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Effect of denervation on ATP consumption rate of diaphragm muscle fibers

Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, Minnesota

Submitted 5 September 2006 ; accepted in final form 4 June 2007

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Denervation (DNV) of rat diaphragm muscle (DIAm) decreases myosin heavy chain (MHC) content in fibers expressing MHC_2X isoform but not in fibers expressing MHC_slow and MHC_2A. Since MHC is the site of ATP hydrolysis during muscle contraction, we hypothesized that ATP consumption rate during maximum isometric activation (ATP_iso) is reduced following unilateral DIAm DNV and that this effect is most pronounced in fibers expressing MHC_2X. In single-type-identified, permeabilized DIAm fibers, ATP_iso was measured using NADH-linked fluorometry. The maximum velocity of the actomyosin ATPase reaction (V_max ATPase) was determined using quantitative histochemistry. The effect of DNV on maximum unloaded shortening velocity (V_o) and cross-bridge cycling rate [estimated from the rate constant for force redevelopment (k_TR) following quick release and restretch] was also examined. Two weeks after DNV, ATP_iso was significantly reduced in fibers expressing MHC_2X, but unaffected in fibers expressing MHC_slow and MHC_2A. This effect of DNV on fibers expressing MHC_2X persisted even after normalization for DNV-induced reduction in MHC content. With DNV, V_o and k_TR were slowed in fibers expressing MHC_2X, consistent with the effect on ATP_iso. The difference between V_max ATPase and ATP_iso reflects reserve capacity for ATP consumption, which was reduced across all fibers following DNV; however, this effect was most pronounced in fibers expressing MHC_2X. DNV-induced reductions in ATP_iso and V_max ATPase of fibers expressing MHC_2X reflect the underlying decrease in MHC content, while reduction in ATP_iso also reflects a slowing of cross-bridge cycling rate.

myosin heavy chain; muscle energetics; muscle plasticity

(1)

where b is the number of half-sarcomeres within a muscle fiber, n is the number of available cross bridges per half-sarcomere, and _fs is the fraction of available cross bridges that are in a strongly bound state.

The rate of ATP consumption during maximum isometric activation (ATP_iso) has been measured in single permeabilized muscle fibers using a NADH-linked fluorometric technique. Several studies have reported that there are intrinsic differences in ATP_iso in muscle fibers expressing different MHC isoforms, with fibers expressing MHC_2X alone or together, with MHC_2B having greater ATP_iso compared with fibers comprising MHC_slow and MHC_2A isoforms (3, 18, 19, 39, 42, 49). These differences in ATP_iso are due in part to differences in fiber MHC content (b·n from Eq. 1), but, even when normalized for MHC content, ATP_iso of fibers expressing MHC_slow is lower than that of fibers expressing fast MHC isoforms (18). During maximum calcium activation, the _fs is comparable across all fiber types, but there are differences in maximum shortening velocity (reflecting cross-bridge cycling rate and g_app) that contribute to the fiber-type differences in ATP_iso (18).

The maximum velocity of the actomyosin ATPase reaction (V_max ATPase) in muscle fibers, as measured by quantitative histochemistry, establishes the upper limit for ATP consumption during work performance for each fiber type in skeletal muscle. The V_max ATPase of diaphragm muscle (DIAm) is higher for DIAm fibers expressing MHC_2X alone or together with MHC_2B compared with MHC_slow and MHC_2A (39, 46). Across all fibers, ATP_iso is substantially less than V_max ATPase, which is expected, since ATP consumption rate increases during shortening and work performance in muscle (Fenn effect) (8, 9). The difference between V_max ATPase and ATP_iso reflects the reserve capacity for ATP consumption in muscle fibers and is higher for fibers expressing MHC_slow and MHC_2A compared with fibers expressing MHC_2X alone or together with MHC_2B (39, 43).

In a previous study (12), our laboratory found that, following 2 wk of unilateral denervation (DNV) of the DIAm, MHC content and maximum specific force were dramatically reduced for DIAm fibers expressing MHC_2X (it should be noted that MHC_2B expression essentially disappeared after DNV) compared with fibers expressing MHC_slow and MHC_2A isoforms. Following DNV, there was also a decrease in maximum unloaded shortening velocity (V_o) of DIAm strips (29, 45, 58) that may reflect either an increase in the relative contribution of type I (MHC_slow) and IIa (MHC_slow) fibers due to atrophy of IIx and/or IIb (MHC_2X and/or MHC_2B), or a slowing of cross-bridge cycling rate (g_app) of individual fibers. In addition, DNV leads to a decrease in V_max ATPase that is more pronounced in DIAm fibers expressing MHC_2X (29, 58), which could also reflect either an increase in the relative contribution of type I and IIa (MHC_slow) fibers or a slowing of single-fiber cross-bridge cycling rate. In the present study, we hypothesized that unilateral DIAm DNV results in a slowing cross-bridge cycling rate and ATP_iso that is more pronounced in fibers expressing MHC_2X.

