Vol. 277, Issue 5, H1690-H1700, November 1999
Role of voltage-sensitive release mechanism in depression of
cardiac contraction in myopathic hamsters
Susan E.
Howlett,
Wei
Xiong,
Cindy L.
Mapplebeck, and
Gregory R.
Ferrier
Cardiovascular Research Laboratories, Department of
Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada
B3H 4H7
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ABSTRACT |
We investigated excitation-contraction (EC)
coupling in isolated ventricular myocytes from prehypertrophic
cardiomyopathic (CM) hamster hearts. Conventional and voltage-clamp
recordings were made with high-resistance microelectrodes, and cell
shortening was measured with a video-edge detector at 37°C.
Contractions were depressed in myocytes from CM hearts, whether they
were initiated by action potentials or voltage-clamp steps. As in
guinea pig and rat, contraction in hamster myocytes could be triggered
by a voltage-sensitive release mechanism (VSRM) or
Ca2+-induced
Ca2+ release (CICR). Selective
activation of these mechanisms demonstrated that the defect in EC
coupling was primarily caused by a defect in the VSRM. However,
activation and inactivation properties of the VSRM were not altered.
When the VSRM was inhibited, the remaining contractions induced by CICR
exhibited identical bell-shaped contraction voltage relations in normal
and CM myocytes. Inward Ca2+
current was unchanged. Thus a defect in the VSRM component of EC
coupling precedes the development of hypertrophy and failure in CM
hamster heart.
congestive heart failure; excitation-contraction coupling
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INTRODUCTION |
CONTRACTILE DYSFUNCTION occurs with many forms of heart
disease and plays a major role in heart failure. Depressed
contractility is observed at the organ and tissue levels (3-5, 10,
11, 22, 26, 28) and more recently has been identified in myocytes isolated from hypertrophied or failed hearts (12, 23, 24). The latter
observations indicate that defects in contractile function may
originate at the cellular level. Thus it is possible that depressed
contractility may reflect, in part, abnormalities in the sequence of
events that link myocyte membrane depolarization to contraction, a
process known as excitation-contraction coupling (EC coupling).
A central step in cardiac EC coupling is the rapid release of
Ca2+ from the sarcoplasmic
reticulum (SR). Membrane depolarization can initiate SR
Ca2+ release by two mechanisms:
1)
Ca2+-induced
Ca2+ release (CICR), whereby a
small influx of Ca2+ through the
sarcolemma leads to a larger release of
Ca2+ from the SR (6); and
2) a voltage-sensitive release
mechanism (VSRM) by which SR Ca2+
release is linked to membrane depolarization and which operates independently of Ca2+ influx (8,
9, 14, 16, 17). A defect in EC coupling in heart disease could reside
in one or both of these mechanisms.
The possibility that a defect in CICR might occur has been explored in
several models of heart disease. Sen et al. (23, 24) showed that the
magnitude of cell shortening is reduced in myocytes from failing
hamster hearts, with no change in the magnitude of
Ca2+ current. This suggests that
the ability of Ca2+ current to
trigger SR Ca2+ release (CICR) may
be compromised in heart failure. Gomez et al. (12) also have
demonstrated that contractions and
Ca2+ transients are reduced in
myocytes from rat hearts with hypertrophy and heart failure (12). This
is associated with a decrease in the ability of
Ca2+ current to activate SR
Ca2+ release, measured as
Ca2+ sparks (12). Thus the results
of these studies suggest that a defect in CICR can contribute to
contractile dysfunction in heart disease.
Whether defects in the VSRM may contribute to depression of
contractility in heart disease has not been established. In fact, the
VSRM is inhibited by experimental conditions widely used in studies of
EC coupling in mammalian heart. Contractions initiated by the VSRM are
inhibited when 1) studies are
conducted at room temperature rather than at 37°C (7, 16),
2) holding or postconditioning potentials near 
40 mV are used (17), and/or
3) cells are dialyzed with patch
pipette solutions that do not contain cAMP or calmodulin to support
phosphorylation (9, 29). As previous studies of EC coupling in heart
disease have utilized one or more of these conditions, it is not known
whether defects in the VSRM might contribute to contractile dysfunction
in heart disease.
This study explores whether changes in the VSRM contribute to
contractile dysfunction in the cardiomyopathic (CM) hamster. The CM
hamster has been selected for study because it is a well-characterized genetic model of cardiomyopathy and congestive heart failure in which
the disease progresses in a characteristic and reproducible fashion (1,
2, 26). Cardiac cell necrosis begins at 40 to 50 days of age (18, 19).
This is followed by hypertrophy of remaining cells, which starts at
about 120 days of age, and progresses to heart failure and premature
death by about one year (18, 19). Previous studies (3, 15) in
multicellular preparations have shown that depression of contractility
in CM hamster heart precedes the onset of hypertrophy and failure. This
suggests that the defect in contractility is an early event rather than
a secondary response to advancing disease. Whether this early decrease
in contractility is accompanied by a defect in EC coupling at the level
of individual myocytes has not been determined. Therefore, this study
examines EC coupling in ventricular myocytes from 80- to 90-day-old
normal and CM hamster hearts. The objectives were 1) to determine whether the early
depression in contractility is accompanied by a defect in EC coupling
in myocytes isolated from CM hearts and
2) to evaluate whether depressed
contractility occurs as a result of changes in the VSRM, CICR, or both
mechanisms of EC coupling.
