DOSE EVALUATION AND PROPOSAL OF LOCAL DIAGNOSTIC REFERENCE LEVELS FOR PAEDIATRIC CARDIAC CATHETERIZATIONS PERFORMED ON A HIGH-SENSITIVITY ANGIOGRAPHIC SYSTEM ALLOWING LOW-DOSE IMAGING.

Radiation doses from paediatric cardiac catheterizations were analysed based on procedure type and patient weight, and local diagnostic reference levels (LDRLs) were proposed. The procedures were performed on a Siemens Artis Q.zen biplane system with high-sensitivity detectors allowing low-dose imaging. Good radiological practice, e.g. adapting dose level and frame rate continuously and minimizing the x-ray field with collimators, was routine during procedures. The median total dose-area-product (DAP) value was 58 µGym2 for diagnostic catheterizations, 48 µGym2 for interventional catheterizations and 33 µGym2 for myocardial biopsies. The median DAP per body weight was 4.0 µGym2 kg-1. The median total fluoroscopy time varied from 6.0 min for myocardial biopsies, to 9.7 and 10.5 min, respectively, for diagnostic and interventional catheterizations. The third quartile of the DAP values in each weight group was proposed as LDRL. LDRLs for fluoroscopy time were determined solely based on procedure type, due to the absence of weight dependence.

A robust multi-objective optimization framework to capture both cellular and intercellular properties in cardiac cellular model tuning: Analyzing different regions of membrane resistance profile in parameter fitting.

Mathematical models of cardiac cells have been established to broaden understanding of cardiac function. In the process of developing electrophysiological models for cardiac myocytes, precise parameter tuning is a crucial step. The membrane resistance (Rm) is an essential feature obtained from cardiac myocytes. This feature reflects intercellular coupling and affects important phenomena, such as conduction velocity, and early after-depolarizations, but it is often overlooked during the phase of parameter fitting. Thus, the traditional parameter fitting that only includes action potential (AP) waveform may yield incorrect values for Rm. In this paper, a novel multi-objective parameter fitting formulation is proposed and tested that includes different regions of the Rm profile as additional objective functions for optimization. As Rm depends on the transmembrane voltage (Vm) and exhibits singularities for some specific values of Vm, analyses are conducted to carefully select the regions of interest for the proper characterization of Rm. Non-dominated sorting genetic algorithm II is utilized to solve the proposed multi-objective optimization problem. To verify the efficacy of the proposed problem formulation, case studies and comparisons are carried out using multiple models of human cardiac ventricular cells. Results demonstrate Rm is correctly reproduced by the tuned cell models after considering the curve of Rm obtained from the late phase of repolarization and Rm value calculated in the rest phase as additional objectives. However, relative deterioration of the AP fit is observed, demonstrating trade-off among the objectives. This framework can be useful for a wide range of applications, including the parameters fitting phase of the cardiac cell model development and investigation of normal and pathological scenarios in which reproducing both cellular and intercellular properties are of great importance.

Pacemaker treatment after Fontan surgery-A Swedish national study.

OBJECTIVE: Fontan surgery is performed in children with univentricular heart defects. Previous data regarding permanent pacemaker implantation frequency and indications in Fontan patients are limited and conflicting. We examined the prevalence of and risk factors for pacemaker treatment in a consecutive national cohort of patients after Fontan surgery in Sweden. METHODS: We retrospectively reviewed all Swedish patients who underwent Fontan surgery from 1982 to 2017 (n = 599). RESULTS: After a mean follow-up of 12.2 years, 13% (78/599) of the patients with Fontan circulation had received pacemakers. Patients operated with the extracardiac conduit (EC) type of total cavopulmonary connection had a significantly lower prevalence of pacemaker implantation (6%) than patients with lateral tunnel (LT; 17%). Mortality did not differ between patients with (8%) and without pacemaker (5%). The most common pacemaker indication was sinus node dysfunction (SND) (64%). Pacemaker implantation due to SND was less common among patients with EC. Pacemaker implantation was significantly more common in patients with mitral atresia (MA; 44%), double outlet right ventricle (DORV; 24%) and double inlet left ventricle (DILV; 20%). In contrast, patients with pulmonary atresia with intact ventricular septum and hypoplastic left heart syndrome were significantly less likely to receive a pacemaker (3% and 6%, respectively). CONCLUSIONS: Thirteen percent of Fontan patients received a permanent pacemaker, most frequently due to SND. EC was associated with a significantly lower prevalence of pacemaker than LT. Permanent pacemaker was more common in patients with MA, DORV, and DILV.

