Molecular and cellular contractile dysfunction of ... - Wiley Online Library

ABSTRACT: The purpose of this study was to determine whether contractile protein alterations are responsible for force deficits in young dystrophic muscle. Contractility of intact extensor digitorum longus muscles and permeabilized fibers from wild-type (wt), dystrophin-deficient (mdx), and dystrophin/utrophin-deficient (mdx:utrn⫺/⫺) mice aged 21 and 35 days was determined. Myosin structural dynamics were assessed by site-directed spin labeling and electron paramagnetic resonance spectroscopy. The principal finding was that force generation was depressed by ⬃20% in mdx muscles, but fiber Ca2⫹-activated force and myosin structure were not different from wt animals, suggesting that contractile proteins are not responsible for the force deficits in those muscles. For mdx:utrn⫺/⫺ mice, muscle and fiber forces were ⬃40% lower than wt and the fraction of strong-binding myosin during contraction was reduced by 13%. These data indicate that contractile protein alterations, in addition to myosin dysfunction, cause force deficit in muscles from young mdx:utrn⫺/⫺ mice. Elucidating the molecular mechanisms underlying muscle weakness at the onset of disease is important for designing treatment strategies. Muscle Nerve 34: 92–100, 2006

MOLECULAR AND CELLULAR CONTRACTILE DYSFUNCTION OF DYSTROPHIC MUSCLE FROM YOUNG MICE DAWN A. LOWE, PhD,1 BRIAN O. WILLIAMS, MS,2 DAVID D. THOMAS, PhD,1 and ROBERT W. GRANGE, PhD2 1

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 420 Delaware Street SE, MMC 388, Minneapolis, Minnesota 55455, USA 2 Department of Human Nutrition, Foods and Exercise, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA Accepted 9 March 2006

Dystrophin is a 427-kDa protein located at the sarcolemma of muscle fibers that, along with the dystrophin– glycoprotein complex, links the cytoskeleton to the extracellular matrix. Primary roles for dystrophin probably include membrane structural integrity, lateral force transmission between fibers, and signal communication between extra- and intracellular spaces. The absence of dystrophin results in Duchenne muscular dystrophy (DMD).16 Within skeletal muscle, the disease is characterized by cycles of fiber degeneration and regeneration, fibrosis, contractures, and weakness. Despite the known geAbbreviations: ANOVA, analysis of variance; DMD, Duchenne muscular dystrophy; EDL, extensor digitorum longus; EPR, electron paramagnetic resonance; Freq50, frequency of half-maximal force; mdx, dystrophin-deficient; mdx:utrn⫺/⫺, dystrophin/utrophin-deficient; pCa50, log of the [Ca2⫹] required for half-maximal activation; Po, maximal isometric tetanic force; Pt, peak twitch force; wt, wild-type Key words: Duchenne muscular dystrophy; electron paramagnetic resonance spectroscopy; force deficit; mdx mouse; myosin Correspondence to: D. A. Lowe; e-mail: [email protected] © 2006 Wiley Periodicals, Inc. Published online 21 April 2006 in Wiley InterScience (www.interscience.wiley. com). DOI 10.1002/mus.20562

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netic cause of the disease, early events in the disease process, such as mechanisms underlying the initiation of muscle weakness, are poorly understood. Previously we reported that muscles from young, maturing dystrophic mice demonstrated muscle weakness compared with muscles from wild-type mice of the same age, despite minimal membrane damage based on uptake of the low-molecularweight fluorescent dye, procion orange.15 More specifically, extensor digitorum longus (EDL) muscles from maturing mice lacking dystrophin (mdx) and mice lacking both dystrophin and utrophin (mdx: utrn⫺/⫺) produced low submaximal and maximal specific force in vitro, indicating that the forcegenerating or force-transmitting capacity of young dystrophic muscle was compromised.15 Analysis of whole-muscle force-generating capacities in vitro involves all processes of muscle contraction from the influx of Na⫹ onward. Therefore, decrements in force generation by maturing dystrophic muscle15 could have resulted from impairments in any of the steps of excitation– contraction coupling to impaired

