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Aims Conflicting data exist supporting differing mechanisms for sustaining ventricular fibrillation (VF), ranging from disorganised multiple-wavelet activation to organised rotational activities (RAs). Abnormal gap junction (GJ) coupling and fibrosis are important in initiation and maintenance of VF. We investigated whether differing ventricular fibrosis patterns and the degree of GJ coupling affected the underlying VF mechanism. Methods and Results Optical mapping of 65 Langendorff-perfused rat hearts was performed to study VF mechanisms in control hearts with acute GJ modulation, and separately in three differing chronic ventricular fibrosis models; compact (CF), diffuse (DiF) and patchy (PF). VF dynamics were quantified with phase mapping and frequency dominance index (FDI) analysis, a power ratio of the highest amplitude dominant frequency in the cardiac frequency spectrum. Enhanced GJ coupling with rotigaptide (n = 10) progressively organised fibrillation in a concentration-dependent manner; increasing FDI (0nM: 0.53±0.04, 80nM: 0.78±0.03, p

The analysis of complex mechanisms underlying ventricular fibrillation (VF) and atrial fibrillation (AF) requires sophisticated tools for studying spatio-temporal action potential (AP) propagation dynamics. However, fibrillation analysis tools are often custom-made or proprietary, and vary between research groups. With no optimal standardised framework for analysis, results from different studies have led to disparate findings. Given the technical gap, here we present a comprehensive framework and set of principles for quantifying properties of wavefront dynamics in phase-processed data recorded during myocardial fibrillation with potentiometric dyes. Phase transformation of the fibrillatory data is particularly useful for identifying self-perpetuating spiral waves or rotational drivers (RDs) rotating around a phase singularity (PS). RDs have been implicated in sustaining fibrillation, and thus accurate localisation and quantification of RDs is crucial for understanding specific fibrillatory mechanisms. In this work, we assess how variation of analysis parameters and thresholds in the tracking of PSs and quantification of RDs could result in different interpretations of the underlying fibrillation mechanism. These techniques have been described and applied to experimental AF and VF data, and AF simulations, and examples are provided from each of these data sets to demonstrate the range of fibrillatory behaviours and adaptability of these tools. The presented methodologies are available as an open source software and offer an off-the-shelf research toolkit for quantifying and analysing fibrillatory mechanisms.

Engineered heart tissue (EHT) strategies, by combining cells within a hydrogel matrix, may be a novel therapy for heart failure. EHTs restore cardiac function in rodent injury models, but more data are needed in clinically relevant settings. Accordingly, an upscaled EHT patch (2.5 cm × 1.5 cm × 1.5 mm) consisting of up to 20 million human induced pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) embedded in a fibrin-based hydrogel was developed. A rabbit myocardial infarction model was then established to test for feasibility and efficacy. Our data showed that hPSC-CMs in EHTs became more aligned over 28 days and had improved contraction kinetics and faster calcium transients. Blinded echocardiographic analysis revealed a significant improvement in function in infarcted hearts that received EHTs, along with reduction in infarct scar size by 35%. Vascularization from the host to the patch was observed at week 1 and stable to week 4, but electrical coupling between patch and host heart was not observed. In vivo telemetry recordings and ex vivo arrhythmia provocation protocols showed that the patch was not pro-arrhythmic. In summary, EHTs improved function and reduced scar size without causing arrhythmia, which may be due to the lack of electrical coupling between patch and host heart.

Background: Atrial fibrillation (AF) and ventricular fibrillation (VF) are complex heart rhythm disorders and may be sustained by distinct electrophysiological mechanisms. Disorganised self-perpetuating multiple-wavelets and organised rotational drivers (RDs) localising to specific areas are both possible mechanisms by which fibrillation is sustained. Determining the underlying mechanisms of fibrillation may be helpful in tailoring treatment strategies. We investigated whether global fibrillation organisation, a surrogate for fibrillation mechanism, can be determined from electrocardiograms (ECGs) using band-power (BP) feature analysis and machine learning.Methods: In this study, we proposed a novel ECG classification framework to differentiate fibrillation organisation levels. BP features were derived from surface ECGs and fed to a linear discriminant analysis classifier to predict fibrillation organisation level. Two datasets, single-channel ECGs of rat VF (n = 9) and 12-lead ECGs of human AF (n = 17), were used for model evaluation in a leave-one-out (LOO) manner.Results: The proposed method correctly predicted the organisation level from rat VF ECG with the sensitivity of 75%, specificity of 80%, and accuracy of 78%, and from clinical AF ECG with the sensitivity of 80%, specificity of 92%, and accuracy of 88%.Conclusion: Our proposed method can distinguish between AF/VF of different global organisation levels non-invasively from the ECG alone. This may aid in patient selection and guiding mechanism-directed tailored treatment strategies.

