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J Appl Physiol 90: 657-664, 2001;
8750-7587/01 $5.00
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Vol. 90, Issue 2, 657-664, February 2001

Reserve capacity for ATP consumption during isometric contraction in human skeletal muscle fibers

Young-Soo Han, David N. Proctor, Paige C. Geiger, and Gary C. Sieck

Departments of Anesthesiology and of Physiology and Biophysics, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Maximum velocity of the actomyosin ATPase reaction (Vmax ATPase) and ATP consumption rate during maximum isometric activation (ATPiso) were determined in human vastus lateralis (VL) muscle fibers expressing different myosin heavy chain (MHC) isoforms. We hypothesized that the reserve capacity for ATP consumption [1 - (ratio of ATPiso to Vmax ATPase)] varies across VL muscle fibers expressing different MHC isoforms. Biopsies were obtained from 12 subjects (10 men and 2 women; age 21-66 yr). A quantitative histochemical procedure was used to measure Vmax ATPase. In permeabilized fibers, ATPiso was measured using an NADH-linked fluorometric procedure. The reserve capacity for ATP consumption was lower for fibers coexpressing MHC2X and MHC2A compared with fibers singularly expressing MHC2A and MHCslow (39 vs. 52 and 56%, respectively). Tension cost (ratio of ATPiso to generated force) also varied with fiber type, being highest in fibers coexpressing MHC2X and MHC2A. We conclude that fiber-type differences in the reserve capacity for ATP consumption and tension cost reflect functional differences such as susceptibility to fatigue.

quantitative histochemistry; immunohistochemistry; muscle biopsy; sodium dodecyl sulfate-polyacrylamide electrophoresis; adenosine triphosphate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MYOSIN HEAVY CHAIN (MHC) is the site of ATP hydrolysis during cross-bridge cycling, and ATP consumption rate during cross-bridge cycling is a major determinant of the mechanical performance of skeletal muscle fibers. This is evident by the close relationship between the maximum velocity of the actomyosin ATPase reaction (Vmax ATPase), measured biochemically, and the fiber-type composition and contractile properties of various skeletal muscles (3).

Several recent studies have used an NADH-linked fluorometric technique to measure the rate of ATP consumption in single permeabilized muscle fibers during maximum isometric activation (ATPiso) (6, 22, 23, 25). In both animal (6, 22, 23) and human (25) studies, muscle fibers expressing the MHCslow isoform were found to have a slower rate ATPiso compared with fibers expressing fast MHC isoforms (MHC2A, MHC2X, and MHC2B). However, ATPiso is submaximal, and therefore this measure does not establish the maximum capacity for ATP hydrolysis (22, 23). It is well established that ATP consumption increases with power output and work performance (11, 12, 22). The Vmax ATPase establishes the upper limit for ATP consumption during work performance for each fiber type in skeletal muscle. In this respect, it is important to establish the range of ATP consumption rates (from ATPiso to Vmax ATPase) because this provides a measure of the reserve capacity for ATP consumption. In the rat diaphragm muscle, we found that the reserve capacity for ATP consumption [calculated as 1- (ratio of ATPiso to Vmax ATPase)] was ~64, ~54, and ~52% for fibers expressing MHCslow, MHC2A, and MHC2X/2B, respectively (23). Unfortunately, values obtained in laboratory animals cannot be necessarily extrapolated to human muscle fibers. Therefore, the purpose of the present study was to determine the reserve capacity for ATP consumption of single permeabilized fibers from the human vastus lateralis (VL) muscle. We hypothesized that the reserve capacity for ATP consumption varies across VL muscle fibers expressing different MHC isoforms.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Muscle biopsies. Needle-biopsy samples (~50-100 mg) were obtained from the superficial portion of the VL muscle in 12 healthy but sedentary volunteers (10 men, 2 women; age 21-66 yr). Muscle samples used for measurement of ATPiso were cleaned of visible fat and connective tissue and placed in a relaxing solution, at 5°C for 24 h, 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+ [-log Ca2+ concentration (pCa) 9.0]; 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 no more than 4 wk before subsequent analysis.

