1 Departments of Pharmaceutical Sciences and Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205; and 2 Department of Pathology, St. Louis University Medical School, St. Louis, Missouri 63104
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ABSTRACT |
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We previously reported that lysoplasmenylcholine (LPlasC) altered the action potential (AP) and induced afterdepolarizations in rabbit ventricular myocytes. In this study, we investigated how LPlasC alters excitation-contraction coupling using edge-motion detection, fura-PE3 fluorescent indicator, and perforated and whole cell patch-clamp techniques. LPlasC increased contraction, myofilament Ca2+ sensitivity, systolic and diastolic free Ca2+ levels, and the magnitude of Ca2+ transients concomitant with increases in the maximum rates of shortening and relaxation of contraction and the rising and declining phases of Ca2+ transients. In some cells, LPlasC induced arrhythmias in a pattern consistent with early and delayed aftercontractions. LPlasC also augmented the caffeine-induced Ca2+ transient with a reduction in the decay rate. Furthermore, LPlasC enhanced L-type Ca2+ channel current (ICa,L) and outward currents. LPlasC-induced alterations in contraction and ICa,L were paralleled by its effect on the AP. Thus these results suggest that LPlasC elicits distinct, potent positive inotropic, lusitropic, and arrhythmogenic effects, resulting from increases in Ca2+ influx, Ca2+ sensitivity, sarcoplasmic reticular (SR) Ca2+ release and uptake, SR Ca2+ content, and probably reduction in sarcolemmal Na+/Ca2+ exchange.
calcium; lipid metabolites; excitation-contraction coupling; heart
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INTRODUCTION |
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AN INCREASE IN
membrane-associated, Ca2+-independent phospholipase
A2 (PLA2) activity in mammalian cardiac
ventricular myocytes occurs during short intervals of hypoxia
(20) or after exposure to interleukin-1, a
proinflammatory cytokine (19). Activation of
membrane-associated, Ca2+-independent PLA2
results in selective hydrolysis of membrane plasmalogen phospholipids
and accumulation of lysoplasmenylcholine (LPlasC; see Ref.
20). Lipid metabolism is altered within seconds of
myocardial ischemia, and significant accumulation of
amphiphilic metabolites such as LPlasC and the structurally similar
compounds lysophosphatidylcholine (LPC) and palmitoylcarnitine have
been demonstrated within 2 min (15, 21). Under these
conditions, the ischemic heart often exhibits
electrophysiological abnormalities, including ventricular arrhythmias
within 1-2 min (15, 21). Perfusion of normoxic
cardiac myocytes with LPC and palmitoylcarnitine causes changes in the
action potential (AP) configuration, suggesting that these amphiphilic
metabolites are potentially arrhythmogenic. Moreover, inhibition of the
accumulation of these lipid metabolites in the ischemic
myocardium is associated with a decrease in arrhythmogenesis (8). Recently, we have demonstrated that LPlasC also
exerts an arrhythmogenic effect on normoxic ventricular myocytes by
altering the AP configuration, thereby inducing early and delayed
afterdepolarizations at concentrations significantly lower than those
reported previously for LPC and palmitoylcarnitine (20).
The arrhythmogenic effect of LPC has been attributed to its ability to exert a nonspecific effect on the biophysical properties of membrane phospholipids, resulting in an inhibition of most cardiac currents (for review, see Refs. 21 and 27). Similar to LPC, palmitoylcarnitine blocks inward-rectifier K+ channels (IK1; see Ref. 22), the Na+ current (23), the L-type Ca2+ channel current (ICa,L; see Ref. 30), and the Na+/K+ pump current (25) in mammalian ventricular myocytes. In contrast, palmitoylcarnitine was also shown to activate a slow-inactivating Na+ current followed by an increase in a transient inward current (31) but lacked effects on IK1 and transient outward K+ currents (32). Nevertheless, these LPC- and palmitoylcarnitine-induced electrophysiological changes could not satisfactorily account for the observed concomitant positive inotropic effect (1, 25) and the increased intracellular Ca2+ (Cai; see Ref. 33). In contrast to LPC and palmitoylcarnitine, little is known about the cellular mechanism underlying LPlasC-induced changes in cardiac contractile and electrical function.
Disturbance of Ca2+ handling such as that elicited by increasing Ca2+ influx or Ca2+ release from sarcoplasmic reticulum (SR), and/or by decreasing Ca2+ efflux, leads to Cai overload that is often associated with cardiac arrhythmias (for review, see Ref. 7). Thus, in this study, we investigated the effects of LPlasC on contraction, Ca2+ transients, and membrane currents to assess its actions on excitation-contraction coupling in adult rabbit ventricular myocytes. We found that LPlasC exerted potent, distinct effects on contractile and electrical functions in ventricular myocytes. These effects could be accounted for by LPlasC-induced alterations in Cai handling.
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MATERIALS AND METHODS |
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Myocyte isolation. The protocol for the use of animals in this study conformed with the National Institutes of Health approved Guide for the Care and Use of Laboratory Animals and was approved by the Institutional Animal Care and Use Committee, University of Arkansas for Medical Sciences.
