© 1999 American Heart Association, Inc.
Original Contribution |
Altered Crossbridge Kinetics in the 
MHC403/+ Mouse Model of Familial Hypertrophic Cardiomyopathy
From the Department of Molecular Physiology and Biophysics (E.B., D.M.), University of Vermont Medical School, Burlington, Vt; Department of Medicine (C.S.), Brigham and Women's Hospital, and Howard Hughes Institute and Department of Genetics (J.G.S.), Harvard Medical School, Boston, Mass; and Cardiology Unit (M.L.), University of Vermont Medical School, Burlington, Vt.
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Abstract |
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Abstract—A mutation in the cardiac ß-myosin heavy chain, Arg403Gln (R403Q), causes a severe form of familial hypertrophic cardiomyopathy (FHC) in humans. We used small-amplitude (0.25%) length-perturbation analysis to examine the mechanical properties of skinned left ventricular papillary muscle strips from mouse hearts bearing the R403Q mutation in the
-myosin heavy chain
(MHC403/+). Myofibrillar disarray with
variable penetrance occurred in the left ventricular
free wall of the MHC403/+ hearts. In resting strips (pCa
8), dynamic stiffness was 
40% greater than in wild-type strips,
consistent with elevated diastolic stiffness
reported for murine hearts with FHC. At pCa 6 (submaximal activation),
strip isometric tension was 3 times higher than for wild-type
strips, whereas at pCa 5 (maximal activation), tension was marginally
lower. At submaximal calcium activation the characteristic frequencies
of the work-producing (b) and work-absorbing
(c) steps of the crossbridge were less in
MHC403/+ strips than in wild-type strips
(b=11±1 versus 15±1 Hz; c= 58±3 versus
66±3 Hz; 27°C). At maximal calcium activation, strip oscillatory
power was reduced (0.53±0.25 versus 1.03±0.18 mW/mm3;
27°C), which is partly attributable to the reduced frequency
b, at which crossbridge work is maximum. The results are
consistent with the hypothesis that the R403Q mutation reduces
the strong binding affinity of myosin for actin. Myosin heads may
accumulate in a preforce state that promotes cooperative activation of
the thin filament at submaximal calcium but blunts maximal tension and
oscillatory power output at maximal calcium. The calcium-dependent
effect of the mutation (whether facilitating or debilitating), together
with a variable degree of fibrosis and myofibrillar disorder, may
contribute to the diversity of clinical symptoms observed in
murine FHC.
Key Words: cardiomyopathy • myosin mutation • mouse • crossbridge kinetics
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAppendix 1References |
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Familial hypertrophic cardiomyopathy (FHC) is a human autosomal dominant disease characterized by ventricular hypertrophy, with myocyte and myofibrillar disarray. The disorder is often devastating, resulting in heart failure and premature death, although many individuals survive to old age.1 2 3 Affected individuals usually experience shortness of breath, angina, and arrhythmia, but many individuals are remarkably asymptomatic.
The heterogeneous effects of FHC mutations are reflected in
the diversity of clinical assessments of ventricular
function.4 5 FHC is a disease of the sarcomere, with
genetic point mutations causing replacement of 1 amino acid for another
in ß-myosin heavy chain (ß-MHC),1 6 myosin essential
light chain,7 myosin regulatory light chain,7
myosin binding protein C,8 -tropomyosin,9
or troponin T.9 Each mutation probably affects the
3-dimensional shape of the mutant protein and its interaction with
regulatory ligands and other sarcomeric proteins in different and
complex ways, resulting in an inconsistent clinical picture of
cardiac function for the general FHC population.
The Arg403Gln (R403Q) missense mutation in the ß-MHC is one of the most extensively characterized FHC mutations.10 11 12 13 The R403Q missense mutation lies on the globular head of ß-MHC near the actin binding interface6 and causes a decrease in affinity for actin, a depression of actin-activated myosin ATPase activity, and a slower actin velocity in motility assays.10 11 12 14 A decreased power output at all loads and a higher stiffness-to-force ratio has also been reported for maximally activated human soleus-skinned fibers containing a ß-MHC R403Q mutation.13
Recently a murine homolog of the R403Q myosin mutation has been
generated using targeted recombination.15 Homozygous
(MHC403/403) mice die within a week or 2 after birth.