Background Survivors of myocardial infarction are at increased risk of late ventricular arrhythmias, with infarct size and scar heterogeneity being key determinants of arrhythmic risk. Gap junctions facilitate the passage of small ions and morphogenic cell signaling between myocytes. We hypothesized that gap junctions enhancement during infarction–reperfusion modulates structural and electrophysiological remodeling and reduces late arrhythmogenesis. Methods and Results Infarction–reperfusion surgery was carried out in male Sprague‐Dawley rats followed by 7 days of rotigaptide or saline administration. The in vivo and ex vivo arrhythmogenicity was characterized by programmed electrical stimulation 3 weeks later, followed by diffusion‐weighted magnetic resonance imaging and Masson's trichrome histology. Three weeks after 7‐day postinfarction administration of rotigaptide, ventricular tachycardia/ventricular fibrillation was induced on programmed electrical stimulation in 20% and 53% of rats, respectively (rotigaptide versus control), resulting in reduction of arrhythmia score (3.2 versus 1.4, P =0.018), associated with the reduced magnetic resonance imaging parameters fractional anisotropy (fractional anisotropy: −5% versus −15%; P =0.062) and mean diffusivity (mean diffusivity: 2% versus 6%, P =0.042), and remodeling of the 3‐dimensional laminar structure of the infarct border zone with reduction of the mean (16° versus 19°, P =0.013) and the dispersion (9° versus 12°, P =0.015) of the myofiber transverse angle. There was no change in ECG features, spontaneous arrhythmias, or mortality. Conclusions Enhancement of gap junctions function by rotigaptide administered during the early healing phase in reperfused infarction reduces later complexity of infarct scar morphology and programmed electrical stimulation–induced arrhythmias, and merits further exploration as a feasible and practicable intervention in the acute myocardial infarction management to reduce late arrhythmic risk.

Cardiac defibrillation through high-energy shocks has remained the most effective life-saving intervention in ventricular arrhythmias. Historically, the pain associated with high-energy shocks has impeded its use in cardioverting atrial fibrillation (AF) through an implantable device in ambulant patients. The advent of optical mapping of arrhythmia mechanisms over the last 30 years has led to a more in-depth understanding of defibrillation mechanisms and to the discovery that low-energy shocks can generate virtual electrode polarizations (VEP) throughout the captured myocardium due to a heterogeneous spread of transmembrane potentials post-shock. VEP have been shown to generate new wavefronts that collide with and extinguish fibrillatory activity. Multiple closely coupled low-energy defibrillation shocks delivered in stages through atrial and coronary sinus defibrillation leads, the so-called multistage defibrillation therapy or multipulse therapy (MPT), have been shown to terminate AF in preclinical animal studies with energies below the pain threshold. MPT generates successive new VEP-induced wavefronts that collide with and eventually extinguishing all fibrillation wavefronts in stages. Multipulse therapy has been shown to be efficacious in reducing AF burden in preclinical animal studies and is now being tested as a potential therapeutic option in reducing AF burden in a clinical study.

