Investigations regarding the role of NOS1AP in the heart using a conditional overexpression mouse model

M. Jänsch1, T. Williams2, J. Schmitt3, L. S. Maier4, O. Ritter1, M. Trum4

1Zentrum für Innere Medizin I, Städt. Klinikum Brandenburg, Brandenburg an der Havel; 2Deutsches Zentrum für Herzinsuffizienz, Universitätsklinikum Würzburg, Würzburg; 3Institut für Pharmakologie und Klinische Pharmakologie, Universitätsklinikum Düsseldorf, Düsseldorf; 4Klinik und Poliklinik für Innere Med. II, Kardiologie, Universitätsklinikum Regensburg, Regensburg;

Background: The QT interval duration (QTc) reflects ventricular de- and repolarization. It may predispose individuals to ventricular tachycardia and sudden cardiac death (SCD) if prolonged, shortened or otherwise unregularly. Genome-wide association studies have linked genetic mutations (single-nucleotide polymorphisms, SNP) in the neuronal nitric oxide synthase 1 adaptor protein (NOS1AP) to variations in QTc and SCD. In addition, about 20 % of families with a clinically proven diagnosis of long QT syndrome (LQTS) host no mutation in any of the 16 genes associated with the LQTS. Nitric oxide (NO) leads to an inhibition of the L-type calcium channel (Cav1.2) via S-nitrosylation and thus affects cardiac electrophysiology. Here, we investigate the relevance of altered expression of NOS1AP, acting as an L-type calcium channel modulator via directing neuronal nitric oxide synthase (NOS1) to Cav1.2, on cardiac electrophysiology. Methods and results: Transgenic FVB.N-Tg(Tre-Nos1ap)Jos mice were mated with a second transgenic FVB.Cg-Tg(Myh6-tTA)6Smbf/J strain (The Jackson Laboratory) to generate mice with conditional overexpression (Tet-off) of NOS1AP in cardiac myocytes. We investigated NOS1AP protein expression and localization using immunocytochemistry. We confirmed the interaction of NOS1AP with NOS1 and Cav1.2. Electrocardiography in NOS1AP overexpressing double transgenic mice showed atrial and ventricular tachycardia both spontaneously and upon programmed stimulation associated with a significant decrease in QTc. Heart rates in NOS1AP overexpressing mice were similar to non-induced animals. Survival was significantly reduced (only 56 % after 12 weeks vs. 100 % in non-induced mice). Induced QTc alterations and accompanied deaths subsided upon re-administration of doxycycline. Whole-cell patch-clamp measurements in isolated adult ventricular myocytes were performed and action potentials were continuously elicited by square current pulses of 1-2 nA amplitude and 1-5 ms duration at a frequency of 1 Hz. The action potential duration at 90 % of repolarization (APD90) was significantly reduced in induced transgenic NOS1AP overexpressing mice compared to control littermates. In addition, we investigated the functional effect of the human SNP rs16847548 (T/C) located within the NOS1AP promoter. The SNP was found to decrease the transcriptional activity of NOS1AP in vitro and therefore, potentially, leading to a decrease in NOS1AP expression in humans. Conclusion: Myocardial overexpression of NOS1AP leads to short QT syndrome with increased susceptibility to atrial and ventricular arrhythmias and cardiac death. In accordance, APD90 is significantly shortened in overexpressing animals. The human SNP rs16847548, which is located in the promoter region of NOS1AP, results in a reduced NOS1AP promoter activity in vitro, hereby providing an explanation for the frequently published elongation of QT intervals. In summary, not only mutations in ion channels themselves but also genetic alterations in the expression of ion channel modulators such as NOS1AP, have an impact on QT intervals and arrhythmogenesis.

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