Muscle samples used for quantitative histochemical measurements of Vmax ATPase were cleaned of visible fat and connective tissue, oriented vertically in embedding medium, frozen in isopentane cooled by liquid nitrogen, and stored at -80°C. No attempt was made to stretch the muscle sample to optimal length before freezing.

Permeabilized single-fiber preparation. Glycerinated fiber bundles were transferred to a relaxing solution containing 10 mM dithiothreitol (DTT) and dissected under a microscope. The dissected single fibers were then transferred to a relaxing solution containing 10 mM DTT and 1% Triton X-100 for 20-30 min to permeabilize the plasma membrane. The permeabilized fibers were again transferred to 50% glycerol relaxing solution before measurements of ATPiso. Permeabilized single fibers, ~3 mm in length, were mounted between force and displacement transducers in a quartz cuvette that was perfused with solutions containing free ionized Ca2+ concentrations of either 1 nM (pCa 9.0) or 100 µM (pCa 4.0) maintained at 15°C. Muscle fiber length was adjusted so that average sarcomere length was 2.5 µm.

Measurement of maximum isometric force and ATPiso. Maximum isometric force (Fmax) and ATPiso were measured concurrently at 15°C in a Gûth Scientific Instruments Muscle Research System (16, 17, 20). The procedures for measuring isometric force in single permeabilized muscle fiber has been previously reported (14, 15, 20, 22, 23). In preliminary studies on human fibers, we confirmed that Fmax is obtained at pCa 4.0 and that no active force was obtained at pCa of 9.0.

The NADH-linked fluorometric technique for measuring ATPiso has been previously described in detail (16, 17, 20, 22, 23). Using this procedure, it was confirmed that mitochondrial ATPases and sarcoplasmic reticulum ATPase make no detectable contribution to the observed ATPase activity (17). Measurements of ATPiso were made while fibers were mounted in the quartz cuvette and perfused with either relaxing (pCa 9.0) or activating (pCa 4.0) solutions. NADH fluorescence was excited at 340 nm using a mercury lamp and an interposed band-pass filter. Emitted fluorescence was measured at 450 nm using a photomultiplier tube. The ATP solutions consisted of relaxing (pCa 9.0) and activating (pCa 4.0) solutions, both containing 5 mM phospho(enol)pyruvate (PEP), 0.2 mM 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
ATP <LIM><OP><ARROW>→</ARROW></OP><UL>ATPase</UL></LIM> ADP<IT>+</IT>P<SUB>i</SUB> (1)

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

<AR><R><C>Pyruvate<IT>+</IT>NADH</C></R><R><C>(fluorescent)</C></R></AR> <LIM><OP><ARROW>→</ARROW></OP><UL>LDH</UL></LIM> <AR><R><C>lactate<IT>+</IT>NAD<SUP><IT>+</IT></SUP></C></R><R><C>(nonfluorescent)</C></R></AR> (3)
where, in reaction 1, ATP is hydrolyzed by actomyosin ATPase to ADP and Pi during detachment of the myosin head from the myosin-binding domain of actin. 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+. For each mole of ADP produced by the actomyosin ATPase-dependent hydrolysis of ATP, 1 mol of NADH is converted to NAD+. Therefore, for a period of time when perfusion of the cuvette was stopped (15 s), the amount of ATP consumed by the ATPiso was determined by measuring the rate of extinction of the NADH fluorescence signal (Fig. 1). Flow through the cuvette was then reinitiated for 1 s and then stopped again, and extinction of the NADH fluorescence signal was remeasured. This cycling continued for ~10 min at each pCa condition. Calibration involved measurements of fluorescence intensity for known amounts of NADH. On the basis of changes in the NADH fluorescence intensity, ATPiso was determined and expressed as nanomoles per cubic millimeter per second. The ATPiso was determined by subtracting the ATP consumption rate measured at a pCa 9.0 from that obtained at pCa 4.0. 


