1625Journal of Cell Science 110, 1625-1634 (1997)Printed in Great Britain © The Company of Biologists Limited 1997JCS1424

Arachidonic acid stimulates protein kinase C-εε redistribution in heart cells

Xu Pei Huang1, YeQing Pi1, Andrew J. Lokuta1, Marion L. Greaser2 and Jeffery W. Walker1

1Department of Physiology and 2Muscle Biology Laboratory, University of Wisconsin, Madison, WI 53706, USA

Arachidonic acid is elevated in a variety of cell types inresponse to extracellular stimuli, and has been hypoth-esized to exert at least some of its intracellular actions viaactivation of protein kinase C. Here we show that arachi-donic acid stimulates a unique pattern of translocation ofthe εε-isoform of protein kinase C in isolated adult ratcardiac myocytes. Using western blot analysis, the majorityof εε-protein kinase C was found in a cytosolic fraction inunstimulated cells. Treatment with 50 µµM arachidonic acidcaused a transient increase of εε-protein kinase C in amembrane fraction within 1 minute, then after 5-20minutes most was found in a filament/nuclear fraction.Immunofluorescence and confocal microscopy of thefilament fraction revealed a striated staining pattern withεε-protein kinase C localized near the Z-line where actinfilaments are anchored and where transverse tubules areclosely apposed to the myofilaments. δδ-Protein kinase C,another isoform highly expressed in these cells, did notredistribute significantly in response to arachidonic acid,

but in response to phorbol ester displayed a predominantlynuclear localization. Arachidonic acid also stimulatedphosphorylation of the thin filament protein, troponin I,consistent with a filament localization for activated PKC.The physiological relevance of these findings was supportedby the observation that 50 µµM arachidonic acid promoteda 2.3-fold enhancement of myocyte twitch amplitude, aneffect that was significantly blocked by the protein kinaseC antagonist chelerythrine. Moreover, the onset of thisphysiological response correlated in time with translocationof εε-protein kinase C to the filaments. The results suggestthat arachidonic acid initiates a redistribution of εε-proteinkinase C to myofilament structures at or near the Z-linewhere this isozyme would be strategically located toregulate myofilament function and excitation-contractioncoupling.

Key words: Cardiac myocyte, Cytoskeleton, PKC isoform

SUMMARY

INTRODUCTION

Protein kinase C (PKC) comprises a large family of at leasteleven related serine/threonine kinases that play a central rolein transmitting signals from the cell surface to the cell interior(Nishizuka, 1995). The reason for such diversity in this familyis unclear, but possibilities include the theory that eachisozyme responds to unique signals (Clerk et al., 1994) or thateach translocates to specific subcellular sites in order to phos-phorylate selected target proteins (Mochly-Rosen, 1995; Kileyet al., 1995). Heart cells are a good model system to test thesepossibilities because: (i) multiple PKC isoforms are present;(ii) PKC may regulate numerous cardiac functions includingcontractility, ionic currents, gene expression and growth; and(iii) microscopic analysis of PKC localization is facilitated bya characteristic ultrastructure and organization of organelles(Forbes and Sperelakis, 1980).

A number of cardiac regulatory proteins have been shownto be phosphorylated by PKC (reviewed by Puceat and Brown,1994), including the myofibrillar proteins troponin and myosin(Venema and Kuo, 1993). Translocation of PKC to cardiacmyofilaments is likely to be a prerequisite for phosphorylation,but current understanding of translocation mechanisms, thePKC isozymes involved, and the nature of subcellularanchoring sites is limited. This is especially true for PKCtranslocation to myofilaments which is likely to occur without

the involvement of the membrane phospholipid phos-phatidylserine. In addition, little is known about the intracel-lular physiological signals that directly initiate PKC redistrib-ution in vivo.

Arachidonic acid (AA) is a central regulatory lipid in mostcell types (Gross, 1992) by virtue of its role as a precursor toeicosanoids (prostaglandins, thromboxanes, etc). In heartmuscle cells, AA is poorly metabolized to these oxygenatedspecies (Freyss-Beguin et al., 1989; Engels, 1990; Gross,1992), so attention has focused on its potential role as anactivator of PKC (Murakami et al., 1986; Damron et al., 1995;Nishizuka, 1995; Blobe et al., 1995). AA and other cis-unsat-urated fatty acids display some selectivity for activation ofspecific PKC isoforms (Koide et al., 1992), and are uniqueamong lipid PKC activators in that they can stimulate kinaseactivity in the absence of phosphatidylserine (Murakami et al.,1986; Koide et al., 1992; Blobe et al., 1995). cis-Unsaturatedfatty acids therefore become good candidates to initiate PKCtranslocation and activation at sites away from membranes.The goal of this work was to examine the effects of arachidonicacid on the subcellular distribution of PKC isoforms in isolatedadult cardiac myocytes. Evidence is presented that AA causesa selective redistribution of ε-PKC from the cytosol to cardiacmyofilaments under the same conditions that it enhancesmyocyte contractility and stimulates phosphorylation oftroponin I (TnI).

