Morphological, biochemical, and electrophysiological characterization of a clonal cell (H9c2) line from rat heart. J Hescheler, R Meyer, S Plant, D Krautwurst, W Rosenthal and G Schultz Circ Res. 1991;69:1476-1486 doi: 10.1161/01.RES.69.6.1476 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1991 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571

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Morphological, Biochemical, and Electrophysiological Characterization of a Clonal Cell (H9c2) Line From Rat Heart Jurgen Hescheler, Rainer Meyer, Sabine Plant, Dietmar Krautwurst, Walter Rosenthal, and Gunter Schultz Morphological, electrophysiological, and biochemical properties of H9c2 cells, a permanent cell line derived from rat cardiac tissue, were studied. Although the lectin binding pattern revealed similar sugar residues in the surface coat of H9c2 cells and isolated rat cardiocytes, heart-specific morphological structures could not be detected in H9c2 cells. Under physiological ionic conditions, H9c2 cells exhibited an outwardly rectifying, transient K' current. When this current component was blocked by Ba2' and Cs+, we observed an inward current through Ca21 channels (15.8±2.2 pA/pF, n=18, measured as Ba21 current) that showed all characteristics of cardiac L-type currents. The activation kinetics were fast, and the current was stimulated by isoproterenol. The effect of isoproterenol was mimicked by forskolin or intracellularly applied cAMP. In radioligand binding experiments, we identified a specific, saturable, stereoselective and reversible high-affinity [3H]-(+)PN 200-110 binding with a dissociation constant Kd=0.53+0.28 nM and a maximal specific binding of Bm.=129.3+16.1 fmollmg protein. There was an additional low-affinity/high-capacity binding site, which is unlikely to be related to a Ca21 channel protein. Signal-transducing G proteins in membranes were characterized by [32P]ADP-ribosylation catalyzed by bacterial toxins and by the use of various antibodies. Cholera toxin substrates of 42 and 45 kd were identified that apparently correlated to Gs a-subunits. Pertussis toxin substrates of 40-41 kd were tentatively identified as Gi aosubunits. The G protein Go was absent or at least extremely low in concentration. (Circulation Research 1991;69:1476-1486)

D uring the last decade, enzymatically dispersed cardiocytes have gained great importance for electrophysiological and biochemical studies (for review see Reference 1). Permanent cell lines are commonly used as model systems for many cell types, but until now studies have not been done on permanent cardiac cell lines. The reason may be the difficulty in establishing such a cell line, because freshly isolated cells are unable to multiply in culture and cardiac tumors are extremely rare.2 Nevertheless, Kimes and Brandt3 succeeded in breeding cells of cardiac origin by selective serial passaging. The established H9c2 cell line is a subclone of the original clonal cell line derived from From the Institut fur Pharmakologie (J.H., D.K., W.R., G.S.), Freie Universitat Berlin, Berlin, Physiologisches Institut der Universitat Bonn (R.M.), Bonn, and II. Physiologisches Institut der Universitat des Saarlandes (S.P.), Homburg/Saar, FRG. Supported by grants from the Deutsche Forschungsgemeinschaft and Sonderforschungsbereich 246 and Fond der Chemischen Industrie. Address for correspondence: Jurgen Hescheler, MD, Institut ffir Pharmakologie, Freie Universitat Berlin, Thielallee 69/73, D-1000 Berlin 33, FRG. Received January 30, 1991; accepted June 26, 1991.

embryonic BDIX rat heart tissue. In their original paper, Kimes and Brandt3 claimed that H9c2 cells had adopted features of skeletal muscle because the cells expressed nicotinic receptors and synthesized a muscle-specific creatine phosphokinase isoenzyme when the mononucleated myoblasts fused. In the present study, we reinvestigated this permanent cardiac cell line, H9c2. We extended the previous investigations of Kimes and Brandt3 by characterizing lectin binding pattern, membrane currents under voltage-clamp conditions, 1,4-dihydropyridine binding sites, and the signal-transducing G proteins. The voltage-dependent Ca2' channel current of these cells showed cardiac-specific characteristics. Therefore, the H9c2 cell line may provide a new tool to study the action of class 4 antiarrhythmic drugs and cardiotonic hormones and neurotransmitters on the Ca2' current. Materials and Methods Culturing Conditions H9c2 cells (ATCC CRL1446, cardiac myoblasts from rat, passage numbers 16-21) purchased from the American Type Culture Collection, Rockville,

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Hescheler et al Characterization of H9c2 From Rat Heart

Md., were grown at a density of about 105 cells/cm2 and cultured as monolayers in Dulbecco's modified Eagle's medium supplemented with fetal calf serum (10% [vol/vol]), glutamine (2 mM), nonessential amino acids (1%), penicillin (100 IU), and streptomycin (100 j,g/ml) under an atmosphere of 5% CO2 in air saturated with water vapor at 37°C. The medium was replaced by fresh medium every 2 days. Light and Electron Microscopy For counting the number of nuclei as well as for determination of lectin binding sites, we used an inverted microscope (IM 35, Zeiss, Oberkochen, FRG) equipped with epifluorescence illumination. Staining of the nuclei was performed on permeabilized and fixed H9c2 cells for 30 minutes in 0.5 mmol/ml 4,6-diamidino-2-phenylindole dihydrochloride (DAPI).4 To characterize the sugar residues in the surface coat, H9c2 cells were incubated with various lectins (50 gtg/ml for 30 minutes at 37°C) that were coupled to rhodamine isothiocyanate (Medac, Hamburg, FRG). For electron microscopy, H9c2 cells were fixed with 2.5% (wt/vol) glutaraldehyde in 100 mM Na+ cacodylate buffer or 0.14 M PIPES for 1 hour at room temperature (pH 7.2), postfixed in 1% (wt/vol) OS04 in 100 mM Na+ cacodylate, dehydrated in a graded series of ethanol, and embedded in Spurr's lowviscosity epoxy resin (ERL-4206, Serva, Heidelberg, FRG). Ultrathin sections (70-100 nm) were cut on an LKB Ultrotome (LKB, Uppsala, Sweden) and stained with 1% (wt/vol) uranyl acetate in 70% (vol/vol) ethanol for 30 minutes at room temperature. The sections were examined using a Siemens 101A electron microscope at 60 or 80 kV.

