Programme Contents

Acute application of n-3 polyunsaturated fatty acids modify calcium sparks in permeabilised rat cardiac myocytes

Animal studies have demonstrated that acute administration of n-3 polyunsaturated fatty acids (PUFAs) prevent ischemia-induced arrhythmias (Billman, et al., 1997). During and following ischemia, the sarcoplasmic reticulum (SR) becomes overloaded with Ca²⁺ and spontaneous release events occur (Daniels et al., 1991). The subsequent rise in cytosolic [Ca²⁺] activates sarcolemmal current which can in turn produce after-depolarisations and arrhythmias. PUFAs can reduce the ionic currents responsible for the cardiac action potential and this is believed to be the mechanism for their cardio-protective effects (Xiao et al., 1997).

Studies on the effects of PUFAs on Ca²⁺ handling have shown that 10 μmol/l of eicosapentaenoic acid (EPA) resulted in a 15% reduction in the amplitude of spontaneous Ca²⁺ waves (Negretti et al., 2000). Also 15 μmol/l EPA was found to reduce both the width and duration of Ca²⁺ sparks by ∼25% (Honen et al., 2003). When PUFAs were applied directly to the SR Ca²⁺ release channel (Ryanodine receptor, RyR) in artificial bilayers, 30-50 μmol/l caused a 50-80% decrease channel activity. It is not clear if the action of PUFA's on cell Ca²⁺ handing is mediated primarily by the sarcolemma or SR.

This study aimed to determine if PUFAs could directly affect the Ca²⁺ release properties of the intact SR. This was done by measuring the properties of Ca²⁺ sparks in permeabilised cardiac myocytes in which sarcolemmal ion currents did not contribute to Ca²⁺ release within the cell.

Sprague-Dawley rats were anesthetized with sodium pentobarbitone (1ml/kg), the hearts were removed and the cardiac ventricular myocytes were isolated by enzymatic digestion. Following isolation, the myocytes were treated with saponin to permeablise the sarcolemma. Ca²⁺ sparks were viewed using confocal microscopy in line scan mode using the Ca²⁺ indicator fluo-3. Fatty acids tested were oleic acid (OA), arachidonic acid (AA), EPA and docosahexaenoic acid (DHA). Images of Ca²⁺ sparks were collected prior to the addition of fatty acids and at 2 min and 5 min following their addition. Sham experiments were performed to ensure SR Ca²⁺ rundown did not occur.

Spark properties did not vary during experiments in both sham and OA (mono unsaturated fatty acid) treated cells indicating that rundown did not occur. However, PUFA's did affect some spark properties. AA at 50 μmol/l, significantly reduced (10%) spark width within 2 min of exposure. EPA at 50 μmol/l significantly reduced spark intensity (21%) within 5 min. Exposure to 50 μmol/l DHA for 2 min reduced intensity by ∼25% and spark mean rate of rise by ∼20%. Following exposure for 5 min, spark frequency reduced by ∼30% and spark width reduced by ∼7%. Even at 30 μmol/l DHA was observed to significantly alter spark properties within 2 min.

The actions of fatty acids on Ca²⁺ sparks in this study were similar to those seen on the open probability of RyRs (Honen et al., 2003). During ischemia, fatty acids are released within the cell by PLA₂. Fatty acid concentrations (all species) up to 0.73 mmol/l have been measured in rat aortic plasma during transient ischemia (Chen et al., 2001). Previously we have shown that 10-20% of membrane fatty acids can release n-3 PUFAs and so it is quite possible for free PUFA levels to reach 70 μmol/l. Therefore under physiological ischemic conditions it is likely that PUFAs could play an important role in protecting myocardium from ischemia by modulating Ca²⁺ handling.

Billman, G.E., Kang, J.X. & Leaf, A. (1997) Lipids 32, 1161-1168.

Chen, L.G., Nohara, R., Hirai, T., Li, X.H., Kataoka, K., Hosokawa, R., Masuda, D., Fujita, M., Taguchi, S. & Sasayama, S. (2001) Japanese Circulation Journal 65, 550-555.

Daniels, M.C.G., Fedida, D., Lamont, C. & ter Keurs, H.E. (1991) Circulation Research 68, 1408-1421.

Honen, B.N., Saint, D.A. & Laver D.R. (2003) Journal of Membrane Biology 196, 95-103.

Negretti, N., Perez, M.R., Walker, D. & O'Neill, S.C. (2000) Joournal of Physiology 523, 367-375.

Xiao, Y.F., Gomez, A.M., Morgan, J.P., Lederer, W.J. & Leaf, A. (1997) Proceedings of the National Academy of Science USA 94, 4182-4187.