1626 X. P. Huang and others

MATERIALS AND METHODS

Polyclonal PKC antibodies were from Gibco BRL (Grand Island,NY), including the set of isozyme specific antibodies and affinitypurified anti-ε-PKC. Goat anti-rabbit or anti-mouse IgGs conjugatedwith either horseradish peroxidase or fluorescein were obtained fromGibco, BRL or Boehringer Mannheim (Indianapolis, IN). MonoclonalPKC antibodies were purchased from Transduction Laboratories Inc(Lexington, KY). The enhanced chemiluminescence (ECL) westernblot detection reagents, nitrocellulose transfer membranes (Hybond,ECL), and high performance luminescence detection film (Hyperfilm-ECL) were from Amersham Corp. (Arlington Heights, IL). Col-lagenase (Class 2) was from Worthington Biochemical Corp.(Freehold, NJ). Phorbol 12-myristate 13-acetate (PMA) was from Cal-biochem (La Jolla, CA). Other chemicals were purchased from SigmaChemical Co (St Louis, MO).

Isolation of cardiac myocytesAdult rat cardiac myocytes were isolated enzymatically by perfusionof isolated heart with collagenase using the method described byHaworth et al. (1989) modified as follows. Female Sprague-Dawleyrats weighing 200-250 g were anesthetized using Metofane. Theexcised heart was retrogradely perfused at 37°C with continuouslyoxygenated Ringer’s solution of the following composition: 118 mMNaCl, 4.8 mM KCl, 25 mM Hepes (adjusted to pH 7.4 with NaOH),2 mM KH2PO4, 1.2 mM MgCl2, 5 mM pyruvate, 11 mM glucose and1 µM insulin. Initial perfusion was for 10-15 minutes with Ringer’ssolution containing 1 mM CaCl2 followed by another 10-15 minuteperfusion with Ca2+-free Ringer’s solution. Finally, 50 ml Ca2+-freeperfusate containing 1 mg/ml collagenase and 0.4 mg/mlhyaluronidase was recirculated through the heart for 10 minutes.CaCl2 was then added to the recirculation solution to a final concen-tration of 0.5 mM and perfusion was continued for an additional 15minutes. The cannulus was removed from the heart and the ventricleswere cut away and diced. This ventricular tissue was added to 20 mlof the recirculated collagenase/hyaluronidase solution containing 25mg of BSA and shaken in a 37°C water bath for 25 minutes. Myocyteswere filtered through a 300 µm Teflon mesh, pelleted at 800 g for 45seconds in a table top centrifuge, washed twice with fresh 0.5 mMCa2+ Ringer’s solution, then resuspended in the same buffer solution.Typically, the yield of cells was at least 106 myocytes per heart, andviability ranged from 70-90% rod-shaped Ca2+ tolerant cells asassessed by microscopic inspection. Preparations displaying less than70% viability were not used.

Measurements of twitch shorteningElectrical field stimulation was carried out in a custom designed 200µl plexiglass chamber with a glass floor and two 10 mm × 2 mmplatinum electrodes along opposite walls. The stimulation protocolwas 0.5 Hz, 1 millisecond duration, 40 V at 20-22°C using a GrassSD9 stimulator (Quincy, MA). The chamber was mounted on a NikonDiaphot inverted microscope, and individual cells were monitoredwith a Model VED 104 video based edge detector (Crescent Elec-tronics, Sandy, UT) and plotted on an X-Y plotter (Plotomatic, Salem,NH).

Labelling of myofibrils in situ with [32P]Pi

Myocytes from a single heart were suspended in 10 ml of phosphate-free Dulbecco’s minimum essential medium containing [32P]Pi (0.5mCi/ml). Cells were incubated for 2 hours at 37°C, and continuouslybubbled with 95% O2, 5% CO2. Samples (1 ml) of these 32P-labelledcell suspensions were further incubated for the indicated time withdifferent test compounds or with the vehicle ethanol. Cells were thenquickly pelleted in a bench-top centrifuge (45 seconds at 800 g),washed twice in Ringer’s solution, and homogenized with skinningsolution (0.5% Triton X-100, 0.5% BSA, 1 mM PMSF, 10 mM ben-zamidine, 0.01 mM leupeptin in relaxing solution) on ice for 10

minutes. These detergent-extracted myofibrils were pelleted andimmediately solubilized in sodium dodecyl sulfate (SDS) samplebuffer by heating for 5 minutes at 100°C.

32P-labelled myofibrillar proteins were separated by 12% SDS-polyacrylamide gel electrophoresis. Gels were stained withCoomassie blue, dried, and subjected to autoradiography. 32P-labelled TnI and myosin light chain-2 (LC2) bands were excisedfrom gels and quantified by liquid scintillation counting. Labellingstoichiometries were calculated by extracting and purifying ATPfrom 32P-labelled myocytes, determining its specific radioactivity(dpm/mole), then using this value to convert dpm in labelled proteinbands to moles of phosphate incorporated. Protein levels (in moles)were determined by densitometric scanning of Coomassie bluestained gels then compared with purified cardiac TnI and LC2 ofknown quantity (in moles).