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METHODS |
Myocyte isolation.
Experiments were conducted on ventricular myocytes isolated from 80- to
90-day-old CM (CHF 146) and genetically matched normal (CHF 148) male
hamsters purchased from Canadian Hybrid Farms (Halls Harbour, Nova
Scotia, Canada). Animals were weighed, injected with heparin (3.3 IU/g), and anesthetized with pentobarbitol sodium (80 mg/kg). The heart
was rapidly cannulated in situ and perfused, retrogradely through the
aorta (8-10 ml/min), with oxygenated (100%
O2; 36.5°C)
Ca2+-free solution containing (in
mM) 120 NaCl, 3.8 KCl, 1.2 KH2PO4, 1.2 MgSO4, 10 HEPES, and 11 glucose (pH 7.4 with NaOH). The heart was removed from the chest and
perfused with Ca2+-free solution
for 7-8 min. Then the heart was perfused with the same solution
supplemented with collagenase A (35 mg/50 ml; Boehringer-Mannheim), trypsin (1 mg/50 ml; Sigma type III), dispase (20 mg/50 ml; neutral protease, Grade II, Boehringer Mannheim), and 50 µM
CaCl2 for 10-15 min. When the
ventricles had become soft, they were minced and washed in a
substrate-rich dissociation solution containing (in mM) 80 KOH, 50 glutamic acid, 30 KCl, 30 KH2PO4,
20 taurine, 10 HEPES, 10 glucose, 3 MgSO4, and 0.5 EGTA (pH 7.4 with
KOH). With this method, ~60-70% of the dissociated cells were
Ca2+-tolerant, rod-shaped
myocytes. There were no apparent differences in cell survival between
normal and CM hearts.
Ventricular myocytes were placed in a modified culture dish (volume,
0.75 ml) in an open-perfusion microincubator (model PDMI-2, Medical
Systems) on the stage of an inverted microscope. Cells were allowed to
adhere to the bottom of the chamber for 15-20 min and were then
superfused (at 37°C) with the HEPES-buffered solution described in
Myocyte isolation supplemented with
2.0 mM Ca2+. Except
where otherwise indicated, experiments were conducted in the presence
of lidocaine (200 µM) or tetrodotoxin (50 µM) to inhibit sodium
current and 4-aminopyridine (4-AP, 2 mM) to inhibit transient outward
current which is prominent in hamster myocytes. Solutions were pumped
through the experimental chamber from a buffer reservoir at a rate of 3 ml/min. The time required to completely replace the bath solution was
~90 s. In some cases, heated extracellular solutions were applied
with a computer-controlled rapid solution switching device (9, 14).
This device allows for a complete change of the extracellular solution
bathing the myocyte in <500 ms, while maintaining temperature at
37°C.
Experimental methods.
Discontinuous single-electrode, voltage-clamp recordings (sample rate
10-16 kHz) were made with an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA). Recordings were made with
high-resistance microelectrodes (18-25 M
, filled with 2.7 M
KCl) to reduce cell dialysis and to avoid buffering intracellular
Ca2+ levels. The bath was grounded
with a 2.7 M KCl-agar bridge to minimize liquid junction potential
changes. Voltage-clamp protocols were generated with pCLAMP software
(Axon Instruments), which also was used to acquire and analyze data on
computer. The output of the switching circuit was continuously
monitored during discontinuous single-electrode voltage clamp to ensure
that adequate settling time for accurate voltage measurement was
maintained. Current and transmembrane voltage were recorded in all
experiments. We had previously confirmed that the voltage measured by
the current-passing electrode is an accurate measurement of the
membrane potential by monitoring membrane potential with a second
independent electrode (8).
Cells were visualized with a closed-circuit television camera with
interlace defeat and partial scan capability (Panasonic, model
1-GP-CD60), and were displayed on a video monitor (Hitachi Densi, model
VM-1220C). Unloaded cell shortening was sampled at 120 Hz with a video
edge detector (Crescent Electronics, Sandy, UT) coupled to the camera.
Current, voltage, and contractions were digitized with a Labmaster
analog-to-digital interface at 125 kHz (TL1-125, Axon Instruments)
and stored on hard disk for subsequent analysis. Detailed descriptions
of specific voltage-clamp protocols are provided in the appropriate
results sections.
Data measurement and analyses.
Current, voltage, and contraction were measured with pClamp analysis
software. The magnitude of inward L-type
Ca2+ current
(ICa,L) was
measured as the difference between the peak inward current and a
reference point at the end of the voltage step. Li et al. (20) have
demonstrated previously that inward current measured in this way
provides an accurate measurement of the magnitude of
ICa,L. Cell
capacitance was estimated by integrating the capacitive transients with
pCLAMP analysis software. Contraction was measured as the difference
between the baseline preceding contraction and the peak of the contraction.
