1 Department of Biochemistry & Biophysics, Cardiovascular Research Institute, University of California, San Francisco, California 94143; and 2 Department of Physiological Sciences, University of Stellenbosch, Matieland, Stellenbosch 7602, South Africa
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ABSTRACT |
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The role played by ADP in modulating cross-bridge function has been difficult to study, because it is hard to buffer ADP concentration in skinned muscle preparations. To solve this, we used an analog of ADP, spin-labeled ADP (SL-ADP). SL-ADP binds tightly to myosin but is a very poor substrate for creatine kinase or pyruvate kinase. Thus ATP can be regenerated, allowing well-defined concentrations of both ATP and SL-ADP. We measured isometric ATPase rate and isometric tension as a function of both [SL-ADP], 0.1-2 mM, and [ATP], 0.05-0.5 mM, in skinned rabbit psoas muscle, simulating fresh or fatigued states. Saturating levels of SL-ADP increased isometric tension (by P'), the absolute value of P' being nearly constant, ~0.04 N/mm2, in variable ATP levels, pH 7. Tension decreased (50-60%) at pH 6, but upon addition of SL-ADP, P' was still ~0.04 N/mm2. The ATPase was inhibited competitively by SL-ADP with an inhibition constant, Ki, of ~240 and 280 µM at pH 7 and 6, respectively. Isometric force and ATPase activity could both be fit by a simple model of cross-bridge kinetics.
skeletal muscle; tension; fatigue; cross-bridge modeling
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INTRODUCTION |
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DURING SKELETAL MUSCLE FATIGUE, several major mechanical and energetic changes take place. These changes include a decrease in the maximal tension (P0), a slower maximal contraction velocity (Vmax), a slower rate of relaxation (15, 16, 24), and a higher tension economy, i.e., more tension generated per ATP hydrolyzed (9, 10). A popular hypothesis is that many of these changes may be due to the effects of increased levels of specific metabolites, including ADP, on muscle cross bridges. However, data to support this hypothesis have not been unambiguous, and many aspects of fatigue remain unexplained.
Studies of fatigue in vivo are complex because numerous metabolites change, making the correlation with the alterations of the properties of the muscle difficult to establish (for review, see Refs. 12 and 20). To delineate the effects of a single metabolite, such as ADP, permeabilized isolated fibers have been studied in vitro (8, 13, 19, 23, 27, 30). Measurements made at 10-15°C suggested that the drop in tension is probably due to the lower pH and higher free inorganic phosphate concentration ([Pi]) observed during fatigue (5, 28, 31, 42), but these effects are much reduced at more physiological temperatures (34, 42, 44, 41). A lower pH is also known to decrease contraction velocity; however, it does not completely account for the drop in Vmax seen during fatigue (34, 42).
According to the accepted model of the actomyosin cross-bridge cycle, ADP, which increases from 20 to 200 µM during fatigue (14), should compete with ATP for the ATP binding site on myosin and thereby slow down cross-bridge detachment (13, 36, 38). This, in turn, should inhibit ATPase activity and contraction velocity and should enhance isometric tension. Together, these effects would increase the economy of contraction. However, to date the observed effects of increased ADP on the tension and ATPase activity, although in the right direction, have been found to be too small to make a significant contribution (4, 8, 13, 29, 30, 40).
In skinned muscle studies, an ATP-regenerating system [with creatine kinase (CK) or pyruvate kinase (PK)] can be used to maintain ATP concentrations within the fiber. However, if one were to add additional ADP to a solution with an ATP-regenerating system, it would quickly be phosphorylated to ATP, leaving little time to observe the effects of increasing ADP on fiber mechanics. To circumvent this problem, investigators have used high concentrations of nucleotides in the absence of a regeneration system and have estimated intracellular concentrations by analyzing the diffusion of nucleotides (8). Photolysis of caged compounds has also been used for instantaneous generation of ADP (29). Also, higher ADP concentrations have been generated by addition of extra creatine (Cr) to the bathing solution (4).
We have recently come up with one solution that facilitates the study of the effects of ADP in skinned muscle preparations while controlling other nucleotide concentrations with a regeneration system. We have found that attachment of a spin label, to the 2' or 3' positions on the ribose of ATP, greatly inhibits interaction with CK or PK. This analog binds to myosin and to actomyosin with an affinity that is equal to that of ADP (11), and mechanical data presented here reconfirmed this conclusion. Thus we were able to use an ATP-regenerating system to maintain a well-defined concentration of ATP within the fiber, avoiding the buildup of ADP from hydrolysis of ATP, and we could then measure the mechanical responses of the fiber over wide ranges of ATP and spin-labeled ADP (SL-ADP) concentrations. We determined the effects of SL-ADP on ATPase activity and tension at varying concentrations of ATP, finding that SL-ADP changes ATPase activity and tension in a competitive manner. The cross-bridge interactions are also affected by lowering of pH and/or accumulation of Pi, discussed above, and under such conditions we hypothesized that the effects of SL-ADP may be different from those obtained at control conditions (i.e., high ATP, low Pi, pH 7).