Production and affinity purification of polyclonalantibodies Anti-ε-PKC directed against the C-terminal peptide of ε-PKC(residues 726-737) was raised in rabbits at the Antibody ProductionFacilities of the University of Wisconsin. The peptide, CKGF-SYFGEDLMP, was synthesized on an Applied Biosystems Model432 instrument, purified by reverse phase HPLC, and its identityconfirmed by amino acid analysis and mass spectrometry(mass/charge, expected 1,493.8, found 1,494.2). For antibody pro-duction, the purified peptide was coupled to keyhole limpet hemo-cyanin using a commercial kit (Pierce, Rockford, IL). Affinitypurification was carried out according to the method described bySharp and Campbell (1989). Briefly, purified ε-PKC was subjectedto SDS-PAGE on mini gels using a single large sample well. Theseparated proteins were then transferred electrophoretically toImmobilon-P membranes. Blot strips containing the ε-PKC bandswere cut from the transfer and blocked with TBS-BSA (3% BSA inTris-buffered solution, 20 mM Tris-HCl, 137 mM NaCl, pH 7.6).Antisera from rabbits were diluted 4-fold in 150 mM NaCl, 15 mMTris-HCl, pH 7.4, and incubated with the blot strips overnight at4°C. The strips were then washed three times for 10-15 minutes in500 mM NaCl, 50 mM Tris-HCl, pH 7.4, followed by a final 10minute wash in 100 mM NaCl, 50 mM Tris-HCl, pH 7.4. Boundantibodies were eluted by treatment of strips with 50 mM glycineHCl, pH 2.5. After a 10 minute incubation with gentle mixing, theeluate was removed and adjusted to pH 7.5 by the addition of 2 MTris-HCl, pH 8.0. Protein concentration was estimated byabsorbance at 280 nm, then BSA and sodium azide were added tofinal concentrations of 2 mg/ml and 0.02%, respectively. Antibodieswere stored at 4°C.

Subcellular fractionation of cardiac myocytes Fractionation of cardiac myocytes was carried out according to theprotocol reported by Gu and Bishop (1994). In brief, isolatedmyocytes in 0.5 mM Ca2+ Ringer’s were incubated with testcompounds for the indicated times at 37°C. Myocytes were quicklypelleted by centrifugation (45 seconds at 800 g) and washed once withfresh Ringer’s solution. Cell pellets were resuspended in relaxingsolution (4 mM MgATP, 100 mM KCl, 10 mM imidazole, 2 mMEGTA, 1 mM MgCl2, 1 mM PMSF, 1 mM sodium orthovanadate, 10mM benzamidine and 0.01 mM leupeptin) containing 20% glyceroland 0.05% Triton X-100. The cell suspension was sonicated for 5minutes on ice to facilitate cell permeabilization and membrane sol-ubilization. A nuclear/filament fraction was separated from the sus-pension by centrifugation at 800 g for 2 minutes. The 800 g super-natant was centrifuged at 100,000 g for 30 minutes at 4°C producinga pellet which was designated the membrane fraction, and the super-natant was designated the cytosol fraction. The 800 g pellet contain-ing nuclei and myofilaments was washed 3 times with relaxingsolution. Microscopic examination of these fractions revealed intactnuclei and striated myofibrillar structures in the nuclear/filament

1627Protein kinase C in heart cells

Fig. 1. Effect of AA on cardiac myocyte twitches. The amplitude ofcell shortening was assessed in single electrically stimulatedventricular myocytes as described in Materials and Methods. (A) 50µM AA in Ringer’s buffer was added by continuous perfusion of thecell chamber (mean ± s.e.m. was 2.3±0.2-fold increase over basal;n=6). (B) 50 µM AA in the presence of 4 µM PKC inhibitorchelerythrine chloride (1.3±0.2-fold increase over basal or a 77%decrease of AA response; n=4). (C) 50 µM AA in the presence of 10µM indomethacin (2.2±0.2-fold increase over basal; n=5). (D) 50µM oleic acid (OA) perfused onto a myocyte in the same manner(2.5±0.3-fold increase over basal; n=7).

fraction, and few such structures in the membrane or cytosol fractions.Immunofluorescence and confocal microscopy was carried out on thisnuclear/filament fraction.

Western blotting Immunoblot analysis was carried out on each of the three subcellularfractions: cytosol, membranes and nuclear/filaments. To facilitate sol-ubilization and loading of protein onto gels, the filament fraction wasfirst extracted with 1% Triton X-100 (in relaxing solution on ice for1 hour) to solubilize bound PKC, then centrifuged at 100,000 g for30 minutes at 4°C to remove insoluble material. Immunoblotsconfirmed that essentially all of the ε-PKC immunoreactivity wasextracted into the Triton X-100 supernatant by this procedure. Forconsistency, the membrane fraction was treated with 1% Triton X-100in an analogous manner, and Triton X-100 was added to the cytosolfraction to a final concentration of 1%. Samples were prepared forwestern blots by measuring protein concentration using the Bicin-coninic acid assay (Sigma Chemical Co) then mixing with SDS-PAGEsample buffer (300 mM Tris-HCl, pH 6.8, 8% (w/v) SDS, 15% (v/v)glycerol, 150 mM dithiothreitol and 0.5% (w/v) bromophenol blue).Extracts of whole cardiac myocytes were prepared by mixing intactmyocytes directly with sample buffer after assaying for protein con-centration. All samples were adjusted to a protein concentration of 1-2 mg/ml in sample buffer, boiled for 5 minutes and then kept at −20°Cbefore western analysis.

Equal amounts of protein (typically 20-30 µg) from each fractionwere electrophoresed on 12% SDS-polyacrylamide gels and thentransferred to nitrocellulose membranes. Blots were then stained with0.1% Ponceau S solution to visualize protein bands and confirm bothconsistent protein loading among wells and complete transfer ofproteins to blots. After blocking nonspecific sites with TBS-T solution(0.1% (v/v) Tween-20 in 20 mM Tris base, 137 mM NaCl adjusted topH 7.6 with 1 M HCl, containing 3% (w/v) BSA) for 1 hour at roomtemperature, the nitrocellulose was incubated with primary antibody(1/1,000-1/2,500 dilution in the TBS-T solution containing 1% BSA)for 1 hour at room temperature with or without appropriate competingpeptide (2 µg/ml). After washing in TBS-T solution 3 times (10minutes each), filters were incubated for 1 hour at room temperaturewith horseradish peroxidase-linked secondary antibody (1/5,000dilution in TBS-T solution containing 1% BSA). Filters were washedagain in TBS-T solution 3 times for 10 minutes. Bound antibody wasdetected by the enhanced chemiluminescence method according to themanufacturer’s (Amersham) instructions. Exposure times ofimmunoblots to Hyperfilm were 0.25-2 minutes.