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interactions between the contractile proteins, actin and myosin. An approach to dissect possible sites of contractile dysfunction is to use permeabilized fibers. Permeabilized fibers do not have intact membranes, so contraction is initiated by exogenously added Ca2⫹. Thus, force generation is independent of neural and other excitation– contraction coupling components and directly reflects the function of the contractile proteins. Previous studies of permeabilized fibers have been conducted on dystrophic muscle of fully grown, mature mice,27,38 but it is not presently known whether fibers from young dystrophic mice are impaired. There is evidence for altered contractile protein function in older dystrophic muscle. Coirault and co-workers directly implicated myosin as a molecular explanation for muscle weakness in mdx mice.3 They calculated that the total number of myosin heads strongly bound to actin and the force generated per myosin head were lower in mdx compared to wildtype diaphragm fibers.3 These calculations, based on Huxley’s mathematical model of muscle contraction, implicate myosin, but direct measurements on myosin are necessary to confirm the calculations. The same group also reported that myosin isolated from mdx compared to wild-type diaphragm muscles moved actin more slowly in an in vitro motility assay,4 again implicating impairment in myosin function in mature dystrophic muscle. In the present study, we used permeabilized fibers to study directly the function of the contractile proteins in dystrophic muscle from young mice. Furthermore, we used electron paramagnetic resonance (EPR) spectroscopy paired with site-specific spinlabeling to detect directly and quantify the structure and function of myosin in muscle fibers.23,32,36,37 Using this technique, we precisely measured the fraction of myosin heads strongly bound to actin, and thus generating force, during a contraction. We have used EPR spectroscopy previously to demonstrate that a major factor underlying age-related and denervated-induced decrements in fiber force generation was a decrease in the strong-binding structural state of myosin.23,25 We hypothesize that a common molecular mechanism explaining a good portion of muscle weakness, in both aging and the early stages of muscular disease, is the failure of myosin to interact strongly with actin and generate force at the molecular level. We tested this hypothesis in the present study by analyzing myosin function, via permeabilized fibers, and myosin structural dynamics, via EPR, from muscles of young mdx, mdx:utrn⫺/⫺, and wild-type mice.


MATERIALS AND METHODS

The purpose of this study was to determine the extent to which alterations in contractile proteins are responsible for decrements in contractile function in young dystrophic muscle. To do this, our studies incorporated measurements of force generation at the level of (1) intact muscle; (2) single cell, that is, permeabilized fibers; and (3) molecule, that is, the contractile protein, myosin. The dystrophic and wild-type (wt; C57BL/6) mice used in this study were obtained from our established colony at Virginia Polytechnic Institute.15 The genotype of each mouse (mdx or mdx:utrn⫺/⫺) was determined by polymerase chain reaction analysis of DNA isolated from a tail-snip as described in detail previously.15 At age 21 or 35 days, mice were anesthetized (2 mg xylazine and 20 mg ketamine/ 100 g body mass, intraperitoneally) and EDL muscles were dissected and studied for contractile capacities as described in what follows. All procedures were approved by the Virginia Polytechnic Institute Animal Care Committee. Animals.

EDL muscles from one hindlimb of 21- and 35-day-old wt (n ⫽ 6 and 6), mdx (n ⫽ 5 and 3), and mdx:utrn⫺/⫺ (n ⫽ 4 and 5) mice were immediately studied in vitro for whole-muscle force capacities as described previously.15 Muscles were incubated at 30°C in an oxygenated (95% O2–5% CO2) physiological salt solution (in mM: 120.5 NaCl, 4.8 KCl, 1.2 MgSO4, 20.4 NaHCO3, 1.6 CaCl2, 1.2 NaH2PO4, 10.0 dextrose, and 1.0 pyruvate). The muscle was attached to a dual-mode servomotor system (Aurora Scientific, Ontario, Canada) via 4-0 suture, and optimal muscle length was determined.15 Peak twitch force (Pt) was elicited by stimulating a muscle with a single squarewave 0.2-ms pulse at 20 V. Maximal isometric tetanic tension was similarly elicited, but at a frequency of 150 Hz. A force–frequency protocol was performed at increasing stimulation frequencies (1, 30, 50, 80, 100, and 150 Hz for a duration of 900 ms at 1-min intervals). The frequency at which each muscle produced half-maximal force (Freq50) was determined by fitting the force–frequency data using TableCurve 2D software (Systat Software, Inc., Point Richmond, California). The sigmoidal curve described by y ⫽ a/1 ⫹ exp(⫺x ⫺ (b/c)) consistently gave the best fit (r2 ⬎ 0.991) and was thus used for analyses of all muscles. At the end of the contractile analyses protocol, wet muscle mass was determined and crosssectional area for the muscle was calculated from its Whole-Muscle Contractile Analyses In Vitro.

