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1Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont; 2Cardiovascular Branch, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland; and 3Dakota Cardiovascular, Physician Corporation, Rapid City, South Dakota
Submitted 30 June 2004 ; accepted in final form 28 October 2004
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ABSTRACT |
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-myosin heavy chain are the most common causes of FHC, and we proposed that "enhanced" mutant actin-myosin function is the causative molecular abnormality. We have studied individuals from families in which members have two, one, or no mutant MYH7 alleles to examine for dose effects. In one family, a member homozygous for Lys207Gln had cardiomyopathy complicated by left ventricular dilatation, systolic impairment, atrial fibrillation, and defibrillator interventions. Only one of five heterozygous relatives had FHC. Leu908Val and Asp906Gly mutations were detected in a second family in which penetrance for Leu908Val heterozygotes was 46% (21/46) and 25% (3/12) for Asp906Gly. Despite the low penetrance, hypertrophy was severe in several heterozygotes. Two individuals with both mutations developed severe FHC. The velocities of actin translocation (Vactin) by mutant and wild-type (WT) myosins were compared in the in vitro motility assay. Compared with WT/WT, Vactin was 34% faster for WT/D906G and 21% for WT/L908V. Surprisingly Vactin for Leu908Val/Asp906Gly and Lys207Gln/Lys207Gln mutants were similar to WT. The apparent enhancement of mechanical performance with mutant/WT myosin was not observed for mutant/mutant myosin. This suggests that Vactin may be a poor predictor of disease penetrance or severity and that power production may be more appropriate, or that the limited availability of double mutant patients prohibits any definitive conclusions. Finally, severe FHC in heterozygous individuals can occur despite very low penetrance, suggesting these mutations alone are insufficient to cause FHC and that uncharacterized modifying mechanisms exert powerful influences.
hypertrophy; cardiomyopathy; motility assay; penetrance
-myosin heavy chain (-MHC), the myosin regulatory and essential light chains, myosin binding protein C, troponin I, troponin C, 
-cardiac actin, and titin (8, 29). The dominant negative expression of these mutant proteins leads to pathological remodeling of the heart that includes myocyte hypertrophy and disarray, myocardial fibrosis, and ischemia, with the end result being an increased risk of arrhythmia, cardiac failure, and sudden death. Although expression of these mutant proteins initiates a cascade of events ultimately leading to pathological left ventricular (LV) hypertrophy, a true mechanistic understanding of this cascade and its initiation are poorly understood (29). Simplistically, the extent to which a mutant protein's function has been altered should correlate with the severity of the FHC phenotype, and therefore characterizing a mutant protein's functional capacity may aid in developing a mechanistic view of this hypertrophic response. However, variations in penetrance that occur for a given mutant protein, even within a given family, suggest that other factors contribute to the development of this disease.
As many as 20% of FHC patients possess one of >70 missense mutations of the gene encoding -MHC (6, 18, 25). Myosin, the molecular motor of the heart, is a hexameric protein consisting of two heavy chains and two associated light chains per heavy chain. Each heavy chain has a globular catalytic head domain that has actin binding and ATP hydrolytic and motor activities. Beyond the globular head, two heavy chains associate in an -helical coiled-coil rod allowing the myosin molecule to polymerize into thick filaments within the sarcomere. Thus each myosin molecule is a double-headed structure, in which both heads are necessary for myosin to exhibit its maximal force and motion generation (32). In most cases, the point mutations are associated with only one -MHC allele, leading to a patient that is heterozygous for the mutation, such that 50% of the expressed myosin will be of the mutant form.
Although earlier reports suggest that diminished mutant myosin function was the primary cause for FHC (3, 4, 28, 31), recent reports from our laboratory suggest that the same mutant myosins have enhanced function (23, 33, 37). These and other data led to numerous hypotheses regarding the etiology of FHC. For example, LV hypertrophy may develop as a compensatory response to a mutant's diminished function (4). Alternately, enhanced function could lead to the hypercontractile state characteristic of FHC (11, 29) and may result in heterogeneous force profiles within the muscle along with depletion of sarcomeric energy reserve (2, 9) leading to myocyte injury, fibrosis, and hypertrophy (15).
