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. 2013 May 24;288(21):14780-7.
doi: 10.1074/jbc.M113.450668. Epub 2013 Apr 11.

Transgenic Mouse α- And β-Cardiac Myosins Containing the R403Q Mutation Show Isoform-Dependent Transient Kinetic Differences

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Free PMC article

Transgenic Mouse α- And β-Cardiac Myosins Containing the R403Q Mutation Show Isoform-Dependent Transient Kinetic Differences

Susan Lowey et al. J Biol Chem. .
Free PMC article

Abstract

Familial hypertrophic cardiomyopathy (FHC) is a major cause of sudden cardiac death in young athletes. The discovery in 1990 that a point mutation at residue 403 (R403Q) in the β-myosin heavy chain (MHC) caused a severe form of FHC was the first of many demonstrations linking FHC to mutations in muscle proteins. A mouse model for FHC has been widely used to study the mechanochemical properties of mutated cardiac myosin, but mouse hearts express α-MHC, whereas the ventricles of larger mammals express predominantly β-MHC. To address the role of the isoform backbone on function, we generated a transgenic mouse in which the endogenous α-MHC was partially replaced with transgenically encoded β-MHC or α-MHC. A His6 tag was cloned at the N terminus, along with R403Q, to facilitate isolation of myosin subfragment 1 (S1). Stopped flow kinetics were used to measure the equilibrium constants and rates of nucleotide binding and release for the mouse S1 isoforms bound to actin. For the wild-type isoforms, we found that the affinity of MgADP for α-S1 (100 μM) is ~ 4-fold weaker than for β-S1 (25 μM). Correspondingly, the MgADP release rate for α-S1 (350 s(-1)) is ~3-fold greater than for β-S1 (120 s(-1)). Introducing the R403Q mutation caused only a minor reduction in kinetics for β-S1, but R403Q in α-S1 caused the ADP release rate to increase by 20% (430 s(-1)). These transient kinetic studies on mouse cardiac myosins provide strong evidence that the functional impact of an FHC mutation on myosin depends on the isoform backbone.

Keywords: Cardiac Myosin Isoforms; Cardiomyopathy; Cardiovascular Disease; Familial Hypertrophic Cardiomyopathy; Kinetics; Myosin; Myosin Heavy Chain Mutations; Transgenic Mice; Transient Kinetics.

Figures

FIGURE 1.
FIGURE 1.
The basic actomyosin contractile cycle. Myosin (M) is dissociated from actin (A) by ATP, which is rapidly hydrolyzed to ADP.Pi (green). Upon rebinding to actin (orange), the initial weak binding states release phosphate (Pi) and undergo a lever arm swing (power stroke) to form the strong binding ADP and rigor states. The primary steps in the cycle with the equilibrium constants for ATP (K1) and ADP (KAD) binding and the rates for AM dissociation (k2) and product release are indicated in schematic form below the graphic representation.
SCHEME 1.
SCHEME 1.
Interactions between S1 and actin with ATP and ADP.
FIGURE 2.
FIGURE 2.
Isolation of His6-tagged S1 using a metal-chelating column. After digestion of crude myosin with α-chymotrypsin, ∼10 mg of S1 was applied to a HiTrap chelating column equilibrated with 0.5 m NaCl, 20 mm HEPES, pH 7.5, and eluted with a step to 30 mm imidazole (peak II) for the nonspecifically bound S1 (verified by a negative Western blot with anti-histidine antibody) and 120 mm imidazole (peak III) for the His6-tagged S1. The inset shows SDS gels of the pooled fractions; the purified S1 in peak III contains the heavy chain and essential light chain. The starting material in this experiment was wild-type α-S1.
FIGURE 3.
FIGURE 3.
Transient rates as a function of ATP and ADP for acto α-S1 dissociation and ADP affinity at 11 °C. A, the maximum rate for the hyperbolic fit of observed rate (kobs) versus ATP is k2 at high concentrations of ATP. Inset, at low ATP concentrations the slope of the linear dependence of observed rate on ATP gives the second order rate constant, K1k2. B, at a constant low concentration of ATP (200 μm), KAD is obtained from the hyperbolic fit of observed rate versus increasing concentrations of ADP.
FIGURE 4.
FIGURE 4.
Fluorescence transients for acto S1 dissociation and ADP affinity at 20 °C. A and C, the time course of fluorescence increase because of dissociation of S1 from pyrene-labeled actin by 10 (blue), 20 (green), and 40 μm ATP (red); the 30 μm trace (black) was omitted for clarity (A). Single exponential fits to the raw data gave the rates plotted at the four ATP concentrations in C. B and D, the increase of fluorescence with time for 0 ADP (red), 250 μm ADP (green), and 500 μm ADP (blue) in the presence of 200 μm ATP (B). The acto S1 was preincubated with the ADP before dissociation by the addition of ATP. The observed rates obtained from single exponential fits of these three and several other ADP concentrations are plotted in D. A hyperbolic fit of kobs to Equation 2 gave the dissociation constant, KAD. These data are from experiment 1, wild-type α-S1.
FIGURE 5.
FIGURE 5.
The ADP release rate from acto S1 at 20 °C. In the absence of added ADP nucleotide, the rates of acto S1 dissociation became too fast to measure at high ATP concentrations. They are approximately >1000 s−1 for both α- and β-S1 isoforms (black and red symbols, respectively). With the addition of a constant concentration of ADP (usually 200 μm, but the same observed rates were obtained with 50 or 100 μm ADP), the rate constants for ADP release (k−AD) for wild-type α-S1 and β-S1 differed by ∼3-fold.

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