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mass, optimal length, and a muscle density of 1.06 g/cm3.28 All contralateral EDL muscles from these mice were dissected and permeabilized for myosin EPR analyses as described in what follows. Permeabilized Fiber Contractile Analyses. From a second set of 21-day-old wt (n ⫽ 7), mdx (n ⫽ 9), and mdx:utrn⫺/⫺ (n ⫽ 3) mice, both EDL muscles were dissected, sutured at the tendons to glass capillary tubes, and stored at ⫺20°C in a permeabilization/ storage buffer [in mM: 2 ethylene-glycol tetraacetic acid (EGTA), 1 MgCl2, 126 K-propionate, 20 imidazole (pH 7.0), 4 Na2ATP with 50% glycerol]. Permeabilized muscles were shipped in this buffer, overnight on wet ice, from Virginia to Minnesota. Samples were at 0°C (i.e., on wet ice) for no longer than 20 hours during the shipment and then immediately stored at ⫺20°C until the experiments were conducted. Although permeabilized/skinned fiber bundles for single-fiber experiments are typically stored at ⫺20°C, it is common practice to place bundles at 0°– 4°C for 24 – 48 hours.21 In addition, results from preliminary experiments on muscles from young wt mice harvested directly in Minnesota and stored at ⫺20°C continually were the same as those from muscles shipped from Virginia. Thus, although shipping muscle samples on wet ice could raise concern, our present and past experiences and reports from the literature suggest that the shipping conditions did not affect sample quality. Force–pCa relations were determined using established methods.1,24 A bundle of approximately 10 fibers, 2 mm in length, was dissected and mounted between two microtweezers, one of which was attached to a force transducer (Scientific Instruments Muscle Research System, Heidelberg, Germany). The fiber bundle and tweezers were moved into a quartz capillary filled with a relaxing solution at 21°C (in mM: 1 EGTA, 1 MgO, 85 KOH, 2 Na2ATP, 10⫺6 CaO, and propionate as the major anion and imidazole to maintain pH at 7.0). An activating solution (relaxing solution plus 10⫺4.5 M CaO) was flowed into the cuvette, fiber-bundle length was adjusted to elicit peak isometric force, and the fibers were relaxed. Bundle cross-sectional area was estimated using a calibrated video system. Force was then measured as fibers were exposed to a gradient of Ca2⫹ buffers prepared at 10⫺9 to 10⫺4.5 M. The [Ca2⫹] of those two end-point buffers were measured fluorometrically (Gemini EM Dual Scanning Microplate Spectrofluorometer; Molecular Devices, Sunnyvale, California) using Fura-2 pentopotassium salt (Molecular Probes, Eugene, Oregon) and a Ca2⫹ calibration buffer kit with magnesium (Molecular Probes).