Electrophysiology For electrophysiological examinations, H9c2 cells were cultured for 2-6 weeks on small glass slides (4x5 mm), until they reached a density of about 103 cells/mm2. The whole glass slide was transferred into a small chamber (volume about 200 ,gl) mounted on an inverted microscope. The chamber was continuously perfused with Tyrode's solution (El) containing (mM) NaCl 140, CaCI2 1.8, MgCl2 1, KCI 5.4, glucose 10, HEPES 10 (pH 7.4) at 36°C. For determination of inward currents, K' currents were blocked by Ba2' and Cs+ in the external solution (E2) containing (mM) NaCl 125, BaCl2 10.8, MgCl2 1, CsCl 5.4, glucose 10, HEPES 10 (pH 7.4) at 36°C. Under these conditions Ba21 was the divalent charge carrier permeating Ca21 channels. Patch electrodes were prepared from Pyrex glass capillaries according to Hamill and coworkers5; the capillaries had an inside tip diameter of 1-3 gm and a resistance of 3-6 MQ when filled with pipette solution I1 containing (mM) K' aspartate 80, KCl 50, MgCl2 1, EGTA 0.1, MgATP 3, HEPES 10 (pH 7.4) or solution 12 containing (mM) CsCl 120, MgCl2 3, MgATP 5, EGTA 10, HEPES 5 (pH 7.4).

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Gigaohm seals were formed by suction with a negative pressure of about -30 cm H20, and wholecell clamp configuration was achieved by disruption of the membrane patch. Under voltage-clamp conditions, cells were step-depolarized from a holding potential (usually -50 mV) to various test potentials (duration, 300 msec; stimulation rate, 0.5 Hz). Ba2+ inward currents through Ca21 channels were determined as peak inward currents, outward currents as current at the end of the 300-msec test pulses. The membrane capacity, measured as current response to a ramp pulse, amounted to 21.1+2.7 pF (mean+SD, n=34). Assuming a cell surface of 1,880 gtm2 (calculated from the microscopically determined cell diameter of spherical cells, 24.5 ±2.8 ,um [mean+SD, n=31], an underestimation by 10-20% has to be taken into account because of membrane enlargements below light microscopical resolution), the specific membrane capacity was 1.2 gF/cm2, which is in quite good agreement with values obtained from other cells.6,7 The input conductance determined as ohmic conductance in the current-voltage relations amounted to 0.5±0.17 nS (mean+SD, n=17, measured under solution E2 supplemented with 5 mM Ni2' and 1 ,uM tetrodotoxin). The following pharmacological tools were used: D600 (1 ,uM; Knoll, Ludwigshafen, FRG), PN 200110 (1 ,M; Sandoz, Basel, Switzerland), Bay K 8644 (1 ,M; Bayer, Leverkusen, FRG), w-conotoxin (0.1 ,uM; Bissendorf, Hannover, FRG); isoproterenol (0.1 ,M; Sigma, Deisenhofen, FRG), and forskolin (1

bLM; Calbiochem, Frankfurt, FRG). Preparation of Membranes and Binding Studies H9c2 cells were harvested with a cell scraper, collected by centrifugation at 600g (4°C, 20 minutes), and disrupted by nitrogen cavitation in a buffer consisting of (mM) NaCl 100, EDTA 0.5, KH2PO4 50 (pH 7.0). Immediately after cell disruption, the EDTA concentration was increased to 3 mM, and 2-mercaptoethanol was added to a final concentration of 15 mM. Nuclei were removed by short centrifugation at 1,000g (4°C), and sedimentation of membranes was achieved by centrifugation at 30,000g (4°C, 15 minutes). Total protein was determined according to Peterson.8 Membranes were stored at -70°C in 10 mM triethanolamine-HCl buffer (pH 7.4). Dihydropyridine binding assays were performed according to Glossmann and Ferry9 with modifications. For the saturation experiments, membranes were incubated (37°C, triplicates) in 0.2 ml of TrisHCI (50 mM, pH 7.4) buffer containing CaCI2 (1 mM), MgCI2 (1 mM), and increasing concentrations of [3H](+)PN 200-110. For competition experiments, increasing concentrations of nonradioactive 1,4-dihydropyridines were added in the presence of 0.25 nM [3H]-(+)PN 200-110. When the binding equilibrium was reached after 30 minutes, 2 ml ice-cold buffer (20 mM Tris-HCl, 10% [wt/vol] polyethylene glycol 6000, 10 mM MgCL2) was added to each tube, and samples

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Circulation Research Vol 69, No 6 December 1991

were rapidly filtered under vacuum through Whatman GF/B glass fiber filters. Filters were washed twice and dried under vacuum; the radioactivity was measured by liquid scintillation counting. Nonspecific binding was determined in the presence of 2 ,M (+)PN 200-110. The specific binding was at least 80% of the total binding. Curves represent best fits as calculated by the iterative, nonlinear least-squares method of De Lean and coworkers.10

[32P]ADP-Ribosylation Pertussis toxin- and cholera toxin-catalyzed [32P]ADP-ribosylation of G proteins, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography of gels were performed as describedl1 l3with the following modifications. Pertussis toxin was preactivated in the presence of 1 mM ATP, and the incubation mixture contained 0.1% (wt/vol) Lubrol PX. To resolve subtypes of G protein a-subunits, polyacrylamide slab gels were composed of 9.6% (wt!vol) acrylamide, 0.25% (wtlvol) bisacrylamide, and 4 M urea.14

