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Am J Physiol Heart Circ Physiol 277: H2290-H2297, 1999;
0363-6135/99 $5.00
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Vol. 277, Issue 6, H2290-H2297, December 1999

Dietary fish oil promotes positive inotropy of ouabain in the rat heart

Jean-Michel Maixent1, Alain Gerbi1, Odile Barbey1, Carole Lan2, Isabelle Jamme4, Henri Burnet3, André Nouvelot4, Samuel Lévy1, Patrick J. Cozzone2, and Monique Bernard2

1 Laboratoire de Recherche Cardiologique, 2 Centre de Résonance Magnétique Biologique et Médicale, Unité Mixte de Recherche 6612, Centre National de la Recherche Scientifique (CNRS), and 3 Equipe Associée 2205, Faculté de Médecine, Université de la Méditerranée, 13005 Marseille; and 4 Laboratoire de Neurosciences Unité de Recherche Associée 1829, CNRS, Caen, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We tested the hypothesis that a fish oil (FO) diet promotes positive inotropy of ouabain without increased toxicity. For 2 mo, two groups of adult male rats were fed 1) a regular food diet supplemented with dietary long-chain polyunsaturated fatty acid from FO or 2) a regular food diet (control). The responsiveness to ouabain was evaluated for the two groups in Langendorff-perfused hearts, by 31P nuclear magnetic resonance spectroscopy, and on purified membrane-bound Na-K-ATPase. The maximum positive inotropy achieved with ouabain was nearly two times higher in the FO than in the control group and was not associated with significant changes in energetics. Alteration of function and energetic metabolism and inhibition of Na-K-ATPase in response to 3 × 10-4 M ouabain were delayed in the FO group. This study demonstrates that dietary FO, by a cardiac membrane incorporation of n-3 polyunsaturated fatty acid, promotes positive inotropy of ouabain without toxicity and changes in cardiac metabolism.

fatty acid; nutrition; sodium-potassium-adenosine 5'-triphosphatase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

DIGOXIN IS CLINICALLY effective in relieving the signs and symptoms of heart failure due to systolic dysfunction (34). The positive inotropic concept could be used in long-term treatment to improve clinical status and prolong survival (7). However, digitalis in the treatment of heart failure has an extremely narrow therapeutic index, and an increased mortality in clinical trials was often reported for inotropic agents (6). Inclusion of fish oil (FO) in the diet may reduce the risk of death from heart disease by various mechanisms (21): decreased heart rate and occurrence of arrhythmias (5, 15, 18) and increased mechanical activity and ejection fraction (17, 27).

The mechanism of cardiac glycoside action in promoting contractility is inhibition of the Na-K-ATPase, which leads to a small (1 mM) increase in intracellular Na+ and Ca2+ by slowing the Na+/Ca2+ exchange rate across membranes (35). If too much Na-K-ATPase is inhibited, toxicity ensues from intracellular Ca2+ overload. Ca2+ overload, contracture, and increased energetic metabolism in response to inotropic drugs are toxic, especially in myocardium from patients with severe heart failure (13). FO supplementation may have a beneficial effect on digitalis therapy by increasing cardiac contractility. The combined use of digitalis and FO on the inotropic effect has not been previously reported, to our knowledge.

The rat is the only mammalian species with a well-characterized biphasic positive inotropic response to ouabain at low external Ca2+ concentrations (12). This biphasic effect is coupled to consecutive inhibition of the alpha 2- and alpha 1-isoforms of the Na-K-ATPase of high and low affinity for ouabain, respectively (1, 12, 22); thus on this model we can correlate the physiological response to alpha 1- and alpha 2-subunit inhibition. Recently, we demonstrated a direct influence of dietary FO on ouabain affinity of Na-K-ATPase isozymes of the rat (9-11).

