1 Department of Biochemistry, Molecular Biology, and Biophysics, 2 Department of Physical Medicine and Rehabilitation, and 3 Center on Aging, University of Minnesota, Minneapolis, Minnesota 55455
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ABSTRACT |
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We tested the hypothesis that low specific tension (force/cross-sectional area) in skeletal muscle from aged animals results from structural changes in myosin that occur with aging. Permeabilized semimembranosus fibers from young adult and aged rats were spin labeled site specifically at myosin SH1 (Cys-707). Electron paramagnetic resonance (EPR) was then used to resolve and quantify the structural states of the myosin head to determine the fraction of myosin heads in the strong-binding (force generating) structural state during maximal isometric contraction. Fibers from aged rats generated 27 ± 0.8% less specific tension than fibers from younger rats (P < 0.001). EPR spectral analyses showed that, during contraction, 31.6 ± 2.1% of myosin heads were in the strong-binding structural state in fibers from young adult animals but only 22.1 ± 1.3% of myosin heads in fibers from aged animals were in that state (P = 0.004). Biochemical assays indicated that the age-related change in myosin structure could be due to protein oxidation, as indicated by a decrease in the number of free cysteine residues. We conclude that myosin structural changes can provide a molecular explanation for age-related decline in skeletal muscle force generation.
spectroscopy; specific tension; force; weakness; aging
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INTRODUCTION |
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SKELETAL MUSCLE FUNCTION diminishes with age. For example, several studies have shown that muscles from hindlimbs of aged rodents produce less force than those muscles from younger animals (10, 14, 19, 23, 26, 27, 41). Part of the reduced ability results from muscle atrophy that occurs with aging. However, if atrophy is accounted for by normalizing to muscle size, force generation is still ~20% lower in muscles of aged animals (10, 14, 19, 23, 26, 27, 41). Recently, this age-related reduction has been shown in permeabilized, single fibers from human and rodent muscles (20, 39). Permeabilized fibers do not have intact membranes, so force generation reflects directly the interactions of myosin and actin, exclusive of other factors in excitation-contraction coupling (e.g., sarcoplasmic reticulum Ca2+ release). Therefore, it appears that the age-related reduction in force-generating capacity is a result, at least in part, of defects in contractile protein.
There is evidence to suggest that changes in myosin structure can compromise muscle function. For example, oxidation of thiol residues in myosin decreases isometric force production, maximal shortening velocity, and ATPase activity in rabbit muscle fibers (25). Because thiols are not involved in the catalytic mechanism of myosin ATPase, this strongly suggests that a structural perturbation in myosin occurs as a result of oxidation, culminating in altered myosin function. Specific structural changes within the myosin molecule have also been implicated in a variety of muscle disorders (30), but the question of age-related myosin structural changes has not been thoroughly investigated. A recent study by Höök and coworkers (16) directly implicates myosin as the protein responsible for the reduced maximal shortening velocity reported in muscle fibers from aged animals. In vitro motility assays showed that myosin molecules extracted from soleus muscle fibers of aged rats have a decreased ability to move actin relative to younger rats (16). Those results suggest that a likely age-related site of defect in muscle contraction is the myosin molecule itself. However, there has been no direct study of the molecular mechanism of an age-related defect in myosin.
Electron paramagnetic resonance (EPR) spectroscopy has been used
extensively for investigating myosin because it is exquisitely sensitive to changes in molecular structure and its high resolution permits the quantitative analysis of distinct structural states (32, 37, 38). The use of site-directed spin labels has
played a vital role in these studies because probes have been attached specifically to a distinct residue within myosin in functional skinned
muscle fibers, providing structural information about a specific domain
within myosin under physiological conditions (for review see Refs.
36 and 38). In several studies, spin labels have been
attached to one of two highly reactive sulfhydryl groups, SH1 (Cys-707)
or SH2 (Cys-697), both of which are located in the catalytic domain of
the myosin head, near the "converter" region that is thought to
couple the active site to the large structural changes that generate
force (5, 6, 8, 12, 24, 37). From these and similar
studies, it has been shown that the myosin head has two primary
structural states: a strong-binding (to actin) structural state that is
rigid and stereospecific and a weak-binding structural state that is
dynamically disordered (Fig. 1).
