades in many medical applications particularly due to its mechanical properties and the high collagen content. In the acellular form it favors faster tissue repair, providing a three-dimensional support for cellular and vascular events observed during tissue repair and due, to a low elastin content, may favor its use as a breast implant cover, resulting in a low possibility of contracture of the biomaterial, preventing the appearance of irregularities during the reconstruction process. Thus, the aim of this study was to evaluate, histomorphologically, the behavior of acellularized bovine pericardium (ABP) as a mammary implant cover in rats. For this purpose, 16 animals were divided into two groups, with eight animals at each biological point: 7 and 15 days after surgery. Of the 16 animals, 32 specimens were obtained: 16 in the experimental group (EG) and 16 in the control group (CG). Throughout this study, none of the studied groups had postoperative complications. Results: The histomorphological results showed, in the two biological points, both in the EG and in the CG, chronic inflammatory infiltrate, leukocyte fibrin exudate, formation of granulation tissue and deposition of collagen fibers, more evident in the EG, regressive along the biological points. At 15 days, the implanted ABP showed initial biointegration with the fibrous capsule and surrounding tissues of the recipient bed. Conclusion: These results indicate that the due to the observed favorable tissue response ABP may be of potential use as a breast implant cover.

Anisotropic Mechanical Response of Bovine Pericardium Membrane Through Bulge Test and In-Situ Confocal-Laser Scanning.

In this work, we present a new experimental setup for the assessment of the anisotropic properties of Bovine Pericardium (BP) membranes. The chemically fixed BP samples have been subjected to a bulge test with in situ confocal laser scanning at increasing applied pressure. The high resolution topography provided by the confocal laser scanning has allowed to obtain a quantitative measure of the bulge displacement; after polynomial fitting, principal curvatures have been obtained and a degree of anisotropy (DA) has been defined as the normalized difference between the maximum and minimum principal curvatures. The experiments performed on the BP membranes have allowed us to obtain pressure-displacement data which clearly exhibit distinct principal curvatures indicating an anisotropic response. A comparison with curvatures data obtained on isotropic Nitrile Buthadiene Rubber (NBR) samples has confirmed the effectiveness of the experimental setup for this specific purpose. Numerical simulations of the bulge tests have been performed with the purpose of identifying a range of constitutive parameters which well describes the obtained range of DA on the BP membranes. The DA values have been partially validated with biaxial tests available in literature and with suitably performed uni-axial tensile tests.

A computational model of rabbit geometry and ECG: Optimizing ventricular activation sequence and APD distribution.

Computational modeling of electrophysiological properties of the rabbit heart is a commonly used way to enhance and/or complement findings from classic lab work on single cell or tissue levels. Yet, thus far, there was no possibility to extend the scope to include the resulting body surface potentials as a way of validation or to investigate the effect of certain pathologies. Based on CT imaging, we developed the first openly available computational geometrical model not only of the whole heart but also the complete torso of the rabbit. Additionally, we fabricated a 32-lead ECG-vest to record body surface potential signals of the aforementioned rabbit. Based on the developed geometrical model and the measured signals, we then optimized the activation sequence of the ventricles, recreating the functionality of the Purkinje network, and we investigated different apico-basal and transmural gradients in action potential duration. Optimization of the activation sequence resulted in an average root mean square error between measured and simulated signal of 0.074 mV/ms for all leads. The best-fit T-Wave, compared to measured data (0.038 mV/ms), resulted from incorporating an action potential duration gradient from base to apex with a respective shortening of 20 ms and a transmural gradient with a shortening of 15 ms from endocardium to epicardium. By making our model and measured data openly available, we hope to give other researchers the opportunity to verify their research, as well as to create the possibility to investigate the impact of electrophysiological alterations on body surface signals for translational research.

Electrocardiographic marker of the cardiac action potential triangulation induced by antiarrhythmic drugs in perfused guinea-pig heart.

