Cellular Biology

Creatine Phosphate Consumption and the Actomyosin Crossbridge Cycle in Cardiac Muscles

Ozgur Ogut, Frank V. Brozovich
https://doi.org/10.1161/01.RES.0000080536.06932.E3
Circulation Research. 2003;93:54-60
Originally published July 11, 2003

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Abstract

To investigate the regulation of the actomyosin crossbridge cycle in cardiac muscles, the effects of ATP, ADP, Pi, and creatine phosphate (CP) on the rate of force redevelopment (ktr) were measured. We report that CP is a primary determinant in controlling the actomyosin crossbridge cycling kinetics of cardiac muscles, because a reduction of CP from 25 to 2.5 mmol/L decreased ktr by 51% despite the presence of 5 mmol/L MgATP. The effects of CP on ktr were not a reflection of reduced ATP or accumulated ADP, because lowering ATP to 1 mmol/L or increasing ADP to 1 mmol/L did not significantly decrease ktr. Therefore, the effect of CP on the actomyosin crossbridge cycle is proposed to occur through a functional link between ADP release from myosin and its rephosphorylation by CP–creatine kinase to regenerate ATP. In activated fibers, the functional link influenced the kinetics of activated crossbridges without affecting the aggregate number of force-generating crossbridges. This was demonstrated by the ability of CP to affect ktr in maximally and submaximally activated fibers without altering the force per cross-sectional area. The data also confirm the important contribution of strong binding crossbridges to cardiac muscle activation, likely mediated by cooperative recruitment of adjacent crossbridges to maximize force redevelopment against external load. These data provide additional insight into the role of CP during pathophysiological conditions such as ischemia, suggesting that decreased CP may serve as a primary determinant in the observed decline of dP/dt.

Force development in striated muscles is a cooperative process of actomyosin (AM) crossbridge cycling and depends on the availability of ATP. The conversion of chemical energy from ATP hydrolysis to mechanical energy supports the transitions between individual states of the AM ATPase during muscle contraction.1 Therefore, the balance between ATP and its hydrolysis byproducts is a primary determinant for the kinetics of the AM crossbridge cycle. However, the in vivo concentrations of ATP under normal and pathophysiological conditions remain above the Km for AM’s ability to generate maximal force or maintain its maximum unloaded velocity of shortening (Vmax). In fact, the half-maximal ATP concentration required to support the AM ATPase is ≈150 μmol/L or less.2,3 This is well below the observed concentrations of ATP in the myocardium under normal or pathophysiological conditions.4–8 These data raise the question of how in vivo ATP concentration affects the kinetics of the AM crossbridge cycle, indirectly suggesting that ATP is not the limiting factor. Alternatively, ADP accumulation may accompany the reduction in ATP, resulting in the decline of cardiac muscle contractility by slowing ADP release from the rate-limiting, ADP-bound state of AM (AM · ADP).9,10 The balance between ATP and ADP concentrations is hypothesized to be a primary factor complicating contractility during cardiomyopathies,11 aortic valve disease,12 and coronary artery disease.13 Although ADP is believed to exert a dominant effect in slowing contractility, the magnitude of ADP accumulation is modest, remaining as low as 27 μmol/L with 30 minutes of low-flow ischemia.4,5,7,14 In myocytes, excessive accumulation of ADP is prevented in part by creatine kinase (CK) catalyzing the rephosphorylation of ADP to ATP using creatine phosphate (CP) as a substrate. This implicates CP as an important factor in maintaining ATP while moderating the build up of ADP concentrations in muscle, consistent with the proposal that the CK reaction is involved in high-energy phosphoryl homeostasis.15,16 However, function of CK in cells may be more complex, indirectly influencing the kinetics of other ATPases as well as the rate of glycolysis and oxidative phosphorylation.17,18 The importance of CK in cardiac muscle contractility is indirectly supported by the large changes in CP concentration observed during ischemia. For example, Cave et al4 demonstrated a decrease in CP from 18.5 to 8.9 mmol/L (52%) within 10 minutes of low-flow ischemia, compared with a 25% decrease in ATP (10.8 to 8.1 mmol/L) during the same time frame. These observed changes in CP concentration are consistently rapid in onset and greater in magnitude than the decline of ATP.4,5 Therefore, it is of interest to determine the contribution of CP to contractility beyond the assumption that it serves as a substrate to maintain intracellular ATP concentration.

Using known in vivo substrate concentrations as a guideline, our experiments focused on the factors controlling force redevelopment in cardiac muscles. Experiments testing the rate of tension redevelopment (ktr19) and Vmax of trabeculae under various substrate conditions were used to monitor overall crossbridge cycling under loaded (ktr) and unloaded (Vmax) conditions. The results suggest that the CP-CK reaction is functionally linked to the AM crossbridge cycle and may serve as a primary substrate that limits force redevelopment under load. We provide additional evidence that ADP release from myosin is the step of the crossbridge cycle coupled with the CK reaction.

