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Cardiovascular Research 2007 73(2):395-403; doi:10.1016/j.cardiores.2006.09.013
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Copyright © 2006, European Society of Cardiology

Differential sensitivities of the NCX1.1 and NCX1.3 isoforms of the Na+–Ca2+ exchanger to {alpha}-linolenic acid

Bradley P. Andera,b,c, Cecilia Hurtadob,c,1, Carla S. Raposob,c, Thane G. Maddafordb,c, Justin F. Denisetb,c, Larry V. Hryshkob,c, Grant N. Piercea,b,c,d and Anton Lukasa,b,c,*

aCanadian Centre for Agri-Food Research in Health and Medicine, Winnipeg, Canada
bInstitute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Winnipeg, Canada
cDepartment of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada
dDepartment of Physiology, Faculties of Medicine and Pharmacy, University of Manitoba, Winnipeg, Canada

* Corresponding author. St. Boniface General Hospital Research Centre, 351 Taché Avenue, Winnipeg, MB, Canada, R2H 2A6. Tel.: +1 204 235 3206; fax: +1 204 231 1151. Email address: gpierce{at}sbrc.ca alukas{at}shaw.ca

Received 19 April 2006; revised 9 September 2006; accepted 18 September 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Dietary intake of {omega}-3 polyunsaturated fatty acids (PUFA) like {alpha}-linolenic acid (ALA) is antiarrhythmic and cardioprotective. PUFA may also be beneficial in hypertension. Altered Na+–Ca2+ exchanger (NCX) activity has been implicated in arrhythmias, hypertension and heart failure and may be a target for PUFA. Thus, we tested the effects of ALA and other distinct fatty acids on the cardiac (NCX1.1) and vascular (NCX1.3) NCX isoforms.

Methods: HEK293 cells stably expressing NCX isoforms were ramped from +60 to –100 mV (over 1600 ms) in the absence and presence of 25 µM oleic acid (OA, {omega}-9), linoleic acid (LA, {omega}-6), ALA ({omega}-3), or eicosapentaenoic acid (EPA, {omega}-3). NiCl2 (5 mM) was used to inhibit and therefore identify the NCX current. The effect of 25 µM ALA on NCX1.1 and NCX1.3 activity was also assessed in adult rat ventricular cardiomyocytes and rabbit aortic vascular smooth muscle cells (VSMC) by measuring [Ca2+]i following substitution of [Na+]o with Li+.

Results: Application of Ni2+ had no effect in non-transfected cells. ALA and EPA (25 µM) reduced the Ni2+-sensitive forward NCX1.1 current (at –100 mV) by 64% and reverse current (at +60 mV) by 57%, and inhibited the Ni2+-sensitive NCX1.3 forward and reverse currents by 79% and 76%, respectively. Neither OA nor LA (25 µM) affected the NCX1.1 currents, but both partially inhibited the forward and reverse mode NCX1.3 currents. Inhibition of NCX1.3 by ALA occurred at a much lower IC50 (~19 nM) than for NCX1.1 (~120 nM). In cardiomyocytes and VSMC, ALA significantly reduced the Li+-induced rise in intracellular [Ca2+].

Conclusions: NCX1.3 is more sensitive to inhibition by ALA than NCX1.1. In addition, only {omega}-3 PUFA inhibits NCX1.1, but several classes of fatty acids inhibit NCX1.3. The differential sensitivity of NCX isoforms to fatty acids may have important implications as therapeutic approaches for hypertension, heart failure and arrhythmias.

KEYWORDS Na/Ca-exchanger; Hypertension; Ion exchangers; Antihypertensive agents; Antiarrhythmic agents


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The Na+–Ca2+ exchanger (NCX) is an electrogenic membrane transporter that transports three Na+ for one Ca2+. The NCX normally functions to maintain Ca2+ homeostasis by removing Ca2+ that enters the cardiomyocyte during each beat, or by restoring resting cytosolic Ca2+ in the vasculature following an agonist response (‘forward mode’). However, the NCX can also operate in reverse mode to bring Ca2+ into the cell. The direction of ion movement is determined by the concentration gradients for Na+ and Ca2+, and membrane potential. Alternative splicing of the NCX1 protein can yield different isoforms that exhibit tissue specific expression [1,2]. These isoforms differ only in a small region near the carboxyl terminus of the large intracellular loop and have different regulatory characteristics. For example, NCX1.1 is prominently expressed in cardiac muscle whereas NCX1.3 is found in smooth muscle and the kidneys [3]. Also, NCX activity is decreased by elevations in [Na+]i (Na+-dependent or I1 inactivation), which can be alleviated by increased [Ca2+]i in NCX1.1, but not in NCX1.3 [4].

