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© 1995 American Heart Association, Inc.
Articles |
Calcium Sensitivity of Isometric Tension Is Increased in Canine Experimental Heart Failure
From the Departments of Medicine and Physiology, University of Wisconsin School of Medicine, Madison.
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Abstract |
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Abstract To examine the role of alterations in myofibrillar function in chronic heart failure, we determined isometric tension–pCa relations in permeabilized myocardium from a canine model of dilated cardiomyopathy (DCM) produced by chronic rapid pacing. In the initial series of experiments, seven dogs were paced at 250 beats per minute for 28.9±7.0 days, resulting in ventricular dilatation and reduced ejection fractions by echocardiography and elevated intracardiac filling pressures. Isometric tension–pCa relations were measured by using mechanically disrupted and permeabilized myocyte-sized preparations obtained from left ventricular biopsies before (n=11) and after (n=10) chronic rapid pacing–induced heart failure. Resting sarcomere length (SL) was set at 2.35 µm, and preparations had low end compliance (SL was 2.23±0.03 µm during maximal activation). Passive tension (2.1±1.0 versus 2.4±0.6 mN/mm2) and maximal Ca2+-activated tension (25.9±9.3 versus 27.8±6.8 mN/mm2) were similar for control and DCM preparations, respectively. However, the calcium sensitivity of isometric tension was increased in failing myocardium (pCa50 5.95±0.11 [DCM] versus 5.83±0.10 [control], P=.001). Treatment of myofibrillar preparations with the catalytic subunit of protein kinase A decreased calcium sensitivity of tension to a greater degree in failing preparations (shift of pCa50 from 6.04±0.06 to 5.75±0.09, n=7) than in nonfailing preparations (5.91±0.08 to 5.74±0.07, n=8), and isometric tension–pCa relations in the two groups were not significantly different after protein kinase A treatment. These data suggest that the increased calcium sensitivity in DCM may be due at least in part to a reduction of the adrenergically mediated phosphorylation of myofibrillar regulatory proteins. This increased calcium sensitivity of isometric tension may partially compensate for decreases in systolic calcium transients in DCM but may also contribute to the diastolic dysfunction that accompanies this condition.
Key Words: dilated cardiomyopathy • Ca2+ sensitivity • isometric tension
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The subcellular mechanisms responsible for the chronic myocardial dysfunction associated with dilated cardiomyopathy (DCM) remain incompletely characterized. The purpose of the present study was to examine the role of altered myofibrillar function in chronic heart failure by determining the relation between isometric tension and activator calcium concentration in permeabilized myocardium obtained from a canine model of DCM produced by chronic rapid pacing. This large animal model shares many of the structural, hemodynamic, and neurohumoral changes seen in human congestive heart failure.1 2 3 4 5 6 In addition, the observation of ventricular dilatation and dysfunction accompanying persistent tachyarrhythmias in humans7 suggests that this model may share pathophysiological mechanisms similar to human DCM.
Using myocyte-sized myofibrillar preparations obtained from transmural ventricular biopsies, we measured isometric tension over a range of calcium concentrations before and after chronic rapid pacing. Because both the density and function of the sarcolemmal ß-adrenergic receptor complex are reduced in this model8 9 10 11 as in human heart failure,12 13 we also examined the role of ß-adrenergic–mediated protein phosphorylation in modulating myofibrillar function. Our results demonstrate an increase in the calcium sensitivity of tension in myopathic preparations without changes in either passive or maximally calcium-activated tension, a finding that may have important implications regarding the mechanism of both systolic and diastolic ventricular dysfunction. In addition, evidence is provided suggesting a role of chronically decreased ß-adrenergic–dependent phosphorylation of myofilament regulatory proteins in mediating this change.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Surgical Preparation
Mongrel dogs were sedated with morphine sulfate (15 mg s.q.) and cepromazine maleate (10 mg s.q.), induced with inhaled isoflurane (3% to 5% by mask), and anesthetized with inhaled isoflurane (1.5%) after being endotracheally intubated and mechanically ventilated. By use of a standard sterile technique, the heart was exposed via a left thoracotomy. Intracardiac pressures were measured by using a micromanometer-tipped catheter (model PC-380, Millar Instruments) inserted into the right ventricle, left atrium, and left ventricle via small stab incisions. Two or three full-thickness core biopsies (
0.5
to 0.75 g tissue) were taken from the left ventricular free wall by
using a custom-made biopsy needle with a 1.8-mm internal diameter.
