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Departments of Anesthesiology and 1 Physiology and Biophysics,2 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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It has been found that maximum specific force (Fmax; force per cross-sectional area) of rat diaphragm muscle doubles from birth to 84 days (adult). We hypothesize that this developmental change in Fmax reflects an increase in myosin heavy chain (MHC) content per half-sarcomere (an estimate of the number of cross bridges in parallel) and/or a greater force per cross bridge in fibers expressing fast MHC isoforms compared with slow and neonatal MHC isoforms (MHCslow and MHCneo, respectively). Single Triton 100-X-permeabilized fibers were activated at a pCa of 4.0. MHC isoform expression was determined by SDS-PAGE. MHC content per half-sarcomere was determined by densitometric analysis and comparison to a standard curve of known MHC concentrations. MHC content per half-sarcomere progressively increased during early postnatal development. When normalized for MHC content per half-sarcomere, fibers expressing MHCslow and coexpressing MHCneo produced less force than fibers expressing fast MHC isoforms. We conclude that lower force per cross bridge in fibers expressing MHCslow and MHCneo contributes to the lower Fmax seen in early postnatal development.
postnatal development; maximum specific force; myosin heavy chain content; force per cross bridge; single fibers
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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IN THE RAT DIAPHRAGM MUSCLE (Diam), early postnatal development is characterized by dramatic transitions in myosin heavy chain (MHC) isoform expression. During the first 3 postnatal wk, expression of the neonatal MHC isoform (MHCneo) decreases, whereas expression of MHC2X and MHC2B isoforms appears only after the second postnatal week and increases thereafter (18-20, 35, 44-47). This postnatal transition from MHCneo to adult fast MHC isoform expression was found to be associated with changes in contractile properties of the rat Diam (18, 31, 35, 43, 44, 46, 47). In particular, it was noted that the increase in maximum specific force (Fmax; force per cross-sectional area) of the rat Diam during early postnatal development was associated with an increase in the relative expression of fast MHC isoforms (18, 35, 44, 46, 47).
Although controversial (8), previous studies in the rat Diam have indicated that fibers expressing MHC2X and MHC2B isoforms have a greater Fmax than fibers expressing the MHCslow and MHC2A isoform (6, 9, 10, 33, 34). In the adult rat Diam, differences in MHC content per half-sarcomere (reflecting the number of cross bridges in parallel) exist across fibers expressing different MHC isoforms, with fibers expressing MHC2X and MHC2B having greater MHC content per half-sarcomere than fibers expressing MHCslow and MHC2A (9). When Fmax of adult Diam fibers was normalized for MHC content per half-sarcomere, fibers expressing all fast MHC isoforms (MHC2A, MHC2X, and MHC2B) still generated greater force than fibers expressing MHCslow (9). This indicated a greater force per cross bridge in rat Diam fibers expressing fast MHC isoforms. Thus the postnatal increase in Fmax of the rat Diam could reflect the emergence of MHC2X and MHC2B expression (18, 35, 46, 47). In the present study, we examined the hypothesis that the postnatal increase in Diam Fmax reflects an increase in MHC content per half-sarcomere and/or a greater force per cross bridge in fibers expressing fast MHC isoforms compared with MHCslow and MHCneo.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Experiments were performed on male Sprague-Dawley rats at postnatal days 0, 14, and 28 (D0, D14, and D28, respectively) and on 84-day-old rats (adults) (n = 8 rats for each age group). Pregnant mothers were received at 14 days gestation, and litter size was culled to eight pups after parturition. To ensure normal body growth, pups from smaller litters were not studied. Animal body weights were measured daily. The pups were housed with their lactating mothers until D21 when they were weaned; thereafter, they were housed two per cage under a 12:12-h light-dark cycle. The adult animals and postweaning pups were fed Purina rat chow and provided with water ad libitum. The Institutional Animal Care and Use Committee of the Mayo Clinic approved all procedures.
