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Departments of
* Cardiovascular Medicine and
Physiology, University of Oxford, Oxford, UK
1Correspondence: Department of Cardiovascular Medicine, University of Oxford, Wellcome Trust Centre of Human Genetics, Oxford OX3 7BN, UK. E-mail: credwood{at}well.ox.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: contractility • muscle disease
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. These syndromes are transmitted by autosomal dominant inheritance. Various attempts have been made to classify the different forms of DAs based on the presence or absence of associated abnormalities. Initially, six forms, labeled I and IIA-IIE were defined (1)
whereas more recently ten different variants (1, 2A, 2B, 3–9) have been identified (2
, 3)
. Of relevance to this study, distal arthrogryposis type 1 (DA1) occurs in 1:10,000 to 1:50,000 births and is a common cause of inherited clubfoot and vertical talus (4)
. Affected individuals also have clenched hands at birth with ulnar deviation and digitotalar dysmorphism, and the shoulders and hips may also be affected. Type 2B (DA2B) is similar to Freeman-Sheldon or whistling face syndrome (now classified as DA2A) and is characterized by distinctive facial abnormalities along with campodactyly and vertical talus (5)
.
The precise cause of these malformations has been unclear. It has been postulated that decreased fetal movement brought about by a variety of means may lead to contractures at birth via the accumulation of additional connective tissue around the joints (6)
. The reduced movement could be due to primary muscular or neuronal disorders, connective tissue abnormalities, or problems within the uterus, such as intrauterine vascular compromise (1
, 6
, 7)
. Recent studies have sought to identify the relevant disease genes. Bamshad’s group identified the pericentromeric region of chromosome 9 to be a disease locus for DA1 (8)
and has since shown that a missense mutation (Arg91Gly) in the TPM2 gene within this region can cause this disease (9)
. TPM2 encodes the muscle regulatory protein ß-tropomyosin, which is expressed in all skeletal muscles. Molecular genetic work has also shown that DA2B can be caused by two mutations in TNNI2, a Arg174Gln missense mutation and a C
T nonsense mutation, resulting in the loss of the C-terminal 26 amino acids (Arg156ter), (9)
and a Arg63His missense mutation in TNNT3 (10)
. These genes encode the fast skeletal isoforms of troponin I and troponin T, respectively, and hence it appears that alteration of the Ca2+-regulation of fast skeletal muscle can be the underlying cause of these disorders.
Skeletal muscle contraction is principally controlled by the concentration of Ca2+ ions surrounding the myofilaments. The Ca2+ concentration signal is sensed by the trimeric troponin complex (subunits C, I and T) and transmitted to the 
-helical coiled coil tropomyosin dimer that lies along the actin filament (11
, 12)
. Both troponin and the tropomyosin dimer are present at ratios of 1:7 with actin. On activation of the muscle, troponin C binds Ca2+ at its two low-affinity, regulatory sites; the subsequent conformational changes within the troponin complex result in both the inhibitory effect of troponin I being removed and a change in the position of the tropomyosin, allowing productive myosin head interaction with actins not directly in contact with troponin I and hence cooperative switch on of the thin filament. Kinetic measurements have suggested that this mechanism involves three states: blocked (in the absence of Ca2+, tropomyosin prevents productive myosin head binding), closed (the tropomyosin position moves in response to Ca2+ binding to allow partial myosin attachment), and myosin-induced (in which myosin head binding has shifted tropomyosin to expose fully the interaction sites on actin) (13
14
15
16
17)
. This has been strongly supported by electron microscopy (EM) reconstruction data of thin filaments from different muscles in the presence and absence of Ca2+ (18
19
20)
.
