The zebrafish atrioventricular canal: A model for mammalian AV node conduction and secondary or follower pacemaker activity

Sensitive, robust, and cost-efficient methods for studies leading to new insights into genetically-mediated heart rhythm disturbances and their treatments are continuing to be developed and refined. Examples include: (i) isolated myocytes obtained from specific heart chambers, (ii) pluripotent and patient-specific stem cell preparations, and (iii) selected ion channel signaling complexes assayed using heterologous expression systems. All of these approaches have been used in mechanistic studies of leading or primary pacemaker activity in mammalian sinoatrial node.1 However, less complete data sets are available concerning the secondary pacemaker activity, or other important intrinsic functional properties (e.g., the long delays that may occur before action potential firing) within the atrioventricular node.2, 3 In this issue of Acta Physiologica, a group consisting of investigators from Albacete, Spain, and Montpellier, France4 have made use of a zebrafish larvae heart preparation and complemented this with analyses of spatially localized changes in intracellular Ca2+, to gain new information concerning the functional roles of (i) the ERG family of K+ channels and (ii) T-type Ca2+ channels in regulating secondary pacemaker activity and “atrioventricular conduction”. Specifically, this group4 took zebrafish larvae that express a ratiometric fluorescent biosensor for Ca2+, and then imaged and analyzed these signals to obtain differences in the sequential atrial versus ventricular Ca2+ transients. This approach yielded new information concerning the conduction (patterns, velocities, delays) and refractory periods within these hearts. Comparisons of results recorded under control conditions and after superfusion with selected compounds (dofetilide, approx. 100 μM for K+ channels; and ML218 approx. 0.3 mM for T-type Ca2+ channels) that are known to be potent (but perhaps not completely selective) ion channel blockers have been used to advantage. Based on this, these authors have revealed the functional roles of these channels in the action potential waveform and regulation of excitation-contraction coupling. Taken together, the extensive sets of data and semiquantitative analyses provide a firm basis for the main conclusion: that both ERG K+ channels and T-type Ca2+ channels contribute significantly to atrioventricular conduction in this zebrafish preparation. This well-planned and rigorous study4 adds significantly to previous work (cited extensively by the authors) that has illustrated the usefulness and applicability of the zebrafish heart for mechanistic studies of cardiovascular function. The zebrafish heart has been used to study essential electrophysiological principles that are known to be involved in pacemaker activity, action potential initiation and conduction, and underlying ion channel pharmacology and biophysics.5-8 Going forward, it will be interesting to continue to combine patch pipette-based recordings of action potentials7 with studies of a number of different transmembrane ionic currents that generate action potential excitability and repolarization, or modulate spontaneous firing or pacemaker activity.8 The application of voltage clamp methods, perhaps complemented with voltage-sensitive dye recordings of heart chamber-specific action potentials and their conduction patterns6 would yield data that could further test the central working hypothesis in this paper.4 A primary reason for this is that a number of the K+ currents (including those generated by ERG channels) that regulate repolarization and action potential duration have distinctly nonlinear ion transfer or I–V characteristics.9 This important inwardly rectifying property is not captured by indirect assays of either action potential duration or chamber-specific Ca2+ transients. Second, (as these authors acknowledge), pharmacological studies in this zebrafish heart preparation continue to raise concerns regarding the requirement for very high concentrations of pharmacological agents. Third, there are also well-known differences between the essential Ca2+ mediated mechanisms responsible for excitation-contraction coupling in the zebrafish larvae heart preparation, compared to those in either the atrium or the ventricle of the adult mammal. A number of these reservations concerning the translational significance of primary data recorded from this zebrafish heart preparation can be captured using the phrase: What Does Physiological Mean?10 For example, it is challenging to carry out semiquantitative studies of Na+/Ca2+ exchange in mammalian heart preparations. This is due to both the small size of this carrier-mediated electrogenic current and the requirement to be able to resolve and reproduce the nonlinear features of its I–V relationship within the physiological range of membrane potentials. Provided that the majority of the Ca2+ that is needed for excitation-contraction coupling enters the zebrafish myocyte on a beat-by-beat basis through Ca2+ channels, the subsequent Ca2+ extrusion by the Na+/Ca2+ exchanger can be identified unambiguously11 and accounted for in semi-quantitatively models.12 Second, although no well-defined Purkinje fiber-mediated conduction system has been identified in the zebrafish heart, previous work has provided functional and morphological evidence for a vestigial conduction system in the ventricle of the zebrafish heart.13, 14 This important finding, combined with available techniques for electrocardiogram recordings, continues to be used to advantage in developmental, physiological, and drug discovery studies.7 Finally it is also notable that neutrophil-mediated inflammatory responses15, 16 have been identified and studied in detail in zebrafish preparations. By taking advantage of the optical transparency of the zebrafish heart and combining related imaging techniques with morpholino-based molecular targeting, mechanistic information concerning the cell-to-cell interactions that regulate inflammatory responses, including reactions to injury will be able to be studied in more detail.17 In summary, it is apparent that the zebrafish heart preparation continues to provide a very useful platform for studying heart physiology and pathophysiology. This paper by Salgado-Almario et al.4 illustrates this, while also providing new insights into atrioventricular conduction, based on established methods for spatially localized measurements of Ca2+ transients and thoughtful signal analysis. W. R. Giles: planned, wrote and edited this manuscript. I thank my former lab personnel and funding agencies. No external funding was needed for this project. No conflicts of interest have been identified.