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Am J Physiol Heart Circ Physiol 287: H91-H99, 2004. First published March 4, 2004; doi:10.1152/ajpheart.01015.2003
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Differential cross-bridge kinetics of FHC myosin mutations R403Q and R453C in heterozygous mouse myocardium

Bradley M. Palmer,1 David E. Fishbaugher,1 Joachim P. Schmitt,2 Yuan Wang,1 Norman R. Alpert,1 Christine E. Seidman,2 J. G. Seidman,2 Peter VanBuren,1 and David W. Maughan1

1Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405; and 2Department of Genetics, Howard Hughes Medical Institute and Harvard Medical School, Boston, Massachusetts 02115

Submitted 28 October 2003 ; accepted in final form 21 February 2004


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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The kinetic effects of the cardiac myosin point mutations R403Q and R453C, which underlie lethal forms of familial hypertrophic cardiomyopathy (FHC), were assessed using isolated myosin and skinned strips taken from heterozygous (R403Q/+ and R453C/+) male mouse hearts. Compared with wild-type (WT) mice, actin-activated ATPase was increased by 38% in R403Q/+ and reduced by 45% in R453C/+, maximal velocity of regulated thin filament (VRTF) in the in vitro motility assay was increased by 8% in R403Q/+ and was not different in R453C/+, myosin concentration at half-maximal VRTF was reduced by 30% in R403Q/+ and not different in R453C/+, and the characteristic frequency for oscillatory work production (b frequency), determined by sinusoidal analysis in the skinned strip at maximal calcium activation, was 27% lower in R403Q/+ and 18% higher in R453C/+. The calcium sensitivity for isometric tension in the skinned strip was not different in R403Q/+ (pCa50 5.64 ± 0.02) and significantly enhanced in R453C/+ (5.82 ± 0.03) compared with WT (5.58 ± 0.02). We conclude that isolated myosin and skinned strips of R403Q/+ and R453C/+ myocardium show marked differences in cross-bridge kinetic parameters and in calcium sensitivity of force production that indicate different functional roles associated with the location of each point mutation at the molecular level.

myosin heavy chain; ATPase; myosin motility; isometric tension; sinusoidal analysis

FAMILIAL HYPERTROPHIC CARDIOMYOPATHY (FHC) is an autosomal dominant disorder caused by a mutant allele for any one of several sarcomeric proteins, including {beta}-myosin heavy chain ({beta}-MHC) (13, 23, 26). Characteristics of the FHC phenotype include various degrees of cardiac hypertrophy, fibrosis, myocyte disarray, contractile dysfunction, and premature death in the absence of hemodynamic factors (23, 26, 31). Of the >67 identified mutant alleles for {beta}-MHC (13), those that code for the missense mutations R403Q and R453C are among the most lethal. Both mutations are associated with early onset of symptoms and a high mortality rate, with average life spans of 30–35 yr (2, 12, 31). The single leading hypothesis to explain the symptoms and prognoses in cases of FHC associated with mutant alleles for {beta}-MHC has been the significant modification of sarcomeric performance due to incorporation of the mutant {beta}-MHC protein (2, 23, 31).

Despite the similar phenotype arising from these two mutations, the R403Q and R453C mutations reside in different functional domains of the myosin molecule, the actin-binding loop and near the nucleotide-binding pocket of myosin (22), respectively. Thus assessing the functional consequences of these mutations may offer some insight into the mechanisms responsible for development of the FHC phenotype. Previous studies characterizing the R403Q mutation in cardiac MHC (MHCR403Q) have been extensive but inconclusive (see Ref. 10 for review) and have not led to an accepted hypothesis as to the functional role of this mutation in the pathogenesis of FHC. The consequences of the R453C mutation have been characterized in only one study (24), which reported reduced function of human cardiac {beta}-MHCR453C expressed by a baculovirus/insect cell system. Given the equivocal results for the R403Q mutation and the paucity of data for the R453C mutation, we undertook a side-by-side comparison of the functional consequences of both mutations in the mouse heart to better elucidate the common FHC-causing mechanisms that stem from the R403Q and R453C mutations.

