Download Brief rapid pacing depresses contractile function via Ca

Am J Physiol Heart Circ Physiol
280: H90–H98, 2001.
Brief rapid pacing depresses contractile function via
Ca2⫹/PKC-dependent signaling in cat ventricular myocytes
YONG GAO WANG, WILLIAM J. BENEDICT, JÖRG HÜSER, ALLEN M. SAMAREL,
LOTHAR A. BLATTER, AND STEPHEN L. LIPSIUS
Department of Physiology, Stritch School of Medicine, Loyola University Chicago
and Cardiovascular Institute, Maywood, Illinois 60153
Received 23 May 2000; accepted in final form 15 August 2000
intracellular calcium; excitation-contraction coupling; stunning; tachyarrhythmias; protein kinase C
which is considered to be another form of myocardial
stunning. The mechanisms by which short periods of
tachyarrhythmia elicit myocardial stunning are much
less understood than those responsible for chronic
tachycardia-induced cardiomyopathy or ischemia-induced myocardial stunning. To date, experimental
studies of RP-induced dysfunction have focused primarily on chronically paced in vivo heart preparations
in an attempt to understand the complex changes that
lead to congestive heart failure. Research into cellular
mechanisms have used myocytes isolated from in vivo
paced heart preparations (27, 34, 37). This approach,
however, makes it difficult to distinguish between the
direct effects of RP per se from the secondary in vivo
changes that can affect contractile function. For example, in vivo models of chronic RP have documented
changes in sympathetic nerve activity, alterations in
the renin-angiotensin system and secretions of atrial
natriuretic factor, reduced coronary perfusion, and
ventricular remodeling (34). The present study indicates that a brief period of RP of ventricular myocytes
depresses contractile function at the myofilament level
via stimulation of a Ca2⫹/protein kinase C (PKC)-dependent signaling mechanism. These findings may be
relevant to the inhibitory effects of paroxysmal ventricular tachyarrhythmia on cardiac contractile function
and the development of pacing-induced cardiomyopathy. Portions of this work have been presented in
abstract form (2).
METHODS
(26, 31) and experimentally (1, 8), chronic
tachycardia can depress cardiac function and lead to
congestive heart failure. Shorter periods (24 h) of rapid
pacing (RP) can depress contractile function without
overt signs of heart failure (40). These early changes in
excitation-contraction (E-C) coupling may be important in the development of myocardial stunning. Classically, myocardial stunning is a reversible depression
in contractile function that follows short periods of
ischemia (6, 25). However, termination of atrial (20) or
ventricular (23, 26) tachyarrhythmias also is followed
by a reversible depression in contractile function,
CLINICALLY
Address for reprint requests and other correspondence: S. L. Lipsius, Loyola Univ. Medical Center, Dept. of Physiology, 2160 S. First
Ave., Maywood, IL 60153 (E-mail: [email protected]).
H90
Single cell isolation procedure. The methods used to isolate
cardiac myocytes have been reported in detail (29). Briefly,
adult cats are anesthetized with pentobarbital sodium (80
mg/kg ip). Isolated hearts were mounted on a Langendorff
perfusion apparatus for enzymatic (0.07% type II collagenase; Worthington) cell isolation. After enzyme treatment,
tissue obtained from the endocardial-midwall region of the
left ventricular free wall was cut into small pieces and incubated in fresh enzyme solution. Isolated ventricular myocytes were stored in a solution of HEPES-Tyrode plus 0.1%
albumin until use on the same day.
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society
http://www.ajpheart.org
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Wang, Yong Gao, William J. Benedict, Jörg Hüser,
Allen M. Samarel, Lothar A. Blatter, and Stephen L.
Lipsius. Brief rapid pacing depresses contractile function
via Ca2⫹/PKC-dependent signaling in cat ventricular myocytes. Am J Physiol Heart Circ Physiol 280: H90–H98,
2001.—The purpose of this study is to determine the effects
of brief rapid pacing (RP; ⬃200–240 beats/min for ⬃5 min) on
contractile function in ventricular myocytes. RP was followed
by a sustained inhibition of peak systolic cell shortening
(⫺44 ⫾ 4%) that was not due to changes in diastolic cell
length, membrane voltage, or L-type Ca2⫹ current (ICa,L).