Immunoblotting Experiments Sodium dodecyl sulfate-polyacrylamide slab gels used for immunoblots were composed as those used to separate [32P]ADP-ribosylated proteins (see above). Immunoblotting, detection of filter-bound antibodies with `251-protein A, and autoradiography of nitrocellulose filters were performed as described.11,15 The sequences of peptides used for immunization, their coupling to keyhole limpet hemocyanine, immunization of rabbits, and the characterization of antisera generated against these peptides (with G proteins purified from various tissues) have been described elsewhere.12'13 Results

Light Microscopy Studies H9c2 cells grown in culture dishes or on glass coverslips developed different forms: polygonal (Figures 1C-lE) and spindle-shaped flat cells, which were attached to the bottom at different spots, as well as spherical (Figures 1A, 1C, and 1E) and angular cells (Figure 1B), which were located above the level of the flat cells and in contact to the bottom by a few long cytoplasmic filopodia. The spindle-shaped cells were frequently arranged in parallel. All cells were mechanically quiescent. Evaluation of DAPI-stained cells revealed 95.3% mononucleated, 4.3% binucleated, 0.35% trinucleated, and 0.05% tetranucleated cells (n=4,033). Most nuclei appeared spherical and contained one to several nucleoli (Figures 1D and 1E; see also Figure 2A). For characterization of the sugar residues of the cell surface, lectins were tested for their ability to bind to

the cells (Table 1). Since both Limaxflavus agglutinin

(Figure 1B) and wheat germ agglutinin (Figure 1C) labeled the cells, sialic acid appears to be a prominent sugar residue. Fluorescence comparable to that achieved with Limax fiavus agglutinin was observed

with concanavalin A (Figure 1A) and Ricinus communis agglutinin-I (not shown), indicating the presence of a-mannose and galactose. The lectins Dolichos biflorus agglutinin, soybean agglutinin, peanut agglutinin, and Ulex europaeus agglutinin-I failed to bind to the cells (Figure 1E). In addition to the homogeneous binding to the cell surface, a spotted fluorescence was obtained in the case of succinylated wheat germ agglutinin and Ricinus communis agglutinin-1 (Figure 1B). In general, the spherical cells exhibited a brighter fluorescence than the polygonal ones, possibly because of differences in geometry. Electron Microscopy Studies At low magnification, isolated H9c2 cells of polygonal type appeared in the cross section widely spread with numerous protrusions (Figure 2A). Cells grown in a confluent layer overlapped in their peripheral parts (Figure 2D) and formed two types of junctions: 1) the membranes of neighboring cells were indented and ran in parallel at a distance of less than 40 nm (not shown); and 2) the membranes were attached to each other (around 14 nm distance), forming spot desmosome-like contacts. Gap junctions were not observed (280 studied sections). The most prominent cell organelles were spherical or lobed nuclei (Figure 2A). In all cell types except the spherical ones, the cytoplasm was interlaced by a network of stress fibers16 (Figure 2B) attached to the cell membrane. Another element of the cytoskeleton was microtubules, which were sometimes arranged as a dense network (Figure 2F). The cells were rich in mitochondria and rough endoplasmic reticulum. Both were often parallel to each other (Figure 2G). Golgi cisternae were regularly found near the cell nucleus. The surface membrane was often enlarged by microvilli (Figure 2C). On the extracellular side, the cell membrane was partially covered by a surface coat (Figure 2H). Caveolae were absent.

Membrane Currents Membrane currents of H9c2 cells were recorded in the whole-cell configuration during voltage-clamp pulses from - 50 mV to various test potentials. With the external solution El and the internal solution I1, cells exhibited outwardly rectifying currents, which developed a transient inactivation during the 300-msec test pulse. The outward current-voltage relation could be approximated by two straight lines with slope conductances of 0.6 and 7.2 nS, negative and positive to -20 mV, respectively (results not shown; for further characterization see Reference 17). No inwardly rectifying K' current could be detected. Inward currents through voltage-dependent Ca2' channels were determined as Ba2` currents under blockade of K' currents (solutions E2 and 12) and Na+ currents (0.1 ,uM tetrodotoxin). H9c2 cells that were cultured for several days on glass coverslips showed no voltage-dependent current under these conditions. The only detectable current was an ohmic component that may have been due to nonspecific cation channels (see

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Hescheler et al Characterization of H9c2 From Rat Heart

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(left) and fluorescence (right) images are shown. Pancl A: Isolated spherical cell. Panel B: Isolated angular cell. Panels C and D: Groups of various cells in different forms. Panel E: Densely grown cell layer with a single spherical cell (arrowhead). Binding of lectins is shown in the fluorescence images: concanavalin A (panel A), Limax flavus agglutinin (panel B), wheat germ agglutinin (panel C), succinylated wheat germ agglutinin (panel D), and Ulex europaeus agglutinin-I (panel E). The bars represent 20 pn. The magnification ofpanels B, D, and E is the same as in panel C. various

Reference 17). However, if the cells were cultured for longer periods of time (e.g., several weeks), voltagedependent calcium current became detectable, suggesting that some kind of autocrine factor is necessary for the expression of this type of channel. The shape of currents (Figure 3A) was similar to that observed in freshly prepared ventricular cardiocytes from the guinea pig.18 The activation occurred with a fast time course (1-2 msec) in contrast to the time course of activation in skeletal muscle (about 50 msec; see Ref-

19 and 20). The inactivation time constant was about 150 msec at 10 mV. Currents recorded at potentials positive or negative to this maximum declined with a slower time course. The current-voltage relation was bell shaped (Figure 3B). The current had an apparent threshold at -28.6t18 mV, a maximum at 8.3±+1.9 mV, and an apparent reversal potential at 46.1 2.2 mV (n=18; estimated according to Reference 21). The maximum mean peak current density (measured at 10 mV) was 15.8±2.2 pA/pF (n=18). The steady-state erences

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±

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Circulation Research Vol 69, No 6 December 1991 TABLE 1. Binding of Various Lectins to H9c2 Cells

Lectin Concanavalin A Dolichos biflorus agglutinin Limax fiavus agglutinin Peanut agglutinin Ricinus communis agglutinin-I

