Molecular Medicine |
From the Department of Molecular Physiology and Biophysics (M.J.T., E.H., D.M.W.), University of Vermont, Burlington, Vt; Howard Hughes Medical Institute and Department of Genetics (M.G., J.G.S.), Harvard Medical School, Boston, Mass; and Howard Hughes Medical Institute (C.E.S.), Brigham and Womens Hospital, Boston, Mass. M.J.T.s present affiliation is Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Conn.
Correspondence to David M. Warshaw, PhD, Department of Molecular Physiology and Biophysics, Given Building, D217, University of Vermont, Burlington, VT 05405. E-mail warshaw{at}salus.med.uvm.edu
| Abstract |
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1 pN) and displacements (
7 nm) without
any differences in event durations. On the basis of the distribution of
mean unitary displacements, this mutation may possibly perturb the
mechanical coordination between the 2 heads of cardiac myosin. Any of
these observations could, alone or possibly in combination, result in
abnormal power output and potentially a stimulus for the
hypertrophic response.
Key Words: familial hypertrophic cardiomyopathy cardiac myosin R403Q mouse model laser trap molecular motor
| Introduction |
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-tropomyosin, and
myosin binding protein-C.3 4 5 6 7 8 9 Although a large portion of
these point mutations have been localized to the ß-MHC gene (n>50
specifically in the motor domain or "head" region),10
few have been characterized in terms of their effect on myosin motor
function to the extent of the R403Q substitution. This mutation causes
malignant disease, with 50% of the affected individuals dying by 40
years of age.6 11 Investigators seeking to characterize the effects of R403Q have worked with muscle fibers from afflicted individuals, myosin purified from human patients, and genetically engineered fragments of myosin. Because ß-MHC is expressed in slow skeletal muscle fibers in addition to adult cardiac tissue,12 Lankford et al13 were able to isolate fibers from the soleus muscles of R403Q FHC patients for mechanical characterization. These fibers exhibited a depressed mechanical state including decreased isometric force generation and lower shortening velocities, resulting in decreased power output and depressed force/stiffness ratios. Is this altered performance due to the inability of R403Q myosin to assemble properly within the sarcomere, or is the myosin motor itself functionally compromised? Recent evidence suggests that in both human fibers and cultured cells, myosin carrying this amino acid change assembles into functional sarcomeric units.14 15 However, Cuda et al16 showed that slow skeletal muscle myosin purified from patients expressing the R403Q mutation produced markedly decreased sliding filament velocities in the in vitro motility assay, suggesting that the motor itself is mechanically compromised.
To obtain larger quantities of purified R403Q MHC for biochemical and
mechanical characterization, various laboratories have used protein
expression systems to produce myosin fragments presenting this
mutation.17 18 19 20 Interestingly, equivalent 403
substitutions expressed in Dictyostelium myosin II, rat
-cardiac, and human ß-cardiac MHCs result in heavy meromyosins
that generate slower sliding velocities in the in vitro motility
assay, depressed actin-activated ATPase rates, and elevated
Kms for actin.17 18 19 20
Although the in vitro motility and ATPase assays provide useful
information concerning the performance of myosins, they are
"ensemble" measurements based on large populations of protein. As
such, they do not provide information regarding the behavior of these
motors at the level of a single molecule.
To understand how the R403Q mutation perturbs myosin function at the molecular level, we performed a single-molecule mechanical assay on native cardiac myosin isolated from homozygous mice expressing only the altered protein.21 Using the mouse model provided an opportunity to examine myosin from animals with a well-characterized phenotype, similar to the disease state experienced by humans (myocyte hypertrophy, slowed relaxation rates, elevated rates of pressure development, interstitial fibrosis, and myofibrillar disarray).21 22 Here we present evidence that the R403Q substitution enhances both hydrolytic and motor function of the mutant cardiac myosin. Furthermore, our interpretation of the data from single-molecule experiments indicates that this mutation may uncouple the mechanical coordination between the 2 "heads" of a cardiac myosin molecule. The results from these single-molecule and ensemble assays together uncover the fundamental perturbations associated with R403Q as well as provide insight into the mechanism of chemomechanical energy transduction in cardiac muscle myosins.
| Materials and Methods |
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100% V1-myosin (ie, 
-MHC
homodimer) 1 week after birth. Control (+/+), heterozygote (+/403), and
homozygote (403/403) mice were bred. 403/403 animals lived only 1 week
and thus were generally euthanized within this time and compared with
age-matched controls. Mice were treated in accordance with the
guidelines of the Animal Care and Use Committee of Harvard
University.
