Proc. Natl. Acad. Sci. USA Vol. 94, pp. 2724–2728, March 1997 Pharmacology Regulation of sodium channel gene expression by class I antiarrhythmic drugs and n 2 3 polyunsaturated fatty acids in cultured neonatal rat cardiac myocytes JING X. KANG*, YUNYUAN LI*, AND ALEXANDER LEAF*†‡ *Department of Medicine, Harvard Medical School and Massachusetts General Hospital, Boston, MA 02114; and †BrocktonyWest Roxbury Veterans Affairs Medical Center, West Roxbury, MA 02132 Contributed by Alexander Leaf, December 30, 1996 ABSTRACT Previous studies have shown that chronic ministration of the class I antiarrhythmic drugs are not fully administration of class I antiarrhythmic drugs, which have understood, one possible explanation comes from the findings definite inhibitory action on the fast Na1 channel, result in reported from the laboratories of Catterall and Duff (2, 3). up-regulation of cardiac Na1 channel expression, and suggest They found that chronic treatment in rats with the class I that this effect may contribute to their deleterious effects antiarrhythmic drug mexiletine resulted in upregulation of during chronic administration. Recent studies have shown cardiac Na1 channel expression, as shown by increase in both that the antiarrhythmic effects of free n 2 3 polyunsaturated the level of mRNA encoding sodium channel a-subunits and fatty acids (PUFA) are associated with an inhibition of the the number of sodium channels per cell. It was proposed that Na1 channel. Whether the PUFA when used chronically will the increased number of sodium channels caused by chronic mimic the effect of the class I drugs on the expression of the treatment with these drugs may itself cause arrhythmias as a Na1 channel is not known. To answer this question, we secondary consequence of therapy (2, 3). These previous determined the level of mRNA encoding cardiac Na1 channels observations indicate the importance of careful reevaluation of and the number of the Na1 channels per cell in cultured the safety of other forms of antiarrhythmic agents and the need neonatal rat cardiac myocytes after supplementation of the of development of a safe and highly effective means of cells with the n 2 3 PUFA eicosapentaenoic acid (EPA), the preventing lethal arrhythmias. class I drug mexiletine, or both EPA and mexiletine for 3–4 Recent studies have shown a role for n 2 3 polyunsaturated days. The number of sodium channels was assessed with a fatty acids (PUFA) in the prevention of fatal ventricular radioligand binding assay using the sodium channel-specific arrhythmias (for review, see ref. 4). We have found that the 3 3 toxin [ H]batrachotoxinin benzoate ([ H]BTXB). The sup- antiarrhythmic effects of the fatty acids are associated with plementation of myocytes with mexiletine (20 mM) induced a their inhibitory action on cardiac excitability automaticity, an 3 y 4-fold increase in [ H]BTXB specific binding to the cells. In important factor influencing the generation and the termina- contrast, chronic treatment with EPA (20 mM) alone did not tion of arrhythmias (5–8). Our electrophysiological study 3 significantly affect [ H]BTXB binding. However, the combi- shows that free PUFA significantly increase the threshold for nation of EPA with mexiletine produced a 40–50% reduction 1 3 gating the fast Na channel (which initiates the action poten- in the [ H]BTXB binding, compared with that seen with tial), hyperpolarizes the resting or diastolic membrane poten- mexiletine alone. RNA isolated from cardiac myocytes was tial, and prolongs the refractory period duration in rat cardiac probed with a 2.5-kb cRNA transcribed with T7 RNA poly- myocytes (8). The increase in threshold for the gating of the merase from the clone Na-8.4, which encodes nucleotides fast Na1 channel indicates that Na1 currents through this 3361–5868 of the -subunit of the R sodium channel a IIA channel are modulated by the PUFA. This has been confirmed subtype. The changes in the level of mRNA encoding sodium by our recent study showing that n 2 3 PUFA inhibited Na1 channel a-subunit were correlated with comparable changes currents in a dose-, time-, and voltage-dependent manner (9). in sodium channel number in the cultured myocytes, indicat- Since sodium channel blockers (i.e., class I antiarrhythmic ing that regulation of transcription of mRNA or its processing drugs) have been shown to increase the level of rat cardiac Na1 and stability is primarily responsible for the regulation of channel when used chronically, the inhibitory action of n 2 3 sodium channel number. These data demonstrate that chronic PUFA on the Na1 currents raises the question of whether EPA treatment not only does not up-regulate the cardiac chronic supplementation with n 2 3 PUFA would produce sodium channel expression but also reduces the mexiletine- 1 induced increase in the cardiac sodium channel expression. upregulation of cardiac Na channels similar to the findings of Duff and Catterall with the class