Journal of Applied Physiology Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 94: 1896-1902, 2003. First published January 31, 2003; doi:10.1152/japplphysiol.00617.2002
8750-7587/03 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
94/5/1896    most recent
00617.2002v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sieck, G. C.
Right arrow Articles by Zhan, W.-Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sieck, G. C.
Right arrow Articles by Zhan, W.-Z.
Vol. 94, Issue 5, 1896-1902, May 2003

Changes in actomyosin ATP consumption rate in rat diaphragm muscle fibers during postnatal development

Gary C. Sieck, Y. S. Prakash, Young-Soo Han, Yun-Hua Fang, Paige C. Geiger, and Wen-Zhi Zhan

Departments of Anesthesiology and Physiology and Biophysics, Mayo Medical School, Rochester, Minnesota 55905


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Early postnatal development of rat diaphragm muscle (Diam) is marked by dramatic transitions in myosin heavy chain (MHC) isoform expression. We hypothesized that the transition from the neonatal isoform of MHC (MHCNeo) to adult fast MHC isoform expression in Diam fibers is accompanied by an increase in both the maximum velocity of the actomyosin ATPase reaction (Vmax ATPase) and the ATP consumption rate during maximum isometric activation (ATPiso). Rat Diam fibers were evaluated at postnatal days 0, 14, and 28 and in adults (day 84). Across all ages, Vmax ATPase of fibers was significantly higher than ATPiso. The reserve capacity for ATP consumption [1 - (ratio of ATPiso to Vmax ATPase)] was remarkably constant (~55-60%) across age groups, although at day 28 and in adults the reserve capacity for ATP consumption was slightly higher for fibers expressing MHCSlow compared with fast MHC isoforms. At day 28 and in adults, both Vmax ATPase and ATPiso were lower in fibers expressing MHCSlow followed in rank order by fibers expressing MHC2A, MHC2X, and MHC2B. For fibers expressing MHCNeo, Vmax ATPase, and ATPiso were comparable to values for adult fibers expressing MHCSlow but significantly lower than values for fibers expressing fast MHC isoforms. We conclude that postnatal transitions from MHCNeo to adult fast MHC isoform expression in Diam fibers are associated with corresponding but disproportionate changes in Vmax ATPase and ATPiso.

myosin heavy chain; fiber types; skeletal muscle; immunohistochemistry


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN SKELETAL MUSCLES SUCH AS the diaphragm muscle (Diam), myosin heavy chain (MHC) functions as a molecular motor, converting chemical (ATP) into mechanical energy, thus driving muscle contraction (5). In adult skeletal muscle, several MHC isoforms exist, and the singular expression of these MHC isoforms in muscle fibers forms the basis for histochemical classification of different fiber types (18, 24, 25, 31). In the neonatal rat Diam, a neonatal isoform of MHC (MHCNeo) is predominantly expressed, most often in combination with MHCSlow or MHC2A isoforms (10, 16, 18, 30, 33). During the first 3-4 postnatal weeks, a dramatic transition in MHC isoform expression occurs in the rat Diam, with the complete disappearance of MHCNeo expression by ~28 days of age (D-28). Concurrently, expression of MHCSlow and MHC2A increases, and expression of MHC2X and MHC2B emerges (16, 18, 33).

The expression of different MHC isoforms is associated with varying contractile properties of muscle fibers (3, 10-12, 24, 27, 28, 32). For example, muscle fibers expressing adult fast MHC isoforms (MHC2A, MHC2X, and MHC2B) have faster maximum shortening velocities than fibers expressing MHCSlow. In developing muscle fibers coexpressing MHC isoforms, Reiser and colleagues (22, 23) reported that maximum shortening velocity correlated with the amount of embryonic MHC (presumably MHCEmb and MHCNeo) isoform expressed. Furthermore, it was suggested that fibers expressing embryonic MHC had maximum shortening velocities intermediate to fibers expressing MHCSlow and adult fast MHC isoforms. This may explain, at least in part, the slower maximum shortening velocity of the rat Diam during early postnatal development, when MHCNeo and MHCSlow contribute predominantly to the MHC isoform composition of the muscle (16, 27, 29, 30, 35). The lower maximum specific force (force normalized for fiber cross-sectional area) and greater fatigue resistance of the neonatal Diam may also be partially explained by the predominant expression of MHCNeo and MHCSlow isoforms (10, 16, 29, 33, 35).