We describe a human and large animal Langendorff experimental apparatus for live electrophysiological studies and measure the electrophysiological changes due to gap junction uncoupling in human and porcine hearts. The resultant ex vivo intact human and porcine model can bridge the translational gap between smaller simple laboratory models and clinical research. In particular, electrophysiological models would benefit from the greater myocardial mass of a large heart due to its effects on far-field signal, electrode contact issues and motion artefacts, consequently more closely mimicking the clinical setting. Porcine (n = 9) and human (n = 4) donor hearts were perfused on a custom-designed Langendorff apparatus. Epicardial electrograms were collected at 16 sites across the left atrium and left ventricle. A total of 1 mM of carbenoxolone was administered at 5 ml/min to induce cellular uncoupling, and then recordings were repeated at the same sites. Changes in electrogram characteristics were analysed. We demonstrate the viability of a controlled ex vivo model of intact porcine and human hearts for electrophysiology with pharmacological modulation. Carbenoxolone reduces cellular coupling and changes contact electrogram features. The time from stimulus artefact to (-dV/dt)max increased between baseline and carbenoxolone (47.9 ± 4.1–67.2 ± 2.7 ms) indicating conduction slowing. The features with the largest percentage change between baseline and carbenoxolone were fractionation + 185.3%, endpoint amplitude − 106.9%, S-endpoint gradient + 54.9%, S point − 39.4%, RS ratio + 38.6% and (-dV/dt)max − 20.9%. The physiological relevance of this methodological tool is that it provides a model to further investigate pharmacologically induced pro-arrhythmic substrates.

Supplemental Digital Content is available in the text., Background: The mechanisms sustaining myocardial fibrillation remain disputed, partly due to a lack of mapping tools that can accurately identify the mechanism with low spatial resolution clinical recordings. Granger causality (GC) analysis, an econometric tool for quantifying causal relationships between complex time-series, was developed as a novel fibrillation mapping tool and adapted to low spatial resolution sequentially acquired data. Methods: Ventricular fibrillation (VF) optical mapping was performed in Langendorff-perfused Sprague-Dawley rat hearts (n=18), where novel algorithms were developed using GC-based analysis to (1) quantify causal dependence of neighboring signals and plot GC vectors, (2) quantify global organization with the causality pairing index, a measure of neighboring causal signal pairs, and (3) localize rotational drivers (RDs) by quantifying the circular interdependence of neighboring signals with the circular interdependence value. GC-based mapping tools were optimized for low spatial resolution from downsampled optical mapping data, validated against high-resolution phase analysis and further tested in previous VF optical mapping recordings of coronary perfused donor heart left ventricular wedge preparations (n=12), and adapted for sequentially acquired intracardiac electrograms during human persistent atrial fibrillation mapping (n=16). Results: Global VF organization quantified by causality pairing index showed a negative correlation at progressively lower resolutions (50% resolution: P=0.006, R2=0.38, 12.5% resolution, P=0.004, R2=0.41) with a phase analysis derived measure of disorganization, locations occupied by phase singularities. In organized VF with high causality pairing index values, GC vector mapping characterized dominant propagating patterns and localized stable RDs, with the circular interdependence value showing a significant difference in driver versus nondriver regions (0.91±0.05 versus 0.35±0.06, P=0.0002). These findings were further confirmed in human VF. In persistent atrial fibrillation, a positive correlation was found between the causality pairing index and presence of stable RDs (P=0.0005,R2=0.56). Fifty percent of patients had RDs, with a low incidence of 0.9±0.3 RDs per patient. Conclusions: GC-based fibrillation analysis can measure global fibrillation organization, characterize dominant propagating patterns, and map RDs using low spatial resolution sequentially acquired data.