NEW FINDINGS: What is the central question of this study? Can the triangular appearance of ventricular action potential, indicating proarrhythmic profile of antiarrhythmic agent, be approximated by specific changes on an electrocardiogram (ECG)? What are the main finding and its importance? The triangulation of the ventricular action potential seen when antiarrhythmic drugs induce a greater lengthening of the late repolarization compared to the initial repolarization in epicardium is closely approximated by a greater prolongation of the T wave upslope relative to the interval between the J point and the start of the T wave (the JTstart interval) on the ECG. These findings may improve the power of ECG assessments in predicting the drug-induced arrhythmia resulting from slowed phase 3 repolarization. ABSTRACT: Antiarrhythmic drugs prescribed to treat atrial fibrillation can occasionally precipitate ventricular tachyarrhythmia through a prominent slowing of the phase 3 repolarization. The latter results in the triangular shape of ventricular action potential, indicating high arrhythmic risk. However, clinically, the utility of triangulation assessments for predicting arrhythmia is limited owing to the invasive nature of the ventricular action potential recordings. This study examined whether the triangulation effect can be detected indirectly from electrocardiogram (ECG) analysis. Epicardial monophasic action potentials and the ECG were simultaneously recorded in perfused guinea-pig hearts. With antiarrhythmics (dofetilide, quinidine, procainamide and flecainide), a prolongation of the initial repolarization seen in the action potential recordings was closely approximated by lengthening of the interval between the J point and the start of the T wave (the JTstart interval) on the ECG, whereas a prolongation of the late repolarization was paralleled by widening of the T wave upslope. Dofetilide, quinidine and procainamide induced a prominent slowing of the phase 3 repolarization in epicardium, leading to triangulation of the action potential. These effects were accompanied by a greater prolongation of the T wave upslope compared to the JTstart interval. Flecainide elicited a proportional prolongation of the initial and the late ventricular repolarization, and therefore failed to induce triangulation, based on analysis of both epicardial action potential and ECG profiles. Collectively, these findings suggest that the ratio between the durations of the T wave upslope and the JTstart interval may represent the ECG metric of the ventricular action potential triangulation induced by antiarrhythmic drugs.

Epicardium-derived cells organize through tight junctions to replenish cardiac muscle in salamanders.

The contribution of the epicardium, the outermost layer of the heart, to cardiac regeneration has remained controversial due to a lack of suitable analytical tools. By combining genetic marker-independent lineage-tracing strategies with transcriptional profiling and loss-of-function methods, we report here that the epicardium of the highly regenerative salamander species Pleurodeles waltl has an intrinsic capacity to differentiate into cardiomyocytes. Following cryoinjury, CLDN6+ epicardium-derived cells appear at the lesion site, organize into honeycomb-like structures connected via focal tight junctions and undergo transcriptional reprogramming that results in concomitant differentiation into de novo cardiomyocytes. Ablation of CLDN6+ differentiation intermediates as well as disruption of their tight junctions impairs cardiac regeneration. Salamanders constitute the evolutionarily closest species to mammals with an extensive ability to regenerate heart muscle and our results highlight the epicardium and tight junctions as key targets in efforts to promote cardiac regeneration.

Comparison on the properties of bovine pericardium and porcine pericardium used as leaflet materials of transcatheter heart valve.

BACKGROUND: In order to obtain the smaller delivery diameter, porcine pericardium had been used as a substitute material of bovine pericardium for the leaflet materials of transcatheter heart valve (THV). However, the differences between them had not been fully studied. Therefore, this study compared the microstructure, biochemical and mechanical properties of two materials and hydrodynamics of THV made by the two materials in detail. METHODS: In this study, firstly, the microstructure of pericardium was analyzed by staining and scanning electron microscope; secondly, the biochemical properties of pericardium after different processes were compared by heat shrinkage temperature test, free amino and carboxyl concentration test, enzyme degradation test, subcutaneous implantation calcification analysis in rats; finally, the mechanical properties were evaluated by uniaxial tensile test before and after the pericardium being crimped, and then, the hydrodynamics of THV was studied according to the ISO5840 standard. RESULTS: Compared with bovine pericardium, after the same process, porcine pericardium showed a looser and tinier fiber bundle, a similar free carboxyl concentration, a lower resistance to enzyme degradation, a significantly lower calcification, bearing capacity and damage after being crimped, a better hydrodynamic and adaption with lower cardiac output and deformation of implantation position. Meanwhile the dehydration process of pericardium almost had preserved all the biochemical advantages of two materials. CONCLUSION: In this study, porcine and bovine pericardium showed some significant differences in biochemical, mechanical properties and hydrodynamics. According to the results, it was presumed that the thinner porcine pericardium might be more suitable for THV of right heart system. Meanwhile, more attention should be taken for the calcification of THV made by the bovine pericardium.

A computationally efficient dynamic model of human epicardial tissue.

We present a new phenomenological model of human ventricular epicardial cells and we test its reentry dynamics. The model is derived from the Rogers-McCulloch formulation of the FitzHugh-Nagumo equations and represents the total ionic current divided into three contributions corresponding to the excitatory, recovery and transient outward currents. Our model reproduces the main characteristics of human epicardial tissue, including action potential amplitude and morphology, upstroke velocity, and action potential duration and conduction velocity restitution curves. The reentry dynamics is stable, and the dominant period is about 270 ms, which is comparable to clinical values. The proposed model is the first phenomenological model able to accurately resemble human experimental data by using only 3 state variables and 17 parameters. Indeed, it is more computationally efficient than existing models (i.e., almost two times faster than the minimal ventricular model). Beyond the computational efficiency, the low number of parameters facilitates the process of fitting the model to the experimental data.

