1660
Ethanol Acutely and Reversibly Suppresses
Excitation-Contraction Coupling
in Cardiac Myocytes
Robert S. Danziger, Makoto Sakai, Maurizio C. Capogrossi,
Harold A. Spurgeon, Richard G. Hansford, and Edward G. Lakatta
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
We used adult rat cardiac myocytes to examine the acute effects of 0.1-5.0% (vol/vol) ethanol
(ETOH) on 1) the cytosolic [Ca2+] (Cai) transient measured as the change in indo 1
fluorescence at 410/490 nm and contraction elicited by electrical stimulation of single cells and
2) the sarcoplasmic reticulum (SR) Ca2+ content in cell suspensions. During stimulation at 1
Hz, clinically relevant ETOH correlations (0.1-0.15% [vol/vol]) caused a 10-15% decrease in
the contraction amplitude, measured by myocyte edge tracking, without decreasing the Caj
transient that initiates contraction. At higher ETOH concentrations (1-5% [vol/vol]), ETOH
caused profound contractile depression and also reduced the magnitude of the Caj transient.
These effects were reversed within minutes of ETOH washout. Addition of norepinephrine (10
,uM) to the bathing solution or an increase in bathing [Ca2+] in the continued presence of
ETOH could also reverse its effects. The relation of the amplitude of the Ca; transient to the
contraction amplitude measured across a range of bathing [Ca2+] was shifted by ETOH, such
that for a given Caj transient a marked reduction in contraction amplitude occurred. In
unstimulated myocyte suspensions, ETOH (1-5% [vol/vol]) caused a concentration-dependent
depletion of SR Ca2+ content, manifested as a diminution in the Caj increase elicited by caffeine
in the presence of extracellular EGTA and no added Ca2+. Thus, in rat cardiac myocytes a
reduction in the myofilament Ca2+ response, possibly due to a decrease in myofilament Ca21
sensitivity, is a mechanism for contractile depression due to clinically relevant ETOH
concentrations. Higher concentrations of ETOH cause further contractile depression, in part,
by inducing SR Ca2' release and depleting the SR of Ca2 , leading to an attenuation of the Caj
transient elicited by electrical stimulation, and by further depressing the myofilament
length-Ca2+ relation. (Circulation Research 1991;68:1660-1668)
It has been established that ethanol (ETOH)
depresses myocardial contractility in humans
and experimental animals, both in situ and in
isolated hearts and cardiac muscle.'-"1 The primary
mechanism(s) of the negative inotropic effect of
ETOH is only partially understood. If ETOH induced metabolic alterations (e.g., a reduction in
cytosolic pH or high-energy phosphates), it could
decrease contractility on this basis. Recent studies,
however, have indicated that ETOH, in concentrations up to 5%, does not deplete high-energy phosphates, decrease cytosolic pH, or increase inorganic
phosphate.7
From the Laboratory of Cardiovascular Science, Gerontology
Research Center, National Institute on Aging, National Institutes
of Health, Baltimore, Md.
Address for reprints: Dr. Edward G. Lakatta, Laboratory of
Cardiovascular Science, Gerontology Research Center, 4940 Eastern Avenue, Baltimore, MD 21224.
Received January 9, 1990; accepted February 22. 1991.
Regulation of myocardial contractility is under the
control of Ca2', which initiates contraction by binding to troponin C.12 The major source and sink of the
excitation-induced cytosolic [Ca21] (Cai) transient in
cardiac muscle is the sarcoplasmic reticulum (SR).13
ETOH may decrease cardiac contractility through
actions at the myofilaments or by alteration of Ca21
transport at the SR. The former possibility would
likely involve a decrease in the myofilament sensitivity to cytoplasmic Ca2+. An action at the SR could be
the result of a decrease in SR Ca2' store or secondary
to a reduction in the effectiveness or size of the
trigger for SR Ca2' release. Studies in isolated subcellular organelles may provide some insight into the
acute contractile depression by ETOH, because isolated canine cardiac microsomes enriched in SR
acutely exhibit decreased Ca21 uptake, increased
passive Ca21 permeability, and decreased maximal
storable Ca21 in response to ETOH.14 Acute addition
of ETOH also reduces Ca2' binding to the regulatory
Danziger et al Ethanol Effects on Cardiac Myocytes
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complex of isolated tropomyosin and decreases actomyosin ATP hydrolysis.15 However, whether ETOH
depletes SR Ca'+ in the intact cardiac myocyte (i.e.,
when the amount of Ca'+ available to SR is regulated
by the sarcolemmal transport mechanisms) or
whether it alters the interaction of Ca'2 with intact
myofibrils is not known.