Immunofluorescence The 800 g pellet which contained nuclei and myofilaments waswashed with relaxing solution 3 times. The nuclear/filament sus-pension was incubated with affinity purified PKC isozyme anti-bodies (1/50) at 4°C for 2 hours. After washing 3 times, themyofibril suspension was then gently smeared over no. 1.5 cover-slips and myofibrils were fixed in cold methanol for 1 minute. Slideswere then incubated with second antibody, fluorescein-conjugatedgoat anti-rabbit IgG (1/10) at 25°C for 1 hour. Unbound secondantibody was washed away with RB (74 mM KCl, 2 mM MgCl2, 1mM EGTA, 1mM NaN3, 5 mM dibasic potassium phosphate, 1 mMdithiothreitol, adjusted to pH 7.0 with HCl) and coverslips weremounted on glass slides in 70% glycerol/RB solution containing p-phenylenediamine (1 mg/ml). Slides were viewed with a NikonDiaphot microscope equipped for phase contrast and epifluorescenceillumination. Images were obtained using a ×40 or ×100 objectiveand a cooled CCD camera controlled via a Macintosh IIfx with aMatrox frame grabber board using IPLABS software. Exposuretimes were typically 0.2 seconds for phase contrast and 2 secondsfor fluorescent images. Some slides were observed under a Bio-Rad2,000 confocal microscope and images were analyzed with AdobePhotoshop software.

RESULTS

Effects of arachidonic acid on myocyte contractilityPerfusion of adult rat ventricular myocytes with 50 µM arachi-donic acid (AA) caused a 2.3-fold increase in electrically-paced twitch amplitude (Fig. 1). This positive inotropic effectinitiated by 50 µM AA typically reached a plateau within 5minutes of initial exposure and had a half-time of 2-3 minutes(Fig. 1C). The vehicle (0.1% ethanol final concentration) waswithout effect on myocyte twitch amplitude (not shown). Pre-treatment of myocytes with the PKC antagonist chelerythrineinhibited the increase in twitch amplitude by 77% (Fig. 1B),whereas chelerythrine had no effect on basal twitch propertiesor on the response of these cells to β-receptor stimulation (Piet al., 1997). Pretreatment with the cyclooxygenase inhibitorindomethacin was without effect on the myocyte response toAA (Fig. 1C). Further evidence that this effect was not causedby a metabolite of AA was provided by the observation thatthe cis-unsaturated fatty acid, oleic acid, gave a similar 2.5-foldincrease in twitch amplitude (Fig. 1D).

1628 X. P. Huang and others

PKC isoforms in adult cardiac myocytesWe used isoform-specific antibodies to determine whichisoforms of PKC were present in our adult rat ventricularmyocytes preparation. Fig. 2A illustrates representativewestern blots of myocyte homogenates using affinity purifiedpolyclonal antibodies. Antibodies were raised against syntheticpeptides derived from unique sequences of each PKC isozyme.A homogenate of adult rat brain was used as a control to showthat antisera recognized appropriate bands for the α, β, ε, δ andζ PKC isozymes (Fig. 2A, left panel). Single bands weredetected with apparent molecular mass 85 kDa (α), 80 kDa (β),93 kDa (ε), 74 kDa (δ), 78 kDa (ζ) in brain homogenates. Inparallel measurements using cardiac myocyte homogenates,positive bands were detected using anti-PKC-ε or anti-PKC-δantibodies, with each band displaying the appropriatemolecular mass, ε (93 kDa) and δ (74 kDa) (Fig. 2A, right

Fig. 2. Immunoblot analysis of cardiac myocytes. (A) Blots of ratbrain (left lane) and cardiac myocyte extracts (right lane) showingPKC isoforms expressed in each tissue. Each lane contained 20-30µg protein and was exposed for 15-30 seconds. (B) Assessment ofanti-ε-PKC cross-reactivity with other proteins in cardiac extracts.Blots were incubated with affinity purified ε-PKC Ab in the absence(left panel) or presence (right panel) of ε-PKC C-terminal peptide,KGFSYFGEDLMP. Blots contained 50 µg of cardiac protein andwere overexposed (2 minutes) to optimize detection of cross-reactivebands.

panel). Similar results were obtained with polyclonal anti-bodies from several sources including affinity purified anti-bodies from GIBCO BRL and Calbiochem, and monoclonalantibodies from Transduction Laboratories. Anti-PKC-αantisera reacted only weakly with cardiac myocyte extracts andthe signal could not be significantly enhanced by overexposureof the blot. This weak positive reaction was consistentlyobserved in a variety of experiments with antibodies fromdifferent sources and with cells from different rat hearts, so itis possible that α-PKC is expressed in these cells albeit at lowlevels.

