K+ current inhibition by amphiphilic fatty acid metabolites in rat ventricular myocytes

Zhi Xu and George J. Rozanski

Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska, 68198-4575

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Fatty acid metabolites accumulate in the heart under pathophysiological conditions that affect beta -oxidation and can elicit marked electrophysiological changes that are arrhythmogenic. The purpose of the present study was to determine the impact of amphiphilic fatty acid metabolites on K+ currents that control cardiac refractoriness and excitability. Transient outward (Ito) and inward rectifier (IK1) K+ currents were recorded by the whole cell voltage-clamp technique in rat ventricular myocytes, and the effects of two major fatty acid metabolites were examined: palmitoylcarnitine and palmitoyl-coenzyme A (palmitoyl-CoA). Palmitoylcarnitine (0.5-10 µM) caused a concentration-dependent decrease in Ito density in myocytes internally dialyzed with the amphiphile; 10 µM reduced mean Ito density at +60 mV by 62% compared with control (P < 0.05). In contrast, external palmitoylcarnitine at the same concentrations had no effect, nor did internal dialysis significantly alter IK1. Dialysis with palmitoyl-CoA (1-10 µM) produced a smaller decrease in Ito density compared with that produced by palmitoylcarnitine; 10 µM reduced mean Ito density at +60 mV by 37% compared with control (P < 0.05). Both metabolites delayed recovery of Ito from inactivation but did not affect voltage-dependent properties. Moreover, the effects of palmitoylcarnitine were relatively specific, as neither palmitate (10 µM) nor carnitine (10 µM) alone significantly influenced Ito when added to the pipette solution. These data therefore suggest that amphiphilic fatty acid metabolites downregulate Ito channels by a mechanism confined to the cytoplasmic side of the membrane. This decrease in cardiac K+ channel activity may delay repolarization under pathophysiological conditions in which amphiphile accumulation is postulated to occur, such as diabetes mellitus or myocardial infarction.

heart; potassium channels; fatty acids; transient outward current

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

FREE FATTY ACIDS are the primary substrate used by the heart to generate cellular ATP (6, 12, 18). The initial steps involved in ATP production via beta -oxidation include the formation of essential intermediates that facilitate transport of fatty acids to the mitochondrial matrix where the enzymes responsible for beta -oxidation are located (1, 6, 12). The two principal intermediates involved in this process are acyl-coenzyme A (CoA), produced by acyl-CoA synthetase at the outer mitochondrial membrane, and acylcarnitine, which is formed at the inner mitochondrial membrane by palmitoylcarnitine transferase I (1, 6, 12). These amphiphilic metabolites, although essential to lipid metabolism, are potentially toxic to the cell at high concentrations but are normally kept at low levels under physiological conditions (1, 6, 12). However, disturbances in beta -oxidation can result in the overproduction of fatty acid metabolites, particularly palmitoylcarnitine and palmitoyl-CoA, which may have deleterious effects on cardiac function. For example, in diabetes mellitus, it is postulated that impaired glucose utilization by the myocardium along with elevations in plasma free fatty acids profoundly increases the rate of beta -oxidation and the subsequent formation of amphiphilic metabolites (6, 12). Moreover, under hypoxic or ischemic conditions, inhibition of electron transport, due to lack of O2, impairs beta -oxidation and also leads to marked accumulation of fatty acid metabolites (1, 7). Indeed, McHowatt et al. (7) have shown that canine ventricular myocytes subjected to hypoxic conditions for 20 min exhibit a 15-fold increase in the total content of long-chain acylcarnitine that is confined mainly to the sarcolemma.

