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Neonatal rat cardiac fibroblasts express three types of voltage-gated K⁺ channels: regulation of a transient outward current by protein kinase C

Department of Pharmacology, Physiology, and Neuroscience, University of South Carolina, School of Medicine, Columbia, South Carolina

Submitted 15 October 2007 ; accepted in final form 12 December 2007

	ABSTRACT

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Cardiac fibroblasts regulate myocardial development via mechanical, chemical, and electrical interactions with associated cardiomyocytes. The goal of this study was to identify and characterize voltage-gated K⁺ (Kv) channels in neonatal rat ventricular fibroblasts. With the use of the whole cell arrangement of the patch-clamp technique, three types of voltage-gated, outward K⁺ currents were measured in the cultured fibroblasts. The majority of cells expressed a transient outward K⁺ current (I_to) that activated at potentials positive to –40 mV and partially inactivated during depolarizing voltage steps. I_to was inhibited by the antiarrhythmic agent flecainide (100 µM) and BaCl₂ (1 mM) but was unaffected by 4-aminopyridine (4-AP; 0.5 and 1 mM). A smaller number of cells expressed one of two types of kinetically distinct, delayed-rectifier K⁺ currents [I_K fast (I_Kf) and I_K slow (I_Ks)] that were strongly blocked by 4-AP. Application of phorbol 12-myristate 13-acetate, to stimulate protein kinase C (PKC), inhibited I_to but had no effect on I_Kf and I_Ks. Immunoblot analysis revealed the presence of Kv1.4, Kv1.2, Kv1.5, and Kv2.1 -subunits but not Kv4.2 or Kv1.6 -subunits in the fibroblasts. Finally, pretreatment of the cells with 4-AP inhibited angiotensin II-induced intracellular Ca²⁺ mobilization. Thus neonatal cardiac fibroblasts express at least three different Kv channels that may contribute to electrical/chemical signaling in these cells.

cardiac fibroblasts

Recent experiments suggest that cardiac fibroblasts also serve to relay electrical signals in the heart. When plated in primary culture, cardiac fibroblasts form physical interactions with cardiac myocytes through establishment of gap-junctional channels (6, 8, 14). With the use of a heterocellular culture model, it was established that fibroblasts are capable of relaying electrical excitation over distances as long as 300 µm between strands of cardiac myocytes (14). However, the ionic mechanisms responsible for this electrical propagation are not well understood. An initial study of neonatal rat cardiac fibroblasts in cell culture identified an outward-rectifying current that exhibited partial inactivation during strong depolarization (20). The ion channel responsible for this current was not identified, nor was the ionic basis for the resting potential of the neonatal fibroblasts (–20 to –40 mV) determined in the study. Recently, Giles and colleagues (4, 23) analyzed ion channels in freshly dissociated adult cardiac fibroblasts and cultured myofibroblasts. Two types of K⁺ currents were measured in the fibroblasts: a voltage-dependent delayed-rectifier current (I_K) (4, 23) and an inward-rectifying current (4). This latter current contributes to the relatively negative resting membrane potential (–50 mV) recorded in the adult fibroblasts. Nonetheless, the disparities between the adult and neonatal fibroblast results were not addressed in these studies.

The goal of the present study was to identify and characterize voltage-gated K⁺ (Kv) channels in neonatal rat ventricular fibroblasts. It is reported that the cultured neonatal fibroblasts express three different types of Kv currents and several Kv -subunit proteins. The majority of cells expressed a transient outward K⁺ current (I_to) with kinetic properties similar to those of transient outward currents measured in excitable cells such as cardiac myocytes and neurons. The fibroblast I_to was inhibited both by the antiarrhythmic agent flecainide and during stimulation of protein kinase C (PKC) by application of phorbol 12-myristate 13-acetate (PMA). A minority of fibroblasts expressed one of two different I_K. Thus neonatal cardiac fibroblasts express several different types of Kv channels that may participate in chemical/electrical signaling.