Bipolar screw-in epicardial pacing leads were fixed to the epicardial
ventricular surface, and the distal leads were tunneled to a
subcutaneous pocket constructed on the animal's back, where they were
connected to a pacemaker (model SX 5984 or 5940, Medtronics, Inc),
modified to pace at 250 beats per minute. The thoracotomy was closed in
layers. Cefazolin (1 g IV) was administered before surgery, and a
suspension of penicillin G procaine and dihydrostreptomycin (600 000
U, 0.75 g IM) was given immediately after surgery. Flunixin meglumine
(25 mg IM or PO once a day, for 3 days) was used as a postoperative
analgesic. After allowing 3 to 5 days for recovery, the pacemaker was programmed to 250 beats per minute. The animals were examined daily for evidence of pulmonary and right heart congestion (ascites). Terminal studies were performed after the dogs developed signs of heart failure or after 4 weeks of pacing. Animals with signs of heart failure received furosemide (20 mg IV) and/or were reprogrammed to a paced heart rate of 210 to 230 beats per minute 1 to 2 days before the terminal studies. Identical sedation, induction, and anesthesia were used for the terminal studies, and cardiac pressures were measured in a similar fashion with the pacemaker off. Repeat biopsies of the left ventricular free wall were obtained before the animals were euthanatized by barbiturate overdose.
Two-dimensional short-axis echocardiograms were obtained at the level of the head of the papillary muscle from anesthetized animals just before the initial surgeries and the terminal studies. Endocardial short-axis contours from three beats were averaged to determine end-diastolic (maximal) and end-systolic (minimal) areas and area ejection fraction.
Myocyte-Sized Myofibrillar Preparations
Ventricular biopsies were washed in ice-cold relaxing solution
and were mechanically disrupted in 10 mL of relaxing solution for 15
seconds by using a tissue homogenizer (Polytron, Brinkmann Instruments)
set at low speed (12 500 rpm). The resulting suspensions of small
clumps of myocytes, single-myocyte–sized preparations, and cell
fragments were centrifuged at 165g for 90 seconds, and the
pellet was resuspended in 5 mL of relaxing solution containing 0.3%
Triton X-100 (Pierce Chemicals) for 6 minutes at 22°C to remove
adherent sarcolemma and sarcoplasmic reticulum (SR). Myofibrillar
preparations were then centrifuged (165g for 90 seconds),
the pellet was resuspended in 5 mL of fresh relaxing solution twice,
and the preparations were stored on ice for use within 8 hours.
Solutions
Relaxing and activating solutions contained (mmol/L) ATP 4, free
Mg2+ 1, imidazole 7, EGTA 7, and creatine phosphate
7. Ionic strength was adjusted to 180 mmol/L with KCl; pH was adjusted
to 7.0 at 22°C. A computer program was used to determine the
concentration of metals and metal ligands in the relaxing and
activating solutions.14 Calcium concentrations were varied
from 10-9 mol/L (relaxing solution) and
10-4.5 mol/L (maximally activating solution), and single
stocks of relaxing and maximally activating solutions were used for
each series of experiments. Submaximally activating solutions were
prepared by mixing appropriate volumes of previously frozen relaxing
and maximally activating solution stocks. Calcium concentrations are
expressed as pCa (-log[Ca2+]). Chemicals were
obtained from Sigma Chemical Co.