The NCX is believed to play a prominent role in the pathophysiology of hypertension, heart failure, and ischemia/reperfusion injury. In hypertension, high salt diets can promote production of cardiotonic steroids that inhibit the Na+–K+ ATPase and thus increase [Na+]i [5]. One possible mechanism for the increased vascular tone in this disease state is removal of this [Na+]i at the expense of Ca2+ entry, via the vascular NCX. Expression [6] and activity [7] of the cardiac NCX are also increased in heart failure. Action potentials in failing hearts are prolonged relative to healthy hearts [8]. The longer depolarization during the plateau of the action potential can promote reverse mode NCX activity. Ca2+ entry through reverse NCX activity plays an important role in augmenting contractility in heart failure [9]. However, the combination of reduced SERCA function and augmented Ca2+ removal via the NCX could enhance the depletion of sarcoplasmic reticulum (SR) Ca2+ stores and further impair contractility.

Drugs targeting the NCX could have potential as treatments for cardiovascular disease in the future [10]. Recently, the long-chain {omega}-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), were shown to inhibit forward and reverse modes of the cardiac NCX1.1 transiently expressed in HEK293 cells [11]. Only long-chain PUFA had an inhibitory effect [11]. Omega-3 PUFA are essential fatty acids that cannot be synthesized in humans and must be consumed in the diet. Their beneficial effect in cardiovascular diseases is typically attributed to inhibition of ion channels [12], although inhibition of NCX1 by {omega}-3 PUFA may constitute another potential mechanism. Clinical studies have reported that {omega}-3 PUFA reduce sudden cardiac death, presumably by preventing lethal arrhythmias. Omega-3 PUFA also have beneficial effects in other disease states, such as hypertension [13,14]. The presence of multiple isoforms of NCX in the body necessitates a further understanding of how NCX1 isoforms and PUFA interact.

In the present study, we examine the effects of ALA, the parent {omega}-3 PUFA, on the cardiac (NCX1.1) and vascular (NCX1.3) isoforms of NCX1 stably transfected and expressed in HEK293 cells. The effects are compared to {omega}-9 monounsaturated fatty acids (MUFA), {omega}-6 and other {omega}-3 PUFA to determine the specificity and selectivity of action of these compounds. We also investigate the effects of ALA on NCX1.1 and NCX1.3 activity in native rat cardiomyocytes and transfected rabbit aortic vascular smooth muscle cells (VSMC), respectively.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
All experiments were approved by the University of Manitoba Protocol Management and Review Committee and conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.1. Western blots
The cardiac (NCX1.1) and vascular (NCX1.3) isoforms of the Na+–Ca2+ exchanger were stably expressed in HEK293 cells as previously described [15]. Control untransfected HEK293 cells, or cell lines expressing NCX1.1 or NCX1.3, were grown to 70% confluency in 175 cm2 flasks. Protein was collected from cells lysed in RIPA buffer (mM): NaCl 150, Tris 50, EDTA 1, EGTA 1, PMSF 1, benzamidine 1, protease inhibitor cocktail 1 µg/ml, 1% Triton-X-100 and 0.5% deoxycholate, pH 7.5. Protein samples (200 µg) were loaded on a 7.5% polyacrylamide gel and transferred at 35 V overnight to a nitrocellulose membrane. Membranes were incubated with antibody (1:500) directed at the intracellular loop of NCX1 (R3F1, Swant, Bellinzona, Switzerland) overnight at 4 °C. Secondary antibody (1:10,000) conjugated to horseradish peroxidase was detected using an ECL Plus chemiluminescence kit and captured on X-ray film.

2.2. Preparation of fatty acid solutions
Fatty acids were conjugated to BSA to increase their solubility in the aqueous bath [16]. Briefly, a 3% BSA solution (fatty acid-free) was prepared in perfusion solution and the desired fatty acid was added to produce a final concentration of 25 mM. An equivalent amount of Na2CO3 was added along with 95% ethanol and double distilled H2O. Ethanol was evaporated and the solution was dialyzed at 4 °C overnight in the perfusion solution. The following day, the solution was brought up to final volume, aliquoted, and stored at –20 °C under N2. The solutions were thawed and added to the bath solution to achieve the desired final concentration.