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Actual buffer pCa values were measured at 10⫺8.8 and 10⫺4.2, respectively. These buffer pCa values were important to validate because our overall, average pCa50 value was 6.26, which is slightly higher than many pCa50 values of permeabilized fibers reported in the literature. However, pCa50 values from young, maturing mice have not been reported, and pCa50 values for adult mice are quite variable, ranging from 5.433 to 6.16.11 Thus, we believe our pCa50 values are reasonable. Small bundles of fibers were used for the force– pCa experiments because individual fibers from these young mice were difficult to dissect and mount due to their small size (⬍20 ␮m diameter). The average diameter of the bundles analyzed was 153 ⫾ 3 ␮m (n ⫽ 105) and did not differ between groups (P ⫽ 0.65). Hill plots of the force–pCa data were used to calculate pCa50 (log of the [Ca2⫹] required for half-maximal activation) and activation threshold (log of the minimum [Ca2⫹] required for activation).12,29 Four to six bundles of fibers from each mouse were analyzed and the data averaged to represent Ca2⫹-activated fiber contractility for that mouse. The experimenter was blinded to the genotype of the mice from which the muscles were obtained until all force–pCa experiments were completed. Contralateral EDL muscles to those studied in vitro from the 21- and 35-day-old wt, mdx, and mdx:utrn⫺/⫺ mice were dissected, tied to glass capillary tubes, permeabilized for spectroscopy,23 and then shipped in this buffer to the University of Minnesota. Muscles were labeled with 0.5 mM 4-(2-iodoacetamido)-2,2,6,6-tetramethyl-1-piperidinyloxy spin label (IASL; Sigma), specifically at Cys 707 (SH1) in the catalytic domain of the myosin head.23,32 Each IASL-labeled EDL muscle was dissected into three bundles for spectroscopy. Each bundle was placed in a capillary tube fixed in a TE102 cavity (4102ST/8838; Bruker Instruments, Billerica, Massachusetts) perpendicular to the magnetic field with one end being attached to a force transducer (SensoNor Ackers 801 strain gauge; Aksjelskapet, Norway).2,23 EPR spectra were collected under conditions of rigor, relaxation, and contraction on an E500 EleXsys spectrometer (Bruker Instruments). The following parameters were used to collect the low-field portion of the EPR spectrum: central peak 3425 gauss; sweep width 38 gauss; peak-to-peak modulation amplitude 5.0 gauss; and microwave power 16 mW. Spectra were analyzed to determine the fraction of myosin heads in the strong-binding structural state during contraction, as Myosin, Molecular Contractile Analyses.

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described previously. For each sample, the spectrum obtained during maximal isometric contraction (VCON) was analyzed as a linear combination of the spectra obtained during rigor and relaxation using the equation: VCON ⫽ xVRIG ⫹ (1 ⫺ x)VREL, where VRIG (rigor) corresponds to all heads in the strongbinding structural state (x ⫽ 1) and VREL (relaxation) corresponds to all heads in the weak-binding structural state (x ⫽ 0), as shown previously.23,32 The values from two or three bundles from each EDL muscle were averaged to represent the fraction of strong-binding myosin during contraction for that muscle. The experimenter was blinded to the genotype of the mice from which the muscles were obtained until all EPR spectroscopy experiments were completed. Statistical Analyses. The effects of muscular dystrophy and age on muscle morphology, whole-muscle contractility, and myosin were evaluated using a twoway analysis of variance (ANOVA; genotype ⫻ age). No significant interactions between genotype and age were found for any of the variables measured, except for Freq50. Several significant main effects were found. When there was a significant effect of genotype, a Tukey post hoc test was performed to determine which of the three groups were different from one another. A one-way ANOVA (genotype) with a Tukey post hoc test was used to evaluate permeabilized fiber contractility. Pearson correlation analysis was performed to test for a relationship between force and myosin. Least-square means were used to calculate the percent differences reported in the text. Significance was accepted at P ⱕ 0.05. All statistical analyses were performed using SigmaStat version 2.03 (Systat Software, Inc., Point Richmond, California). Values are expressed as mean ⫾ SE unless otherwise stated. RESULTS Morphological Data. All mice demonstrated increased body mass with age from 21 to 35 days, with a mean increase of 66% (Fig. 1, top). There was also a significant main effect of genotype on body mass with mdx mice being 21% and 36% heavier than wt and mdx:utrn⫺/⫺ mice, respectively. There was no difference in body mass between wt and mdx:utrn⫺/⫺ mice. EDL muscle mass increased 85% from 21 to 35 days, independent of genotype (Fig. 1, middle). EDL muscle mass differed according to genotype with muscles from the mdx:utrn⫺/⫺ mice weighing 25%