	METHODS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Adult male Sprague-Dawley rats (initial body weight 300 g) were randomly assigned to either control (CTL; n = 6) or DNV (n = 7) groups. The animals were housed in separate cages under a 12:12-h light-dark cycle, fed Purina Rat Chow, and provided water ad libitum. Body weights were monitored every 3rd day during the experimental period. Surgical procedures were performed under aseptic conditions, and recovery of animals from surgery was carefully monitored. All procedures were approved by the Institutional Animal Care and Use Committee of the Mayo Clinic.

Unilateral DIAm DNV.   The procedures for unilateral DNV of the rat DIAm have been previously described in detail (16, 29, 58). Briefly, animals were anesthetized by intramuscular injection of ketamine (60 mg/kg) and xylazine (2.5 mg/kg). After a midline incision, the right phrenic nerve was exposed beneath the sternomastoid muscle. The phrenic nerve was then transected, and an 2 cm length from the distal end was removed to prevent DIAm reinnervation and to minimize any residual neurotrophic effects emanating from the remaining nerve stump, which we estimated would persist for 1–2 days based on the rate of axoplasmic transport. The wound was closed with 6-0 silk sutures, and the surgical wounds were treated topically with Neosporin ointment (containing aerosporin, neomycin, and bacitracin). Typically, animals recovered from the anesthetic within 1 h. In a previous study, our laboratory demonstrated that arterial blood-gas levels were normal in animals after DNV, indicating that ventilation is not compromised by unilateral DIAm paralysis (29).

In the present study, the effects of unilateral DNV were assessed after 2 wk. This time period was chosen based on previous studies demonstrating an effect of DNV on DIAm fiber size, on MHC content in single DIAm fibers, on DIAm force, and velocity of shortening (10, 12, 29, 58, 59).

Measurement of V_max ATPase in single fibers.   The quantitative histochemcial procedure for measuring the V_max ATPase in type-identified muscle fibers has been previously described in detail (2, 43, 46, 58). Briefly, segments from the right midcostal DIAm were stretched to optimal length (1.5 times resting excised muscle length) (32) before being rapidly frozen in isopentane cooled by liquid nitrogen. The muscle segments were mounted, and serial transverse sections were cut at 10-µm thickness using a cryostat kept at –20°C (model 2800E Frigocut, Reichert-Jung).

To determine V_max ATPase in single muscle fibers, the reaction product of the actomyosin ATPase reaction (amount of P_i liberated by ATP) was measured. First, free P_i in the muscle fiber sections was reacted with a lead ammonium citrate/acetate complex to form a lead phosphate precipitate. The lead phosphate precipitate was then converted to a lead sulfide precipitate by reaction with sodium sulfide. The concentration of the lead sulfide precipitate in muscle fiber sections was then determined by densitometry using the Lambert-Beer equation:

(2)

where [NBT-dfz] is the nitroblue tetrazolium-diformazan concentration; 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, our laboratory demonstrated that the actomyosin ATPase reaction is not contaminated by other ATPases (e.g., those associated with the sarcolemma and sarcoplasmic reticulum) and that the reaction is linear for at least 9 min (2, 46). Accordingly, a single end-point reaction time of 4 min was selected to limit OD measurements to <1.0 OD unit and thereby minimize measurement error in the image processing system (see below). To determine V_max ATPase, ATP concentration in the incubation media was varied from 0.0 to 5.0 mM, and four replicate OD measurements (in four separate sections) of free P_i were obtained for each single fiber at 0.0, 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mM ATP (a total of 28 serial sections) at 22°C. This was necessary since 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 (OD₅₅₀/min) and ATP concentration was performed to determine V_max ATPase and the apparent Michaelis-Menten rate constant of the actomyosin ATPase reaction (2, 46). Based on the stoichiometry of P_i sulfide exchange in the histochemical reaction, V_max ATPase was expressed as millimoles of P_i per liter per minute. However, this unit was converted to nanomoles per millimeter cubed per second to be compatible with that used in the ATP_iso (19).

