This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, J. Articles by Wang, Z. Search for Related Content PubMed PubMed Citation Articles by Wang, J. Articles by Wang, Z. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Related Collections Electrophysiology Arrythmias-basic studies Ischemic biology - basic studies Ion channels/membrane transport Lipid and lipoprotein metabolism

(Circulation. 2001;104:2645.)
© 2001 American Heart Association, Inc.

Brief Rapid Communications

Phospholipid Metabolite 1-Palmitoyl-Lysophosphatidylcholine Enhances Human Ether-a-Go-Go-Related Gene (HERG) K⁺ Channel Function

Jingxiong Wang, MD; Huizhen Wang, MD; Hong Han, MD, PhD; Yiqiang Zhang, MSc; Baofeng Yang, MD, PhD; Stanley Nattel, MD; Zhiguo Wang, PhD

From the Research Center, Montreal Heart Institute, Montreal, Canada (J.W., H.W., H.H., Y.Z., Z.W.); the Department of Medicine, University of Montreal, Montreal, Canada (J.W., Y.Z., Z.W.); the Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada (S.N.); and the Department of Pharmacology, Harbin Medical University, Harbin, Heilongjiang, China (B.Y.).

Correspondence to Zhiguo Wang, Research Center, Montreal Heart Institute, 5000 Belanger East, Montreal, PQ H1T 1C8 Canada. E-mail wzmail{at}canada.com

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background— Lysophosphatidylcholine (LPC), a naturally occurring phospholipid metabolite, accumulates in the ischemic heart and causes extracellular K⁺ accumulation and action potential shortening. LPC has been incriminated as a biochemical trigger of lethal cardiac arrhythmias, but the underlying mechanisms remain poorly understood.

Methods and Results— We studied the effect of 1-palmitoyl-LPC (Pal-LPC) on currents resulting from human ether-a-go-go-related gene (HERG) expression in human embryonic kidney (HEK) cells using whole-cell patch-clamp techniques. Bath application of Pal-LPC consistently and reversibly increased HERG current (I_HERG). The effects of Pal-LPC were apparent as early as 3 minutes after application of the drug, reached maximum within 10 minutes, and were reversible on washout. Pal-LPC increased I_HERG at voltages between -20 and +30 mV, with greater effects at stronger depolarization. However, Pal-LPC did not affect the voltage-dependence of I_HERG activation. In contrast, Pal-LPC significantly shifted the inactivation curve toward more positive potentials, causing a mean 20.0±2.2 mV shift in half-inactivation voltage relative to control.

Conclusions— Our results indicate that apart from being a well-recognized target for drug inhibition, I_HERG can also be enhanced by natural substances. An increase in I_HERG by Pal-LPC may contribute to K⁺ loss, abnormal electrophysiology, and arrhythmia occurrence in the ischemic heart.

Key Words: lysophosphatidylcholines • ion channels • patch-clamp techniques

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Lysophosphatidylcholine (LPC) is a naturally occurring intracellular phospholipid metabolite that is present in a variety of mammalian tissues.¹ LPC accumulates rapidly in the heart during cardiac ischemia and in diabetic cardiomyopathy.² It has been incriminated as a biochemical trigger of lethal cardiac arrhythmias,^3–8 and it promotes abnormal rhythmic activity, delayed afterdepolarizations, triggered activity, and intramyocardial reentry. Action potential duration (APD) shortening by LPC has been documented in rabbit atrial and ventricular cells,⁴ guinea pig ventricular cells,⁵ and canine ventricular cells⁶ and Purkinje fibers.³ LPC levels correlate with the occurrence of arrhythmias in ischemic and diabetic hearts.^7–9

