Plasmalogen-Derived Lysolipid Induces a Depolarizing Cation Current in Rabbit Ventricular Myocytes

From the Department of Physiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Va.

Correspondence to Dr Clive M. Baumgarten, Department of Physiology, Medical College of Virginia, Box 980551, Richmond, VA 23298-0551. E-mail baumgart{at}hsc.vcu.edu

	Abstract

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Abstract—Plasmalogen rather than diacyl phospholipids are the preferred substrate for the cardiac phospholipase A₂ (PLA₂) isoform activated during ischemia. The diacyl metabolite, lysophosphatidylcholine, is arrhythmogenic, but the effects of the plasmalogen metabolite, lysoplasmenylcholine (LPLC), are essentially unknown. We found that 2.5 and 5 µmol/L LPLC induced spontaneous contractions of intact isolated rabbit ventricular myocytes (median times, 27.4 and 16.4 minutes, respectively) significantly faster than lysophosphatidylcholine (>60 and 37.8 minutes, respectively). Whole-cell recordings revealed that LPLC depolarized the resting membrane potential from –83.5±0.2 to –21.5±1.0 mV. Depolarization was due to a guanidinium toxin–insensitive Na⁺ influx. The LPLC-induced current reversed at –18.5±0.9 mV and was shifted 26.7±4.2 mV negative by a 10-fold reduction of bath Na⁺ (Na⁺/K⁺ permeability ratio, 0.12±0.06). In contrast, block of Ca²⁺ channels with Cd²⁺ and reducing bath Cl^– failed to affect the current. The actions of LPLC were opposed by lanthanides. Gd³⁺ and La³⁺ were equally effective inhibitors of the LPLC-induced current and equally delayed the onset of spontaneous contractions. However, the characteristics of lanthanide block imply that Gd³⁺-sensitive, poorly selective, stretch-activated channels were not involved. Instead, the data are consistent with the view that lanthanides increase phospholipid ordering and may thereby oppose membrane perturbations caused by LPLC. Plasmalogens constitute a significant fraction of cardiac sarcolemmal choline phospholipids. In light of their subclass-specific catabolism by phospholipase A₂ and the present results, it is suggested that LPLC accumulation may contribute to ventricular dysrhythmias during ischemia.

Key Words: plasmalogen • lysophosphatidylcholine • ischemia • lanthanide • lysoplasmenylcholine

	Introduction

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Altered membrane phospholipid metabolism is one of the earliest manifestations of myocardial ischemia. Phospholipase A₂ (PLA₂) is activated,^{1 2 3} and lysophosphatidylcholine (LPC), a product of diacyl phospholipid catabolism, increases in tissue^{4 5 6} and coronary effluents.^{7 8 9} In addition to diacyl phospholipids, cardiac cell membranes contain plasmalogen phospholipids. Plasmalogens constitute 57% of choline phospholipids in canine sarcolemma¹⁰ and >35% of total choline phospholipids in rabbit and human hearts.¹¹ In contrast to the ester-linked fatty acid in diacyl phospholipids, the sn-1 position in plasmalogens contains an alkenyl ether moiety. Because of this structural feature, plasmalogens are metabolized differently from their diacyl counterparts. For instance, a Ca²⁺-independent PLA₂ rapidly activated during ischemia is reported to have a 16-fold higher reaction velocity with choline plasmalogen substrate than with phosphatidylcholine.² This PLA₂ isoform also is present in human heart and is claimed to represent >95% of total PLA₂ activity.¹² In addition, the rate of lysoplasmalogen removal from membranes is substantially slower than that of LPC.¹³

The abundance of plasmalogens in cardiac sarcolemma, their selective targeting by PLA₂, and their slow clearance suggest that subclass-specific alterations in phospholipid metabolism may be important during ischemia. The effects of LPC on cardiac electrophysiology and ion transport processes have been well described (for review, see Reference 3³ ) and include decreased K⁺ conductance,^{14 15} modified voltage dependence and kinetics of Na⁺ current,^{16 17 18} decreased Ca²⁺ conductance,¹⁴ and inhibition of Na⁺,K⁺-ATPase.¹⁹ On the other hand, the influence of lysoplasmalogens on cardiac electrophysiology is essentially unknown.

We sought to determine whether lysoplasmenylcholine (LPLC), a lysoplasmalogen, possessed properties that potentially are arrhythmogenic and to characterize the effects of LPLC on membrane potential (E_m) and ionic currents in ventricular myocytes. At comparable concentrations, LPLC induced spontaneous contractions significantly faster than LPC, depolarized E_m, and elicited a [Na⁺]_o-dependent guanidinium toxin–insensitive current that was blocked by the lanthanides, Gd³⁺ and La³⁺. Among other effects, lanthanides increase membrane lipid ordering^{20 21} and may oppose perturbations induced by LPLC. These results suggest that accumulation of LPLC in the sarcolemma may contribute to ventricular dysrhythmias during ischemia.

