Long-Term Fish Oil Supplementation Induces Cardiac Electrical Remodeling by Changing Channel Protein Expression in the Rabbit Model

Clinical trials and epidemiological studies have suggested that dietary fish oil (FO) supplementation can provide an anti-arrhythmic benefit in some patient populations. The underlying mechanisms are not entirely clear. We wanted to understand how FO supplementation (for 4 weeks) affected the action potential configuration/duration of ventricular myocytes, and the ionic mechanism(s)/molecular basis for these effects. The experiments were conducted on adult rabbits, a widely used animal model for cardiac electrophysiology and pathophysiology. We used gas chromatography - mass spectroscopy to confirm that FO feeding produced a marked increase in the content of n-3 polyunsaturated fatty acids in the phospholipids of rabbit hearts. Left ventricular myocytes were used in current and voltage clamp experiments to monitor action potentials and ionic currents, respectively. Action potentials of myocytes from FO-fed rabbits exhibited much more positive plateau voltages and prolonged durations. These changes could be explained by an increase in the L-type Ca current (ICaL) and a decrease in the transient outward current (Ito) in these myocytes. FO feeding did not change the delayed rectifier or inward rectifier current. Immunoblot experiments showed that the FO-feeding induced changes in ICaL and Ito were associated with corresponding changes in the protein levels of major pore-forming subunits of these channels: increase in Cav1.2 and decrease in Kv4.2 and Kv1.4. There was no change in other channel subunits (Cav1.1, Kv4.3, KChIP2, and ERG1). We conclude that long-term fish oil supplementation can impact on cardiac electrical activity at least partially by changing channel subunit expression in cardiac myocytes.


Introduction
Clinical trials and epidemiological studies have suggested that dietary fish oil (FO) supplementation can provide an antiarrhythmic benefit in some patient populations [1]. One of the largest trials, the GISSI Prevenzione trial, showed that patients that survived recent (,3 months) myocardial infarction when receiving FO supplementation had a reduced mortality rate [2]. There was no reduction in the risk for non-fatal myocardial infarction. The reduced mortality could be attributed, at least partly, to a protection against sudden cardiac death by the FO supplementation [2].
The mechanism(s) underlying the anti-arrhythmic effect of FO supplementation has been under investigation for years. It has been proposed that this anti-arrhythmic effect is mainly due to a direct suppression of Na (I Na ) and L-type Ca (I CaL ) currents in cardiac myocytes by the active ingredients of FO, n23 polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA or C22:6,n23) and eicosapentaenoic acid (EPA or C20:5,n23) [3]. This is similar to a combination of class I and class IV anti-arrhythmic mechanisms. There are several issues with this proposed anti-arrhythmic mechanism for fish oil or n-3 PUFAs. First, the acute current-suppressing effects observed in tissue bath experiments cannot explain why clinically it takes ,3 months for FO supplementation to manifest the protective effect [2]. Second, n26 PUFAs (i.e. arachidonic acid) have similar current-suppressing effects in tissue bath experiments [4,5]; yet they do not provide anti-arrhythmic protection. Third, although acute exposure to DHA or EPA can suppress I Na in neonatal rat cardiomyocytes [4] or in heterologous expression systems [6], experiments of feeding adult animals with an FO-rich diet for weeks have not shown any I Na reduction [7].
To more closely mimic the clinical situation, it is important to study the effects of dietary FO supplementation in animal models after long-term (weeks to months) FO feeding. To gain insights into the ionic mechanisms for the anti-arrhythmic effects of FO supplementation, it is necessary to study how such treatment can impact on ion channels that are involved in shaping the action potential configuration and duration in the heart. Since clinically the protective effects of FO supplementation lag behind the beginning of FO regimen by about 3 months [2], the involvement of changes in gene expression must be taken into consideration. Therefore, to provide a molecular basis for such changes in ion channel function, it is necessary to examine the expression level of relevant ion channel subunit proteins in cardiac myocytes.
Our goals were to understand: (a) how FO feeding for 4 weeks could affect the action potential configuration and duration in ventricular myocytes, (b) how FO feeding affected the function of ion channels that are important determinants of action potential properties, and (c) whether changes in ion channel function involved alterations in gene expression. We chose a popular animal model, rabbit, in our experiments. Rabbits have been widely used to study cardiac electrophysiology, pathology and pharmacology. Our data confirm that FO feeding induces an 'electrical remodeling' in rabbit ventricular myocytes by altering channel gene expression.