Single-cell transcriptomics defines heterogeneity of epicardial cells and fibroblasts within the infarcted murine heart.

In the adult heart, the epicardium becomes activated after injury, contributing to cardiac healing by secretion of paracrine factors. Here, we analyzed by single-cell RNA sequencing combined with RNA in situ hybridization and lineage tracing of Wilms tumor protein 1-positive (WT1+) cells, the cellular composition, location, and hierarchy of epicardial stromal cells (EpiSC) in comparison to activated myocardial fibroblasts/stromal cells in infarcted mouse hearts. We identified 11 transcriptionally distinct EpiSC populations, which can be classified into three groups, each containing a cluster of proliferating cells. Two groups expressed cardiac specification markers and sarcomeric proteins suggestive of cardiomyogenic potential. Transcripts of hypoxia-inducible factor (HIF)-1α and HIF-responsive genes were enriched in EpiSC consistent with an epicardial hypoxic niche. Expression of paracrine factors was not limited to WT1+ cells but was a general feature of activated cardiac stromal cells. Our findings provide the cellular framework by which myocardial ischemia may trigger in EpiSC the formation of cardioprotective/regenerative responses.

Depolarization of the Atrial Subepicardium in Rats with Experimentally Induced Pulmonary Hypertension.

Using an experimental model of pulmonary hypertension in rats (monocrotaline in a dose of 60 mg/kg), we revealed an additional focus of early excitation in the zone where the pulmonary veins enter the left atrium, in addition to the main focus in the sinoatrial node. Pulmonary hypertension leads to the formation of regions of early activation in the right and left atria and a significant change in the sequence of atrial depolarization. Propagation of independent excitation waves in the right and left atria increases heterogeneity of depolarization and leads to the formation of atrial arrhythmias.

Gap Junctional Communication via Connexin43 between Purkinje Fibers and Working Myocytes Explains the Epicardial Activation Pattern in the Postnatal Mouse Left Ventricle.

The mammalian ventricular myocardium forms a functional syncytium due to flow of electrical current mediated in part by gap junctions localized within intercalated disks. The connexin (Cx) subunit of gap junctions have direct and indirect roles in conduction of electrical impulse from the cardiac pacemaker via the cardiac conduction system (CCS) to working myocytes. Cx43 is the dominant isoform in these channels. We have studied the distribution of Cx43 junctions between the CCS and working myocytes in a transgenic mouse model, which had the His-Purkinje portion of the CCS labeled with green fluorescence protein. The highest number of such connections was found in a region about one-third of ventricular length above the apex, and it correlated with the peak proportion of Purkinje fibers (PFs) to the ventricular myocardium. At this location, on the septal surface of the left ventricle, the insulated left bundle branch split into the uninsulated network of PFs that continued to the free wall anteriorly and posteriorly. The second peak of PF abundance was present in the ventricular apex. Epicardial activation maps correspondingly placed the site of the first activation in the apical region, while some hearts presented more highly located breakthrough sites. Taken together, these results increase our understanding of the physiological pattern of ventricular activation and its morphological underpinning through detailed CCS anatomy and distribution of its gap junctional coupling to the working myocardium.

Impact on Mechanical Properties of 10 versus 20 Minute Treatment of Human Pericardium with Glutaraldehyde in OZAKI Procedure.

PURPOSE: The aim of this study was to analyze the effects of 10-minute (standard term) versus 20-minute treatment with glutaraldehyde (GA) on mechanical stability and physical strength of human pericardium in the setting of the OZAKI procedure. METHODS: Leftover pericardium (6 patients) was bisected directly after the operation, and one-half was further fixed for 10 additional minutes. Uniaxial tensile tests were performed and ultimate tensile strength (UTS), ultimate tensile strain (uts), and collagen elastic modulus were evaluated. RESULTS: Both treatments resulted in similar values of uniaxial stretching-generated elongations at rupture (10 minutes 25 ± 7 % vs. 20 minutes: 22 ± 5 %; p = 0.05), UTS (5.16 ± 2 MPa vs. 6.54 ± 3 MPa; p = 0.59), and collagen fiber stiffness (elastic modulus: 31.80 ± 15.05 MPa vs. 37.35 ± 15.78 MPa; p = 0.25). CONCLUSION: Prolongation of the fixation time of autologous pericardium has no significant effect on its mechanical stability; thus, extending the intraoperative treatment cannot be recommended.

