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Am J Physiol Cell Physiol 282: C835–C844, 2002. First published December 5, 2001; 10.1152/ajpcell.00482.2001.

Influence of myosin isoforms on contractile properties of intact muscle fibers from Rana pipiens GORDON J. LUTZ, SHASHANK R. SIRSI, SARAH A. SHAPARD-PALMER, SHANNON N. BREMNER, AND RICHARD L. LIEBER Biomedical Sciences Graduate Group, Departments of Orthopaedics and Bioengineering, University of California, Veterans Affairs Medical Center, and Veterans Medical Research Foundation, San Diego, California 92161 Received 10 October 2001; accepted in final form 14 November 2001

Lutz, Gordon J., Shashank R. Sirsi, Sarah A. Shapard-Palmer, Shannon N. Bremner, and Richard L. Lieber. Influence of myosin isoforms on contractile properties of intact muscle fibers from Rana pipiens. Am J Physiol Cell Physiol 282: C835–C844, 2002. First published December 5, 2001; 10.1152/ajpcell.00482.2001.—The myosin heavy chain (MHC) and myosin light chain (MLC) isoforms in skeletal muscle of Rana pipiens have been well characterized. We measured the force-velocity (F-V) properties of single intact fast-twitch fibers from R. pipiens that contained MHC types 1 or 2 (MHC1 or MHC2) or coexpressed MHC1 and MHC2 isoforms. Velocities were measured between two surface markers that spanned most of the fiber length. MHC and MLC isoform content was quantified after mechanics analysis by SDS-PAGE. Maximal shortening velocity (Vmax) and velocity at half-maximal tension (VP 50) increased with percentage of MHC1 (%MHC1). Maximal specific tension (Po/CSA, where Po is isometric tension and CSA is fiber cross-sectional area) and maximal mechanical power (Wmax) also increased with %MHC1. MHC concentration was not significantly correlated with %MHC1, indicating that the influence of %MHC1 on Po/CSA and Wmax was due to intrinsic differences between MHC isoforms and not to concentration. The MLC3-to-MLC1 ratio was not significantly correlated with Vmax, VP 50, Po/CSA, or Wmax. These data demonstrate the powerful relationship between MHC isoforms and F-V properties of the two most common R. pipiens fiber types.

population of muscle cells, with a wide range of contractile properties, is a hallmark of vertebrate skeletal muscle. The differences in force-velocity (F-V) properties among muscle fiber types permit muscles to perform a wide range of mechanical tasks. Functional differences among fiber types have been correlated with myosin heavy chain (MHC) and myosin light chain (MLC) isoforms. Thus differential expression of MHC and MLC isoforms is an important determinant of functional diversity in mus-

cular systems and is matched to the specific motor requirements of an organism. The greatest emphasis in single-fiber mechanical studies has been on determining the influence of MHC and MLC isoforms on maximal shortening velocity (Vmax). Mechanical studies of single skinned fibers (sarcolemma permeabilized or removed) have demonstrated a correlation between MHC isoforms and Vmax (2, 3, 16, 24, 39, 40, 44; for reviews see Refs. 5 and 42). With the use of the in vitro motility assay, where the potential influences of thin-filament regulatory proteins and muscle activation are avoided, the correlation between speed and MHC isoforms has been corroborated (6, 18). The influence of MLC isoforms on Vmax is more controversial and may be variable among vertebrates. Several studies have established a significant effect of the molar ratio of essential (alkali) light chains [MLC3to-MLC1 ratio (MLC3/MLC1)] on Vmax (2, 14, 25, 37, 44; for reviews see Refs. 5, 36, and 42). Independent support for the role of MLC3/MLC1 in regulating myosin kinetics has been documented in a genetic model of fish (7) and with the in vitro motility assay (26). However, in human skinned fast-twitch fibers, MLC3/ MLC1 had no influence on Vmax (23, 24, 46). In any case, the influence of MLC3/MLC1 appears to be restricted to Vmax, inasmuch as there has been no demonstration of an effect at higher relative forces or on maximum specific tension. The influence of MHC isoforms on the remainder of the F-V relation (i.e., velocities below Vmax) and on maximum specific tension remains controversial. Studies on a variety of species representing several vertebrate classes have documented a correlation between MHC isoform and maximum specific tension. However, other studies have reported no clear relationship (for reviews see Refs. 5, 36, and 42). A complicating factor in these studies is that differences in myosin density may exist between fiber types, and this may obscure the effect of MHC isoforms on maximum specific tension (12, 45). A limitation to the aforementioned contractile studies is that the experiments have been performed al-

Address for reprint requests and other correspondence: G. J. Lutz, Dept. of Orthopaedics, University of California, San Diego, School of Medicine, Veterans Affairs Medical Centers (9151), 3350 La Jolla Village Dr., San Diego, CA 92161 (E-mail: [email protected]).