Protein Purification and Storage
To extract myosin, hearts were homogenized in
high-salt buffer (1:5 wt:vol, 0.3 mol/L KCl, 0.15 mol/L
K2HPO4, 0.01 mol/L
Na4PO7, 0.001 mol/L
MgCl2, and 0.002 mol/L DTT, pH 6.8) for 20
minutes.25 The homogenate was cleared of
cellular debris by ultracentrifugation (60 minutes,
150 000g, 4°C with Beckman TLA 120.1). The supernatant
was then diluted by
50 times in 2 mmol/L DTT to precipitate
filamentous myosin, which was pelleted by subsequent
ultracentrifugation (20 minutes, 50 000g,
4°C, with Beckman SW41-Ti). The pellet was resuspended in myosin
buffer (in mol/L, imidazole 0.025, MgCl2 0.004,
DTT 0.01, EGTA 0.001, and KCl 0.3, at pH 7.4) and stored in 50%
glycerol at -20°C. Using this procedure, a 20-mg heart typically
produced 0.2 mg of whole myosin. Myosin was purified from +/+, +/403,
and 403/403 mice, with only the +/+ and 403/403 myosin used in the
single-molecule experiments. All experiments were performed within 1
week of purification.
Chicken gizzard smooth muscle myosin was purified and thiophosphorylated as previously described26 and then used in the average force assay described below.
ATPase Assays
High-salt Ca2+- and
NH4+-ATPase activities were
measured in either Ca2+ assay buffer (10 mmol/L
Tris [pH 8.0], 0.23 mol/L KCl, and 2.5 mmol/L CaCl2) or
NH4+ assay buffer (0.4 mol/L
NH4Cl, 2 mmol/L EDTA, 25 mmol/L Tris [pH 8.0],
0.2 mol/L sucrose, 1 mmol/L DTT, and 1 mg/mL BSA) at 25°C.
Actin-activated ATPase activity was measured at multiple actin
concentrations (5 to 100 µmol/L) in actin buffer at 20°C (pH
7.4). ATPase activity was determined using 2 mmol/L MgATP
(
1.8 mmol/L free Mg2+). Inorganic
phosphate (Pi) concentrations at fixed time
points were determined colorimetrically, using a
malachite green phosphate indicator.27 For the
actin-activated assay, values of Pi
released s1xhead1
versus [actin] were plotted and fitted to Michaelis-Menten kinetics
(V=Vmaxx[actin]/Km+[actin]),
with Vmax and
Km fit parameters using
Tablecurve 2D version 4 from SPSS.
Motility Assays
Actin filament velocities (vactin)
were measured in low-salt actin buffer (in mol/L, imidazole 0.025,
MgCl2 0.004, DTT 0.01, EGTA 0.001, and KCl 0.025,
at pH 7.4) for each cardiac myosin (+/+, +/403, and 403/403), as
previously described.26 28 The relative average
isometric force (Favg) of myosin was
measured in a "mixture assay" as previously
described26 (see Results for description). Details of
the optical trap instrumentation and experimental procedures for the
single molecule assay have been published elsewhere.29 30 31
This assay provides estimates of the unitary displacements
(d) and forces (F) of myosin. The estimates of
d, F, and event durations
(ton) were obtained by mean-variance (MV)
analysis.30 The assay was performed in 1
µmol/L MgATP actin buffer.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
| Results |
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ATPase Activity
Actin-activated ATPase activity of all 3 cardiac myosin
species was assessed at multiple actin concentrations (see Materials
and Methods). Both 403/403 and +/403 myosins hydrolyzed ATP at rates
2.3 and 1.8 times faster, respectively, than control (Figure 2
, Table 1
). These results were somewhat
unexpected, as all previous studies have shown that this substitution
produces a substantial reduction in actin-activated ATPase rate
relative to control values.17 18 19 20 The acceleration in
Vmax was also accompanied in both cases by
a
4-fold elevation of Km for actin as
previously observed.17 18 19 20 To reveal whether or not
the inherent enzymatic activity (ie, in the absence of actin) was
altered in proteins with the 403 mutation, we also performed
"high-salt" Ca2+- and
NH4+-ATPase assays (see
Materials and Methods). As indicated in Table 1
, there were no
significant differences in the rates obtained during these
experiments.