Differences between means were tested either with a Student's
t-test (with a Bonferroni correction
for multiple comparisons) or with a two-way repeated-measures ANOVA.
Post hoc comparisons were made with a Tukey's Studentized range test.
All statistical analyses were performed with SigmaStat (Jandel) or with
SAS (SAS Institute). Nonlinear curve-fitting procedures were conducted with Sigmaplot (Jandel). Data are means ± SE. The value
n represents the number of myocytes
sampled; no more than two replicates (myocytes) were collected from
cells from the same heart.
Sources of drugs and chemicals.
Lidocaine, 4-AP, tetrodotoxin, and cadmium were purchased from Sigma
Chemical (St. Louis, MO). Concentrated stock solutions of all drugs
were dissolved in distilled water.
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RESULTS |
Electrophysiological and contractile properties of ventricular
myocytes from normal and CM hamsters.
Initially, conventional (non-voltage clamp) recordings were made to
examine the characteristics of contractions initiated by action
potentials in cells from normal and CM hearts. Figure 1 shows representative original recordings
from normal (Fig. 1A) and CM (Fig.
1B) myocytes. Mean data are shown in
Table 1. Resting membrane potentials (RMP)
near 88 mV were recorded from both normal and CM myocytes (Table
1). Action potentials were characterized by a rapid upstroke, a marked
first phase of repolarization followed by a relatively brief plateau at
negative membrane potentials (Fig. 1,
A and
B). Action potential duration (APD)
was not significantly different at 30 mV
(APD30mV) or 80 mV
(APD80mV) between myocytes from
normal and CM hearts (Table 1). In contrast, mean peak amplitude of
contraction recorded from CM myocytes was only 21% of the amplitude observed in normal myocytes (Table 1,
P < 0.05). Although the magnitude of
contraction was depressed in CM myocytes, there were no significant
differences in time to peak contraction, half-relaxation time, or cell
length (Table 1).
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Fig. 1.
Electrophysiological and contractile recordings from normal and
cardiomyopathic (CM) hamster myocytes.
A: action potential
(top) and contraction
(bottom) recorded from a normal
hamster ventricular myocyte. Action potentials were initiated by trains
of 5 stimuli (1-4 nA) delivered at a frequency of 2 Hz. Recordings
shown are average of 5 traces. B:
representative action potential and contraction recorded from a CM
myocyte. Action potential configuration appeared similar in normal and
CM cells. However, magnitude of contractions was greatly reduced in CM
cells compared with normal. C:
recordings of membrane current (top)
and contraction (bottom) under
voltage-clamp conditions in a normal myocyte. Voltage-clamp
protocol is above (inset). A 200-ms voltage step from
60 to 0 mV initiated both inward current and contraction.
D: membrane current and contraction
recorded from a CM myocyte. Contraction was reduced in amplitude
although magnitude of inward current appeared similar in normal and CM
myocytes. Mean data are tabulated in Table 1.
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Differences in EC coupling might be related to differences in
electrical activity or to differences in subsequent events coupling electrical activity to contraction. In the present case, it is unlikely
that the difference in contractions between normal and CM myocytes is
related to changes in action potential configuration because we
observed no significant differences in this parameter. To eliminate the
effects of minor differences in action potential configuration,
additional experiments were conducted under voltage-clamp conditions.
Figure 1, C and
D, shows representative recordings of
membrane currents and contractions from normal and CM myocytes. Sodium
currents and transient outward currents were inhibited with lidocaine
(200 µM) and 4-AP (2 mM), respectively. A schematic of the
voltage-clamp protocol is illustrated in Fig. 1,
C and D. Each test step was preceded by a
train of 10 conditioning pulses to 0 mV to provide a consistent history
of regular activation. After the last conditioning pulse, the cell was
repolarized to a postconditioning potential
(vPC) of
60 mV for 300 ms. Then a test step to 0 mV was delivered to
activate contraction under voltage-clamp conditions. Recordings of
current are shown at top and contraction below (Fig. 1,
C and
D). Our results show that under
voltage-clamp conditions the difference in contraction between normal
and CM myocytes persisted. Mean peak contraction in CM myocytes was
only 30% of the magnitude observed in normal cells (Table 1,
P < 0.05). Time to peak and
half-relaxation times were not significantly different under
voltage-clamp conditions, despite the large change in peak contraction.
Thus depression of contraction persisted in myocytes from CM hamsters
when membrane potential was controlled by voltage-clamp. Furthermore,
reduction in peak contraction is not likely caused by changes in the
magnitude of ICa,L, because
peak inward current was not significantly different (Table 1).
Initiation of contraction by the VSRM and CICR in hamster
ventricular myocytes.