In previous studies (14, 27, 31), our laboratory employed a quantitative histochemical procedure to determine the maximum velocity of the actomyosin ATPase reaction (Vmax ATPase) in muscle fibers expressing different MHC isoforms. In the rat Diam, we found that fibers expressing MHC2X and MHC2B isoforms display significantly higher Vmax ATPase compared with fibers expressing MHCSlow and MHC2A isoforms. In other studies (14, 27, 28), we used an NADH-linked fluorometric procedure to determine the ATP consumption rate of single permeabilized muscle fibers during maximum isometric activation (ATPiso). In the rat Diam, we found that ATPiso was highest in fibers expressing MHC2X and MHC2B and lowest in fibers expressing MHCSlow and MHC2A. We found that, for all fibers, ATPiso was substantially less than Vmax ATPase. This is not surprising because it is well known that ATP consumption rate increases during shortening and work performance in muscle (7, 8). The reserve capacity for ATP consumption of muscle fibers can be defined as the difference between ATP consumption during isometric contraction vs. the maximum velocity of the actomyosin ATPase reaction. According to the Fenn effect (7), the ATP consumption of muscle fibers during shortening will be intermediate to these two extremes, peaking during isovelocity shortening at a value corresponding to maximum power output. We found that the reserve capacity for ATP consumption of Diam fibers expressing MHC2X and MHC2B isoforms was lower than that of fibers expressing MHCSlow and MHC2A. This difference in reserve capacity for ATP consumption may account, at least in part, for their greater fatigability of Diam fibers expressing MHC2X and MHC2B (9, 26). In the present study, we hypothesized that the postnatal transition from MHCNeo to adult fast MHC isoform expression in Diam fibers is accompanied by an increase in Vmax ATPase and ATPiso and a reduction in the reserve capacity for ATP consumption.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

All experimental procedures were approved by the Institutional Animal Care and Use Committee at Mayo and were in strict accordance with the American Physiological Society Animal Care Guidelines. Experiments were performed on male Sprague-Dawley rats at postnatal days 0, 14, 28 (D-0 to D-28), and 84 (adults). Pregnant mothers were received at 14 days gestation, and after parturition the litter size was culled to eight pups. Pups were not studied from smaller litters to ensure comparable postnatal growth of the pups. Body weights of the pups were measured daily. At D-21, the pups were weaned, and thereafter they were housed two per cage. The animals were randomly assigned to two experimental groups. In one group (n = 6 at each age), Vmax ATPase of Diam fibers was determined by using a quantitative histochemical procedure. In the second group, ATPiso of single permeabilized Diam fibers was determined during maximum Ca2+ activation by using an NADH-linked fluorometric technique. At the prescribed day, rats were anesthetized by intramuscular injection of ketamine (60 mg/kg) and xylazine (2.5 mg/kg). The Diam was then rapidly excised, and segments were dissected from the right midcostal region.

Measurement of Vmax ATPase. The quantitative histochemical procedure for measuring Vmax ATPase in type-identified muscle fibers has been previously described (1, 31). Muscle segments were stretched to optimal length (1.5 times resting excised muscle length) (21) and then rapidly frozen in isopentane cooled by liquid nitrogen. From frozen muscle segments, serial transverse sections were cut at 10-µm thickness by use of a cryostat kept at -20°C (model 2800E Frigocut, Reichert-Jung). In the histochemical actomyosin ATPase reaction, the amount of inorganic phosphate ions (Pi) liberated by the hydrolysis of ATP is determined in a two-step process: 1) the liberated Pi is reacted with a lead ammonium citrate-acetate complex to form a lead phosphate precipitate in the tissue section; and 2) the lead phosphate precipitate is converted into a brown-colored lead sulfide precipitate by reaction with sodium sulfide. The concentration of the lead sulfide precipitate in muscle fibers is then determined from optical density (OD) measurements by using the Lambert-Beer equation
[NBT-dfz] = OD / <IT>kl</IT>
where OD is the optical density of the muscle fiber measured at 550 nm (the peak absorbency wavelength for the lead sulfide precipitate), k is the molar extinction coefficient for the lead sulfide precipitate (1,450 mol-1 · cm-1), and l is the path length for light absorbency (10-µm section thickness). In previous studies, we determined that the actomyosin ATPase reaction is linear for at least a 9-min period (1, 31). A single endpoint reaction time of 4 min was selected as well as the 10-µm section thickness to limit OD measurements to <1.0 OD units and thereby reduce measurement errors. The Vmax ATPase was determined in alternate cross sections of the same fibers by varying the ATP concentration in the incubation media from 0.0 to 5.0 mM (4 replicate sections each at 0.0, 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mM ATP). This was necessary because sufficient ATP could not be added to the incubation medium to avoid substrate-limiting the actomyosin ATPase reaction. A Lineweaver-Burk transformation relating velocity of the actomyosin ATPase reaction (OD570 min-1) and ATP concentration (Fig. 1) was performed to determine Vmax ATPase and the apparent Michaelis-Menten rate constant of the actomyosin ATPase reaction (1, 31). On the basis of the stoichiometric relationships of the Pi sulfide exchange in the histochemical reactions, Vmax ATPase was expressed as millimoles Pi per liter per minute.


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 1.   Lineweaver-Burk plot displaying the dependence of the velocity (V) of the actomyosin ATPase reaction on ATP concentration of the incubation medium for diaphragm muscle (Diam) fibers expressing different myosin heavy chain (MHC) isoforms. The maximum velocity of the actomyosin ATPase reaction (Vmax ATPase) was determined from the y-intercept of this relationship. OD, optical density.