Furthermore, force is generated on transition of the myosin head from
the weak-binding to the strong-binding structural state. The
combination of EPR and site-directed spin labeling provides a unique
opportunity to resolve and quantify distinct structural states of
myosin in an intact biological system. For example, structural changes
within the myosin head have been reported in functioning skeletal
muscle fibers (2, 4, 6, 12, 24, 37). Of direct relevance
to the present study is the observation that, with the use of rabbit
psoas fibers labeled at SH1 with iodoacetamide spin label (IASL), at
any given moment during a maximal isometric contraction, 27% of the
myosin heads are in the strong-binding (force-producing) structural
state (24).
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In the present study, we have taken advantage of the high resolution of EPR spectroscopy to investigate age-related alterations in the distribution of myosin structural states during muscle contraction. That is, we have used this sensitive biophysical technique, which has been shown to resolve weak- and strong-binding structural states in spin-labeled muscle fibers (24), to probe the underlying molecular mechanism of aging in skeletal muscle. We hypothesized that the reduced force-generating ability of fibers from aged animals is due to a decreased population of myosin heads in the strong-binding (force generating) structural state during muscle contraction relative to fibers from younger animals.
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METHODS |
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Animals.
Fischer 344 × Brown Norway F1 hybrid male rats, aged
8-12 mo (young adult; n = 6) and 32-36 mo
(aged; n = 6), were obtained from the aging colony
maintained by the National Institutes on Aging. The animals were housed
locally for 1 wk in pathogen-free conditions and received food and
water ad libitum. Animals were anesthetized with pentobarbital sodium
(55 mg/kg ip), tissues were carefully dissected, and then animals were
euthanized with an overdose of pentobarbital sodium.
Tissue preparation and spin labeling.
Immediately after dissection, semimembranosus muscles were submerged in
a relaxing buffer [7 mM EGTA, 0.016 mM CaCl2, 5.6 mM
MgCl2, 80 mM KCl, 20 mM imidazole (pH 7.0), 14.5 mM
creatine phosphate, 4.8 mM ATP] on ice and pinned to approximately
resting length. Bundles of semimembranosus muscles fibers, ~25 mm
long and ~2 mm diameter, were formed and tied onto glass rods. Fibers were permeabilized by placing the bundles in glycerination buffer consisting of 25% glycerol, 0.5% Triton X-100, 9 mM EGTA, and 75%
rigor buffer [60 mM potassium propionate, 25 mM MOPS (pH 7.0), 6 mM
MgCl2, 1 mM EGTA, 1 mM NaN3] for 24 h at
4°C with gentle shaking; buffer was refreshed four times throughout
this period. Bundles were then placed in a storage buffer (50%
glycerol-50% rigor buffer) for 24 h at 4°C with gentle shaking
and four changes of buffer. Final storage was at 20°C in fresh
storage buffer containing 0.1 mM dithiothreitol. The permeabilization
procedure was similar to that used previously for rabbit fibers
(24).
Characterization of spin-labeled fibers.
Myosin ATPase activities of myofibrils were measured in high salt to
determine the extent and specificity of myosin SH1 labeling by IASL, as
described previously (21, 29). First, myofibrils were
prepared from IASL-labeled and unlabeled fibers by brief homogenization
in 10 mM MOPS (pH 7.0) at 0°C. Protein concentration was then
determined using the bicinchoninic acid protein assay, with BSA as a
standard (Pierce, Rockford, IL). ATPase activities of the myofibrils
were determined spectrophotometrically at 25°C by measuring the
production of Pi (18).
K+/EDTA-ATPase was measured in a solution containing 0.60 M
KCl, 5 mM EDTA, and 50 mM MOPS (pH 7.5).