	Materials and Methods

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Cardiac Myocyte Isolation
Freshly isolated ventricular myocytes were obtained from New Zealand White rabbits (2.0 to 3.0 kg) by a collagenase-protease digestion procedure. Briefly, spontaneously beating hearts were perfused via the aorta with an oxygenated modified Tyrode's solution containing (mmol/L) NaCl 130, KCl 5.4, CaCl₂ 0.75, MgCl₂ 3.5, NaH₂PO₄ 0.4, taurine 20, creatine 10, glucose 10, and HEPES 5, titrated to pH 7.25 with 1 mol/L NaOH. Subsequently, the perfusate was switched to a Tyrode's solution containing 1 mg/mL collagenase (type II, Worthington Biochemical Corp), 0.1 mg/mL pronase E (type XIV, Sigma Chemical Co), and 80 µmol/L Ca²⁺. After collection, myocytes were stored in a modified Kraft-Brühe solution containing (mmol/L) potassium glutamate 80, KH₂PO₄ 10, KCl 2.5, MgSO₄ 1.8, EGTA 0.5, taurine 10, glucose 11, and HEPES 10, titrated to pH 7.2 with 1 mol/L KOH. A typical isolation yielded 60% to 70% viable cells, and all patch-clamp recordings were made within 8 hours of isolation. Cells selected for study were rod-shaped with well-defined regular striations and bleb-free borders and remained quiescent in control bath solution.

Bath and Pipette Solutions
Myocytes were placed in a 0.2-mL flowing bath mounted on the stage of an inverted microscope (Diaphot, Nikon) and perfused at 1 to 2 mL/min. The standard bath solution contained (mmol/L) NaCl 140, KCl 5, CaCl₂ 1.8, MgCl₂ 1, and HEPES 5, titrated to pH 7.4 with 1 mol/L NaOH. The pipette (internal) solution contained (mmol/L) potassium aspartate 140, CaCl₂ 0.062, K₂EGTA 5, MgCl₂ 5, Na₂ATP 5, Na₃GTP 0.4, and HEPES 5, titrated to pH 7.1 with 1 mol/L KOH. For experiments in low bath Na⁺, 126 mmol/L NaCl was replaced with equimolar N-methyl-D-glucamine (NMDG) chloride. For experiments in low bath Cl^-, 135.5 mmol/L NaCl was replaced with equimolar sodium isethionate. Ca²⁺-free bath solution was made by omitting CaCl₂ and adding 1.8 mmol/L Na₂EGTA. Bath solution was made hypertonic by adding 150 mmol/L mannitol (relative osmolarity, 1.5). Tetrodotoxin (TTX), saxitoxin (STX) (Calbiochem), and chloride salts of lanthanides (Gd³⁺ and La³⁺) and Cd²⁺ were dissolved directly in the bath solution. LPLC dissolved in CHCl₃ (Serdery Research) was evaporated to dryness under an N₂ atmosphere to limit oxidation, redissolved in bath solution, and sonicated for 2 minutes. Concentrations indicated for LPLC are based on nominal formula weight calculated from the sn-1 analysis and the plasmalogen content provided by the supplier. Synthetic LPC (Sigma), prepared as a 10 mmol/L stock solution and kept frozen, was added to the bath solution and sonicated for 2 minutes. All experiments were conducted at room temperature (23°C).

Electrophysiology
For whole-cell recordings, pipettes were fabricated from 7740 or 7052 glass, coated with Sylgard 184 (Dow Corning), and fire-polished. The pipette resistance ranged from 1 to 3 M. An Ag/AgCl₂ pellet connected to the bath via a 0.15 or 3 mol/L KCl agar bridge served as the ground electrode. The diffusion potential between pipette and bath solutions was 13.1±0.2 mV, and all voltages were corrected by this amount.

A List EP-7 amplifier (List-Medical) was used to patch-clamp myocytes. Voltage- and current-clamp protocols and data acquisition were controlled by custom programs written in ASYST (Keithly). Ionic currents were elicited by 200-ms voltage pulses from a holding potential of –83 mV to potentials ranging from –113 to +37 mV. The current output was filtered at 2 kHz (–3 dB, 8-pole Bessel, Frequency Devices) and digitized at 10 kHz (12 bits). The quasi–steady-state current, taken as the average of the last 16.7 ms of each voltage step, is plotted in the current-voltage (I-V) relationships. Series resistance averaged 7.7±2.5 M (n=26), and 40% was compensated electronically.

The resting membrane and action potentials were recorded under current-clamp conditions. Membrane voltage was filtered at 0.5 or 1 kHz, digitized at 1 or 3 kHz, and reproduced on a strip-chart recorder (Gould). Action potentials were evoked by 0.7- to 1.5-nA current pulses, 3 to 5 ms in duration, applied at 0.5 Hz.