In the present study we tested the hypothesis that an administration of dietary FO with an incorporation of n-3 polyunsaturated fatty acids (PUFAs) could promote positive inotropy of ouabain. The purpose of this study was therefore to investigate in rats fed dietary FO 1) the changes in inotropic and toxic effects of ouabain in a heart model in which high- and low-affinity positive inotropy of ouabain exist, respectively, and 2) the correlation with the changes in activity and the protein abundance of alpha 2- and alpha 1-isoforms of Na-K-ATPase and energetic metabolism.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Twenty-one male Wistar rats weighing ~150 g were included in the study. We designed our study with a control group that consisted of rats matched for age and weight with treated rats. One group was treated daily over the 8 wk of the study with the n-3 fatty acid-enriched FO by oral administration at a dose of 0.5 g/kg. This diet was enriched with 180 mg of eicosapentaenoic acid (EPA) plus 120 mg of docosahexaenoic acid (DHA) per gram of oil and was supplemented with the antioxidant alpha -tocopherol (4 mg/g of oil). The diet of the control animals was not enriched with fatty acids. All animals were fed normal rat chow (A03, U.A.R, Villemoisson sur Orge, France) and water ad libitum for 8 wk. The investigation conforms to the Guide for the Care and Use of Laboratory Animals [DHHS Publ. No. (NIH) 85-23, revised 1996, Office of Science and Health Reports, Bethesda, MD 20892]. All investigations in this project were conducted under a license for animal research granted by the French Ministry of Agriculture. Twelve animals were used for isolated heart preparation and eight for membrane isolation.

Isolated heart preparation. Hearts were quickly removed from rats intraperitoneally anesthetized with pentobarbital sodium, perfused at a constant temperature of 37°C and a constant pressure of 100 mmHg, and paced at a frequency ~20% above the spontaneous heart rate (22). The perfusion medium was a modified Krebs-Henseleit buffer containing (in mM) 118 NaCl, 5.85 KCl, 25 NaHCO3, 1.2 MgSO4, 11 glucose, and 0.5 CaCl2. The buffer was gassed with 95% O2-5% CO2 and buffered to pH 7.4. A drainage cannula was inserted into the apex of the left ventricular cavity through a left atrial incision to vent the Thebesian flow. Isovolumic left ventricular pressure was measured by insertion of a water-filled latex balloon into the left ventricle. End-diastolic pressure (EDP) was set to 10 mmHg at the beginning of heart perfusion and was allowed to evolve during the protocol; developed pressure (dP), the first pressure derivative (+dP/dt), and dP/dt/Psys (where Psys is systolic pressure) (22) were monitored using a Gould Statham P23db pressure transducer, a Gould pressure amplifier, and a Gould differentiator.

The stability of the preparation during perfusion with 0.5 mM Ca2+ over the total experimental time (72 min) was checked in control experiments (data not shown). Past experiences (12, 22) have shown also that the preparation was stable at low external Ca2+ perfusion. Furthermore, Pilati and Paradise (31) previously showed that the reduction in Ca2+ to 0.5 mM did not protect against ouabain-induced toxicity.

31P magnetic resonance spectroscopy experiments. The perfused hearts were placed in a 20-mm sample tube and inserted into a 31P probe that was seated in the bore of a superconducting wide-bore (89 mm), 4.7-T magnet (Oxford Instruments) (4). 31P spectra were generated at 81 MHz with a Bruker-Nicolet WP-200 spectrometer. Field homogeneity was adjusted using the water 1H signal. 31P spectra were accumulated for 4 min by averaging data obtained from 344 free induction decays by using a pulse angle of 45° and a recycle time of 0.7 s, with a spectral width of 6,000 Hz and 2,048 data points. Before Fourier transformation, the free induction decay was multiplied by an exponential function that generated a 20-Hz line broadening. The positions and areas of the resonances were determined using the NMRi software package (NMRi, Syracuse, NY). Integrals of resonances were converted to concentrations by comparison with a standard reference. Values for intracellular pH (pHi) were derived from the chemical shift of the Pi resonance by use of a standard titration curve. The chemical shifts were referred to the resonance position of phosphocreatine (PCr).