For densitometric measurements, microscopic images of the same region of the muscle in alternate serial sections were digitized at 8-bit resolution (256 gray levels) into a 1,024 x 1,024 array of picture elements (pixels) using a video image-processing system (Metamorph). The imaging system was calibrated for densitometry using a set of neutral density filters ranging from 0.02 to 2.00 OD units. In a previous study, our laboratory determined that, for OD values 1.0, measurement errors were <1.5% (32). Before digitizing images of the muscle sections, light intensity of the microscope was adjusted to optimize the full use of the 256 gray level range of the video camera but to avoid light saturation. Subsequently, light intensity of the microscope was not adjusted.

Determination of MHC isoform expression in single fibers by immunohistochemistry.   MHC immunohistochemistry was used to determine MHC isoform expression in those single fibers in which V_max ATPase was measured. The methods employed for detecting MHC expression (or coexpression) based on immunohistochemistry have been previously described in detail (33). Briefly, alternate serial sections of the same muscle region as those used for measuring the actomyosin ATPase reaction were reacted with mouse primary antibodies against different MHC isoforms. Pairs of mouse IgG or IgM primary antibodies were used, e.g., anti-MHC_slow (Novocastra, IgG; 1;100), anti-MHC_2A (Blau A4.74, IgG; 1:1), anti-MHC_2B (BFF3, IgM, purified from mouse hybridoma, German Collection of Microorganisms and Cell Culture; 1:1) and anti-MHC_neo (Novocastra; 1:10). To determine MHC_2X expression, only a single antibody was used, i.e., anti-MHC_all-2X (BF-35, IgG, purified from mouse hybridoma, German Collection of Microorganisms and Cell Culture; 1:1). The primary antibodies were diluted in PBS containing 0.5% bovine serum albumin (5 mg/ml), and muscle sections were incubated in these primary antibodies for 2 h at room temperature. The sections were then 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. To assess the specificity of the reactivity against primary antibodies, alternate sections were incubated with only the secondary antibodies. The muscle sections were imaged with an Olympus FluoView confocal microscope. The imaging system was calibrated for morphometry using a stage micrometer.

Single-fiber preparation.   Two weeks after DNV, the rats were reanesthetized, and the right side of the DIAm was rapidly excised. Muscle fiber bundles were stretched to 1.5 times resting excised muscle length, pinned on cork, 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, 10^–9 M free Ca²⁺ (pCa 9), 15 mM creatine phosphate, and 1 mg/ml creatine phosphokinase; imidazole was used to maintain the pH at 7.00 ± 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 immediately at –20°C for 2–3 wk (5).

Before single-fiber dissection, a fiber bundle was placed in relaxation solution containing 10 mM DTT and dissected under a dissecting microscope. The dissected single fibers were then transferred to relaxing solution containing 10 mM DTT and 1% Triton X-100 to permeabilize the plasma membrane for 20 min. The permeabilized single fibers were again transferred to 50% glycerol relaxing solution before measurements of muscle contractile properties and ATP consumption rate. These measurements were performed at 15°C.

Measurement of ATP_iso.   ATP_iso was measured using an NADH-linked fluorescence technique (17, 19, 31, 39). The ATP solutions contained the same composition as the relaxing and activating solutions without creatine phosphate and creatine phosphokinase, plus 5 mM phospho(enol)-pyruvate (PEP), 0.2 mM reduced NADH, 100 U/ml pyruvate kinase (PK), and 140 U/ml lactate dehydrogenase (LDH). The NADH-linked enzymatic assay involves the following reaction:

(3)

(4)

(5)

In these reactions, the ATP hydrolyzed by actomyosin ATPase (Eq. 3) is regenerated by the biochemical reaction of ADP and PEP, which is catalyzed by PK (Eq. 4). This reaction is coupled to the reduction of pyruvate to lactate, which is catalyzed LDH and the oxidation of NADH to NAD⁺ (Eq. 5). For each mole of ATP regenerated by these coupled reactions, 1 mol of NADH is oxidized to NAD⁺. Most important in the quantification of ATP consumption is the fact that NADH is fluorescent, while NAD⁺ is nonfluorescent. Thus the rate of decrease in NADH fluorescence signal at 450 nm is proportional to the rate of ATP consumption (actomyosin ATPase activity). The reactions are optimized so that PK and LDH activities are not rate limiting, and there are no competing reactions for ADP.