FHC patients with two mutant sarcomeric alleles have recently been identified by ourselves and others (6, 10, 18, 21, 25, 27). The existence of such patients provides an opportunity to compare clinical phenotypes and mechanical capacity of mutant -MHC associated with individuals having one or two mutant alleles. With two mutant alleles, expression of mutant myosin in which both heavy chains carry a mutation will exist, which may help define the impact of the mutation on myosin molecular function. In addition, the penetrance and severity of the disease may be gene-dose dependent, which would result in patients with a more severe clinical phenotype. Here we have identified several patients with point mutations, with one being novel, in both -MHC alleles. These patients were uniformly of a severe FHC phenotype. Mutant myosin prepared from biopsy of the upper portion of the biceps muscle obtained from these patients and family members was characterized for the effect of the mutation on myosin's ability to move actin in a motility assay.
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MATERIALS AND METHODS |
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Clinical studies included two-dimensional echocardiography, 12-lead electrocardiography, and in selected cases, cardiac magnetic resonance imaging. An individual was considered positive for the FHC phenotype if LV wall thickness was >13 mm by echocardiography in the absence of another cause for cardiac hypertrophy. Genetic analysis was performed on genomic DNA extracted from whole blood (PureGene; Gentra) as per the manufacturer's instructions, and genotype was determined by direct sequencing and confirmed by restriction enzyme digestion when possible. Restriction enzyme cleavage was predicted by using MapDraw (SeqMan; DNAStar). Penetrance (%) was defined as (phenotype and genotype positive individuals/genotype positive individuals) x 100.
Biopsy and myosin preparation.
Skeletal muscle myosin was isolated from samples of human biceps tissue (
10 mg) obtained through a small incision over the upper portion of the biceps muscle in the nondominant arm under local anesthesia. The biceps muscle is made up of 40–60% slow fibers (19) that express the cardiac -MHC isoform. Therefore, the isolated myosin will be a mixture of both normal and mutant heavy chains as well as fast and slow skeletal myosin isoforms. This concern was mitigated by comparison of motility results for myosins isolated from the biceps versus cardiac and soleus tissues (see RESULTS). Myosin purification from small tissue samples for use in the in vitro motility assay has been previously described in detail (22, 23, 33). With the limited sample size, the quantity of purified myosin only allowed in vitro motility experiments to be performed.
In vitro motility assay. Standard methods were used for the in vitro motility assay with special care taken to remove all nonfunctional myosin (23, 35). This last step is critical so that the internal load contributed by nonfunctional myosin is eliminated. Actin prepared from chicken skeletal muscle was fluorescently labeled by incubation in tetramethylrhodamine isothiocyanate-phalloidin overnight (24, 35). Assays were carried out at 30°C using a 30-µl chamber to which the following solutions were added and removed (23) 1) 100 µg/ml myosin; 2) bovine serum albumin; 3) 1 µM unlabeled actin in actin buffer (in mM: 25 KCl, 25 imidazole, 1 EGTA, 4 MgCl2, and 10 dithiothreitol, plus oxygen scavengers, pH 7.4); 4) actin buffer with 1 mM MgATP; 5) 6 x 30 µl washes with actin buffer; 6) 10 nM labeled actin; and 7) 1 mM MgATP in actin buffer with 0.375% methylcellulose. Step 3 and a comparable step in the initial myosin isolation procedure remove denatured, rigor-like, nonfunctional myosin that might act as a load to the free movement of actin filaments in the motility assay (13, 23). Actin movement was visualized, recorded, and digitally analyzed to determine actin filament velocity or velocity of actin translocation (Vactin) (35, 36).