The results can be explained by a simple model in which SL-ADP competes with ATP at the end of the power stroke. An adequate fit to the data required that cross bridges in force-generating states were capable of rapid exchange with pre-power stroke cross bridges. Thus the transition from pre-power stroke cross bridges to force-generating cross bridges can be reversed by binding of SL-ADP, which occurs at the end of the power stroke.
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METHODS |
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Fiber mechanics. Rabbits (New Zealand White) were heavily sedated and euthanized according to the guidelines of the Institutional Animal Care and Use Committee for the University of California, San Francisco. Psoas fibers were harvested and chemically skinned as described previously (7). For mechanical experiments, single fibers were dissected from a bundle of fibers on a cold stage while still immersed in the skinning solution. A single fiber was then mounted between a solid-state force transducer and a rapid motor for changing fiber length, as previously described (7), using Duco cement (Dencon Consumer Products, Danvers, MA) diluted in acetone. The apparatus had two temperature-controlled wells, and fibers could be rapidly switched between solutions. The fiber was then lowered into a relaxing solution, and fiber length and thickness were measured. The length of the unfixed portion of the fiber between the arms varied from 3 to 6 mm, fiber diameter ranged from 50 to 80 µm, and initial sarcomere length ranged from 2.2 to 2.4 µm.
A common protocol was to first immerse the fiber in a relaxing solution (well 1) and allow complete perfusion of CK and phosphocreatine (PCr) or PK and phosphoenol pyruvate (PEP) and nucleotides (~3 min). The fiber was then switched to an activating solution in well 2, and mechanical measurements were made. After measurements, the fiber was returned to well 1 and the solution in well 2 was replaced by the same fresh or another appropriate experimental buffer. Mechanical measurements were repeated, and the fiber was returned to the relaxing solution. The solution in well 2 was changed again, and mechanical measurements were repeated for a last time under control conditions to determine stability of the fiber. A 10% decline in P0 was set as our performance criterion. Damaged fibers were discarded. All fiber experiments were done at 10°C in both wells, and the bathing solutions were continuously stirred.Solutions. The basic rigor buffers contained (in mM) 120 potassium acetate (KAc), 5 MgCl2, 1 EGTA, and either 50 MOPS (pH 7) or 50 MES (pH 6). Phosphate (3-54 mM) was added to this solution, keeping the ionic strength constant by changing KAc (35). A relaxing solution was achieved by adding 0.05-0.5 mM ATP and either 20 mM PCr with 1 mg/ml CK or 20 mM PEP with 1 mg/ml PK. These solutions were made fresh on each experimental day. Activating solution was achieved at pCa 4.5. A range of SL-ADP concentrations was used (0.1-2 mM) to examine the effects of the ADP analog on mechanical fiber properties. The concentration of free Mg2+ varied in our experiments from 3.8 to 4.9 mM. Variation of Mg2+ in this range had no perceptible effect on fiber function. For some experiments involving measurements of ATPase activity at very low [ATP], trace contamination of ADP in the SL-ADP, ~0.1-0.5%, was removed by treatment of the stock solution of SL-ADP with 1 mg/ml myofibrils and 0.02 mg/ml adenylate kinase for 10 min at room temperature. This incubation hydrolyzes ADP to AMP but does not affect SL-ADP. The activity of the adenylate kinase was then inhibited by adding 100 µM diadenosine pentaphosphate, the myofibrils were removed by centrifugation, and the purified SL-ADP was used. All reagents were purchased from Sigma (St. Louis, MO).
Enzymatic studies.
In addition to mechanical experiments, the ATPase activities of rabbit
psoas myofibrils [prepared basically as described by Etlinger et al.
(18)] were measured by direct determination of NADH
depletion linked to ADP production, described in more detail previously
(33). Activating solutions contained 10 mM PEP, 0.25 mM
-NADH, 0.07 mg/ml lactate dehydrogenase (LDH), and 0.2 mg/ml
pyruvate kinase. The activities of the enzymes used were (in
µmol · min
1 · mg
protein
1) 200 CK (pH 7, 37°C), 430 PK (pH 7.6, 37°C),
and 940 LDH (pH 7.5, 37°C).