Heterozygous (MHC403/+) mice survive for at least 1
year, although their hearts exhibit histopathology and dysfunction that
resemble human FHC, including compromised exercise
capacity.15 Spindler et al16 used
1 of the MHC403/+ lines to obtain additional mechanical
and energetic information from hearts perfused with the Langendorff
method. Although no evidence of systolic dysfunction was
apparent, diastolic function was significantly impaired
(decreased rate of left ventricular relaxation, increased
end-diastolic pressure). 31P nuclear
magnetic resonance measurements showed a reduced availability of
high-energy phosphates (lower phosphocreatine (PCr) content, higher
inorganic phosphate content) that may have contributed to the observed
diastolic dysfunction. Spindler et
al16 hypothesized that altered actin-myosin binding
kinetics of the R403Q crossbridges underlie the decreased rate of left
ventricular relaxation and increased
end-diastolic pressure.
To gain further insight into the functional consequences of the R403Q
myosin defect at the sarcomere level, we used the
MHC403/+ mouse model to examine isometric force,
oscillatory power, and crossbridge kinetics derived from dynamic
stiffness measurements in skinned left ventricular
papillary muscle strips. At submaximal calcium activation (pCa 6),
isometric tension of MHC403/+ strips is significantly
greater than that of wild-type mice, whereas at maximal calcium
activation (pCa 5), isometric tension and oscillatory power are less.
Crossbridge rate constants are depressed, consistent with the
hypothesis that the R403Q mutation reduces the strong binding affinity
of myosin for actin. The kinetic data suggest that myosin heads
accumulate in a preforce state that promotes cooperative activation of
the thin filament at submaximal calcium but blunts tension and
oscillatory power output at maximal calcium.
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Animals, Dissection, and Preparation for Ultrastructural Analysis
Fourteen heterozygous
MHC403/+ mutant
mice15 and 15 MHC+/+ wild-type
(control) mice were used in the study (males, 24 to 36 weeks old). The
mice, treated according to the guidelines of the Animal Care and Use
Committee of the University of Vermont, were euthanized by cervical
dislocation and their hearts removed. Twelve mutant hearts and 13
wild-type hearts earmarked for mechanical studies (see below) were
placed immediately in a standard Krebs solution18
containing 30 mmol/L 2,3-butanedione monoxime (BDM) and gassed
with 95% O2, 5% CO2. BDM
protects the myocardial tissue from cutting injury.18 The
remaining hearts (2 mutant and 2 wild type) were fixed, stained with
osmium tetroxide, and embedded using conventional
methods.19 Thin sections were contrasted with lead and
uranyl acetate, and micrographs were taken on a scanning transmission
electron microscope (JEOL 1210) operated at 60 kV. Fibrosis was quantified in 3 mutant and 2 wild-type hearts. Fixed hearts were cut transversely at the midventricular level, and the apical portions (containing sections of the free wall and papillary muscles) were embedded in glycolmethacrylate. Sections were cut at 2 µm and stained with Mason's trichrome stain for collagen. An Olympus BX-50 microscope (Olympus Corp) was used to view the slides. Images (magnification x400) were collected with a true-color video camera (DXC-960 MD/LLP; Sony) and camera adapter (CMA-D2; Sony) coupled with an Olympus viewing screen and remote control unit (RM-930; Sony). Images were stored, manipulated, and analyzed with a Sun SPARCstation 5 computer (Sun Microsystems) using IMIX/IMAGIST Version 8 software (Integrated Microanalyzer for Imaging Software, Princeton Gamma-Tech). The TrueColor module of the imaging software was used for area-extraction analysis. Trichrome-stained collagen was imaged in blue, and all other material in red. Fibrosis was defined as the area in blue divided by the total area imaged (blue plus red), expressed as a percentage.
Strip Preparation
Left ventricular papillary muscles were dissected in
BDM-Ringer's solution to yield thin strips (diameter, 0.125
mm; length, 1.5 mm), which were tethered with silk, transferred
to a vessel containing relaxing solution, and stretched just taut.
