Functional effects of the hypertrophic cardiomyopathy R403Q mutation are different in an alpha- or beta-myosin heavy chain backbone.
The R403Q mutation in the beta-myosin heavy chain (MHC) was the first mutation to be linked to familial hypertrophic cardiomyopathy (FHC), a primary disease of heart muscle. The initial studies with R403Q myosin, isolated from biopsies of patients, showed a large decrease in myosin motor function, leading to the hypothesis that hypertrophy was a compensatory response. The introduction of the mouse model for FHC (the mouse expresses predominantly alpha-MHC as opposed to the beta-isoform in larger mammals) created a new paradigm for FHC based on finding enhanced motor function for R403Q alpha-MHC. To help resolve these conflicting mechanisms, we used a transgenic mouse model in which the endogenous alpha-MHC was largely replaced with transgenically encoded beta-MHC. A His(6) tag was cloned at the N terminus of the alpha-and beta-MHC to facilitate protein isolation by Ni(2+)-chelating chromatography. Characterization of the R403Q alpha-MHC by the in vitro motility assay showed a 30-40% increase in actin filament velocity compared with wild type, consistent with published studies. In contrast, the R403Q mutation in a beta-MHC backbone showed no enhancement in velocity. Cleavage of the His-tagged myosin by chymotrypsin made it possible to isolate homogeneous myosin subfragment 1 (S1), uncontaminated by endogenous myosin. We find that the actin-activated MgATPase activity for R403Q alpha-S1 is approximately 30% higher than for wild type, whereas the enzymatic activity for R403Q beta-S1 is reduced by approximately 10%. Thus, the functional consequences of the mutation are fundamentally changed depending upon the context of the cardiac MHC isoform.
Lowey
Susan
S
Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT 05405, USA. lowey@physiology.med.uvm.edu
Lesko
Leanne M
LM
Rovner
Arthur S
AS
Hodges
Alex R
AR
White
Sheryl L
SL
Low
Robert B
RB
Rincon
Mercedes
M
Gulick
James
J
Robbins
Jeffrey
J
D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002312:Cardiomyopathy, Hypertrophic / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D051379:Mice; D008822:Mice, Transgenic; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D018995:Myosin Heavy Chains / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011441:Propylthiouracil / Q000494:pharmacology; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D024723:Ventricular Myosins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D020033:Protein Isoforms; D011441:Propylthiouracil; D001120:Arginine; D017301:Ca(2+) Mg(2+)-ATPase; D024723:Ventricular Myosins; D018995:Myosin Heavy Chains