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In Vitro Motility Assay
The in vitro motility assay measures the ability of a population
of myosin motors to propel actin under unloaded conditions. As with the
ATPase data, the 403/403 and +/403 myosins demonstrated 60% and 16%
higher vactin, respectively, when compared
with +/+ myosin (see Table 1
). Previous investigations of the
R403Q mutation in other myosin systems have reported a decline in
filament motility to as little as 20% of control
values.17 18 19 20 Motility experiments contributing to
the data in Table 1
were performed on numerous hearts from
multiple litters, with the 403 mutants demonstrating
consistently higher velocities.
In the first week after birth, a rapid myosin isoform shift occurs
within the mouse heart from being predominantly ß-MHC to being
predominantly
-MHC. Because the hearts used in this work were
obtained during this period, altered cardiac MHC content could
contribute to the observed functional differences. To test this
hypothesis, we performed in vitro motility on purified myosin from
mouse hearts ranging from 4 to 8 days old. These experiments, in
conjunction with SDS-PAGE of corresponding samples, revealed that any
isoform shifts were complete by 4 days after birth and that myosins
isolated from 4- to 8-day-old hearts were functionally
indistinguishable (data not shown).
Measurement of Relative Isometric Force
Relative levels of isometric force
(Favg) produced by the +/+ and 403/403
myosins were determined using the in vitro motility "mixture"
protocol26 (Figure 3
, Table 1
). In these
experiments, each "fast" cardiac myosin (+/+ or 403/403) was mixed
with different proportions of a "slow" reference myosin (ie,
chicken gizzard smooth muscle myosin, for which
vactin=1.6 µm/s). By analyzing how
the cardiac myosinbased actin filament velocity slowed with the
addition of the slower smooth muscle myosin (see Figure 3
), an
estimate of the relative Favg for each
cardiac myosin was obtained. The observed relationships between the
fraction of fast myosin versus sliding velocity were fitted to a model
(solid lines, Figure 3
) based on the mechanical interaction of 2
myosins having independent force-velocity
relationships.26 In the context of this model, a
straight-line fit indicates that the 2 myosin species generate equal
force (dashed line, Figure 3
). The relationships shown in Figure 3
were
concave down (ie, extended below the dashed line) for
both the +/+ and 403/403:smooth muscle myosin mixtures, suggesting that
smooth muscle myosin generates 3.3 and 1.5 times greater average force
compared with the +/+ and 403/403 cardiac myosins, respectively. The
enhanced force-generating capacity of smooth muscle myosin relative to
either V1 or V3
rabbit cardiac myosin has been reported previously.32
Using smooth muscle myosin as a common reference, it appears that the
403/403 myosin produces 2.2-fold higher
Favg when compared with +/+ myosin (see
Table 1
).
|
Unitary Mechanical Measurements
To understand the enhanced force and motion-generating ability of
403/403 myosin at the molecular level, we measured the mechanics of
single cardiac myosin molecules in the optical trap (see Materials and
Methods).30 For these experiments, only preparations
providing homogenous populations of MHC were assayed (ie, +/+ and
403/403). Representative unitary displacement and force
records are shown in Figure 4
. Both
displacement and force events appeared as rapid deflections from
baseline (indicated by arrowheads in Figure 4
). MV
analysis (see Materials and Methods) was used to estimate
d, F, and average event durations
(ton) from the time series data. Under the
lightly loaded conditions of the optical trap (ie, low trap stiffness),
d values produced by the +/+ and 403/403 myosins were
comparable at
7 nm (see Table 2
). Likewise, under high load,
F values produced by +/+ and 403/403 myosins were also very
similar at
1 pN (see Table 2
). Interestingly, +/+ and 403/403
myosins produced similar ton for
displacement (
70 ms) and force events (
175 ms; see Table 2
).
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The only potential difference between +/+ and 403/403 myosins was found
in the distribution of mean displacement event amplitudes, as shown in
Figure 5
(see also d* in Table 2
). Each data point represents the mean event amplitude
estimated by MV analysis of a displacement time series
record containing tens to hundreds of events generated by a single
myosin molecule. Therefore, the entire distribution represents
behavior of multiple independent myosin molecules. In this plot, the
+/+ distribution was best fit by 2 gaussian curves with individual
peaks at 5.2 and 9.2 nm (see Figure 5
legend). In contrast, the
403/403 distribution was well fit by a single peak at 6.3 nm (see
Figure 5
legend).