Experimental protocol. The basic experimental protocol consisted of a 12-min control period followed by five successive 12-min periods of ouabain perfusion at different concentrations (from 10-7 M for the 1st 12-min period to 3 × 10-4 M for the 5th period). Temperature was maintained at 37°C throughout the protocol. Function and 31P-NMR spectra were registered simultaneously every 4 min on the same hearts. Values at the end of the 12-min perfusion period of each dose are presented in Table 1 and Figs. 1-3.

Tissues and membrane preparations. Hearts were rapidly removed and infused on the apparatus as described above for only 1 min with a cold (4°C) modified Krebs-Henseleit buffer. Left ventricle and septum were frozen in liquid nitrogen and stored at -80°C until use. Na-K-ATPase analysis was performed on the microsomal membrane fraction, with a yield in enzyme of 50%, rather than with purified sarcolemmal membranes, because the latter preparation has a poor enzyme yield (<10%). Frozen tissues were homogenized, and preparations consisted of Na-K-ATPase-enriched membrane microsomal fractions according to a previously described procedure (9). Protein yield was consistently 2% for animals in control and FO groups. Na-K-ATPase activity was determined using the coupled assay method with or without ouabain, as previously described (22). Assays were carried out with vesicles permeabilized by treatment with SDS (0.1 mg/mg protein) for 30 min at 20°C. The relative proportions of alpha 1- and alpha 2-isozymes were inferred from ouabain affinities as estimated from dose-response curves on permeabilized membranes (10). The number of independent sites used to model the data was chosen according to the Schwarz criterion (9).

The expression of alpha -isoforms of Na-K-ATPase was assessed by immunologic detection with specific antibodies by SDS-PAGE and Western blotting, as previously described (3, 9). Briefly, microsomal membrane preparations of the two groups (control and FO) were diluted in three volumes of buffer containing 0.5 M Tris · HCl (pH 6.8), 0.1% glycerol, 10% SDS, and 1% bromophenol blue supplemented with 1% beta -mercaptoethanol. SDS-PAGE was carried out with a Miniprotean II Cell Apparatus on 4-15% gradient ready gels (Bio-Rad, Ivry sur Seine, France) for 90 min at 100 V. Proteins were then transferred to nitrocellulose (Hybond, Amersham, Les Ulis, France) in a transfer buffer containing 192 mM glycine, 24 mM Tris, 0.1% SDS, and 10% methanol at 4°C for 60 min at 200-mA constant current. After incubation in PBS (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, pH 7.5, supplemented with 3% low-fat milk) overnight at 4°C to minimize nonspecific binding, the resulting nitrocellulose blots were probed with antibodies specific for the various Na-K-ATPase isoforms. We used an anti-rat polyclonal alpha 1-antibody provided by E. Feraille (Hôpital Cantonal, Geneva, Switzerland) and an anti-rat monoclonal alpha 2-antibody (McB2) provided by K. Sweadner (Harvard University, Boston, MA). Membranes were then washed four times with PBS supplemented with 0.1% Tween 20 and incubated with peroxidase-conjugated anti-rabbit or anti-mouse IgG (Amersham) for 15 min at 37°C. After the washing step was repeated four times with PBS alone, antigen-antibody reaction was detected by chemiluminescence. These blots were exposed to X-ray films (enhanced chemiluminescence, Hyperfilm) for various times to ensure that chemiluminescent signals were within the linear range of the film. At least three independent blots were analyzed, with reproducible results by quantitative densitometry. Autoradiogram bands were scanned with a scanning densitometer (Arcus, Agfa Gevaert, Morbel, Belgium) in transparency mode at a resolution of 150 pixels/in. The scans were processed on a Macintosh II running the public domain software Image written by Wayne Rasband (National Institutes of Health, Bethesda, MD).

Fatty acids were analyzed as methyl esters on a Varian model 3300 gas chromatograph equipped with a flame ionization detector by use of a Spirawax capillary column (25 m × 0.2 mm ID). The temperature program scanned from 150 to 210°C at 1.5°C/min. Peak areas of the resulting chromatogram were measured with an integrator (model D 2000, Merck). After extraction of free acids according to the method of Folch et al. (8), fatty acid methyl esters were prepared according to Hagenfeldt (14). As an internal standard, nonadecanoic acid was added to the mixture before methylation.