During RP, baseline and peak intracellular Ca2⫹ concentration ([Ca2⫹]i) increased markedly. After RP, Ca2⫹ transients
were similar to control. The effects of RP on cell shortening
were not prevented by 1 ␮M calpain inhibitor I, 25 ␮M
5
G
L-N -(1-iminoethyl)-orthinthine, or 100 ␮M N -monomethylL-arginine. However, RP-induced inhibition of cell shortening
was prevented by lowering extracellular [Ca2⫹] (0.5 mM)
during RP or exposure to chelerythrine (2–4 ␮M), a protein
kinase C (PKC) inhibitor, or LY379196 (30 nM), a selective
inhibitor of PKC-␤. Exposure to phorbol ester (200 nM phorbol 12-myristate 13-acetate) inhibited cell shortening (⫺46 ⫾
7%). Western blots indicated that cat myocytes express
PKC-␣, -␦, and -⑀ as well as PKC-␤. These findings suggest
that brief RP of ventricular myocytes depresses contractility
at the myofilament level via Ca2⫹/PKC-dependent signaling.
These findings may provide insight into the mechanisms of
contractile dysfunction that follow paroxysmal tachyarrhythmias.
PACING-INDUCED CONTRACTILE DYSFUNCTION
state times (60–90 s), and then held at 300–250 ms (200–240
beats/min) for 5 min. Progressive shortening of the pacing
cycle length was used to allow APD to accommodate to the
changing cycle length. If electromechanical alternans appeared, the pacing cycle length was increased a few milliseconds to a cycle length at which alternans did not appear.
After pacing at the shortest cycle length, we progressively
lengthened the pacing cycle length in reverse order back to
control (1,000 ms) (see Fig. 1). At least 3 min of pacing at
cycle lengths ⱕ 300 ms was required to elicit consistent
RP-induced inhibition of cell shortening. Measurements of
peak cell shortening were determined as an average of the
last five beats at each pacing cycle length using a custom
software program (LabView). The RP protocol was always
accomplished by stimulated action potentials. Because RP
induces sustained changes in cell shortening, in some experiments a single cell could not serve as its own control. In this
case, RP-induced inhibition of cell shortening was studied in
two groups of cells from the same hearts: control and test
group cells.
Measurement of intercellular Ca2⫹ concentration. Single
ventricular myocytes were loaded with fluorescent Ca2⫹ indicator by exposure to 5 ␮M acetoxymethyl esters (AM) of
indo 1 (indo 1-AM; Molecular Probes, Eugene, OR) for 20 min
at 20°C. For fluorescence measurements, a coverslip of cells
was mounted on the stage of an inverted microscope. Indo-1
fluorescence was excited at 360 nm, and the Ca2⫹-dependent
changes of fluorescence emitted from single cells was recorded simultaneously at 405 nm (F405) and 485 nm (F485),
respectively, with photomultiplier tubes. Changes of the intracellular Ca2⫹ concentration ([Ca2⫹]i) are expressed as
changes in the ratio (R ⫽ F405/F485).
Analysis of PKC isoenzyme expression by Western blotting.
Acutely isolated cat ventricular myocytes were centrifuged
(1,000 g for 10 min) and resuspended in lysis buffer [20 mM
Tris 䡠 HCl (pH 7.5) containing 0.5 mM EGTA, 0.5 mM EDTA,
10 mM mercaptoethanol, 0.5% Triton X-100, 1 mM sodium
vanadate, 10 ␮g/ml leupeptin, 10 ␮g/ml aprotonin, and 1 mM
Pefabloc]. After sonication and repeat centrifugation, we assessed the protein content of the supernatant fraction using
a bicinchoninic acid assay (Pierce, Rockford, IL), and 100–
300 ␮g of extracted protein were separated by SDS-PAGE
and Western blotting. Separated proteins were probed with
specific monoclonal antibodies to PKC-␣, -␤, -␦, and -⑀ (Transduction Laboratories, Lexington, KY). Protein bands were
visualized using an enhanced chemiluminescence method
(Amersham, Arlington Heights, IL). Similarly prepared tissue extracts of the rat brain (50 ␮g) and cat brain (50 ␮g)
served as positive controls.
