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Departments of 1 Anesthesiology and 2 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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We hypothesize that 1) the
effect of denervation (DNV) is more pronounced in fibers expressing
fast myosin heavy chain (MHC) isoforms and 2) the effect of
DNV on maximum specific force reflects a reduction in MHC content per
half sarcomere or the number of cross bridges in parallel.
Studies were performed on single Triton X-100-permeabilized fibers
activated at a pCa (
log Ca2+ concentration) of 4.0. MHC
content per half sarcomere was determined by densitometric analysis of
SDS-PAGE gels and comparison to a standard curve of known MHC
concentrations. After 2 of wk DNV, the maximum specific force of fibers
expressing MHC2X was reduced by ~40% (MHC2B
expression was absent), whereas the maximum specific force of fibers
expressing MHC2A and MHCslow decreased by
only ~20%. DNV also reduced the MHC content in fibers expressing
MHC2X, with no effect on fibers expressing
MHC2A and MHCslow. When
normalized for MHC content per half sarcomere, force generated by DNV
fibers expressing MHC2X and MHC2A was decreased
compared with control fibers. These results suggest the force per cross
bridge is also affected by DNV.
inactivity; skinned fibers; myosin heavy chain content; force per cross bridge
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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PREVIOUSLY, OUR LABORATORY HAS SHOWN that unilateral denervation (DNV) of the diaphragm muscle (Diam) results in a marked reduction in maximum specific force and a decrease in maximum shortening velocity in muscle bundles (25, 31, 46, 48). In addition, DNV results in a decrease in the cross-sectional area of Diam fibers expressing 2X and 2B myosin heavy chain isoforms (MHC2X and MHC2B, respectively), whereas fibers expressing MHC2A and MHCslow display a slight hypertrophy (25, 46-48).
The reduction in Diam maximum specific force after DNV is not unique, but it has been reported for other muscles after disuse (9, 27) as well as with hindlimb unloading-induced muscle atrophy (10), induced muscle injury (24), and sarcopenia (4). Moreover, as in the DNV Diam, muscle adaptations to disuse are fiber type specific. For example, hindlimb unloading has a larger effect on the predominantly slow soleus muscle compared with the predominantly fast gastrocnemius (11) and extensor digitorum longus muscles (10). In addition, after spinal transection, maximum specific force decreased significantly in the medial gastrocnemius muscle but was maintained in soleus muscle (27). Although these responses to disuse could be attributed to physiological differences in activation history and functional requirements of different muscles, they also lend support to the concept that morphological and contractile adaptations to muscle disuse are fiber type specific.
Previous studies from our laboratory indicate that maximum specific force differs with MHC isoform expression in single fibers from normal rat Diam (13, 33, 34). Fibers expressing MHC2X and MHC2B isoforms generate the greatest amount of force compared with fibers expressing MHC2A and MHCslow. When normalized for MHC content per half sarcomere, or the number of cross bridges in parallel per half sarcomere (i.e., force per cross bridge), differences in maximum specific force across fibers expressing fast MHC isoforms in the rat Diam were eliminated. However, fibers expressing MHCslow still produced less force per cross bridge than did fast fibers when normalized for MHC content per half sarcomere. These results prompt us to consider whether fibers expressing fast and slow MHC isoforms in the rat Diam may be differentially affected by a period of inactivity induced by unilateral DNV.
In the present study, the effects of DNV on maximum specific force generated by rat Diam single fibers expressing different MHC isoforms were examined. We hypothesized that DNV decreases maximum specific force primarily in fibers expressing MHC2X and MHC2B with little or no effect on fibers expressing MHC2A and MHCslow. We further hypothesized that the DNV-induced decrease in maximum specific force is due to a reduction 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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Experiments were performed on 20 adult male Sprague-Dawley rats (body weight ~300 g). The animals were assigned to either control (n = 10) or DNV (n = 10) groups. Animals were housed in separate cages under a 12:12-h light-dark cycle, fed with Purina rat chow, and provided with water ad libitum. Body weights were monitored daily in all groups. Surgical procedures were performed under aseptic conditions, and recovery of animals from surgery was carefully monitored. The Institutional Animal Care and Use Committee of the Mayo Clinic approved all procedures.