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.

mechanics; maximal shortening velocity; fiber type; maximal power; specific tension

EXPRESSION OF A HETEROGENEOUS

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MYOSIN ISOFORM PROPERTIES

most exclusively on skinned muscle fibers. In contrast, relatively few studies have assessed the influence of MHC or MLC isoforms on F-V properties of intact single fibers. Although amphibian muscle has long been favored for intact single-fiber contractile studies, its use in studies of myosin isoform properties has been limited by a lack of clear definition of MHC and MLC isoforms. By far, the most comprehensive studies of the influence of myosin isoforms on F-V properties of intact amphibian fibers is the detailed work on Xenopus laevis by Lannergren and coworkers (19–21). They demonstrated a nearly 10-fold range in Vmax across the five major fiber types in the following order: type 1 ⬎ type 2 ⬎ type 3 ⬎ type 4 ⬎ type 5. Fiber types were classified primarily by their nondenatured myosin banding pattern on native-polyacrylamide gels, along with very limited analysis of MHC and MLC isoforms on SDS-polyacrylamide gels (19). The five major fiber types identified in these experiments are likely to be comprised of as many as five different MHC isoforms, although the specific relationship between fiber type and MHC isoform remains unresolved. Thus, although direct evidence is limited, differences in MHC isoforms appear to be correlated with the differences in Vmax in X. laevis. The influence of the ratio of essential MLCs (MLC3/MLC1) on F-V properties was less clear, because only the velocity at half-maximal tension, rather than Vmax, was considered. Significant differences in maximal specific tension and maximal mechanical power (Wmax) were also reported in the following order: type 1 ⬎ type 2 ⬎ type 3 ⬎ type 4 ⬎ type 5 (19). These data indicate that MHC isoforms have a significant influence on maximal isometric tension and Wmax. We recently performed a detailed analysis of MHC isoforms in the full range of fiber types of Rana pipiens at the protein and mRNA levels (29, 31; for review see Ref. 30). We showed that four major fiber types (type 1, type 2, type 3, and tonic) distinguished by myofibrillar ATPase reactivity and immunohistochemistry contained four unique MHC isoforms. Single-fiber analysis showed that MHC1, MHC2, and MHC3 migrated as separate bands on SDS-polyacrylamide gels, while the tonic type comigrated with MHC1. The MLC isoforms in these fiber types were also characterized, and their migration pattern on SDS-polyacrylamide gels was established (27). These definitions of MHC and MLC isoforms provide a foundation for the determination of their influence on contractile function in intact single fibers. In this report, we measured the relationship between MHC and MLC isoform content and F-V properties of intact R. pipiens single fibers. Analysis was restricted to fibers that contained type 1 or 2 or coexpressed type 1 and 2 MHCs, inasmuch as these are the predominant MHCs expressed in the hindlimb musculature of R. pipiens (28). A spot-follower device was used to measure the precise F-V relation of the central 78% of single fibers, and myosin isoforms were determined after mechanics analysis by quantitative SDS-PAGE. We found that differences in MHC isoforms were correlated with differences in F-V properties across the AJP-Cell Physiol • VOL

full extent of the F-V relation, from maximal isometric specific tension (Po/CSA, where Po is isometric tension and CSA is cross-sectional area) to Vmax. There was no significant independent influence of MLC3/MLC1 on the F-V relation. Increased percentage of MHC1 (%MHC1) resulted in higher shortening velocity and specific tension, thus yielding a significant influence on Wmax production. These differences in contractile properties at the single-fiber level are discussed in the context of the functional importance of MHC isoform expression on muscle performance during jumping. METHODS

Animals. Adult male R. pipiens frogs (Charles Sullivan, Nashville, TN) were housed at room temperature (⬃20°C) in 20-gallon aquaria containing dry, dark surfaces and circulating filtered water. Live crickets were provided twice weekly, and the frogs were kept for up to 2 mo before experimentation. Single-fiber dissection. Frogs were killed by double pithing, and both anterior tibialis (AT) muscles were removed and pinned in Sylgard-lined glass dishes containing Ringer solution. The Ringer solution contained (in mM) 120.0 NaCl, 3.0 KCl, 4.0 NaPO4, 3.0 MgSO4, and 4.0 CaCl2, at pH 7.1. Single-fiber dissections were carried out at room temperature under a stereomicroscope (model MZ12.5, Leica, Deerfield, IL) equipped with a transmitted light base for dark-field imaging. The medial portion of the AT toward its origin was thinned to a shallow layer of cells that ran between the central and medial aponeuroses. This portion of the AT was chosen, because it contained predominantly type 1, type 1-2, and type 2 fibers and because the tendons were relatively robust. For single-fiber isolation, small bundles (2–4 fibers) were taken from the thinned muscle and transferred to a shallow glass-bottomed dish. Individual intact fibers were isolated and cleaned of debris using microscissors, fine forceps, and 30-gauge needles. Fibers were tested periodically throughout the dissection with point stimulation. Fibers were considered viable for mechanical analysis if they responded to point stimulations along their full length. Small holes were punched in both tendons for mounting in the mechanics chamber. Single-fiber mechanical recording system. Single fibers were placed in a Ringer solution-filled anodized aluminum chamber with a glass bottom insert. The chamber was mounted on a translation stage under a stereomicroscope (model MZ8, Leica). Ringer solution was constantly refreshed with a miniperistaltic pump (Harvard Apparatus, Holliston, MA). Fibers were mounted between a force transducer (model 405A, Aurora Scientific, Ontario, Canada) and a highspeed length controller (model 318B, Aurora Scientific) by securing the tendons to fine titanium wires (127 ␮m; Aldrich, Milwaukee, WI) protruding from the force transducer and motor arm using 9-0 black monofilament nylon (Ashaway Line and Twine, Ashaway, NJ). The force transducer and motor were mounted independently on xyz-translation stages to permit precise fiber alignment. Chamber temperature was regulated by direct contact between the chamber plate and a flow-through aluminum bar with temperature-controlled circulating water (model RTE101, Thermo Neslab, Portsmouth, NH). The microscope was equipped with a charge-coupled device videocamera (Techni-Quip, Livermore, CA) mounted in the phototube. The video system included a videocassette recorder, video micrometer (Techni-Quip), monitor (Sony), and video capture card (model RS170, Scion, Frederick MD).

