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Departments of Anesthesiology and of Physiology and Biophysics, Mayo Clinic and Foundation, Rochester, Minnesota 55905
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In the present study, myosin heavy
chain (MHC) content per half sarcomere, an estimate of the number of
cross bridges available for force generation, was determined in rat
diaphragm muscle (Diam) fibers expressing different MHC
isoforms. We hypothesize that fiber-type differences in maximum
specific force [force per cross-sectional area (CSA)] reflect the
number of cross bridges present per CSA. Studies were performed on
single, Triton X-100-permeabilized rat Diam fibers. Maximum
specific force was determined by activation of single Diam
fibers in the presence of a high-calcium solution (pCa, 
log
Ca2+ concentration of 4.0). SDS-PAGE and Western blot
analyses were used to determine MHC isoform composition and MHC content
per half sarcomere. Differences in maximum specific force across fast MHC isoforms were eliminated when controlled for half-sarcomere MHC
content. However, the force produced by slow fibers remained below that
of fast fibers when normalized for the number of cross bridges
available. On the basis of these results, the lower force produced by
slow fibers may be due to less force per cross bridge compared with
fast fibers.
skinned fibers; force per cross bridge; fiber morphometry
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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PREVIOUS STUDIES IN LIMB MUSCLES have reported significant differences in maximum specific force across fibers expressing different myosin heavy chain (MHC) isoforms in humans (1, 19, 34) and in rats (2, 7). In the rat diaphragm muscle (Diam), fiber-type differences in specific force have also been reported (7, 27, 28). Fast fibers expressing the MHC2X isoform, either alone or in combination with the MHC2B isoform, produce greater specific force than fibers expressing MHC2A and MHCslow isoforms. Maximum specific force in single skeletal muscle fibers is dependent on the number of cross bridges per half sarcomere, the average force per cross bridge, and the fraction of cross bridges in the force-generating state. In the present study, MHC content per half-sarcomere fiber volume was used as an estimate of the number of cross bridges per half sarcomere.
It is likely that the number of cross bridges per half sarcomere varies across fiber types due in part to differences in mitochondrial volume density (27). Fibers expressing MHC2A and MHCslow isoforms have greater mitochondrial volume densities compared with fibers expressing MHC2X and/or MHC2B isoforms (27). This suggests that fibers expressing MHC2X and MHC2B isoforms have greater MHC content per half sarcomere compared with fibers expressing MHC2A and MHCslow isoforms. Therefore, the purpose of the present study was to test the hypothesis that differences in maximum specific force across fiber types in the rat Diam are due to differences in MHC content per half sarcomere.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Tissue Preparation and Single-Fiber Dissection
Ketamine (60 mg/kg) and xylazine (2.5 mg/kg) were injected intramuscularly into adult male Sprague-Dawley rats (body wt of ~300 g), and the right side of the Diam was excised. Muscle fiber bundles were then stretched ~20% to optimal length, (Lo), pinned on cork, and placed for 24 h in a relaxing solution consisting of 59.0 mM potassium acetate, 6.7 mM magnesium acetate, 5.6 mM NaATP, 10 mM EGTA, 2.0 mM dithiothreitol (DTT), 15.0 mM creatine phosphate, 1 mg/ml phosphocreatine kinase (PCK), and 50 mM imidazole for a total ionic strength of 200 mM at a pH of 7.0 at 5°C. The fiber bundles were then stored in relaxing solution containing 50% glycerol (vol/vol) for up to 3 wk. Before single-fiber dissection was started, a fiber bundle was placed in relaxing solution containing 1% Triton X-100 to permeabilize the plasma membrane. While in the skinning solution (~20 min), single fibers were dissected under a dissecting microscope. Before force measurements were made, the single fibers were transferred from the skinning solution to a relaxing solution (pCa 9.0).MHC Concentration Measurements
