This Article Abstract Full Text (PDF) Methods Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tyska, M. J. Articles by Warshaw, D. M. Search for Related Content PubMed PubMed Citation Articles by Tyska, M. J. Articles by Warshaw, D. M. Related Collections Contractile function Myocardial cardiomyopathy disease Animal models of human disease Genetically altered mice

(Circulation Research. 2000;86:737.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Single-Molecule Mechanics of R403Q Cardiac Myosin Isolated From the Mouse Model of Familial Hypertrophic Cardiomyopathy

M. J. Tyska, E. Hayes, M. Giewat, C. E. Seidman, J. G. Seidman, D. M. Warshaw

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 Women’s 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—Familial hypertrophic cardiomyopathy (FHC) is an inherited cardiac disease that can result in sudden death in the absence of any overt symptoms. Many of the cases documented to date have been linked with missense mutations in the ß-myosin heavy chain gene. Here we present data detailing the functional impact of one of the most deadly mutations, R403Q, on myosin motor function. Experiments were performed on whole cardiac myosin purified from a mouse model of FHC to eliminate potential uncertainties associated with protein expression systems. The R403Q mutant myosin demonstrated 2.3-fold higher actin-activated ATPase activity, 2.2-fold greater average force generation, and 1.6-fold faster actin filament sliding in the motility assay. The force- and displacement-generating capacities of both the normal and mutant myosin were also characterized at the single molecule level in the laser trap assay. Both control and mutant generated similar unitary forces (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

Top Abstract Introduction Materials and Methods Results Discussion References

 
Familial hypertrophic cardiomyopathy (FHC) is an inherited cardiac disease that frequently results in the sudden death of young and otherwise healthy individuals.^{1 2} Although the clinical manifestation of FHC is widely varied, common features include asymmetric septal hypertrophy, potential outflow tract obstruction, myocyte disarray, interstitial fibrosis, and arrhythmia.^{1 2 3} Approximately 70% of all FHC cases documented to date have been linked to single point mutations in various contractile proteins of the cardiac sarcomere. These include ß-myosin heavy chain (MHC), myosin regulatory light chain, myosin essential light chain, troponin-T, troponin-I, -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 K_ms 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
FHC Mouse Model
Mice expressing the R403Q mutation were created using recombinant techniques, as previously described,^{23 24} and expressed 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 (P_i) concentrations at fixed time points were determined colorimetrically, using a malachite green phosphate indicator.²⁷ For the actin-activated assay, values of P_i released s^–1xhead^–1 versus [actin] were plotted and fitted to Michaelis-Menten kinetics (V=V_maxx[actin]/K_m+[actin]), with V_max and K_m fit parameters using Tablecurve 2D version 4 from SPSS.

Motility Assays
Actin filament velocities (v_actin) 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 (F_avg) 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 (t_on) 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
Protein Purification
Whole cardiac myosin was successfully purified from hearts excised from 1-week-old neonatal mice using standard high-salt extraction procedures as outlined in Materials and Methods. PAGE analysis of freshly purified samples (see Figure 1) revealed no differences in purity between +/+ and 403/403 myosins. Moreover, contaminating proteins and proteolysis were minimal and thus unlikely to account for any phenotypic differences observed in the various functional assays.

View larger version (30K): [in this window] [in a new window]   Figure 1. SDS-PAGE analysis of purified whole cardiac control (+/+) and mutant (403/403) myosin samples. A 4% to 20% polyacrylamide gradient gel stained with Coomassie blue. -MHC, essential light chain (LC1), and regulatory light chain (LC2) are indicated next to their respective bands. Total protein loads were 5 µg/lane.

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 V_max was also accompanied in both cases by a 4-fold elevation of K_m 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.

View larger version (17K): [in this window] [in a new window]   Figure 2. Actin-activated ATPase measurements for +/+ (), +/403 (), and 403/403 () mouse cardiac myosin. Each data point represents mean±SEM at a given actin concentration. Data were fitted to a Michaelis-Menten kinetic model. Vmax and Km±SE of the fit parameters are indicated in each case. Three 403/403, two +/403, and four +/+ hearts were used to generate these data. *P<0.05 vs +/+.

View this table: [in this window] [in a new window]   Table 1. Summary of Ensemble Measurements

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 v_actin, 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 (F_avg) 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 v_actin=1.6 µm/s). By analyzing how the cardiac myosin–based actin filament velocity slowed with the addition of the slower smooth muscle myosin (see Figure 3), an estimate of the relative F_avg 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 F_avg when compared with +/+ myosin (see Table 1).