Materials and Methods

Trabecula Preparation

Mice were purchased from Jackson Labs (Bar Harbor, Maine) and their care and use were in accordance with the guidelines of the American Physiological Society using a protocol approved by the Institutional Animal Care and Use Committee of Case Western Reserve University. Mice were killed by CO2 asphyxiation, the heart was rapidly removed and placed in cold Ca2+-free physiological saline solution (in mmol/L, NaCl 140, KCl 4.7, Na2HPO4 0.3, MOPS 2, EGTA 0.5, EDTA 0.2, MgCl2 1.2, and glucose 5.6, pH adjusted to 7.4 with 1 N KOH),20 and the left ventricle was exposed. Trabeculae were immediately excised, and their ends were fixed with a small volume (<1 μL) of 1% glutaraldehyde in 50% glycerol,21 followed by attachment of aluminum T-clips to the ends. Trabeculae were skinned for 45 minutes at 4°C in pCa9 (1 nmol/L free Ca2+) solution containing 1% vol/vol Triton X-100 (in mmol/L, MgATP 5 [free], EGTA 5, potassium methane sulfonate 25, MgCl2 6.9 [1 mmol/L free Mg2+], CP 25, BES 25, and glutathione 2, 1% vol/vol Triton X-100, pH 7). After skinning, the tissues were transferred to pCa9 relaxing solution (1 nmol/L free Ca2+, 5 mmol/L MgATP [free], 10 mmol/L EGTA, 56.5 mmol/L potassium methane sulfonate, 7.2 mmol/L MgCl2 [1 mmol/L free Mg2+], 25 mmol/L CP, and 25 mmol/L BES, pH 7) and mounted on the stage of a computer-controlled mechanics workstation. One end of the tissue was hooked to a length controller (Model 308B, Aurora Scientific) and the other to a force transducer (Akers AE801). The trabeculae were stretched to an average sarcomere length of ≈2.2 μm, as determined by laser diffraction,22 and the length of the muscle was recorded as Lo. Trabeculae were activated by a pCa4 solution (in mmol/L, free Ca2+ 0.1, MgATP 5 [free], EGTA 10, potassium methane sulfonate 35.8, MgCl2 7.0 [1 mmol/L free Mg2+], CP 25, and BES 25, pH 7). When specifically indicated, CK (20 U/mL; Sigma Chemicals) was added to the solutions before experiments. Temperature during experiments was controlled to 15±0.1°C. Solutions of varying CP, MgATP, MgADP, Pi, or Ca2+ were mixed according to a program calculating the amount of stock solutions required for a given set of free ion concentrations at 15°C and an ionic strength of 200 mmol/L.20 Binding constants of the species present were corrected for temperature and ionic strength. For experiments testing the effect of CP at submaximal Ca2+ concentrations, a Ca2+ electrode (Orion) was used to ensure reproducible Ca2+ concentrations across solutions. Force generated from the fibers was normalized to the maximum force per cross-section (Fmax) by measuring the width and thickness of the tissues and assuming an elliptical cross-section. In experiments testing ktr during submaximal activation (pCa5.4), tissues were activated by saturating Ca2+ (pCa4) at completion of the measurements to determine the relative force produced at pCa5.4 versus pCa4.

Mechanical Measurements

Mechanical measurements predicated on controlling the force or muscle length were programmed in steps into the 600A Digital Controller (Aurora Scientific). Experiments controlling the length of the preparation used a feedback loop based on muscle, not sarcomere length. The software was interfaced to the length controller and force transducer by a 16-bit A/D:D/A, 200-kHz interface. Maximum sampling frequency of recording was 10 kHz.

Rate of Force Redevelopment

Experiments were done as described19 with an adjustment to maximize crossbridge detachment in cardiac muscle (M. Regnier, University of Washington, Seattle, Wash, personal communication, 2001). The protocol involved decreasing Lo by 20% during a 5-ms ramp, a subsequent step change in length to 1.05 Lo for 2 ms followed by restoration of length to Lo. With this protocol, force routinely fell to and redeveloped from zero. ktr was determined by single exponential fits to the force redevelopment over time. Data are presented as average±SEM.

Maximum Unloaded Shortening Velocity

Vmax was determined by a series of isotonic contractions between 3% and 80% of Fmax. Forces routinely fell to the set isotonic level within 3 to 5 ms. The velocity of shortening for each isotonic contraction was determined from the slope of a linear fit to the length trace between 20 and 25 ms of the protocol. Velocities were normalized to muscle length (ML) per second, plotted as a function of force, and fit by the Hill equation2,23 to determine the extrapolated shortening velocity at zero force. Data are presented as average±SEM.