Soybean agglutinin Ulex europaeus agglutinin-I Wheat germ agglutinin

Specificity a-mannose, a-glucose a-N-acetylgalactosamine N-acetylneuraninic acid

Binding + +

galactosyl(P-1,3)Nacetylgalactosamine galactose

+

a/P-N-acetylgalactosamine

*

L-fructose

(N-acetylglucosamine)n

++

sialic acids Succinylated wheat germ agglutinin (N-acetylglucosamine), + +, Strong; +, medium; +, weak; - no binding. About 100 cells were analyzed for each lectin. *No distinct fluorescent signal from the membrane, only faint fluorescence from the whole cell body.

inactivation curve was determined by clamping the cells from various prepotentials to the test potential of 0 mV. The inward currents measured during the test pulse were normalized to the maximum current and plotted versus the holding potential (Figure 3D, triangles). Original recordings for current tracings are shown in Figure 3C. The average inactivation curve measured from 18 cells showed a half-maximal value at -21.3 mV and a steepness of 7.2. From the data shown in panel B, the average activation curve of the inward current was evaluated and normalized in the same way as the inactivation curve (Figure 3D, triangles). It reached 50% at -3.6 mV and had a steepness of 6.9.

Effects of Ca21 Channel Blockers and

f3Adrenergic Agonists The inward current of H9c2 cells was further characterized using pharmacological tools (mean+SD of the Ba21 current densities are summarized in Table 2). In the experimental results shown in Figure 4, cells were voltage-clamped from -50 to 0 mV. Superfusion with the inorganic Ca2' channel blocker Cd2` (0.1 mM) resulted in a complete blockage of inward currents (panel A). Similar effects were seen with 0.1 mM Ni2' and 1 mM Co24 (not shown). A Ca24 channel blocker of the phenylalkylamine type, D600 (1 ,uM), and a Ca2' channel blocker of the dihydropyridine type, PN 200-110 (1 ,uM), partially reduced the inward current (panels C and D). An increase was seen with 1 ,uM Bay K 8644, a Ca24 channel agonist of the dihydropyridine type (panel B). (o-Conotoxin, which was previously reported to block N-type Ca24 channels of neuronal but not L-type Ca24 channels of muscular origin,22-24 did not affect the Ba24 current of H9c2 cells (0.1 ,uM; see Table 2). The L-type Ca24 current of cardiac cells is augmented by ,B-adrenergic agonists via cAMP-dependent phosphorylation (for review see Reference 25). To test whether the current through Ca24 channels in H9c2 cells is also sensitive to ,3-adrenergic stimulation, cells were superfused with 0.1 ,uM isoproterenol (Figure 5, left panel). Isoproterenol caused about a twofold incre-

+

ment of the inward current within 2 seconds. The effect was fully reversible after washing out the agonist. In two of 27 cells, a reversible inhibition of the Ba24 inward currents was observed. The reason for this

inhibitory effect is unknown. To elucidate whether cAMP is involved in the ,-adrenergic stimulation of the Ba24 current in H9c2 cells, the adenylyl cyclase stimulator forskolin26,27 (1 ,uM) was applied from the outside (Figure 5, right panel). The observed increment was comparable to that caused by isoproterenol. An increment of the inward currents was also observed when cAMP (0.1 mM) was intracellularly infused via the patch pipette (see Table 2). At the applied concentrations, the effects of isoproterenol, forskolin, and cAMP were not additive. 1, 4-Dihydropyridine Binding Sites Among radiolabeled 1,4-dihydropyridines, which are commonly used to determine specific binding to the a1-subunit of voltage-dependent Ca24 channels, [3H]-(+)PN 200-110 is known as a radioligand with high affinity for the dihydropyridine-sensitive Ca2+ channels, especially in heart (Kd.l nM) and brain and with lower affinity in skeletal muscle.9,28,29 In saturation experiments with [3H]-(+)PN 200-110, we detected high- and low-affinity binding sites (Figure 6A) with Bmax values of 129.3+16.1 and 986.3+113.0 fmol/mg protein and Kd values of 0.53+0.28 and 31.7±2.0 nM, respectively (mean+SEM, n=4). In competition experiments with 0.25 nM [3H]-(+)PN 200-110 (Figure 6B), we measured IC50 values of 0.31+0.16, 87.0+8.6, and 6.7±1.4 nM for (+)PN 200-110, (-)PN 200-110, and nimodipine, respectively. We calculated an affinity ratio of 280 for (-)PN 200-110 versus (+)PN 200-110 and of 22 for nimodipine versus (+)PN 200-110. Similar highaffinity Kd and Bmax values and affinity ratios for the (+) and (-) isomers of PN 200-110 have been reported for rat heart membranes and rat heart primary cell cultures.3031 Pattern of G Proteins We also investigated the pattern of membranous signal-transducing heterotrimeric G proteins.32 Their

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Hescheler et al Characterization of H9c2 From Rat Heart

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4~~~~~~~~~~~~~~~~~~~~~~~~~~~54 FIGURE 2. Transmission-electron microscopic images of H9c2 cells. Panel A: Section through the cell body of a flattened cell; prominent elements are the nucleous with nucleoli (n), mitochondria (dark spots), vacuoles, and cytoskeletal fibers. Bar, 5 p.m. Panel B: Cytoskeletal fibers. Bar, 1 pm. Panel C: Microvilli on the cell surface. Bar, 1 um. Panel D: Cross section through a densely grown cell layer. The upper and lower cells are connected by a mechanical cell contact (arrowhead). Bar, 1 gm. Panel E: Mechanical cell contact shown at higher magnification. Bar, 0.2 pm. Panel F: In some regions of the cells numerous microtubules appear as a network of thin lines. Bar, 2 p.m. Single microtubules are illustrated in the inset (bar, 0.1 ,m). Panel G: Rough endoplasmic reticulum is often in parallel with mitochondria. Bar, 1 pm. Panel H: The membrane is partly covered by a fuzzy coat. Bar, 0.6 pm.

