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 2

Assessment of total and mutant CALM protein expression.

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Assessment of total and mutant CALM protein expression. (A) Whole Wester...
(A) Whole Western blot of lysates from LV of adult Calm1+/+, Calm1N98S/+, and Calm1N98S/N98S littermate mice probed with an antibody recognizing the C-terminal part of CALM (listed as binding within region aa 100). Values on the right are in kilodaltons. Samples were normalized to GAPDH protein expression. Densitometry with ImageJ (NIH) was used to calculate the intensity of the protein bands. (B) Normalized intensities of the CALM protein bands. Data are displayed as scatter dot plots with lines representing the median and interquartile ranges from 11 biological replicates per genotype. The Kruskal-Wallis test and the Wilcoxon rank sum test were used to compare normalized intensities among genotypes. *P ≤ 0.001 vs. Calm1+/+; †P = 0.018 vs. Calm1N98S/+. (C) 3D model of Ca2+-bound CALM (Protein Data Bank code 1exr) highlighting the position of the amino acid sequence within the C-terminal region bracketing position 98 (green color). Shown are the amino acid compositions of the N98- and S98-harboring peptides that were obtained by enzymatic cleavage of wild-type or mutated CALM, respectively. Red spheres denote Ca2+ ions bound to EF-hand Ca2+-chelating loops within the C- and N-terminal lobes of CALM. (D) Wild-type and mutant CALM protein levels (normalized to the total protein amount per sample). N = 4 per genotype. (E) Relative abundance of mutant over total CALM protein in LV of adult Calm1+/+, Calm1N98S/+, and Calm1N98S/N98S male littermate mice. Data are displayed as scatter dot plots with horizontal lines denoting the median and interquartile ranges from 4 mice each per genotype. The Kruskal-Wallis test and the Wilcoxon rank sum test were used to compare CALM protein levels among genotypes. (D and E): *P = 0.030 vs. Calm1+/+; †P = 0.030 vs. Calm1N98S/+.