© 1995 American Heart Association, Inc.
Articles |
Protein Kinase A Does Not Alter Economy of Force Maintenance in Skinned Rat Cardiac Trabeculae
From the Section on Cardiology (P.P. de T.), Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC, and the Institute for Cardiovascular Research (G.J.M.S.), Department of Physiology, Free University of Amsterdam (Netherlands).
Correspondence to Pieter P. de Tombe, PhD, Section on Cardiology, Department of Internal Medicine, Bowman Gray School of Medicine, Wake Forest University, Medical Center Blvd, Winston-Salem, NC 27157-1045. E-mail pdetombe@sarc.card.wfu.edu.
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Abstract Recent mechanical, biochemical, and energetic experiments have suggested that catecholamines may increase the cycling rate of cross-bridges independent of changes in intracellular calcium. An increased rate of cross-bridge cycling is expected to result in decreased economy of force maintenance. The present study tested this hypothesis directly by measuring the rate of ATP consumption in skinned cardiac trabeculae as a function of steady state force. Rat cardiac trabeculae were skinned with Triton X-100. Resting sarcomere length was measured by laser diffraction, and ATP consumption was assessed by an enzyme-coupled optical technique. Force-[Ca2+] relations were fit to a modified Hill equation. Force dependency of the rate of ATP consumption was analyzed by multiple linear regression analysis. ß-Adrenergic stimulation was mimicked by incubation of the skinned muscle preparation with the catalytic subunit of protein kinase A (PKA). Treatment with PKA (3 µg/mL, 40 minutes) induced a significant (65±23%, P=.01) increase in [Ca2+] required for half-maximal steady state force, whereas the steepness of the force-[Ca2+] relation was not affected. The rate of ATP consumption was linearly correlated with steady state force, regardless of PKA treatment status (P<.001). However, neither the slope nor the intercept was affected by PKA treatment. Hence, PKA treatment did not affect either the maximum rate of ATP consumption or the economy of force maintenance. These results suggest that ß-adrenergic stimulation does not alter the rate-limiting step of cross-bridge cycling during isometric contraction in myocardium.
Key Words: myofibrillar proteins • actomyosin ATPase activity • cross-bridges • ß-adrenergic stimulation
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Stimulation of ß-adrenergic receptors by catecholamines has a profound effect on myocardial function, such as increased peak force development, decreased time to peak force, and enhanced rate of force relaxation.1 ß-Adrenergic stimulation causes activation of a cAMP-dependent protein kinase that catalyzes phosphorylation of several key proteins that are intimately involved in calcium handling by the cardiac myocyte.2 3 Thus, phosphorylation of sarcolemmal calcium channels leads to an enhanced flux of calcium ions into the cell during the plateau phase of the action potential,4 phosphorylation of phospholamban leads to an enhanced rate of calcium uptake by the sarcoplasmic reticulum,3 5 and phosphorylation of troponin I leads to reduced affinity of the contractile proteins to calcium.3 6 7 Therefore, it has been suggested that the inotropic effects of ß-adrenergic stimulation are due, to a large extent, to alterations in calcium handling by the cardiac myocyte.8 9 10
Recent studies of both mechanical11 12 13 14 and biochemical15 16 properties of isolated myocardium, however, have suggested that part of the inotropic effects of ß-adrenergic stimulation may be due to a direct effect on the cardiac cross-bridge cycle that is independent of the effects of ß-adrenergic stimulation on calcium handling. This proposition is challenged by other investigators17 18 19 who found no direct effect of ß-adrenergic stimulation on the mechanical properties of isolated myocardium. In addition, it has been reported that the application of isoproterenol results in a reduced economy of force generation in studies using myothermal measurements on isolated papillary muscles.20 21 This observation also suggests that ß-adrenergic stimulation causes an increase in the cycling rate of cross-bridges.21 However, interpretation of myothermal experiments in isolated myocardium is complicated by uncertainties in the partitioning of total energy consumption between that used for excitation-contraction coupling (ie, calcium handling) and that used for the contractile process (ie, cross-bridge cycling).22 These uncertainties are of particular concern in studies on the effects of ß-adrenergic stimulation, since marked alterations in calcium handling are known to occur under these conditions.8 9 10 Because of these uncertainties, it is unclear at present whether ß-adrenergic stimulation alters the economy of the contractile process in myocardium.
