Original Article Ventricular arrhythmias, increased cardiac calmodulin kinase II expression, and altered repolarization kinetics in ANP receptor deficient mice Paulus Kirchhof a, *, Larissa Fabritz a , Ana Kilic ´ b , Frank Begrow b , Günter Breithardt a , Michaela Kuhn b a Department of Cardiology and Angiology, Hospital of the University of Münster, and Institute for Arteriosclerosis Research at the University of Münster, Münster 48129, Germany b Institute of Pharmacology and Toxicology, Hospital of the University of Münster, Münster 48129, Germany Received 30 October 2003; received in revised form 18 February 2004; accepted 11 March 2004 Abstract Cardiac hypertrophy is associated with ventricular arrhythmias and sudden death. The molecular mechanisms that predispose the hypertrophied heart to arrhythmias are not well understood. In mice, deletion of the gene coding for the atrial natriuretic peptide receptor, guanylyl cyclase A (GC-A–/–), causes arterial hypertension, cardiac hypertrophy and sudden death. We used this mouse model to study molecular mechanisms of arrhythmias in the hypertrophied heart. Right and left ventricular monophasic action potential durations (APD) were recorded in isolated, Langendorff-perfused hearts during pacing from the right atrium and ventricle. The atrioventricular (AV) node was ablated to provoke bradycardia. Intracellular Ca 2+ transients were measured in isolated INDO-1 loaded ventricular myocytes. Cardiac expression of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) was analyzed by western blotting. Polymorphic ventricular arrhythmias (pVT) occurred spontaneously after mechanical AV block in 20/45 hearts from 12-month-old GC-A–/– mice (P < 0.05), but neither in age-matched GC-A+/+ hearts nor in hearts from 3-month-old mice of either genotype. Triggered activity preceded pVT.APD were prolonged and systolic Ca i 2+ levels were increased in GC-A–/– hearts independently of age. In 12-month-old GC-A–/– hearts only, dispersion of APD and expression levels of CaMKII were increased. CaMKII expression was particularly increased in hearts with pVT. Direct inhibition of CaMKII activation by KN93 (0.5 or 2 μM) or inhibition of Ca 2+ /calmodulin-dependent activation of CaMKII by W-7 (25 μM) suppressed pVT in GC-A–/– hearts (P < 0.05) while prolonging APD. The combination of increased CaMKII activity and altered action potential characteristics facilitates ventricular arrhythmias in hypertrophic GC-A–/– hearts. © 2004 Elsevier Ltd. All rights reserved. Keywords: Hypertrophy; Arrhythmias; Action potential; Triggered activity; Ca 2+ /calmodulin-dependent kinase II; Knockout mouse; Guanylyl cyclase A; Atrial natriuretic peptide; Sudden death 1. Introduction Ventricular hypertrophy is associated with ventricular ec- topy and sudden death [1,2]. It is known that hypertrophy prolongs the ventricular action potential [3,4], but the mo- lecular mechanisms of arrhythmias in the hypertrophied heart are not well understood. An early and pathognomonic marker in the development of cardiac hypertrophy is the increased cardiac expression and release of atrial and B-type natriuretic peptides (ANP and BNP) [5]. At the same time the vasodilative and natriuretic effects of these peptides are markedly attenuated, suggesting a downregulation or im- paired responsiveness of their receptor, guanylyl cyclase A (GC-A) [6,7]. Targeted deletion of the GC-A gene in mice causes marked arterial hypertension and global cardiac hy- pertrophy [8]. Of note, sudden death has been reported in GC-A deficient mice [9]. To examine whether a chronic inhibition of the ANP/BNP/GC-A system could contribute to arrhythmogenesis in the hypertrophied heart, we analyzed Abbreviations: APD, action potential duration; ANP, atrial natriuretic peptide; BNP, B-type natriuretic peptide; CaMKII, Ca 2+ /calmodulin- dependent protein kinase II; GC-A, guanylyl cyclase A; pVT, polymorphic ventricular tachycardia. * Corresponding author. Tel.: +49-251-8347638; fax: +49-251-8347864. E-mail address: kirchhp@uni-muenster.de (P. Kirchhof). Journal of Molecular and Cellular Cardiology 36 (2004) 691–700 www.elsevier.com/locate/yjmcc © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2004.03.007