Sinus node dysfunction in patients with Fontan circulation: could heart rate variability be a predictor for pacemaker implantation?

Sinus node dysfunction (SND) causes significant morbidity in patients after Fontan surgery. Heart rate variability (HRV) reflects the autonomic regulation of the heart, and changes in HRV have been associated with SND in adults. We aimed to study whether changes in HRV could be detected in 24-h electrocardiographic (ECG) recordings in Fontan patients with SND. We compared HRV results from two patient groups; patients with Fontan circulation who later required a pacemaker due to severe SND (n = 12) and patients with Fontan circulation and SND, without indication for pacemaker treatment (n = 11), with two control groups; patients with Fontan circulation without SND (n = 90) and healthy controls (n = 66). The Poincaré plot index SD2 (representing changes in heart rate over 24-h) and the very low-frequency (VLF) HRV component were significantly higher in both SND groups, both compared with healthy controls and patients with Fontan circulation without SND. In SND patients with pacemakers, SD2 and VLF were slightly reduced compared to SND patients without pacemaker (p = 0.06). In conclusion, in Fontan patients with SND the HRV is significantly higher compared to healthy controls and Fontan patients without SND. However, in patients with severe SND requiring pacemaker, SD2 and VLF tended to be lower than in patients with SND without pacemaker, which could indicate a reduced diurnal HRV in addition to the severe bradycardia. This is a small study, but our results indicate that HRV analysis might be a useful method in the follow-up of Fontan patients regarding development of SND.

Mechanistic basis of an epistatic interaction reducing age at onset in hereditary spastic paraplegia.

Many genetic neurological disorders exhibit variable expression within affected families, often exemplified by variations in disease age at onset. Epistatic effects (i.e. effects of modifier genes on the disease gene) may underlie this variation, but the mechanistic basis for such epistatic interactions is rarely understood. Here we report a novel epistatic interaction between SPAST and the contiguous gene DPY30, which modifies age at onset in hereditary spastic paraplegia, a genetic axonopathy. We found that patients with hereditary spastic paraplegia caused by genomic deletions of SPAST that extended into DPY30 had a significantly younger age at onset. We show that, like spastin, the protein encoded by SPAST, the DPY30 protein controls endosomal tubule fission, traffic of mannose 6-phosphate receptors from endosomes to the Golgi, and lysosomal ultrastructural morphology. We propose that additive effects on this pathway explain the reduced age at onset of hereditary spastic paraplegia in patients who are haploinsufficient for both genes.

Key differences between 13 KRAS mutation detection technologies and their relevance for clinical practice.

INTRODUCTION: This study assessed KRAS mutation detection and functional characteristics across 13 distinct technologies and assays available in clinical practice, in a blinded manner. METHODS: Five distinct KRAS-mutant cell lines were used to study five clinically relevant KRAS mutations: p.G12C, p.G12D, p.G12V, p.G13D and p.Q61H. 50 cell line admixtures with low (50 and 100) mutant KRAS allele copies at 20%, 10%, 5%, 1% and 0.5% frequency were processed using quantitative PCR (qPCR) (n=3), matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) (n=2), next-generation sequencing (NGS) (n=6), digital PCR (n=1) and Sanger capillary sequencing (n=1) assays. Important performance differences were revealed, particularly assay sensitivity and turnaround time. RESULTS: Overall 406/728 data points across all 13 technologies were identified correctly. Successful genotyping of admixtures ranged from 0% (Sanger sequencing) to 100% (NGS). 5/6 NGS platforms reported similar allelic frequency for each sample. One NGS assay detected mutations down to a frequency of 0.5% and correctly identified all 56 samples (Oncomine Focus Assay, Thermo Fisher Scientific). One qPCR (Idylla, Biocartis) and MALDI-TOF (UltraSEEK, Agena Bioscience) assay identified 96% (all 100 copies and 23/25 at 50 copies input) and 92% (23/25 at 100 copies and 23/25 at 50 copies input) of samples, respectively. The digital PCR assay (KRAS PrimePCR ddPCR, Bio-Rad Laboratories) identified 60% (100 copies) and 52% (50 copies) of samples correctly. Turnaround time from sample to results ranged from ~2 hours (Idylla CE-IVD) to 2 days (TruSight Tumor 15 and Sentosa CE-IVD), to 2 weeks for certain NGS assays; the level of required expertise ranged from minimal (Idylla CE-IVD) to high for some technologies. DISCUSSION: This comprehensive parallel assessment used high molecular weight cell line DNA as a model system to address key questions for a laboratory when implementing routine KRAS testing. As most of the technologies are available for additional molecular biomarkers, this study may be informative for other applications.