Unilateral DNV. The procedure for unilateral DNV has been previously described in detail (17, 25, 46-48). Briefly, animals were anesthetized by intramuscular injection of ketamine (60 mg/kg) and xylazine (2.5 mg/kg). The right phrenic nerve was then exposed in the lower neck at a point beneath the sternomastoid muscle. The phrenic nerve was transected, and a portion (~10-20 mm) of the distal end was removed to prevent reinnervation of the Diam and to minimize neurotrophic effects emanating from the remaining nerve stump. The wound was then sutured and treated with topical antibiotics. At the end of the 2-wk DNV period, inactivity of the right Diam was verified by the absence of electromyograph activity.
Tissue preparation and single-fiber dissection.
After 2 wk, the animals were reanesthetized, and the right side of the
Diam was rapidly excised. Muscle fiber bundles were stretched 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, 15.0 mM creatine phosphate, 1 mg/ml
phosphocreatine kinase, and 50 mM imidazole for a total ionic strength
of 200 mM at a pH of 7.0 at 5°C. Fiber bundles were later stored in
relaxing solution containing 50% glycerol (vol/vol) for up to 3 wk. To
permeabilize the plasma membrane before single-fiber dissection, fiber
bundles were placed in relaxing solution containing 1% Triton X-100.
Single fibers were dissected under a dissecting microscope while in the skinning solution (~20 min). The single fibers were then transferred from the skinning solution to a relaxing solution [log
Ca2+ concentration (pCa) 9.0] before force measurement.
Single-fiber mechanical measurements. The computer program described by Fabiato and Fabiato (7) was used to determine free ionic Ca2+ concentration in the activating and relaxing solutions used for force measurements, with stability constants listed by Godt and Lindley (15). The solutions contained the following (in mM): 10.0 EGTA, 1.0 free Mg2+, 5.0 MgATP, 15.0 creatine phosphate, 50.0 imidazole, and 2.0 dithiothreitol and 1 mg/ml phosphocreatine kinase with a total ionic strength of 150 mM. The relaxing and activating solutions had a pCa of 9.0 and 4.0, respectively.
Dissected fibers were attached, via small stainless steel hooks, between a force transducer (model AE-801, Aksjeselskapet), with a resonant frequency of 5 kHz, and a servo-motor (model G120DT, General Scanning), with a step time of 800 µs. To maintain noncompliant attachments of fibers to the force transducer and servo-controlled motor, the fiber ends were fixed in 5% glutaraldehyde and anchored using aluminum foil T clips. The attached fiber was then mounted horizontally in a temperature-controlled flow-through acrylic chamber (volume 120 µl) positioned on the stage of an inverted microscope (model IMT-2, Olympus). First-order laser diffraction (He-Ne laser, model LSC 30D, UDT Sensors) was used to monitor sarcomere length (set at 2.5 µm). During experiments, Brenner cycling (2), as modified by Sweeney et al. (38), was used to stabilize sarcomere length. LabView-based software and a data acquisition board were used to record signals. The length of the muscle fiber (~2.0 mm) was measured with a reticule in the microscope eyepiece [×10 Olympus Plan 10, 0.30 numerical aperture (NA)]. The XY fiber diameter was measured with a ×40 objective (Olympus LWD CD Plan 40, 0.55 NA). The ×40 objective was also used to measure the XZ fiber diameter (depth) 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. Previously, we showed a 20% error in the depth measurement using this method when compared with the direct Z-axis measurement of the fiber using confocal microscopy (13). Therefore, a correction factor for Z-axis distortion was established and used to calculate fiber cross-sectional area directly from fiber width (XY) and depth (XZ) measurements. Fiber cross-sectional area was measured while the fiber was mounted on the stage of an inverted microscope at a sarcomere length of 2.5 µm. Using the ×40 0.55-NA objective, the XY measurements were accurate within 0.5 µm, whereas the Z-axis distortion (~20%) was corrected according to previous