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RESULTS |
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The proband, who presented with palpitations, had been diagnosed with FHC at the age of 47 yr. Both parents are assumed to be heterozygous, and there is no historical evidence that they were related. His father died in his mid-40s with a history strongly suggestive of FHC, but his mother died at the age of 92 yr. The proband has survived to his 70s, but his FHC had been complicated by chronic atrial fibrillation (AF), multiple episodes of syncope, and therapeutic interventions with a defibrillator-cardioverter device. Serial echocardiograms demonstrate progressive LV dilatation and systolic impairment and reduction in septal thickness, severe left atrial (LA) enlargement, and the development of pulmonary hypertension. No other family member was homozygous and nine were heterozygous, including a sister who was subsequently diagnosed with FHC at the age of 80 yr. She has a history of frequent presyncope and syncope and a septal wall thickness of 38 mm with midventricular obstruction. There was no history of hypertension. Eight other family members heterozygous for K207Q had not developed LV hypertrophy at the ages of 46, 43, 40, 18, 15, 14, 13, and 9 yr (20% penetrance over the age of 18 yr). Five of these individuals have a resting sinus bradycardia, suggesting an alternative phenotypic expression (Table 1).
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-MHC, a conservative substitution L908V, and D906G, a charge-changing substitution. Both the 906 and 908 residues are conserved across all mammalian cardiac isoforms (1). Two descendants have both L908V and D906G and several dozen others have either L908V or D906G. Both L908V/D906G compound heterozygotes developed FHC, but the penetrance for L908V/wild type (WT) was 46% (21/46), and 25% (3/13) for D906G/WT (P value not significant). The two double heterozygotes both had prominent mid-LV cavity hypertrophy, moderate-to-severe LA enlargement, and paroxysmal AF. LV phenotype in penetrant heterozygotes (both for the D906G and the L908V) varied from mild LV hypertrophy to severe LV hypertrophy with LV outflow obstruction. Sudden death and defibrillator discharges were also reported for several individuals with proven or obligate heterozygous genotypes.
In vitro motility assay.
The effects of the MYH7 mutations on myosin mechanical function were assessed by characterizing skeletal muscle myosin from biceps muscles. This approach was similar to that previously used by ourselves and other investigators (3, 23). The biceps express -MHC as well as fast skeletal myosin isoforms. To control for any differences in actin filament velocities resulting from differential expression of the fast skeletal, normal, and mutant -MHC rather than an effect due to the mutant myosin itself, we compared the results from our previous studies of the L908V mutation isolated from both heart and soleus muscle biopsies to data for the L908V myosin from the biceps (Fig. 3) (3, 23).
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-MHC (WT/WT) moved actin with a Vactin of 1.4 ± 0.1 µm/s, whereas myosin from biceps containing the L908V/WT mutant moved actin 21% faster (P < 0.05, Figs. 3 and 4). The increased Vactin for L908V agrees qualitatively with our previous observation of faster Vactin for the L908V when isolated from either heart (in which no skeletal myosin is expressed) or soleus tissue samples (Fig. 3) (3, 23). Thus any confounding effects due to the known mixture of fast and slow myosin isoforms that are normally expressed in the biceps (19) are minimal, allowing the biceps to serve as a valid tissue preparation for assessing mutant myosin mechanical performance.