Inhibitory effects on sodium current¹⁰ and inwardly rectifying K⁺ current (I_K1) ¹¹ explain the conduction slowing and membrane depolarization produced by LPC, but the mechanisms underlying APD shortening remain incompletely understood. Reduced I_K1 should, if anything, increase APD. A recent study by Goldhaber et al¹² reported that LPC (20 µmol/L) decreases tissue K⁺ content by 15%, an effect associated with gradual APD shortening and increased K⁺ efflux. During acute myocardial ischemia, increased K⁺ efflux in the face of maintained Na⁺/K⁺ pumping results in extracellular K⁺ accumulation, a key arrhythmogenic factor. Although increased K⁺ efflux can account for some of the important arrhythmogenic effects of LPC, how LPC increases K⁺ efflux is essentially unknown. Increased K⁺ conductance could account for APD abbreviation and could contribute to increased K⁺ efflux; however, LPC is not known to increase cardiac K⁺-currents.

The human ether-a-go-go-related gene (HERG) encodes the rapid delayed rectifier K⁺ current (I_Kr),¹³ a critically important cardiac repolarizing current in most animals, including humans.¹⁴ Apart from being a molecular target for mutations that generate long-QT syndrome, HERG is also a prime pharmacological target for a wide range of drugs that block the HERG current (I_HERG) exclusively; this blockade either confers antiarrhythmic efficacy or produces proarrhythmic cardiotoxicity.¹⁵ The present study was designed to investigate whether LPC can modulate I_HERG and, if so, to characterize the biophysical components of this action.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Cell Culture
HEK293 cells stably expressing HERG (a kind gift from Drs Zhou and January, University of Wisconsin, Madison¹⁶) were seeded in a 25-cm², triangular, cell-culture flasks and grown in Dulbecco’s modified eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, 200-µmol/L G418, 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells subcultured to 85% confluency were harvested by trypsinization and stored in Tyrode solution containing 0.5% BSA at 4°C. Electrophysiological recordings were conducted within 10 hours of storage.

Whole-Cell Patch-Clamp Recording
Patch-clamp techniques have been described in detail elsewhere.¹⁴ Currents were recorded by whole-cell voltage-clamp with an Axopatch-200B amplifier (Axon Instruments). Borosilicate glass electrodes had tip resistances of 1 to 3 M when filled with (in mmol/L): GTP 0.1, potassium aspartate 110, KCl 20, MgCl₂ 1, Mg-ATP 5, HEPES 10, and phosphocreatine 5 (pH 7.3). The extracellular solution contained (in mmol/L): NaCl 136, KCl 5.4, MgCl₂ 1, HEPES 5, glucose 10, and CaCl₂ 1 (pH 7.4). Experiments were conducted at 36±1°C. Junction potentials were zeroed before formation of the membrane-pipette seal. Series resistance and capacitance were compensated, and leak currents were subtracted. 1-Palmitoyl-LPC (Pal-LPC; Sigma) was dissolved directly into the bath solution at the desired concentrations immediately before each experiment. 1-Palmitoyl-lysophosphatidylglycerol (Avanti Polar Lipid) and membrane-permeable ceramide (Sigma) were used for negative controls.

Data Analysis
Group data are expressed as mean±SEM. Comparisons among groups were made by ANOVA (F test), Bonferroni-adjusted t tests were used for multiple group comparisons, and paired t tests were used for single comparisons. A 2-tailed P<0.05 indicated statistically-significant difference. Nonlinear least-square curve-fitting was performed with CLAMPFIT in pCLAMP 8.0 or Graphpad Prism.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Bath application of Pal-LPC reversibly increased I_HERG (Figures 1A and 1B) in a concentration-dependent manner (Figure 1D). Pal-LPC increased I_HERG at voltages between -20 and +30 mV (Figure 1B), and the F-test indicated the effect was voltage-dependent (P<0.01, Figure 1C). However, the half-maximum activation voltage, which was determined by the tail currents, was not affected by Pal-LPC (from control to LPC: -26±3 to -24±3 mV, P>0.05), nor was the slope factor k (from control to Pal-LPC: 8±1 to 9±1 mV, P>0.05). The effects of Pal-LPC were apparent (37% the maximum) as early as 3 minutes after application of the drug, and they reached maximum within 10 minutes (Figure 1E). Under our experimental conditions, 2.5 minutes are required for the solution to circulate into the bathing chamber, and this implies that Pal-LPC effects took place within 0.5 to 1 minute of exposure. Neither 1-palmitoyl-lysophosphatidylglycerol (5 µmol/L) nor membrane-permeable ceramide (50 µmol/L) produced significant effects on I_HERG (Figures 1F and 1G).