Tissue preparation and single-fiber dissection. Animals were injected intramuscularly with ketamine (60 mg/kg) and xylazine (2.5 mg/kg), and the Diam was excised. The Diam was cut into muscle bundles, stretched ~20% beyond resting length, pinned on cork, and placed in a relaxing solution at 5°C 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 (CrP), 1 mg/ml phosphocreatine kinase (PCK), and 50 mM imidazole. An ionic strength of 150 mM and a pH of 7.0 were adjusted with propionic acid. After 24 h, fiber bundles were transferred to relaxing solution containing 50% glycerol (vol/vol) and stored for up to 3 wk. On the day of the study, fiber bundles were dissected and placed into relaxing solution containing 1% Triton X-100 for 20 min to permeabilize the plasma membrane. While in this skinning solution, single fibers were dissected under a stereo microscope. Because of the technical difficulty of dissecting single fibers in the neonatal Diam, muscle bundles (~5-10 fibers) were used at D0. The single fibers or neonatal bundles were then transferred from the skinning solution to a relaxing solution (pCa 9.0).
Single-fiber mechanical measurements. The activating (high Ca2+) and relaxing (low Ca2+) solutions were prepared using the computer program described by Fabiato and Fabiato (7) with stability constants listed by Godt and Lindley (11). Solutions contained the following (in mM unless otherwise specified): 10.0 EGTA, 1.0 free Mg2+, 5.0 NaATP, 15.0 CrP, 50.0 imidazole, 2.0 DTT, and PCK at 1 mg/ml with a total ionic strength of 150 mM. The ionic strength and pH of 7.0 were adjusted with propionic acid. Relaxing solution had a pCa of 9.0, and the activating solution had a pCa of 4.0.
Noncompliant attachments of the fibers to a force transducer and a servo-controlled motor were maintained by fixing the fiber ends in a 5% gluteraldehyde solution. To further reduce compliance and allow for fiber mounting, aluminum foil T clips were attached at the fixed ends of the fiber. Fibers were mounted on two stainless steel hooks in a temperature-cooled flow-through acrylic chamber (volume = 120 µl) on the stage of an Olympus IMT-2 inverted microscope. One end of the fiber was attached to a servo-motor (General Scanning, G120DT) with a step time of 800 µs, and the other end of the fiber was attached to a force transducer (Aksjeselskapet, AE-801) with a resonant frequency of 5 kHz. A reticule in the eyepiece of the inverted microscope was used to measure the length [×10 Olympus Plan 10, 0.30 numerical aperture (NA)], width (xy-plane), and depth (xz-plane) of fibers (×40 Olympus LWD CD Plan 40, 0.55 NA). The depth measurements were made by setting the microscope fine focus to zero while focusing on the top of the fiber and then focusing through the fiber to the bottom. In a previous study, we obtained a correction factor to account for z-axis distortion due to the optics used (9). First-order laser diffraction (He-Ne laser, UDT Sensors, LSC 30D) was used to set the sarcomere length to 2.5 µm, the optimal length (Lo) for force development. Sarcomere length was stabilized during experiments with Brenner cycling (4) as modified by Sweeney et al. (40). Force signals were digitized at 1 kHz using a Lab-View data-acquisition board (National Instruments). Initially, fibers were perfused with a pCa 9.0 solution to obtain a baseline force measurement. While the fiber was kept in the same location, the perfusate was switched to a pCa 4.0 solution to maximally activate fibers. Fibers were exposed to the pCa 4.0 solution until maximum force was stable, and fibers were then reexposed to the pCa 9.0 solution to verify that force returned to the original baseline level. Fmax (N/cm2) was calculated by dividing the maximum isometric force during pCa 4.0 activation by the corrected fiber cross-sectional area. Force per half-sarcomere MHC content (N/µg MHC content) was obtained by dividing the maximum isometric force by MHC content per half-sarcomere (see below). Muscle fiber stiffness was determined from sinusoidal length oscillations (0.2% Lo) at 2 kHz, during activation at pCa 4.0 in the presence and absence (rigor solution) of ATP. Stiffness measurements under both conditions were normalized for fiber cross-sectional area. Rigor stiffness was assumed to reflect full recruitment of all available cross bridges. Thus the fraction of cross bridges in the strongly bound force-generating state was estimated from the ratio of fiber stiffness during rigor solution compared with activation at pCa 4.0 (with ATP) (3).Determination of MHC content per half-sarcomere. MHC content per half-sarcomere in single rat Diam fibers was determined as previously described (9). Briefly, the first step in determining MHC content per half-sarcomere involved an accurate determination of the volume of each fiber segment. Single fibers or neonatal bundles (~1.5-2.5 mm in length) were fixed in 4% paraformaldehyde for 30 s and then placed on a microscope stage (Nikon Optiphot-2) with a MTI CCD72 camera. With the use of a Nikon Plan ×20 lens (0.5 NA), a fiber image was projected onto a video screen. From this projected image, the number of sarcomeres in series was counted and fiber cross-sectional area was determined from width and depth measurements. Because force measurements were obtained at a sarcomere length of 2.5 µm, fiber cross-sectional area was normalized to this sarcomere length. This was accomplished by measuring the sarcomere length in the fixed fiber segment, dividing this by 2.5 µm, and multiplying this ratio by the fiber cross-sectional area. From the number of sarcomeres in series and the fiber volume measurements, the volume of a half-sarcomere was determined.