measurements made with confocal microscopy (13). Baseline force was measured while fibers were perfused with a pCa 9.0 solution. After maximal activation in pCa 4.0 solution, the fiber was again perfused with a pCa 9.0 solution to verify that force returned to its original baseline level. The isometric force generated at pCa 4.0 was divided by fiber cross-sectional area to determine maximum specific force (N/cm2). Maximum isometric force was also divided by the estimated value of MHC content per half sarcomere (see MHC content per half sarcomere measurements) to determine the force per half-sarcomere MHC content (N/µg MHC content). Sinusoidal length oscillations (0.2% Lo) at 2 kHz were used to determine muscle fiber stiffness during activation at a pCa 4.0 in the presence and absence (rigor solution) of ATP. Stiffness measurements under both conditions were normalized for fiber cross-sectional area. Fiber stiffness during rigor was assumed to reflect full recruitment of all available cross bridges. The ratio of fiber stiffness during rigor solution compared with activation at pCa 4.0 (with ATP) thus reflected the fraction of cross bridges in the strongly bound force-generating state (3).MHC content per half sarcomere measurements. MHC concentration in rat Diam single fibers was determined as previously described (13). An accurate determination of the volume of each fiber segment was the initial step in determining MHC content per half sarcomere. Single fibers were fixed in 4% paraformaldehyde for 30 s and imaged using a microscope (Nikon Optiphot-2 with ×20 0.5-NA objective) with a MTI CCD72 camera. The number of sarcomeres in series was counted from this projected image, and width and depth measurements were used to determine fiber cross-sectional area. Fiber cross-sectional area was normalized to a sarcomere length of 2.5 µm, because force measurements were obtained at this sarcomere length. The volume of a half sarcomere was determined from the number of sarcomeres in series and the fiber-volume measurements.
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. The samples were denatured by boiling for 2 min. Gradient gels were prepared using a modified procedure by Suguira and Murakami (37). 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 (1)] 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. (26). Identification of MHC isoforms by migration patterns was confirmed by Western blot analysis as previously described (13, 14). Briefly, rat Diam bundles were run on SDS-PAGE and transferred to nitrocellulose overnight at 1 A. The nitrocellulose sheet was divided into five sections, and one segment was stained with colloidal gold to ensure adequate protein transfer and visualize protein band migration. One of the following mouse monoclonal or polyclonal antibodies was used to stain the four additional segments: NCL (Novocastra, IgG), which reacts with MHCslow; SC.71 (ATTC, 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. Isoform specificity of these antibodies was previously determined (21, 28). The nitrocellulose segments were 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). The MHC concentration of rat Diam fibers was determined electrophoretically by comparison with a standard curve of known concentrations of purified rabbit MHC (Sigma Chemical M-3889). The standard concentrations of MHC were verified by measuring protein concentrations using the Bradford method (1). This technique has been previously described (13). A high-resolution scanner (Microtek ScanMaker 5, 600 dpi) was used to image the gels after silver staining. After background subtraction, the density of delineated electrophoretic bands was measured, and the brightness-area product (BAP) was determined. The relationship between the BAP and MHC concentration was linear across a range from 0.01 to 0.25 µg/µl. From the standard curve, the MHC concentration of the single fiber was determined. The MHC content per half sarcomere was determined by multiplying the MHC concentration of the single fiber by the half-sarcomere volume of the fiber.Statistical analysis.
