Editorial |
From the Department of Physiology, University of Wisconsin Medical School, Madison, Wis.
Correspondence to Richard L. Moss, PhD, 1300 University Ave, Madison, WI 53706. E-mail rlmoss{at}physiology.wisc.edu
Key Words: hypertrophic cardiomyopathy myosin contraction kinetics
| Introduction |
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Although several FHC mutations have been identified using gene mapping approaches, little is known about the effects of most of the mutations on myofibrillar function or integrated contractile function in the context of human intact cardiac myocytes, muscle strips, or working hearts. However, in the case of one of these mutations, R403Q in the human ß-myosin heavy chain (MHC), recent studies4 5 have taken advantage of the fact that mammalian slow muscle MHC is identical to cardiac ß-MHC to obtain muscle that was heterozygous for the R403Q mutation. Biopsies from soleus muscles of individuals yielded fibers on which mechanical measurements could be made. Fibers from R403Q heterozygotes exhibited less force and slower shortening velocities than fibers from healthy homozygous individuals. The R403Q mutation has also been found to slow sliding velocities in in vitro motility assays of myosin from human soleus muscles and in other myosins with the R403Q substitution.6 In the context of these results, concentric hypertrophy is a plausible compensatory mechanism for reestablishing the work capacity and power output of heterozygous R403Q hearts toward normal.
| R403Q Mutation Increases Force and Speeds Actin-Activated Cycling Kinetics of Myosin |
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-MHC expressed with the
R403Q mutation, as well as measurements of the force-generating
properties of single myosin molecules from homozygous mice expressing
the mutation. This intriguing mouse model was previously developed by
Seidman and colleagues, who also characterized cardiac function in
heterozygous animals, and found that rates of pressure development were
accelerated and rates of relaxation were slowed compared with healthy
homozygous controls.8 In contrast to these earlier
studies, Tyska et al7 found that ensembles of R403Q myosin
yielded in vitro sliding velocities, actin-activated ATPase
activities, and average forces that were greater than control values,
whereas the ATPase activity of R403Q myosin alone (in the absence of
actin) was not different from control. These results suggest that the
R403Q mutation accelerates the kinetics of myosin interaction with
actin, an idea that is further supported by the authors findings that
unitary force and mean step size in single molecules did not differ
between R403Q and control myosins. However, when the authors measured
the average duration of force generating events
(ton), no differences were observed between
R403Q and control myosins. As the authors point out, the solution to
this conundrum might be found in the significantly different
concentrations of MgATP used to assess in vitro motility and function
of single myosin molecules. It is likely that the rate of MgADP
dissociation determines crossbridge detachment rate in the in vitro
motility assay where [MgATP] is high (mmol/L), whereas the rate of
MgATP association determines crossbridge detachment rate in the optical
trap assay (µmol/L MgATP). Thus, the rate of MgADP release might be
faster for R403Q than for wild-type myosin, which is suggested by its
greater ATPase activity, but the temporal resolution of the optical
trap assay is insufficient to allow the increase in [MgATP] that
would be required to detect the difference. Such a technical limitation
in assessing ton could certainly explain
the apparent similarity in turnover kinetics between single molecules
of R403Q and wild-type myosins but also suggests that if firm
conclusions are to be drawn, alternative methods for assessing the
rates of MgADP release in solution should be used in studies of the
mutation. | Implications of Results for Mechanism of Myocardial Hypertrophy |
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-MHC (by
4 days of age) suggests that the transition is too
rapid or too great to be offset by the compensating effects of
myocardial hypertrophy.
Although faster crossbridge cycling in humans and animals expressing
the R403Q mutation could account for the faster rate of rise in
pressure (+dP/dt) that has been observed in vivo8 and the
faster rate of force development in muscle strips,9 faster
kinetics do not straightforwardly explain the slower rate of relaxation
of pressure (or myocardial force) observed in these same individuals.
However, as discussed by the authors, the greater average force
observed in R403Q myosin is most likely the result of a greater
fraction of the duty cycle of myosin being spent in the
force-generating state. This explanation seems plausible because
ton is similar in R403Q and wild-type
myosins, but total cycle time is shorter in R403Q myosin. Thus, the
fraction of crossbridges bound to the thin filament at any given time
should be greater for R403Q myosin, which in turn would be expected to
enhance crossbridge-induced cooperative activation of the thin filament
and slow the rate of relaxation. A cooperative mechanism similar to
this has recently been proposed as an explanation for the slower rates
of relaxation observed in mouse hearts expressing significant amounts
of ß-tropomyosin on a normal
-tropomyosin
background.10 11
| Discrepancies With Earlier Studies |
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-MHC, which has faster kinetics than ß-MHC normally
expressed in humans.14 Although it might be possible to
address this point directly by expressing the R403Q mutation on a mouse
ß-MHC background, questions could still be raised owing to the 3- to
4-fold faster kinetics of mouse ß-MHC compared with human
ß-MHC.15 Tyska et al7 are certainly aware
of this issue and make the important point that they have observed
similar gain of function in cardiac myosin obtained by biopsy from FHC
patients,16 although this work has yet to be published,
and the nature of the underlying mutation is unclear. | Looking to the Future |
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-MHC expressing the R403Q mutation. Such a gain of function is
likely to stimulate the development of compensatory
hypertrophy via pathways that somehow differ from the
pathways involved in hypertrophy as a response to a loss of
function, ie, reduced power-generating capabilities. At the present
time, the mechanism for the enhancement of myosin function is not known
for certain, although it seems likely that the rate of
actin-activated nucleotide turnover by myosin is
accelerated by the R403Q mutation. New experimental approaches will be
required to resolve this point, which would be most directly done by
assessing MgADP dissociation rates from R403Q and wild-type myosin.
