Skip Navigation

Cardiovascular Research 2006 70(3):509-520; doi:10.1016/j.cardiores.2006.02.022
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Verkerk, A. O.
Right arrow Articles by Coronel, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Verkerk, A. O.
Right arrow Articles by Coronel, R.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2006, European Society of Cardiology

Incorporated sarcolemmal fish oil fatty acids shorten pig ventricular action potentials

Arie O. Verkerka,b, Antoni C.G. van Ginnekena,b, Géza Bereckia,b, Hester M. den Ruijtera, Cees A. Schumachera, Marieke W. Veldkampa, Antonius Baartscheera, Simona Casinia, Tobias Opthofa, Robert Hovenierc, Jan W.T. Fioleta, Peter L. Zockd and Ruben Coronela,*

aExperimental and Molecular Cardiology Group, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
bDepartment of Physiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
cDepartment of Nutrition, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
dDepartment of Human Nutrition, the Wageningen Centre for Food Sciences, Wageningen University, Wageningen, The Netherlands

* Corresponding author. Academic Medical Center, Rm M0-108, Department of Experimental Cardiology, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Tel.: +31 20 5663267; fax: +31 20 6975458. Email address: R.Coronel{at}amc.uva.nl

Received 13 September 2005; revised 10 February 2006; accepted 21 February 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Background Omega-3 polyunsaturated fatty acids ({omega}3-PUFAs) from fish oil reduce the risk of sudden death presumably by preventing life-threatening arrhythmias. Acutely administered {omega}3-PUFAs modulate the activity of several cardiac ion channels, but the chronic effects of a diet enriched with fish oil leading to {omega}3-PUFA-incorporation into the sarcolemma on membrane currents are unknown.

Methods Pigs received a diet either rich in {omega}3-PUFAs or in {omega}9-fatty acids for 8weeks. Ventricular myocytes (VMs) were isolated and used for patch-clamp studies.

Results {omega}3-VMs contained higher amounts of {omega}3-PUFAs and had a shorter action potential (AP) with a more negative plateau than control VM. In {omega}3 VMs, L-type Ca2+ current (ICa,L) and Na+–Ca2+ exchange current (INCX) were reduced by approximately 20% and 60%, respectively, and inward rectifier K+ current (IK1) and slow delayed rectifier K+ current (IKs) were increased by approximately 50% and 70%, respectively, compared to control. Densities of rapid delayed rectifier K+ current, Ca2+-activated Cl– current, and Na+ current (INa) were unchanged, although voltage-dependence of INa inactivation was more negative in {omega}3 VMs.

Conclusions A fish oil diet increases {omega}3-PUFA content in the ventricular sarcolemma, decreases ICa,L and INCX, and increases IK1 and IKs, resulting in AP shortening. Incorporation of {omega}3-PUFAs in the sarcolemma may have consequences for arrhythmias independent of circulating {omega}3-PUFAs.

KEYWORDS Ion channels; Ion exchangers; Membrane potential; Repolarization; Ca2+ transients; Nutrition; Fatty acids


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Increased consumption of fish oil, rich in omega-3 ({omega}3) polyunsaturated fatty acids (PUFAs), reduces cardiovascular mortality, especially sudden cardiac death, in patients with healed myocardial infarction [1,2]. In rats fed a fish oil-rich diet the number of ischemia/reperfusion related arrhythmias is reduced [3]. The mechanisms underlying the anti-arrhythmic effect of a diet rich in {omega}3-PUFAs are unknown. Acutely administered {omega}3-PUFAs reversibly modulate a variety of cardiac ion channels and exchangers [4]. Long term electrophysiological effects of {omega}3-PUFA intake leading to incorporation of {omega}3-PUFAs into the sarcolemma have not been studied. Incorporation of {omega}3-PUFAs into the phospholipid bilayer affects sarcolemmal biophysical properties [5].

We hypothesize that incorporation of {omega}3-PUFAs in the absence of circulating fatty acids has electrophysiological effects on various cardiac ion channels and transporters. Therefore, we studied ion channel characteristics of cardiac ventricular myocytes (VMs) isolated from pigs fed for 8weeks with a diet rich in either fish oil ({omega}3-PUFA) or in {omega}9 fatty acids (high oleic sunflower oil, HOSF) as a control. We show that incorporated sarcolemmal {omega}3-PUFAs alone result in action potential (AP) shortening, caused by combined effects on various Ca2+ and K+ ion channels and the Na+–Ca2+ exchanger. Therefore, incorporation of {omega}3-PUFAs into the sarcolemma may affect reentrant and triggered arrhythmias.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
2.1 Cell preparation
The investigation conforms to the Guide for the Care and Use of Laboratory Animals (NIH Publication 85-23, 1996). Male pigs (7weeks old) received a diet rich in {omega}3-PUFA or HOSF for 8weeks. Table 1 summarizes the composition of these diets. Average body weight after 8 weeks of diet was similar in {omega}3-PUFA and HOSF fed animals (55.4±1.8 vs. 52.3±1.4kg, mean±S.E.M., n=8).


View this table:
[in this window]
[in a new window]

  		Table 1 Composition of HOSF and {omega}3 diet (g/100g feed (% total dietary energy))

 
After the feeding period, pigs were sedated with ketamine (500mg, i.m. Nimatek; Animal Health), azaperon (160mg, i.m. Stresnil; Janssen-Cilag) and atropine (0.5mg, i.m.; Centrafarm) and anaesthetized with pentobarbital (20mg/kg, i.v. Nembutal; Ceva Sante Animale). A sidebranch of the circumflex artery was cannulated and the perfused myocardium was moved to a perfusion setup. Left midmyocardial VMs were enzymatically isolated as described previously [6]. Small aliquots of cell suspension were put in a recording chamber on the stage of an inverted microscope. Cells were allowed to adhere for 5min before superfusion was initiated. The temperature was 35–36°C, except for Na+ current recordings (22–23°C). Quiescent, rod-shaped cross-striated cells and smooth surface were selected for measurements.