Ca2+/K+-ATPase was measured in a solution
containing 10 mM CaCl2, 0.60 M KCl, and 50 mM MOPS (pH
7.5). Reactions were started by the addition of 5 mM ATP and quenched
by 34% citrate. The fraction of myosin heads labeled at SH1
(fSH) by IASL was determined from the fractional inhibition
of K+/EDTA-ATPase activity: fSH = [1 (KL/KUL)]/0.95, where
KL is the K+/EDTA activity of
labeled myofibrils and KUL is the
K+/EDTA activity of unlabeled myofibrils (21,
24).
Single-fiber contractile measurements. Individual fiber segments (~2 mm long) from permeabilized bundles were isolated and studied at 25°C, as described in detail previously (39). Fiber segments were mounted in relaxing buffer, sarcomere lengths were set to 2.5 µm, diameter was measured at three places along the length of the fiber, and then maximal isometric force was determined. Four to six unlabeled fibers from each animal were studied.
After force measurements, each fiber was solubilized in 20 µl of sample buffer [24 mM EDTA, 60 mM Tris (pH 6.8), 1% SDS, 5%EPR spectroscopy. IASL-labeled fiber bundles were prepared for spectroscopy by attaching 7.0 silk suture to each end of the bundle and pulling the bundle into a glass capillary tube (1.3 mm ID and 1.5 mm OD) that was filled with storage buffer. The capillary tube was then fixed in a TE102 cavity (model 4102ST/8838, Bruker Instruments, Billerica, MA) designed to hold the tube perpendicular to the magnetic field. One suture was attached to a force transducer (model 801 strain gauge, SensoNor Ackers, Aksjelskapet, Norway), and the other was stabilized to hold the bundle isometrically (11). The capillary tube inside the cavity was in line with tubing and a peristaltic pump, such that a continuous supply of fresh buffer was delivered to the fibers at the rate of 0.2 ml/min. All EPR experiments were conducted at 22°C with room temperature buffers. Force was monitored throughout the duration of EPR spectra collection. This ensured that spectra of the labeled fibers were collected under fully relaxed and maximally activated conditions.
Once the cavity was mounted in the spectrometer (ESP 300 or E500 EleXsys spectrometer, Bruker Instruments), fibers were perfused with rigor buffer and X-band EPR spectra were collected (24). Under our conditions, the central peak of the IASL spectrum is located at a magnetic field strength of 3,425 gauss. Therefore, to collect the entire IASL spectrum of our muscle fibers, the magnetic field 120 gauss about that peak was scanned using a peak-to-peak modulation amplitude of 2.0 gauss and a microwave power of 16 mW. A representative spectrum is shown in Fig. 2.
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EPR data analyses.
The 38-gauss low-field EPR spectra were analyzed to determine the
fraction of myosin heads in the strong-binding structural state
(x) during muscle contraction. For each bundle, the spectrum obtained during maximal isometric contraction (VCon) was
analyzed as a linear combination of the spectra obtained during rigor
and relaxation using
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(1) |
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(2) |
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Statistical analyses. Values are means ± SE. Student's t-tests were used to compare results of fibers/bundles from young adult animals with those from aged animals. P < 0.05 was considered significant.
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RESULTS |
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Single-fiber contractile properties.
Cross-sectional area of semimembranosus fiber segments from young adult
and aged animals was not different (5,863 ± 673 and 5,303 ± 692 µm2, respectively, P = 0.56).
However, fibers from young adult animals generated greater force
and specific tension (0.995 ± 0.119 mN and 171.9 ± 7.4 kN/m2) than those from aged animals (0.659 ± 0.078 mN
and 125.7 ± 6.6 kN/m2, P 0.025). Fibers
from spin-labeled muscle bundles generated about half as much specific
tension as the unlabeled fibers: 91.7 ±13.2 (n = 10)
and 69.4 ± 6.2 kN/m2 (n = 8) for
fibers from young adult and aged animals, respectively. This reduction
in contractility is consistent with previous reports that SH1 labeling
alters the kinetics of the myosin ATPase cycle, decreasing the rate of
the ATP hydrolysis step and increasing the rate of the product release
step (8, 21). The magnitude of effect we observed, i.e.,
~50% reduction in tension, is in agreement with other reports of
myosin SH1 labeling effects on fiber contractility (8, 13, 21,
22).