Experimental Apparatus
The experimental apparatus has been previously
described15 16 and consisted of a stainless steel plate
with two glass-bottomed solution troughs (100 µL) mounted on the
mechanical stage of an inverted microscope (Zeiss). Single-cell–sized
myofibrillar fragments were selected on the basis of size (length, 100
to 150 µm; diameter, 20 to 30 µm) and uniformity of striation
pattern and were attached via borosilicate glass pipettes (tip
diameter, 5 to 10 µm) to a force transducer (model 403, Cambridge
Technology; sensitivity, 20 mV/mg; resolution, <50 µg; resonant
frequency, 600 Hz) and a piezoelectric translator (Physik Instrumente
GmbH & Co) by using a small drop of silicone adhesive (Dow-Corning).
Both the force transducer and the piezoelectric translator were mounted
to a micromanipulator for fine positioning during myocyte attachment.
After curing for 45 minutes, the myofibrillar preparations were tightly
adherent to the pipettes, forming a low-compliance attachment.
Force and myocyte length (piezoelectric translator position) were
recorded during experiments on a storage oscilloscope (model NIC-310,
Nicolet Instruments) and saved on magnetic disk for later analysis.
Preparations were monitored by photomicroscopy (magnification, x1690)
via a CCD camera (Panasonic, model WV-B1600) and recorded on videotape
(JVC, model HR-s6600u super-VHS videotape recorder). Average sarcomere
length was determined by measuring the span of at least 7 and more
typically 10 to 15 adjacent sarcomeres on the calibrated video screen
and was set to 2.35 µm in relaxing solution. Sarcomere length and
striation uniformity was verified after maximal calcium activation, and
myofibrillar preparations with significant internal shortening (>0.15
µm per sarcomere) during maximal activation were rejected.
Preparation depth was measured after the completion of the experiment
by rotating the cell 90° in the solution trough, and cross-sectional
area was calculated with an elliptical geometry assumed.
Tension-pCa Measurements
Tension-pCa relations were determined at room temperature
(22°C to 23°C). Active tension was calculated as the difference
between the total tension developed at any given calcium concentration
and passive tension measured in relaxing solution. Tension was first
determined at maximal Ca2+ activation; thereafter,
maximal Ca2+-activated tension was redetermined
after every four or five submaximal activations. Active tensions were
normalized by assuming a linear decrease in maximal tension with each
activation, but the cell was rejected if maximal tension declined by
>30% over the course of the experiment.
Calcium sensitivity of tension was determined by least-squares
regression using the Hill transformation to linearize the sigmoidal
tension-pCa relations17 :
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Protein Kinase A Treatment
A subset of myofibrillar preparations were incubated at 22°C
to 23°C for 40 minutes with relaxing solution containing the
catalytic subunit of porcine cardiac protein kinase A (PKA, 3.0
µg/mL, Sigma Chemical Co) and 6 mmol/L dithiothreitol to maximally
phosphorylate the PKA-dependent sites of myofibrillar proteins. This
protocol has been shown (using [
-32P]ATP
autoradiography) to phosphorylate rat cardiac troponin I and C protein
in vitro and reduces myofibrillar calcium sensitivity of isometric
tension in permeabilized rat myocytes to an extent similar to that
found with the exposure of intact rat myocytes to a high concentration
of isoproterenol.16 Tension-pCa relations were determined
in these myofibrillar preparations both before and after treatment with
PKA.