FIGURE 1. Body and extensor digitorum longus (EDL) muscle masses of wild-type (wt), dystrophin-deficient (mdx), and dystrophin/utrophin-deficient (mdx:utrn⫺/⫺) mice aged 21 and 35 days that were used in the whole-muscle contractility and myosin experiments. (Top) Body mass was affected by genotype (P ⫽ 0.002) and age (P ⬍ 0.001), with mdx being significantly different than both wild-type and mdx:utrn⫺/⫺ mice. (Middle) EDL muscle mass was affected by genotype (P ⫽ 0.021) and age (P ⬍ 0.001), with mdx:utrn⫺/⫺ being significantly different than both wild-type and mdx. (Bottom) EDL muscle mass normalized to body mass was affected by genotype (P ⫽ 0.014) but not age (P ⫽ 0.436), with mdx and mdx:utrn⫺/⫺ being significantly different than wildtype. No interactions between genotype and age were detected for any variable (P ⱖ 0.120). Values are expressed mean and standard error.

and 28% less than muscles from wt and mdx mice, respectively. The ratio of EDL muscle mass to body mass did not change as the mice aged from 21 to 35 days (Fig. 1, bottom). However, this ratio was affected by mus-

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cular dystrophy, independent of age. Both mdx and mdx:utrn⫺/⫺ EDL muscle mass to body mass ratios were ⬃16% less than that for the wt, indicating that, relative to body size, dystrophic EDL muscles were undersized. Whole-Muscle Contractility. Overall, the ability of intact muscle to generate force for all genotypes increased with age with mean increases of 33% for peak twitch force (Pt) (Fig. 2, top) and 28% for maximal isometric tetanic force (Po) (Fig. 2, middle). Force generation was also affected by genotype. Pt was 36% lower in mdx:utrn⫺/⫺ mice compared with wt mice. Both dystrophic genotypes had lower force values compared to wt mice (Fig. 2, middle). The mdx:utrn⫺/⫺ mice generated 45% and 30% less Po than muscles from wt and mdx mice, respectively, and muscle from mdx mice generated 21% less Po than muscles from wt mice. Absolute isometric tetanic forces (means ⫾ SE) generated by EDL muscles from 12-day-old wt, mdx, and mdx:utrn⫺/⫺ mice were 12.8 ⫾ 0.5, 9.7 ⫾ 0.9, and 6.6 ⫾ 0.6 g, and for 35-day-old wt, mdx, and mdx:utrn⫺/⫺ mice were 25.1 ⫾ 2.0, 22.8 ⫾ 1.5, and 10.5 ⫾ 1.5 g, respectively. The relationship between force production and stimulation frequency was affected by genotype, but the effect was dependent upon age (Fig. 2, bottom). At age 35 days, Freq50 was lower for muscles from mdx and mdx:utrn⫺/⫺ mice than for wt mice (P ⱕ 0.002). Freq50 increased significantly with age for wt mice and decreased with age for mdx mice. Permeabilized Fiber Contractility. Only fibers of EDL muscles from 21-day-old mice were evaluated in these experiments. The lack of dystrophin and utrophin significantly affected the ability of permeabilized fibers to generate maximal Ca2⫹-activated force (P ⫽ 0.035). Fibers from mdx:utrn⫺/⫺ mice generated 40% lower Ca2⫹-activated force than fibers from wt mice (67 ⫾ 4 mg vs. 120 ⫾ 12 mg). There were no differences between fibers from mdx (113 ⫾ 14 mg) and wt or mdx:utrn⫺/⫺ mice. These data suggest that the contractile apparatus in muscle from young mdx: utrn⫺/⫺, but not mdx mice, is compromised. The sensitivity of the contractile apparatus to 2⫹ Ca was assessed by generating Hill plots of force produced at various [Ca2⫹]. The pCa50 for all EDL fibers tested from 21-day-old mice was 6.26 ⫾ 0.04 and was not different between wt and dystrophic groups (P ⫽ 0.220). Activation threshold was also not affected by genotype (P ⫽ 0.586) and equaled 6.89 ⫾ 0.07 for all fibers tested. These data indicate that the thin-filament regulatory proteins are not

