Omega-3 fatty acids do not alter P-wave parameters in electrocardiogram or expression of atrial connexins in patients undergoing coronary artery bypass surgery.

AIMS: We previously reported omega-3 polyunsaturated fatty acids (n-3PUFAs) supplementation does not reduce atrial fibrillation (AF) following coronary artery bypass graft (CABG) surgery. The aim of the present study is to evaluate the impact of n-3 PUFAs on electrocardiogram (ECG) atrial arrhythmic markers and compare with expression of gap-junction proteins, Connexins. METHODS AND RESULTS: Subset of clinical trial subjects with right atrial sampling during CABG surgery included. Twelve-lead ECG performed at recruitment and at surgery [after supplementation with n-3 PUFA (∼1.8 g/day) or matched placebo] for ∼14 days. Electrocardiograms analysed for maximum P-wave duration (P-max) and difference between P-max and minimum P-wave duration, P-wave dispersion (PWD). Right atrial specimens analysed for expression of Connexins 40 and 43 using real-time quantitative polymerase chain reaction (qPCR) and western blot. Serum levels of n-3 PUFA at baseline, at surgery, and atrial tissue levels at surgery collated from file. Postoperative AF was quantified by analysing data from stored continuous electrograms. A total of 61 patients (n-3 PUFA 34, Placebo 27) had ECG analysis and AF burden, of which 52 patients (26 in each group) had qPCR and 16 (8 in each group) had western blot analyses for Connexins 40 and 43. No difference between the two groups in ECG parameters or expression of Connexin 40 or 43. P-wave dispersion in the preoperative ECG independently predicted occurrence of AF following CABG surgery. CONCLUSIONS: Omega-3 polyunsaturated fatty acids supplementation does not alter pro-arrhythmic P-wave parameters in ECG or connexin expression in human atrium with no effect on the incidence of AF following CABG surgery.

Mechanisms by which cytoplasmic calcium wave propagation and alternans are generated in cardiac atrial myocytes lacking T-tubules-insights from a simulation study.

This study investigated the mechanisms underlying the propagation of cytoplasmic calcium waves and the genesis of systolic Ca(2+) alternans in cardiac myocytes lacking transverse tubules (t-tubules). These correspond to atrial cells of either small mammals or large mammals that have lost their t-tubules due to disease-induced structural remodeling (e.g., atrial fibrillation). A mathematical model was developed for a cluster of ryanodine receptors distributed on the cross section of a cell that was divided into 13 elements with a spatial resolution of 2 µm. Due to the absence of t-tubules, L-type Ca(2+) channels were only located in the peripheral elements close to the cell-membrane surface and produced Ca(2+) signals that propagated toward central elements by triggering successive Ca(2+)-induced Ca(2+) release (CICR) via Ca(2+) diffusion between adjacent elements. Under control conditions, the Ca(2+) signals did not fully propagate to the central region of the cell. However, with modulation of several factors responsible for Ca(2+) handling, such as the L-type Ca(2+) channels (Ca(2+) influx), SERCA pumps (sarcoplasmic reticulum (SR) Ca(2+) uptake), and ryanodine receptors (SR Ca(2+) release), Ca(2+) wave propagation to the center of the cell could occur. These simulation results are consistent with previous experimental data from atrial cells of small mammals. The model further reveals that spatially functional heterogeneity in Ca(2+) diffusion within the cell produced a steep relationship between the SR Ca(2+) content and the cytoplasmic Ca(2+) concentration. This played an important role in the genesis of Ca(2+) alternans that were more obvious in central than in peripheral elements. Possible association between the occurrence of Ca(2+) alternans and the model parameters of Ca(2+) handling was comprehensively explored in a wide range of one- and two-parameter spaces. In addition, the model revealed a spontaneous second Ca(2+) release in response to a single voltage stimulus pulse with SR Ca(2+) overloading and augmented Ca(2+) influx. This study provides what to our knowledge are new insights into the genesis of Ca(2+) alternans and spontaneous second Ca(2+) release in cardiac myocytes that lack t-tubules.

Three-dimensional electron microscopic reconstruction of intracellular organellar arrangements in vascular smooth muscle--further evidence of nanospaces and contacts.