Statistical Methods
Results are given as mean±SD. Echocardiographic and hemodynamic
data were compared by using paired t tests. Tension-pCa
relations were linearized by using the Hill transformation and compared
by using multiple linear regression analysis as an alternative to
ANCOVA, with a dummy variable coding for the presence or absence of
heart failure.18 Differences were accepted as significant
at P<.05. Statistical analyses were performed by using
commercially available software (SYSTAT, Inc).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For the initial series of experiments, 10 dogs were instrumented and underwent rapid ventricular pacing. Two dogs died during the induction of anesthesia during the terminal study, and a third dog was excluded because of pacemaker lead failure. The remaining 7 dogs were rapidly paced for 28.9±7.0 days. Five dogs had signs of congestive heart failure at the time of the terminal study (lethargy, anorexia, ascites, and/or rales). Echocardiographic data are provided in Table 1
. Chronic rapid pacing produced a dilated
cardiomyopathy characterized by a 99% increase in
end-diastolic short-axis cross-sectional area, a 199%
increase in end-systolic area, and a 53% reduction in area ejection
fraction. Chronic rapid pacing also resulted in elevated cardiac
filling pressures, consistent with the syndrome of congestive heart
failure (Table 1).
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Isometric Tension-pCa Relations
Tension-pCa relations were determined with myofibrillar
preparations obtained from transmural left ventricular biopsies taken
before (control, n=11 preparations) and after development of DCM
secondary to chronic rapid pacing (DCM, n=10 preparations). As shown in
Table 2, the dimensions of the single-myocyte–sized
preparations were similar in the two groups. There was no difference in
passive tension before or after pacing. Likewise, there was no change
in the maximal tension-generating capability of the myofibrils after
the development of heart failure. Sarcomere length measurements
demonstrated little internal shortening during maximal activation
(mean, 0.12 µm per sarcomere, or 5%), demonstrating that the
attachment of the preparations to the experimental apparatus had low
compliance. Photomicrographs of a representative myocyte-sized
myofibrillar preparation in both relaxing (pCa 9.0) and maximally
activating (pCa 4.5) solutions show preserved sarcomere pattern
resolution and uniformity with minimal internal shortening after
maximal activation (Fig 1).
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Mean tension-pCa relations for the seven dogs are provided in Fig 2 and demonstrate a significant increase in the
Ca2+ sensitivity of isometric tension after the
development of dilated cardiomyopathy due to chronic rapid pacing. The
tension-pCa relations before and after chronic rapid pacing are shown
in the top panel, with tensions normalized to the maximal
Ca2+-activated tension for each preparation. The
same data, linearized by using the Hill transformation, are plotted in
the bottom panel. An increase in pCa50 (the x
intercept of the Hill transformation and the calcium concentration
resulting in half-maximal tension) was observed after the development
of DCM (5.83±0.10 [control] versus 5.95±0.11 [DCM],
P<.001). There was no significant change in the Hill
coefficient after the chronic rapid pacing (Table 3).
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Role of PKA-Mediated Myofilament Phosphorylation
ß-Adrenergic simulation of cardiac muscle decreases myofibrillar
calcium sensitivity of isometric tension, an effect that is likely
mediated via PKA-dependent phosphorylation of troponin I and/or C
protein.16 19 Because both ß-adrenergic receptor density
and adenylate cyclase activity are downregulated in this model of heart
failure,8 9 10 11 decreased PKA-dependent phosphorylation of
myofilament regulatory proteins is one possible mechanism underlying
the observed increase in calcium sensitivity of isometric tension. This
hypothesis was examined in two ways. First, the possibility of a
differential effect of acutely elevated sympathetic tone during the
open-chest surgery on myofibrillar calcium sensitivity of tension in
normal and failing myocardium was examined in a smaller second series
of experiments. If the increase in calcium sensitivity of tension seen
with heart failure was mediated by a reduction in PKA-dependent
myofilament protein phosphorylation, nonphysiological elevations in
circulating catecholamines would have the effect of amplifying this
difference (because ß-adrenergic receptor function is decreased in
heart failure). Dogs in this second series of experiments were
pretreated with both oral and intravenous ß-adrenergic antagonists
before the initial surgical procedure, but not before the terminal
study. By acutely blocking ß-adrenergic receptors before the control
studies, any differential effect of elevated sympathetic tone or
increased circulating catecholamines associated with surgery on
myofibrillar PKA-dependent phosphorylation in the two groups should be
minimized. A second approach more directly examined the degree of
PKA-dependent phosphorylation of myofibrillar proteins by determining
the effects of in vitro treatment of the myofibrillar preparations with
the catalytic subunit of PKA on calcium sensitivity of isometric
tension.