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| Discussion |
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Is this enhanced functional state unique to the in vitro assays performed here? In situ functional measurements conducted in +/403 mouse hearts indicate that these muscles are indeed hypercontractile and display a significantly elevated +dP/dt.22 This finding is consistent with the faster velocities and higher forces reported here for the 403 mutation. Blanchard et al,33 studying papillary muscles from +/403 mouse hearts, also observed elevated isometric tension at submaximal activation. However, isometric tension was equal to controls at maximal activation, a result that may be related to the fiber having a contractile system with intact regulatory proteins. The in vitro experiments reported here were all performed using unregulated filamentous actin.
This report is the first to document a "gain of function" resulting
from the R403Q substitution. This is in direct contrast to earlier
reports from other groups.17 18 19 20 One possible explanation
for this discrepancy may relate specifically to using the mouse as a
model system. For example, the functional impact of this mutation may
vary when expressed in MHC backbones from different species. However,
the enhanced motor function may not be unique to mouse cardiac myosin,
as preliminary data from chicken gizzard smooth muscle myosin
expressing the R403Q mutation suggest that actin-activated
ATPase and vactin are accelerated in this
case as well.34 As the majority of previous studies
have been performed on expressed myosin fragments (eg, from the
baculovirus system), it is worth noting that investigators have had an
exceedingly difficult time trying to express striated muscle myosin
fragments at high yield.17 This leads to the possibility
that the myosin fragments expressed with the 403 mutation in these in
vitro systems may be compromised for reasons other than the single
amino acid substitution at position 403. The fact that native whole
cardiac myosin purified for this study actually demonstrated enhanced
performance highlights this possibility. It should also be
noted that in the human disease state, the 403 mutation is found in
V3-cardiac myosin (ie, ßß-MHC homodimer), the
isoform predominantly expressed in adults. In contrast, our mouse
hearts are
100% V1-myosin (ie, 
-MHC
homodimer). Although primary sequences of the
- and ß-MHCs are
remarkably similar, minor variations in functionally significant
regions35 are thought to contribute to the different
characteristics of the 2 isoforms.36 Therefore, it is
conceivable that the same mutation presented in a ß-MHC might
have distinct consequences. However, we have also demonstrated similar
enhanced function in myosin isolated from cardiac biopsy samples from
human FHC patients.37
In humans, the FHC disease state is a heterozygous condition, where only 1 allele contains the mutation. Given that myosin consists of 2 heavy chains and that the expression of the normal and mutant heavy chain appears to be equal,12 then one would expect myosin to assemble in vivo as a mixture containing 25% +/+ homodimers, 50% +/403 heterodimers, and 25% 403/403 homodimers. The enhanced function demonstrated in the ATPase and in vitro motility assays was clearly evident, not only for the 403/403 homodimer, but also for myosin from the +/403 heterozygote. If only the 403/403 homodimers contributed to the observed increase in ATPase activity for myosin from the heterozygote mouse, then only a 31% increase would have been predicted as compared with the 80% observed. This suggests that only 1 of the MHCs within a molecule needs to possess the R403Q substitution in order for the enzymatic and mechanical phenotypes to be significantly altered. Given that 75% of the myosin molecules in the human condition will contain at least 1 mutant MHC, it is not surprising that the heterozygous individual will present the clinical manifestations of the disease.
On the basis of the enhanced vactin and
Favg observed for a population of 403/403
myosin motors, is it possible to understand the molecular mechanism by
which this mutation exerts its effects? At the single-molecule
level,
![]() | (1) |
![]() | (2) |
![]() | (3) |
Perhaps kinetic differences do exist between +/+ and 403/403 myosin but
were not resolved under the 1 µmol/L MgATP conditions of the
optical trap assay. This potential difficulty stems from the fact that
there are 2 crossbridge cycle transitions that contribute to the event
durations: MgADP release from myosin and the subsequent rebinding of
MgATP.36 38 Under the 1 mmol/L MgATP conditions of
the in vitro motility assay, the ATP rebinding rate is exceedingly
high, and thus MgADP release governs the attached duration (ie,
ton), in turn limiting
vactin.36 39 40 41 At 1
µmol/L MgATP, the MgATP rebinding rate is several times slower than
ADP release,36 41 raising the possibility that a
difference in MgADP release rate between +/+ and 403/403 could exist
but would be obscured by the low-MgATP assay conditions. This
possibility may explain why, under both loaded and unloaded conditions,
there were no measurable differences in the
ton values for +/+ and 403/403
myosins (see Table 2
). Unfortunately, because of the rapid
kinetics of the
-MHC,36 performing experiments at
1 mmol/L MgATP in an effort to resolve this issue would produce
ton values comparable with the temporal
resolution of our instrument (
2 to 5 ms).