Therefore, in the present study, we directly measured the rate of ATP consumption in chemically permeabilized (ie, skinned) isolated rat cardiac trabeculae by means of an enzyme-coupled optical technique. Adopting this approach allowed us to circumvent many of the problems that hamper myothermal measurements on intact isolated myocardium. This is because the skinning procedure removes virtually all membranous structures from the isolated muscle preparation, thus leaving the contractile proteins as the only source of energy consumption (ie, conversion of ATP into ADP). Hence, in this preparation the amount of ATP hydrolyzed can be correlated directly to the amount of force that is developed, thereby allowing for an unambiguous determination of the economy of force maintenance. To mimic ß-adrenergic stimulation, skinned trabeculae were exposed to the catalytic subunit of protein kinase A (PKA). This form of the protein kinase does not require cAMP for activity and catalyzes phosphorylation of the same subunits of the contractile proteins as observed with ß-adrenergic receptor stimulation in intact myocardium.3 14 18
In the present study, we found a significant reduction in the sensitivity of the contractile system to calcium ions after PKA treatment, consistent with previously reported studies.7 14 18 However, in contrast to previous myothermal studies,20 21 we found no effect of PKA treatment on the economy of force maintenance. Therefore, our results suggest that ß-adrenergic stimulation does not affect the rate-limiting step of cross-bridge cycling during isometric contraction in myocardium.
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Preparation
Rats, 250 to 350 g in weight and of either sex, were anesthetized with pentobarbital (50 mg/kg IP), and the hearts were rapidly excised. All procedures that were used in the present study were in accordance with institutional guidelines regarding care and use of laboratory animals. After excision, the heart was immediately perfused with a Tyrode's solution containing 20 mmol/L 2,3-butanedione monoxime (BDM) and placed in a dissection dish beneath a binocular microscope equipped with an ocular micrometer (
10-µm resolution).
Treatment with BDM has been shown to protect myocardium from cell
contracture and muscle damage during dissection.23 Thin,
unbranched, and uniform trabeculae were carefully dissected from the
free wall of the right ventricle. The preparations were 1.78±0.08 mm
in length, 199±13 µm in width, and 162±11 µm in thickness
(mean±SEM, n=10; measured at 2.2- to 2.3-µm sarcomere length). After
dissection, the trabeculae were transferred to a dish containing cold
standard relaxing solution to which 1% (vol/vol) Triton X-100 was
added in order to chemically permeabilize the preparation. The
composition of this standard relaxing solution was as follows (mmol/L):
Na2ATP 7.3, MgCl2 10.6, EGTA 20,
phosphocreatine 10, and N,N-bis[2
hydroxyethyl]-2-aminoethanesulfonic acid (BES) 100, pH 7.1, adjusted
with KOH; ionic strength, 200 mmol/L (adjusted with KCl). The
preparations were left in this solution for 2 hours to allow
solubilization of virtually all membranous structures. Next, a skinned
trabecula was attached to aluminum T clips24 and mounted
in the experimental setup. Treatment of intact myocardium with BDM may affect the phosphorylation status of myofilament proteins.25 However, preliminary experiments showed no effect of BDM pretreatment on the force-[Ca2+] relation of chemically permeabilized cardiac trabeculae (G.J.M. Stienen, unpublished data, 1994). Furthermore, it has been previously shown that the addition of inhibitors of the sarcoplasmic reticulum calcium ATPase (such as cyclopiazonic acid) does not significantly affect the rate of ATP consumption at full contractile activation.26 Hence, it appears that this potentially confounding enzyme activity is removed by the 2-hour skinning procedure. To suppress any residual mitochondrial ATPase or ATP synthase activity, both sodium azide (5 mmol/L) and oligomycin B (10 µmol/L) were added to all solutions.