Fig. 1. Short-term rapid pacing (RP)
inhibits contraction of ventricular
myocytes. A: starting at a pacing cycle
length (CL) of 1,000 ms, CL was progressively decreased (descending), held
at 250 ms for 5 min, and then progressively lengthened (ascending) back to
control (1,000 ms). After RP, cell shortening was inhibited at each pacing CL.
B: original recordings of action potentials (AP) and cell shortening recorded
from a myocyte stimulated at 1,000 ms
before (control) and after RP. After RP,
peak cell shortening was decreased
without changes in resting cell length.
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Recording techniques. Ventricular myocytes selected for
study were elongated and relaxed and exhibited regular
striations and normal action potential configurations when
stimulated. Action potentials and ionic currents were recorded in the whole cell configuration (15) using a perforated
(nystatin; 150 ␮g/ml)-patch method (17). Cells were superfused at 35 ⫾ 1°C with external solutions containing (in mM)
137 NaCl, 5.4 KCl, 2.0 CaCl2, 1.0 MgCl2, 5 HEPES, and 11
glucose, which were titrated with NaOH to pH 7.35 and
bubbled with 100% O2. The internal pipette solution contained (in mM) 120 potassium glutamate, 20 KCl, 1.0 MgCl2,
3 Na2ATP, and 5 HEPES; pH 7.2. Isolation of the L-type
Ca2⫹ current (ICa,L) was accomplished by replacing intrapipette K⫹ with cesium (Cs⫹) and adding 5 mM CsCl to external solutions to block K⫹ currents. Access resistance stabilized at 10–15 M⍀ within 5–10 min of forming a gigaseal.
Action potentials (bridge) and ionic currents (discontinuous single-electrode voltage clamp) were recorded with an
Axoclamp-2A amplifier (Axon Instruments) at a sampling
rate of 8–10 kHz. A second oscilloscope was used to monitor
the duty cycle to ensure complete settling of the voltage
transient between samples. Unloaded cell shortening was
monitored using a video-based edge detector (Crescent Electronics), which uses a single-raster line-scanning technique
to detect edge motion at one or both ends of a cell. Computer
software (pCLAMP version 7) was used to generate voltageclamp protocols as well as acquire and analyze voltage and
current signals. Voltage and current traces were sampled by
a 12-bit resolution analog-to-digital converter using a Pentium III computer. Data were stored on hard disk and Axotape (Axon Instruments) for later analysis. Computer software (LabView; National Instruments) was used to analyze
cell shortening and action potential duration (APD) at 90%
repolarization (APD90). In voltage-clamp protocols, ICa,L was
activated by depolarizing steps to 0 mV from a holding
potential of ⫺40 mV, which inactivates fast Na⫹ current and
T-type Ca2⫹ current. ICa,L was measured as the difference
between peak and steady-state current without compensation for leak currents. Data are presented as means ⫾ SE.
Data obtained from two different groups of cells from the
same heart were statistically analyzed using two-tailed unpaired Student’s t-test, with significance at P ⬍ 0.05. Data
obtained from a single cell serving as both control and test
were analyzed for statistical significance using two-tailed
paired Student’s t-test at P ⬍ 0.05.
RP protocol. The RP protocol consisted of the following:
cells were electrically stimulated through the recording pipette at a cycle length of 1,000 ms (60 beats/min) until APD
and cell shortening reached steady state, paced at progressively shorter cycle lengths (700, 500, and 400 ms) for steady-
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PACING-INDUCED CONTRACTILE DYSFUNCTION
Drugs. The drugs used included the following: chelerythrine (Alexis, San Diego, CA), calpain inhibitor I (Calbiochem,
La Jolla, CA), L-N5-(1-iminoethyl)-orthinthine (L-NIO)
(Alexis, San Diego, CA), NG-monomethyl-L-arginine (LNMMA) (Alexis), and LY-379196 (generously provided by Dr.