Advances in the isolation of Ca'+-tolerant adult
cardiocytes and the more recent development of
Ca'+-sensitive fluorescent probes, which permit the
detection of changes in Cai, provide opportunities for
further study of the cellular mechanisms that underlie the cardiac depressant effect of ETOH. The
present study used these Ca'+ probes in rat cardiac
myocytes, with simultaneous measurement of contraction. In some experiments, it was determined
whether the acute ETOH-mediated depression of
contractility could be attributed to a decrease in SR
Ca'+ release, measured either as diminution of the
amplitude of the Caj transient in response to electrical stimulation or a diminution in the caffeineinduced increase in Cai. In other experiments, it was
determined whether ETOH alters the myofilament
responsiveness to Ca2', measured as the relation of
contraction amplitude to that of the Caj transient
that initiates contraction.
Materials and Methods
Enzymatically isolated rat cardiac myocytes were
prepared via collagenase digestion as previously described16 and plated in Petri dishes perfused with
HEPES-buffered solution containing (mM) NaCl
137, MgSO4 1.2, KCl 5.0, NaH2PO4 1.2, HEPES 20,
D-glucose 11, and CaCl2 1.0 (pH adjusted to 7.4 with
NaOH).
In the initial experiments, the effect of ETOH
(0.10-5% [vol/vol]) on the contractile response to
electrical stimulation of single cells not loaded with
Ca2 indicators was determined. Cells were in 1 mM
bathing [Ca2] (Ca0) at 37°C and were stimulated
continually at 1 Hz via field stimulation (2-5-msec
pulses). Contraction amplitude was measured via
video edge-motion detection as described previously16 and was recorded 1 minute before ETOH exposure after 3-5 minutes of ETOH exposure, and 5
minutes after ETOH washout. In a subset of these
cells, norepinephrine (10 ,lM) was added in the
continued presence of ETOH. In another series of
experiments, the Caj transient and the contraction in
single myocytes were simultaneously measured, as
recently described,17 before and after a broad concentration range of ETOH (0.1-3.0 [vol/vol]). Briefly,
single myocytes bathed in a HEPES-buffered medium containing 1 mM Ca. at 23°C were loaded with
the membrane-permeant acetoxymethyl ester derivative (AM form) of the Ca2 probe indo 1 (4 ,M),
which was dissolved in dimethyl sulfoxide mixed with
fetal calf serum and pluronic F-127 (BASF Wyandotte Corp., Wyandotte, Mich.), a dispersing agent.18
Indo 1 fluorescence was excited by epi-illumination
with 3.8-,usec flashes of 350+5 nm light. Paired
1661
photomultipliers collected indo 1 emission by simultaneously measuring spectral windows of 391-434
and 457-507 nm selected by band-pass interference
filters. The ratio of indo 1 emission at the two
wavelengths was calculated as a measure of Caj using
a pair of fast-integrator sample-and-hold circuits
under the control of a VAX 11/730 computer (Digital
Equipment Corp., Maynard, Mass.). Cell length was
simultaneously monitored using red light (650-750
nm) to form a bright field image of the cell, which was
projected onto a photodiode array (1024 SAQ Starlight Sensor, EG&G Reticon, Inc., Sunnyvale, Calif.)
with a 5 -msec time scan rate. Cells were stimulated at
0.5 or 1.0 Hz. Ten to 20 twitch contractions and the
associated Caj transients were signal-averaged before
and after ETOH.
In a third series of experiments, the Caj of suspensions of myocytes was monitored by recording the
fluorescence of cell suspensions loaded with quin 2
using a modified fluorometer (model A2, Farrand
Optical Co. Inc., New York) as described previously.19 Briefly, the cells were loaded by exposure to 40
,uM quin 2-AM for 20 minutes at 37°C. For these
studies, the incubation medium comprised 117 mM
NaCl, 1 mM MgSO4, 5.4 mM KCl, 25 mM HEPESTris (pH 7.4), 1.2 mM NaH2P04, 20 mM glucose, 1
mM CaCl2 and 5 mg/ml bovine serum albumin (Fraction 5, Miles Laboratories, Inc., Elkhart, Ind.). Creatine phosphate and creatinine kinase were present
at 10 mM and 20 units/ml, respectively. Excitation of
fluorescence was at 333 nm, and emission was collected at wavelengths >480 nm. Temperature was
maintained at 37°C, and the cell suspension was
stirred magnetically in the cuvette of the instrument
and maintained under an atmosphere of 100% 02.
To validate comparisons between cuvettes of cells,19
the minimal fluorescence with MnCl2 (0.1 mM) and
Triton X-100 (0.1%) and the maximal fluorescence
achieved with a molar excess of diethylenetriaminepentaacetate (5 mM)/CaCl2 (6 mM) were measured.
Materials
Indo 1-AM and quin 2-AM were obtained from
Calbiochem Corp., La Jolla, Calif. All other chemicals were of laboratory grade obtained from standard sources.