Experimental Apparatus
The skinned trabecula was attached to a displacement generator
(Ling Dynamic Systems 101) on one end and to a force transducer element
(AE 801, SensoNor) on the other end (see Fig 1
). The
natural frequency of the force transducer was 2 kHz. The muscle
preparation could be transferred manually between several baths to
expose the trabecula to the various solutions that were used in the
present study (see Table 1). The bath that was used
for the ATPase assay had quartz windows to allow transmission of
near-UV light (340 nm) for the measurement of NADH absorbance (see
below). The volume of this bath was 30 µL and was continuously
stirred by motor-driven vibration of a membrane positioned at the
bottom of the bath (see Fig 1). The baths were milled in aluminum
blocks (anodized) and mounted on top of an aluminum base, through
which water was circulated to allow temperature control of all
solutions (20±1°C). The force and displacement generator position
(ie, muscle length) signals were filtered at 1 kHz (corner frequency;
slope, -12 dB per octave). The NADH absorbance signal was filtered at
2.5 Hz (corner frequency; slope, -12 dB per octave). The data were
recorded on a chart recorder and sampled via an A/D convertor on
computer disk (M280, Olivetti). Samples were collected at a rate of
five per second for 5 minutes. In addition, force and muscle length
signals were also collected at a higher sample rate (1 kHz for 0.5
seconds) during a rapid release step, which we applied to the muscle at
the end of a contraction to accurately measure the zero force level in
the ATPase assay bath. Sarcomere length of the preparation was measured
in relaxing solution by means of a He-Ne laser (model 1125,
Uniphase).
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Measurement of ATPase Activity
The ATPase activity of the skinned trabecula was measured
on-line by means of an enzyme-coupled assay as described in detail
previously.26 27 28 29 Briefly, formation of ADP by the
trabecula was stoichiometrically coupled first to the synthesis of
pyruvate and ATP from phospho(enol)pyruvate (PEP), a reaction that is
catalyzed by the enzyme pyruvate kinase, and subsequently to the
synthesis of lactate, a reaction that is catalyzed by the enzyme
lactate dehydrogenase (LDH) and during which NADH is oxidized to
NAD+. The breakdown of NADH was determined photometrically
by measuring the absorbance of 340-nm near-UV light obtained from a
75-W xenon lamp (XBO75, Osram) that was projected through the bath just
beneath the preparation (see Fig 1
). The transmitted light was passed
through a beam splitter (78150, Oriel) and then projected onto two
UV-enhanced photo diodes (UV 100, United Detector Technology) via 340-
and 400-nm interference filters, respectively (models 35-2989 and
35-3201, Ealing Electro-optics). The ratio of light intensity at 340
nm, which is sensitive to the NADH concentration in the bath, and the
light intensity at 400 nm, which serves as a reference signal, was
obtained by means of an analog divider. The output of the divider was
applied to the input of a logarithmic amplifier to obtain a voltage
that was linear proportional to the NADH concentration in the bath.
The first time derivative of this signal, which is proportional to the
rate of ATP consumption in the assay bath, was determined off-line by
linear regression of the sampled data using custom-designed software. A
small decline in the absorbance signal in the absence of the muscle
fiber in the bath, caused by an ATPase contamination in the LDH enzyme
preparation and by photo bleaching of NADH, was subtracted off-line
from the absorbance data throughout the recording period. After each
recording in the assay bath, the NADH absorbance signal was calibrated
by multiple injections of 0.50 nmol ADP (0.050 µL of 10 mmol/L ADP
solution) by use of a stepper motor-controlled calibration pipette.
When this method was used, the standard error of the first time
derivative of the NADH absorbance signal, determined during a period of
20 seconds, corresponded to 0.1 pmol/s. Since the isometric ATPase
activity during contractions at saturating [Ca2+]
levels typically amounted to 25 pmol/s, this translates to a
signal-to-noise ratio of 250 under these conditions. During
contractions at submaximal activation, where the ATPase activity of the
skinned trabecula was lower, a signal-to-noise ratio of at least 25 was
achieved by appropriately increasing the time the preparation was
activated so as to allow linear regression to be performed over a
longer time period.