Fibers were then transferred to 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. To denature the fibers, samples were boiled for 2 min. A modification of the procedure by Sugiura and Murakami (39) was used to prepare gradient gels. The separating gel contained 5-8% acrylamide (pH 8.8) with 25% glycerol, and the stacking gel contained 3.5% acrylamide (pH 6.8) (8 × 10 cm, 0.75 mm thick, Hoefer SE250). Control samples of Diam bundles were loaded at a 1:200 dilution of SDS samples buffer [~9.0 ng/µl MHC concentration determined by Bradford method (2)] to compare migration patterns of the MHC isoforms. Volumes of 10 µl per lane were loaded onto the gel. After electrophoresis at 16 mA for ~18 h, gels were silver stained according to the procedure described by Oakley et al. (23). The migration patterns of the MHC isoforms were confirmed with Western analysis as previously described (9). Increasing volumes of a known concentration of purified rabbit MHC (Sigma M-3889) were loaded onto the gel to establish a standard curve for each gel. MHC concentrations were verified using the Bradford method (2), as previously described (9). Images of the silver-stained gels were obtained using a high-resolution scanner (Microtek ScanMaker 5, 600 dpi). The migration band corresponding to each MHC isoform was delineated, and the brightness-area product (BAP) was determined after local background subtraction. The BAP and MHC concentration relationships were linear across a range from 0.01 to 0.25 µg/µl. MHC concentration of the single fiber or the neonatal bundle was determined from the standard curve. The MHC concentration of the single fiber was then multiplied by the half-sarcomere volume of the fiber to determine the MHC content per half-sarcomere.Immunohistochemical determination of MHC isoform expression in Diam fibers. D0 frozen muscle samples were cut into five serial transverse sections at 10 µm thickness and reacted with antibodies against different MHC isoforms. This technique has been previously described (37, 47). Briefly, pairs of mouse IgG or IgM primary antibodies were used in the majority of samples, e.g., anti-MHCslow (Novocastra, IgG), anti-MHC2A [Blau A4.74 (16), IgG or Blau N1.551 (16), IgM], and anti-MHCneo (Novocastra, IgG). The primary antibodies were diluted in PBS with 0.5% bovine serum albumin (5 mg/ml) and reacted with the muscle samples for ~2 h at room temperature. Samples were washed in PBS, and Cy3- or Cy5-conjugated secondary antibodies (goat anti-mouse IgG or goat anti-mouse IgM) were applied for 45 min at room temperature. Double labeling of MHC isoform expression in the same section with antibody pairs was achieved in this manner with minimal cross-reactivity. Confirmation of this was obtained by adding the alternate secondary antibody to a muscle section incubated with only IgG or IgM primary antibody. In addition, nonspecific reactivity of all primary antibodies was not a factor, as indicated by sections incubated with only the secondary antibodies. Coexpression of different MHC isoforms within single fibers could be determined with these methods.
Statistical analysis.
