Download Effect of pH, phosphate, and ADP on relaxation of myocardium after

Effect of pH, phosphate, and ADP on relaxation
of myocardium after photolysis of diazo 2
S. J. SIMNETT, E. C. JOHNS, S. LIPSCOMB, I. P. MULLIGAN, AND C. C. ASHLEY
University Laboratory of Physiology, Oxford OX1 3PT, United Kingdom
muscle; heart; calcium; guinea pig; cross bridges
is poorly understood. Studies of relaxation of the intact heart are complicated by
the geometry of the organ, the auxotonicity of relaxation, and the beat-to-beat modulation of the inotropic
state because of changes in the degree of ventricular
filling and catecholamine stimulation. We have studied
cardiac myofilaments in isolation from other cellular
components to determine the kinetics of myofilament
relaxation and to examine how these kinetics are
altered by changes in the concentration of the byproducts of ATP hydrolysis.
During ischemia there is a decrease in cardiac output
and a change in the rates of relaxation and activation.
These changes, caused by a combination of alterations
in the functions of the cellular membranes and the
myofilaments, are brought about, at least in part, by
increases in the intracellular concentrations of metabolites such as ADP and Pi and by a decrease in the
intracellular pH (pHi ) (2, 3, 14, 16, 18). It has been
proposed that the fall in pHi plays a major role in this
decline of contractile function in intact cardiac tissue.
However, the attenuation of force in hypoxic conditions,
and those mimicking ischemia, cannot be wholly attributed to an increasing acidity, because the majority of
CARDIAC MUSCLE RELAXATION
the decline in contractile force occurs before the onset of
acidosis (2, 10, 17). The time course of the increase in Pi
concentration ([Pi]) is, however, very similar to that of
force degradation (10, 17).
Much effort has been made to elucidate the effect of
these metabolites on individual cellular processes such
as Ca21 uptake by the sarcoplasmic reticulum (SR).
However, little is known about any effects of these
metabolites on myofibrillar relaxation. Until recently,
relaxation was induced by moving surface membranepermeabilized preparations (skinned fibers) into solutions containing relatively high concentrations of Ca21
buffers such as EGTA. These protocols produce relaxations that are dependent on, and limited by, diffusion
and equilibration of the buffers. Use of the photolabile
caged Ca21 chelator diazo 2 circumvents this problem.
The affinity of diazo 2 for Ca21 rapidly changes after
exposure to ultraviolet light (1). Initially, the Ca21
affinity is low [dissociation constant (Kd ) 5 2.2 µM]; it
increases on photolysis (Kd 5 0.073 µM), producing a
rapid decrease in the free Ca21 concentration within a
skinned muscle fiber (4, 25, 30–32, 35).
In this study we have used this photolysis method to
investigate the effect of increases in the concentrations
of metabolites, which accumulate during ischemia and
hypoxia (MgADP, Pi, and H1 ), on the rate of myofilament relaxation. The chemically skinned trabecular
preparation, in which the cellular membranes have
been rendered nonfunctional, permits effects on the
myofilaments (including possible changes in the Ca21
affinity of troponin C) to be examined separately from
changes in the SR and surface membrane. This study
provides insight into changes in the cardiac myofilament relaxation processes that occur during ischemia.
Additionally, information is provided about the control
of cross-bridge transitions when the free Ca21 is rapidly
(#2 ms) removed from the muscle system.
METHODS
Guinea pig preparation. Female albino Dunkin-Hartley
guinea pigs (200–300 g) were killed by cervical dislocation in
accordance with institutional guidelines. Their hearts were
removed and placed in an iced, oxygenated modified Tyrode
solution (5.0 mM HEPES; pH 7.35; see Solutions). Trabeculae
were dissected from the right ventricle and placed in a bath of
light paraffin oil maintained at 8°C. Trabeculae with a
uniform diameter of 100–200 µm and a length of 2–3 mm
were selected, and aluminum T clips were attached at either
end. The trabeculae were transferred to the photolysis apparatus and suspended between two stainless steel hooks (100
µm in diameter).
The trabeculae were skinned by immersion in a ‘‘skinning’’
solution (see Solutions) for 30 min at 12°C and then transferred to a ‘‘relaxing’’ solution. The length of each trabecula
0363-6135/98 $5.00 Copyright r 1998 the American Physiological Society
H951
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Simnett, S. J., E. C. Johns, S. Lipscomb, I. P. Mulligan,
and C. C. Ashley. Effect of pH, phosphate, and ADP on
relaxation of myocardium after photolysis of diazo 2. Am. J.
Physiol. 275 (Heart Circ. Physiol. 44): H951–H960, 1998.—
The aim of this study was to examine the effect of the
metabolites H1, ADP, and Pi on the rate of cardiac relaxation.
We used guinea pig right ventricular trabeculae that had
been chemically skinned, allowing the myofilaments to be
studied in isolation. Laser-flash photolysis of the caged Ca21
chelator diazo 2, causing a rapid fall in intracellular Ca21,
enabled investigation of relaxation independently of the
rate of Ca21 diffusion. On the photolysis of diazo 2, the
trabeculae relaxed biphasically with exponential rate constants (k1 and k2 ) of 10.07 and 4.23 s21, respectively, at 12°C
and 18.35 and 2.52 s21, respectively, at a nominal 20°C.
Increasing the concentration of both protons (pH 7.2–6.8) and
MgADP (0.5–3.4 mM) slowed the two phases of the relaxation
transients. Raising the concentration of Pi from the control
level of 1.36 mM to 15.2 mM increased the rate of both phases,
with relaxation becoming monoexponential at 19.4 mM Pi
(with a k of 20.31 s21 at 12°C). Cardiac muscle was compared
with skeletal muscle under identical conditions; in cardiac
muscle 19.4 mM Pi increased the rate of relaxation, whereas
in skeletal muscle this concentration of Pi slowed relaxation.