Results
Contractile Measurements in Individual Myocytes
The threshold concentration necessary for ETOH
to depress contractility varied from 0.1% to 0.2%
(vol/vol) (see below). Figure 1 shows a representative
chart recording of reversible twitch depression by
threshold and higher concentrations of ETOH in
myocytes not loaded with fluorescent Ca2+ probes.
Table 1 depicts the average effects of a 5-minute
exposure to ETOH on the following parameters:
resting cell length, maximum velocity of cell shortening during the twitch (VS), maximum extent of cell
shortening (ES), and contraction duration, that is,
1662
Circulation Research Vol 68, No 6 June 1991
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FIGURE 1. Recordings showing examples of the
suppression of the twitch amplitude in single
ventricular myocytes of rats by 0.15% (vol/vol)
ethanol (ETOH) (panel A) and 3% (iol/vol)
ETOH (panel B). ETOH was present in the bath
during the time indicated. Recovery of contractile
function occurs on ETOH washout. Panel C
illustrates reversal of ETOH twitch depression by
10 pM norepinephrine (NE). The ETOH concentration was 4% (vol/vol), and it was present
during the period between the arrows.
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the time from stimulus to half twitch relaxation. At
0.1% (vol/vol) ETOH, ES and VS were decreased by
15% (Table 1). This result is strikingly similar to the
TABLE 1. Effect of Ethanol and Ethanol Plus Norepinephrine on
Twitch Parameters of Single Cardiac Myocytes of Rats
VS
ES
RL
ETOH concentration n (% control) (% control) (% control)
0.15%
ETOH
6 88-+-5
85 +2
100+0.2
After ETOH
3 96+7
103+3
100+0.1
ETOH+NE (10 ,M) 3 308+106
198+62
97+2
1%
ETOH
10 68±7
66+7
101+0.3
After ETOH
5 164+36
199+44
100+0.6
ETOH+NE (10 ,uM) 5 152+26
156+32
99+0.7
3%
ETOH
10 53+9
49+9
101+0.4
After ETOH
5 128+33
154±42
101+0.7
ETOH+NE (10 ,uM) 5 127+33
114±36
97+1
5%
ETOH
10
0
0
101 +-0.5
After ETOH
5 127±15
135±17
103±3
ETOH+NE (10tlM) 5
8+8
6+6
100+1
Values are mean+SEM. ETOH, ethanol; VS, maximum velocity
of cell shortening during twitch; ES, maximum extent of cell
shortening; RL, resting cell length; NE, norepinephrine. Cells
were stimulated at 1 Hz at 37C. In all cells measurements were
made 5 minutes after exposure to ETOH and 5 minutes after
washout in half the cells. In the remainder of cells, NE was added
after 5 minutes of ETOH, and measurements were taken after an
additional 5 minutes of ETOH plus NE. Before NE, VS, ES, and
RL averaged 135±28 ,um/sec, 7+0.8% of RL, and 107±8 gm,
respectively.
depression of contraction by this concentration of
ETOH in intact ferret cardiac muscle.20 VS and ES
decreased to 68% and 66% of control, respectively,
in 1% (vol/vol) and to 53% and 49%, respectively, in
3% (vol/vol) ETOH. The contraction was abolished
in 5% ETOH. ETOH (1% [vol/vol] or greater) also
caused a small increase in resting cell length. After
washout of the higher ETOH concentration (1% or
greater), VS, ES, and contraction duration not only
recovered, but at 5 minutes after washout, the twitch
contraction amplitude and velocity were greater than
they were before ETOH exposure (Table 1). In many
cells this overshoot in twitch amplitude was accompanied by the occurrence of spontaneous contractile
waves in diastole (not shown). A similar overshoot in
contraction strength has been observed after washout
of high concentrations of ETOH in isolated perfused
rat hearts.21
Because previous studies911 in vivo indicated that
reflex sympathetic stimulation could compensate for
ETOH-induced myocardial suppression in situ, a subset of cells was exposed to norepinephrine (10 ,uM)
during exposure to ETOH (Figure iB). Norepinephrine caused marked increases in VS and ES during
0.1% (vol/lvol) ETOH exposure, reversed the effect of
1% and 3% (vol/vol) ETOH, and partially restored the
contraction in 5% (vol/vol) ETOH (Table 1).
Ca2' and Contraction
Threshold concentrations of ETOH for contractile
depression did not markedly reduce the Ca, transient,
measured as the change in indo 1 fluorescence after
electrical stimulation (Figure 2). In this regard, it is
important to note that this concentration of ETOH
Measurements of Cytosolic
Danziger et al Ethanol Effects on Cardiac Myocytes
ETOH 0.1%
twitch amplitudes as a function of the peak systolic
indo 1 ratios measured across a range of Ca0. The
data points in ETOH are displaced downward to the
right, and this is reversed on ETOH washout.