Animal preparation
The investigation conforms with 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). The animal protocol is reviewed by IACUC of VCU annually (IACUC protocol # 10294). Twenty-three young adult (2-2.5 months) male New Zealand White rabbits were included in this study. Ten (FO group) were fed complete content of FO soft gel (NatureMade, ,120 mg/kg/d of DHA+EPA) for 4 weeks. The others (control group) were not given any fat supplementation. FO-fed rabbits did not show any signs of pathology. Their weight gain during 4-week FO feeding was the same as control rabbits during the same period: at the end of 4 weeks, body weights of control rabbits increased from 2.3660.05 to 2.9060.09 kg, while those of FO-fed rabbits increased from 2.3860.07 to 2.8860.05 kg (p.0.05). Furthermore, the FO-fed rabbits did not show any signs of myocardial hypertrophy: mean values of cell capacitance were160615 and 15966 pF for control and FO myocytes (n = 20 and 33, p.0.05).
For myocyte isolation, the aorta was cannulated and the heart was mounted on a Langendorff apparatus for enzyme treatment (see below). If the heart was used for biochemical experiments, it was dissected into different regions of ,565 mm chunks, snap frozen in liquid nitrogen and stored at 280uC until experiments.

Single myocyte preparation
The heart was perfused retrogradely through the aorta sequentially with the following oxygenated solutions warmed to Figure 1. Fish oil (FO) feeding increased the n-3 PUFA content in phospholipids (PLs) of rabbit hearts. Lipids were extracted from ventricular myocardium and analyzed by thin-layer chromatography (TLC), or transmethylated followed by characterization/quantification by gas chromatography-mass spectroscopy (GC-MS). (A) Image of a representative TLC plate sprayed with 29,79-dichlorofluorescence which made lipid spots fluorescent under UV. The loaded PL standard and lipid samples with their dilutions (Dilu) are labeled on the bottom. The positions of PL, cholesterol (CHL) and free fatty acids (FFA) spots are marked on the right. Spot intensities were quantified by densitometry, and the percentages of the three components are listed. (B) Superimposed chromatograms of fatty acids from a control rabbit (gray trace) and an FO-fed rabbit (black trace). Peak amplitudes were normalized by the first peak (butylated hydroxytoluene, antioxidant in solvents at 50 ug/ml). The peaks of interest, C20:4,n26 (arachidonic acid, AA), C20:5,n23 (eicosapentaenoic acid, EPA) and C22:6,n23 (docosahexaenoic acid, DHA) are labeled. (C) Data summary. Areas under the peaks of AA, EPA and DHA were normalized by the total areas of peaks above threshold and averaged over three samples from each group. doi:10.1371/journal.pone.0010140.g001 37uC: (1) normal Tyrode's (composition given below), 4-5 min, to monitor the regularity/strength of heart beats, (2) nominally Cafree Tyrode's supplemented with 0.1% BSA, 6-7 min, to wash out Ca, (3) same solution as (2) but with collagenase (Worthington type II, 0.5 mg/ml), 30 min, to digest the extracellular matrix, and (4) KB solution, 3 min, to stop enzyme action. LV base was minced and the tissue was very gently shaken in KB to release single myocytes. The cell suspension was filtered through a 500-um nylon mesh and stored at room temperature in KB. Experiments were done in #8 hr after cell isolation.

Patch clamp experiments
Cell suspension was added to a poly-lysine-coated coverslip placed in the bath mounted on an inverted Nikon microscope. After allowing cell attachment for 3-5 min, cells were superfused with normal Tyrode's solution at 3461uC. We used pipettes with tip resistance ,1-2 MV. Patch clamp recordings of whole cell currents were controlled by pClamp 10 via Digidata 1440A, using an Axopatch 200B amplifier. Series resistance was compensated up to 95%. Pipette tip potential was zeroed before making seal, and a liquid junction potential of 10 mV between the pipette and bath solutions (pipette side negative) was corrected during data analysis. Currents were low-pass filtered at 1 kHz (Frequency Devices) and stored for off-line analysis.