The pathophysiological accumulation of fatty acid intermediates in cardiac myocytes has been proposed to elicit a wide range of adverse electrophysiological, biochemical, and mechanical effects. In particular, long-chain acylcarnitines, such as palmitoylcarnitine, have been shown to produce marked electrophysiological changes in vitro that affect repolarization and excitability and that favor the generation of triggered rhythms from afterdepolarizations (1). For instance, extracellular palmitoylcarnitine applied to isolated cardiac preparations has been shown to produce biphasic effects on action potential duration and to reduce the maximal velocity of phase 0, resting membrane potential, and action potential amplitude (1). Several studies exploring the ionic mechanisms for these electrophysiological changes have shown that palmitoylcarnitine is capable of modulating several ionic currents, including L-type Ca2+ (ICa; see Refs. 3 and 22), fast Na+ (INa; see Refs. 15 and 23), inward rectifier K+ (16), and transient inward (23) currents. Changes in one or more of these currents are likely to underlie the overall electrophysiological effects of fatty acid metabolites on the myocardium and help explain the ionic mechanisms of arrhythmogenesis in diseased hearts (1).

The present investigation examined the effects of two major amphiphilic fatty acid metabolites, palmitoylcarnitine and palmitoyl-CoA, on K+ currents of rat ventricular myocytes that participate in the control of refractoriness and excitability of the heart: the transient outward (Ito) and inward rectifier (IK1) currents. Our data suggest that palmitoylcarnitine and palmitoyl-CoA selectively downregulate Ito density and delay recovery from inactivation by a mechanism that is confined to the cytoplasmic side of the membrane. These amphiphiles had no effect on IK1 and were not mimicked by internal application of palmitate or carnitine alone.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Isolation of ventricular myocytes and recording techniques. Male Sprague-Dawley rats weighing 150-200 g were given an overdose of pentobarbital sodium (100 mg/kg ip), and single ventricular myocytes were dissociated from excised, perfused hearts by a collagenase digestion procedure described previously (13, 14, 24). Isolated myocytes from both ventricles were suspended in Dulbecco's modified Eagle's medium and stored in an incubator at 37°C until used, usually within 8 h of isolation. For electrophysiological recordings, aliquots of myocytes were transferred to a cell chamber mounted on the stage of an inverted microscope and perfused with standard external solution containing (in mM) 138 NaCl, 4.0 KCl, 1.2 MgCl2, 1.8 CaCl2, 0.5 CdCl2, 10 glucose, and 5 HEPES, pH 7.4.

Ionic currents were recorded using the whole cell configuration of the patch-clamp technique. Specifically, borosilicate glass pipettes were heat polished to an internal tip diameter of 1-2 µm and filled with a standard pipette solution containing (in mM) 135 KCl, 3 MgCl2, 10 HEPES, 3 Na2-ATP, 10 EGTA, and 0.5 Na-GTP, pH 7.2 adjusted with KOH. Filled pipettes with resistances of 2-4 MOmega were coupled to a patch-clamp amplifier (Axopatch 1C, Axon Instruments), and, after whole cell recording conditions were established, series resistance was compensated and whole cell capacitance was calculated as the area under the capacitive transient divided by the amplitude (-5 mV) of an applied test pulse (13, 14, 24). A computer program (pCLAMP, Axon Instruments) controlled command potentials and acquired current signals, which were sampled at 4 kHz by an analog-to-digital converter and which were stored on the hard disk of a 486 computer. All experiments were done at room temperature (22-24°C).

