In the last decade the zebrafish has emerged
In the last decade, the zebrafish has emerged as a valuable vertebrate model to systematically dissect the genetic underpinnings of both vertebrate heart development and function [8,9], as well as distinct cardiac diseases such as congenital heart disease , cardiomyopathies [11,12] and cardiac arrhythmias [, , ]. Thus, in search for novel regulators of the vertebrate heart rate, reduced hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN) currents were identified as a cause of severe bradycardia in the zebrafish mutant line slow mo (slo) further underlining the crucial role of HCN currents in regulating the vertebrate heart rate in vivo . Recently, we showed that reduced heart rate in the zebrafish mutant line schneckentempo (ste) is caused by a homozygous mutation of the dihydrodilipoyl succinyltransferase (DLST) gene which leads to impaired citric Solasodine australia cycle function, consecutively to reduced ATP production and finally to reduced cardiac pacemaker activity . Furthermore, characterization of an Islet1-deficient zebrafish mutant line as well as Shox2-specific knock-down studies deciphered an essential role of Shox2-Islet1 signaling in cardiac rhythm control [18,19].
Next to these newly identified regulators of the vertebrate heart beat other important proteins such as the sodium‑potassium-exchanger protein (Na+/K+-ATPase) are considered to play a role in myocardial impulse formation and propagation [, , ]. Furthermore, Na+/K+-ATPase single nucleotide polymorphisms (SNP) are associated with prolonged QT interval in several human genomic wide-association studies (GWAS) insinuating a crucial role of this ion pump protein in cardiac repolarization [23,24].
Na+/K+-ATPase transports 2 K+−ions in and 3 Na+-ions out of the cell in an energy-dependent manner and thereby enables proper membrane and action potentials in cardiomyocytes [25,26]. Due to the electrochemical gradient driven interplay of Na+/K+-ATPase with the sodium-calcium exchanger (NCX), Na+/K+-ATPase pump function influences intracellular calcium homeostasis and consecutively myocardial contraction. Furthermore, inhibition of the Na+/K+-ATPase α-subunit by cardiac glycosides is considered to impact on the refractory period of human myocardium and atrioventricular conduction [, , ]. However, the definite role and function of the Na+/K+-ATPase in the regulation of the vertebrate heart rate is still unclear.
Using a forward genetic approach , we here characterized the embryonic-lethal recessive ENU-induced zebrafish mutant hiphop (hip), which shows irregular and reduced heart rate. By positional cloning, gene inactivation studies as well as in vitro Na+/K+-ATPase ion pump current assays we found, that a missense mutation in the zebrafish Na+/K+-ATPase α1-subunit (atp1a1a.1) significantly inhibits its ion transport capacity. As demonstrated by in vivo electrocardiogram (ECG) and electrical cardiac stimulation maneuvers, Na+/K+-ATPase hip mutation result in prolonged QT interval as well as prolonged myocardial refractoriness. To the best of our knowledge, this is the first in vivo study, demonstrating the essential role of Na+/K+-ATPase in heart rate regulation by influencing myocardial repolarization.
Materials and methods
Discussion In the present study, the electrophysiological impact of reduced Na+/K+-ATPase function regarding heart rate control was investigated in a genetic in vivo model of severe bradycardia using the zebrafish mutant hiphop. By positional cloning we identified the hip missense-mutation to reside within the 7th transmembrane domain of the α1-subunit of the Na+/K+-ATPase (ATP1A1A.1) leading to reduced Na+/K+-ATPase pump currents in vitro. As shown by ECG as well as external pacemaker stimulation prolonged myocardial repolarization and refractoriness represent the relevant bradycardia causing mechanism exerted by reduced intrinsic Na+/K+-ATPase pump currents. To the best of our knowledge, this is the first in vivo study describing a long-QT mediated bradycardia as a consequence of reduced Na+/K+-ATPase function.