Research Article

Cellular and Molecular Life Sciences

, Volume 69, Issue 20, pp 3457-3479

Functional characterization of the human α-cardiac actin mutations Y166C and M305L involved in hypertrophic cardiomyopathy

  • Mirco MüllerAffiliated withInstitute for Biophysical Chemistry, OE 4350, Hannover Medical School
  • , Antonina Joanna MazurAffiliated withDepartment of Anatomy and Molecular Embryology, Ruhr-UniversityDepartment of Cell Pathology, Faculty of Biotechnology, University of Wroclaw
  • , Elmar BehrmannAffiliated withDepartment of Physical Biochemistry, Max-Planck-Institute for Molecular Physiology
  • , Ralph P. DiensthuberAffiliated withInstitute for Biophysical Chemistry, OE 4350, Hannover Medical School
  • , Michael B. RadkeAffiliated withInstitute for Biophysical Chemistry, OE 4350, Hannover Medical School
  • , Zheng QuAffiliated withDepartment of Anatomy and Molecular Embryology, Ruhr-University
  • , Christoph LittwitzAffiliated withDepartment of Physiology, Stritch School of Medicine, Loyola University Chicago
  • , Stefan RaunserAffiliated withDepartment of Physical Biochemistry, Max-Planck-Institute for Molecular Physiology
  • , Cora-Ann SchoenenbergerAffiliated withMaurice E. Müller Institute for Structural Biology, Biozentrum, University of Basel
    • , Dietmar J. MansteinAffiliated withInstitute for Biophysical Chemistry, OE 4350, Hannover Medical School
    • , Hans Georg MannherzAffiliated withDepartment of Anatomy and Molecular Embryology, Ruhr-UniversityDepartment of Physical Biochemistry, Max-Planck-Institute for Molecular Physiology Email author 

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Abstract

Inherited cardiomyopathies are caused by point mutations in sarcomeric gene products, including α-cardiac muscle actin (ACTC1). We examined the biochemical and cell biological properties of the α-cardiac actin mutations Y166C and M305L identified in hypertrophic cardiomyopathy (HCM). Untagged wild-type (WT) cardiac actin, and the Y166C and M305L mutants were expressed by the baculovirus/Sf9-cell system and affinity purified by immobilized gelsolin G4–6. Their correct folding was verified by a number of assays. The mutant actins also displayed a disturbed intrinsic ATPase activity and an altered polymerization behavior in the presence of tropomyosin, gelsolin, and Arp2/3 complex. Both mutants stimulated the cardiac β-myosin ATPase to only 50 % of WT cardiac F-actin. Copolymers of WT and increasing amounts of the mutant actins led to a reduced stimulation of the myosin ATPase. Transfection of established cell lines revealed incorporation of EGFP- and hemagglutinin (HA)-tagged WT and both mutant actins into cytoplasmic stress fibers. Adenoviral vectors of HA-tagged WT and Y166C actin were successfully used to infect adult and neonatal rat cardiomyocytes (NRCs). The expressed HA-tagged actins were incorporated into the minus-ends of NRC thin filaments, demonstrating the ability to form hybrid thin filaments with endogenous actin. In NRCs, the Y166C mutant led after 72 h to a shortening of the sarcomere length when compared to NRCs infected with WT actin. Thus our data demonstrate that a mutant actin can be integrated into cardiomyocyte thin filaments and by its reduced mode of myosin interaction might be the basis for the initiation of HCM.

Keywords

Cardiac actin Actin expression Hypertrophic cardiomyopathy Myosin Tropomyosin