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Fibers were activated by direct bipolar electrical stimulation through a pair of platinum plate electrodes that straddled the fiber along its full length. Stimulus pulses were generated with a bipolar stimulator (Pulsar 6bp, FHC, Bedowin, NJ) and amplified with a power amplifier (model 6826A, Hewlett-Packard, Palo Alto, CA). To improve the accuracy of the mechanical measurements, a spot-follower system was used to measure length changes within a defined region of the fiber that constituted 78% of the overall fiber length. Two surface markers consisting of black cat hair (⬃200 ⫻ 50 ␮m) were adhered to the upper surface of the fiber within ⬃0.75 mm of each myotendonous junction. The markers were coated with silicon grease to promote adherence to the fiber and were oriented perpendicular to the long axis of the fiber. Light was directed onto the upper surface of the fiber, and the position of the markers was detected with a 2,048-element linear photodiode array (PDA; model S2048, Reticon) oriented parallel to the long fiber axis in the phototube of the microscope. The position of each marker was detected as a sharp decrease in light intensity from the video output signal of the PDA. Marker positions were monitored continuously during contractions and converted to fractional length change (see below). To improve signal-to-noise ratio, a removable white background was inserted below the fiber. On the basis of the dimensions of the markers and their deviation from perpendicular, we calculated systematic error in fractional change in distance between the markers during F-V measurements to be ⬍2%. We also attempted to measure the length transients in smaller segments along the fiber length by placing additional markers between the end-most markers. Typically, four additional markers were applied at ⬃1-mm increments. Unfortunately, in most cases, we could not apply a full set of markers that were each perfectly perpendicular to the fiber axis. We determined that, during contractions, slight angulation of markers, coupled with lateral fiber movement, could result in relatively large systematic errors in our measurement of the change in distance between successive markers and, hence, in determination of segment shortening velocity. Therefore, all F-V measurements were based on the velocity between the end-most markers (see F-V measurements). However, the remaining internal markers served as useful landmarks for dividing the fiber into segments for morphological and sarcomere length (SL) measurements before the mechanics experiments and for measurements of myosin content after the mechanics experiments (see below). Computer control and software. Data were acquired and mechanical experiments were controlled with a four-channel, 5-MHz analog-to-digital/digital-to-analog board (model PCI 6110E, National Instruments, Austin, TX) interfaced to a Pentium personal computer using LabVIEW 5.11 and NIDAQ 6.7 software (National Instruments). Output from the surface marker PDA was acquired at 4 MHz, yielding a complete scan every 0.52 ms. Output signals from the force transducer, motor, and stimulator were acquired on the remaining input channels. Custom LabVIEW software was used to trigger the stimulator and control output to the motor. Resting SL, fiber length, and diameter measurements. After the fiber was mounted and aligned in the chamber, surface markers were applied as described above. Passive SL was measured with laser diffraction in each of the ⬃1-mm segments. A helium-neon laser beam (Melles Griot, Irvine, CA) was directed through the fiber from below, and the diffraction lines were imaged directly onto a PDA (model S2048, Reticon) placed 30 mm above the fiber. The PDA was mounted so that it could be rotated away from the fiber when AJP-Cell Physiol • VOL

not in use. SL was calculated from the distance between the two first-order diffraction lines using the standard diffraction equation. Fiber length was adjusted to yield an average resting SL (SLo) of ⬃2.35 ␮m. Because the laser beam had a diameter of ⬃0.8 mm, SLo provided an accurate estimate of the average SL within the region between the end-most markers. The length of the entire fiber (Lf) was measured using the video system. Only a rough estimate of the exact insertion points of the fiber on each tendon was possible, because, in many cases, fibers curved slightly around the connective tissue at the insertion, and the view of the insertion was obstructed by its tendon. Average diameter of each segment was measured in two orthogonal views (a 45° mirror was used for the side view) from digitized video images. The entire length of each segment was imaged, and diameters were measured along the full length of the segments with cursors, and CSA was calculated from the two diameters with the assumption of an elliptical shape. The position of each marker relative to the fiber ends was measured using the video system. F-V measurements. All experiments were performed at 25°C. Fibers were activated with supramaximal 0.3-ms voltage pulses (25% above twitch threshold) with frequency of 200 Hz. A series of isovelocity contractions were performed to obtain F-V curves, with contractions separated by 4 min. Examples of typical contractions are shown in Fig. 1. For each contraction, the fiber was stimulated and held isometric at Lf for 100 ms, while tetanic force reached a plateau (Po). The fiber was then given a high-velocity [20 fiber lengths/s (L/s)] small release step to unload the series elastic component, which was followed immediately by a constant-velocity shortening ramp. The ramp amplitude was 12–15% of Lf. The amplitude of the rapid release step was adjusted in each contraction to cause force to drop to a level that could be maintained during the ensuing ramp. Force typically stabilized within 5–10 ms after the step. Contractions were performed in increments of 1.0 L/s over most of the velocity range and increments of 0.5 L/s near the highest and lowest velocities. Fractional length change was calculated as the change in distance between the peaks corresponding to the end-most surface markers on the PDA (units of pixels) divided by the pixel distance under static conditions with the fiber held at Lf. Shortening velocity was taken as fractional length change divided by time, normalized to an SL of 2.35 ␮m (in L/s). Force and length records were read over the shortening period, beginning ⬃5–10 ms after the high-velocity step (when force had stabilized), for 5–20 ms (depending on ramp velocity; Fig. 1). When ramp velocity was high, the records were read for a shorter duration. This corresponded to a shortening range of 2–7.5% for the lowest and highest velocities, respectively. Velocity and force were nearly constant over this shortening range in all contractions. Force (P) was taken as the average value over the same time period used to calculate velocity. SL change during contractions was calculated directly from the fractional length change and SLo. Analysis and curve fitting of F-V data. F-V data were fit with the standard Hill equation (Eq. 1) by least-squares analysis over the region 0.05 ⬍ P/Po ⬍ 0.78 as V ⫽ 共P *o ⫺ P兲b/共P ⫹ a兲