Solutions
Three bathing solutions were used: a relaxing solution, a
preactivating solution with low calcium-buffering capacity, and an
activating solution. The composition of these solutions is shown in
Table 1. In addition, all solutions contained 0.9 mmol/L NADH, 100
mmol/L BES, 5 mmol/L sodium azide, 10 mmol/L PEP, 4 mg/mL pyruvate
kinase (500 U/mg), 0.24 mg/mL LDH (870 U/mg), 10 µmol/L oligomycin B,
0.2 mmol/L
P1,P5-di(adenosine-5')pentaphosphate
(A2P5), and 100 µmol/L leupeptin. The
ionic strength of the solutions was kept at 200 mmol/L by adding the
appropriate amount of potassium propionate (KProp). The pH was adjusted
to 7.1 at 20°C with KOH. The compositions were calculated by using
the methods described by Fabiato and Fabiato.30 The free
Mg2+ and MgATP concentrations were calculated at 1
and 5 mmol/L, respectively. To achieve a range of free
[Ca2+], activating and relaxing solutions were
appropriately mixed, with an apparent stability constant of the
Ca2+-EGTA complex of 106.58
assumed. All chemicals were of the highest purity available (Sigma
Chemical Co).
Experimental Protocol
The protocol consisted of two series of measurements: a first
series under control conditions (pre-PKA) and a second series after
exposure of the trabecula to the catalytic subunit of PKA
(post-PKA).
During each series of measurements, the trabecula was incubated in the
relaxing solution for 4 minutes, in the preactivating solution for 3
minutes, in the activating solution for 2 minutes, and from there
back into the relaxing solution. Before the first activation-relaxation
cycle, sarcomere length in the preparation, as measured in relaxing
solution, was adjusted to 2.2 to 2.3 µm. Then, after a first
activation at saturating [Ca2+] (50 µmol/L
free [Ca2+], pCa 4.3), sarcomere length was
readjusted to 2.2 to 2.3 µm. It was found that after this
readjustment, resting sarcomere length remained stable throughout the
experiment. Next, a second activation was performed at the saturating
[Ca2+], which served as a first force and ATP
consumption rate reference. The next five to six contractures were
carried out at a range of intermediate [Ca2+] that
also included the relaxing solution (pCa 9). These measurements were
then followed by a final control contracture at saturating
[Ca2+]. In each contracture, after active force
had become stable and after sufficient data were collected to allow
reliable calculation of the rate of ATP consumption, the trabecula was
quickly (2 milliseconds) released by 25% muscle length; this
release caused an immediate drop in force to the zero level. During
this time, force and muscle length signals were sampled at a faster
rate to allow accurate off-line determination of the zero force level.
This approach was used to allow a correction for small variations in
the baseline of the force signal caused by variations in the amount of
fluid in the ATPase assay bath. Only after the preparation was fully
relaxed by returning it back into the relaxation solution was muscle
length returned to the original length before the release (see also Fig 2), allowing for a measurement of passive force that was
subtracted from total force during the contraction in order to obtain
the active developed force.
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After the pre-PKA series, the trabecula was incubated for 40 minutes in standard relaxing solution, to which was added 3 µg/mL catalytic subunit of PKA (prepared from porcine heart, Sigma) and 6 mmol/L dithiothreitol.14 18 In two trabeculae, we used 100 µg/mL PKA instead of 3 µg/mL PKA; results from these two trabeculae were similar to those in which we used 3 µg/mL PKA. Hence, it appeared that the effect of 3 µg/mL PKA treatment for 40 minutes was already saturated. After the treatment with PKA, the trabecula was incubated for an additional 15 minutes in standard relaxing solution without added PKA or dithiothreitol. We found no changes in resting sarcomere length after PKA treatment; readjustment of muscle length, therefore, was not required at this stage of the experiment. After PKA treatment, a second experimental series (post-PKA) of force and ATPase measurements was performed by using the above-described protocol.