The specificity of PKC isoform recognition on western blotswas tested with synthetic peptides corresponding to theisoform-specific sequences against which the antibodies wereraised. In all cases, preabsorption of antibody with the appro-priate peptide prior to exposure to the blot, resulted in loss ofthe band corresponding to PKC. This shows that the antibodyrecognized the expected epitopes on the blots and supports astraightforward interpretation of the western blots of Fig. 2A.We focused our further investigations on ε- and δ-PKCisoforms. To reduce uncertainty associated with antibodycross-reactivity with other proteins in cardiac myocyte extracts,we raised rabbit polyclonal antibodies to the ε-PKC peptide inour own laboratory, then affinity purified the antibodies usingpurified PKC-ε expressed in Sf9 cells. The quality of westernblots obtained with this antibody preparation is illustrated inFig. 2B. The antibody recognized a band in cardiac myocytehomogenates that corresponded in mobility to ε-PKC and thisbinding was blocked by preabsorption with the ε-PKC peptide,indicating specific targeting of the expected epitopes on theblots.

Translocation of εε-PKC in adult cardiac myocytesTranslocation was initially monitored by western blotanalysis of three subcellular myocyte fractions: cytosol,membranes and myofilaments/nuclei (hereafter myofilamentsor filaments). In myocytes not stimulated by PKC activators,most of the ε-PKC and the δ-PKC was detected in the cytosol,with the remainder distributed among the membrane andfilament fractions. Incubation of myocytes in Ringer’ssolution with 50 µM AA resulted in a redistribution of ε-PKCthat was characteristic in both its pattern and time course (Fig.3). A rapid increase of membrane associated ε-PKC wasobserved 1 minute after stimulation with 50 µM AA. Afterfive minutes, membrane ε-PKC had decreased back to theunstimulated control level or below and remained at that levelfor up to 20 minutes (the longest time tested). Myofilamentassociated ε-PKC increased steadily to a peak 5 minutes afterstimulation with AA. The increase in filament associated ε-PKC was 2 to 3-fold over the unstimulated control level, andwas undiminished after 20 minutes of exposure to AA. Theε-PKC immunoreactivity in the cytosol fraction decreasedconcomitantly with increases in the membrane and filamentfractions (Fig. 3B). Incubation of cardiac myocytes with 100nM PMA under the same conditions caused ε-PKC redistri-bution to a similar extent and with a similar time course tothat observed with AA (Fig. 3A). Overall, the results showthat AA induced a transient increase in membrane associatedε-PKC lasting less than 5 minutes and a sustained increase inε-PKC translocation to the cardiac myofilament fractionlasting at least 20 minutes.

1629Protein kinase C in heart cells

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Fig. 3. Immunoblot analysis of ε-PKC translocation. (A) Time-course of ε-PKC translocation induced by 100 nM PMA. (B) Time-course of ε-PKC translocation induced by 50 µM AA. Each point represents mean ± s.e.m. for 3 separate experiments. (C) Blots showing redistribution ofε-PKC immunoreactivity in cytosol, membranes and myofilaments/nuclei following incubation of intact myocytes with 100 nM PMA (lanes 4-6), or 50 µM AA (lanes 7-9) for 5 minutes. (D) Blots showing similar data in response to 50 µM OA. Each well in C and D contained 25 µgprotein. Control represents untreated cells (lanes 1-3). Purified ε-PKC (0.1 µg) as an internal reference is shown in lane 10 (C) and lane 1 (D).

Immunofluorescence microscopy In order to confirm that ε-PKC was binding to filaments andnot to nuclei or membranes contaminating the filamentfraction, we visualized antibody staining in the filamentfraction by immunofluorescence microscopy. Filamentsisolated from unstimulated myocytes showed only diffusestaining with affinity purified anti ε-PKC antibodies (Fig. 4).After 20 minutes of incubation with 50 µM AA, the filamentfraction showed a strong cross-striation pattern of staining withε-PKC antibodies (Fig. 4). Nuclei associated with many of thefilament structures were clearly visible under phase contrastoptics but no staining of nuclei was observed with ε-PKC anti-bodies. To ensure that the ε-PKC binding occurred throughoutthe entire cell thickness and was not limited to surface staining,we carried out serial Z-section image analysis with a confocalmicroscope. Sectional images showed a similar cross-striationpattern in all 15 of the 1 µm thick layers of myocytes pretreatedwith AA, indicating ε-PKC binding to myofilaments through-out the cells (Fig. 5A). In addition, filament staining was notdue to non-specific binding of primary or secondary antibodiesbecause inclusion of the epitope peptide eliminated the cross-striation staining pattern (Fig. 5B). Thus, immunofluorescenceconfirms the results obtained with western blotting thatexogenous AA induces a large scale redistribution of the ε-isoform of PKC to cardiac myofilaments.

Translocation of δδ-PKC in adult cardiac myocytes To further examine the specificity of the effects of AA on ε-PKC translocation, we investigated the translocation behaviorof δ-PKC under the same conditions. Our results showed that50 µM AA had little effect on the distribution of δ-PKC in adultcardiac myocytes assessed by western blots (Fig. 6A). Bycontrast, stimulation of myocytes with 100 nM PMA resultedin a substantial redistribution of δ-PKC, with increases in boththe filament fraction (2.2±0.2-fold) and membrane fraction

(1.4±0.2-fold), and a concomitant decrease in the cytosolfraction (Fig. 6). Immunofluorescence microscopy using anti-δ-PKC antibodies revealed diffuse staining in filamentfractions from unstimulated cells or cells stimulated with 50µM AA, consistent with western blot analysis using thisantibody. Immunofluorescence of filament fractions from cellsstimulated with 100 nM PMA showed strong staining in thenuclei and little cross-striation staining (Fig. 6C). Thus, evenwhen δ-PKC translocation is maximally stimulated by additionof PMA, it tends to associate more with nuclei than withfilaments. These results provide a further indication that theobserved effects of AA on ε-PKC translocation to the myofil-aments are specific for the ε-isoform because the δ-isoform islargely insensitive to AA treatment and when activated byPMA has a preference for translocation to sites other than themyofilaments (i.e. membranes and nuclei).

