Investigating the utility of adult zebrafish ex vivo whole hearts to pharmacologically screen hERG channel activator compounds

Abstract

There is significant interest in the potential utility of small-molecule activator compounds to mitigate cardiac arrhythmia caused by loss of function of hERG1a voltage-gated potassium channels. Zebrafish ( Danio rerio) have been proposed as a cost-effective, high-throughput drug-screening model to identify compounds that cause hERG1a dysfunction. However, there are no reports on the effects of hERG1a activator compounds in zebrafish and consequently on the utility of the model to screen for potential gain-of-function therapeutics. Here, we examined the effects of hERG1a blocker and types 1 and 2 activator compounds on isolated zkcnh6a (zERG3) channels in the Xenopus oocyte expression system as well as action potentials recorded from ex vivo adult zebrafish whole hearts using optical mapping. Our functional data from isolated zkcnh6a channels show that under the conditions tested, these channels are blocked by hERG1a channel blockers (dofetilide and terfenadine), and activated by type 1 (RPR260243) and type 2 (NS1643, PD-118057) hERG1a activators with higher affinity than hKCNH2a channels (except NS1643), with differences accounted for by different biophysical properties in the two channels. In ex vivo zebrafish whole hearts, two of the three hERG1a activators examined caused abbreviation of the action potential duration (APD), whereas hERG1a blockers caused APD prolongation. These data represent, to our knowledge, the first pharmacological characterization of isolated zkcnh6a channels and the first assessment of hERG enhancing therapeutics in zebrafish. Our findings lead us to suggest that the zebrafish ex vivo whole heart model serves as a valuable tool in the screening of hKCNH2a blocker and activator compounds.

Utility of Zebrafish Models of Acquired and Inherited Long QT Syndrome

“…Rescue of the gain-of-function reggae zERG mutant cardiac phenotype by terfenadine block ( Hassel et al, 2008 ) further demonstrated the targeted action of hERG-specific drugs in zebrafish. Indeed isolated zERG ( zkcnh6a ) channels have subsequently been shown to present similar pharmacological sensitivity to blocker compounds, such as terfenadine, as observed in hERG ( hKCNH2 ) channels ( Hull et al, 2019 ). Further developments to incorporate high-throughput zebrafish larvae screens revealed that bradycardia and susceptibility to 2:1 block provided accurate detection of QT prolonging compounds with high sensitivity and specificity ( Burns et al, 2005 ; Mittelstadt et al, 2008 ; Peal et al, 2011 ; Letamendia et al, 2012 ) as shown in Table 1 .…”

“…Direct comparison of action potentials recorded from adult zebrafish ventricular cells with those from human papillary muscle and murine ventricular strips revealed the closely associated morphology of zebrafish and human ventricular action potential ( Nemtsas et al, 2010 ). As a result, measurements of APD in adult and larval (e.g., 3 dpf) zebrafish hearts report values of ∼140–230 ms (see Table 1 ), which are remarkably dependent on temperature ( Rayani et al, 2018 ), but reflect the duration of the human ventricular action potential reasonably well ( Alday, 2014 ; Vornanen and Hassinen, 2016 ; Hull et al, 2019 ; Shi et al, 2020 ; Zhao et al, 2020 ). The QT interval measured from ECG recordings in zebrafish demonstrates comparable resting heart rate and conduction intervals with humans and the QT interval has a near linear relationship with the RR interval ( Milan et al, 2006b ), features that are imperative for a model examining LQTS that involves delayed ventricular repolarization and prolongation of the APD and QTc interval.…”

“…A consistent feature of zebrafish cardiac repolarization is the prominent role of I Kr in phase 3 ventricular repolarization. I Kr is conveyed by zERG channels from the zkcnh6a gene ( Langheinrich et al, 2003 ; Scholz et al, 2009 ; Leong et al, 2010 ; Vornanen and Hassinen, 2016 ; Hull et al, 2019 ). The substantial genetic and pharmacological evidence for the importance of zERG channels and I Kr as a critical driving force behind phase 3 repolarization suggests that zebrafish bear great potential as a model of both acquired and inherited LQTS as is discussed in more detail below.…”

“…In adult zebrafish hearts, hERG blockers prolong the APD and increase the QT interval ( Milan et al, 2009 ; Tsai et al, 2011 ; Genge et al, 2016 ; Hull et al, 2019 ) while hERG activator compounds reduce APD ( Hull et al, 2019 ; Shi et al, 2020 ; see Table 1 ). Studies using adult zebrafish hearts also permit more detailed study of markers of cardiac arrhythmogenicity, such as electrical instability and beat-to-beat variability, which is an important consideration given that although APD and QT prolongation provide a substrate for the initiation of TdP, there is evidence that QT prolongation alone correlates poorly with TdP ( Mattioni et al, 1989 ; Shimizu et al, 1995 ; Fossa et al, 2002 ; Fenichel et al, 2004 ; Belardinelli et al, 2006 ).…”

“…Indeed, triangulation of the action potential (often quantified as APD 90 :APD 30 ), which represents prolongation of late phase repolarization or abbreviation of earlier phases, is associated with development of TdP ( Hondeghem et al, 2001 ) as a result of increased risk for the generation of EADs and triggered activity ( Frommeyer et al, 2011 ) and an increased temporal dispersion of repolarization which promotes re-excitation by current flow ( Hondeghem et al, 2001 ). In adult zebrafish hearts, hERG activator compounds reduce APD with a selective effect on late phase repolarization resulting in reduced triangulation ( Hull et al, 2019 ; Shi et al, 2020 ), indicating that zebrafish may be suited to screening for therapeutic compounds to ameliorate hERG channel loss of function.…”