Data Analysis and Statistics
Current- and voltage-clamp data were analyzed with custom programs written in ASYST and plotted using SigmaPlot (SPSS). Except as noted, results are reported as mean±SEM, and n corresponds to the number of cells. For spontaneous activity experiments, multiple comparisons of median response times for each lysolipid and experimental protocol were made with Dunn's method after a 1-way ANOVA on ranks. Comparisons of patch-clamp data were made by the Student-Newman-Keuls method after ANOVA or by a Student t test where indicated. Statistics were computed using SigmaStat 2.0 (SPSS), and P<0.05 was considered significant.

	Results

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Evaluation of the Arrhythmogenic Potential of LPLC
On exposure to exogenous LPC, myocytes contract spontaneously and die.²² To determine whether LPLC possesses potentially arrhythmogenic properties similar to those described for LPC, the effects of LPLC and LPC on ventricular myocytes were compared. Figure 1 is a survival plot of the fraction of myocytes remaining quiescent over time after exposure to different concentrations of LPLC or LPC in physiological bath solution. In this experiment, myocytes were intact; ie, they were not dialyzed by a patch pipette. Induction of spontaneous contractions depended on both the amount and type of lysolipid used. At the lowest concentrations, 2.5 and 5 µmol/L, LPLC induced spontaneous contractions significantly faster than LPC. Spontaneous activity developed with median times of 27.4 minutes (n=36) and 16.4 minutes (n=64) for 2.5 and 5 µmol/L LPLC, respectively, compared with >60 minutes (n=29) and 37.8 minutes (n=48) for LPC. However, there were no significant differences in the median times to development of spontaneous activity with 10 µmol/L lysolipids (LPLC, 8.2 minutes, n=62; LPC, 8.7 minutes, n=15). The end point was 60 minutes, and under control conditions, all myocytes remained quiescent for the duration of the protocol (n=20, data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 1. Lysolipid subclass affects the induction of spontaneous contractions in intact (ie, undialyzed) myocytes. First, viable myocytes in a field were identified on the basis of morphological criteria (see Materials and Methods) and their quiescence for at least 10 minutes in control bath solution. Then, myocytes were exposed to LPLC (solid symbols) or LPC (open symbols), and fields of cells were observed visually for an additional 60 minutes. The fraction of cells remaining quiescent is plotted at selected times. At 2.5 and 5 µmol/L, LPLC induced spontaneous activity significantly faster than LPC (n=29 to 64). However, median times for 10 µmol/L lysolipids were not significantly different (n=15 to 64). In contrast, all myocytes perfused with control solution remained quiescent (n=20, not shown). Median times were compared by Dunn's method after ANOVA on ranks.

Ultimately myocyte contraction depends on Ca²⁺. Therefore, the effect of a Ca²⁺-free bathing solution on the time to spontaneous contraction was evaluated for the 2 highest concentrations of LPLC. A Ca²⁺-free bathing solution prevented the occurrence of spontaneous contractions in 5 µmol/L LPLC, and cells remained quiescent for the entire 60-minute exposure (n=14). In contrast, adding 10 µmol/L LPLC to Ca²⁺-free bathing solution caused cells to promptly round up and die rather than simply contract (n=12). This suggests that the role of Ca²⁺ is complex.

Electrophysiological Effects of LPLC on Ventricular Myocytes
To investigate the mechanism underlying the spontaneous contractions observed after exposure to LPLC, E_m was recorded under current-clamp conditions. Figure 2 shows results from a representative myocyte. Under control conditions, E_m was stable and well-polarized at –83.5±0.2 mV (n=17). Within 7 minutes of exposure to 10 µmol/L LPLC, E_m underwent a small depolarization (typically 5 mV) followed by an abrupt sustained depolarization to –21.5±1.0 mV (n=11). The mean time to the large depolarization in LPLC was dose dependent (31.1±7.4 [n=3], 9.0±1.5 [n=11], and 3.0±0.5 [n=6] minutes for 2.5, 5, and 10 µmol/L LPLC, respectively). In contrast, myocytes under control conditions remained well polarized for at least 60 minutes (n=5), the maximum time tested.

View larger version (7K): [in this window] [in a new window]   Figure 2. LPLC induced membrane depolarization in a dialyzed myocyte under current clamp. After 6 minutes of exposure to 10 µmol/L LPLC, Em depolarized by 18 mV and then abruptly depolarized to –24 mV. Hyperpolarizing current pulses (75 pA) were applied at points a to c. At point b, hyperpolarizing current pulses partially restored Em. Input resistance increased 5-fold after the sustained depolarization (compare point c with point a). This excludes gigaseal deterioration as the cause of depolarization. Depolarization did not immediately cause contraction, because myocytes were dialyzed with EGTA-containing pipette solution. Break at VC indicates suppression of voltage-clamp command pulses. Break in record (*) was for 2 minutes during control period.

