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

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.

Cryo-EM structure of a human cytoplasmic actomyosin complex at near-atomic resolution

“…Protein expression and purification. G-actin (γ 1-actin, ACTG1 from Homo sapiens) was recombinantly expressed using the baculovirus/Sf9-cell system and purified as described previously 31,32 . Afterwards, the sample was polymerized to F-actin by increasing the salt concentration to 100 mM KCl and 2 mM MgCl 2 .…”

Distribution of formins in cardiac muscle: FHOD1 is a component of intercalated discs and costameres

“…Cardiomyocytes from 1 to 5 days old rats were isolated according to the protocol described by (Przygodzki et al, 2005;Müller et al, 2012). Neonatal rats were killed in accordance with the guidelines of the European community (86/609/EEC).…”

“…Selective adhesion of non-cardiomyocyte cells was performed; the non-attached cardiomyocytes were collected by gentle centrifugation and seeded in 35 mm tissue culture dishes coated with 10 g/ml laminin and 10 g/ml fibronectin to a final density of 3.5 × 10 5 cells and cultivated in DMEM/M199 medium at 37 • C and 5% CO 2 . ARCs isolated from adult rat hearts as described (Müller et al, 2012) were generously supplied by Prof. Jaquet (St. Josef Hospital, Ruhr-University, Bochum).…”

Small-Molecule Inhibitors of Myosin Proteins

“…The remaining, 'unconventional' myosins function in such processes as intracellular transport and tethering (e.g., regulation of exocytosis/secretion by myosins 1c/1e, Va/Vb, VI, VII and X), cell division, cell motility, actin cytoskeletal organization and cellular signaling [15]. Myosins have also been implicated in several human diseases, such as hypertrophic cardiomyopathy [16,17], Griscelli syndrome [18], deafness [19,20] and cancer [21,22]. Therefore, inhibitors of specific myosins could act as a valuable tool both in characterizing many intracellular processes and also in developing targeted treatments for diseases involving myosin overproduction/malfunction.…”