Statistical analysis. Values are means ± SE. Statistical analysis (contractility, energetic metabolism, pH, and ouabain inhibition of Na-K-ATPase) was performed using a two-way ANOVA with Tukey's test for multiple comparisons (SigmaStat statistical software). P < 0.05 was considered statistically significant. Analysis of time to arrhythmias was done with the Proc Life Test of the Statistical Analysis System program incorporating various probability tests: Wilcoxon rank test, likelihood ratio test, and log rank test. The relationship between PCr and ouabain concentration in control and FO groups was analyzed by regression analysis (38). The significance of the slopes was tested by an ANOVA procedure, and the slopes of the two regression lines were compared using Student's t-test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General features of control and FO-supplemented rats. Two months of FO supplementation did not produce any significant differences in body weight [515 ± 7 g for control (n = 11) vs. 528 ± 9 g for FO (n = 10)] or cardiac ventricular and septum weights (1.1 ± 0.1 g for control vs. 1.2 ± 0.2 g for FO) of the rats. Thus it is unlikely that a stress and a change in calorie intake produced only by an oral administration of FO would affect the results.

Inotropic effect and energetic metabolism variations. Contractile performance during the control period (1st 12-min perfusion) in isolated perfused hearts from the two groups of rats is displayed in Table 1. The contractile values were consistent with those of a 0.5 mM Ca2+ perfusion. There were no statistically significant differences between the two groups. Dietary treatment with FO significantly improved the positive inotropic effect of ouabain regardless of the index of contractility chosen (Table 1). No significant changes in diastolic pressure and heart rate were observed (data not shown).

                              
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  		Table 1.   Baseline contractile values of isolated hearts and changes from baseline values with increasing ouabain doses

Typical examples of magnetic resonance spectroscopy spectra in response to low-dose (10-7 M) and high-dose ouabain (10-4 M) are displayed in Fig. 1; peak assignments are given in the Fig. 1 legend. Typically, the resonance area of PCr decreases when the ouabain dose is increased. Quantitative analysis of PCr is given in Fig. 2 before ouabain infusion and during the positive inotropic effects of ouabain. The baseline level of PCr was significantly higher in hearts of the FO group (Fig. 2). PCr levels were significantly decreased compared with baseline values, while the ouabain dose was increased in both groups, inasmuch as the slopes of the two regressions were significantly different from zero (Fig. 2). However, during ouabain perfusion, there were no significant differences in PCr levels between the control and the FO group (there were no significant differences in their mean slopes). ATP levels and pHi (Fig. 1) with ouabain perfusion were not significantly different from baseline values and were not different between the two groups (quantitative data not shown). Therefore, the higher inotropic response to ouabain in the FO than in the control group was not associated with significant changes in high-energy phosphates or in pHi.


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  		Fig. 1.   31P magnetic resonance spectra of rat hearts of control animals (A) and animals fed diets supplemented with fish oil (B) and treated with different doses of ouabain. Spectra were acquired over 4-min periods. For 10-7 and 10-4 M ouabain, spectra were recorded from 8 to 12 min of perfusion. For 3 × 10-4 M ouabain, spectra were recorded from 1 to 4 min of perfusion. Spectra resonance assignments are Pi, phosphocreatine (PCr), and alpha -, beta -, and gamma -isoforms of ATP. ppm, Parts per million.



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  		Fig. 2.   Effect of sequential additions of increasing concentrations of ouabain (10-7-10-4 M) on intracellular PCr levels of hearts of control rats (C) and rats fed a diet supplemented with fish oil (FO). Values (means ± SE of 7 for C group and 5 for FO group) are calculated from 31P magnetic resonance spectra recorded during last 4 min of 12-min perfusion period. * P < 0.05, FO vs. C. Relationship between PCr and ouabain concentration has been analyzed by regression methods. For C and FO groups, PCr decreased significantly with ouabain concentration (P < 0.01), but slopes of 2 regression lines were not significantly different between groups (P < 0.25).