We have set out to understand how the subtle mutations that cause DA alter the control of skeletal muscle contractility and how this may lead to the disease state. The ability of recombinant mutant troponin/tropomyosin to regulate reconstituted actomyosin ATPase has been compared to WT, and the function of DA mutant troponins has been tested in exchanged fast skeletal muscle fibers. Our data suggest that all four reported mutations cause increased contractility and are likely to produce a "hypercontractile" muscle in vivo. We suggest that this aberrant thin filament regulation in developing skeletal muscles causes contractures, which may be responsible for the development of the observed limb defects.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Nine base pairs encoding Met-Ala-Ser were inserted 5' to the initiator methionine codon of the ß-tropomyosin construct. A clone corresponding to TNNT3 was generated by PCR using a skeletal muscle cDNA mixture (Clontech) as template and also cloned into pMW172 using NdeI and HindIII. pMW172 expression constructs encoding each of the reported DA mutations in troponin T, troponin I, and ß-tropomyosin were made from the WT constructs using a two-step PCR protocol for site-directed mutagenesis.
The pMW172 constructs were used to transform the E. coli strain BL21(DE3)pLysS and large-scale cultures were grown and overexpression was induced according to standard methods (22)
. Human recombinant fast skeletal troponin T and troponin C were purified, according to our established protocols for the cardiac isoforms of these proteins (23)
. Inclusion bodies containing recombinant troponin I were isolated using a proprietary extraction buffer (Novagen), and the crude protein (CP) was solubilized in 6M urea, 1 mM EDTA, 20 mM MOPS pH 6.5 and 1 mM 2-mercaptoethanol. Troponin I was subsequently purified using sequential cation exchange and hydroxyapatite chromatography. Bacterial cell lysates containing recombinant human Ala-Ser ß-tropomyosin were heated to 95°C, before clarification by centrifugation at 33,200 g for 10 min. The resulting supernatant was fractionated by reducing the pH to 4.8 and recombinant protein purified by anion exchange chromatography. Each mutant was purified by the same method as used for the corresponding WT protein.
Actin-tropomyosin-activated myosin ATPase assays
Whole troponin complexes were reconstituted from the individual recombinant subunits by stepwise dialysis and gel filtration using a Superdex 200 column (GE Healthcare), as described previously for human cardiac troponin (24)
. Rabbit skeletal muscle actin (25)
and myosin subfragment-1 (S-1) (26)
were prepared using standard methods. Actin-activated myosin ATPase assays were carried out as described previously using 0.5 µM myosin S1, 3.5 µM actin, 1 µM ß-tropomyosin, and 0.5 µM reconstituted troponin in 5 mM PIPES pH 7.0, 3.87 mM MgCl2, 1 mM dithiothreitol at 37°C (23
, 27)
. The free calcium concentration was set using 1 mM EGTA, and an appropriate concentration of CaCl2 was calculated by the WinMAXC program (28)
to give a pCa range from 9.0 to 5.5. The data were fitted to the Hill equation using KaleidaGraph (Synergy Software) and the mean pCa50 and nH calculated from the derived pCa50 and nH values from 4 independent experiments.
Exchange of human troponin into rabbit psoas fibers
Human fast skeletal troponin was used to replace the endogenous complex in rabbit-skinned psoas fibers according to the whole troponin replacement method of Brenner (29)
with our previously described modifications (30)
. Briefly, small bundles of glycerinated psoas fibers (100 µm x 4 mm) were attached at one end to an Akers 801 piezoelectric force transducer and at the other to a static wire hook. The fibers were then skinned in relaxing solution (pCa9) containing 1% vol/vol Triton X-100 for 2 min. Fibers were brought to rigor by washes in prerigor solution followed by rigor solution to ensure complete removal of ATP (see (30)
for details of solutions). The fibers were then incubated in 5 mg/ml whole troponin solution (either WT or mutant) for 2 h at 20°C. Following this, the fibers were washed in exchange buffer and relaxing solution to remove excess protein. SDS-PAGE analysis of exchanged fibers and immunoblotting showed that WT troponin accounted for 55 ± 3% of total troponin; the exchange of mutant troponins was not significantly different. For WT troponin-replaced fibers, the recovered maximum force after exchange was 88 ± 19% of maximum force before replacement. For pCa-force measurements, initial sarcomere length was set to 2.5 µm, and fibers were cycled between relaxing solution and solution of increasing Ca2+ concentration by means of a rotating bath mechanism (31)
.