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  		Fig. 1.   Typical trace of the simultaneous measurement of force and ATP consumption rate during maximum isometric activation (ATPiso) using the NADH-linked fluorometric procedure in a single permeabilized fiber from the human vastus lateralis muscle [slow myosin heavy chain (MHCslow) expression]. Fiber size: length, 3.0 mm; diameter, 78.3 µm.

In a subset of permeabilized muscle fibers, the temperature dependence of the ATPiso was determined by obtaining measurements at 15, 20, and 25°C. The temperature coefficient (Q10) for the ATPiso was then calculated over this temperature range.

Gel electrophoretic determination of MHC isoform expression in single fibers. After completion of the force and ATPiso measurements, the MHC isoform composition of the muscle fiber was determined by SDS-PAGE using a previously described procedure (14, 15, 22, 23). Briefly, 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 a pH of 6.8. The sample was denatured by boiling for 2 min, and 10-µl samples [~125 ng as determined by the Lowry method (19)] were loaded per lane. The gels were silver stained to visualize the MHC migration bands. Two mixed muscle fiber samples were run on each gel to compare the migration patterns of identified MHC isoforms. In the case of coexpression of MHC isoforms within a single fiber, the relative expression of each MHC isoform was determined by densitometric analysis.

Quantitative histochemical measurement of fiber Vmax ATPase. The quantitative histochemical procedure for measuring the Vmax ATPase in type-identified muscle fibers has been previously described in detail (4, 24). Serial cross sections of muscle fibers were cut at 10-µm thickness using a cryostat kept at -20°C, and alternate sections were used to determine MHC isoform expression and the Vmax ATPase. In four alternate transverse sections, immunoreactivity against antibodies specific for anti-MHCslow (NCL), anti-MHC2A (SC-71), and anti-MHCall-2X (BF-35) (MHCall-2X means all but the MHC2X isoform) was evaluated. Primary antibodies were diluted in PBS (pH 7.4) containing 0.5% bovine serum albumin and were then applied to the muscle sections for ~12 h at room temperature in a humidified chamber. Slides were then washed in PBS and incubated with a fluorescein-conjugated secondary antibody (goat anti-mouse IgG) for ~60 min at room temperature in a humidified chamber. The slides were then washed in PBS, coverslipped with Permount, and viewed through a microscope (model BH2, Olympus) equipped with epifluorescence. An additional four alternate sections were stained for myofibrillar ATPase (mATPase) after preincubation at pH 4.3, 4.6, 9.0, and 10.4 (after 4% paraformaldehyde fixation) (7, 24).

The Vmax ATPase was measured in a series of 28 alternate sections of the same muscle fibers. The values for ATPase in a given fiber were the average of measurements across the four sections at a given ATP concentration, and the same fiber was measured at each of seven ATP concentrations (see below). In a previous study, our laboratory verified that the quantitative histochemical method is specific for the Vmax ATPase (4) and that the deposition of the reaction product is localized at the site of cross-bridge cycling (i.e., A band). In this procedure, an image-processing system (MegaVision 1024 XM), mounted on an Olympus BH-2 microscope and calibrated for microdensitometry using a set of neutral density filters [0.1-2.0 optical density (OD) units], was used. Microscopic images of muscle fiber cross sections were digitized at 8-bit resolution into a 1,024 × 1,024 picture element (pixel) array using a video scanner and then stored in a computer file. In the digitizing procedure, 16 separate scans were averaged to reduce electronic noise, and a shading algorithm was employed to reduce errors attributed to uneven illumination of the tissue. Previously, our laboratory estimated that, when using this imaging system, the measurement error for microdensitometry was <4% (4, 24). The boundaries of individual fibers within the digitized images were delineated, and the average OD of all pixels within the fiber was calculated.