Relaxing solution contained 5 mmol/L MgATP, 40 mmol/L PCr,
240 U/mL creatine kinase (CK), 1 mmol/L free
Mg2+, 0.11 mmol/L
CaCl2, 5 mmol/L EGTA, and 20 mmol/L BES
buffer (pH 7.0); ionic strength was adjusted to 190 mmol/L with
added sodium methane sulfonate. The strips were demembranated (skinned)
by adding 1% wt/vol Triton x100, incubated overnight at 4°C,
transferred to detergent-free relaxing solution (with 50% wt/vol
glycerol and 10 µg/mL leupeptin), and stored at –20°C. Strips were
used within 1 week of dissection (generally the day after
dissection).
The tethered strips were placed in relaxing solution in a second
vessel, where small aluminum clips were used to isolate a uniform
segment (0.7 mm in length). The clipped segment was cut free
and transferred to a 30-µL drop of relaxing solution in a
glass-bottom aluminum chamber filled with mineral oil. The strip was
attached to a strain gauge and piezoelectric motor, described
elsewhere.19 Analogue displacement and tension signals
were monitored by a thermal strip chart recorder and digital
storage oscilloscope. Oil temperature was maintained at 27°C or
37°C (±0.5°C) by a Peltier-effect thermoelectric device
(Cambion; Cambridge Thermionic Corp).
The skinned strips were stretched (incrementally, by 0.05-µm steps
per sarcomere) to a sarcomere spacing of 2.2 µm (estimated
with an inverted microscope and filar micrometer). Strip
tension (mN/mm2) was calculated by dividing force
by the cross-sectional area obtained by multiplying the width imaged
from the top by the width imaged from the side (using a small mirror).
The mean diameter (average of top and side dimensions) of mutant strips
(133±6 µm) was slightly larger than that of wild-type strips
(119±3 µm; P<0.05).
The skinned strips were activated incrementally by Ca2+ by exchanging equal volumes of relaxing solution for activating solution (pCa 4.5) to attain pCa values of 7, 6, 5.75, 5.5, and 5. Activating solution had the same ionic composition as relaxing solution, except the total concentration of CaCl2 was 5.03 mmol/L (pCa 4.5). Solutions were formulated by solving equations describing the ionic equilibria.20
Sinusoidal Analysis and Strip Viscoelasticity
Small-amplitude sinusoidal length perturbation analysis
(sinusoidal analysis) was used to model strip viscoelastic
properties and to obtain information about R403Q-induced changes in
crossbridge kinetics. Sinusoidal length perturbations of 0.25% strip
length (peak-to-peak) were applied at 42 discrete frequencies (0.125 to
100 Hz) using a microcomputer and 16-bit data acquisition board
(DT2838, Data Translation Inc.). The length and force signals from the
servomotor and strain gauge were digitized, and the elastic and viscous
components of dynamic stiffness were calculated from the change in
tension and length at each frequency (Figure 1
, left and caption). Nyquist diagrams
were constructed (Figure 1, right) by plotting the viscous
modulus versus the elastic modulus at each frequency. Details of the
method have been described previously.21 22
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F) is divided by strip cross-sectional area
(Ac) to yield tension
(
ei
Statistics
Significance of differences between MHC403/+
(mutant) and MHC+/+ (wild-type) groups for any specific
parameter was assessed using an unpaired Student
t test. Data are presented as mean±SEM, unless
otherwise indicated.
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Results |
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Morphometric Analysis
Electron micrographs of left-ventricular sections from the
MHC403/+ mouse hearts (Figure 2B and 2C) displayed sarcomeres that
appeared to be identical to those of wild-type hearts (Figure 2A). In 1 of 2 mutant mouse hearts examined (Figure 2B),
myofibrils were disarrayed, especially where myofibrils attach to the
intercalated disk (arrow). The disarray is similar to that reported
previously on both the light15 and
electron23 microscopic levels. In the other mutant mouse
heart (Figure 2C), all areas examined appeared to be normal.
These results indicate variable penetrance of the morphological
phenotype, reminiscent of the diversity of
ventricular myocyte disarray and left atrial enlargement in
male mice reported previously.15
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We assessed the extent and regional variability of fibrosis in representative hearts from both mutant and wild-type strains. Two of 3 mutant hearts examined showed evidence of fibrosis (blue-stained collagen), but the extent of fibrosis varied considerably from region to region in both the papillary and free wall muscles. In sections from 1 mutant heart, fibrosis was 4.41±1.44% (n=14 regions sampled) for papillary muscle and 1.13±0.76% (n=18) for ventricular wall. In another, fibrosis was absent in the papillary muscle but present in the ventricular wall (1.13±3.25%; n=18). In the third mutant heart, there was no evidence of fibrosis. Sections from both wild-type hearts also exhibited little (<0.6%) or no fibrosis.