Solution compositions were calculated according to a computer program (32)
using the affinity constants of Smith and Martell (33)
. All contained a final concentration of free 1 mM Mg2+, 5 mM MgATP, 10 mM creatine phosphate, 7 mM EGTA, 10 mM imidazole, pH 7.0. Ionic strength was made up to 0.18 mM using potassium propionate. Activating solutions were made by adding CaCl2 to give final pCa values from 6.4 to 4.0. Relaxing solution was the same, with no added calcium.
Measurement of the affinity of tropomyosin for actin
The binding of tropomyosin to actin was measured by cosedimentation. 0.25–12 µM of recombinant Ala-Ser ß-tropomyosin and 21 µM of native rabbit skeletal actin were cosedimented by centrifugation at 436,000 g in a Beckman TLA100 rotor at 20°C in 200 mM NaCl, 10 mM Tris HCl pH 7.5, 3.87 mM MgCl2, and 0.5 mM dithiothretol. Pellets and supernatants were analyzed using Coomassie blue-stained 10.5% SDS-polyacrylamide gels and quantified using an FC8800 geldoc chemiluminescence densitometry program (Alpha Inotech). All calculated tropomyosin concentrations were adjusted by percentage pellet recovery, which was independently calculated using the same densitometry system. Apparent binding constants Kapp were determined by fitting the data to the Hill equation using KaleidaGraph (Synergy Software).
Circular dichroism measurements.
Thermal stability measurements were made by following the molar ellipticity of Ala-Ser ß-tropomyosin at 222 nm as a function of temperature in buffer containing 0.5 M NaCl, 10 mM sodium phosphate pH 7.5, 1 mM EDTA and 0.5 mM dithiothretol using a Jasco J720 spectropolarimeter equipped with variable temperature bath (cell path length 1 cm). Data were obtained at 1.0°C intervals from 10 to 90°C using a protein concentration of 0.1 mg/ml. The molar ellipticity was normalized (10°C=1, 90°C=0) to give fraction folded and fitted to a double exponential equation:
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corresponds to the proportion of each state of unfolding, 
H is the enthalpy change of denaturation for each transition, while the Tm gives the median temperature (in K) for each transition. These parameters were used to calculate the free energy (G in kcal/mol) of the tropomyosin helix at 20°C using the following equation: G = 1 (H1–293 S1) + 2 (H2–293 S2) where S is the entropy calculated at the Tm of each transition (34)
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 24)
. ß-tropomyosin was expressed with a Met-Ala-Ser N-terminal leader sequence, reduced to Ala-Ser in the mature protein. This modification has been used for recombinant -tropomyosin and shown to mimic the N-terminal acetylation of the native protein, which is necessary for normal head-to-tail interactions and binding to actin (35)
. SDS-PAGE analysis of the recombinant proteins is shown in Fig. 1
. Our sequential dialysis and gel filtration protocol for troponin reconstitution reproducibly resulted in the purification of complexes of 1:1:1 subunit stoichiometry, this being true for WT and all mutant troponins.