Chris Vlahos, Eli Lilly Research Laboratories). Generally,
cells under study were exposed to drugs for ⬃5 min before the
RP protocol was performed unless stated otherwise.
RESULTS
Figure 1, A and B, shows the effects of electrical
pacing on unloaded cell shortening (contraction) of
single ventricular myocytes. In Fig. 1A, a myocyte was
initially paced at a cycle length of 1,000 ms (60 beats/
min). Cycle length was progressively decreased to 250
ms (240 beats/min), maintained there for 5 min, and
then progressively returned to 1,000 ms (see METHODS).
After RP at 250 ms for 5 min, peak (systolic) cell
shortening was decreased at each pacing cycle length.
Diastolic cell length was not significantly different
before and after RP. Figure 1B shows an original recording of action potential configuration and cell shortening before and after RP in the same ventricular
myocyte. RP-induced inhibition of cell shortening were
typically accompanied by a decrease in APD90. In 26
cells, RP decreased APD90 by ⫺9 ⫾ 3% (P ⬍ 0.05).
However, RP-induced changes in APD90 were variable
among cells. In other words, it was not uncommon for
RP to exert little to no effect on APD90 at the same time
that it markedly decreased cell shortening. This is
reflected in the finding that RP-induced changes in
peak cell shortening and APD90 did not show a significant correlation (r ⫽ ⫺0.17; P ⫽ 0.406). Experiments
described below will show that RP-induced inhibition
of cell shortening was, in fact, independent of membrane voltage. In a total of 43 cells obtained from eight
hearts, RP decreased peak cell shortening by ⫺44 ⫾ 4%
(P ⬍ 0.001).
Fig. 3. L-type Ca2⫹ current (ICa,L) is unchanged after RP. Original
recordings of ICa,L before (left) and after RP (right) in the same
ventricular myocyte. RP had no discernible effects on holding current, peak Ca2⫹ current amplitude, or time course of inactivation.
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Fig. 2. RP-induced inhibition of contraction is independent of
changes in membrane voltage. Cell shortening was elicited by either
AP or constant voltage-clamp pulses (clamp) before and after RP in
the same cells. After RP, cell shortening was inhibited when stimulated by either AP or clamp pulses, showing that RP-induced inhibition of contraction is not due to changes in membrane potential
induced by RP.
RP-induced shortening of APD90 could result from
activation of ATP-sensitive K⫹ channels, indicating
cellular hypoxia. However, exposure to 5 ␮M glibenclamide, a specific inhibitor of ATP-sensitive K⫹ channels (11), failed to prevent RP-induced shortening of
APD90 [control ⫺16 ⫾ 7% (n ⫽ 6) vs. glibenclamide
⫺14 ⫾ 6% (n ⫽ 7)]. This result is consistent with
reports that RP of unloaded cardiac myocytes does not
cause hypoxia (45). RP-induced changes in cell shortening and APD90 may result from a nonspecific timedependent rundown in cell viability. This possibility
was examined by stimulating cells at 1,000 ms without
RP for the same time period as the RP protocol (⬃15
min). There were no discernable changes in cell shortening or APD90.
To determine whether changes in membrane voltage
induced by RP (such as shortening of APD) may be
responsible for inhibition of cell shortening, we used
constant depolarizing voltage-clamp pulses to trigger
cell shortening before and then after RP in the same
myocytes. During voltage clamp, each cell was held at
its respective resting membrane voltage (⫺75 to ⫺80
mV) and stepped to ⫹10 mV for 200 ms at 1 Hz. Cell
shortening was measured before and after RP when
triggered by either action potentials or voltage-clamp
pulses at 1 Hz. The graph in Fig. 2 summarizes the
results obtained in a total of eight cells obtained from
two hearts. Generally, the amplitude of cell shortening
under control conditions was smaller, although not
significantly (P ⫽ 0.5), when triggered by a voltageclamp pulse than by an action potential. Nevertheless,
RP inhibited cell shortening to a similar extent when
triggered by action potentials (⫺52 ⫾ 8%) or by depolarizing voltage-clamp pulses (⫺48 ⫾ 10%). These findings indicate that potential changes in membrane voltage induced by RP are not responsible for the
inhibition of contractility induced by RP.