	MATERIALS AND METHODS

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Isolation and culture of cardiac ventricular fibroblasts and myocytes. Animal protocols were approved by the Institutional Animal Care and Use Committee of the University of South Carolina, Columbia. Neonatal rat ventricular fibroblasts and myocytes were isolated and cultured as described previously (9, 27). In brief, heart ventricles were removed from neonatal pups (3–4 days old), minced into 1-mm³ pieces, and subjected to collagenase (type B; Boehringer Mannheim) dissociation (9, 27). Cells were separated by using selective attachment procedures and were cultured in DMEM (GIBCO), supplemented with either 10% newborn bovine serum and 5% fetal bovine serum (fibroblasts) or 8% horse serum and 5% newborn bovine serum (myocytes). Cells were maintained in a humidified atmosphere of 5% CO₂ at 37°C. Fibroblasts (patch clamping and immunoblot analysis) were used at passage 2, whereas myocytes (immunoblot analysis) were harvested on days 3–4 of primary culture. An antibody against the discoidin domain receptor 2 (DDR2) was used for the positive identification of cardiac fibroblasts. DDR2 is a collagen receptor expressed in fibroblasts but not in endothelial cells, smooth muscle cells, or cardiac myocytes (9).

Recording procedure and measurement of Kv currents. The patch-clamp method (12) was used to record whole cell K⁺ currents with the use of a L/M EPC 7 (Adams & List Scientific) and an Axopatch 200 (Axon Instruments) amplifier. Our procedure for measurement and analysis of Kv currents has been described previously (26). All experiments were conducted on isolated, noncoupled fibroblasts at room temperature (22–24°C). For Kv current measurement, cells were placed in Tyrode solution consisting of (in mM) 132 NaCl, 5 KCl, 1 MgCl₂, 1 CaCl₂, 5 dextrose, 5 HEPES, pH 7.4 (with NaOH; 280 mosM). In some experiments, a TEA external solution was used with 132 mM TEA substituted for NaCl. Pipettes were made from Prism glass capillaries (Dagan) and had resistances of 1–2 M when filled with internal solution. Electrodes contained (in mM) 50 KCl, 60 K-glutamate, 2 MgCl₂, 1 CaCl₂, 11 EGTA, 3 ATP, 10 HEPES, pH 7.3 (with KOH; K⁺ concentration = 140 mmol/l; 280 mosM). The ratio of EGTA/CaCl₂ in this solution sets the free intracellular Ca²⁺ concentration to 10 nM.

K⁺ currents were recorded with 12-bit analog/digital converters (Axon Instruments). Data were sampled at 2.5 KHz, filtered at 1 KHz with a low-pass Bessel filter (Frequency Devices), and stored on personal computers. Fibroblasts were maintained at a holding potential of –80 mV, and 100-ms voltage steps were applied to the indicated potentials. Linear leak and capacity transients were removed from the test currents by using a digital P/4 protocol. Use of this procedure was justified because no voltage- or time-dependent conductances were detected in the voltage range of –80 to –120 mV. Cell membrane capacitance values were read from the amplifier dial, and currents were normalized to these measurements. Series resistance was compensated to give the fastest possible capacity transient without producing oscillations. Because of the small size of the K⁺ currents in the cultured fibroblasts (100–600 pA), voltage errors resulting from uncompensated series resistance were minimal. Averaged current values presented are means ± SE. Where appropriate, statistical significance was estimated by Student's t-test for unpaired observations.