2.3. Whole cell patch clamping of HEK293 cells
HEK293 cells were enzymatically removed from culture dishes with 0.25% Trypsin–EDTA and placed in a chamber on an inverted microscope. Cells were perfused with (mM): NaCl 137, KCl 5, MgCl2 1, CaCl2 1.5, HEPES 10, D-glucose 10, pH 7.4. Glass pipettes (1.8–2.2 M{Omega}) were filled with (mM): NaCl 5, CsOH 100, KCl 5, MgCl2 2, TEA–Cl 20, HEPES 10, D-glucose 8, Na2ATP 1, EGTA 5, CaCl2 4.94, pH 7.2. Pipette capacitance was corrected following gigaseal formation and cell capacitance was measured and corrected following whole cell access. Pipettes were connected to the headstage of an Axopatch-1D amplifier (Axon Instruments, Foster City, CA, USA) and grounded via a Ag–AgCl wire in an agar bridge. The cells were held at –40 mV and then ramped from +60 to –100 mV over 1600 ms. Dose–response curves were obtained for ALA in NCX1.1 and NCX1.3 cell lines at up to 100 µM. To compare different classes of fatty acids, currents were recorded before and after addition of 25 µM oleic acid (OA, 18:1{omega}-9), linoleic acid (LA, 18:2{omega}-6), ALA (18:3{omega}-3) or EPA (20:5{omega}-3), introduced from a separate reservoir. NiCl2 (5 mM), an NCX inhibitor, was used to assess the total NCX current. Experiments were performed at 23±2 °C. Current records were acquired and analyzed using pClamp 9.2 software (Axon Instruments).

2.4. In situ Na+–Ca2+ assessment of NCX1.1 and NCX1.3
Cardiomyocytes were isolated from adult male Sprague–Dawley rats by perfusing the hearts with collagenase and hyaluronidase as previously described [17]. Isolated cardiomyocytes were suspended in M199, seeded on laminin coated coverslips and maintained in an incubator until use for experiments.

Cultured VSMC were adenovirally transfected with the vascular isoform of the NCX (NCX1.3) to test the effects of ALA on the NCX in intact cells. Rabbit aortic VSMC were grown in culture using the explant technique [18]. Recombinant adenovirus expressing canine NCX1.3 [19] was used to infect cultured VSMC using a multiplicity of infection (MOI) of 150 viral particles/cell after 2 days of incubation in DMEM–5% FBS [20]. Cells were infected for 48 h before use. VSMC were incubated with fura-2 in order to measure [Ca2+]i as described below. The effects of ALA and KB-R7943 treated groups were compared to controls.

Intracellular [Ca2+] was measured using the fluorescent Ca2+ indicator fura-2 as described previously [15]. Once loaded with fura-2, cells were washed with HEPES buffer and incubated in 25 µM ALA for 30 min. Cells were excited by dual wavelength (340 and 380 nm) and emission was recorded at 505 nm with a Photon Technology International spectrofluorometer. The ratio of Ca2+-saturated (340 nm) to Ca2+-free (380 nm) fura-2 represented the change in [Ca2+]i. Reverse NCX activity was stimulated by substituting Na+ in the bathing solution with Li+. Li+-stimulated reverse NCX activity was compared between control, ALA and KB-R7943 treated groups.

2.5. Statistical analysis
Statistical analysis of data was performed using SigmaStat software (version 2.03, SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) and Student–Newman–Keuls post hoc tests were used to compare currents following fatty acid and Ni2+ treatment within the groups. A Student's t-test was used to compare reversal potentials and IC50 values for ALA inhibition of NCX activity. P<0.05 was considered significant. All results are expressed as mean±SEM.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Western blots
NCX1 protein was detectable only in cells transfected with and expressing NCX1.1 or NCX1.3 (Fig. 1). The band for NCX1.1 appeared at the reported weight of 120 kDa, while that of NCX1.3 was slightly lower, since it contains 36 fewer amino acids. No band was visible in the protein from non-transfected HEK293 cells, indicating a lack of endogenous NCX1 in these cells.


