1Medical Pharmacology and Physiology, College of Medicine, 2Biomedical Sciences, College of Veterinary Medicine, 3Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, Missouri; and Molecular Imaging Research Center, Departments of 4Physiology and 5Radiology, Michigan State University, East Lansing, Michigan
Submitted 17 December 2004 ; accepted in final form 25 January 2005
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ABSTRACT |
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muscle energetics; muscle relaxation; magnetic resonance spectroscopy
The importance of limiting ADP accumulation in skeletal muscle is implied by the apparent lack of such a limit on another product of ATP hydrolysis, inorganic phosphate (Pi). The accumulation of Pi during high-energy demands is close to being proportional to the decline in phosphocreatine (PCr), and can be as great as 40 mM (24). In contrast, ADP accumulation in healthy muscle has been estimated to rise to no greater than 300 µM even during intense, fatiguing contraction regimens (4). The physiological consequences of ADP accumulation are related to a direct effect of ADP on the kinetics of various enzymes. For example, millimolar concentrations of ADP inhibit the peak rate of myosin cross-bridge cycling (6), and may cause calcium leakage from the sarcoplasmic reticulum (SR) (17). In addition, ADP accumulation will cause a decline in the free energy available from the hydrolysis of ATP (
GATP) because
GATP is a function of the ratio of ADP and Pi to ATP as defined below.
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Direct quantification of the GATP is impossible because the ADP present in muscle is largely bound to proteins and not metabolically active or free to participate in enzymatic reactions (18). The myofibrillar proteins actin and myosin both bind ADP (2) and account for most of the ADP measured in acid extracts of muscle tissue, making a direct measurement of free ADP (20300 µM) from the total, chemically measured ADP (>600 µM) unreliable. Thus the free ADP in skeletal muscle is typically calculated from the stoichiometry and assumed equilibrium of the creatine kinase reaction (16). In principle, free ADP can be directly measured by high-resolution in vivo 31P-NMR spectroscopy (31P-NMRS; e.g., Ref. 7) because the ADP bound to macromolecules is substantially line broadened and not observed in high-resolution spectra. However, phosphorus NMR is a relatively insensitive technique, and the small ADP accumulation expected in normal skeletal muscle has not been directly observed. Therefore, the limit of free ADP accumulation normally observed in skeletal muscle has prevented any direct assessment of free ADP.
A transgenic knockout model of AK deficiency (AK1/) has been recently developed (12), and provides a tool to investigate the metabolic and functional consequences of AK deficiency in skeletal muscle. Because AK is an important part of the metabolic circuit involved in the management of ADP, consequences of an inordinate accumulation of ADP would be expected if energy demands could be sufficiently elevated. The basal metabolic phenotype of AK1/ gastrocnemius muscle was reported to be similar to the wild-type (WT) controls, although an increase in mitochondrial markers and mitochondrial volume has been reported in a small superficial portion of the gastrocnemius muscle (9, 13). The tetanic contractile performance of AK/ muscle was also examined at extreme energy demands, and whereas the overall pattern of muscle fatigue was similar to WT controls, there was a marked slowing of muscle relaxation in AK/ muscles (9). Remarkably, AK1/ muscle exhibited an accumulation of up to 1.5 mM ADP (as measured in perchloric acid extracts) during energetically demanding, repetitive tetanic contractions, whereas the WT control muscles exhibited no such accumulation of ADP (9). An accumulation of free ADP of this magnitude would be expected to severely impair muscle relaxation (7), and slow cross-bridge cycling (6, 34). The main purpose of this study was to determine whether the increase in ADP observed in contracting AK1/ muscle is in fact free, representing an energetic challenge to muscle larger than previously considered possible in intact muscle. This was accomplished using 31P-NMR because only the free ADP is discernible using this technique. A secondary objective was to determine whether the reported elevations in mitochondrial content in AK1/ would result in an enhanced rate of PCr recovery after demanding contractions. The results indicate that free ADP accumulation observed by 31P-NMRS is transiently as high as 1.7 mM in contracting AK1/ gastrocnemius muscle, consistent with the amount of ADP accumulation estimated from the chemical measurements reported previously (9). In addition, the results show that the PCr recovery rate constant is significantly faster in muscle of AK1/ compared with WT control mice, consistent with increased mitochondrial capacity.