Results

Initial experiments demonstrated that ktr was largely insensitive to MgATP concentration (1 to 10 mmol/L) in the presence of 25 mmol/L CP, resulting in similar rates at all ATP concentrations tested (average 24.6±1.4 s−1 across MgATP concentrations; Table 1). In fact, the 1 mmol/L MgATP tested is well below concentrations observed with extended periods of ischemia (5.3 mmol/L4), increased cardiac work (7.5 mmol/L14), or even myocardial infarction (3.7 μmol/g wet weight or ≈4.63 mmol/L, assuming 80% of muscle wet weight is water5). This suggests that physiological concentrations of MgATP are unlikely to play a role in defining ktr.

View this table:
Table 1.

Rates of Force Redevelopment in the Presence of Varying MgATP or CP

We next determined the effect of CP on the rate of force redevelopment. In the presence of 5 mmol/L MgATP, changes in CP concentration under saturating Ca2+ concentrations (pCa4) were not expected to affect ktr because of sufficient MgATP supply. However, decreasing CP from 25 to 2.5 mmol/L at pCa4 resulted in a 51% decrease in ktr (23.9 to 11.6 s−1; Figure 1). This effect was also observed in fibers activated with submaximal Ca2+ concentrations. In the presence of 25 mmol/L CP, ktr decreased by 30.5% when the free Ca2+ concentration was decreased from pCa4 to pCa5.4. This was accompanied by a 20.2% decrease in Fmax (Figure 2). Decreasing CP concentration additionally from 25 to 10 mmol/L at pCa5.4 caused a relative 18% drop in ktr, similar to the 20% decline observed when CP was decreased from 25 to 10 mmol/L CP at pCa4 (Table 1). These data suggest that the relationship between ktr and CP concentration is similar in submaximally and maximally Ca2+-activated cardiac muscle fibers.

Figure1

Figure 1. Force redevelopment under load for mouse trabeculae at different CP concentrations. A, Typical force response after the length release-restretch procedure to determine the rate of force redevelopment. The experiment was done in the presence of 5 mmol/L MgATP and 25 mmol/L CP. B, Relationship between ktr and CP concentration in the absence (•) and presence (○) of 1 mmol/L MgADP. The addition of 1 mmol/L MgADP did not slow ktr but rather increased ktr at all CP concentrations tested. Data points are average±SEM from a minimum 3 trabeculae with a minimum of 6 measurements from each fiber.

Figure2

Figure 2. Effect of CP concentration on ktr during submaximal contractions. To determine if CP could affect ktr rates under submaximal Ca2+ concentrations, experiments were done to determine the ktr and Fmax at pCa5.4 in the presence of either 10 or 25 mmol/L CP. Compared with pCa4, ktr and force both declined when Ca2+ concentration was lowered to pCa5.4. The force per cross-sectional area generated at pCa5.4 was 76.5±4.9% (10 mmol/L CP) and 79.8±3.6% (25 mmol/L CP) of the force generated at pCa4/25 mmol/L CP. At submaximal Ca2+ concentrations, changes in CP concentration influenced the kinetics of force redevelopment, with ktr at pCa5.4 being 56.8±2.4% (10 mmol/L CP) or 69.5±1.0% (25 mmol/L CP) of ktr at pCa4/25 mmol/L CP.

An alternate explanation for the observed decline of ktr with decreasing CP would be MgADP accumulation from insufficient rephosphorylation by CK. Therefore, the effect of CP concentration on ktr was tested with the addition of 1 mmol/L total MgADP (Figure 1). After the addition of 1 mmol/L MgADP, Fmax increased from 110±14 mN/mm2 (n=8) to 152±21 mN/mm2 (n=7; Figure 3). This observation is consistent with the concentration of MgADP rising locally at the crossbridges, in turn increasing the population of the strongly bound, AM · ADP state to account for the increase in Fmax.10,24 However, at all CP concentrations tested, the additional 1 mmol/L MgADP did not slow ktr but rather resulted in small increases (Figure 1). This suggests that the decline in ktr attributable to decreasing CP was not caused by MgADP accumulation. Nonetheless, it remained possible that the loss of myofibrillar CK activity after Triton X-100 permeabilization additionally compromised the ATP:ADP balance. Using a modification of a published protocol,25 the activity of the myofibrillar CK fraction in the skinned cardiac muscle fibers was measured to be 6.7±0.1 U/min per mg protein (n=3, data not shown), in agreement with previously published data.26 These results did not indicate irreversible loss of myofibrillar CK enzyme during tissue preparation, implying that additional CK in the fibers would not effectuate any change in contractility. This was confirmed by experiments testing ktr in the presence or absence of additional CK (20 U/mL). At pCa4 with 10 or 25 mmol/L CP, the addition of 20 U/mL CK did not change the measured ktr values (Figure 4), suggesting that the endogenous myofibrillar CK activity in the fibers was not a limiting factor for the experiments.