Transgenic mice overexpressing desmocollin-2 (DSC2) develop cardiomyopathy associated with myocardial inflammation and fibrotic remodeling.

BACKGROUND: Arrhythmogenic cardiomyopathy is an inherited heart muscle disorder leading to ventricular arrhythmias and heart failure, mainly as a result of mutations in cardiac desmosomal genes. Desmosomes are cell-cell junctions mediating adhesion of cardiomyocytes; however, the molecular and cellular mechanisms underlying the disease remain widely unknown. Desmocollin-2 is a desmosomal cadherin serving as an anchor molecule required to reconstitute homeostatic intercellular adhesion with desmoglein-2. Cardiac specific lack of desmoglein-2 leads to severe cardiomyopathy, whereas overexpression does not. In contrast, the corresponding data for desmocollin-2 are incomplete, in particular from the view of protein overexpression. Therefore, we developed a mouse model overexpressing desmocollin-2 to determine its potential contribution to cardiomyopathy and intercellular adhesion pathology. METHODS AND RESULTS: We generated transgenic mice overexpressing DSC2 in cardiac myocytes. Transgenic mice developed a severe cardiac dysfunction over 5 to 13 weeks as indicated by 2D-echocardiography measurements. Corresponding histology and immunohistochemistry demonstrated fibrosis, necrosis and calcification which were mainly localized in patches near the epi- and endocardium of both ventricles. Expressions of endogenous desmosomal proteins were markedly reduced in fibrotic areas but appear to be unchanged in non-fibrotic areas. Furthermore, gene expression data indicate an early up-regulation of inflammatory and fibrotic remodeling pathways between 2 to 3.5 weeks of age. CONCLUSION: Cardiac specific overexpression of desmocollin-2 induces necrosis, acute inflammation and patchy cardiac fibrotic remodeling leading to fulminant biventricular cardiomyopathy.

Drip 'n Ship Versus Mothership for Endovascular Treatment: Modeling the Best Transportation Options for Optimal Outcomes.

BACKGROUND AND PURPOSE: There is uncertainty regarding the best way for patients outside of endovascular-capable or Comprehensive Stroke Centers (CSC) to access endovascular treatment for acute ischemic stroke. The role of the nonendovascular-capable Primary Stroke Centers (PSC) that can offer thrombolysis with alteplase but not endovascular treatment is unclear. A key question is whether average benefit is greater with early thrombolysis at the closest PSC before transportation to the CSC (Drip 'n Ship) or with PSC bypass and direct transport to the CSC (Mothership). Ideal transportation options were mapped based on the location of their endovascular-capable CSCs and nonendovascular-capable PSCs. METHODS: Probability models for endovascular treatment were developed from the ESCAPE trial's (Endovascular Treatment for Small Core and Anterior Circulation Proximal Occlusion With Emphasis on Minimizing CT to Recanalization Times) decay curves and for alteplase treatment were extracted from the Get With The Guidelines decay curve. The time on-scene, needle-to-door-out time at the PSC, door-to-needle time at the CSC, and door-to-reperfusion time were assumed constant at 25, 20, 30, and 115 minutes, respectively. Emergency medical services transportation times were calculated using Google's Distance Matrix Application Programming Interface interfaced with MATLAB's Mapping Toolbox to create map visualizations. RESULTS: Maps were generated for multiple onset-to-first medical response times and door-to-needle times at the PSCs of 30, 60, and 90. These figures demonstrate the transportation option that yields the better modeled outcome in specific regions. The probability of good outcome is shown. CONCLUSIONS: Drip 'n Ship demonstrates that a PSC that is in close proximity to a CSC remains significant only when the PSC is able to achieve a door-to-needle time of ≤30 minutes when the CSC is also efficient.