Ouabain intoxication. Typical examples of spectra averaged during the 4th min of perfusion with 3 × 10-4 M ouabain are shown in Fig. 1. No dramatic changes were observed in spectra during this period of time. However, consistent with the narrow therapeutic index of digitalis, toxicity was developing rapidly with the perfusion of 3 × 10-4 M ouabain. Ouabain intoxication is shown in Fig. 3 as a function of the time of perfusion of 3 × 10-4 M ouabain. The time course of ouabain intoxication was significantly different between the two groups for dP (Fig. 3A) and energetics (Fig. 3, C and D) but was only suggested for EDP (Fig. 3B; P = 0.07). In the FO group, at the beginning of the perfusion of 3 × 10-4 M ouabain, there was a further increase in dP above the inotropic level of 10-4 M (Fig. 3A); in the control group the intoxication by 3 × 10-4 M ouabain was evidenced by early decreased contractility (P < 0.001). Therefore, with the FO diet the maximal inotropic effect was increased. As a sign of earlier toxicity in the control group, the increase in EDP (contracture) occurred earlier in the control group (Fig. 3B). Toxicity in the control group is also shown by higher depletions in PCr (P < 0.05; Fig. 3C) and ATP (P < 0.01; Fig. 3D) than in the FO group. The time to arrhythmia was not significantly different between the control and the FO group (145 ± 46 and 300 ± 95 s, respectively).


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  		Fig. 3.   Time course of changes in function and energetics during ouabain intoxication (3 × 10-4 M ouabain). Values are means ± SE of 7 for C group and 5 for FO group. Relative increased inotropic effect is represented by left ventricular developed pressure (dP) expressed as percentage above dP obtained at 10-4 M ouabain. EDP, end-diastolic pressure. PCr and ATP values are calculated from 31P magnetic resonance spectra. * P < 0.05; dagger  P < 0.01; Dagger  P < 0.001, FO vs. C.

Fatty acid composition of myocardial membranes. Fatty acid analysis of hearts of rats fed an FO diet showed that the proportion of EPA (20:5) and DHA (22:6) rose significantly while the proportion of n-6 fatty acids declined (compared with the control group; Table 2). A specific incorporation was evidenced by a 12-fold increase in EPA in cardiac membranes after 2 mo of FO dietary supplementation. As a consequence, the ratio of n-6 to n-3 PUFA was reduced.

                              
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  		Table 2.   Effects of dietary FO supplementation on composition of total fatty acids in cardiac membranes

Na-K-ATPase. Table 3 compares the ouabain-sensitive Na-K-ATPase activity in microsomal vesicles from control animals and animals fed FO. Dietary FO had no effect on overall Na-K-ATPase activity. The alpha 1- and alpha 2-isoforms of Na-K-ATPase in the rat heart are known to exhibit a biphasic dose-response inhibitory curve with low and high affinities for ouabain, respectively. Consistently, the dose-response curves presented in Fig. 4 are biphasic and best modeled by assuming two affinities rather one affinity. Inhibition from 3 × 10-5 to 5 × 10-4 M ouabain was found to differ in control and FO-treated groups. This corresponds to a significantly lower ouabain affinity of the alpha 1-isoform (by a factor of 4.8) in the FO group than in the control group. Figure 4 and Table 3 show that FO treatment did not significantly change the affinity of the alpha 2-isoform and the contributions of the alpha 1- and alpha 2-isoforms to Na-K-ATPase activity. To exclude possible changes in protein expression, immunodetection by Western blot analysis was performed. As shown in Fig. 5 and evaluated by densitometric scanning in Fig. 6, the membrane abundance of alpha 1- and alpha 2-isoforms was similar in the FO and the control group. These findings confirmed our results showing that the specific activity of the two isozymes was unchanged by the FO diet (Table 3).