ICa,L plays a critical role in triggering cell shortening
and may influence APD90. We therefore used voltage
clamp to determine whether RP inhibited ICa,L. ICa,L
was measured by voltage clamping the same cell before
and then after imposing the RP protocol. As shown in
Fig. 3, in cells obtained from three hearts, RP had no
PACING-INDUCED CONTRACTILE DYSFUNCTION
H93
Fig. 4. Intracellular Ca2⫹ concentration ([Ca2⫹]i) measurements recorded
from a ventricular myocyte before, during, and after RP. Top: stimulated AP.
Bottom: [Ca2⫹]i transients. During RP,
baseline and peak [Ca2⫹]i increased.
After RP, baseline and peak [Ca2⫹]i
returned to control values. Vm, membrane voltage; F405 and F480, fluorescence at 405 and 480 nm, respectively.
[Ca2⫹]i is significantly elevated, and, after RP, SR Ca2⫹
release and reuptake are not significantly changed. In
addition, neither during nor after RP did we observe
any signs that cells were Ca2⫹ overloaded or damaged.
In other words, RP never induced Ca2⫹-mediated afterdepolarizations, Ca2⫹ waves, or spontaneous Ca2⫹
transients. Also, cells did not exhibit blebbing or become granular as a result of RP.
RP-induced increases in Ca2⫹ influx could, in principle, activate a number of Ca2⫹-mediated signaling
pathways that may underlie RP-induced inhibition of
cell shortening. Therefore, we tested the role of Ca2⫹
influx by lowering the extracellular Ca2⫹ concentration
([Ca]o) from 2 to 0.5 mM during the RP protocol. Measurements of cell shortening were obtained in two
groups of cells (control and low [Ca]o) from the same
two hearts before and after RP when the [Ca]o ⫽ 2 mM.
In control cells, RP elicited a typical decrease in cell
shortening (3.6 ⫾ 0.8 vs. 1.8 ⫾ 0.4 ␮m; ⫺48%) (n ⫽ 5).
Figure 5, A–C, shows selected recordings of action
potentials and cell shortening before, during, and after
RP, when the [Ca]o was reduced to 0.5 mM during RP.
Comparing cell shortening before and after RP, it is
Fig. 5. RP-induced inhibition of contraction is dependent on Ca2⫹ influx. AP and
cell shortenings recorded before (A), during (B), and after (C) RP at a CL of 300 ms.
During RP, the extracellular calcium concentration ([Ca]o) was reduced from 2 to
0.5 mM. Lowering [Ca]o during RP prevented RP-induced inhibition of cell shortening.
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affect on peak ICa,L density (control 8.9 ⫾ 1.8 vs. after
RP 9.0 ⫾ 1.9 pA/pF; n ⫽ 13) or the rapid (␶1) and slow
(␶2) time constant of ICa,L inactivation (control ␶1 ⫽ 5 ⫾
0.8 and ␶2 ⫽ 44 ⫾ 3 ms vs. after RP ␶1 ⫽ 6 ⫾ 0.8 and
␶2 ⫽ 45 ⫾ 5 ms; n ⫽ 4).
A key step in cardiac E-C coupling is Ca2⫹ release
from the sarcoplasmic reticulum (SR). Therefore, fluorescent microscopy and the Ca2⫹-sensitive dye indo
1-AM were used to determine whether RP altered the
handling of intracellular Ca2⫹. Figure 4 shows typical
measurements of [Ca2⫹]i obtained before, during, and
after RP. This cell was rapidly paced at 225 beats/min
for 6 min. During RP, baseline and peak [Ca2⫹]i were
prominently elevated compared with control, resulting
in an increase in mean [Ca2⫹]i. After RP baseline,
[Ca2⫹]i and the peak Ca2⫹ transient amplitude returned to control values. Moreover, there was no
change in diastolic [Ca2⫹]i or the time course of relaxation of the Ca2⫹ transient, suggesting that SR Ca2⫹
uptake was unchanged. In five cells obtained from two
hearts, during RP baseline, [Ca2⫹]i increased from
0.33 ⫾ 0.03 (control) to 0.41 ⫾ 0.03 (⫹24%; P ⬍ 0.05).