2.2 Electrophysiology
2.2.1 Data acquisition and analysis
Membrane potentials and currents were recorded in the whole-cell configuration of the patch-clamp technique (Axopatch 200B Clamp amplifier, Axon Instruments Inc.) using patch pipettes (1–3M{Omega}, borosilicate glass). Voltage control, data acquisition, and analysis were accomplished using custom software. Potentials were corrected for liquid junction potential, except for INa measurements where it was 0.2mV. Membrane currents and potentials were low-pass filtered (1 kHz) and digitized (2 kHz), except for AP and INa measurements, 5 and 20kHz, respectively. Cell membrane capacitance (Cm) was estimated as described previously [7]. Series resistance was compensated for by at least 80%.

2.2.2 Current clamp experiments
APs were elicited at 0.2 to 6Hz by 3ms, 1.5 x threshold current pulses through the patch pipette. We analyzed resting membrane potential (RMP), maximal upstroke velocity, stimulation threshold, maximal AP amplitude, plateau amplitude (defined as the potential difference between RMP and potential 50ms after the upstroke), and AP duration at 20%, 50%, and 90% repolarization. Parameters from 10 consecutive APs were averaged.

2.2.3 Voltage-clamp experiments
Na+ current (INa), L-type Ca2+ current (ICa,L), T-type Ca2+ current (ICa,T), Ca2+-activated Cl– current (ICl(Ca)), inward rectifier K+ current (IK1), slow delayed rectifier K+ current (IKs), rapid delayed rectifier K+ current (IKr), and Na+–Ca2+ exchange current (INCX) were measured with the solutions indicated below and by voltage-clamp protocols shown in the appropriate figures. Voltage-dependence of (in)activation was determined by fitting a Boltzmann function (y=A/[1+exp{(V – V1/2)/k}]) to the individual curves, yielding half-maximal voltage (V1/2) and slope factor k. The time constants of recovery from inactivation were determined using a double-exponential function (INa=[Af x exp(– t/{tau}f)]+[As x exp(– t/{tau}s)), where t is the recovery time interval, {tau}f and {tau}s are the time constants of fast and slow components, and Af and As are the fractions of the fast and slow components. Current densities were calculated by dividing current amplitudes by Cm.

2.2.4 Solutions
Standard pipette solution contained (mmol/L): K-gluconate 125, KCl 20, K2-ATP 5, HEPES 10; pH 7.2 (KOH). EGTA (10mmol/L) was added to the pipette solution for INa, ICa,T, ICa,L, IKr and IKs measurements. For INa, ICa,T and ICa,L measurements, CsCl replaced all K-gluconate and KCl in the pipette solution. Furthermore, for INa measurements K2-ATP was replaced by Na2-ATP (2mmol/L) and NaCl (3mmol/L). Standard Tyrode's solution contained (mmol/L): NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 1.0, glucose 5.5, HEPES 5.0; pH 7.4 (NaOH). ICl(Ca)was measured as the transient outward current sensitive to 0.2mmol/L 4,4'diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). For ICa,T and ICa,L measurements, TEA-Cl and CsCl replaced NaCl and KCl of the bath solution, respectively. For INa measurements, NaCl was reduced to 7mmol/L and KCl was replaced by CsCl (133mmol/L). For INa, IKr, and IKs measurement, the bath solution included 5µmol/L nifedipine. IKr was measured as 5µmol/L E-4031-sensitive current while IKs was measured as 90µmol/L chromanol 293B-sensitive current in the presence of 5µmol/L E-4031. pH of both extracellular and pipette solution was adjusted with CsOH and NMDG-OH for INa and ICa measurements, respectively. For INCX recording, the pipette solution contained (mmol/L): CsCl 145, NaCl 5, Mg-ATP 10, TEA 10, HEPES 10, EGTA 20, CaCl2 10; pH 7.2 (NMDG-OH). To suppress membrane currents other than INCX, the following blockers were added to a K+-free Tyrode's solution (mmol/L): BaCl2 1, CsCl 2, nifedipine 0.005, ouabain 0.1, DIDS 0.2. INCX was measured as 10mmol/L Ni2+-sensitive current during a descending voltage ramp protocol [8].

2.2.5 Drugs
All drugs used for cellular measurements were obtained from Sigma-Aldrich (MO, USA), except for E-4031 (Eisai Inc., NJ, USA) and chromanol 293B (Tocris Cookson Inc. MO, USA). DIDS was freshly prepared as 0.5mol/L stock solution in DMSO. Nifedipine and chromanol 293B, respectively, were prepared as 5mmol/L and 0.1mol/L stock solutions in ethanol. E-4031 was prepared as 5mmol/L stock solution in distilled water. All stock solutions were diluted appropriately before use. DIDS and nifedipine were stored in the dark.

2.3 Cytosolic Ca2+ measurements
Intracellular Ca2+([Ca2+]i) was measured in indo-1 loaded VMs as described previously [9]. VMs were stimulated at 2Hz with field stimulation. Dual wavelength emission of indo-1 was recorded ((405–440)/(505–540) nm, excitation at 340nm) and free [Ca2+]i was calculated [9].