Ito was evoked by 500-ms depolarizing pulses to test potentials (Vm) between -40 and +60 mV (0.2 Hz). The holding potential for this protocol was -80 mV, and a 100-ms prepulse was applied to -60 mV to inactivate INa. For each test pulse, the amplitude of Ito was measured as the difference between peak outward current and the current level at the end of the depolarizing clamp pulse. In addition to measuring current-voltage (I-V) relations of Ito, voltage-dependent steady-state parameters and kinetics of Ito recovery from inactivation were also determined. First, steady-state activation parameters were derived from recorded I-V relations by first calculating the conductance at each Vm, normalized to maximum conductance at +60 mV (G/Gmax), and plotting these values as a function of Vm (2, 24). Data were fitted by a Boltzmann distribution to determine the steady-state activation parameters V1/2 and k according to the relation
<IT>G</IT>/<IT>G</IT><SUB>max</SUB> = 1/{1 + exp [(<IT>V</IT><SUB>1/2</SUB> − <IT>V</IT><SUB>m</SUB>)/<IT>k</IT>]}
where V1/2 is the voltage at half-maximal activation, and k the slope factor at Vm = V1/2. Steady-state inactivation was examined by applying 500-ms prepulses from -100 to 0 mV before pulsing to +60 mV (0.1 Hz), and inactivation curves were constructed by plotting normalized current against prepulse voltage. These data were also fitted by a Boltzmann distribution to derive the steady-state inactivation parameters V1/2 and k. Finally, recovery of Ito from inactivation was measured by delivering two identical 500-ms depolarizing pulses from -80 to +60 mV and varying the interpulse interval from 50 to 900 ms. Reactivation curves were constructed by plotting the ratio of peak Ito elicited by the second pulse relative to the first against interpulse interval and were fitted to a single exponential to derive the time constant of recovery.

IK1 was also measured in some myocytes with 100-ms test pulses applied from a prepulse potential of -30 mV (holding potential -80 mV). I-V data were obtained by changing Vm from -120 to -40 mV (0.2 Hz), and the amplitude of IK1 was measured at the end of each test pulse. Raw measurements of both Ito and IK1 were normalized as current densities by dividing measured current amplitude by whole cell capacitance (pA/pF). Finally, action potentials were elicited in some myocytes in current-clamp mode using 5-ms suprathreshold depolarizing current pulses delivered at 1 Hz. For these experiments, myocytes were superfused with room temperature external solution without added Cd2+.

Experimental protocols and statistical analyses. Three different experimental protocols for voltage-clamp analyses were used in the present investigation. In most experiments, modulation of K+ currents by intracellular compounds was examined by using pipette solutions containing different concentrations of palmitoylcarnitine, palmitoyl-CoA, palmitate, or carnitine. For each cell, a dialysis period of 15 min was allowed, after establishing whole cell recording conditions, to achieve equilibration between the pipette solution and the cell cytoplasm. Second, to determine if extracellular amphiphiles modulate K+ channel function, other experiments were conducted in which palmitoylcarnitine or palmitoyl-CoA was applied extracellularly for 15 min during whole cell recordings. Finally, in some experiments, myocytes were preconditioned in external solution containing amphiphile for up to 4 h before recording K+ currents.

All results are expressed as a means ± SE. Statistical comparisons of two groups were made using a Student's t-test, whereas comparisons of more than two groups were carried out by analysis of variance. When a significant difference among groups was indicated by the initial analysis, individual paired comparisons were made using a modified Bonferroni t-test (19). Differences were considered significant at P < 0.05.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Intra- versus extracellular actions of palmitoylcarnitine. Previous experiments have shown that fatty acid metabolites of myocardial beta -oxidation are generated intracellularly and that palmitoylcarnitine mainly accumulates in the sarcolemma under pathophysiological conditions (1, 7). Therefore, we first examined the impact of intracellular palmitoylcarnitine by adding this amphiphile to the pipette solution to give a final concentration from 0.5 to 10 µM. Figure 1A compares raw traces of Ito recorded at potentials from -40 to +60 mV in a control myocyte (Cm = 135 pF; traces at top) and another dialyzed for 15 min with 5 µM palmitoylcarnitine (Cm = 152 pF). At each test potential, Ito amplitude was less in the palmitoylcarnitine-dialyzed myocyte compared with control. Figure 1B summarizes the effects of different pipette concentrations of palmitoylcarnitine on the mean I-V relation of Ito after 15 min of internal dialysis compared with time controls. There was a marked, concentration-dependent decrease in Ito density as reflected by a downward shift in the mean I-V relation. This is further illustrated in Fig. 1C, which plots maximum Ito density (at +60 mV), expressed as a percentage of the mean density of time controls, at each pipette concentration of palmitoylcarnitine tested. Note that, in myocytes dialyzed with 5 and 10 µM palmitoylcarnitine, maximum Ito density was significantly reduced by 43 and 62% from control, respectively (P < 0.05).


