Thin-filament length correlates with fiber type in human skeletal muscle

Am J Physiol Cell Physiol 302: C555–C565, 2012. First published November 9, 2011; doi:10.1152/ajpcell.00299.2011.

Thin-filament length correlates with fiber type in human skeletal muscle David S. Gokhin,1 Nancy E. Kim,1 Sarah A. Lewis,1 Heinz R. Hoenecke,2 Darryl D. D’Lima,2 and Velia M. Fowler1 1

Department of Cell Biology, The Scripps Research Institute, and 2Shiley Center for Orthopaedic Research and Education, Scripps Clinic, La Jolla, California

Submitted 15 August 2011; accepted in final form 8 November 2011

Gokhin DS, Kim NE, Lewis SA, Hoenecke HR, D’Lima DD, Fowler VM. Thin-filament length correlates with fiber type in human skeletal muscle. Am J Physiol Cell Physiol 302: C555–C565, 2012. First published November 9, 2011; doi:10.1152/ajpcell.00299.2011.—Force production in skeletal muscle is proportional to the amount of overlap between the thin and thick filaments, which, in turn, depends on their lengths. Both thin- and thick-filament lengths are precisely regulated and uniform within a myofibril. While thick-filament lengths are essentially constant across muscles and species (⬃1.65 ␮m), thin-filament lengths are highly variable both across species and across muscles of a single species. Here, we used a high-resolution immunofluorescence and image analysis technique (distributed deconvolution) to directly test the hypothesis that thin-filament lengths vary across human muscles. Using deltoid and pectoralis major muscle biopsies, we identified thin-filament lengths that ranged from 1.19 ⫾ 0.08 to 1.37 ⫾ 0.04 ␮m, based on tropomodulin localization with respect to the Z-line. Tropomodulin localized from 0.28 to 0.47 ␮m further from the Z-line than the NH2-terminus of nebulin in the various biopsies, indicating that human thin filaments have nebulinfree, pointed-end extensions that comprise up to 34% of total thinfilament length. Furthermore, thin-filament length was negatively correlated with the percentage of type 2X myosin heavy chain within the biopsy and shorter in type 2X myosin heavy chain-positive fibers, establishing the existence of a relationship between thin-filament lengths and fiber types in human muscle. Together, these data challenge the widely held assumption that human thin-filament lengths are constant. Our results also have broad relevance to musculoskeletal modeling, surgical reattachment of muscles, and orthopedic rehabilitation. actin; length-tension curve; myosin heavy chain; nebulin; tropomodulin

muscle arises from cross-bridge interactions between myosin (thick) filaments and actin (thin) filaments in the sarcomeres of the myofibrillar lattice. A sarcomere’s force-generating capacity is described by the sliding filament model, which states that the degree of myofilament overlap determines the amount of force that can be actively produced (25). Therefore, myofilament lengths are among the chief determinants of the shape of the length-tension curve (13, 19, 46, 56), which quantitatively describes a sarcomere’s force output as a function of myofilament overlap and establishes a sarcomere’s operating length range (9, 17, 18, 24, 56). Myofilaments are polymeric, but their lengths are strictly controlled during sarcomere assembly and maintenance and are highly uniform within a myofibril (38). Given that whole muscle contractile performance can be accurately predicted from sarcomere-level myofilament geometry (58), obtaining high-resolution measurements of myofilament lengths in human muscles to construct muscle-specific, length-tension rela-

FORCE GENERATION IN SKELETAL

Address for reprint requests and other correspondence: V. M. Fowler, Dept. of Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., CB163, La Jolla, CA 92037 (e-mail: [email protected]). http://www.ajpcell.org

tionships has profound clinical significance. For example, accurate length-tension properties are critical when setting muscle length during surgical tensioning procedures (32) and when assigning the intrinsic force-generating properties of the muscular components of musculoskeletal models (7, 8). Studies in animals have shown that, to the best of our knowledge, thick filaments have a constant length of ⬃1.65 ␮m across different skeletal muscles (19, 47, 55), although a slightly shorter thick-filament length of ⬃1.58 ␮m has also been recently reported (39). However, high-resolution immunofluorescence (IF) and electron microscopy (EM) studies in a host of vertebrate species have all shown that thin-filament lengths are highly variable across skeletal muscles, ranging from ⬃0.95 to ⬃1.30 ␮m, with slow-twitch muscles generally having longer thin filaments than fast-twitch muscles (6, 14, 19). These observations have suggested that slow-twitch muscles have longer optimal sarcomere lengths and wider sarcomere length operating ranges than fast-twitch muscles, which has been directly demonstrated by measurements of sarcomere length-tension relationships in perch muscles (19). Two key molecules involved in thin-filament length regulation are nebulin and tropomodulin (Tmod). Nebulin is a giant (⬃600 –900 kDa) elongated actin-binding protein with its COOH-terminus anchored in the Z-line and its NH2-terminal M1M2M3 domain oriented toward the thin-filament pointed end, which has been proposed to function as a molecular ruler to specify thin-filament lengths by providing a template along which thin filaments polymerize (29, 30, 36, 38, 42). In contrast, Tmod (⬃42 kDa) is an actin-capping protein that regulates actin monomer association/dissociation at the thinfilament pointed end, thereby specifying thin-filament lengths in striated muscles (20, 35, 40, 57). Recently, Littlefield and colleagues (6, 14) made the surprising discovery that nebulin does not extend all the way to the thin-filament pointed ends, resulting in a nebulin-free, pointed-end extension beyond nebulin’s NH2-terminal M1M2M3 domain. This and other studies have suggested that nebulin mechanically and chemically stabilizes an ⬃0.9- to 1.0-␮m-long core region of the thin filament, whereas Tmod regulates the length of a relatively more dynamic, nebulin-free, pointed-end extension (2, 6, 14, 35, 38, 48). The nebulin-stabilized thin-filament core and nebulin-free/Tmod-capped, pointed-end extension are concatenated to establish a bipartite actin filament whose length reflects total in vivo thin-filament length. To date, studies of thin-filament lengths in humans have been limited to an unspecified leg muscle (56) and extensor carpi radialis brevis (34) measured from thin-section EM and quadriceps measured from IF images of myofibrils (45, 46). EM is unsuitable for performing large numbers of thin-filament length measurements, because it is highly labor intensive, and the potent fixatives and embedding procedures involved can