SL-ADP. SL-ADP was synthesized according to Crowder and Cooke (11). Both thin-layer chromatography (TLC) and mechanical measurements revealed that SL-ADP was very slowly phosphorylated to SL-ATP by CK. Enzyme activity was tested by incubating the SL-ADP with the enzyme in rigor buffer at 10°C. At specified times an aliquot was removed and spotted on a precoated high-performance TLC aluminum plate (silica gel, 60 F254, 0.2 nm; Merck, Darmstadt, Germany) along with appropriate controls. The plate was well dried and developed in an isopropanol:NH4OH:H2O solution (6:3:1 vol/vol) at room temperature. After development, the spots corresponding to SL-ADP and SL-ATP were scraped and extracted in 600 mM KCl and 50 mM MOPS, pH 7.5, and the presence and quantity of SL-nucleotide was measured by electron paramagnetic resonance (EPR) spectroscopy (ER/200D; IBM Instruments, Danbury, CT). A 1 mM solution of SL-ADP was approximately half phosphorylated, 50 ± 12% (n = 4), in 15 min at 10°C by 1 mg/ml CK. This rate is only 0.06% of the activity of CK for ADP under similar conditions. For our mechanical experiments performed at 10°C within 90 s of adding SL-ADP, the conversion rate was slow enough that negligible SL-ATP would be generated within the fiber. In addition, during the course of the experiments we discovered that SL-ADP was not phosphorylated by the PK-PEP system, within the accuracy of our measurements (again TLC, EPR, and mechanical measurements). Thus we switched to the PK-PEP system, which gave results identical to those of the CK-PCr system but allowed SL-ADP solutions to be used for a longer time.
ATP regeneration system. To verify the capability of 1 mg/ml PK to maintain ATP concentration and eliminate ADP, we measured force (n = 6) at three conditions: in the presence of 150 µM ATP, 3 mM Pi, and 20 mM PEP, pH 7. We compared force generated with 1 mg/ml PK vs. 2 mg/ml PK, which gave forces of 0.093 ± 0.003 and 0.092 ± 0.002 N/mm2, respectively; we also compared force generated with 1 mg/ml PK and then with the addition of 1 mM ADP, which resulted in forces of 0.096 ± 0.003 and 0.094 ± 0.005 N/mm2, respectively. In the latter comparison, a small drop in force was observed that was expected as the PK rapidly rephosphorylated the added ADP to ATP, thus making more ATP available to the fiber. We estimated internal [ADP] to be <25 µM, based on a calculation of the production, consumption, and diffusion of ADP according to the equations of Ref. 8. Thus, in the experiments described here, competition from internally generated ADP is much less than that from added SL-ADP.
Data reduction.
Force data for each condition (in N/mm2) were averaged and
are expressed as means ± SD; n represents the number
of fibers used, when reported. In the case of parameters based on fits,
the ±95% confidence interval is given (i.e., when reporting
inhibition or dissociation constants). Mean force data were fit by
assuming that the effect of SL-ADP could be expressed as a simple
binding isotherm
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(1) |
Model.
Simple three- and four-state models of cross-bridge kinetics were
analyzed using commercially available software (Berkeley Madonna, vers.
8.0.1; R. I. Macey and G. F. Oster). The kinetic rates were
determined from the experimental data obtained for force and ATPase
activity in our isometric fibers and myofibrils. For a four-state
model, cross bridges are assumed to be in one of four states:
state 1, detached from actin, or attached weakly without
generating force; state 2, attached to actin in the power stroke; state 3, attached to actin in the state achieved
following binding of SL-ADP at the myosin nucleotide site; or
state 4, attached to actin with no nucleotide at the myosin
site. States 2, 3, and 4 all generate
force. We envision the following cross-bridge cycle. Cross bridges
start in state 1, having ATP or ADP Pi at the
nucleotide site and do not generate force. The transition to
state 2 involves some conformational change in myosin,
associated with the release of Pi that produces
actin-attached force-generating cross bridges. State 2 is in
reality a mixture of different states, and ADP release and binding
involve one of these, which is state 3 in the model. Release
of ADP from state 3 leads to the rigor state, state
4. The binding of ATP in state 4 returns the cross
bridge to state 1. The force generated by the model is a
function of the populations in the force-generating states,
states 2, 3, and 4, and the amount of
force generated by a cross bridge in each one of these states. The
magnitudes of the relative forces generated by cross bridges in
states 2, 3, and 4 are not known. We
assume here that the transitions among states 2,
3, and 4 do not change the conformation of the cross bridge so that all these states generate the same force. Release
of ADP from force-generating cross bridges does not change the strain
on the cross bridge, so rigor states (state 4) maintain the
same tension as state 2 or 3. However, the effect
of relaxing this assumption did not change the conclusions. At high
levels of ATP, the population of states 3 and 4 is small, and the fraction of force-generating cross bridges is
given by the steady-state distributions of states 1 and
2. For a three-state model, states 2 and 3 were combined. As we will discuss later, a four-state model was
more applicable (see Fig. 5). ATPase activity was calculated as the
flux from state 4 back to state 1. The model was
not sensitive to the exact values of the rate constants connecting
states 1, 2, and 3, as long as certain
conditions were satisfied. In particular, the reverse transitions had
to be more rapid than the flux through the states, or P' would not be a
constant. The values of the rate constants k12,
k21, k23, and
k32 were set so that the ATPase activity was 1 s1 and the fraction of the non-force state, state
1, was 50% at high [ATP]. The assumption that 50% of the cross
bridges generate force is arbitrary, but the exact value is not
crucial, and the data could be fit equally well by assuming other
values, e.g., 25%. In the simulations shown, all four rate constants
were set to 3 s
1. However, the conclusions were similar
if they were varied between 1 and 10, keeping the flux through them at
the assumed ATPase rate of 1 s
1, by using variable ratios
of forward to backward rate constants to keep the flux constant.