Furthermore, this study has used a unique and important mouse model of
a human familial hypertrophic cardiomyopathy to
study mechanisms of altered function; ie, homozygous expression of this
mutation in mouse
-MHC has yielded pure preparations of mutant
myosin for studies of force-generating and kinetic properties. Looking
to the future, there is a growing need to study the R403Q mutation in
the context of regulated thin filaments from the heart, both to
investigate the effects of cardiac regulatory proteins on actin
activation of myosin turnover kinetics and to determine whether
variations in regulatory protein content of the thin filament might
account for qualitative differences in results obtained by different
investigators studying the R403Q mutation. Ultimately, experiments that
assess force and sliding velocities of human ß-MHC and regulated thin
filaments from the heart will be useful in providing validation of
mouse models of FHC and the subsequent use of these models in studies
of interventions designed to slow or reverse development of the
hypertrophic phenotype.
| Footnotes |
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| References |
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2. Vikstrom KL, Leinwand LA. Contractile protein mutations and heart disease. Curr Opin Cell Biol.. 1996;8:97105.[Medline] [Order article via Infotrieve]
3.
Bonne G, Carrier L, Richard P, Hainque B, Schwartz K.
Familial hypertrophic cardiomyopathy: from
mutations to functional defects. Circ Res. 1998;83:580593.
4. Lankford EB, Epstein ND, Fananapazir L, Sweeney HL. Abnormal contractile properties of muscle fibers expressing ß-myosin heavy chain mutations in patients with hypertrophic cardiomyopathy. J Clin Invest. 1995;95:14091414.
5. Cuda G, Fananapazir L, Zhu WS, Sellers JR, Epstein ND. Skeletal muscle expression and abnormal function of ß-myosin in hypertrophic cardiomyopathy. J Clin Invest. 1993;91:28612865.
6. Cuda G, Fananapazir L, Epstein ND, Sellers JR. The in vitro motility activity of ß-cardiac myosin depends on the nature of the ß-myosin heavy chain gene mutation in hypertrophic cardiomyopathy. J Muscle Res Cell Motil. 1997;18:275283.[Medline] [Order article via Infotrieve]
7.
Tyska, MJ, Hayes E, Giewat M, Seidman CE, Seidman JG,
Warshaw DM. Single-molecule mechanics of R403Q cardiac myosin isolated
from the mouse model of familial hypertrophic
cardiomyopathy. Circ Res. 2000;86:737744.
8.
Georgakopoulos D, Christe ME, Giewat M, Seidman CM,
Seidman JG, Kass DA. The pathogenesis of familial hypertrophic
cardiomyopathy: early and evolving effects from
an
-cardiac myosin heavy chain missense mutation. Nat
Med.. 1999;5:327330.[Medline]
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9.
Blanchard E, Seidman C, Seidman JG, LeWinter M,
Maughan D. Altered crossbridge kinetics in the
MHC403/+ mouse model of familial hypertrophic
cardiomyopathy. Circ Res. 1999;84:475483.
10.
Wolska BM, Keller RS, Evans CC, Palmiter KA, Phillips
RM, Muthuchamy M, Oehlenschlager J, Wieczorek DF, deTombe PP,
Solaro RJ. Correlation between myofilament response to
Ca2+ and altered dynamics of contraction and
relaxation in transgenic cardiac cells expressing ß-tropomyosin.
Circ Res. 1999;84:745751.
11.
Moss RL. Plasticity in the dynamics of myocardial
contraction: Ca2+, crossbridge kinetics, or
molecular cooperation. Circ Res. 1999;84:862865.
12. Homsher E, Kim B, Bobkova A, Tobacman LS. Calcium regulation of thin filament movement in an in vitro motility assay. Biophys J. 1996;70:18811892.[Medline] [Order article via Infotrieve]
13.
Solaro RJ, Rarick HM. Troponin and tropomyosin:
proteins that switch on and tune in the activity of cardiac
myofilaments. Circ Res. 1998;83:471480.
14.
Schiaffino S, Reggiani C. Molecular diversity of
myofibrillar proteins: gene regulation and functional significance.
Physiol Rev. 1996;76:371423.
15. SantAna Periera J, Moss RL. Kinetic differences of cardiac myosins with identical loop 1. Biophys J.. 2000;78:273a. Abstract.
16. Palmiter KA, Alpert NR, Fananapazir L, Warshaw DW. Single cardiac myosin molecules from familial hypertrophic cardiomyopathy patients exhibit enhanced mechanical performance in the laser trap assay. Circulation. 1999;100(suppl I):I-193I-194.
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