2.4 Lipid analyses
Lipids from food, heart samples and myocytes were extracted with the method of Folch et al. [10]. Phospholipids from plasma and heart were isolated with aminopropyl bonded phase columns (Bond Elut; Varian BV). Saponification and methylation of the phospholipids with boron trifluoride (Pierce, IL, USA) was performed and the formed fatty acid methyl esters were subjected to capillary gas chromatography using a Chrompack column (Fused Silica, Chrompack), a flame ionization detector and H2 as carrier gas. Fatty acid methyl esters were expressed as fraction of the total amount.

2.5 Statistics
Data are mean±S.E.M. A t-test or in two-way Repeated Measures ANOVA followed by pairwise comparison using the Student–Newman–Keuls test was used. P<0.05 defined statistical significance.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
3.1 Heart, cell, and membrane properties
3.1.1 Morphology
Fig. 1A summarizes basic heart and isolated VMs morphology characteristics of HOSF and {omega}3-PUFAs fed animals. Mean heart weight and left ventricular wall thickness were not different between the HOSF and {omega}3 group. Cell length was similar, but cell width and Cm was significantly larger in {omega}3 VMs compared to HOSF.


Figure 1
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Heart, cell, and membrane properties. (A) Morphological characteristics of heart and isolated VMs. n, number of hearts/myocytes (*P<0.05 unpaired t-test). (B and C) PUFA composition of VM membranes measured in biopsies (B) and isolated myocytes (C). n, number of hearts (*P<0.05 unpaired t-test).

 
3.1.2 PUFA composition
Fig. 1B summarizes the most prominent differences of cell phospholipid fraction in the two groups. In biopsies obtained from intact {omega}3 hearts as well as in isolated VMs, the proportion of {omega}3-PUFAs was increased at the expense of {omega}6-PUFAs. Thus, {omega}3-PUFAs from diet were incorporated in the cell membrane. Moreover, PUFA compositions in biopsies and isolated myocytes were similar (Fig. 1B), indicating that the cell isolation procedures did not affect the composition of the sarcolemma.

3.2 Action potentials and [Ca2+]i-transients
Fig. 2A shows representative APs at 1Hz from a HOSF and {omega}3 VM. The {omega}3 AP has a more negative plateau potential and is considerably shorter than HOSF AP. Fig. 2B summarizes AP characteristics of HOSF and {omega}3 VMs. On average, {omega}3 VMs show a 10% more negative AP plateau potential and a 30% shorter AP at both 50% and 90% repolarization. No significant differences in RMP, excitability threshold, maximal upstroke velocity or maximal AP amplitude are observed. The AP shortening in {omega}3 VMs is particularly clear at physiological heart rates, i.e. at 3Hz, and slower; the more negative AP plateau potential is present at all pacing frequencies (Fig. 2C). Fig. 2D shows typical [Ca2+]i-transients and Fig. 2E summarizes [Ca2+]i-transient characteristics of HOSF and {omega}3 VMs. No significant differences in diastolic [Ca2+]i and [Ca2+]i-transient amplitudes are observed. The duration of [Ca2+]i-transient at both 50% and 90% of the transient decrease is significantly shorter in {omega}3 VMs.


Figure 2
View larger version (28K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Dietary {omega}3-PUFAs shorten action potentials (APs) and intracellular Ca2+-transients. (A) Representative APs of a HOSF and an {omega}3 VM at 1Hz. Inset shows maximal upstroke velocities (arrows). (B) Average AP parameters of HOSF and {omega}3 VMs. ST=stimulation threshold, Vmax=maximal upstroke velocity, RMP=resting membrane potential, APA=maximal AP amplitude, Pla=AP plateau amplitude, APD20, APD50, and APD90=AP duration at 20%, 50%, and 90% repolarization. *P<0.05 in unpaired t-test. (C) Stimulus frequency-dependency of APD90 and Pla (inset) in HOSF and {omega}3 VMs. *P<0.05 in ANOVA followed by Student–Newman–Keuls test. (D) Typical example [Ca2+]i-transients of a HOSF and an {omega}3 VM. (E) Average [Ca2+]i-transient parameters of HOSF- and {omega}3 VMs. DC=diastolic [Ca2+]i, TA=transient amplitude, TD20, TD50, and TD90=transient duration at 20%, 50%, and 90% transient decrease. *P<0.05 in unpaired t-test.

 
3.3 Na+ current
Fig. 3A shows representative INa recordings. Mean INa densities (Fig. 3B) are not different between HOSF and {omega}3 VMs. Peak INa averages – 26.6±1.8 and – 26.1±2.3pA/pF in HOSF and {omega}3 VMs, respectively. Also voltage dependency of INa activation (Fig. 3C) does not differ significantly between HOSF and {omega}3 VMs. V1/2 and k are – 36.9±0.6mV and 4.8±0.3mV (HOSF) and – 38.4±0.5mV and 4.9±0.4mV ({omega}3), respectively. Half-maximal inactivation voltages, based on current densities following a 1000-ms depolarizing prepulse, are – 79.1±0.8mV (HOSF) and – 83.5±0.9 ({omega}3) (P<0.05). In HOSF and {omega}3 VMs, k's of inactivation are identical (– 4.6±0.1mV (HOSF) vs. – 4.9±0.2mV ({omega}3)). Recovery from inactivation (Fig. 3D) is not significantly different between HOSF and {omega}3 VMs. Double-exponential fits revealed that the time constants of fast and slow components of recovery ({tau}fast and {tau}slow, respectively) are 32.1±4.1 and 1490±205ms in HOSF and 37.5±3.6 and 1420±372ms in {omega}3 (both n.s.). Slow inactivation of INa (Fig. 3E) also is not significantly different between HOSF and {omega}3 VMs.