Genetically engineered heterozygous mice bearing alleles for the point mutations {alpha}-MHCR403Q (R403Q/+) and {alpha}-MHCR453C (R453C/+) develop hypertrophic cardiomyopathy (3, 15, 25) and offer highly controlled MHC expression systems with which to study the functional consequences of these FHC mutations. In intact left ventricular (LV) studies, diastolic function was significantly reduced in the R403Q/+ and R453C/+ mice compared with their wild-type (WT) littermates (19, 25, 27; unpublished observations), which in part may reflect the impaired calcium handling observed in isolated myocytes (8, 25). In contrast, LV systolic function in the R403Q/+ mouse has been characterized by an earlier and higher peak pressure (3, 15, 19, 27) and enhanced free wall fractional shortening (15) compared with WT mice, which would imply that the R403Q mutation directly enhances force generation in the mouse heart.

This study examines the functional consequences of the myosin R403Q and R453C mutations at the molecular level and in skinned myocardium, a level of structural organization just below that at which the FHC phenotype emerges. We report actin-activated ATPase in solution, thin filament velocity in the myosin in vitro motility assay, and isometric tension and sinusoidal analysis of skinned myocardial strips dissected from WT, R403Q/+, and R453C/+ male mice. Our primary aim was to identify those functional changes that are shared by R403Q/+ and R453C/+ compared with WT mice and those that are different, and we rationalized that changes in common are more likely to contribute to the emergence of FHC. Our results in R403Q/+ compared with WT mice were consistent with the changes reported previously with this mouse model (1, 28). Remarkably, changes observed in the R453C/+ mice differed significantly from those in the R403Q/+ mice, not only in the magnitude of the changes, but also in the direction of many of the changes. These opposite trends in cross-bridge kinetics relative to those of WT mice imply strikingly different functional roles for the locations of each point mutation in the MHC and further imply distinctly different mechanisms or triggers by which these mutations ultimately cause the cardiac phenotype of FHC.


   METHODS
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 ABSTRACT
 METHODS
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All procedures were reviewed and approved by the University of Vermont and Harvard University Institutional Animal Care and Use Committees. All reagents were purchased from Sigma (St. Louis, MO) except where noted.

Isolation of contractile proteins. Mouse cardiac myosin was purified from frozen LVs of R403Q/+ and R453C/+ mice aged 6–9 wk and respective WT littermates (18, 29). Myosin protein concentrations were assessed using a Bradford-method protein assay (Bio-Rad, Hercules, CA). Figure 1 illustrates that myosin isolated from these mouse hearts resulted in samples with slight contamination of actin and tropomyosin, as expected from previous reports (18). Beef cardiac troponin and tropomyosin were isolated according to the method of Potter (21) with slight modification (29). Actin was purified from chicken pectoralis by standard techniques (11, 20). Regulated thin filaments (RTFs) were reconstituted from isolated tropomyosin, troponin, and actin in low-salt buffer (in mM: 25 KCl, 25 imidazole, 5 MgCl2, 10 DTT, and 2 EGTA, pH 7.4) as previously described (29). RTFs were labeled with rhodamine-phalloidin at a 1:1 actin-to-phalloidin ratio.



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  		Fig. 1. SDS-PAGE of myosin isolated from wild-type (WT) mouse hearts and hearts from heterozygous mice bearing alleles for point mutations {alpha}-MHCR403Q (R403Q/+) and {alpha}-MHCR453C (R453C/+). Lanes are marked as molecular weight standards (left) and as genotype. Technique used to isolate myosin resulted in samples with high myosin yields, similar stoichiometry with light chains, and minimal contamination of actin (~42 kDa) and tropomyosin (~36 kDa).