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-MHC in which Vactin was 1.9 ± 0.1 µm/s or 34% faster than control (P < 0.005, Fig. 4). In contrast, L908V/D906G -MHC had Vactin similar to controls (1.5 ± 0.1 µm/s). Thus the effect on Vactin of the individual mutations do not appear to be additive and, in fact, may even cancel each other. A similar result was observed for the K207Q/K207Q mutation with Vactin of 1.5 µm/s being similar to normal values. However, because myosin from a K207Q heterozygote was not available for comparison, it is not possible to compare this double mutant with a K207Q/WT. It is important to note that due to the limited number of patients (L908V/D906G, n = 2; K207Q/K207Q, n = 1), it may not be possible to draw any statistical conclusions. For each of the samples obtained from these patients, duplicate motility experiments were performed and the average Vactin reported for an individual. |
DISCUSSION |
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10% of FHC patients (16, 30). Animal models exist that demonstrate that the cardiac pathologies of the heterozygote and homozygote "malignant" sarcomeric protein mutations result in significantly different cardiomyopathies (7, 17). To our knowledge, no nonpenetrant homozygote or double heterozygotes have been reported in humans or homozygous transgenic animal models, consistent with a view that gene dosage also affects penetrance (7, 17). The considerable variation of hypertrophy and other manifestations of FHC in individuals heterozygous for the same mutation is well described (14). Notably, in our pedigrees there were some severe expressions of FHC despite the very low penetrance, suggesting that uncharacterized modifying mechanisms exert powerful influences. It is reasonable to expect that the effects of these modifying factors are most prominent when a mutant protein's functional abnormalities are mildest. Accordingly, the degree of penetrance may be the most appropriate phenotypic correlate of the severity of a mutant's functional effect.
The D906G is reported here for the first time. We previously found no amino acid changing sequence abnormalities after screening many exons encoding the motor region of -MHC in 100 normal controls (200 alleles) (18). This screening included exon 23, encoding D906 and is consistent with the view that the D906G is a mutation and not a polymorphism, and that there may be no amino acid changing polymorphisms in -MHC's entire motor region.
Assaying myosin's molecular mechanics allows us to test the hypothesis that the extent of alterations to mutant myosin function may be related to one or more measurements of the severity of the FHC cardiac phenotype, such as the degree of penetrance, magnitude of hypertrophy, or incidence of sudden death. If so, then measurable parameters of myosin molecular motor function may contribute to the prognostic evaluations for FHC patients. Therefore, we isolated myosin from the biceps muscle of patients with the following mutations: L908V/WT, D906G/WT, L908V/D906G, and K207Q/K207Q and assessed their ability to propel actin filaments in the in vitro motility assay. This assay serves as a molecular model system to characterize the actomyosin interaction that relates to unloaded shortening in muscle. Of note, the L908V/WT and the D906G/WT mutations enhanced rather than compromised myosin's unloaded velocity-generating capacities. The enhanced mechanical performance of the L908V/WT confirms our previous studies (23) of this mutation from human cardiac and soleus samples. This earlier study (23) was unique in that the same human biopsy samples from patients with either the L908V or the R403Q mutation that were initially characterized and found to have diminished actin filament velocities (3) in fact had enhanced function once extreme care was exercised to eliminate modified or "dead" myosin (for detailed discussion see Refs. 13 and 23). The same isolation procedure described in Palmiter et al. (23) was used in the present study.
The L908V and D906G are located in the S2-segment of the myosin rod, far from the motor domain in which ATP hydrolysis and actin binding occur. This raises the important question of how the mutations in the S2 segment can affect the mechanical performance of the motor domain. The potential for long distance communication between the catalytic site and domains either proximal or distal to this site is possible through structural elements within the myosin motor domain (for examples see review in Ref. 34). Another explanation is that muscle myosin needs both heads to generate maximum force and motion (32) and that coordination of the heads requires the region of the coiled-coil rod near the S2-segment to unwind or breathe (12). It is possible that the L908V and the D906G mutations exert their effect through alterations to head-head interactions by virtue of their effect on S2-segment flexibility.