Figure 3
View larger version (32K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Effect of dietary {omega}3-PUFAs on Na+ current (INa). (A) Representative INa of a HOSF and {omega}3 VM. (B) Peak current–voltage (I–V) relationships of INa. (C) Voltage-dependence of (in)activation. Inactivation was approximately – 4mV shifted in {omega}3 VMs (*P<0.05). Solid lines: Boltzmann fits of the average data. (D) Recovery from inactivation. Solid lines: double-exponential functions of the average data (P=n.s. in ANOVA). (E) Development of slow inactivation of INa. Solid lines: mono-exponential functions of the average data. Insets: protocols used.

 
3.4 Ca2+ currents
3.4.1 T-type Ca2+ current
Fig. 4A shows representative ICa traces. The depolarizing steps from – 90 and – 50mV to – 20mV elicit the time- and voltage-dependent inward currents typical of ICa. ICa,T is obtained by digital subtraction of ICa traces elicited by stepping from holding potentials of – 90 and – 50mV. However, in both HOSF and {omega}3 VMs, the ICa traces from – 90 and – 50mV are overlapping (Fig. 4A), indicating that ICa,T was virtually absent in both cell types, in agreement with previous findings in adult VMs of other large mammals [11].


Figure 4
View larger version (28K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Dietary {omega}3-PUFAs reduce L-type Ca2+ current (ICa,L) density. (A) Current traces elicited by depolarizing steps to – 20mV from – 90 (ICa,L and ICa,T) and – 50mV (ICa,L). ICa.T was obtained by subtracting currents evoked from a HP of – 50mV from those with the same depolarization but evoked from a HP of – 90mV. Note that ICa.T was absent. (B) Peak I–V relationships of ICa,L. *P<0.05 in ANOVA followed by Student–Newman–Keuls test. (C) Voltage-dependence of ICa,L (in)activation. Solid lines: Boltzmann fits of the average data. Activation and inactivation properties did not differ between HOSF and {omega}3 VMs, however, at leg25mV, where ‘incomplete inactivation’ occurs, ICa,L was significantly smaller in {omega}3 VMs (*P<0.05 in ANOVA followed by Student–Newman–Keuls test).

 
3.4.2 L-type Ca2+ current
ICa,L is defined as ICa elicited from – 50mV and is significantly smaller in {omega}3 than HOSF VMs (Fig. 4A and B). For example, at 0 mV ICa,L density averaged – 7.6±0.6 (HOSF) and – 6.2±0.4pA/pF ({omega}3), indicating a 20% reduction of ICa,L in {omega}3 VMs. The voltage-dependence of ICa,L activation and inactivation are shown in Fig. 4C. Activation V1/2 averaged – 11.5±0.7 and – 11.1±0.9mV (P=n.s.). and slope factors are 6.2±0.2 and 6.2±0.1mV (P=n.s., HOSF and {omega}3 VMs, respectively). Inactivation voltage-dependence was assessed with 500-ms prepulses followed by 500-ms test pulses to 0mV (Fig. 4C, inset). Inactivation V1/2 averages – 28.3±1.1 (HOSF) vs. – 28.4±1.6mV ({omega}3, P=n.s.); slope factors are – 4.8±0.2 and – 4.6±0.1mV (P=n.s.). Fig. 4C shows that the steady-state inactivation curve (or availability curve) rises positive to +10mV, due to ‘incomplete inactivation’ [12]. Interestingly, in {omega}3 VMs, this ‘incomplete inactivation’ is significantly smaller than in HOSF VMs at potentials of +25mV and more positive.

3.5 Cl– currents
Fig. 5A shows superimposed current traces recorded in absence and presence of 0.2mmol/L DIDS. By digitally subtracting the two traces (Fig. 5B), the DIDS-sensitive ICl(Ca) is obtained [7]. ICl(Ca) was found in all VMs, and exhibits similar current densities and I–V relationships in both groups (Fig. 5C). No DIDS-sensitive steady-state currents are observed, excluding the presence of persistently activated Cl– currents [13].


Figure 5
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Dietary {omega}3-PUFAs do not alter Ca2+-activated Cl– current (ICl(Ca)). (A) Current traces elicited by depolarizing voltage steps from – 40 to 40mV in absence and presence of 0.2mmol/L DIDS. Inset: protocol used. (B) DIDS-sensitive current (ICl(Ca)). (C) Average I–V relationships of ICl(Ca).

 
3.6 K+ currents
3.6.1 Inward rectifier K+ current
IK1 is defined as steady-state current at the end of hyperpolarizing voltage-clamp steps from – 40mV [6]. The steady-state current is blocked by 2mmol/L Ba2+, and no time-dependent currents are observed in the presence of Ba2+ (Fig. 6A), excluding the contribution of the non-selective cation pacemaker current If to the steady-state current. Fig. 6B shows mean I–V relationships of IK1. The I–V relationships have a reversal potential of – 88mV, which is close to EK and the RMP, and demonstrates inward rectification, characteristic for IK1. Both inward and outward IK1 components are significantly larger in {omega}3 VMs. Maximum outward IK1 density at – 70mV was increased by 54% in {omega}3 VMs (3.1±0.3 to 2.0±0.1pA/pF).