View larger version (35K): [in this window] [in a new window]   Figure 1. A, IHERG recordings in control conditions, 10 minutes after LPC application, and after 10 minutes of LPC washout. B, IHERG step current-voltage relations in control conditions (Ctl, ), 10 minutes after LPC (5 µmol/L; ) application, and after 10 minutes of LPC washout (W/O; ). *P<0.05, **P<0.01, and ***P<0.001, LPC versus control (n=10 cells). C, Ratio of IHERG in the presence of LPC over control (P<0.05 by ANOVA for the voltage-dependence of current increase between -30 and +20 mV). D, Concentration-dependence of LPC on IHERG at 0 mV (n=12). E, Time-course (time after LPC application) of LPC effects (n=10), expressed as ratios of LPC/control, normalized to 15 minutes. F and G, Raw and mean data of IHERG before and after application of 1-palmitoyl-lysophosphatidylglycerol (LP-G; n=6) or membrane-permeable ceramide (C2; n=6).

Pal-LPC significantly shifted the inactivation curve toward more positive potentials (Figures 2A and 2B), causing a mean 20.0±2.2 mV shift in half-maximum activation voltage for inactivation relative to control. Figure 2C shows Pal-LPC-induced percent change in currents at +20 mV after prepulses to different voltages, and it indicates that Pal-LPC did not alter currents when inactivation was fully removed by prepulses to more negative voltages but it significantly increased current at prepulse voltages allowing for inactivation. The inactivation time constants determined by monoexponential fit to the decaying currents recorded at +20 mV (Figure 2A) were 4.8±0.4 and 6.9±0.9 ms for control and Pal-LPC, respectively (P<0.05). The recovery time constants measured by the inward currents during the hyperpolarizing prepulses were not significantly altered by Pal-LPC (from 1.9±0.1 to 1.7±0.2 ms, P>0.05).

View larger version (35K): [in this window] [in a new window]   Figure 2. A, IHERG recordings (with voltage protocol in inset) before and 10 minutes after LPC application. B, IHERG inactivation voltage-dependence. The tail current (Itail) during the test pulse to +20 mV at various prepulse potentials was normalized to the value for a prepulse to -120 mV. Symbols are mean±SEM of 13 experiments, and curves are Boltzmann fits. Half-maximum activation voltage for inactivation was shifted by LPC from -40.9±3.4 to -21.2±1.8 mV (P<0.001). C, Ratio of IHERG with LPC over control (P<0.001 for voltage-dependence by ANOVA). *P<0.05, **P<0.01, and ***P<0.001 for LPC versus control.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this study, we demonstrated that Pal-LPC increases I_HERG in HEK293 cells. The Pal-LPC-induced increase in I_HERG is reversible and occurs within minutes of extracellular application. The current-enhancing effect is in large measure due to a substantial depolarizing shift in I_HERG inactivation, such that more current is available at any given voltage within the activation range.

Intracellular K⁺ loss and accompanying extracellular K⁺ accumulation impairs cardiac electrical activity and can also promote cell death through apoptosis,¹⁷ thereby contributing to the pathophysiology of the ischemic myocardium. ATP-sensitive K⁺ current (I_KATP) contributes significantly to the rapid increase in cellular K⁺ efflux and APD shortening during myocardial ischemia and hypoxia. However, during the first 10 minutes of ischemia in intact hearts, cytosolic ATP concentrations remain 2 orders of magnitude greater than the ATP concentration, thus causing half-maximal blockade of I_KATP in excised membrane patches.¹⁸ It is conceivable that membrane conductances other than I_KATP may contribute to APD abbreviation and loss of intracellular K⁺.