Applying 75-pA hyperpolarizing pulses initially caused partial repolarization after depolarization in LPLC (Figure 2, point b) demonstrating the bistability of E_m. However, E_m spontaneously depolarized again 1 minute later. With longer exposure to LPLC, hyperpolarizing current pulses failed to restore E_m (Figure 2, point c), and contracture of the myocyte followed shortly thereafter. Dialysis with an EGTA-containing pipette solution prevented contraction on depolarization and is likely to have delayed the contracture. Depolarization was not due to loss of seal resistance. The hyperpolarizations elicited by 75-pA pulses after LPLC-induced depolarization (Figure 2, point c) were greater than those under control conditions (Figure 2, point a). This indicates that input resistance was greater after the LPLC-induced depolarization than before and was consistent with the voltage dependence of membrane resistance rather than loss of the pipette-membrane seal.

The sustained depolarization in LPLC usually was irreversible. Up to 25 minutes of washout of LPLC in bath solution containing 5 mg/mL albumin, which hastens lysolipid extraction from the membrane,²³ failed to restore the resting potential in 4 of 5 cells.

Characterization of Ionic Current Underlying LPLC-Induced Membrane Depolarization
The effects of LPLC on the ionic currents are illustrated in Figure 3A and 3B. The steady-state I-V relationships under control conditions () and after a 9-minute exposure to 5 µmol/L LPLC () are plotted in Figure 3C. Exposure to LPLC caused a counterclockwise rotation of the control I-V relationship, and a pronounced inward current was observed at the physiological resting potential (–83 mV). Figure 3D shows the I-V relationship of the LPLC-induced current obtained by subtracting the steady-state current under control conditions from that in LPLC. The reversal potential (E_rev) of the LPLC-induced current was –18.5±0.9 mV (n=9) and indicated that a poorly selective permeation pathway or multiple ion channels were involved. The LPLC-induced current exhibited variable amounts of rectification at negative potentials, and the conductance increased with time in LPLC. Washout attempts for up to 15 minutes failed to restore the control I-V relationship.

View larger version (16K): [in this window] [in a new window]   Figure 3. Effect of LPLC on steady-state membrane currents. A and B, Families of membrane currents were obtained under control conditions (A) and after a 9-minute exposure to 5 µmol/L LPLC (B). Bars indicate zero current levels. LPLC induced a net inward current at the holding potential and increased steady-state currents. C, I-V relationships under control conditions () and with LPLC () are shown. Im indicates membrane current. D, The LPLC-induced difference current (Idiff) I-V relationship, obtained by subtracting the current in the control condition from that after LPLC exposure, reversed at –18.5±0.9 mV, indicating a poorly selective ionic current.

Ionic Basis of the LPLC-Induced Current
To identify the charge carriers responsible for the LPLC-induced current, ion channels were blocked pharmacologically, and ion substitution experiments were performed. Figure 4 shows the effects of a 10-fold reduction in bath Na⁺ and 1 µmol/L STX on the E_m profile and ionic currents from a representative myocyte under control conditions and after brief exposure to 5 µmol/L LPLC. In Figure 4A, the control resting E_m was near –80 mV. Reducing bath Na⁺ from 140 to 14 mmol/L caused a modest but statistically significant depolarization of 2.6±0.5 mV (P=0.016, n=15) that was reversible. After returning to physiological bath Na⁺, LPLC caused an abrupt membrane depolarization to –23 mV. Lowering bath Na⁺ resulted in nearly complete recovery of the control resting potential in the continued presence of LPLC (range, –65 to –80 mV). This suggests that the inward current is carried, at least in part, by Na⁺. However, guanidinium toxin–sensitive Na⁺ channels were not involved. Blockade of Na⁺ channels with 1 µmol/L STX failed to inhibit the membrane depolarization in LPLC on returning to 140 mmol/L bath Na⁺. Pretreating myocytes with 10 µmol/L TTX before LPLC exposure also failed to prevent or significantly delay membrane depolarization in LPLC (10 µmol/L LPLC+TTX, 4.6±1.5 minutes, n=3; 10 µmol/L LPLC, 3.0±0.5 minutes, n=6; P=0.283).

View larger version (12K): [in this window] [in a new window]   Figure 4. LPLC-induced depolarization was [Na+]o dependent but insensitive to guanidinium toxins. A, After depolarization in 5 µmol/L LPLC, reducing [Na+]o from 140 to 14 mmol/L reversibly restored Em (n=3). In contrast, 1 µmol/L STX (n=3) and 10 µmol/L TTX (n=3, not shown) failed to restore Em or prevent LPLC-induced depolarization. B, LPLC-induced currents from the same cell as in panel A are shown. The LPLC-induced current was [Na+]o dependent but insensitive to STX. Low [Na+]o caused a –26.7±4.2 mV (n=6) shift in Erev and reduced the inward current at negative potentials. Similar results were obtained with 10 µmol/L TTX (n=4, data not shown). The shift in Erev of the LPLC-induced current is equivalent to that expected for a channel with a PNa/PK ratio of 0.12±0.06 (n=6).