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HUMAN THIN-FILAMENT LENGTH HETEROGENEITY

introduce shrinkage artifacts. Moreover, the spatial resolution of conventional IF with standard confocal microscopy is insufficient for achieving high-precision quantitation. To circumvent these obstacles, in this study, we used an IF-based superresolution computational fluorescence microscopy approach known as distributed deconvolution (DDecon) (37) to measure thin-filament lengths in human skeletal muscle biopsies. Under optimal imaging conditions, this method can measure thinfilament lengths in myofibrils with a precision as high as 10 –20 nm (35, 37). We identified thin-filament length heterogeneities of up to 0.18 ␮m that vary across biopsies of the same muscle from different patients and different muscles from the same patient, establishing that human thin-filament lengths are both patient and muscle specific. Thus, to maximize the accuracy of length-tension relationships of human muscles for clinical applications, thin-filament lengths must be measured on a case-by-case basis. Furthermore, consistent with previous observations (6, 14), thin-filament lengths were independent of the localization of the NH2-terminus of nebulin, extending past the NH2-terminus of nebulin from 0.28 to 0.47 ␮m. In contrast, thin-filament lengths were negatively correlated with type 2X myosin heavy chain (MHC) isoform levels and significantly shorter in type 2X MHC-positive fibers than in type 1 MHCpositive fibers in the same muscle. This provides direct evidence that human muscle fibers coordinate sarcomere length operating ranges (determined by thin-filament lengths) and fiber types (determined by MHC content). MATERIALS AND METHODS

Muscle biopsies. Deltoid and pectoralis major muscle biopsies (⬃1 cm3) were obtained incidentally from individuals undergoing total shoulder arthroplasty due to osteoarthritis at Scripps Green Hospital, La Jolla, CA. None of the patients had acute muscle injury or disease that would have compromised the integrity of the biopsy. Each muscle biopsy was taken from a single location within the muscle. This study was approved by the Scripps Clinic Institutional Review Board, and all patients provided written, informed consent before participating in this study. Antibodies. For immunostaining, primary antibodies were affinitypurified rabbit polyclonal anti-human Tmod1 [R1749bl3c, 3.1 ␮g/ml (14)], rabbit polyclonal antiserum to chicken Tmod4 preadsorbed by passage through a Tmod1 Sepharose column [R3577bl3c, 1:25 (14)], affinity-purified rabbit polyclonal anti-nebulin M1M2M3 domain (R1357L, 9.3 ␮g/ml; a generous gift from Carol C. Gregorio, University of Arizona, Tucson, AZ), mouse monoclonal anti-␣-actinin (EA53, 1:100; Sigma-Aldrich, St. Louis, MO), mouse monoclonal anti-type-1 MHC (BA-D5, 1:100; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), and mouse monoclonal anti-type 2X MHC (6H1, 1:100; Developmental Studies Hybridoma Bank). Secondary antibodies were Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200, Invitrogen, Carlsbad, CA) and Alexa Fluor 647-conjugated goat anti-mouse IgG (1:200, Invitrogen). For Western blotting, primary antibodies were rabbit polyclonal antiserum to residues 340 –359 of a human Tmod1 peptide (PA2211, 1:5,000; Open Biosystems, Huntsville, AL), rabbit polyclonal antiserum to chicken Tmod4 preadsorbed by passage through a Tmod1 Sepharose column (R3577bl3c, 1:2,500), and mouse monoclonal anti-GAPDH (1D4, 1:5,000; Novus Biologicals, Littleton, CO). Secondary antibodies were horseradish peroxidase-conjugated goat anti-rabbit IgG (1: 10,000, Invitrogen) and horseradish peroxidase-conjugated goat antimouse IgG (1:10,000, Invitrogen). IF and confocal imaging. Each muscle biopsy was divided into two to five fiber bundles (⬃3 mm ⫻ 3 mm ⫻ 1 cm), depending on the size