Single fiber segments (~1.5-2.5 mm in length) were fixed in 4% paraformaldehyde for 30 s and placed on a microscope stage (Nikon Optiphot-2) with a MTI CCD72 camera. The fiber image was projected onto a video screen, and the total number of sarcomeres was counted using a Nikon Plan ×20 lens [0.5 numerical aperture (NA)]. Fibers were then placed in 25 µl of SDS sample buffer containing 62.5 mM Tris · HCl, 2% (wt/vol) SDS, 10% (vol/vol) glycerol, 5% 2-mercaptoethanol, and 0.001% (wt/vol) bromophenol blue at a pH of 6.8. Boiling for 2 min denatured the samples. Gradient gels were prepared with the use of a modified procedure by Sugiura and Murakami (31). The stacking gel contained a 3.5% acrylamide concentration (pH 6.8), and the separating gel contained 5-8% acrylamide (pH 8.8) with 25% glycerol (8 × 10 cm, 0.75 mm thick; Hoefer SE250). To compare migration patterns of the MHC isoforms, control samples of Diam bundles in a 1:200 dilution of SDS sample buffer [~9.0 ng/µl MHC concentration determined by the Bradford method (3)] were run on the gels. Sample volumes of 10 µl were loaded per lane. The gels were silver stained according to the procedure described by Oakley et al. (25).Identification of MHC isoforms by migration patterns was confirmed by Western blot analysis. Rat Diam bundles were run on SDS-PAGE and transferred to nitrocellulose. After overnight transfer at 1 A, the nitrocellulose sheet was divided into five sections. One nitrocellulose segment was stained with colloidal gold to visualize protein bands and ensure adequate protein transfer. The four other segments were stained with one of the following mouse monoclonal or polyclonal antibodies: NCL (Novocastra, IgG), which reacts with MHCslow; SC.71 (American Type Culture Collection, IgG), which reacts with MHC2A; BF-F3 (Schiaffino, IgM), which reacts with MHC2B; and BF-35 (Schiaffino, IgG), which reacts with all but the MHC2X isoform. The specificity of these isoforms was previously determined (17, 26). Each nitrocellulose segment was stained with a biotinylated secondary antibody specific to IgG (NCL, SC.71, BF-35) or IgM (BF-F3) and visualized with alkaline phosphatase (Vectastain ABC kit, Vector Labs).
To determine the MHC concentration of rat Diam fibers,
increasing volumes of a known concentration of purified rabbit myosin [Sigma M-3889, concentrations verified with the Bradford method (3)] were loaded on the gels. Gels were silver stained,
and a high-resolution scanner (Microtek ScanMaker 5, 600 dpi) was used
to image the gels. The brightness-area product (BAP) of each rabbit
myosin sample was determined from the area and average brightness of
each densitometric band following subtraction of local background. A
linear relationship between the BAP, or densitometric measurement of
electrophoretic bands, and the myosin content in the rabbit myosin
samples was used to determine the myosin content in rat
Diam single fibers (Fig. 1).
The MHC concentration in the entire Diam fiber was
calculated by dividing the MHC content determined from the myosin
standard curve by the fiber volume. This concentration was then
multiplied by the half-sarcomere volume of the fiber, normalized to
Lo of 2.5 µm/sarcomere.
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Extent of myosin extraction.
A potential source of error in this study is incomplete extraction of
MHC from fiber samples. To estimate the extent of MHC extraction from
single fibers, the Bradford method (3) for protein
quantification was used to measure the amount of total protein in rat
Diam single fibers. The Bradford method has the advantage
of minimal interference from most commonly used biochemical reagents,
and the use of the microassay procedure allows detection of 1-10
µg of protein. Bovine 
-globulin was used as the protein standard
for the assay, and the absorbance measured at 595 nm was used to
determine the concentration of protein in Diam single fibers. The expected amount of MHC in rat Diam fibers was
then determined from generally accepted assumptions about the amount of
myosin in muscle protein.