Densitometric measurements on single muscle fibers were performed from microscopic images digitized at 8-bit resolution (256 gray levels) into a 1,024 × 1,024 array of picture elements (pixels) by using a video image-processing system (Metamorph). The image-processing system was calibrated for densitometry by using a set of neutral density filters ranging from 0.02 to 2.00 OD units. At OD values of <= 1.0, measurement errors were <1.5%, whereas at OD values >1.0 measurement errors were ~3% (31). Repeated measurements of the same image field (0.4 OD units) were found to be reproducible with a confidence of >99% (31). The light intensity of the microscope was adjusted before images of the muscle sections were digitized to utilize nearly the full 256 gray-level range of the video scanner but to avoid light saturation. Subsequently, light intensity of the microscope was not adjusted.

MHC immunohistochemistry. Muscle cross sections were reacted with mouse primary antibodies against different MHC isoforms as previously described (31). Briefly, to detect MHC isoform coexpression, pairs of mouse IgG or IgM primary antibodies were used, e.g., anti-MHCSlow (Novocastra, IgG; 1:100), anti-MHC2A (Blau A4.74, IgG; 1:1), anti-MHC2B (BFF3, IgM, purified from mouse hybridoma, German Collection of Microorganisms and Cell Culture; 1:1), and anti-MHCNeo (Novocastra; 1:10). However, to determine MHC2X expression, only a single antibody was used, e.g., anti-MHCAll-2X (BF-35, IgG, purified from mouse hybridoma, German Collection of Microorganisms and Cell Culture; 1:1). Primary antibodies were diluted in PBS containing 0.5% bovine serum albumin (5 mg/ml), and the muscle section was incubated in these primary antibodies for ~2 h at room temperature. Thereafter, the sections were washed in PBS and reacted with Cy3- and Cy5-conjugated secondary antibodies (goat anti-mouse IgG or goat anti-mouse IgM; 1:200) for 3-4 h at room temperature. Sections incubated with only the secondary antibodies served as controls for nonspecific reactivity of all primary antibodies. The slides were imaged with a Bio-Rad (MRC500/600) confocal system mounted on an Olympus BH2 microscope. The imaging system was calibrated for morphometry by use of a stage micrometer.

Single fiber preparation. Muscle fibers used for measurement of ATPiso were cut into ~3-mm-wide bundles, stretched and pinned to optimal length on cork, and placed for 24 h in a relaxing solution at 5°C consisting of 85 mM K+, 1 mM free Mg2+, 5 mM MgATP, 7 mM EGTA, propionate as the major anion, and 10-9 M free Ca2+ (pCa 9); imidazole was used to maintain the pH at 7.0 ± 0.02 and to adjust the ionic strength to 150 mM. The fiber bundles were then transferred to relaxing solution containing 50% glycerol (vol/vol) and stored at -20°C for 2-4 wk. Glycerinated fiber bundles were transferred to a relaxing solution containing 10 mM dithiothreitol, and from Diam in D-14, D-28, and adult animals, single fibers were dissected under a dissecting microscope. At D-0, it was not possible to reliably dissect single fibers, so at this age small fiber bundles (~5 fibers) were dissected. The dissected fibers were transferred to relaxing solution containing 10 mM dithiothreitol and 1% Triton X-100 for 20-30 min to permeabilize the plasma membrane. The permeabilized fibers were then transferred to 50% glycerol relaxing solution before measurements of ATPiso.

Measurement of ATPiso. The NADH-linked fluorometric technique used to measure ATPiso in single permeabilized fibers has been previously described (13, 14, 20, 27). Permeabilized fibers were mounted at optimal length (2.5 µm) between force and displacement transducers in a quartz cuvette that was perfused with solutions containing a free ionized Ca2+ concentration of either 1 nM (pCa 9.0) or 100 µM (pCa 4.0) maintained at 15°C. The ATP solutions consisted of relaxing solution and activating solution, containing 5 mM phospho(enol)pyruvate (PEP), 0.2 mM reduced B-nicotinamide adenine dinucleotide (NADH), 100 U/ml pyruvate kinase (PK), 140 U/ml lactate dehydrogenase (LDH), and 0.2 mM P1,P5-di(adenosine-5')pentaphosphate. The NADH-linked enzymatic assay involves the following reactions
(1)

ADP + PEP <LIM><OP><ARROW>→</ARROW></OP><UL> PK</UL></LIM> pyruvate + ATP (2)

(3)
In reaction 1, ATP is hydrolyzed by the actomyosin ATPase to ADP and Pi during detachment of the myosin head from the myosin-binding domain of actin (15). In reaction 2, ATP is regenerated from ADP and PEP by PK. In reaction 3, the resulting pyruvate is converted to lactate by LDH, which results in stoichiometric conversion of fluorescent NADH to nonfluorescent NAD+. The amount of ADP produced by the actomyosin ATPase is determined by measuring the rate of decrease in NADH fluorescence signal (at 450 nm) after stopping the flow in the quartz cuvette. Previous work on single permeabilized fibers has shown that mitochondrial ATPase and sarcoplasmic reticulum ATPase make no detectable contribution to the observed ATPase activity (17). A mercury lamp was used as the excitation light source, and excitation wavelength was restricted to 340 nm by using a band-pass filter. Emitted fluorescence (limited to 450 nm by use of an interference cutoff filter) was detected by using a photomultiplier positioned perpendicular to the axis of excitation, and the photomultiplier gain was fixed during ATPiso measurements.