a-subunits are substrates of ADP-ribosylating bacterial toxins (i.e., cholera toxin and pertussis toxin). In H9c2 cell membranes, cholera toxin mainly [32P]ADP-ribosylated a 42-kd protein and much less

a 45-kd protein. In cholate extract from brain membranes used for comparison, cholera toxin [32P]ADPribosylated equally well 45- and 42-kd proteins. The data suggest that H9c2 cells contain two forms of the

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Circulation Research Vol 69, No 6 December 1991

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A

B !-20,- 40;-60FIGURE 3. Electrophysiological characterization of the Ca2' inward current. Panel A: Recordings of inward currents during 200-msec voltage-clamp pulses from -80 mVto the indicated test potentials. The dotted line corresponds to zero current. Panel B: Current-voltage relation of the Ba21 inward current. Mean ±SD are shown (n=18). Panel C: Original recordings of inward currents measured during voltage-clamp pulses from various holding potentials to 0 mV Panel D: Steady-state activation (circles) and inactivation (trtiangles) curves of 18 cells are shown (mean ±SD). Inward currents were measured during 200-msec voltage-clamp pulses from -80 mVto the test potentials varying from -60 to 50 mV

010.2 +10 nA

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D

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G, a-subunit.33 Pertussis toxin catalyzed [32P]ADPribosylation of a 40/41-kd doublet in H9c2 cell and brain membranes. Only the latter preparation contained a 39-kd pertussis toxin substrate. For identification of pertussis toxin substrates, the two preparations were probed with antisera generated against synthetic peptides that were derived from sequences of cDNAs encoding G protein a-subunits (Figure 7). Since modification of G protein a-subunits by bacterial toxins changes their mobility in ureacontaining sodium dodecyl sulfate gels,14 the relative molecular masses of [32P]ADP-ribosylated G protein a-subunits (see Figure 8) differ from those of the unmodified a-subunits detected by the various antibodies (see Figure 7). The acoon peptide antiserum, which reacts with a-subunits of Gi, GO, and Gs, recognized 40-kd proteins in membranes of H9c2 cell and 3941-kd proteins in cholate extract from membranes of porcine brain. The a,common antiserum did not detect

proteins corresponding to the 42- and 45-kd cholera toxin substrates; this observation may be due to the low abundance of GS a-subunits compared with that of GJQ, a-subunits. The a1i,,om0m peptide antiserum, which recognizes the a-subunits of Gi1, Gc, or G3, recognized 40-kd proteins in membranes of H9c2 cells and in cholate extract from membranes of porcine brain. The finding that the A,cmmon peptide antiserum and the aW peptide antiserum failed to detect a doublet that may account for the 40/41-kd pertussis toxin substrates found in either preparation is most likely due to the poor resolution of these proteins. An

-Cd A _-9, .

pA

TABLE 2. Modification of the Ca2' Current of H9c2 Cells by Various Agents

Ca2' Before drug application

applied.

Con

A

50ms

J

}100

Con

current

After drug Concentration application 1.0 g.M 17.5±+1.2 29.1+2.1 (n=5) 0.1 mM 15.7+2.1 Cd2+ 0.4+0.8 (n=5) 0.6 +0.3 (n=4) 1.0 ,uM 15.0+2.4 D600 1.0 gM 14.1+1.9 0.9+0.4 (n=6) PN 200-110 0.1 ,.M 14.6+2.2 13.7±2.1 (n=6) co-CT 0.1 ,uM 12.9+1.6 25.0+1.5 (n=7) ISP 1.0 ,uM 16.5+1.6 30.6+1.5 (n =3) Forsk 0.1 mM 13.9±2.3 25.9±2.1 (n=4) cAMP (intra) Values (given in pAlpF) represent mean+SD. o-CT, co-conotoxin; ISP, isoproterenol; Forsk, forskolin; intra, intracellularly

Agent Bay K 8644

D0

100

B

D_ 100

pA

PN 200-1

100 pA

i Con

FIGURE 4. Pharmacological characterization of Ca2' inward current. Pulses from -50 to 0 mV were applied. Dotted lines correspond to zero current. Con, control currents. Cells were superfused with Cd2` (0.1 mM, panel A), Bay K 8644 (1 /m, panel B), D600 (1 gM, panel C), or PN 200-110 (1 M, panel D). Tetrodotoxin (0.1 M) was present in all experiments.

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Hescheler et al Characterization of H9c2 From Rat Heart

pA0

pA:

Con ISP

Con

Forsk. u-i

lOOms FIGURE 5. Modulation of Ca2' inward current by a f-adrenergic agonist and forskolin. Dotted lines correspond to zero current. Holding potential was -50 mV An approximately twofold increment occurred in the presence of isoproterenol (0.1 ,MtM, ISP, left panel) or forskolin (1 gI, Forsk, right

panel). Con, control currents.

a% peptide antiserum, which specifically reacts with the 40-kd G2 a-subunit, detected a 40-kd protein in either preparation. The a0 peptide antiserum, which specifically reacts with the two forms of the Go a-subunit, failed to detect proteins in the H9c2 membranes but clearly detected proteins of 39 and 40 kd in brain cholate extract. Discussion In the present study we show that H9c2 cells lack morphological properties of freshly prepared cardiocytes. The cells did not express gap junctions, caveolae, T tubules, or myofibrils with organized sarcomeres, but were rich in rough endoplasmic reticulum, and the cell surface was often enlarged by microvilli. H9c2 cells did not form confluent, multinuclear cells (myotubes) as were observed regularly by Kimes and Brandt.3 Since the cell density was high enough to allow formation of myotubes, the difference between our cells and those of Kimes and Brandt3 may point to a dedifferentiation of this cell line caused by increased passage number. Whether the dedifferen-