Consistent with the lack of effect on E_m, STX also failed to inhibit the LPLC-induced current (Figure 4B). In contrast, a 10-fold reduction in bath Na⁺ shifted E_rev of the LPLC-induced current by –26.7±4.2 mV (n=6). Based on the shift in E_rev and assuming that only K⁺ and Na⁺ contribute to the LPLC-induced conductance and that NMDG remains impermeant, the Na⁺/K⁺ permeability (P_Na/P_K) ratio was 0.12±0.06 (n=6).

Although spontaneous contractions observed in LPLC ultimately must reflect modified intracellular Ca²⁺ homeostasis, blockade of voltage-dependent Ca²⁺ channels by 100 µmol/L Cd²⁺ failed to inhibit the LPLC-induced current (n=3, data not shown). Other investigators reported that LPLC²⁴ and long-chain acylcarnitines,²⁵ structural analogues of LPC, inhibit Ca²⁺ channels. Consequently, it is unlikely that an LPLC-induced increase in the Ca²⁺ current is responsible for the observed depolarization of E_m.

The LPLC-induced current was also insensitive to changes in bath Cl^–. After the sustained membrane depolarization in LPLC, a 10-fold reduction in bath Cl^– (from 150 to 15 mmol/L) failed to inhibit the current or shift E_rev of the LPLC-induced current (E_rev=1.4±1.8 mV, n=3, P=0.547).

Lanthanides Inhibit the LPLC-Induced Current
Ion substitution studies indicated that the LPLC-induced current was a poorly selective cationic current. This raised the possibility that LPLC modulates cationic stretch-activated ion channels (SACs), which poorly distinguish between Na⁺ and K⁺.^{26 27} Moreover, recent preliminary studies have shown that cardiac cell volume regulation is disrupted by lysolipids, including LPLC.²⁸ To investigate the possibility that SACs contributed to the LPLC-induced current, Gd³⁺ and La³⁺ were used. Gd³⁺ is a moderately selective and potent (K_0.5, 2 µmol/L) blocker of SACs that exhibits cooperative binding,^{26 29} whereas La³⁺ is devoid of SAC-blocking activity in this concentration range.²⁹ In rabbit ventricular myocytes, Gd³⁺ blocks all of the cation SAC current elicited by cell swelling.²⁶ Figure 5 shows the effect of both lanthanides on the LPLC-induced current. Gd³⁺ (100 µmol/L) inhibited the LPLC-induced current by 80±8% at –83 mV (n=7). The effect of 100 µmol/L La³⁺ was indistinguishable from that achieved with equimolar Gd³⁺. La³⁺ inhibited the current by 81±8% (n=6). Because the LPLC-induced current tended to increase with time, the inhibition reported is based on the average of the LPLC-induced current before application and after washout of the lanthanide from the bath.

View larger version (12K): [in this window] [in a new window]   Figure 5. Lanthanides (Gd3+ and La3+, ) blocked the LPLC-induced current (). Gd3+ (100 µmol/L) inhibited 80.2±8.3% (n=7) (A) and La3+ (100 µmol/L) inhibited 80.7±8.3% (n=6) (B) of the LPLC-induced current at -83 mV. The effect of the lanthanides was indistinguishable.

Figure 6 shows the dose-response relationship for inhibition of the LPLC-induced current by Gd³⁺. Assuming 1:1 binding, the IC₅₀ was 23.5±5.1 µmol/L at –83 mV. The Hill equation with a variable coefficient did not result in a statistically better fit to the data. The inhibition achieved with 100 µmol/L La³⁺ () is also shown. The failure of the LPLC-induced pathway to discriminate between La³⁺ and Gd³⁺, the lower than expected potency of Gd³⁺, and the lack of apparent cooperativity in the Gd³⁺ dose-response relationship suggest that SACs were not responsible for the LPLC-induced current. This conclusion was confirmed by showing that osmotic shrinkage of myocytes (relative osmolarity, 1.5) failed to inhibit the current (n=2) or reverse the sustained membrane depolarization (n=5) observed in LPLC (data not shown). Osmotic shrinkage itself does not appear to activate a mechanosensitive current in rabbit ventricular myocytes.²⁶

View larger version (14K): [in this window] [in a new window]   Figure 6. Dose-response relationship for inhibition of the LPLC-induced current by Gd3+ (). Inhibition was measured at –83 mV by using the average LPLC-induced current before and after lanthanide exposure. The data were well described by a 1:1 binding relationship with an IC50 of 23.5 µmol/L. Data for inhibition by 100 µmol/L La3+() is also shown.

Lanthanides Delay Spontaneous Activity in LPLC
Consistent with electrophysiological studies, pretreatment of myocytes with 100 µmol/L Gd³⁺ or La³⁺ significantly delayed the median time to development of spontaneous contractions in LPLC (Figure 7). In contrast, Ca²⁺ channel blockade with 100 µmol/L Cd²⁺ failed to delay spontaneous activity in LPLC. Although spontaneous activity was not completely inhibited with Gd³⁺ or La³⁺ pretreatment, neither was the LPLC-induced current. The unblocked current may have contributed to the eventual development of spontaneous activity.