 
Actin-activated ATPase assay. Actin-activated ATPase was measured using 100 nM myosin over 5–40 µM actin at 2 mM ATP, 25°C, and pH 7.4. Pi concentration at fixed time points was measured using a malachite green phosphate indicator (9). The Pi concentration released per minute was recorded, converted to units of s–1 (V), and plotted against actin concentration ([actin]). Individual V-[actin] relations were fitted to the following equation describing Michaelis-Menten kinetics: V = Vmax[actin]/(Km + [actin]), where Vmax is maximal rate of actin-activated ATPase and Km is [actin] at one-half of Vmax.

In vitro motility assay. The in vitro motility assay has been previously described in detail (29, 30). Briefly, whole myosin (50 µg/ml) was adhered to a nitrocellulose-coated glass coverslip. The velocity of fluorescently labeled RTFs (VRTF) in the presence of 2 mM ATP and various pCa was measured (30) for a minimum of four hearts in each group. Typically >250 thin filament velocities were averaged to determine a single data point. Mean values for each pCa point were used to fit the VRTF-pCa relation to the Hill equation: VRTFmax [Ca2+]n/([Ca2+]50n + [Ca2+]n), where VRTFmax is maximal VRTF, [Ca2+]50 is calcium concentration at one-half activation, pCa50 is the negative logarithm of [Ca2+]50, and n is the Hill coefficient.

The apparent affinity of myosin for the RTF was determined at pCa 5 by varying the myosin concentration adhered to the motility surface from 10 to 100 µg/ml. The normalized VRTF-myosin relation was fit to a Hill equation: [myosin]n/([myosin]50n + [myosin]n), where [myosin]50 is myosin concentration at one-half activation.

Solutions for skinned myocardial strips. Solutions were formulated by solving equations describing ionic equilibria (4). Relaxing solution (pCa 8) consisted of 5 mM EGTA, 5 mM ATP, 1 mM Mg2+, 0.25 mM Pi, 240 U/ml creatine kinase, and 40 mM phosphocreatine (190 meq ionic strength, pH 7.0). Activation solution was the same as relaxing solution, with pCa 4. Storage solution was the same as relaxing solution, with 10 µg/ml leupeptin and 50% (wt/vol) glycerol. Skinning solution was the same as relaxing solution, with 10 µg/ml leupeptin, 1% (wt/vol) Triton X-100, and 50% (wt/vol) glycerol.

Preparation and experimentation of skinned myocardial strips. WT, R403Q/+, and R453C/+ male mice aged 10–20 wk were killed by cervical dislocation. Hearts were excised rapidly and placed in 95% O2-5% CO2-bubbled Krebs solution containing 30 mM 2,3-butanedione monoxime (16). RVs were trimmed away, and LV papillary muscles were removed. Apical halves of four LVs from each group were stained with Masson's trichrome and hematoxylin-and-eosin (American Histolabs, Gaithersburg, MD).

Papillary muscles were dissected to yield at least two thin strips (~100–140 µm diameter, ~600 µm long) with longitudinally oriented parallel fibers, as described previously (1, 16). The strips were skinned for 18 h at 4°C and stored at –20°C for ≤4 days. At the time of study, aluminum T clips were attached to the ends of a strip ~200 µm apart. The strip was mounted between a piezoelectric motor (Physik Instrumente, Auburn, MA) and a strain gauge (SensorNor, Horten, Norway), lowered into a 30-µl droplet of relaxing solution maintained at 27°C, and incrementally stretched to and maintained at 2.2 µm sarcomere length as determined by a filar micrometer (1).