Calcium Dependency of Isometric Tension and Oscillatory
Power
Figure 3 illustrates the calcium
dependency of isometric force and oscillatory power output (per
cross-sectional area) in MHC403/+ and wild-type strips
at 27°C. After steady-state isometric tension reached a maximal level
at each calcium concentration, small-amplitude (0.25%, peak to peak)
sinusoidal length perturbations were used to generate oscillatory work.
At or near saturating calcium concentrations (pCa 5 to 5.75),
MHC403/+ strips tended to generate less isometric
tension and oscillatory power than wild-type strips (Figure 3A and 3C; Table 1), although values were
highly variable within each group. Differences between group means
were marginal (0.06<P<0.08).
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) and 
) strips from left
ventricular papillary muscle at 27oC.
In panel B, isometric tension is expressed as a fraction of that at pCa
5, and the resulting pCa-tension relationship is fitted by least
squares to the Hill equation as follows:
Tx/Tmax=xn/(an
+ xn), where Tx
is the isometric tension at [Ca2+]=x (in
mol/L), Tmax is the maximum isometric
tension generated at [Ca2+]=10–5 mol/L,
a=[Ca2+] at half-maximal tension
(pCa50=-log a), and n is the Hill
coefficient (cooperativity index). Net power was calculated at the
frequency of maximum power output (6 to 12 Hz; see text).
pCa-log [Ca2+] (in mol/L). Significance levels,
mutant versus wild type, are as follows: ***P<0.001,
P=0.05 to 0.075. Data shown are
mean±SEM.
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At submaximal calcium concentration (pCa 6), MHC403/+
strip tension was significantly higher (2.5 times;
P<0.001) than wild-type tension, but oscillatory power was
not significantly different from wild-type power (Figure 3A
and 3C; Table 1); at the lowest calcium concentrations (pCa 7 to 8),
there was no difference between mutant and wild-type strips (Figure 3A and 3C; Table 1). Similar results were obtained at
37°C (Table 1).
Tensions were normalized with respect to their values at pCa 5, and the
Hill equation (Figure 3, caption) was fit by least squares to
data from each strip. The MHC403/+ strips required less
calcium (1/3 µmol/L) to achieve half activation than control
strips (Figure 3B; Table 1); that is, calcium sensitivity
increased. The pCa-tension relationship is very steep (Hill
coefficient, n, is 6), indicating a highly cooperative
activation mechanism. Thus, over a limited range of calcium
concentrations, small differences in [Ca2+]
make large differences in tension and power output. The
MHC403/+ strips appeared to have slightly steeper
pCa-tension relationships (ie, higher Hill coefficient n)
than wild-type strips, but the difference was not significant (Table 1).
We confirmed the adequacy of the PCr/CK MgATP-regenerating system by
examining the PCr and CK dependency of the frequency of maximum
oscillatory power output (fmax) under
conditions of maximal activation (pCa 5; [CK], 240 U/mL; perturbation
amplitude, 0.25%). Since fmax declines
sharply with substrate depletion (data not shown),
fmax is a sensitive indicator of a lack of
[MgATP] in the core of the strip. For wild-type strips at 40
mmol/L PCr, fmax was 13±1 Hz at 27°C and
15±1 Hz at 37°C. At 33 mmol/L PCr,
fmax was 12±1 Hz at 27°C and 14±1 Hz at
37°C, a reduction of 5% at both temperatures, which suggests that
33 mmol/L PCr represents a slight substrate limitation. At
20 mmol/L PCr, fmax was 10±1 Hz at
37°C and 7.5±1 Hz at 27°C, a drop of 22% and 50%,
respectively. The more severe drop suggests greater substrate
limitations at 20 mmol/L PCr, especially at 37°C. Similar
parameter values were obtained when CK concentration was
doubled, from 240 to 580 U/mL, indicating that the lower [CK] is
adequate. Similar results were obtained from mutant strips, although
fmax of mutant strips (10±1 Hz, n=12)
tended to be lower than that of wild-type strips (12±1 Hz, n=12;
P=0.068). These results suggest that 40 mmol/L PCr and
240 U/mL CK constitutes an adequate MgATP regenerating system at
27°C, with slightly less regenerating capacity at 37°C. This
conclusion was supported by the absence of any correlation between
fmax and strip diameter (range, 77 to
163 µm) at 40 mmol/L PCr and 240 U/mL CK.