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Specificity of spin labeling.
High salt concentration (e.g., 0.6 M KCl) activates myosin ATPase, and
the resulting activities yield information about two highly reactive
sulfhydryl groups, SH1 (Cys-707) and SH2 (Cys-697) in the catalytic
domain of myosin (13, 17, 28, 33). Specifically, when SH1
is modified (labeled), K+/EDTA-ATPase activity is inhibited
and Ca2+/K+-ATPase activity is increased.
SH2 modification also inhibits K+/EDTA-ATPase activity but
does not activate Ca2+/K+-ATPase activity. When
both SH1 and SH2 are modified, K+/EDTA- and
Ca2+/K+-ATPase activities are severely reduced.
In the present experiments, Ca2+/K+-ATPase
activities increased and K+/EDTA-ATPase activities
decreased with IASL labeling in fibers from young adult and aged
animals (Fig. 5), indicating that the spin label was highly specific for SH1, with little or no SH2 labeling.
The extent of SH1 labeling by IASL was greater in fiber bundles from
young adult animals than in those from aged animals: fSH = 0.96 ± 0.03 and 0.63 ± 0.09, respectively (P = 0.007). The difference in the extent
of labeling appears to be due to the lower K+/EDTA-ATPase
activity of unlabeled fibers in muscles from aged animals than in
muscles from younger animals (P = 0.001; Fig. 5).
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EPR spectroscopy.
Representative 38-gauss low-field EPR spectra are shown in Fig.
6. The end-point spectra, i.e., the rigor
spectrum in which all heads are in the strong-binding structural state
and the relaxation spectrum in which all heads are in the weak-binding
structural state, were the same for fibers from young adult and aged
animals, even though the fibers from the aged animals were not SH1
labeled as extensively. Thus there is no age dependence in the
conformations of the two structural states (weak and strong binding) as
detected by EPR, so any difference observed between the EPR spectra of young and old rats in contraction (Fig. 6) must be due to a difference in the distribution between these states.
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DISCUSSION |
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We have used site-specific EPR spectroscopy to investigate structural states of the myosin head during contraction in fibers from a rat hindlimb muscle. Furthermore, we investigated how those structural states differ in fibers from aged rats. Our main finding was that age-related decrements in specific tension can be explained in part by changes in myosin structure.
Most previous EPR studies of spin-labeled myosin have been conducted on purified proteins or filaments isolated from rabbit skeletal muscle (5, 29) or on fibers from rabbit psoas muscle (2, 6, 8, 12, 24, 31, 37). The rabbit psoas muscle has been preferred for EPR fiber studies because it is a large fusiform muscle that is easily exposed in vivo from end to end. This makes it possible to form bundles of fibers >10 cm long, which is advantageous for EPR spectroscopy. However, there is little information regarding biological aging of rabbits. Instead, rodents have been the model of choice for aging studies, and hindlimb muscles from these animals have been studied the most extensively in terms of age-related functional changes in skeletal muscle. Thus our first goal was to adapt methodologies of spin labeling and EPR spectroscopy designed for rabbit psoas fibers to fibers from a much smaller rodent hindlimb muscle.
A rat thigh muscle, the semimembranosus, was chosen for the present study because, like rabbit psoas muscle, it is a relatively large muscle that is easily exposed and contains fibers arranged parallel with the long axis of the muscle. It was fairly easy to form bundles containing fibers up to 2 cm long, making it a candidate for EPR spectroscopy. We also chose the semimembranosus muscle because it is composed primarily of type II fibers (3) and, in general, type II fibers show greater age-related functional changes than type I fibers. However, different researchers using various rodent species, assorted animal ages, a variety of hindlimb muscles, and different muscle preparations (i.e., whole muscle in vivo, in situ, or in vitro preparations or single-fiber preparations) have not always found age-related decreases in specific tension. Therefore, it was essential that we first identified a rat hindlimb muscle that showed the age-related reduction in specific tension and was suitable for EPR spectroscopy. It was important for us to use the same muscle and the same muscle preparation for our muscle function (specific tension) experiments and for our structural (EPR) experiments so that we were able to directly assess the relationship between the age-related muscle functional changes and myosin structural changes.