Papillary muscle strips were calcium activated by exchange of equal volumes of bathing solution for activating solution, thereby incrementally increasing the free calcium concentration from pCa 8.0 to 4.5. Individual recordings of normalized isometric tension (Piso) were fit to the Hill equation: [Ca2+]n/([Ca2+]50n + [Ca2+]n). At each calcium concentration, sinusoidal perturbations of amplitude 0.125% of strip length were applied at 42 discrete frequencies from 0.125 to 100 Hz. The two components of the recorded tension transients that were in phase and 90° out of phase with respect to the imposed sinusoidal length perturbations have been termed the elastic and viscous moduli, which are, respectively, the real and imaginary parts of the complex modulus used to represent the tension response. Details of the method have been described previously (1, 6, 7).

Analysis. Values are means ± SE. Data from isolated myosin were tested for significant differences between R403Q/+ mice and WT littermate controls and between R453C/+ mice and WT littermate controls using Student's t-test at P < 0.05, P < 0.01, and P < 0.001. In the case of VRTFmax, where >250 measured velocities were averaged to provide a single data point, the Z-test was applied between groups, and significance was reported at P < 0.05. Data from skinned myocardial strips were tested for significant differences among the R403Q/+, R453C/+, and WT mice, which were not necessarily littermate controls, by one-way ANOVA followed by Duncan's post hoc analysis reported at P < 0.05 and P < 0.01.


   RESULTS
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 RESULTS
DISCUSSION
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Characteristics of mouse models. Data describing the 10- to 20-wk-old mice used in the present study are presented in Table 1. Body mass, RV mass, LV mass, and LV-to-body mass ratio were not different between the WT, R403Q/+, and R453C/+ groups. Histolographic analysis showed that two of the four R403Q/+ apexes and three of the four R453C/+ apexes underwent slight, although noticeable, fibrosis and myocyte disarray compared with WT controls (Fig. 2). The LVs of the R403Q/+ and R453C/+ mice, therefore, displayed early signs of tissue remodeling without significant hypertrophy, in agreement with measures made previously in these mice of this age (15, 25; unpublished observations). Although slight fibrosis and myocyte disarray may contribute marginally (if at all) to the viscoelastic properties of the relaxed skinned strips, the properties of isolated myosin will not be affected by fibrosis or disarray, because these factors are removed during isolation.


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  		Table 1. Animal characteristics

 


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  		Fig. 2. Myocardial histology and fibrosis. A and B: normal myocyte alignment (A) and fibrosis (B) characterize WT myocardium. C and D: most extreme examples of myocyte disarray (C) and fibrosis (D) in R403Q/+ myocardium show only slight onset of remodeling. E and F: most extreme examples of myocyte disarray (E) and fibrosis (F) in R453C/+ myocardium also show only slight onset of cardiac remodeling at 10–20 wk of age.

 
Actin-activated ATPase in isolated myosin. Figure 3 illustrates the measures of actin-activated ATP hydrolysis over 5–40 µM actin. The maximal rate of actin-activated ATP hydrolysis (Vmax) was significantly (P < 0.05) increased by 38% in the R403Q/+ mice (3.13 ± 0.35 s–1) compared with WT littermate controls (2.27 ± 0.25 s–1) and was significantly (P < 0.01) decreased by 45% in the R453C/+ mice (1.23 ± 0.25 s–1) compared with WT controls (2.23 ± 0.30 s–1). An increase in Vmax was reported previously for myosin isolated from R403Q/+ mice compared with WT controls (28). These measures for Vmax strongly suggest a fundamental difference in cross-bridge kinetics of the myosin isolated from the R403Q/+ and R453C/+ mouse hearts. The actin concentration at which one-half of Vmax was achieved (Km) was not statistically different between R403Q/+ (8.5 ± 2.8 µM actin) and WT (6.9 ± 2.5 µM actin) mice and was also not different between R453C/+ (7.3 ± 4.6 µM actin) and WT (6.3 ± 2.7 µM actin) mice.