Background Clinical identification of fibrillation drivers remains challenging in both atrial and ventricular fibrillation (VF). In this study, we developed novel tools using granger causality (GC) analysis for quantifying the causal relationship between neighbouring fibrillatory signals. We tested whether it was adaptable to low resolution, limited coverage and sequentially acquired data for quantifying global organisation of VF and mapping regions with stable rotational drivers (RDs). Methods Eighteen Sprague-Dawley rat hearts were perfused ex vivo for optical mapping studies. VF with differing degrees of organisation was induced with carbenoxolone (10–50μM, n=8), or prior maturation of patchy ventricular fibrosis (n=10) generated by ischaemia-reperfusion. After phase mapping, the data was downsampled to 25% of full resolution to develop validated GC-based tools. The causality pairing index (CPI), a global measure of organisation, quantified propagational effects between all neighboring signals. Low-resolution GC-vector maps localized areas harboring RDs and quantified the prevalence of RDs over time using a novel index called circular interdependence value (CIV). These GC-based tools were then adapted to analyze low-resolution multi-electrode electrograms of sixteen persistent-AF (psAF) patients presenting for a first ablation procedure. Results A spectrum of fibrillatory organisation and mechanisms in VF was observed. In rat VF there was a positive correlation between CPI and the number of stable RDs (R2=0.41, p=0.004), and CIV showed a significant difference in driver vs non-driver regions (0.91±0.05 vs 0.35±0.06, p=0.0002). Similarly, in psAF patients, there was a positive correlation between CPI and the number of stable RDs (R2=0.56, p≤0.001). GC vector mapping showed that 8/16 of patients had at least one RD area, and 8/16 had chaotic activity with no RDs. Conclusion Mechanisms of myocardial fibrillation occurs along a spectrum between organized activity with discrete areas harboring RDs and disorganised myocardial activation with no RDs. GC maps can be utilised for identifying regions localising RDs with sequential mapping in limited spatial resolution and coverage. In psAF GC-based analysis accurately identified specific fibrillatory mechanisms from low-resolution mapping. GC vector mapping holds potential for development with human fibrillation data as a mapping tool for driver guided ablation. Acknowledgement/Funding BHF Programme Grant PG/16/17/32069

Background The underlying mechanism of ventricular fibrillation (VF) remains unclear. There are both experimental and clinical data to support the existence of rotational drivers (RDs), though other opposing studies suggest that VF is the result of disorganized myocardial activation. Abnormal electrical coupling between cardiomyocytes through gap junctions (GJ) has been considered an important factor in the genesis and maintenance of VF and pre-treatment with GJ couplers, rotigaptide (RTG), has been shown to reduce VF inducibility. Purpose We hypothesized that the degree of GJ coupling determines the underlying mechanism of VF, and that changes in GJ coupling can shift or modify the predominant mechanism of fibrillation along the spectrum between disorganised activity and organised drivers. We proposed that increased organisation of VF is critical to its termination. Methods Thirty Sprague-Dawley rat hearts were explanted, perfused ex-vivo and acute VF was induced with burst pacing and 30μM pinacidil. Optical mapping of transmembrane potential was performed at baseline and the effects of GJ coupling on VF dynamics were studied in an acute VF model by perfusing with increasing concentrations of a GJ uncoupler; carbenoxolone (0–50μM, CBX, n=10) or a GJ coupling-enhancer; RTG (0–80nM, n=10). A chronic diffuse fibrosis model (n=10) was generated with 4 weeks of in-vivo angiotensin infusion (500nm/kg/min). Fibrillation dynamics were quantified using phase analysis, phase singularity (PS) tracking and our novel method of global fibrillation organisation quantification, frequency dominance index (FDI), which is a power ratio of highest amplitude dominant frequency in the frequency spectrum. Results RTG increased average rotations per RD (Baseline: 2.86±0.10 vs 80nM: 5.66±0.43, p5 rotations. FDI increased with RTG (0.53±0.04 vs 0.78±0.3, p Conclusion The degree of GJ coupling is a key determinant of the underlying mechanism of VF. RTG organised fibrillation and stabilised RDs in a concentration-dependent manner whilst CBX disorganised VF. Enhancing GJ coupling with RTG in diseased hearts with fibrosis can terminate VF and may be a potential therapeutic target in acute VF. Acknowledgement/Funding BHF Programme Grant PG/16/17/32069