Baseline ATP consumption rate was determined at a pCa of 9.0 (relaxing condition), and ATPiso was then determined at pCa 4.0 (maximum Ca2+ activation). During these measurements, flow through the cuvette was stopped for 15 s, and the rate of extinction of the NADH fluorescence signal was recorded. This cycling of flow every 15 s through the cuvette was regulated by using a computer-controlled peristaltic pump. Calibration involved measurements of fluorescence intensity for known amounts of NADH. On the basis of changes in the NADH fluorescence intensity, ATPiso was determined by subtracting the baseline ATP consumption rate measured at pCa 9.0 from that obtained at pCa 4.0.

Gel electrophoretic determination of MHC isoform expression in single fibers. After the completion of ATPiso measurements, MHC isoform composition of the single Diam fiber (or fiber bundle at D-0) was determined by SDS-PAGE by using a previously described procedure (10-12, 27, 28). Fibers were placed in 25 µl of SDS sample buffer containing 62.5 mM Tris · HCl, 2% (wt/vol) SDS, 10% (vol/vol) glycerol, and 0.001% (wt/vol) bromophenol blue at pH 6.8. Samples were denatured by boiling for 2 min, and 10-µl volumes of sample were loaded per lane. Diam bundles in a 1:200 dilution of SDS sample buffer (~9.0 ng/µl as determined by the Bradford method; Ref. 4) were run on each gel to compare the migration patterns of identified MHC isoforms. The gels were silver stained to visualize the MHC migration bands after the procedure by Oakley et al. (19). In the case of coexpression of MHC isoforms within a single fiber, the relative expression of each MHC isoform was determined by densitometric analysis.

Statistical analysis. For measurements of Vmax ATPase of Diam fibers, the linearity of the 1/V vs. 1/[ATP] relationship (where V is velocity and [ATP] is ATP concentration) for each fiber was determined by regression analysis. On the basis of the number of ATP concentrations assessed, R2 >0.658 represented a statistically significant linear regression (P < 0.05). The relationships between 1/V and 1/[ATP] were linear for all fibers analyzed in this study (R2 values ranged from 0.88 to 0.98). An analysis of variance was used to evaluate maturational changes in Vmax ATPase and ATPiso of Diam fibers expressing different MHC isoforms. When appropriate, post hoc analyses (unpaired Student's t-test) were also performed. In all cases, statistical significance was established at the 0.05 level. All data are represented as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Body weight and muscle length. During postnatal development, body weights of rats increased rapidly (D-0: 6.5 ± 0.2 g; D-14: 29.6 ± 1.3 g; D-28: 64.9 ± 4.2 g; and D-84: 305.4 ± 15.6 g). Accompanying these changes in body weight, Diam fiber length increased by more than threefold from D-0 to adulthood (D-0: 6.0 ± 0.1 mm; D-14: 8.6 ± 0.1 mm; D-28: 11.4 ± 0.4 mm; D-84: 18.5 ± 0.6 mm).

Developmental transitions in MHC isoform expression. Immunohistochemical analyses provided qualitative information regarding the distribution of MHC isoform expression within single Diam fibers at different postnatal ages (Fig. 2). However, with coexpression of MHC isoforms, immunohistochemistry could not determine the relative amounts of each MHC isoform expressed. In addition, coexpression of the MHC2X isoform could not be unambiguously determined on the basis of immunohistochemistry.


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 2.   MHC isoform expression of Diam fibers was identified by immunohistochemistry. The proportion of Diam fibers expressing different MHC isoforms was then determined for each postnatal age group: days 0, 14, and 28 (D-0, D-14, and D-28; A, B, and C, respectively) and day 84 (adult; D). Values are means ± SE.

At D-0, most Diam fibers expressed MHCNeo, either alone or in combination with MHC2A and/or MHCSlow, whereas a small proportion of Diam fibers singularly expressed MHCSlow. At D-14, singular expression of MHCNeo in Diam fibers was not detected, but coexpression of MHCNeo with MHC2A and/or MHCSlow continued to be observed (Fig. 2). The proportion of Diam fibers singularly expressing MHCSlow remained relatively low at this age. By D-28, MHCNeo was not expressed in the rat Diam, and the pattern of MHC isoform expression generally appeared to be similar to that observed in adults (Fig. 2). However, in contrast to the adult Diam, the proportion of fibers expressing MHC2A at D-28 was greater, whereas fewer fibers expressed MHC2X. In addition, there were no fibers singularly expressing MHC2B at D-28.