Fatty acid analysis
Lipids were extracted from tissue samples using a procedure modified from that of Folch [8]. All solvents contained butylated hydroxytoluene (BHT, 50 ug/ml) to prevent lipid oxidation. Briefly, tissue (0.3-0.7 g) were minced in methanol (6 ml) and homogenized with polytron grinder for 2 min. Chloroform (12 ml) was added and the mixture was further homogenized for 2 min. Then 0.88% (w/v) KCl 4.5 ml was added and the mixture was vortexed vigorously for 2 min. After the phases separated, the lower organic phase was further extracted with 5 ml of methanol plus 0.88% KCl (1:1, v/v). The final organic phase was dried down under a stream of nitrogen, reconstituted in 100 ul chloroform and stored at 280uC.
The phospholipid (PL) component was separated from the other components in the lipid extracts using thin-layer chromatography [9]. Samples along with a PL standard (PL mix from soybean, Supelco) of different dilutions in 10 ul total volume were applied to preparative silica gel G plates (20620 cm, silica gel thickness = 0.5 mm). The mobile phase was hexane/diethyl ether/acetic acid (85:15:1, by vol). After the mobile phase reached within 1 inch to the top, the plate was dried by baking on a hotplate at ,120uC for 5 min, sprayed with 29,79-dichlorofluorescene (in 95% methanol, 0.1% w/v), and the lipid spots were visualized/ quantified under UV (ChemiImager model 4400, a-Innotech).
To make the fatty acids volatile at ,200uC (for separation by gas chromatography), the lipid extracts (25 ul) were transmethylated by boiling in excess (500 ul) boron trifluoride (BF 3 , in methanol 14% w/v) in a boiling water bath for 2-3 min. Methyl esters of long-chain fatty acids were extracted using 1.5 ml hexane/water (3:2, v/v). The organic phase was dried down under a stream of nitrogen, reconstituted in 25 ul of hexane and stored at 280uC.
The above methylated fatty acid samples were analyzed by gas chromatography -mass spectroscopy (GC-MS, Shimadzu, model QP5050A). We used a GC capillary column (Omegawax250, Supelco) designed for the separation of long-chain polyunsaturated fatty acids. The injection volume was 1 ul with a split ratio of 30:1. The oven temperature was set a 200uC and the carrier gas (helium) was set at a total flow rate of 1 ml/min. The following methylated fatty acid standards (Supelco) were used to confirm peaks identity: C20:4,n-6, C22:6,n-3, C18:1,n-9, and C18:3,n-3.

Immunoblot analysis
Except for Kv1.4, the protein levels of channel subunits were quantified from membrane-enriched fraction prepared using PLoS ONE | www.plosone.org procedures modified from those described by Takimoto [10]. Frozen tissue chunks were pulverized under liquid nitrogen, and homogenized in 10 vol of buffer (0.25 M sucrose, 1 mM EDTA [pH 7.4]). This and all the following procedures took place in the presence of the protease inhibitor cocktail at 4uC or on ice. The homogenate was centrifuged at 3,500 rpm for 10 min to pellet nuclei and debris. The supernatant was centrifuged at 30,000 rpm for 1 hr to pellet the membranes. The membrane pellet was washed with a solution (Tris-HCl 20 mM [pH 7.4], EDTA 1 mM) and referred to as post-nuclear membrane fraction. The postnuclear membrane fraction was rehomogenized in a Tritoncontaining lysis buffer (Tris-HCl 20 mM [pH 7.5], NaCl 0.2 M, EDTA 1 mM, Triton X-100 1%) using Dounce grinder. The mixture was centrifuged at 17,000 rpm for 1 hr, and the supernatant (Triton extract) was used for immunoblotting.
Initial attempts to detect Kv1.4 in the membrane-enriched fraction failed, although the antibody detected a strong ,100 kDa fuzzy band in whole tissue lysate (WTL) of rat brain (representing glycosylated Kv1.4) and a faint band of a similar size in WTL of rabbit hearts (see below). Therefore, the Kv1.4 data reported here were from WTL prepared using the procedures described by O'Rourke et al [11]. Briefly, frozen tissue chunks were pulverized in 10 vol of lysis buffer (in mM: NaCl 145, MgCl 2 0.1, HEPES 15, EGTA 10, pH 7, Triton X-100 0.5, with protease inhibitor cocktail), and solubilized for 30 min on ice. The above was homogenized by tip sonicator (2 of 15-s bursts), and then centrifuged to pellet nuclei and debris. The supernatant was used for immunoblotting. Importantly, the effect of FO feeding on Cav1.2 protein measured in the same set of hearts was similar between these two methods of protein preparation (see below).