MHC concentration reproducibility. Error in the calculation of MHC concentration can be introduced by the gel-loading technique. To determine the reproducibility of the electrophoretic technique, rabbit psoas fibers (~15-20 mm in length) were dissected and placed in 4% paraformaldehyde to maintain striation spacing as indicated above. The number of sarcomeres and the fiber width and length were measured on a microscope stage (Nikon Optiphot-2) with a MTI CCD72 camera (see above). Depth measurements were adjusted according to the correction factor established using the confocal microscope. The long psoas fiber was then placed in 100 µl of SDS sample buffer and boiled for 2 min to denature the protein. The sample was then loaded in multiple wells on a gel in 10-µl volumes per lane. The gels were stained, and BAP values were compared across wells. A percent coefficient of variation for the BAP measurements from each psoas fiber was determined.
Fiber volume measured by confocal microscopy. Accurate measurement of fiber volume is necessary to compare MHC concentration and maximum specific force across fibers expressing different MHC isoforms. Therefore, confocal microscopy was used as a gold standard for fiber volume measurement. A total of 10 rat Diam fibers were measured using both confocal microscopy and conventional light microscopy (see below) to establish a correction factor for errors introduced in the measurement of fiber depth using conventional light microscopy (i.e., z-axis distortions). All subsequent measurements of fiber volume were done with conventional light microscopy (see Single-Fiber Mechanical Measurements) and corrected for the measurement error in fiber depth.
Single rat Diam fibers were placed in a 1% solution of a fixable mitochondrion-selective probe, MitoTracker Red (CMXRos, Molecular Probes, M-7512) for 30 s and then fixed in a 4% paraformaldehyde solution. The sole purpose of this procedure was to stain the entire cell for volume measurements without introducing additional artifacts common with membrane dyes. After fixation, single fibers were transferred to glycerol relaxing solution in a hemocytometer well with a depth of 100 µm (Fisher Scientific, 0267110). A small plastic coverslip was placed over the well, and the apparatus was positioned on the stage of a confocal microscope (Bio-Rad MRC 600). Complete staining of the fiber membrane was verified by comparing measurements made in the xy plane (parallel to the microscope stage) using fluorescence with measurements of the xy plane in transmission mode. A series of optical slices was obtained for each single fiber using a ×40 oil-immersion lens (1.3 NA). The aperture size of the confocal microscope was adjusted to obtain an optical slice thickness of ~2.0 µm, and, on average, 20-40 optical slices were collected through the entire z-axis distance of the fiber. From the reconstructed set of optical slices, measurements of fiber diameter in the xz plane (plane vertical to the microscope stage) were obtained. The set of optical slices was then used to calculate fiber cross-sectional area (CSA). Although measurements in the xy axis are relatively accurate (~0.3 µm resolution using the ×40 objective), distortions in the z axis are an inherent problem of light microscopy and can be related to a number of factors (28). To estimate z-axis distortion using the confocal microscope (×40 objective, 1.3 NA), 10-µm fluorescently labeled beads embedded in mounting medium were optically sectioned (0.6-µm step size), and the difference in measured z-axis diameter was compared with the diameter specified by the manufacturer. An elongation of the z axis was consistently observed, and the average error was ~8%, which would introduce an error of ~17% in volume measurements, if uncorrected. It is likely that with muscle fiber diameters ranging from 30 to 100 µm and a z-axis slice of 2 µm the measurement error would be less.Single-Fiber Mechanical Measurements