With patients heterozygous for either the L908V or the D906G mutations, the predominant mutant myosin specie is a heterodimeric myosin with one normal and one mutant head, assuming random association of the -MHC. Therefore, the 20–35% enhanced Vactin for these mutations may not be the maximum possible effect. Indeed, myosin from a transgenic mouse homozygous for the R403Q FHC mutation had a 60% enhancement in Vactin compared with a 16% increase for the heterozygote (33). On the basis of these findings and on the observed severe clinical phenotype, myosin from patients expressing both L908V and D906G in separate heavy chains was expected to alter Vactin to a greater extent than myosin heterozygous for either mutation. However, Vactin was similar to normal controls (Fig. 4). A trivial explanation for the lack of an effect is due to the limited number of patients (n = 2), which prevents any statistically definitive statements from being made. This, too, was the case for the single patient in which both MYH7 alleles code for the K207Q mutation (Fig. 4), a mutation that exists in a surface loop that spans the entrance to the nucleotide-binding pocket. Therefore, the true effect of having a double mutation on myosin function cannot be determined at this time, although the severity of the clinical phenotype for these patients strongly suggests that a mutational effect does exist for these myosins. The paucity of data on double mutants is due to the apparent rarity of such patients, and the probability that the same double mutations are present in other FHC pedigrees must be exceedingly low.
In the present study, Vactin was the only measured mechanical index due to the limited tissue sample size. Studies of a limited number of mutant myosins in which careful isolation procedures were exercised (see above) have demonstrated an increased Vactin, suggesting that enhanced Vactin may contribute to the pathological abnormality leading to FHC (13, 23, 33, 37). However, the enhanced Vactin may not be universal for all myosin mutations associated with FHC, given that numerous other mutations have yet to be characterized. In fact, given the limited data set reported here for the compound mutations, enhanced function could not be determined and raises concern that Vactin may not be the mechanical index of choice. The normal functioning of the heart, though, is critically dependent on its ability to generate power in which power is defined as the product of force and velocity. Therefore, changes in Vactin alone may not represent the fundamental molecular abnormality that ultimately results in the FHC phenotype but rather alterations to power output. Given that muscle force and velocity are interdependent mechanical parameters, it is only possible to measure a muscle's peak power output once the relationship between muscle velocity and load (i.e., force) has been determined. Power producing capacities for mutant myosins have yet to be determined in any previous studies, and it may be possible that a mutant myosin exhibits increased Vactin, yet generates either reduced or greater power than normal myosin. Our future goal is to define the force-velocity relationship for mutant myosins at the molecular level in the laser trap. Such experiments may provide greater insight to the mutations' effects on myosin function and better define how different mutations, e.g., mutations in -MHC's actin binding region may result in functional abnormalities distinct from those in other structural domains of the myosin molecule. Finally, a more precise description of myosin power production may uncover how the primary insult in myosin function leads to the development of the FHC phenotype, which is more severe in patients with double -MHC mutations.
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FOOTNOTES |
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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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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-cardiac myosin depends on the nature of the -myosin heavy chain gene mutation in hypertrophic cardiomyopathy. J Muscle Res Cell Motil 18: 275–283, 1997.[CrossRef][Web of Science][Medline]
-myosin in hypertrophic cardiomyopathy. J Clin Invest 91: 2861–2865, 1993.[Web of Science][Medline]
-myosin heavy chain gene. A 908Leu—-Val mutation and a 403Arg—-Gln mutation. Circulation 86: 345–352, 1992.-myosin heavy chain genes. Hum Genet 102: 299–304, 1998.[CrossRef][Web of Science][Medline]
-myosin mutations. Genet Test 7: 21–27, 2003.[CrossRef][Web of Science][Medline]
-cardiac myosin from patients with familial hypertrophic cardiomyopathy exhibit enhanced mechanical performance at the single molecule level. J Muscle Res Cell Motil 21: 609–620, 2000.[CrossRef][Web of Science][Medline]
-myosin heavy chain gene have a severe form of familial hypertrophic cardiomyopathy. J Mol Cell Cardiol 32: 1575–1583, 2000.[CrossRef][Web of Science][Medline]
-myosin heavy chain and in the cardiac myosin binding protein C genes in a family with hypertrophic cardiomyopathy. J Med Genet 36: 542–545, 1999.-myosin that are responsible for familial hypertrophic cardiomyopathy. Implication for the clinical outcome. J Clin Invest 98: 2866–2873, 1996.[Web of Science][Medline]
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