These observations demonstrate that before the onset of hypertrophy and
heart failure, myocytes from CM hamsters exhibit a defect in EC
coupling. This defect might be related to changes in the VSRM, CICR, or
both mechanisms of contraction. The VSRM has not previously been
measured in hamster myocytes. Therefore, we first established whether a
VSRM with characteristics similar to those described in other species
(8, 17) is present in hamster ventricular myocytes. We used a
voltage-clamp protocol which we have shown in previous studies with
guinea pig and rat myocytes to separate CICR and VSRM components of EC
coupling (8, 17). This protocol, shown in Fig.
2, utilized sequential test steps from
70 mV to 40 and then 0 mV to activate VSRM and CICR contractions respectively. Test steps were preceded by a series of 10 conditioning pulses to 0 mV, followed by a 4-s step to a vPC of 70
mV. During the 4-s interval, extracellular solution was changed rapidly
at 37°C by a computer-controlled switching device. When myocytes
were exposed to control solution, steps to 40 and 0 mV each
elicited contractions (Fig. 2A). The
step to 0 mV activated
ICa,L, however,
the step to 40 mV activated little inward current. Figure
2B shows the effects of a rapid change
to solution containing 100 µM
Cd2+, 3 s in advance of the test
steps. Cd2+ had no effect on
contraction activated by the step to 40 mV but strongly
inhibited both current and contraction initiated by the step to 0 mV
(Fig. 2B). These results closely
resemble results previously shown for guinea pig and rat ventricular
myocytes (8, 17); contractions activated by a step to 40 mV are
initiated by the VSRM and are not affected by
ICa,L blockade,
whereas contractions initiated by the step to 0 mV represent CICR and
are abolished by inhibition of
Ca2+ current.
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Fig. 2.
Voltage-sensitive release mechanism (VSRM) contractions are resistant
to blockade of L-type Ca2+ current
(ICa,L) by
Cd2+.
Top inset: schematic of
voltage-clamp protocol. A:
representative traces of contraction
(top) and membrane current
(bottom) recorded from a normal
hamster ventricular myocyte under control conditions. A voltage step to
40 mV elicited a VSRM contraction, and a second step to 0 mV
elicited ICa,L
and a contraction. B: rapid
application of 100 µM Cd2+ 3 s
in advance of activation steps strongly inhibited both
ICa,L and
ICa,L contraction
but had no effect on VSRM contraction triggered by step to 40
mV. Data were recorded in presence of 50 µM tetrodotoxin and 2 mM
4-aminopyridine (4-AP) to inhibit sodium current and transient outward
current, respectively. Similar results were obtained in 9 normal
myocytes.
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In guinea pig and rat ventricular myocytes, phasic VSRM contractions
initiated by a step to 40 mV exhibit steady-state inactivation (17). In the present study, we determined the steady-state inactivation properties of VSRM contractions in hamster ventricular myocytes. The
voltage-clamp protocol is shown schematically in Fig.
3A. The
protocol utilized a train of 10 conditioning pulses followed by
repolarization to a
vPC for 600 ms. A
test step to 35 mV was utilized to activate the VSRM. The test
step was preceded by a 10- ms repolarization to 65 mV.
Steady-state inactivation was assessed by changing the
vPC in 5-mV steps
from 35 to 70 mV. Representative recordings of
contraction are shown in Fig. 3B. When
the vPC was
35 mV, no contraction was observed in response to the activation
step. However, as the
vPC was made more
negative, contraction gradually appeared and increased in amplitude.
Figure 3C shows a graph of the
amplitude of contraction plotted as a function of
vPC. Phasic VSRM
contractions were fully available when steps were made from 70
mV and almost completely inactivated at membrane potentials near
35 mV. The data in Fig. 3C were
fitted with a Boltzmann function of the following form
y = (a b)/{1 + exp[(v vh)/k]} + b, where
a is maximum contraction,
b is minimum contraction,
v is the
vPC,
vh is the
half-inactivation voltage, and k is
the slope factor. In this example, from a normal hamster ventricular
myocyte, vh and
k were 51.2 mV and 4.46 mV,
respectively. These values are close to values reported previously for
the VSRM in guinea pig and rat myocytes (9, 17). Thus the inactivation properties, negative activation voltage and
Cd2+ insensitivity indicate that a
VSRM is operative in hamster ventricular myocytes.
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Fig. 3.
VSRM contractions in hamster ventricular myocytes exhibit
steady-state inactivation.
A: a schematic of voltage-clamp
protocol. After last conditioning pulse, cells were repolarized to a
postconditioning potential
(vPC) of
35 mV for 600 ms. The
vPC was
changed in 5 mV steps to potentials between 70 and
35 mV. After a 10-ms return to 65 mV, VSRM contractions
were measured with a test step to 35 mV.
B: representative recordings
from a normal ventricular myocyte. Amplitude of contractions increased
progressively as
vPC was
made more negative. C: a
steady-state inactivation curve was constructed by plotting magnitude
of contraction as a function of
vPC.
Steady-state inactivation curve was fitted with a Boltzmann function.
In this example from a normal myocyte, values of half inactivation
(vh) and
slope (k) were 51.2 mV
and 4.46 mV, respectively.
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Roles of the VSRM and CICR in contraction of CM hamster ventricular
myocytes.
