Calmodulin mutations are associated with arrhythmia syndromes in humans. Exome sequencing previously identified a de novo mutation in CALM1 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 Calm1 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_Ca.L significantly more in Calm1^N98S/+ versus Calm1^+/+ ventricular myocytes, increasing late 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 Calm1^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_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.