Figure 6
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Dietary {omega}3-PUFAs enhances slow delayed rectifier K+ current (IKs) and inward rectifier K+ current (IK1) but not rapid delayed rectifier K+ current (IKr). (A) Representative current traces elicited by a hyperpolarizing voltage step from – 40 to – 100mV in control conditions and in presence of 2mmol/L Ba2+. (B) Average I–V relationships of IK1. (C) Representative current traces elicited by a voltage step from – 50 to 0mV in control conditions and in presence of 5µmol/L E-4031. E-4031-sensitive current is defined as IKr. (D) Representative current traces elicited by a voltage step from – 50 to 40mV in presence of 5µmol/L E-4031 and in the combined presence of 90µmol/L chromanol-293B and E-4031. Chromanol 293B-sensitive current is defined as IKs. (E) Average I–V relationships of IKr. (F) Average I–V relationships of IKs. *P<0.05 in ANOVA followed by Student–Newman–Keuls test.

 
3.6.2 Delayed rectifier K+ currents
Fig. 6C and D shows superimposed current traces recorded upon depolarizing steps from – 50 to 0mV (Fig. 6C) and from – 50 to 40mV (Fig. 6D) under control conditions, in the presence of E-4031, and in the combined presence of E-4031 and chromanol 293B to discriminate between IKr and IKs, respectively. IKr is defined as E-4031-sensitive current, and IKs as chromanol 293B-sensitive current. No change in mean IKr amplitude (Fig. 6E) is observed. Voltage-dependence of activation of IKr assessed by normalizing tail current amplitudes to maximum current is similar in HOSF and {omega}3 (V1/2 and k, respectively, – 36.8±2.0 and 10.0±1.4mV (HOSF) and – 37.1±2.3 and 8.1±1.6mV ({omega}3)). IKs density is significantly larger in {omega}3 VMs (Fig. 6F). IKs density at 40mV is increased by 70% in {omega}3 VMs (1.16±0.2 in HOSF vs. 1.98±0.4pA/pF in {omega}3 VMs). The increase in density is accompanied by a tendency to shift the activation voltage-dependence toward more negative potentials. V1/2 of IKs activation was 34.3±6.5 and 16.0±7.8mV (P=0.07) in HOSF and {omega}3, respectively; k of IKs activation was identical (16.1±1.7 vs. 19.7±4.5mV) in both groups.

3.7 Na+–Ca2+ exchange current
Fig. 7A shows representative current traces in response to a descending voltage ramp protocol (Fig. 7A, inset) in the absence and presence of 10mmol/L Ni2+. INCX is measured as the Ni2+-sensitive current [8]. In {omega}3 VMs (Fig. 7B), the current density of INCX is significantly reduced to 60% for the reverse (outward) mode (at +80mV; 1.3±0.2 ({omega}3) vs. 2.3±0.3 (HOSF) pA/pF) and 31% for the forward (inward) mode (at – 120mV; – 0.46±0.14 ({omega}3) vs. – 1.5±0.4 (HOSF) pA/pF).


Figure 7
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Dietary {omega}3-PUFAs reduce Na+–Ca2+ exchange current (INCX) density. (A) Typical current traces elicited by ramp protocol (inset) in absence and presence of 10mmol/L NiCl2, and the Ni2+-sensitive current (INCX). (B) Average I–V relationships of INCX. *P<0.05 ANOVA followed by Student–Newman–Keuls test.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
Chronic feeding of a large mammal with fish oil leads to incorporation of {omega}3-PUFAs into the ventricular sarcolemma and at the same time induces various electrophysiological effects in the absence of circulating fatty acids. It decreases ICa,L and INCX, increases IK1 and IKs, and causes AP shortening.

4.1 Comparison with previous studies of remodeling of membrane currents by {omega}3-PUFAs
We have studied the electrophysiological effects of incorporated sarcolemmal {omega}3-PUFAs, rather than the acute direct interaction between circulating {omega}3-PUFAs and the ion channels in the sarcolemma. We isolated the myocytes in the presence of lipid-free albumin, which removes loosely bound {omega}3-PUFAs from the cell surface [14]. Moreover, the experiments were performed with a PUFA-free superfusion solution. The cellular electrophysiological effects of {omega}3-PUFAs administered by diet are unknown.

A decrease in INa density after acute administration of {omega}3-PUFAs has been observed in isolated rat myocytes and HEK cells [15,16]. It was accompanied by a negative shift in voltage-dependence of inactivation. This increased the stimulation threshold and decreased Vmax in cultures of neonatal myocytes [16]. In our study, no reduction INa density was found (Fig. 3B) and the shift in voltage-dependence of inactivation (Fig. 3C) was considerably smaller than that observed after acute administration of {omega}3-PUFAs [15]. As a consequence, we did not observe a significant change in stimulation threshold or upstroke velocity (Fig. 2B).

ICa,L was smaller in {omega}3 than in HOSF VMs (Fig. 4B), in agreement with effects of acute administration of {omega}3-PUFAs [17]. Voltage-dependence of activation and inactivation of ICa,L were not significantly different between the two groups (Fig. 4C). When the protocol used for determining inactivation was extended to more positive potentials, a ‘U-shaped curve’ was seen in both groups (Fig. 4C). The current increase at potentials positive to +10mV is thought to be caused by reduced Ca2+ current-dependent inactivation [12], and was significantly smaller than in {omega}3 VMs. It suggests that the inward current caused by reopening of ICa,L channels during the late plateau phase, is smaller. This might subsequently shorten AP (Fig. 2), and, more importantly, reduce the propensity to early afterdepolarizations (EADs) in {omega}3 VMs.

IK1 density was larger in {omega}3 VMs (Fig. 6B). The increased IK1 may result in earlier repolarization and a more stable resting membrane potential [18], the latter potentially protects against delayed afterdepolarizations (DADs).