We then determined whether contractions initiated by the VSRM and CICR
differed between myocytes from normal and CM hearts. Figure
4 shows representative recordings of
contractions and membrane currents. In Fig.
4A, the traces recorded from a normal
myocyte demonstrate that sequential test steps to 40 and 0 mV
activated VSRM and
ICa,L
contractions, respectively. Figure
4B shows recordings from a CM myocyte.
The magnitude of the VSRM contraction initiated by the step to
40 mV was much smaller in the CM myocyte. The contraction
initiated by the step to 0 mV was also smaller in this example, but the
relative difference was not as great as for the VSRM contraction. The
magnitude of peak inward current with the step to 0 mV was slightly
larger in the CM myocyte.
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Fig. 4.
VSRM contractions are reduced in magnitude in ventricular myocytes from
CM hamsters. Top inset: schematic of voltage-clamp protocol.
After last conditioning pulse, cell was repolarized to a
vPC of 65
mV. Sequential 200-ms test steps to 40 and 0 mV were utilized to
activate VSRM and
ICa,L
contractions, respectively. A:
representative recordings of contractions
(top) and currents
(bottom) initiated by steps to
40 and 0 mV in a normal myocyte.
B: VSRM contraction was absent, and
ICa,L contraction
was reduced in amplitude in CM cell.
ICa,L was of
similar magnitude in normal and CM myocytes. Mean data are shown in
Fig. 5.
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Mean data for contractions and currents from normal and CM myocytes are
shown in Fig. 5. The data are from
experiments conducted with the voltage-clamp protocol shown in Fig. 4.
Figure 5A shows that VSRM contractions
were markedly reduced in myocytes from CM hamsters compared with
myocytes from normal hamsters (P < 0.05). The amplitude of contraction in CM cells was 33% of the
contraction in normal myocytes. The mean data in Fig.
5B show that
ICa,L
contractions also were reduced in CM cells, but only to 63% of the
amplitude of myocytes in normal animals. This difference was not
statistically significant. Figure 5, C
and D, shows that there were no
significant differences in inward current between normal and CM
myocytes for either step.
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Fig. 5.
Comparison of mean magnitudes of contractions and currents elicited by
test steps to 40 and 0 mV in myocytes from normal and CM hearts.
Voltage-clamp protocol was same as in Fig. 4.
A: amplitude of VSRM contractions
(step to 40 mV) was significantly reduced in cells from CM heart
compared with normal. B: magnitude of
small inward current elicited by test step to 40 mV was similar
in normal and CM myocytes. C:
amplitude of
ICa,L
contractions (step to 0 mV) was slightly reduced in CM myocytes
compared with normal, although this difference was not statistically
significant. D: magnitude of
ICa,L was similar
in cells from normal and CM hearts. * Significantly different
from normal (P < 0.05);
n = 11 normal and 14 CM myocytes.
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Inactivation and activation properties of VSRM in normal and CM
hamster myocytes.
A possible explanation for the reduction in amplitude of VSRM
contractions in CM cells would be a shift in the voltage dependence of
steady-state inactivation. Therefore, we determined steady-state inactivation curves for the VSRM in myocytes from normal and CM hearts.
Experiments were conducted with the voltage-clamp protocol described
for Fig. 3. Figure 6 shows mean
steady-state inactivation curves. The curves show that the maximum
amplitude of VSRM contractions was significantly reduced in CM myocytes
compared with normal (Fig. 6A). When
the data were normalized as shown in Fig.
6B, it was clear that there was no
shift in the steady-state inactivation properties. Curves were fitted
with Boltzmann functions as described for Fig. 3. Mean values for
k were 4.16 ± 0.49 mV in normal
myocytes and 4.65 ± 1.1 mV in CM myocytes
(n = 6-8). Mean values for
vh were
49.9 ± 1.3 mV (n = 8) in
normal and 49.4 ± 0.7 mV in CM myocytes
(n = 6). Neither
vh nor
k values were significantly different between normal and CM myocytes. These data show that the change in
magnitude of VSRM contractions in CM myocytes cannot be attributed to a
change in steady-state inactivation properties.
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Fig. 6.
Steady-state inactivation properties of VSRM contractions are similar
in cells from normal and CM hearts. Voltage-clamp protocol is shown in
Fig. 3. A: mean amplitudes of
contractions plotted as a function of
vPC are reduced
significantly in cells from CM hearts compared with normal.
B: data shown in
A were normalized to maximum
contraction. Normalized steady-state inactivation relations demonstrate
that neither vh
nor k differed between 2 groups. Lines
represent Boltzmann functions fitted to mean data. Mean values for
k were 4.16 ± 0.49 mV in normal
myocytes and 4.65 ± 1.1 mV in CM myocytes
(n = 6-8; not significant, ns).
Mean values for
vh were
49.9 ± 1.3 mV in normal and 49.4 ± 0.7 mV in CM
myocytes (n = 6-8; ns). Error
bars on curves in B have been omitted
for clarity. * Significantly different from normal
(P < 0.05).