Data analysis
For patch clamp recordings, the experimental protocol and methods of data analysis are described in text or figure legends. Data were analyzed using Clampfit of pClamp10. Statistical analysis of data from patch clamp experiments, GC-MS analysis, and densitometry was done using SigmaStat (v 2.1). Multiple group data were analyzed by one-way ANOVA and, if p,0.05, followed by pair-wise comparisons. The t-test was used for comparison between two groups. Statistical significance is noted as: *** p,0.001, ** p,0.01, * p,0.05.

1.
Fish oil feeding enriches n-3 PUFA content in the phospholipids of rabbit heart Fig. 1A shows that phospholipids (PLs) accounted for $90% of the lipids extracted from both control and FO-fed rabbit hearts. To reduce the risk of oxidation of long-chain polyunsaturated acids, which would invalidate the downstream analysis (as shown in Fig. 2, due to changes in the fatty acid alkyl chain properties), we bypassed the TLC procedure of PL purification and directly subjected lipid extracts to transmethylation and GC-MS analysis. Fig. 1B shows that we could unequivocally identify C20:4,n26 (arachidonic acid or AA) and two n23 PUFA peaks: C20:5,n23 (eicosapentaenoic acid or EPA) and C22:6,n23 (docosahexaenoic acid or DHA). FO feeding for 4 weeks markedly increased the content of EPA (from 0.1960.02 to 2.9660.12%) and DHA (from 0.2860.05 to 4.4160.35%) in rabbit ventricles (Fig. 1C). There was also a slight reduction in AA (from 16.161.7 to 12.060.5%), although the difference did not reach p,0.05.
2. Fish oil feeding elevates the action potential plateau voltage and prolongs the action potential duration of rabbit ventricular myocytes  normal Tyrode's to Na-and Ca-free Tyrode's while monitoring the disappearance of I Na and I CaL (to remove interference from I Na , I Ca and Na/Ca exchanger current, I NCX , in the measurement of I K ), (5) 20-25 min, recording I K , (6) 25-30 min, washing in dofetilide 1 uM while monitoring the change in I K , and (6) 30-35 min, recording dofetilide-insensitive currents.
Action potentials were elicited by passing suprathreshold 2-ms current pulses via the patch pipette. We tested the effects of FO feeding on action potential configuration and duration at CLs of 0.3, 0.5, 1 and 2 s, to simulate heart rates of bradycardiatachycardia. For each of the CLs, a train of action potentials was elicited till the configuration and duration reached a steady state (requiring 36-60 action potentials at the CL of 2 s, 72-120 action potentials at the CL of 0.3 s). The order in which the CLs were applied was random among myocytes to avoid the issues of usedependent changes in the action potential parameters. The last 10 action potentials of a train were averaged and used to measure the resting membrane potential, the action potential plateau height, and the action potential duration. Fig. 3A depicts representative APs recorded from a control and an FO myocyte, each subjected to stimulation at 4 CLs. Fig. 3B presents data summary. FO feeding markedly elevated the AP plateau height and prolonged APD at all 4 CLs. The degree of APD prolongation was modest at CL 0.3 s, but became more profound at longer CLs. On the other hand, FO feeding did not affect the resting membrane potential.

Fish oil feeding reduces I to current density and I to subunit expression in rabbit ventricular myocytes
We quantified the peak amplitude of I to at +50 mV to avoid interference from I Na and I Ca , because this voltage was close to the apparent reversal potentials of Na and Ca currents. The voltage clamp protocol is diagrammed in the inset of Fig. 4A. From a holding voltage (V h ) of 280 mV, a 2-s conditioning pulse to 2110 mV was applied to remove I to inactivation [12], so that a fully available I to along with other overlapping currents was recorded during the subsequent test pulse to +50 mV (solid trace). Then a 2-s conditioning pulse to 210 mV was applied to maximally inactivate I to [12], so that a current trace without I to was recorded during the step to +50 mV (dotted trace). The difference current between the two represents fully available, isolated I to (lower traces of Fig. 4A). FO feeding decreased the peak I to current densities from 8.761.0 to 5.260.5 pA/pF (Fig. 4B, first panel). I to inactivation followed a double exponential time course (double exponential fits in Fig. 4A, lower panel). FO feeding did not change the time course of I to inactivation (Fig. 4B, second to fourth panels).
To understand why the I to peak current density was reduced in FO-fed rabbit ventricle, we used immunoblotting to quantify the protein levels of I to channel subunits. Rabbit cardiac I to has 2 components: Kv4.x-based (Kv4.2 and Kv4.3 as pore-forming subunits, KChIP2 as auxiliary subunit) and Kv1.4-based channels [13]. We used Kv4.2, Kv4.3 and Kv1.4 Abs raised against rat sequences, that are 94%, 98% and 100% identical in rabbit sequences. The KChIP2 Ab was also raised against a rat sequence. We could not find information on rabbit KChIP2 in the NCBI database. Fig. 5 shows validation of these Abs as tools to detect target proteins in the rabbit heart, with rat brain or rat heart proteins as positive controls. Fig. 6A and 6B depict immunoblot images of I to channel subunits in five control and four FO-fed rabbit hearts. We used a-actin as the internal control to correct for variations in protein loading among lanes. Fig. 6C presents data summary of densitometry quantification. Among the four I to channel subunits examined, Kv4.2 and Kv1.4 protein levels in FO-fed rabbit ventricle dropped to 0.3460.14 and 0.4060.05 of control. There was no change in the protein level of Kv4.3 or KChIP2.