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METHODS |
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In situ contractions. Muscle contractions of the mouse hindlimb muscles were elicited via direct electrical stimulation of the sciatic nerve, accessed by an incision on the lateral portion of the right leg, near the hip. Electrodes were placed in contact with the nerve and shielded from surrounding tissue to prevent extraneous stimulation. The sciatic nerve was crushed by tying a knot above the point of contact with the electrodes to eliminate antidromic propagation. The plastic shielding of the electrode wires was secured to the mouse limb with adhesive to prevent any movement of the electrodes. Stimulation voltage was determined to be supramaximal by test stimuli before the experiment. Tetanic contractions were elicited via 100-ms trains of 0.05-ms pulses at a frequency of 150 Hz, applied at a rate of 2 tetani/s for 32 s. This is the same frequency and duration of contraction that previously elicited large changes in total chemically measured ADP (9).
The mouse was secured in the head-down position, such that the lower right limb could be positioned adjacent to the NMR coil in a custom-built probe. Movement of the lower limb was minimized by tying the patellar tendon to Plexiglas supports. The achilles tendon was tied to a wheatstone load cell for recording muscle tension. The muscle was then stretched by adjusting the position of the load cell to a length that yielded maximum tetanic tension.
31P-NMR spectroscopy.
31P-NMR spectra [162 MHz, 10,000 Hz sweep width, 1,024 complex points, repetition time (TR), 1 s] were acquired on a Bruker AM400 spectrometer via a 0.5 x 0.8-cm saddle-shaped surface coil positioned over the gastrocnemius muscle. Spectra acquired at rest and after stimulation were the average of 128 and 32 scans, respectively. To minimize motion artifacts during the 2 tetani/s stimulation, acquisition of single scans was triggered by the stimulator to occur 200 ms after the start of every other tetanus (i.e., TR 1 s). These scans were retrospectively added in blocks of eight, yielding a time resolution of 8 s (16 contractions) during the stimulation. All spectra were filtered by a 25-Hz exponential and zero filled to 2,048 points before Fourier transformation and manual phase correction. Finally, spectra from 89 animals at corresponding time intervals during the stimulation were added together. Four complete series of combined spectra during stimulation with 89 animals each were thus obtained. All spectra are presented with a common (2.52 ppm) shift for the PCr peak. The time course of PCr recovery after stimulation was determined in individual animals. For this analysis, PCr concentrations were estimated using the method of natural line shapes (11). The time constant for PCr recovery () was estimated from a single exponential fit (25) immediately after 64 contractions at 120 tetani/min. Intracellular pH was estimated from the chemical shift of Pi, as done previously (23). The concentrations of ATP, ADP, IMP, and PCr were calculated using the known content of ATP measured from chemical extracts in the gastrocnemius muscle of these mice [5.84 µmol/g wet wt (9)]. The peak integral of the
-ATP peak at rest, and the sum of the peak integrals of the
-ATP, IMP, and
-ADP peaks during contractions, were considered quantitatively equivalent to the chemically measured concentration of ATP (5.84 µmol/g wet wt), assuming that no substantial loss of nucleotides occurred during contractions (21). Thus metabolite concentrations were estimated on the basis of relative peak integrals.
Analysis of the amount and location of 31P-NMRS of ADP in muscle may potentially be confounded by the changes in the chemical shift of the ADP peak with pH, and by differences in T1, and hence in the relative signal saturation that occurs as a result of the short TR interval used in this study (1 s). Therefore, a series of 31P-NMR spectra (242 MHz, sweep width 48,000 Hz, 1,024 complex points, TR 0.28 s, 1,024 scans/spectrum) were acquired on a Varian Unity 600 MHz spectrometer from solutions maintained at 37°C containing the following constituents (in mM): 8 ATP, 0.5 ADP, 1 Pi, 25 PCr, and 1 Mg, at an ionic strength of 0.25 M; ionic strength was balanced using acetate as the anion and Tris as the cation. The change in pH was buffered by (in mM) 50 MES, pH 6 and 6.5, 50 MOPS, pH 7, and 50 TES, pH 7.5 and 8. The apparent T1 of each peak was estimated from the exponential rise in peak intensity with increased TR. In solution, and at the higher magnetic field strength, we were able to maximize the signal-to-noise ratio to examine both pH and the pattern of relative saturation of ATP and ADP.