MHC isoform expression was also determined in 15 fiber bundles at D-0 and in 175 single fibers (combined total for Diam at D-14, D-28, and D-84) by SDS-PAGE and Western blot analysis (Fig. 3). As mentioned above, the MHC isoform composition of single Diam fibers could not be determined at D-0 by SDS-PAGE, because of the technical difficulty in dissecting single fibers. Instead, bundles of ~5 fibers were dissected and used for determination of MHC isoforms at D-0. In these neonatal Diam fiber bundles, MHCNeo was the predominant isoform expressed, comprising ~65% of the total MHC expression. Expression of MHCSlow and MHC2A constituted ~19% and ~16% of total MHC, respectively.


View larger version (37K):
[in this window]
[in a new window]
  		Fig. 3.   In single Diam fibers, MHC isoform expression was determined by electrophoretic migration patterns. Examples of MHC isoform expression in single fibers from adult and D-14 rat Diam are shown.

By D-14, when single fibers could be reliably dissected, considerable coexpression of MHC isoforms was observed (46 of 62 fibers sampled), with only 16 of 62 fibers displaying singular expression of MHCSlow. No other singular expression of MHC isoforms was observed in Diam fibers at D-14. Among those fibers coexpressing MHC isoforms, 14 fibers coexpressed MHCNeo and MHC2A; 10 fibers coexpressed MHCNeo, MHCSlow, and MHC2A; and 22 fibers coexpressed MHCNeo, MHC2A, and MHC2X. In contrast to the Diam at D-0, MHC2X expression was detected in single fibers at D-14, and MHC2B expression was detected in the whole Diam but not in any of the single fibers sampled.

At D-28, all of 49 fibers that were sampled expressed only a single isoform. Of these fibers, 17 fibers expressed MHCSlow, 22 fibers expressed MHC2A, and 10 fibers expressed MHC2X. Although MHC2B isoform expression was detected in the whole Diam at D-28, it was not detected in any fibers dissected. In the adult group, 64 fibers were dissected, of which 20 fibers expressed MHCSlow, 10 fibers expressed MHC2A, 13 fibers expressed MHC2X, and 21 fibers coexpressed MHC2X and MHC2B.

Vmax ATPase of Diam fibers. The Vmax ATPase of Diam fibers during development varied with MHC isoform expression (Fig. 4). In D-0 Diam, Vmax ATPase was comparable across fibers expressing MHCNeo, either alone or in combination with other MHC isoforms, but slightly higher than that of fibers singularly expressing MHCSlow. In D-14 Diam, Vmax ATPase of fibers coexpressing MHCNeo and MHCSlow increased compared with D-0, whereas Vmax ATPase of fibers singularly expressing MHCSlow did not change.


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 4.   Vmax ATPase and ATPiso of rat Diam fibers at different postnatal ages: D-0, D-14, and D-28 (A, B, and C, respectively) and adult (D). Values are means ± SE.

By D-28, Vmax ATPase varied considerably across fibers expressing different MHC isoforms, with a rank order similar to that found in adult fibers, i.e., lowest for fibers expressing MHCSlow and highest for fibers expressing MHC2X and MHC2B. The actomyosin ATPase activity of fibers expressing MHC2A was intermediate (Fig. 4; P < 0.05).

ATPiso of Diam fibers. The ATPiso of Diam fibers progressively increased from D-0 to D-28, with little change thereafter (Fig. 4). This increase was associated with a transition from MHCNeo to MHC2X and/or MHC2B isoform expression. In adult rat Diam fibers, ATPiso was highest for fibers expressing MHC2X and/or MHC2B, which was about threefold higher than that of fibers expressing MHCSlow (Fig. 4). The ATPiso of fibers singularly or predominantly expressing MHCNeo was similar to that of adult fibers expressing MHCSlow and only about half of that of adult fibers expressing MHC2X and/or MHC2B (Fig. 4).

Reserve capacity for ATP consumption of Diam fibers. The reserve capacity for ATP consumption of Diam fibers was calculated as [1 - (ratio of ATPiso to Vmax ATPase)]. Despite marked changes in both ATPiso and Vmax ATPase, the reserve capacity for ATP consumption remained remarkably constant across age groups (55-60%) (Table 1). However, at D-28 and in adults, the reserve capacity for ATP consumption was slightly higher for fibers expressing MHCSlow compared with fibers expressing MHC2X isoform (Table 1).

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Summary of the average reserve capacity (%) for ATP consumption of Diam fibers at different postnatal ages


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results of the present study clearly demonstrate that there are developmental differences in isometric and isovelocity ATP consumption rates of Diam fibers regardless of MHC isoform expression. Furthermore, these results demonstrate that isometric ATP consumption rate in single muscle fibers does not reflect the maximum enzymatic capacity for ATP consumption (maximum velocity of the actomyosin ATPase reaction) and that ATP consumption rate increases in relation to power output of the fibers, as predicted by the Fenn effect. There was no a priori basis for any prediction with respect to developmental changes in this "reserve capacity" for ATP consumption, which is fundamental to support increasing work performance. To the best of our knowledge, no previous study has reported the ATP consumption rates of neonatal muscle fibers during isometric or isovelocity conditions; therefore, these results are completely novel.