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FIGURE 2. Top panel: Chart recording of the electrically
stimulated twitch in a single rat myocyte loaded with indo
1-AM before, during, and after exposure to a low concentration (0.1% [vol/voll) of ethanol (ETOH). Bottom panel:
Tracings depicting, on an expanded time scale, the indo 1
fluorescence transients and contractions at times A, B, and C
in upper tracing. ETOH causes a 10% reduction in contractile
amplitude, but the fluorescence transients before, during, and
after ETOH exposure are superimposable.
does not affect the indo 1 fluorescence ratio or alter
the Ca2' response of the indo 1 ratio. This was
determined by addition to ETOH to droplets of indo
1-free acid at diastolic (100 nM) and systolic (1,000
nM) Ca' levels. A similar result (i.e., contractile
depression at 0.15% ETOH without a reduction in
the cytosolic Ca> transient), measured via aequorin
luminescence, has been recently observed in intact,
isolated ferret heart muscle.20
Higher concentrations of ETOH (3% [vol/vol])
elicited a reversible decline in twitch amplitude and a
reduction in the amplitude of the Caj transient that
leads to myofilament shortening (Figure 3). Note that
ETOH also causes a decrease in resting indo 1
fluorescence accompanied by an increase in the
resting cell length; both changes are reversible on
ETOH washout. Figure 4 shows that an increase in
Cao can partially reverse this ETOH-induced twitch
depression. A spectrum of twitch and Caj transients
produced by changing Ca0 (0.25-8 mM) in the absence or presence of ETOH is depicted in panels A
and B. Contraction amplitudes associated with a Caj
transient of the same amplitude in the presence and
absence of ETOH are depicted in panel C. For a
given Caj transient, the twitch is substantially depressed in the presence of ETOH. This suggests that
ETOH affects the myofilament-Ca2` interaction.
This is shown clearly in panel D, which depicts the
ETOH Addition to Unstimulated Myocytes
Monitoring the spontaneous focal propagated
wave frequency that occurs in some resting rat myocytes16 allows further inferences to be made on the
mechanism of the decrease in the Caj transient by
ETOH. These contractile waves result from a spontaneous localized release of Ca2' from the SR, which
is thought to propagate by diffusion, and regenerative
Ca2`-induced Ca2' release at the wave front.22 One
determinant of the frequency of contractile wave
occurrence is the extent to which the SR is Ca2+loaded.13,23 Figure 5 shows that ETOH initially increases the wave frequency by 50%. The initial
concomitant decrease in wave amplitude is attributable to the increased frequency.23 Eventually, as the
cell apparently becomes Ca2'-depleted, the amplitude decreases further. The increase in wave frequency suggests that a primary effect of ETOH on
the SR is to increase Ca2' release similar to that
described previously for caffeine,23 although an increase in the sarcolemmal Ca21 permeability due to
ETOH could also produce a similar result. Enhanced
SR Ca2' release subsequently leads to SR Ca21
depletion.
The hypothesis that ETOH leads to SR Ca'
release was tested in indo 1-loaded myocytes. Figure
6 shows that addition of 3% (vol/vol) ETOH to an
unstimulated cell leads to a spontaneous rapid increase in indo 1 fluorescence followed by smaller
oscillations, during which the resting fluorescence
remains elevated. This is followed by a gradual
decrease in resting indo 1 fluorescence. Additional
experiments with suspensions of cardiac myocytes
loaded with quin 2 allow further quantitative insights
to be made into the ETOH-induced depletion of SR
Ca`. Figure 7A illustrates a protocol used to assess
an increase in Caj by SR Ca>2 release induced by the
acute addition of caffeine. Figure 7B shows the effect
of the addition of ETOH on resting Caj and on the
subsequent caffeine response. Figure 8 shows that
the fluorescence transient induced by the addition of
caffeine after ETOH (1-5% [vol/vol]) is decreased in
a dependent manner. In these experiments any contribution to the Caj increase by caffeine via sarcolemmal influx is prevented by prior addition of EGTA to
the cuvette. Thus, the net increase in fluorescence
after the addition of caffeine is due to a release of
Ca21 from the SR.
Discussion
The present study shows that acute ETOH exposure reversibly depresses contraction in single cardiac
myocytes, just as in intact isolated myocardium and in
the heart in situ.1-5,7-11 The results indicate that the
effect of ETOH in amounts routinely ingested by
man, that is, those leading to clinically relevant
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Circulation Research Vol 68, No 6 June 1991
1664
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FIGURE 3. Panel A: Tracings showing that 3%
(vol/vol) ethanol (ETOH) produces a reversible
decline in the cytosolic Ca2+ transient, measured
as the transient increase in indo 1 fluorescence
ratio (410/490 nm) in response to electrical stimulation, depresses twitch amplitude, and subsequently abolishes the twitch. (Contraction proceeds in a downward direction). The time scale is
as follows: a, control; b and c, during acute
ETOH exposure; d and e, after ETOH removal.