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FIGURE 2. In vitro contractility analyses of intact EDL muscles from wild-type (wt), dystrophin-deficient (mdx), and dystrophin/ utrophin-deficient (mdx:utrn⫺/⫺) mice aged 21 and 35 days. (Top) Peak twitch specific force (Pt) was affected by genotype (P ⫽ 0.001) and age (P ⫽ 0.006), with mdx:utrn⫺/⫺ mice being significantly different than wild-type. (Middle) Maximal isometric specific force (Po) was affected by genotype (P ⬍ 0.001) and age (P ⫽ 0.001) with mdx and mdx:utrn⫺/⫺ being significantly different than wild-type and mdx:utrn⫺/⫺ being significantly different than mdx. No interactions between genotype and age were detected for Pt or Po (P ⱖ 0.545). (Bottom) Frequency of stimulation that produced half-maximal force (Freq50) was affected by genotype but the effect was dependent on age (significant interaction, P ⫽ 0.004). Therefore, pairwise comparisons were made and those significant results are depicted in the bar graphs. *Significantly different from 35-day wt; #significantly different from corresponding genotype at 21 days.

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involved in the compromised contractility of dystrophic muscle in young mice. Myosin Contractility. To directly assess whether myosin is the contractile element compromised in dystrophic muscle, we performed EPR spectroscopy of a spin probe specifically located on the myosin catalytic domain. The fraction of myosin heads in the strong-binding (force-generating) structure during a maximal isometric contraction was affected by genotype, independent of age (Figs. 3 and 4). The fraction of strong-binding myosin in fibers from mdx: utrn⫺/⫺ mice was 13% lower than that in fibers from wt mice, indicating that myosin structural dynamics is compromised in young mouse muscle lacking both dystrophin and utrophin. To further explore

FIGURE 4. Fraction of strong-binding myosin heads during a maximal isometric contraction in fibers of EDL muscles from wild-type (wt), dystrophin-deficient (mdx), and dystrophin/utrophin-deficient (mdx:utrn⫺/⫺) mice aged 21 and 35 days. The fraction was affected by genotype (P ⫽ 0.027) but not age (P ⫽ 0.058), with mdx:utrn⫺/⫺ being significantly different than wildtype. No interactions between genotype and age were detected (P ⫽ 0.767). Values are expressed as mean and standard error.

the relationship between force generation (Po) and myosin structural distribution, a correlation analysis between these two variables was conducted. There was a significant correlation between Po and strongbinding myosin (r ⫽ 0.487, P ⫽ 0.009), supporting the concept that dystrophy-related deficits in Po are due, in part, to alterations in myosin structure during contraction. DISCUSSION

FIGURE 3. Electron paramagnetic resonance (EPR) spectra of spin-labeled fibers from a wild-type mouse (wt), aged 35 days, and a mouse lacking dystrophin and utrophin (mdx:utrn⫺/⫺), aged 21 days. Spectra were obtained during conditions of rigor (red), maximal isometric contraction (blue), and relaxation (black). In the wt sample, the fraction of myosin heads strongly bound to actin was 0.391 ⫾ 0.061 (mean ⫾ SD), and in the dystrophic sample the fraction equaled 0.237 ⫾ 0.047. These two particular samples were the extremes, that is, those with the highest and lowest fractions, and were selected so that differences could be observed. The larger fraction of strong-binding myosin in the wt sample can be visualized as the greater area between the relaxation and contraction spectra (hatched area), relative to that area in the mdx:utrn⫺/⫺ spectra.


The main finding of the present study is that weakness exhibited by EDL muscles of young mdx:utrn⫺/⫺ mice was due to contractile protein dysfunction, only part of which could be attributed to altered myosin structural dynamics. Weakness exhibited by EDL muscles of young mdx mice, by contrast, is due to factors upstream of contractile protein interaction. Muscle weakness is characteristic in children and adolescents with DMD,6 but in addressing the mechanisms of this weakness the research has been primarily focused on the characteristics of adult dystrophic mouse models. To obtain varied levels of contractile capacity we employed two ages and two dystrophic genotypes. The first signs of dystrophy are evident at age 21 days in mdx mice, whereas in the mdx:utrn⫺/⫺ mice the disease process begins about 7 days earlier.8,14 By age 35 days, peak fiber degeneration and regeneration are evident in both dystrophic mouse models.8,14 As noted in our whole-muscle contractile data (Fig. 1), we did obtain graded responses for both Pt and Po among the genotypes at age 21 and 35 days. A po-