The sarcoplasmic reticulum (SR) of smooth muscle is crucial for appropriate regulation of Ca(2+) signalling. In visceral and vascular smooth muscles the SR is known to periodically lie in close register, within a few nanometres, to the plasma membrane. Recent work has focussed on reconstructions of the ultrastructural arrangement of this so-called peripheral SR that may be important for the genesis of phenomena such as Ca(2+) sparks. Here, we turn our attention to vascular smooth muscle and explore the 3-dimensional (3D) ultrastructural positioning of SR found deeper in the cell that is involved in the propagation of Ca(2+) waves. We use digital reconstruction and volume rendering of serial electron microscopic sections from isolated resistance arteries, pressurized in vitro to mimic cellular geometric conformations anticipated in vivo, to map SR positioning. We confirm that these central portions of SR are in close register with mitochondria and the nucleus with all three organelles tightly enveloped by a myofilament/cytoskeletal lattice. Nanospacings between the SR and individual mitochondria are visible and in three dimensions as the SR contorts to accommodate these organelles. Direct connection of the SR and nuclear membranes is confirmed. Such 3D positioning of centrally located SR further informs us of its likely role in the manifestation of spatiotemporal Ca(2+) dynamics: signal encoding may be facilitated by spatially directed release of Ca(2+) to influence several processes crucial to vascular smooth muscle and resistance artery function including myofilament activation by Ca(2+) waves, mitochondrial respiration and gene transcription.

The sarcoplasmic reticulum and arrhythmogenic calcium release.

There is much evidence showing that some lethal ventricular arrhythmias arise from waves of Ca(2+) release from the sarcoplasmic reticulum (SR) that propagate along cardiac cells. The purpose of this review is to discuss the mechanism of production of these waves and how they depend on the properties of the SR Ca(2+) release channel or ryanodine receptor (RyR). The best-known method of producing Ca(2+) waves is by increasing the Ca(2+) content of the cell by either increasing Ca(2+) influx or decreasing efflux. Once SR Ca(2+) content reaches a threshold level a Ca(2+) wave is produced. Altering the properties of the RyR affects the threshold level of Ca(2+) required to produce a wave. Patients with a mutation in the RyR suffer from catecholaminergic polymorphic ventricular tachycardia, and this may be due to a decrease in the SR Ca(2+) threshold for wave production. Heart failure has also been suggested to result in Ca(2+) waves due to a leak of Ca(2+) through the RyR. We review the finding that these changes in RyR function will only result in Ca(2+) waves in the steady state if some other mechanism maintains the SR Ca(2+) content. The review concludes with a description of potential mechanisms for treating arrhythmias produced by Ca(2+) waves.

Caveolae and sarcoplasmic reticular coupling in smooth muscle cells of pressurised arteries: the relevance for Ca2+ oscillations and tone.

OBJECTIVE: A close association of caveolae and sarcoplasmic reticulum (SR) has been suggested to be important for contractile activation of smooth muscle. Here, we investigate the presence of such arrangements in pressurised resistance arteries and examine the influence of two agents purported to disrupt caveolae and/or SR conformations by different mechanisms of action. METHODS: Rat mesenteric small arteries (RMSA) were mounted on a pressure myograph and the functional (lumen diameter and Ca(2+) oscillations) and ultrastructural effects of the phosphatase inhibitor calyculin-A (cal-A), or the cholesterol binding agent methyl-beta-cyclodextrin (mbetacd), examined by light and electron microscopy. RESULTS: Smooth muscle cells of RMSA exhibited a prominent peripheral SR that often encircled individual caveolae. The peripheral SR on occasion was observed to make contact with centrally located SR allowing for a structural association of caveoale-SR-myofilaments. Cal-A maximally constricted RMSA and disrupted the regular SR-caveolae appearance such that concentrated swirls of SR not enveloping caveolae were evident. Mbetacd treatment, in contrast, inhibited agonist contractility and reduced the appearance of caveolae whilst peripheral SR apposition to the plasmalemma could still be observed. Treatment with either agent inhibited agonist-mediated smooth muscle Ca(2+) oscillations. CONCLUSION: We present data that supports a structural arrangement of caveolae and underlying peripheral SR in smooth muscle cells of pressurised resistance arteries that serves to regulate Ca(2+) oscillations and contractile activation.

Sarcoplasmic reticulum calcium content fluctuation is the key to cardiac alternans.