(1)

where P/Po is relative tension, a and b are constants with dimensions of force and velocity, respectively, and P*o is the intercept on the force axis (8, 17). The parameter a/P*o describes the degree of curvature of the F-V relation, where higher values indicate less curvature. Velocity at P/Po ⫽ 0.05 and P/Po ⫽ 0.50 were calculated from Eq. 1 and designated VP 05 and VP 50, respectively. Maximal specific tension was

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Fig. 1. Sarcomere length transients (A) and force production (B) of an intact single frog muscle fiber during a series of isovelocity contractions over a range of shortening velocities. Sarcomere length transients were obtained by measuring the fractional length change between two surface markers, one near each end of the fiber, throughout the contractions. Sarcomere length was calculated directly from the fractional length change and the average initial sarcomere length between the markers (see METHODS). Fiber shortening velocities were 3.0, 6.0, and 12.5 lengths/s (L/s) for traces a, b, and c, respectively. The period over which length and force were measured for force-velocity (F-V) curves is indicated by thickened lines in the respective traces. In practice, ⬃13–15 such contractions were used to construct the F-V relationship (cf. Fig. 2). Stimulus began at time 0 and continued throughout the period shown. C and D: traces from A and B on a zoomed scale to demonstrate that force and velocity were quite constant over the period used in the analysis of F-V curves. Experiments were performed at 25.0°C.

calculated as Po/CSA (in kN/m2), where CSA was taken as the weighted average of the segments between the first and last surface markers. Mechanical power was calculated directly from the F-V fit as the product of specific tension (P/CSA) and velocity (in W/kg). Vmax was calculated by fitting the data with Eq. 1 over the region 0 ⬍ P/Po ⬍ 0.78 and calculating the intercept at zero force. Vmax was also calculated using linear regression of the F-V data in the region 0 ⬍ P/Po ⬍ 0.075. SDS-PAGE analysis of MHC and MLC isoforms. After mechanical experiments, fibers were pinned at ⬃Lf to pieces of wax while in the chamber and then removed from the chamber and stored at ⫺80°C. Fibers were freeze-dried and cut into the segments delineated by the surface markers. In most cases, one or more of the markers had fallen off during freezing. The position of the lost markers on the freeze-dried fibers was determined from previous video images of the fiber in the mechanics chamber. Freeze-dried fiber segments were placed into microfuge tubes and suspended in 10 ␮l of SDSPAGE sample buffer consisting of 100 mM dithiothreitol, 2% SDS, 80 mM Tris-base (pH 6.8), 10% glycerol, and 0.012% (wt/vol) bromphenol blue. Samples were boiled (2 min) and stored at ⫺80°C for up to 3 wk before they were loaded onto gels. MHC and MLC isoforms were measured in each segment using SDS-PAGE, with 10% of the sample volume used for analysis of MHCs and the remaining 90% for MLCs. SDSPAGE of MHC isoforms was carried out as previously described (29). Gels were silver stained, and MHC bands were quantified by densitometry (PDI, Bio-Rad, Hercules, CA). The total amount of MHC in each segment was determined by comparison with a standard prepared from semimembranosus (SM) muscle on the same gels. The SM standard was prepared by isolating the myofibrillar fraction from SM muscle containing no external tendon, as previously described (28). Protein content in the myofibrillar fraction was determined (Pierce, Rockford, IL) and suspended in SDS-PAGE AJP-Cell Physiol • VOL

sample buffer to a concentration of 0.125 mg protein/ml. Because myosin represents ⬃43% of myofibrillar protein (47) and MHC ⬃85% of myosin mass, the SM standard contained ⬃0.045 mg MHC/ml. Ten microliters of a fivefold dilution of the SM standard were loaded per lane, which was equivalent to 0.091 ␮g MHC/lane. Molar concentration of MHC in each segment was calculated as follows: M/V 䡠 MW, where M is the mass of MHC estimated from the gel, V is segment volume taken from its dimensions in the mechanical chamber (normalized to SL ⫽ 2.35 ␮m), and MW is the molecular weight of MHC (205,000 g/mol). SDS-PAGE of MLC isoforms was carried out as previously described (27). Gels were silver stained, and MLC3/MLC1 in each segment was calculated from the band densities and molecular mass values using densitometry and NIH Image software (version 1.62). Statistical analysis. To quantify the relative influence of various parameters on peak power production, multiple linear regression was implemented. Independent variables were screened for normality and skew to justify the use of the parametric analysis. Two separate regression analyses were performed. First, the three mechanical parameters used to calculate maximal power were allowed to enter a multiple regression model. This was performed to understand the extent to which each of these parameters was related to maximum power. In an independent analysis, 15 mechanical and structural parameters (see RESULTS) were allowed to enter the multiple regression model. This was performed to understand which parameters were overall more highly correlated with maximum power production. Significance level (␣) was set to 0.05. RESULTS