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tential contributor to this decreased force production is replacement of functional contractile tissue with fat and connective tissue. However, even at 35 days of age, despite the initial cycles of degeneration–regeneration, this should be minimal. Indeed, in adult mdx mice, diaphragm contractile dysfunction precedes collagen accumulation.5 This progression suggests that additional molecular mechanisms underlie contractile dysfunction in dystrophic muscle, particularly in young mice prior to cycles of degeneration–regeneration. Our main finding that contractile dysfunction occurred in the muscle of very young dystrophic mice was shown by significant main effects of genotype for indices of force generation at levels of the whole muscle, permeabilized fibers, and myosin. There were no significant interactions between age and genotype for most of the contractile parameters tested, indicating that alterations in contractile function did not change during growth from 21 to 35 days despite the increases in muscle size and use during this period. This suggests that the underlying mechanisms leading to decreased force generation occurred before 21 days of age and that the normal increases in contractile function that occurred with growth from 21 to 35 days were not affected by the lack of dystrophin. Similarly, in the mdx:utrn⫺/⫺ mice, contractile protein function was already affected by age 21 days and did not get worse (or better) as the mice aged to 35 days. The only parameter in which there was a significant interaction between genotype and age was Freq50. Our data show that, between 21 and 35 days of age, normal muscle generated half-maximal force at an increased frequency of stimulation, probably reflecting shifts toward faster sarcoplasmic reticulum Ca2⫹-ATPase or myosin heavy-chain isoforms. However, as mdx muscle aged from 21 to 35 days, Freq50 decreased, whereas that of mdx:utrn⫺/⫺ muscle did not change. Both a slower Ca2⫹-ATPase10 and decreased Ca2⫹ sequestration by calsequestrin-like proteins7 have been reported for young mdx mice. Myosin isoform expression is the same for EDL muscles from 9 –12-day-old wt, mdx, and mdx:utrn⫺/⫺ mice,15 and does not shift from faster to slower isoforms in mdx mice until age 3– 4 months.34 Thus, the depressed Freq50 in young dystrophic EDL muscles is more likely due to slowed Ca2⫹ uptake or sequestration rather than to changes in myosin heavy chain isoform expression. The whole-muscle contractile deficits we measured were similar to findings in our previous report on young dystrophic mouse muscle15 and also similar in magnitude to deficits reported in adult dystro-

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phic mouse muscle. For example, Po deficits of 20%– 40% have been reported for EDL muscles from adult mdx mice aged 11–24 weeks9,22,26,31 and Po deficits of 35%–50% for EDL and soleus muscles from mdx: utrn⫺/⫺ mice aged ⬃11 weeks.9 In the current study, EDL muscles from young mdx and mdx:utrn⫺/⫺ mice aged 3–5 weeks had Po deficits of ⬃20% and 45%, respectively. These deficits could have resulted from any combination of events that have been reported to occur in adult dystrophic muscle, such as a loss of intracellular Ca2⫹ homeostasis,13 mitochondrial alterations,20 sarcoplasmic reticulum dysfunction,10,18 or faulty contractile protein function. It is unclear which if any of these events occurs first, whether the events are additive, or whether one event causes another event, particularly in muscle from young dystrophic mice. In earlier work we showed that the sarcolemma of fibers from maturing dystrophic muscle is resistant to acute mechanical injury, indicating that membrane damage is not a primary initiating factor in the onset of muscular dystrophy.15 We tested Ca2⫹-activated force generation by permeabilized fibers to establish the contribution of contractile protein dysfunction to the overall muscle force-generating deficit in young dystrophic muscle. Bundles of fibers were used to assess Ca2⫹-activated force rather than single fibers because fibers from these young mice are very small (⬍20 ␮m in diameter). Bundles, however, were not optimal due to difficulties in measuring their cross-sectional areas and because, following permeabilization, interfiber spacing increased. Due to these factors, we compared absolute Po values between genotypes. Bundle diameters were roughly measured and the averages were not different between groups, supporting the use of absolute force measures. In addition, to help overcome these limitations, we analyzed four to six bundles per mouse and used the average of these bundles for comparisons between genotypes. Despite these precautions, the force values we reported seem low compared with values reported in the literature for single fibers. Several aspects should be considered when making this comparison, however. For example, force levels by single fibers or fiber bundles have not been reported from very young mice, and comparisons between maturing and mature responses should be performed with caution. Second, the variable interfiber spacing that is typical for bundles of fibers is not optimal for transmitting lateral force, which clearly contributes to the overall force generated by muscle samples larger than a single fiber.17 Last, we have recently used a method to normalize force by myosin content, specifically to avoid erroneous cross-sectional area measures of fi-