For the second series of experiments, six dogs were instrumented as described in "Materials and Methods." However, these dogs were pretreated with propranolol 2 mg/kg PO 1 hour before the initial surgery and were subsequently treated with propranolol (1 mg/kg IV) immediately after the induction of anesthesia. This dose of the oral and intravenous ß-adrenergic blockers completely attenuates the chronotropic response to a 5 µg IV bolus of isoproterenol in anesthetized dogs (data not shown). In this series, three dogs died with the induction of anesthesia before the terminal studies. The remaining three dogs were paced at 250 beats per minute for 31.7±1.2 days. Terminal studies were also performed as outlined in "Materials and Methods," and animals did not receive ß-adrenergic antagonists before the terminal studies.
As with the initial series of experiments, echocardiographic data were
consistent with the development of DCM after chronic rapid pacing.
End-diastolic cross-sectional areas increased from 8.8±1.8
to 22.2±1.6 cm2 (P=.013), end-systolic areas
increased from 4.2±0.6 to 17.3±0.6 cm2
(P=.003), and mean area ejection fraction decreased by 57%
(from 0.51±0.9 to 0.22±0.4, P=.016). There was a trend
toward increased intracardiac filling pressures after chronic pacing,
which did not reach statistical significance. Characteristics of the
myofibrillar preparations used in this group of experiments are
presented in Table 4. Again, there were no
differences in either passive or maximal
Ca2+-activated tension before and after development
of heart failure. Of note, maximal Ca2+-activated
tension was significantly greater in both groups compared with maximal
tensions in the first series of experiments. This was likely due at
least in part to less compliant attachments to the experimental
apparatus, which we infer from the observation that sarcomere lengths
at maximal tension in both groups were significantly greater than those
in the first series of experiments.
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As shown in Fig 3, acute ß-adrenergic blockade before
control studies did not abolish the increase in calcium sensitivity of
isometric tension seen after chronic rapid pacing. As in the first
series of experiments, calcium sensitivity of tension was greater
(pCa50 6.03±0.03 [DCM] versus 5.94±0.06 [control],
P<.005) after the development of heart failure. Again,
there was no change in the Hill coefficient (Table 5).
The pCa50 values both before and after pacing were
significantly greater when compared with the original series of
experiments. This may also be due at least in part to the less
compliant attachments achieved in the latter series of experiments,
since calcium sensitivity of tension as well as maximal tension
increases with increasing sarcomere length.20 21
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Although acute ß-adrenergic blockade of the dogs before the initial
surgery did not attenuate the difference in calcium sensitivity of
tension seen with heart failure, chronic reductions of
ß-adrenergic–dependent (PKA-dependent) phosphorylation of
myofibrillar regulatory proteins could potentially explain this
observation. To test this hypothesis, a subset of myofibrillar
preparations from both series of experiments (obtained from the last
three dogs from the first experimental series and all three dogs from
the second series) were treated with the catalytic subunit of PKA.