of the biopsy. Fiber bundles were dissected from biopsies, stretched, secured onto wooden dowels using 3– 0 silk sutures, and immersed in ice-cold buffer (100 mM KCl, 5 mM MgCl2, 5 mM EGTA, 10 mM imidazole, 5 mM ATP, pH 7.0), supplemented with protease inhibitor cocktail (1:1,000, Invitrogen). Adequately stretched sarcomeres were those with H-zones that were sufficiently wide to resolve Tmod doublets at the pointed ends of the thin filaments extending from neighboring I-Z-I arrays (in practice, sarcomere lengths of ⬎3.0 ␮m). Muscle fiber bundles were stretched for 24 h at 4°C, then fixed for 24 h at 4°C in 4% paraformaldehyde in the same buffer, cryoprotected in 15% sucrose followed by 30% sucrose in the same buffer, embedded in OCT medium, frozen on a metal block chilled in liquid N2, and sectioned into 12-␮m-thick cryosections. Sections were mounted on slides, washed in PBS ⫹ 0.1% Triton X-100 (PBST), permeabilized for 15 min in PBS ⫹ 0.3% Triton X-100, and blocked overnight at 4°C in 4% BSA ⫹ 1% goat serum in PBST, as previously described (14). Sections were labeled with primary antibodies diluted in blocking buffer overnight at 4°C, washed in PBST, and then labeled with a fluorophore-conjugated secondary antibody mixture in blocking buffer for 2 h at room temperature. The secondary antibody mixture was supplemented with rhodamine-phalloidin (1:100, Invitrogen) to stain F-actin. Tissues were then washed again in PBST, preserved in Gel/Mount aqueous mounting medium (Sigma-Aldrich), and coverslipped. Images of single optical sections were collected on a Bio-Rad Radiance 2100 laser-scanning confocal microscope mounted on a Nikon TE2000-U microscope using a ⫻100/1.4 numerical apertureoil objective lens (zoom 3). Image sampling was performed randomly throughout the entire longitudinal cryosection of the biopsy. Bio-Rad LaserSharp 2000 software was used for image collection. Images were processed with Adobe Photoshop (version CS4), and image figures were constructed in Adobe Illustrator (version CS4). DDecon. DDecon is a superresolution light microscopy technique that computes thin-filament lengths with a precision of 10 –20 nm by directly measuring the peak positions of fluorescent antibody-labeled Tmod1 or Tmod4 with respect to ␣-actinin at the Z-line, from line scans of fluorescence intensities along myofibrils (37). Both Tmod1 and Tmod4 cap the pointed ends of the thin filaments in mammalian skeletal muscle and are faithful markers for the thin-filament pointed (free) ends that are localized at the edges of the H-zone (1, 11, 14). We also used DDecon to measure the position of the NH2-terminal M1M2M3 domain of nebulin, which is located slightly proximal to the Z-line with respect to the Tmod at the pointed ends (6), and to measure the breadth of the F-actin (phalloidin) signal across the Z-lines of adjacent half-sarcomeres (I-Z-I arrays) (37). In this study, we used a DDecon plugin originally developed for ImageJ by Ryan S. Littlefield (University of Washington, Seattle, WA) (14) and modified by Rohan Anil (University of California-San Diego, La Jolla, CA). The DDecon plugin generates the best fit of a model intensity distribution function for a given thin-filament component (Tmod, nebulin M1M2M3 domain, F-actin) to an experimental one-dimensional myofibril fluorescence intensity profile (line scan) obtained for each fluorescent probe (anti-Tmod, phalloidin, anti-nebulin M1M2M3) (37). These probe-specific model distributions are applied to line scans of a repeating series of three to five thin-filament arrays along a given myofibril to calculate each probe’s average distance from the Z-line for an individual myofibril. Model fitting is optimized by an iterative fitting procedure that minimizes the error between the observed line scan intensities and the modeled intensities, using a multivariate line-fitting algorithm, as described previously (37). Image regions containing adequately stretched sarcomeres were identified visually based on the presence of Tmod or nebulin M1M2M3 doublets and clear gaps in the phalloidin signal (H-zones). Line scans were background corrected by subtracting the average of the intensities of the minima in the line scan, and then the fluorescence peaks associated with each I-Z-I array were identified computationally, using appropriate model distributions for Tmod, phalloidin, and nebulin M1M2M3 (37). Distances in micrometers were calculated by

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converting pixel sizes into micrometers based on the magnification factor for each image (25.6 pixels/␮m). For whole biopsy-wide average thin-filament length measurements, line scans were performed on over 100 individual myofibrils from each muscle biopsy and averaged to yield probe-specific distances from the Z-line (i.e., thin-filament lengths) for each muscle biopsy from each patient. In some experiments, cryosections were stained with antibodies against type 1 or type 2X MHC, along with Tmod4 and rhodamine-phalloidin, and thin-filament lengths were only measured in type 1 or type 2X MHC-positive fibers. Western blotting. Muscle tissues were snap-frozen in liquid N2, crushed on dry ice using a mortar and pestle, solubilized in an equal volume of 2⫻ SDS sample buffer, and boiled for 5 min. Proteins were separated via SDS-PAGE on 4 –20% Tris-glycine gradient mini-gels (Invitrogen) for 70 min at 200 V and transferred to nitrocellulose (pore size ⫽ 0.2 ␮m; Fisher Scientific, Pittsburgh, PA), as described previously (14). Blots were stained with 0.2% Ponceau S in 3% TCA to verify protein transfer, heated for 1 h at 65°C in PBS, blocked overnight in 4% BSA in PBS at 4°C, and then incubated in primary antibodies diluted in Blitz buffer (4% BSA, 10 mM NaHPO4, 150 mM NaCl, 1 mM EDTA, 0.2% Triton X-100, pH 7.4) for 4 h at room temperature (14). After washing in PBST, blots were incubated in secondary antibodies diluted in Blitz ⫹ 2% goat serum for 1–2 h at room temperature. After washing again in PBST, protein bands were detected using ECL (Fisher Scientific) and quantified in ImageJ. Only film exposures in the linear response range were quantified. Recombinant Tmod proteins used to demonstrate antibody specificity on Western blots were human Tmod1, rat Tmod2, mouse Tmod3, and

Table 1. Subject information Patient No.

Sex

Age, yr

1 2 3 4

M F F M

57 71 56 81

M, male; F, female.

mouse Tmod4, prepared as described previously (59). Ten nanograms of each Tmod were loaded on the gels. MHC isoform composition. Proteins were separated via SDSPAGE on 8% Tris-glycine mini-gels, adapted from methods described previously (26). SDS-PAGE was performed for 16 h at 140 V. The upper tank buffer was supplemented with 1 mM DTT, and the lower tank buffer was one-sixth the concentration of the upper tank buffer. After protein separation, gels were stained using a SilverQuest Silver Staining Kit (Invitrogen), and densitometry was performed in ImageJ to compute the distribution of types 1, 2A, and 2X MHCs. Statistics. Data are presented as means ⫾ SD, and statistical significance was defined as P ⬍ 0.05. Differences between two groups were detected using Student’s t-test. Differences between more than two groups (with a single independent variable) were detected using one-way ANOVA with post hoc Fishers paired least significant difference tests. Differences between more than two groups (with two independent variables) were detected using two-way ANOVA with post hoc Fishers paired least significant difference tests. Relationships between pairs of