Single, Triton X permeabilized fibers were mounted between force and displacement transducers in a quartz cuvette that was perfused with solutions containing a free ionized Ca²⁺ of either 1 nM (relaxing state) or 100 µM (maximal Ca²⁺ activation). For adjusting optimum sarcomere length and measuring a diameter of single fibers in this system, a charge-coupled device video camera system (COHU, solid-state camera) was used. Using an image-processing system digitized a video image of the single fiber. Sarcomere length was adjusted to 2.5 µm, and fiber diameter was measured at three points along the length of the fiber. In addition, fiber depth (Z-axis diameter) was also estimated. Based on these measurements, fiber cross-sectional areas were calculated. Flow of solution through the cuvette was flushed every 15 s, providing fresh constituents necessary to couple ATP hydrolysis to NADH consumption. The rate of decline in NADH fluorescence during a 15-s period was measured and used to calculate ATP consumption rate based on a prior calibration of the system at known NADH concentrations.

Reserve capacity for ATP consumption of DIAm fibers.   The difference between V_max ATPase and ATP_iso reflects the reserve capacity for ATP consumption. The reserve capacity was calculated as [1 – (ratio of ATP_iso to V_max ATPase)], based on fiber types. Since V_max ATPase and ATP_iso assays were performed at different temperature (22 vs. 15°C), the measures of ATP_iso were adjusted to 22°C based on the Q₁₀ of ATP_iso so that comparisons with V_max ATPase could be made. The procedures for this correction have been previously described in detail (19). Briefly, in a subset of permeabilized DIAm fibers, the temperature dependence (temperature coefficient Q₁₀) of ATP_iso was determined by obtaining measurements at 15, 20, and 25°C for 10 single fibers. The Q₁₀ was 1.3, regardless of fiber type.

Measurement of single-fiber mechanical properties.   A computer program described by Fabiato and Fabiato (7), with stability constants listed by Godt and Lindley (15), was used to prepare a high Ca²⁺ (pCa 4.0) and a low Ca²⁺ (pCa 9.0) relaxing solution. With an ionic strength of 150 mM at 15°C, both high and low Ca²⁺ solutions contained the following: 1.0 mM free Mg²⁺, 10 mM EGTA, 15 mM CrP, 50 mM imidazole, 2 mM DTT, 5 mM NaATP, and creatine phosphokinase at 1 mg/ml.

A 5% gluteraldehyde solution was used to chemically fix the ends of the fiber to maintain the noncompliant attachment of the fiber to the force transducer and the servo-controlled motor. To further reduce compliance and allow for fiber mounting, aluminum foil T-clips were attached to the fixed end of the fiber. On an Olympus IMT-2 inverted microscope, fibers were mounted on two stainless steel hooks between a force transducer (Aksjeselskapet, AE-801) with a resonant frequency of 5 kHz and a servo-motor (General Scanning, G120DT), with a step time of 800 µs in a flow-through acrylic chamber (volume, 120 µl). Fiber length (10x Olympus Plan 10, 0.30 numerical aperture), width (XY plane), and depth (XZ plane) (40x Olympus LWD CD Plan 40, 0.55 numerical aperture) were measured using a reticule in the eye piece of the inverted microscope. As stated in a previous study (13), a correction factor (0.867) was used to account for the distortion of the Z-axis due to the optics. Using first-order laser diffraction (He-Ne laser, UDT Sensors, LSC 30D), the fiber sarcomere length was set to 2.5 µm, where the muscle fiber was at the optimal fiber length (L_o). Brenner cycling (4), as modified by Sweeney (51), was used to maintain the sarcomere length during the experiment. LabView (National Instruments) based software and data acquisition boards were used to record signals. The experimental setup was used to measure the following: 1) the _fs, 2) V_o, and 3) the rate constant for force redevelopment (k_TR).

_fs.   The method employed to estimate the _fs has been previously described in detail (3, 4, 13, 14, 34–37, 39, 52). Briefly, permeabilized fibers were exposed to a pCa 9.0 solution to determine baseline force. Fibers were then maximally activated using pCa 4.0 solution. During maximum activation, muscle fiber stiffness was determined by imposing small-amplitude (<0.2% L_o), high-frequency (2 kHz) sinusoidal length perturbations and measuring the resulting changes in force. The measurement of muscle stiffness assumes that these small-amplitude, high-frequency length perturbations do not disrupt cross-bridge binding and exceed normal cross-bridge cycling rate. In this case, each strongly bound cross bridge contributes incrementally to the measured stiffness. In the present study, fiber stiffness was measured during maximal Ca²⁺ activation (pCa 4.0) in the presence or absence (rigor) of ATP. It was assumed that stiffness during rigor conditions represents the maximum number of strongly bound cross bridges. Accordingly, the ratio of fiber stiffness in the presence and absence of ATP provided an estimate of _fs during maximal Ca²⁺ activation.

