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 Müller
  • Antonina Joanna Mazur
  • Elmar Behrmann
  • Ralph P. Diensthuber
  • Michael B. Radke
  • Zheng Qu
  • Christoph Littwitz
  • Stefan Raunser
  • Cora-Ann Schoenenberger
  • Dietmar J. Manstein
  • Hans Georg Mannherz
Research Article

DOI: 10.1007/s00018-012-1030-5

Cite this article as:
Müller, M., Mazur, A.J., Behrmann, E. et al. Cell. Mol. Life Sci. (2012) 69: 3457. doi:10.1007/s00018-012-1030-5

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 

Supplementary material

18_2012_1030_MOESM1_ESM.doc (52 kb)
Supplementary material 1 (DOC 51.5 kb)
18_2012_1030_MOESM2_ESM.pdf (286 kb)
Supplementary material 2 (PDF 285 kb)
18_2012_1030_MOESM3_ESM.pdf (577 kb)
Supplementary material 3 (PDF 576 kb)
18_2012_1030_MOESM4_ESM.pdf (205 kb)
Supplementary material 4 (PDF 204 kb)
18_2012_1030_MOESM5_ESM.pdf (1001 kb)
Supplementary material 5 (PDF 0.97 mb)

Copyright information

© Springer Basel AG 2012

Authors and Affiliations

  • Mirco Müller
    • 1
  • Antonina Joanna Mazur
    • 2
    • 6
  • Elmar Behrmann
    • 3
  • Ralph P. Diensthuber
    • 1
  • Michael B. Radke
    • 1
  • Zheng Qu
    • 2
  • Christoph Littwitz
    • 4
  • Stefan Raunser
    • 3
  • Cora-Ann Schoenenberger
    • 5
  • Dietmar J. Manstein
    • 1
  • Hans Georg Mannherz
    • 2
    • 3
  1. 1.Institute for Biophysical Chemistry, OE 4350Hannover Medical SchoolHannoverGermany
  2. 2.Department of Anatomy and Molecular EmbryologyRuhr-UniversityBochumGermany
  3. 3.Department of Physical BiochemistryMax-Planck-Institute for Molecular PhysiologyDortmundGermany
  4. 4.Department of Physiology, Stritch School of MedicineLoyola University ChicagoChicagoUSA
  5. 5.Maurice E. Müller Institute for Structural Biology, BiozentrumUniversity of BaselBaselSwitzerland
  6. 6.Department of Cell Pathology, Faculty of BiotechnologyUniversity of WroclawWroclawPoland

Personalised recommendations