Complex Arrhythmia Syndrome in a Knock-In Mouse Model Carrier of the N98S <i>Calm1</i> Mutation.

Abstract

Calmodulin mutations are associated with arrhythmia syndromes in humans. Exome sequencing previously identified a de novo mutation in <i>CALM1</i> resulting in a p.N98S substitution in a patient with sinus bradycardia and stress-induced bidirectional ventricular ectopy. The objectives of the present study were to determine if mice carrying the N98S mutation knocked into <i>Calm1</i> replicate the human arrhythmia phenotype and to examine arrhythmia mechanisms. Mouse lines heterozygous for the Calm1^N98S allele (Calm1^N98S/+) were generated using CRISPR/Cas9 technology. Adult mutant mice and their wildtype littermates (Calm1^+/+) underwent electrocardiographic monitoring. Ventricular de- and repolarization was assessed in isolated hearts using optical voltage mapping. Action potentials and whole-cell currents and [Ca²⁺]_i, as well, were measured in single ventricular myocytes using the patch-clamp technique and fluorescence microscopy, respectively. The microelectrode technique was used for in situ membrane voltage monitoring of ventricular conduction fibers. Two biologically independent knock-in mouse lines heterozygous for the Calm1^N98S allele were generated. Calm1^N98S/+ mice of either sex and line exhibited sinus bradycardia, QT_c interval prolongation, and catecholaminergic bidirectional ventricular tachycardia. Male mutant mice also showed QRS widening. Pharmacological blockade and activation of β-adrenergic receptors rescued and exacerbated, respectively, the long-QT phenotype of Calm1^N98S/+ mice. Optical and electric assessment of membrane potential in isolated hearts and single left ventricular myocytes, respectively, revealed β-adrenergically induced delay of repolarization. β-Adrenergic stimulation increased peak density, slowed inactivation, and left-shifted the activation curve of <i>I</i>_Ca.L significantly more in Calm1^N98S/+ versus Calm1^+/+ ventricular myocytes, increasing late <i>I</i>_Ca.L in the former. Rapidly paced Calm1^N98S/+ ventricular myocytes showed increased propensity to delayed afterdepolarization-induced triggered activity, whereas in situ His-Purkinje fibers exhibited increased susceptibility for pause-dependent early afterdepolarizations. Epicardial mapping of Calm1^N98S/+ hearts showed that both reentry and focal mechanisms contribute to arrhythmogenesis. Heterozygosity for the <i>Calm1</i>^N98S mutation is causative of an arrhythmia syndrome characterized by sinus bradycardia, QRS widening, adrenergically mediated QTc interval prolongation, and bidirectional ventricular tachycardia. β-Adrenergically induced <i>I</i>_Ca.L dysregulation contributes to the long-QT phenotype. Pause-dependent early afterdepolarizations and tachycardia-induced delayed afterdepolarizations originating in the His-Purkinje network and ventricular myocytes, respectively, constitute potential sources of arrhythmia in Calm1^N98S/+ hearts.