Single adult ventricular myocytes were isolated from the hearts of adult rabbits (either sex, 2-3 kg), as described previously (20). Isolated ventricular myocytes were harvested and plated in 60-mm culture dishes (Falcon) for 2 h or overnight in culture medium composed of 60% medium-199 (GIBCO, Grand Island, NY) and 36% Earle's balanced salt solution containing (in mM) 116 NaCl, 4.7 KCl, 0.9 NaH2PO4, 0.8 MgSO4, 26 NaHCO3, 5.6 glucose, and 4% FBS (pH 7.40 in 5% CO2-95% air at 37°C; GIBCO), as described previously (17). Rod-shaped cells with clear striations were used for experiments, and there was no significant difference in the response to lipid metabolites between freshly isolated and primary cultured myocytes (within 24 h). All experiments were carried out at 35-37°C.Measurement of cell shortening.
Unloaded cell shortening (CS) or contraction of myocytes was elicited
in normal Tyrode solution containing (in mM) 140 NaCl, 5.4 KCl, 1 CaCl2, 0.8 MgCl2, 10 HEPES/Tris, and 5.6 glucose (pH = 7.40 at 37°C; 290 mosmol/kgH2O)
through field stimulation with bipolar platinum electrodes at a
frequency of 0.5 Hz with 1- to 2-ms voltage pulses. Cells were then
superfused with normal Tyrode solution containing ethanol, followed by
solutions with different concentrations of LPlasC. The stimulating
electrode was placed near the suction pipette in the perfusion chamber
to minimize its damage to cells during long periods of stimulation. The
video signal was fed into the video motion detector (Crescent
Electronics, Sandy, UT) connected to a video monitor through a
charge-coupled device video camera mounted to a microscope. The analog
voltage output was calibrated to indicate actual micrometers of cell
motion and recorded on a personal computer using pClamp software (Axon Instruments). Measured parameters of contractile function in myocytes included peak magnitude of CS, maximum rates of contraction
(+dL/dtmax) and relaxation
(dL/dtmax), and rise and decay
times between 10 and 90% of peak CS.
Measurement of intracellular free
Ca2+ concentration.
Ventricular myocytes seeded on 25-mm coverslips in culture medium were
loaded for 30 min in a culture incubator at 37°C with 2 µM
fura-PE3-AM (TEFLABS, Austin, TX), a cell-permeable form of fura-PE3
that is a new analog of fura 2 and is retained inside the cell longer
than fura 2. Myocytes were then transferred to a recording/perfusion
chamber (Harvard Apparatus, Holliston, MA) on the stage of an inverted
microscope (model TE300; Nikon, Irving, TX) and superfused with normal
Tyrode solution. Fluorescent measurements were made through a ×40
long-working-distance ultraviolet (UV) objective (Nikon Fluor with
numerical aperture of 1.3). Fura-PE3-loaded cells were alternately
excited with UV light at 340- and 380-nm wavelengths via a filter
wheel, controlled by a spectrophotometry unit (Cairn Research) at
60-75 Hz. The emitted fluorescence signal at 510 nm was collected
through an adjustable diaphragm and a photomultiplier tube (Cairn) to
the spectrophotometer control unit. The signals were sampled at
200-300 Hz using pClamp software (Axon Instruments) and stored in
a personal computer for later calibration and analysis. After
subtraction of the background signal, fluorescent signals were recorded
as the ratio (R or f340/f380) of the
fluorescent intensity when excited at 340 nm (f340) to that
when excited at 380 nm (f380). Because of difficulties with the in vivo calibration procedure, many results were represented as
f340/f380. The measured parameters of the
Ca2+ transient included peak magnitude, maximum rates of
the rising phase (+dR/dtmax) and the declining
phase (dR/dtmax), and the rise and decay times
between 10 and 90% of peak amplitude. In some experiments with
successful in situ calibrations, cytosolic free Ca2+
concentrations were determined using the equation (13)
[Ca+]i = Kd ×
× (R
Rmin)/(Rmax
R) where
Kd is the apparent dissociation constant of 224 nM at 37°C and
is the ratio of f380,free to f380,bound measured under Rmin and
Rmax conditions, respectively. Rmin is the
minimum fluorescent ratio in Ca2+-free solutions containing
3 µM ionomycin and 10 mM EGTA, and Rmax is the maximum
intensity ratio in perfusion buffer solution containing 3 µM
ionomycin and 2 mM CaCl2. In some experiments, myocyte
contraction was recorded simultaneously with Ca2+
transients when the cells were illuminated with a halogen lamp (Nikon)
through a long-wavelength pass filter (640 nm; Chroma).
Electrophysiological measurements.