The delayed rectifier K+ currents, IKr and IKs, underlie repolarization of cardiac APs [18]. In our study, IKs, showed relatively the largest increase (70%) by {omega}3-PUFAs (Fig. 6F). The effects on IKs are similar to those after acute administration of docosahexanoic acid [19]. The increase in repolarizing current IKs contributes importantly to AP shortening (Fig. 2).

INCX was reduced both in the reversed and forward mode by the dietary {omega}3-PUFAs (Fig. 7), in line with the acute administration of {omega}3-PUFAs on INCX expressed in HEK cells [20]. The NCX maintains the Ca2+ balance of the VM by transport of Ca2+ across the membrane in exchange for Na+ [21]. Its activity is called "forward" when Ca2+ is transported outward. During the early phase of a cardiac AP, the NCX is briefly in reversed mode resulting in an outward current. During the plateau and final repolarizing phases of the AP, however, the NCX is in forward mode, resulting in depolarizing current [21]. The decreased INCX thus results in less depolarizing current during the final repolarizing phase of the AP, causing AP shortening. Moreover, INCX carries the transient inward current responsible for the DADs [21]. Decreased INCX may therefore result in decreased propensity to develop DADs.

The mechanism by which dietary {omega}3-PUFAs decrease INCX and ICa,L, increase IKs and IK1, and leave other ionic currents unaltered remains to be elucidated. Evidence is accumulating that {omega}3-PUFAs alter different microdomains of the plasma membrane, such as lipid rafts and caveolae, thereby directly influencing protein function [4,5], but changes in PKC activation may also play a role [22]. The present study indicates that the role of changes in channel gating is limited, since activation and inactivation properties were hardly affected by dietary {omega}3-PUFAs. Further experiments are required to study the mechanism by which PUFAs alter ionic current densities.

4.2 Comparison with previous studies of action potential duration remodeling by {omega}3-PUFAs
Dietary {omega}3-PUFAs shortens ventricular APs of pigs (Fig. 2). To our knowledge, no other experimental findings on effects of {omega}3-PUFAs administered by diet on APs are available. Our findings are in line with the observation that rabbits on a diet enriched with {alpha}-linolenic acid (an {omega}3-PUFA) have shorter QTc intervals [23], but disagree with observations in healthy humans where dietary supplementation of {omega}3-PUFAs did not alter QTc intervals [24]. Acutely administered {omega}3-PUFAs also reduce AP duration in rabbit [23] and guinea pig [25] VMs. Interestingly, acute administration of {omega}3-PUFAs (>10µM) reduces [25], whereas lower concentrations prolong AP duration in rat VMs [17].

AP duration in rats and mice is determined by the transient outward K+ current (Ito1), a current that is lacking in porcine ventricle [26]. It determines the plateau potential rather than AP duration in other (larger) mammalian species. The observed AP shortening in response to dietary {omega}3-PUFAs agrees with our findings of decreased inward currents, i.e. ICa,L and INCX and increased repolarizing currents, i.e., IKS and IK1 (see above).

4.3 Comparison with previous studies of altered [Ca2+]i by {omega}3-PUFAs
The AP shortening induced by dietary {omega}3-PUFAs is likely due to changes in ICa,L, INCX, IKS, and IK1 densities. However, these currents were measured with EGTA in the pipette solution, while the APs were recorded without EGTA. In addition to changes in current densities, changes in [Ca2+]i, might also contribute to the AP shortening by modulation of membrane currents [27]. Diastolic [Ca2+]i and [Ca2+]i-transient amplitudes, however, were similar in HOSF and {omega}3 VMs (Fig. 2). This agrees with results in rats [28], but contrasts with data obtained following acute administration of {omega}3-FUFAs. There, a rapid decrease in diastolic [Ca2+]i and negative inotropic effect ensued [25,29]. We observed a shorter [Ca2+]i-transient in {omega}3 VMs. Whether this occurred due to altered SR function [28] or to AP shortening by itself [24] is not known. The earlier occurrence of phase-3 repolarization by itself would reduce [Ca2+]i via modulation of the NCX and cause a shorter [Ca2+]i-transient [25].

4.4 (Patho)physiological implications
Our results suggest that incorporated sarcolemmal {omega}3-PUFAs, as a result of a fish oil-rich diet, may have distinct consequences for arrhythmogenesis. The increase in K+ current density and decrease of INCX and ICa,L most likely contribute importantly to AP shortening and may facilitate rather than prevent reentrant arrhythmias [30]. On the other hand, AP shortening may also reduce the occurrence of triggered activity based on EADs [31]. EADs constitute an important cellular mechanism for triggered activity, which may trigger life-threatening ventricular arrhythmias in patients with heart failure and/or long QT-syndromes [32]. The occurrence of EADs is likely to be further prevented by the reduction of ICa,L by dietary {omega}3-PUFAs [17,18]. The {omega}3-PUFAs-induced reduction of EADs is supported by preliminary results in rabbit hearts where the {omega}3-PUFA diet reduced the occurrence of Torsades de Pointes arrhythmias [33], which are importantly due to EADs [34]. AP shortening, increase of IK1, and decrease of INCX potentially protects against DADs evoked by high heart rates in Ca2+-overload conditions [18]. Finally, the negative shift of the INa inactivation curve indicates that at a certain degree of depolarization (that occurs in acute myocardial ischemia) fewer Na+ channels are available, causing conduction slowing and block. This may be potentially proarrhythmic [35].