Four replicate sections of the same muscle fibers were reacted at 22°C in an incubation medium containing one of a series of ATP concentrations (0.0, 0.5, 0.75, 1.0, 2.0, 4.0, 5.0 mM ATP). The use of several ATP concentrations was necessary because sufficient ATP could not be dissolved in the incubation medium to avoid substrate limiting the Vmax ATPase (4). In the Vmax ATPase, the Pi ions produced by the enzymatic hydrolysis of ATP were precipitated within the muscle fiber cross section by complexing with lead ions (lead ammonium citrate-acetate complex) to form a lead phosphate precipitate. The precipitated lead ions were subsequently exchanged for sulfide ions, by reaction with sodium sulfide, to form a brown-colored lead sulfide precipitate. The concentration of the lead sulfide precipitate in the muscle section was then measured by microdensitometry using the Lambert-Beer equation and a molar extinction coefficient of 1,450 mol/cm for lead sulfide (4, 24).

To determine the Vmax ATPase, a Lineweaver-Burke transformation of the data was performed (Fig. 2). On the basis of stoichiometric relationships of the Pi-sulfide exchange in the histochemical reactions, the Vmax ATPase was expressed as millimoles of Pi precipitated per liter of tissue (fiber cross-sectional area × 10-µm thickness) per minute (mM Pi/min). However, this unit of millimoles of Pi per minute was converted to nanomoles per cubic millimeter per second to be compatible with that used in the ATPiso. The Vmax ATPase for at least 20 fibers singularly expressing each MHC isoform was determined for each muscle biopsy. In the case of MHC isoform coexpression, fewer fibers were sampled due to the relative paucity of such fibers.


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  		Fig. 2.   Lineweaver-Burke plot displaying the dependence of the velocity (V) of the maximum velocity of the actomyosin ATPase reaction (Vmax ATPase) on ATP concentration in the incubation medium for 3 human vastus lateralis muscle fibers expressing different (MHC) isoforms. Slope and y-intercept of each line were calculated by linear regression analysis, from which the Vmax ATPase reaction was determined. OD570nm, optical density at 570 nm. Brackets denote concentration.

Statistical analysis. Values are reported as means ± SE. A one-way ANOVA with repeated-measures design was used to independently assess fiber-type differences in histochemical Vmax ATPase and ATPiso. Post hoc analysis (Neuman-Keuls) was performed when appropriate. Statistical significance was accepted when P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Electrophoretic determination of MHC isoform expression in single fibers. The VL muscle displayed three distinct MHC migration bands. On the basis of Western blot analysis, these MHC migration bands were found to correspond with the expression of MHCslow, MHC2A, and MHC2x isoforms. To evaluate MHC isoform expression in single VL fibers, a larger number of fibers were sampled in addition to those used in the mechanical and energetic studies (see Classification of fiber types in muscle cross sections). Among human VL fibers, MHCslow (n = 64) and MHC2A isoforms (n = 65) were found to be singularly expressed. However, the MHC2X isoform was not found to be singularly expressed and was coexpressed predominantly with the MHC2A isoform (n = 24; Fig. 3). Within fibers coexpressing MHC2X and MHC2A, the relative expression of each isoform ranged from 20 to 80%, but the mean relative expression was 54.8 ± 1.2% MHC2X and 45.2 ± 1.2% MHC2A. In these preliminary studies, it was determined that the fiber-type composition of the VL muscle could not be characterized from a single biopsy. It was estimated that up to seven biopsies would be required, and such repeated biopsies were not possible in these subjects. In addition, there was a relatively low abundance of fibers expressing the MHC2X isoform in the biopsies that were obtained. Indeed, it was necessary to obtain biopsies from 12 subjects to obtain a sufficient sample of fibers expressing the MHC2X isoform. For these reasons, it was not possible to characterize the overall population of this or other fiber types in the VL of subjects. This raised the important issue of whether across-subject variability may have influenced the results. For fibers expressing the MHC2X isoform, this issue could not be addressed. However, in comparing values for fibers expressing MHCslow and MHC2A isoforms, there were no significant differences across subjects. The coefficient of variation for ATPiso of fibers expressing MHC2A across subjects was 6.26%.