We conclude that the mechanism of relaxation differs between cardiac and skeletal muscle. This study is a direct
demonstration of the effects of ATP metabolites on cardiac
myofilament processes during relaxation.
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CAGED CA21 CHELATOR AND RELAXATION
Fig. 1. Protocol for photolysis procedure for a single trabecular
relaxation transient. Once steady-state force has been reached,
trough containing diazo 2 is lowered pneumatically, leaving trabeculae suspended in air; 400 ms later laser is fired. This produces a 20-ns
pulse of light with a wavelength of 347 nm (,100 mJ), which causes
photolysis of diazo 2, rapid uptake of Ca21, and relaxation of
trabeculae. Temperature 5 12°C; trabecular diameter 5 145 µm;
maximum steady-state force (Pmax ) 5 20 mN · mm22. Length 5
3.5 mm.
length of the preparation was exposed to the laser pulse and
was optimized each time.
Solutions. The composition of the experimental solutions
was calculated using a program written in Fortran 77, using
equilibrium constants from Smith and Martell (37). Adjustments were made to take into account the experimental
temperatures employed (12°C in all but one set of experiments). The program corrected the ionic strength (to 0.15 M)
using potassium propionate. The pH was set to an appropriate value (7.2 except in the experiments investigating the
effect of pH) by the addition of a small amount of 5 M
potassium hydroxide. All solutions contained 60 mM N, Nbis[2-hydroxyethyl]-2-aminoethanesulfonic acid (BES), 5 mM
MgATP, and 1 mM free Mg21. EGTA (10 mM) was added to the
relaxing and activating (pCa 4.5) solutions except the zeroEGTA relaxing solution. Creatine phosphokinase (15 U/ml)
and creatine phosphate (10 mM) were added to all solutions
except those containing added ADP. Contaminant Pi was
assessed by a phosphate assay kit available from Sigma
(Poole, UK). The method of Jaworek and Welsch (15) was
employed to assess contaminant ADP levels. Leupeptin (0.5
mM) was added to the relaxing solution to prevent deterioration of the fibers by Ca21-activated proteases. The skinning
solution was prepared by the addition of 1% (vol/vol) Triton
X-100 to relaxing solution. All reagents were obtained from
Sigma, except for BES, which was obtained from Calbiochem
(Cambridge, UK), and were of analytical grade. The modified
(no Ca21 ) Tyrode solution (pH 7.35) was oxygenated and
contained (in mM) 134 NaCl, 5.4 KCl, 1.2 MgSO4, 11.1
glucose, and 5.0 HEPES. Omission of Ca21 from the Tyrode
solution enhanced the viability of the trabeculae after dissection.
Curve fitting. The relaxation transients were fitted with
two exponential processes, k1 and k2, using the curve-fitting
program P.Fit (Biosoft, Cambridge) and the equation
Y 5 A exp(2k1 x) 1 B exp(2k2 x) 1 C
(1)
where Y is force, X is time, and A and B represent the relative
proportions of the fast phase (rate constant k1 ) and slow
phase (rate constant k2 ) processes, respectively. Both the
individual and mean curves were well fitted by a double
exponential. Mean relaxation transients were obtained by
normalizing and then averaging all of the individual transients. To ensure the closest possible fit, the data were fitted
several times using different initial settings for the parameters. The fitting process did not take the standard deviation
of each point into consideration; therefore, the standard
errors given for the curve-fitting parameters are for the
fitting process only. The standard error of any point from the
mean curve was ,3%. The constant C expresses the remnant
(% tension) after the curve-fitting process has been completed.
Diazo 2 solutions. Diazo 2 was initially a gift from Drs. R. Y.
Tsien and S. R. Adams (University of California, San Diego);
it was later custom synthesized by Molecular Probes (Eugene,
OR). The experimental diazo 2 solution contained 2 mM diazo
2 and had no added EGTA. The Ca21 concentration of the
diazo 2 solution was adjusted to produce activations that
were ,95% of Pmax by adding 10-µl aliquots of 20 mM CaCl2.
Effect of pH on diazo 2 kinetics. Ca21 kinetics at different
pH values were investigated by monitoring the decrease in
relative fluorescence of the Ca21 indicator fluo 3 after photolysis of 400 µM diazo 2. The change in relative fluorescence of
fluo 3 (excitation wavelength 488 nm, emission wavelength
525 nm) was followed using a laser-scanning confocal microscope (LSCM; MRC-600, BioRad) with a time resolution of 2
ms. The LSCM was equipped with a flash lamp; both pieces of
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was adjusted to produce passive force; sarcomere length was
,2.2 µm (measured using a light microscope).
Frog muscle preparation. Rana temporaria were obtained
from Blades Biological (Edenbridge, UK) and stored at 2°C in
shallow water, without food, for a maximum period of a week.
Frogs were stunned and killed by cervical dislocation according to institutional guidelines. Single muscle fibers were
dissected from the semitendinosus muscle and placed in a
bath of light mineral oil at 8°C. The fibers were treated as the
trabeculae except that they were chemically skinned for 12
min, as opposed to 30 min, at 12°C.
Apparatus and experimental protocol. The apparatus designed by Ferenczi (11) allows rapid changes of the solution
that bathes each muscle fiber. The laser-flash photolysis
technique and the triggering and recording instrumentation
were essentially the same as those described previously by
that author (11). Experiments were carried out at 12°C,
except during the study investigating temperature effects,
when a nominal value of 20°C was selected. Initially, a
steady-state activation was reached at 90–100% of the maximal activation (maximum steady-state force, Pmax ) achieved
in a pCa 4.5 solution containing 10 mM EGTA (see Solutions).