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1483

tiation of H9c2 cells is a progressive process or arrested at this stage needs to be demonstrated by further studies. To classify H9c2 cells by morphological criteria, they have to be compared with other types of cultured heart cells. According to the literature, cardiocytes of the adult, neonatal, or embryonic heart undergo various changes when cultured for longer periods of time (see, e.g., References 34 and 35). Soon after the isolation procedure heart cells transform from rod-shaped to rounded cells with disorganized ultrastructural features, that is, sarcomeric arrangement is lost, former Z lines appear as focal densities, and mitochondria have compact cristae and are often found in the numerous blebs at the surface. In addition, microvilli are found on the surface of these cells. After the fifth day in culture, nontransformed cells start to reorganize ultrastructure; the cells spread out and build up new myofibrils with complete sarcomeres. This is accompanied by formation of new intercalated disks and T tubules. During this phase of redifferentiation, embryonic cultured cardiocytes36,37 build up stress fibers that are progressively replaced by myofibrils. In our study, H9c2 cells did not develop myofibrils but only stress fibers. Thus, the only cultured cells of mammalian heart comparable to H9c2 cells are immature embryonic cardiocytes with stress fibers. It may be concluded that H9c2 cells have lost nearly all morphological characteristics of cultured cardiocytes and have dedifferentiated to a high degree. In contrast to these discrepancies between H9c2 and freshly prepared cardiocytes, the composition of the surface coat is greatly preserved. The lectins soybean agglutinin and Dolichos biflorus agglutinin, known to preferentially bind to the cell poles of adult

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FIGURE 6. 1,4-Dihydropyridine binding sites. Panel A: Specific [3H]-(+)PN 200-110 binding to membranes of H9c2 cells. The inset shows a Scatchard analysis. B, bound; F, free. Panel B: Inhibition of [3HI-(+)PN200-110 binding by (+)PN 200-110 (filled circles), (-)PN 200-110 (open circles), and nimodipine (triangles). One offour experiments which yielded similar results is shown. Values represent mean ±SEM of triplicates. Downloaded from http://circres.ahajournals.org/ at University of Virginia on June 26, 2014

Circulation Research Vol 69, No 6 December 1991

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ai

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FIWURE 7. G protein a.subunits in membranes of H9c2 cells and cholate extract from porcine brain membranes. H9c2 cell membranes (150 ggprotein) or cholate extract from porcine brain membranes (30 ggprotein) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresks in the presence of urea and transferred onto nitrocellulose filters. Filters were incubated with an ac0,mm0npeptide antivermm AS8 (acom..Q. an a- (mmvop peptide antiserum AS19 (a, aan a,2 peptide antiserum AS64 (a,), and an a,, peptide antiserum AS6 (a,,). Dilutions of antisera were 1: 150 to 1 :300. After the incubation with 1251-protein A, filter-

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current, which is characterized by a slow time course of inactivation (using Ba` as charge carrier) and by its sensitivity to organic Ca` channel blockers of the dihydropyridine and phenylalkylamine type.1 Furthermore, the open probability is enhanced by f-adrenergic agonists acting via G, and cAMP-dependent phosphorylation (for review see Reference 25). All these properties of cardiac Ca> channels were found in H9c2 cells, opposing the statement of Kimes and Brandt3 that H9c2 cells have adopted all features of

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proteins in the 40-kd region was blocked or considerably reduced if the antisera had been incubated with the respective peptide (5 vg/ml) used for immunization. The experiment was performed in duplicate. DF, dye front.

guinea pig cardiocytes, did not bind to adult rat cardiocytes nor to H9c2 cells. Similarly, the reaction of peanut agglutinin was negative in both H9c2 cells and freshly isolated adult rat heart cells.38 The only difference occurred in the case of Ulex europaeus agglutinin-I, which binds to isolated rat cardiocytes to a small extent but failed to bind to H9c2 cells. Another cell type-specific parameter is voltagedependent Ca21 channels (for review see References 39 and 40). Cardiac cells exhibit an L-type Ca2+

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autoradiography. One of three experiments that yielded comparable results is shown. As was tested in various systems (membranes and purified pro-

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FIGURE 8. Cholera and pertussis toxin substrates in H9c2 cell membranes and cholate extract from porcine brain membranes. Cholate extract from porcine brain membranes was prepared as described. 1" H9c2 cell membranes (100 gg protein) or cholate extract from porcine brain membranes (30 gg protein) were incubated with ['2P]NAD and, if indicated, with cholera toxin (CT) orpertussis toxin (PT). [32P/ADP-ribosylated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of urea and visualized by autoradiography. One of three experiments that yielded comparable results is shown. DF, dye front.

+

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Hescheler et al Characterization of H9c2 From Rat Heart

a skeletal muscle cell line. The L-type Ca2+ current of skeletal muscle activates more than one order of magnitude more slowly and inactivates very slowly with a time constant of about 1 second.41 The difference between the Ca2' current of cardiac and skeletal muscle has been related to the different mechanisms of depolarization-contraction coupling. Whereas in cardiac cells the L-type Ca2+ current represents the major pathway for entry of extracellular Ca2+ and subsequently triggers the release from the sarcoplasmic reticulum, depolarization of the skeletal muscle releases Ca2+ from the sarcoplasmic reticulum without the requirement for entry of extracellular Ca2`. The dihydropyridine-sensitive L-type Ca2' current of other cell types, such as smooth muscle, neuronal, and endocrine cells, exhibits kinetics similar to that found in H9c2 cells but lacks a regulation by cAMP-dependent phosphorylation.39O40 A specific feature of the neuronal and endocrine Ca2' current is its sensitivity toward (a-conotoxin.22-24 Another argument for the similarity of H9c2 cells to cardiocytes comes from binding studies. 1,4-Dihydropyridine binding sites showed all heart-specific characteristics, that is, much lower capacity and higher affinity than in skeletal muscle tissues.28 The use of the enantiomeric pair of (+) and (-)PN 200-110 in our binding assays reveals strong stereoselectivity for the pure radioligand [3H]-(+)PN 200110. Low-affinity binding sites for the 1,4-dihydropyridines, some of which are discussed to be functionally relevant in heart, have been reported by several laboratories.42,43 However, in saturation experiments, we found that EGTA (1 mM) added to the incubation medium or heating of membranes (56°C, 10 minutes) strongly diminished the [3H](+)PN 200-110 high-affinity binding site but did not affect the Kd or Bmax values of the low-affinity binding site (unpublished data). Therefore, we consider the high-affinity [3H]-(+)PN 200-110 binding site reported here as the 1,4-dihydropyridine-sensitive Ca24 channel.4445 From their pattern of G proteins, H9c2 cells show all characteristics of a striated muscle cell.46 H9c2 cells possess two forms of the G, a-subunit and two forms of the Gi a-subunit, one of which apparently corresponds to the a-subunit of GQ2. G, plays an important role in the signal transduction of cardiotonic hormones since G, proteins functionally couple ,B-adrenergic receptors to adenylate cyclase and possibly also directly to Ca2+ channels.25,32 G proteins of the Gi family are involved in the hormonal inhibition of adenylate cyclase and stimulation of K+ channels in atrial cardiocytes. H9c2 cells lack GO, which is apparently involved in the inhibition of Ca2+ current in neuronal and endocrine cells (for review see Reference 47). In line with this observation we found no hormone or neurotransmitter (e.g., acetylcholine, epinephrine, somatostatin) that significantly reduced the amplitude of the Ca2+ channel current in H9c2 cells. The data are consistent with