LPC, a class of lysophospholipids that accumulate in the cell membrane during acute myocardial ischemia, decreases peak but enhances sustained Na⁺ current, resulting in a noninactivated component in ventricular myocytes of various species.^10,19,20 LPC also reduces I_K1, ¹¹ the major determinant of the membrane resting potential in cardiac cells. These findings can account for the conduction slowing and membrane depolarization caused by LPC, but they do not explain how LPC produces APD shortening ^3–6 and K⁺ loss.¹² Our observation regarding Pal-LPC effects on I_HERG may explain, at least in part, the latter observations. Because Pal-LPC increased I_HERG significantly in the range of plateau voltages (-10 to +10 mV), it is quite possible that Pal-LPC promotes the repolarization of cardiac action potentials or APD shortening during acute ischemia and that Pal-LPC-induced I_HERG enhancement could also contribute to K⁺-efflux.

Many class III drugs produce their antiarrhythmic effects by acting on HERG/I_Kr currents. In addition, a wide spectrum of non-antiarrhythmic agents can cause proarrhythmic effects (acquired long-QT syndrome) by inhibiting I_HERG. ¹⁵ In the present study, we found that I_HERG can also be enhanced. Furthermore, the mechanism of enhancement involved a substantial positive shift in I_HERG inactivation voltage-dependence. The rapid inactivation mechanism of I_HERG is a distinct property and, to our knowledge, this is the first time that a shift in I_HERG inactivation voltage-dependence has been shown to underlie a potentially significant endogenous pathophysiological mechanism. In addition to the potential intrinsic significance of this observation, it may open up new possibilities in exploring the regulation of HERG properties by membrane lipids.

Under ischemic conditions, LPC (normally present at 0.5% to 3.5% of total membrane phospholipids), increases to an extent that varies among different species and different tissue compartments. The typical LPC concentration range is 100 to 200 µmol/L. ²¹ Assuming that 90% of LPC protein-bound, the free concentration of LPC is 10 to 20 µmol/L. LPC, mainly Pal-LPC,^21–23 accumulation during cardiac ischemia is thought to occur predominantly in the extracellular space.²⁴ Thus, the concentration of Pal-LPC (5 µmol/L) used in this study is likely relevant to pathophysiological situations.

	Acknowledgments

 
This work was supported by the Canadian Institutes of Health Research (CIHR) and the Heart and Stroke Foundation of Quebec. Dr Wang is a research scholar of the Heart and Stroke Foundation of Canada (HSFC). Drs H. Han and H. Wang are research fellows of the HSFC and CIHR, respectively. The authors thank XiaoFan Yang for excellent technical support.

Received September 27, 2001; revision received October 11, 2001; accepted October 11, 2001.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Hatch GM, O K, Choy PC. Regulation of phosphatidylcholine metabolism in mammalian hearts. Biochem Cell Biol.. 1989; 67: 67–77.[Medline] [Order article via Infotrieve]
   
2. Makino N, Dhalla KS, Elimban V, et al. Sarcolemmal Ca²⁺ transport in streptozotocin-induced diabetic cardiomyopathy in rats. Am J Physiol.. 1987; 253: E202–E207.[Abstract/Free Full Text]
   
3. Corr PB, Cain ME, Witkowski FX, et al. Potential arrhythmogenic electrophysiological derangements in canine Purkinje fibers induced by lysophosphoglycerides. Circ Res.. 1979; 44: 822–832.[Abstract/Free Full Text]
   
4. Fazekas T, Nemeth M, Papp JG, et al. Electrophysiological changes induced by lysophosphatidylcholine, an ischaemic phospholipid catabolite, in rabbit atrial and ventricular cardiac cells. Acta Physiol Hung.. 1992; 79: 261–272.[Medline] [Order article via Infotrieve]
   
5. Liu E, Goldhaber JI, Weiss JN. Effects of lysophosphatidylcholine on electrophysiological properties and excitation-contraction coupling in isolated guinea pig ventricular myocytes. J Clin Invest.. 1991; 88: 1819–1832.
   