Strip Viscoelasticity and Crossbridge Model Parameters
Sinusoidal length perturbation analysis was used to
investigate actomyosin crossbridge function. The force response to a
small-amplitude sinusoidal length change is also sinusoidal (Figure 1, left), although the amplitude and phase relationship of the
force response to the length change varies dramatically with frequency
in calcium-activated strips. The resulting dynamic stiffness
plot (Figure 1, right) exhibits components that have
counterparts in the time domain of step-length perturbation
analysis (see Appendix, part 1), but, because of the
high signal-to-noise ratio, the unique signatures of the individual
components in the frequency domain (see below), and the ease of
extracting information about oscillatory work and power, we chose to
use the sinusoidal length perturbation method to probe crossbridge
function.
In the frequency domain (sinusoidal analysis), the expression
for complex stiffness (tension divided by fractional change in length)
is y(f)=A
(i2
f/)k-B
if/(b+if)+C
if/(c+if), for f<1
kHz, where i=
, =1 Hz (by
definition), and k is a unitless exponent proportional to
the phase angle 
of the Nyquist plot (Figure 1).
Coefficients A, B, and C (in
N/m2) define the magnitude of the complex
stiffness of components (or processes) A, B, and C (note that
B and C are of opposite sign), and kinetic
parameters b and c (in Hz) define the
characteristic frequencies of processes B and C, respectively. Maximum
oscillatory work production is produced by the crossbridges at
frequency b; maximum oscillatory work absorption, at
frequency c. The process A term A
(i2f/)k plots
as a straight line in the Nyquist diagram (see Appendix, part 2),
whereas the process B and C terms [B
if/(b+if) and C
if/(c+if)] plot as semicircles of
opposite sign (Figure 4). These
distinctive features allowed us to readily distinguish processes A, B,
and C and to derive model parameters from curve fits of
y(f) to the data (Table 2 and Figure 5).
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Figure 4 (top panels) shows Nyquist plots of
y(f) fit to average data from
MHC403/+ (left) and wild-type (right) strips. (An
exemplar fit to data from 1 strip is illustrated in Figure 1
[right].) Components A, B, and C
deconvolved from each Nyquist plot and averaged are shown in Figure 4 (bottom).
Parameter A is a measure of the amplitude of the
viscoelastic response of passive elements of the strip, on a per
cross-sectional area basis. At 27°C, A at pCa 8 to 6 was
greater in MHC403/+ than in wild-type strips, but at pCa
5.75 to 5, A was not significantly different (Table 2). At 37°C, A of MHC403/+ was
marginally greater at pCa 8 and 7.
Values for parameter B and C reflect
both the number and unitary stiffness of cycling crossbridges. At pCa
5.5 and 5, B and C of MHC403/+
strips were 20% less than that of wild-type strips (although the
reduction was highly variable), whereas at pCa 6, B and
C were 50% more (Figure 5, 27°C). Similar
differences between mutant and wild-type strips were observed at 37°C
(data not shown). In general, the calcium dependency of B
(Figure 5A) and C (Figure 5B) mimics the
pCa-tension relationship (Figure 3A).
The apparent rate constants characterizing the strain-induced
transitions between crossbridge states underlying processes
B and C (Figure 6)
are the characteristic frequencies b and c
multiplied by 2.21 Essentially, 2b
is the apparent rate constant of the work-producing step of the
crossbridge cycle, and 2c is the apparent rate constant
of the work-absorbing step. At pCa 6 (submaximal calcium
activation), the values of 2b and 2c were
significantly less in MHC403/+ than in wild-type strips
(Figure 5C). The value of 2b of
MHC403/+ strips was also significantly reduced at higher
calcium concentration (pCa<6), although not quite to the same extent.