The results of the present study also demonstrated that the transition from MHCNeo to adult fast MHC isoform expression during early postnatal development of the rat Diam is associated with a proportionate increase in both Vmax ATPase and ATPiso. At early postnatal ages, Vmax ATPase and ATPiso were fairly uniform across fibers, most likely reflecting the coexpression of MHCNeo with MHCSlow and MHC2A isoforms. With the expression of MHC2X and MHC2B, fiber type differences in both Vmax ATPase and ATPiso emerged. At each age and for each MHC isoform, Vmax ATPase was higher than ATPiso. Although the reserve capacity for ATP consumption was slightly higher for fibers expressing MHCSlow (and possibly MHCNeo), this reserve capacity remained remarkably constant across postnatal age groups.

Developmental transitions in MHC isoform expression. Postnatal transitions in MHC isoform expression in rat Diam fibers observed in the present study was similar to that reported previously (16, 34, 35). At birth, MHCNeo expression predominated, usually coexpressed with other MHC isoforms. With subsequent development, MHCNeo expression decreased, disappearing completely by D-28. At D-14, MHC2X and MHC2B isoform expression was not detected in single fibers, whereas at D-28 MHC2X was detected, but MHC2B was not. These observations were consistent with our previous observations on single dissected rat Diam fibers at these ages (10).

Maximum velocity of actomyosin ATPase reaction. Previously, we noted that Vmax ATPase varies across Diam fibers expressing different MHC isoforms (6, 27, 28). The results of the present study confirmed these previous observations and further demonstrated that these MHC isoform-specific differences in Vmax ATPase of Diam fibers are apparent even at very early postnatal ages. Among Diam fibers expressing MHCSlow, Vmax ATPase did not increase with age. This also appeared to be the case for Diam fibers expressing MHC2A. The Vmax ATPase of Diam fibers expressing MHC2X at D-28 was only slightly lower than that of fibers expressing this isoform in the adult Diam. These results indicate that the Vmax ATPase of different MHC isoforms in Diam fibers is remarkably stable across early postnatal development.

Isometric ATP consumption rate. Recent studies, using an NADH-linked fluorescence technique, have clearly demonstrated that, in single permeabilized muscle fibers, MHC isoform expression is associated with differences in ATPiso (2, 27, 28). The present study demonstrated that, during the early postnatal transition in MHC isoform expression, there was an increase in ATPiso of Diam fibers. Overall, fibers expressing fast MHC isoforms (MHC2A, MHC2X, and MHC2B) have higher rates of ATP consumption than fibers expressing MHCSlow (2, 27, 28). Similar to Vmax ATPase, the ATPiso of Diam fibers expressing MHCSlow and MHC2A did not change across postnatal development. The ATPiso of Diam fibers expressing MHC2X and/or MHC2B was also similar between D-28 and adults. Thus both Vmax ATPase and ATPiso appear to be intrinsic properties of MHC isoforms.

Reserve capacity for ATP consumption. The observation that Vmax ATPase is substantially higher than ATPiso in skeletal muscle fibers is consistent with previous reports in the adult rat Diam (27) and human vastus lateralis muscle (14) and indicates a substantial reserve capacity for ATP hydrolysis throughout postnatal development. As mentioned above, the reserve capacity for ATP consumption was remarkably constant across age groups, averaging from ~55 to 60%.

In 1923, Fenn observed that energy utilization of skeletal muscle increases in proportion to work (Fenn effect) (7). Therefore, as muscle fibers reach maximum power during shortening, ATP consumption rate should increase (27). Consistent with the Fenn effect, we previously reported that the maximum rate of ATP consumption was achieved at a shortening velocity corresponding to peak power output of Diam fibers (27). Although the maximum ATP consumption rates achieved at peak power output were closer to Vmax ATPase, they were still lower. This may reflect differences in the number of cross bridges contributing to the measured ATP consumption rate during active shortening vs. those contributing to the measurements of Vmax ATPase.

In conclusion, the present study demonstrates that during early postnatal development of the rat Diam, the transition from MHCNeo to adult fast MHC isoform expression is accompanied by an increase in both Vmax ATPase and ATPiso. Furthermore, the results of the present study demonstrate that MHC isoform expression is an important determinant of both Vmax ATPase and ATPiso in rat Diam fibers regardless of age.


    ACKNOWLEDGEMENTS

The authors acknowledge the contributions of Rebecca Macken and Megan Bayrd in these studies.

This research was supported by National Heart, Lung, and Blood Institute Grants HL-34817 and HL-37680.


    FOOTNOTES

Address for reprint requests and other correspondence: G. C. Sieck, Dept. of Physiology & Biophysics, 4-184 W. Joseph, Mayo Medical School, 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.