The preparation was transferred to a relaxing solution and
then to the diazo 2 solution via a second relaxing solution that
contained no Ca21 buffer (zero-EGTA solution) for ,60 s. The
experimental procedure for photolysis of diazo 2 in a single
trabecula and the resultant, typical relaxation transient are
shown in Fig. 1. The trough containing the diazo 2 solution
was lowered to leave the fiber suspended in air; 400 ms later
the laser was fired. There was a decline of ,5% in developed
force before the laser fired. The photolysis of diazo 2 caused a
rapid chelation of Ca21 and a very fast relaxation (Fig. 1).
Usually two or three relaxation transients could be obtained
from the same preparation without apparent deterioration of
the Pmax value; however, we limited ourselves to two per
trabeculae as a precaution against any damage. The full
H953
CAGED CA21 CHELATOR AND RELAXATION
equipment were controlled by a Macintosh Ilex (Apple Computers) equipped with a NB-M10 16A/D-D/A board (National
Instruments). The composition of the solutions was 122 mM
KCl, 10 mM NaCl, 1 mM MgCl2, 10 mM HEPES, and 20 µM
fluo 3. The initial ‘‘free’’ Ca21 was calculated as 66 nM, and the
final free Ca21 after 70% diazo 2 photolysis was calculated as
5 nM. The experiments were performed at three different pH
values, 6.5, 7.0, and 7.5 (see Fig. 10, A, B, and C, respectively).
The decline in relative fluorescence fitted closely to a single
exponential curve (with rates of 0.3, 0.6, and 0.44 ms21,
respectively). Both decreasing and increasing the pH from 7.0
slowed the uptake of Ca21 by diazo 2 after photolysis.
RESULTS
Trabecular relaxation rates after photolysis of diazo 2
and effect of temperature. The rate of relaxation of
skinned trabeculae from the guinea pig was investigated using the caged Ca21 chelator diazo 2 (Fig. 1).
After the photolysis of diazo 2, the trabeculae relaxed
with an average half-time of 72.4 6 2.6 ms (mean 6 SE;
n 5 8) at 12°C. The mean relaxation transient was well
fitted with a double exponential curve fit (Fig. 2) with
the fitting values shown in Table 1.
Ideally, these experiments would have been performed at the body temperature of the guinea pig
(37.2–40°C). However, to enable .70% photolysis to
occur at 2 mM diazo 2, the trabeculae were photolysed
in air rather than in the experimental trough (25, 31).
After removal from the trough, the temperature of a
fiber suspended in air will decrease rapidly toward the
dew point because of evaporation (11). This would
result in an increase in the ionic strength of the
solutions within the fiber and result in a decline in
maximal force production in the 400 ms between the
trough dropping and the laser firing. Thus the majority
of experiments were carried out at the dew point of the
laboratory (12°C) with little or no evaporation, cooling,
or change in ionic strength occurring before photolysis.
In some experiments the trough temperature was set to
20°C and the relaxation transient on photolysis was
investigated. Given that some evaporative loss and
cooling would have occurred, a 5% decline in Pmax was
observed. The average relaxation transient at a nominal 20°C (see Fig. 2) was well fitted by double exponential processes with the parameters shown in Table 1.
Comparison of the parameters indicated that increasing the temperature greatly increased the rate and
proportion of the fast phase (k1 ), whereas the rate of the
slow phase (k2 ) decreased only slightly. The mean
half-time of relaxation decreased to 53.4 6 1.84 (SE) ms
(n 5 8) with the increase in temperature to a nominal
20°C.
It has been shown that muscle activation is cooperative after photolysis of the caged Ca21 molecules nitr-5
or DM-nitrophen (4). Figure 3 shows the half-time of
Table 1. Curve-fitting parameter values for relaxation transient in guinea pig skinned trabeculae
Control
20°C
pH 6.8
0.5 mM MgADP
0.8 mM MgADP
3.4 mM MgADP
k1 , s21 6 SEM
A, % 6 SEM
k2 , s21 6 SEM
B, % 6 SEM
C, % 6 SEM
n
10.07 6 0.3
18.35 6 0.25
3.94 6 0.64
6.69 6 0.12
6.00 6 0.16
2.36 6 0.05
49.2 6 2.84
92.29 6 1.01
57.4 6 0.65
80.09 6 2.12
58.87 6 2.68
70.6 6 2.58
4.23 6 0.12
2.52 6 0.39
0.68 6 0.01
0.64 6 0.23
1.91 6 0.09
0.53 6 0.07
50.82 6 2.91
15.1 6 0.63
45.5 6 0.55
27.5 6 2.68
45.19 6 2.57
29.21 6 1.71
0.32 6 0.07
21.85 6 0.66
1.98 6 1.2
24.21 6 4.49
21.05 6 0.24
23.35 6 1.03
8
8
8
7
7
7
Parameter values (n 5 no. of trabeculae) were used to fit relaxation transients with double exponential curves. SE were derived from curve
fit of mean data; they are not SE from mean transients themselves. A and B, relative proportions of processes k1 and k2 ; C, remnant (% tension)
after curve-fitting procedure has been completed (see Eq. 1).
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Fig. 2. Average relaxation transients and their double exponential
curve fits (dashed lines) at 12°C (control, n 5 8) and nominal 20°C
(n 5 8) from skinned trabeculae, showing effect of temperature on
speed of relaxation on photolysis of 2 mM diazo 2. Average transients
were well fitted with double exponential curve fits; parameters are
shown in Table 1. Average trabecular diameter 5 151.7 6 10.7 (SE)
µm (n 5 14).
Fig. 3. Graph of half-time of relaxation against postphotolysis force
level in skinned trabeculae expressed as percentage of Pmax (value
was obtained at saturating Ca21 levels). Each trabecula was activated to 90–100% of Pmax and then relaxed to differing degrees by
varying energy of laser output. Graph was fitted with a linear
regression line (P.Fit) with the equation y 5 0.0065x 1 62.2; r 5
20.038, r2 5 0.001. Dashed lines, 95% confidence limits. Each data
point is an individual experimental transient; 2 mM diazo was used
in each case.