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  		Fig. 3. Actin-activated ATPase (V) for mutant myosins compared with WT myocardium. A: mean maximal rate of ATP hydrolysis (Vmax) was significantly increased by 38% in R403Q/+ mice compared with WT littermates. Actin concentration ([actin]) at one-half of Vmax (Km) was not significantly different between R403Q/+ and WT mice. B: Vmax in R453C/+ mice was significantly reduced by 45% compared with WT littermates, and Km was not different between R453C/+ and WT mice.

 
VRTF-pCa relations in isolated myosin. The VRTF-pCa relations in myosin isolated from the R403Q/+ and R453C/+ myocardium are presented in Fig. 4, A and B. VRTFmax was significantly (P < 0.05) faster (by ~8%) in the R403Q/+ mice than in WT littermate controls (Table 2). The faster VRTFmax in the R403Q/+ mice was similar to that observed for unregulated actin and myosin isolated from the hearts of R403Q/+ mouse pups (28). In contrast, VRTFmax in the R453C/+ mice was similar to that in WT controls (Table 2). There was no significant difference in the calcium sensitivity for VRTFmax between the R403Q/+ mice and WT controls or between the R453C/+ mice and WT controls.



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  		Fig. 4. Actin regulated filament velocity (VRTF) in in vitro motility assay. A: as illustrated with VRTF-pCa relation, myosin isolated from R403Q/+ mice demonstrated ~8% increase in maximal VRTF compared with WT littermate controls without a significant difference in calcium sensitivity. B: no alteration in calcium-regulated VRTF was observed for myosin of R453C/+ myocardium. C: surface concentration of myosin required to achieve half-maximal VRTF was significantly lower in myocardium isolated from R403Q/+ mice than from WT littermate controls. Therefore, affinity for thin filament was higher in myosin with R403Q mutation than in WT controls, and myosin strong-binding activation of the thin filament was possibly enhanced compared with WT controls. D: R453C/+ myosin strong-binding activation of the regulated thin filament was similar to that in WT littermate controls.

 

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  		Table 2. Functional characteristics measured by in vitro motility assay of myosin isolated from R403Q/+, R453C/+, and WT mouse hearts

 
VRTF-myosin relations. The sensitivity of VRTF to myosin concentration of R403Q/+ and R453C/+ mice is presented in Fig. 4, C and D. The surface myosin concentration required to achieve one-half of VRTFmax ([myosin]50) was significantly lower in the R403Q/+ mice than in the WT littermate controls (Table 2). These data indicate that the apparent affinity of myosin for the RTF was enhanced with the R403Q mutant myosin isolated from the R403Q/+ hearts. Myosin isolated from the R453C/+ hearts demonstrated an apparent affinity for RTF that was similar to that from WT littermate controls (Table 2).

Piso-pCa relation in skinned myocardial strips. Normalized Piso was significantly higher in R453C/+ than in WT mice over a wide range of calcium concentrations from pCa 6 to 5.25 and was higher in R403Q/+ than in WT mice at pCa 5.5 (Fig. 5A). The sensitivity of Piso to calcium concentration as measured by pCa50 was significantly greater in R453C/+ (5.82 ± 0.03) than in WT (5.58 ± 0.02) and R403Q/+ (5.64 ± 0.02) mice, which were not different from each other. The Hill coefficients for WT (3.46 ± 0.18), R403Q/+ (3.83 ± 0.29), and R453C/+ (3.27 ± 0.23) mice were not significantly different among the groups.



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  		Fig. 5. Characteristics of isometric tension (Piso)-pCa relations. A: calcium sensitivity of Piso was significantly enhanced in R453C/+ compared with WT and R403Q/+ mice. Only at pCa 5.5 was normalized Piso greater for R403Q/+ mice than for WT controls. Curves represent fits to mean values displayed. B: maximum calcium-activated Piso was not different among the 3 groups. See METHODS for definitions of Hill parameters and RESULTS for values. *P < 0.05, R403Q/+ or R453C/+ vs. WT. #P < 0.05, R453C/+ vs. R403Q/+.