View larger version (12K): [in this window] [in a new window]   Figure 7. Consistent with patch-clamp studies, pretreatment with 100 µmol/L La3+ or Gd3+, but not Cd2+, delayed development of spontaneous activity in 5 µmol/L LPLC. Times for 25%, 50%, and 75% of myocytes to develop spontaneous activity are plotted. Median times were 16.4 minutes (n=36) for LPLC alone and 17.4 (n=19), 53.0 (n=38), and 55.4 (n=35) minutes for cells treated with LPLC+Cd2+, LPLC+La3+, and LPLC+Gd3+, respectively. The 75th percentile (not shown) was >60 minutes for both lanthanides.

Effects of LPLC on the Ventricular Action Potential
The LPLC-induced alterations in membrane current were expected to have profound effects on the action potential. Figure 8 shows representative recordings from 2 different cells under control conditions and within 5 minutes of exposure to 1 µmol/L LPLC. In control myocytes, the resting E_m was –82.1±0.7 mV (n=7), and action potential duration, measured to 90% repolarization (APD₉₀), was 319.2±19.2 ms (n=7). After a switch to bathing media supplemented with LPLC, APD₉₀ increased to 505.0±32.5 ms (n=7) when measured just before induction of the sustained depolarization described earlier (see Figure 2). LPLC also caused a small but statistically significant reduction in action potential amplitude from 128.3±1.4 to 122.5±1.6 mV(n=7, P=0.009), predominantly resulting from a modest depolarization of resting E_m from –82.5±0.7 to –78.3±1.2 mV (n=7, P=0.01). Subsequently, resting E_m underwent an abrupt and sustained depolarization to –22.3±1.6 mV, behavior similar to that found in unstimulated myocytes (Figure 2). This was associated with loss of excitability, although an occasional Ca²⁺-dependent upstroke could be elicited (Figure 8, LPLC). In the examples shown, resting E_m recovered during washout, but APD₉₀ appeared shorter than under control conditions (Figure 8, Wash).

View larger version (10K): [in this window] [in a new window]   Figure 8. Effect of LPLC on ventricular action potentials from 2 cells. Within 3 to 5 minutes of exposure to 1 µmol/L LPLC, APD90 increased from 349.7±43.6 ms (control) to 505.0±32.5 ms (LPLC) (n=7). Action potential amplitude also was reduced from 128.3±1.4 to 122.5±1.6 mV (n=7); this reduction was primarily due to a 4.2-mV depolarization of resting Em. Longer exposure to LPLC resulted in depolarization and usually loss of excitability. In some cases, a Ca2+-dependent upstroke could be elicited (arrow). In 2 of 7 myocytes, the effects of LPLC were reversible on washout (Wash), although APD90 became shorter than control.

	Discussion

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Accumulation of lysolipids after activation of PLA₂ during ischemia has been recognized as an arrhythmogenic factor for over 3 decades. Previous efforts have focused on the actions of LPC, a product of diacyl lipid catabolism. The present study is the first to characterize and compare the effects of plasmalogen-derived LPLC with those of LPC in intact ventricular myocytes. We found that 2.5 and 5 µmol/L LPLC induced spontaneous contractions faster than LPC. Electrophysiological studies demonstrated that the contractions were the result of an LPLC-induced depolarization due to Na⁺ influx via a poorly selective pathway. Replacement of Na⁺ with NMDG hyperpolarized E_m and caused a negative shift in E_rev of the LPLC-induced current. On the other hand, lowering bath Cl^– failed to affect E_rev. The LPLC-induced current was not the result of altered Na⁺ channel properties. The guanidinium toxins, TTX and STX, did not affect the I-V relation in LPLC. In contrast, both Gd³⁺ and La³⁺ substantially inhibited the LPLC-induced current. These lanthanides did not act through blockade of SACs or Ca²⁺ channels. Closing SACs by osmotic shrinkage and blockade of Ca²⁺ channels with Cd²⁺ failed to inhibit the LPLC-induced current. Consistent with electrophysiological studies, both Gd³⁺ and La³⁺, but not blockade of Ca²⁺ channels with Cd²⁺, significantly delayed the time to development of spontaneous activity in LPLC.

Differences Between LPLC and LPC
Although LPLC and LPC are both choline phospholipids, important differences in the actions of these lysolipids have emerged. LPLC does not induce a guanidinium toxin–sensitive steady-state current. In contrast, exposure to 9 to 25 µmol/L LPC for up to 2 hours modifies cardiac Na⁺ channel gating and causes long-lasting bursts of openings far negative to the normal voltage range for Na⁺ channel activation.¹⁶ This generates a sustained depolarizing current and substantial Na⁺ influx at the resting potential. More acute effects of LPC noted in whole-cell studies include a modest depolarizing shift in the peak amplitude of macroscopic Na⁺ current within 16 minutes of exposure to 10 µmol/L LPC.¹⁸ This differs from the hyperpolarizing shift reported with prolonged LPC exposure in the single-channel studies.¹⁶ However, a slowing of macroscopic inactivation kinetics is consistent with the effect if LPC on single Na⁺ channels.¹⁸ A similar study evaluating the effects of 10 µmol/L LPC on Na⁺ current in guinea pig ventricular myocytes also reported a slowing of inactivation kinetics within 3 minutes.¹⁷ However, the effects of LPC on the voltage dependence of activation were not reported in that study.

