Vol. 275, Issue 3, H951-H960, September 1998
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
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ABSTRACT |
The aim of this study was to examine the effect
of the metabolites H+, 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
Ca2+ chelator diazo 2, causing a
rapid fall in intracellular Ca2+,
enabled investigation of relaxation independently of the rate of
Ca2+ diffusion. On the photolysis
of diazo 2, the trabeculae relaxed biphasically with exponential rate
constants (k1 and
k2) of 10.07 and 4.23 s
1, respectively,
at 12°C and 18.35 and 2.52 s1, 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 s1 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.
muscle; heart; calcium; guinea pig; cross bridges
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INTRODUCTION |
CARDIAC MUSCLE RELAXATION 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 by-products 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 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
Ca2+ 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 membrane-permeabilized preparations (skinned
fibers) into solutions containing relatively high concentrations of
Ca2+ 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 Ca2+ chelator diazo 2 circumvents this problem. The affinity of diazo 2 for
Ca2+ rapidly changes after
exposure to ultraviolet light (1). Initially, the
Ca2+ affinity is low
[dissociation constant
(Kd) = 2.2 µM]; it increases on photolysis
(Kd = 0.073 µM), producing a rapid decrease in the free
Ca2+ 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
H+), 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
Ca2+ 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 Ca2+ is rapidly (
2
ms) removed from the muscle system.
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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 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
Ca2+ 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 Ca2+
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 length of the preparation was exposed to
the laser pulse and was optimized each time.
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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 Ca2+, and
relaxation of trabeculae. Temperature = 12°C; trabecular diameter = 145 µm; maximum steady-state force
(Pmax) = 20 mN · mm2.
Length = 3.5 mm.
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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,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid (BES), 5 mM MgATP, and 1 mM free
Mg2+. EGTA (10 mM)
was added to the relaxing and activating (pCa 4.5) solutions except the
zero-EGTA 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 Ca2+-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
Ca2+) 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 Ca2+ 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
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(1)
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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
Ca2+ 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.
Ca2+ kinetics at different pH
values were investigated by monitoring the decrease in relative
fluorescence of the Ca2+ 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 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"
Ca2+ was calculated as 66 nM, and
the final free Ca2+ 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 ms1, respectively).
Both decreasing and increasing the pH from 7.0 slowed the uptake of
Ca2+ by diazo 2 after photolysis.
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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 Ca2+
chelator diazo 2 (Fig. 1). After the photolysis of diazo 2, the trabeculae relaxed with an average half-time of 72.4 ± 2.6 ms (mean ± SE; n = 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.
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Fig. 2.
Average relaxation transients and their double exponential curve fits
(dashed lines) at 12°C (control, n = 8) and nominal 20°C (n = 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 = 151.7 ± 10.7 (SE)
µm (n = 14).
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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 ± 1.84 (SE) ms (n = 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 Ca2+
molecules nitr-5 or DM-nitrophen (4). Figure
3 shows the half-time of 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
Ca2+ indicator fluo 3 together
with 2 mM diazo 2 in single myocytes (20); it was shown that the free
Ca2+ declined rapidly and
uniformly from an assumed free
Ca2+ 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 ± 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).
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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 Ca2+ 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 = 0.0065x + 62.2;
r =  0.038,
r2 = 0.001. Dashed lines, 95% confidence limits. Each data point is an individual
experimental transient; 2 mM diazo was used in each case.
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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 ± 1.8 (SE)%,
(n = 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 + 0.58 mM HPO24 at pH 7.2 as the
principal species, 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 increases in
[Pi] (from 1 to 5.5 mM) there is a decline in the half-time of relaxation [from 63 ± 4.2 (SE) (n = 7) to 44 ± 4.3 (SE) ms (n = 6)]; however,
further increases in Pi have
little effect on the half-time (Fig. 5).
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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 = 165 ± 19.6 (SE) µm (n = 25).
Control level of Pi = 1.36 mM.
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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.
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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 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.
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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 = 0.15 mM and pH = 7.2. Temperature = 12°C; average fiber
diameter = 102.4 ± 10.8 (SE) µm; and average fiber length = ~3
mm (n = 12).
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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 ±5.0% 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 ± 1.0 (SE)% (n = 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-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).
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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 = 145.6 ± 15.2 (SE) µm
(n = 29).
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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 (±SE). Half-maximal
activation occurs at MgADP concentration of 1.5 mM. Points are fitted
to a parabola.
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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 ± 1.92 (SE)% (n = 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 half-time of the mean relaxation
transient at 12°C increased from 72.4 ± 2.6 (SE) ms (n = 8) at pH 7.2 to 260.9 ± 25.1 (SE) ms (n = 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).
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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 = 12°C; average trabecular
diameter = 171.5 ± 10.7 (SE) µm
(n = 16).
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One explanation for the results seen in Fig. 9 is that there may be a
change in the kinetics of Ca2+
uptake by diazo 2 with decreasing pH. However, the results 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).
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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).
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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 ± 2.6 (SE) ms at 12°C
and 53.4 ± 1.8 (SE) ms at 20°C. These relaxation rates are
significantly faster than those of electrically stimulated guinea pig
trabeculae (half-time = ~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
would imply that the activity of the SR
Ca2+ 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 s1 and 4 s1 in skinned trabeculae at
12°C) is compared with the rate of relaxation of rigor cross
bridges after photorelease of caged ATP in the absence of
Ca2+ (~0.45
s1 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
Ca2+ 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 s1 (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 rate-limiting 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
[AM'ADP · 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 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 = 22,000 M1 · cm1
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 force-generating 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 the force-generating state in cardiac muscle
as in skeletal muscle. Another possibility is that the cross-bridge
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,
non-force-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 Ca2+
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-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
Ca2+ 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 ischemiathe
time 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 ± 2.6 (SE) ms
(n = 8) at pH 7.2 to 260.9 ± 25.1 (SE) ms (n = 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 Ca2+
and force during acidosis and found that although the relaxation rate
decreased, the Ca2+ transient was
prolonged. They suggested that this might be the result of a decrease
in the level of Ca2+ 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 Ca2+
off-rate from troponin C, assuming that no change in the
Ca2+ 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
s1 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.
 |
(2) |
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
H+ 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 force-generating 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 cross-bridge cycling.
The mechanism of proton action has not been clearly defined. In
physiological systems, the two types of
Pi are the diprotonated (H2PO4)
and the monoprotonated (HPO24) forms. In skeletal muscle it has been suggested (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
H+ is thought to be independent of
Pi (10, 18, 28). This proposal is
confirmed by the results of this study, in which
H+ decreased the cardiac
relaxation rate and Pi increased
it.
The effects of Pi, ADP, and
H+ on
Pmax and
Ca2+ sensitivity have been
documented in skinned cardiac muscle; they are qualitatively the same
as in skinned skeletal muscle. Pi
and protons decrease Pmax and
Ca2+ 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.
| |
ACKNOWLEDGEMENTS |
Experiments with fluo 3 were performed by Drs. W. Zhang, E. Niggli,
and H. Oetliker at the University of Bern, Switzerland.
| |
FOOTNOTES |
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.
| |
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Abstract
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