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Effect of DA mutations on the Ca2+-regulation of actin -activated myosin ATPase
The functional effects of mutant troponin I, troponin T, and Ala-Ser ß-tropomyosin were compared to WT by assay of thin-filament activation of skeletal muscle myosin S-1 ATPase activity. WT and mutant troponin complexes were reconstituted from recombinant human fast skeletal troponin subunits; no difference in the efficacy of reconstitution was noted between WT and any mutant troponin. Thin filaments were assembled using rabbit skeletal actin and myosin S-1, WT or mutant Ala-Ser ß-tropomyosin and WT or mutant troponin. Actin cosedimentation assays were carried out, as described previously (23)
and showed that binding of WT and mutant troponin were indistinguishable (data not shown). WT troponin and Ala-Ser ß-tropomyosin regulated ATPase activity in a Ca2+-dependent, cooperative manner with pCa50 of 7.29 ± 0.02 (n=4) and Hill coefficient (nH), a measure of cooperativity, 2.58 ± 0.28 (n=4) (Fig. 2
). Each mutant troponin or tropomyosin also conferred Ca2+-dependent, cooperative regulation with no statistically significant differences in either pCa50 or nH; however, each mutant did have the striking effect of increasing ATPase activity throughout the pCa range (Fig. 2)
. Thus, we have documented this by comparing the maximally activated ATP activities at pCa5.5 for WT and each mutant and also the maximally inhibited rates at pCa9 (Fig. 3
). WT troponin/tropomyosin gave a maximally activated rate of 4.50 ± 0.01 s–1 (n=12); each DA mutant gave enhanced activation of ATPase activity, the most marked being produced by the ß-tropomyosin Arg91Gly mutant (6.40±0.08 s–1, n=6, P<0.001) with the troponin mutants giving a more moderate but significant (P<0.01, n=8), increase (TnI Arg156ter 6.04±0.1 s–1, TnI Arg174Gln = 5.02±0.08 s–1, TnT Arg63His=5.02±0.07 s–1) (Fig. 3A
). Under relaxing conditions at pCa 9.0, WT troponin and Ala-Ser ß-tropomyosin gave an inhibited activity of 1.43 ± 0.03 s–1 (n=12) and significant (P<0.01) increases in this rate were observed using 3 of the DA mutant proteins (TnI Arg156ter 2.39±0.08 s–1, TnT Arg63His=2.08±0.08 s–1, ßTM Arg91Gly 3.14±0.01 s–1). TnI Arg174Gln gave a small, nonsignificant (P>0.05) increase (Fig. 3B
).
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The disease caused by the DA mutations is autosomal dominant, and it would be anticipated that the fast skeletal thin filaments of affected individuals contain equal proportions of the relevant WT and mutant protein. There have been no direct biopsy analyses of WT and DA mutant protein ratios reported; in the few cases in which studies have been carried out on contractile protein mutants that cause cardiomyopathy, close to 1:1 ratios have usually been reported; for example, in the case of missense -tropomyosin hypertrophic cardiomyopathy (HCM)-causing mutants (36)
. ATPase experiments were therefore repeated using WT and mutant protein in a 1:1 mix in an attempt to reflect more accurately the in vivo ratio. The Ca2+-regulation of ATPase activity by 1:1 mixes showed no significant differences from WT in either pCa50 or nH but gave increases in rate of ATPase at all Ca2+ concentrations intermediate to the 100% mutant and WT levels (Fig. 2)
. In activating conditions, all mutants in a 1:1 mix gave significantly enhanced ATPase rates compared with WT. At pCa9, all except the TnI Arg174Gln mutant produced significantly higher inhibited activity compared with WT; this mutant gave a small but nonsignificant increase (1.47±0.06 s–1) (Fig. 3B
).
The effect of DA mutations on the Ca2+ regulation of force generation in chemically skinned rabbit psoas fibers
To investigate the effect of the DA troponin mutants on force generation, reconstituted human skeletal troponin was used to displace the endogenous troponin complex in rabbit-skinned psoas fibers using the method of Brenner (29)
. Using this technique, we incorporated WT human skeletal troponin at 55 ± 3% of total troponin in these fibers, and exchange of the mutant troponins was not significantly different from WT. A section of a force trace from a typical experiment (in this case using troponin containing Arg174Gln TnI) is shown in Fig. 4
. The maximum Ca2+-activated force produced by fibers containing WT human troponin was 0.079 ± 0.010 N mm–2 and that were produced by fibers containing either Arg174Gln TnI or Arg63His TnT was not significantly different (Fig. 5
). However, fibers containing the truncation mutant of TnI, Arg156ter, gave markedly enhanced maximum force (0.154±0.025 N mm–2; P<0.05). In all three cases, incorporation of mutant troponin did not result in a change in resting tension at pCa9 (illustrated for Arg174Gln TnI in Fig. 4
). The Ca2+-sensitivity of force generation was also tested (Fig. 6
). WT-exchanged fibers had a pCa50 of force generation of 5.26 ± 0.11 with Hill coefficient, nH, of 2.27. All three mutants tested showed enhanced Ca2+-sensitivity with pCa50s of 5.69 ± 0.05 (Arg156ter TnI), 5.62 ± 0.08 (Arg174Gln TnI), and 5.49 ± 0.05 (Arg63His TnT).
