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CARDIOVASCULAR |
1 Department of Medicine, University of Manchester, Core Technology Facility, Grafton Street, Manchester M13 9NT, UK
2 Inst. de Medicina Experimental, Facultad de Medicina, University Central de Venezuela, Caracas, Ap. 50587 Venezuela
3 Centro Nacional de Diálisis y Transplante Renal, Hospital Universitario de Caracas, Venezuela
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Abstract |
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(Received 19 March 2007;
accepted after revision 10 May 2007;
first published online 17 May 2007)
Corresponding author S. C. O'Neill: Department of Medicine, University of Manchester, Core Technology Facility, Grafton Street, Manchester M13 9PT, UK. Email: mdssssco{at}manchester.ac.uk
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Introduction |
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n–3 PUFAs ought also to have effects via intracellular signalling pathways that can alter cardiac contractility and affect diastolic function in ischaemic hearts. Many protein kinases are inhibited by n–3 PUFAs, e.g. calmodulin-dependent protein kinase (CaM kinase II), protein kinase C (PKC), MAPK as well as protein kinase A (PKA) (Mirnikjoo et al. 2001). However, the case with PKA is complicated as phosphodiesterase is also inhibited, allowing cAMP levels to rise (Picq et al. 1996). Given this, it is difficult to predict whether PKA will be activated or inhibited. In the context of myocardial ischaemia, however, the contractile function of the heart is compromised, leading to diastolic dysfunction. Any changes in PKA activity, and e.g. phospholamban phosphorylation, would clearly be of importance to the degree of diastolic dysfunction.
Here, we report experiments that show activation of the PKA pathway by application of the n–3 PUFA EPA. We propose this may have two beneficial effects: (i) a positive lusitropic effect will help to reduce diastolic dysfunction during ischaemia, and (ii) a reduction of myofilament Ca2+ sensitivity may promote cell survival in ischaemia by preserving ATP.
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Methods |
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The bathing solution was as follows (mmol l–1): NaCl, 134; KCl, 4; Hepes, 10; glucose, 11; MgCl2, 1; CaCl2, 1; titrated to pH 7.4 with NaOH. Solutions with 50% Na+ content had 67 mM N-methyl-D-glucamine added to replace Na+. EPA was prepared in ethanol as a 33 mmol l–1 stock solution and stored under a N2 atmosphere for up to 1 week. Fatty-acid-free bovine serum albumin was added (2 mg ml–1) to the washout solution to ensure rapid and complete removal of fatty acid from the solution (Kang & Leaf, 1994; Kang et al. 1995). In voltage-clamp experiments, the holding potential was –40 mV and cells were depolarized for 200 ms to 0 mV at 0.5 Hz, the bathing solution contained 5 mmol l–1 4-aminopyridine and 0.1 mmol l–1 BaCl2 to inhibit outward currents. The use of H89 caused a substantial reduction in the L-type Ca2+ current, to retain a large enough current for analysis, we raised external Ca2+ to 3 mmol l–1.
Preparation of cardiac tissue for PKA assay and cAMP measurements
Langendorff hearts were perfused with the solution (mmol l–1): NaCl, 134; KCl, 4; Hepes, 10; glucose, 11; MgCl2, 1.2; CaCl2, 1; titrated to pH 7.4 with NaOH, used as control. Hearts were perfused with control solution, EPA (10 µmol l–1) or isoprenaline (1 µmol l–1) at 37°C. Left ventricular pressure was measured using a balloon in the left ventricle attached to a pressure transducer, the frequency was set at 4 Hz by stimulating electrodes in the left ventricular wall. After 20 min perfusion (sufficient for the positive lusitropic effect of EPA and isoprenaline to be observed) tissue was taken for PKA and cAMP determinations. (1) 1 g of heart tissue was homogenized in 5 ml of cold extraction buffer (25 mmol l–1 Tris HCl at pH 7.4, 0.5 mmol l–1 EDTA, 0.5 mmol l–1 EGTA, 10 mmol l–1
-mercaptoethanol, 1 µg ml–1 leupeptin, 1 µg ml–1 aprotinin, 200 mmol l–1 phenylmethylsulfonyl fluoride (PMSF)).The lysate was centrifuged for 5 min at 600 g (4°C) and the supernatant saved. The supernatant was taken and PKA activity was determined by a spectrophotometric assay (Roskoski, 1983). (2) Heart tissue was cut in fine pieces; weighed and 10 µl of 3-isobutyl-1-methylxanthine (IBMX; 100 µM) was added. Heart samples were frozen in liquid nitrogen. Frozen samples were ground to a fine powder and homogenized in 10 vol. 0.1 M HCl, centrifuged at 600 g and assayed immediately. cAMP was measured using a direct cyclic AMP enzyme immunoassay kit (Assay Designs). A standard cAMP curve was fitted using a four-parameter logistic equation (Sigmaplot 10 program).
