Congruously our findings of voltage electrode clamping assay
Congruously, our findings of voltage-electrode-clamping assays indicate that hipN851K mutation mediates partial loss of Na+/K+-ATPase pump currents, also confirming our thesis that hipN851K mutation acts as a hypomorph. As demonstrated here by our external electrical stimulation studies and ECG recordings, partial loss of Na+/K+-ATPase function results in prolonged myocardial repolarization as well as refractoriness in hip mutants. It is known from previous electrophysiological studies in zebrafish that impaired cardiac repolarization, either by mutations in channels or by drugs, can in addition to prolonged QT interval also cause bradycardia and AV block [, , , ]. Thus, it can be assumed that intrinsic sinus node impulses or propagated impulses from the atrium to the ventricle come up against prolonged atrial or ventricular repolarization leading to cardiac unexcitability and consequently to bradycardia, as observed in pediatric patients with atrioventricular block due to long QT syndrome or in patients with sinus exit block [69,70]. Hence, we conclude that atrioventricular conduction disturbances in hip mutants are rather based on ventricular refractoriness than on impaired electrical conduction of the AV node itself.
Accordingly, electrophysiological studies in humans show that inhibition of Na+/K+-ATPase pump function by methyldigoxin hampers selective myocardial electrical stimulation due to prolonged myocardial repolarization and prolonged refractoriness , thereby further confirming that Na+/K+-ATPase currents are an essential modulator of cardiac repolarization in the vertebrate heart. Congruously, Glitsch and colleagues could show that treatment of isolated ventricular cells with cardiac glycosides increases tandospirone duration in a concentration dependent manner . Furthermore, several large genome wide-association studies in humans demonstrated that SNP in Na+/K+-ATPase are associated with prolonged QT intervals [23,24]. For instance, Pfeufer et al. analyzed genome-wide data from different population-based cohorts with >15.000 Europeans. They found new loci associated with genes playing an essential role in cardiac electrophysiology. More specifically, in chromosome 1q24.2 the strongest signal was within ATP1B1. At this locus, no further gene candidates were identified, suggesting that ATP1B1 is an essential regulator of the QT interval. Accordingly, it is well known from human studies that ATP1B1 regulates cellular calcium concentration via interaction with sodium-potassium exchanger (NCX), which essentially contributes to repolarization current in cardiomyocytes [72,73].
Somatic ATP1A1 mutations in humans can lead to secondary hypertension caused by impaired electrophysiological properties of aldosterone-producing adenoma cells. As shown by whole cell as well as patch clamping studies ATP1A1 mutations result in reduced Na+/K+ATPase activity and finally in cellular depolarization similar to our hip Na+/K+-ATPase pump current assay .
However, the electrophysiological mechanisms leading to impaired myocardial repolarization and refractoriness mediated by reduced Na+/K+-ATPase pump currents have never been studied in vivo due to a lack of suitable animal models. Furthermore, present studies on pharmacological Na+/K+-ATPase inhibition by cardiac glycosides, and their influence on myocardial impulse formation and propagation in mammals, have been limited, since cardiac glycosides are also considered to stimulate the parasympathetic nerve system . In contrast, in zebrafish embryos functional autonomic nervous system is not established at the time, when bradycardia is even established . Thus, the zebrafish mutant hiphop displays an optimal genetic model to decipher the essential electrophysiological underpinnings of the Na+/K+-ATPase in regulating the vertebrate heart rate in vivo.
Limitations Action potential duration (APD) is widely considered to correlate with myocardial refractory period under physiological conditions. We here demonstrate that prolonged repolarization in hip mutants is accompanied by suspended response to external electrical stimulation suggesting that impaired cardiac excitability is a result of QT prolongation. However, alternative electrophysiological pathomechanisms such as postrepolarization refractoriness (PRR), in which recovery of excitability lags behind full repolarization caused by reduced membrane potential (RMP) [77,78] might also contribute to bradycardia in hip mutants. Since electrocardiogram does not directly represent action potential of a cardiomyocyte, the complex interplay of RMP, APD and PRR as shown in cardiomyocytes of several mouse models for atrial disorders [79,80] can only deciphered partially by our experimental setting.