Myocardial Strain Measured by Epicardial Transducers-Comparison Between Velocity Estimators.

This study describes results from an experimental ultrasound system with miniature transducers sutured directly onto the epicardial surface and used to measure heart contractions continuously. This system was used to find velocity distributions through the myocardium. The resulting velocities were used to track the motion of four layers at different depths through the myocardium and to find the regional strain in each of the four layers. Velocities inside the myocardium vary from the epicardial to the endocardial borders. Conventional velocity estimators based on Doppler and on time delay estimation were modified to better handle these variations. Results from four different velocity estimators were tested against a simulation model for ultrasound echoes from moving tissue and on ultrasound recordings from five animals. We observed that the tested velocity estimators were able to reproduce the myocardial velocity distributions, track the myocardial layer motion and estimate strain at different positions inside the myocardium for both simulated and real ultrasound recordings. The most accurate results were obtained when the digitized ultrasound scanlines were upsampled by a factor of 10 before applying cross-correlation to estimate time delays. A modified Doppler algorithm allowing the velocity to vary linearly with time throughout the duration of the pulse packet (constant acceleration Doppler) was found to be better at capturing rapidly changing velocities compared with conventional Doppler processing. The best results were obtained using upsamling and time delay estimation, but the long computation time required by this method may make it best suited in a laboratory setting. In a real-time system, the computationally quicker constant acceleration Doppler may be preferred.

lncRNA expression profiles and associated ceRNA network analyses in epicardial adipose tissue of patients with coronary artery disease.

Epicardial adipose tissue (EAT) contributes to the pathophysiological process of coronary artery disease (CAD). The expression profiles of long non-coding RNAs (lncRNA) in EAT of patients with CAD have not been well characterized. We conducted high-throughput RNA sequencing to analyze the expression profiles of lncRNA in EAT of patients with CAD compared to patients without CAD. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were executed to investigate the principal functions of the significantly dysregulated mRNAs. We confirmed a dysregulated intergenic lncRNA (lincRNA) (LINC00968) by real-time quantitative PCR (RT-qPCR). Subsequently, we constructed a ceRNA network associated with LINC00968, which included 49 mRNAs. Compared with the control group, lncRNAs and genes of EAT in CAD were characterized as metabolic active and pro-inflammatory profiles. The sequencing analysis detected 2539 known and 1719 novel lncRNAs. Then, we depicted both lncRNA and gene signatures of EAT in CAD, featuring dysregulation of genes involved in metabolism, nuclear receptor transcriptional activity, antigen presentation, chemokine signaling, and inflammation. Finally, we identified a ceRNA network as candidate modulator in EAT and its potential role in CAD. We showed the expression profiles of specific EAT lncRNA and mRNA in CAD, and a selected non-coding associated ceRNA regulatory network, which taken together, may contribute to a better understanding of CAD mechanism and provide potential therapeutic targets.Trial registration Chinese Clinical Trial Registry, No. ChiCTR1900024782.

Recapturing embryonic potential in the adult epicardium: Prospects for cardiac repair.

Research into potential targets for cardiac repair encompasses recognition of tissue-resident cells with intrinsic regenerative properties. The adult vertebrate heart is covered by mesothelium, named the epicardium, which becomes active in response to injury and contributes to repair, albeit suboptimally. Motivation to manipulate the epicardium for treatment of myocardial infarction is deeply rooted in its central role in cardiac formation and vasculogenesis during development. Moreover, the epicardium is vital to cardiac muscle regeneration in lower vertebrate and neonatal mammalian-injured hearts. In this review, we discuss our current understanding of the biology of the mammalian epicardium in development and injury. Considering present challenges in the field, we further contemplate prospects for reinstating full embryonic potential in the adult epicardium to facilitate cardiac regeneration.

Cell Sheet Comprised of Mesenchymal Stromal Cells Overexpressing Stem Cell Factor Promotes Epicardium Activation and Heart Function Improvement in a Rat Model of Myocardium Infarction.