Ventricular myocytes were perfused with normal Tyrode solution and
patch-clamped using perforated-patch (16) or conventional whole cell patch techniques (14) with a patch-clamp
amplifier (Axopatch 200A; Axon Instruments; see Refs. 17
and 18). APs of myocytes were measured in normal Tyrode solution and
K+-rich pipette solutions only in perforated-patch clamp
configurations, as described previously (18, 20). The
current-voltage (I-V) relationship of the
steady-state membrane current was obtained by applying 300-ms voltage
step pulses to potentials between 120 and +80 mV from the holding
potential in 20-mV increments at 0.2 Hz or a voltage ramp between
120
and +80 mV at a rate of 1 V/s.
Synthesis of LPlasC. LPlasC was prepared by alkaline hydrolysis of bovine heart choline glycerophospholipids, as described previously (9). The LPlasC product was isolated by column chromatography on a 2.5 × 60-cm column of silica using a stepwise gradient elution procedure. According to our previous study (20), the majority of experiments in this study used 1 µM LPlasC, which elicited apparent cardiac effects, unless otherwise indicated.
Chemicals.
Most reagents were purchased from Sigma Chemical (St. Louis, MO) and
were added directly when needed. Stock solutions of lipid metabolites
(102 M) were prepared in 100% ethanol. The final
concentration of ethanol in extracellular solutions was <0.01% and
had no significant effect (<10%) on measured parameters.
Statistics. In all experiments, data in response to LPlasC were compared with the steady-state control before treatment in each individual cell and thus expressed as a ratio or percentage of each control value before combining for statistical analysis. Values are presented as means ± SE. Statistical significance was evaluated by the two-tailed Student's paired t-test or, when more than two conditions were compared, by one- or two-way ANOVA with Duncan's multiple-range test. Differences with P < 0.05 were considered significant.
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RESULTS |
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Effects of LPlasC on contraction of intact ventricular myocytes.
We first examined the effect of LPlasC on the unloaded contractile
function of intact ventricular myocytes under physiological conditions.
Figure 1 shows that LPlasC increased
contractility, which reached a plateau in 5 min and was followed by a
small decrease. In combined data, LPlasC elicited a maximum positive
inotropic effect (2.59 ± 0.44-fold increase, n = 25) in 3-8 min (varied from cell to cell), followed by a decline
(83.9 ± 4.3% of the maximum, n = 18). After the
baseline to the control level was offset, Fig. 1C shows
superimposed CS before, during, and after exposure to 1 µM LPlasC, as
shown in Fig. 1B, each of which was obtained by averaging
five to six shortening traces. The first derivative of contraction
traces (Fig. 1C, inset) shows that
+dL/dtmax and
dL/dtmax were increased
dramatically during exposure to LPlasC. Combined data in Fig.
1E show that LPlasC caused a 3-fold and 4.3-fold increase in
+dL/dtmax and
dL/dtmax, respectively, concomitant with reductions in the rise time and decay time of contraction. Normalized traces (to each peak amplitude) as shown in Fig.
1D confirmed that LPlasC had a more profound effect on
dL/dtmax than
+dL/dtmax, and combined data show 41 and 18% increases in
dL/dtmax and
+dL/dtmax, respectively (Fig.
1E). Thus LPlasC elicited positive inotropic and lusitropic
effects in adult rabbit ventricular myocytes. Figure 1 also shows that,
after removal of LPlasC, a rebound stimulation of contraction was
observed before returning to the control level. Nine of twenty-five
tested myocytes showed the rebound activation of contraction (1.32 ± 0.11-fold of the amplitude before LPlasC removal), whereas nine
cells did not (also see Fig. 2). The remaining cells did not survive
during LPlasC exposure or washout because of arrhythmia and/or
contracture.
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Effects of LPlasC on the Ca2+
transient in fura-PE3-loaded ventricular myocytes.
The effect of LPlasC on Cai handling in fura-PE3-loaded
myocytes was examined under conditions similar to those described for
CS. Figure 2 shows results from
simultaneous measurements of CS (Fig. 2A) and the
Ca2+ transient (Fig. 2B) in a myocyte. LPlasC
caused rapid increases in systolic and diastolic Ca2+
levels, and the magnitude of the Ca2+ transient,
accompanied by a fourfold increase in CS, an inotropic response similar
to that observed in non-fura-PE3-loaded cells. The time course for the
increase in Ca2+ transients appears to be more rapid than
that of the positive inotropy (: 63.8 vs. 91.2 s), suggesting
that LPlasC increases Ca2+ cycling and alters the
Ca2+ sensitivity of contractile machinery. Upon removal of
LPlasC, Ca2+ transients and contraction partially recovered
without a rebound activation. Steady-state Ca2+ transients
and contraction before and during LPlasC exposure were superimposed and
shown in Fig. 2, D and C, respectively. It is
noticeable in Fig. 2C that an aftercontraction was
developing 8 min after exposure to LPlasC compared with the control. In
addition, Fig. 2D, inset, shows that LPlasC
increased +dR/dtmax and
dR/dtmax of the Ca2+ transient.
The LPlasC-induced increases in systolic and diastolic f340/f380 were 0.041 ± 0.004 and
0.019 ± 0.002 (n = 29), respectively, which
approximated 340 and 80 nM of free Cai, respectively.