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Fig. 1.   Internal dialysis with palmitoylcarnitine. A: superimposed traces of transient outward current (Ito) at -40 to +60 mV (20-mV steps) are shown for a control myocyte (Cm = 135 pF) and a myocyte dialyzed for 15 min with 5 µM palmitoylcarnitine added to the pipette solution (PCp; Cm = 152 pF). B: mean current-voltage (I-V) relations of Ito in myocytes dialyzed for 15 min with pipette solution containing 0-10 µM palmitoylcarnitine. [PC]p, pipette palmitoylcarnitine concentration. No. of myocytes are in parentheses. C: concentration-response of Ito to pipette palmitoylcarnitine. Maximum Ito density measured at +60 mV is expressed as a percentage of the mean density of control, untreated cells (26.5 pA/pF; n = 15 myocytes). * P < 0.05 compared with time control myocytes.

To determine whether the effects of palmitoylcarnitine on Ito were specific to the cytoplasmic side of the membrane, two additional series of experiments were conducted. First, palmitoylcarnitine was applied extracellularly for 15 min during whole cell recording of Ito with standard, internal solution (i.e., without palmitoylcarnitine). Figure 2A summarizes the effect of 10 µM extracellular palmitoylcarnitine on mean I-V relations. For these experiments, data were recorded from the same cell before (baseline) and 15 min after adding this amphiphile to the external solution. As Fig. 2A illustrates, external application of palmitoylcarnitine for 15 min did not significantly alter the mean I-V relation of Ito. In a second series of related experiments, myocytes were preconditioned with 10 µM palmitoylcarnitine for 4 h before recording Ito. Figure 2B shows that even external palmitoylcarnitine exposure for several hours did not significantly alter Ito density, which contrasts markedly with data obtained during only 15 min of internal dialysis (Fig. 1).


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Fig. 2.   External application of palmitoylcarnitine. A: comparison of mean I-V relations recorded from the same cell before (baseline) and 15 min after adding 10 µM palmitoylcarnitine to the external solution (PCo). B: mean I-V relations of Ito of two separate groups of myocytes incubated with or without 10 µM palmitoylcarnitine for 4 h before recording currents. Vm, membrane potential. No. of myocytes are in parentheses.

To assess the significance of intracellular amphiphile on the overall electrophysiology of myocytes, action potentials were recorded in current-clamp mode during internal dialysis with palmitoylcarnitine. Figure 3 shows examples of action potentials recorded at 1 Hz from a Cm (Fig. 3, left) and a myocyte dialyzed with 5 µM palmitoylcarnitine, illustrating a markedly longer action potential duration in the latter. This difference is summarized in Table 1, which compares action potential parameters from control and palmitoylcarnitine-dialyzed myocytes. Note that action potential duration was significantly increased at 25, 50, and 90% of repolarization in the palmitoylcarnitine-dialyzed myocytes, whereas action potential amplitude was significantly less than control.


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Fig. 3.   Action potentials from myocytes dialyzed with palmitoylcarnitine. Action potentials were recorded in current-clamp mode at 1 Hz from a control myocyte (left) and a myocyte dialyzed with 5 µM palmitoylcarnitine (PCp; right). For these recordings, the external solution did not contain Cd2+. See Table 1 for summary data.