Background Ventricular fibrosis is known to play a critical role in initiation and maintenance of ventricular fibrillation (VF). Post myocardial infarction the quantity of fibrosis negatively correlates with survival. There is a lack of data on how the quantity and degree of fibrosis influences the mechanisms of VF itself. VF mechanisms remain debated, there are data to support both critical areas sustaining rotational drivers (RDs) and the contrary hypothesis of disorganized myocardial activation driving VF. Purpose We hypothesized that the underlying mechanism of VF is influenced by the spatial distribution and quantity of ventricular fibrosis. Methods Thirty-five Sprague-Dawley rats underwent permanent left anterior descending (LAD) ligation (n=11), 20mins LAD territory ischaemia-reperfusion (n=13) or in-vivo angiotensin infusion (500ng/kg/min, n=11) to generate compact (CF), patchy (PF) and diffuse fibrosis (DF) models respectively. After a 4-week maturation period, the hearts were explanted, Langendorff perfused and VF induced with burst pacing and 30μM pinacidil. Fibrillation dynamics were quantified using phase analysis, phase singularity (PS) tracking and our novel method of global fibrillation organisation quantification, frequency dominance index (FDI), which is a power ratio of highest amplitude dominant frequency in the frequency spectrum. Results Ventricular fibrosis for each group was characterized and quantified (CF: 22.3±3.2%, PF: 18.4±4.2%, DF: 5.8±1.3%, p=0.046). VF was driven predominantly by disorganised activity in CF, PSs were detected 26±7% of time comparative to 51.2±4% in DF and 69.5±8% in PF group (p=0.001). PF stabilised RDs, average maximum rotations for a single RD in PF were 31.6±7.1 comparative to 12.5±1.7 in DF and 6.4±1.1 in CF, p Conclusion VF mechanisms occur along a spectrum between organised activity sustained by discrete drivers and disorganised myocardial activation. The underlying VF mechanism can differ significantly dependent on the quantity and pattern of fibrosis. Patchy fibrosis stabilises RDs with localization to discrete areas and sustains an organised form of VF comparative to CF where VF is largely disorganised. Characterising the degree and pattern of fibrosis in patient groups vulnerable to VF might be beneficial in identifying patients with suitable targets for ablation. Acknowledgement/Funding BHF Programme Grant PG/16/17/32069

Background Mechanisms that maintain ventricular fibrillation (VF) remain debated. There are data supporting both critical areas sustaining rotational drivers (RDs) and the contrary hypothesis of disorganised myocardial activation driving VF. Abnormal electrical coupling between cardiomyocytes through gap junctions (GJ) and ventricular fibrosis are major factors in disease related remodelling, and have been implicated in initiation and maintenance of VF. In this study we hypothesised that the mechanism of VF is directly altered by fibrosis and GJ coupling, and that modulating these factors shifts or changes the predominant mechanism of fibrillation along the spectrum between disorganised activity and organised drivers. Methods VF optical mapping was performed in fifty-five perfused rat hearts with differing degrees and patterns of chronic ventricular fibrosis [compact (CF, n=11), patchy (PF, n=13) and diffuse (DF, n=11)], and acute GJ coupling modulation with rotigaptide (RTG, GJ coupling enhancer, 0–80nM, n=10) or carbenoxolone (CBX, GJ uncoupler, 0–50μM, n=10). Fibrillation dynamics were quantified using phase analysis, RD tracking and our novel method of global fibrillation organisation quantification, frequency dominance index (FDI), which is defined as the power ratio of highest amplitude dominant frequency in the frequency spectrum. Results In the fibrosis group, VF was driven predominantly by disorganised activity in CF, RDs were detected 26±7% of time comparative to 51.2±4% in DF and 69.5±8% in PF group (p=0.001). PF stabilised RDs, average maximum rotations for a single RD in PF were 31.6±7.1 comparative to 12.5±1.7 in DF and 6.4±1.1 in CF, p Conclusion VF mechanisms occur along a spectrum between organised activity with discrete drivers and disorganised myocardial activation. The degree of GJ coupling and ventricular fibrosis are key determinants of the underlying mechanism of VF. Enhanced GJ coupling and patchy fibrosis organised fibrillation and stabilised RDs, whilst GJ uncoupling and compact fibrosis disorganised VF. This study presents a unifying explanation for the numerous mechanisms reported for sustaining fibrillation. Characterising the degree and pattern of fibrosis in patient groups vulnerable to VF might be beneficial in identifying patients with targetable substrate, and GJ modulation might be a potential therapeutic target. Conflict of interest Nil

This chapter will explore the anatomy of the heart and its vasculature, discussing key structures and their functional role within the organ’s contractility. A solid anatomical understanding will aid the learning of this complex organ’s role in the body and the consequences of morphological abnormalities such as malformations. Finally, the potential role of three-dimensional (3D) printing will be discussed as a novel technique of visualising anatomical structures.