V_o.   While fibers were maximally activated, V_o was determined using the slack test (6, 28) during which fiber length was rapidly shortened using length steps ranging from 5 to 11% of L_o. After each length step, the time required for the fiber to redevelop force (slack time) provides a measure of V_o. A linear regression of the different length steps vs. slack times was used to determine V_o (expressed as muscle length/s).

k_TR.   Cross-bridge cycling rate was estimated for each fiber by measuring the rate constant for force redevelopment (k_TR) using a method previously described in detail (5, 13, 14, 34, 35, 37, 39, 41, 52). Briefly, during maximum Ca²⁺ activation, fiber length was rapidly shortened (by 20% of L_o) and then restretched to L_o. This resulted in detachment of all cross bridges, and force was reduced to baseline (zero). Thereafter, as cross bridges reattached, force redeveloped, and the rate of rise of force reflected cross-bridge cycling rate within the fiber. A computer algorithm for least squares fit of a first-order exponential was used to determine the k_TR. Since k_TR depends on both f_app and g_app (Eq. 3), this relationship can be used to estimate these parameters.

(6)

Equations 7 and 8 describe how an approximation of f_app is obtained from the values of _fs and k_TR (4, 41):

(7)

(8)

By subtracting the value of f_app from k_TR as in Eq. 9, an approximation of g_app can be obtained:

(9)

By using the equations listed above, the cross-bridge cycling kinetics for single fibers expressing different MHC isoforms in the rat DIAm can be examined.

Determination of MHC isoform expression in single fibers by gel electrophoresis.   MHC isoform expression in single fibers was determined post hoc by SDS-PAGE (13, 14). The single fibers were placed in 25 µl of SDS sample buffer containing 62.5 mM Tris·HCl, 2% (wt/vol) SDS, 10% (vol/vol) glycerol, 5% 2-mercaptoethanol, and 0.001% (wt/vol) bromophenol blue at a pH of 6.8. The samples were denatured by boiling for 2 min. A modified procedure by Sugiura and Murakami (50) was used to prepare the gradient gels. The stacking gel contained a 3.5% acrylamide concentration (pH 6.8), and the separating gel contained 5–8% acrylmide (pH 8.8) with 25% glycerol (8 x 10 cm, 0.75 mm thick; Hoefer SE 250). Ten-microliter samples were loaded per lane. The gels were stained with silver, according to the procedure described by Oakley et al. (30). Control samples of DIAm bundles in a 1:200 dilution of SDS sample buffer were run with the single fibers for comparison of migration patterns of the MHC isoforms. Western blot analysis was also periodically performed to confirm this identification of MHC isoforms by migration patterns (13, 14, 53).

Measurement of MHC concentration in single fibers.   The methods used to measure MHC concentration in single DIAm fibers have been previously described in detail (13, 14, 53). Briefly, known concentrations of purified rabbit MHC (Sigma M-3889) were separated on the same gels as those used for detecting MHC in single fibers. The gels were silver stained, and a high-resolution scanner (Microtek ScanMaker 5, 600 dpi) was used for densitometric analysis. The brightness area product of each rabbit MHC sample was determined from the area and average brightness of each band following subtraction of local background. Based on the linear relationship between brightness area product and MHC concentration in these standard samples, the MHC concentration in single DIAm fibers was determined.

Statistical analysis.   The effects of unilateral DIAm DNV on V_max ATPase, ATP_iso, _fs, V_o, and k_TR were analyzed by using a two-way ANOVA. When appropriate, post hoc analysis (Student's t-test) was also used. Differences were considered as significant at P < 0.05. All data are presented as means ± SE.

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
MHC isoform expression in DIAm fibers.   On the basis of differences in electrophoretic migration, four MHC isoforms were identified: MHC_slow, MHC_2A, MHC_2X, and MHC_2B. In single fibers from CTL DIAm, MHC_slow, MHC_2A, and MHC_2X were all observed to be singularly expressed, while expression of the MHC_2B isoform was only observed together with MHC_2x (Fig. 1). The expression of four different MHC isoforms was also detected by immunohistochemistry, although the coexpression of MHC_2X and MHC_2B isoforms could not be unambiguously identified.