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RESULTS |
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SL-ADP increases isometric tension.
In analyzing the effect of SL-ADP on isometric tension, we activated a
fiber and allowed it to rise to a stable peak force (P0).
We then added SL-ADP and allowed the tension increase to reach a stable
value. A sample data set is presented in Fig.
1, showing where we added 0.5 mM of
SL-ADP to a fiber activated in 50 µM ATP. The fiber could then be
returned to a bath that lacked SL-ADP, and tension returned to control
values, showing that the increase was reversible.
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Comparison with previous results. In a number of previous investigations the effect of ADP on fiber tension was measured by addition of relatively high millimolar concentrations of ADP to fibers activated in millimolar ATP (8, 13, 27, 29, 30, 40). These investigations have found that tension increases by ~10% upon addition of 1 mM ADP to fibers activated in 3-5 mM ATP. To determine whether SL-ADP would have the same effect on tension as reported in these earlier studies, we added 1 mM SL-ADP or 1 mM ADP to fully activated fibers in 3 mM ATP and 3 mM Pi, in the absence of an ATP-regenerating system (pH 7, 10°C). The average increase in tension was 12 ± 5% for SL-ADP or 10 ± 2.5% for ADP (n = 3). The value we obtained for ADP is similar to the results of previous investigations and is similar to that for SL-ADP. The equivalence of the force increase found for SL-ADP and ADP further shows that the presence of the label on the ribose does not alter the binding of the analog to myosin in active fibers, as initially reported by Crowder and Cooke (11) in an EPR spectra analysis. Moreover, mant-ADP, a fluorescent analog that is similar in structure to SL-ADP, also binds to myosin similarly to ADP (45).
SL-ADP decreases isometric ATPase activity.
The effect of SL-ADP on the ATPase activity was determined by using
myofibrils from psoas muscle. The myofibrils were first cross-linked
lightly with glutaraldehyde to prevent shortening so that they were a
reasonable model of an isometric muscle fiber (22). The
ATPase activity was determined as a function of concentration for both
ATP and SL-ADP. Our data show that SL-ADP acted as a competitive
inhibitor of the binding of the substrate ATP (Fig. 4). Competitive inhibition is described
by the equation
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(2) |
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DISCUSSION |
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The main findings of the present study are that SL-ADP increased isometric tension and decreased ATPase rate in skinned rabbit psoas muscle fibers. In current models of cross-bridge interaction, ATP is thought to dissociate the rigor state cross bridges (nucleotide-free myosin heads), which occur upon release of ADP from force-generating states in the cross-bridge cycle (17, 21, 36). The binding of ADP, preventing cross-bridge dissociation in vivo, would be expected to increase tension and decrease ATP utilization rate. In general, as discussed in the Introduction, experimental studies have confirmed these predictions, as do the results presented here. The measurements described here explore the effects of the competition of ATP with an ADP analog over a wider range of concentrations of both nucleotides, allowing us to show that the ADP analog is a pure competitor with ATP and to determine how this competition changes with changing conditions. In particular, we address the question of the role of ADP in muscle fatigue. Finally, we present a model that describes nucleotide competition in isometric fibers.