The computer program described by Fabiato and Fabiato (8) with stability constants listed by Godt and Lindley (13) was used to determine the activating and relaxing solutions used for force measurements. The solutions contained the following (in mM): 10.0 EGTA, 1.0 free Mg2+, 5.0 MgATP, 15.0 creatine phosphate, 50.0 imidazole, 2.0 DTT, and PCK at 1 mg/ml with a total ionic strength of 150 mM. The relaxing solution had a pCa of 9.0, and the activating solution had a pCa of 4.0.The ends of the fibers were fixed by exposing them to a 5% glutaraldehyde solution to maintain noncompliant attachments of the fibers to a force transducer and servo-controlled motor (see below). Aluminum foil T clips attached to the fiber ends further reduced compliance. The fiber was mounted horizontally on two small stainless steel hooks in a temperature-controlled flow-through acrylic chamber (volume = 120 µl) located on the stage of an inverted microscope (Olympus IMT-2). The fiber was attached at one end to a force transducer (Aksjeselskapet, AE-801) with a resonant frequency of 5 kHz, and the other end was attached to a servo-motor (General Scanning, G120DT) with a step time of 800 µs. Sarcomere length was set at 2.5 µm and monitored by first-order laser diffraction (He-Ne laser, UDT Sensors, LSC 30D). Brenner cycling (5) as modified by Sweeney et al. (32) was used in an effort to stabilize sarcomere length during experiments. Signals were recorded with LabView-based software and a data acquisition board. A reticule in the microscope eyepiece was used to measure the length (×10 Olympus Plan 10, 0.30 NA) of muscle fiber segments (usually 1.5-2.5 mm). A ×40 objective (Olympus LWD CD Plan 40, 0.55 NA) was used to measure the xy fiber diameter. The xz fiber diameter (depth) was also measured with the ×40 objective by setting the microscope fine focus control to zero while focusing on the top of the fiber and focusing through to the bottom of the fiber. In 10 fibers, the xy- and xz-axis measurements obtained with this inverted microscope system were directly compared with measurements obtained with the confocal microscope (see above). No consistent differences in xy-axis measurements were observed. However, differences in xz-axis measurements were observed between the two systems, with the xz fiber diameter measured using the inverted system being ~20% shorter than the xz diameter measured using the confocal system. On the basis of these differences, a correction factor for z-axis distortion was established and used to calculate fiber CSA.
A baseline force measurement was obtained while fibers were perfused with a pCa 9.0 solution. The perfusate was then switched to a pCa 4.0 solution (in the same flow-through chamber) to maximally activate the fibers. After maximal activation was achieved, the fiber was again perfused with a pCa 9.0 solution to verify that force returned to its original baseline level. Maximum specific force (N/cm2) was calculated by dividing the maximum isometric force by the corrected fiber CSA (see above). Force per half-sarcomere MHC content (N/µg MHC content) was obtained by dividing maximum isometric force by the estimated value of MHC content per half sarcomere.
Muscle fiber stiffness was determined using sinusoidal length oscillations (0.2% Lo) at 2 kHz and normalized for fiber CSA. The ratio of fiber stiffness during maximal activation in a rigor solution (pCa 4.0 without ATP) vs. a normal pCa 4.0 (with ATP) solution was used to determine the fraction of cross bridges in the strongly bound force-generating state (4).
Statistical Analysis
One-way ANOVA was performed to compare fiber CSA, maximum specific force, MHC content per half sarcomere, force per half-sarcomere MHC content, and the fraction of cross bridges in the force-generating state across fibers expressing different MHC isoforms. When appropriate, a Student's t-test with Bonferroni correction was used to compare between fiber types. A P < 0.05 was used to indicate statistical significance. Reproducibility of MHC content values was assessed with analysis of the coefficient of variation across BAP measurements.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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MHC Concentration Measurements
Several factors are involved in the analysis of MHC content per half sarcomere in single fibers. These include determining the MHC concentration by comparison to a standard calibration curve, evaluating the extent of myosin extraction, assessing the reproducibility of the electrophoretic technique, and obtaining accurate measurement of fiber volume.MHC isoform expression in rat Diam single fibers was readily identified by SDS-PAGE and Western analysis (Fig. 1). The majority of single fibers analyzed in this study expressed a single MHC isoform (~70%), whereas coexpression of the MHC2B and MHC2X isoforms occurred in ~30% of the rat Diam fibers. These results are in agreement with previously published results in the rat Diam (11, 30). In the present study, single fibers expressing the MHC2B isoform alone were not identified.