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  		Fig. 3.   MHC isoform expression in human vastus lateralis muscle fibers was identified by SDS-PAGE. A mixed muscle sample of human vastus lateralis muscle fibers was included for comparison of electrophoretic migration patterns.

Classification of fiber types in muscle cross sections. The patterns of immunoreactivity against specific MHC antibodies in the VL muscle generally corresponded with the histochemical classification of fiber types. VL fibers classified histochemically as type I displayed immunoreactivity for the anti-MHCslow antibody, and fibers classified histochemically as type IIa displayed immunoreactivity for the anti-MHC2A antibody. However, the anti-MHCsll-2X antibody (BF-35), specific for all MHC isoforms except for MHC2X, was less reactive with fibers classified histochemically as type IIb, indicating expression of the MHC2X isoform. In these fibers, immunoreactivity for the MHCall-2X antibody varied from faint to moderate, and most of these fibers were also immunoreactive for the anti-MHC2A antibody in varying degrees. Therefore, these immunohistochemical results were consistent with the coexpression of MHC2A and MHC2X.

Fmax (n = 46). Fmax of human VL fibers was significantly lower for fibers expressing MHC2A (n = 21; range 10-20 N/cm2) compared with fibers expressing MHCslow (n = 19; range 12-22 N/cm2; Table 1; P < 0.05). For fibers coexpressing MHC2X and MHC2A (n = 6), there was a considerable range in Fmax (11-27 N/cm2). Unfortunately, there were an insufficient number of fibers sampled in this group to determine whether Fmax depended on the relative expression of MHC2X and MHC2A.

                              
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  		Table 1.   Cross-sectional area, maximum isometric force, and tension cost of human vastus lateralis muscle fibers at 15°C

ATPiso (n = 46). The ATPiso of human VL fibers expressing the MHCslow isoform was significantly lower than that of fibers expressing MHC2A as well as fibers coexpressing the MHC2X and MHC2A (P < 0.05; Fig. 4). The ATPiso of single fibers singularly expressing MHC2A was also significantly lower than that of fibers coexpressing MHC2X and MHC2A (P < 0.05; Fig. 4).


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  		Fig. 4.   Comparison of ATPiso and Vmax ATPase for human vastus lateralis fibers expressing different MHC isoforms. * Significantly from fibers expressing MHCslow, P < 0.05 + Significantly different from fibers expressing MHC2A, P < 0.05.

Vmax ATPase (n = 525). The Vmax ATPase of VL muscle fibers expressing MHCslow was significantly lower than that of fibers expressing MHC2A either alone or coexpressed with MHC2X (P < 0.05; Fig. 4). The Vmax ATPase of fibers expressing MHC2A alone was significantly lower than that of fibers coexpressing MHC2X and MHC2A (P < 0.05; Fig. 4).

Reserve capacity of ATP consumption. For human VL muscle fibers, the reserve capacity for ATP consumption was calculated as
Reserve capacity for ATP consumption<IT>=1−</IT>(ratio of ATP<SUB>iso</SUB> to<IT> V</IT><SUB>max</SUB> ATPase)
In fibers singularly expressing MHCslow or MHC2A, the reserve capacity for ATP consumption was 56 and 52%, respectively. For fibers coexpressing MHC2X and MHC2A, the reserve capacity for ATP consumption was only 39% (P < 0.05 compared with fibers singularly expressing MHCslow or MHC2A).