Figure3

Figure 3. Determination of Vmax. A, Typical isotonic contraction used to determine the velocity of contraction at various percentages of the maximum Ca2+-activated force (3% to 80%). The velocity of shortening for each isotonic contraction was determined from the slope of a linear fit to the length trace corresponding to 20 and 25 ms of the isotonic contraction protocol. B, In the presence of 5 mmol/L MgATP and 25 mmol/L CP, both Fmax and Vmax were increased by the addition of 1 mmol/L MgADP. Furthermore, 5 mmol/L CP did not appreciably decrease Vmax or Fmax compared with 25 mmol/L CP. Data are presented as average±SEM from 7 (5 mmol/L CP and 25 mmol/L CP+1 mmol/L MgADP) and 8 (25 mmol/L CP) fibers.

Figure4

Figure 4. Effect of exogenous CK on ktr. Contribution of exogenous CK to ktr was tested by duplicate experiments at pCa4 with (filled bars) or without (open bars) an additional 20 U/mL CK added to the solution bath. Although ktr varied with CP concentration as expected, the addition of exogenous CK did not affect ktr for either of the conditions tested. When normalized to the ktr at pCa4 in the presence of 25 mmol/L CP and 20 U/mL CK, relative ktr at the other conditions was 102.2±1.4% (pCa4/25 mmol/L CP), 84.6±0.8% (pCa4/10 mmol/L, CP/20 U/mL CK), and 80.8±1.2% (pCa4/10 mmol/L CP).

Because CP was shown to influence contractility under load, we also tested the effect of CP on unloaded contractility. Experiments measured the Vmax of fibers under different CP concentrations, either in the presence or absence of 1 mmol/L MgADP. For muscles activated at pCa4 with 5 mmol/L MgATP and 25 mmol/L CP, Vmax was 4.9±0.3 ML/s (n=8, Figure 3). With the addition of 1 mmol/L MgADP, Vmax increased to 6.4±0.7 ML/s (n=7, P=0.03). By contrast, decreasing CP from 25 to 5 mmol/L did not have a significant effect on Vmax (4.3±0.3 ML/s, n=7, P=0.09), despite a 37.7% decrease in ktr under those conditions. Similar to previous reports,27 the decrease of CP from 25 to 5 mmol/L did not significantly change Fmax.

To determine the crossbridge step coupled with CK activity, ktr experiments were also done in the presence of varying Pi. In agreement with previously published data,28,29 we observed an ≈3 fold increase in ktr with increasing Pi in the presence of 25 mmol/L CP (23.9±0.4 to 62.3±1.0 s−1, Figure 5). The changes in ktr were accompanied by decreases in Fmax (Table 2), consistent with a low or no force producing AM ADP Pi state, rapidly isomerizing to a force producing AM ADP Pi* step before Pi release.28–30 Additional Pi titrations were also done without CP. Although the effect on Fmax was unchanged (Table 2), ktr no longer showed a dose-dependent response to Pi, with rates remaining between 13.2 and 15.0 s−1 (Figure 5). This strongly suggests that the increase in ktr with increasing Pi requires CP.

Figure5

Figure 5. Increased rate of force redevelopment in the presence of Pi is CP-dependent. In the presence of 25 mmol/L CP (•), exogenous Pi increased ktr in a dose-dependent manner. However, exclusion of CP from the Pi titrations depressed ktr without dose dependence (○), suggesting that CP-CK controlled a rate-limiting step in the Pi-dependent increase of ktr.

View this table:
Table 2.

Rates of Force Redevelopment and Forces per Cross-Sectional Area in the Presence of Varying Pi

Discussion

In this study, we provide evidence that CP utilization by CK is functionally linked and tightly coupled with the AM crossbridge cycle, exerting control on the kinetics of the crossbridge cycle despite adequate, physiological ATP concentrations. We found that ktr in cardiac muscle is strongly dependent on CP concentration, resulting in a decline of ktr with declining CP during both maximal and submaximal Ca2+-activated contractions (Figures 1 and 2⇑; Table 1). Our results also suggest that the CP-CK system regulates the kinetics of the activated AM crossbridges without affecting the aggregate number of active crossbridges. This was supported by data demonstrating that a significant change in ktr can be observed without commensurate changes in Fmax. For example, when CP was decreased from 25 to 10 mmol/L at pCa5.4, ktr decreased by 18% despite only a 4% decrease in Fmax (Figure 2). This was also true at saturating Ca2+ (pCa4), because a decrease of CP from 25 to 5 mmol/L decreased ktr by 38% (Figure 1; Table 1) without a significant decrease in Fmax (Figure 3).

To address the hypothesis that there is a functional link between the CP-CK reaction and the AM cycle, three substrate variables known to contribute to crossbridge kinetics were varied. First of all, declining ktr with declining CP may be indicative of a decrease in ATP, suggesting that CK was unable to maintain adequate ATP concentrations and thereby slowed the rate of crossbridge cycling. However, changes of MgATP concentration within the physiological range did not affect force redevelopment. This was demonstrated by the relative insensitivity of ktr to concentrations of MgATP ranging from 1 to 10 mmol/L (Table 1) and additionally supported by published data demonstrating an insensitivity of ktr,29 Fmax,2,3 and Vmax2 to MgATP concentrations >1 mmol/L. In healthy individuals, mean total ATP values in the heart may range between 7.2 and 7.7 μmol/g wet weight. Assuming water content of 80% per gram of wet muscle weight, this calculates to an ATP concentration of ≈9 mmol/L.6,31 Models of myocardial infarction have shown a 42% decrease in ATP content from 6.4 to 3.7 μmol/g wet weight,5 or from 8 to 4.6 mmol/L. Therefore, ATP concentrations even after infarction remain well above the 1 mmol/L MgATP tested in our experiments, strongly suggesting that variations in ATP would not be a limiting factor in the decline of contractility when CP concentration decreases during infarction.