A two-way ANOVA was used to make comparisons of fiber cross-sectional
area, Fmax, 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 across developmental ages. At D28 and in the adult rat
Diam, where singular MHC isoform expression occurs, comparisons were made across fibers expressing different MHC isoforms. At D14, coexpression of MHCslow, MHC2A,
MHC2X, and MHCneo occurs along with singular
expression of MHCslow. At this developmental time point,
single fibers were grouped according to the dominant MHC isoform
expressed. The relative proportion of MHC isoforms within single fibers
at D14 was reported (Table 1). At D0, the majority of fibers expressed the MHCneo isoform (~90%
according to immunohistochemical data); therefore, these fibers were
considered relatively uniform, and a comparison across fibers within
this developmental time point was not made. When appropriate, a
Student's t-test with Bonferroni correction was used for
post hoc analyses. Values are reported as means ± SE. Statistical
significance was indicated by a P value of <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 in 16 neonatal bundles and 127 single fibers by SDS-PAGE and Western blot
analysis (Fig. 1). At D0, the
MHCneo, MHCslow, and MHC2A isoforms
were all expressed but in different proportions. The MHC isoform
composition of single Diam fibers could not be determined directly at D0 because of the technical difficulty of dissecting the
extremely small and fragile fibers at this age. However,
immunohistochemical analysis provided insight into the MHC isoform
composition of single Diam fibers at D0 (Fig.
2). In the neonatal Diam,
MHCneo was the predominant isoform expressed, comprising
~67% of total MHC expression and being expressed in ~92% of all
fibers. Expression of MHCslow constituted ~18% of total
MHC, and singular expression was noted in only ~8% of all fibers at
D0. Expression of MHC2A accounted for ~15% of total MHC,
and there were no fibers that singularly expressed MHC2A at
D0. Even by D14, no Diam fibers were found that singularly
expressed MHC2A. Expression of MHC2X was not
detected until D14, and then singular expression of MHC2X was not observed by single fiber SDS-PAGE. Overall, it appeared that
most Diam fibers continued to coexpress MHC isoforms at
D14, with the exception of ~10% of all fibers that singularly
expressed MHCslow. On the basis of single-fiber SDS-PAGE,
the relative MHC isoform composition of single Diam fibers
was evaluated, and the relative coexpression of MHC isoforms was found
to vary at D14. There was a predominant expression of one isoform
(e.g., >40% of total MHC), and this formed the basis for
categorization of Diam fibers at D14 (Table 1). Fibers
predominantly expressing MHC2A exhibited a variety of
coexpression patterns as described in Table 1.
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Cross-sectional area.
Accurate cross-sectional area measurements were important for the
measurement of MHC content per half-sarcomere and Fmax. Fiber cross-sectional area was therefore measured while the fiber was
mounted on the stage of an inverted microscope at a set sarcomere length of 2.5 µm. The xy-axis measurements were made using
the ×40, 0.55-NA objective and were accurate within 0.5 µm, whereas the z-axis distortion (~20%) was corrected according to
previous measurements made with confocal microscopy (9).
In the present study, cross-sectional area measurements were made on 13 rat Diam bundles and 246 single fibers. A dramatic increase
in the cross-sectional area of Diam fibers occurred during
early postnatal development (Fig.
3A). The average
cross-sectional area of D0 Diam fibers was determined from
images of D0 muscle cross sections used for immunohistochemistry. The
cross-sectional area of individual D0 fibers was ~200
µm2. The cross-sectional areas of fibers singularly
expressing MHCslow at D0 were not significantly different
from those of fibers coexpressing MHCslow together with
MHCneo and MHC2A. Fibers predominantly
expressing MHC2X at D14 had a slightly greater
cross-sectional area than fibers predominantly expressing
MHC2A, MHCslow, or MHCneo, although the difference was not significant. At D28, the cross-sectional area of
fibers singularly expressing the MHC2X isoform was
significantly greater than that of fibers singularly expressing
MHCslow and MHC2A isoforms. However, the
difference in cross-sectional area between fibers expressing the
MHC2X isoform and other MHC isoforms at D28 was not as
pronounced as that seen in the adult rat Diam (Fig.
3A).