Isometric tension cost (n = 46). For a measurement of ATP cost for generating force, the isometric tension cost of human VL fibers was determined by dividing the ATPiso by the corresponding isometric force. The tension cost of human VL fibers expressing the MHCslow isoform was significantly lower than that of fibers expressing MHC2A (P < 0.05; Fig. 5). There were an insufficient number of fibers sampled in this group to determine whether tension cost depended on the relative expression of MHC2X and MHC2A. Comparison of tension cost determined at 15°C between human VL fibers and rat diaphragm fibers in our laboratory's previous study (22) is shown in Fig. 5. The relationship between Vmax ATPase and tension cost is displayed in Fig. 6.


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  		Fig. 5.   Comparison of tension cost determined at 15°C between vastus lateralis muscle fibers and rat diaphragm fibers expressing MHCslow, MHC2A, and MHC2X/2A isoforms. Human vastus lateralis fibers expressing MHC2A/2X were compared with the rat diaphragm fibers expressing MHC2X/2B. * Significantly different from fibers expressing MHCslow, P < 0.05.



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  		Fig. 6.   Comparison of Vmax ATPase and tension cost for human vastus lateralis fibers expressing different MHC isoforms. * Significantly different from fibers expressing MHCslow, P < 0.05.

Temperature dependence of ATPiso (n = 6). The dependence of ATPiso was determined by comparing measurements at 15, 20, and 25°C. As temperature increased, ATPiso of fibers also increased. The Q10 value of the ATPiso was 1.72 ± 0.16 (Fig. 7).


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  		Fig. 7.   Relationship between temperature and ATPiso was measured in 6 human vastus lateralis muscle fibers. Values represent individual measurements on 6 human vastus lateralis muscle fibers from 2 subjects.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study examined both maximal (Vmax ATPase) and submaximal (ATPiso) values for ATP consumption in human VL muscle fibers. The Vmax ATPase establishes the upper limit for ATP consumption during work performance for each fiber type in skeletal muscle (23). In this respect, it is important to determine the range of ATP consumption rates (from Vmax ATPase to ATPiso), because this provides a measure of the reserve capacity for ATP consumption. The present study reports important new information in this regard. The reserve capacity for ATP consumption was lower for fibers expressing MHC2X (coexpressed with MHC2A) compared with fibers expressing MHC2A and MHCslow. This is consistent with the lower energy efficiency of fibers expressing the MHC2X isoform as reflected by the higher tension cost of these fibers.

Similar to previous reports (10, 30), we found that three MHC isoforms were expressed in the VL muscle of healthy adult humans, MHCslow, MHC2A, and MHC2X. Other studies evaluating MHC isoform expression in the human VL muscle also found three isoforms, although MHC2B expression was reported rather than MHC2X (18, 25). Because there is now strong evidence to indicate that MHC2B is not expressed in human fibers, it is likely that expression of MHC2B was confused with MHC2X expression. On the basis of single-fiber gel electrophoresis and Western blot analysis, it appears that MHCslow and MHC2A are singularly expressed in VL fibers, whereas the MHC2X isoform is only coexpressed, predominantly with MHC2A. The coexpression of MHC2X and MHC2A in human VL muscle fibers has also been reported in other studies (10, 30). The pattern of MHC isoform expression in single human VL muscle fibers, as determined by SDS-PAGE, generally corresponded with the pattern of immunoreactivity against specific MHC antibodies, as well as the histochemical classification of fiber types based on the pH lability of myofibrillar mATPase staining. These results are consistent with other studies showing a relationship between histochemical fiber-type classification and MHC isoform composition in human muscle fibers (1, 10, 30).