View larger version (21K): [in this window] [in a new window]   Figure 3. Measurements of relative isometric force for +/+ (bottom) and 403/403 (top) mouse cardiac myosin. These curves represent the effect of varying the proportion of fast cardiac myosin (, 403/403; , +/+) to slow smooth muscle myosin on actin filament sliding velocity. The relative measure of isometric force (Po) is given by the ratio of Po slow myosin to Po fast myosin in each case (shown on each plot). A concave down curve signifies that the slower myosin produces higher Favg, a nearly straight line indicates that slow and fast myosins produce comparable Favg (dashed line shown on both plots), and a concave up curve indicates that the faster myosin produces higher Favg. By dividing out the common reference myosin, Po(Smooth), from the ratios provided by the curve fits (shown on each plot±SE of the fit), one can deduce that 403/403 produced 2.2-fold higher Favg when compared with the purified +/+ myosin. Three 403/403 and two +/+ hearts were used to generate these data. *P<0.05 vs +/+.

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 (t_on) 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 t_on for displacement (70 ms) and force events (175 ms; see Table 2).

View larger version (41K): [in this window] [in a new window]   Figure 4. Representative unitary displacement and force traces from +/+ and 403/403 mouse cardiac myosin. A, Time series are raw unitary displacement data from +/+ and 403/403 cardiac myosin. Rapid deflections with low variance relative to the baseline noise represent unitary actomyosin interactions (indicated by arrowheads). B, Unitary force data (F) are shown with accompanying residual displacement time series (RD). These traces represent the motion of the trapped bead that was not suppressed by the feedback circuit. Reductions of the variance in RD indicate unitary force events (indicated by arrowheads). Scale bars are given to indicate calibration.

View this table: [in this window] [in a new window]   Table 2. Summary of Unitary Mechanical Data

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).

View larger version (20K): [in this window] [in a new window]   Figure 5. Scatter plots representing the distributions of MV histogram displacement fits from +/+ (left) and 403/403 (right) mouse cardiac myosin. In these representations, each open circle indicates the mean value from a single MV histogram fit generated from 1 displacement record (+/+, n=57 records; 403/403, n=34 records). Therefore, each circle entered into these plots represents 50 to 100+ unitary events. Six +/+ hearts and seven 403/403 hearts were used to obtain these data. Bar histograms below each scatter plot are frequency distributions for the same data. In each case, data were fit with both single and double gaussian functions. Best fit (displayed on each plot) was chosen as the fit that produced the highest F statistic. Parameters from the fits were as follows: +/+ first peak amplitude, 11.8 (mean, 5.2; SD, 1.24); second peak amplitude, 9.8 (mean, 9.2; SD, 1.3); and 403/403 amplitude, 8.17 (mean, 6.3; SD, 2.6). Means are also indicated above each gaussian peak in the figure.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The ensemble measurements presented here reveal that the R403Q mutation associated with the mouse model of FHC results in a gain of hydrolytic and mechanical function; ie, actin-activated ATPase rates, v_actin, and F_avg were all elevated (Table 1, Figures 2 and 3). Interestingly, the enhanced performance was only a property of assays involving the interaction of actin and myosin, as the intrinsic ATPase rates measured under high-salt conditions were not significantly different from those of controls (see Table 1). This finding can be explained, because the R403 amino acid is thought to be located on the putative actin-binding interface of myosin.¹⁰ Therefore, only measurements involving the formation of the actomyosin complex should be perturbed. Indeed, this result has also been shown by others.¹⁷

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 v_actin 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 v_actin and F_avg 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)

where d is the unitary displacement and t_on is the displacement duration. In addition,

(2)

where F represents the unitary force developed by a single molecule and f_iso is the isometric "duty cycle" or fraction of the total cycle time (t_cycle) that the motor spends strongly bound to actin exerting F,³⁰ written as

(3)

Assuming these molecular definitions for v_actin and F_avg, one can then determine whether the mutation has its effect by changing either the inherent mechanical (ie, a change in d or F) and/or kinetic (ie, a change in t_on or f_iso) properties of myosin. Given the results of unitary mechanical measurements, we believe that both F and d produced by the 403/403 myosin were similar to that of the +/+ myosin. If so, then based on Equations 1 and 2, in the absence of any change in mechanical properties, the enhanced average force and velocity should be due to a change in the cycling kinetics of the myosin molecule. However, there were no apparent differences in the t_on estimated for both unitary displacements and forces for the 403/403 and +/+ myosins.