Phosphorylation of myofilament proteins To investigate the consequences of PKC translocation tomyofilaments, we monitored myofibrillar protein phosphoryla-tion in cardiac myocytes. Myocytes were pre-labelled with[32P]Pi then stimulated with 50 µM AA or 100 nM PMA for5 minutes. Filaments were isolated, separated by SDS-PAGE,and gels subjected to autoradiography. Compared to unstimu-lated control cells, both 50 µM AA and 100 nM PMA resultedin significant (P<0.01) increases in phosphorylation of TnI, amajor thin filament associated protein (Fig. 7). The identity ofTnI on gels was established by its molecular mass (30 kDa),by comparison with purified standards, and by western blotswith anti-TnI antibodies (not shown). Calculations of stoi-chiometry based upon measurements of the specific activity ofcellular ATP, gave values of 0.13 mol P/mol TnI in unstimu-lated myocytes. After 50 µM AA treatment phosphorylation ofTnI increased by 1.6±0.1-fold (n=6), and after 100 nM PMAby 2.2±0.1-fold (n=4). Increases in TnI phosphorylation by AA

1630 X. P. Huang and others

Fig. 4. Phase contrast (A,C,E) and fluorescent(B,D,F) images of material in the filamentfraction after decoration with anti-ε-PKC andfluorescein-conjugated second antibody. (A,B) Myofilaments from control (untreated)myocytes. ×1,000. (C,D) Myofilaments after50 µM AA. ×1,000. (E,F) Myofilaments after50 µM AA. ×400.

Fig. 5. Confocal images of material from the filament fraction afterstimulation with 50 µM AA. Samples were decorated with anti-ε-PKC in the absence (A) or presence (B) of C-terminal ε-PKCpeptide, KGFSYFGEDLMP.

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and PMA were prevented by coincubation of myocytes with10 µM PKC inhibitor chelerythrine (Fig. 7). These data arecompatible with the ability of AA and PMA to translocateactive PKC to myofilaments in cardiac myocytes. 32P incor-poration into LC2 after AA or PMA treatment was morevariable and no consistent increase in phosphorylation of thisprotein was detected under our assay conditions.

DISCUSSION

The results of this study show that AA can stimulate PKCredistribution in freshly isolated cardiac myocytes from adultrat ventricle. The effects of AA appeared to be selective for theε-isozyme of PKC over another isozyme, δ, found at highlevels in these cells. The characteristic pattern of ε-PKCtranslocation included a transient increase in the membranefraction, followed by a sustained increase in the filamentfraction. Binding of ε-PKC to cardiac myofilaments wasdetected by both immunoblotting and by immunofluorescencemicroscopy. This pattern of redistribution was similar for twodifferent activators of PKC, AA and PMA, indicating that it isan intrinsic property of activated ε-PKC rather than beingdependent upon the nature of the activator. In contrast, analysisof δ-PKC revealed a difference in behavior depending on thePKC activator used. AA had no apparent effect on δ-PKCredistribution, while PMA increased its association withmembranes and with nuclei. Thus, under our experimental con-ditions δ-PKC was not detectably influenced by AA, butretained its responsiveness to the PKC activator PMA. Thisfinding is consistent with in vitro studies which showed thatAA is a good activator of ε-PKC but a poor activator of δ-PKC(Koide et al., 1992).

A consensus has yet to be reached among different labora-tories concerning which isozymes are present in adult ratcardiac myocytes. Most investigators agree upon the presenceof ε-PKC and δ-PKC (Koide et al., 1992; Kohout and Rogers,

1631Protein kinase C in heart cells

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Fig. 6. (A) Summary of δ-PKC translocation in cardiac myocytes. Intact cells were incubated with 50 µM AA or 100 nM PMA at 37°C for 5minutes, then fractionated into cytosol, membranes and filaments/nuclei and subjected to immunoblot analysis. Control refers to untreated cells.Data represent mean ± sem for 5 experiments. Statistical significance was evaluated by Student’s t-test comparing band intensity to the controlband in the same subcellular fraction (*P<0.05, **P< 0.01). Phase contrast (B) and fluorescent (C) images of material in the filament fractionafter decoration with anti-δ-PKC and fluorescein-conjugated second antibody. Myofilaments after 100 nM PMA. ×1,000.

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1993; Rybin and Steinberg, 1994; Puceat et al., 1994), butBogoyevitch et al. (1993) found only the ε-form. α-PKC hasbeen detected in adult myocardium by some (Kohout and

Fig. 7. AA mediated protein phosphorylation in cardiac myocytes.Myocytes pre-labelled with [32P]Pi were incubated with (A) 0.1%ethanol vehicle, (B) 100 nM PMA, (C) 50 µM AA, (D) 100 nMPMA plus 10 µM chelerythrine and (E) 50 µM AA plus 10 µMchelerythrine. Myofilament fractions were isolated, proteinconcentration measured and subjected to gel electrophoresis andautoradiography (Materials and Methods). Less than 5% variation inprotein loading from well to well was confirmed by scanning actinon Coomassie blue stained gels. 10 µM chelerythrine consistentlyreduced basal phosphorylation of light chain 2 (LC2). Similar resultswere obtained in 4 separate experiments.