These results indicate that, during RP, time-averaged
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PACING-INDUCED CONTRACTILE DYSFUNCTION
Fig. 6. Inhibition of protein kinase C (PKC)-␤
abolishes RP-induced inhibition of contraction. A: control recordings of AP and cell
shortening before and after RP. RP markedly
inhibited cell shortening. B: recordings of AP
and cell shortening from another myocyte in
the presence of LY-379196, a selective inhibitor of PKC-␤. RP-induced inhibition of cell
shortening was abolished.
tory (43, 44) have shown that both L-NIO and L-NMMA
inhibit NO-mediated processes in cat atrial myocytes.
In cells from the same three hearts, L-NIO (control
⫺47 ⫾ 10% vs. L-NIO ⫺48 ⫾ 8%; n ⫽ 11) or L-NMMA
(control ⫺69 ⫾ 22% vs. L-NMMA ⫺60 ⫾ 21%; n ⫽ 6)
both failed to prevent RP-induced decreases in cell
shortening, suggesting that NO is not mediating the
inhibitory effects of RP on contractility.
Activation of PKC is an important Ca2⫹-dependent
signaling mechanism, which is known to be associated
with negative inotropic effects in the heart (38, 41, 42).
We therefore examined the role of PKC signaling by
performing the RP protocol in two groups of cells from
the same four hearts in the absence and presence of
2–4 ␮M chelerythrine, a specific nonselective PKC
inhibitor (16). In the test cell group, chelerythrine
alone had no significant effect on peak cell shortening.
When compared with control cells, however, chelerythrine significantly attenuated RP-induced decreases in
cell shortening (control ⫺59 ⫾ 7%, n ⫽ 8, vs. chelerythrine ⫺14 ⫾ 3%, n ⫽ 9) (P ⬍ 0.001).
In the transgenic mouse, overexpression of PKC-␤
decreases myofilament Ca2⫹ responsiveness and contractility, possibly via phosphorylation of troponin I
(38). Therefore, to assess more specifically the role of
the Ca2⫹-dependent isoenzyme PKC-␤, we tested the
effects of 30 nM LY-379196, a selective inhibitor of
PKC-␤ (33). At 30 nM, the specificity of LY-379196 for
inhibition of PKC-␤ is more than an order of magnitude
greater than for inhibition of other PKC isoenzymes
(33). Figure 6, A and B, shows original recordings of
action potentials and cell shortenings obtained from
two different myocytes. In a control cell (Fig. 6A), RP
markedly decreased cell shortening (⫺81%) and
slightly shortened APD90 (⫺7%). In the test cell group,
LY-379196 alone elicited a small increase in basal cell
shortening (⫹18 ⫾ 9%; n ⫽ 7). In a cell exposed to
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evident that RP failed to decrease cell shortening
(4.7 ⫾ 0.6 vs. 4.6 ⫾ 0.7 ␮m; ⫺3 ⫾ 4%) (n ⫽ 6). In
addition, measurements of cell shortening during RP
in normal [Ca]o (2.9 ⫾ 0.5 ␮m) and low [Ca]o (1.3 ⫾ 0.4
␮m) (P ⬍ 0.05) indicate that cell shortening was significantly smaller in cells paced in low [Ca]o. This is
consistent with a reduced Ca2⫹ influx during RP in low
[Ca]o. Moreover, the records (Fig. 5B) show that lowering [Ca]o did not interfere with excitation at high
frequencies of stimulation. These results, in conjunction with those in Fig. 4, indicate that RP-induced
increases in Ca2⫹ influx and subsequent elevation of
[Ca2⫹]i is an essential factor in the mechanism by
which RP inhibits cell shortening.