To address the possible influence of ADP in our experiments, the effect of CP on ktr was measured in the presence of 1 mmol/L MgADP. The chosen ADP concentration is ≈6- to 10-fold above known levels of intracellular total ADP during ischemia or increased cardiac5,7,14 and ≈3 to 4 times the estimated Ki for inhibition of fiber velocity by MgADP.10 The addition of MgADP resulted in an expected rise in Fmax (Figure 3),10,24 suggesting that MgADP reached the contractile apparatus and increased the population of strong binding AM · ADP crossbridges. Interestingly, 1 mmol/L MgADP accelerated ktr at various CP concentrations (Figure 1), possibly through cooperative activation of adjacent thin filament regulatory units by strong binding crossbridges in the AM · ADP state. This raised the possibility that the observed acceleration in the kinetics of the crossbridge cycle reflected by ktr may also result in an increase of Vmax. In agreement with this prediction, the addition of 1 mmol/L MgADP accelerated Vmax (Figure 3). Coupled with the measured concentrations of total ADP during ischemia or myocardial infarction, the data suggest that the increase of ADP under physiological or pathophysiological conditions in cardiac muscles would not slow the AM cycle.32 Therefore, the slowing of ktr with decreased CP is not reasonably explained by a rise in ADP. However, it remained possible that Triton X-100 skinning of mouse myocardium resulted in complete removal of the endogenous myofibrillar CK and therefore reduced the overall activity of the CP-CK reaction. Although we observed that myofibrillar CK activity was retained after skinning, results of experiments measuring ktr in the presence or absence of an additional 20 U/mL CK were not different (Figure 4). This suggests that the CK enzyme content was not a rate-limiting factor in the experiments.

The improvement of contractility (ktr and Vmax) with the addition of 1 mmol/L MgADP was likely through cooperative recruitment of adjacent crossbridges, in agreement with previous experiments using either strong binding, N-ethyl-maleimide-modified myosin S1 subfragments, or alternate myosin substrates (dATP33,34). The data here support the findings that maximal cardiac muscle activation depends not only on Ca2+ but also on myosin crossbridge attachment.33,34 Although the ADP results suggested that strong binding crossbridges may serve to activate adjacent regulatory units, this effect likely has an optimal window whereby additional increases in ADP concentrations (or the ADP:ATP ratio) would be inhibitory to the rate of the AM crossbridge cycle. Higher ADP concentrations competing with ATP for AM binding would thereby limit crossbridge detachment.24 This is supported by our observations that in the presence of 5 mmol/L MgATP, increasing ADP concentrations from a nominal level to 2 mmol/L or greater decreases ktr by at least 19% or more (data not shown). This agrees with experiments by Tesi et al28 demonstrating a 23% decrease in ktr when MgADP was increased to 3 mmol/L.

With the demonstration of the functional link between CP consumption and the AM crossbridge cycle, it remains of interest to determine the crossbridge steps involved. The two steps of the AM cycle that share common substrates with CK are the release of ADP from myosin and the reintroduction of ATP to myosin (Figure 6). Using CK knockout mice, Ventura-Clapier et al35 demonstrated that rigor crossbridges were more effectively detached with active CP-CK and ATP versus ATP alone. It was therefore suggested that ATP from CP-CK is the preferential substrate for detachment of myosin from the AM state. This would be consistent with the assumption that all crossbridges resulting from ATP depletion are in the AM state. However, Martin and Barsotti36 demonstrated that crossbridges in cardiac muscle formed by ATP depletion include a significant population of AM · ADP crossbridges in addition to the expected, rigor AM population. Therefore, to relax muscles from the rigor state, the AM · ADP must move to the AM state before ATP-induced crossbridge detachment. Therefore, the ability of CP-CK to effectively reduce experimental rigor does not require CK-derived ATP to serve as a preferential myosin substrate. Rather, CK seems to accelerate the release of ADP from AM · ADP to establish the AM state for subsequent ATP binding and myosin detachment (Figure 6). This hypothesis would be in agreement with the increase in relaxation times for CK knockout mice being a reflection of impaired ADP release from the AM · ADP state of the crossbridge cycle.37 This hypothesis is also in agreement with biochemical data suggesting that ADP release from AM · ADP, not ATP binding to AM, is the rate-limiting step of the crossbridge cycle.9 These data support a functional link between CP-CK and the AM · ADP state of the crossbridge cycle, facilitating ADP release from AM · ADP to decrease the relative attachment time of this strong binding state.

