Cardiac Enrichment of Mutant Calmodulin Protein in a Murine Model of a Human Calmodulinopathy

Heterozygosity for missense mutations in one of 3 seemingly redundant calmodulin (CaM)-encoding genes can cause life-threatening ventricular arrhythmias, suggesting that small fractions of mutant CaM protein suffice to cause a severe phenotype. However, the exact molar ratios of wildtype to mutant CaM protein in calmodulinopathy hearts remain unknown. The aim of the present study was to directly quantitate mutant versus wildtype CaM transcript and protein levels in hearts of knock-in mice harboring the p.N98S mutation in the Calm1 gene. We found that the transcripts from the mutant Calm1 allele were the least abundantly expressed Calm transcripts in both hetero- and homozygous mutant hearts, while mutant hearts accumulate high levels of N98S-CaM protein in a Calm1N98S allele dosage-dependent manner, exceeding those of wildtype CaM protein. We further show that the severity of the electrophysiological phenotype incrementally increases with the graded increase in the mutant-to-wildtype CaM protein expression ratio seen in homozygous versus heterozygous mutant mice. We finally show a decrease in N98S-CaM protein degradation, suggesting that mutant CaM stabilization contributed to its enrichment in the heart. Our results support a novel mechanism by which a mutation in a single Calm gene can give rise to a severe phenotype. Overall design: To assess the effect of hetero- and homozygosity for the Calm1N98S allele on CaM gene expression, we performed RNA-sequencing (RNA-seq) to quantify expression levels of Calm1, Calm2, and Calm3 transcripts in male hearts (left ventricles) of Calm1+/+, Calm1N98S/+ and Calm1N98S/N98S littermate mice at 2 to 4 months of age.