Similar to the previous results of Stienen and colleagues (25) for the human rectus abdominis and VL muscles, we found that ATPiso varied across fibers expressing different MHC isoforms in human VL muscle. These results are also in general agreement with our laboratory's previous observations in the rat diaphragm muscle (22, 23). However, the values for ATPiso in human muscle fibers reported by Stienen and colleagues were lower than those found in the present study. These investigators measured ATPiso at 20°C rather than 15°C. The dependence of ATPiso on temperature was measured in both studies; a Q10 of 1.72 was found in the present study vs. a Q10 of 2.34 in the study of Stienen et al. Even when corrected for differences in temperature, the ATPiso values found in the present study were ~30-40% higher than those reported by Stienen et al. It should be noted that Stienen and colleagues measured NADH concentration by absorbency rather than fluorometry, and these technical differences may have accounted for the discrepancies in reported values.

31P-nuclear magnetic resonance (NMR) spectroscopy has also been used to measure ATP consumption in human muscle fibers in vivo on the basis of the dynamics of creatine phosphate content. Using this procedure, Blei and colleagues (5) reported an average ATP consumption rate of 0.15 ± 0.03 nmol · mm-3 · s-1 during single-twitch stimulation in the human forearm flexor musculature, whereas Turner and colleagues (27) reported an average value of 4.4 ± 0.4 nmol · mm-3 · s-1 in the human adductor pollicis muscle during maximum isometric activation. After corrections for the higher in vivo temperature were made, the ATPiso for VL muscle fibers in the present study would range from 1.02 ± 0.07 nmol · mm-3 · s-1 for fibers expressing MHCslow to 3.06 ± 0.15 nmol · mm-3 · s-1 for fibers coexpressing MHC2X and MHC2A. Using the NADH-linked fluorometric procedure, only the ATP consumption related to the actomyosin ATPase was measured, whereas in the 31P-NMR spectroscopy method the total ATP consumption of activated fibers was measured, including other membrane ATPases. These differences may account for the slightly higher ATP consumption measured using 31P-NMR spectroscopy, although several other factors may have also contributed.

In the present study, Vmax ATPase was highest for fibers coexpressing MHC2X and MHC2A and lowest for fibers expressing MHCslow. These results are consistent with the previous study of Castro et al. (8) on human VL muscle fibers using an identical technique. These results are also generally consistent with differences in the Vmax ATPase of fibers expressing different MHC isoforms in the rat diaphragm muscle (23).

The reserve capacity for ATP consumption in single muscle fibers was estimated by the following calculation: 1 - [ratio of the ATPiso to the Vmax ATPase]. In a previous study in the rat diaphragm muscle, our laboratory found that the reserve capacity for ATP consumption ranged from ~64% for fibers expressing MHCslow to ~52% for fibers expressing fast MHC isoforms (23). In the human VL, we found that fibers singularly expressing MHCslow and MHC2A had a greater reserve capacity (56 and 52%, respectively) compared with fibers coexpressing MHC2X and MHC2A (39%). It should be noted that both the ATPiso and the Vmax ATPase for human VL fibers were substantially lower than values for fibers expressing the same MHC isoforms (MHCslow, MHC2A, and MHC2X) in the rat (22-24). Together, these results indicate that ATPiso represents only submaximal energy utilization compared with the Vmax ATPase in muscle fibers. The Vmax ATPase in a muscle fiber is determined by the product of the ATP consumed per MHC molecule (i.e., the quantal contribution from a single myosin cross bridge) times the number of available cross bridges (i.e., MHC concentration). Similarly, ATPiso is determined by the ATP consumed per MHC molecule times the number of cross bridges in the force-generating state. In both cases, MHC concentration becomes an important determinant of ATP consumption. Recently, our laboratory reported that, in the rat diaphragm muscle, fibers expressing MHCslow and MHC2A have lower MHC concentrations compared with fibers expressing MHC2X and MHC2B (14). Furthermore, our laboratory found that, during maximum isometric activation, the fraction of cross bridges in the force-generating state was comparable across fiber types (14). Thus, with a lower MHC concentration, lower ATPiso and Vmax ATPase would be expected for fibers expressing MHCslow and MHC2A. However, when we normalized our Vmax ATPase for previously reported myofibrillar volume densities in the VL muscle (28), fiber-type differences in ATPiso and Vmax ATPase persisted. Thus it is likely that the lower ATPiso and Vmax ATPase values seen in VL fibers expressing the MHCslow isoform primarily reflect phenotypic differences in the capacity for ATP consumption of MHCslow vs. MHC2A or MHC2X molecules. The lower ATPiso and Vmax ATPase of fibers expressing MHCslow are also likely to be reflected by a slower maximum rate constant for cross-bridge detachment compared with fibers expressing MHC2A or MHC2X (23).

