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

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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.

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Acknowledgments

The authors thank Prof. Pott (Bochum) for supplying ARCs and Dipl. Biol. S. Fister and Dr. H. Milting (Herz- und Diabeteszentrum NRW, Bad Oeyenhausen) for the human cardiac actin clones, and Prof. Claycomb (New Orleans, Lousiana, USA) for supplying the HL-1 cells. Mass spectrometry analysis was kindly performed at the Hannover Medical School (MHH) Core Unit Mass Spectrometry/Proteomics by A. Pich. This work was supported by the ‘Deutsche Forschungsgemeinschaft’ Grant MA 807/17-2 (to H.G.M.), MA 1081/11-2 (to D.J.M), RA 1781/1-1 (to S.R.), the ‘Fonds der Chemischen Industrie’ Grant 684052 (to E.B.), the Hannover Biomedical Research School (to M.B.R.), the Deutsche Akademische Ausstauschdienst (DAAD) and FoRUM der Ruhr-Universität Bochum (to A.J.M.), the Schweizer National Fond (to C.A.S.), and the Max-Planck Society (to S.R. and E.B.).

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  1. Antonina Joanna Mazur

    Present address: Department of Cell Pathology, Faculty of Biotechnology, University of Wroclaw, 51-148, Wroclaw, Poland

Authors and Affiliations

  1. Institute for Biophysical Chemistry, OE 4350, Hannover Medical School, 30625, Hannover, Germany

    Mirco Müller, Ralph P. Diensthuber, Michael B. Radke & Dietmar J. Manstein

  2. Department of Anatomy and Molecular Embryology, Ruhr-University, Universitätsstrasse 150, 44780, Bochum, Germany

    Antonina Joanna Mazur, Zheng Qu & Hans Georg Mannherz

  3. Department of Physical Biochemistry, Max-Planck-Institute for Molecular Physiology, 44227, Dortmund, Germany

    Elmar Behrmann, Stefan Raunser & Hans Georg Mannherz

  4. Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Chicago, USA

    Christoph Littwitz

  5. Maurice E. Müller Institute for Structural Biology, Biozentrum, University of Basel, 4046, Basel, Switzerland

    Cora-Ann Schoenenberger

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Correspondence to Hans Georg Mannherz.

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M. Müller and A. J. Mazur contributed equally to this work.

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Müller, M., Mazur, A.J., Behrmann, E. et al. Functional characterization of the human α-cardiac actin mutations Y166C and M305L involved in hypertrophic cardiomyopathy. Cell. Mol. Life Sci. 69, 3457–3479 (2012). https://doi.org/10.1007/s00018-012-1030-5

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  • DOI: https://doi.org/10.1007/s00018-012-1030-5

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