A B C D EkDa

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- LC2

Rogers, 1993; Puceat et al., 1994) but not all investigators(Rybin and Steinberg, 1994; Bogoyevitch et al. 1993). Onestudy using the sensitive reverse transcriptase-PCR methodprovided evidence for the presence of mRNA for the η isoform(Kohout and Rogers, 1993). Our observations that the pre-dominant PKC isoforms in adult ventricular myocytes are ε andδ, that expression of α-PKC is significantly lower, and that βand ζ isoforms are not detectable are in general agreement withprevious investigations (Puceat and Brown, 1994). Because α-PKC was near the detection limit of our methods, we could notreliably evaluate its redistribution in response to AA.

In addition to the observation that the ε- and δ-PKCisozymes responded differently to AA, our results also suggestthat these isozymes translocated to distinct subcellular sites: εto myofilaments and δ to the nucleus. Translocation of δ-PKCto membranes also occurred in response to phorbol ester andwas more sustained than was translocation of ε-PKC tomembranes. A similar localization of ε-PKC to a cross-striation pattern, and of δ-PKC to the nucleus following PMAtreatment was recently described in neonatal cardiac myocytes(Disatnik et al., 1994; Johnson et al., 1996). Translocation ofthese PKC isoforms thus appears similar in adult and neonatalmyocytes at least at the level of resolution afforded byimmunofluoresence microscopy. An intriguing question raisedby these results is the nature of binding sites for ε- and δ-PKCin the various subcellular compartments. Mochly-Rosen andcoworkers have characterized PKC anchoring sites in non-cardiac cells that appear to be specific for β-PKC, an isoformnot expressed in adult cardiac myocytes. These binding siteswere called RACKs (an acronym for receptors for activated Ckinase) (Mochly-Rosen et al., 1991). The RACKS for ε- andδ-PKC have yet to be fully characterized in any tissue. A

1632 X. P. Huang and others

number of laboratories have suggested that PKC may bindspecifically to actin filaments (Zalewski et al., 1991; Blobe etal., 1996), including a recent report that this property is uniqueto ε-PKC (Prekeris et al., 1996). This may account for thecross-striation pattern observed here and in other studies (Liuet al., 1989; Mochly-Rosen et al., 1990; Disatnik et al., 1994;Johnson et al., 1996). However, this may not be the whole storybecause our high magnification images showed most ε-PKClocalized at or near the Z-line rather than all along the lengthof the actin filaments. Other cytoskeletal elements such asintermediate filaments (Spudich et al., 1992) and focal contacts(Jaken et al., 1989) have been suggested to be anchoring sitesfor β- and α-PKC, respectively.

This pattern of ε-PKC binding is intriguing for anotherreason. In cardiac muscle, the T-tubule membranes penetratedeep into the myofibrils near the Z-lines (Forbes and Sperelakis,1980), which places two major proteins involved in excitation-contraction coupling (i.e. L-type Ca2+ channels and ryanodinereceptors) in the immediate vicinity of the Z-lines. Thissuggests that translocation of ε-PKC to these sites in cardiacmyocytes may allow this kinase to regulate not only myofila-ment properties but also excitation-contraction coupling. Therole that T-tubular proteins play in anchoring ε-PKC to thefilaments remains to be determined. However, preliminarystudies show that exhaustively skinned cardiac myocytesretain the ability to bind purified ε-PKC in the presence of AA,suggesting that the myofilaments are sufficient for anchoring ε-PKC. Moreover, such a myofilament location is consistent withour finding that TnI, a major thin filament protein in cardiacmuscle, is phosphorylated by the lipids that induced transloca-tion (see also Liu et al., 1989; Damron et al., 1995; Venema andKuo, 1993).

While the stoichiometry of TnI phosphorylation is low(approximately 1/10 of a mole of phosphate per mole of TnIprotein), low stoichiometries are often observed in studies ofprotein phosphorylation by PKC (Liu et al., 1989; Lowndes etal., 1990; Blobe et al., 1996). TnI phosphorylation levels canbe increased by inclusion of protein phosphatase inhibitorsduring cell stimulation (Damron et al., 1995) or duringmyofibril isolation (Venema and Kuo, 1993). Even so, itremains to be demonstrated that a given PKC site on TnI is sto-ichiometrically phosphorylated following stimulation ofendogenous signaling pathways. The preferred localization ofε-PKC to the Z-line ends of the actin filaments may provide anexplanation for substoichiometric phosphorylation of cardiacthin filament proteins in this and other studies (Liu et al., 1989;Blobe et al., 1996).