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MHC content per half-sarcomere. MHC content per half-sarcomere was determined in 16 rat Diam bundles and 127 single fibers. During early postnatal development, MHC content per half-sarcomere did not change, with the exception of fibers predominantly expressing MHC2X (Fig. 3B). However, from D28 to adulthood, there was a dramatic increase in MHC content per half-sarcomere across all fibers. This increase in MHC content per half-sarcomere between D28 and adulthood was most pronounced in fibers expressing MHC2X.
In Diam bundles at D0, MHC content per half-sarcomere was comparable with that found in single fibers at D14, regardless of MHC isoform expression. However, even at D14, fibers predominantly expressing MHC2X tended to have greater MHC content compared with other fibers. If corrected for the fact that MHC2X expression accounted for only ~50% of total MHC expression at D14 (Table 1), it is likely that MHC2X content per half-sarcomere was higher even at this early age. This is supported by the fact that the MHC content per half-sarcomere of fibers singularly expressing MHC2X at D28 was significantly higher than that found for D14 fibers. This is in contrast to the switch from coexpression to singular expression of MHC2A and MHCslow between D14 and D28, when MHC content per half-sarcomere remained constant.Fmax.
Fmax was measured in 13 rat Diam bundles and
246 single fibers and found to increase dramatically with postnatal
development (Fig. 4). Fmax of
Diam bundles at D0 was significantly lower than that at all
other developmental ages. The Fmax of fibers predominantly expressing the MHC2X at D14 was significantly greater than
that of fibers predominantly expressing MHC2A and
MHCneo, as well as that of fibers singularly expressing
MHCslow. These differences in Fmax were even
more pronounced at D28, when fibers singularly expressed MHC isoforms.
At D28, fibers expressing MHC2X generated the greatest
Fmax followed in rank order by fibers expressing MHC2A and MHCslow. However, it should be noted
that Fmax did not significantly increase between D14 and
D28 for any fiber type. In contrast, between D28 and adulthood,
Fmax increased by ~30% across all fibers, regardless of
MHC isoform expression. Continuing the pattern first noted at D14,
fibers expressing MHC2X generated the greatest
Fmax in the adult Diam compared with fibers
expressing MHCslow and MHC2A isoforms.
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Force per half-sarcomere MHC content.
Maximum force values of rat Diam bundles and single fibers
were normalized for MHC content per half-sarcomere to determine the
effect of cross-bridge number on Fmax. With the exception of fibers predominantly expressing MHC2X, there was no
change in force per half-sarcomere MHC content between D0 and D14 (Fig. 5). At D14, the force per half-sarcomere
MHC content of fibers predominantly expressing MHC2X was
significantly greater than that of fibers predominantly expressing
MHC2A and MHCneo, as well as that of fibers
singularly expressing MHCslow. Between D14 and D28, the
force per half-sarcomere MHC content of fibers expressing MHCslow did not change. In contrast, the force per
half-sarcomere MHC content of fibers expressing MHC2A
increased significantly. The force per half-sarcomere MHC content of
fibers expressing MHC2X tended to increase between D14 and
D28, but this change was not significant. By D28, fibers expressing
fast MHC isoforms generated greater force per half-sarcomere MHC
content compared with fibers expressing MHCslow. This
pattern was even more pronounced in the adult Diam. From
D28 to adulthood, there was an increase in force per half-sarcomere MHC
content across all fibers.
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Fraction of cross bridges in the force-generating state. To evaluate a possible change in the recruitment of cross bridges during postnatal development, the ratio of fiber stiffness during activation in pCa 4.0 and rigor (pCa 4.0 without ATP) solutions was used as an estimate of the fraction of cross bridges in the force-generating state. The fraction of cross bridges in the force-generating state in neonatal bundles was 0.69 ± 0.02, and the average value in D14 single fibers was 0.79 ± 0.04. The average fraction of cross bridges in the force-generating state in D28 and adult fibers was 0.75 ± 0.02 and 0.75 ± 0.02, respectively. All values represent means ± SE. No significant differences in the fraction of cross bridges in the force-generating state were found across fibers expressing different MHC isoforms or across developmental time points in the rat Diam.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The results of the present study provide new information concerning the progressive increase in Fmax during early postnatal development at the level of single muscle fibers. Between D0 and D28 in the rat Diam, differences in single fiber Fmax emerge, such that fibers expressing fast MHC isoforms generate greater Fmax compared with fibers expressing MHCslow. These fiber-type differences in Fmax cannot be attributed to differences in MHC content per half-sarcomere. The results of the present study suggest that force per cross bridge is higher for fibers expressing fast MHC isoforms.