In ischemic myocardial stunning, elevation of [Ca2⫹]i
is thought to activate the Ca2⫹-sensitive protease calpain, resulting in degradation of contractile myofibrillar protein (9, 13). To determine whether a similar
mechanism may operate in the contractile dysfunction
induced by RP, we performed the RP protocol in cells
exposed to calpain inhibitor I, a cell-permeant inhibitor
of calpain (30). The experiments were performed by
either exposing cells to 1 ␮M calpain inhibitor I acutely
(5–10 min) or by incubating cells in the inhibitor for up
to 1 h before the RP protocol was initiated. Because
similar results were obtained with the two methods,
the data have been pooled. Calpain inhibitor I failed to
affect RP-induced inhibition of cell shortening (control
⫺37 ⫾ 11% vs. calpain inhibitor I ⫺39 ⫾ 7%; n ⫽ 6 cells
from 2 hearts).
In cardiac myocytes, constitutive nitric oxide (NO)
synthase activity is stimulated by RP-induced elevation of [Ca2⫹]i (21), and NO can inhibit cardiac contractility (5, 21). To assess the potential role of NO, we
performed two series of experiments to test the effects
of 25 ␮M L-NIO and 100 ␮M L-NMMA, both inhibitors
of NO synthase (28). Previous work from this labora-
PACING-INDUCED CONTRACTILE DYSFUNCTION
H95
LY-379196 (Fig. 6B), RP-induced inhibition of cell
shortening was abolished, whereas APD90 still shortened (⫺13%). In cells obtained from the same two
hearts, LY-379196 blocked RP-induced inhibition of
cell shortening (control ⫺69 ⫾ 11%, n ⫽ 5, vs. LY379196 ⫺9 ⫾ 4%, n ⫽ 7) (P ⬍ 0.001). To further
evaluate the role of PKC activation, we directly stimulated phorbol ester-sensitive PKC isoenzymes by exposure to 0.2 ␮M phorbol 12-myristate 13-acetate
(PMA) for 5 min. PMA consistently and significantly
decreased peak cell shortening (⫺46 ⫾ 7%; P ⬍ 0.01;
n ⫽ 5) much the same as RP (data not shown).
Exactly which PKC isoenzymes are expressed in the
heart is somewhat controversial (39), and feline cardiac PKC isoenzyme expression has not been previously evaluated. Therefore, cellular extracts of cat ventricular myocytes were probed with monoclonal
antibodies specific for PKC-␣, -␤, -␦, and -⑀ , the major
PKC isozymes reported to be present in ventricular
myocytes of several different species, including humans (37). As shown in Fig. 7, PKC-␣ (A), PKC-␦ (C),
and PKC-⑀ (D) were all detected in cell extracts of
freshly isolated myocytes obtained from four hearts. In
addition, as shown in Fig. 7B, an 80-kDa band was
detected, which comigrated with PKC-␤ isolated from
both the cat and rat brain. These results are consistent
with the presence of PKC-␤ (as well as PKC-␣, -␦, and
-⑀) in adult cat ventricular myocytes.
DISCUSSION
The main finding of the present study is that RP of
ventricular myocytes for just a few minutes elicits
depression of systolic contractile function via activation of a Ca2⫹/PKC-dependent signaling mechanism.
Moreover, the depression in contractile function that
follows brief RP cannot be explained by alteration in
the basic handling of intracellular Ca2⫹ during E-C
coupling. More specifically, RP-induced inhibition of
contraction was not associated with changes in 1) the
trigger for SR Ca2⫹ release, i.e., Ca2⫹ influx via ICa,L; 2)
SR Ca2⫹ release as determined from peak intracellular
Ca2⫹ transient amplitude; or 3) reuptake of SR Ca2⫹,
as determined from relaxation of the intracellular
Ca2⫹ transient. Baseline levels of [Ca2⫹]i also were
unchanged after RP, indicating that diastolic [Ca2⫹]i
was unaffected. This is consistent with our finding that
RP did not significantly affect diastolic cell length.