 
Maximum calcium-activated Piso was not found to be different between the groups (Fig. 5B). These data suggest that {alpha}-MHCR403Q and {alpha}-MHCR453C do not significantly affect the number of cross bridges available, the unitary force per cross bridge, or the cross-bridge duty ratio, i.e., ton/(ton + toff), at maximum calcium activation.

Characteristics of cross-bridge kinetics in skinned myocardial strips. Cross-bridge kinetics in the skinned myocardial strips were characterized using sinusoidal length perturbation analysis over a range of pCa. The complex modulus recorded for each strip was characterized by the parameters of the following mathematical expression (1, 6, 7, 14)

(1)
where {omega} = 2{pi} x frequency of perturbation and {alpha} = 1 s–1 (a constant). The parameters A and k describe the passive response of the muscle (A process), B and b describe the kinetic-dependent work-generating response of the cross bridges (B process), and C and c describe the kinetic-dependent work-absorbing response of the cross bridges (C process).

Figure 6A displays a Nyquist plot, i.e., viscous vs. elastic modulus, of the mean complex modulus observed for the WT group at maximum calcium activation. The constitutive A, B, and C processes have also been plotted in Fig. 6A, where the contribution of each parameter given in Eq. 1 has been depicted graphically. The Nyquist plot of the mean complex modulus for the R403Q/+ group (Fig. 6B) provided qualitative evidence of reduced magnitudes for the B and C processes compared with WT controls, as has been shown previously (1). In contrast, the mean complex modulus for the R453C/+ group (Fig. 6C) displayed greater magnitudes for the B and C processes than that for the WT group. These and other differences between the groups were also demonstrated quantitatively as reported below.



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  		Fig. 6. Nyquist plots of complex modulus ({circ}) for each group and constitutive A, B, and C processes (solid lines) at maximal calcium activation. A: mean complex modulus for WT group is plotted such that each point corresponds to a specific frequency from 0.125 to 100 Hz. A process is displayed as a straight line in the 1st quadrant. Value of A indicates magnitude of passive viscoelastic stiffness and is displayed as length of arrow between origin and A process at frequency 2{pi}–1 Hz. Relative contribution of viscosity to passive viscoelastic stiffness is represented by k{pi}/2 and is displayed as the angle at which A process lies with respect to abscissa. B (and C) indicates magnitude of B process (and C process) and is displayed as diameter of the semicircle drawn in the 3rd (and 1st) quadrant(s). (Extrapolation of C process for frequencies >100 Hz is provided by dotted line.) Value of b (and c) indicates frequency in Hz at which B process (and C process) exhibits the most negative (and positive) viscous modulus and the greatest oscillatory work generation (and absorption) by cross bridges. B: Nyquist plot of mean complex modulus for R403Q/+ mice and corresponding B and C processes illustrates a reduced response of cross bridges to sinusoidal analysis compared with WT controls. C: Nyquist plot for R453C/+ mice illustrates an increased response of cross bridges to sinusoidal analysis compared with WT and R403Q/+ mice.

 
The values for A in all three groups were directly proportional to activation by calcium (Fig. 7A), indicating that the magnitude of the passive viscoelastic stiffness of the muscle was dependent on the number of cross bridges attached at any one time. The significantly higher values of A for the R453C/+ than for the R403Q/+ mice at pCa 5.5 and 5.25 are likely reflections of the greater calcium sensitivity observed in the R453C/+ group. The values for k in all three groups were also directly proportional to activation by calcium (Fig. 7B), where the relative contribution of the viscous property of the passive elements became greater with the number of cross bridges. The reduced value for k in the R453C/+ group compared with WT and R403Q/+ groups at submaximal pCa suggested that the passive elements of the R453C/+ muscle did not absorb work to as high a degree as those of the other groups. At maximum calcium activation, however, the values for A and k were similar between WT, R403Q/+, and R453C/+ groups.