Tissue preparation for Western blots
SR vesicles were isolated from tissue harvested as above for Western blot analysis (Korge & Campbell, 1995). SR preparation was lysed in 150 µl of ice-cold RIPA buffer (1x PBS, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS) with freshly added protease and phosphatase inhibitors (100 µg ml–1 PMSF, 10 µg ml–1 aprotinin, 2 mmol l–1 sodium orthovanadate), incubated on ice for 30 min and centrifuged at 15 000 g for 20 min at 4°C.
Total protein concentrations were measured by Bradford's method. Whole cell lysate (60 µg) was added to 10 µl of 5x electrophoresis sample buffer and separated by SDS/PAGE under reducing conditions on a 15% separation gel with a 4% stacking gel in a Miniprotean II camera (Bio-Rad).
Proteins were electrophoretically transferred to nitrocellulose membrane (16 h, 4°C). Unspecific binding was blocked by incubation in Blotto buffer (1xTBS, 5% nonfat milk and 0.05% Tween 20). Proteins in the membrane were then immunoblotted with antibodies Anti-Phospholamban Phospho-Specific (Ser16; Calbiochem), according to the manufacturer's protocols.
Blots were incubated in a chemiluminescence reagent (Pierce) according to data sheet and exposed to a K-Omat X-ray film (Kodak). The densities of the bands were evaluated using Quantity One software in a G-800 Densitometer (Bio-Rad).
All experiments in the UK were done at room temperature (25°C) and in accordance with the Animal Scientific Procedures Act (1986). All experiments in Venezuela were done according to guidelines of the Universidad Central de Venezuela. All statistics quoted are means ± S.E.M. Student's t tests (one- and two-tailed, where appropriate) were used to test significance.
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Results |
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The Western blot in Fig. 3B shows that phosphorylation of phospholamban at Ser16 in SR vesicles increases in EPA and in 1 µmol l–1 isoprenaline (positive control). In control, tissue band density was 7.4 ± 2.2 densitometric units µg–1 protein (n = 7); in EPA it was 32.8 ± 8.8 (n = 10, P < 0.01). In isoprenaline, the average density was 89.9 ± 12.5 (n = 3, P < 0.01).
We decided to determine and compare PKA activity in rat hearts in control and after perfusion with EPA once the lusitropic effect developed, using isoprenaline as a positive control in other hearts. A typical example of records obtained for PKA activity is shown in Fig. 3C; both EPA and isoprenaline increased the rate of fall of absorbance in the PKA assay. On average, EPA increased PKA activity by 21.0 ± 7.3% (P = 0.016; n = 11), and activity was increased in isoprenaline by 21.1 ± 9.1% (P = 0.042; n = 11). The control test showed an average activity for PKA of 244 ± 41.3 UI (mg protein)–1 (n = 11).
With such an increase of PKA activity we would expect there also to be an increase of cAMP therefore we set out to measure [cAMP] in the same tissue samples used in Fig. 3C. The values measured were 60.4 ± 7.9 pmol ml–1 (n = 14) in the control, 33.0 ± 5.0 pmol ml–1 (P < 0.01; n = 17) in EPA, and 90.9 ± 14.0 pmol ml–1 (P < 0.02; n = 10) in isoprenaline.