Although RP generally shortened APD90, there was no
significant correlation between changes in APD90 and
peak contraction. In addition, the use of voltage-clamp
pulses to elicit contraction also directly demonstrated
that RP-induced inhibition of contraction is independent of RP-induced changes in action potential configuration. These results lead to the conclusion that RPinduced inhibition of contractile function occurs at the
level of the contractile myofilaments. In contrast to the
present results, chronically paced hearts, with (1, 8) or
without (40) signs of heart failure, exhibit contractile
dysfunction, which is associated with significant alterations in [Ca2⫹]i regulation and E-C coupling. At the
cellular level, ventricular myocytes isolated from
hearts chronically paced in vivo exhibit more positive
resting membrane potentials, triangular action potential configurations, prolonged APD, decreased ICa,L
density, and alterations in cytoarchitecture; all of
which are thought to contribute to contractile dysfunction (34). In contrast, various models of myocardial
stunning (10, 12, 13, 32) exhibit impaired contractile
function that is not associated with changes in basic
E-C coupling mechanisms. For example, in ischemic
myocardial stunning, ICa,L density and SR Ca2⫹ release and reuptake are unchanged, whereas the responsiveness to Ca2⫹ is attenuated, indicating that
contractility is affected at the myofilament level.
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Fig. 7. PKC isoenzyme expression in
cat ventricular myocytes. Detergentextracted cellular proteins from isolated cat ventricular myocytes (100–
300 ␮g) and cat brain (50 ␮g) and rat
brain (50 ␮g) were separated by SDSPAGE and Western blotting. The resulting blot was probed with mouse monoclonal antibodies generated against
human PKC-␣ (A), PKC-␤ (B), PKC-␦
(C), and PKC-⑀ (D). Bands were detected by an enhanced chemiluminescence protocol. The position of molecular weight standards is depicted to the
left of each blot.
H96
PACING-INDUCED CONTRACTILE DYSFUNCTION
With regard to our initial explanation, increased
expression of PKC-␤ is consistently associated with the
contractile dysfunction of cardiomyopathic and failing
hearts. For instance, in streptozotocin-induced myopathic hearts, the expression of PKC-␤ is preferentially
increased over other PKC isoenzymes (18). In failing
human hearts, expression of PKC-␤ was significantly
increased compared with nonfailing hearts (3). Moreover, blocking PKC-␤ with LY-333531, a selective
PKC-␤ blocker and close analog of LY-379196, indicated that PKC-␤ contributed the greatest amount to
the total increase in PKC activity in failing hearts.
Transgenic expression of PKC-␤ in adult mice hearts
caused mild and progressive ventricular hypertrophy
(4, 42). In addition, troponin I is a substrate for PKC
phosphorylation, and phosphorylation of troponin I decreases myofilament Ca2⫹ responsiveness (19, 41). In
transgenic mouse hearts, the specific overexpression of
PKC-␤ decreases myofilament Ca2⫹ responsiveness
and contractility, presumably via phosphorylation of
troponin I (38). The present results therefore suggest
that by raising [Ca2⫹]i, RP activates a Ca2⫹/PKC-dependent signaling mechanism, possibly via PKC-␤,
which subsequently depresses myofilament Ca2⫹ responsiveness. Moreover, the early stimulation of PKC
signaling by RP may set in motion further downstream
signaling mechanisms that lead to the pathological
changes characteristic of chronic tachycardia-induced
cardiomyopathy. Further studies, however, involving
direct measurements of PKC activation and/or translocation as well as protein phosphorylation of troponin
I will be required to prove our hypothesis.
We thank Rachel Gulling and Alan Furguson for expert technical
assistance with this study and Dr. Chris Vlahos, Eli Lilly and
Company Research Laboratories, for generously providing compound LY-379196.
Support was provided by the National Heart, Lung, and Blood
Institute Grants HL-27652 (to S. L. Lipsius), HL-34328 and HL63711 (to A. M. Samarel), and HL-51941 and HL-62231 (to L. A.
Blatter), the American Heart Association National Center (to L. A.
Blatter), and the Deutsche Forschungsgemeinschaft (to J. Hüser).
L. A. Blatter is an Established Investigator of the American Heart
Association.
Present address of J. Hüser: PH-R Molecular Screening Technology, Bayer AG, 42096 Wuppertal, Germany.
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