The resting indo 1 fluorescence ratio decreases
during ETOH exposure (compare b vs. c), and
this is accompanied by an increase in resting
length. All ETOH effects are reversible after
ETOH removal (d and e). Note the break in the
time scale after c and before e. Panel B: Tracings
of contractions and indo 1 transients on an
expanded time scale at times a-e in panel A.
c
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108.
250 msec
ETOH plasma concentrations (0. 1- 0. 15 % [vol/vol]),
is to depress contraction in the absence of a reduction in the Caj transient. A similar result for clinically
relevant ETOH concentration (0.15%) has recently
been observed in intact isolated heart. In that study,21
contractile depression was observed not only in the
absence of a reduction in the Ca, transient but also in
the absence of a change in the transmembrane action
potential configuration. These results suggest that a
reduced myofilament response to Ca2+ underlies the
contractile depression at clinically relevant ETOH
concentrations. The specific mechanism by which
ETOH alters myofilament response to Ca2+ in the
intact cell cannot be determined from the present
experiments. However, it may be related to decreased Ca2' binding to the myofilaments.1' Because
ETOH in any concentration used in the present study
does not cause a reduction of pH or an increase in
inorganic phosphate,7 these are not likely a cause of
a decreased myofilament Ca2+ response effected by
ETOH.
At higher ETOH concentrations (1-5% [vol/vol]),
contractile depression is the result, in part, of both a
decreased Caj transient and an altered Ca24myofilament interaction during an electrically stimulated contraction. Thus, these alterations, in addition
to sarcolemmal membrane effects,24-26 appear to play
a role in the "general anesthetic" effects of high
concentrations of ETOH. The elucidation of the
mechanisms for the contractile depression by high
concentrations of ETOH and the observation that
profound contractile depression at the cellular level
is reversible may be of significance with respect to the
quest for more optimal modalities of cardioplegia.
The present results suggest that even very high (5%
[vol/vol]) concentrations of ETOH do not cause a
generalized increase in sarcolemmal ion permeability, because if this were the case, Ca2' ions would
flow along their electrochemical gradient and produce cytosolic Ca2' overload; clearly this does not
occur.
The addition of high concentrations of ETOH to
single unstimulated myocytes caused an increase in
indo 1 fluorescence. That this ETOH effect (an
increase in Caj in unstimulated cells) resulted from
Ca2' release from SR is suggested by the diminished
effect of a subsequent addition of caffeine (an increase in Caj in cell suspensions). That ETOH initially increases spontaneous focal propagated contractile wave frequency in resting cells may be a
functional manifestation of its enhancement of SR
Ca24 release. However, the possibility that the SR
Ca24 pump is simultaneously inhibited14 and contributes to SR Ca24 depletion cannot be excluded on the
Danziger et al Ethanol Effects
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FIGURE 5. Sequential sample tracings of the occurrence of
spontaneous focal propagated contractile waves in unstimulated myocytes bathed in 1 mM [Ca2"] before and during
superfusion with 3% (vol/vol) ethanol (ETOH). Note that
ETOH initially increases the wave frequency. The wave amplitude then decreases, and discrete contractile waves are no
longer detected.
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FIGURE 4. Panels A and B: Tracings showing indo 1
fluorescence ratio (410/490 nm) and cell length in a myocyte
bathed in varying [Ca2+] and stimulated at 1 Hz in steady
state before (panel A) and during (panel B) exposure to 3%
(vol/vol) ethanol (ETOH). The bathing [Ca2"] (mM) is the
number adjacent to each fluorescence or length transient.
During ETOH exposure for a fluorescence transient of the
same amplitude present in control, the contraction amplitude
is decreased. Panel C: Tracings showing that fluorescence
transients of equal magnitude before and during ETOH
exposure produce a markedly depressed contraction during
ETOH. Panel D: Plots showing twitch amplitudes measured
in a given bathing [Ca2"] in panels A and B as a function of
the corresponding peak fluorescence transient achieved after
each excitation. Note that the points in ETOH are markedly
shifted downward and to the right.