Myosin extraction. One possible limitation in this study is incomplete extraction of MHC from the muscle samples. For this reason, the Bradford method (3) for protein quantification was used to determine the amount of total protein in rat Diam single fibers and the amount of MHC was estimated on the basis of several established assumptions. The total protein concentration in 37 rat Diam single fibers with an average diameter of 70 µm was 647 ± 76 µg/µl. It is commonly assumed that total protein and myofibrillar protein are 20 and 12% of muscle wet weight, respectively, and myosin is ~43% of myofibrillar protein (35). This results in a myosin concentration in single rat Diam fibers of 167 ± 20 µg/µl (~26% of total protein). Finally, MHC accounts for ~85% of the myosin mass, resulting in a MHC concentration of 142 ± 17 µg/µl in rat Diam fibers (~22% of total protein). The MHC concentration in rat Diam fibers of similar size determined by electrophoresis and densitometric analysis in the present study was 131 ± 8 µg/µl. Therefore, the MHC concentrations in single rat Diam fibers as determined in this study are within 10% of expected values for MHC concentration, based on the generally accepted assumptions listed above.
In the present study, single fibers were fixed in 4% paraformaldehyde in an attempt to stabilize sarcomere length for fiber volume measurements. To determine if fixation altered the extraction of myosin from single fibers, a long rabbit psoas fiber was dissected and cut into two segments. Only one segment was fixed in 4% paraformaldehyde for 30 s, and both segments were measured for fiber volume and run on gels. The ratio of MHC concentration for the unfixed to fixed segments of 6 psoas fibers was 1.01 ± 0.02 (values are means ± SE).MHC concentration reproducibility.
Accurate determination of MHC concentration in single fibers depends on
consistent and reproducible electrophoretic analysis. For this reason,
repeated electrophoretic measurements of rabbit psoas fibers were
performed. Reproducibility determinations of BAP values from multiple
samples of the same fiber were made using 24 rabbit psoas fibers. Psoas
fiber sample volumes of 10 µl were repeatedly loaded on a gel in
consecutive lanes (Fig. 2A).
The BAP values were compared across the gel, and a coefficient of variation was established for each psoas fiber. The average coefficient of variation ± SE for all 24 psoas fibers was 1.49 ± 0.30%
(Fig. 2B).
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Fiber volume measurements. Determination of maximum specific force and MHC content per half sarcomere is dependent on highly accurate measurements of fiber volume. Single-fiber volume measurements differ considerably depending on the shape assumption applied in the calculations. A subset of single fibers was measured on both a confocal microscope and an inverted microscope system used for single fiber mechanical measurements. Single-fiber measurements made on the two microscopes were comparable in the xy plane. However, the z axis as measured by the inverted microscope was underestimated compared with z-axis values obtained with the confocal microscope. The ratio of depth measured with the confocal to the inverted microscope was 1.19 ± 0.06 (values are means ± SE). Therefore, when fibers were measured on the inverted microscope system, a 20% correction factor in the z axis, or fiber depth, was included in the CSA calculation. The fiber depth was ~87% of the fiber diameter based on these calculations.
Average CSA measurements for each MHC isoform were determined. CSA measurements of Diam fibers coexpressing MHC2B/2X (3,940 ± 275 µm2) and MHC2X alone (3,234 ± 410 µm2) were significantly greater (P < 0.05) than fibers expressing MHC2A (1,583 ± 254 µm2) and MHCslow (1,253 ± 96 µm2). Values are reported as averages ± SE.MHC content per half sarcomere.
MHC concentration was determined in 74 rat Diam fibers. MHC
content per half sarcomere, determined from the MHC concentration and
half-sarcomere volume of each fiber, was significantly higher in fibers
expressing the MHC2X isoform either alone or in combination with the MHC2B isoform compared with fibers expressing the
MHC2A and MHCslow isoforms (Fig.