Data Processing and Statistical Analysis
Sigmoidal force-[Ca2+] relations were fit
by a nonlinear fit procedure31 to a modified Hill
equation:
 | (1) |
for
the pre- and post-PKA series were next subjected to paired Student's
t test; ie, the fit parameters were treated statistically as
if they were obtained by direct measurement.33 In
addition, for the purpose of displaying the data that were obtained
from all trabeculae, steady state force data were normalized to steady
state force measured during the final control contracture of the
pre-PKA series.
To assess the impact of PKA treatment on the economy of force
maintenance, the rate of ATP consumption as a function of developed
force was analyzed by multiple linear regression34 using
the following as a model:
 | (2) |
, ß, 
, and 
are regression coefficients.
In addition, Equation 2 was extended with additional dummy variables to
allow for interexperiment variation for both the and ß
parameters.34 Note that a significant coefficient ß in
Equation 2 would indicate a significant correlation between F and
ATPase irrespective of PKA treatment status, whereas a significant
coefficient would indicate an effect of PKA treatment on the
coefficient ß (ie, economy of force maintenance). For the purpose of
this analysis, steady state force was not corrected for the slight
deterioration during the experiment as was described above, since
steady state force and the rate of ATP consumption decreased
proportionally during the course of the experiments. To ensure that
this approach did not bias the analysis of the data, we also
performed the statistical tests by using the corrected steady state
force data; both methods of analysis resulted in similar levels of
statistical significance, and the choice of analysis did not alter
our conclusions. For the purpose of displaying the data that were
obtained from all trabeculae, steady state force and the rate of ATP
consumption were first normalized to their respective values measured
at the final control contracture during the pre-PKA series;
subsequently, data for the normalized rate of ATP consumption were
collected in 10% wide normalized steady state force bins. Commercially available software was used for all statistical analyses (SYSTAT). Data are presented as mean±SEM; a value of P<.05 was considered significant.
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Fig 2
shows original recordings obtained during a typical
contraction-relaxation cycle of a skinned trabecula. The left panels
show NADH absorbance (top tracing), muscle length (middle tracing), and
force (bottom tracing) recorded at the slower sample rate (5 Hz). After
transfer of the muscle preparation to the activating solution
(indicated by the upward arrow), force rapidly increased to a steady
state with a concomitant accelerating decrease of NADH absorbance. The
decline of NADH absorbance became linear during the later phase of the
contracture when force was constant, a result that indicates that the
rate of ATP consumption was also constant at that time. A period of
steady state force and constant rate of ATP consumption was observed in
every contracture and in all trabeculae; off-line measurements of these
parameters were confined to this time period. Just before transfer of
the muscle preparation back to the relaxing solution, at a time
indicated by the asterisk in Fig 2, muscle length was rapidly reduced
to quickly slacken the muscle preparation to allow accurate
determination of the zero force level in the ATP assay bath. The method
is illustrated in greater detail in the right panels of Fig 2, which
show muscle length (top tracing) and force (bottom tracing) sampled at
a faster rate (1000 Hz) during the time of the quick release. After a
rapid decline, force remained at the baseline level while the muscle
shortened to take up the slack induced by the rapid release.
After transfer of the trabecula back to the relaxing solution (down
arrow), recording of NADH absorbance in the ATP assay bath was
continued for some time to allow a determination of the background
decrease in NADH absorbance that is due to contaminant ATPase activity
and photo bleaching. As is evident from the data shown in Fig 2, the
decline in this background signal was small. This was the case in all
experiments performed. Nevertheless, for accurate calculation of the
rate of ATP consumption during the contracture, all NADH absorbance
data were corrected for this background decline (see also "Materials
and Methods"). After assessment of the background decline, the NADH
absorbance signal was calibrated by rapid injection of 0.5 nmol ADP
into the ATP assay bath. As evident from the data shown in Fig 2, this
caused a rapid decline in NADH absorbance to a new steady state within
<3 seconds, indicating that the bath was sufficiently stirred to
ensure a state of rapid equilibrium throughout the ATP assay bath at
all times.