Interplay among distinct Ca2+ conductances drives Ca2+ sparks/spontaneous transient outward currents in rat cerebral arteries.

KEY POINTS: Distinct Ca2+ channels work in a coordinated manner to grade Ca2+ spark/spontaneous transient outward currents (STOCs) in rat cerebral arteries. The relative contribution of each Ca2+ channel to Ca2+ spark/STOC production depends upon their biophysical properties and the resting membrane potential of smooth muscle. Na+ /Ca2+ exchanger, but not TRP channels, can also facilitate STOC production. ABSTRACT: Ca2+ sparks are generated in a voltage-dependent manner to initiate spontaneous transient outward currents (STOCs), events that moderate arterial constriction. In this study, we defined the mechanisms by which membrane depolarization increases Ca2+ sparks and subsequent STOC production. Using perforated patch clamp electrophysiology and rat cerebral arterial myocytes, we monitored STOCs in the presence and absence of agents that modulate Ca2+ entry. Beginning with CaV 3.2 channel inhibition, Ni2+ was shown to decrease STOC frequency in cells held at hyperpolarized (-40 mV) but not depolarized (-20 mV) voltages. In contrast, nifedipine, a CaV 1.2 inhibitor, markedly suppressed STOC frequency at -20 mV but not -40 mV. These findings aligned with the voltage-dependent profiles of L- and T-type Ca2+ channels. Furthermore, computational and experimental observations illustrated that Ca2+ spark production is intimately tied to the activity of both conductances. Intriguingly, this study observed residual STOC production at depolarized voltages that was independent of CaV 1.2 and CaV 3.2. This residual component was insensitive to TRPV4 channel modulation and was abolished by Na+ /Ca2+ exchanger blockade. In summary, our work highlights that the voltage-dependent triggering of Ca2+ sparks/STOCs is not tied to a single conductance but rather reflects an interplay among multiple Ca2+ permeable pores with distinct electrophysiological properties. This integrated orchestration enables smooth muscle to grade Ca2+ spark/STOC production and thus precisely tune negative electrical feedback.

Swedish children with hereditary angioedema report good overall health and quality of life despite symptoms.

AIM: Few studies have been published on children with hereditary angioedema (HAE), an autosomal dominant disease caused by mutations on chromosome 11. This study explored various aspects of the disease in the Swedish paediatric population. METHODS: A retrospective questionnaire was sent to all 36 Swedish children known to have HAE, and a physician carried out follow-up telephone interviews. RESULTS: Most of the questionnaires were completed by the parents of 31 (86%) children with HAE, with or without their input, at a median age of nine years (range 1-17), and the physician also interviewed 29. HAE symptoms were experienced by 23 children, including abdominal attacks (96%), skin swelling (78%) and swelling in the mouth and/or upper airways (52%). Psychological stress was the most common trigger for abdominal attacks and trauma and sports triggered skin swelling. The majority (n = 19) had access to complement-1 esterase inhibitor concentrate at home. Current health and quality of life were generally rated as good, independent of whether the child had experienced HAE symptoms or not. CONCLUSION: Most children with HAE had experienced abdominal attacks and skin swelling, but their overall health and quality of life were generally perceived to be good.

Ultrasensitive Detection of Multiplexed Somatic Mutations Using MALDI-TOF Mass Spectrometry.

Multiplex detection of low-frequency mutations is becoming a necessary diagnostic tool for clinical laboratories interested in noninvasive prognosis and prediction. Challenges include the detection of minor alleles among abundant wild-type alleles, the heterogeneous nature of tumors, and the limited amount of available tissue. A method that can reliably detect minor variants <1% in a multiplexed reaction using a platform amenable to a variety of throughputs would meet these requirements. We developed a novel approach, UltraSEEK, for high-throughput, multiplexed, ultrasensitive mutation detection and used it for detection of mutant sequence mixtures as low as 0.1% minor allele frequency. The process consisted of multiplex PCR, followed by mutation-specific, single-base extension using chain terminators labeled with a moiety for solid phase capture. The captured and enriched products were then identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. For verification, we successfully analyzed ultralow fractions of mutations in a set of characterized cell lines, and included a direct comparison to droplet digital PCR. Finally, we verified the specificity in a set of 122 paired tumor and circulating cell-free DNA samples from melanoma patients. Our results show that the UltraSEEK chemistry is a particularly powerful approach for the detection of somatic variants, with the potential to be an invaluable resource to investigators in saving time and material without compromising analytical sensitivity and accuracy.