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  		Fig. 7. Calcium dependence of curve-fit parameters determined by sinusoidal analysis. A: significantly higher values of A for R453C/+ than for R403Q/+ mice at pCa 6 and 5.75 reflect greater calcium sensitivity in R453C/+ mice. This parameter of passive viscoelastic stiffness was not different among the 3 groups at maximum pCa. B: values for k were variable at submaximal pCa but were similar in all 3 groups at maximum pCa. C: values for B at almost all submaximal pCa were higher in R453C/+ than in WT and R403Q/+ mice and at maximum pCa were lower in R403Q/+ than in WT mice. D: values for b were higher in R453C/+ than in WT and R403Q/+ mice and lower in R403Q/+ than in WT mice. E: values for C were higher in R453C/+ than in WT and R403Q/+ mice and lower in R403Q/+ than in WT mice. F: values for c were not different among the 3 groups at any pCa. See Eq. 1 and RESULTS for definitions. *P < 0.05, R403Q/+ or R453C/+ vs. WT. #P < 0.05, R453C/+ vs. R403Q/+.

 
The parameter B represents the magnitude of the active work-generating response of cross bridges and, according to Kawai and colleagues (6, 7), is proportional to the number of cross bridges in the strongly bound (force-producing) state of the actin-myosin-ADP-Pi (AMDP) complex. The values for B near maximum pCa were lower in the R403Q/+ than in the WT group, and, at almost all pCa, values for B were higher in the R453C/+ than in the WT and R403Q/+ groups (Fig. 7C). These results for B suggest that the number of cross bridges residing in the strongly bound AMDP state was lower in the R403Q/+ mice and higher in the R453C/+ mice than in the WT controls. The parameter b, which is proportional to the rate of transition between the weakly and strongly bound states of the AMDP complex, was likewise found to be significantly reduced in the R403Q/+ group and significantly enhanced in the R453C/+ group compared with WT controls at maximal as well as at some submaximal pCa values (Fig. 7D). These results for B and b indicate that the R403Q and R453C mutations produce significantly different numbers of cross bridges in the strongly bound AMDP state as well as significantly different rates of transition between weakly and strongly bound states.

The parameter C represents the magnitude of the active work-absorbing response of cross bridges and has been interpreted to be proportional to the number of cross bridges in the strongly bound state of the actin-myosin-ATP (AMT) complex (6, 7). The values for C were lower in the R403Q/+ mice than in the WT controls and higher in the R453C/+ than in the WT and R403Q/+ mice (Fig. 7E). These results for C suggest that the number of cross bridges residing in the AMT complex were lower in the R403Q/+ and higher in the R453C/+ mice than in the WT controls. The parameter c (Fig. 7F), which is proportional to the rates of transition between the strongly and weakly bound states of the AMT complex (6, 7), was not different between WT, R403Q/+, and R453C/+ groups at any pCa value.


   DISCUSSION
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 ABSTRACT
 METHODS
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 DISCUSSION
GRANTS
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The prognoses for individuals with FHC due to a mutant allele for {beta}-MHC vary from good (e.g., N232S, G256E, V606M, and L908V) to intermediate (R249Q, V606M, G741R, and Q903K) to poor (R403Q, R453C, R719Y, and R723G) (2, 13, 23, 26, 31). On the basis of structural analyses of MHC, the various mutations reside in or close to the actin-binding interface (R403Q and V606M), the nucleotide-binding pocket (N232S, R249Q, G256E, and R453C), the converter domain (R719Y and R723G), the lever arm (G741R), or the rod (Q903K and L908V) of the MHC (10, 22). There appears to be no correlation between the location of a specific mutation in the {beta}-MHC and the lethality of its phenotype. Nevertheless, on the basis of the differential locations of the R403Q and R453C mutations, we anticipated that the functional consequences of these highly malignant mutations on MHC performance would be quite variable.