Comparison of fiber cross-sectional area, 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 and between control and DNV
fibers was done by two-way ANOVA. A Student's t-test with
Bonferroni correction was used as a post hoc analysis to compare
between fiber types when appropriate. A power analysis was performed
for each parameter to determine the minimal change from control values
that could be detected using the number of animals per experimental
group (n = 10) at a 
level of 0.8. Statistical significance was indicated by a P value <0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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MHC isoform expression.
In the present study, MHC isoform expression was determined by SDS-PAGE
and Western blot analysis in ~300 Diam fibers. The adult
rat Diam is a mixed muscle expressing four MHC isoforms (Fig. 1). In a previous systematic
analysis of MHC isoform expression in single fibers of the rat
Diam, coexpression of MHC isoforms was detected in ~30%
of fibers (primarily the MHC2X and MHC2B coexpression) (35). In the present study, 22% of the 155 control Diam fibers examined expressed the
MHCslow isoform, 20% expressed MHC2A, 29%
expressed MHC2X, and 29% coexpressed the MHC2B
and MHC2X isoforms. Singular expression of the
MHC2B isoform was not detected. Although these results are
consistent with our laboratory's previous report (35), it
should be noted that, because the smaller, more fragile fibers
expressing MHC2A and MHCslow were more
difficult to dissect, the fiber sampling process was biased in the
present study. As a result, dissection was often selective to obtain a sufficient number of fibers expressing each MHC isoform for statistical analysis. These values were not meant to characterize the fiber-type distribution of the Diam as a whole; instead, they only
represent the proportion of single fibers sampled in the present study.
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Cross-sectional area.
Accurate cross-sectional area measurements were important for the
determination of MHC content per half sarcomere and maximum specific
force. In control rat Diam fibers, fiber-type differences in cross-sectional area were observed (Fig.
2A). The average
cross-sectional area of Diam fibers coexpressing
MHC2B and MHC2X was significantly greater than
fibers singularly expressing MHC2X,
MHC2A, and MHCslow isoforms. Similarly,
fibers expressing the MHC2X isoform had significantly greater cross-sectional areas than fibers expressing MHC2A
and MHCslow. However, no significant differences in
cross-sectional area were found between fibers expressing
MHC2A and MHCslow isoforms.
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MHC content per half sarcomere. MHC content per half sarcomere was determined in 74 control rat Diam fibers. Fibers coexpressing MHC2B and MHC2X had the greatest MHC content per half sarcomere (Fig. 2B). Fibers expressing MHC2X either alone or in combination with MHC2B had significantly higher (~3-fold) MHC content per half sarcomere compared with fibers expressing MHC2A and MHCslow. No significant difference in MHC content per half sarcomere was found between fibers expressing MHCslow and MHC2A isoforms.
MHC content per half sarcomere was determined in 51 DNV rat Diam fibers (Fig. 2B). After DNV, the MHC content per half sarcomere in fibers expressing MHC2X decreased by ~50%. In contrast, no significant changes in MHC content per half sarcomere were found in fibers expressing MHCslow and MHC2A isoforms. As a result, MHC content per half sarcomere did not differ across fibers expressing different MHC isoforms in the DNV Diam.Maximum specific force.
Maximum specific force was measured in 82 control rat Diam
fibers (Fig.
3A). The
highest maximum specific forces were generated by fibers coexpressing
the MHC2B and MHC2X isoforms, followed by
fibers singularly expressing MHC2X, MHC2A, and
MHCslow isoforms. Fibers expressing the
MHC2X isoform produced significantly greater force than
fibers expressing MHC2A and MHCslow, with no
significant difference between MHC2A and
MHCslow fibers.
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Force per half-sarcomere MHC content. Maximum force values of rat Diam fibers were normalized for MHC content per half sarcomere to evaluate the effect of cross-bridge number on maximum specific force. When controlled for MHC content, differences in maximum specific force were eliminated across control Diam fibers expressing fast MHC isoforms (Fig. 3B). However, fibers expressing the MHCslow generated significantly less force per half-sarcomere MHC content (~50%) than fibers expressing fast MHC isoforms.