basis of these experiments. The subsequent decreases in resting Ca, and wave frequency following
their initial increases after higher ETOH concentra-
tions in single myocytes likely occurs because Ca2`
removed from the cytosol, and probably that removed from the cell via sarcolemmal efflux mechanisms, depletes the SR of Ca`+. Thus, the present
results suggest that a prominent effect of high concentrations of ethanol, like that of the myocardial
depressant halothane,27 is to decrease the amount of
Ca2+ stored within the SR. The reversal of ETOH
contractile depression by norepinephrine (Figure 1B
and Table 1) or by increased Ca. (Figure 4) suggests
that, if increased Ca2+ is available to the SR, it can be
pumped into the SR and that this compensates for a
direct depletion of SR Ca2+ content by ETOH (even
in the presence of an alteration by ETOH of the
myofilament response to Ca2`). In the case of norepinephrine, the reversal of the ETOH effect by a
direct cAMP-dependent stimulation of the SR Ca2+
pump may also be involved.28-30
The reversibility of profound contractile depression of Ca2+ ions or after ETOH washout indicates
that, under the conditions of the present study,
generalized membrane damage due to protein denaturation does not occur. A similar reversibility of
ETOH effect in cardiac plasma membranes has previously been observed.31 On washout of high ETOH
concentrations, an overshoot in contraction amplitude was observed, often associated with spontaneous contractile oscillations occurring in the interstimulus interval, a sign of cell Ca2 overload.32 A similar
overshoot in myocardial contraction amplitude occurs in intact perfused rat hearts.21 In that model the
magnitude of the contractile overshoot after ETOH
washout varied directly with the extent of contractile
expression during ETOH exposure. This apparent
Ca21 overload after washout of high concentrations
of ETOH may be related, in part, to an increase in
Ca2' binding of cardiac plasma membrane lipoproteins by ETOH.33
Although the present study does not specifically
address the effect of ETOH on sarcolemmal ion flux,
Circulation Research Vol 68, No 6 June 1991
1666
1.681
1.681
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0.-.
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FIGURE 6. Tracings showing that
the addition of 3% (vol/vol) ethanol
(ETOH) to a representative unstimulated myocyte leads to a rapid
increase in indo 1 fluorescence ratio
followed by fluorescence oscillations
about an elevated resting value.
(The inset shows an amplified and
filtered tracing of the initial ETOHinduced events). After additional
time in ETOH, the average fluorescence decreases.
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the observed decrease in Ca, with time after the initial
ETOH-induced increase in individual resting cells in
conjunction with a decrease in SR Ca>2 stores suggests
that a net cellular Ca' efflux occurs; that is, either Ca'
influx into the cell is inhibited, or efflux is enhanced.
Studies in mechanically skinned single cardiac cell
fragments indicate that a large Ca>2 release from the
SR can be triggered by a small quantity of Ca2+13: in
intact cells, the Ca>2 trigger is Ca>2 flux via L-type Ca>2
channels.34-38 A reduction in the magnitude of L-type
sarcolemmal Ca>2 current by ETOH39A40 likely contributes to the decrease of SR Ca>2 release and contractile
amplitude elicited by electrical stimulation in the presence of a high concentration of ETOH. When the Ca0
is increased or when norepinephrine is added to the
bathing solution, an enhanced Ca2' trigger for Ca2'
release would also be expected to occur. Thus, the
effect of these perturbations (reversal of the ETOH
effect) could occur via this mechanism, in addition to
their other effects (Ca2+ loading of the cell and stimulation of SR Ca24 pumping). The inexcitability of most
cells in 5% ETOH likely occurs because of total action
potential failure.39,40 ETOH also inhibits Na-K pump
activity in cultured rat heart cells.41 In summary, multiple effects of ETOH underlie its depressant effects on
cardiac contractile function. At clinically relevant
ETOH concentrations, this appears to occur as a result
of a depression of the myofilament response to Ca2`. At
higher ETOH concentrations, impairments of excitation-contraction coupling mechanisms occur at both
B
A
1004
W
Z
\
20
WI
U.)
WI
0
0
WO0
60
U0
3 min
+
t
CJ
Q
0
WZ-1
1
40-
Ul)
W
Z
cc
x
W
U.
c
W
U.
x
0
U.
FIGURE 7. Tracings from experiments with suspensions of
quin 2-loaded myocytes to assess whether ethanol (ETOH)
depletes Ca>2 content of the sarcoplasmic reticulum. Protocols
for the introduction of quin 2 into the cells and for the study of
fluorescence are given in "Materials and Methods. " Panel A:
Addition of caffeine after EGTA to control, resting myocytes
gives an increase in fluorescence that is used as an index of
Ca>2 content of the sarcoplasmic reticulum. DTPA, diethylenetriaminepentaacetic acid. Panel B: Subsequent addition of
caffeine after ETOH exposure leads to a reduced Ca, transient
compared with control in panel A.
20}
0
13
5
% ETOH
FIGURE 8. Graph showing the diminution of the fluorestransient in response to caffeine. Ethanol (ETOH)
data were previously obtained in experiments of the type
shown in Figure 7, using three different preparations of cells.