View larger version (31K): [in this window] [in a new window]   Fig. 1. Electrophoretic determination of myosin heavy chain (MHC) isoform expression in single rat diaphragm muscle (DIAm) fibers. Most DIAm fibers in control (CTL) displayed singular expression of MHCslow, MHC2A, and MHC2X isoforms. Expression of MHC2B in DIAm fibers was always observed, together with MHC2X (coexpression; top). Following DNV, expression of MHC2B in DIAm fibers was not detected, and the incidence of coexpression of MHCslow with MHC2A, and MHC2A with MHC2X, isoforms increased (not shown). The concentration of MHC extracted from single DIAm fibers was determined by comparison to known concentrations of commercially available MHC run in the same gel (bottom). The dimensions of the single fiber (cross-sectional area and length – number of sarcomeres in series) were measured, and MHC content per half-sarcomere was calculated based on these measurements.

Effects of DNV on DIAm fiber cross-sectional area.   In CTL DIAm, the cross-sectional area of fibers expressing MHC_slow and MHC_2A isoforms was significantly smaller than those expressing MHC_2X alone or together with MHC_2B (Fig. 2; P < 0.05). After 2 wk of DNV, the cross-sectional area of fibers expressing MHC_slow and MHC_2A was slightly greater than CTL, while the cross-sectional areas of fibers expressing MHC_2X were significantly smaller (Fig. 2; P < 0.05).

View larger version (12K): [in this window] [in a new window]   Fig. 2. Cross-sectional areas of DIAm fibers expressing MHC2X decreased following 2 wk of denervation (DNV). In CTL DIAm, fibers expressing MHCslow and MHC2A were smaller than those expressing MHC2X alone or together with MHC2B. Following 2 wk of DNV, cross-sectional areas of fibers expressing MHCslow and MHC2A increased slightly, whereas fibers expressing MHC2X atrophied. Expression of MHC2B was not detected in single fibers after DNV. However, the incidence of coexpression of MHCslow and MHC2A, and MHC2A and MHC2X, isoforms increased in the DNV DIAm. *Significant difference (P < 0.05) compared with fibers expressing MHCslow. #Significant difference (P < 0.05) between CTL and DNV.

View larger version (11K): [in this window] [in a new window]   Fig. 3. The maximum velocity (Vmax) of the actomyosin ATPase reaction in single DIAm fibers expressing different MHC isoforms was measured using a quantitative histochemical technique. In CTL DIAm, Vmax was significantly higher in fibers expressing MHC2X alone or together with MHC2B compared with fibers expressing MHCslow and MHC2A. After 2 wk of DNV, there was significant decrease in Vmax across all fibers, but this effect was most pronounced in fibers expressing MHC2X. *Significant difference (P < 0.05) compared with fibers expressing MHCslow. #Significant difference (P < 0.05) between CTL and DNV.

View larger version (15K): [in this window] [in a new window]   Fig. 4. A: the ATP consumption rate during maximum isometric activation (ATPiso) of single permeabilized rat DIAm fibers expressing MHC2X decreased after DNV. ATPiso (measured at 15°C) varied with MHC isoform expression in CTL fibers, whereas no significant differences across DNV fibers expressing different MHC isoforms were observed. There was a significant decrease in ATP consumption rate of DNV fibers expressing MHC2X compared with CTL. B: effect of DNV on ATP consumption rate per MHC content. ATPiso was normalized to MHC content to evaluate the effect of cross-bridge number on ATP consumption rate. Significant differences were found in ATPiso per MHC content across fibers expressing different MHC isoforms in CTL DIAm. DNV resulted in significant decrease in ATPiso per MHC content of fibers expressing MHC2X. *Significant difference (P < 0.05) from fibers expressing MHCslow. #Significant difference (P < 0.05) from CTL within a fiber type.

Reserve capacity.   In CTL DIAm, the difference between V_max ATPase and ATP_iso, reflecting the reserve capacity for ATP consumption, was higher for fibers expressing MHC_slow and MHC_2A compared with fibers expressing MHC_2X alone or together with MHC_2B (Fig. 5; P < 0.05). After 2 wk of DNV, the reserve capacity for ATP consumption was significantly reduced across all DIAm fibers, but to a greater extent in fibers expressing MHC_2X (Fig. 5; P < 0.05).

View larger version (12K): [in this window] [in a new window]   Fig. 5. Effect of DNV on reserve capacity for ATP consumption. Reserve capacity was lower for fibers expressing MHC2X and MHC2X/2B compared with fibers expressing MHCslow and MHC2A in CTL DIAm. DNV reduced reserve capacity in all fiber types, but to a greater extent in fibers expressing MHC2X. *Significant difference (P < 0.05) from fibers expressing MHCslow. #Significant difference (P < 0.05) from CTL within a fiber type.