After DNV, force per half-sarcomere MHC content decreased in fibers expressing MHC2A and MHC2X isoforms (Fig. 3B). However, no significant change in force per half-sarcomere MHC content was found for fibers expressing MHCslow. As a result, fast fibers exhibited force per cross-bridge values equivalent to that of slow fibers, and thus fiber-type differences in force per cross bridge were eliminated after DNV.Fraction of cross bridges in the force-generating state. To evaluate a possible change in the recruitment of cross bridges after DNV, the ratio of fiber stiffness during activation in pCa 4.0 and rigor (pCa 4.0) solutions was used as an estimate of the fraction of cross bridges in the force-generating state (Fig. 3C). In 38 control and 57 DNV rat Diam fibers, no significant differences in the fraction of cross bridges in the force-generating state were found across fibers expressing different MHC isoforms. In addition, after DNV, there was no change in the fraction of cross bridges in the force-generating state compared with control fibers.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In the present study, we found that 2 wk after DNV of the rat Diam, there is a significant reduction of cross-sectional area and MHC content per half sarcomere in fibers expressing the MHC2X isoform, whereas there was little or no effect on fibers expressing MHC2A and MHCslow isoforms. Expression of the MHC2B isoform was not detected in Diam fibers after 2 wk of DNV. In addition, maximum specific force was reduced in all DNV Diam fibers, but it was reduced to the greatest extent in fibers expressing the MHC2X isoform with less reduction of maximum specific force in fibers expressing the MHC2A and MHCslow isoforms. When normalized for changes in MHC content per half sarcomere, the force generated by Diam fibers expressing fast MHC isoforms was reduced by DNV, with no effect on the force normalized for MHC content per half sarcomere in fibers expressing MHCslow. These observations suggest that DNV of the rat Diam results in MHC isoform-specific adaptations with an effect on both cross-bridge number and the force-generating capacity of individual cross bridges.
MHC isoform expression. In the present study, 2 wk of DNV affected gene regulation of MHC isoform expression, as evidenced by the absence of the MHC2B isoform and increase in MHC2A and MHCslow coexpression. The absence of MHC2B expression is in agreement with previous results in the rat Diam showing an overall decrease in the proportion of type IIb fibers (19, 31, 47) and a transition from type II (fast) to type I (slow) fibers in the rat Diam after 1 wk of DNV (43). It is possible that the loss of MHC2B expression is due to a transition from fast to slow isoforms, which, along with initial hypertrophy, is characteristic of the early stages of DNV (17, 36, 44).
The increased incidence of hybrid fibers (MHC2A and MHCslow coexpressing fibers) is also characteristic of the early stages of DNV. In a study by Gauthier and Hobbs (12) in the rat Diam, an increase in the number of type IIc fibers (coexpressing the MHCslow and MHC2A isoforms) occurred after 4 wk of DNV of the rat Diam. The increased incidence of MHC2A and MHCslow coexpression during the early stages of DNV may be an early indicator of phenotypic transition from slow to fast fiber types that has been reported in the rat Diam after chronic DNV (5, 12). In a study by Carraro et al. (5), it was reported that, whereas many Diam fibers were immunoreactive for an anti-slow MHC antibody even after 16 mo of DNV, singular expression of the MHCslow isoform was no longer present.Fiber cross-sectional area. Two weeks of DNV resulted in a significant reduction in the cross-sectional area of Diam fibers expressing the MHC2X isoform. This is in agreement with previous studies in the rabbit Diam after 1 and 4 wk of DNV (46) and in the hamster and rat Diam after 2 wk of DNV (25, 48). Previous studies from our laboratory (17, 31, 47) indicated a significant hypertrophy of fibers expressing MHCslow and MHC2A isoforms, whereas only the cross-sectional area of MHCslow fibers increased significantly in the present study. Failure to see a significant hypertrophy in MHC2A fibers in the present study could reflect a sampling bias in the dissection of single fibers or differences in technique for measuring fiber cross-sectional area. In previous studies, fiber cross-sectional area was measured from histochemically stained transverse sections of frozen muscle samples. In addition, the extent of hypertrophy and atrophy seen in individual fibers may vary slightly from animal to animal, depending on prior activity levels and the influence of humoral factors.