Fig 3 shows the relation between steady state force and
free [Ca2+] in the bathing solution. The data
obtained during the pre-PKA series are indicated by open circles; the
post-PKA data are indicated by solid circles. The top panel shows data
obtained from the representative muscle preparation (compare
with Fig 2), and the bottom panel shows the average data obtained in
all muscle preparations. The solid lines indicate the best fit of the
force-[Ca2+] coordinates to a modified Hill
equation. The average fit parameters obtained in all 10 trabeculae are
shown in Table 2. Consistent with previous
reports,7 14 18 treatment with PKA in skinned myocardium
induced a rightward shift in the force-[Ca2+]
relation. PKA treatment resulted in a 65±23% increase in the
EC50 parameter, indicating that PKA treatment induced a
significant reduction in the sensitivity of the muscle preparation to
calcium ions. However, neither maximum steady state force at saturating
[Ca2+] nor the slope of the
force-[Ca2+] relation (as indexed by the Hill
coefficient) was significantly affected by PKA treatment. Passive force
in the relaxing solution was 4.2±0.54 and 4.3±0.52
mN/mm2, respectively (P=.9).
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indicates pre-PKA series; 
, post-PKA series. Data were fit to a
modified Hill equation as indicated by the solid lines. Average fit
parameters are shown in Table 2
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Fig 4 shows the relation between the rate of ATP
consumption by the skinned muscle preparation and the level of steady
state force. The data obtained during the pre-PKA series are indicated
by open circles; the post-PKA data are indicated by solid circles. The
top panel shows data obtained from the representative muscle
preparation; the bottom panel shows the pooled normalized data obtained
in all muscle preparations collected in bins. Although there was a
significant and linear relation between the rate of ATP consumption and
steady state force, this relation was not affected by PKA treatment.
This was also observed in the pooled normalized data (Fig 4,
bottom panel), where it is seen that the data from the pre-PKA and
post-PKA series cluster close to a common regression line. Multiple
linear regression analysis using the non-normalized data from all
muscle preparations confirmed this conclusion. The regression
coefficients obtained by this analysis are presented in Table 3. Steady state force significantly affected the rate of
ATP consumption regardless of the PKA treatment status (coefficient
ß), whereas treatment with PKA resulted in only a small upward
elevation of the relation (coefficient ) and a small decrease in the
slope of the relation (coefficient ), neither of which was
significant. Finally, the standard error of the slope of the relation
between steady state force and rate of ATP consumption was 1.8% in the
pre-PKA series and 2.5% in the post-PKA series. Thus, although PKA
treatment in skinned myocardium significantly affected the
force-[Ca2+] relation, it did not affect the
economy of force maintenance.