                              
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  		Table 3.   Effects of dietary FO supplementation on Na-K-ATPase activity and ouabain sensitivity in cardiac membranes



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  		Fig. 4.   Ouabain inhibition of Na-K-ATPase activity in cardiac membranes from rats of C () and FO (open circle ) groups. Data (n = 4 in each group) were analyzed by 2-way ANOVA and showed a statistically significant effect of ouabain concentration [F(8,108) = 50.9, P < 0.001] and diet [F(1,108) = 14.3, P < 0.001] as well as an interaction effect between ouabain concentration and diet [F(8,108) = 2.7, P < 0.01]. Post-ANOVA comparisons (Tukey's test) showed that Na-K-ATPase activity in C group was not significantly different from that in FO group when ouabain concentration was <= 10-5 M. Conversely, Na-K-ATPase activity was significantly higher in FO group than in C group when ouabain concentration was >= 3 × 10-5 M. Two lines represent theoretical curves with assumption of a 2-site model fit as described in METHODS. Estimated IC50 and contributions of low- and high-affinity Na-K-ATPase isoforms are reported in Table 3. Values are means ± SE of average of 12 determinations. * P < 0.05.



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  		Fig. 5.   Immunoblot analysis of alpha 1- and alpha 2-subunits of Na-K-ATPase in cardiac membranes from C rats and rats fed a diet supplemented with FO. Samples of microsomal cardiac membranes were electrophoresed on 4-15% polyacrylamide gradient gel, blotted onto nitrocellulose, and probed with isoform-specific anti-rat alpha 1- and alpha 2-antibodies. Subunits were detected by enhanced chemiluminescence method. Lane 1, prestained SDS-PAGE standards from Amersham used to localize 100-kDa isoform between beta -galactosidase (116 kDa) and phosphorylase B (97.4 kDa); lane 2, C group; lane 3, FO group. alpha 1-Isoform was detected with 10 µg of protein and alpha 2-isoform with 100 µg of protein. Antiserum dilutions were 1:250 for alpha 1-subunit and 1:20 for alpha 2-subunit.



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  		Fig. 6.   Densitometric analysis of autoradiograms for Na-K-ATPase alpha 1-subunit (A) and alpha 2-subunit (B). Values are means ± SD (n = 4).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our objective was to compare a group of hearts with incorporation of n-3 PUFA with a group without n-3 PUFA incorporation. We choose a 2-mo-duration diet, inasmuch as most of the studies have been done with this dosage, which corresponds to a plateau incorporation (2, 32) in membranes of most tissues. The effect of the diet was demonstrated by the specific incorporation of EPA and DHA into the cardiac membranes of treated animals compared with control animals. Furthermore, the baseline characteristics of the two groups, such as the body weight and the functional characteristics, were very similar (Table 1).

In this study, dietary supplementation with FO significantly promoted the positive inotropic response to ouabain without contracture. During ouabain intoxication, this supplementation delayed the alteration of contractility (dP) and energy metabolism. This dietary effect was expressed in the heart by incorporation of the n-3 fatty acids in the phospholipids from cell membranes, as previously evidenced in the rat heart (37), and by a lower ouabain affinity of the alpha 1-isoform of Na-K-ATPase, as previously shown in the rat diabetic heart (9). Dietary FO also increased cytosolic PCr levels (P < 0.05) but did not change ATP and pHi, protein expression of Na-K-ATPase, overall Na-K-ATPase activity, contribution of alpha 1- and alpha 2-isozymes, and affinity of alpha 2-isozymes. These findings on a combined use of two well-studied therapies, digitalis and FO supplements, to promote the contractile force in a whole heart have not been previously reported.

Ouabain and digoxin are cardiotonic drugs used in clinics primarily to produce a positive inotropic effect for the treatment of congestive heart failure. Unfortunately, cardiac toxicity limits their full inotropic use. We combined a dietary FO with digitalis to enhance ouabain contractility without toxicity. Antiarrhythmic effects of n-3 PUFAs have been well documented through their direct effects on Na+, Ca2+, and K+ channels (16, 23). A lower incidence of ventricular fibrillation after ischemic and reperfusion stress has been shown in isolated hearts from adult rats fed a diet rich in PUFA (28, 29). Such n-3 fatty acid protective effects against cardiac signs of toxicity could be extended to the present study. However, in the present experiments, the potentiation of positive inotropy was associated with delayed signs of ouabain toxicity, and the same type of arrhythmia and similar levels of contracture occurred at the end of toxic ouabain perfusion.

