Integration of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G)
Constanze Erdmann1, Roua Hassoun2,3, Sebastian Schmitt4
1Department of Anatomy and Molecular Embryology, Medical Faculty, Ruhr-University Bochum, D-44780 Bochum, Germany.
Insights
Mutant cardiac actins, linked to cardiomyopathy, interact differently with actin-binding proteins and are modified by MICAL-1. These variants show distinct cellular incorporation patterns, impacting cardiomyocyte structure.
Area of Science:
- Cardiovascular Biology
- Molecular Cell Biology
- Protein Biochemistry
Background:
- Mutations in cardiac α-actins are associated with cardiomyopathies, including hypertrophic and dilated forms.
- Understanding how these mutant actins interact with cellular proteins is crucial for elucidating disease mechanisms.
Purpose of the Study:
- To investigate the interactions of human mutant cardiac α-actins (p.A295S, p.R312H, p.E361G) with actin-binding and -modifying proteins.
- To analyze the polymerization dynamics and cellular incorporation of these mutant actins.
Main Methods:
- Expression and purification of recombinant human cardiac α-actin variants using the baculovirus/Sf21 system.
- Biochemical assays to assess Ca2+-sensitivity of myosin-subfragment1 ATPase activity.
- Analysis of actin polymerization stimulated by Arp2/3 complex, mDia3, and MICAL-1.
- Cell-based studies using MDCK cells and neonatal rat cardiomyocytes to examine actin variant incorporation.
Main Results:
- Purified mutant actins retained native states but exhibited altered Ca2+-sensitivity.
- Arp2/3 complex and mDia3 differentially modulated actin polymerization rates and extents.
- MICAL-1 induced oxidation and de-polymerization of mutant actins at varying rates.
- Cell transfection revealed distinct localization preferences for wild-type and mutant actins within cellular actin networks.
- Cardiomyocyte transduction showed specific incorporation sites for mutants, with p.E361G favoring minus ends.
Conclusions:
- Cardiac α-actin mutations associated with cardiomyopathy affect interactions with key regulatory proteins.
- MICAL-1's modification of mutant actins highlights its role in actin dynamics during cellular stress or development.
- Differential cellular incorporation of actin variants provides insights into the structural basis of distinct cardiomyopathy phenotypes.
Abstract:
The human mutant cardiac α-actins p.A295S or p.R312H and p.E361G, correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by the baculovirus/Sf21 insect cell system and purified to homogeneity. The purified cardiac actins maintained their native state but showed differences in Ca2+-sensitivity to stimulate the myosin-subfragment1 ATPase. Here we analyzed the interactions of these c-actins with actin-binding and -modifying proteins implicated in cardiomyocyte differentiation. We demonstrate that Arp2/3 complex and the formin mDia3 stimulated the polymerization rate and extent of the c-actins, albeit to different degrees. In addition, we tested the effect of the MICAL-1 monooxygenase, which modifies the supramolecular actin organization during development and adaptive processes. MICAL-1 oxidized these c-actin variants and induced their de-polymerization, albeit at different rates. Transfection experiments using MDCK cells demonstrated the preferable incorporation of wild type and p.A295S c-actins into their microfilament system but of p.R312H and p.E361G actins into the submembranous actin network. Transduction of neonatal rat cardiomyocytes with adenoviral constructs coding HA-tagged c-actin variants showed their incorporation into microfilaments after one day in culture and thereafter into thin filaments of nascent sarcomeric structures at their plus ends (Z-lines) except the p.E361G mutant, which preferentially incorporated at the minus ends.
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