Figure 1
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  		Fig. 1 Western blot of total protein extracts from non-transfected HEK293 cells (NT-HEK293), or HEK293 cells expressing NCX1.1 or NCX1.3.

 
3.2. NCX current in control HEK293 cells and cells stably transfected with NCX1.1 or NCX1.3
Non-transfected HEK293 cells have no native NCX activity as confirmed by the absence of an effect of Ni2+ application during the voltage ramp protocol (Fig. 2A). In contrast, HEK293 cells expressing the NCX1.1 (Fig. 2B) or NCX1.3 (Fig. 2C) genes displayed a sizeable current, which was sensitive to Ni2+. The Ni2+-sensitive current, which represents only NCX current, was calculated by subtracting the current remaining after 5 mM NiCl2 exposure from the total measured current. The reversal potential for the NCX was not significantly different in NCX1.1 and NCX1.3 expressing cells (8.09±2.48 mV and 4.57±2.73 mV, respectively) under control conditions. Also, maximal inward and outward currents measured at –100 and +60 mV were similar in both cell lines. When normalized for cell capacitance, forward and reverse mode NCX1.1 activity was –11.21±0.87 pA/pF and 7.39±0.52 pA/pF, respectively, and NCX1.3 activity was –12.16±0.80 pA/pF and 9.26±0.84 pA/pF in the respective transport modes. Also shown are representative NCX1.1 and NCX1.3 current recordings following addition of ALA (25 µM) to the bath (Fig. 2B and C). In both cases, ALA inhibited both forward and reverse mode NCX activity to cause a decrease in the recorded current.


Figure 2
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  		Fig. 2 Representative current records in response to a voltage ramp from +60 to –100 mV. Incubation of control HEK293 cells with the NCX blocking ion Ni2+ (5 mM) has no effect on the current demonstrating the absence of NCX in these cells (A). In contrast, HEK293 cells expressing NCX1.1 (B) or NCX1.3 (C) show sizeable Ni2+-sensitive currents, which can be partially blocked by application of ALA. For each panel, currents are recorded from the same cell, prior to and following treatments.

 
3.3. Dose response of ALA on NCX1 isoforms
Dose–response curves for ALA inhibition were constructed in HEK293 cells expressing NCX1.1 or NCX1.3 and subjected to voltage ramp protocols. ALA inhibited the reverse mode NCX1.1 activity (measured at +60 mV) with an IC50 of 0.11±0.02 µM (Fig. 3A), which was similar to the IC50 for forward mode inhibition (0.12±0.02 µM, measured at –100 mV) (Fig. 3B). ALA also inhibited the reverse (Fig. 3C) and forward (Fig. 3D) mode activities of NCX1.3, similar to its effects on NCX1.1. However, NCX1.3 was five to seven times more sensitive to ALA inhibition than NCX1.1. ALA inhibited the reverse and forward modes of NCX1.3 activity at an IC50 of 0.017±0.009 µM and 0.021±0.009 µM, respectively. Thus, inhibition of NCX1 by ALA appears to be non-mode selective for NCX1.


Figure 3
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  		Fig. 3 Application of ALA (0–100 µM) to HEK293 cells expressing the cardiac isoform of the NCX produced a dose-dependent inhibition of reverse (A) and forward mode (B) NCX1.1 current (n=10). The dose-dependent inhibition is also observed in cells expressing the vascular NCX isoform. ALA inhibited the NCX1.3 reverse (C) and forward mode (D) activity at lower concentrations (n=6–11). Maximal inhibition of NCX1 was determined with 100 µM ALA and all other currents measured in response to each ALA dose were compared to this value. Reverse and forward measurements of NCX1 activity were taken at +60 and –100 mV, respectively.

 
3.4. Effects of different classes of fatty acids on NCX1.1 activity
A concentration of 25 µM was chosen to test the effects of different classes of fatty acids on the exchanger. As shown in Fig. 4A, ALA inhibited NCX1.1, but only significantly at the most positive and negative potentials tested. Eicosapentaenoic acid significantly inhibited both modes of exchange activity at most potentials, except those close to the convergence point of the traces (Fig. 4B). NCX1.1 activity was not completely blocked by {omega}-3 PUFA, since addition of 5 mM NiCl2 produced further inhibition. In contrast, the MUFA, OA (Fig. 4C), and the {omega}-6 PUFA, LA (Fig. 4D), had no effect on forward or reverse mode NCX1.1 activity. At membrane potentials that the NCX1.1 would encounter in the heart (–85 to +30 mV), only EPA significantly altered exchanger activity, although ALA did display a trend towards inhibition. A summary of the inhibitory effects of the different fatty acids on NCX1.1 forward and reverse modes, measured at –100 and +60 mV, respectively, is shown in Fig. 5. The {omega}-3 PUFA, ALA and EPA, inhibited forward and reverse modes of NCX activity, while OA and LA had no effect.