First, we found that type II fibers from semimembranosus muscles of aged rats generated 27% less specific tension than fibers from young adult rats. Several studies have reported age-related reductions in specific tension in rodent muscles composed predominantly of type II fibers (10, 14, 19, 23, 41). Also, a study on single fibers from vastus lateralis muscles of young and older men showed an age-related reduction in specific tension in type IIA but not type I fibers (20). Our results on type II rat semimembranosus fibers are consistent with these previous reports of age-related reductions in contractility, specifically of type II fibers. Therefore, our first criterion was met, i.e., fibers from the semimembranosus muscle showed the age-related decrement in specific tension.
Next, to confirm that quantitative and reliable EPR spectroscopy was feasible on rat muscle, we compared our results on IASL-labeled semimembranosus fibers from young adult rats with previous results from our laboratory on IASL-labeled rabbit psoas fibers. We found that the specificity and extent of labeling myosin SH1 with IASL was very similar between fibers from rabbits and young rats. IASL-EPR spectra of young rat semimembranosus fibers (Figs. 2 and 3) are essentially identical to those obtained from rabbit psoas fibers (24). In addition, our sensitivity to detect and quantify the two structural states of myosin heads in fibers from rats was equivalent to that in fibers from rabbits. Lastly, our finding that fibers from young adult rats have 31.6% of myosin heads in the strong-binding structural state during maximal isometric contraction is similar to the 27% found in rabbit psoas fibers (24). Thus we identified a rat hindlimb muscle suitable for EPR spectroscopy in which specific tension was reduced with age. Because semimembranosus muscle fibers fit these criteria, we used this muscle to further investigate a potential underlying molecular mechanism to explain why skeletal muscle contractile function declines with age.
The major finding in the present study is that the age-related reduction in specific tension can be explained by changes in the structure of myosin during contraction (Fig. 8). Because clear isoclinic points were observed (Figs. 3 and 7), even though no structural differences were detected in the end-point spectra of relaxation or rigor (Fig. 6), it is clear that the only detected difference between young and aged fibers is in the mole fraction x of myosin heads in the strongly bound structural state. We found that, during a maximal isometric contraction, 30 ± 0.8% fewer myosin heads are in the strong-binding, i.e., force-generating, structural state in fibers from aged animals (x = 0.22) than in fibers from younger animals (x = 0.32; Figs. 7 and 8). Because it is usually proposed that the force generated during contraction is directly proportional to x and because the decrease in x (30 ± 0.8%) is in good agreement with the decrease in specific tension (27 ± 0.8%; Fig. 8), we propose that the decrement in force-generating capacity of fibers from aged animals is a direct result of a reduced fraction of myosin heads in the strong-binding structural state. In other words, the number of force-generating cross bridges per unit area during a muscle contraction is reduced with age.
Alternative suggestions have been made for mechanisms underlying the age-related reduction in specific tension of skeletal muscle studied in vitro, but little evidence has been shown in support of these suggestions. The most predominant suggestion has been that there is a decrease in myofibrillar protein content per unit area in muscle of aged animals, implying that the total number of cross bridges per unit area is decreased. In support of this suggestion are the observations that connective tissue, fat, and lipofuscin molecules are increased in muscle of aged animals relative to muscle of young animals (1, 7, 9). However, increased amounts of these substances, and hence decreased amounts of contractile proteins, are not of sufficient magnitude to explain the 10-30% reduction in specific tension that is commonly reported in muscle of aged animals. To explain the reduction of force in skinned muscle fibers, the only other plausible explanations are that 1) the fraction of strong-binding myosin molecules (x) changes, as our results indicate, or 2) x does not change but the force per strong-binding myosin head decreases. Our results suggest that explanation 1 is correct and explanation 2 can, therefore, be ruled out. In principle, this should be confirmed in the future by single-molecule force measurements. These alternative mechanistic explanations are the only ones that can explain the decrease of force in maximally activated skinned fibers (20, 39), in which the neural and excitation-contraction coupling components of muscle activation are eliminated. Of course, our conclusions do not contradict reports of additional effects of aging on these membrane-related factors (15).