Elevated Troponin Levels in Acute Stroke Patients Predict Long-term Mortality.

BACKGROUND: Elevated plasma levels of troponin in acute stroke patients are common and have in several studies been shown to predict in-hospital and short-term mortality. Little is, however, known about the long-term prognosis of these patients. The aim of this study was to determine patient characteristics and 5-year mortality in patients with acute stroke and troponin elevation on admission. METHODS: A retrospective cohort study of all consecutive patients with acute stroke and a plasma troponin I (TnI) analyzed on admission to Danderyd Hospital between January 1, 2005, and January 1, 2006 (n = 247). Patient characteristics were obtained from the Swedish National Stroke Register, Riksstroke, as well as hospital records. Mortality data were obtained from the Swedish Cause of Death Register. RESULTS: There were 133 patients (54%) with TnI less than .03 µg/L (normal), 74 patients (30%) with TnI .03-.11 µg/L (low elevation), and 40 patients (16%) with TnI greater than .11 µg/L (high elevation). TnI elevations were associated with a higher age, prior ischemic stroke, chronic heart failure, renal insufficiency, stroke severity, and ST segment elevation or depression on admission. The rate of hyperlipidemia decreased with increasing TnI. Adjusted for age and comorbidity, elevated TnI values on admission had a significantly and sustained increased mortality over the 5-year follow-up, with a hazard ratio of 1.90 (95% confidence interval, 1.33-2.70). CONCLUSIONS: Troponin elevation in patients with acute stroke, even when adjusted for several possible confounders, is associated with an almost 2-fold increased risk of 5-year mortality.

Strategies for validation and testing of DNA methylation biomarkers.

DNA methylation is a stable covalent epigenetic modification of primarily CpG dinucleotides that has recently gained considerable attention for its use as a biomarker in different clinical settings, including disease diagnosis, prognosis and therapeutic response prediction. Although the advent of genome-wide DNA methylation profiling in primary disease tissue has provided a manifold resource for biomarker development, only a tiny fraction of DNA methylation-based assays have reached clinical testing. Here, we provide a critical overview of different analytical methods that are suitable for biomarker validation, including general study design considerations, which might help to streamline epigenetic marker development. Furthermore, we highlight some of the recent marker validation studies and established markers that are currently commercially available for assisting in clinical management of different cancers.

A multiplexed transcription activator-like effector system for detecting specific DNA sequences.

Transcription activator-like effectors (TALEs), originating from the Xanthomonas genus of bacteria, bind to specific DNA sequences based on amino acid sequence in the repeat-variable diresidue (RVD) positions of the protein. By altering these RVDs, it has been shown that a TALE protein can be engineered to bind virtually any DNA sequence of interest. The possibility of multiplexing TALEs for the purposes of identifying specific DNA sequences has yet to be explored. Here, we demonstrate a system in which a TALE protein bound to a nitrocellulose strip has been utilized to capture purified DNA, which is then detected using the binding of a second distinct TALE protein conjugated to a protein tag that is then detected by a dot blot. This system provides a signal only when both TALEs bind to their respective sequences, further demonstrating the specificity of the TALE binding.

Fitting membrane resistance along with action potential shape in cardiac myocytes improves convergence: application of a multi-objective parallel genetic algorithm.

Fitting parameter sets of non-linear equations in cardiac single cell ionic models to reproduce experimental behavior is a time consuming process. The standard procedure is to adjust maximum channel conductances in ionic models to reproduce action potentials (APs) recorded in isolated cells. However, vastly different sets of parameters can produce similar APs. Furthermore, even with an excellent AP match in case of single cell, tissue behaviour may be very different. We hypothesize that this uncertainty can be reduced by additionally fitting membrane resistance (Rm). To investigate the importance of Rm, we developed a genetic algorithm approach which incorporated Rm data calculated at a few points in the cycle, in addition to AP morphology. Performance was compared to a genetic algorithm using only AP morphology data. The optimal parameter sets and goodness of fit as computed by the different methods were compared. First, we fit an ionic model to itself, starting from a random parameter set. Next, we fit the AP of one ionic model to that of another. Finally, we fit an ionic model to experimentally recorded rabbit action potentials. Adding the extra objective (Rm, at a few voltages) to the AP fit, lead to much better convergence. Typically, a smaller MSE (mean square error, defined as the average of the squared error between the target AP and AP that is to be fitted) was achieved in one fifth of the number of generations compared to using only AP data. Importantly, the variability in fit parameters was also greatly reduced, with many parameters showing an order of magnitude decrease in variability. Adding Rm to the objective function improves the robustness of fitting, better preserving tissue level behavior, and should be incorporated.