The response to caffeine, after ETOH addition, is presented
as a percentage of the unattenuated response to caffeine in
control cells.
cence
Danziger et al Ethanol Effects on Cardiac Myocytes
the SR and myofilaments and probably (though not
investigated in the present study) at the sarcolemma as
well. In this regard, the ETOH effects resemble those
of the local anesthetic, halothane.27 Each of these
ETOH effects is reversible within minutes after washout and can be overcome in the continued presence of
ETOH by an increase in Ca0 or by the addition of
norepinephrine. The present results, thus, also suggest
a cellular mechanism by which reflex adrenergic stimulation of the heart serves to compensate for an ETOH
depression of cardiac function.9-11
References
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1. Mason DT, Spann JF, Miller RR, Lee G, Arbogast R, Segel
LD: Effect of acute ethanol on the contractile state of normal
and failing cat papillary muscles. Eur J Cardiol 1978;714:
311-316
2. Horwitz LD, Atkins JM: Acute effects of ethanol on left
ventricular performance. Circulation 1974;49:124-128
3. Regan TJ, Koroxenidis G, Moschos CB, Oldewurtel HA,
Lehan PH, Hellems HK: The acute metabolic and hemodynamic responses of the left ventricle to ethanol. J Clin Invest
1966;45:270-280
4. Kobayashi M, Furukawa Y, Chiba S: Effects of ethanol and
acetaldehyde on the isolated, blood-perfused canine atrium.
Arch Int Pharmacodyn 1979;239:109-120
5. Lochner A, Cowley R, Brink AJ: Effect of ethanol on metabolism and function of perfused rat heart. Am Heart J 1969;78:
770-780
6. Hirota Y, Bing OHL, Abelmann WH: Effect of ethanol on
contraction and relaxation of isolated rat ventricular muscle. J
Mol Cell Cardiol 1976;8:727-732
7. Schulman SP, Lakatta EG, Weiss RG, Wolff MR, Hano 0,
Gerstenblith G: Contractile, metabolic, and electrophysiologic
effects of ethanol in the isolated rat heart. J Mol Cell Cardiol
(in press)
8. Auffermann W, Wu S, Parmley WW, Higgins CB, Sievers R,
Wikman-Coffelt J: Reversibility of acute alcohol cardiac
depression: 31-P NMR in hamsters. FASEB J 1988;3:256-263
9. Delgado CE, Fortunin NJ, Ross RS: Acute effects of low doses
of alcohol on left ventricular function by echocardiography.
Circulation 1975;51:535-540
10. Kalbaek H, Gjorup T, Brynjolf I, Christensen NJ, Godtfredsen
J: Acute effects of alcohol on left ventricular function in
healthy subjects at rest and during upright exercise. Am J
Cardiol 1985;55:164-167
11. Child JS, Kovick RB, Levisman JA, Pearce ML: Cardiac
effects of acute ethanol ingestion unmasked by autonomic
blockade. Circulation 1979;59:120-125
12. Katz AM: Physiology of the Heart. New York, Raven Press,
Publishers, 1977
13. Fabiato A: Time and calcium dependence of activation and
inactivation of calcium induced release of calcium from the
sarcoplasmic reticulum of a skinned canine cardiac Purkinje
cell. J Gen Physiol 1985;85:247-289
14. Swartz MH, Repke DI, Katz AM, Rubin E: Effects of ethanol
on calcium binding and calcium uptake by cardiac microsomes.
Biochem Pharmacol 1974;23:2369-2376
15. Puszkin S, Rubin E: Effects of ADP, ethanol and acetaldehyde
on the relaxing complex of human muscle and its adsorption by
polystyrene particles. Arch Biochem Biophys 1976;177:574-584
16. Capogrossi MC, Kort A, Spurgeon HA, Lakatta EG: Single
adult rabbit and rat cardiac myocytes retain the Ca24- and
species-dependent systolic and diastolic contractile properties
of intact muscle. J Gen Physiol 1986;88:589-613
17. Spurgeon HA, Stern MD, Baartz G, Raffaeli S, Hansford RG,
Talo E, Lakatta EG, Capogrossi MC: Simultaneous measurements of Ca2' contraction and potential in cardiac myocytes.