Combined data in Fig. 2E show that LPlasC increased the
magnitude of the Ca2+ transient approximately twofold
and doubled +dR/dtmax and
dR/dtmax. It is also worth mentioning that the
declining phase of the Ca2+ transient in the presence of
LPlasC was better fit by a biexponential function, whereas it was best
fit by a single exponential in control conditions. The
LPlasC-associated initial rapid phase (i.e.,
dR/dtmax) and later slow phase of the
f340/f380 decline were more rapid and slower
than the decay time constant in control, respectively. As a
consequence, LPlasC increased the area under the Ca2+
transient and the decay time (Fig. 2E).
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Arrhythmiogenic effect of LPlasC.
In many cells, LPlasC induced arrhythmias with different patterns and
severity that varied from cell to cell. For example, 1 µM LPlasC
elicited mild arrhythmias in one cell (Fig.
5A), whereas it induced more
severe arrhythmias in another cell (Fig. 5B). In Fig.
5A, inset, potentiated contractions were preceded
by early aftercontractions and oscillations. In contrast, primarily
delayed aftercontractions were observed in the cell shown in Fig.
5B. Figure 5C shows Ca2+ transients
during LPlasC-induced arrhythmias; an apparent increase in systolic
Cai was followed by Ca2+ transients in a
pattern consistent with early and delayed aftercontractions 2 min after
exposure to LPlasC. Figure 5C also shows that SR
Ca2+ load was reduced after the arrhythmia but gradually
returned to the control level upon removal of LPlasC. When the
concentration of LPlasC was increased to 10 µM, severe arrhythmias
and contracture occurred in <1 min in all four cells tested (data not
shown).
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Effects of LPlasC on ICa,L in patch-clamped ventricular
myocytes.
We next examined whether LPlasC-elicited increases in systolic
Cai and postcaffeine Ca2+ influx result from an
enhancement of ICa,L. In a perforated-patch configuration, ICa,L was monitored before,
during, and after exposure to LPlasC in normal Tyrode solution. Figure
6A shows that the I-V curve of peak ICa,L
was increased ~10% (measured at +10 mV) in 20 s and reached a
maximum (~20%, measured at 0 mV) after 80-130 s of exposure to
1 µM LPlasC. In addition, it seemed that LPlasC had a profound
stimulatory effect at potentials between 20 and 0 mV; e.g., it
shifted the maximum peak ICa,L in response to
depolarizing pulses from +10 to 0 mV. Figure 6A also shows
that LPlasC gradually increased peak outward current (measured at +60
mV) and caused a leftward shift in the zero-current potential. The
LPlasC-induced changes in ICa,L were reversible
after washout (data not shown); however, this was preceded by a
transient increase in current amplitude (rebound stimulation of
ICa,L; Fig. 6A). Superimposed selected current traces (measured at
10, 0, and +10 mV) recorded before, during, and after exposure to LPlasC are shown in Fig. 6B. Summarized data show that LPlasC increased
ICa,L by 22 ± 3% (n = 5)
within 1-2 min, whereas its rebound during LPlasC removal was
34 ± 6% (n = 3) above the control level. The
LPlasC-induced increase in ICa,L and rebound
stimulation are consistent with its inotropic effects, as shown in Fig.
1A.
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Effects of LPlasC on steady-state membrane currents.
Figure 8A demonstrates that
nearly identical quasi-steady-state I-V
relationships were generated using voltage-pulse or voltage-ramp protocols in a ventricular myocyte. With the use of the voltage-ramp protocol, Fig. 8B shows I-V
relationships of the steady-state membrane current before, during, and
after exposure to 1 µM LPlasC. Results show that, after 5 min of
exposure, LPlasC caused a 60-80% increase in the steady-state
outward current in the voltage range between +40 and +80 mV without
altering IK1 (measured between 70 and
110
mV). Similar to its effect on the peak outward current, the effect of
LPlasC on the steady-state outward current was irreversible. Comparable
results were obtained using the perforated-patch configuration in
which 1 µM LPlasC increased the steady-state outward current (measured at +60 mV) 2.1 ± 0.2-fold without significant change in
IK1 (n = 4).
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Effects of LPlasC on AP and cell contraction in patch-clamped
ventricular myocytes.
Figure 9 shows selected traces obtained
from continuous, simultaneous recordings of AP (A) and
contraction (B) in a myocyte before, during, and after
exposure to LPlasC. Exposure for 3 min to 0.1 µM LPlasC caused 35 and
16% prolongations of AP duration at 25 and 75% of repolarization,
respectively (Fig. 9A), concomitant with a small increase in
systolic shortening (Fig. 9B). A subsequent increase in the
concentration of LPlasC to 1 µM resulted in a substantially prolonged
AP duration accompanied by a ~10-mV depolarization of diastolic
membrane potential and a 20% increase in the magnitude of contraction
at 40 s. When the diastolic membrane potential depolarized
dramatically toward 20 mV in the presence of LPlasC (e.g., an
afterdepolarization was observed at 45 s), contraction became
smaller. The basal (diastolic) level of contraction was increased
gradually with time during exposure to LPlasC, consistent with CS
measured in intact cells (Fig. 1A) and the increased
diastolic Cai (Fig. 2B). Upon rapid removal of
LPlasC, AP partially recovered, whereas cell contraction was
transiently increased by 134% before returning to the control level,
similar to that shown in Fig. 1A. Similar results were
observed in three other experiments.