MHC isoform expression. A decrease in MHCneo expression and a corresponding increase in fast MHC isoform expression characterize early postnatal development in the rat Diam. Furthermore, coexpression of MHCneo with adult MHC isoforms is characteristic of Diam fibers through the first 2 postnatal wk. At D14, singular expression of MHC isoforms was limited to MHCslow; however, by D28, most Diam fibers singularly expressed MHC isoforms. These results are in general agreement with previous observations (18-20, 35, 36, 44-47). However, the present study provides important new information regarding the relative coexpression of MHC isoforms in single fibers during postnatal development. Such information can only be obtained by single-fiber analysis. Unfortunately, at D0, it was not possible to reliably dissect the extremely small (~200 µm2) and fragile fibers in the rat Diam. However, the predominant expression of MHCneo at this time point agrees with previous results from our laboratory (18, 35, 36, 44-47). At D14, although there was typically a predominant MHC isoform expressed (i.e., >40% of total MHC), no fibers were found that coexpressed MHC isoforms in only trace amounts (i.e., <10%). Indeed, it was common for three MHC isoforms (e.g., MHCneo, MHCslow, and MHC2A) to be coexpressed in relatively equal amounts. It remains unresolved whether MHC isoforms are coexpressed equally along the length of fibers or whether local regions [e.g., a "nuclear domain" (13, 25)] comprise predominantly a single isoform.
Fiber cross-sectional area. At D0 and D14, the cross-sectional areas of Diam fibers were relatively homogeneous (<10% coefficient of variation). By D28, the cross-sectional area of Diam fibers expressing the MHC2X isoform was ~30% greater than that of fibers expressing MHC2A and MHCslow isoforms. In adults, Diam fibers expressing MHC2X were three times larger than fibers expressing MHC2A and MHCslow. These results are consistent with previous reports of postnatal growth of Diam fibers (46, 47).
Coexpression of MHC isoforms during postnatal development may confound fiber-type differences in cross-sectional area. At D14, Diam fibers predominantly expressing MHC2X were slightly larger than fibers predominantly expressing other MHC isoforms. This difference was not significant, however, perhaps due to the extent of MHCneo and MHC2A coexpression in these fibers (~50% of total MHC; Table 1). As shown in Fig. 3A, changes in Diam fiber cross-sectional area were relatively minor until D28, when significant hypertrophy of fibers singularly expressing MHC2X occurred. There was no significant growth of fibers expressing MHC2A and MHCslow isoforms between D14 and D28. Most impressive was the dramatic but disproportionate growth of Diam fibers from D28 to adulthood (D84). However, this increase in fiber cross-sectional area in the rat Diam from D28 to adulthood cannot be explained by transitions in MHC isoforms, since such transitions were already complete by D28.MHC content per half-sarcomere. MHC content per half-sarcomere in rat Diam fibers increased with postnatal development, with the greatest increase in fibers expressing the MHC2X isoform. At D14, there was no significant difference in MHC content per half-sarcomere across fibers expressing different MHC isoforms. However, the substantial coexpression of MHCneo and MHC2A in D14 fibers predominantly expressing MHC2X may have diluted any isoform-dependent difference in MHC content per half-sarcomere in these fibers. By D28, MHC content per half-sarcomere in fibers singularly expressing MHC2X was ~50% greater than that in fibers singularly expressing MHC2A and MHCslow isoforms. It is important to note that MHC content per half-sarcomere in fibers expressing MHC2A and MHCslow did not increase significantly until adulthood when a significant increase in MHC content per half-sarcomere occurred across all fiber types. Like the dramatic increase in cross-sectional area between D28 and adulthood, this increase in MHC content per half-sarcomere across all fiber types cannot be attributed to transitions in MHC isoform expression.