Figure6

Figure 6. Proposed AM crossbridge cycle. Modified from Reference 19, the schematic describes the functional link between the AM crossbridge cycle and CK. CK is demonstrated to play a large part in limiting AM crossbridge cycling at steps dependent on ADP release. This was shown to be relevant for the release of ADP from AM · ADP (shown), as well as from detached M · ADP (Figure 5).

However, the observed effect of Pi on ktr suggested that AM · ADP was not the sole state coupled with CK activity. It is known that exogenous Pi leads to a shift in the distribution of crossbridges from strong-binding, high-force producing states (AM · ADP, AM) to weak binding states (AM · ADP · Pi) in equilibrium with detached states (A+M · ADP · Pi).1,28,38 In our experiments in the presence of 25 mmol/L CP, increasing Pi increased ktr, consistent with a rapid AM · ADP · Pi to AM · ADP · Pi* isomerization.28,29 Surprisingly, the ability of exogenous Pi to increase ktr was abolished when CP was omitted (Figure 5), suggesting that a crossbridge step involved in the Pi-dependent increase of ktr was rate-limited by CP. If the detached A+M · ADP · Pi state is followed by Pi release from myosin, then the subsequent ADP release from detached myosin may also depend on the CP-CK reaction. This is indirectly supported by the expectation that the AM · ADP state would be sparsely populated under high Pi, explaining the observed decrease of Fmax and therefore necessitating involvement of the detached M · ADP state. Therefore, we propose that ADP release either from the bound AM · ADP or the detached M · ADP states are coupled with CK activity.

In myocytes, CK is colocalized to the A band of sarcomeres in proximity to myosin.35,39,40 The localization of CK may have additional implications during myocardial ischemia, because the enzyme has been found to diffuse away from the A band toward the I band, possibly compromising the relationship between CK activity and the AM crossbridge cycle.41,42 Furthermore, the clinical importance of CP-CK is exemplified by the observed decrease in CK enzyme activity and CP content in failing versus nonfailing hearts.12,13,43,44 This is in addition to the compromise of the CP:ATP ratio in heart failure, which serves as a predictor of cardiovascular mortality.11,45 The experiments described herein support the hypothesis that a change in CK activity, either through changes in substrate concentration or changes in the specific activity of CK, will affect the crossbridge cycle. Therefore, the demonstrated functional link between CK activity and the AM crossbridge cycle may have additional implications for the observed deficits in cardiac muscle contractility under pathophysiological conditions.

Acknowledgments

This research was supported by American Heart Association Post-Doctoral grant 0120359B (to O.O.) and NIH grants HL44181 and HL64137 (to F.V.B.). The authors thank Dr Albert Rhee for critical reading of the manuscript.

Footnotes

  • Original received February 4, 2003; resubmission received March 31, 2003; revised resubmission received May 27, 2003; accepted May 28, 2003.