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Analysis of the effect of the Arg91Gly mutation on ß tropomyosin structure and actin binding
We carried out analysis of the effect of the mutation on the coiled-coil structure of tropomyosin and its actin binding properties, alterations in which may help to explain the alteration in regulatory function. The affinity of Ala-Ser ß-tropomyosin for actin was measured by cosedimentation. Both WT and mutant displayed cooperative binding, and the data were fitted to the Hill equation (Fig. 7
A). WT bound with a calculated dissociation constant Kapp of 1.89 x 10–6 M and nH of 2.58. The affinity of the DA mutant was significantly lower: Kapp was 7.72 x 10–6 M and nH of 3.47.
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The impact of the Arg91Gly mutation on the coiled-coil structure of Ala-Ser ß-tropomyosin was examined by monitoring the molar ellipticity at 222 nm as a function of temperature (Fig. 7B
). The transition of the Arg91Gly protein from coiled-coil to separate strands took place at a markedly lower temperature range than WT, indicating that the mutation had a significant impact on the coiled-coil stability. The Tm of the complete transition was 45.5°C for WT and 38.5°C for the DA mutant. Previous analyses of tropomyosin unfolding have fitted the data to either two or three transitions (34
, 37)
; both our WT and mutant data fitted well to a two-step model (Fig. 7B
). Using the constants derived from these fits, the calculated G of folding at 20°C were calculated as –5.04 and –4.10 kcalmol–1 for WT and Arg91Gly ß-TM, respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Each of the four mutants had similar effects on the regulation of ATPase activity and the three troponin mutants all increased the Ca2+-sensitivity of force generation, despite the mutations altering different parts of troponin-tropomyosin. The two TnI mutations affect the C-terminal region of the protein with the Arg156ter mutant missing the C-terminal 27 amino acids. This region has previously been shown to be required for full inhibitory activity of TnI in studies of C-terminal truncations of both the fast skeletal protein (38)
and the cardiac isoform (39)
. The Arg63His TnT mutation affects an amino acid within the extended N-terminal T1 domain of the protein, which does not participate in direct interaction with either TnI and TnC but lies along the tropomyosin coiled coil. Residues 92–110 of the cardiac TnT sequence have been suggested to bind to the overlap region of tropomyosin (40)
; these correspond to 61–79 in fast skeletal TnT, and hence Arg63 is likely to interact with tropomyosin in this structurally important region. The Arg91 residue of ß-tropomyosin is located within the third of seven pseudorepeat regions, each of which has been proposed to contribute to the binding to actin (41)
. We hypothesize that the DA mutations in TnT and ß-tropomyosin cause changes in the equilibria governing the blocked
closed myosin-induced transitions described by the three-state steric blocking model (14)
, in favor of the myosin-induced open state. The TnI mutations may also have an impact on these equilibria via altered interactions of the troponin head with tropomyosin but also are likely to have a direct effect on the inhibitory role of TnI. In the measurements of thin-filament-activated myosin S-1 ATPase activity, these thin-filament perturbations are expressed as an increase in rate; in the fiber studies, the troponin mutations result in significant increases in Ca2+ sensitivity. The likely main reason is the oriented actin-myosin lattice array present in the intact sarcomeres which gives an added element of cooperativity, as attached myosin heads can increase the transition of neighboring troponin-tropomyosin complexes from closed to the myosin-induced open state, effectively causing increased Ca2+ sensitivity. Furthermore, crossbridge strain affects the affinity of myosin for adenine nucleotides, and this would have subtle effects on ATPase rates in the fibers (12
, 42
, 43)
. The stoichiometry of incorporation of human troponin into the fibers was close to the expected 1:1 WT/mutant ratio, although it should be borne in mind that the exchange of exogenous subunits does not always occur evenly across the thin filaments (44)
, a factor that may have some effect on the degree of the observed changes.