The present study demonstrates the anticipated variability, exhibited as a differential effect of {alpha}-MHCR403Q and {alpha}-MHCR453C, in heterozygous mouse myocardium on the cross-bridge kinetic parameters reflected by actin-activated ATPase, VRTF in the in vitro motility assay, and the complex modulus due to sinusoidal length perturbations. Several of the parameters used to characterize actin-activated ATPase, VRTF and the complex modulus in the R403Q/+ and R453C/+ mice, were qualitatively and statistically different from each other over a wide range of experimental conditions and often with opposite trends compared with WT controls (Figs. 3, 6, and 7). Because the R403Q and R453C mutations reside in the actin-binding interface and near the nucleotide-binding pocket of the MHC (22), respectively, the kinetic differences found in the present study likely reflect the influence of each mutation in the cross-bridge cycle.

We can begin to understand the functional consequences of each mutation in the cross-bridge cycle and in the role each plays in initiating FHC by examining the differential results in the context of present cross-bridge theory.

Vmax for actin-activated ATPase reflects the maximum rate of myosin-actin cross-bridge cycling in solution. We found that Vmax was faster in R403Q/+ and slower in R453C/+ mice than in WT controls. It is widely believed that the rate-limiting step for actin-activated ATPase in solution is the rate of Pi release (5, 17). Our results therefore suggest that the R403Q point mutation effectively increases and the R453C point mutation decreases the rate of Pi release.

VRTF measured in the in vitro motility assay is sensitive to the reciprocal of ton of the cross-bridge cycle (28). Because the rate of ADP release is considered to be the rate-limiting step to determine ton and, therefore, VRTF, an enhanced VRTF for myosin isolated from the R403Q/+ myocardium most likely reflects an increased rate of ADP release induced by the R403Q mutation. A similar finding and conclusion were reported using unregulated actin filaments (28). No difference in VRTFmax between R453C/+ and WT mice suggests no difference in ton or in the rate of ADP release due to the R453C mutation.

The myosin concentration at which VRTFmax is half-activated reflects the apparent affinity of myosin for RTF. We found that the apparent myosin affinity for RTF is significantly enhanced by the R403Q mutation. The increase in apparent affinity could be due to an actual increase in strong binding activation of the thin filament by the R403Q/+ myosin. The R453C/+ myosin, however, demonstrated no difference in apparent affinity for RTF compared with WT control.

The complex modulus due to sinusoidal length perturbations reflects the frequency-dependent redistribution of the myosin-actin cross bridges between weakly bound and strongly bound states (6, 7). According to the model proposed by Kawai et al. (7), the magnitude and frequency of the B process are reciprocally related to the rate of Pi release as well as other factors. The observed decrease in parameters B and b for the R403Q/+ myocardium, as reported previously (1), and an increase in B and b for the R453C/+ myocardium compared with WT control are consistent with an increase in the rate of Pi release due to the R403Q mutation and a decrease in the rate of Pi release due to the R453C mutation.

The present data suggest that the R403Q and R453C mutations lead to the FHC phenotype through differential effects on the cross-bridge cycle. Although the detailed mechanisms that underlie the R403Q/+ and R453C/+ phenotypes are somewhat speculative, our results from multiple assays suggest a differential effect on the rate of Pi release. Further investigation of the dependency of the cross-bridge kinetics on Pi concentration in the vicinity of the myosin head should help elucidate the underlying mechanisms responsible for the differential effects seen in the myosin mutations examined in this study. The challenge remains, however, to account for the proximal mechanisms, possibly in combination with whole heart studies, which lead to the phenotype of FHC.


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This study was funded by National Heart, Lung, and Blood Institute Grant HL-59408-3.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. M. Palmer, 127 HSRF Beaumont Ave., Dept. of Molecular Physiology and Biophysics, Univ. of Vermont, Burlington, VT 05405 (E-mail: palmer{at}physiology.med.uvm.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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