Fig. 1. Confocal fluorescence microscopy of human skeletal muscle thin-filament components. Shown are examples of longitudinal cryosections of pectoralis major muscle from patient 1 that were immunostained for tropomodulin (Tmod) 1 (A), Tmod4 (B), or the nebulin M1M2M3 domain (C), phalloidin stained for F-actin, and immunostained for ␣-actinin to visualize Z-lines. White rectangles indicate myofibrils that are magnified in Fig. 2. Bars, 5 ␮m. AJP-Cell Physiol • doi:10.1152/ajpcell.00299.2011 • www.ajpcell.org

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Fig. 2. Line-scan analysis of fluorescence intensity of thin-filament components during distributed deconvolution (DDecon) analysis. Higher magnification views of the boxed myofibrils in Fig. 1 are shown. Longitudinal cryosections of pectoralis major muscle from patient 1 were immunostained for Tmod4 (A) or the nebulin M1M2M3 domain (B), phalloidin stained for F-actin, and immunostained for ␣-actinin to visualize Z-lines. Each probe’s corresponding fluorescence intensity profile used for DDecon analysis is shown below each myofibril. Note that the nebulin M1M2M3 domain is localized more proximally with respect to the Z-line compared with Tmod4. Z, Z-line; P, thin-filament pointed end. Bars, 1 ␮m.

continuous variables were analyzed using linear regression. Statistical analysis was performed in Microsoft Excel and StatPlus:mac. RESULTS

Human thin-filament lengths are heterogeneous across muscles and individuals. To ascertain thin-filament length variability in human skeletal muscles across muscles and individuals, we obtained deltoid and pectoralis major muscle biopsies from

four patients undergoing total shoulder arthroplasty (Table 1). Biopsies were immunostained with antibodies against ␣-actinin, a key structural component of the Z-line, and Tmod isoforms 1 and 4, which are the only Tmod isoforms that localize to the pointed ends of the thin filaments and define the periphery of the H-zone in chickens and mammals (1, 11, 14) (Figs. 1 and 2). Biopsies were also stained with fluorescent phalloidin to visualize F-actin, which comprises the thin-

Table 2. Average thin-filament lengths in human muscle biopsies determined by distributed deconvolution analysis of fluorescence images Tmod Muscle

Deltoid

Pectoralis major

Patient No.

Mean ⫾ SD, ␮m

Minimummaximum, ␮m

1 2 3 4 1 2 3 4

1.22 ⫾ 0.05a 1.19 ⫾ 0.08b 1.34 ⫾ 0.05c 1.19 ⫾ 0.07b 1.37 ⫾ 0.04d 1.24 ⫾ 0.05e 1.31 ⫾ 0.10f 1.28 ⫾ 0.05g

1.07–1.32 1.00–1.34 1.23–1.51 1.05–1.35 0.98–1.42 1.09–1.41 1.26–1.68 1.02–1.38

F-actin (phalloidin) n

Mean ⫾ SD, ␮m


136 141 149 131 131 132 146 117

1.17 ⫾ 0.04a 1.18 ⫾ 0.05a 1.27 ⫾ 0.07b 1.30 ⫾ 0.06c 1.22 ⫾ 0.03d 1.25 ⫾ 0.04b 1.26 ⫾ 0.03b 1.27 ⫾ 0.04b

1.07–1.25 1.07–1.31 1.05–1.40 1.11–1.40 1.12–1.30 1.12–1.35 1.19–1.33 1.15–1.36

Nebulin M1M2M3 n

Mean ⫾ SD, ␮m


n

151 141 112 167 185 132 120 124

0.87 ⫾ 0.07a 0.86 ⫾ 0.03a 0.90 ⫾ 0.06b 0.87 ⫾ 0.02a 0.90 ⫾ 0.04b 0.92 ⫾ 0.05b 1.02 ⫾ 0.07c 0.86 ⫾ 0.04a

0.70–0.99 0.78–0.92 0.76–1.12 0.80–0.91 0.78–0.99 0.82–1.05 0.71–1.11 0.73–0.98

157 148 155 142 126 137 164 177

n, No. of myofibrils. Tmod, tropomodulin. a,b,c,d,e,f,g| Different superscripted letters within a single column reflect significantly different values, as determined by two-way ANOVA with post hoc Fishers paired least significant difference tests. AJP-Cell Physiol • doi:10.1152/ajpcell.00299.2011 • www.ajpcell.org


filament I-Z-I arrays that extend from one Tmod stripe to an opposing Tmod stripe on the opposite side of an ␣-actininstained Z-line (Figs. 1 and 2) (37). Using DDecon to perform line-scan analysis and measure the breadth of the phalloidin signal and the positions of Tmod1 and Tmod4 with respect to the Z-line (14, 37), we found that thin-filament lengths were highly variable both across individuals and across different muscles within the same individual. Thin-filament lengths determined by Tmod1 vs. Tmod4 localization were statistically indistinguishable (data not shown), and, therefore, these data were pooled and collectively treated as “Tmod” values. Numerical values for thin-filament lengths ranged from 1.19 ⫾ 0.08 to 1.37 ⫾ 0.04 ␮m based on Tmod localization, and from 1.17 ⫾ 0.04 to 1.30 ⫾ 0.06 ␮m based on the breadth of F-actin (Table 2; Fig. 3). Interestingly, the two younger patients (patients 1 and 3) had longer thin-filament lengths than the two older patients (patients 2 and 4). Furthermore, as expected from the literature (36, 38), thin-filament lengths within individual myofibrils were highly uniform (coefficient of variation ⫽ 1.4%). Generally, lengths determined by Tmod localization were somewhat greater than those determined by F-actin breadth, consistent with previous DDecon measurements (6, 14), but some exceptions were observed (Table 2; Fig. 3). Unexpectedly, lengths determined by Tmod localization did not correlate with those determined by F-actin breadth (data not shown), suggesting the possible occurrence of actin polymerization and/or depolymerization after biopsy excision. To confirm that both Tmod1 and Tmod4 are indeed expressed in skeletal muscle and that our Tmod1 antibodies did not cross-react with Tmod4 (and vice versa), we performed Western blots on skeletal muscle lysates using antibodies raised against Tmod1 and Tmod4. Both deltoid and pectoralis major muscles coexpressed Tmod1 and Tmod4, and both anti-Tmod1 and anti-Tmod4 antibodies specifically recognized their respective Tmod isoforms with no cross-reaction with other Tmod isoforms (Fig. 4A). Normalization of Tmod1 and Tmod4 levels to a GAPDH loading control indicated that the relative levels of Tmod1 and Tmod4 were also highly variable