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, t_on), in turn limiting v_actin.^{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 t_on 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 t_on values comparable with the temporal resolution of our instrument (2 to 5 ms).

Despite the lack of a difference in t_on from unitary force measurements, a kinetic change can still explain why F_avg 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 F_avg was higher. Because t_cycle=(ATPase rate)^-1, a higher ATPase activity means a shorter overall cycle time, which, in the absence of a change in t_on, would result in an increase in f_iso (see Equation 3). As indicated by Equation 2, an increase in f_iso can explain the higher F_avg 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, v_actin, or F_avg 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 (F_avg), velocity (v_actin), and ATPase (V_max) 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

 
This work was supported by funds from the National Heart, Lung, and Blood Institute of the NIH. We thank Donald Gaffney for superb computer support and Guy Kennedy for the design and support of the laser trap.

Received September 16, 1999; accepted December 14, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Maron BJ. Hypertrophic cardiomyopathy. Lancet. 1997;350:127–133.[Medline] [Order article via Infotrieve]
2. Davies MJ, McKenna WJ. Hypertrophic cardiomyopathy: pathology and pathogenesis. Histopathology. 1995;26:493–500.[Medline] [Order article via Infotrieve]
3. Solomon SD, Wolff S, Watkins H, Ridker PM, Come P, McKenna WJ, Seidman CE, Lee RT. Left ventricular hypertrophy and morphology in familial hypertrophic cardiomyopathy associated with mutations of the beta-myosin heavy chain gene. J Am Coll Cardiol. 1993;22:498–505.[Abstract]
4. Watkins H, MacRae C, Thierfelder L, Chou YH, Frenneaux M, McKenna W, Seidman JG, Seidman CE. A disease locus for familial hypertrophic cardiomyopathy maps to chromosome 1q3. Nat Genet. 1993;3:333–337.[Medline] [Order article via Infotrieve]
5. Geisterfer-Lowrance AA, Kass S, Tanigawa G, Vosberg HP, McKenna W, Seidman CE, Seidman JG. A molecular basis for familial hypertrophic cardiomyopathy: a beta cardiac myosin heavy chain gene missense mutation. Cell. 1990;62:999–1006.[Medline] [Order article via Infotrieve]
6. Watkins H. Multiple disease genes cause hypertrophic cardiomyopathy. Br Heart J. 1994;72:S4–S9.
7. Bonne G, Carrier L, Richard P, Hainque B, Schwartz K. Familial hypertrophic cardiomyopathy: from mutations to functional defects. Circ Res. 1998;83:580–593.[Abstract/Free Full Text]
8. Vikstrom KL, Leinwand LA. Contractile protein mutations and heart disease. Curr Opin Cell Biol. 1996;8:97–105.[Medline] [Order article via Infotrieve]
9. Marian AJ, Kelly D, Mares AJ, Fitzgibbons J, Caira T, Qun T, Hill R, Perryman MB, Roberts R. A missense mutation in the beta myosin heavy chain gene is a predictor of premature sudden death in patients with hypertrophic cardiomyopathy. J Sports Med Phys Fitness. 1994;34:1–10.[Medline] [Order article via Infotrieve]
10. Rayment I, Holden HM, Sellers JR, Fananapazir L, Epstein ND. Structural interpretation of the mutations in the beta-cardiac myosin that have been implicated in familial hypertrophic cardiomyopathy. Proc Natl Acad Sci U S A. 1995;92:3864–3868.[Abstract/Free Full Text]
11. Epstein ND, Cohn GM, Cyran F, Fananapazir L. Differences in clinical expression of hypertrophic cardiomyopathy associated with two distinct mutations in the beta- myosin heavy chain gene: a 908Leu–Val mutation and a 403Arg–Gln mutation. Circulation. 1992;86:345–352.[Abstract/Free Full Text]