Preparation of cell lysates and Western blot analysis. To prepare cell lysates for Western blot analysis, cardiac fibroblast and myocyte cultures were placed into a lysis buffer [50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 50 mM dithiothreitol, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 1 mM EGTA, 0.25% sodium deoxycholate, 2 µg/ml aprotinin, and protease inhibitor cocktail (Roche)]. Lysates were immediately transferred to precooled Microfuge tubes and were sonicated to reduce viscosity. The protein content of the cell preparations was determined by using a protein assay kit (Pierce). For Western blot analysis of Kv channels (26), equal amounts of protein were separated by electrophoresis on 10% SDS polyacrylamide gels by using a standard cell (Bio-Rad) run overnight. The running buffer contained 25 mM Tris, 193 mM glycine, pH 8.3, and 0.1% SDS. Proteins were transferred to polyvinylidene difluoride membranes by using a Trans-Blot apparatus (Bio-Rad). The transfer buffer contained 25 mM Tris, 192 mM glycine, pH 8.5, and 20% methanol. For immunodetection, membranes were first blocked in TBS containing 0.1% Tween 20, bovine serum albumin, and 0.025% Na-azide for 60 min at room temperature. Antibodies to the Kv -subunits (rat anti-Kv1.2, -Kv1.4, -Kv1.5, -Kv1.6, -Kv2.1, and -Kv4.2) were incubated with the membranes overnight at 4°C. After primary antibody treatment, the membranes were washed with TBS-0.1% Tween 20 and were incubated with a secondary antibody (horseradish peroxidase-conjugated goat anti-rat IgG; Cell Signaling Technology). Immunoreactive bands were visualized on X-ray film (Kodak) by using the enhanced chemiluminescence method (Pierce). Each immunoblot result was confirmed on three separate cell cultures.

Intracellular Ca²⁺ mobilization. Fibroblast ANG II-stimulated Ca²⁺ release was measured as described previously (27). Cultured fibroblasts were incubated in a rotating water bath for 30 min in buffer containing 10 µM fluo 4-AM and 0.01% Pluronic (Molecular Probes). The cells were then washed several times without the dye and were incubated for 30 min to facilitate the deesterification of the dye. ANG II-induced intracellular Ca²⁺ release was measured by using AttoFluor-CARV-1 (BD Biosciences) (27) and Elements (Nikon) imaging systems. The AttoFluor system was interfaced to a Carl Zeiss Axiovert 200 inverted microscope and an ORCA ER 1394 12-bit charge-coupled device firewire camera. The Elements system was interfaced to a Leica DM IL inverted microscope (Vashaw Scientific) and CoolSNAP EZ camera (Photometrics). Fluo 4 fluorescence was excited by using an X-Cite (EFOS) metal-halide fluorescence illumination system (Photonic Solutions) with a green fluorescent protein excitation/emission cube (492/520 nm). Images were captured by using Kinetic Imaging AQM6 (Kinetic Imaging) and NIS Elements (Nikon) software. Intracellular Ca²⁺ was determined as the average intensity of the gray pixels represented by the fluorescent signal.

Drugs and antibodies. 4-aminopyridine (4-AP), flecainide, PMA, forskolin, and ANG II were purchased from Sigma (St. Louis, MO). Bisindolylmaleimide I was obtained from Calbiochem (San Diego, CA). Antibodies to the Kv1.2, Kv1.4, Kv1.5, Kv1.6, and Kv2.1 -subunits were purchased from Alomone Labs (Jerusalem, Israel). Kv4.2 and Kv1.5 antibodies were obtained from Chemicon (Billerica, MA).

	RESULTS

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Neonatal cardiac fibroblasts express an I_to. Figure 1 shows an example of an I_to measured in the neonatal rat cardiac fibroblasts. All recordings for this study were obtained from cultured fibroblasts at passage 2. These cells were identified by their large, flat, spindle shape and through labeling with an antibody to the collagen receptor DDR2 (9). The cell membrane capacity of the fibroblasts ranged from 22 to 82 pF, with a mean ± SE value of 48 ± 2 pF (n = 60 cells). Consistent with previous studies (4, 27), no voltage-dependent inward Ca²⁺ currents were measured in the cardiac fibroblasts. In addition, voltage steps applied from the holding potential of –80 mV to potentials ranging from –120 through –90 mV failed to elicit Ba²⁺-sensitive inward-rectifier currents (Fig. 1). The Ba²⁺-sensitive current measured at –120 mV was 0.06 ± 0.05 pA/pF (n = 5 cells). However, small tetrodotoxin-sensitive Na⁺ currents were observed in 24 out of 67 fibroblasts analyzed (for example, see Fig. 2).