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across muscles and individuals (Fig. 4, B–E). We observed no obvious relationship between relative Tmod isoform levels and thin-filament lengths. Thin-filament pointed ends extend beyond the NH2-terminus of nebulin in human muscle. The giant protein nebulin coextends with actin along thin filaments, with its COOH-terminus anchored in the Z-line and its NH2-terminal M1M2M3 domain oriented toward the thin-filament pointed ends. Despite interaction between the nebulin M1M2M3 domain and Tmods in vitro (43), the nebulin M1M2M3 domain does not colocalize with Tmods in chicken, mouse, and rabbit muscles in vivo (6, 14). Specifically, the nebulin M1M2M3 domain localizes more proximal to the Z-line with respect to Tmods, with the M1M2M3 domain located at the same distance from the Z-line in different muscles, which leads to a nebulin-free, pointed-end extension whose length determines total in vivo thin-filament length (6, 14). To determine whether this phenomenon occurs in human muscle, we immunostained biopsies with antibodies against the nebulin M1M2M3 domain. As observed in chicken, mouse, and rabbit muscle (6, 14), the nebulin M1M2M3 domain localized proximal to the thin-filament pointed ends in human muscle, which can be observed in the merged fluorescence images of the nebulin M1M2M3 domain and F-actin (Figs. 1 and 2). By contrast, Tmod signals were located just distal to the edge of the F-actin at the H-zone at the position of the thin-filament pointed ends, consistent with previous studies in chickens, mice, rats, and rabbits (1, 6, 11, 14) (Figs. 1 and 2). DDecon measurements revealed that the localization of the nebulin M1M2M3 domain ranged from 0.86 ⫾ 0.03 to 1.02 ⫾ 0.07 ␮m from the Z-line, considerably less than the distance of Tmod from the Z-line, which ranged from 1.19 ⫾ 0.08 to 1.37 ⫾ 0.04 ␮m, as stated above (Table 2; Fig. 3). By subtracting the localization of the nebulin M1M2M3 domain from the total thin-filament length determined by Tmod localization, we calculated that nebulin-free, pointed-end extensions vary in length from 0.28 to 0.47 ␮m in human muscles and can occupy up to 34% of total in vivo thin-filament length. Furthermore, as expected from previous studies on rabbits and

Fig. 3. Thin-filament lengths as determined by Tmod localization and F-actin breadth and the position of the NH2-terminal M1M2M3 domain of nebulin. Data are shown for individual biopsies and as pooled data. Note the more proximal position of the nebulin M1M2M3 domain with respect to the Z-line compared with Tmod. Error bars reflect means ⫾ SD of myofibril counts shown in Table 2. Pat., patient.


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Fig. 4. Human skeletal muscles coexpress Tmod1 and Tmod4. A: Western blots of muscle tissue lysates probed with antibodies against Tmod1 and Tmod4. GAPDH was used for normalization. Note that native human Tmod1 (hTmod1) and Tmod4 migrate slightly lower and higher than purified recombinant hTmod1 and mouse Tmod4 (mTmod), respectively, which were used to verify the specificity of our Tmod isoform-specific antibodies. 10 ng of each Tmod1– 4 were loaded on gels. rTmod2, rat Tmod2. B–E: quantitation of Tmod1 (B and C) and Tmod4 (D and E) protein levels in the deltoid (B and D) and pectoralis major (C and E) muscles from each patient, with normalization to GAPDH. Error bars reflect means ⫾ SD of n ⫽ 3 replicates. a,b,c,d | Different letters above bars within a single graph reflect significantly different values, as determined by one-way ANOVA with post hoc Fishers paired least significant difference tests. a.u., Arbitrary units.

mice (6, 14), the localization of the nebulin M1M2M3 domain did not correlate with thin-filament length, as determined by Tmod localization (data not shown). Thin-filament length correlates with type 2X MHC protein levels in human muscle. Studies comparing thin-filament lengths across muscles of fish, mice, rabbits, and chickens have suggested that thin-filament length varies coordinately with fiber type; that is, fast-twitch muscles have shorter thin-filament lengths, whereas slow-twitch muscles have longer thinfilament lengths (6, 14, 19, 29, 37, 44, 49). To determine whether this relationship exists in human muscle, we separated skeletal muscle lysates using SDS-PAGE, measured MHC isoform distributions, and examined thin-filament length as a function of MHC isoform composition. The MHC isoform distributions of the deltoid and pectoralis major were similar, with type 1 (slow) MHC predominating in both muscle types, but the proportion of type 1 MHC was significantly greater in the pectoralis major (Fig. 5, A and B). When thin-filament length was plotted vs. the percentage of total MHC for each MHC isoform for each biopsy, we found that the proportion of

type 2X MHC was negatively correlated with thin-filament length (Fig. 5C). By contrast, no significant correlation was observed between either type 1 or 2A MHCs and thin-filament length (Fig. 5C). Therefore, human skeletal muscle appears to exhibit an explicit relationship between thin-filament lengths and type 2X fiber types. To directly test whether thin-filament lengths vary in a fiber-type-dependent manner in human muscle, we measured thin-filament lengths in type 1 or type 2X MHC-positive fibers that were identified by immunostaining with antibodies against type 1 or type 2X MHC, respectively. In both deltoid and pectoralis major muscle biopsies from patient 1, thin-filament lengths based on Tmod localization were 0.17 to 0.25 ␮m longer in type 1 MHC-positive fibers than in type 2X MHCpositive fibers (Table 3; Fig. 6). Similarly, thin-filament lengths based on F-actin breadth were 0.16 – 0.29 ␮m longer in type 1 MHC-positive fibers than in type 2X MHC-positive fibers (Table 3; Fig. 6). Interestingly, thin-filament lengths in type 1 MHC-positive fibers were more heterogeneous than in type 2X MHC-positive fibers (Table 3; Fig. 6), which