It is not surprising that the ATPiso of muscle fibers was only a fraction of the Vmax ATPase (i.e., maximum capacity for ATP consumption). In 1923, Fenn observed that energy utilization of skeletal muscle increases in proportion to work (Fenn effect; Refs. 11, 12). Thus, as muscle fibers reach maximum power during shortening, ATP consumption rate should increase (22). In a previous study on the rat diaphragm muscle, our laboratory found that the maximum rate of ATP consumption was achieved at a shortening velocity corresponding to peak power output of fibers (22). Although the maximum rate of ATP consumption achieved at peak power output was closer to the Vmax ATPase, it was still less. 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.

The Fmax values for human VL muscle fibers measured in the present study were comparable to those reported in previous studies in single fibers (9, 18, 25, 29) as well as whole human muscle in vivo (13). We found that the Fmax of VL fibers expressing MHCslow was slightly greater than that for fibers expressing MHC2A. In contrast, Stienen et al. (25) reported that VL fibers expressing MHCslow generated lower Fmax compared with fibers expressing fast MHC isoforms. Larsson and Moss (18) reported no significant differences in Fmax across human VL muscle fibers expressing different MHC isoforms. In the rat diaphragm muscle, our laboratory found that fibers expressing MHCslow generated lower Fmax compared with fibers expressing fast MHC isoforms (14, 15, 22, 23).

The tension cost (the ratio of ATPiso to isometric force; Fig. 5) of human VL muscle fibers reported in the present study is in general agreement with that reported by Steinen et al. (25). Fibers expressing MHCslow had the lowest values of tension cost followed by fibers expressing MHC2A and fibers coexpressing MHC2X and MHC2A. Therefore, fibers expressing MHCslow are the most energy efficient. Compared with values of tension cost reported for rat diaphragm muscle fibers (22), the tension cost of human VL muscle fibers was significantly lower. These results generally agree with the principle that energetic costs of generating muscular force are lower in larger animals (26).

In conclusion, measurement of submaximal and maximal rates of ATP consumption in the present study indicates that a substantial reserve capacity for ATP consumption exists in human muscle fibers. In addition, fiber-type differences in the reserve capacity for ATP consumption exist, with fibers expressing MHC2X (coexpressed with MHC2A) displaying a significantly lower reserve capacity compared with fibers singularly expressing MHC2A and MHCslow. These measurements provide new information that is important in determining the balance between energy supply and demand. Certainly this reserve capacity for ATP consumption becomes important under conditions where ATP production may be insufficient to meet the demands for cross-bridge cycling. Such an energetic imbalance has been suggested as an underlying mechanism of muscle fatigue. The lower reserve capacity for ATP consumption, together with the higher ATP consumption rates, may explain, at least in part, the greater fatigue susceptibility of fibers expressing MHC2X (21). Under conditions of greater workloads, as energy utilization increases in proportion to work (Fenn effect; Refs. 11, 12), reserve capacity for ATP consumption decreases and susceptibility to fatigue increases (2). Therefore, these novel results regarding the reserve capacity of ATP consumption in human VL muscle fibers have important functional implications.


    ACKNOWLEDGEMENTS

We thank Dr. Janet L. Vittone, Y. H. Fang, Heidi M. Hinderaker, A. Iyanoye, Rebecca Macken, and J. Sieck for their assistance in these studies.


    FOOTNOTES

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 9 June 2000; accepted in final form 31 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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