If EPA does indeed activate PKA we should be able to measure other effects of PKA, e.g. an increase of L-type Ca2+ current. Previous work has shown that EPA inhibits L-type Ca2+ channels (Xiao et al. 1997; Negretti et al. 2000) but if EPA also activates PKA leading to more L-type current, the extent of inhibition would appear reduced. In Fig. 4A we see inhibition of L-type current by 5 µmol l–1 EPA; current was reduced by 38.7 ± 3.9% (n = 18). Repeating this in 10 µmol l–1 H89, to inhibit PKA, current inhibition by EPA significantly increased to 63.3 ± 3.4% (n = 12, P < 0.00005). In H89 we raised external Ca2+ to 3 mmol l–1 to compensate for the reduction of L-type current (the current fell in H89 by 72.7 ± 0.8%, n = 2). This extra EPA-dependent inhibition in H89 indicates that activation of PKA by EPA, indeed, increases L-type Ca2+ current.
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Repeating this experiment in H89 to inhibit PKA (open and filled squares represent EPA and control, respectively, in H89) EPA no longer shifts the relationship (P > 0.09); we conclude therefore that the separation in the absence of H89 is due to phosphorylation of phospholamban by PKA. It is also worth noting that H89 alone causes a substantial shift of the curves to longer time constants of decay. This, in addition to the reduction of L-type Ca2+ current by H89, indicates that under normal conditions there is a resting level of PKA activity (also shown in Fig. 3B) that is inhibited by H89.
As mentioned before, under voltage-clamp EPA leads to a slower decay of the Ca2+ transient (Fig. 5A). The experiment in Fig. 6, however, shows, the cell still relaxes faster; in the upper panel, three averaged contractions are shown under voltage clamp. The amplitude is clearly reduced by application of EPA (5 µmol l–1); however, normalization shows that time to peak and relaxation are faster in EPA. Due to the complicated shape of contraction, we could not fit exponentials, so to quantify this effect of EPA we normalized contraction amplitude and calculated the area under the curve. Briefer contractions will have a smaller area, although the increased rate of relaxation will be underestimated due to the more rapid rate of rise of contraction in EPA. On average, this integral decreased to 78.2 ± 8.4% of control (P < 0.05, n = 6). This suggests an additional effect of EPA on the contractile filaments, as faster relaxation results in spite of a slower fall of Ca2+.
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Discussion |
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Throughout this study, EPA increases the rate of fall of the Ca2+ transient and relaxation of contraction (irrespective of the effect on Ca2+ transient and contraction amplitudes). In intact hearts this is associated with increased phosphorylation of phospholamban at the PKA-specific site and increased PKA activity. Is there any evidence that n–3 PUFAs activate PKA? In fact the literature suggests the opposite; in vitro n–3 PUFAs inhibit the PKA catalytic subunit (Mirnikjoo et al. 2001). However, there is also evidence that n–3 PUFAs inhibit phosphodiesterase (Dubois et al. 1993; Picq et al. 1996) and increase cAMP levels (Picq et al. 1996). Although this should overcome some direct PKA inhibition, it appears not to take place in our study where we find cAMP levels actually fall. However, our measurements show that 10 µmol l–1 EPA increases PKA activity (Fig. 3), so how can this be explained? Recently it has been shown that reactive oxygen species (ROS) can activate PKA by forming a disulphide bridge between cysteine groups linking regulatory subunits together, leading to activation of the kinase (Brennan et al. 2006). Of course, for this to be important here it is necessary that n–3 PUFAs lead to production of ROS and indeed this is the case (Maziere et al. 1999; Arita et al. 2001). Thus, perhaps by acting on mitochondrial respiration, ROS are formed, PKA is activated and the changes of contractile behaviour we report take place. ROS formation may also account for the fall of [cAMP] in EPA as it is known that adenylate cyclase is inhibited by ROS (Persad et al. 1998). Such a sequence of events remains to be confirmed experimentally.