4.5 Limitations of the study
We studied porcine VMs. The configuration of the human AP is very much like that of pig, but this does not exclude differences in the underlying membrane currents and thereby of the overall effects {omega}3-PUFAs have [18]. Further studies are required to clarify the effects of dietary {omega}3-PUFAs on Ito1 (because the latter is lacking in porcine ventricle [26]) and on human VM electrophysiology.

Although feeding pigs with fish oil leads to major electrophysiological alterations due to incorporation of {omega}3-PUFAs into the membrane alone, additional effects of circulating {omega}3-PUFAs may be important as well.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
Incorporated {omega}3-PUFAs in the absence of circulating {omega}3-PUFAs causes AP shortening. Based on the underlying changes in the profile of ionic sarcolemmal currents, we speculate that a fish oil-rich diet may have pro-arrhythmic as well as anti-arrhythmic consequences. This may explain why {omega}3-PUFAs seem protective against arrhythmias in patients with healed infarction (and with heart failure), but inefficient in patients with acute ischemia (angina pectoris). Thus, the impact of a fish oil enriched diet may depend on the pathophysiological setting.


   Acknowledgements
 
We thank J. Zegers for kindly providing the data-acquisition program and F.J. Wilms-Schopman, C.N. Belterman, D. Bakker, and W.L. ter Smitte for their valuable help. This study was funded by the Wageningen Centre for Food Sciences, an alliance of major Dutch food industries, Maastricht University, TNO Nutrition and Food Research, and Wageningen University and Research Centre, with financial support by the Dutch government, and by the Netherlands Heart Foundation (2003B079) and SEAFOODplus program of the European Union (506359).


