Hypertrophic cardiomyopathy-linked variants of cardiac myosin-binding protein C3 display altered molecular properties and actin interaction

Sahar Da'as, Khalid Adnan Mohamed A. Fakhro, Angelos Thanassoulas, Navaneethakrishnan Krishnamoorthy, Alaaeldin Saleh, Brian L. Calver, Bared Safieh-Garabedian, Egon Toft, George Nounesis, F. Anthony Lai, Michail Nomikos

Research output: Contribution to journalArticle

Abstract

The most common inherited cardiac disorder, hypertrophic cardiomyopathy (HCM), is characterized by thickening of heart muscle, for which genetic mutations in cardiac myosin-binding protein C3 (c-MYBPC3) gene, is the leading cause. Notably, patients with HCM display a heterogeneous clinical presentation, onset and prognosis. Thus, delineating the molecular mechanisms that explain how disparate c-MYBPC3 variants lead to HCM is essential for correlating the impact of specific genotypes on clinical severity. Herein, five c-MYBPC3 missense variants clinically associated with HCM were investigated; namely V1 (R177H), V2 (A216T), V3 (E258K), V4 (E441K) and double mutation V5 (V3 + V4), all located within the C1 and C2 domains of MyBP-C, a region known to interact with sarcomeric protein, actin. Injection of the variant complementary RNAs in zebrafish embryos was observed to recapitulate phenotypic aspects of HCM in patients. Interestingly, V3- and V5-cRNA injection produced the most severe zebrafish cardiac phenotype, exhibiting increased diastolic/systolic myocardial thickness and significantly reduced heart rate compared with control zebrafish. Molecular analysis of recombinant C0–C2 protein fragments revealed that c-MYBPC3 variants alter the C0–C2 domain secondary structure, thermodynamic stability and importantly, result in a reduced binding affinity to cardiac actin. V5 (double mutant), displayed the greatest protein instability with concomitant loss of actin-binding function. Our study provides specific mechanistic insight into how c-MYBPC3 pathogenic variants alter both functional and structural characteristics of C0–C2 domains leading to impaired actin interaction and reduced contractility, which may provide a basis for elucidating the disease mechanism in HCM patients with c-MYBPC3 mutations.

Original languageEnglish
Pages (from-to)3933-3948
Number of pages16
JournalBiochemical Journal
Volume475
Issue number24
DOIs
Publication statusPublished - 14 Dec 2018

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Cardiac Myosins
Hypertrophic Cardiomyopathy
Actins
Carrier Proteins
Display devices
Zebrafish
Complementary RNA
Mutation
Injections
Thermodynamics
Recombinant Proteins
Muscle
Myocardium
Proteins
Thermodynamic stability
Embryonic Structures
Genes
Heart Rate
Genotype
Phenotype

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Hypertrophic cardiomyopathy-linked variants of cardiac myosin-binding protein C3 display altered molecular properties and actin interaction. / Da'as, Sahar; Fakhro, Khalid Adnan Mohamed A.; Thanassoulas, Angelos; Krishnamoorthy, Navaneethakrishnan; Saleh, Alaaeldin; Calver, Brian L.; Safieh-Garabedian, Bared; Toft, Egon; Nounesis, George; Anthony Lai, F.; Nomikos, Michail.

In: Biochemical Journal, Vol. 475, No. 24, 14.12.2018, p. 3933-3948.

Research output: Contribution to journalArticle

Da'as, S, Fakhro, KAMA, Thanassoulas, A, Krishnamoorthy, N, Saleh, A, Calver, BL, Safieh-Garabedian, B, Toft, E, Nounesis, G, Anthony Lai, F & Nomikos, M 2018, 'Hypertrophic cardiomyopathy-linked variants of cardiac myosin-binding protein C3 display altered molecular properties and actin interaction', Biochemical Journal, vol. 475, no. 24, pp. 3933-3948. https://doi.org/10.1042/BCJ20180685
Da'as, Sahar ; Fakhro, Khalid Adnan Mohamed A. ; Thanassoulas, Angelos ; Krishnamoorthy, Navaneethakrishnan ; Saleh, Alaaeldin ; Calver, Brian L. ; Safieh-Garabedian, Bared ; Toft, Egon ; Nounesis, George ; Anthony Lai, F. ; Nomikos, Michail. / Hypertrophic cardiomyopathy-linked variants of cardiac myosin-binding protein C3 display altered molecular properties and actin interaction. In: Biochemical Journal. 2018 ; Vol. 475, No. 24. pp. 3933-3948.
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abstract = "The most common inherited cardiac disorder, hypertrophic cardiomyopathy (HCM), is characterized by thickening of heart muscle, for which genetic mutations in cardiac myosin-binding protein C3 (c-MYBPC3) gene, is the leading cause. Notably, patients with HCM display a heterogeneous clinical presentation, onset and prognosis. Thus, delineating the molecular mechanisms that explain how disparate c-MYBPC3 variants lead to HCM is essential for correlating the impact of specific genotypes on clinical severity. Herein, five c-MYBPC3 missense variants clinically associated with HCM were investigated; namely V1 (R177H), V2 (A216T), V3 (E258K), V4 (E441K) and double mutation V5 (V3 + V4), all located within the C1 and C2 domains of MyBP-C, a region known to interact with sarcomeric protein, actin. Injection of the variant complementary RNAs in zebrafish embryos was observed to recapitulate phenotypic aspects of HCM in patients. Interestingly, V3- and V5-cRNA injection produced the most severe zebrafish cardiac phenotype, exhibiting increased diastolic/systolic myocardial thickness and significantly reduced heart rate compared with control zebrafish. Molecular analysis of recombinant C0–C2 protein fragments revealed that c-MYBPC3 variants alter the C0–C2 domain secondary structure, thermodynamic stability and importantly, result in a reduced binding affinity to cardiac actin. V5 (double mutant), displayed the greatest protein instability with concomitant loss of actin-binding function. Our study provides specific mechanistic insight into how c-MYBPC3 pathogenic variants alter both functional and structural characteristics of C0–C2 domains leading to impaired actin interaction and reduced contractility, which may provide a basis for elucidating the disease mechanism in HCM patients with c-MYBPC3 mutations.",
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