F-V properties of intact single fibers (n ⫽ 12) were measured with a spot-follower device that continuously tracked the distance between two surface markers that

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were placed within 0.80 ⫾ 0.05 and 0.71 ⫾ 0.09 mm of the moving and fixed ends of the fiber, respectively. Resting fiber length was 6.97 ⫾ 0.12 mm. Thus markers spanned 78.7 ⫾ 0.9% of the fiber length. Sample records of a series of contractions from one fiber are shown in Fig. 1. Experiments were designed so that shortening ramps were performed over the plateau region of the length-tension curve (optimal myofilament overlap), which, in frog muscle, occurs between SL of 2.25 and 2.05 ␮m (13). On average, contractions started from an SLo of 2.35 ⫾ 0.01 ␮m. During the isometric phase, SL decreased from 2.35 to 2.23 ⫾ 0.07 ␮m. Further sarcomere shortening occurred during the high-velocity step and before force stabilization. Force and velocity records were read immediately after force stabilized. On average, sarcomeres shortened during the period used to construct F-V curves from 2.13 ⫾ 0.04 to 2.05 ⫾ 0.04 ␮m, and thus shortening velocity was indeed measured over the plateau of the lengthtension curve. All fibers maintained excellent integrity throughout the F-V protocol, inasmuch as isometric tension (Po) declined by only 3.1 ⫾ 0.6% from the first to the last contraction (range 0.9–8.4%). Representative F-V curves for a relatively fast- and slow-twitch fiber are shown in Fig. 2A. F-V data for all fibers were well fit by the standard Hill hyperbolic equation (Eq. 1) over the range 0.05 ⬍ P/Po ⬍ 0.78. Vmax values obtained by the Hill fit over the range 0 ⬍ P/Po ⬍ 0.78 and linear regression over the range 0 ⬍ P/Po ⬍ 0.075 were not significantly different (P ⫽ 0.51; Fig. 2A, inset). Thus only Vmax from the Hill fit is reported. Power-velocity curves, calculated directly from the F-V fits, produced reliable fits of the powervelocity data (Fig. 2B). After mechanical experiments, MHC and MLC isoform contents were determined using quantitative SDS-PAGE. Representative MHC and MLC gels are shown in Fig. 3. For each fiber, MHC and MLC isoforms were measured in each segment between the end-most markers, and data were averaged. Fibers contained only type 1, 2, or 1 and 2 (coexpressed) MHCs. For MLCs, we measured the molar ratio of essential light chains, MLC3/MLC1, inasmuch as this ratio has been shown in previous studies to correlate with Vmax. The relationship between %MHC1 and MLC3/MLC1 is shown in Fig. 4. Linear regression showed that although MLC3/MLC1 increased with %MHC1, the relationship was not highly correlated (r2 ⫽ 0.097) and did not reach statistical significance (P ⫽ 0.32). This weak correlation agreed with previous examination of a larger data set from 35 single AT fibers (27). The extreme variability in MLC3/MLC1 between fibers was not due to intrinsic variability in the quantification technique, which was shown to have a coefficient of variation ⬍15% (27). Also, we typically found the coefficient of variation of %MHC1 to be ⬍1.0% over a 10-fold range of sample concentration (including the range of band densities of single fibers in this study; data not shown). Analysis of the F-V data revealed that Vmax increased significantly with %MHC1 but was not signifAJP-Cell Physiol • VOL

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Fig. 2. F-V and power-velocity relationships for a relatively fast (F) and a slow (E) intact frog muscle fiber. A: F-V curves fit with the standard Hill equation (Eq. 1) over the range 0.05 ⬍ P/Po ⬍ 0.78. As expected, the Hill fit deviates significantly from the data points in the high-force region of the curves (dashed lines). Inset: high-velocity region of the F-V data in A. Two methods were used to calculate maximal shortening velocity (Vmax): Hill fit over the range 0 ⬍ P/Po ⬍ 0.78 (where P/Po is relative tension; solid line) and linear regression fit over the region P/Po ⬍ 0.075 (dashed line). Vmax was taken as the intercept of the respective lines on the velocity axis. Force is shown in relative units (P/Po). B: power-velocity curves calculated directly from the F-V fits in A. Data points were calculated as the product of force and velocity in A. Experiments were performed at 25.0°C.

icantly correlated with MLC3/MLC1 (Fig. 5, Table 1). Similarly, VP 05 and VP 50 increased with %MHC1, but we found no correlation between MLC3/MLC1 and either VP 05 or VP 50. Po/CSA also increased significantly with %MHC1 (Fig. 6, Table 1), while MLC3/MLC1 was not significantly correlated with Po/CSA (Table 1). From the regression analysis in Fig. 6, Po/CSA increased by 22.3% as %MHC1 increased from 0 to 100%. Measurements of myosin concentration were used to determine whether the significant increase in Po/CSA with %MHC1 could be attributed to a greater amount of myosin per unit volume, rather than actual differences in MHC isoform properties. The amount of MHC in each segment between the end-most fiber markers was measured using quantitative SDS-PAGE, and segment volumes were calculated as the product of CSA and segment length (see METHODS). For each fiber, the

