Po-02-211 the abundance of mutant calmodulin protein determines the severity of the arrhythmia phenotype in a mouse model of a human calmodulinopathy

Single-point missense mutations in the 3 calmodulin (CaM) genes (CALM1, CALM2, CALM3) which encode the identical amino acid sequence, cause LQTS and/or CPVT. All 3 CaM genes are expressed in the heart. It remains unclear how a mutation in only 1 of 6 CaM alleles can give rise to severe arrhythmia. To test the hypothesis that mutant CaM levels are disproportionally increased compared to wildtype (WT) CaM levels in CaM mutant mouse hearts and that the severity of the phenotype critically depends on the magnitude of the mutant/WT CaM ratio. Adult Knock-in mice heterozygous or homozygous for the p.N98S CaM mutation in CALM1 (Calm1N98S/+ and Calm1N98S/N98S mice, respectively) and wildtype littermates (Calm1+/+) were subjected to telemetric ECG recording. Left ventricular tissues were used to quantify the absolute levels of WT and mutant CaM peptide using C13 and N15–labeled synthetic peptides and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The 24-h plots of heart rate and QTc interval revealed Calm1N98S allele dosage-dependent sinus bradycardia and QTc prolongation (Figure A; p<0.05; 10 Calm1+/+, 9 Calm1N98S/+ and 9 Calm1N98S/N98S mice). Arrhythmia scores were significantly elevated in Calm1N98S/N98S mice of either sex versus Calm1+/+ and Calm1N98S/+ mice (lower left panel Figure A). In addition, episodes of spontaneous bidirectional ventricular tachycardia occurred in 5 of 9 male and 6 of 9 female Calm1N98S/N98S mice but in none of 10 Calm1+/+ or 9 Calm1N98S/+ mice (lower right panel Figure A; p<0.05). Total CaM peptide levels significantly increased in a Calm1N98S allele dosage-dependent manner (left panel Figure B; Calm1+/+ vs Calm1N98S/+ vs Calm1N98S/N98S: 32.0±1.9 vs 43.0±1.7vs 55.9 ±1.5 pg; n=4 per genotype; P<0.05, one way-ANOVA). Intriguingly, the fraction of mutant CaM peptide increased while that of WT CaM decreased as Calm1N98S allele number increased (all p<0.05, one way-ANOVA; Figure B). Our results suggest that the p.N98S CaM mutation causes alterations in the synthesis and/or degradation of both mutant and WT CaM protein, determining the severity of arrhythmogenic calmodulinopathy. Such changes in CaM protein homeostasis may explain the apparent dominate negative phenotype of CaM gene mutations.