Figure 4
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  		Fig. 4 Summary of the inhibitory effects of different classes of fatty acids (25 µM) on NCX1.1. Only ALA (A, n=8) and EPA (B, n=10) inhibited the NCX1.1 current during the voltage ramp protocol. ALA significantly inhibited only at the extreme voltages, whereas EPA inhibited the current throughout the range from +60 to –100 mV. In both cases, NCX activity was further inhibited by 5 mM Ni2+ solution, demonstrating that {omega}-3 PUFA only partially inhibit the NCX. The monounsaturated fatty acid, OA (C, n=14), and {omega}-6 polyunsaturated fatty acid, LA (D, n=10), had no effect on current. *P<0.05 vs. CTRL.

 

Figure 5
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  		Fig. 5 Relative magnitude of NCX current recorded after addition of 25 µM ALA, EPA, LA or OA to HEK293 cells expressing NCX1.1. Forward and reverse modes of NCX activity were measured at –100 and +60 mV, respectively. Total NCX current at these two potentials was calculated by subtracting the current remaining after 5 mM NiCl2 from the pre-NiCl2 current. The current remaining following application of each fatty acid was normalized to controls by dividing it by the pre-treatment, Ni2+-sensitive (NCX) current. Standard error for the control group represents the error associated with the mean of the control and was calculated according to the rules for propagation of error. *P<0.05 vs. CTRL.

 
3.5. Effects of different classes of fatty acids on NCX1.3 activity
We also tested different classes of fatty acids in HEK293 cells expressing the vascular NCX1.3 at a concentration of 25 µM. Similar to NCX1.1, ALA and EPA inhibited both modes of NCX1.3 activity, but to an even greater extent (Fig. 6A and B). However, the MUFA, OA, inhibited the forward and reverse modes of NCX1.3 at most potentials (Fig. 6C), in contrast to its lack of effect on NCX1.1. The {omega}-6 PUFA, LA, significantly inhibited NCX1.3 only at –100 mV, although a trend towards inhibition was seen at other potentials (Fig. 6D). Over the physiological range of potentials in the vasculature (–60 to –30 mV [21]), {omega}-3 PUFA (ALA and EPA) significantly inhibited NCX1.3, while OA inhibited current only at –60 mV. Fig. 7 summarizes the inhibitory effects of the various fatty acids on forward and reverse modes of NCX1.3 activity, measured at –100 and +60 mV, respectively. All fatty acids tested inhibited forward and reverse modes of NCX activity.


Figure 6
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  		Fig. 6 Summary of the inhibitory effects of different classes of fatty acids (25 µM) on NCX1.3. ALA (A, n=11), EPA (B, n=8), OA (C, n=6), or LA (D, n=6), was added to the bath and the voltage ramped from +60 to –100 mV. Both {omega}-3 PUFA and OA inhibited forward and reverse NCX activity across a range of potentials, but LA only significantly inhibited NCX activity at –100 mV. Application of Ni2+ produced further inhibition of the exchanger in all cases, demonstrating an incomplete block by fatty acids. *P<0.05 vs. CTRL.

 

Figure 7
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  		Fig. 7 Relative magnitude of NCX current recorded following addition of 25 µM ALA, EPA, LA or OA to HEK293 cells expressing NCX1.3. Forward and reverse modes of NCX activity were measured at –100 and +60 mV as described for Fig. 5. *P<0.05 vs. CTRL.