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DISCUSSION |
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LPlasC accumulates after activation of a membrane-associated, Ca2+-independent PLA2 in ventricular myocytes during short intervals of hypoxia and is a potent arrhythmogenic lipid metabolite (20). With the use of multiple technical approaches, the present study demonstrated that 1) LPlasC elicits positive inotropic, positive lusitropic, and arrhythmogenic effects in adult ventricular myocytes, 2) LPlasC-induced changes in contractile function are paralleled by its effects on intracellular free Ca2+ concentration and SR function, 3) LPlasC increases Ca2+ influx and ICa,L, and 4) LPlasC-induced changes in contractile function are accompanied by anticipated effects on AP. The increased Ca2+ influx, SR Ca2+ content, and SR Ca2+ release lead to AP prolongation and augmented Ca2+ transients, thereby increasing contractility and/or inducing arrhythmias.
LPlasC increases the systolic and diastolic state of cell contraction with maximum effects in 5-6 min, followed by a small decrease. Simultaneous recordings in fura-PE3-loaded cells showed that LPlasC-induced increases in systolic and diastolic free Ca2+ levels precede its effect on contraction. LPlasC increases ICa,L, and the first electrically elicited Ca2+ transient after SR Ca2+ has been emptied by caffeine, supporting our hypothesis that LPlasC-induced inotropic effects are initiated by an increase in Ca2+ influx via ICa,L. In addition, the time course for CS to reach a quasi-steady state was longer than that for the Ca2+ transient during exposure to LPlasC (Fig. 2A), suggesting an incremental increase in the Ca2+ sensitivity of contractile proteins. The LPlasC-induced left shift of the CS-Cai trajectory during early relaxation supports this suggestion and is consistent with an increase in myofilament Ca2+ sensitivity demonstrated previously by others using adult rat ventricular myocytes (26).
Our data also show that LPlasC increased ICa,L by ~12%, smaller than the ~100% increase in the Ca2+ transient of regular twitch and the 43% increase in the first postcaffeine electrically evoked Ca2+ transient. Several possibilities could account for this difference in the LPlasC-induced changes in these parameters. First, ICa,L might be underestimated because the observed concomitant increase in outward currents masks the true magnitude of the increased ICa,L. Second, LPlasC-induced prolongation of ICa,L inactivation, increases in channel availability and ICa,L window currents, and reduction in ICa,L steady-state inactivation during depolarization would enhance Ca2+ influx and thereby increase Ca2+ transients to values greater than the measured increase in ICa,L. Third, LPlasC amplifies calcium-induced calcium release by increasing available SR Ca2+ release and/or the Ca2+ content of SR (11). This is supported by our data showing increases in the magnitude of caffeine-induced Ca2+ transient, fractional Ca2+ release, and +dR/dtmax elicited by LPlasC. Fourth, LPlasC reduces Ca2+ efflux via sarcolemmal Na+/Ca2+ exchange, thereby increasing net Ca2+ gain in each cycle (11). This possibility is supported by our data showing that LPlasC increases the diastolic Ca2+ and shortening level. Fifth, LPlasC decreases Cai buffering power, i.e., the same Ca2+ influx and SR Ca2+ release results in a greater increase in Cai. Studies in rat ventricular myocytes have suggested that a decrease in Ca2+ buffering power at elevated Cai, such as that induced by caffeine, increases the decay rate of free Ca2+ (10). This seems unlikely because our data showed that LPlasC slows the decay rate of the caffeine-induced Ca2+ transient. Taken together, LPlasC increases Ca2+ influx via L-type Ca2+ channels, SR function, and myofilament Ca2+ responses.
The LPlasC-induced elevation in diastolic Cai could account
for the increased diastolic state of contraction. The return of Cai to baseline during diastole depends primarily on SR
Ca2+ uptake (contributing 70% and being a fast component)
and the normal mode of Na+/Ca2+ exchange (28%
and a slow component) in rabbit ventricular myocytes (2,
3). Our data showed that LPlasC increases
dCa2+/dtmax, attributable to an
enhanced SR Ca2+ uptake, but prolongs the slow phase of the
Ca2+ transient, attributable to a reduced
Na+/Ca2+ exchange activity. These changes were
paralleled by a LPlasC-induced shift in the decay phase of the
Ca2+ transient from a single to a double exponential
function. In addition, LPlasC caused a 60% increase in the time
constant of the decay phase of the caffeine-induced Ca2+
transient, an indirect measure of sarcolemmal
Na2+/Ca2+ exchange resulting from the absence
of SR uptake function (2). Moreover, in the presence of
10-15 mM caffeine, the steady state of free Cai during
LPlasC exposure was ~20 nM higher than that in control (Fig.