There have been few previous reports on the biochemistry of specifically the ß isoform of tropomyosin because of difficulty in obtaining a pure preparation of the protein from native /ß mixtures. Here, we report the overexpression and purification of human ß-tropomyosin with an N-terminal Met-Ala-Ser leader, which is processed to give an Ala-Ser tag in bacteria. The ability of unmodified recombinant tropomyosin to undergo normal head-to-tail interactions and to bind actin is much reduced because of the absence of the usual acetylation of the N-terminal methionine found in eukaryotic native tropomyosins (45)
. The presence of the dipeptide tag has been reported to mimic the posttranslational modification in the case of recombinant -tropomyosin, and has been shown to restore its near-normal actin binding and regulatory properties (35
, 46
, 47)
. Human Ala-Ser-ß-tropomyosin bound to F-actin cooperatively (nH=2.6) with Kapp of 
1.9 x 10– (6)
M. This is somewhat weaker than the affinity reported for Ala-Ser--tropomyosin (8.3x10–7 M (35)
), which may reflect that the binding by ßß is weaker than that of dimers or that the N-terminal tag is a less effective mimic of N-terminal acetylation for ß-tropomyosin. The Arg91Gly mutation caused an approximately four-fold decrease in actin affinity, an unexpectedly large effect given that only a single residue in a region outside of the overlapping termini was mutated. The residue is located within the third of seven pseudorepeat regions and the deletion of this entire repeat (residues 89–123) in rabbit -tropomyosin resulted in a 26-fold drop in actin affinity (48)
. The mutation from Arg to Gly also causes a pronounced destabilization of the coiled-coil (Fig. 7B
). Glycine is well known to reduce stability of helices, and furthermore, the removal of the positively charged arginine from the "g" position of the heptad repeat will result in the loss of favorable interactions with nearby glutamate residues (e.g., Glu96 in the "e" position of the opposing strand). Recent studies by Hitchcock-DeGregori and coworkers have examined the effect on the stability of the -tropomyosin coiled-coil of mutation of several alanine residues thought to confer molecular flexibility and to enable efficient actin binding. It was found that stabilizing the tropomyosin structure at either the N- or C-terminus drastically reduced the affinity for actin (49
50
51)
. These data contrast with our finding that the Arg91Gly mutation in ß-tropomyosin destabilizes the coiled coil but also reduces actin binding, therefore indicating that the reported relationship between these two parameters is not always observed.
The Arg91Gly mutation in ß-tropomyosin that causes DA type 1A gives a greater increase in ATPase activity than mutations in the troponin complex, which cause the phenotypically more severe DA type 2B (2)
. This paradoxical observation can be explained by the analysis of the tissue distribution of the disease causing proteins involved. Fast skeletal troponin is the predominant isoform of the distal fast-twitch skeletal muscle fibers (52)
. Fast-twitch tropomyosin, however, is made up of both -tropomyosin and ß-tropomyosin, with the ß isoform present at no more than 50% of total tropomyosin; moreover, studies have suggested that the isoforms preferentially dimerize such that a considerable majority of the tropomyosin regulating the thin filament in vivo is an -ß-heterodimer (53
, 54)
. This means that the Arg91Gly mutant ß-tropomyosin would only constitute at most 25% of the total tropomyosin in vivo, and hence, the severe structural and functional effects caused by the Arg91Gly mutation measured in this study would be diluted. A direct analysis of the effect of Arg91Gly mutant ß -tropomyosin on -ß-heterodimer function was not attempted in this study because of the practical difficulties in preparing heterodimers and in quantitating the /, /ß, and ß/ß distribution of the formed dimers. We also note that it is conceivable that small changes in the effects of the troponin mutations may be observed if they were to be reconstituted in thin filaments with /ß instead of ß/ß-tropomyosin.