View larger version (18K): [in this window] [in a new window]   Fig. 1. Measurement of a transient outward K+ current (Ito) in neonatal rat ventricular fibroblasts. Top left: currents recorded during voltage steps, given in 10-mV increments, to potentials ranging from –30 to +50 mV. Top right: peak current vs. voltage relationship for Ito (n = 20 cells). Currents were normalized to cell membrane capacity. Middle left: activation and inactivation curves for fibroblast Ito. Vm, membrane potential; EK, Nernst equilibrium potential, gK, conductance; gKmax, maximum conductance; IKmax, maximum current. For activation, conductance was determined by dividing peak current amplitude at each potential by driving force for K+ (Vm – EK). Continuous lines represent best fits of the equation, gK = gKmax{1 + exp[–(Vm – V1/2)/k]}, where V1/2 is half-maximal voltage required for activation and k gives steepness of voltage dependence to data points. Inactivation curves were obtained by using a two-pulse protocol. Currents obtained at +50 mV were normalized and plotted as a function of the 1-s prepulse potential. Data were fit with equation IK = IK(max)/{1 + exp[(Vm – V1/2)/k]}, where IK is the delayed-rectifier K+ current and k is slope. Each point represents mean ± SE conductance obtained in 15–18 fibroblasts. Middle right: immunoblots obtained with fibroblast (F) and myocyte (M) cell lysates using anti-Kv4.2 and anti-Kv1.4 Abs. Bottom: currents recorded during voltage steps, given in 10-mV increments, to potentials ranging from –120 to –90 mV. Ba2+-sensitive inward-rectifier currents were not identified in fibroblasts. Con, control.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Effect of K+ channel blockers on fibroblast Ito. Ito recorded during voltage steps applied to +50 mV in absence (Con) and presence of 1 mM 4-aminopyridine (4-AP), 100 µM flecainide (Flec), and 1 mM BaCl2. Bottom right: percent decrease in Ito measured at +50 mV with K+ channel blockers. Each bar represents mean ± SE decrease in Ito measured in 4–6 fibroblasts.

I_to in cardiac muscle are composed primarily of either Kv4.x or Kv1.4 -subunits (10, 19). To identify the Kv -subunit responsible for the cardiac fibroblast I_to, cell lysates were collected and immunoblot analysis was performed with anti-Kv4.2 and anti-Kv1.4 antibodies. As shown in Fig. 1, an immunoreactive band corresponding to the 75 kDa Kv4.2 -subunit was readily identified in lysates obtained from cultured cardiac ventricular myocytes but not from fibroblast lysates. In contrast, both the myocytes and fibroblasts expressed the 97 kDa Kv1.4 -subunit.

The pharmacological properties of I_to are summarized in Fig. 2. Surprisingly, addition of 0.5 and 1 mM concentrations of the K⁺ channel blocker 4-AP failed to inhibit the current. However, the antiarrhythmic agent flecainide (100 µM) produced almost 70% block of I_to when measured at +50 mV. Previous studies have demonstrated that Ba²⁺ inhibits some I_to (22). Addition of 1 mM BaCl₂ resulted in a partial block of the cardiac fibroblast I_to (Fig. 2). The inability of 4-AP to inhibit I_to made this drug useful for separating the I_to from delayed-rectifier K⁺ channels also found in the fibroblasts (see below).

Neonatal cardiac fibroblasts express I_K. Two types of I_K were measured in the fibroblasts (Fig. 3). These currents could be distinguished by their different current-activation kinetics and the voltage dependence of channel activation. The first current, defined as the fast delayed rectifier (I_Kf), activated with a time constant () of 2.4 ± 1 ms at +50 mV (n = 8 cells). This current was present in 22% (15 out of 67) of the cells examined and displayed an activation V_1/2 of –14 mV (Fig. 3). The second current, defined as the slow delayed rectifier (I_Ks) activated with a of 6.0 ± 1 ms at +50 mV (n = 6 cells). This current was present in 19% (13 out of 67) of the fibroblasts and had an activation V_1/2 of +18 mV (Fig. 3). Although a complete steady-state inactivation curve was not obtained for these currents, both I_Kf and I_Ks were completely inactivated at a holding potential of –40 mV.