Statistical analysis. Student's t-test was used to determine significant differences in performance at 60 contractions and the time constants for PCr recovery between AK1/ and WT control groups. A P value of <0.05 was considered significant.
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RESULTS |
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Figure 6 shows the time course of PCr recovery in muscles after the stimulation in AK/ and WT mice. The fitted time constant () for PCr was significantly faster in AK/ (1.9 ± 0.2 min) compared with WT (2.7 ± 0.2 min) (P < 0.05). The recovery rate of PCr is an index of the oxidative capacity of muscle (25); therefore, this result is consistent with previous reports of increased intermyofibrillar mitochondria (12, 13) and increased citrate synthase activity (9) in the superficial gastrocnemius of AK/ mice.
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DISCUSSION |
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Accurate in vivo quantitation of ADP by 31P-NMRS is difficult for the following reasons: 1) the mouse muscle is very small and the signal available from 31P is not very sensitive, 2) the ADP accumulation is transient, so the signal-to-noise ratio cannot be increased by averaging the signal from an individual experiment over long periods of time, and 3) the longitudinal relaxation constant, or T1, of ADP is unknown in the intracellular environment and impossible to directly determine, given the transient nature of ADP accumulation. To overcome the problem of the low signal-to-noise ratio and transient nature of ADP accumulation, we added spectra from multiple animals at the same time point. Because the calculations of ADP content are dependent on the relative peak integrals of -ATP and
-ADP, and these peak integrals could be influenced by the relative T1 relaxation rates, in vitro experiments were performed using a series of different TRs. This revealed that the peak difference between
-ATP and
-ADP was 24% at 1 s (cf. Fig. 2). In other words, a comparison of the two peaks would underestimate ADP by 24%. However, the difference in vivo is likely less or even completely eliminated for two reasons. First, the T1 relaxation constants in vitro were measured at a higher field strength (600 MHz) than the experiments in vivo (400 MHz). Relatively shorter T1 relaxation constants are expected at lower field strengths (14). Second, relaxation rates in vivo are expected to be shorter than in vitro because of interactions with intracellular proteins. Therefore, in the present experiments, the peak integrals were not adjusted for apparent differences in T1 relaxation rates. Nevertheless, the calculated free ADP content using 31P-NMRS (1.7 mM) is in agreement with the ADP accumulation measured previously from muscle extracts (1.5 ± 0.1 mM; Ref. 9) and supports the accuracy of our calculations.
Free ADP.
The free ADP in tissue has normally been calculated from the known stoichiometry and assumed equilibrium of the CK reaction (16). The ADP measured from muscle extracts is mostly metabolically inactive because it is bound to intracellular proteins such as actin and is not free to participate in biochemical reactions (33). The difficulty in measuring free ADP directly with 31P-NMRS is not due to the presence of bound ADP, because the 31P-NMRS signal from bound ADP would be very broad and indistinguishable from background noise (22). The primary problems with observing free ADP with 31P-NMRS are that it never accumulates to levels necessary to be distinguishable from background noise (0.51 mM) as well as the lack of resolution between the chemical shift from ATP and ADP (Fig. 1). Direct measurement of free ADP by 31P-NMRS has been performed in a hypoxic coronary artery model using 31P-NMRS; however, this requires scans to be averaged over a 30-min time period to maximize the signal-to-noise ratio (8). Free-ADP accumulation in skeletal muscle has been suggested to rise to 0.3 mM (4) under high-energy demands, but this accumulation is transient due to the effectiveness of ATP synthesis pathways in skeletal muscle and to the rapid decline in energy demands due to fatigue. Thus this represents the first report of directly resolved free ADP by 31P-NMRS in the intact contracting skeletal muscle.
ADP and free energy of ATP.