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the finding that adult cardiac myocytes from rat do not (or at very low levels) express G0.46,48 In conclusion, the present study demonstrates that H9c2 cells show morphological characteristics similar to those of immature embryonic cardiocytes but have preserved several elements of the electrical and hormonal signal pathway found in adult cardiac cells. Therefore, this cell line may be useful as a model for cardiocytes in aspects of transmembrane signal transduction. Acknowledgments Part of the experiments were performed in the laboratory of Dr. Trautwein (Homburg/Saar). We thank Dr. T. Plant (Homburg/Saar) for carefully reading the manuscript and competent discussion. The skillful technical work of D. Steimer, M. Bigalke, and I. Reinsch is also acknowledged.

References 1. Piper M, Isenberg G (eds): Isolated Adult Cardiomyocytes. Boca Raton, Fla, CRC Press, 1989, vols I and II 2. Auclair MC, Freyss-Beguin M: Heart cells in culture: Methods and applications. Biol Cell 1980;37:95-208 3. Kimes BW, Brandt BL: Properties of a clonal muscle cell line from rat heart. Exp Cell Res 1976;98:367-381 4. Williamson DH, Fennel DJ: The use of fluorescent DNAbinding agent for detecting and separating yeast mitochondrial DNA, in Prescott D (ed): Methods in Cell Biology. New York, Academic Press, Inc, 1974, vol 12, pp 335-351 5. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth J: Improved patch-clamp technique for high resolution current recording from cells and cell free membrane patches. Pflugers Arch 1981;391:85-100 6. Isenberg G, Klockner U: Calcium tolerant ventricular myocytes prepared by preincubation in a "KB medium." Pflugers Arch 1982;395:6-18 7. Rorsman P, Trube G: Calcium and delayed potassium currents in mouse pancreatic ,B-cells under voltage clamp conditions. J Physiol (Lond) 1986;381:531-550 8. Peterson GL: Determination of total protein. Methods Enzymol 1983;91:95-119 9. Glossmann H, Ferry DR: Assay for calcium channels. Methods Enzymol 1985;109:513-550 10. Lean De A, Munson PJ, Rodbard D: Simultaneous analysis of families of sigmoidal curves: Application to bioassay, radioligand assay, and physiological dose-response curves. Am J Physiol 1978;235:E97-E102 11. Rosenthal W, Koesling D, Rudolph U, Kleuss C, Pallast M, Yajima M, Schultz G: Identification and characterization of the 35-kDa , subunit of guanine nucleotide-binding proteins by an antiserum raised against transducin. Eur J Biochem 1986;158:255-263 12. Rosenthal W, Hescheler J, Hinsch K-D, Spicher K, Trautwein W, Schultz G: Cyclic AMP-independent, dual regulation of voltage-dependent Ca2' currents by LHRH and somatostatin in a pituitary cell line. EMBO J 1988;7:1627-1633 13. Hinsch K-D, Rosenthal W, Spicher K, Binder T, Gausepohl H, Frank R, Schultz G, Joost HG: Adipocyte plasma membranes contain two Gi subtypes but are devoided of G.. FEBS Lett 1988;238:191-196 14. Ribeiro-Neto FAP, Rodbell M: Pertussis toxin induces structural changes in Ga proteins independently of ADPribosylation. Proc Natl Acad Sci U S A 1989;86:2577-2581 15. Offermanns S, Schafer R, Hoffmann B, Bombien E, Spicher K, Hinsch KD, Schultz G, Rosenthal W: Agonist-sensitive binding of a photoreactive GTP analog to a G-protein a-subunit in membranes of HL-60 cells. FEBS Lett 1990;260:14-18 16. Dlugosz AA, Antin PB, Nachmias VT, Holtzer H: The relationship between stress fiber-like structures and nascent myo-