Tension-pCa relations were determined before and immediately after PKA
treatment, and the decrease in calcium sensitivity of isometric tension
was interpreted as a measure of baseline PKA-dependent phosphorylation
(ie, a greater decrease in calcium sensitivity of tension implies
reduced phosphorylation before PKA exposure, whereas a smaller decrease
suggests greater baseline phosphorylation). Calcium sensitivity of
isometric tension was significantly reduced (Fig 4,
Table 6) after PKA treatment both in control
preparations (5.91±0.08 versus 5.74±0.07 [after PKA],
P<.005, n=8) and DCM preparations (6.04±0.06 versus
5.75±0.09 [after PKA], P<.001, n=7). After incubation
with PKA, tension-pCa relations did not differ significantly in the two
groups (P=.932 by multivariate regression analysis; this
analysis of the data would detect a difference in pCa50
between the two groups of 0.04 pCa units, with an 
of .05 and a
power of 0.80). Further, the shift in pCa50 after PKA
exposure was significantly greater in the DCM preparations (0.30±0.08
versus 0.16±0.10 pCa units for the control preparations,
P=.01). These data suggest that reduced levels of
PKA-dependent myofibrillar phosphorylation can explain in large part
the observed increase in isometric calcium sensitivity of tension seen
in the DCM preparations and that the difference in myofilament protein
phosphorylation is unlikely to be solely due to acute elevations in
sympathetic tone associated with open-chest surgery.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Calcium Sensitivity of Isometric Tension
The present study demonstrates an increase in myofibrillar calcium sensitivity of isometric tension in a canine model of DCM produced by chronic rapid pacing, likely due at least in part to chronic reductions in ß-adrenergic–mediated (PKA-dependent) phosphorylation of myofilament regulatory proteins. Previous studies of myofibrillar calcium sensitivity of isometric tension in this model and in other forms of heart failure have yielded conflicting results. Increased calcium sensitivity of isometric tension has been reported in several small animal models of heart failure.22 23 However, one preliminary report found no significant difference in myofibrillar tension-pCa relations obtained from skinned ventricular strips from dogs with chronic rapid pacing–induced heart failure compared with control myocardium.24 In addition, Perreault et al25 observed significant reductions in peak twitch tension in intact ventricular trabeculae obtained from dogs with chronic rapid pacing–induced heart failure compared with control animals, despite similar peak intracellular calcium transients measured with the Ca2+ indicator aequorin. This finding was interpreted by the authors as indicating a reduced myofibrillar calcium sensitivity of tension.
Several possible methodological explanations exist for these differences. First, the phosphorylation state of myofilament regulatory proteins may be altered during the isolation and preparation of myocardial tissue. Our method of rapidly disrupting and subsequently washing myocardial specimens in relaxing solution likely removes endogenous phosphatases that may otherwise be activated during isolation of trabeculae or ventricular strips. Shortening of central sarcomeres during calcium activation, at the expense of end compliance associated with attachment of myocardium to the experimental apparatus, is common in studies using multicellular cardiac preparations21 26 and may represent another potential explanation for the discrepant results in that sarcomere length was not monitored during activation in these previous studies. The relation between peak twitch tension and peak intracellular calcium observed by Perreault et al25 in intact trabeculae is not necessarily correlated with steady state measurements of myofilament calcium sensitivity in permeabilized myocardium, as the peak of the systolic intracellular calcium transient occurs much earlier in the twitch than the peak of tension.
In human DCM, conflicting results have also been reported. A recent report by Schwinger et al27 demonstrated increased calcium sensitivity of isometric tension in permeabilized papillary muscle strips obtained from cardiomyopathic human hearts. However, several investigators have found no difference in tension-pCa relations from permeabilized ventricular myocardium obtained from failing human hearts compared with nonfailing human myocardium.28 29 30 We have been able to determine tension-pCa relations in several myofibrillar preparations obtained from failing human myocardium at the time of heart transplantation by using methods similar to those used in the present study (data not shown). It is possible that these techniques, combined with careful monitoring of sarcomere length and homogeneity during activations, can resolve these apparently conflicting experimental observations and clarify the relevance of the present study to human DCM.
PKA-Dependent Myofibrillar Protein Phosphorylation
Given the desensitizing effects of the ß-adrenergic system on
myofibrillar tension development and the downregulation of the
sarcolemmal components of this hormone–second messenger system in
heart failure, it seems plausible that the differences in calcium
sensitivity of isometric tension demonstrated in the present study
are due to differences in myofilament PKA-dependent phosphorylation.