Cell therapy of the post-infarcted myocardium is still far from clinical use. Poor survival of transplanted cells, insufficient regeneration, and replacement of the damaged tissue limit the potential of currently available cell-based techniques. In this study, we generated a multilayered construct from adipose-derived mesenchymal stromal cells (MSCs) modified to secrete stem cell factor, SCF. In a rat model of myocardium infarction, we show that transplantation of SCF producing cell sheet induced activation of the epicardium and promoted the accumulation of c-kit positive cells in ischemic muscle. Morphometry showed the reduction of infarct size (16%) and a left ventricle expansion index (0.12) in the treatment group compared to controls (24-28%; 0.17-0.32). The ratio of viable myocardium was more than 1.5-fold higher, reaching 49% compared to the control (28%) or unmodified cell sheet group (30%). Finally, by day 30 after myocardium infarction, SCF-producing cell sheet transplantation increased left ventricle ejection fraction from 37% in the control sham-operated group to 53%. Our results suggest that, combining the genetic modification of MSCs and their assembly into a multilayered construct, we can provide prolonged pleiotropic effects to the damaged heart, induce endogenous regenerative processes, and improve cardiac function.

Epicardial Ablation Biophysics and Novel Radiofrequency Energy Delivery Techniques.

Important physiologic and anatomic differences exist between the epicardium and endocardium, particularly of the ventricles, and these differences affect ablation biophysics. Absence of passive convective effects conferred by circulating blood as well as the presence of epicardial fat and vessels and absence of intracavitary ridges and structures affect ablation lesion size when performing epicardial catheter-based ablation, whether using radiofrequency or cryothermal energy. Understanding differential effects in each environment is important in informing strategies to increase ablation lesion depth. When using actively cooled radiofrequency ablation, local impedance can be altered to selectively augment energy delivery.

Tough pNAGA hydrogel hybridized porcine pericardium for the pre-mounted TAVI valve with improved anti-tearing properties and hemocompatibility.

The rate of adoption of transcatheter aortic valve implantation (TAVI) is increasing rapidly, due to the procedure being less invasive. However, TAVI still faces problems relating to durability, the potential incidence of thrombosis, and the inconvenience of storage in glutaraldehyde (Glut) solution. In this work, a tough hydrogel poly(N-acryloyl glycinamide) (pNAGA) is hybridized with Glut-crosslinked porcine pericardium (Glut-PP) via in situ polymerization and glycerolization, so as to obtain dry leafet material for the fabrication of a pre-mounted bioprosthetic heart valve (BHV). The tensile strength, anti-shearing, and anti-tearing properties of the valve are significantly improved by the process of hydrogel hybridization. Following a period of dry-state compression as a simulation for the crimping process of pre-mounted TAV, pNAGA/Glut-PP showed full recovery without structural damage when fully rehydrated. The introduction of pNAGA also improved the blood compatibility of the tissue, with less clot formation and fewer blood cells adhering to the surface of pNAGA/Glut-PP than is found with Glut-PP. Subcutaneous implantation in rats showed that pNAGA/Glut-PP induced a decreased inflammatory response compared with Glut-PP. These results indicate that the strategy for hybridization with hydrogel could be a potential method for preparing pre-mounted TAVs with an improved performance.

Spatial-Temporal Signals and Clinical Indices in Electrocardiographic Imaging (I): Preprocessing and Bipolar Potentials.

During the last years, Electrocardiographic Imaging (ECGI) has emerged as a powerful and promising clinical tool to support cardiologists. Starting from a plurality of potential measurements on the torso, ECGI yields a noninvasive estimation of their causing potentials on the epicardium. This unprecedented amount of measured cardiac signals needs to be conditioned and adapted to current knowledge and methods in cardiac electrophysiology in order to maximize its support to the clinical practice. In this setting, many cardiac indices are defined in terms of the so-called bipolar electrograms, which correspond with differential potentials between two spatially close potential measurements. Our aim was to contribute to the usefulness of ECGI recordings in the current knowledge and methods of cardiac electrophysiology. For this purpose, we first analyzed the basic stages of conventional cardiac signal processing and scrutinized the implications of the spatial-temporal nature of signals in ECGI scenarios. Specifically, the stages of baseline wander removal, low-pass filtering, and beat segmentation and synchronization were considered. We also aimed to establish a mathematical operator to provide suitable bipolar electrograms from the ECGI-estimated epicardium potentials. Results were obtained on data from an infarction patient and from a healthy subject. First, the low-frequency and high-frequency noises are shown to be non-independently distributed in the ECGI-estimated recordings due to their spatial dimension. Second, bipolar electrograms are better estimated when using the criterion of the maximum-amplitude difference between spatial neighbors, but also a temporal delay in discrete time of about 40 samples has to be included to obtain the usual morphology in clinical bipolar electrograms from catheters. We conclude that spatial-temporal digital signal processing and bipolar electrograms can pave the way towards the usefulness of ECGI recordings in the cardiological clinical practice. The companion paper is devoted to analyzing clinical indices obtained from ECGI epicardial electrograms measuring waveform variability and repolarization tissue properties.