References

  1. ↵
    Hibberd MG, Trentham DR. Relationships between chemical and mechanical events during muscular contraction. Annu Rev Biophys Biophys Chem. 1986; 15: 119–161.
    OpenUrlCrossRefPubMed
  2. ↵
    Cooke R, Bialek W. Contraction of glycerinated muscle fibers as a function of the ATP concentration. Biophys J. 1979; 28: 241–258.
    OpenUrlCrossRefPubMed
  3. ↵
    Takashi R, Putnam S. A fluorimetric method for continuously assaying ATPase: application to small specimens of glycerol-extracted muscle fibers. Anal Biochem. 1979; 92: 375–382.
    OpenUrlCrossRefPubMed
  4. ↵
    Cave AC, Ingwall JS, Friedrich J, Liao R, Saupe KW, Apstein CS, Eberli FR. ATP synthesis during low-flow ischemia: influence of increased glycolytic substrate. Circulation. 2000; 101: 2090–2096.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Bottomley PA, Weiss RG. Noninvasive localized MR quantification of creatine kinase metabolites in normal and infarcted canine myocardium. Radiology. 2001; 219: 411–418.
    OpenUrlPubMed
  6. ↵
    Yabe T, Mitsunami K, Inubushi T, Kinoshita M. Quantitative measurements of cardiac phosphorus metabolites in coronary artery disease by 31P magnetic resonance spectroscopy. Circulation. 1995; 92: 15–23.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Tian R, Christe ME, Spindler M, Hopkins JC, Halow JM, Camacho SA, Ingwall JS. Role of MgADP in the development of diastolic dysfunction in the intact beating rat heart. J Clin Invest. 1997; 99: 745–751.
    OpenUrlPubMed
  8. ↵
    Liao R, Nascimben L, Friedrich J, Gwathmey JK, Ingwall JS. Decreased energy reserve in an animal model of dilated cardiomyopathy: relationship to contractile performance. Circ Res. 1996; 78: 893–902.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Siemankowski RF, White HD. Kinetics of the interaction between actin, ADP and cardiac myosin-S1. J Biol Chem. 1984; 259: 5045–5053.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Cooke R, Pate E. The effects of ADP and phosphate on the contraction of muscle fibers. Biophys J. 1985; 48: 789–798.
    OpenUrlCrossRefPubMed
  11. ↵
    Neubauer S, Horn M, Cramer M, Harre K, Newell JB, Peters W, Pabst T, Ertl G, Hahn D, Ingwall JS, Kochsiek K. Myocardial phosphocreatine-to-ATP ratio is a predictor of mortality in patients with dilated cardiomyopathy. Circulation. 1997; 96: 2190–2196.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Conway MA, Allis J, Ouwerkerk R, Niioka T, Rajagopalan B, Radda GK. Detection of low phosphocreatine to ATP ratio in failing hypertrophied human myocardium by 31P magnetic resonance spectroscopy. Lancet. 1991; 338: 973–976.
    OpenUrlCrossRefPubMed
  13. ↵
    Hardy CJ, Weiss RG, Bottomley PA, Gerstenblith G. Altered myocardial high-energy phosphate metabolites in patients with dilated cardiomyopathy. Am Heart J. 1991; 122: 795–801.
    OpenUrlCrossRefPubMed
  14. ↵
    Saupe KW, Spindler M, Tian R, Ingwall JS. Impaired cardiac energetics in mice lacking muscle-specific isoenzymes of creatine kinase. Circ Res. 1998; 82: 898–907.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Bessman SP, Carpenter LC. The creatine-creatine phosphate energy shuttle. Annu Rev Biochem. 1985; 54: 831–862.
    OpenUrlCrossRefPubMed
  16. ↵
    Wallimann T, Wyss M, Brdiczka D, Nicolay K, Eppenberger HM. Intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands: the “phosphocreatine circuit” for cellular energy homeostasis. Biochem J. 1992; 281: 21–40.
    OpenUrlFREE Full Text
  17. ↵
    Erecinska M, Wilson DF. Regulation of cellular energy metabolism. J Membr Biol. 1982; 70: 1–14.
    OpenUrlCrossRefPubMed
  18. ↵
    Balaban RS. Regulation of oxidative phosphorylation in the mammalian cell. Am J Physiol. 1990; 258: C377–C389.
    OpenUrlPubMed
  19. ↵
    Brenner B, Eisenberg E. Rate of force generation in muscle: correlation with actomyosin ATPase activity in solution. Proc Natl Acad Sci U S A. 1986; 83: 3542–3546.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Ogut O, Brozovich FV. Determinants of the contractile properties in the embryonic chicken gizzard and aorta. Am J Physiol. 2000; 279: C1722–C1732.
    OpenUrl
  21. ↵
    Chase P, Kushmerick MJ. Effects of pH on contraction of rabbit fast and slow skeletal muscle fibers. Biophys J. 1988; 53: 935–946.
    OpenUrlCrossRefPubMed
  22. ↵
    Ogut O, Granzier H, Jin J-P. Acidic and basic troponin T isoforms in mature fast-twitch skeletal muscle and effect on contractility. Am J Physiol. 1999; 276: C1162–C1170.
    OpenUrlPubMed
  23. ↵
    Hill AV. The heat of shortening and the dynamic constants of muscle. Proc R Soc Lond B Biol Sci. 1938; 126: 136–195.
    OpenUrlFREE Full Text
  24. ↵
    Lu Z, Moss RL, Walker JW. Tension transients initiated by photogeneration of MgADP in skinned skeletal muscle fibers. J Gen Physiol. 1993; 101: 867–888.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Cook PF, Kenyon GL, Cleland WW. Use of pH studies to elucidate the catalytic mechanism of rabbit muscle creatine kinase. Biochemistry. 1981; 20: 1204–1210.
    OpenUrlCrossRefPubMed
  26. ↵
