BACKGROUND: Calmodulinopathies are rare inherited arrhythmia syndromes caused by dominant heterozygous variants in CALM1 , CALM2 , or CALM3 , which each encode the identical CaM (calmodulin) protein. We hypothesized that antisense oligonucleotide (ASO)–mediated depletion of an affected calmodulin gene would ameliorate disease manifestations, whereas the other 2 calmodulin genes would preserve CaM level and function. METHODS: We tested this hypothesis using human induced pluripotent stem cell–derived cardiomyocyte and mouse models of CALM1 pathogenic variants. RESULTS: Human CALM1 ^F142L/+ induced pluripotent stem cell–derived cardiomyocytes exhibited prolonged action potentials, modeling congenital long QT syndrome. CALM1 knockout or CALM1-depleting ASOs did not alter CaM protein level and normalized repolarization duration of CALM1 ^F142L/+ induced pluripotent stem cell–derived cardiomyocytes. Similarly, an ASO targeting murine Calm1 depleted Calm1 transcript without affecting CaM protein level. This ASO alleviated drug-induced bidirectional ventricular tachycardia in Calm1 ^N98S/+ mice without a deleterious effect on cardiac electrical or contractile function. CONCLUSIONS: These results provide proof of concept that ASOs targeting individual calmodulin genes are potentially effective and safe therapies for calmodulinopathies.

The mechanisms of J wave syndrome (JWS) are incompletely understood. Here, we showed that the concomitant activation of small-conductance calcium-activated potassium (SK) current ( I _KAS ) and inhibition of sodium current by cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) recapitulate the phenotypes of JWS in Langendorff-perfused rabbit hearts. CyPPA induced significant J wave elevation and frequent spontaneous ventricular fibrillation (SVF), as well as sinus bradycardia, atrioventricular block, and intraventricular conduction delay. I _KAS activation by CyPPA resulted in heterogeneous shortening of action potential (AP) duration (APD) and repolarization alternans. CyPPA inhibited cardiac sodium current ( I _Na ) and decelerated AP upstroke and intracellular calcium transient. SVFs were typically triggered by short-coupled premature ventricular contractions, initiated with phase 2 reentry and originated more frequently from the right than the left ventricles. Subsequent I _KAS blockade by apamin reduced J wave elevation and eliminated SVF. β-Adrenergic stimulation was antiarrhythmic in CyPPA-induced electrical storm. Like CyPPA, hypothermia (32.0°C) also induced J wave elevation and SVF. It facilitated negative calcium-voltage coupling and phase 2 repolarization alternans with spatial and electromechanical discordance, which were ameliorated by apamin. These findings suggest that I _KAS activation contributes to the development of JWS in rabbit ventricles.

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 and [Ca ²⁺ ] _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. 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 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. Conclusions: 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.

Apamin-sensitive small-conductance Ca²⁺-activated K⁺(SK) current ( I_KAS) is encoded by Ca²⁺-activated K⁺channel subfamily N ( KCNN) genes. I_KASimportantly contributes to cardiac repolarization in conditions associated with reduced repolarization reserve. To test the hypothesis that I_KASinhibition contributes to drug-induced long QT syndrome (diLQTS), we screened for KCNN variants among patients with diLQTS, determined the properties of heterologously expressed wild-type (WT) and variant KCNN channels, and determined if the 5-HT₃receptor antagonist ondansetron blocks I_KAS. We searched 2,306,335 records in the Indiana Network for Patient Care and found 11 patients with diLQTS who had DNA available in the Indiana Biobank. DNA sequencing discovered a heterozygous KCNN2 variant (p.F503L) in a 52-yr-old woman presenting with corrected QT interval prolongation at baseline (473 ms) and further corrected QT interval lengthening (601 ms) after oral administration of ondansetron. That patient was also heterozygous for the p.S38G and p.P2835S variants of the QT-controlling genes KCNE1 and ankyrin 2, respectively. Patch-clamp experiments revealed that the p.F503L KCNN2 variant heterologously expressed in human embryonic kidney (HEK)-293 cells augmented Ca²⁺sensitivity, increasing I_KASdensity. The fraction of total F503L-KCNN2 protein retained in the membrane was higher than that of WT KCNN2 protein. Ondansetron at nanomolar concentrations inhibited WT and p.F503L SK2 channels expressed in HEK-293 cells as well as native SK channels in ventricular cardiomyocytes. Ondansetron-induced I_KASinhibition was also demonstrated in Langendorff-perfused murine hearts. In conclusion, the heterozygous p.F503L KCNN2 variant increases Ca²⁺sensitivity and I_KASdensity in transfected HEK-293 cells. Ondansetron at therapeutic (i.e., nanomolar) concentrations is a potent I_KASblocker.NEW & NOTEWORTHY We showed that ondansetron, a 5-HT₃receptor antagonist, blocks small-conductance Ca²⁺-activated K⁺(SK) current. Ondansetron may be useful in controlling arrhythmias in which increased SK current is a likely contributor. However, its SK-blocking effects may also facilitate the development of drug-induced long QT syndrome.