Fish oil feeding increases I CaL current density and I CaL subunit expression in rabbit ventricular myocytes
We estimated the peak I CaL by the difference between the inward peak and the current level at the end of a 500 ms pulse from V h 250 mV to 0 mV, where the maximal I CaL occurred. Representative current traces from a control and an FO myocyte are superimposed in the top panel of Fig. 7A. The maximal peak I CaL density was markedly increased by FO feeding (from 7.560.6 to 10.760.7 pA/pF, Fig. 7A, lower panel). We also characterized the voltage-dependence of I CaL inactivation (Fig. 7B), time course of I CaL recovery from inactivation at 250 mV (Fig. 7C), and the time course of I CaL inactivation during depolarization to 0 mV (Fig. 7D). FO feeding did not induce any detectable changes in these parameters of I CaL gating kinetics.
We used immunoblot experiments to test whether there was a corresponding change in the pore-forming subunit of the L-type Ca channels, Cav1.2. The Cav1.2 Ab was raised against a fusion protein containing partial rabbit Cav1.2 sequence (aa 1507-1733). This Ab recognized a fuzzy band of ,240 kDa in rabbit ventricles, as expected for glycosylated Cav1.2. FO-feeding caused a significant increase in Cav1.2 protein level in rabbit ventricles (Fig. 8B, 1.6660.14 vs 1.0060.18, p,0.05).
Interestingly, a Cav1.1 Ab raised against purified dihydropyridine receptor protein isolated from rabbit skeletal muscle t-tubules could detect a ,83 kDa sharp band in rabbit ventricles (Fig. 8).
There was no change in the Cav1.1 protein level in FO-fed rabbit ventricles, supporting the selectivity of FO-feeding in modulating the Cav1.2 protein level.
To check whether the method of sample preparation influenced the results of immunoblot analysis, we compared Cav1.2 quantification in membrane-enriched fraction and in whole-tissue lysate prepared from the same set of hearts. Fig. 9 confirms that immunoblot analysis of both sample preparations reached the same conclusion: FO feeding increased the Cav1.2 protein level in rabbit ventricles.
5. Fish oil feeding does not affect delayed or inward rectifier current of rabbit ventricular myocytes Fig. 10A shows that in the presence of dofetilide, little or no outward tail current could be detected in either the control or the FO myocyte. Therefore, under our recording conditions the delayed rectifier (I K ) current in rabbit ventricular myocytes was mainly the rapid component (I Kr ). FO feeding did not affect the I K current density or the voltage-dependence of I K activation in rabbit ventricular myocytes (Fig. 10B). FO feeding did not alter the protein level of ERG1 (asubunit of I Kr channels) in rabbit ventricles (Fig. 10C). The background current-voltage relationship in the negative voltage range mainly reflects the inward rectifier (I K1 ) current. Fig. 10D shows that FO feeding did not affect I K1 in rabbit ventricular myocytes.