View larger version (26K): [in this window] [in a new window]   Fig. 3. Measurement of IK in neonatal rat ventricular fibroblasts. Top left: slow IK (IKs) recorded during voltage steps, given in 10-mV increments, to potentials ranging from –20 to +40 mV. Top right: fast IK (IKf) recorded during voltage steps, given in 10-mV increments, to potentials ranging from –30 to +50 mV. Bottom left: activation curves for fibroblast IK. For activation, conductance was determined by dividing peak current amplitude at each potential by driving force for K+ (Vm – EK). Continuous lines represent best fits of Boltzmann equations to data. Each point represents mean ± SE conductance obtained in 6 (IKs) and 8 (IKf) fibroblasts. Bottom right: immunoblots obtained with fibroblast (F) and myocyte (M) cell lysates by using anti-Kv2.1, anti-Kv1.2, and anti-Kv1.5 Abs.

The pharmacological properties of the I_K are shown in Figs. 4 and 5. In contrast to I_to, both I_Kf and I_Ks were strongly blocked by 1 mM 4-AP (Figs. 4 and 5). For I_Ks, this block occurred in a voltage-dependent manner with greater block at –20 mV when compared with +50 mV (Fig. 5). The effect of 132 mM TEA external solution was also examined on the currents. Whereas TEA partially inhibited I_Ks, it caused no change in the amplitude of I_Kf (Figs. 4 and 5). Additionally, we tested for, but found no evidence of, block of I_Ks by 100 nM -dendrotoxin (Fig. 5). Finally, unlike I_to, there was no evidence of inhibition of I_Kf during application of 100 µM flecainide (%change = –5 ± 5; n = 3 cells; Fig. 4).

View larger version (13K): [in this window] [in a new window]   Fig. 4. Effect of K+ channel blockers on fibroblast IKf. Left and top right: IKf recorded during voltage steps applied to +50 mV in absence (Con) and presence of 1 mM 4-AP, 100 µM Flec, and TEA external solution. Bottom right: percent decrease in IKf measured at –20 or +50 mV with 1 mM 4-AP or TEA external solution. Each bar represents mean ± SE inhibition of IKf measured in 3–5 fibroblasts.

View larger version (14K): [in this window] [in a new window]   Fig. 5. Effect of K+ channel blockers on fibroblast IKs. Left and top right: IKs recorded during voltage steps applied to +50 mV in absence (Con) and presence of 1 mM 4-AP, 100 nM -dendrotoxin (DTX), and TEA external solution (TEA). Bottom right: percent decrease in IKs measured at –20 or +50 mV with 1 mM 4-AP or TEA external solution. Each bar represents mean ± SE inhibition of IKs measured in 3–5 fibroblasts. *Significant difference (P < 0.05) between results in a column and corresponding column.

View larger version (14K): [in this window] [in a new window]   Fig. 6. Regulation of fibroblast Ito by protein kinase C (PKC). Top: Ito recorded during voltage steps applied to +50 mV in absence (Con) and presence of 100 nM phorbol 12-myristate 13-acetate (PMA) and 2 µM forskolin (Fors). Bottom left: IKf recorded during voltage steps applied to +50 mV in absence (Con) and presence of PMA. Bottom right: Percent change in K+ currents measured with PMA and Fors. Some cells were pretreated with bisindolylmaleimide I before PMA. Each bar represents mean ± SE change obtained in 3–6 fibroblasts. *Significant decrease (P < 0.05).

View larger version (50K): [in this window] [in a new window]   Fig. 7. 4-AP inhibits ANG II-induced Ca2+ mobilization in fibroblasts. Top: Ca2+ transients measured during application of 1 µM ANG II in absence (Con) and presence of 1 mM 4-AP. Fluorescence was measured at time 0 and 30 s after addition of ANG II. Bottom: plot of ratio of whole cell fluorescence intensity measured before (F0) and after (F) ANG II application. Each column represents mean ± SE intensity measured in a total of 20–30 cells obtained in 3 different experiments. Cells were pretreated for 5 min with Flec, PMA, and 4-AP. *Significant difference (P < 0.05) between results in a column and corresponding columns.