H954
CAGED CA21 CHELATOR AND RELAXATION
Fig. 4. Effect of Pi on rate of relaxation of skinned trabeculae from
guinea pig after photolysis of diazo 2. Transients were well fitted with
double exponential curve fits (dashed lines) with constants shown in
Table 2. Average trabecular diameter 5 165 6 19.6 (SE) µm (n 5 25).
Control level of Pi 5 1.36 mM.
Fig. 5. Effect of Pi on half-time of skinned trabecular relaxation.
Each data point is average of at least 5 values; points are fitted with a
parabolic curve.
increases in [Pi] (from 1 to 5.5 mM) there is a decline in
the half-time of relaxation [from 63 6 4.2 (SE) (n 5 7) to
44 6 4.3 (SE) ms (n 5 6)]; however, further increases in
Pi have little effect on the half-time (Fig. 5).
The relaxation transients were well fitted with double
exponential curve fits (shown by the dashed lines in
Fig. 4). Attempts were made to fit the curves with a
single exponential curve, but the results did not give a
good fit as judged by both the sum of the squares and by
eye. However, the mean relaxation transient at 19.4
mM Pi was an exception. The results of the curve-fitting
procedure show that the initial effect of Pi was to
increase k1. Further additions of Pi produced no other
change in k1 but increased the proportion of the relaxation associated with it (A). This occurred at the
expense of k2, and the relaxation was eventually characterized by a single exponential. Thus raising the concentration of Pi produced a general increase in the effect of
the fast phase of trabecular relaxation, until at 19.4
mM the relaxation became monoexponential. The effect
of further Pi addition (.19.4 mM) on the relaxation
transients could not be investigated—the degradation
in Pmax was such that the relaxation transients were too
small to be analyzed accurately at this ionic strength.
Effect of Pi on rate of relaxation of single skinned
skeletal muscle fibers. In contrast to its effect on the
relaxation rate of skinned trabeculae seen here, it was
shown previously that in semitendinosus muscle
skinned fibers from the frog Pi produces a gradual
slowing of the relaxation transient (32). To compare the
effect of Pi on the relaxation rate in the two muscle
types, the experiment was repeated in frog semitendinosus muscle fibers using the same solutions and conditions as for the cardiac experiments. The results (Fig. 6)
confirm that in frog skeletal muscle Pi slows the
relaxation rate. This indicates that the differing effect
of Pi in these cardiac and skeletal muscle preparations
is caused by biological differences as opposed to variations in the experimental conditions (e.g., ionic strength
or pH). The relaxation transients were well fitted with
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relaxation of fully activated fibers (90–100% of Pmax )
relaxed to differing levels by variation of the laser beam
energy. The graph indicates that, over the range investigated, the rate of relaxation is constant and independent of the degree of relaxation (Fig. 3). Thus cardiac
relaxation does not appear to be cooperative, or at least
the step(s) is not rate limiting, unlike the activation
process in muscle (4). We have previously shown (4)
that, in single frog fibers relaxed by diazo 2 photolysis,
the rate of relaxation is also independent of the degree
of prephotolysis force. Finally, experiments used the
Ca21 indicator fluo 3 together with 2 mM diazo 2 in
single myocytes (20); it was shown that the free Ca21
declined rapidly and uniformly from an assumed free
Ca21 concentration of ,2 µM to 100 nM on 70% diazo 2
photolysis, as was suggested by initial simulations
(Fig. 3 in Ref. 25). Recent time-resolved measurements
indicate that there is a ,1% sarcomere length change
in single frog skinned fibers on diazo 2 photolysis
producing .80% relaxation from Pmax with a half-time
of 67.4 6 4.2 (SE) ms at 5°C (B. Hoskins, S. Lipscomb,
P. J. Griffiths, and C. C. Ashley, unpublished observations), implying that the relaxation transient is a good
measure of the deactivation of the force-generating unit
(cross bridges).
Effect of Pi on rate of relaxation of skinned cardiac
trabeculae. Increasing total [Pi] to 5.67 mM produced a
decline in Pmax of 30.6 6 1.8 (SE)%, (n 5 7; data not
shown). The relationship between the decline in tension and log [Pi] is linear and is consistent with
previous reports (14, 18, 28). [Pi] in the control solutions was determined to be 1.36 mM (0.36 mM H2PO4 1
at pH 7.2 as the principal species,
0.58 mM HPO22
4
calculated from the solution program). The precise
origin of this Pi (ATP or creatine phosphate) was not
investigated.
The effect of Pi on the rate of trabecular relaxation
after the photolysis of diazo 2 is shown in Fig. 4.
Elevating [Pi] caused an increase in the rate of relaxation. The effect of Pi is twofold: with relatively small
H955
CAGED CA21 CHELATOR AND RELAXATION
double exponential curves; the parameters are shown
in Table 2. The constants indicate that, in skeletal
muscle, Pi slows the rate of k1 with a slight decrease
in A.
Effect of ADP on rate of relaxation of skinned cardiac
trabeculae. An assay to determine the concentration of
ADP in the experimental solutions (see METHODS )
showed the concentration in the control solution to be
0.05 mM. The levels in the high-ADP solutions were all
found to be within 65.0% error of the assay. The
concentration of MgADP (the active species) in each
solution was calculated using the solution program (see
METHODS ). Raising the concentration of MgADP to 3.4
mM caused an increase in Pmax of 80.2 6 1.0 (SE)%
(n 5 7) at 3.4 mM MgATP; this is consistent with
previous reports (14).
Addition of MgADP produced a significant slowing of
the trabecular relaxation rate, after photolysis of
diazo 2, at all concentrations investigated (see Fig. 7).