First published January 31, 2003;10.1152/japplphysiol.00617.2002

Received 9 July 2002; accepted in final form 23 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Blanco, CE, and Sieck GC. Quantitative determination of calcium-activated myosin adenosine triphosphatase activity in rat skeletal muscle fibres. Histochem J 24: 431-444, 1992[ISI][Medline].

2.   Bottinelli, R, Canepari M, Reggiani C, and Stienen GJM Myofibrillar ATPase activity during isometric contraction and isomyosin composition in rat single skinned muscle fibres. J Physiol 481: 663-675, 1994[ISI][Medline].

3.   Bottinelli, R, Schiaffino S, and Reggiani C. Force-velocity relations and myosin heavy chain isoform compositions of skinned fibres from rat skeletal muscle. J Physiol 437: 655-672, 1991[Abstract/Free Full Text].

4.   Bradford, MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976[ISI][Medline].

5.   Cooke, R. The actomyosin engine. FASEB J 9: 636-642, 1995[Abstract].

6.   Edman, KAP The velocity of unloaded shortening and its relation to sarcomere length and isometric force in vertebrate muscle fibres. J Physiol 291: 143-159, 1979[Abstract/Free Full Text].

7.   Fenn, WO. A quantitative comparison between the energy liberated and the work performed by the isolated sartorius muscle of the frog. J Physiol 58: 175-203, 1923.

8.   Fenn, WO. The relation between the work performed and the energy liberated in muscular contraction. J Physiol 58: 373-395, 1924.

9.   Fournier, M, and Sieck GC. Mechanical properties of muscle units in the cat diaphragm. J Neurophysiol 59: 1055-1066, 1988[Abstract/Free Full Text].

10.   Geiger, PC, Cody MJ, Macken RL, Bayrd ME, and Sieck GC. Mechanisms underlying increased force generation by rat diaphragm muscle fibers during development. J Appl Physiol 90: 380-388, 2001[Abstract/Free Full Text].

11.   Geiger, PC, Cody MJ, Macken RL, and Sieck GC. Maximum specific force depends on myosin heavy chain content in rat diaphragm muscle fibers. J Appl Physiol 89: 695-703, 2000[Abstract/Free Full Text].

12.   Geiger, PC, Cody MJ, and Sieck GC. Force-calcium relationship depends on myosin heavy chain and troponin isoforms in rat diaphragm muscle fibers. J Appl Physiol 87: 1894-1900, 1999[Abstract/Free Full Text].

13.   Guth, K, and Wojciechowski R. Perfusion cuvette for the simultaneous measurement of mechanical, optical and energetic parameters of skinned muscle fibres. Pflügers Arch 407: 552-557, 1986[ISI][Medline].

14.   Han, YS, Proctor DN, Geiger PC, and Sieck GC. Reserve capacity of ATP consumption during isometric contraction in human skeletal muscle fibers. J Appl Physiol 90: 657-664, 2001[Abstract/Free Full Text].

15.   Huxley, AF. Muscle structure and theories of contraction. Prog Biophysics Biophys Chem 7: 255-318, 1957.

16.   Johnson, BD, Wilson LE, Zhan WZ, Watchko JF, Daood MJ, and Sieck GC. Contractile properties of the developing diaphragm correlate with myosin heavy chain phenotype. J Appl Physiol 77: 481-487, 1994[Abstract/Free Full Text].

17.   Kerrick, WG, Potter JD, and Hoar PE. The apparent rate constant for the dissociation of force generating myosin cross-bridges from actin decreases during Ca2+ activation of skinned muscle fibres. J Muscle Res Cell Motil 12: 53-60, 1991[ISI][Medline].

18.   LaFramboise, WA, Daood MJ, Guthrie RD, Butler-Browne GS, Whalen RG, and Ontell M. Myosin isoforms in neonatal rat extensor digitorum longus, diaphragm, and soleus muscles. Am J Physiol Lung Cell Mol Physiol 259: L116-L122, 1990[Abstract/Free Full Text].

19.   Oakley, BR, Kirsch DR, and Morris NR. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem 105: 361-363, 1980[ISI][Medline].

20.   Perkins, WJ, Han YS, and Sieck GC. Skeletal muscle force and actomyosin ATPase activity reduced by a nitric oxide donor. J Appl Physiol 83: 1326-1332, 1997[Abstract/Free Full Text].

21.   Prakash, YS, Fournier M, and Sieck GC. Effects of prenatal undernutrition on developing rat diaphragm. J Appl Physiol 75: 1044-1052, 1993[Abstract/Free Full Text].

22.   Reiser, PJ, Kasper CE, Greaser ML, and Moss RL. Functional significance of myosin transitions in single fibers of developing soleus muscle. Am J Physiol Cell Physiol 254: C605-C613, 1988[Abstract/Free Full Text].

