Enrichment of mutant calmodulin protein in a murine model of a human calmodulinopathy
Wen-Chin Tsai, Chiu-Fen Yang, Shu-Yu Lin, Suh-Yuen Liang, Wei-Chung Tsai, Shuai Guo, Xiaochun Li, Susan Ofner, Kai-Chien Yang, Tzu-Ching Meng, Peng-Sheng Chen, Michael Rubart
Wen-Chin Tsai, Chiu-Fen Yang, Shu-Yu Lin, Suh-Yuen Liang, Wei-Chung Tsai, Shuai Guo, Xiaochun Li, Susan Ofner, Kai-Chien Yang, Tzu-Ching Meng, Peng-Sheng Chen, Michael Rubart
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Research Article Cardiology Cell biology

Enrichment of mutant calmodulin protein in a murine model of a human calmodulinopathy

Abstract

Heterozygosity for missense mutations in 1 of 3 seemingly redundant calmodulin-encoding (CALM-encoding) genes can cause life-threatening arrhythmias, suggesting that small fractions of mutant CALM protein suffice to cause a severe phenotype. However, the exact molar ratios of wild-type to mutant CALM protein in calmodulinopathy hearts remain unknown. The aim of the present study was to quantitate mutant versus wild-type CALM transcript and protein levels in hearts of knockin 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 accumulated high levels of N98S-CALM protein in a Calm1N98S allele dosage-dependent manner, exceeding those of wild-type CALM protein. We further show that the severity of the electrophysiological phenotype incrementally increased with the graded increase in the mutant/wild-type CALM protein expression ratio seen in homozygous versus heterozygous mutant mice. We finally show a decrease in N98S-CALM protein degradation, suggesting that mutant CALM stabilization contributed to its enrichment in the heart. Our results support what we believe to be a novel mechanism by which a mutation in a single Calm gene can give rise to a severe phenotype.

Authors

Wen-Chin Tsai, Chiu-Fen Yang, Shu-Yu Lin, Suh-Yuen Liang, Wei-Chung Tsai, Shuai Guo, Xiaochun Li, Susan Ofner, Kai-Chien Yang, Tzu-Ching Meng, Peng-Sheng Chen, Michael Rubart

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Figure 7

Progressive CDI decrement of cardiac ICa.L with increase in Calm1N98S allele number.

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Progressive CDI decrement of cardiac ICa.L with increase in Calm1N98S al...
(A) Exemplar traces of whole-cell L-type Ca2+ current (ICa.L) recorded in left ventricular cardiomyocytes in response to 400 ms step depolarizations to +10 mV. Traces were normalized to their respective peaks to facilitate comparison of decay kinetics. Pipette solution contained 5 mM ryanodine and 10 mM BAPTA to restrict Ca2+ elevations to those in the nanodomains of individual L-type channels (6). CDI is evident as the accelerated decay in Ca2+ current as compared with Ba2+ in all 3 genotypes. Ca2+ currents exhibit progressively slower decays from Calm1+/+ to Calm1N98S/+ to Calm1N98S/N98S cardiomyocytes, while decay kinetics of Ba2+ currents are almost indistinguishable among genotypes. (B and C) Scatter dot plots of the fraction of peak ICa.L remaining at 50 ms after the peak (r50) using Ca2+ versus Ba2+ as charge carriers (B) and dot plots of f50 = (r50,Ca – r50,Ba)/r50,Ba, a measure of CDI (C). Horizontal lines superimposed on dots represent the median and interquartile ranges; n = 21 cells from N = 4 mice (Calm1+/+); n = 16, N = 5 (Calm1N98S/+); n = 14, N = 4 (Calm1N98S/N98S); *P ≤ 0.001 vs. Calm1+/+; †P = 0.004 vs. Calm1N98S/+. P values from a repeated measures linear model with independent terms of genotype. A compound symmetric covariance matrix was used, which assumes equal correlation from repeated measurements of the same experimental unit.