Differential Effects of Various Eicosanoids on the Production or Prevention of Arrhythmias in Cultured Neonatal Rat Cardiac Myocytes

doi:10.1016/S0090-6980(97)00122-6

Prostaglandins

Volume 54, Issue 2, August 1997, Pages 511-530

Prostaglandins

https://doi.org/10.1016/S0090-6980(97)00122-6 Get rights and content

Abstract

To identify the arrhythmogenic and the antiarrhythmic eicosanoids, cultured, spontaneously beating, neonatal rat cardiac myocytes were used to examine the effects of various eicosanoids added to the medium superfusing the cells at different concentrations on the contraction of the myocytes. Superfusion of the myocytes with the prostaglandins (PGD₂, PGE₂, PGF_2α) or the thromboxane (TXA₂)-mimetic, U 46619, induced reversible tacharrhythmias characterized by an increased beating rate, chaotic activity and contractures. These effects are concentration-dependent. PGF_2α and U 46619 were much more potent than PGD₂ or PGE₂ in the production of tachyarrhythmias. Prostacyclin (PGI₂) induced a marked reduction in the contraction rate of the cells with a slight increase in the amplitude of the contractions and showed a protective effect against the arrhythmias induced by PGF_2α and TXA₂ (U 46619). PGE₁ exerted a dose-dependent dual effect on the contraction of the myocytes. At low concentrations (<2 μM), PGE₁ reduced the contraction rate of the cells with an increase in the amplitude of the contractions and effectively terminated the tachyarrhythmias induced by arrhythmogenic agents, such as isoproterenol, ouabain and U 46619. At higher concentrations (>5 μM), PGE₁ caused cell contractures and chaotic activity. In contrast, the lipoxygenase products [leukotriene (LT) B₄, LTC₄, LTD₄

LTE₄] of arachidonic acid (AA) had no significant effect on the myocyte contractions.

The eicosanoids derived from eicosapentaenoic acid (EPA), including both the cyclooxygenase products (PGD₃, PGE₃, PGF_3α, TXB₃) showed lesser effects on the contraction of the myocytes. The lipoxygenase products (LTB₅, LTC₅, LTD₅ & LTE₅), as with the AA metabolites showed little effect on the contraction of cardiac myocytes. The arrhythmias induced by the arrhythmogenic prostaglandins and thromboxane A₂ could be suppressed by the nonmetabolizable AA analog eicosatetraynoic acid (ETYA) or free AA and EPA, indicating a distinction in the effect on cardiac arrhythmia between the precursor fatty acids (AA & EPA) themselves and their metabolites. In conclusion, the major arrhythmogenic eicosanoids are the cycloxygenase products of AA, whereas those products of EPA are much less or not effective; PGE₁, PGI₂, ETYA and EPA have antiarrhythmic effects.

Introduction

The polyunsaturated fatty acids (PUFAs), arachidonic acid (C20:4n-6; AA) and eicosapentaenoic acid (C22:5n-3; EPA), are metabolized through cyclooxygenase and lypoxygenase pathways to form eicosanoids, including prostaglandins (PGs), thromboxanes (TXs), prostacyclins (PGIs) and leukotrienes (LTs). These eicosanoids are known to have a variety of biological effects. A number of AA metabolites have been implicated in cardiac arrhythmias 1, 2, 3, 4, 5, 6, 7, 8. For example, thromboxane A₂ has been reported to be arrhthmogenic, and prostacyclin (PGI₂) has a protective effect against cardiac myocytes together with inhibitors of the cyclooxygenase and lypoxygenase, exhibited an antiarrhythmic effect, but when added without the inhibitors AA induced arrhythmias in some cases, suggesting that certain AA metabolites are arrhythmogenic. Unlike AA, EPA alone always showed an antiarrhythmic effect, indicating that EPA metabolites, if formed, may be much less or not arrhythmogenic, or may even be antiarrhythmic. Although an association of eicosanoids with cardiac arrhythmias has been reported 1, 2, 3, 4, 5, 6, 7, 8, some basic concerns as to the primary effect of each eicosanoids on the contraction of cardiac myocytes, the differences in the effects of AA and EPA metabolites, their relation to arrhythmias (arrhythmogenic or antiarrhythmic) and the relative potencies of their action have not been defined. Thus, a study designed to comprehensively assess, in a defined system, the effects of each of the major cyclooxygenase and lypoxygenase metabolites of AA and EPA on the contraction of cardiac myocytes and to address their differential effects and relative potency may provide important insight into the relationship between eicosanoids and cardiac arrhythmias.