Similar differences between mutant and wild-type strips were observed
at 37°C (data not shown). The corresponding characteristic
frequencies b and c for mutant versus wild-type
strips at pCa 6 were as follows: b=11±1 versus 15±1 Hz and
c= 58±3 versus 66±3 Hz at 27°C, and b=22±3
versus 27±1 Hz and c= 85±4 versus 94±3 Hz at 37°C (all
P<0.05, mutant versus wild-type strips). These results
demonstrate that kinetic processes directly attributable to crossbridge
function are altered in the MHC403/+ cardiac strips (see
Appendix, part 3).
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Discussion |
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We compared complex stiffness moduli of strips from
MHC403/+ and wild-type mouse hearts, which yielded
information about crossbridge kinetics at myocardial calcium
concentrations (pCa 8 to 5) that encompass the
physiological range (ie, pCa 7 to
624 ; probably higher in exercising mice). The
frequency range used in the sinusoidal analysis (0.125 to 100
Hz) also encompassed the normal heart rate of the mouse (10 Hz, ie,
600 bpm; higher in exercising mice). To distinguish changes in passive
and active properties of the strips and to characterize R403Q-induced
changes in crossbridge recruitment and kinetics, we partitioned the
complex stiffness modulus into 3 components (or processes): A, B, and
C. (In relaxed muscles, A reflects the change in passive force borne
primarily by filaments connecting the thick filaments to the Z line
when sarcomeres are stretched or released by an externally applied
strain. With increasing calcium, an ever-increasing proportion of this
change in passive force, together with the active force [B and C], is
borne by the thin filaments.)
The magnitude of component A (ie, the value of parameter
A) reflects the extent to which passive structural elements
contribute to the dynamic stiffness of the fiber. At low calcium
concentrations (pCa 7 to 8, at which changes in passive viscoelasticity
are expected to be most apparent), parameter A
of MHC403/+ strips was 24% to 44% larger than that of
wild-type strips. Part of the passive stiffness at the 2.2-µm
sarcomere length is that of interstitial
collagen,25 the accumulation of which (fibrosis)
would contribute to A, both by increasing the passive
stiffness of the strip and by simply occupying a greater fraction of
the cross-sectional area (parameters A,
B, and C are normalized to strip cross-sectional
area).
The papillary muscles from the MHC403/+ hearts showed
significant but variable fibrosis, as assessed by collagen stain.
In 1 mutant animal, the papillary muscle showed more evidence of
fibrosis (4% to 5%) than the free wall (1% to 2%). In the second
mutant animal, the papillary muscle showed less evidence of fibrosis
(<1%) than the free wall (2% to 3%). In the third mutant animal,
the papillary muscle and free wall had no evident fibrosis. The
wild-type control animals showed little or no evidence of fibrosis. In
those mutant hearts that showed fibrosis, the extent varied
considerably, even between adjacent regions within the same papillary
muscle or free wall. The within-group variation is reminiscent of the
pronounced variability of myofibrillar disarray (Figure 2). On
the basis of this limited study, we provisionally conclude that both
the increase in coefficient A and its variability can be at
least partly attributed to the variable increase in collagen
content.
The magnitude of processes B and C (ie, the B and
C values) reflect the number of force-producing crossbridges
as well as stiffness per crossbridge (time-averaged unitary stiffness).
Because B and C are normalized to strip
cross-sectional area, the marginal reduction and variability of
B and C at maximal activation (Figure 5A and 5B) may reflect a fibrosis-related variable reduction in
cross-sectional area occupied by the myofibrils. Alternatively, reduced
B and C may indicate a fundamental change in crossbridge stiffness or
redistribution of crossbridge states. (Since the imposed length
perturbations was small [<0.25% of the sarcomere length, or <4 nm
per half sarcomere], the viscoelastic properties
represented by B and C are those of a
steady-state population of crossbridges undergoing relatively small
fluctuations in strain about some mean position.)
We assumed that changes in the rate constants of the exponential
responses B and C (Figure 5C and 5D) reflected changes in
crossbridge kinetics due to the R403Q mutation. Our method does not
allow us to assess the degree to which fibrosis (in the form of
additional parallel elasticity) directly affects, if it affects at all,
the elementary crossbridge rate constants. However, our model does take
into account an effect of fibrosis on strip kinetics through the
passive viscoelastic A term of
y(f), such that an increased passive
viscoelasticity will reduce the frequency
(fmax) at which power output is maximal.
This prediction is consistent with the tendency of
fmax to be less in R402Q strips than in
wild-type strips (see Results section).