Am I Physiol 1990;258(Heart Circ Physiol 27):H574-H586
1667
18. Poenie M, Alderton J, Steinhardt R, Tsien R: Calcium rises
abruptly and briefly throughout the cell at the onset of
anaphase. Science 1986;233:886-889
19. Hansford RG, Lakatta EG: Ryanodine releases calcium from
sarcoplasmic reticulum in calcium-tolerant rat cardiac myocytes. J Physiol (Lond) 1987;390:454-467
20. Guarnieri T, Lakatta EG: Mechanism of myocardial contractile depression by clinical concentrations of ethanol. J Clin
Invest 1990;85:1462-1467
21. Schulman SP, Gerstenblith G, Lakatta EG: Functional and
arrhythmogenic effects of ethanol withdrawal in the isolated
rat heart (abstract). Clin Res 1989;37:522A
22. Stern MD, Capogrossi MC, Lakatta EG: Propagated contractile waves in single cardiac myocytes modeled as regenerative
calcium induced calcium release from the sarcoplasmic reticulum (abstract). Biophys J 1984;45(pt 2):94a
23. Kort AA, Capogrossi MC, Lakatta EG: Frequency amplitude,
and propagation velocity of spontaneous Ca24-dependent contractile waves in intact adult rat cardiac muscle and isolated
myocytes. Circ Res 1985;57:844-855
24. Taraschi TF, Rubin E: Biology of disease: Effects of ethanol
on the chemical and structural properties of biologic membranes. Clin Invest 1985;52:120-131
25. Polimeni PI, Otten MD, Hoeschen LE: In vivo effects of
ethanol on the rat myocardium: Evidence for a reversible,
non-specific increase of sarcolemmal permeability. J Mol Cell
Cardiol 1983;15:113-122
26. Katz AM: Effects of ethanol on ion transport in muscle
membranes. Fed Proc 1982;41:2456-2458
27. Wheeler DM, Rice RT, Hansford RG, Lakatta EG: The effect
of halothane on the free intracellular calcium of isolated rat
heart cells. Anesthesiology 1988;69:578-583
28. Kirchberger MA, Tada M, Repke DI, Katz AM: Cyclic
adenosine 3'5'-monophosphate dependent protein kinase
stimulation of calcium uptake by canine cardiac microsomes. J
Mol Cell Cardiol 1972;4:673-680
29. Tada M, Ohmori F, Repke DI, Katz AM: Mechanism of the
stimulation of Ca24-dependent ATPase of cardiac sarcoplasmic reticulum by adenosine 3'5'-monophosphate-dependent
protein kinase. J Biol Chem 1979;254:319-326
30. Watanabe AM: Recent advances in knowledge about beta
adrenergic receptors: Application to clinical cardiology. JAm
Coll Cardiol 1983;1:82-89
31. Williams JW, Tada M, Katz AM, Rubin E: Effect of ethanol
and acetaldehyde on the (Na++K+)-activated adenosine triphosphatase activity of cardiac plasma membranes. Biochem
Pharmacol 1975;24:27-32
32. Capogrossi MC, Stern MD, Spurgeon HA, Lakatta EG:
Spontaneous Ca24 release from the sarcoplasmic reticulum
limits Ca24-dependent twitch potentiation in individual cardiac myocytes: A mechanism for maximum inotropy in the
myocardium. J Gen Physiol 1988;91:133-155
33. Ohnishi ST, Obzansky DM, Price HL: The increase in calcium
binding of cardiac plasma membrane lipoprotein caused by
general anesthetics and alcohol. Can J Physiol Pharmacol
1980;58:525-530
34. Talo A, Stern MD, Spurgeon HA, Isenberg G, Lakatta EG:
Sustained subthreshold-for-twitch depolarization in rat single
ventricular myocytes causes sustained calcium channel activation and sarcoplasmic reticulum calcium release. J Gen Physiol
1990;90:1085-1103
35. Talo A, Spurgeon HA, Lakatta EG: The relationship of
calcium current, sarcoplasmic reticulum function and contraction in single rat ventricular myocytes excited in the rested
state, in Clark WA, Decker RS, Borg TK (eds): Biology of
Isolated Adult Cardiac Myocytes. New York, Elsevier Science
Publishing Co, Inc, 1988, pp 362-365
36. London B, Krueger JW: Contraction in voltage-clamped,
internally perfused single heart cells. J Gen Physiol 1986;88:
475-505
37. Callewaert G, Cleemann L, Morad M: Epinephrine enhances
Ca24 current-regulated Ca24 release and Ca24 reuptake in rat
ventricular myocytes. Proc Natl Acad Sci USA 1988;85:
2009-2013
1668
Circulation Research Vol 68, No 6 June 1991
38. Beuckelmann DJ, Wier WG: Mechanism of release of calcium
from sarcoplasmic reticulum of guinea-pig cardiac cells. J
Physiol (Lond) 1988;405:233-255
39. Takeda R, Momose Y, Nakanishi S: Effects of ethanol on
calcium and potassium currents in single bullfrog atrial cells.
Jpn J Pharmacol 1984;36:422- 424
40. Ikeda K, Hachisuka M, Goto H: Effects of ethanol on membrane currents and contractility in frog atrial cells. J Physiol
Soc Jpn 1984;48:113-120
41. McCall D, Ryan K: The effect of ethanol and acetaldehyde on
(Na+ ) sodium pump function in cultured rat heart cells. J Mol
Cell Cardiol 1987;19:453-463
KEY WORDS * ethanol * single isolated cardiac myocytes
contractility * cytosolic calcium * fluorescent Ca`- probes
indo 1 * quin 2
-
.
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Ethanol acutely and reversibly suppresses excitation-contraction coupling in cardiac
myocytes.
R S Danziger, M Sakai, M C Capogrossi, H A Spurgeon, R G Hansford and E G Lakatta
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Circ Res. 1991;68:1660-1668
doi: 10.1161/01.RES.68.6.1660
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