The remaining experiments in this study sought to demonstrate a role for PKA in the positive lusitropic effect of EPA. Phospholamban is an important PKA target protein, and in Fig. 3B we show that EPA increased phospholamban phosphorylation at Ser16 (the PKA-specific site). This would contribute to the positive lusitropic effect of EPA we report.
Phosphorylation of the L-type Ca2+ channel by PKA increases its open probability (Brum et al. 1984) leading to more current. However, EPA also inhibits the L-type Ca2+ channel (Xiao et al. 1997; Macleod et al. 1998; Negretti et al. 2000). This inhibition might disguise the effect of PKA on current amplitude. Our approach to this has been to compare EPA-induced inhibition of L-type current in control and with PKA inhibited. Inhibition of PKA should leave only direct inhibition without any indirect increase of current due to phosphorylation. One problem with this is that H89 reduced L-type current (not shown) perhaps due to loss of baseline phosphorylation of L-type channels or direct inhibition of the channel as has been recently shown in rat myocytes (Bracken et al. 2006). To allow us to compare current inhibition, we raised bathing Ca2+ to 3 mmol l–1 (the inhibition of L-type current by EPA in 3 mmol l–1 Ca2+ is not significantly different from that in 1 mmol l–1 Ca2+; not shown). EPA inhibited about 40% of L-type current in control and over 60% with PKA inhibited (Fig. 4), indicating an increase of L-type current through PKA-dependent channel phosphorylation compensating for some of the direct inhibition by EPA.
One possible complication for this experiment is the selectivity of H89, as it also inhibits other kinases, e.g. CaM kinase II. However, even at the relatively high concentrations used here we would expect only minor inhibition of this enzyme (Chijiwa et al. 1990). Similarly, PK C is inhibited only slightly by 10 µmol l–1 H89 (Chijiwa et al. 1990). It seems fair therefore to conclude that the effects of EPA in H89 differ from those in the absence of H89 due to inhibition of PKA. This also seems fair in the light of the direct measurements of activation of PKA and PKA-dependent phosphorylation (Fig. 3) we also report.
The evidence so far suggests PKA is activated in the presence of EPA, why then, is the rate of decay of the Ca2+ transient reduced in voltage clamp (Fig. 5) and increased when the action potential stimulates contraction (Fig. 2)? One obvious difference is that the Ca2+ transient amplitude is reduced in voltage clamp, but increases in current clamp (cf. Figs 5 and 2). Underlying this are the electrophysiological effects of EPA. Under voltage clamp, the negative inotropic effect follows inhibition of L-type current whereas under current clamp the longer action potential leads to a larger Ca2+ transient. One consequence of a smaller Ca2+ transient would be reduced activation of CaM kinase II, as less activating Ca2+ would be bound to calmodulin. CaM kinase II also phosphorylates phospholamban increasing SERCA activity (Le Peuch et al. 1979). Here again, we have two counteracting effects: increased PKA-dependent phosphorylation of phospholamban and a probable decrease of CaM-kinase-II-dependent phosphorylation. In order to distinguish between these effects, we compared the rate of decay of the Ca2+ transient over a range of amplitudes. In this way we hoped to remove Ca2+ transient amplitude as a variable. In Fig. 5B, we see that in EPA the time constant of decay is smaller at all Ca2+ transient amplitudes, i.e. Ca2+ falls faster than in the control. This shows the effect of EPA on PKA activity without the confusing influence of altering Ca2+ transient amplitude. The second approach we made to this question was simply to inhibit PKA. As can also be seen in Fig. 5B, in H89 there is almost complete overlap between the points collected in the presence and absence of EPA. Again, a resting level of PKA-dependent phosphorylation is suggested by the upward shift of the curves in H89 in agreement with recent data in rat myocytes (Bracken et al. 2006). We conclude therefore that the lack of a positive lusitropic effect in EPA under voltage clamp, e.g. in Fig. 5A, results from the smaller amplitude of the Ca2+ transient under these conditions. As has been shown earlier (Schouten, 1990; Bassani et al. 1995), the smaller size of the transient reduces its rate of recovery, probably through the level of phosphorylation of phospholamban at the CaM-kinase-II-specific site, although CaM-kinase-II-independent mechanisms may also exist (Valverde et al. 2005).