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Fig. 5. Correlation between thin-filament length and myosin heavy chain (MHC) isoform composition in human skeletal muscles. A: muscle tissue lysates were separated on 8% SDS-PAGE gels and silver-stained. A sample separation of pectoralis major muscle from patient 1 is shown, and the positions of type 1, 2A, and 2X MHC are labeled. B: MHC isoform distributions. Error bars reflect means ⫾ SD of n ⫽ 4 muscles. *P ⬍ 0.05, as determined by Student’s t-test. C: correlation between thin-filament length (based on Tmod localization) and the percentages of type 1, 2A, and 2X MHC. Note that the proportion of type 2X MHC (R2 ⫽ 0.56; P ⬍ 0.01), but not type 1 (R2 ⫽ 0.14; P ⬎ 0.5) or type 2A (R2 ⫽ 0.07; P ⬎ 0.5) MHC is significantly correlated with thin-filament length.

suggests that fiber type is a stronger predictor of thin-filament length in type 2X MHC-positive (fast) fibers than in type 1 MHC-positive (slow) fibers. Note that the average biopsy-wide thin-filament length (Table 2; Fig. 3) was not a simple weighted average of the thin-filament lengths of the type 1 and type 2X MHC-positive fibers within the muscle (Table 3; Fig. 6), as thin-filament lengths in type 2A MHC-positive were not considered. Furthermore, some mixed fibers may coexpress multiple MHC isoforms. Together, these data demonstrate that thin-filament lengths are fiber type-specific within a human skeletal muscle biopsy. These data also confirm that faster fiber types are associated with shorter thin-filament lengths in human muscle. DISCUSSION

Biomechanical implications of thin-filament length heterogeneity. This study is the first to demonstrate that human skeletal muscle thin-filament lengths can vary across specific muscles and within individuals. In our series of muscle biopsies, thin-filament lengths based on Tmod localization ranged from 1.19 to 1.37 ␮m, a spread of nearly 0.2 ␮m (Table 2; Fig. 7). By assuming a constant thick-filament length of 1.65 ␮m and bare zone width of 0.2 ␮m (18, 19, 56), we constructed hypothetical sarcomere length-tension curves based on our measured thin-filament lengths (Fig. 7). Optimum sarcomere length starts at twice thin-filament length and ends at twice thin-filament length plus

bare zone width, and maximum sarcomere length for force production is twice thin-filament length plus thick-filament length (9, 17, 18, 24, 56). Hence, thin-filament lengths of 1.19 –1.37 ␮m correspond to optimum sarcomere lengths of 2.38 –2.58 to 2.74 –2.94 ␮m, respectively, and maximum sarcomere lengths for force production of 4.03 and 4.39 ␮m, respectively (Fig. 7). These predictions are analogous to those that were experimentally verified in fast- and slow-perch muscles with 0.94- vs. 1.24-␮m-long thin filaments, respectively (19), and in the narrower and left-shifted sarcomere lengthtension curves measured in muscles from nebulin-null mice and nemaline myopathy patients with shorter thin filaments (13, 46). To experimentally verify our calculations, future physiological experiments will necessitate that isometric length-tension curves be collected in conjunction with thinfilament length measurements on human muscles. Our data highlight the importance of measuring length-tension properties in the same biopsy from which the thin-filament length measurements are collected. Namely, the thin-filament length heterogeneity that we observe across human muscles argues that the length-tension properties of a muscle biopsy from one individual cannot be accurately predicted from the myofilament lengths of a matched muscle biopsy from another individual. The relatively constant position of the NH2-terminal M1M2M3 domain of nebulin with respect to the Z-line in

Table 3. Thin-filament lengths in type 1 and type 2X MHC-positive fibers in patient 1 Tmod Muscle

Deltoid Pectoralis major

MHC Antibody Reactivity

Type Type Type Type

1 (slow) 2X (fast) 1 (slow) 2X (fast)

F-actin (phalloidin)

Mean ⫾ SD, ␮m


n

Mean ⫾ SD, ␮m


n

1.27 ⫾ 0.07 1.10 ⫾ 0.09 1.40 ⫾ 0.10 1.15 ⫾ 0.08

1.07–1.38 0.91–1.33 1.14–1.50 1.01–1.27

101 87 94 80

1.21 ⫾ 0.07 1.05 ⫾ 0.11 1.35 ⫾ 0.08 1.06 ⫾ 0.09

1.06–1.37 0.86–1.29 1.22–1.47 0.93–1.37

77 83 50 61

n, No. of myofibrils. MHC, myosin heavy chain. P ⬍ 0.001 for all thin-filament length comparisons (type 1 vs. type 2X MHC), as determined by Student’s t-tests. AJP-Cell Physiol • doi:10.1152/ajpcell.00299.2011 • www.ajpcell.org

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Fig. 6. Average thin-filament lengths in type 1 and type 2X MHC-positive fibers in deltoid and pectoralis major muscles in patient 1, as determined by Tmod localization and F-actin breadth. Note longer thin filaments in type 1 MHC-positive fibers and shorter thin filaments in type 2X MHC-positive fibers. Error bars reflect means ⫾ SD of myofibril counts shown in Table 3.