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Another possible mechanism for the reduction in amplitude of VSRM
contractions in CM cells is a shift in the voltage dependence of
activation of the VSRM. Activation of VSRM contractions occurs at more
negative membrane potentials than
ICa,L
contractions. Therefore, the initial rising phase of the
contraction-voltage relationship is determined primarily by the VSRM.
Figure 7 shows mean contraction-voltage
relationships for normal and CM myocytes determined from a
vPC of 60
mV. In normal myocytes, contractions were first observed with steps to
50 mV, increased to reach a peak at 0 mV and decreased only
slightly at membrane potentials as positive as +80 mV (Fig.
7A). The magnitude of contractions was greatly reduced in CM myocytes (P < 0.05), however, contraction first appeared and reached a maximum
over the same range of membrane potentials as in normal myocytes (Fig.
7A). This suggests that there is
little difference in voltage dependence of activation of the VSRM
between normal and CM myocytes. Indeed, when contraction voltage
relationships were normalized as shown in Fig.
7B, the curves for normal and CM
myocytes were superimposable between 60 and +20 mV. These
potentials include the range over which the VSRM activates.
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Fig. 7.
Mean contraction-voltage determined from a
vPC of 60
mV in myocytes from normal and CM hearts. After a train of 10 conditioning pulses to 0 mV, cells were repolarized to a
vPC of 60
mV. Test steps (200 ms) to membrane potentials between 60 and
+80 mV were utilized to activate contractions and currents.
Contraction-voltage relations were obtained by plotting amplitude of
contraction as a function of test step potential.
A: in normal myocytes, contractions
first appeared near 50 mV, became maximal near 10 mV, and
declined slowly, even with depolarization to very positive potentials.
Shape of contraction-voltage relations was similar in cells from normal
and CM hearts, but magnitude of contraction was greatly reduced in CM
myocytes. B: contractions were
normalized to amplitude of contraction at 0 mV in normal and CM
myocytes. Normalized contraction-voltage relations were similar in two
groups. * Significantly different from normal
(P < 0.05).
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The contraction-voltage relationships determined from a
vPC of 60
mV include components of contraction initiated both by the VSRM and by
CICR linked to
ICa,L. To
evaluate the contribution of CICR alone, we also determined
contraction-voltage relations from a
vPC of 40
mV to inactivate the VSRM. Figure
8A shows
that identical, bell-shaped contraction-voltage relations were observed in myocytes from normal and CM hearts. Figure
8B shows that normalized curves also
were superimposable. These data demonstrate that the voltage dependence
of activation of CICR contractions also is not altered in CM cells.
Thus CICR does not contribute to the decrease in magnitude of
contractions in curves determined from a
vPC of 60
mV (Fig. 7A).
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Fig. 8.
Mean contraction-voltage relations determined from a
vPC of 40
mV in myocytes from normal and CM hearts. Voltage-clamp protocol was
similar to that described in legend to Fig. 7, except that test steps
were made from a
vPC of 40
mV. A: contraction-voltage relations
determined from a
vPC of 40
mV were bell-shaped with a peak near 0 mV in both normal and CM
myocytes. Contraction-voltage relations were virtually identical in
normal and CM cells. B: contractions
normalized to amplitude of contraction at 0 mV also were similar in
normal and CM myocytes.
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Figure 9 shows current-voltage
(I-V) relations corresponding to the
experiments shown in Figs. 7 and 8. Current data have been normalized
to cell capacitance, which was not significantly different between the
two groups (capacitance = 172.2 ± 7.6 pF, 153.0 ± 13.7 pF for
n = 11 normal and
n = 14 CM cells, respectively). Figure
9A shows that
I-V relationships were identical in
normal and CM myocytes when the
vPC was 60
mV. I-V relationships also were
identical when the
vPC was 40
mV as shown in Fig. 9B. There was a
small increase in inward current observed when
I-V relations were determined from a
vPC of 60
mV compared with a
vPC of 40 mV. This current might represent T-type
Ca2+ current and/or
Na+-Ca2+
exchange current in response to release of SR
Ca2+. We assessed the magnitude of
this current by subtracting I-V relations determined with a
vPC of 40
mV from those determined with a
vPC of 60
mV. Figure 9C shows that magnitude and
voltage dependence of the difference current was the same in normal and CM cells.
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Fig. 9.
Mean current-voltage (I-V) relations
for inward currents in normal and CM hearts. Data are from experiments
illustrated in Figs. 7 and 8. A:
I-V relations determined with test
steps from a vPC
of 60 mV. In both normal and CM myocytes, inward current first
appeared near 40 mV, increased to a maximum near 0 mV, and
declined with steps to more positive voltages.
I-V relations determined in normal and
CM cells were similar. B:
I-V relations determined with test
steps from a vPC
of 40 mV also were not significantly different in normal and CM
cells. C: currents determined from a
vPC of 40
mV were subtracted from currents determined from a
vPC of 60
mV to illustrate difference currents. Difference currents were also of
similar magnitude in normal and CM myocytes. Data have been normalized
to cell capacitance.