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On average, maximum steady state force at saturating [Ca2+] was 52.8 mN/mm2 in the pre-PKA series (Table 2
). This amount of steady state force, considering the
range of sarcomere lengths used in the present study (2.2 to 2.3
µm), is comparable to that found in previous studies using skinned
isolated rat myocardium.26 28 32 The average rate of ATP
consumption accompanying this level of steady state force was 0.41
mmol/(secondsxL fiber volume) (Table 3). When a myosin head
concentration of 0.15 mmol/L is assumed,35 this would
amount to an ATP hydrolysis rate of 2.7 s-1 per
cross-bridge. This value is comparable to previous reports using
rat26 28 36 and guinea pig35 myocardium but
slightly higher than that found in pig myocardium.37 Treatment with PKA at 3 µg/mL for 40 minutes in relaxing solution resulted in a significant rightward shift of the force-[Ca2+] relation, such that the EC50 parameter of the fit to the modified Hill equation increased from 4.16 to 6.82 µmol/L. These values translate into a rightward shift of 0.22 pCa units (-log[Ca2+]), which is comparable to the shift found previously by Strang et al14 (0.18 pCa units) and Hofmann and Lange18 (0.16 pCa units) after exposure of single skinned rat cardiac myocytes for 40 minutes to 3 µg/mL PKA. In these studies, it was demonstrated that treatment with PKA resulted in substantial phosphorylation of the troponin I and C-protein subunits of the contractile proteins. Likewise, ß-adrenergic stimulation in intact myocardium has been shown to be correlated with phosphorylation of these same contractile protein subunits.3 Thus, it is reasonable to assume that treatment with PKA in skinned myocardium mimics the "downstream" effects of catecholamine ß-receptor stimulation on contractile protein function in myocardium. The great similarity between the present study in skinned cardiac trabeculae and the previous studies on skinned myocytes14 18 with regard to the effect of PKA exposure on the force-[Ca2+] relation strongly suggests that PKA exposure induced troponin I and C-protein phosphorylation in the present study as well. This conclusion is strengthened further by the observation that increasing the PKA concentration to 100 µg/mL did not result in a greater rightward shift of the force-[Ca2+] relation. Nevertheless, it should be noted that the extent of phosphorylation of the contractile proteins induced by PKA treatment in the present study may differ from that in intact myocardium after ß-adrenergic stimulation. Finally, which of the phosphorylation sites on the contractile proteins is responsible for the shift in the force-[Ca2+] relation, ie, troponin I or C-protein, will require further studies.
There was a linear relation between steady state force and rate of ATP
consumption, which is consistent with previous
observations.7 28 Despite its significant effect on the
sensitivity of force generation of the muscle preparation to calcium
ions, however, PKA exposure did not affect the relation between steady
state force and rate of ATP consumption (Fig 4 and Table 3). Thus,
treatment with PKA did not appear to alter the maximum rate of ATP
consumption nor the economy of force maintenance in the present
study. Previous mechanical measurements have suggested a direct
accelerating effect of ß-adrenergic stimulation on the cardiac
actomyosin interaction. In isolated papillary muscle, ß-agonist
stimulation resulted in a significant (23% to 49%) increase in the
frequency of minimum stiffness during barium
contracture.11 12 Likewise, Strang et al14
have demonstrated a 41% increase in the maximum velocity of shortening
in single isolated rat myocytes after exposure to PKA, although this
finding is the subject of debate.17 18 19 An increase in the
rate of cross-bridge cycling is expected to increase the rate of ATP
consumption for a given level of steady state force and thereby lead to
a reduction in the economy of force maintenance.21 38 The
results of the present study, however, provided no indication of an
altered economy of isometric force maintenance after treatment with
PKA. The standard error of the slope of the relation between steady
state force and rate of ATP consumption was 1.8% in the pre-PKA series
and 2.5% in the post-PKA series. Therefore, it is unlikely that we
would have been unable to detect an effect of PKA treatment if it were
as large as some of the mechanical measurements indicate.
How is it possible that the mechanical parameters of cardiac contraction are affected by PKA treatment but the energetic parameters are not? One solution to this conundrum may be found in the fact that the mechanical measurements inherently rely on changes in sarcomere length. That is, the measurement of dynamic stiffness only probes transitions between cross-bridge states that are either directly or indirectly dependent on crossbridge strain.39 Likewise, it is probable that the maximum velocity of shortening is determined by the rate of cross-bridge detachment under conditions of low strain.38 It is possible that phosphorylation of troponin I or C-protein alters the strain dependency of cross-bridge state transitions without affecting the intrinsic rates of cross-bridge kinetics. Since cross-bridge strain was constant during the isometric contractures that we examined, this may explain why we found no effect of contractile protein phosphorylation on the economy of force maintenance in the present study. This hypothesis would also be consistent with previous biochemical studies,15 16 which suggested an increase in ATPase activity after ß-adrenergic stimulation, since it is likely that cross-bridges in vitro resemble those cycling under conditions of low strain.