                              
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Table 1.   Action potential parameters of myocytes dialyzed with 5 µM palmitoylcarnitine

Effects of intracellular palmitoyl-CoA, palmitate, and carnitine. A second major fatty acid metabolite that accumulates under pathophysiological conditions in the myocardium, palmitoyl-CoA (1, 6, 12), was also examined in experiments similar to those described for palmitoylcarnitine. In general, internal dialysis with palmitoyl-CoA also reduced Ito density (Fig. 4), whereas extracellular application for 15 min had no effect (data not shown). Figure 4 summarizes the response of Ito in myocytes dialyzed for 15 min with 5 or 10 µM palmitoyl-CoA. When compared on an equimolar basis with pipette palmitoylcarnitine, the effects of internal palmitoyl-CoA were smaller in magnitude over the same duration of dialysis. Thus, with 10 µM palmitoyl-CoA in the pipette solution, maximum Ito density (measured at +60 mV) was significantly reduced but by only 37% from control. However, when compared with either amphiphile, neither palmitate nor carnitine alone significantly affected Ito density when added to the pipette solution. This is shown in Fig. 5, A and B, which compares I-V relations in myocytes dialyzed for 15 min with 10 µM palmitate (Fig. 5A) or 10 µM carnitine with time controls. Although there was a downward shift in the I-V relation in palmitate-dialyzed cells (Fig. 5A), this change did not reach significance under these experimental conditions.


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Fig. 4.   Internal dialysis with palmitoyl-CoA. Mean I-V relations of Ito are shown for myocytes dialyzed for 15 min with pipette solution containing 5 or 10 µM palmitoyl-CoA and time-matched controls (open circle ). [PCoA]p, pipette palmitoyl-CoA concentration. No. of myocytes are in parentheses. * P < 0.05 compared with control.


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Fig. 5.   Dialysis with palmitate and carnitine. A: mean I-V relations of Ito for myocytes dialyzed for 15 min with pipette solution containing 10 µM palmitate and time-matched controls (open circle ). B: mean I-V relations of Ito for myocytes dialyzed for 15 min with pipette solution containing 10 µM carnitine. No. of myocytes are in parentheses. [Palmitate]P and [L-carnitine]P, pipette concentrations of palmitate and L-carnitine, respectively.

Amphiphile-mediated alterations in recovery from inactivation. In addition to analyzing the effects of palmitoylcarnitine and palmitoyl-CoA on Ito density, we also compared time- and voltage-dependent properties of this current with and without internal amphiphile. Figure 6 summarizes the effects of intracellular palmitoylcarnitine (A) and palmitoyl-CoA (B) on the recovery of Ito from inactivation. Note that internal dialysis with both compounds delayed recovery from inactivation compared with time control cells but that only palmitoylcarnitine had a significant effect at the highest concentration tested (10 µM; see Fig. 6, insets). However, neither compound had a significant effect on the voltage dependence of steady-state activation or inactivation (Table 2).


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Fig. 6.   Delayed recovery of Ito from inactivation with intracellular amphiphiles. Recovery from inactivation was measured in myocytes dialyzed with palmitoylcarnitine (A) or palmitoyl-CoA (B) for 15 min. Time controls are shown by open circle  in A and B. Insets summarize the measured rate of recovery from inactivation (tau , in ms) at each concentration of amphiphile tested. I2/I1, ratio of peak Ito elicited by the second pulse relative to the first. * P < 0.05 compared with the untreated group; n, no. of myocytes.

                              
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Table 2.   Voltage dependence of Ito steady-state activation and inactivation in myocytes dialyzed for 15 min with palmitoylcarnitine or palmitoyl-CoA

Sensitivity of IK1 to intracellular palmitoylcarnitine. To examine whether intracellular amphiphiles influenced other K+ currents to the same extent as Ito, we additionally analyzed IK1 as a function of the concentration of palmitoylcarnitine in the pipette solution. Figure 7A compares raw traces of IK1 (-120 to -20 mV) recorded from the same control (traces on top) and palmitoylcarnitine-dialyzed myocytes from which examples in Fig. 1A were obtained. These traces show that a 5 µM concentration of the amphiphile added to the pipette solution did not clearly alter the amplitude of IK1, as measured at the end of each test pulse. This is further illustrated in Fig. 7B, which compares mean I-V relations of IK1 in myocytes dialyzed with 0-10 µM palmitoylcarnitine for 15 min. The lack of effect of palmitoylcarnitine on IK1 contrasts markedly with the reduction in Ito density observed with the same pipette concentrations. Moreover, in a small subset of myocytes (n = 5), internal dialysis with 10 µM palmitoyl-CoA also did not change IK1 (data not shown).