In the context of a simple 3-state crossbridge scheme (Figure 6), process B represents transitions between a preforce
state X1 and a postforce state
X2, the apparent rate constant of which is
2b. For a stepwise change in average crossbridge strain
of <4 nm [y(f)],
2b=k12+k21,
where k12 and
k21 are the apparent forward and backward
unidirectional rate constants, respectively. Process C
represents transitions between another postforce state
X3 (Figure 6, asterisk) and the preforce state
X1. The apparent rate constant of this transition
is
2c=k31+k13,
where the ks are the unidirectional rate constants. Assuming
that bridges in the preforce state X1 are not
force bearing over the range of frequencies used (0.1 to 100 Hz), our
mechanical measurements cannot distinguish myosin in the preforce state
from detached (or weakly attached; see below) myosin; thus,
X1 constitutes a lumped state.
Rat -myosin that contains the R403Q mutation has an elevated
Km for actin-activated MgATPase
activity,11 which implies a reduced actin-binding
affinity. Reductions of 2b and 2c (Figure 5C and 5D), or, more specifically, reductions of
k12 and k13,
are consistent with a reduced actin-binding affinity. The
elevation of tension and dynamic stiffness (ie, increased B)
of MHC403/+ strips at submaximal activation (pCa 6;
Figures 3A, 3B, 5A, and 5B) is consistent with a
reduction in k12 if some preforce bridges
promote thin-filament activation (eg, if some are in the "open"
state; see Reference 3030 ). Because the decrease in 2b
(Figure 5C) offsets the increase in B (Figure 5A), oscillatory power (which is proportional to the product
of 2b and amplitude B; see Figure 4
legend) remains unchanged (Figure 3B). The reduced peak tension
and dynamic stiffness of MHC403/+ strips at maximal
calcium concentration (pCa 5, Figures 3A, 3B, 5A, and 5B)
are at least qualitatively consistent with the postulated
reduction of bridges in state X2.
Implications of MHC403/+ Mouse Studies and Relevance
to Human ß-MHC403/+ Disease
The present studies provide a possible explanation for the
diversity of clinical presentations characteristic of the
R403Q FHC mutation. Contractile function of murine
MHC403/+ strips may be enhanced or depressed, depending
on calcium concentration. Below 50% calcium activation
(pCa<5.8, Table 1), tension is increased because of enhanced
thin-filament activation; above 50% activation (pCa more than
5.8), tension is depressed. Fibrosis is likely to reduce
MHC403/+ strip tension by a variable extent,
depending on the progression of the disease. Near-maximal or at-maximal
activation oscillatory power is reduced, for reasons explained above.
It is tempting to speculate that both peak tension and power reserve
are compromised during heavy exercise,15 when
systolic calcium may approach maximal levels (pCa5.5).
The major findings of the present study are (1) the elevated passive dynamic stiffness of relaxed myocardium, (2) the enhanced tension at submaximal calcium activation, (3) the reduced oscillatory power at maximal calcium activation, and (4) the depressed kinetics of MHC403/+ strips compared with wild-type strips. Because the mutant animal was heterozygous, the observed differences from wild-type animals are probably less than would be observed for homozygous mutants, if they lived to a comparable age. In any case, the observed differences in strip mechanics are at least partly responsible for altered cardiac function. An elevated passive dynamic stiffness and increased calcium sensitivity of active tension in MHC403/+ strips, for example, would be expected to increase tone during diastole, a prediction that is consistent with evidence that diastolic function is impaired in MHC403/+ mouse hearts.16 17 Several human clinical studies also report impaired relaxation of the left ventricle in FHC, with improved performance by calcium-channel blockers.26 27 28 Although the results of these clinical studies have been used to argue for diastolic calcium overload in FHC, impaired relaxation could just as well reflect greater tone due to increased calcium sensitivity.
Near maximal or at maximal calcium activation (pCa5.5), the reduced
force production and oscillatory power output due to depressed
crossbridge kinetics and reduced actomyosin content (fibrosis), may
impair systolic reserve during strenuous exercise. Thus,
the calcium-dependent effect of the mutation (whether facilitating or
debilitating), together with a variable degree of fibrosis and
myofibrillar disorder, may contribute to the diversity of clinical
symptoms observed in murine FHC.