The final observation we make here relates to myofilament Ca2+ sensitivity. EPA greatly reduces the amount of cell shortening for a given rise of intracellular Ca2+ (Fig. 8). This too, may result from activation of PKA as phosphorylation of troponin I lowers myofilament Ca2+ sensitivity (Zhang et al. 1995). Under physiological conditions this helps allow faster relaxation to accommodate higher frequencies during -adrenergic stimulation. Under ischaemic conditions, therefore, two effects of n–3 PUFAs, i.e. the faster relaxation we report here and the lower resting [Ca2+]i we have previously reported (O'Neill et al. 2002), could combine to combat diastolic dysfunction during myocardial ischaemia that may develop due, for example, to loss of ATP and consequent slowing of SERCA activity (Halow et al. 1999; Overend et al. 2001).
The benefit of a positive lusitropic effect
Several lines of evidence presented here either suggest (Figs 4, 5 and 7) or show directly (Fig. 3) that EPA causes an activation of PKA. The resulting phosphorylations lead to changes of the contractile properties of the cells that may benefit ischaemic myocardium. The rate of relaxation is increased in the presence of EPA in intact hearts (Fig. 3) and isolated myocytes (Fig. 1). Diastolic dysfunction is an early consequence of myocardial ischaemia, a positive lusitropic effect would benefit the heart by preserving pump function. In addition, reduced myofilament Ca2+ sensitivity would limit energy consumption thereby preserving ATP levels. This might enable the cell to survive a period of ischaemia with less damage. Evidence that reduced Ca2+ sensitivity of the myofilaments might, indeed, be beneficial comes from a study showing that an inhibitor of actin–myosin interaction, BDM, limits the infarct size following coronary artery occlusion (Garcia-Dorado et al. 1992). It is also worth noting that fish oil supplementation of the diet leads to less ischaemic damage following coronary artery ligation in rabbits (Ogita et al. 2003) and reduces the fraction of isolated rat ventricular myocytes that die during simulated ischaemia/reperfusion (Takeo et al. 1998).
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References |
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Bassani RA, Mattiazzi A & Bers DM (1995). CaMKII is responsible for activity-dependent acceleration of relaxation in rat ventricular myocytes. Am J Physiol Heart Circ Physiol 268, H703–H712.
Bracken N, Elkadri M, Hart G & Hussain M (2006). The role of constitutive PKA-mediated phosphorylation in the regulation of basal I (Ca2+) in isolated rat cardiac myocytes. Br J Pharmacol 148, 1108–1115.[CrossRef][Medline]
Brennan JP, Bardswell SC, Burgoyne JR, Fuller W, Schroder E, Wait R, Begum S, Kentish JC & Eaton P (2006). Oxidant-induced activation of type I protein kinase A is mediated by RI subunit interprotein disulfide bond formation. J Biol Chem 281, 21827–21836.
Brum G, Osterrieder W & Trautwein W (1984). -Adrenergic increase in the calcium conductance of cardiac myocytes studied with the patch clamp. Pflugers Arch 401, 111–118.[CrossRef][Medline]
Capogrossi MC, Houser SR, Bahinski A & Lakatta EG (1987). Synchronous occurrence of spontaneous localised calcium release from the sarcoplasmic reticulum generates action potentials in rat cardiac ventricular myocytes at normal resting membrane potential. Circ Res 61, 498–503.
Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Naito K, Toshioka T & Hidaka H (1990). Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem 265, 5267–5272.
Dubois M, Picq M, Nemoz G, Lagarde M & Prigent AF (1993). Inhibition of the different phosphodiesterase isoforms of rat heart cytosol by free fatty acids. J Cardiovasc Pharmacol 21, 522–529.[Medline]
Eisner DA, Nichols CG, O'Neill SC, Smith GL & Valdeolmillos M (1989). The effects of metabolic inhibition on intracellular calcium and pH in isolated rat ventricular cells. J Physiol 411, 393–418.