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Fig. 7.   Effect of intracellular palmitoylcarnitine on inward rectifier K+ current (IK1). A: superimposed traces of IK1 at -120 to -20 mV (10-mV increment) are shown from the same control (traces on top) and palmitoylcarnitine-dialyzed myocytes from which the examples in Fig. 1A were obtained. B: mean I-V relations of IK1 in ventricular myocytes dialyzed for 15 min with 0-10 µM palmitoylcarnitine. No. of myocytes are in parentheses.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Modulation of cardiac ion channels by amphiphilic metabolites. Recent studies have shown that palmitoylcarnitine and a structurally related phospholipid metabolite, lysophosphatidylcholine, are capable of modulating a variety of ion channels that control refractoriness and excitability of the myocardium. For example, action potential recordings from avian cardiomyocytes suggest that relatively high concentrations of extracellular palmitoylcarnitine (100-300 µM) increase ICa (3), whereas voltage-clamp data from isolated guinea pig myocytes indicate that lower palmitoylcarnitine concentrations (1-25 µM) are inhibitory to this current whether administered extra- or intracellularly (22). Sato et al. (15) report that external palmitoylcarnitine (0.5-50 µM) decreases INa in guinea pig ventricular myocytes, whereas in rabbit myocytes Wu and Corr (23) have shown that this same amphiphile elicits a slow-inactivating Na+ and transient inward current, an effect that was observed with either extracellular or internal application. These latter studies thus provide evidence that lipid metabolites may have complex actions on Na+ conductance that may contribute to the genesis of arrhythmias under pathophysiological conditions.

In the present study, we show that palmitoylcarnitine and palmitoyl-CoA (0.5-10 µM) decrease Ito density (Figs. 1 and 4) and prolong its recovery from inactivation (Fig. 6). This change in K+ channel activity in the intact cell would be expected to delay repolarization and possibly act in concert with increased Na+ conductance at plateau potentials (23) to elicit automatic or triggered responses. In support of this hypothesis, we found that internal palmitoylcarnitine increased action potential duration (Fig. 3 and Table 1), although we did not attempt to separate the relative contributions of Na+ and K+ channels to this overall change. The decrease in action potential amplitude observed in palmitoylcarnitine-dialyzed myocytes (Table 1) is consistent with a decrease in INa, as reported by Sato et al. (15) in guinea pig ventricular myocytes.

We also show that the amphiphile-mediated decrease in Ito density was greatest during intracellular application, a result that correlates with findings that fatty acid metabolites are generated intracellularly (1) and may distribute preferentially in the intact cell, with palmitoylcarnitine accumulating mainly in the sarcolemma (7) and palmitoyl-CoA in the mitochondrial matrix (1). Although our data suggest that palmitoylcarnitine causes a greater reduction in Ito density than palmitoyl-CoA, this difference may be due to limitations of the recording technique more than a true difference in sensitivity to these amphiphiles. That is, given the marked difference in molecular weight of the amphiphiles studied in our model (palmitoyl-CoA-1006; palmitoylcarnitine-436), it is likely that the rate of internal dialysis with palmitoyl-CoA and its intracellular concentration were less than those for palmitoylcarnitine (10) over the 15-min dialysis period used in our experiments. This limitation therefore precludes a more quantitative analysis of the sensitivity of Ito channels to intracellular palmitoylcarnitine and palmitoyl-CoA.