Molecular mechanisms for fish oil supplementationinduced electrical remodeling
Long-term FO supplementation can affect membrane protein function by at least three mechanisms that are not mutually exclusive. First, incorporation of polyunsaturated acyl chains of n23 PUFAs into membrane phospholipids will alter the membrane material properties [14]. The increase in membrane fluidity and decrease in lateral pressure may reduce the energy costs of conformational changes in membrane proteins that are critical for their function. Second, an increase in the n23 PUFA content in the membrane lipid bilayer may facilitate phase  separation between PUFA-rich/sphingomyelin and cholesterolpoor disordered lipid domains and PUFA-poor/sphingomyelin and cholesterol-rich ordered lipid domains (lipid rafts) [15]. This can lead to changes in the function and modulation of membrane proteins associated with lipid rafts [16]. Third, PUFAs can modulate gene expression [17] by regulating the activity of transcription factors directly (e.g. sterol regulatory element-binding proteins) or indirectly by binding to nuclear receptors (e.g. peroxisome proliferators-activated receptors) [18]. Our observations that long-term FO feeding altered I CaL and I to channel subunit expression in rabbit hearts suggest that the third mechanism, altered gene expression, is at work. Fig. 11 shows that acute application of n23 PUFA (DHA, 10 mM) markedly suppressed the peak amplitudes of I CaL and I to in rabbit ventricular myocytes. These observations are similar to previous reports of tissue bath experiments [5,19]. Acute effects of n23 PUFAs on membrane channels in the heart are likely to occur in vivo transiently after ingestion of FO, when the plasma level of n23 PUFAs is high. Importantly, the effects of chronic FO feeding on I CaL (enhancement) differ from that of acute DHA application (suppression), while both treatments similarly suppress the I to amplitude. Based on these observations, we suggest that dietary FO supplementation in humans will have both chronic (sustained) and acute (transient) effects, and the two aspects may antagonize or complement each other.

Clinical Implications
We show that ventricular myocytes isolated from FO-fed rabbits, relative to myocytes from control rabbits, had much more positive plateau heights and longer action potential durations tested at cycle lengths of 0.3-2 s. Our observations were consistent Figure 10. Fish oil feeding did not alter the delayed rectifier (I K ) or inward rectifier (I K1 ) current in rabbit ventricular myocytes. (A) Representative current traces recorded from a control and an FO myocyte, before and during exposure to 1 uM dofetilide (DOF). Inset: protocol (V h 250 mV, V t to 230-+40 mV in 5 mV steps for 5 s, V r to 240 mV for 5 s, interpulse interval 30 s). (B) Summary of voltage dependence of I K activation (main graph) and I K current amplitude (inset). The voltage-dependence of activation was analyzed by normalizing the I K tails to the maximal I K tail following V t to +40 mV (fraction activated), and the relationship between 'fraction activated' and 'V t ' was fit with a Boltzmann function: fraction activated = 1/[1+exp((V 0.5 2V t )/k)], where V 0.5 and k are half-maximum activation voltage and slope factor, respectively. The V 0.5 and k values are (mV): 24.562 and 5.261.0 for control myocytes, 24.761.2 and 5.660.4 for FO myocytes. The I K current amplitude was measured from peak tail current following V t to +40 mV (numbers of cells analyzed in parentheses). (C) Immunoblot analysis of ERG1 protein level in control and FO rabbit LV (same set of hearts as shown in Figs. 6 and 8). Shown on top are Mean6SE of ERG1-immunoreactive band intensities corrected for loading (divided by CB stain) and normalized by the mean value of control rabbits (p = 0.524). (D) Background current-voltage relationship was analyzed using the following voltage clamp protocol. From V h 250 mV, test pulses to V t of 0 to 2120 mV in 10 mV steps for 500 ms were applied once every 5 s. Currents were measured at the end of the test pulses. doi:10.1371/journal.pone.0010140.g010 Figure 11. Effects of acute exposure to DHA (10 uM) on I CaL and I to in rabbit ventricular myocytes. I CaL and I to were measured as described for Fig. 7A and Fig. 4A, respectively, before and during exposure to DHA. (A) Superimposed current traces. (B) Summary of % reduction of peak I CaL and I to in DHA. doi:10.1371/journal.pone.0010140.g011 with a previous report showing that FO feeding in rabbits for 30 days caused a prolongation of the plateau phase of monophasic action potentials recorded from the ventricles of Langendorffperfused hearts [20].
Our observations are different from a previous study in the pig model [7]. FO feeding in pigs caused a decrease in action potential plateau height and a shortening of APD. These changes were accounted for by a decrease in I CaL and I NCX , along with an increase in the slow delayed rectifier (I Ks ) and I K1 [7]. The differences in FO feeding-induced cardiac electrical remodeling between rabbit and pig models may be due to a combination of factors: differences in cellular and membrane environment of ion channels in cardiac myocytes which can impact the effects of n-3 PUFA enrichment, differences in gene regulation, and differences in the animal diets. These species variations serve as a cautionary note about generalizing animal studies to the clinical situation.
They also suggest that the effects of FO supplementation on the cardiac electrical activity in people can vary due to differences in genetic makeup and conditions of the heart.