The isolated, cultured, neonatal rat cardiac myocytes which grow as multicellular syncytia with cell-to-cell communication and beat spontaneously, synchronously and rhythmically provide an appropriate preparation for assessing the arrhythmogenic or antiarrhythmic effects of an agent 9, 10, 11, 12, 13. Cultured cells can be placed in direct, uniform contact with a defined concentration of specific agents without confounding effects of possible neurogenic or circulating humoral factors. Moreover, cultured myocytes have been shown to develop arrhythmias similar to that in the in vivo heart [13]. Using this preparation, we have examined the effects of different concentrations of individual metabolites of AA or EPA added to the medium superfusing the cells on their ability to produce or prevent tachyarrhythmias. We will use the descriptive term “tachyarrhythmia” to describe the increased beating rate, chaotic, asynchronous activity and contractures of the myocytes observed in this study. Here we report the differential effects of these eicosanoids and their relative potencies in this preparation.

Section snippets

Cell Isolation and Culture

Cardiac myocytes were isolated from 1-day-old neonatal Sprague-Dawley rats using the Neonatal Cardiomyocyte Isolation System (Worthington Biochemical Corp, New Jersey). The aortic root and atria were physically removed before isolation. The isolated cells were placed on petri dishes with culture medium (F-10 Nutrient Mixture [Ham], 10% horse serum, 5% fetal bovine serum, 50 μg/ml streptomycin and 50 units/ml penicillin G) and cultured at 37°C in air with 5% CO₂ added and 98% relative humidity

Materials

Neonatal Cardiomyocyte Isolation System Kit was purchased from Worthington Biochemical Corp., New Jersey. Eicosanoids were obtained from Cayman Chemical Company, Ann Arbor, MI. Fatty acids were obtained from Sigma Chemical Co., St Louis, Eicosanoids and fatty acids were dissolved in ethanol and stored under a nitrogen atmosphere at −70°C. The final concentration of ethanol was 0.1% or less and had no effect on myocyte contractions.

Statistics: Statistical methods used were the Student “t” test

Effects of Cyclooxygenase Metabolites of AA and of EPA on Contractions of Myocytes

Table 1 summarizes the results on all of the cyclooxygenase metabolites tested. The major AA cyclooxygenase metabolites including PGD₂, PGE₂, PGF_2α, U46619 (a TXA₂ mimetic) and PGI₂ have been tested at different concentrations (0.01 μM – 10 μM). Perfusion of the myocytes with low concentrations of PGD₂, PGE₂, PGF_2α or U46619 quickly (1–2 minutes) produced tachyarrhythmias, characterized by a regular fast rhythm (at least >25% of the control) with a reduction in the contraction amplitude (50–70

Discussion

With a defined system, we have characterized the contractile responses of cardiac myocytes to various exogenous eicosanoids. The results reveal distinct effects on the myocyte contraction between prostaglandins and leukotrienes, between AA metabolites and EPA metabolites, and between the precursor fatty acids (AA & EPA) and their products (eicosanoids).

Although the involvement of eicosanoids in cardiac arrhythmias has been recognized, attention has been paid mainly to thromboxane A₂ (TXA₂) and

Acknowledgements

This study was supported by a NIH grant from National Institute of Diabetes and Digestive and Kidney Diseases (R01-DK 38165). We are grateful to Amy Howard for her assistance with the cell culture.

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Differential Effects of Various Eicosanoids on the Production or Prevention of Arrhythmias in Cultured Neonatal Rat Cardiac Myocytes

Abstract

Introduction

Section snippets

Cell Isolation and Culture

Materials

Effects of Cyclooxygenase Metabolites of AA and of EPA on Contractions of Myocytes

Discussion

Acknowledgements

Prog. Lipid Res.

J. Mol. Cell Cardiol.

Life Science

Prostaglandins

Biochem. Biophy. Res. Comm.

Europ. J. Pharmacol.

J. Mol. Cell Cardiol.

Prog. Lipid Res.

Europ. J. Pharmacol.

Europ. J. Pharmacol.

Eicosanoids and cardiac arrhythmias

Biomed. Biochim. Acta.

Endogenous chemical mediators of ventricular arrhythiias in ischemic heart disease

Cardiovasc. Res.