_fs.

View this table: [in this window] [in a new window]   Table 3. Effect of DNV on fs in single diaphragm muscle fibers

View larger version (13K): [in this window] [in a new window]   Fig. 6. A: effect of DNV on maximum unloaded shortening velocity (Vo). Vo was significantly higher in fibers expressing MHC2X alone or together with MHC2B isoforms compared with fibers expressing MHCslow and MHC2A in CTL DIAm. DNV resulted in significant decrease in Vo across all fibers, but this effect was more pronounced in fibers expressing MHC2X. B: effect of DNV on cross-bridge cycling rate (kTR). Significant differences in kTR were found across all fibers expressing different MHC isoforms in both CTL and DNV DIAm. DNV resulted in significant decrease in kTR across all fibers. *Significant difference (P < 0.05) from fibers expressing MHCslow. #Significant difference (P < 0.05) from CTL within a fiber type.

f_app and g_app.   In CTL DIAm, f_app and g_app varied across fibers expressing different MHC isoforms, with a rank order of MHC_2X and MHC_2B coexpress > MHC_2X > MHC_2A > MHC_slow (Fig. 7; P < 0.05). After 2 wk of DNV, both f_app and g_app slowed across all DIAm fibers (Fig. 7; P < 0.05), but this slowing was more pronounced for fibers expressing MHC_2X (Fig. 7; P < 0.05).

View larger version (13K): [in this window] [in a new window]   Fig. 7. Effect of DNV on apparent rate constants for cross-bridge attachment (fapp) and detachment (gapp). Significant differences in both fapp and gapp were found across all fibers expressing different MHC isoforms in CTL DIAm. DNV resulted in significant decrease in both fapp and gapp across all fibers. *Significant difference (P < 0.05) from fibers expressing MHCslow. #Significant difference (P < 0.05) from CTL within a fiber type.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

Effect of DNV on DIAm MHC isoform expression and cross-sectional area.   In agreement with previous studies, we found that DNV affected the expression of different MHC isoforms. Most striking was the markedly reduced expression of the MHC_2B isoform and the increased incidence of coexpression of MHC isoforms. In the rate DIAm, the MHC_2B isoform is typically coexpressed in varying proportion with the MHC_2X isoform (10–12). Clearly, MHC isoform coexpression affected ATP_iso, since values in fibers coexpressing MHC isoforms were generally intermediate to those observed in fibers singularly expressing MHC isoforms. However, MHC isoform transitions and coexpression alone cannot explain the results of the present study, since both the ATP_iso of fibers singularly expressing the MHC_2X isoform were reduced following DNV.

Previous studies in several species have reported a transient hypertrophy of DIAm fibers following unilateral DNV (16, 20, 38, 47, 48), with fiber-type-dependent differences in morphological adaptations appearing after 1–2 wk (56–59). Following DNV, DIAm fibers expressing MHC_slow and MHC_2A remain slightly hypertrophied, whereas fibers expressing MHC_2X display selective atrophy. Similar selective fiber-type-dependent atrophy of DIAm fibers has been noted in a number of other physiological and pathological conditions, such as corticosteroid treatment (25, 40, 54), hypothyroidism (11, 16, 44), heart failure (21), and under-nutrition (24, 26, 27, 40). A decrease in MHC content per half-sarcomere (reduced number of cross bridges, n in Eq. 1) should result in a reduction in ATP_iso, which was observed in DIAm fibers expressing MHC_2X following DNV. Similarly, we found that a decrease in MHC content per half-sarcomere induced by DNV (10) or hypothyroidism (11) is associated with reduced maximum isometric force generated by DIAm fibers expressing MHC_2X.

