Islands of spatially discordant APD alternans underlie arrhythmogenesis by promoting electrotonic dyssynchrony in models of fibrotic rat ventricular myocardium

Fibrosis and altered gap junctional coupling are key features of ventricular remodelling and are associated with abnormal electrical impulse generation and propagation. Such abnormalities predispose to reentrant electrical activity in the heart. In the absence of tissue heterogeneity, high-frequency impulse generation can also induce dynamic electrical instabilities leading to reentrant arrhythmias. However, because of the complexity and stochastic nature of such arrhythmias, the combined effects of tissue heterogeneity and dynamical instabilities in these arrhythmias have not been explored in detail. Here, arrhythmogenesis was studied using in vitro and in silico monolayer models of neonatal rat ventricular tissue with 30% randomly distributed cardiac myofibroblasts and systematically lowered intercellular coupling achieved in vitro through graded knockdown of connexin43 expression. Arrhythmia incidence and complexity increased with decreasing intercellular coupling efficiency. This coincided with the onset of a specialized type of spatially discordant action potential duration alternans characterized by island-like areas of opposite alternans phase, which positively correlated with the degree of connexinx43 knockdown and arrhythmia complexity. At higher myofibroblast densities, more of these islands were formed and reentrant arrhythmias were more easily induced. This is the first study exploring the combinatorial effects of myocardial fibrosis and dynamic electrical instabilities on reentrant arrhythmia initiation and complexity

Optogenetics enables real-time spatiotemporal control over spiral wave dynamics in an excitable cardiac system

Propagation of non-linear waves is key to the functioning of diverse biological systems. Such waves can organize into spirals, rotating around a core, whose properties determine the overall wave dynamics. Theoretically, manipulation of a spiral wave core should lead to full spatiotemporal control over its dynamics. However, this theory lacks supportive evidence (even at a conceptual level), making it thus a long-standing hypothesis. Here, we propose a new phenomenological concept that involves artificially dragging spiral waves by their cores, to prove the aforementioned hypothesis in silico, with subsequent in vitro validation in optogenetically modified monolayers of rat atrial cardiomyocytes. We thereby connect previously established, but unrelated concepts of spiral wave attraction, anchoring and unpinning to demonstrate that core manipulation, through controlled displacement of heterogeneities in excitable media, allows forced movement of spiral waves along pre-defined trajectories. Consequently, we impose real-time spatiotemporal control over spiral wave dynamics in a biological system

Dynamic loading of human engineered heart tissue enhances contractile function and drives a desmosome-linked disease phenotype

The role that mechanical forces play in shaping the structure and function of the heart is critical to understanding heart formation and the etiology of disease but is challenging to study in patients. Engineered heart tissues (EHTs) incorporating human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have the potential to provide insight into these adaptive and maladaptive changes. However, most EHT systems cannot model both preload (stretch during chamber filling) and afterload (pressure the heart must work against to eject blood). Here, we have developed a new dynamic EHT (dyn-EHT) model that enables us to tune preload and have unconstrained contractile shortening of >10%. To do this, three-dimensional (3D) EHTs were integrated with an elastic polydimethylsiloxane strip providing mechanical preload and afterload in addition to enabling contractile force measurements based on strip bending. Our results demonstrated that dynamic loading improves the function of wild-type EHTs on the basis of the magnitude of the applied force, leading to improved alignment, conduction velocity, and contractility. For disease modeling, we used hiPSC-derived cardiomyocytes from a patient with arrhythmogenic cardiomyopathy due to mutations in the desmoplakin gene. We demonstrated that manifestation of this desmosome-linked disease state required dyn-EHT conditioning and that it could not be induced using 2D or standard 3D EHT approaches. Thus, a dynamic loading strategy is necessary to provoke the disease phenotype of diastolic lengthening, reduction of desmosome counts, and reduced contractility, which are related to primary end points of clinical disease, such as chamber thinning and reduced cardiac output

Decreased repolarization reserve increases defibrillation threshold by favoring early afterdepolarizations in an in silico model of human ventricular tissue

Cardiolog

Response by Feola et al. to letter regarding article, 'Localized optogenetic targeting of rotors in atrial cardiomyocyte monolayers'