human muscle argues that human thin-filament length heterogeneity specifically arises from variations in the lengths of the nebulin-free, Tmod-capped pointed-end extensions of the thin filaments and not from variations in the lengths of the nebulinstabilized cores of the thin filaments (Fig. 7). This is consistent with observations in chickens, mice, and rabbits that also identified a constant position of the nebulin M1M2M3 domain ⬃0.9 –1.0 ␮m from the Z-line, but a variable position of Tmod, distal to the nebulin M1M2M3 domain and located ⬃0.3 ␮m further from the Z-line (6, 14). Thus thin filaments are nonuniform, bipartite structures across vertebrate species, and the actin dynamics of the thin-filament pointed-end extensions are expected to ultimately dictate thin-filament lengths and, in turn, sarcomere length-tension relationships. Furthermore, the inability of human nebulin to span the entire thin filament indicates that human nebulin, like chicken, mouse, and rabbit nebulin (6, 14), cannot function as a molecular ruler in vivo (38). The discovery of substantial thin-filament length heterogeneity complicates the field of musculoskeletal modeling for studying stresses imposed on the musculoskeletal system, predicting movement in the contexts of sports medicine and rehabilitation, and optimizing surgical tendon transfers. In musculoskeletal models, the length-tension properties assigned to muscles have been based on constant myofilament lengths measured from limited numbers of frog and/or human muscles (7, 8, 17, 18, 34, 47). However, the existence of thin-filament

length heterogeneity across individuals and muscles casts doubt on the accuracy of models that assume constant thinfilament lengths. For example, a sarcomere length on the descending limb of a length-tension curve constructed from short thin filaments may correspond to the ascending limb of a length-tension curve constructed from long thin filaments (9, 17, 18, 24, 56). While substantial effort has been invested to comprehensively catalogue the widely variable extrinsic architectural characteristics of human muscles, such as their fiber lengths and physiological cross-sectional areas (33), no equivalent attempt has been made to catalogue the intrinsic myofilament geometries of human muscles. Our data emphasize the need to improve the accuracy of future musculoskeletal models by incorporating both extrinsic and intrinsic muscle characteristics. Considerations for measurement precision and accuracy. Previous DDecon measurements have consistently found that thin-filament lengths based on Tmod localization correlate well with those based on F-actin breadth, but that Tmod lengths are consistently slightly longer (6, 14, 35, 37). This is because the difference between the Gaussian distribution and the point spread of light under the confocal microscope may underestimate the F-actin breadth across the I-Z-I arrays (37). However, in the pectoralis major of patient 2 and the deltoid of patient 4, we made the surprising and counterintuitive observation that lengths based on Tmod localization were actually less than those based on F-actin breadth (Table 2). Given this discrepancy with previous studies, several factors have led us to conclude that lengths based on Tmod localization are more accurate than lengths based on F-actin breadth in human muscle biopsies. The first factor is the methodological observation that lengths based on Tmod localization are less sensitive to background fluorescence subtraction than lengths based on F-actin breadth. For example, excessive background subtraction in an F-actin line scan leads to an underestimate of thin-filament length, which can be explained by “cutoff” of the signal at the base of the F-actin fluorescence intensity profile (Fig. 2). Conversely, inadequate background subtraction results in an overestimate of F-actin breadths and corresponding thin-filament lengths. In contrast, the position of the peak of the Tmod fluorescence intensity profile with respect to the Z-line is mostly unaffected by background subtraction in specimens with high-quality IF staining and, thus, provides a higher precision measurement (Fig. 2). The second factor arguing for the superiority of length measurements based on Tmod localization is biological. Namely, Tmod is a dynamic cap, and the injury imposed on the muscle tissue by the biopsy excision procedure may lead to calpain-mediated proteolysis of myofibrillar components (15, 16, 31), including Tmod, followed by Tmod dissociation from the thin-filament pointed ends. This can potentially lead to actin monomer addition and artifactually increased thin-filament lengths. Indeed, numerous studies in cultured cardiomyocytes have shown that Tmod levels are inversely proportional to thin-filament lengths (20, 35, 38). Furthermore, the existence of nebulin-free pointed-end extensions in human muscle, as shown here, indicates that the distal segments of human thin filaments are likely less chemically and mechanically stable and more dynamic than their proximal segments near the Z-line (2, 13, 48). Therefore, a subset of phalloidin-stained F-actin filaments in our biopsies may be variably uncapped and subject



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Fig. 7. Hypothetical sarcomere length-tension curves of human skeletal muscles. A: model diagrams of sarcomeres with the minimum and maximum biopsy-wide thin-filament lengths measured in this study (1.19 and 1.37 ␮m, respectively) depict constantlength, nebulin-coated, thin-filament cores and variable-length, nebulin-free, pointed-end extensions that are capped by Tmod. Note that, for clarity, the sarcomere lengths are equal. However, at resting sarcomere lengths in vivo, the thin-filament pointed ends coincide with the thick-filament bare zone (38). Green, actin; red, Tmod; purple, nebulin; yellow, nebulin M1M2M3 domain; black, Z-line; gray, thick filament. B: length-tension curves were constructed based on thin-filament lengths of 1.19 and 1.37 ␮m, a constant thick-filament length of 1.65 ␮m, and a bare zone width of 0.2 ␮m (9, 17–19, 24, 56).

to artifactual shortening or elongation, and, hence, their lengths may not reflect actual in vivo thin-filament lengths. Using Tmod as a thin-filament pointed end marker enables us to avoid this potentially confounding effect, because we only measure lengths of intact thin filaments capped by Tmod. Thus we recommend that future IF-based thin-filament length measurements use Tmod as the “gold standard” probe. Coordination of thin-filament lengths and fiber types. Previous studies in animals have suggested that thin-filament lengths and fiber types vary coordinately, with fast-twitch muscles having shorter thin filaments, and slow-twitch muscles having longer thin filaments (6, 14, 19, 29, 37, 44, 49). This study is the first to directly test this hypothesis in human tissue. Our findings that thin-filament length is negatively correlated with the proportion of type 2X MHC in a biopsy (Fig. 5) and that type 2X MHC-positive fibers have shorter thin filaments than type 1 fibers (Table 3 and Fig. 6) support the idea that thin-filament lengths are coordinated with fiber types, as increasing levels of type 2X MHC are associated with a faster fiber phenotype in mammalian muscle (51, 52, 54). Furthermore, as shorter thin filaments result in narrower length-tension curves and, hence, narrower sarcomere length operating ranges (13, 19, 46), our data predict that faster twitch muscles have correspondingly narrower sarcomere length-operating ranges. This experimental prediction has been validated via length-tension analysis of perch muscles (19), but additional experiments will be required to determine whether this prediction can be extended to mammalian muscles and to substantiate whether type 1 or type 2A