Effect of DNV on ATP_iso and cross-bridge cycling rate.   MHC is the site of ATP hydrolysis during cross-bridge cycling, and ATP consumption rate is a major determinant of muscle fiber mechanical performance, as evidenced by the close relationship between V_max ATPase (measured biochemically) and fiber-type composition and contractile properties of various skeletal muscles (1). We and others have used an NADH-linked fluorometric technique to measure ATP consumption rates in single permeabilized muscle fibers during maximum isometric activation (i.e., ATP_iso) (3, 19, 39, 42). In general, in different muscles, it has been shown that fibers expressing MHC_slow have slower ATP_iso compared with fibers expressing MHC_2A, MHC_2X and MHC_2B isoforms. However, ATP_iso is submaximal and does not establish maximum capacity for ATP hydrolysis (19, 39, 42). Furthermore, ATP consumption increases with power output and work performance (Fenn effect) (8, 9, 39). Thus, as fibers shorten and reach maximum power, ATP consumption rate increases (39). We have previously established in the rat DIAm that the maximum rate of ATP consumption is achieved at a shortening velocity corresponding to peak power output of DIAm fibers. Accordingly, V_max ATPase establishes the upper limit for ATP consumption for each fiber type. The difference in ATP consumption rates from ATP_iso to V_max ATPase provides a measure of the reserve capacity for ATP consumption in muscle fibers. The difference in ATP consumption rates from ATP_iso to V_max ATPase provides a measure of the reserve capacity for ATP consumption in muscle fibers. In this study, we found that the reserve capacity for ATP consumption was 65, 55, and 50% for fibers expressing MHC_slow, MHC_2A, and MHC_2X alone or together with MHC_2B, respectively. DNV significantly reduced the reserve capacity for ATP consumption across all DIAm fibers, but to a greater extent in fibers expressing MHC_2X.

According to Eq. 1, a decrease in ATP_iso could result from a decrease in the number of cross bridges per half-sarcomere (n, MHC content per half-sarcomere), a decrease in g_app (as reflected by a slowing of cross-bridge cycling, k_TR or V_o), or a decrease in the _fs. Following 2 wk of DNV, there was a reduction in MHC content per half-sarcomere (n) in DIAm fibers expressing MHC_2X. When ATP_iso was normalized for this DNV-induced reduction in MHC content, ATP consumption per MHC molecule was still decreased in fibers expressing MHC_2X. Following 2 wk of DNV, cross-bridge cycling rate was slower in fibers expressing MHC_2X (as reflected by slower k_TR and V_o); thus a decrease in g_app could also account for the reduction in ATP_iso in DIAm fibers expressing MHC_2X. Finally, the _fs was unaffected by DNV. Together, these results support the conclusion that DNV reduces ATP_iso in DIAm fibers expressing MHC_2X by reducing MHC content and by slowing cross-bridge cycling rate (i.e., g_app).

The underlying mechanism(s) by which DNV affects selective changes in MHC content and cross-bridge cycling in DIAm fibers expressing MHC_2X is unclear. One possibility is that the removal of neurotrophin influence following DNV leads to a selective atrophy of DIAm fibers expressing MHC_2X, and that the reduction in MHC content per half-sarcomere leads to altered myofilament lattice spacing that, in turn, affects cross-bridge cycling rate and ATP consumption.

Clinical relevance.   Although infrequent, DIAm DNV may occur during cardiac or thoracic surgery. However, the DNV model also relates to other physiological and pathological conditions that are associated with decreased muscle mass, reduced MHC content, and/or muscle weakness, such as sarcopenia, corticosteroid treatment, heart failure, chemotherapy, under-nutrition, and hypothyroidism. The significance of the results of the present study should not be narrowly limited to DNV. What is important is that DNV induces a decrease in MHC concentration in muscle fibers. Since MHC is the site of ATP hydrolysis during muscle activation and cross-bridge cycling, a decrease in MHC concentration should result in a decrease in ATP consumption during muscle activation (i.e., a decrease in ATP_iso). Indeed, a decrease in ATP_iso was observed, but to a greater extent than that predicted by the decrease in MHC concentration alone: ATP_iso normalized for MHC concentration was still reduced in type IIx fibers. Thus factors in addition to the decrease in MHC concentration must also play a role. Indeed, DNV was found to induce a slowing of V_o and k_TR, indicating a slowing of cross-bridge cycling rate that was more pronounced in type IIx fibers.

With corticosteroid treatment (25, 54, 55), hypothyroidism (11, 16, 44), heart failure (21), and under-nutrition (24, 26, 27, 40), there is also DIAm weakness and selective atrophy of fiber expressing MHC_2X alone or together with MHC_2B. Thus selective effects on fibers expressing MHC_2X and/or MHC_2B may extend to other models where atrophy and muscle weakness are found. Whether or not there are changes in MHC concentration that affect ATP consumption rate has not been explored in these conditions.

	GRANTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The study was supported by funding from the National Institutes of Health (AR51173 and HL57680 to G. C. Sieck), and the Mayo Foundation.

	ACKNOWLEDGMENTS

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The authors gratefully acknowledge the technical assistance of Yun-Hua Fang, Rebecca Macken, and Thomas Keller.

	FOOTNOTES

 

Address for reprint requests and other correspondence: G. C. Sieck, Dept. of Physiology and Biomedical Engineering, 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.

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