4B). Thus these results support the notion that LPlasC
decreases Ca2+ efflux via sarcolemmal
Na+/Ca2+ exchange.
An intriguing finding was rebound stimulation of contraction (Figs. 1 and 9) and ICa,L (Fig. 6) observed upon removal of LPlasC in some myocytes. The phenomenon of rebound ICa,L stimulation has been shown during withdrawal of ACh (28, 29) and carbachol (CCh), a muscarinic agonist (5). This rebound activation of ICa,L was suggested to be responsible for the initiation of delayed afterdepolarizations in cat atrial myocytes (29) and to result from an increase in cAMP that is mediated by nitric oxide-induced cGMP-mediated inhibition of phosphodiesterase (29). In contrast, a study in ferret right ventricular myocytes showed that a cGMP-dependent pathway is not involved in the rebound ICa,L stimulation observed upon removal of CCh (5). In the present study, the rebound activation of contraction and ICa,L upon LPlasC removal was paralleled by a rapid recovery of AP duration from the preceding shortened AP duration. Interestingly, we have not observed any rebound stimulation of Ca2+ transients under the same experimental conditions. Comparable changes in ICa,L were obtained using perforated- and conventional patch-clamp recordings; however, the role of intracellular second messengers in LPlasC removal-induced rebound could not be completely excluded and requires further investigation.
LPlasC at 1 µM also elicits arrhythmias in intact myocytes, fura-loaded cells, and patch-clamped cells. The pattern of arrhythmia is consistent with a combination of early and delayed afterdepolarizations. LPlasC-induced arrhythmias apparently result from an increase in free Cai and membrane potential depolarization. Both early and delayed afterdepolarizations can be elicited by Cai overload (for review, see Ref. 7). Our data show that LPlasC increases SR Ca2+ release and could thereby cause depolarization of membrane potential and delayed afterdepolarization, as demonstrated by others (24). Thus LPlasC-induced Cai overload could account for its arrhythmogenic effect.
It is also worth mentioning that the LPlasC-induced increase in
ICa,L was detected in 1-2 min and followed
by a decline, whereas contraction and Ca2+ transients
continue to rise to a plateau in 5-6 min, followed by a reduction.
The discrepancy in the time course of LPlasC-induced changes in these
two parameters could have resulted from a disturbance of the cell
membrane in patch-clamped myocytes, which becomes more severe during
LPlasC exposure. For example, the arrhythmias induced by LPlasC are
more severe and occur more often in perforated-patch-clamped cells than
in those with the conventional whole cell configuration or in intact
myocytes. Combined effects of lipid metabolites and ionophores
(nystatin and amphotericin B) could account for the disruption of
membrane stability. In some cells, increases in peak and steady-state
outward currents were observed during prolonged exposure to LPlasC
(Fig. 6, A and B). This could have been mediated by Cl currents and/or a nonselective current because the
zero-current potential was shifted from
70 mV to potentials between
20 and 0 mV with a relatively linear I-V
relationship, a current similar to that reported by others
(6). Thereafter, myocytes rarely recovered. In addition,
the glibenclamide-sensitive ATP-sensitive K+ current
(IK,ATP) was observed in some cells during and
after exposure to LPlasC and could have been responsible for the
observed inexcitability of myocytes with an extremely short AP duration and a membrane potential of approximately
80 mV (unpublished data). LPlasC-associated changes in AP configuration are
determined by the net balance of its effects on all membrane currents.
Because of the high membrane resistance that exists at the negative
slope between
60 and
20 mV of the I-V curve
in rabbit ventricular myocytes, a small increase in inward currents
(e.g., ICa,L or nonspecific current) can
depolarize membrane potential. On the other hand, an increased outward
K+ current (e.g., IK,ATP) can drive
the membrane potential toward K+ equilibrium
potential. This could account for the observed unstable AP
configurations and delayed afterdepolarizations induced by LPlasC.
Lipid metabolites, including LPlasC, have been suggested to alter the
lipid microenvironment of ion channels (15). The distinct
physical and chemical properties of LPlasC and its distribution in
membrane phospholipids could alter lipid-protein interactions, thereby
leading to alterations in gating properties of ion channels and/or
transporters (15). Although measurements of membrane currents and APs in patch-clamped cells provide important information, lipid metabolite-induced disturbances in cell membrane structure and
function make the correlation with physiological measurements in intact
cells more difficult.
In comparison with other amphiphilic metabolites, LPlasC exerts
distinct effects on cardiac contractile and electrical properties at 1 µM, a concentration comparable to those observed in the plasma of
animal models of myocardial ischemia or in coronary sinus
effluent from patients with ischemic myocardium
(21). Similar to LPlasC, palmitoylcarnitine (but not LPC)
has been reported to elicit a transient increase in
ICa,L in guinea pig ventricular muscle measured in normal Tyrode solution, consistent with its positive inotropic effect (1). In contrast, a study in rabbit ventricular
myocytes suggested that 5 µM palmitoylcarnitine decreases
ICa,L 10 min after exposure (30).