MHC content per half sarcomere. Muscle atrophy has been attributed to a decrease in protein content in a variety of models of inactivity, such as hindlimb unweighting (10), muscle injury (24), and sarcopenia (4). Previous studies in the rat Diam after DNV have examined changes in overall protein content (40) or the relative contribution of each MHC isoform to the total area of the Diam (43). These studies did not differentiate changes in total protein from myofibrillar protein concentration or changes in contractile proteins from noncontractile material such as connective tissue protein or interstitial fluid volume. Without such information, the effect of DNV on the contractile machinery itself cannot be determined. In addition, previous DNV studies in the Diam or limb muscles have not examined changes in MHC content, or even myofibrillar protein, at the level of single fibers. By determining MHC content in a single fiber, confounding effects, such as changes in extracellular components of the muscle cross-sectional area, can be avoided. In this manner, we can ascertain the direct effect of changes in MHC content on force production.
In the present study, MHC content per half sarcomere decreased in fibers expressing the MHC2X isoform after 2 wk of DNV. This agrees with previous studies in the rat Diam, in which overall protein content declined after 6 days of DNV (45) and the relative contribution of fibers expressing MHC2X and MHC2B isoforms decreased after 2 wk of DNV (31, 47). In this regard, it is important to note that MHC content did not decrease in fibers expressing MHC2A or MHCslow isoforms after 2 wk of DNV. This seems to contradict previous studies in rat limb muscles that indicated that, after DNV, the protein content of slow muscle fibers decreased to a greater extent than that of fast muscles (22, 39). However, in these studies the unloading effects of muscle paralysis confound the effects of DNV on limb muscles. For example, it has been shown that the influence of hindlimb unloading is more pronounced in the soleus muscle (predominantly type I fibers) compared with the gastrocnemius or extensor digitorum longus (predominantly type IIx and IIb fibers) (10, 11). Similarly, MHC isoform transitions after DNV differ between slow and fast muscle. For example, relative expression of MHC2X increased in the slow rat soleus muscle but decreased in the fast extensor digitorum longus and gastrocnemius muscles after DNV (22). It is likely that the effect of DNV on MHC isoform expression varies depending on muscle activation and loading history, making fiber-type comparison across muscles problematic. Activation history further distinguishes the Diam from limb muscles. Compared with limb muscles, the duty cycle (proportion of time active vs. inactive) of the Diam is very high (~40% compared with ~2% for the extensor digitorum longus muscle and ~14% for the soleus muscle) (18, 29). Quiet breathing is accomplished by the recruitment of fatigue-resistant motor units in the Diam comprising fibers that express MHCslow and MHC2A isoforms (30, 32). Only during short-duration expulsive motor behaviors of the Diam (e.g., coughing) is it necessary to recruit the more fatigable fast motor units comprising fibers that express MHC2X and MHC2B isoforms. It could be argued that this unique activation pattern would make the Diam particularly susceptible to fiber-type-specific adaptations induced by inactivity.Maximum specific force. A decrease in muscle maximum specific force has been previously observed in a number of disuse models (9, 27), including Diam DNV (25, 48). The results of the present study extend these previous observations by exploring effects of DNV on single Diam fibers expressing different MHC isoforms. Although the maximum specific force of all Diam fibers was reduced after 2 wk of DNV, the maximum specific force of fibers expressing MHC2X decreased to the greatest extent. These results are consistent with the more pronounced effect of DNV on the cross-sectional area and MHC content per half sarcomere of fibers expressing MHC2X.