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|
The results shown in Figs. 7 and 8 demonstrate that the amplitudes of
VSRM contractions, but not CICR contractions, are decreased in myocytes
from CM hearts. This suggests that the defect in contraction observed
when both mechanisms are available is mediated primarily by the VSRM.
If this is correct, selective inhibition of the VSRM should eliminate
the difference in contraction between myocytes from CM and normal
hearts. We have shown previously that VSRM contractions are inhibited
selectively when preceded by conditioning pulses to 40 mV,
rather than 0 mV (17). Both CICR and the VSRM contribute to contraction
when contraction-voltage relations are determined from a
vPC of 60
mV. We therefore determined the effects of changing conditioning pulse
voltage on these contraction-voltage relations in cells from normal and
CM hearts. Figure 10 shows mean contraction-voltage and I-V relations.
Figure 10A shows that
contraction-voltage relations in normal cells were greatly reduced when
conditioning pulse amplitude was changed from 0 mV to 40 mV
(P < 0.05). In myocytes from CM
hamsters (Fig. 10B), contractions
with conditioning pulses to 0 mV were smaller than in normal myocytes.
In addition, changing the conditioning pulse voltage to 40 mV
had much less effect (Fig. 10B).
Figure 10, A and
B, also show that contraction amplitudes were almost identical in myocytes from normal and CM animals, when VSRM contractions were inhibited by conditioning pulses
to 40 mV. Figure 10, C and
D, show
I-V relations for normal and CM
myocytes, respectively. In myocytes from both normal and CM hearts,
peak inward current was larger with conditioning pulses to 40 mV
than 0 mV. For each conditioning pulse voltage, the magnitudes of
currents were similar in normal and CM myocytes.
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Fig. 10.
Effect of conditioning pulse amplitude on contraction-voltage relations
in normal and CM myocytes. Voltage-clamp protocol was similar to that
described in legend to Fig. 7, except that voltage of conditioning
pulse train was varied between 0 and 40 mV.
A: contraction-voltage relations
determined from a
vPC of 60
mV in normal myocytes. When VSRM contractions were inhibited by
conditioning pulses to 40 mV, contraction in normal myocytes was
significantly reduced. B: when
conditioning pulse amplitude was changed from 0 to 40 mV,
amplitude of peak inward current was slightly increased in normal
cells. C: when VSRM contractions were
inhibited by conditioning pulses to 40 mV, there was no
difference between contraction in normal and CM myocytes.
D: amplitude of peak inward current
was slightly increased in CM myocytes when conditioning pulse amplitude
was changed from 0 to 40 mV. * Significantly different
from normal (P < 0.05).
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|
| |
DISCUSSION |
The objectives of this study were 1)
to determine whether there is a defect in EC coupling in myocytes
isolated from CM hamsters before the development of hypertrophy and
failure, and 2) to evaluate whether
the VSRM, CICR or both mechanisms of EC coupling contribute to this
defect. The present investigation demonstrates that a defect in EC
coupling is present in CM myocytes at this early stage of the disease.
Furthermore, this malfunction in EC coupling originates primarily from
a defect in the VSRM.
Previous studies in whole heart and in isolated cardiac tissues have
shown that contractility is depressed in CM hamster heart (3-5,
10, 11, 15, 22, 26, 28). Furthermore, several studies in tissues from
young (80-85 day old) CM hamsters indicate that depression of
contractility precedes the onset of hypertrophy and heart failure (3,
15). The results of our study demonstrate that a corresponding defect
is present in isolated myocytes from 80- to 90-day-old CM hamsters.
Although contractions were depressed in myocytes from CM heart, we
found no significant change in action potential configuration.
Furthermore, the difference in contraction persisted even when possible
changes in action potential duration were eliminated with voltage-clamp
techniques. These results indicate that a defect in EC coupling
contributes to the decrease in contractility observed in myocytes from
CM hearts.
Both CICR and the VSRM contribute to EC coupling in myocytes from
normal heart. Therefore, a defect in EC coupling in CM myocytes could
occur as a result of changes in either one or both mechanisms. Our
results showed that when the VSRM was inactivated by depolarization, the remaining CICR contractions were of similar magnitude in normal and
CM myocytes. Contraction-voltage relations for CICR were bell-shaped and reached similar peak amplitudes in normal and CM myocytes. Thus
CICR was virtually unchanged in myocytes from young CM hamsters compared with normal.
We also investigated the role of the VSRM in EC coupling in CM heart.
Our results showed that, when the VSRM was inhibited by preceding test
steps with a train of conditioning pulses to 40 mV (17),
contractions were of similar magnitude in normal and CM cells. In
contrast, when the VSRM was available, contractions were much smaller
in CM cells than in normal cells. These observations indicate that a
defect in the VSRM is responsible for the decrease in contraction in
myocytes from CM hearts. Our results also showed that normalized
activation and inactivation curves from the two groups were
superimposable. Thus the reduction in magnitude of VSRM contractions
was not due to changes in voltage-dependent activation or inactivation
of the VSRM. These results further suggest that the defect in the VSRM
must be related to a change in the efficacy of the VSRM as a trigger,
rather than changes in activation or inactivation.