The physiological significance of AA effects on PKC redis-tribution was supported by the finding that AA enhanced theamplitude of electrically stimulated twitches in a PKC-dependent manner. The concentration of AA used (50 µM) isalso considered to be within the physiological range (Gong etal., 1995). This observation is novel in itself because it showsan enhancement of contractility by AA in adult mammalianmyocytes which is largely PKC-dependent. AA stimulated ε-PKC translocation to myofilaments with a similar time courseto its effects on enhancing myocyte twitch amplitude. Thetransient association of ε-PKC with membranes preceeded thephysiological changes induced by AA, so phosphorylation ofPKC substrates during this membrane association could alsoplay a role. However, the enhanced twitch amplitude depended

upon the continued presence of AA which is not compatiblewith the transient nature of ε-PKC binding to the membranefraction. It is therefore more likely that the sustained translo-cation to filaments was necessary for the physiological effects.Bond and coworkers have also observed that AA enhancedtwitch amplitude in adult rat myocytes, but the role of PKCwas not evaluated (Damron et al., 1995). Curiously, an almostcomplete inhibition of intracellular Ca2+ transients by AA wasalso reported (Damron and Bond, 1993). We found noinhibitory actions of AA in our experiments and the reasonsfor this discrepency are presently unknown. The positiveinotropic nature of the AA response and its characteristic timecourse were similar to that observed for another PKC activator,diC8, in the same cells (Pi et al., 1997).

It is difficult to compare the results we obtained in adultmyocytes to studies on neonatal cardiac cells in culture. Theprimary reason for this is that, unlike adult ventricularmyocytes, neonatal myocytes display the electrophysiologicalcharacteristics of pacemaker cells (Wetzel and Klitzner, 1996).In addition, neonatal cells express distinct patterns of proteinsinvolved in contraction and excitation-contraction coupling(Mahony, 1996), and even express additional PKC isoforms notfound in adult myocytes (Rybin and Steinberg, 1994; Puceat etal., 1994; Mochly-Rosen, 1995). Nevertheless, the demonstra-tion by Johnson et al. (1996) that a peptide capable of blockingε-PKC translocation to myofilaments in neonatal cells alsoblocked PMA- and agonist-stimulated changes in twitchfrequency (chronotropy), is compatible with our suggestionthat ε-PKC translocation is physiologically relevant in cardiaccells. That the biological consequences of ε-PKC stimulationand translocation in the present work differ from those ofJohnson et al. (1996) is likely a reflection of fundamental dif-ferences in the cellular physiology of adult and neonatal heartcells.

Is AA a physiological activator of PKC in myocardial cells?Evidence that AA and other cis-unsaturated fatty acids may beimportant messengers comes from the finding that intracellu-lar levels of these fatty acids are elevated by a variety of extra-cellular stimuli including neurohormones (Rogers and Lokutta,1994), oxygen stress (Gross, 1992; Freyss-Beguin et al., 1989;Engels et al., 1990), and mechanical loading (Sodashima andIzumo, 1993). cis-Unsaturated fatty acids are known to bepotent activators of PKC in vitro (Koide et al., 1992; Blobe etal., 1995), and several studies have implicated PKC as a majormediator of cis-unsaturated fatty acid effects in cardiac cells(Damron and Bond, 1993; Damron et al., 1995) and other celltypes (Murakami et al., 1986; Zhang and Dorman, 1993). Aintriguing suggestion from studies of PKC in vitro is that cis-unsaturated fatty acids act synergistically with diacylglycerolsto activate the enzyme (Shinomura et al., 1991) and to anchorthe enzyme to subcellular sites (Prekeris et al., 1996) even inthe absence of phosphatidylserine. We postulate that such aphospholipid independent mechanism is necessary for translo-cation and anchoring of ε-PKC to cardiac myofilaments.

We cannot rule out the possibility that some of the effectsof AA are due to oxidized metabolites such as prostacyclinswhich can be derived from AA in cardiac muscle (albeit onlyat low levels) (Engels et al., 1990). We found thatindomethacin, an inhibitor of the major AA metabolic enzymecyclooxygenase, was without effect on the myocyte responseto AA. In addition, another cis-unsaturated fatty acid, oleic

1633Protein kinase C in heart cells

acid, which is not a precursor to the myriad of AA metabolites,mimicked the effects of AA on contractility and ε-PKC translo-cation. We therefore favor the interpretation that the observedeffects are due to AA itself. AA may also influence cardiaccontractility by other mechanisms such as by modulation ofionic channels (Kim, 1992). Polyunsaturated fatty acids havebeen shown to exert a variety of electrophysiological effects onneonatal (Kang et al., 1995) and adult myocytes (Pepe et al.,1994), but AA was much less effective in this regard comparedto omega-3 polyunsaturated fatty acids (Hallaq et al., 1992;Pepe et al., 1994).

In summary, we have provided evidence that AA can modifycardiac myocyte twitch characteristics in a PKC-dependentmanner. We also found that AA stimulated a selective redistri-bution of the ε-isoform of PKC to unique subcellular sites at asimilar concentration and with a similar time course to itseffects on myocyte twitch amplitude. AA-induced transloca-tion of PKC at or near the Z-line places the ε-isoform in astrategic location to regulate myofilament properties and T-tubular proteins involved in excitation-contraction coupling.

This work was funded by NIH grant P01 HL55438 (J.W.W. andM.L.G.). X.P.H. was supported by an American Heart Association ofWisconsin Postdoctoral Fellowship (94-POST-28), A.J.L. by an NIHPostdoctoral Fellowship, and J.W.W. by a Research Career Develop-ment Award (K04 HL03119). We thank Drs Hector Valdivia and TerryRogers for helpful discussions, and Drs Rogers and Peter Blumbergfor providing ε-PKC. Some of this work has been presented in pre-liminary form to the Biophysical Society (Huang and Walker, 1996)and American Society for Cell Biology (Huang et al., 1996).

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(Received 7 February 1997 - Accepted 1 May 1997)