	DISCUSSION

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There have been several previous reports of voltage-gated ion currents in isolated cardiac ventricular fibroblasts. In the initial study conducted with neonatal rat fibroblasts (20), membrane depolarization to potentials positive to –30 mV elicited an I_to that decayed during the first 500 ms of the voltage step. Because the goal of this study was to examine myocyte-fibroblast gap-junctional channels, characterization of the ionic properties of this current was not reported (20). More recently, Giles and colleagues (4, 23) examined K⁺ channels in both freshly isolated adult rat ventricular fibroblasts and cultured adult myofibroblasts. Two types of K⁺ currents were measured in the adult fibroblasts: I_K (4, 23) and an inward-rectifying current (4). The I_K measured in the freshly isolated myocytes displayed very slow COOH terminus-type inactivation, which activated with a of 19 ms at +50 mV and underwent steady-state inactivation with a V_1/2 of –24 mV in physiological external solution (23). Qualitative RT-PCR revealed comparatively high levels of Kv1.6 transcripts and low levels of Kv1.1, Kv1.2, and Kv1.5 transcripts (4) Although not identical to either of the delayed-rectifier channels measured in the neonatal fibroblasts, the adult fibroblast I_K is most similar to I_Ks measured in the present study. However, whereas I_K was measured in 90% of the freshly isolated adult fibroblasts (23), I_Ks was found in only 19% of the neonatal fibroblasts. Furthermore, our immunoblot analysis identified Kv1.2, Kv1.5, and Kv2.1 -subunits in the neonatal fibroblasts but not Kv1.6 -subunits. The fast kinetic and pharmacological (4-AP sensitivity, TEA insensitivity) properties of I_Kf are consistent with the expression of Kv1.5 (10). In contrast, the TEA sensitivity and -dendrotoxin insensitivity of I_Ks suggest that Kv2.1 may be at least partially responsible for this current (10).

I_to are found extensively in excitable tissues such as the heart, smooth muscle, and central nervous system, where they give rise to rapidly inactivating (A-type) K⁺ currents (10). Therefore, it was surprising to identify these channels in cardiac fibroblasts, a cell type traditionally regarded as nonexcitable (3, 14). Expression studies have demonstrated that Kv1.4, Kv3.3, Kv3.4, and Kv4.x -subunits can each form A-type channels (10). In the myocardium, I_to can be separated into two different components, the slow component termed I_to,s and the fast component termed I_to.f (19, 25). I_to,s appears to be formed by Kv1.4, whereas the Kv4.x family of channels are the primary -subunits responsible for I_to,f (19, 25). In the present study, I_to was identified in the majority of neonatal cardiac fibroblasts analyzed. Similar to the cardiac myocyte I_to, the fibroblast current was blocked by flecainide and BaCl₂ (22, 26). However, the channel was resistant to block by 4-AP at a concentration of 1 mM. The presence of Kv1.4 subunits in the fibroblast cell lyates suggests that this protein, and not the Kv4.2 subunit, is responsible for the fibroblast I_to. Because I_to were not previously identified in adult cardiac fibroblasts or myofibroblasts (4, 23), our results indicate that developmental factors may be important in determining the electrical properties of the cardiac fibroblasts.

A-type K⁺ currents are regulated via PKA-, PKC- and tyrosine kinase-mediated phosphorylation (25). Application of phorbol esters, to stimulate PKC, suppresses I_to in cardiac ventricular myocytes (1) and hippocampal neurons (13). Stimulation of PKC has been shown to have a biphasic action on Kv1.4 channels expressed in Xenopus oocytes (18). Initially, PKC produces a small increase in the Kv1.4 K⁺ current, which is followed by a more pronounced inhibitory effect (18). Recently, Hagiwara et al. (11) demonstrated that endothelin, acting via PKC, causes a strong inhibition of the Kv1.4 channel. In the present study, application of PMA to the neonatal fibroblasts caused a relatively small (30%) but specific decrease in I_to. This inhibition was prevented by pretreatment of the cells with the PKC inhibitor bisindolylmaleimide I, suggesting that current reduction is mediated by PKC. Although PKC-mediated phosphorylation is known to stimulate neonatal cardiac fibroblast proliferation and adhesion (3, 7, 17), this is the first study to demonstrate a regulatory action of PKC on ion channels in these cells.