Our molecular explanation that the age-related decrement in specific tension is the result of a reduction in the fraction of myosin heads in the strong-binding structural state has been suggested previously (10, 26, 27). The present study provides the first direct evidence supporting this hypothesis. It was previously proposed that myosin might favor the low-force, i.e., weak-binding, state in muscles from aged animals, because those muscles might have a higher intracellular Pi concentration than muscles from younger animals (10, 26). However, a subsequent study (27) found no difference in Pi levels or intracellular pH between muscles from young and old mice. Our results on skinned fibers show that, independent of any age-related changes in intracellular solutes, a substantial force reduction can be explained by a shift in the distribution between myosin structural states. This shift could be due to a fraction of "dead" myosin heads that never enter the strong-binding structural state or a change in kinetics that decreases the fraction of time that each myosin head spends in this state.
What might cause this shift in the myosin structural distribution? We speculate that there is an age-related posttranslational modification of myosin that affects x. Our high-salt ATPase data support this proposal. The fractional inhibition of K+/EDTA-activated ATPase (fSH) showed that in vitro modification by IASL occurred at nearly all the SH1 sites in fibers from young animals but only ~60% of the SH1 sites were modified in fibers from aged animals. This suggests that the other ~40% of the SH1 sites in fibers from aged animals, i.e., those that were unreactive with IASL, were already modified in vivo, posttranslationally, by some unknown mechanism. Previous studies have shown that modification of SH1 decreases muscle force generation (13, 22, 25) and maximal shortening velocity (13, 25), but these effects of SH1 blocking cannot completely explain the functional and EPR effects in the present study, because all our measurements were on labeled fibers having no remaining reactive SH1 sites. We propose that the effects of aging are due, at least in part, to modification of SH groups other than SH1, as suggested by the lack of Ca2+-ATPase activation in aged animals before labeling (Fig. 5). Cysteine modification is an attractive model, since oxidative modification of proteins is thought to play a significant role in cellular aging (34). Specific modification of cysteine residues in muscle proteins has not been studied thoroughly, although it has recently been implicated in the age-related dysfunction of the sarcoplasmic reticulum Ca2+-ATPase protein (40). Thus we propose that there is an age-related loss of free cysteines in skeletal muscle myosin, which reduces the fraction of myosin molecules in the strong-binding structural state and thus reduces muscle force. Further direct experimental evidence is needed to test this proposal.
In conclusion, we have shown that site-specific EPR spectroscopy of spin-labeled muscle is sensitive enough to resolve and quantify structural states of the myosin head in fibers from a rat hindlimb muscle. Using this high-resolution biophysical technique, we have shown that the steady-state fraction of myosin heads in the strong-binding (force generating) structural state during a maximal isometric contraction is reduced in fibers from aged animals, by a fraction (30%) that is comparable to the decrease in specific tension (27%). Therefore, changes in the structure of myosin provide a molecular explanation for the reduction in muscle specific tension that occurs with age. We further speculate that the change in myosin structure is a result of a biochemical posttranslational modification of cysteine residues.
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ACKNOWLEDGEMENTS |
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We thank Leslie E. W. LaConte for help with the EPR spectroscopy and Janice Shoeman for technical assistance.
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FOOTNOTES |
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This work was supported by National Institute on Aging (NIA) Grant AG-18156 and a grant from the American Heart Association, Minnesota Affiliate (L. V. Thompson), National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-32961 and a grant from the Muscular Dystrophy Association (D. D. Thomas), NIA National Research Service Award Fellowship AG-05815 and Training Grant AG-00198 (D. A. Lowe), and National Institute of General Medical Sciences Training Grant in Molecular Biophysics GM-08277 (J. T. Surek).
Address for reprint requests and other correspondence: D. A. Lowe, Dept. of BMBB, University of Minnesota, Jackson Hall 6-155, 321 Church St., Minneapolis, MN 55455 (dl{at}ddt.biochem.umn.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.
Received 31 July 2000; accepted in final form 21 September 2000.
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