As previously shown (9), these results provide further support for a lower MHC content per half-sarcomere in fibers expressing MHC2A and MHCslow compared with fibers expressing MHC2X. These findings are consistent with a higher mitochondrial volume density in fibers expressing MHC2A and MHCslow isoforms compared with fibers expressing MHC2X and MHC2B isoforms (33).Fmax. Fmax increased during postnatal development in the rat Diam across all fiber types. These results are consistent with the increase in Fmax of Diam fiber bundles reported in previous studies (18, 35, 46, 47). Obviously, these results at the single-fiber level are an important extension of these previous observations. Of particular interest, Fmax increased dramatically from D0 to D14 across all MHC isoforms, whereas fiber cross-sectional area and MHC content per half-sarcomere remained constant. Thus fiber-type differences in Fmax precede fiber-type differences in cross-sectional area and MHC content per half-sarcomere in the developing rat Diam.
The results indicating fiber-type differences in specific force in the rat Diam, even at D14, are consistent with previous reports from this laboratory (9, 33, 34) as well as other studies in the rat Diam (6) and in limb muscles (1, 22). However, it should be pointed out that some controversy does exist as to whether fiber-type differences in specific force exist across all muscles (8). A number of studies examining fiber-type differences in specific force have compared fibers from a predominantly fast-twitch muscle with fibers from a predominantly slow-twitch muscle. Differences in physiological function, activation history, and load can influence the contractile properties of these muscles. Few studies have examined fiber-type differences in specific force within the same muscle to eliminate such confounding factors. Previous studies from our laboratory indicated a strong correlation between postnatal changes in Fmax of rat Diam fiber bundles and the relative expression of fast MHC isoforms (18, 35). However, these studies also warned that factors other than changes in the relative expression of fast MHC isoforms must also influence the postnatal changes in Fmax. For example, Johnson and colleagues (18) found very little change in the relative expression of fast MHC isoforms between D3 and D7 and between D21 and adulthood, whereas Fmax of Diam fiber bundles displayed the greatest increase during these time periods. Similarly, MHC isoform expression did not change significantly between D28 and adulthood in the present study; however, single-fiber Fmax increased dramatically during this time period. In fact, the greatest transition in MHC isoform expression in Diam fibers occurs between D14 and D28, yet Fmax of single fibers does not significantly increase during this time period. Thus the increase in Fmax during postnatal development of rat Diam fibers does not appear to be solely dependent on MHC isoform expression.Force per MHC content. In the present study, normalizing maximum Diam fiber force for half-sarcomere MHC content did not eliminate differences in force associated with MHC isoform expression or postnatal development. At D28 and in adults, force per half-sarcomere MHC content was greater in fibers expressing MHC2A and MHC2X isoforms compared with fibers expressing MHCslow. These results are consistent with our previous observations in the adult rat Diam (9). These results indicate that fibers expressing MHC2A and MHC2X isoforms have greater force per cross bridge than fibers expressing MHCneo and MHCslow isoforms.
