Characterisation of re-entrant circuit (or rotational activity) in vitro using the HL1-6 myocyte cell line

Charles Houston,Konstantinos N Tzortzis,Caroline Roney,Andrea Saglietto,David S Pitcher,Chris D Cantwell,Rasheda A Chowdhury,Fu Siong Ng,Nicholas S Peters,Emmanuel Dupont

doi:10.1016/j.yjmcc.2018.05.002

Charles Houston, Konstantinos N Tzortzis + Show 8 more

Open Access

https://doi.org/10.1016/j.yjmcc.2018.05.002

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Abstract

Fibrillation is the most common arrhythmia observed in clinical practice. Understanding of the mechanisms underlying its initiation and maintenance remains incomplete. Functional re-entries are potential drivers of the arrhythmia. Two main concepts are still debated, the “leading circle” and the “spiral wave or rotor” theories. The homogeneous subclone of the HL1 atrial-derived cardiomyocyte cell line, HL1-6, spontaneously exhibits re-entry on a microscopic scale due to its slow conduction velocity and the presence of triggers, making it possible to examine re-entry at the cellular level.We therefore investigated the re-entry cores in cell monolayers through the use of fluorescence optical mapping at high spatiotemporal resolution in order to obtain insights into the mechanisms of re-entry.Re-entries in HL1-6 myocytes required at least two triggers and a minimum colony area to initiate (3.5 to 6.4 mm2). After electrical activity was completely stopped and re-started by varying the extracellular K+ concentration, re-entries never returned to the same location while 35% of triggers re-appeared at the same position. A conduction delay algorithm also allows visualisation of the core of the re-entries. This work has revealed that the core of re-entries is conduction blocks constituted by lines and/or groups of cells rather than the round area assumed by the other concepts of functional re-entry. This highlights the importance of experimentation at the microscopic level in the study of re-entry mechanisms.

Highlights

We found that 1) the number of triggers and re-entrant circuits depends on colony size, 2) the triggers appear more stable while re-entries never reappeared at the same location, 3) the core of the re-entries is a simple round small mass of cells and connected lines(s) of conduction block, 4) the periodicity of the re-entry depends on the size of the core and 5) the cells constituting the core are either inactive or are able to display Ca2+ transients but no detectable action potentials
The aim of the trigger quantification experiments was to determine if a minimum threshold for number of triggers or colony area exists for re-entry formation in HL-1 subclone 6 (HL1-6) monolayers
An expected result is that re-entry/rotational activity required sufficient triggers and colony area in which to form a stable circuit

Summary

Introduction

There must be a vulnerable substrate that enables fibrillation to perpetuate [1,2]. The triggering event is thought to arise from two possible sources: either directly from focal ectopic activity or from re-entry [3,4]. Focal activity may originate from propagation of after-depolarisations from individual myocytes. Some regions of the heart such as infarct border zones or the thin cardiac tissue at the pulmonary veins may allow this abnormal activity to overcome the effects of source-sink mismatch that would be experienced elsewhere in the myocardium [3,6]. It is difficult to identify the specific triggering event, since micro re-entry and propagated after-depolarisations cannot be distinguished from each other at the macroscopic level of clinical studies [7]

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