I Na1 blocker, mexiletine (2, 3). Whether or not this effect occurs may determine the According to the Cardiac Arrhythmia Suppression Trial (1), long-term outcome (efficacy and safety) of the antiarrhythmic the commonly used class I antiarrhythmic drugs that act by 1 therapy with the PUFA. At present, feeding studies in rats inhibiting cardiac Na channels are not safe or effective, since (10–12) and the clinical trials (13–15), unlike the Cardiac chronic treatment with the drugs (encainide or flecainide) Arrhythmia Suppression Trial (1), have indicated an antiar- produced a poorer outcome with a higher mortality than rhythmic not arrhythmogenic effect in animals and humans placebo despite their suppression of premature ventricular chronically supplemented with n 2 3 PUFA. Thus, we hypoth- complexes with short term use. Although the mechanisms esize that chronic supplementation with n 2 3 PUFA may not responsible for these deleterious effects during chronic ad- increase or perhaps may suppress overexpression of cardiac Na1 channel. This study was intended to test this hypothesis. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: BTXB, batrachotoxinin benzoate; EPA, eicosapenta- enoic acid; PUFA, polyunsaturated fatty acids. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA ‡To whom reprint requests should be addressed at: Massachusetts 0027-8424y97y942724-5$2.00y0 General Hospital, East 4th Floor, 149 13th Street, Charlestown, MA PNAS is available online at http:yywww.pnas.org. 02129. 2724 Downloaded by guest on September 30, 2021 Pharmacology: Kang et al. Proc. Natl. Acad. Sci. USA 94 (1997) 2725 We have used cultured neonatal rat cardiac myocytes to specific binding. Under these conditions nonspecific binding examine the effect of chronic supplementation of the cells with comprised 20–30% of total binding at 30 nM [3H]BTXB. The eicosapentaenoic acid (EPA, an n 2 3 PUFA), mexiletine (a dissociation constant (Kd) and maximal binding (Bmax) were class I drug), or combination of EPA and mexiletine on the determined by Scatchard analysis (18). level of Na1 channel a-subunit mRNA by Northern blot Northern Blot Analysis. Rat cardiac Na1 channel mRNA hybridization and the density of Na1 channels by radioligand was determined by Northern analysis similar to that described binding assay. We found that chronic EPA treatment not only by Duff and Catterall (3). Total cellular RNA was isolated fails to upregulate the cardiac sodium channel expression but using RNeasy Total RNA Kits (Qiagen, Chatsworth, CA). The also reduces the mexiletine-induced increase in the cardiac concentration of the RNA is determined by measuring the A260 sodium channel expression. of an aliquot of the final preparation. RNA (10–20 mg for each sample) was electrophoresed in 1% agarose gels containing 2.2 MATERIALS AND METHODS M formaldehyde. The RNA in the gel was transferred by capillary elution to nitrocellulose filters, which were baked in Cell Isolation and Culture. Cardiac myocytes were isolated vacuum to fix the RNA to the filters. Completeness of the from 1-day-old neonatal Sprague–Dawley rats using the Neo- transfer was verified by inspection of the ethidium bromide- natal Cardiomyocyte Isolation System (Worthington). The stained gels. A labeled cRNA probe was made from a linear- aortic root and atria were physically removed before isolation. ized plasmid Na-8.4 (generously provided by W. A. Catterall, The isolated cells were placed on Petri dishes with culture University of Washington) containing an insert spanning medium (F-10 nutrient mixturey10% horse serumy5% fetal nucleotides 3361–5868 of the a-subunit of the RIIA rat brain bovine serumy50 mg/ml streptomyciny50 units/ml penicillin G) sodium channel (3), using Riboprobe in vitro Transcription 32 and cultured at 378C in air with 5% CO2 added and 98% System (Promega) with T7 RNA polymerase and [a- P]UTP. relative humidity in a tissue culture incubator (model 3123, Prehybridization, hybridization, and autoradiography were Forma Scientific, Marietta, OH). The culture medium was performed according to standard procedures (19). Radioac- changed every other day. After 48 hr in culture, cells exhibited tive signals (the autoradiographic band at 8.5 kb) were quan- regular spontaneous contractions. Cells were used for exper- tified using a laser densitometer. iments after 3–6 days of culture. To examine the specificity of the effect on sodium channel Drug Treatment Protocols. After 3 days in culture, when the mRNA, nitrocellulose blots were subsequently probed with myocytes normally form multicellular colonies or a monolayer cDNA probes labeled with [a-32P]dCTP using a random of spontaneously beating cells, the culture medium was re- primer technique from the cDNA clone encoding the consti- moved and replaced in the individual Petri dishes with fresh tutively expressed b-actin (a 700-bp fragment of the full-length medium supplemented with 20 mM EPA, 20 mM mexiletine, 20 cDNA).