ANG II-induced Ca²⁺ mobilization and the physiological role of fibroblast K⁺ channels. Hormones such as ANG II and bradykinin stimulate Ca²⁺ mobilization in adult (16) and neonatal (24, 27) cardiac fibroblasts by enhancing phospholipase C (PLC) activity. The subsequent increase in 1,4,5-trisphosphate production leads to the release of Ca²⁺ from internal stores (16, 24). Pretreatment of neonatal fibroblasts with 4-AP in our study significantly inhibited ANG II-induced Ca²⁺ transients. Previous studies have shown that Kv channel blockers, such as 4-AP and TEA, can modulate intracellular Ca²⁺ release in anterior pituitary cells by depolarizing the cell resting membrane potential (28, 29). It is unclear whether 4-AP acts through a similar mechanism in the fibroblasts or whether one or more Kv channels might be directly linked to Ca²⁺ mobilization.

When plated together in cell culture, cardiac fibroblasts regulate the phenotypic properties of the associated cardiomyocytes through mechanical, chemical, and electrical interactions. For example, coculture of fibroblasts with myocytes dramatically enhances the expression of cytokines such as IL-6, IL-10, and tumor necrosis factor-, compared with cells cultured separately (2, 15). These factors in turn alter the structural and contractile properties of the myocytes (2, 15). Establishment of electrical contact between adjacent fibroblasts and myocytes may be of critical importance in the release of these cytokines and growth factors. Although the resting membrane potential of isolated neonatal rat cardiac fibroblasts is between –20 and –40 mV, the resting potential of cells electrically coupled to ventricular myocytes approaches that of the connected myocyte (–60 to –80 mV) (20). At these negative potentials, activation of the K⁺ currents identified in the present study could influence the electrical properties of both the fibroblasts and myocytes. Future studies will be required to determine whether the subsequent release of cytokines alters the expression and functional properties of Kv, transient receptor potential (21), and other fibroblast ion channels.

Limitations of the study. There were a number of limitations of this study. First, correlations were made between Kv currents measured during whole cell patch-clamp recording and specific Kv -subunits identified via immunoblots. Although the Kv antibodies used in this study were previously shown to detect Kv subunits in cardiac myocyte preparations (26), it is possible that the antibodies labeled nonspecific protein bands in the fibroblasts. In addition, some Kv subunits may have been present but not detected under the conditions of the assay. A second limitation involved the Ca²⁺-imaging experiments. As shown in Fig. 7, 4-AP inhibited ANG II-induced Ca²⁺ release in the majority of fibroblasts. In contrast, 4-AP-sensitive, I_K were measured in only 40% of the cells (Figs. 4 and 5). Therefore, it cannot be ruled out that 4-AP acted through a nonspecific mechanism (ANG II antagonism, PLC inhibition, etc.) to decrease Ca²⁺ release in the cells. In addition, it is possible that 4-AP inhibited I_to under the conditions of the Ca²⁺-imaging experiments but not during the whole cell recording experiments (10 nM free Ca²⁺, –80 mV holding potential, etc.).

	GRANTS

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This work was supported by US Public Health Service Award HL-45789 and grants from the American Heart Association.

	ACKNOWLEDGMENTS

 
We thank Cheryl Cook and Kathryn J. Long for preparing the neonatal cardiac fibroblasts used in the study.

	FOOTNOTES

 

Address for reprint requests and other correspondence: K. B. Walsh, Dept. of Pharmacology, Physiology, and Neuroscience, Univ. of South Carolina, School of Medicine, Columbia, SC 29208 (e-mail: walsh{at}med.sc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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