title: Development of a medium-high throughput electrophysiology method to study cellular heterogeneity in the rabbit heart
Creators: name: Lachaud, Quentin
Publisher: name: University of Glasgow
Year: 2019
Subjects: subject: Heterogeneity, action potential, contraction kinetics, FluoVolt, hERG, L-type calcium channel, high-throughput.

description: Sudden cardiac death (SCD) is a prominent cause of death worldwide today, mainly occurring as a result of coronary heart disease, cardiomyopathies, and inherited or induced arrhythmia syndromes. Survival following sudden cardiac arrest (SCA) has improved in the past decades, but the majority of cases of SCD remain unwitnessed. Although advances have been made towards the investigation of the mechanisms behind SCD, it remains a poorly understood phenomenon. Environmental factors have been identified and associated with increased arrhythmic risk, and most prominently, drug-induced arrhythmias constitute a serious hurdle to both cardiac and non-cardiac drug development. The past decade has seen pro-arrhythmic screening of new compounds become routine, and develop into a major point of interest for drug development. Specifically, the onset of drug-induced polymorphic ventricular tachycardia, such as torsade de pointes (TdP), is of particular interest to cardiac research. The concept of electrophysiological heterogeneity in cardiac muscle holds exciting potential for explaining the pathophysiology of TdP, but quantifying cellular heterogeneity using conventional methods is a challenge. This work developed and refined a fluorescence-based, medium/high-throughput electrophysiological assay to process large cell populations (~50-500 cells) from single hearts. Using this novel approach, transmural electrophysiological differences were found between regions of individual hearts, replicating published work with a 3 to 4-fold reduction in hearts sampled, and additionally providing a previously unknown quantification of cellular heterogeneity in isolated cardiomyocyte populations, in both healthy and failing rabbit hearts. Further classification of electrophysiological differences within smaller regions of the ventricle yielded evidence of repolarisation gradients across the myocardium, with vast overlap in repolarisation duration, challenging the dogma of region-specific repolarisation duration. Lastly, by specifically blocking hERG channels and L-type calcium channels in cardiac subregions (sub-epicardial apex and base) strong evidence was found for heterogeneous electrophysiology response amongst isolated cell populations. Specifically, sub-epicardial action potential shortening using nifedipine was strongly APD dependent, whereby baseline AP duration determined the extent of APD shortening via drug-induced ICa-L blockade. Sub-epicardial AP prolongation mediated via IKr block using dofetilide also produced non-homogeneous cell response in the form of two distinct population responses: (i) The majority (~85%) was made up of normal responding cells, experiencing ~20-30ms AP prolongation not dependent on baseline APD (P&lt;0.001), and (ii) hyper-responding cells (~15% of total) experiencing &gt;100ms AP prolongation, beyond the pacing cycle length (&gt;500ms) without any evidence of early-afterdepolarisations. Large experimental samples of AP parameters gathered in this study can provide real-world data parameter space ranges for mathematical model development, showing that ion channel conductance ranges used today to predict drug responses at the organ level may be too restrictive, or inaccurate. Iterative model adjustment using large experimental datasets can help constrain models and improve their predictive power, saving time by reducing computational power required.