Previous studies have suggested that the Diam mechanical and morphological changes induced by unilateral DNV stem from passive stretching and consequent mechanical loading of muscle fibers imposed by the continuous activation of the intact contralateral side of the Diam (8, 44). In a previous study, our laboratory directly assessed this theory by measuring muscle fiber length changes in the sternal and midcostal regions of the Diam before and after DNV using sonomicrometry (46). It was speculated that, because fibers in these two regions of the Diam have a different orientation, continued activation of the intact contralateral side after DNV would impose a different pattern of passive stretching and mechanical loading on fibers in these two regions. Thus, if passive stretching and mechanical loading were the major factors affecting DNV- induced morphological and contractile changes, then differential adaptations to DNV should have been seen in these two regions. However, the morphological and contractile changes observed after DNV in the sternal and midcostal Diam regions were similar. Furthermore, in both regions the passive stretch imposed by continuous activation of the intact contralateral side did not exceed Lo; thus passive mechanical loading was minimal. From these results, we concluded that passive length changes and mechanical stress are not the main determinants of the morphological and contractile adaptations induced by unilateral DNV. It is possible that Diam inactivity per se could account for the morphological and contractile changes induced by DNV. To address this possibility, we assessed the influence of Diam inactivity imposed by cervical spinal cord hemisection at C2 (25, 47). Both DNV and spinal cord hemisection induce complete paralysis of the Diam, but these two models differ with respect to neurotrophic influence, which is completely disrupted after DNV. Despite the similarity in the Diam inactivity imposed by these two models, the morphological and contractile adaptations observed after 2 wk differed significantly. Spinal hemisection resulted in only a small decrease in maximum specific force and very little change in the relative expression of MHC isoforms (25, 47). These results indicated that neurotrophic influence may have a greater impact on the morphological and contractile properties of Diam fibers than inactivity per se. Disruption of neurotrophic influence after unilateral DNV may indeed explain the fiber-type-specific impact of DNV on the rat Diam. As mentioned previously, more fatigable motor units comprising MHC2X and MHC2B fibers are rarely recruited and only for short-duration high-force behaviors of the Diam (30, 32). Fibers expressing MHC2X and MHC2B isoforms are the largest fibers in the Diam, and they continue to maintain their size despite their relative inactivity. Therefore, under normal conditions of inactivity, these fibers display a unique resistance to disuse atrophy. One explanation could be a greater dependence of fibers expressing MHC2X and MHC2B isoforms on basal neurotrophic influence to maintain their size during periods of inactivity. If such fibers had a greater dependence on neurotrophic influence under normal conditions, then the removal of neurotrophic influence with DNV would have a greater impact, as was observed in the present study. In support of this theory, previous studies have indicated that the influence of neurotrophic factors may vary with fiber type and MHC isoform expression. In a study by Davis and Kiernan (6), DNV of the extensor digitorum longus muscle resulted in a greater atrophy of type IIb (histochemical classification of fibers expressing the MHC2B isoform) fibers compared with type IIa (histochemical classification of fibers expressing the MHC2A isoform) fibers. With the addition of nerve extract, the atrophy of type IIb fibers was reversed with no effect on the cross-sectional of type IIa fibers. This indicates a selective atrophy of type IIb fibers after DNV may be due to the removal of a neurotrophic influence. Therefore, it likely that innervation per se exerts a trophic influence on fibers expressing MHC2X and MHC2B isoforms in the Diam.Force per MHC content. In control rat Diam fibers, when maximum force was normalized for MHC content per half sarcomere, differences in force generation across fibers expressing fast MHC isoforms were eliminated. In contrast, Diam fibers expressing MHCslow generated less force than fibers expressing fast MHC isoforms even after normalization for MHC content per half sarcomere. Because MHC content per half sarcomere estimates the number of cross bridges in parallel, these results indicate that, in the normal rat Diam, force per cross bridge is comparable across fibers expressing fast MHC isoforms but lower in fibers expressing MHCslow. After 2 wk of DNV, maximum force normalized for MHC content per half sarcomere was unchanged for Diam fibers expressing MHCslow but significantly reduced for fibers expressing fast MHC isoforms. Thus the force per cross bridge of fast fibers appeared to be differentially affected by DNV. These results clearly indicate that, something other than a reduction in the number of available cross bridges (MHC content per half sarcomere) is causing the reduction of maximum specific force induced by DNV.