An increased energetic metabolism subsequent to inotropic effects could be deleterious. Thus an increase in contractile force could be associated with a change in the production and utilization of high-energy phosphates. The changes in PCr levels with an increasing ouabain dose in both groups were consistent with previous studies in which high concentrations of ouabain were associated with an inotropic effect in the rat (19, 25) and can be attributed to changes in heart work and intracellular Ca2+ levels. In contrast, changes in PCr at 10-4 M ouabain were not significantly different in both groups, although inotropy was nearly twofold higher in FO-treated than in control animals. Thus an improved myocardial efficiency after n-3 PUFA incorporation could be evidenced, since the work of the heart was improved. The severity of cardiac injury has been shown to be influenced by the magnitude of ATP reduction during Na-K-ATPase inhibition by ouabain (30). Inasmuch as the reduction in high-energy phosphorylated metabolites between the two groups after exposure to 3 × 10-4 M ouabain differed, this can be related to delayed cardiac injury after FO supplementation in the present study.

Inasmuch as ATPase activity and fatty acid membrane composition have been studied with microsomes and not with purified sarcolemmal membranes, we cannot establish a clear correlation between the activity of the enzyme and its microenvironment in the membrane. However, the aim of the present study was to assess the effect of dietary FO supplementation on membrane expression of Na-K-ATPase isozymes. Indeed, the basis for the improved ouabain efficacy after a dietary PUFA supplementation could be related to effects on Na-K-ATPase as a digitalis receptor. A particular biphasic inotropic response to ouabain was related in the rat to specific and consecutive inhibition of the two Na-K-ATPase isoforms. Ouabain recognized one high- and one low-affinity site in the membrane preparation in the two groups. The alpha 2-isozyme of high affinity (IC50 ~0.3 µM ouabain) was not modified by the FO treatment (Fig. 4), and its related positive inotropy was not modified by the FO treatment (Table 1). On the other hand, the positive inotropy related to the low-affinity site was higher with than without FO treatment. To promote ouabain contractility associated with the low-affinity component, the incorporation of n-3 fatty acids into membranes induced by dietary FO should have induced more inhibition of the alpha 1-isoform of Na-K-ATPase (35). A direct alteration of the capacity of the Na+ pump or isoform-specific transcription changes may be excluded, inasmuch as the overall Na-K-ATPase activity (Table 3) and alpha -subunit expression of the two isoforms are unchanged. Interestingly, a shift by a factor of 4.8 in ouabain affinity appears specific to the alpha 1-isozyme (IC50 ~340 µM ouabain) and is in agreement with our previous results obtained in rat brain supplemented with FO (11). The relationship between the drug-induced inhibition of Na-K-ATPase activity in the membrane and the inotropic effect of ouabain occurred over the same range of ouabain concentrations between 10-7 and 3 × 10-4 M. A correlation (r = 0.933, P < 0.05) between the extent of Na-K-ATPase inhibition and the increased positive inotropy in the control group supports the idea that cardiac Na-K-ATPase occupancy would be the primary target of ouabain (data not shown). However, a higher inotropic effect with ouabain in the FO group was associated with a paradoxically lower inhibition of Na-K-ATPase, which suggests an increased efficiency of ouabain in the FO group. The n-3 diet-dependent changes mediating the increased ouabain response must occur in other stages of ouabain-induced inotropy. FO may have modified the ouabain-induced increase in intracellular Na+ as a result of changes in the conformation of the enzyme or one or several pathways involved in Ca2+ homeostasis, such as Na+/Ca2+ exchange or sarcoplasmic Ca2+-ATPase (15). Interestingly, the n-3 fatty acids have been found also to interact with the cardiac Ca2+ channels and modify the inward Ca2+ fluxes (16). It may also be speculated that an increased Ca2+ influx during the systolic phase occurs with FO supplementation, which is similar to the effect of a combination of BAY K 8644 and digitalis (36).