Ca(V)3.2 channels and the induction of negative feedback in cerebral arteries.

RATIONALE: T-type (CaV3.1/CaV3.2) Ca(2+) channels are expressed in rat cerebral arterial smooth muscle. Although present, their functional significance remains uncertain with findings pointing to a variety of roles. OBJECTIVE: This study tested whether CaV3.2 channels mediate a negative feedback response by triggering Ca(2+) sparks, discrete events that initiate arterial hyperpolarization by activating large-conductance Ca(2+)-activated K(+) channels. METHODS AND RESULTS: Micromolar Ni(2+), an agent that selectively blocks CaV3.2 but not CaV1.2/CaV3.1, was first shown to depolarize/constrict pressurized rat cerebral arteries; no effect was observed in CaV3.2(-/-) arteries. Structural analysis using 3-dimensional tomography, immunolabeling, and a proximity ligation assay next revealed the existence of microdomains in cerebral arterial smooth muscle which comprised sarcoplasmic reticulum and caveolae. Within these discrete structures, CaV3.2 and ryanodine receptor resided in close apposition to one another. Computational modeling revealed that Ca(2+) influx through CaV3.2 could repetitively activate ryanodine receptor, inducing discrete Ca(2+)-induced Ca(2+) release events in a voltage-dependent manner. In keeping with theoretical observations, rapid Ca(2+) imaging and perforated patch clamp electrophysiology demonstrated that Ni(2+) suppressed Ca(2+) sparks and consequently spontaneous transient outward K(+) currents, large-conductance Ca(2+)-activated K(+) channel mediated events. Additional functional work on pressurized arteries noted that paxilline, a large-conductance Ca(2+)-activated K(+) channel inhibitor, elicited arterial constriction equivalent, and not additive, to Ni(2+). Key experiments on human cerebral arteries indicate that CaV3.2 is present and drives a comparable response to moderate constriction. CONCLUSIONS: These findings indicate for the first time that CaV3.2 channels localize to discrete microdomains and drive ryanodine receptor-mediated Ca(2+) sparks, enabling large-conductance Ca(2+)-activated K(+) channel activation, hyperpolarization, and attenuation of cerebral arterial constriction.

Cri du chat syndrome and primary ciliary dyskinesia: a common genetic cause on chromosome 5p.

Cri du chat syndrome (CdCS) and primary ciliary dyskinesia (PCD) are rare diseases that present with frequent respiratory symptoms. PCD can be caused by hemizygous DNAH5 mutation in combination with a 5p segmental deletion attributable to CdCS on the opposite chromosome. Chronic oto-sino-pulmonary symptoms or organ laterality defects in CdCS should prompt an evaluation for PCD.

The role of Purkinje-myocardial coupling during ventricular arrhythmia: a modeling study.

The Purkinje system is the fast conduction network of the heart which couples to the myocardium at discrete sites called Purkinje-Myocyte Junctions (PMJs). However, the distribution and number of PMJs remains elusive, as does whether a particular PMJ is functional. We hypothesized that the Purkinje system plays a role during reentry and that the number of functional PMJs affect reentry dynamics. We used a computer finite element model of rabbit ventricles in which we varied the number of PMJs. Sustained, complex reentry was induced by applying an electric shock and the role of the Purkinje system in maintaining the arrhythmia was assessed by analyzing phase singularities, frequency of activation, and bidirectional propagation at PMJs. For larger junctional resistances, increasing PMJ density increased the mean firing rate in the Purkinje system, the percentage of successful retrograde conduction at PMJs, and the incidence of wave break on the epicardium. However, the mean firing of the ventricles was not affected. Furthermore, increasing PMJ density above 13/[Formula: see text] did not alter reentry dynamics. For lower junctional resistances, the trend was not as clear. We conclude that Purkinje system topology affects reentry dynamics and conditions which alter PMJ density can alter reentry dynamics.