Ford DA, Hazen SL, Saffitz JE & Gross RW (1991). The rapid and reversible activation of a calcium-independent plasmalogen-selective phospholipase A2 during myocardial ischemia. J Clin Invest 88, 331–335.[Medline]
Garcia-Dorado D, Theroux P, Duran JM, Solares J, Alonso J, Sanz E, Munoz R, Elizaga J, Botas J & Fernandez-Aviles F (1992). Selective inhibition of the contractile apparatus. A new approach to modification of infarct size, infarct composition, and infarct geometry during coronary artery occlusion and reperfusion. Circulation 85, 1160–1174.
Halow JM, Figueredo VM, Shames DM, Camacho SA & Baker AJ (1999). Role of slowed Ca2+ transient decline in slowed relaxation during myocardial ischemia. J Mol Cell Cardiol 31, 1739–1748.[CrossRef][Medline]
Hazen SL, Ford DA & Gross RW (1991). Activation of a membrane-associated phospholipase A2 during rabbit myocardial ischemia which is highly selective for plasmalogen substrate. J Biol Chem 266, 5629–5633.
Horn R & Marty A (1988). Muscarinic activation of ionic currents measured by a new whole-cell recording method. J Gen Physiol 92, 145–159.
Kang JG & Leaf A (1994). Effects of long chain polyusaturated fatty acids on the contraction of neonatal rat cardiac myocytes. Proc Natl Acad Sci U S A 91, 9886–9890.
Kang JX, Xiao YF & Leaf A (1995). Free, long-chain polyunsaturated fatty acids reduce membrane electrical excitability in neonatal rat cardiac myocytes. Proc Natl Acad Sci U S A 92, 3997–4001.
Korge P & Campbell KB (1995). Regulation of calcium pump function in back inhibited vesicles by calcium-ATPase ligands. Cardiovasc Res 29, 512–519.[CrossRef][Medline]
Kris-Etherton PM, Harris WS & Appel LJ (2002). Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease. Circulation 106, 2747–2757.
Le Peuch CJ, Haiech J & Demaille JG (1979). Concerted regulation of cardiac sarcoplasmic reticulum calcium transport by cyclic adenosine monophosphate dependent and calcium–calmodulin-dependent phosphorylations. Biochemistry 18, 5150–5157.[CrossRef][Medline]
Macleod JC, MacKnight ADC & Rodrigo GC (1998). The electrical and mechanical response of adult guinea pig and rat ventricular myocytes to omega-3 polyunsaturated fatty acids. Eur J Pharmacol 356, 261–270.[CrossRef][Medline]
Maziere C, Conte MA, Degonville J, Ali D & Maziere JC (1999). Cellular enrichment with polyunsaturated fatty acids induces an oxidative stress and activates the transcription factors AP1 and NFkappaB. Biochem Biophys Res Commun 265, 116–122.[CrossRef][Medline]
McHowat J, Liu S & Creer MH (1998). Selective hydrolysis of plasmalogen phospholipids by Ca2+-independent PLA2 in hypoxic ventricular myocytes. Am J Physiol Cell Physiol 274, C1727–C1737.
Mirnikjoo B, Brown SE, Kim HF, Marangell LB, Sweatt JD & Weeber EJ (2001). Protein kinase inhibition by omega-3 fatty acids. J Biol Chem 276, 10888–10896.
Nair SS, Leitch J, Falconer J & Garg ML (1999). Cardiac (n–3) non-esterified fatty acids are selectively increased in fish oil-fed pigs following myocardial ischemia. J Nutr 129, 1518–1523.
Negretti N, Perez MR, Walker D & O'Neill SC (2000). Inhibition of sarcoplasmic reticulum function by polyunsaturated fatty acids in intact, isolated myocytes from rat ventricular muscle. J Physiol 523, 367–375.