The molecular mechanisms underlying the specific effects of amphiphilic metabolites on Ito or other ion channels have not been defined thoroughly. One possibility is that long-chain acylcarnitines indirectly affect ion channels by incorporating into the membrane and altering local membrane fluidity by changing the packing of phospholipid molecules (1, 22, 23). However, it is equally possible that these compounds directly interact with the channel protein to alter gating kinetics or probability of channel opening (23). The process by which lipids directly alter protein function, i.e., palmitoylation, has not been examined thoroughly for cardiac ion channels but has been studied in relation to turnover of the alpha -subunit of Gs, which couples the beta -adrenergic receptor to adenylyl cyclase (21). Whether or not Ito channels are downregulated via palmitoylation requires further study, but our finding that palmitoylcarnitine and palmitoyl-CoA, which are effective palmitoylating compounds (21), only reduced Ito during internal dialysis suggests that a specific interaction with the Ito channel protein is a plausible mechanism.

Our data also suggest that intracellular fatty acid metabolites may selectively downregulate Ito channels, since we found no effect of either palmitoylcarnitine or palmitoyl-CoA on IK1. This latter finding differs however with the work of Kiyosue and Arita (5) and Shen and Pappano (16) who show that extracellular lysophosphatidylcholine and palmitoylcarnitine reduce IK1 in guinea pig ventricular myocytes in addition to inhibiting the current carried by the Na+-K+ pump (16). The differences between our results and those of others (16) are likely to be species-related, since all studies used the same concentration of palmitoylcarnitine (10 µM).

Pathogenic conditions responsible for accumulation of fatty acid metabolites. A number of pathophysiological states are associated with altered fatty acid metabolism that potentially can lead to intracellular accumulation of toxic intermediates, either as a result of an excess or reduced rate of beta -oxidation. For example, diabetes mellitus is a pathophysiological state that is characterized by a marked decrease in Ito density (17, 20, 24) and where excess beta -oxidation has been proposed to cause an accumulation of amphiphilic metabolites (6, 12). During insulin deficiency, there are significant increases in plasma free fatty acids and in myocardial triglyceride content plus a dramatic fall in oxidative metabolism of glucose (6, 12). Indeed, in the diabetic heart, glucose oxidation is nearly abolished, and the contribution of fatty acids to overall ATP production can exceed 90% of the total ATP produced (6, 12). Therefore, under these conditions, it is proposed that an enhanced rate of beta -oxidation may not keep pace with an oversupply of exogenous and endogenous fatty acids, thereby causing fatty acid intermediates to accumulate (6, 12). A similar set of conditions may also exist in the failing, nondiabetic heart, which exhibits a profound increase in fatty acid utilization (9) and which is also characterized by a significant decrease in Ito density (4, 8, 11, 13, 14). However, it is uncertain to what extent fatty acid metabolites contribute to the depressed contractile function and electrophysiological abnormalities that characterize the diabetic and failing heart.

Depressed beta -oxidation is a second mechanism by which fatty acid metabolites can accumulate in the myocardium and elicit deleterious effects (1, 6). For example, in the hypertrophied heart, fatty acid oxidation is reduced compared with normal and is mediated by a decrease in fatty acid transport in the mitochondria secondary to a reduction in myocardial carnitine content (6). Under hypoxic or ischemic conditions, inhibition of electron transport, due to lack of O2, also impairs beta -oxidation and is associated with a marked accumulation of fatty acid metabolites (1, 7). This latter pathogenic condition may be worsened upon reperfusion of the myocardium where fatty acid oxidation may be greater than control (6).

In conclusion, our data suggest that palmitoylcarnitine and palmitoyl-CoA selectively downregulate Ito channels in rat ventricular myocytes by a mechanism that is confined to the cytoplasmic side of the membrane. The increased content of these amphiphiles in the sarcolemma, particularly palmitoylcarnitine, may underlie the contractile and electrical abnormalities in the heart during pathophysiological states that influence fatty acid metabolism, such as diabetes mellitus and myocardial infarction.

    ACKNOWLEDGEMENTS

This work was supported by a Research Award from the American Diabetes Association.

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: G. J. Rozanski, Dept. of Physiology and Biophysics, Univ. of Nebraska College of Medicine, 600 South 42nd St., Omaha, NE 68198-4575.

Received 16 March 1998; accepted in final form 3 September 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Cell Physiol 275(6):C1660-C1667
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