    Gregor M, Mejsnar J, Janovska A, Zurmanova J, Benada O, Mejsnarova B. Creatine kinase reaction in skinned rat psoas muscle fibers and their myofibrils. Physiol Res. 1999; 48: 27–35.
    OpenUrlPubMed
  27. ↵
    Kentish JC. The effects of inorganic phosphate and creatine phosphate on force production in skinned muscles from rat ventricle. J Physiol. 1986; 370: 585–604.
    OpenUrlPubMed
  28. ↵
    Tesi C, Colomo F, Nencini S, Piroddi N, Poggesi C. The effect of inorganic phosphate on force generation in single myofibrils from rabbit skeletal muscle. Biophys J. 2000; 78: 3081–3092.
    OpenUrlCrossRefPubMed
  29. ↵
    Regnier M, Homsher E. The effect of ATP analogs on posthydrolytic and force development steps in skinned skeletal muscle fibers. Biophys J. 1998; 74: 3059–3071.
    OpenUrlCrossRefPubMed
  30. ↵
    Nosek TM, Fender KY, Godt RE. It is diprotonated inorganic phosphate that depresses force in skinned skeletal muscle fibers. Science. 1987; 236: 191–193.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Bottomley PA, Hardy CJ, Roemer PB. Phosphate metabolite imaging and concentration measurements in human heart by nuclear magnetic resonance. Magn Reson Med. 1990; 14: 425–434.
    OpenUrlPubMed
  32. ↵
    Chase PB, Kushmerick MJ. Effect of physiological ADP concentrations on contraction of single skinned fibers from rabbit fast and slow muscles. Am J Physiol. 1995; 268: C480–C489.
    OpenUrlPubMed
  33. ↵
    Swartz DR, Moss RL. Strong binding of myosin increases shortening velocity of rabbit skinned skeletal muscle fibres at low levels of Ca2+. J Physiol. 2001; 533: 357–365.
    OpenUrlCrossRefPubMed
  34. ↵
    Regnier M, Martyn DA, Chase PB. Calcium regulation of tension redevelopment kinetics with 2-deoxy-ATP or low [ATP] in rabbit skeletal muscle. Biophys J. 1998; 74: 2005–2015.
    OpenUrlCrossRefPubMed
  35. ↵
    Ventura-Clapier R, Kuznetsov AV, d’Albis A, van Deursen J, Wieringa B, Veksler VI. Muscle creatine kinase-deficient mice, I: alterations in myofibrillar function. J Biol Chem. 1995; 270: 19914–19920.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Martin H, Barsotti RJ. Relaxation from rigor of skinned trabeculae of the guinea pig induced by laser photolysis of caged ATP. Biophys J. 1994; 66: 1115–1128.
    OpenUrlPubMed
  37. ↵
    Steeghs K, Benders A, Oerlemans F, de Haan A, Heerschap A, Ruitenbeek W, Jost C, van Deursen J, Perryman B, Pette D, Bruckwilder M, Koudijs J, Jap P, Veerkamp J, Wieringa B. Altered Ca2+ responses in muscles with combined mitochondrial and cytosolic creatine kinase deficiencies. Cell. 1997; 89: 93–103.
    OpenUrlCrossRefPubMed
  38. ↵
    Eisenberg E, Hill TL, Chen Y. Cross-bridge model of muscle contraction. Biophys J. 1980; 29: 195–227.
    OpenUrlCrossRefPubMed
  39. ↵
    Bessman SP, Yang WC, Geiger PJ, Erickson-Viitanen S. Intimate coupling of creatine phosphokinase and myofibrillar adenosinetriphosphatase. Biochem Biophys Res Commun. 1980; 96: 1414–1420.
    OpenUrlCrossRefPubMed
  40. ↵
    Saks VA, Ventura-Clapier R, Huchua ZA, Preobrazhensky AN, Emelin IV. Creatine kinase in regulation of heart function and metabolism, I: further evidence for compartmentation of adenine nucleotides in cardiac myofibrillar and sarcolemmal coupled ATPase-creatine kinase systems. Biochim Biophys Acta. 1984; 803: 254–264.
    OpenUrlPubMed
  41. ↵
    Otsu N, Hirata M, Tuboi S, Miyazawa K. Immunocytochemical localization of creatine kinase M in canine myocardial cells: most creatine kinase M is distributed in the A-band. J Histochem Cytochem. 1989; 37: 1465–1470.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    Otsu N, Yamaguchi I, Komatsu E, Miyazawa K. Changes in creatine kinase M localization in acute ischemic myocardial cells: immunoelectron microscopic studies. Circ Res. 1993; 73: 935–942.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    Rajagopalan B, Blackledge MJ, McKenna WJ, Bolas N, Radda GK. Measurement of phosphocreatine to ATP ratio in normal and diseased human heart by 31P magnetic resonance spectroscopy using the rotating frame-depth selection technique. Ann N Y Acad Sci. 1987; 508: 321–332.
    OpenUrlPubMed
  44. ↵
    Neubauer S, Krahe T, Schindler R, Horn M, Hillenbrand H, Entzeroth C, Mader H, Kromer EP, Riegger GA, Lackner K, et al. 31P magnetic resonance spectroscopy in dilated cardiomyopathy and coronary artery disease: altered cardiac high-energy phosphate metabolism in heart failure. Circulation. 1992; 86: 1810–1818.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    Nascimben L, Ingwall JS, Pauletto P, Friedrich J, Gwathmey JK, Saks V, Pessina AC, Allen PD. Creatine kinase system in failing and nonfailing human myocardium. Circulation. 1996; 94: 1894–1901.
    OpenUrlAbstract/FREE Full Text
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  • Creatine Phosphate Consumption and the Actomyosin Crossbridge Cycle in Cardiac Muscles
    Ozgur Ogut and Frank V. Brozovich
    Circulation Research. 2003;93:54-60, originally published July 11, 2003
    https://doi.org/10.1161/01.RES.0000080536.06932.E3
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