Other laboratories have shown that LPC induces a nonselective current in guinea pig ventricular myocytes.^{22 30} As in the present case, the conductance induced by LPC increased with time and was insensitive to bath Cl^–. The LPC-induced current reversed near 0 mV, 20 mV positive to that observed for LPLC. This difference may be explained by the higher P_Na/P_K ratio estimated for the LPC-induced current³⁰ (0.79) compared with that estimated in the present study for the LPLC-induced current (0.12). Interestingly, membrane permeability was greater for NMDG than for Na⁺ (NMDG/Na⁺ permeability ratio, 1.13) after LPC treatment.³⁰ This sharply contrasts with the results of the present study. Equimolar replacement of Na⁺ by NMDG shifted E_rev of the LPLC-induced current >25 mV in a negative direction. The negative shift of E_rev precludes the possibility that NMDG is more permeant than Na⁺ in the present case. However, an independent determination of the NMDG permeability was not made.

Another difference is that the median time for development of LPLC-induced spontaneous activity was less than half that required for LPC at 2.5 and 5 µmol/L. We did not determine the time required for LPC to induce depolarization of myocytes. Nevertheless, the mean time to depolarization in 5 µmol/L LPLC also was less than half that reported previously for LPC under similar conditions.²² The mechanism responsible for the faster onset of action of LPLC compared with LPC is unknown. One possibility is that LPLC may partition into the sarcolemma faster than LPC. Although no data are available comparing the partitioning kinetics of these amphiphiles, the sn-1 vinyl-ether bond in plasmalogens is more lipophilic than the acyl ester contained in LPC. However, the remainder of the molecules are identical. Differences in the rates of lysolipid catabolism must also be considered. Catabolism of LPLC is substantially slower than that of LPC and requires distinct enzyme isoforms.¹³ Thus, even with equal partitioning into the sarcolemma, slower catabolism is expected to lead to higher sarcolemmal levels of LPLC.

Effects of Lysolipids and Lanthanides on Membranes
The electrophysiological effects of LPLC could result directly from lysolipid interaction with individual proteins or secondarily from changes in the membrane biophysical properties. Lysolipids increase membrane fluidity in model membranes³¹ and biomembranes.³² An increase in membrane fluidity is correlated with an increased membrane permeability to a number of solutes³³ and could result in nonspecific ion fluxes. Interestingly, plasmalogen-derived lysolipids are more potent effectors of membrane fluidity than are sn-1 acyl ester lysolipids.³¹ This difference in potency may help explain the shorter time to onset and lower bath concentrations required for the effects of LPLC.

Inhibition of the effects of LPLC by lanthanides is consistent with the idea that LPLC acts through changes in membrane fluidity. Lanthanides prevent increases in membrane fluidity, such as those caused by lysolipids.^{31 32} At concentrations comparable to those used in the present study, La³⁺ was previously found to be maximally effective at reducing membrane fluidity in cardiac sarcolemma.²⁰ In another study,²¹ Gd³⁺ was comparable to La³⁺ in reducing membrane fluidity in model membrane systems. However, details of the membrane phospholipid composition may alter the sensitivity to lanthanides.³⁴ It should be noted that lanthanides exert a number of other effects on membranes and their proteins. Consequently, the inhibition of the LPLC-induced current by lanthanides is not sufficient to establish that the LPLC-induced current results from an increase in membrane fluidity.

Other effects of lanthanides should also be considered. Lanthanide inhibition of the LPLC-induced current cannot be explained by inhibition of the Ca²⁺ current. Although lanthanides block the Ca²⁺ current, Cd²⁺, a more selective inhibitor of the Ca²⁺ current, failed to block the LPLC-induced current. Moreover, pretreating myocytes with Gd³⁺ or La³⁺, but not Cd²⁺, significantly delayed the median time for development of spontaneous contractions in LPLC.

Lanthanides were also not likely to be acting through blockade of SACs. Gd³⁺ and La³⁺ were equally effective at inhibiting the LPLC-induced current, even though Gd³⁺ is more potent than La³⁺ at blocking SACs. Gd³⁺ inhibited the LPLC-induced current with an IC₅₀ of 23.5 µmol/L, nearly 14-fold higher than the IC₅₀ for block of SACs in this preparation.²⁶ Gd³⁺ appears to block all of the cation SAC current elicited by cell swelling in this preparation.²⁶ In addition, raising bath osmolarity to close SACs failed to affect E_rev of the LPLC-induced current. On the other hand, the possibility that lysolipids activate poorly selective mechanosensitive channels that are insensitive to Gd³⁺ and unaffected by cell swelling or shrinkage cannot be excluded. Such channels might explain the Na⁺-dependent depolarization and provide a Ca²⁺ influx pathway.