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fibrils in cultured cardiac myocytes. J Cell Biol 1984;99: 2268-2278 Sipido K, Marban E: L-type calcium channels, potassium channels, and novel nonspecific cation channels in a clonal muscle cell line derived from embryonic rat ventricle. Circ Res 1991;69:1487-1499 McDonald T, Cavalie A, Trautwein W, Pelzer D: Voltagedependent properties of macroscopic and elementary calcium channel current in guinea pig ventricular myocytes. Pflugers Arch 1986;406:437-447 Tanabe T, Mikami A, Numa S, Beam K: Cardiac-type excitation-contraction coupling in dysgenic skeletal muscle injected with cardiac dihydropyridine receptor cDNA. Nature 1990; 344:451-453 Cognard C, Lazdunski M, Romey G: Different types of Ca' channels in mammalian skeletal muscle cells in culture. Proc Natl Acad Sci U S A 1986;83:517-521 Isenberg G, Klockner U: Calcium currents of isolated bovine ventricular myocytes are fast and of large amplitude. Pflugers Arch 1982;395:30-41 Rivier J, Galyean R, Gray WR, Azimi-Zonooz A, McIntosh JM, Cruz LJ, Olivera BM: Neuronal calcium channel inhibitors: Synthesis of w-conotoxin GVIA and effects on 45Ca uptake by synoptosomes. J Biochem 1987;262:1194-1198 McCleskey EW, Fox AP, Feldman DH, Cruz LJ, Olivera BM, Tsien RW, Yoshikami D: w-Conotoxin: Direct and persistent blockade of specific types of calcium channels in neurons but not muscle. Proc Natl Acad Sci U S A 1987;84:4327- 4331 Feldman DH, Olivera BM, Yoshikami D: Omega Conus geographus toxin: A peptide that blocks calcium channels. FEBS Lett 1987;214:295-300 Trautwein W, Hescheler J: Regulation of cardiac L-type calcium current by phosphorylation and G-proteins. Annu Rev Physiol 1990;52:257-274 Daly J: Forskolin, adenylate cyclase, and cell physiology: An overview. Adv Cyclic Nucleotide Res 1984;17:81-89 Seamon K, Daly J: Forskolin: Its biological and chemical properties. Adv Cyclic Nucleotide Prot Phos 1986;20:1-150 Glossmann H, Striessnig J: Structure and pharmacology of voltage-dependent calcium channels. ISI Atlas Sci Pharmacol 1988;2:202-210 Godfraind T, Miller R, Wibo M: Calcium antagonism and calcium entry blockade. Pharmacol Rev 1986;38:321-416 Lee RL, Roeske WR, Yamamura HI: High affinity specific [3H](+)PN200-110 binding to dihydropyridine receptors associated with calcium channels in rat cerebral cortex and heart.

Life Sci 1984;33:721-732 31. Kokobun S, Prod'hom B, Becker C, Porzig H, Reuter H: Studies on Ca channels in intact cardiac cells: Voltage dependent effects and cooperative interactions of dihydropyridine enantiomers. Mol Pharmacol 1986;30:571-584 32. Birnbaumer L, Abramowitz J, Brown A: Receptor-effector coupling by G proteins. Biochim Biophys Acta 1990;1031: 163-224 33. Gilman A: G proteins: Transducers of receptor-generated signals. Annu Rev Biochem 1987;56:615-649

34. Moses RL, Claycomb WC: Disorganization and reestablishment of cardiac muscle cell ultrastructure in cultured adult rat ventricular muscle cells. J Ultrastruct Res 1982;81:358-374 35. Nag AC, Cheng M, Fischmen DA, Zak R: Long-term cell culture of adult mammalian cardiac myocytes: Electron microscopic and immunofluorescent analyses of myofibrillar structure. J Mol Cell Cardiol 1983;15:301- 317 36. Legato JM: Ultrastructural characteristics of the rat ventricular cell growth in tissue culture, with special reference to sarcomerogenesis. J Mol Cell Cardiol 1972;4:299-317 37. Perissel B, Charbonne F, Moalic JM, Malet P: Initial stages of trypsinized cell culture of cardiac myoblasts: Ultrastructural data. J Mol Cell Cardiol 1980;12:63-75 38. Stegemann M, Meyer R, Haas HG, Robert-Nicoud M: The cell surface of isolated cardiac myocytes: A light microscope study with use of fluorochrome coupled lectins. J Mol Cell Cardiol 1990;22:787-803 39. Tsien RW, Lipscombe D, Madison DV, Bley KR, Fox AP: Multiple types of neuronal calcium channels and their selective modulation. Trends Neurosci 1988;11:431-437 40. Bean BP: Classes of calcium channels in vertebrate cells. Annu Rev Physiol 1989;51:367-384 41. McCleskey EW: Calcium channels and intracellular calcium release are pharmacologically different in frog skeletal muscle.

JPhysiol (Lond) 1985;361:231-249 42. Hamilton SL, Yatani A, Brush K, Schwartz A, Brown AM: A comparision between the binding and electrophysiological effects of dihydropyridines on cardiac membranes. Mol Pharmacol 1986;31:221-231 43. Vaghy PL, Galube I, Grupp G, Grupp J, Williams J, Baik J, Schwartz A: A proposed pharmacological role for dihydropyridine binding sites in heart and coronary smooth muscle, in Fleckenstein A, Van Breeran C, Grob R, Hoffmeister F (eds): Bayer-Symposium IX: Cardiovascular Effects of DihydropyridineType Calcium-Antagonists and Agonists. Berlin, SpringerVerlag, 1985, pp 156-184 44. Glossmann H, Zernig G, Graziadei I, Moshammer T: Non L-type Ca2' channel linked receptors for 1,4-dihydropyridines and phenylalkylamines, in Traber J, Gispen WH (eds): Nimodipine and Central Nervous System Function: New Vistas: International Workshop, Cologne, FRG, April 12-14. Stuttgart/New York, Schattauer-Verlag, 1989, pp 51-67 45. Zernig G: Widening potential for Ca2' antagonists: Non-Ltype Ca21 channel interaction. Trends Pharmacol Sci 1990;11: 38-44 46. Asano T, Semba R, Kamiya N, Ogasawara N, Kato K: G0, a GTP-binding protein: Immunochemical and immunohistochemical localization in the rat. J Neurochem 1988;50: 1164-1169 47. Schultz G, Rosenthal W, Hescheler J, Trautwein W: Role of G proteins in calcium channel modulation. Annu Rev Physiol 1990;52:275-292 48. Asano T, Kamiya N, Semba R, Kato L: Ontogeny of the GTP-binding protein G. in rat brain and heart. J Neurochem 1988;51:1711-1716 KEY WORDS * cardiocytes * H9c2 cells * lectins * glycokalix * calcium current * dihydropyridine binding * G proteins

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