Complex Arrhythmia Syndrome in a Knock-In Mouse Model Carrier of the N98S Calm1 Mutation

Abstract

Background: 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. Methods: 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 as well as [Ca 2+ ] i were measured in single ventricular myocytes using the patch-clamp technique and fluorescence microscopy, respectively. The microelectrode technique was employed for in situ membrane voltage monitoring of ventricular conduction fibers. Results: 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 electrical 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/+ vs 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. Conclusions: Heterozygosity for the Calm1 N98S mutation is causative of an arrhythmia syndrome characterized by sinus bradycardia, QRS widening, adrenergically mediated QT c 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.

Network Pharmacology-based Systematic Analysis of Molecular Mechanisms of Dingji Fumai Decoction for Ventricular Arrhythmia

“…is a regulator of voltage-dependent L-type calcium channels, its mutations are related to congenital arrhythmia 55 . Heterozygosity for the CALM1 mutation is causative of an arrhythmia syndrome 56 .…”

Network Pharmacology-Based Systematic Analysis of Molecular Mechanisms of Dingji Fumai Decoction for Ventricular Arrhythmia

“…CALM1 is a regulator of voltage-dependent L-type calcium channels; its mutations are related to congenital arrhythmia [ 54 ]. Heterozygosity for the CALM1 mutation is causative of an arrhythmia syndrome [ 55 ]. Moreover, it can lead to catecholaminergic polymorphic ventricular tachycardia, idiopathic ventricular fibrillation, long QT syndrome, and even sudden death [ 56 ].…”

De novo mutations in childhood cases of sudden unexplained death that disrupt intracellular Ca 2+ regulation

“…Mouse models have been made to study the mechanisms of these three mutations. All three available knockin mouse models showed Ca 2+ dysregulation, often with physiological phenotypes mimicking each other: in Ryr2 R420W/ R420W mice, the amplitude of the depolarization-induced Ca 2+ transient was significantly reduced while the decay time from the peak was prolonged (37); in CALM1 N98S/+ knockin mice, the degree to which β-adrenergic stimulation increased local Ca 2+ and slowed inactivation exceeded that in wild-type littermates (57); in both CALM1 N98S/+ and Ryr2 S2246L/+ mice, epinephrineinduced ventricular tachycardia was observed (38,57).…”