12. Cuda G, Fananapazir L, Zhu WS, Sellers JR, Epstein ND. Skeletal muscle expression and abnormal function of beta-myosin in hypertrophic cardiomyopathy. J Clin Invest. 1993;91:2861–2865.
13. Lankford EB, Epstein ND, Fananapazir L, Sweeney HL. Abnormal contractile properties of muscle fibers expressing beta-myosin heavy chain gene mutations in patients with hypertrophic cardiomyopathy. J Clin Invest. 1995;95:1409–1414.
14. Becker KD, Gottshall KR, Hickey R, Perriard JC, Chien KR. Point mutations in human beta cardiac myosin heavy chain have differential effects on sarcomeric structure and assembly: an ATP binding site change disrupts both thick and thin filaments, whereas hypertrophic cardiomyopathy mutations display normal assembly. J Cell Biol. 1997;137:131–140.[Abstract/Free Full Text]
15. Vybiral T, Deitiker PR, Roberts R, Epstein HF. Accumulation and assembly of myosin in hypertrophic cardiomyopathy with the 403 Arg to Gln beta-myosin heavy chain mutation. Circ Res. 1992;71:1404–1409.[Abstract/Free Full Text]
16. Cuda G, Fananapazir L, Epstein ND, Sellers JR. The in vitro motility activity of beta-cardiac myosin depends on the nature of the beta-myosin heavy chain gene mutation in hypertrophic cardiomyopathy. J Muscle Res Cell Motil. 1997;18:275–283.[Medline] [Order article via Infotrieve]
17. Sweeney HL, Straceski AJ, Leinwand LA, Tikunov BA, Faust L. Heterologous expression of a cardiomyopathic myosin that is defective in its actin interaction. J Biol Chem. 1994;269:1603–1605.[Abstract/Free Full Text]
18. Fujita H, Sugiura S, Momomura S, Sugi H, Sutoh K. Functional characterization of Dictyostelium discoideum mutant myosins equivalent to human familial hypertrophic cardiomyopathy. Adv Exp Med Biol. 1998;453:131–137.[Medline] [Order article via Infotrieve]
19. Roopnarine O, Leinwand LA. Functional analysis of myosin mutations that cause familial hypertrophic cardiomyopathy. Biophys J. 1998;75:3023–3030.[Abstract/Free Full Text]
20. Sata M, Ikebe M. Functional analysis of the mutations in the human cardiac beta- myosin that are responsible for familial hypertrophic cardiomyopathy: implication for the clinical outcome. J Clin Invest. 1996;98:2866–2873.[Medline] [Order article via Infotrieve]
21. Geisterfer-Lowrance AA, Christe M, Conner DA, Ingwall JS, Schoen FJ, Seidman CE, Seidman JG. A mouse model of familial hypertrophic cardiomyopathy. Science. 1996;272:731–734.[Abstract]
22. Georgakopoulos D, Christe ME, Giewat M, Seidman CM, Seidman JG, Kass DA. The pathogenesis of familial hypertrophic cardiomyopathy: early and evolving effects from an alpha-cardiac myosin heavy chain missense mutation. Nat Med. 1999;5:327–330.[Medline] [Order article via Infotrieve]
23. Hasty P, Ramirez-Solis R, Krumlauf R, Bradley A. Introduction of a subtle mutation into the Hox-2.6 locus in embryonic stem cells. Nature. 1991;350:243–246. (Correction. 1991;353:94).[Medline] [Order article via Infotrieve]
24. Valancius V, Smithies O. Testing an "in-out" targeting procedure for making subtle genomic modifications in mouse embryonic stem cells. Mol Cell Biol. 1991;11:1402–1408.[Abstract/Free Full Text]
25. Nguyen TT, Hayes E, Mulieri LA, Leavitt BJ, ter Keurs HE, Alpert NR, Warshaw DM. Maximal actomyosin ATPase activity and in vitro myosin motility are unaltered in human mitral regurgitation heart failure. Circ Res. 1996;79:222–226.[Abstract/Free Full Text]
26. Warshaw DM, Desrosiers JM, Work SS, Trybus KM. Smooth muscle myosin cross-bridge interactions modulate actin filament sliding velocity in vitro. J Cell Biol. 1990;111:453–463.[Abstract/Free Full Text]
27. Lanzetta PA, Alvarez LJ, Reinach PS, Candia OA. An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem. 1979;100:95–97.[Medline] [Order article via Infotrieve]