The aim of this work was to investigate whether beat-to-beat alternation in the amplitude of the systolic Ca(2+) transient (Ca(2+) alternans) is due to changes of sarcoplasmic reticulum (SR) Ca(2+) content, and if so, whether the alternans arises due to a change in the gain of the feedback controlling SR Ca(2+) content. We found that, in rat ventricular myocytes, stimulating with small (20 mV) depolarizing pulses produced alternans of the amplitude of the Ca(2+) transient. Confocal measurements showed that the larger transients resulted from propagation of Ca(2+) waves. SR Ca(2+) content (measured from caffeine-evoked membrane currents) alternated in phase with the alternans of Ca(2+) transient amplitude. After a large transient, if SR Ca(2+) content was elevated by brief exposure of the cell to a Na(+)-free solution, then the alternans was interrupted and the next transient was also large. This shows that changes of SR Ca(2+) content are sufficient to produce alternans. The dependence of Ca(2+) transient amplitude on SR content was steeper under alternating than under control conditions. During alternation, the Ca(2+) efflux from the cell was also a steeper function of SR Ca(2+) content than under control. We attribute these steeper relationships to the fact that the larger responses in alternans depend on wave propagation and that wave propagation is a steep function of SR Ca(2+) content. In conclusion, alternans of systolic Ca(2+) appears to depend on alternation of SR Ca(2+) content. This, in turn results from the steep dependence on SR Ca(2+) content of Ca(2+) release and therefore Ca(2+) efflux from the cell as a consequence of wave propagation.

Omega-3 fatty acid supplementation does not reduce risk of atrial fibrillation after coronary artery bypass surgery: a randomized, double-blind, placebo-controlled clinical trial.

BACKGROUND: Omega-3 polyunsaturated fatty acids (n-3 PUFA) have been reported to reduce the risk of sudden cardiac death presumed to be due to fatal ventricular arrhythmias, but their effect on atrial arrhythmias is unclear. METHODS AND RESULTS: Patients (n=108) undergoing coronary artery bypass graft surgery were randomly assigned to receive 2 g/d n-3 PUFA or placebo (olive oil) for at least 5 days before surgery (median, 16 days; range, 12 to 21 days). Phospholipid n-3 PUFA were measured in serum at study entry and at surgery and in right atrial appendage tissue at surgery. Echocardiography was used to assess left ventricular function and left atrial dimensions. Postoperative continuous ECG monitoring (Lifecard CF) for 5 days or until discharge, if earlier, was performed with a daily 12-lead ECG and clinical review if patients remained in the hospital beyond 5 days. Lifecard recordings were analyzed for episodes of atrial fibrillation (AF) > or =30 seconds (primary outcome). Clinical AF, AF burden (% time in AF), hospital stay, and intensive care/high dependency care stay were measured as secondary outcomes. One hundred three patients completed the study (51 in the placebo group and 52 in the n-3 PUFA group). There were no clinically relevant differences in baseline characteristics between groups. n-3 PUFA levels were higher in serum and right atrial tissue in the active treatment group. There was no significant difference between groups in the primary outcome of AF (95% confidence interval [CI], -6% to 30%, P=0.28) in any of the secondary outcomes or in AF-free survival. CONCLUSIONS: Omega-3 PUFA do not reduce the risk of AF after coronary artery bypass graft surgery. Clinical Trial Registration- www.ukcrn.org.uk. Identifier: 4437.

Protein kinase A is activated by the n-3 polyunsaturated fatty acid eicosapentaenoic acid in rat ventricular muscle.

During cardiac ischaemia antiarrhythmic n-3 polyunsaturated fatty acids (PUFAs) are released following activation of phospholipase A2, if they are in the diet prior to ischaemia. Here we show a positive lusitropic effect of one such PUFA, eicosapentaenoic acid (EPA) in the antiarrhythmic concentration range in Langendorff hearts and isolated rat ventricular myocytes due to activation of protein kinase A (PKA). Several different approaches indicated activation of PKA by EPA (5-10 micromol l(-1)): the time constant of decay of the systolic Ca2+ transient decreased to 65.3 +/- 5.0% of control, Western blot analysis showed a fourfold increase in phospholamban phosphorylation, and PKA activity increased by 21.0 +/- 7.3%. In addition myofilament Ca2+ sensitivity was reduced in EPA; this too may have resulted from PKA activation. We also found that EPA inhibited L-type Ca2+ current by 38.7 +/- 3.9% but this increased to 63.3 +/- 3.4% in 10 micromol l(-1) H89 (to inhibit PKA), providing further evidence of activation of PKA by EPA. PKA inhibition also prevented the lusitropic effect of EPA on the systolic Ca2+ transient and contraction. Our measurements show, however, PKA activation in EPA cannot be explained by increased cAMP levels and alternative mechanisms for PKA activation are discussed. The combined lusitropic effect and inhibition of contraction by EPA may, respectively, combat diastolic dysfunction in ischaemic cardiac muscle and promote cell survival by preserving ATP. This is a further level of protection for the heart in addition to the well-documented antiarrhythmic qualities of these fatty acids.