Creatine Phosphate Consumption and the Actomyosin
Crossbridge Cycle in Cardiac Muscles
Ozgur Ogut, Frank V. Brozovich
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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. (Circ Res. 2003;93:54-60.)
Key Words: creatine phosphate 䡲 actomyosin 䡲 crossbridge cycle 䡲 cardiac muscle
F
orce 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 ␮mol/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 ␮mol/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
Original received February 4, 2003; resubmission received March 31, 2003; revised resubmission received May 27, 2003; accepted May 28, 2003.
From the Departments of Physiology and Biophysics (O.O., F.V.B.) and Medicine (F.V.B.), Case Western Reserve University School of Medicine,
Cleveland, Ohio.
Correspondence to Dr Frank V. Brozovich, Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Room
E538, Cleveland, OH 44106. E-mail [email protected]
© 2003 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000080536.06932.E3
54
Ogut and Brozovich
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
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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 ␮L) 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 ␮m, 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.
Creatine Kinase Affects the Crossbridge Cycle
55
TABLE 1. Rates of Force Redevelopment in the Presence of
Varying MgATP or CP
MgATP or CP,
mmol/L
With 25
mmol/L CP,
ktr, s⫺1
With 5
mmol/L MgATP,
ktr, s⫺1
1
21.6⫾0.6
ND
2.5
27.2⫾0.7
11.6⫾0.1
5
23.9⫾0.4
14.9⫾0.2
10
25.8⫾0.5
19.2⫾0.2
15
ND
23.3⫾0.4
25
ND
23.9⫾0.4
ND indicates not determined.
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
␮mol/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.
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
56
Circulation Research
July 11, 2003
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 (䢇) and presence
(䡩) 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.
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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.
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
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.
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.
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
Ogut and Brozovich
Creatine Kinase Affects the Crossbridge Cycle
57
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.
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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 dosedependent 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.
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
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).
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
Figure 5. Increased rate of force redevelopment in the presence
of Pi is CP-dependent. In the presence of 25 mmol/L CP (䢇),
exogenous Pi increased ktr in a dose-dependent manner. However, exclusion of CP from the Pi titrations depressed ktr without dose dependence (䡩), suggesting that CP-CK controlled a
rate-limiting step in the Pi-dependent increase of ktr.
58
Circulation Research
July 11, 2003
TABLE 2. Rates of Force Redevelopment and Forces per
Cross-Sectional Area in the Presence of Varying Pi
5 mmol/L
MgATP⫹25 mmol/L CP
5 mmol/L MgATP
ktr, s⫺1
Force, mN/mm2
0
23.9⫾0.4
110⫾14
ND
ND
1
ND
ND
15.0⫾0.5
108⫾17
2.5
29.9⫾0.5
57⫾14
13.2⫾0.5
69⫾15
Pi, mmol/L
ktr, s⫺1
Force, mN/mm2
5
46.3⫾0.7
27⫾8
14.8⫾0.6
49⫾6
10
61.2⫾2.4
27⫾8
14.5⫾0.4
44⫾6
15
62.3⫾1.0
23⫾11
ND
ND
ND indicates not determined.
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supported by published data demonstrating an insensitivity of
ktr, 29 F max , 2,3 and V max 2 to MgATP concentrations
⬎1 mmol/L. In healthy individuals, mean total ATP values in
the heart may range between 7.2 and 7.7 ␮mol/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 ␮mol/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 ⬇6to 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 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).
(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-ethylmaleimide-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
Ogut and Brozovich
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crossbridge detachment. Therefore, the ability of CP-CK to
effectively reduce experimental rigor does not require CKderived 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.
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.
Creatine Kinase Affects the Crossbridge Cycle
59
Acknowledgments
This research was supported by American Heart Association PostDoctoral 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.
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Creatine Phosphate Consumption and the Actomyosin Crossbridge Cycle in Cardiac
Muscles
Ozgur Ogut and Frank V. Brozovich
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Circ Res. 2003;93:54-60; originally published online June 5, 2003;
doi: 10.1161/01.RES.0000080536.06932.E3
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