   Notes
 
Time for primary review 27 days


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

  1. GISSI-Prevenzione Investigators. Dietary supplementation with n – 3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial. Lancet (1999) 354:447–455.[CrossRef][ISI][Medline]
  2. Burr M.L., Fehily A.M., Gilbert J.F., Rogers S., Holliday R.M., Sweetnam P.M., et al. Effects of changes in fat, fish, and fibre intakes on death and myocardial reinfarction: diet and reinfarction trial (DART). Lancet (1989) 2(8666):757–761.[ISI][Medline]
  3. McLennan P.L. Relative effects of dietary saturated, monounsaturated, and polyunsaturated fatty acids on cardiac arrhythmias in rats. Am J Clin Nutr (1993) 57:207–212.[Abstract/Free Full Text]
  4. Leaf A., Xiao Y.-F., Kang J.X., Billman G.E. Prevention of sudden cardiac death by n – 3 polyunsaturated fatty acids. Pharmacol Ther (2003) 98:355–377.[CrossRef][ISI][Medline]
  5. Ma D.W.L., Seo J., Switzer K.C., Fan Y.-Y., McMurray D.N., Lupton J.R., et al. n – 3 PUFA and membrane microdomains: a new frontier in bioactive lipid research. J Nutr Biochem (2004) 15:700–706.[CrossRef][ISI][Medline]
  6. Veldkamp M.W., Verkerk A.O., van Ginneken A.C.G., Baartscheer A., Schumacher C., de Jonge N., et al. Norepinephrine induces action potential prolongation and early afterdepolarizations in ventricular myocytes isolated from human end-stage failing hearts. Eur Heart J (2001) 22:955–963.[Abstract/Free Full Text]
  7. Verkerk A.O., Tan H.L., Ravesloot J.H. Ca2+-activated Cl– current reduces transmural electrical heterogeneity within the rabbit left ventricle. Acta Physiol Scand (2004) 180:239–247.[CrossRef][ISI][Medline]
  8. Hinde A.K., Perchenet L., Hobai I.A., Levi A.J., Hancox J.C. Inhibition of Na/Ca exchange by external Ni in guinea-pig ventricular myocytes at 37°C, dialysed internally with cAMP-free and cAMP-containing solutions. Cell Calcium (1999) 25:321–331.[CrossRef][ISI][Medline]
  9. Baartscheer A., Schumacher C.A., van Borren M.M.G.J., Belterman C.N.W., Coronel R., Fiolet J.W.T. Increased Na+/H+-exchange activity is the cause of increased [Na+]i and underlies disturbed calcium handling in the rabbit pressure and volume overload heart failure model. Cardiovasc Res (2003) 57:1015–1024.[Abstract/Free Full Text]
  10. Folch J., Lees M., Sloane Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem (1957) 226:497–509.[Free Full Text]
  11. Perez-Reyes E. Molecular physiology of low-voltage-activated T-type calcium channels. Physiol Rev (2003) 83:117–161.[Abstract/Free Full Text]
  12. Tillotson D. Inactivation of Ca conductance dependent on entry of Ca ions in molluscan neurons. Proc Natl Acad Sci (1979) 76:1497–1500.[Abstract/Free Full Text]
  13. Clemo H.F., Stambler B.S., Baumgarten C.M. Swelling-activated chloride current is persistently activated in ventricular myocytes from dogs with tachycardia-induced congestive heart failure. Circ Res (1999) 84:157–165.[Abstract/Free Full Text]
  14. Kang J.X., Leaf A. Effects of long-chain polyunsaturated fatty acids on the contraction of neonatal rat cardiac myocytes. Proc Natl Acad Sci (1994) 91:9886–9890.[Abstract/Free Full Text]
  15. Leifert W.R., McMurchie E.J., Saint D.A. Inhibition of cardiac sodium currents in adult rat myocytes by n – 3 polyunsaturated fatty acids. J Physiol (1999) 520:671–679.[Abstract/Free Full Text]
  16. Xiao Y.-F., Kang J.X., Morgan J.P., Leaf A. Blocking effects of polyunsaturated fatty acids on Na+ channels of neonatal rat ventricular myocytes. Proc Natl Acad Sci (1995) 92:11000–11004.[Abstract/Free Full Text]
  17. Xiao Y.-F., Gomez A.M., Morgan J.P., Lederer W.J., Leaf A. Suppression of voltage-gated L-type Ca2+ currents by polyunsaturated fatty acids in adult and neonatal rat ventricular myocytes. Proc Natl Acad Sci (1997) 94:4182–4187.[Abstract/Free Full Text]
  18. Roden D.M., Balser J.R., George A.L. Jr., Anderson M.E. Cardiac ion channels. Annu Rev Physiol (2002) 64:431–475.[CrossRef][ISI][Medline]
  19. Doolan G.K., Panchal R.G., Fonnes E.L., Clarke A.L., Williams D.A., Petrou S. Fatty acid augmentation of the cardiac slowly activating delayed rectifier current (IKs) is conferred by hminK. FASEB J (2002) 16:1662–1664.[Abstract/Free Full Text]
  20. Xiao Y.-F., Ke Q., Chen Y., Morgan J.P., Leaf A. Inhibitory effect of n – 3 fish oil fatty acids on cardiac Na+/Ca2+ exchange currents in HEK293t cells. Biochem Biophys Res Commun (2004) 321:116–123.[CrossRef][ISI][Medline]
  21. Blaustein M.P., Lederer W.J. Sodium/calcium exchange: its physiological implications. Physiol Rev (1999) 79:763–854.[Abstract/Free Full Text]
  22. Judé S., Roger S., Martel E., Besson P., Richard S., Bougnoux P., et al. Dietary long-chain omega-3 fatty acids of marine origin: a comparison of their protective effects on coronary heart disease and breast cancers. Prog Biophys Mol Biol (2006) 90:299–325.[CrossRef][ISI][Medline]
  23. Ander B.P., Weber A.R., Rampersad P.P., Gilchrist J.S., Pierce G.N., Lukas A. Dietary flaxseed protects against ventricular fibrillation induced by ischemia–reperfusion in normal and hypercholesterolemic rabbits. J Nutr (2004) 134:3250–3256.[Abstract/Free Full Text]
  24. Geelen A., Brouwer I.A., Zock P.L., Kors J.A., Swenne C.A., Katan M.B., et al. (n – 3) Fatty acids do not affect electrocardiographic characteristics of healthy men and women. J Nutr (2002) 132:3051–3054.[Abstract/Free Full Text]
  25. Macleod J.C., Macknight A.D.C., Rodrigo G.C. The electrical and mechanical response of adult guinea pig and rat ventricular myocytes to {omega}3 polyunsaturated fatty acids. Eur J Pharmacol (1998) 356:261–270.[CrossRef][ISI][Medline]
  26. Li G.-R., Du X.-L., Siow Y.L., K O., Tse H.-F., Lau C.P. Calcium-activated transient outward chloride current and phase 1 repolarization of swine ventricular action potential. Cardiovasc Res (2003) 58:89–98.[Abstract/Free Full Text]
  27. Carmeliet E. Intracellular Ca2+ concentration and rate adaptation of the cardiac action potential. Cell Calcium (2004) 35:557–573.[CrossRef][ISI][Medline]
  28. Leifert W.R., Dorian C.L., Jahangiri A., McMurchie E.J. Dietary fish oil prevents asynchronous contractility and alters Ca2+ handling in adult rat cardiomyocytes. J Nutr Biochem (2001) 12:365–376.[CrossRef][ISI][Medline]
  29. Negretti N., Perez M.R., Walker D., O'Neill S.C. Inhibition of sarcoplasmic reticulum function by polyunsaturated fatty acids in intact, isolated myocytes from rat ventricular muscle. J Physiol (2000) 523:367–375.[Abstract/Free Full Text]
  30. Janse M.J., Wit A.L. Electrophysiological mechanisms of ventricular arrhythmias resulting from myocardial ischemia and infarction. Physiol Rev (1989) 69:1049–1169.[Free Full Text]
  31. Ducceschi V., Di Micco G., Sarubbi B., Russo B., Santangelo L., Iacono A. Ionic mechanisms of ischemia-related ventricular arrhythmias. Clin Cardiol (1996) 19:325–331.[ISI][Medline]
  32. Janse M.J. Electrophysiological changes in heart failure and their relationship to arrhythmogenesis. Cardiovasc Res (2004) 61:208–217.[Abstract/Free Full Text]
  33. Dujardin K, Dumotier B, Hondeghem L. Dietary supplementation with n-3 polyunsaturated fatty acids: a far less toxic alternative and perhaps superior antiarrhythmic therapy to amiodarone? Circulation 2005;112(Suppl. S):U554–U554 2368 [abstract].
  34. Yan G.X., Wu Y., Liu T., Wang J., Marinchak R.A., Kowey P.R. Phase 2 early afterdepolarization as a trigger of polymorphic ventricular tachycardia in acquired long-QT syndrome: direct evidence from intracellular recordings in the intact left ventricular wall. Circulation (2001) 103:2851–2856.[Abstract/Free Full Text]
  35. Preliminary report: effect of encainide and flecainide on mortality in a randomized trial of arrhythmia suppression after myocardial infarction. The Cardiac Arrhythmia Suppression Trial (CAST) Investigators. N Engl J Med (1989) 321:406–412.[Abstract]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Verkerk, A. O.
Right arrow Articles by Coronel, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Verkerk, A. O.
Right arrow Articles by Coronel, R.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?