LPlasC (1 µM) appears to have more profound effects on
ICa,L than palmitoylcarnitine (5 µM). In
contrast to other studies, we found that most myocytes could not
survive in LPlasC at concentrations of >3 µM for 3 min in
patch-clamp experiments. Thus LPlasC appears to be more potent at
causing arrhythmias and cell death than LPC (6) and
palmitoylcarnitine (30).
In summary, the present data show that LPlasC causes increases in ICa,L, intracellular free Ca2+, and myofilament Ca2+ sensitivity, prolongs AP duration, and augments contractility. Such changes are often followed by early and/or delayed afterdepolarizations and sustained membrane depolarization, resulting from an abnormal AP duration that is probably mediated by intracellular Ca2+ overload. Myocytes would eventually become inexcitable because of substantial depolarization or hyperpolarization, probably resulting from LPlasC-induced nonspecific currents or IK,ATP, respectively. Such rapid, dramatic electrophysiological and mechanical changes in the heart function could occur under many pathophysiological conditions, including ischemia/reperfusion, hypoxia, cytokine-related cardiac dysfunction, and sudden cardiac death.
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ACKNOWLEDGEMENTS |
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We thank Meei-Yueh Liu and Kerrey A. Roberto for excellent technical assistance.
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FOOTNOTES |
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This work was supported in part by the American Heart Association/Heartland Affiliate and National Heart, Lung, and Blood Institute Grant R01HL-62226.
Address for reprint requests and other correspondence: S. J. Liu, Dept. of Pharmaceutical Sciences, Univ. of Arkansas for Medical Sciences, 4301 West Markham St., MS 522-3, Little Rock, AR 72205 (E-mail: sliu{at}uams.edu).
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.
First published November 27, 2002;10.1152/ajpcell.00465.2002
Received 3 October 2002; accepted in final form 22 November 2002.
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REFERENCES |
---|
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---|
1.
Aomine, M,
Arita M,
and
Shimada T.
Effects of L-propionylcarnitine on electrical and mechanical alterations induced by amphiphilic lipids in isolated guinea pig ventricular muscle.
Heart Vessels
4:
197-206,
1988[Medline].
2.
Bassani, JWM,
Bassani RA,
and
Bers DM.
Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms.
J Physiol
476:
279-293,
1994[Abstract].
3.
Bassani, RA,
Bassani JWM,
and
Bers DM.
Mitochondrial and sarcolemmal Ca2+ transport reduce [Ca2+]i during caffeine contractures in rabbit cardiac myocytes.
J Physiol
453:
591-608,
1992[Abstract].
4.
Bers, DM.
Calcium fluxes involved in control of cardiac myocyte contraction.
Circ Res
87:
275-281,
2000
5.
Bett, GCL,
Dai S,
and
Campbell DL.
Cholinergic modulation of the basal L-type calcium current in ferret right ventricular myocytes.
J Physiol
542:
107-117,
2002
6.
Caldwell, RA,
and
Baumgarten CM.
Plasmalogen-derived lysolipid induces a depolarizing cation current in rabbit ventricular myocytes.
Circ Res
83:
533-540,
1998
7.
Carmeliet, E.
Cardiac ionic currents and acute ischemia: from channels to arrhythmias.
Physiol Rev
79:
917-1017,
1999
8.
Corr, PB,
Creer MH,
Yamada KA,
and
Sobel BE.
Prophylaxis of early ventricular fibrillation by inhibition of acylcarnitine accumulation.
J Clin Invest
83:
927-936,
1989[ISI][Medline].
9.
Creer, MH,
and
Gross RW.
Reversed-phase high-performance liquid chromatographic separation of molecular species of alkyl ether, vinyl ether, and monoacyl lysophospholipids.
J Chromatogr Sci
338:
61-69,
1985.
10.
Diaz, ME,
Trafford AW,
and
Eisner DA.
The role of intracellular Ca buffers in determining the shape of the systolic Ca transient in cardiac ventricular myocytes.
Pflügers Arch
442:
96-100,
2001[ISI][Medline].
11.
Eisner, DA,
Choi HS,
Diaz ME,
O'Neill SC,
and
Trafford AW.
Integrative analysis of calcium cycling in cardiac muscle.
Circ Res
87:
1087-1094,
2000
12.
Frampton, JE,
Orchard CH,
and
Boyett MR.
Diastolic, systolic and sarcoplasmic reticulum [Ca2+] during inotropic interventions in isolated rat myocytes.
J Physiol
437:
351-375,
1991[Abstract].
13.
Grynkiewicz, G,
Poenie M,
and
Tsien RY.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J Biol Chem
260:
3440-3450,
1985[Abstract].
14.
Hamill, OP,
Neher E,
Sakmann B,
and
Sigworth FJ.