Comparisons of the half-times (Fig. 8) show that for
each subsequent increase of the MgADP concentration
there is a significant difference from the previous one,
and the relationship between half-time and concentration of MgADP showed a saturation, with a half-
Fig. 7. Effect of MgADP on rate of skinned trabecular relaxation
after photolysis of 2 mM diazo 2. Average transients were well fitted
with double exponential curve fits (dashed lines) with parameters
shown in Table 1. Average trabecular diameter 5 145.6 6 15.2 (SE)
µm (n 5 29).
maximal effect at an MgADP concentration of 1.5 mM.
The mean tension transients (Fig. 7) were well fitted
with double exponential curve fits; the parameters used
for the curve-fitting procedure (Table 1) reveal that
MgADP decreases the speed of the fast phase (k1 ).
Effect of pH on rate of relaxation of skinned cardiac
trabeculae. Decreasing the pH of the experimental
solutions from 7.2 to 6.8 reduced the maximal activated
tension to 66.77 6 1.92 (SE)% (n 5 8) of the control; this
is consistent with previous reports (18).
A decrease in pH caused a marked decline in the rate
of relaxation after the photolysis of diazo 2. The halftime of the mean relaxation transient at 12°C increased
from 72.4 6 2.6 (SE) ms (n 5 8) at pH 7.2 to 260.9 6
25.1 (SE) ms (n 5 10) at pH 6.8 (Fig. 9). The mean
transients were well fitted with double exponential
curves (dashed lines in Fig. 9); the rate constants are
shown in Table 1. The rate constants show that there is
a decrease in both the fast (k1 ) and slow (k2 ) phases of
the relaxations, with little alteration in the percentage
of the relaxations associated with each phase (A and B,
respectively).
One explanation for the results seen in Fig. 9 is that
there may be a change in the kinetics of Ca21 uptake by
diazo 2 with decreasing pH. However, the results
Table 2. Curve-fitting parameter values for relaxation transient in skinned cardiac and skeletal muscle
Control
5.67 mM Pi
15.2 mM Pi
19.4 mM Pi
Frog control
Frog 19.4 mM Pi
k1 , s21 6 SEM
A, % 6 SEM
k2 , s21 6 SEM
B, % 6 SEM
C, % 6 SEM
n
13.39 6 0.60
22.86 6 0.14
22.37 6 1.08
20.31 6 1.25
22.61 6 0.29
16.1 6 0.06
81.68 6 6.62
63.48 6 1.01
78.42 6 6.10
101 6 2.4
93.40 6 0.62
81.5 6 0.16
5.48 6 0.78
5.82 6 0.13
7.79 6 1.01
24.2 6 6.65
39.3 6 1.01
27.36 6 6.08
1.65 6 0.21
1.29 6 0.02
10.3 6 0.46
22.3 6 0.12
0.41 6 0.16
20.11 6 0.09
0.11 6 0.28
20.30 6 0.17
0.11 6 0.45
20.48 6 0.10
7
6
5
7
6
6
Parameter values (n 5 no. of muscle fibers or trabeculae) were used to fit relaxation transients with double exponential curves. SE were
derived from curve fit of mean data; they are not SE from mean transients themselves.
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Fig. 6. Effect of 19.4 mM Pi on rate of relaxation of single skinned
semitendinosus muscle fibers from frog on photolysis of 2 mM diazo 2.
Transients were well fitted with double exponential curve fits (dashed
lines) with constants shown in Table 2. Slowing of relaxation rate
seen in this skeletal muscle preparation is in contrast to acceleration
of relaxation rate seen in trabecular preparation (Fig. 4). Experiments were carried out using same solutions as those used for the
trabecular experiments in Fig. 4, i.e., ionic strength 5 0.15 mM and
pH 5 7.2. Temperature 5 12°C; average fiber diameter 5 102.4 6
10.8 (SE) µm; and average fiber length 5 ,3 mm (n 5 12).
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CAGED CA21 CHELATOR AND RELAXATION
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Fig. 8. Effect of increasing MgADP concentration [MgADP] on speed
of skinned trabecular relaxation after photolysis of 2 mM diazo 2.
Each point is average of at least 7 points (6SE). Half-maximal
activation occurs at MgADP concentration of 1.5 mM. Points are
fitted to a parabola.
reported in Fig. 10 indicate that the changes in diazo 2
kinetics are not significant over the time scale of these
relaxation transients (see METHODS ).
DISCUSSION
Trabecular relaxation rates after photolysis of diazo 2
and effect of temperature. After the photolysis of diazo
2, the half-time of relaxation of skinned trabeculae
from the guinea pig was 72.4 6 2.6 (SE) ms at 12°C and
53.4 6 1.8 (SE) ms at 20°C. These relaxation rates are
significantly faster than those of electrically stimulated
guinea pig trabeculae (half-time 5 ,276 ms at 20°C;
J. C. Kentish, personal communication). This suggests
that in intact preparations under normal conditions the
transfer of cross bridges from an activated to a relaxed
state is not a rate-limiting factor for relaxation and
Fig. 10. Relative decrease in fluorescence of 20 µM fluo 3 after
photolysis of 400 µM diazo 2 at 3 pH values, 6.5 (A), 7.0 (B), and 7.5
(C). Photolysis occurred at time 0. Data were fitted with a single
exponential curve fit (solid line).
Fig. 9. Effect of decreasing pH on relaxation of skinned trabeculae
from guinea pig after photolysis of 2 mM diazo 2. Average transients
were well fitted with double exponential curve fits (dashed lines) with
parameters shown in Table 1. Temperature 5 12°C; average trabecular diameter 5 171.5 6 10.7 (SE) µm (n 5 16).
would imply that the activity of the SR Ca21 pump
plays the major role in relaxation, as suggested by Luo
and colleagues (23).