We previously have reported that the VSRM is regulated by the adenylate
cyclase-protein kinase A cascade and also by
calcium-calmodulin-dependent kinase in guinea pig ventricular myocytes
(9, 29). Thus it is possible that the defect in the VSRM in CM hamster
myocytes might represent a defect in phosphorylation through one or
both of these pathways. Therefore, it will be important to investigate the role of these and other regulatory mechanisms in causing the decreased effectiveness of the VSRM as a trigger for contraction in CM heart.
In the present study we found that
ICa,L was of
similar magnitude in myocytes from normal and CM heart. Previous
studies have reported either no difference (23) or a decrease (13, 20) in the magnitude of
ICa,L in CM
myocytes compared with normal myocytes. It is not clear why different
observations have been reported in these studies. However, the studies
reporting a decrease in current were conducted with
Ca2+ as the charge carrier and
with holding potentials of 40 or 50 mV. With the use of
depolarized holding potentials intracellular Ca2+ levels are expected to
increase. Myocytes from CM hamsters also have been
reported to have elevated Ca2+
levels (24, 28). It is possible that
Ca2+ inhibition of
Ca2+ current may have been greater
in CM hamster myocytes, especially when depolarized holding potentials
were utilized. This may have contributed to reduction of
ICa,L in these
studies. The study of Sen et al. (24) used
Ba2+ as the charge carrier that
would be expected to eliminate
Ca2+ inhibition of
ICa,L. The
present study utilized a holding potential of 80 mV, which also
may have minimized elevation of cytosolic Ca2+ levels and thereby inhibition
of ICa,L. In
addition, other differences in experimental conditions may have
contributed to differences in observations in these studies.
In this study we also observed an increase in inward current in
I-V relations determined from a
vPC of 60
mV compared with I-V relations
determined from a
vPC of 40
mV. This might represent Ca2+
current through T-type Ca2+
channels (21). Previous studies have reported that the magnitude of
T-type Ca2+ current is increased
in myocytes from 200- to 300-day-old hamster heart compared with normal
(25) myocytes. In the present study, we found no increase
in the magnitude of putative T-type
Ca2+ current in cells from young,
prehypertrophic normal and CM hearts. Thus our results suggest that
changes in contractile function develop before an increase in T-type
Ca2+ current. Furthermore, it is
highly unlikely that T-type Ca2+
current contributes to activation of VSRM contractions in the heart. We
have observed VSRM contractions in myocytes from rat heart (17) where
T-type Ca2+ current is absent
(27). Furthermore, VSRM contractions are not abolished by
Ni2+ (14), at concentrations in
excess of those required to inhibit T-type
Ca2+ current (27).
Although the central question in this paper was to assess the
contribution of the VSRM to contractile dysfunction in CM hamster heart, this study also characterizes the VSRM in hamster ventricular myocytes. We found that the VSRM in hamster myocytes exhibited characteristics very similar to those described in rat and guinea pig
ventricular myocytes in previous studies (8, 9, 17). VSRM contractions
were not blocked by 100 µM Cd2+,
a concentration of Cd2+ which
blocked both
ICa,L and CICR
contractions. Furthermore, VSRM contractions in hamster myocytes
exhibited steady-state inactivation properties with
vh values near
50 mV and k values near 4 mV. These values are similar to those reported previously for the VSRM in
guinea pig and rat myocytes (17). In addition, when the VSRM was
inactivated (vPC = 40 mV), contraction-voltage relations were bell-shaped as
observed in guinea pig ventricular myocytes under similar conditions
(8, 17). When the VSRM was available (vPC = 60
mV), contraction-voltage relations became sigmoidal with a threshold
near 60 mV and a plateau near 10 mV as in guinea pig and
rat ventricular myocytes (8, 17). Thus the properties of the VSRM are
very similar in ventricular myocytes from different species.
In summary, the results of this study demonstrate that a defect in EC
coupling is present in CM myocytes early in the development of disease.
This abnormality in EC coupling is not due to a change in CICR but
originates primarily from a defect in the VSRM component of
contraction. This raises the possibility that defects in the VSRM might
contribute to the development of cardiac hypertrophy and/or heart failure.
| |
ACKNOWLEDGEMENTS |
The authors thank Peter Nicholl, Isabel Redondo, and Claire Guyette
for excellent technical assistance.
| |
FOOTNOTES |
This work was supported by grants from the Medical Research Council of
Canada and the Heart and Stroke Foundation of Nova Scotia. W. Xiong is
supported by an Izaak Walton Killam Memorial Scholarship.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. E. Howlett
and G. R. Ferrier, Dept. of Pharmacology, Dalhousie Univ., Halifax,
Nova Scotia, Canada B3H 4H7 (E-mail: susan.howlett{at}dal.ca;
gregory.Ferrier{at}dal.ca).
Received 8 January 1999; accepted in final form 3 June 1999.
| |
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Am J Physiol Heart Circ Physiol 277(5):H1690-H1700
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ABSTRACT
INTRODUCTION