MHC levels correlate with human thin-filament length over a more diverse assortment of muscle fiber types and with increased sample sizes. The molecular mechanism linking fiber types (MHC isoforms) and thin-filament lengths (actin dynamics) remains elusive. Despite evidence that de novo thin- and thick-filament assembly occur independently (10, 12, 23, 28, 41, 50), thinfilament lengths in mature skeletal myofibrils appear to correlate with the molecular weight of the giant protein titin (6), which links thick filaments to Z-lines by spanning across the half-sarcomere, serving as both the principal scaffold on which thick filaments assemble and the main determinant of resting sarcomere length (27). Thus cross talk between thin and thick filaments appears to be important for coordinating sarcomere structure and thin-filament lengths in mature myofibrils (36). Our data suggest that MHC isoforms may play a role in this process. For example, it is conceivable that the particular MHC isoforms expressed in muscle fibers preferentially enhance or inhibit actin dynamics at the thin-filament pointed ends via their specific actomyosin cross-bridge activities (36, 38). Such a model would be consistent with previous observations that actomyosin contractility regulates actin dynamics in cardiomyocytes (53), and that myosin can directly affect actin polymerization (5, 22). Alternatively, expression of fiber-type-specific tropomyosin isoforms (i.e., slow vs. fast tropomyosins) and their differential stabilization of actin filaments may explain the coordination of fiber types and thin-filament lengths (3, 4, 21). Additional studies are required to dissect the mo-


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lecular mechanism underlying the relationship between fiber types and thin-filament lengths. ACKNOWLEDGMENTS We gratefully acknowledge Rohan Anil for updating, streamlining, and debugging the DDecon software, James Boyd for coordinating biopsy tissue transfer, and Roberta B. Nowak and Ciara Kamahele-Sanfratello for helpful discussions and technical assistance. We also thank Richard L. Lieber for providing initial encouragement and continuing enthusiasm for this study. GRANTS This work was supported by National Institutes of Health (NIH)/National Heart, Lung, and Blood Institute vascular biology training grant T32HL007195 (to D. S. Gokhin), NIH/National Center for Research Resources pilot award UL1-RR025774 from the Scripps Translational Science Institute (to V. M. Fowler), and NIH/National Institute of Child Health and Human Development/National Institute of Neurological Disorders and Stroke pilot award R24-HD050837 from the National Skeletal Muscle Research Center at the University of California-San Diego (to V. M. Fowler). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: D.S.G., D.D.D., and V.M.F. conceived and designed the research; D.S.G., N.E.K., S.A.L., and H.R.H. performed experiments; D.S.G., N.E.K., and S.A.L. analyzed data; D.S.G. interpreted results of experiments; D.S.G. prepared figures; D.S.G. drafted manuscript; D.S.G. and V.M.F. edited and revised manuscript; D.S.G., N.E.K., S.A.L., H.R.H., D.D.D., and V.M.F. approved final version of manuscript. REFERENCES 1. Almenar-Queralt A, Lee A, Conley CA, Ribas de Pouplana L, Fowler VM. Identification of a novel tropomodulin isoform, skeletal tropomodulin, that caps actin filament pointed ends in fast skeletal muscle. J Biol Chem 274: 28466 –28475, 1999. 2. Bang ML, Li X, Littlefield R, Bremner S, Thor A, Knowlton KU, Lieber RL, Chen J. Nebulin-deficient mice exhibit shorter thin filament lengths and reduced contractile function in skeletal muscle. J Cell Biol 173: 905–916, 2006. 3. Broschat KO. Tropomyosin prevents depolymerization of actin filaments from the pointed end. J Biol Chem 265: 21323–21329, 1990. 4. Broschat KO, Weber A, Burgess DR. Tropomyosin stabilizes the pointed end of actin filaments by slowing depolymerization. Biochemistry 28: 8501–8506, 1989. 5. Carlier MF, Valentin-Ranc C, Combeau C, Fievez S, Pantoloni D. Actin polymerization: regulation by divalent metal ion and nucleotide binding, ATP hydrolysis and binding of myosin. Adv Exp Med Biol 358: 71–81, 1994. 6. Castillo A, Nowak R, Littlefield KP, Fowler VM, Littlefield RS. A nebulin ruler does not dictate thin filament lengths. Biophys J 96: 1856 – 1865, 2009. 7. Delp SL, Anderson FC, Arnold AS, Loan P, Habib A, John CT, Guendelman E, Thelen DG. OpenSim: open-source software to create and analyze dynamic simulations of movement. IEEE Trans Biomed Eng 54: 1940 –1950, 2007. 8. Delp SL, Loan JP, Hoy MG, Zajac FE, Topp EL, Rosen JM. An interactive graphics-based model of the lower extremity to study orthopaedic surgical procedures. IEEE Trans Biomed Eng 37: 757–767, 1990. 9. Edman KA. The relation between sarcomere length and active tension in isolated semitendinosus fibres of the frog. J Physiol 183: 407–417, 1966. 10. Epstein HF, Fischman DA. Molecular analysis of protein assembly in muscle development. Science 251: 1039 –1044, 1991. 11. Fowler VM, Sussmann MA, Miller PG, Flucher BE, Daniels MP. Tropomodulin is associated with the free (pointed) ends of the thin filaments in rat skeletal muscle. J Cell Biol 120: 411–420, 1993. 12. Fritz-Six KL, Cox PR, Fischer RS, Xu B, Gregorio CC, Zoghbi HY, Fowler VM. Aberrant myofibril assembly in tropomodulin1 null mice leads to aborted heart development and embryonic lethality. J Cell Biol 163: 1033–1044, 2003.

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