 
3.6. Effects of ALA on reverse mode NCX activity in cardiomyocytes and VSMC
We also examined the effects of PUFA, specifically ALA, on NCX activity in rat cardiomyocytes (NCX1.1) or rabbit VSMC (NCX1.3). Due to the difficulties of recording NCX current directly in these cells, extracellular Li+ substitution of Na+ was used to assess NCX activity (reverse mode). In VSMC expressing NCX1.3, ALA significantly reduced the rise in intracellular Ca2+ induced by substitution of Li+ for [Na+]o from 108.7±0.8% to 102.2±0.6% of baseline values (Fig. 8). Therefore, ALA also inhibits reverse mode NCX1.3 activity in native cells. In cardiomyocytes, 25 µM ALA significantly reduced the rise in [Ca2+]i that accompanies Li+ substitution from 149.8±5.6% to 101.6±0.5% of baseline values (Fig. 8). The NCX inhibitor, KB-R7943, blocked the exchanger in a similar manner to ALA. Thus, ALA also inhibits reverse mode Na+–Ca2+ exchange in cardiomyocytes (NCX1.1).


Figure 8
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  		Fig. 8 Changes in intracellular Ca2+ following substitution of Li+ for Na+ in the bath solution for rabbit VSMC expressing NCX1.3 or native rat ventricular cardiomyocytes (RVC). Application of 25 µM ALA (white) or KB-R7943 (grey) significantly attenuated the Li+-triggered increase in [Ca2+]i in both cell types compared to pre-treatment (black). *P<0.001 vs. CTRL VSMC, #P<0.001 vs. CTRL RVC.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Our study demonstrates that the parent {omega}-3 PUFA, ALA, significantly inhibits both the forward and reverse mode of NCX1.1 activity in a similar manner to the long-chain {omega}-3, EPA. We also report, for the first time, that the vascular NCX isoform, NCX1.3, is inhibited by several classes of fatty acids with a profile different than NCX1.1. Notably the vascular NCX1.3 was six times more sensitive to ALA than the cardiac NCX1.1. Also, MUFA, and to some degree {omega}-6 PUFA, inhibited NCX1.3 at 25 µM, whereas neither class inhibited the cardiac NCX1.1. Only {omega}-3 PUFA inhibited the NCX1.1 isoform at this concentration.

ALA inhibited the reverse mode of NCX1.1 with an IC50 of 0.11±0.02 µM. Although a dose response was not performed for EPA, Figs. 4, 5, 6 and 7GoGoGo would suggest EPA and ALA inhibited the NCX to a similar degree. If that is the case, the IC50 of ALA is eight times lower than that reported for EPA on NCX1.1 activity in acutely transfected HEK293 cells [11]. One possibility for this discrepancy is the different delivery vehicles used. We found that suspending fatty acids in ethanol, as is commonly utilized [11,22–25], does not provide optimal delivery. The mixing with BSA produces a more physiological and better dissolution of the fatty acids [16]. The identification of such low IC50 values further increases the likelihood that these {omega}-3 PUFA may act in vivo to alter NCX function.

In normal individuals, the free plasma concentrations of {omega}-3 PUFA attainable in the body are in the high nanomolar range, and low micromolar concentrations are certainly possible with dietary supplementation [26,27]. Our data indicate that such concentrations could significantly inhibit NCX activity, especially NCX1.3. The IC50 values for inhibition of both NCX1.1 and NCX1.3 are lower than those reported for the inhibition of ion channels by {omega}-3 PUFA (2.1 µM for ICa-L [22], 6.0–26.6 µM for INa [23,24]). Thus, the protective effect of {omega}-3 PUFA may reflect a greater contribution of NCX inhibition at low plasma concentrations and inhibition of both the NCX and ion channels at higher plasma levels. This suggestion presumes that the effects of the fatty acids in our heterologous system duplicate those in native cells. Our results in native rat cardiomyocytes and rabbit VSMC offer initial support for this premise.

Experimentally, inhibitors of NCX show promise as treatments for arrhythmias [28,29] and heart failure [30]. We previously reported that the dietary inclusion of high ALA content flaxseed exerts a marked antiarrhythmic effect in rabbits [31], and these results are consistent with other studies in animal models [32]. The beneficial effects of {omega}-3 PUFA in humans are also evident. The GISSI-Prevenzione study [33] found that 0.9 g/day EPA/DHA (1:2 ratio) significantly decreased all-cause mortality (35%), cardiovascular mortality (30%) and sudden cardiac death (45%), suggesting that {omega}-3 PUFA exert their benefits through reduction in lethal arrhythmias and sudden cardiac death.