It is usually hypothesized that during relaxation,
cross bridges enter weakly bound states via the same
pathway as that followed in actively contracting muscle.
However, if the rate of relaxation after photolysis of
diazo 2 (rate constants of 10 s21 and 4 s21 in skinned
trabeculae at 12°C) is compared with the rate of
CAGED CA21 CHELATOR AND RELAXATION
two exponentials. Increasing the temperature increased k1 and the proportion of the relaxation associated with this phase. At body temperature myofilament
relaxation may well be monoexponential. It was impossible to test this theory, because if the temperature of
the fiber was the body temperature of a guinea pig
(,38°C) there would be a very large degradation of
force during the 400 ms in which the trabecula is
suspended in air before the laser firing. It is not
possible to conduct the experiments with the fibers in
solution when the diazo 2 concentration is 2 mM,
because the ‘‘optical filter’’ of the diazo 2 (molar extinction coefficient 5 22,000 M21 ·cm21 at 370 nm) surrounding the fiber results in an inadequate degree of diazo 2
photolysis. Photolysis of 2 mM diazo 2 in single myocytes under paraffin oil is certainly possible (9).
Any uncertainties about the validity of inferring that
the kinetic behavior of skinned trabeculae is the same
as that of equivalent unskinned preparations were
previously dispelled by Saeki and colleagues (34), who
showed that the cross-bridge dynamics of skinned and
intact trabeculae from the ferret were not significantly
different at 20°C.
Effect of Pi on rate of relaxation of skinned cardiac
trabeculae. The assay of the phosphate concentration in
the experimental solutions found the level of Pi in the
control solutions, presumably caused by the breakdown
of either creatine phosphate or ATP, to be very similar
to that found in myocytes under normal conditions (,1
mM; Refs. 14 and 17). Thus any effects of this contaminant Pi on the myofilaments in the control conditions
will be a good approximation of those in intact muscle
cells under normal metabolic conditions. The highest
[Pi] investigated here (19.4 mM) is similar to that
reached within 2 min of cardiac ischemia (16).
The results showing that Pi increased the rate of
trabecular relaxation were unexpected because in skeletal muscle it decreases the rate of relaxation (32). One
set of experiments (effect of 19.4 mM Pi on the speed of
relaxation) was repeated in frog semitendinosus skinned
muscle fibers, using the same experimental solutions
and conditions as for the cardiac experiments. It was
revealed that the observed discrepancy between cardiac and skeletal systems was not caused by experimental variations, e.g., the ionic strength or pH of the
solutions, but was the result of real differences between
the muscle types.
It has previously been shown that cardiac tissue is
more sensitive to Pi than skeletal muscle is (14, 18); the
slope of the graph of cardiac force against log [Pi] is
twice as steep as that of skeletal muscle (18). The
reason for this discrepancy has not yet been fully
elucidated. The model of Pate and Cooke (33) predicts
that the slope of the relationship is proportional to the
fraction of cross bridges in the main force-generating
cross-bridge state (AM · ADP in their model), suggesting that in cardiac muscle there are more forcegenerating cross bridges. However, to account for the
observed difference in the slope of the force-log [Pi]
relationship between cardiac and skeletal muscle fibers
there would have to be twice as many cross bridges in
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relaxation of rigor cross bridges after photorelease of
caged ATP in the absence of Ca21 (,0.45 s21 in guinea
pig trabeculae; Ref. 5), then it is apparent that the
process of relaxation is distinct from that of the equivalent transition during relaxation from rigor. This is
despite the fact that both processes apparently involve
the binding of ATP and transfer of cross bridges to the
nonforce, weakly bound state. Our result suggests that
the mechanism by which cross-bridge force decays
when Ca21 is rapidly removed from the thin filament is
either inherently different from cross-bridge detachment during sustained muscle contraction or that the
same pathway is differently regulated (I. P. Mulligan,
R. E. Palmer, S. J. Simnett, S. Lipscomb, and C. C.
Ashley, unpublished observations).
The average relaxation transient and the individual
transients were well fitted with two exponential processes. The rate constants of the two phases were 10.07
and 4.23 s21 (Table 1); these are consistent with reports
of the relaxation rate initiated by diazo 2 photolysis in
single skinned ventricular myocytes from the rat at pH
7.0–7.1 and 20–21°C (30, 38). In frog skeletal muscle,
the relaxation transient after the photolysis of diazo 2
can also be well fitted to two exponential processes;
however, the rate constant of each phase is slightly
greater than that in cardiac muscle at the same temperature (31). Although the two rate constants of the
faster and slower processes are not greatly different,
they suggest the possibility that there are two populations of cross bridges and that they relax (pass to a
nonforce, weakly bound state) by two distinct pathways. It has previously been suggested that the ratelimiting step in the cross-bridge cycle is the dissociation
of ADP from the attached cross bridges (33, 36); it is
possible that one of the rate constants represents cross
bridges passing through this cross-bridge transition.
The other rate constant may represent cross bridges in
states before the isomerization step [AM8ADP · Pi to
AM · ADP · Pi, where A is actin and M is myosin, in the
model of Pate and Cooke (33)] relaxing through the
reversal of this or via alternative reaction steps to the
relaxed state.
From comparisons of the control relaxation transients, it is apparent that the rate of relaxation varied
somewhat between different sets of experiments. However, in each set of experiments the experimental
conditions (e.g., control or 0.5 mM MgADP) were selected randomly and in most cases it was sufficient to
obtain two relaxation transients under different conditions from each fiber. Comparisons of these individual
transients always produced the same results as the
mean results, regardless of the order in which they
were performed.
Increasing the temperature of the trabeculae to a
nominal 20°C accelerated the relaxation rate by 35%. It
was not possible to measure the temperature of the
trabeculae at the time of photolysis, but from the
results of Ferenczi (11) it would be expected that the
fiber temperature decreased by 2–3°C in the 400 ms
between the trough being lowered and the laser firing.