Muscle properties at pH 7. The current results support and extend the results of a number of previous investigations, all of which showed that increased [ADP] potentiates isometric tension (8, 13, 27, 29, 30, 40, 23). In most of these investigations high concentrations of ADP competed against high concentrations of ATP in the absence of an ATP-regenerating system. We have advanced these observations by showing that this effect is also seen over a wider range of nucleotide concentrations, including more physiological (lower) concentrations of ADP. The potentiation of tension measured here at these lower ADP concentrations is in rough agreement with that seen earlier when the concentrations are extrapolated to the higher values previously used. We are in agreement with one study that showed that at high levels of ATP, low levels of ADP have little or no effect (4). We also agree with a study where addition of 0.7 mM ADP resulted in ~7% force increase (23). In the present study, the ATPase activity of isometric myofibrils was inhibited by SL-ADP. The values for Km for ATP and for Ki, 29 and 240 µM at pH 7, respectively, are both slightly higher than those observed by Sleep and Glyn (22, 39) using ADP instead of SL-ADP (Km = 17 µM and Ki = 170 µM).
Effects of lower pH and/or increased Pi.
At pH 6, a pH that is approached during severe fatigue, there was a
decrement in tension relative to that found at pH 7 of ~50%, as has
previously been shown (5, 16, 23, 28, 31, 32). The
addition of SL-ADP produced the same absolute increase in fiber tension
as was found at pH 7 (Table 1 or Fig. 2), which a simple model,
described below, could explain. K
Fatigue. In general, during fatigue in vivo, average [ADP] is observed to rise to ~200 µM and average [ATP] to not fall below 2-3 mM (see Ref. 20 for review). The effects of ADP seen in vitro in skinned fibers and myofibrils under similar conditions have been too small to produce a significant effect on the physiological properties of fatigued fibers in vivo. Our results generally agree with this conclusion. Our data using SL-ADP predict that if the ADP concentration were raised from 20 µM to 500 µM and the ATP level was decreased from 5 mM to 2 mM, the effect of ADP would be a 4% increase in force at pH 7. At pH 6, the relative effect of SL-ADP is greater, but it is a weaker competitor with ATP, so the relative effect on tension would be about the same as at pH 7. Thus the rise in ADP seen during fatigue can only play a significant role if the actual changes in the concentrations of the nucleotides are greater in the interior of the myofibrils than what has been inferred from measurements averaged over whole muscles. Is this possible? The ATP and ADP concentrations have been mostly measured in whole muscle using NMR or biopsy homogenates (14, 20). Both of these methods produce an average value of each nucleotide distributed throughout the fiber and in many different fibers that may not be equally activated or equally fatigued. Recent analyses by one of us (C. Karatzaferi) of single human muscle fibers using maximal intensity exercise (25, 26) have shown a large variability in nucleotide content of the different fiber types at rest and postexercise, with much lower levels of ATP and near depletion of PCr in some postexercise fast fiber fragments. Moreover, it has been calculated that ADP could rise to as high as 3.0 mM during contraction, when the PCr store is depleted (43). Our measurements show that if myofibrillar ADP reached 1 mM with 3 mM ATP, the effect would be an approximately further twofold increase in force to ~8% at either pH 7 or pH 6. Although an increase in the [ADP] may increase the tension economy by potentiating tension and inhibiting the ATPase activity, the effect on the tension, described above, will still be modest, and the effect on ATPase activity will be even smaller. Thus another mechanism must also operate to produce the twofold increase in tension economy seen in fatigue (9, 10).
Modeling cross-bridge kinetics.
Force and ATPase activity in isometric fibers as a function of ATP and
SL-ADP were analyzed by using a simple model to determine values for
the relevant kinetic parameters governing the binding of nucleotides in
the fiber (Fig. 5). Cross bridges are
assumed to be in one of four states, described in Fig. 5. The effect of SL-ADP is to increase the rate of the transition from the rigor state,
state 4, back into state 3. As the concentration
of SL-ADP increases, the proportion of cross bridges in state 4 decreases, effectively decreasing the rate of ATP binding by decreasing
the concentration of state 4. The effect of increasing the
rate of this transition will be to increase force and inhibit the
ATPase activity, as observed. The model allows us to quantify these
phenomena and to draw connections between force and ATPase activity.
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ACKNOWLEDGEMENTS |
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We thank Marija Matuska for synthesizing SL-ADP.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-32145 (R. Cooke). C. Karatzaferi is a recipient of an American Heart Association Fellowship.
Address for reprint requests and other correspondence: R. Cooke, Box 0448, Dept. of Biochemistry & Biophysics, Univ. of California, San Francisco, CA 94143-0448 (E-mail: cooke{at}cgl.ucsf.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 November 27, 2002;10.1152/ajpcell.00291.2002
Received 24 June 2002; accepted in final form 21 November 2002.
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