Permeabilization of muscle fibers results in alterations in lattice spacing as a result of changing fiber volume (16). Previous studies have shown that modulation of the filament lattice by the addition of osmotically active polymers such as dextran to the bathing medium can alter the force-generating capacity of skinned fibers (16, 23). On the basis of these studies, when the filament lattice is compressed below its in situ value, movement of the S1 fragment of the cross bridge is hindered, resulting in force inhibition (23). It is possible that variations in the lateral spacing of the filament lattice may occur with DNV and thus affect the probability of cross-bridge attachment. In this manner, a shift in the lateral spacing of myosin filaments in Diam fibers expressing fast MHC isoforms could reduce the force per cross bridge in these fibers. However, the possibility that alteration of the filament lattice affects additional sites within the sarcomere cannot be ruled out. For example, variations in lattice spacing may alter the structural protein titin. Titin is thought to be primarily responsible for the passive mechanical properties and elasticity of skeletal muscle (42), as well as the positional stability of thick filaments during isometric contraction (20). Various isoforms of titin exist in skeletal muscle, and these isoforms differ in their extent of elasticity and contribution to resting tension (41). On the basis of a study by Wang et al. (41), the differential expression of titin isoforms in fast and slow skeletal muscles dictates the elasticity, compliance, and the elastic limit of the sarcomere. Because of this variability in elastic properties, it is possible that titin isoforms, which are most likely associated with MHC isoform expression, are differentially affected by DNV. Diam DNV occurs occasionally during surgery; however, the real clinical application for this model relates to other physiological and pathological conditions associated with reduced maximum specific force. For example, reduced maximum specific force occurs with sarcopenia, corticosteroid treatment, exposure to a zero-gravity environment, chemotherapy, and hypothyroidism. Examining the possible mechanisms underlying the reduction in maximum specific force after Diam DNV provides valuable information about muscle weakness in response to altered muscle activity. The results of the present study clearly demonstrate that after DNV, structural and functional properties of Diam fibers are differentially affected, depending on MHC phenotype. The underlying mechanism(s) by which DNV exerts these differential effects remains unresolved, although an effect on MHC content per half sarcomere appears to contribute at least in part. Still other mechanisms, such as force per cross bridge, must contribute. Adaptations to DNV in the rat Diam appear to be most pronounced in fast fibers, an effect that may serve to protect the essential ventilatory functions of the Diam. For example, ventilatory behavior of the Diam primarily involves recruitment of only type S motor units (type I fibers) (31), which are less adaptive to DNV, and therefore the decrements after DNV may be limited to nonventilatory behaviors requiring the recruitment of fast-twitch (type II fibers) motor units.| |
ACKNOWLEDGEMENTS |
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We thank Dr. Wen-Zhi Zhan for assistance with these studies.
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FOOTNOTES |
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This research was supported by National Heart, Lung, and Blood Institute Grants HL-34817 and HL-37680.
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. Section 1734 solely to indicate this fact.
Received 25 July 2000; accepted in final form 17 October 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Significantly different from pre-DNV value,
P < 0.05. B: MHC content per half sarcomere in
Ctl and DNV rat Diam fibers expressing different MHC
isoforms. Values are means ± SE. * Significantly different
from fibers expressing MHCslow, P < 0.05. + Significantly different from fibers expressing
MHC2A, P < 0.05. # Significantly different from fibers expressing
MHC2X, P < 0.05. 