3A). MHC
content values for fibers expressing the MHCslow and
MHC2A isoforms were not significantly different. The MHC
content values per half-sarcomere values in fibers expressing the fast
MHC2B/2X isoform were approximately threefold higher than
the values for the fibers expressing MHC2A and
MHCslow isoforms.
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Maximum Specific Force
Maximum specific force was evaluated in 96 rat Diam fibers. Peak force was measured for each fiber and normalized for CSA (in µm2) (Fig. 3B). Fibers coexpressing the MHC2B and MHC2X isoforms exhibited the highest specific force values, followed by fibers expressing the MHC2X, MHC2A, and MHCslow isoforms. Thus differences in specific force exist across fibers expressing fast MHC isoforms. The specific force of slow fibers was significantly less than that produced by fibers expressing MHC2B/2X and MHC2X isoforms but not significantly different from fibers expressing the MHC2A isoform. Slow and MHC2A fibers produced ~60% of the force produced by fibers expressing the MHC2B/2X isoform.Force Per Half-Sarcomere MHC Content
To determine the effect of cross-bridge number on maximum specific force, peak force values of rat Diam fibers were normalized for the amount of MHC per half-sarcomere volume (Fig. 3C). Differences in specific force across fibers expressing fast MHC isoforms were eliminated when controlled for MHC content. However, a significant difference in force per half-sarcomere MHC content was found between fibers expressing fast and slow MHC isoforms. Slow fibers produced ~50% less force per half-sarcomere MHC content than fibers expressing fast MHC isoforms.Fraction of Cross Bridges in the Force-Generating State
An estimate of the fraction of cross bridges in the force-generating state was determined from the ratio of fiber stiffness in pCa 4.0 and rigor solution (pCa 4.0) (Fig. 4). The fraction of cross bridges in the force-generating state was evaluated in 41 rat Diam single fibers. No significant differences were found across fibers expressing different MHC isoforms.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Maximum specific force of rat Diam fibers differs with MHC isoform expression. This difference in specific force could not be attributed to the fraction of cross bridges in the force-generating state. At least among fibers expressing fast MHC isoforms, results from this study indicate that differences in specific force are attributed to differences in MHC content per half sarcomere. However, fibers expressing the MHCslow isoform generated less force than fibers expressing fast MHC isoforms even after they were normalized for the number of cross bridges per half sarcomere. These results provide new evidence that the lower force per MHC content of slow fibers is due to a lower force per cross bridge compared with fast fibers.
MHC Concentration Measurements
Myosin extraction. The Bradford method (3) for protein quantification was used to determine the extent of MHC extraction from single Diam fibers in the present study. The expected MHC content determined from total protein measurements with the Bradford micro-assay (142 ± 17 µg/µl) and the actual MHC content in single fibers determined by electrophoresis and densitometry (131 ± 8 µg/µl) were in close agreement. These results indicate myosin extraction is not a limitation in the quantification of MHC content in the present study. Nevertheless, it is impossible to ensure 100% myosin extraction by any method, and the possibility of incomplete extraction cannot be ruled out. However, any limitation in the extraction procedure would be consistent across all Diam fibers and would not affect the major finding of this study regarding MHC content differences across fibers expressing different MHC isoforms.
The results from the present study are in good agreement with previous estimates of myosin concentration in skeletal muscle (Table 1). A previous study by Krasner and Kushmerick (18) allows direct comparison of total protein measurements in single fibers. Krasner and Kushmerick used the method of Lowry et al. (23) to determine the amount of total protein in rabbit psoas single fibers with bovine serum albumin as the protein standard. These investigators reported 1 µg/mm fiber length of total protein for rabbit psoas fibers compared with the results from the present study of 1.6 µg/mm fiber length in rat Diam fibers (Table 1). However, this comparison does not take fiber volume into consideration. Calculating the volume for a psoas fiber that is 85 µm in diameter (assuming a cylindrical CSA) and 4 mm in length results in 176 µg/µl of total protein per fiber volume. The myosin and MHC concentrations for a fiber this size would be 45 and 39 µg/µl, respectively [assuming that myofibrillar protein is 12% of total protein, ~43% of myofibrillar protein is myosin, and 85% of myosin is MHC (35)]. These values are significantly lower than those reported in the present study (see RESULTS).