Again, the relaxation transients were closely fitted by
H957
H958
CAGED CA21 CHELATOR AND RELAXATION
bridge states and resulting in the observed increase in
isometric tension.
An increase in MgADP caused a decrease in the
speed of skinned trabecular relaxation after the photolysis of diazo 2 (4, 35). This was a graded effect, with the
relaxation half-time increasing with each addition of
MgADP (to concentrations well above physiological
levels); it is consistent with the effect of raised MgADP
concentrations on the relaxation of skinned skeletal
muscle fibers following diazo 2 photolysis (R.E. Palmer,
personal communication).
There are at least two possible mechanisms by which
MgADP may be acting. It could, by end product inhibition, decrease the rate of MgADP release from the
several AM · ADP states, or it could compete with ATP
for the myosin nucleotide binding site, acting as a
competitive inhibitor. The situation regarding competitive inhibition appears to be inconclusive. Cooke and
Pate (8) found that, in skeletal muscle, the effect of
MgADP on maximum unloaded shortening velocity was
consistent with a competitive inhibition of the MgATP
for the nucleotide binding site. This contrasts with the
results of Lipscomb and colleagues (21), who investigated the effect of altered MgATP concentrations on the
slowing of force relaxation produced by a rise in MgADP.
This group reported that, in frog skeletal muscle,
increasing the MgATP levels from 5 to 15 mM did not
affect the slowing of relaxation produced by MgADP on
photolysis of diazo 2. It was concluded that MgADP and
MgATP binding are noncompetitive at the single nucleotide binding site identified by crystallography (21).
Investigations by Lu and colleagues (22) into the effect
of caged ADP release on isometric force were inconclusive as to the mechanism by which MgADP was acting.
If MgADP was acting as a competitive inhibitor, it
would be expected that the relationship of inverse of
the velocity of relaxation (represented by half-time)
against the concentration of MgADP would be a straight
line. However, as shown in Fig. 8, the data points are
best fitted by a parabolic curve, suggesting that MgADP
is acting as a partial competitive inhibitor. It is possible
that the mechanism of action of MgADP is dependent
on the degree of force being produced by the cross
bridges, i.e., whether they are in an isometric or
isotonic situation.
Increases in the concentration of MgADP are known
to occur during ischemia in the heart; the concentration
rises from a resting level of ,0.05 mM up to a maximum of 1–2 mM (16). The buildup of MgADP, although
not as large as that of Pi, would cause a change in
contractile function. MgADP has two favorable effects;
it increases both Pmax and the apparent Ca21 sensitivity
of the myofilaments. These properties of MgADP would
tend to increase the inotropic state of the heart. However, offsetting this, MgADP decreases the maximal
speed of both contraction and relaxation. Therefore, the
presence of MgADP would tend to decrease cardiac
output. The importance of any pathological MgADP
buildup is debatable, because levels do not rise significantly in the first 5–10 min of ischemia—the time
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the force-generating state in cardiac muscle as in
skeletal muscle. Another possibility is that the crossbridge cycle Pi release step is closer to equilibrium in
cardiac tissue and hence is more susceptible to perturbation by changes in steady-state [Pi].
The data from the curve fitting of the relaxation
transients reveal that the addition of small concentrations of Pi increases the speed of k1, with a slight decline
in the proportion of A. Further additions of Pi caused no
further changes in k1, but increased A at the expense of
B, until the relaxation became monoexponential at 19.4
mM Pi. This suggests that Pi has a greater effect on the
latter part of the tension decline, whereas the initial
decay of force is largely unaffected. Also, it implies that
increasing [Pi] reduces the number of cross bridges that
relax via the slow phase. If relaxation can be described
by two exponentials, this suggests that there may be
two distinct populations of cross bridges (A and B) that
relax by different pathways with rate constants k1 and
k2, respectively. Thus the fact that at 19.4 mM Pi the
relaxation becomes monoexponential suggests that, at
high [Pi], all the cross bridges relax by the same
pathway.
However, although there is a clear difference between
cardiac and skeletal muscle in the effect that raised Pi
has on the relaxation transient as judged by the
changes in half-times, it may be difficult to make a clear
interpretation of this change in terms of the constants
k1 and k2. This is because the values of these rate
constants are too similar to determine for certain that
they represent two distinct populations. Nevertheless,
the experimental finding that cardiac and skeletal
muscle respond in very different ways to a raised [Pi]
under identical conditions remains clear.
Whatever the mechanism by which Pi increases the
rate of relaxation (positive lusitropic effect), this effect
may be beneficial in cardiac ischemia and hypoxia, in
which there is a large accumulation of Pi (up to 20 mM,
Ref. 16; see also Ref. 6) and energy supply for relaxation
is limited.
Effect of ADP on rate of relaxation of skinned cardiac
trabeculae. The release of MgADP is thought to occur
toward the end of the cross-bridge power stroke and to
be closely followed by MgATP binding and cross-bridge
detachment (or transition into a weakly bound, nonforce-generating state). It has been suggested that this
transition (or perhaps the MgADP isomerization step)
is the rate-limiting step for cross-bridge cycling and is
slow so as to match the ATPase rate.
The increase in Pmax with increasing MgADP concentrations seen here is consistent with previous reports,
both in cardiac (14) and skeletal (13, 14, 33) muscle.