Relevance of the rat model to humans. Rats have only two cardiac isozymes, whereas human hearts possess three isoforms (35), but the respective role of each human isoform in contractility and toxicity to digitalis remains undefined. Resistance to ouabain of the rat alpha 1-isoform results from a change in two amino acids in the H1-H2 transmembrane domain of alpha 1. It has been shown that mutation of the sheep alpha 1-isoform sensitive to ouabain, which is similar to the human alpha 1-isoform, confers the rat resistance to the alpha 1-isoform (26). The difference in ouabain sensitivity between rats and humans appears to be dependent only on these two mutations. Nevertheless, the responsiveness of the alpha 2-isozyme to ouabain is similar in humans and rats. However, the common point between this rat model and humans is the narrow therapeutic index: a specific dose corresponds to a significant inotropic effect, and a small increase in this dose will induce toxicity. The calculation of the therapeutic index in our rat heart model is 3 for the ratio of 40% inotropism to onset of toxic effect (3 × 10-4 M/10-4 M). Such a calculation in the human corresponds to a ratio of 2 (34); thus under these conditions the rat model is not far from the human model. Potentiation of the inotropic effect in the rat may be related to limited toxicity and increased inotropic-to-toxic ratio or therapeutic index. The effect of FO is to increase the therapeutic index essentially by decreasing the inotropic dose of ouabain; a similar effect could well be observed in humans with different ouabain concentrations.

We used a MaxEPA dose (0.5 g · kg-1 · day-1), which corresponds to a daily dose of 90 mg of EPA and is equivalent to 6.3 g in a human with a body weight of 70 kg. A review of most clinical trials with FO supplements shows that they have been conducted with daily doses of FO that vary from 1.5 to 6.7 g of EPA (24). In humans the follow-up of the dietary manipulation and the compliance with the FO supplementation are usually assessed by the fatty acid composition of red blood cells; to our knowledge, no data are available for human cardiac membranes. Leaf et al. (20), in a large clinical study (551 patients), used a daily dietary supplement of 8 g of n-3 fatty acids for 6 mo, which approaches the intake of Greenland Inuits. EPA and DHA in red blood cells increased from 0.5 to 4.9% and from 4.1 to 8.3%, respectively, after 6 mo of supplementation. These red blood cell increases in EPA and DHA were similar in proportion to those found in rat cardiac membranes after FO supplementation in our study.

Possible clinical implications. A potentiation of digitalis-induced stimulation of the force of contraction by n-3 PUFAs is of potential interest in the management of congestive heart failure. It is possible to increase membrane incorporation of n-3 PUFAs by oral administration to provide long-term clinical management for patients with hypertriglyceridemia (33). Serious adverse effects of potent inotropic agents in heart failure (vasoconstriction, increased heart rate, and development of deleterious arrhythmia) have never been related to the use of n-3 PUFAs in cardiovascular disease (21). Further studies are required to demonstrate a possible superiority of digoxin combined with n-3 PUFAs in the management of cardiac failure.

In conclusion, dietary supplementation with FO improves the ouabain efficiency on myocardial contractility in isolated hearts. Improving contractility induced by digitalis may represent an additional mechanism whereby FOs exert their cardioprotective action in rats and possibly in humans.


    ACKNOWLEDGEMENTS

This work was supported in part by French Ministry of Health Grants UF 1638 and UF 1673, Ministère de la Recherche et de la Technologie Grant 650994, and Centre National de la Recherche Scientifique Grants Unité Mixte de Recherche 6612 and Unité de Recherche Associée 1829 and by the Assistance Publique-Hôpitaux de Marseille.


    FOOTNOTES

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

Address for reprint requests and other correspondence: J. M. Maixent, Laboratoire de Biochimie, Faculté de Pharmacie, 27 Bld. J. Moulin, 13005 Marseille, France (E-mail: Hematim{at}pharmacie.univ-mrs.fr).

Received 22 February 1999; accepted in final form 23 July 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 277(6):H2290-H2297
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society



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