The degree of ADP accumulation we report here represents, at the very least, a significant transient energetic challenge in the AK1/ muscle compared with the WT control muscle that is apparent from the calculated GATP [46 and 53 kJ/mol, respectively (9)]. A decline in the energy available from ATP would be expected to affect ATPases that depend on a large portion of the total energy available from ATP. The management of calcium in skeletal muscle is an energetically expensive process due to the high concentration of calcium inside the SR (
1 mM) and the relatively low concentration in the cytosol (
50100 nM at rest). The energy required to support this calcium gradient is defined as 2RT ln ([Ca2+]sr/[Ca2+]cyt), where [Ca2+]sr is the SR-free [Ca2+] and [Ca2+]cyt is the cytosolic [Ca2+], and at resting concentrations it is approximately 51 to 48 kJ/mol. At resting adenine nucleotide, and Pi concentrations, the
GATP is approximately 65 kJ/mol in the AK1/ and WT muscle. Thus, if there were no limit on SR calcium loading, and the cytosolic calcium were maintained at resting levels, the resting
GATP of 65 kJ/mol would be sufficient to support SR free calcium concentration of
15 mM (Fig. 8), which is
15 times what the physiological SR free-calcium load is estimated to be (5, 10, 26, 30). Thus at rest there is an excess of energy available to maintain the expected calcium gradient between the cytosol and the SR. However, if the minimum cost to maintain the resting calcium gradient is 48 kJ/mol, the
GATP of 46 kJ/mol measured in AK1/ muscle suggests that there is insufficient energy available to support this gradient. Research on the consequences of a depressed energy state on muscle calcium management has suggested that if the
GATP dropped below this limit, the failure of normal calcium management would lead to a lack of muscle relaxation (7), or in the case of cardiac muscle, impaired contractile reserve (31, 32). Remarkably, we (9) observed similar tetanic force production in the AK1/ muscles compared with WT muscle, albeit with delayed but nearly complete muscle relaxation. We believe that there are a few possible reasons that full muscle relaxation can occur when the energy available is presumed insufficient to maintain the resting calcium gradient. First, a
G of 43 kJ/mol is required to maintain a ratio of
1:4,000 between the cytosol and the SR, which works out to be 250 nM [Ca2+]cyt to 1 mM [Ca2+]sr (Fig. 8). An increase in the [Ca2+]cyt of this magnitude may not result in any significant force production and thus allow full muscle relaxation (35). Furthermore, if a decline in the SR calcium occurred, the energy available would support lower cytosolic calcium concentrations. A decline in the free calcium content of the SR may occur by calcium phosphate precipitation during fatigue; however, this process likely requires
12 min to occur, and therefore cannot explain our findings (1). Another possible mechanism to reduce cytosolic calcium and allow full relaxation is an increased calcium uptake by other organelles in the cell, such as the mitochondria, to effect a lower cytosolic-to-[Ca2+]sr ratio (3, 15). However, due to the relatively small amount of calcium taken up by the mitochondria, this does not appear to be a very reasonable explanation (15).
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It is worth mentioning that the increase in ADP, and hence the energetic challenge discussed above is likely very transient in nature. As contractions proceed, the force of contractions fall until a force is achieved that can be supported by the rate of ATP synthesis, and ADP accumulation would likely fall. The transient nature of this large ADP accumulation is even evident from an apparent reduction in the signal from -ADP observed from contractions 34-48 to contractions 49-64 in Fig. 5A. Thus, whereas the energetic challenge of calcium sequestration illustrated in Fig. 8 should still be valid even for the transiently high ADP, we do not know what the consequences of a more sustained ADP accumulation would be. It is also interesting to consider that the direct measure of free ADP observed in this study is an average of the free ADP present in the whole gastrocnemius muscle within the field of the NMR coil, and not necessarily representative of the highest free-ADP accumulation in select muscle fibers. Thus the energetic impairment in certain sections of the muscle due to an inordinate ADP accumulation may in fact be more dramatic than what we discuss here.