The {omega}-3 PUFA, ALA, exerted greater effects on NCX1.3 compared to NCX1.1. The vascular/renal NCX1.3 isoform was six times more sensitive to ALA than the cardiac NCX1.1, with an IC50 of 0.021±0.009 µM and 0.017±0.009 µM for the forward and reverse modes, respectively. Other NCX inhibitors, such as SEA0400, also display preferential inhibition of the vascular over the cardiac NCX isoform [34,35]. The greater sensitivity of NCX1.3 to fatty acids suggests that {omega}-3 PUFA may offer a potential new treatment for hypertension. Several reports demonstrate that {omega}-3 PUFA lower blood pressure in hypertensive animal models [36,37] and humans [13]. The ability to lower blood pressure is not specific to a particular class of fatty acids, although fish oils appeared to produce the largest effects [38]. The heightened sensitivity of NCX1.3 to inhibition by fatty acids was also associated with a loss of specificity to different classes of fatty acids. The {omega}-3 PUFA, {omega}-9 MUFA, and to some degree {omega}-6 PUFA, all inhibited the forward and reverse modes of NCX1.3 activity, albeit at the higher concentration of 25 µM.

A comparison of fatty acids found that {omega}-3 PUFA were the most effective in delaying the rise in systolic blood pressure that occurs with age in spontaneously hypertensive rats (SHR) [37]. The {omega}-9 and {omega}-6 fatty acids also delayed the blood pressure rise, but to a lesser extent. We found a similar rank order of potency based on their inhibition of NCX. In humans, the ability of {omega}-3 PUFA to lower blood pressure is related to the severity of hypertension. High doses (3–4 g/day) of fish oil decreased systolic blood pressure by 2–3 mm Hg in mild to moderate hypertensive patients [13,14]. However, a 3 mm Hg decrease in systolic blood pressure may be sufficient to reduce mortality from coronary heart disease by 5% and stroke by 8% [39]. In contrast, {omega}-3 PUFA did not affect blood pressure in normotensive Wistar Kyoto rats [40], or in normotensive patients [41]. These findings suggest that the effects of {omega}-3 PUFA on the vasculature become manifest only when the systolic blood pressure is elevated.

At 25 µM, {omega}-3 PUFA inhibited the total Ni2+-sensitive NCX current by only 50–80% in our study. Thus, sufficient reserves of NCX would still exist to allow normal cellular function. In fact, heterozygous NCX knockout mice, in which NCX levels are ~50% of normal, fail to show major functional consequences [42]. However, in pathological states such as heart failure, this level of NCX inhibition could affect function due to greatly increased NCX expression and/or activity, or a greater role of NCX in the excitation–contraction coupling process. Alterations in ionic gradients associated with various pathologies could also influence NCX function. For example, increased [Na+]i associated with hypertension would shift the reversal potential for NCX to more negative values and result in greater inhibition of {omega}-3 PUFA on reverse mode NCX activity, a strategy that seems to reduce blood pressure [34].

Finally, our data indicate that {omega}-3 PUFA derived from either fish (EPA and DHA) or plant sources (ALA) inhibit the NCX. Clearly, ALA directly inhibited NCX1.1 and NCX1.3 yet its elongation to EPA and DHA does not occur in our HEK293 cells. Our results argue against the suggestion that ALA itself is inactive and must be elongated to EPA or DHA to produce a biological effect.

In summary, our data show that short and long-chain {omega}-3 PUFA inhibit NCX1.1 and NCX1.3 activity, but the NCX1.3 isoform is much more sensitive. Conversely, MUFA or {omega}-6 PUFA of the same chain length have no effect on the cardiac NCX1.1 at 25 µM, but inhibit the vascular NCX1.3. In pathological conditions, such as heart failure and particularly hypertension, where inhibition of the NCX can restore cardiac function or vascular tone, increased dietary intake of {omega}-3 PUFA, including ALA from plant sources such as flaxseed, may be beneficial.


   Acknowledgements
 
This research was supported by grants from the Heart and Stroke Foundations of Manitoba and New Brunswick, Canadian Institutes for Health Research and St. Boniface Hospital and Research Foundation. Dr. Hurtado and Mr. Ander were Trainees of the Heart and Stroke Foundation of Canada. Dr. Hryshko is supported by a Canada Research Chair.


   Notes
 
1 Current address: Howard Hughes Medical Institute, Department of Biological Chemistry, University of California, Los Angeles, United States. Back

Time for primary review 19 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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