Although one might suspect that cAMP concentration is reduced in
chronic heart failure because of the downregulation of the sarcolemmal
ß-adrenergic receptor/G-protein/adenylate cyclase complex, several
studies have reported similar concentrations of cAMP31 32
and PKA activity31 in failing and nonfailing human
myocardium. However, evidence also suggests the existence of several
physiologically distinct intracellular pools of cAMP in heart
muscle,19 33 34 and it has been proposed that a relatively
small pool of cAMP critical to inotropic regulation is altered in heart
failure without an overall change in total myocardial cAMP
content.31 Myofibrillar phosphorylation status may also
depend on the activity of PKA site-specific phosphatases, although
little is known concerning phosphatase activity in heart failure.
However, phosphatase type 1 (a phosphatase active on PKA-dependent
serine residues) is itself regulated by the ß-adrenergic system via
phosphorylation of phosphatase 1 inhibitor.35 More work is
needed to clarify the physiological role in heart failure of changes in
the concentration, activity, and localization of these intracellular
"second messengers" of the ß-adrenergic system.
Calcium sensitivity of tension could potentially be altered in this model by changes in the phosphorylation state or isoform content of several other myofilament regulatory proteins. For instance, increased expression of a fetal isoform of troponin T has been found in failing human myocardium,36 although this isoform shift might be expected to decrease isometric calcium sensitivity of tension.37 Both troponin T and troponin I can be phosphorylated by PKC (the latter at sites distinct from those phosphorylated by PKA).38 39 Margossian et al40 reported a decrease in myosin light chain 2 content in ventricular tissue from myopathic human hearts. In addition, myosin light chain 2 is the substrate for a specific Ca2+-calmodulin–dependent protein kinase.41 Although the effects of alterations in the content, isoform distribution, and phosphorylation status of these other myofibrillar regulatory proteins remain incompletely characterized in myocardium, they represent potential alternative molecular explanations for the increased calcium sensitivity of tension with heart failure. Our finding that tension-pCa relations in DCM and control preparations were identical after in vitro treatment with the catalytic subunit of PKA suggests that the difference in calcium sensitivity of tension observed in the present study might be in large part due to differences in ß-adrenergic–mediated phosphorylation. However, it is also possible that maximum phosphorylation of PKA-dependent sites on myofilament regulatory proteins minimizes the effects of other biochemical alterations on the calcium sensitivity of tension.
Physiological Implications
Because maximal Ca2+-activated tension was
unchanged and myofibrillar calcium sensitivity of isometric tension was
increased after chronic rapid pacing, our results suggest that
alterations in excitation-contraction coupling might be responsible for
systolic dysfunction in this model of heart failure. This conclusion is
consistent with recent work suggesting alterations in the structure and
function of the SR in failing canine myocardium after chronic rapid
pacing. Using a microsomal fraction enriched in terminal cisternae,
Cory et al42 found that the activities of both the SR
Ca2+ pump and the Ca2+ release
channel are reduced in this model. Vatner et al43
demonstrated a decrease in myocardial SR Ca2+
release channel density 1 day after the initiation of rapid pacing, a
finding that correlated with reductions in postextrasystolic
potentiation in conscious dogs. Also, Williams et al44
recently reported a relation between reductions in SR
Ca2+-ATPase mRNA expression and elevations in left
ventricular end-diastolic pressure in dogs after chronic
rapid pacing.