Cardiolog

Localized Optogenetic Targeting of Rotors in Atrial Cardiomyocyte Monolayers

BACKGROUND: Recently, a new ablation strategy for atrial fibrillation has emerged, which involves the identification of rotors (ie, local drivers) followed by the localized targeting of their core region by ablation. However, this concept has been subject to debate because the mode of arrhythmia termination remains poorly understood, as dedicated models and research tools are lacking. We took a unique optogenetic approach to induce and locally target a rotor in atrial monolayers. METHODS AND RESULTS: Neonatal rat atrial cardiomyocyte monolayers expressing a depolarizing light-gated ion channel (Ca2+-translocating channelrhodopsin) were subjected to patterned illumination to induce single, stable, and centralized rotors by optical S1-S2 cross-field stimulation. Next, the core region of these rotors was specifically and precisely targeted by light to induce local conduction blocks of circular or linear shapes. Conduction blocks crossing the core region, but not reaching any unexcitable boundary, did not lead to termination. Instead, electric waves started to propagate along the circumference of block, thereby maintaining reentrant activity, although of lower frequency. If, however, core-spanning lines of block reached at least 1 unexcitable boundary, reentrant activity was consistently terminated by wave collision. Lines of block away from the core region resulted merely in rotor destabilization (ie, drifting). CONCLUSIONS: Localized optogenetic targeting of rotors in atrial monolayers could lead to both stabilization and destabilization of reentrant activity. For termination, however, a line of block is required reaching from the core region to at least 1 unexcitable boundary. These findings may improve our understanding of the mechanisms involved in rotor-guided ablation

QRS prolongation after premature stimulation is associated with polymorphic ventricular tachycardia in nonischemic cardiomyopathy: Results from the Leiden Nonischemic Cardiomyopathy Study

BACKGROUND Progressive activation delay after premature stimulation has been associated with ventricular fibrillation in nonischemic cardiomyopathy (NICM). OBJECTIVES The objectives of this study were (1) to investigate prolongation of the paced QRS duration (QRSd) after premature stimulation as a marker of activation delay in NICM, (2) to assess its relation to induced ventricular arrhythmias, and (3) to analyze its underlying substrate by late gadolinium enhancement cardiac magnetic resonance imaging (LGE-CMR) and endomyocardial biopsy. METHODS Patients with NICM were prospectively enrolled in the Leiden Nonischemic Cardiomyopathy Study and underwent a comprehensive evaluation including LGE-CMR, electrophysiology study, and endomyocardial biopsy. Patients without structural heart disease served as controls for electrophysiology study. RESULTS Forty patients with NICM were included (mean age 57 14 years; 33 men [83%]; left ventricular ejection fraction 300/0 13%). After the 400 -ms drive train and progressively premature stimulation, the maximum increase in QRSd was larger in patients with NICM than in controls (35 18 ms vs 23 12 ms; P=.005) and the coupling interval window with QRSd prolongation was wider (47 23 ms vs 31 14 ms; P=.005). The maximum paced QRSd exceeded the ventricular effective refractory period, allowing for pacing before the offset of the QRS complex in 20 of 39 patients with NICM vs 1 of 20 controls (P CONCLUSION QRSd is a simple parameter used to quantify activation delay after premature stimulation, and its prolongation is associated with the inducibility of polymorphic ventricular tachycardia and with the pattern of myocardial fibrosis in biopsies

Identification of Functional Variant Enhancers Associated with Atrial Fibrillation

Rationale: Genome-wide association studies have identified a large number of common variants (single-nucleotide polymorphisms) associated with atrial fibrillation (AF). These variants are located mainly in noncoding regions of the genome and likely include variants that modulate the function of transcriptional regulatory elements (REs) such as enhancers. However, the actual REs modulated by variants and the target genes of such REs remain to be identified. Thus, the biological mechanisms by which genetic variation promotes AF has thus far remained largely unexplored. Objective: To identify REs in genome-wide association study loci that are influenced by AF-associated variants. Methods and Results: We screened 2.45 Mbp of human genomic DNA containing 12 strongly AF-associated loci for RE activity using self-transcribing active regulatory region sequencing and a recently generated monoclonal line of conditionally immortalized rat atrial myocytes. We identified 444 potential REs, 55 of which contain AF-associated variants (P<10-8). Subsequently, using an adaptation of the self-transcribing active regulatory region sequencing approach, we identified 24 variant REs with allele-specific regulatory activity. By mining available chromatin conformation data, the possible target genes of these REs were mapped. To define the physiological function and target genes of such REs, we deleted the orthologue of an RE containing noncoding variants in the Hcn4 (potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4) locus of the mouse genome. Mice heterozygous for the RE deletion showed bradycardia, sinus node dysfunction, and selective loss of Hcn4 expression. Conclusions: We have identified REs at multiple genetic loci for AF and found that loss of an RE at the HCN4 locus results in sinus node dysfunction and reduced gene expression. Our approach can be broadly applied to facilitate the identification of human disease-relevant REs and target genes at cardiovascular genome-wide association studies loci