6. Saffitz JE, Corr PB, Lee BI, et al. Pathophysiologic concentrations of lysophosphoglycerides quantified by electron microscopic autoradiography. Lab Invest.. 1984; 50: 278–286.[Medline] [Order article via Infotrieve]
   
7. Man RY. Lysophosphatidylcholine-induced arrhythmias and its accumulation in the rat perfused heart. Br J Pharmacol.. 1988; 93: 412–416.[Medline] [Order article via Infotrieve]
   
8. Corr PB, Yamada KA, Creer MH, et al. Lysophosphoglycerides and ventricular fibrillation early after onset of ischemia. J Mol Cell Cardiol.. 1987; 19: 45–53.
   
9. Kinnaird AA, Choy PC, Man RY. Lysophosphatidylcholine accumulation in the ischemic canine heart. Lipids.. 1988; 23: 32–35.[Medline] [Order article via Infotrieve]
   
10. Undrovinas AI, Fleidervish IA, Makielski JC. Inward sodium current at resting potentials in single cardiac myocytes induced by the ischemic metabolite lysophosphatidylcholine. Circ Res.. 1992; 71: 1231–1241.[Abstract/Free Full Text]
    
11. Kiyosue T, Arita M. Effects of lysophosphatidylcholine on resting potassium conductance of isolated guinea pig ventricular cells. Pflugers Arch.. 1986; 406: 296–302.[Medline] [Order article via Infotrieve]
    
12. Goldhaber JI, Deutsch N, Alexander LD, et al. Lysophosphatidylcholine and Cellular Potassium Loss in Isolated Rabbit Ventricle. J Cardiovasc Pharmacol Ther.. 1998; 3: 37–42.
    
13. Sanguinetti MC, Jiang C, Curran ME. A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the I_Kr potassium channel. Cell.. 1995; 81: 299–307.[Medline] [Order article via Infotrieve]
    
14. Wang Z, Fermini B, Nattel S. Rapid and slow components of delayed rectifier outward current in human atrial myocytes. Cardiovasc Res.. 1994; 28: 1540–1545.[Medline] [Order article via Infotrieve]
    
15. Taglialatela M, Castaldo P, Pannaccione A, et al. Human ether-a-go-go related gene (HERG) K⁺ channels as pharmacological targets: present and future implications. Biochem Pharmacol.. 1998; 55: 1741–1746.[Medline] [Order article via Infotrieve]
    
16. Zhou Z, Gong Q, Ye B, et al. Properties of HERG channels stably expressed in HEK 293 cells studied at physiological temperature. Biophys J.. 1998; 74: 230–241.[Abstract/Free Full Text]
    
17. Yu SP, Choi DW. Ions, cell volume, and apoptosis. Proc Natl Acad Sci U S A.. 2000; 97: 114–117.
    
18. Weiss JN, Venkatesh N, Lamp ST. ATP-sensitive K⁺ channels and cellular K⁺ loss in hypoxic and ischaemic mammalian ventricle. J Physiol.. 1992; 447: 649–673.[Abstract/Free Full Text]
    
19. Burnashev NA, Undrovinas AI, Fleidervish IA, et al. Ischemic poison lysophosphatidylcholine modifies heart sodium channels gating inducing long-lasting bursts of openings. Pflugers Arch.. 1989; 415: 124–126.[Medline] [Order article via Infotrieve]
    
20. Sato T, Kiyosue T, Arita M. Inhibitory effects of palmitoylcarnitine and lysophosphatidylcholine on the sodium current of cardiac ventricular cells. Pflugers Arch.. 1992; 420: 94–100.[Medline] [Order article via Infotrieve]
    