Between D0 and D14, force per half-sarcomere MHC content increased by ~50%, but only in Diam fibers predominantly expressing MHC2X. Of interest is the fact that, at D14, the force per half-sarcomere MHC content was not greater in Diam fibers predominantly expressing the MHC2A isoform. However, between D14 and D28, the force per half-sarcomere MHC content in Diam fibers predominantly expressing the MHC2A isoform increased dramatically, approximating that of fibers expressing MHC2X. The underlying basis for this difference in the development of force-generating capacity in fibers expressing fast MHC isoforms remains unresolved. It is clear from the results of the present study that factors other than MHC isoform expression contribute to both fiber-type and postnatal differences in Fmax. One possibility is that the lateral spacing of the filament lattice may vary with fiber-type and postnatal development and thus affect the probability of cross-bridge attachment necessary for force generation. It is also possible that there are developmental transitions in the expression of structural proteins important in maintaining sarcomere stability and thus force generation. For example, titin is a large structural protein in skeletal muscle fibers that provides a major contribution to passive mechanical properties (14, 42) as well as positional stability of thick filaments during isometric contraction (15). It has been demonstrated that different isoforms of titin exist in fast-twitch vs. slow-twitch skeletal muscles, and these isoforms differ in their elasticity (41). Differences in titin isoform expression in rat Diam fibers have not been explored. It is also unknown whether there are postnatal transitions in titin isoform expression. It is possible that changes in titin isoform expression could contribute to the differences in Fmax across Diam fibers and the changes in Fmax during postnatal development.Influence of cross-bridge cycling kinetics on specific force. On the basis of Huxley's two-state model of cross-bridge cycling (17), cross bridges cycle between a force-generating state, in which cross bridges are strongly bound to actin, and a non-force-generating state, in which cross bridges are detached from actin. It might be expected that, with slower cross-bridge cycling kinetics and a longer duty cycle for cross-bridge attachment, there would be an increase in the fraction of strongly bound cross bridges and a greater Fmax (12). To evaluate this possibility in the present study, the fraction of strongly bound cross bridges was estimated from measurements of muscle fiber stiffness. However, the fraction of strongly bound cross bridges during maximum Ca2+ activation did not change during early postnatal development, and thus this could not account for the lower Fmax generated by fibers expressing MHCslow and MHCneo. These results are consistent with our laboratory's previous studies in the adult rat Diam, in which no difference in the fraction of strongly bound cross bridges during maximum Ca2+ activation was found across fibers expressing different MHC isoforms (9, 34) despite an approximately twofold difference in cross-bridge cycling kinetics (as estimated by the rate of force redevelopment after rapid release and restretch) (34). In a previous study on the superfast swim bladder muscle in toadfish, Rome and colleagues (26) reported that the superfast cross-bridge cycling kinetics of this muscle was associated with a lower fraction of strongly bound cross bridges during maximum activation and reduced specific force. However, these investigators also reported that white (fast-twitch) muscle fibers in the toadfish actually generated slightly greater specific force than red (slow-twitch) muscle fibers despite an approximately threefold difference in cross-bridge cycling kinetics.
Force per cross bridge has been estimated from in vitro motility assays measuring unitary force. The results of previous studies examining unitary force in cardiac V1 and V3 myosins have been controversial, with some studies reporting no difference in force (5, 28, 38) and others reporting that V3 myosin generates greater force than V1 (21, 27). These discrepant results could be due to incorrect orientation of myosin molecules in the in vitro system or differences in the actual number of myosin molecules involved in the force measurement. These are all problems inherent to the in vitro assay, and further work is needed to determine potential differences in unitary force between myosin molecules.Functional significance. Neonatal rats demonstrate increased respiratory rates and lower tidal volumes compared with adults (24). The reliance on rapid, shallow breaths relates in part to the distortion of the highly compliant neonatal chest wall. It may also reflect the inability of the neonatal Diam to generate sufficient force to accommodate deeper breaths. During development, the increase in Diam force-generating capacity coincides with the development of a more stabile chest wall. The Diam becomes more heterogeneous after the emergence of MHC2X isoform expression, and this coincides with the capacity for differential recruitment of motor units (31). In the adult Diam, quiet breathing is accomplished by the recruitment of fatigue-resistant motor units comprising fibers that express MHCslow and MHC2A isoforms (29, 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. During early postnatal development (up to D14), polyneuronal innervation of Diam fibers exists (31), and this limits the efficacy of differential motor unit recruitment. It is likely that the coincidence of developmental changes in innervation pattern, MHC isoform expression, and force-generating capacity in Diam fibers supports the emergence of a wider range of functional requirements and motor behaviors characteristic of the adult.
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ACKNOWLEDGEMENTS |
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We thank Dr. W. Z. Zhan for assistance in 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 10 August 2000; accepted in final form 16 October 2000.
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REFERENCES |
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TOP
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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, MHCneo; 
,
MHCslow; 
, MHC2A;
, MHC2X. Values represent means ± SE.
*Significantly different (P < 0.05) from fibers
expressing MHCslow. #Significantly different
(P < 0.05) from fibers expressing MHC2A.
+Significantly different (P < 0.05) from D14 values.
=Significantly different (P < 0.05) from day
28 (D28) values.