28. Warshaw DM, Desrosiers JM, Work SS, Trybus KM. Mechanical interaction of smooth muscle crossbridges modulates actin filament velocity in vitro. Prog Clin Biol Res. 1990;327:815–826.[Medline] [Order article via Infotrieve]
29. Finer JT, Simmons RM, Spudich JA. Single myosin molecule mechanics: piconewton forces and nanometre steps. Nature. 1994;368:113–119.[Medline] [Order article via Infotrieve]
30. Guilford WH, Dupuis DE, Kennedy G, Wu J, Patlak JB, Warshaw DM. Smooth muscle and skeletal muscle myosins produce similar unitary forces and displacements in the laser trap. Biophys J. 1997;72:1006–1021.[Abstract/Free Full Text]
31. Dupuis DE, Guilford WH, Wu J, Warshaw DM. Actin filament mechanics in the laser trap. J Muscle Res Cell Motil. 1997;18:17–30.[Medline] [Order article via Infotrieve]
32. Harris DE, Work SS, Wright RK, Alpert NR, Warshaw DM. Smooth, cardiac and skeletal muscle myosin force and motion generation assessed by cross-bridge mechanical interactions in vitro. J Muscle Res Cell Motil. 1994;15:11–19.[Medline] [Order article via Infotrieve]
33. Blanchard E, Seidman C, Seidman JG, LeWinter M, Maughan D. Altered crossbridge kinetics in the alphaMHC403/+ mouse model of familial hypertrophic cardiomyopathy. Circ Res. 1999;84:475–483.[Abstract/Free Full Text]
34. Yamashita H, Lowey S, Trybus KM. Functional consequences of myosin heavy chain mutations implicated in familial hypertrophic cardiomyopathy. Biophys J. 1997;72:A339.
35. McNally EM, Kraft R, Bravo-Zehnder M, Taylor DA, Leinwand LA. Full-length rat alpha and beta cardiac myosin heavy chain sequences: comparisons suggest a molecular basis for functional differences. J Mol Biol. 1989;210:665–671.[Medline] [Order article via Infotrieve]
36. Palmiter KA, Tyska MJ, Dupuis DE, Alpert N, Warshaw DM. Kinetic differences determined at the single molecule level account for the functional diversity of rabbit cardiac myosin isoforms. J Physiol (Lond). 1999;519:669–678.[Abstract/Free Full Text]
37. Palmiter KA, Alpert NR, Fananapazir L, Warshaw DM. Single cardiac myosin molecules from familial hypertrophic cardiomyopathy patients exhibit enhanced mechanical performance in the laser trap assay. Circulation. 1999;100(suppl I):I-193–I-194. Abstract.
38. Lauzon AM, Tyska MJ, Rovner AS, Freyzon Y, Warshaw DM, Trybus KM. A 7-amino-acid insert in the heavy chain nucleotide binding loop alters the kinetics of smooth muscle myosin in the laser trap. J Muscle Res Cell Motil. 1998;19:825–837.[Medline] [Order article via Infotrieve]
39. Siemankowski RF, White HD. Kinetics of the interaction between actin, ADP, and cardiac myosin-S1. J Biol Chem. 1984;259:5045–5053.[Abstract/Free Full Text]
40. Siemankowski RF, Wiseman MO, White HD. ADP dissociation from actomyosin subfragment 1 is sufficiently slow to limit the unloaded shortening velocity in vertebrate muscle. Proc Natl Acad Sci U S A. 1985;82:658–662.[Abstract/Free Full Text]
41. Marston SB, Taylor EW. Comparison of the myosin and actomyosin ATPase mechanisms of the four types of vertebrate muscles. J Mol Biol. 1980;139:573–600.[Medline] [Order article via Infotrieve]
42. Tyska MJ, Dupuis DE, Guilford WH, Patlak JB, Waller GS, Trybus KM, Warshaw DM, Lowey S. Two heads of myosin are better than one for generating force and motion. Proc Natl Acad Sci U S A. 1999;96:4402–4407.[Abstract/Free Full Text]
43. Spindler M, Saupe KW, Christe ME, Sweeney HL, Seidman CE, Seidman JG, Ingwall JS. Diastolic dysfunction and altered energetics in the alphaMHC403/+ mouse model of familial hypertrophic cardiomyopathy. J Clin Invest. 1998;101:1775–1783.[Medline] [Order article via Infotrieve]
44. Kammermeier H. High energy phosphate of the myocardium: concentration versus free energy change. Basic Res Cardiol. 1987;82(suppl 2):31-6–31-36.