23.   Reiser, PJ, Moss RL, Giulian GG, and Greaser ML. Shortening velocity and myosin heavy chains of developing rabbit muscle fibers. J Biol Chem 260: 14403-14405, 1985[Abstract/Free Full Text].

24.   Schiaffino, S, Ausoni S, Gorza L, Saggin I, Gundersen K, and Lomo T. Myosin heavy chain isoforms and velocity of shortening of type 2 skeletal muscle fibres. Acta Physiol Scand 134: 575-576, 1988[ISI][Medline].

25.   Schiaffino, S, Gorza L, Sartore S, Saggin L, Ausoni S, Vianello M, Gundersen K, and Lomo T. Three myosin heavy chain isoforms in type 2 skeletal muscle fibres. J Muscle Res Cell Motil 10: 197-205, 1989[ISI][Medline].

26.   Sieck, GC, Fournier M, Prakash YS, and Blanco CE. Myosin phenotype and SDH enzyme variability among motor unit fibers. J Appl Physiol 80: 2179-2189, 1996[Abstract/Free Full Text].

27.   Sieck, GC, Han YS, Prakash YS, and Jones KA. Cross-bridge cycling kinetics, actomyosin ATPase activity and myosin heavy chain isoforms in skeletal and smooth respiratory muscles. Comp Biochem Physiol B Biochem Mol Biol 119: 435-450, 1998[Medline].

28.   Sieck, GC, and Prakash YS. Cross-bridge kinetics in respiratory muscles. Eur Respir J 10: 2147-2158, 1997[Abstract].

29.   Sieck, GC, Wilson LE, Johnson BD, and Zhan WZ. Hypothyroidism alters diaphragm muscle development. J Appl Physiol 81: 1965-1972, 1996[Abstract/Free Full Text].

30.   Sieck, GC, and Zhan WZ. Denervation alters myosin heavy chain expression and contractility of developing rat diaphragm muscle. J Appl Physiol 89: 1106-1113, 2000[Abstract/Free Full Text].

31.   Sieck, GC, Zhan WZ, Prakash YS, Daood MJ, and Watchko JF. SDH and actomyosin ATPase activities of different fiber types in rat diaphragm muscle. J Appl Physiol 79: 1629-1639, 1995[Abstract/Free Full Text].

32.   Sweeney, HL, Kushmerick MJ, Mabuchi K, Gergely J, and Sreter FA. Velocity of shortening and myosin isozymes in two types of rabbit fast-twitch muscle fibers. Am J Physiol Cell Physiol 251: C431-C434, 1986[Abstract/Free Full Text].

33.   Watchko, JF, and Sieck GC. Respiratory muscle fatigue resistance relates to myosin phenotype and SDH activity during development. J Appl Physiol 75: 1341-1347, 1993[Abstract/Free Full Text].

34.   Zhan, WZ, Miyata H, Prakash YS, and Sieck GC. Metabolic and phenotypic adaptations of diaphragm muscle fibers with inactivation. J Appl Physiol 82: 1145-1153, 1997[Abstract/Free Full Text].

35.   Zhan, WZ, Watchko JF, Prakash YS, and Sieck GC. Isotonic contractile and fatigue properties of developing rat diaphragm muscle. J Appl Physiol 84: 1260-1268, 1998[Abstract/Free Full Text].

J APPL PHYSIOL 94(5):1896-1902
8750-7587/03 $5.00 Copyright © 2003 the American Physiological Society


This article has been cited by other articles:


Home page
 J. Appl. Physiol.Home page
J. G. Ryall, J. D. Schertzer, T. M. Alabakis, S. M. Gehrig, D. R. Plant, and G. S. Lynch
Intramuscular {beta}2-agonist administration enhances early regeneration and functional repair in rat skeletal muscle after myotoxic injury
J Appl Physiol, July 1, 2008; 105(1): 165 - 172.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
C. B. Mantilla and G. C. Sieck
Key aspects of phrenic motoneuron and diaphragm muscle development during the perinatal period
J Appl Physiol, June 1, 2008; 104(6): 1818 - 1827.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
C. B. Mantilla, R. V. Sill, B. Aravamudan, W.-Z. Zhan, and G. C. Sieck
Developmental effects on myonuclear domain size of rat diaphragm fibers
J Appl Physiol, March 1, 2008; 104(3): 787 - 794.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
G. C. Sieck, W.-Z. Zhan, Y.-S. Han, and Y. S. Prakash
Effect of denervation on ATP consumption rate of diaphragm muscle fibers
J Appl Physiol, September 1, 2007; 103(3): 858 - 866.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
P. C. Geiger, J. P. Bailey, C. B. Mantilla, W.-Z. Zhan, and G. C. Sieck
Mechanisms underlying myosin heavy chain expression during development of the rat diaphragm muscle
J Appl Physiol, December 1, 2006; 101(6): 1546 - 1555.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
94/5/1896    most recent
00617.2002v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sieck, G. C.
Right arrow Articles by Zhan, W.-Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sieck, G. C.
Right arrow Articles by Zhan, W.-Z.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online