Our results suggest a possible mechanism related to papillary muscle
dysfunction to explain mitral regurgitation in FHC.
During diastole, the mitral valve is open and the chordae
tendinae that attach the papillary muscles to the valve leaflets are
slack. With the onset of systole, the mitral valve closes and the
chordae are passively stretched. At the same time, the papillary
muscles actively contract, which helps prevent the valve leaflets from
prolapsing into the atrium. The papillary muscles attached to the
chordae will be stretch-activated, thereby developing
additional tension and doing work (proportional to
parameter B, Figure 5A) that helps
maintain the valve in the closed position. Precise timing of
contraction and the additional benefit of prolonged activation that is
independent of electrical activity may be critical in maintaining
mitral competence, especially at very rapid heart rates. If so,
papillary muscle function will benefit from an enhanced oscillatory
power output, especially at heart rates near
fmax (mutant heart, 13±1 Hz; wild-type
heart, 15±1 Hz at 37°C), ie, heart rates during strenuous exercise.
Clearly, further work is required to establish whether
MHC403/+ mouse hearts in fact exhibit mitral
regurgitation.
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Acknowledgments |
|---|
This research was supported by National Institutes of Health Grant R01 HL52087. We thank Louis Mulieri, David Kass, Jeff Moore, Matt Tyska, David Warshaw, Peter VanBuren, and Norman Alpert for their input to the discussion. We thank Bill Barnes, Peter Pak, Hiroyuki Sumino, and Douglas Taatjes for technical help.
|
Footnotes |
|---|
Reprint requests to David W. Maughan, PhD, Department of Molecular Physiology and Biophysics, D215 Given Bldg, University of Vermont, Burlington, VT 05405.
This manuscript was sent to Leslie A. Leinwand, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Preliminary results were presented at the Scientific Conference on the Molecular Biology of the Normal, Hypertrophied, and Failing Heart, Snowbird Conference, August 1996.
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Appendix 1 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAppendix 1References |
|---|
1. Response to a Stepwise Stretch
In the response to a stepwise stretch,31 32 force increases concurrently with stretch, then falls abruptly after the stretch is complete (rapidly at first, then more slowly, with an exponential rate constant 2
c). An even slower rise in
force develops with an exponential rate constant 2b (the
stretch activation response), which finally gives way to another slow
fall in force. The initial and final declines in force together
resemble conventional stress-relaxation, which can be approximated (for
t>1 ms) by the term At–k, where
A is a coefficient (in mN/mm2, if
expressed as a tension or stiffness) and k is a unitless
exponent. For very small amplitudes (<0.125% strip length), the
response to a step decrease in length (ie, a release) mirrors that of a
step increase (stretch); that is, the response is
"linear."32 The amplitude coefficients are
positive with stretch and negative with release.
2.
A(i2f/)k
In the presence of 30 mmol/L BDM, the number of attached
force-generating crossbridges is sharply reduced.33 When
30 mmol/L BDM was added to activating solution in our experiments,
coefficients B and C became very small, and the Nyquist plot
approximated a straight line with a slope (k value) that was
similar to that seen in the rigor state (data not given). This
observation supports our use of the term
A(i2f/)k
in the Nyquist plot to characterize process A, which appears to differ
in magnitude (coefficient A) but not in form (straight line,
constant k) among relaxed, active, and rigor
states.
3. Depressed Kinetic Response of the MHC403/+
Strips
Additional tests suggest that the depressed kinetic response of
the MHC403/+ strips is not due to substrate limitation.
Kinetic parameters b and c, which are
sensitive to [MgATP],34 were significantly reduced
by lowering the [PCr] to 20 mmol/L, but a reduction from 40 to
33 mmol/L produced only a marginal reduction of b and
c in both mutant and wild-type strips (<5% at 27°C and
<10% at 37°C). Doubling the concentration of CK (to 580 U/mL) had
no effect on b and c, confirming saturation at
240 U/mL. These results provide additional support that 40 mmol/L
PCr and 240 U/mL CK are adequate (with slightly less regenerating
capacity at 37°C). This conclusion is supported by the absence of any
correlation between any of the kinetic constants of
y(f) and strip diameter (range, both
groups, 77 to 163 µm).
Received June 8, 1998; accepted December 11, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAppendix 1References |
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