MgADP also increases the apparent Ca21 sensitivity of
the myofibrils (14). As the MgADP concentration is
increased, the free energy of the AM · ADP state decreases relative to the A · M state, making the release of
MgADP (and subsequent cross-bridge cycling) less energetically favorable (33). Thus raised levels of MgADP
would be expected to retard cross-bridge dissociation,
increasing the population of high force-generating cross-
CAGED CA21 CHELATOR AND RELAXATION
Protons are thought to be released at several stages
of the cross-bridge cycle (see Eq. 2, in which the states
within the box are considered to be non-force-generating, weakly bound cross bridges), and thus they could
slow relaxation by a mass action (concentration) effect
on any one of the transitions with which they are
involved. However, it would appear that simple mass
action effects of H1 on these steps (ADP release and Pi
rebinding) would speed up and not slow down the rate
of relaxation (Eq. 2) by enhancing Pi rebinding or ADP
release. Metzger and Moss (24), investigating the kinetics of force redevelopment after rapid shortening in
skeletal muscle, revealed that pH modulates the forcegenerating step of the cross-bridge cycle in a manner
that is not compatible with mass action effects. It has
been shown that the maximum ATPase rate is decreased in acidosis (7, 13); any slowing of enzymatic
activity may suggest a reduction in the rate of crossbridge cycling.
The mechanism of proton action has not been clearly
defined. In physiological systems, the two types of Pi
are the diprotonated (H2PO2
4 ) and the monoprotonated
)
forms.
In
skeletal
muscle it has been sug(HPO22
4
gested (27) that the active form is diprotonated Pi and
that the myofibrillar effect of protons is caused by
an increase in the proportion of this species. However,
this has been disputed by other studies in skeletal
and cardiac muscle (10, 18). It is thought that Pi is
released as the diprotonated form and is converted to
the monoprotonated form with the formation of a
hydrogen ion; thus it may be expected that proton
effects occur via a shift in this equilibrium. There
is controversy as to whether this is true in skeletal
muscle (28); in cardiac muscle the action of H1 is
thought to be independent of Pi (10, 18, 28). This
proposal is confirmed by the results of this study, in
which H1 decreased the cardiac relaxation rate and Pi
increased it.
The effects of Pi, ADP, and H1 on Pmax and Ca21
sensitivity have been documented in skinned cardiac
muscle; they are qualitatively the same as in skinned
skeletal muscle. Pi and protons decrease Pmax and Ca21
sensitivity, whereas ADP has the opposite effect. The
mechanism by which these metabolites affect contractile function is not entirely clear. There have been
suggestions that the effect of Pi on Pmax, is a result of a
decline in the free energy of hydrolysis of ATP, which is
proportional to [ATP]/([ADP] · [Pi]) (33). However, increases in [Pi] reduce Pmax, whereas increases in ADP
levels have the opposite effect (8, 14). It is most
probable that Pi and ADP act by altering the equilibrium of certain cross-bridge transitions.
(2)
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during which the majority of decline in contractile
function occurs (16). MgADP will, however, play a role
in the contractile state of the heart during long periods
of ischemia.
Effect of pH on rate of relaxation of skinned cardiac
trabeculae. It has previously been shown that hydrogen
ions produce a slowing of the rate of skeletal muscle
relaxation after photolysis of diazo 2 (31). Here it has
been shown that acidosis also decreases the relaxation
rate of cardiac muscle, with the average half-time for
relaxation increasing from 72.4 6 2.6 (SE) ms (n 5 8) at
pH 7.2 to 260.9 6 25.1 (SE) ms (n 5 8) at pH 6.8. The
demonstrated decline in Pmax is in agreement with
previous studies (13, 18).
Prior investigations into the rate of relaxation during
acidosis in intact preparations have been contradictory.
Fry and Poole-Wilson (12) recorded a decline in the
speed of guinea pig papillary muscle relaxation with
increasing acidosis, whereas in ferret papillary muscle
the reverse was observed (3, 29). Allen and colleagues
(3) made simultaneous measurements of Ca21 and force
during acidosis and found that although the relaxation
rate decreased, the Ca21 transient was prolonged. They
suggested that this might be the result of a decrease in
the level of Ca21 binding to troponin C during acidosis
(Ref. 3; see also Ref. 7). It is possible that these effects
are caused by changes in phospholamban or other SR
proteins, as opposed to direct effects on the myofilaments. This is because the present results indicate a
decrease in relaxation rate with decreased pH rather
than the increase required if the relaxation rate was
determined solely by the Ca21 off-rate from troponin C,
assuming that no change in the Ca21 on-rate is associated with the well-described pH-induced decrease in
affinity (19).
Whatever the reason, it is unlikely that changes in
diazo 2 kinetics are causing the observed change in
relaxation rate. Although the kinetics are slightly
slower at pH 6.5 compared with pH 7.5, they are still
greater than ,300 s21 and thus 30-fold faster than the
fast phase of the relaxation transient. Thus diazo 2
kinetics are not considered to be rate limiting in these
experiments.
The trabecular relaxations after the photolysis of
diazo 2 were well fitted with double exponential curve
fits. Acidosis slows the rate of both the fast and the slow
transitions, with little effect on the proportions of the
two phases. The lack of shift in the proportions of the
two phases suggests that there is no alteration in the
distribution of cross bridges. This supports the notion
that the decline in steady-state force is caused by a
reduction in the amount of force produced by each
strongly bound cross bridge.
H959
H960
CAGED CA21 CHELATOR AND RELAXATION
Experiments with fluo 3 were performed by Drs. W. Zhang, E.
Niggli, and H. Oetliker at the University of Bern, Switzerland.
This work was supported by grants from the British Heart
Foundation (BHF) and the Wellcome Trust; a BHF Intermediate
Fellowship was awarded to I. P. Mulligan.
Present address of I. P. Mulligan: Dept. of Cardiovasc. Med., John
Radcliffe Hosp., Oxford OX3 9DU, UK.
Address for reprint requests: C. C. Ashley, Univ. Lab. of Physiology,
Parks Rd., Oxford OX1 3PT, UK.
Received 26 November 1996; accepted in final form 1 May 1998.
18.
19.
20.
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