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eters (Vmax, VP 50, VP 05, P*o, a/P*o, %MHC1, MLC3/ MLC1, Po, CSA, Po/CSA, velocity at Wmax, P/Po at Wmax, MHC mass, volume, and MHC concentration) showed that %MHC1 had the highest correlation with Wmax (r2 ⫽ 0.846). DISCUSSION

Fig. 3. Myosin heavy and light chain (MHC and MLC) isoforms determined by SDS-PAGE. A: MHC isoforms in segments of one fiber determined on an SDS-polyacrylamide gel (silver stained) after mechanical analysis. Each lane corresponds to a different segment along the length of the fiber (a–f), and the segments together comprised the entire length between the end-most surface markers. Type 1 MHC (MHC1), calculated as a percentage of total MHC, is indicated below each lane. Total MHC in each segment was quantified by comparison with a semimembranosus (SM) muscle standard that contained a known amount of MHC (0.091 ␮g/lane). B: SDS-PAGE of MLCs in each of the fiber segments in A. Ratio of MLC3 to MLC1 (MLC3/MLC1) calculated from this gel (silver stained) is indicated below each sample lane. Molecular masses of gel markers (left-most lane) were 45, 31, 21.5, and 14.5 kDa.

amount of MHC per unit volume was taken as the weighted average of the segments. Linear regression showed that the amount of MHC was directly proportional to fiber volume (r2 ⫽ 0.973; Fig. 7A), providing confidence that MHC mass and fiber volume measurements were accurately determined. On average, fiber MHC concentration was 197.7 ⫾ 10.9 ␮M, which was in close agreement with previous estimates of MHC concentration in single muscle fibers (12, 45). The relationship between %MHC1 and MHC concentration (Fig. 7B) was poorly correlated (r2 ⫽ 0.113) and did not reach statistical significance (P ⫽ 0.28). Thus the increase in Po/CSA with %MHC1 was not explained by greater myosin concentration, indicating that the effect of MHC isoform on Po/CSA was more likely to result directly from differences in isoform properties (see DISCUSSION). Wmax increased significantly with %MHC1 (Fig. 8, Table 1), while no significant correlation was found between MLC3/MLC1 and Wmax (Table 1). From the regression analysis in Fig. 8, Wmax increased by 61.3% as %MHC1 increased from 0 to 100%. Wmax is the product of Po/CSA, P/Po, and velocity at the point of peak power. Multiple regression analysis showed that Po/CSA accounted for 70.4% of the variability in Wmax, while velocity and P/Po explained an additional 17.1 and 11.6% of the variability, respectively. A more general multiple regression analysis of 15 different paramAJP-Cell Physiol • VOL

Because of their robust stability when isolated from whole muscle, single intact frog fibers continue to serve as a valuable experimental model for studies of contractile function and high-resolution myosin crossbridge properties. However, the lack of precise definition and detection of myosin isoforms in anuran skeletal muscle have placed a significant limitation on their use in determining the influence of myosin isoforms on contractile properties in intact cells. We took advantage of the recent detailed description of MHC and MLC isoforms in the various fiber types of R. pipiens (27, 29) to determine their influence on the dynamic properties of isolated intact single fibers. Contractile properties were measured in fibers that contained type 1, 2, or 1 and 2 MHCs, which are the predominant MHC isoforms expressed in hindlimb muscles of R. pipiens (28). Overall, the mechanical analysis showed that Vmax, VP 05, VP 50, Po/CSA, and Wmax were strongly correlated with %MHC1. We found no significant influence of MLC3/MLC1 on any mechanical parameters. Mechanical experiments were performed using a spot-follower device that permitted F-V properties to be precisely measured between two surface markers that spanned the central 78% of the fiber length. The surface marker technique has significant advantages over whole fiber F-V measurements. First, systematic errors in estimating fiber length are eliminated. Fiber length can be difficult to measure accurately because of inherent ambiguity in determining exactly where fibers insert onto tendons. Also, measurement of fiber CSA is improved, because fiber diameter can be accurately measured throughout the region of interest, whereas morphology at the fiber ends may be variable

Fig. 4. Relationship between percentage of MHC1 (%MHC1) and MLC3/MLC1 in single fibers. Data are shown for each of the fibers used in mechanical analysis (n ⫽ 12).

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Fig. 5. Shortening velocity as a function of %MHC1 and MLC3/MLC1. A–C: linear regression analysis showed that Vmax, velocity at 5% of maximum isometric tension (VP 05), and velocity at 50% of maximum isometric tension (VP 50) increased with %MHC1. D–F: Vmax, VP0 5, and VP 50 were not significantly correlated with MLC3/MLC1. Data points are individual fibers (n ⫽ 12), and regression coefficients are provided in Table 1.

compared with the central portion. Also, the end-most regions of fibers are known to have mechanical properties that are significantly different from the rest of the fiber (9, 11). Exclusion of the end regions should produce more reliable SL and velocity measurements and, thus, more meaningful correlations between contractile behavior and myosin isoforms.