O'Neill SC & Eisner DA (1990). A. mechanism for the effects of caffeine on Ca2+ release during diastole and systole in isolated rat ventricular myocytes. J Physiol 430, 519–536.
O'Neill SC, Perez MR, Hammond KE, Sheader EA & Negretti N (2002). Direct and indirect modulation of rat cardiac sarcoplasmic reticulum function by n–3 polyunsaturated fatty acids. J Physiol 538, 179–184.
Ogita H, Node K, Asanuma H, Sanada S, Takashima S, Minamino T, Soma M, Kim J, Hori M & Kitakaze M (2003). Eicosapentaenoic acid reduces myocardial injury induced by ischemia and reperfusion in rabbit hearts. J Cardiovasc Pharmacol 41, 964–969.[CrossRef][Medline]
Overend CL, Eisner DA & O'Neill SC (2001). Altered cardiac sarcoplasmic reticulum function of intact myocytes of rat ventricle during metabolic inhibition. Circ Res 88, 181–187.
Persad S, Panagia V & Dhalla NS (1998). Role of H2O2 in changing beta-adrenoceptor and adenylyl cyclase in ischemia-reperfused hearts. Mol Cell Biochem 186, 99–106.[CrossRef][Medline]
Picq M, Dubois M, Grynberg A, Lagarde M & Prigent AF (1996). Specific effects of n–3 fatty acids and 8-bromo-cGMP on the cyclic nucleotide phosphodiesterase activity in neonatal rat cardiac myocytes. J Mol Cell Cardiol 28, 2151–2161.[CrossRef][Medline]
Roskoski R Jr (1983). Assays of protein kinase. Methods Enzymol 99, 3–6.[Medline]
Schouten VJA (1990). Interval dependence of force and twitch duration in rat heart explained by Ca2+ pump inactivation in sarcoplasmic reticulum. J Physiol 431, 427–444.
Swan JS, Dibb KM, Negretti N, O'Neill SC & Sitsapesan R (2003). Effects of eicosapentaenoic acid on cardiac SR Ca2+-release and ryanodine receptor function. Cardiovasc Res 60, 337–346.
Takeo S, Nasa Y, Tanonaka K, Yabe K, Nojiri M, Hayashi M, Sasaki H, Ida K & Yanai K (1998). Effects of long-term treatment with eicosapentaenoic acid on the heart subjected to ischemia/reperfusion and hypoxia/reoxygenation in rats. Mol Cell Biochem 188, 199–208.[CrossRef][Medline]
Valverde CA, Mundina-Weilenmann C, Said M, Ferrero P, Vittone L, Salas M, Palomeque J, Vila PM & Mattiazzi A (2005). Frequency-dependent acceleration of relaxation in mammalian heart: a property not relying on phospholamban and SERCA2 a phosphorylation. J Physiol 562, 801–813.
Xiao YF, Gómez AM, Morgan JP, Lederer WJ & Leaf A (1997). Suppression of voltage-gated L-type Ca2+ currents by polyunsaturated fatty acids in adult and neonatal rat ventricular myocytes. Proc Natl Acad Sci U S A 94, 4182–4187.
Xiao YF, Kang JX, Morgan JP & Leaf A (1995). Blocking effects of polyunsaturated fatty acids on Na+ channels of neonatal rat ventricular myocytes. Proc Natl Acad Sci U S A 92, 11000–11004.
Xiao YF, Ke Q, Wang SY, Auktor K, Yang Y, Wang GK, Morgan JP & Leaf A (2001). Single point mutations affect fatty acid block of human myocardial sodium channel alpha subunit Na+ channels. Proc Natl Acad Sci U S A 98, 3606–3611.
Zhang R, Zhao J, Mandveno A & Potter JD (1995). Cardiac troponin I phosphorylation increases the rate of cardiac muscle relaxation. Circ Res 76, 1028–1035.
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).
), in EPA (10 µmol l–1, 
) and isoprenaline (1 µmol l–1, 
).
, The effect of EPA. The effect of EPA is lost when H89 (10 µmol l–1) is present (
, EPA; 