Enhanced mitochondrial capacity. In addition to the findings regarding free ADP, the faster rate of PCr recovery in AK/ muscles after the extreme energy demands used in this study indicate that AK/ resulted in an adaptive increase in mitochondrial capacity. This is in line with reports of increased citrate synthase activity (9) and mitochondrial volume by EM-morphometric analysis (13). While the increase in oxidative capacity was not evident in significantly improved force production with fatigue in this study (Fig. 7) or in others (9, 12), enhanced fatigue resistance may be more clear in much less demanding conditions.
In summary, we have demonstrated by 31P-NMRS that the chemically measured ADP accumulation of 1.5 mM in AK1/ muscle represents free and metabolically active ADP. This is the first direct observation of free ADP in contracting skeletal muscle and is severalfold greater than the normal limit of ADP accumulation. Finally, we estimate that the energetic consequence of this large ADP accumulation on calcium uptake may be that cytosolic calcium is elevated but still maintained below force-generating levels, thus permitting full muscle relaxation.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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2. Beinfeld MC and Martonosi AN. Effect of F-actin upon the binding of ADP to myosin and its fragments. J Biol Chem 250: 78717878, 1975.[Abstract]
3. Bruton J, Tavi P, Aydin J, Westerblad H, and Lannergren J. Mitochondrial and myoplasmic [Ca2+] in single fibres from mouse limb muscles during repeated tetanic contractions. J Physiol 551: 179190, 2003.
4. Chase PB and Kushmerick MJ. Effect of physiological ADP concentrations on contraction of single skinned fibers from rabbit fast and slow muscles. Am J Physiol Cell Physiol 268: C480C489, 1995.
5. Chen W, London R, Murphy E, and Steenbergen C. Regulation of the Ca2+ gradient across the sarcoplasmic reticulum in perfused rabbit heart. A 19F nuclear magnetic resonance study. Circ Res 83: 898907, 1998.
6. Cooke R and Pate E. The effects of ADP and phosphate on the contraction of muscle fibers. Biophys J 48: 789798, 1985.[Abstract]
7. Dawson MJ, Gadian DG, and Wilkie DR. Mechanical relaxation rate and metabolism studied in fatiguing muscle by phosphorus nuclear magnetic resonance. J Physiol 299: 465484, 1980.[Abstract]
8. Fisher MJ and Dillon PF. Direct determination of ADP in hypoxic porcine carotid artery using 31P NMR. NMR Biomed 1: 121126, 1988.[Medline]
9. Hancock CR, Janssen E, and Terjung RL. Skeletal muscle contractile performance and ADP accumulation in adenylate kinase deficient mice. Am J Physiol Cell Physiol 288: C1287C1297, 2005.
10. Hasselbach W and Oetliker H. Energetics and electrogenicity of the sarcoplasmic reticulum calcium pump. Annu Rev Physiol 45: 325339, 1983.[CrossRef][ISI][Medline]
11. Heineman FW, Eng J, Berkowitz BA, and Balaban RS. NMR spectral analysis of kinetic data using natural lineshapes. Magn Reson Med 13: 490497, 1990.[ISI][Medline]
12. Janssen E, Dzeja PP, Oerlemans F, Simonetti AW, Heerschap A, de Haan A, Rush PS, Terjung RR, Wieringa B, and Terzic A. Adenylate kinase 1 gene deletion disrupts muscle energetic economy despite metabolic rearrangement. EMBO J 19: 63716381, 2000.
13. Janssen E, De Groof A, Wijers M, Fransen J, Dzeja PP, Terzic A, and Wieringa B. Adenylate kinase 1 deficiency induces molecular and structural adaptations to support muscle energy metabolism. J Biol Chem 278: 1293712945, 2003.
14. Krssak M, Mlynarik V, Meyerspeer M, Moser E, and Roden M. 1H NMR relaxation times of skeletal muscle metabolites at 3 T. Magma 16: 155159, 2004.[Medline]
15. Lannergren J, Westerblad H, and Bruton JD. Changes in mitochondrial Ca2+ detected with Rhod-2 in single frog and mouse skeletal muscle fibres during and after repeated tetanic contractions. J Muscle Res Cell Motil 22: 265275, 2001.[CrossRef][ISI][Medline]
16. Lawson JW and Veech RL. Effects of pH and free Mg2+ on the Keq of the creatine kinase reaction and other phosphate hydrolyses and phosphate transfer reactions. J Biol Chem 254: 65286537, 1979.[Abstract]
17. Macdonald WA and Stephenson DG. Effects of ADP on sarcoplasmic reticulum function in mechanically skinned skeletal muscle fibres of the rat. J Physiol 532: 499508, 2001.