This article has been cited by other articles:

				A. Belus, N. Piroddi, B. Scellini, C. Tesi, G. D Amati, F. Girolami, M. Yacoub, F. Cecchi, I. Olivotto, and C. Poggesi The familial hypertrophic cardiomyopathy-associated myosin mutation R403Q accelerates tension generation and relaxation of human cardiac myofibrils J. Physiol., August 1, 2008; 586(15): 3639 - 3644. [Abstract] [Full Text] [PDF]

				S. Lowey, L. M. Lesko, A. S. Rovner, A. R. Hodges, S. L. White, R. B. Low, M. Rincon, J. Gulick, and J. Robbins Functional Effects of the Hypertrophic Cardiomyopathy R403Q Mutation Are Different in an {alpha}- or {beta}-Myosin Heavy Chain Backbone J. Biol. Chem., July 18, 2008; 283(29): 20579 - 20589. [Abstract] [Full Text] [PDF]

				B. M. Palmer, Y. Wang, P. Teekakirikul, J. T. Hinson, D. Fatkin, S. Strouse, P. VanBuren, C. E. Seidman, J. G. Seidman, and D. W. Maughan Myofilament mechanical performance is enhanced by R403Q myosin in mouse myocardium independent of sex Am J Physiol Heart Circ Physiol, April 1, 2008; 294(4): H1939 - H1947. [Abstract] [Full Text] [PDF]

				S. Morimoto Sarcomeric proteins and inherited cardiomyopathies Cardiovasc Res, March 1, 2008; 77(4): 659 - 666. [Abstract] [Full Text] [PDF]

				M. Krenz, S. Sadayappan, H. E. Osinska, J. A. Henry, S. Beck, D. M. Warshaw, and J. Robbins Distribution and Structure-Function Relationship of Myosin Heavy Chain Isoforms in the Adult Mouse Heart J. Biol. Chem., August 17, 2007; 282(33): 24057 - 24064. [Abstract] [Full Text] [PDF]

				E. P. Debold, J. P. Schmitt, J. B. Patlak, S. E. Beck, J. R. Moore, J. G. Seidman, C. Seidman, and D. M. Warshaw Hypertrophic and dilated cardiomyopathy mutations differentially affect the molecular force generation of mouse {alpha}-cardiac myosin in the laser trap assay Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H284 - H291. [Abstract] [Full Text] [PDF]

				T. Kubo, J. R. Gimeno, A. Bahl, U. Steffensen, M. Steffensen, E. Osman, R. Thaman, J. Mogensen, P. M. Elliott, Y. Doi, et al. Prevalence, Clinical Significance, and Genetic Basis of Hypertrophic Cardiomyopathy With Restrictive Phenotype J. Am. Coll. Cardiol., June 26, 2007; 49(25): 2419 - 2426. [Abstract] [Full Text] [PDF]

				O. Agbulut, A. Huet, N. Niederlander, M. Puceat, P. Menasche, and C. Coirault Green Fluorescent Protein Impairs Actin-Myosin Interactions by Binding to the Actin-binding Site of Myosin J. Biol. Chem., April 6, 2007; 282(14): 10465 - 10471. [Abstract] [Full Text] [PDF]

				P. Richard, E. Villard, P. Charron, and R. Isnard The Genetic Bases of Cardiomyopathies J. Am. Coll. Cardiol., October 27, 2006; 48(9_Suppl_A): A79 - A89. [Abstract] [Full Text] [PDF]

				N. Frey, K. Brixius, R. H. G. Schwinger, T. Benis, A. Karpowski, H. P. Lorenzen, M. Luedde, H. A. Katus, and W. M. Franz Alterations of Tension-dependent ATP Utilization in a Transgenic Rat Model of Hypertrophic Cardiomyopathy J. Biol. Chem., October 6, 2006; 281(40): 29575 - 29582. [Abstract] [Full Text] [PDF]

				J. P. Schmitt, E. P. Debold, F. Ahmad, A. Armstrong, A. Frederico, D. A. Conner, U. Mende, M. J. Lohse, D. Warshaw, C. E. Seidman, et al. Cardiac myosin missense mutations cause dilated cardiomyopathy in mouse models and depress molecular motor function PNAS, September 26, 2006; 103(39): 14525 - 14530. [Abstract] [Full Text] [PDF]

				J. James, L. Martin, M. Krenz, C. Quatman, F. Jones, R. Klevitsky, J. Gulick, and J. Robbins Forced Expression of {alpha}-Myosin Heavy Chain in the Rabbit Ventricle Results in Cardioprotection Under Cardiomyopathic Conditions Circulation, May 10, 2005; 111(18): 2339 - 2346. [Abstract] [Full Text] [PDF]

N. R. Alpert, S. A. Mohiddin, D. Tripodi, J. Jacobson-Hatzell, K. Vaughn-Whitley, C. Brosseau, D. M. Warshaw, and L. Fananapazir Molecular and phenotypic effects of heterozygous, homozygous, and compound heterozygote myosin heavy-chain mutations Am J Physiol Heart C