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ber bundles.30 When this is done, similar force values can be obtained for whole muscles and permeabilized fiber bundles. Unfortunately, we could not apply a similar analysis to the samples from the current study because these fiber bundles were not kept after the experiments. However, when absolute force values of fiber bundles from those recent experiments30 are compared with the absolute force values reported here, they are very similar. These aspects strongly suggest that the seemingly low force values reported herein are a result of using bundles of fibers instead of single fibers. Most results on permeabilized adult mdx fibers have shown little if any decrement in Ca2⫹-activated force.19,27,35,38 Our results with young mice were comparable as Ca2⫹-activated force generation by mdx fibers was not different from that by wt animals. Similarly, our EPR data on mdx fibers revealed that myosin structural dynamics were not significantly altered. Collectively these results indicate that forcegeneration deficits observed at the whole-muscle level in mdx mice result from detrimental alterations upstream of actin–myosin interaction, that is, at some earlier steps in excitation– contraction coupling. The force loss does not seem to be explained by changes in the content of calcium-handling proteins, which were not different between wt, mdx, and mdx:utrn⫺/⫺ mice at 21 days, or to a decreased sarcoplasmic reticulum Ca2⫹ release rate (Grange, unpublished observations). However, whether Ca2⫹ transients are reduced in young mdx compared to wt muscles is yet to be determined. There were strikingly different results obtained on fiber force-generating capacity between the two dystrophic mouse models. Although permeabilized fibers from mdx mice did not have force deficits, fibers from mdx:utrn⫺/⫺ mice had 40% deficits. The 40% decrement in maximal Ca2⫹-activated fiber force explained essentially all of the whole-muscle Po loss in those muscles. Sensitivity of the contractile apparatus to Ca2⫹ was not affected, so it can be surmised that the thin-filament regulatory proteins were not detrimentally altered during the early stages of muscular dystrophy, similar to what has been reported for adult dystrophic mouse muscle.35 Therefore, the remaining contractile proteins to be investigated in young mdx:utrn⫺/⫺ muscle were the contractile proteins, myosin and actin. We directly assessed myosin function by placing a spin probe specifically on the catalytic domain of the myosin head in permeabilized fibers and then used EPR spectroscopy to monitor changes in that spin probe as muscle fibers proceeded from a state of rigor, to relaxation, and then to maximal isometric contrac-


tion. We found that myosin was detrimentally affected in EDL muscle fibers from mdx:utrn⫺/⫺ mice. During a maximal contraction, the fraction of myosin heads strongly bound to actin and generating force was 13% lower than in wt mice. Therefore, only about one-third of the force deficit by intact muscle from young mdx:utrn⫺/⫺ mice can be attributed to dysfunctional myosin dynamics. Perturbations in actin should next be assessed. Another possibility is that the amount of actin or myosin molecules (per muscle area) in mdx:utrn⫺/⫺ muscle is reduced, corresponding to the myofibrillar loss that is suggested by histological assessments of dystrophic muscle. In summary, although DMD is characterized by the lack of dystrophin, at present it is not clear how its absence leads to pathophysiological outcomes such as muscle weakness, especially during early maturation. Delineating fundamental molecular events that occur at the onset of the disease has implications for treatment strategies, early diagnoses, and tracking disease progression. Our results reflect the complexity of the dystrophic pathophysiology, because for young mdx:utrn⫺/⫺ mice we could attribute only a fraction of the muscle weakness to alterations in myosin, and thus other events must also contribute. This research was supported by grants from the Muscular Dystrophy Association (R.W.G. and D.D.T.) and NIH grants AR049881 (R.W.G.) and AG20990 (D.A.L.).

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