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4µg MHC per half sarcomere.
This value is less than the MHC content per half sarcomere reported for
MHC2B fibers (70 µm in diameter) in the present study.
Differences in myosin concentrations can be attributed to the
comparison of different species as well as the use of different protein
assays and different protein. The method of Lowry et al.
(23) used by Krasner and Kushmerick is less sensitive and
subject to greater interference from common reagents than the Bradford
method (3). Although the extraction buffer used by Krasner
and Kushmerick was not described, the SDS content in the present study
(2% vol/vol) is high compared with that used in other studies
(12).
Fiber morphometry. In the present study, a rigorous effort was made to ensure the accurate determination of CSA and volume of rat Diam fibers. A major concern with these measurements relates to optical distortion in the z-axis, which can be due to a variety of factors, including the refractive mismatches going from the objective lens to the muscle fiber in the perfusion chamber. To address this issue, we assessed z-axis distortions under optimal conditions of confocal microscopy. We then compared measurements of fiber diameter in the z-axis between the inverted microscope system and the confocal microscope. As might be expected, the inverted microscope system used to measure fiber mechanics introduced significantly greater z-axis distortion. Although the confocal system introduced a z-axis error of up to 8% elongation, we selected to use this measurement as a gold standard and to correct the z-axis measurements to this standard with the inverted microscope. With the use of this method, the z-axis measurement of all fibers was corrected in the same fashion.
Previous studies in the rat Diam reported differences in CSA across fibers expressing different MHC isoforms that were in agreement with the present study (7). Slight differences in the reported CSA values could be attributed to the methods used for measuring fiber morphology. In the study by Eddinger and Moss (7), the fiber width and depth were measured and the CSA was calculated as an ellipse. However, unlike the present study, measurement error in the z-axis was not considered. Previous studies from our laboratory reported smaller CSA values across all fiber types in the rat Diam (30). These measurements were made on cross sections of muscle fibers without accounting for possible shrinkage of the frozen samples.MHC content per half sarcomere. In the present study, MHC content per half sarcomere was greater in fibers expressing the MHC2B/2X and MHC2X isoforms compared with fibers expressing the MHC2A and MHCslow isoforms. These results are in agreement with preliminary results previously reported from this laboratory (27). Normalizing force for MHC content eliminated differences in specific force across fibers expressing fast MHC isoforms. In contrast, slow fibers did not produce force values equivalent to those of fast fibers, even when normalized for the number of cross bridges available. The lower force per MHC content produced by slow fibers indicates that the number of cross bridges is not the sole determinant of specific force differences between slow and fast fibers.