Our laboratories and others have previously analyzed the effects of mutations in cardiac thin-filament proteins, which cause either HCM or dilated cardiomyopathy (DCM) (55)
. A distinct pattern has emerged from these studies: the HCM mutations in troponin and -tropomyosin have been largely shown to increase Ca2+ sensitivity, whereas DCM-causing mutations in the same proteins reduce Ca2+ sensitivity (24
, 47
, 56)
. Hence, it is likely that the HCM mutations initially give rise to increased contractility and DCM to lower, and these changes provide separate stimuli for generation of the two different diseases of cardiac muscle. The qualitative effects of the DA mutant proteins more closely resemble those of the HCM mutants, although the DA mutants show Ca2+ sensitivity changes only in the fiber experiments. Interestingly, the Arg63His mutation in fast skeletal TnT occurs at the equivalent position to a known HCM-causing mutation in the cardiac sequence. This mutation Arg94Leu is somewhat unusual in that it produces the myocyte disarray characteristic of HCM and causes sudden death but does not generate significant ventricular hypertrophy (57)
. The magnitude of some of the effects seen with the DA mutants is greater than that seen with HCM mutations, suggesting a more severe disturbance of skeletal muscle function in DA than likely could be compatible with life if cardiac muscle were affected to a similar extent.
We propose that if the observed functional changes caused by the DA mutant proteins are manifest in vivo in affected individuals, the markedly altered contractile regulation will cause increased tension in the developing muscles. The chronic effect of this will be the contractures and limb defects observed in these syndromes. Thus the development of the skeletal abnormalities is an active process, rather than a passive one, as had been previously proposed (7)
. This is in keeping with the demonstration that the contractures develop in utero in the second trimester in previously straight limbs at a time when fast-twitch muscle isoforms are being expressed at a high level (9)
. These disorders are generally not progressive after birth, and indeed, some of the symptoms can be treated with simple physical therapy (1)
. The proximal limb skeletal muscle performance in affected adults is generally normal, as is muscle histology (e.g., of the quadriceps muscle). The likely interpretation of these findings is that it is only the distal muscles that are predominantly fast-twitch and so are susceptible to contracture, whereas the altered regulation at the myofilament level may be compensated for in muscles with mixed fast and slow troponin and tropomyosin isoforms, for example, by changes in Ca2+ handling.
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ACKNOWLEDGMENTS |
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Received for publication July 25, 2006. Accepted for publication September 29, 2006.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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- tropomyosin, Asp175Asn and Glu180Gly, on Ca2+ regulation of thin-filament motility. Biochem. Biophys. Res. Commun. 236,760-764[CrossRef][Medline]This article has been cited by other articles:
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  M. Bamshad, A. E. Van Heest, and D. Pleasure Arthrogryposis: A Review and Update J. Bone Joint Surg. Am., July 1, 2009; 91(Supplement_4): 40 - 46. [Full Text] [PDF]
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 M. Ohlsson, S. Quijano-Roy, N. Darin, G. Brochier, E. Lacene, D. Avila-Smirnow, M. Fardeau, A. Oldfors, and H. Tajsharghi New morphologic and genetic findings in cap disease associated with {beta}-tropomyosin (TPM2) mutations Neurology, December 2, 2008; 71(23): 1896 - 1901. [Abstract] [Full Text] [PDF]
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 J. Ochala, M. Li, M. Ohlsson, A. Oldfors, and L. Larsson Defective regulation of contractile function in muscle fibres carrying an E41K {beta}-tropomyosin mutation J. Physiol., June 15, 2008; 586(12): 2993 - 3004. [Abstract] [Full Text] [PDF]
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P. B. Chase Tropomyosin in the groove? Molecular insights into an inherited myopathy J. Physiol., June 15, 2007; 581(3): 889 - 889. [Full Text] [PDF]
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), 50% mutant/50% WT (
), B) Arg174Gln troponin I: 100% mutant (
) ß-tropomyosin. B) The temperature dependence of unfolding of WT and Arg91Gly ß-tropomyosin. Fraction folded as measured by the relative ellipticity at 222 nm as a function of temperature, in 500 mM NaCl, 10 mM NaPO4 pH 7.0, 1 mM EDTA, and 0.5 mM DTT. WT (