Despite the lack of a difference in ton
from unitary force measurements, a kinetic change can still explain why
Favg was elevated in the case of the
mutant. Assuming that ATPase differences measured in solution (see
Table 1
) reveal information about cycling kinetics under load as
well, the increased ATPase rate measured for 403/403 myosin may help to
explain why Favg was higher. Because
tcycle=(ATPase
rate)-1, a higher ATPase activity means a
shorter overall cycle time, which, in the absence of a change in
ton, would result in an increase in
fiso (see Equation 3
). As indicated
by Equation 2
, an increase in fiso
can explain the higher Favg produced by
403/403 myosin.
Interestingly, another potential functional difference between 403/403
and +/+ reveals itself on closer inspection of the single-molecule
data. The scatter plots shown in Figure 5
demonstrate that the
mutation has a profound effect on the distributions of displacement
amplitudes. In this representation, the +/+ distribution of
displacements is better fit by a bimodal distribution, whereas the
403/403 scatter plot is better described by a single gaussian (see
legend to Figure 5
). Using a similar scatter plot
analysis, Tyska et al42 recently described a
molecular-level comparison of single- and double-headed myosins. These
data indicate that double-headed muscle myosins coordinate their action
to produce twice the force and motion in the optical trap assay when
compared with their single-headed counterparts. Given this evidence, we
propose that the bimodal distribution shown for +/+ myosin arises from
the functional relationship between the 2 heads of cardiac myosin. It
is likely that the bimodal distribution shown for +/+ myosin
represents the action of both heads (peak at 9.2 nm, Figure 5
) or,
at times, a single cardiac myosin head (peak at 5.2 nm,
Figure 5
). The shift in mass toward the lower distribution may
indicate that 403/403 myosin is behaving more "single-headed" than
the +/+ control, with a single peak at 6.3 nm (Figure 5
). This
is consistent with the mean value we have reported for
single-headed smooth and skeletal muscle myosins.42 At
present, the precise mechanism linking potential changes in
head-head coordination to observed changes in ATPase activity,
vactin, or Favg
is unclear and requires further investigation.
How could the enhanced function observed in the in vitro assays result in a disease state in vivo? One potential explanation may relate to the abnormally high levels of energy consumption associated with the increased ATPase activity for the 403/403 mutant. This is consistent with the nuclear magnetic resonance spectroscopic data of Spindler et al,43 demonstrating perturbations in the levels of high-energy phosphate compounds in +/403 mouse hearts. The authors reasoned that this would ultimately result in a decreased free energy for ATP hydrolysis within the cell,43 and, because other cellular ATPases have high free energy demands (eg, sarcoplasmic/endoplasmic reticulum Ca2+ ATPase pump),44 they run the risk of becoming "energy starved." This energetic imbalance could be one possible stimulus leading to cardiac hypertrophy. A second possibility relates to the higher force levels produced by the 403 mutant myosin. If the cardiac sarcomere is designed to function with tolerance for a normal range of physiological force development, then abnormally high levels of average force could be the origin of sarcomeric and myocyte disarray seen in FHC-afflicted hearts.21
The in vitro functional analysis presented here has revealed that the R403Q mutation present in the mouse model of FHC may exert its effect through a combination of kinetic perturbations that alter ensemble force (Favg), velocity (vactin), and ATPase (Vmax) and possibly the suppression of the native level of head-head coordination in mouse V1-cardiac myosin. Through these effects, the R403Q mutation creates a stronger, faster myosin so that under any load, the power output of these hearts should be augmented beyond the tolerance of a normal cardiac sarcomere. The in vivo significance of the functional alterations reported here is highlighted by the fact that these effects are likely the fundamental stimuli for the hypertrophic response in the R403Q mouse model of FHC.
| Acknowledgments |
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Received September 16, 1999; accepted December 14, 1999.
| References |
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