Specific Force
Fiber-type differences in specific force reported in the present study confirm previous results in rat Diam (7, 27, 29). However, fiber-type differences in specific force remain controversial. This may be because previous studies have examined fiber-type differences in specific force by comparing fibers from a predominantly fast muscle with fibers from a predominantly slow muscle. For example, a previous study by Mounier and colleagues (24) reported that fibers from the primarily slow rat soleus muscle generate less specific force than fibers from the primarily fast plantaris muscle. In contrast, Gardetto and colleagues (10) found no significant differences in specific force when slow-twitch fibers from rat soleus muscle were compared with fast-twitch fibers from the gastrocnemius muscle. Clearly, differences in physiological function may confound comparisons of specific force across fiber types from different muscles. However, comparisons of fiber-type differences in specific force within the same muscle are also conflicting. For example, a study by Bottinelli and colleagues (1) found that slow fibers from the human vastus lateralis muscle developed less specific force than fast fibers. In contrast, Greaser and colleagues (15) reported no significant differences in specific force between fast and slow fibers from the rabbit plantaris muscle. One possible explanation for this discrepancy in previous studies lies in the assessment of muscle fiber morphometry.Force Per Half-Sarcomere MHC Content
Differences in mitochondrial volume density across fiber types suggest the number of cross bridges available for force generation contributes to fiber-type differences in specific force. Previous studies examining the effects of hindlimb suspension, a condition reported to induce muscle atrophy, support the role of MHC content in determining specific force. For example, a study by Gardetto and colleagues (10) resulted in a 28% decline in peak tetanic force following 2-wk hindlimb suspension of the soleus muscle. These investigators attributed the decrease in force to a loss in the number of cross bridges per CSA. In a similar study by Fitts and colleagues (9), the soleus muscle demonstrated a decline in peak specific tension of ~50% after hindlimb suspension, which was attributed to reduced muscle size and loss in contractile protein content.Fraction of Cross Bridges in the Force-Generating State
Results from the present study indicate no difference in the ratio of stiffness during maximal Ca2+ activation to rigor in rat Diam fibers expressing different MHC isoforms. Values reported here agree with results previously published in skinned fibers (14, 16). Values for the ratio of stiffness during maximal Ca2+ activation to rigor in skinned fibers are slightly higher than those previously reported for intact fibers (20), which can be attributed to the expansion of the filament lattice that occurs with skinning and subsequent osmotic compression of the fiber in solution.In the present study, the ratio of stiffness during maximal Ca2+ activation to rigor was used as an estimate of the fraction of cross bridges in the force-generating state. This method assumes that myosin affinity for actin is high in the rigor state and that all myosin heads are attached to actin (6, 21, 33). However, the contribution of myofilament compliance to the overall fiber compliance was not accounted for in the present study. Recent studies indicate myofilament compliance contributes ~40-60% to the total fiber (16, 20). Thus the ratio of stiffness in maximal Ca2+ to rigor determined in the present study overestimates the fraction of cross bridges contributing to force during an isometric contraction. There is no evidence to suggest myofilament compliance should vary across fiber types. When myofilament compliance is accounted for in the present study as ~40% of the overall fiber compliance (with ~30% due to thin filament compliance), the fraction of cross bridges in the force-generating state is reduced to ~0.45. This is in agreement with the value of 0.43 for the fraction of cross bridges attached to actin reported by Linari and colleagues (20).
In conclusion, results from this study indicate that differences in specific force across fast MHC isoforms in the rat Diam are eliminated when controlled for MHC content. However, differences in specific force between fast and slow fibers persist when normalized for the number of cross bridges per half sarcomere. This fiber-type difference in force per MHC content cannot be attributed to the fraction of cross bridges in the force-generating state. Therefore, the lower force values produced by slow fibers compared with fast fibers is most likely due to a difference in the amount of force produced per cross bridge. Although these results relate specifically to the Diam, they apply across all muscles, since MHC content per half sarcomere is likely to be an important determinant of force generation for any muscle fiber. A decrease in MHC content per half sarcomere may be an important factor in sarcopenia regardless of physiological (e.g., aging, response to unloading) or pathological (cancer, chemotherapy, bed rest) condition. Therefore, an understanding of the contribution of myosin content to differences in specific force has important implications for both physiological and pathological conditions. Future studies are needed to assess force per cross bridge and its role as a determinant of specific force.
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. C. Sieck, Anesthesia Research, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905 (E-mail: sieck.gary{at}mayo.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 12 October 1999; accepted in final form 25 February 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Bottinelli, R,
Canepari M,
Pellegrino A,
and
Reggiani C.
Force-velocity properties of human skeletal muscle fibres: myosin heavy chain isoform and temperature dependence.
J Physiol (Lond)
495:
573-586,
1996
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, Known amounts
of myosin loaded in 25-ng increments corresponding to lanes
6, 7, and 8 in A. 
, Rat
Diam single fibers loaded in 10-µl volumes and analyzed
for MHC content on the basis of the standard curve.