21. Liu SY, Yu CH, Hays JA, et al. Modification of heart sarcolemmal phosphoinositide pathway by lysophosphatidylcholine. Biochim Biophys Acta.. 1997; 1349: 264–274.[Medline] [Order article via Infotrieve]
    
22. Sargent CA, Vesterqvist O, Ogletree ML, et al. Effects of endogenous and exogenous lysophosphatidylcholine in isolated perfused rat hearts. J Mol Cell Cardiol.. 1993; 25: 905–913.[Medline] [Order article via Infotrieve]
    
23. Van Bilsen M, Van der Vusse GJ. Phospholipase-A2-dependent signaling in the heart. Cardiovasc Res.. 1995; 30: 518–529.[Medline] [Order article via Infotrieve]
    
24. McHowat J, Corr PB. Thrombin-induced release of lysophosphatidylcholine from endothelial cells. J Biol Chem.. 1993; 268: 15605–15610.[Abstract/Free Full Text]
    

This article has been cited by other articles:

				D. Heitzmann and R. Warth Physiology and Pathophysiology of Potassium Channels in Gastrointestinal Epithelia Physiol Rev, July 1, 2008; 88(3): 1119 - 1182. [Abstract] [Full Text] [PDF]

				I. M. Fearon OxLDL enhances L-type Ca2+ currents via lysophosphatidylcholine-induced mitochondrial reactive oxygen species (ROS) production Cardiovasc Res, March 1, 2006; 69(4): 855 - 864. [Abstract] [Full Text] [PDF]

				H. Chapman, C. Ramstrom, L. Korhonen, M. Laine, K. T. Wann, D. Lindholm, M. Pasternack, and K. Tornquist Downregulation of the HERG (KCNH2) K+ channel by ceramide: evidence for ubiquitin-mediated lysosomal degradation J. Cell Sci., November 15, 2005; 118(22): 5325 - 5334. [Abstract] [Full Text] [PDF]

				T. Saito, T. Sato, T. Miki, S. Seino, and H. Nakaya Role of ATP-sensitive K+ channels in electrophysiological alterations during myocardial ischemia: a study using Kir6.2-null mice Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H352 - H357. [Abstract] [Full Text] [PDF]

				T. Schilling, F. Lehmann, B. Ruckert, and C. Eder Physiological mechanisms of lysophosphatidylcholine-induced de-ramification of murine microglia J. Physiol., May 15, 2004; 557(1): 105 - 120. [Abstract] [Full Text] [PDF]

				J. Wang, H. Wang, Y. Zhang, H. Gao, S. Nattel, and Z. Wang Impairment of HERG K+ Channel Function by Tumor Necrosis Factor-{alpha}: ROLE OF REACTIVE OXYGEN SPECIES AS A MEDIATOR J. Biol. Chem., April 2, 2004; 279(14): 13289 - 13292. [Abstract] [Full Text] [PDF]

				J. Tamargo, R. Caballero, R. Gomez, C. Valenzuela, and E. Delpon Pharmacology of cardiac potassium channels Cardiovasc Res, April 1, 2004; 62(1): 9 - 33. [Abstract] [Full Text] [PDF]

				Y. Zhang, H. Han, J. Wang, H. Wang, B. Yang, and Z. Wang Impairment of Human Ether-a-Go-Go-related Gene (HERG) K+ Channel Function by Hypoglycemia and Hyperglycemia. SIMILAR PHENOTYPES BUT DIFFERENT MECHANISMS J. Biol. Chem., March 14, 2003; 278(12): 10417 - 10426. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, J. Articles by Wang, Z. Search for Related Content PubMed PubMed Citation Articles by Wang, J. Articles by Wang, Z. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Related Collections Electrophysiology Arrythmias-basic studies Ischemic biology - basic studies Ion channels/membrane transport Lipid and lipoprotein metabolism