For all contractions combined, fiber velocity measured from the servomotor and velocity measured between the surface markers were highly correlated (r2 ⫽ 0.998). However, fiber velocity was 4.6 ⫾ 0.7% greater (P ⬍

Table 1. Linear regression variables describing influence of %MHC1 and MLC3/MLC1 on contractile function of intact single muscle fibers %MHC1

Vmax VP 05 VP 50 Po/CSA Wmax

r2

0.373 0.603 0.753 0.595 0.860

MLC3/MLC1 P

r2

P

0.035 0.003 0.0003 0.003 ⬍ 0.0001

0.072 0.184 0.016 0.139 0.075

0.400 0.164 0.694 0.239 0.390

MLC3/MLC1, ratio of myosin light chain (MLC) 3 to MLC 1; MHC1, myosin heavy chain type 1; Vmax, maximal shortening velocity; VP 05 and VP 50, velocity at P/Po of 0.05 and 50 (where P is force and Po is tetanic force); CSA, cross-sectional area; Wmax, maximum mechanical power. AJP-Cell Physiol • VOL

Fig. 6. Maximal specific isometric tension as a function of %MHC1 in intact single frog fibers. Data points are individual fibers (n ⫽ 12), and linear regression coefficients are provided in Table 1.

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Fig. 7. MHC content in single frog fibers. A: MHC mass was directly proportional to fiber volume (n ⫽ 11). B: MHC concentration increased slightly with %MHC1, but the correlation was weak and did not reach statistical significance.

0.0001) than velocity between the surface markers (data not shown). One explanation for the difference is that fiber ends are thought to be stronger than the central part of the fiber (9). Thus, for a given force level, velocity is likely to be faster at the fiber ends than in the central region. To account for the full 4.6% difference, the fiber ends (which constitute ⬃22% of fiber length) would have to shorten 22% faster than the central portion. Differences in velocity of this magnitude along fibers are reasonable, inasmuch as it has been shown that differences in Vmax of up to 40% (at 2–3°C) occur between successive segments of fibers (10, 11). However, it is also possible that fiber length was systematically underestimated because of difficulty in determining fiber insertions. The lack of an influence of MLC3/MLC1 on Vmax (Fig. 5) is in contrast to several previous studies of skinned single fibers (for reviews see Refs. 5, 36, and 42). For example, in rodent fast-twitch fibers that contained only MHCIIB, MLC3/MLC1 was strongly correlated with increased Vmax, and the differences were as large as the differences in Vmax between fibers containing MHCIIA, MHCIIX, and MHCIIB (2). In contrast, our results agree with those of human fast-twitch fibers, which showed no discernable influence of MLC3/MLC1 on Vmax (23, 24, 46). However, the MHCIIB isoform AJP-Cell Physiol • VOL

was not studied in human fibers (it is not expressed), but MHCIIB was the isoform that showed the greatest effect with MLC3/MLC1 function in the rodent (5). The lack of a modulatory effect of MLC3/MLC1 on Vmax in R. pipiens is somewhat surprising, given the fact that MLC3/MLC1 varied nearly fivefold (0.24–1.19), which was nearly identical to the magnitude and variability within rodent MHCIIB fibers (2). It remains to be seen whether the somewhat greater MLC3/MLC1 values in fibers containing only MHC1 from other muscles in R. pipiens (27) are correlated with increased Vmax. We are not aware of any previous studies of MLC3/MLC1 on Vmax in intact single fibers; thus the extent to which skinning may cause differences between our results and those from skinned fibers remains unanswered. It will be useful to see if the steep relationship between Vmax and MLC3 holds up in intact fibers of rodents. There appears to be a consensus that the effect of MLC3/MLC1 is restricted to Vmax and does not extend to loaded fibers (4, 5, 42). Thus the functional significance of variability in MLC3/MLC1 is questionable, since muscles do not generate either force or power when shortening at Vmax. Whether MHC isoforms affect Po/CSA in striated muscle remains controversial (5, 36, 42). We found that Po/CSA increased by 22.3% as %MHC1 increased from 0 to 100% (Fig. 6). The MHC isoform effect did not appear to result from an increased number of cross bridges per half sarcomere, inasmuch as MHC concentration was unrelated to %MHC1 (Fig. 7B). Thus it appears that intrinsic differences in cross-bridge function between type 1 and 2 MHC isoforms resulted in differences in Po/CSA. Further experiments are required to assess whether the specific influence of MHC isoforms on Po/CSA was due to differences in force per cross bridge or the fraction of cross bridges in the strongly bound force-generating state (duty cycle) (5, 12, 42, 45). Kinetic measurements of single myosin molecules suggest that differences in force-generating capacity between myosin isoforms are the result of different duty cycles, rather than differences in unitary force production (15).

Fig. 8. Maximal mechanical power (Wmax) as a function of %MHC1 in intact single frog fibers. Data points are individual fibers (n ⫽ 12), and linear regression coefficients are provided in Table 1.