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
Pflügers Arch
391:
85-100,
1981[ISI][Medline].
15.
Hazen, SL,
and
Gross RW.
Principles of membrane biochemistry and their application to the pathophysiology of cardiovascular disease.
In: The Heart and Cardiovascular System, edited by Fozzard HA,
Haber E,
Jennings RB,
Katz AM,
and Morgan HE.. New York: Raven, 1992, p. 839-860.
16.
Horn, R,
and
Marty A.
Muscarinic activation of ionic currents measured by a new whole-cell recording method.
J Gen Physiol
92:
145-159,
1988[Abstract].
17.
Liu, S,
and
Schreur KD.
G protein-mediated suppression of L-type Ca2+ current by interleukin-1 in cultured rat ventricular myocytes.
Am J Physiol Cell Physiol
268:
C339-C349,
1995
18.
Liu, SJ,
and
Kennedy RH.
1-Adrenergic activation of L-type Ca current in rat ventricular myocytes: perforated patch-clamp recordings.
Am J Physiol Heart Circ Physiol
274:
H2203-H2207,
1998
19.
McHowat, J,
and
Liu S.
Interleukin-1 stimulates phospholipase A2 activity in adult rat ventricular myocytes.
Am J Physiol Cell Physiol
272:
C450-C456,
1997
20.
McHowat, J,
Liu S,
and
Creer MH.
Selective hydrolysis of plasmalogen phospholipids by Ca2+-independent PLA2 in hypoxic ventricular myocytes.
Am J Physiol Cell Physiol
274:
C1727-C1737,
1998
21.
McHowat, J,
Yamada KA,
Wu J,
Yan GX,
and
Corr PB.
Recent insights pertaining to sarcolemmal phospholipid alterations underlying arrhythmogenesis in the ischemic heart.
J Cardiovasc Electrophysiol
4:
288-310,
1993[ISI][Medline].
22.
Sato, T,
Arita M,
and
Kiyosue T.
Differential mechanism of block of palmitoyl lysophosphatidylcholine and of palmitoylcarnitine on inward rectifier K+ channels of guinea-pig ventricular myocytes.
Cardiovasc Drugs Ther
7:
575-584,
1993[ISI][Medline].
23.
Sato, T,
Kiyosue T,
and
Arita M.
Inhibitory effects of palmitoylcarnitine and lysophosphatidylcholine on the sodium current of cardiac ventricular cells.
Pflügers Arch
420:
94-100,
1992[ISI][Medline].
24.
Schlotthauer, K,
and
Bers DM.
Sarcoplasmic reticulum Ca2+ release causes myocyte depolarization: underlying mechanism and threshold for triggered action potentials.
Circ Res
87:
774-780,
2000
25.
Shen, JB,
and
Pappano AJ.
Palmitoyl-L-carnitine acts like ouabain on voltage, current, and contraction in guinea pig ventricular cells.
Am J Physiol Heart Circ Physiol
268:
H1027-H1036,
1995
26.
Spurgeon, HA,
DuBell WH,
Stern MD,
Sollott SJ,
Ziman BD,
Silverman HS,
Capogrossi MC,
Talo A,
and
Lakatta EG.
Cytosolic calcium and myofilaments in single rat cardiac myocytes achieve a dynamic equilibrium during twitch relaxation.
J Physiol
447:
83-102,
1992[Abstract].
27.
Ten Eick, RE,
Whalley DW,
and
Rasmussen HH.
Connections: heart disease, cellular electrophysiology, and ion channels.
FASEB J
6:
2568-2580,
1992
28.
Wang, YG,
Hüser J,
Blatter LA,
and
Lipsius SL.
Withdrawal of acetylcholine elicits Ca2+-induced delayed afterdepolarizations in cat atrial myocytes.
Circulation
96:
1275-1281,
1997
29.
Wang, YG,
and
Lipsius SL.
Acetylcholine elicits a rebound stimulation of Ca2+ current mediated by pertussis toxin-sensitive G protein and cAMP-dependent protein kinase A in atrial myocytes.
Circ Res
76:
634-644,
1995
30.
Wu, J,
and
Corr PB.
Influence of long-chain acylcarnitines on voltage-dependent calcium current in adult ventricular myocytes.
Am J Physiol Heart Circ Physiol
263:
H410-H417,
1992
31.
Wu, J,
and
Corr PB.
Palmitoyl carnitine modifies sodium currents and induces transient inward current in ventricular myocytes.
Am J Physiol Heart Circ Physiol
266:
H1034-H1046,
1994
32.
Xu, Z,
and
Rozanski GJ.
K+ current inhibition by amphiphilic fatty acid metabolites in rat ventricular myocytes.
Am J Physiol Cell Physiol
275:
C1660-C1667,
1998
33.
Yu, L,
Netticadan T,
Xu Y-J,
Panagia V,
and
Dhalla NS.
Mechanisms of lysophosphatidylcholine-induced increase in intracellular calcium in rat cardiomyocytes.
J Pharmacol Exp Ther
286:
1-8,
1998