In contrast to the present results, it was reported that the poorly selective LPC-induced current in guinea pig myocytes is not inhibited by 15 µmol/L Gd³⁺.³⁰ However, these experiments were performed in a PO₄^3–-containing bathing solution. Published stability constants for gadolinium phosphate indicate that virtually no free Gd³⁺ is available in phosphate-buffered media.^{34 35} As a result, the ability of Gd³⁺ to block the LPC-induced nonselective current in guinea pig myocytes remains an open question.

Pathophysiological Relevance
The kinetics of onset and the dose dependence of LPLC underscore its importance as an ischemic metabolite that induced a potentially arrhythmogenic inward current and prolonged APD₉₀ by extending the plateau at a partially depolarized level. The highest concentration of LPLC used in the present study (10 µmol/L) was less than or equal to the lowest LPC concentrations reported to have effects on ion channels. For instance, 20 to 100 µmol/L LPC¹⁵ or up to 200 µmol/L LPC¹⁴ was used to inhibit inward rectifier K⁺ current. Effects on Na⁺ current have been described using 10 to 50 µmol/L LPC,^{17 18} and exposure to between 9 and 25 µmol/L for up to 2 hours has been used for single-channel studies.¹⁶ Inhibition of the Na⁺,K⁺-ATPase was reported with a similar range of LPC concentrations.¹⁹

Although LPLC induced spontaneous depolarization and contraction at lower concentrations than LPC, the extent of myocardial LPLC accumulation during myocardial ischemia remains uncertain. Many studies examining lysolipid content after ischemia did not report membrane LPLC content.^{5 7 36 37} In rats,³⁸ rabbits,³⁸ dogs,⁴ and humans,³⁹ no increases in total LPLC were found for up to 24 hours after myocardial ischemia. This is not surprising for the rat, in which choline plasmalogen content is <2% of choline phospholipids compared with 36% in humans.¹¹ The remaining studies may not shed light on the question at hand because lysoplasmalogen content was normalized to total myocardial phospholipid content. Sarcolemma represents only 2% to 8% of the total cardiac membrane phospholipid content,^{3 40} and large increases in sarcolemmal lysoplasmalogen content could go undetected in total lipid measurements. In contrast to the situation in heart, significant increases in both LPLC and LPC have been reported in rabbit proximal tubule cell membranes after 10 minutes of hypoxia.⁴¹ Clear answers regarding the extent of LPLC accumulation in the ischemic heart will have to await a more detailed biochemical analysis of subclass-specific lysolipid content of sarcolemmal membranes.

One question that can be raised is whether exogenous application of lysolipids mimics the in vivo situation during ischemia. PLA₂ activity and the formation of lysolipids occurs intracellularly. Nevertheless, substantial amounts of lysolipids are found in venous and lymphatic effluents and have access to the outer surface of the sarcolemma as do lysolipids added to the bath. Plasma lysolipid levels in coronary sinus effluents in humans are 100 µmol/L within 2 minutes of ischemia induced by rapid atrial pacing and reach 178 µmol/L.⁸ Similar values for lysolipids have been reported in plasma from cats⁷ and cardiac lymph from dogs⁴² during the first 10 to 15 minutes of ischemia. Plasma lysolipids are likely derived from both ischemic myocytes⁴⁰ and vascular endothelium,⁴³ although the contribution from each lysolipid subclass is unknown. The concentrations of lysolipid in extracellular fluid are much higher than those used in the present study. However, the majority of lysolipids in plasma are bound to protein.²³ In a protein-free bathing solution, the uptake of 10 µmol/L [¹⁴C]LPC in rat ventricular myocytes after a 60-minute incubation period⁴⁴ was comparable to tissue levels found during ischemia in cats,⁴⁵ rabbits,³⁸ dogs,^{5 46} and humans.³⁹

Electrophysiological studies focusing on LPC have ignored the potential contribution of lysolipids derived from plasmalogens. Nevertheless, plasmalogens are abundant in heart and are selectively degraded to lysolipids by PLA₂ during ischemia, and LPLC is slowly catabolized after its production. In view of the LPLC-induced current, membrane depolarization, and changes in action potential configuration observed in the present study, it is reasonable to propose that sarcolemmal accumulation of LPLC may contribute to ventricular dysrhythmias during ischemia.

	Acknowledgments

 
This study was supported by National Heart, Lung, and Blood Institute grant HL-46764. Work contained herein was submitted by Ray A. Caldwell to the graduate faculty of the Medical College of Virginia, the Health Sciences Division of Virginia Commonwealth University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the field of physiology.

	Footnotes

 
Presented previously in abstract form (Biophys J. 1996;70:A391).

Received December 22, 1997; accepted June 4, 1998.

	References

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