18. Martonosi AN, Gouvea MA, and Gergely J. Studies on actin III. G-F transformation of actin and muscular contraction (experiments in vivo). J Biol Chem 235: 17071710, 1960.[ISI][Medline]
19. Meyer RA and Terjung RL. Differences in ammonia and adenylate metabolism in contracting fast and slow muscle. Am J Physiol Cell Physiol 237: C111C118, 1979.[Abstract]
20. Meyer RA, Dudley GA, and Terjung RL. Ammonia and IMP in different skeletal muscle fibers after exercise in rats. J Appl Physiol 49: 10371041, 1980.
21. Meyer RA and Terjung RL. AMP deamination and IMP reamination in working skeletal muscle. Am J Physiol Cell Physiol 239: C32C38, 1980.
22. Meyer RA, Kuchmerick MJ, and Brown TR. Application of 31P-NMR spectroscopy to the study of striated muscle metabolism. Am J Physiol Cell Physiol 242: C1C11, 1982.
23. Meyer RA, Brown TR, and Kushmerick MJ. Phosphorus nuclear magnetic resonance of fast- and slow-twitch muscle. Am J Physiol Cell Physiol 248: C279C287, 1985.[Abstract]
24. Meyer RA, Brown TR, Krilowicz BL, and Kushmerick MJ. Phosphagen and intracellular pH changes during contraction of creatine-depleted rat muscle. Am J Physiol Cell Physiol 250: C264C274, 1986.
25. Meyer RA. A linear model of muscle respiration explains monoexponential phosphocreatine changes. Am J Physiol Cell Physiol 254: C548C553, 1988.
26. Posterino GS and Lamb GD. Effect of sarcoplasmic reticulum Ca2+ content on action potential-induced Ca2+ release in rat skeletal muscle fibres. J Physiol 551: 219237, 2003.
27. Rall JA. Energetics of Ca2+ cycling during skeletal muscle contraction. Fed Proc 41: 155160, 1982.[ISI][Medline]
28. Rall JA. Energetic aspects of skeletal muscle contraction: implications of fiber types. Exerc Sport Sci Rev 13: 3374, 1985.[Medline]
29. Roman BB, Meyer RA, and Wiseman RW. Phosphocreatine kinetics at the onset of contractions in skeletal muscle of MM creatine kinase knockout mice. Am J Physiol Cell Physiol 283: C1776C1783, 2002.
30. Somlyo A, McClellan G, Gonzalez-Serratos H, and Somlyo A. Electron probe X-ray microanalysis of post-tetanic Ca2+ and Mg2+ movements across the sarcoplasmic reticulum in situ. J Biol Chem 260: 68016807, 1985.
31. Tian R and Ingwall JS. Energetic basis for reduced contractile reserve in isolated rat hearts. Am J Physiol Heart Circ Physiol 270: H1207H1216, 1996.
32. Tian R, Halow JM, Meyer M, Dillmann WH, Figueredo VM, Ingwall JS, and Camacho SA. Thermodynamic limitation for Ca2+ handling contributes to decreased contractile reserve in rat hearts. Am J Physiol Heart Circ Physiol 275: H2064H2071, 1998.
33. Veech RL, Lawson JW, Cornell NW, and Krebs HA. Cytosolic phosphorylation potential. J Biol Chem 254: 65386547, 1979.[Abstract]
34. Westerblad H, Dahlstedt AJ, and Lannergren J. Mechanisms underlying reduced maximum shortening velocity during fatigue of intact, single fibres of mouse muscle. J Physiol 510: 269277, 1998.
35. Wetzel P and Gros G. Decay of Ca2+ and force transients in fast- and slow-twitch skeletal muscles from the rat, mouse and Etruscan shrew. J Exp Biol 201: 375384, 1998.