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The relationship between MHC isoforms and Po/ CSA, including factors that complicate the interpretation of results in single skinned fibers, has been comprehensively discussed (5, 12, 36, 42, 45). There is general consensus that, in skinned fibers of mammals, Po/CSA is greater in fast-twitch fibers (types IIA, IIX, and IIB) than in slow-twitch fibers (type I). However, because of significant swelling of fibers and other factors specific to skinned fibers, measurements of Po/CSA in skinned fibers are unreliable, especially when optical measurements are used to determine CSA (see Table 7 in Ref. 42 and Table 3 in Ref. 5). Importantly, by measuring myosin density and fiber mass in single fibers after mechanics experiments, the direct dependence of isometric force on MHC isoform can be determined (12, 45). Significant differences in Po/CSA among mammalian fiber types have also been documented in intact fibers using motor unit (1) and whole muscle mechanical measurements (38). In a comprehensive series of studies, Lannergren and colleagues (19–21) measured the contractile properties of single intact fibers representing the full range of fiber types from X. laevis at 20°C. Fiber types were classified after mechanics analysis on the basis of native isomyosin bands and limited analysis of MHC and MLC isoforms with SDS-PAGE (19). Five major fiber types and several subtypes were identified in X. laevis and appeared to contain as many as five MHC isoforms (20, 22, 41, 43). However, a one-to-one correspondence between fiber type and MHC isoform is not yet certain. Importantly, it was shown with SDS-polyacrylamide gels that type 1 and 2 fibers (subclassified as types 1n and 2n) in X. laevis contained unique MHC isoforms (19). The differences in contractile properties between type 1 and 2 fibers in R. pipiens are similar to the differences between type 1n and 2n fibers in X. laevis. From linear regression analysis, we calculate that Vmax, VP 50, Po/ CSA, and Wmax increased by 21, 34, 22, and 61% as %MHC1 increased from 0 to 100% in R. pipiens fibers (Figs. 5, 6, and 8). Lannergren (19) showed an increase of 41, 50, 32, and 93% for these same mechanical parameters in type 1n compared with type 2n fibers. Because Lannergren’s studies on X. laevis were performed at 20°C and our experiments on R. pipiens at 25°C, direct comparison of absolute values is problematic. We used Q10 values of 1.16, 1.63, and 1.88 for Po/CSA, Vmax, and Wmax, respectively, to extrapolate the mechanics of type 1 fibers in R. pipiens to 20°C (34). With the use of these Q10 values and the linear regression analysis stated above, Po/CSA and Wmax of R. pipiens were 15.7 and 12.3% less, respectively, for type 1 than type 1n fibers in X. laevis. In contrast, Vmax was only 2.8% greater in R. pipiens than in X. laevis type 1n fibers. Thus, overall, the contractile properties of the fastest fiber types in these two anuran species are very similar. More importantly, both studies demonstrate that MHC isoforms have a significant influence on the full F-V relationship and maximal mechanical power production in intact fibers. Because of the functional significance of mechanical power production on in vivo motor performance, it was of interest to determine in more detail how MHC isoforms AJP-Cell Physiol • VOL

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influenced power production. From a strict mechanical point of view, the largest influence on Wmax was Po/CSA, which accounted for 70% of the variability in the data. Surprisingly, when the influence of 15 mechanical and structural parameters on Wmax was determined, %MHC1 was by far the best predictor of Wmax. Thus the MHC isoform has a profound influence on Wmax in R. pipiens single fibers, even more so than Vmax or Po/CSA, parameters traditionally associated with power production in muscle. A potential problem with regression models is the preferential entry of parameters that are measured with greater accuracy and resolution than others. On the basis of difficulties associated with quantitative measurements of %MHC1 in single fibers, we believe that the %MHC1 parameter actually represents one of the more difficult parameters to quantify. This makes its dominance in the regression analysis even more impressive. Importance of MHC isoforms on muscle performance during jumping. During jumping, frogs generate high levels of mechanical power in their hindlimb extensor muscles, and jump distance is directly proportional to the peak power produced (33, 35). Fiber shortening velocity has been measured in the muscle fibers of the SM muscle (a large hindlimb extensor muscle) during maximal jumps at 25°C in R. pipiens (32–34). On average, shortening velocity during jumping was 3.94 L/s (normalized to SL ⫽ 2.35 ␮m), and fibers shortened mostly over the plateau of the length-tension curve (33). From the power-velocity curves of single fibers (such as those in Fig. 2), we calculated that at 3.94 L/s fibers generated ⬃100% of their maximal power (range 96.1–100%; data not shown). Accordingly, power produced at the in vivo jump velocity of 3.94 L/s increased by 57% as %MHC1 increased from 0 to 100%. The strong influence of MHC isoforms on mechanical power production is reflected in their overall distribution in the various hindlimb muscles of R. pipiens. We previously compared the MHC isoform content in the large extensor muscles involved in jumping with other muscles in the hindlimb (28). We found that the large extensor muscles contained ⬃90% MHC1, while the other muscles not involved in jumping were predominantly composed of MHC2. The contractile data in this study, along with this MHC isoform distribution data, demonstrate the importance of MHC isoform expression in muscular system design. In the case of R. pipiens, the muscular system is designed to generate high levels of mechanical power in the hindlimb extensor muscles during jumping. We are grateful to Dustin Robinson and Haiyan Yu for excellent technical assistance and the University of California, San Diego, Physics Department Electronics Shop for valuable assistance with PDA design. This study was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants AR-40050, AR-45631, and AR-46469 and a grant from the Department of Veterans Affairs. REFERENCES 1. Bodine SC, Roy RR, Eldred E, and Edgerton VR. Maximal force as a function of anatomical features of motor units in the cat tibialis anterior. J Neurophysiol 57: 1730–1745, 1987. 2. Bottinelli R, Betto R, Schiaffino S, and Reggiani C. Unloaded shortening velocity and myosin heavy chain and alkali

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