Although increased calcium sensitivity of tension could in part compensate for abnormalities of the systolic calcium transient in failing myocardium, it may have deleterious effects on diastolic function. To the extent that calcium dissociation from the myofilament limits the rate of myocardial relaxation,45 increased myofibrillar calcium sensitivity could contribute to the prolongation of twitch duration and impaired rates of relaxation described in this model of heart failure.25 46 Interestingly, Parker et al47 found that although the time constant of isovolumic relaxation (an index of myocardial relaxation in the intact ventricle) is prolonged in cardiomyopathic human hearts relative to normal patients, the responsiveness of this index of relaxation to ß-adrenergic stimulation by intracoronary infusion of dobutamine appears to be preserved. Since the responsiveness of the maximal rate of systolic pressure rise (dP/dtmax, an index of systolic function) to intracoronary dobutamine in failing hearts was dramatically attenuated in the same study, the authors hypothesized that "the lusitropic pathway distal to cAMP is more sensitive to cAMP than is the inotropic pathway." Similar observations concerning preserved lusitropic responsiveness to ß-adrenergic stimulation have been reported in chronic rapid pacing–induced heart failure.48 The findings of the present study offer a molecular explanation for this phenomena, as reduced levels of PKA-dependent myofibrillar phosphorylation result in greater decreases in calcium sensitivity of tension (and potentially greater increases in relaxation rate) after subsequent exposure to saturating concentrations of activated PKA.
Potential Limitations
The use of myocyte-sized myofibrillar preparations obtained from
relatively small ventricular biopsies provided several advantages,
including the ability to determine tension-pCa relations serially in
the same dogs before and after the development of DCM and the ability
to accurately monitor sarcomere length even during maximal activations.
However, this preparation has several potential limitations. First,
only one to two successful preparations could be obtained per
experiment because of the finite useful life of the isolated
permeabilized preparations, the time needed to select and mount each
preparation, and the relatively stringent criteria for a successful
preparation in terms of internal sarcomere shortening and decline of
maximal force. A second limitation relates to maximal
Ca2+-activated tensions, which were somewhat less
and more variable than those reported from some studies using
permeabilized cardiac trabeculae.49 Although the maximal
tensions and corresponding standard deviations obtained in the
present study are comparable to those previously reported in our
laboratory for mechanically disrupted myocyte-sized myofibrillar
preparations obtained from rat myocardium15 and for
enzymatically digested and detergent-permeabilized rat cardiac
myocytes16 and values obtained in single myocytes by other
investigators,50 it is possible that a real difference in
maximal Ca2+-activated tension in this model was not
appreciated.
In addition, myofibrillar calcium sensitivity of isometric tension may be altered by permeabilization of the sarcolemma. A recent study by Gao et al49 determined tension-pCa relations in intact ventricular trabeculae during tonic activation by tetani after exposure to ryanodine by use of the fluorescent calcium indicator fura 2. They found that the calcium sensitivity of tension was greater in the intact trabeculae than in the same trabeculae after subsequent permeabilization with Triton X-100. It is possible that mechanical disruption and permeabilization has similar effects on calcium sensitivity of tension. However, control and myopathic tissues were prepared in an identical manner in the present study, and a differential effect of the preparation process on myopathic tissue seems unlikely.
Tension-pCa relations were determined at a relatively long sarcomere length, whereas myofibrils in situ shorten significantly under physiological loading conditions. Alterations in the length dependence of tension has recently been reported in myofibrillar preparations from failing human hearts,27 and a blunted Frank-Starling mechanism has been observed in vivo in dogs with chronic rapid pacing–induced cardiomyopathy.51 In addition, differences in loading conditions may account for some of the abnormalities of myocardial relaxation observed in this model of heart failure.46 By measuring tension-pCa relations at a long sarcomere length, we hoped to maximize differences between failing and nonfailing myocardium. Future work is needed to characterize the length dependence of calcium sensitivity of isometric tension and the relation between myofibrillar calcium sensitivity of tension and relaxation in this model of heart failure.
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Acknowledgments |
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This study was supported by National Institutes of Health grant K08-HL02969 (Dr Wolff) and the Rennebohm Foundation. We gratefully acknowledge the excellent technical assistance of Scott Stoker and Catherine Kidd.
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Footnotes |
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Reprint requests to Matthew R. Wolff, MD, H6/315 Clinical Science Center, University of Wisconsin, 600 Highland Ave, Madison, WI 53972. E-mail mrw@parc.medicine.wisc.edu.
Received October 28, 1994; accepted February 13, 1995.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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