Our results of an unaltered economy of force maintenance following ß-adrenergic stimulation differ from previous reports using myothermal measurements on isolated, intact, and twitching papillary muscles.20 21 In those experiments, application of isoproterenol resulted in a reduced economy of force generation, which has been interpreted by these investigators to indicate an enhanced cross-bridge cycling rate. However, interpretation of myothermal experiments in isolated myocardium has been limited because of several factors. First, uncertainties exist in the partitioning of total energy consumption between that used for activation (calcium handling) and that used for the contractile process (ie, cross-bridge cycling).22 Second, developed force during a twitching contraction in intact myocardium is not constant. Hence, there is uncertainty with regard to the force parameter to which the rate of ATP consumption is correlated. Alpert and colleagues20 21 have used developed force integrated over time. However, it is well known that stimulation of ß-adrenergic receptors leads to a profound reduction of the duration of the twitch contraction.1 Although the mechanism underlying the reduction of twitch duration is unknown, it may be related in part to alterations in the kinetics of calcium binding to troponin C, a phenomenon that is unrelated to the cross-bridge cycling rate. A reduction in economy of developed force due to a reduction in force time integral, therefore, may not necessarily be the result of an increase in cross-bridge cycling rate. It should be noted that the present study on skinned trabeculae was performed at 20°C, whereas the myothermal measurements on intact isolated myocardium were made at a higher temperature.20 21 Hence, different steps in the cross-bridge cycle may be rate limiting at the different temperatures studied, and these different steps may exhibit a different sensitivity toward PKA-induced protein phosphorylation.
Sarcomere length of the preparations was measured in relaxing solution.
During the contracture, however, sarcomere length is expected to be
less because of internal shortening of the preparation at the expense
of damaged ends contained within the aluminum T clips. Although we did
not measure active sarcomere length in the present study, we
estimate that the magnitude of internal shortening is 0.05 to 0.1
µm (P.P. de Tombe, unpublished data, 1994, and References 26 and 2826 28 ).
Therefore, at full activation actual sarcomere length would be between
2.1 and 2.25 µm (compared with 2.2 to 2.3 µm at rest).
Nevertheless, during the steady state of the contracture, ie, during
the time to which our measurements were confined, sarcomere length is
still expected to have been constant, albeit at a slightly shorter
length than estimated from the relaxed sarcomere length. The impact of
this confounding factor was further minimized by assessing the effects
of PKA treatment in each individual muscle preparation compared with
data obtained before the exposure to PKA. In addition, neither
sarcomere length nor passive force in relaxing solution was affected by
PKA treatment, making a change in the properties of the end compliance
unlikely. It has been shown that the compliance of the damaged end
region of an isolated trabecula is highly nonlinear.40
Hence, the small differences in steady state force between the pre- and
post-PKA series would not be expected to have significantly affected
active sarcomere length. Finally, Kentish and Stienen26
have shown that the contribution of myocardium underneath the T clips
to the total rate of ATP consumption is negligible. Apparently, this
tissue has either been rendered noncontractile, or the aluminum T clip
prevents efficient exchange of the bathing solution with the underlying
tissue. Because of these considerations, we do not expect that our
conclusions are affected by end compliance.
In conclusion, treatment of skinned rat myocardium with the catalytic subunit of PKA significantly reduced the sensitivity of the preparation to [Ca2+] for steady state force. Neither the maximum rate of ATP consumption nor the economy of force maintenance, however, was affected by this treatment. These results suggest that ß-adrenergic stimulation does not affect the rate-limiting step of cross-bridge cycling during isometric contraction in myocardium.
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Acknowledgments |
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This study was supported in part by grants from the Whitaker Foundation for Biomedical Research (Dr de Tombe), the American Heart Association National Center (94-006380, Dr de Tombe), National Institutes of Health (HL-52322, Dr de Tombe), and the Netherlands Heart Foundation (Dr Stienen). We thank I.A. van Graas for excellent technical assistance.
Received December 9, 1994; accepted February 13, 1995.
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References |
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