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(Circulation Research. 1999;84:964-972.)
© 1999 American Heart Association, Inc.

Rapid Communication

Manipulation of Cellular Excitability by Cell Fusion

Effects of Rapid Introduction of Transient Outward K⁺ Current on the Guinea Pig Action Potential

Uta C. Hoppe¹, David C. Johns¹, Eduardo Marbán, Brian O'Rourke

From the Section of Molecular and Cellular Cardiology, Department of Medicine, Johns Hopkins University, Baltimore, Md.

Correspondence to David C. Johns, PhD, Section of Molecular and Cellular Cardiology, The Johns Hopkins University, 844 Ross Bldg, 720 Rutland Ave, Baltimore, MD 21205. E-mail djohns{at}jhmi.edu

	Abstract

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Abstract—To investigate the still-undetermined role of the Ca²⁺-independent transient outward current (I_to1) on repolarization of the cardiac action potential, we used cell fusion to introduce I_to1 into guinea pig cardiomyocytes, which normally lack this current. This technique enables the rapid delivery of premade functional ion channels to cardiomyocytes within hours of isolation, thus eliminating the action potential alterations that complicate prolonged cell culture. Chinese hamster ovary (CHO) cells stably expressing Kv4.3 (CHO-Kv4.3) were loaded with a fluorescent dye and fused to guinea pig cardiomyocytes using polyethylene glycol. As controls, nontransfected CHO cells were fused using the same protocol. Myocytes fused with CHO-Kv4.3 cells exhibited a robust I_to1 (16.5±2.6 pA/pF at +40 mV; 37°C; n=19), whereas controls had none. I_to1 accelerated the early repolarization velocity (r=-0.68; 3 ms after the overshoot) and progressively suppressed the voltage of the plateau phase (r=-0.90) with increasing I_to1 density. Reduction of the action potential duration to 50% repolarization (r=-0.76) and to 90% repolarization (r=-0.65) also correlated well with I_to1 density. Thus, I_to1 exerted a significant effect on the early repolarization phase and abbreviated action potential duration. Cell fusion is a valuable and generalizable technique to introduce preformed membrane proteins into native cells.

Key Words: transient outward current • action potential • cell fusion • repolarization

	Introduction

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Sudden cardiac death is the single largest killer in the United States. Among the multiple pathological conditions that can lead to the triggering of lethal arrhythmias, heart failure, the single largest predisposing condition, is characterized by a number of changes in the currents that shape the action potential. The Ca²⁺-independent transient outward current (I_to1) has been shown to be decreased in both humans with heart failure and in animal models of heart failure.^{1 2 3} Nevertheless, the role of I_to1 in shaping the action potential is still unclear, making it difficult to assess whether this change is adaptive, maladaptive, or simply a marker of the disease process. It also is unclear whether the loss of I_to1 leads to the increased risk of malignant arrhythmias associated with heart failure.

In most species studied to date, I_to1 is encoded either by Kv4.2 or Kv4.3, or by a combination of the two.^{4 5 6 7} To probe specifically the role of Kv4.x channels in cardiac electrophysiology, we previously used an adenovirus that encoded a truncated form of Kv4.2 as a dominant-negative suppressor of I_to1. However, further elucidation of the contribution of I_to1 to the action potential was complicated by culture-induced changes in the normal action potential.⁸ To circumvent this problem, in the present study, we have adapted standard protocols for somatic cell fusion⁹ to deliver premade functional Kv4.3 ion channels to acutely dissociated guinea pig ventricular myocytes, which under physiological conditions lack an endogenous I_to1.¹⁰

The creation of heterokaryons was first used to demonstrate rapid mixing of membrane components.^{11 12 13} Further applications have included delivery of macromolecules to mammalian cells, manipulation of Na⁺/K⁺-ATPase isoforms in embryonic myocytes, investigation of the dynamics of organelle turnover and processing, and studies of a dominant regulator of skeletal muscle differentiation.^{9 14 15 16 17 18} From these previous studies, we reasoned that this technique would be useful in delivering ion channel proteins to heart cells to assess the influence of a given channel type on cardiac repolarization. Preliminary reports have been published.^{19 20}

	Materials and Methods

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The present investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication 85-23, revised 1985)

Plasmid Constructs
The expression plasmid for Kv4.3 (pCGI-Kv4.3) was generated by cloning the coding sequence from rat Kv4.3 (kindly supplied by Dr Bernardo Rudy, New York University, New York, NY) into the vector pCGI (formerly pGFP-IRES).⁶ The vector pEGFP-N3 (Clontech) was used for control transfections. The coding sequence for the human CD8 was polymerase chain reaction (PCR)-amplified from the plasmid pEBO-CD-Leu2 (No. 59564, American Type Culture Collection [ATCC], Manassas, Va) and cloned into pCGI in place of the EGFP sequence (pC8I).

Transfections
Transient transfections were performed using lipofectamine (Life Technologies) following the manufacturer's recommendations. Briefly, 2.5x10⁵ Chinese hamster ovary (CHO)-K1 cells (CCL61, ATCC) were plated the day before transfection, and transfections were completed using pEGFP or pEGFP and pC8I (0.75 µg each) for 4 to 6 hours. For stable transfections, the same protocol was followed except the plasmid vector (pCGI-Kv4.3) was first linearized with AseI. Cells that had stably integrated the plasmid were selected using geneticin sulfate (500 µg/mL; G418, Life Technologies). Robust expressors were selected using a fluorescence-activated cell sorter (FACS) followed by colony isolation and further analysis.

Flow Cytometry
Flow cytometry was performed using a Facstar (Becton Dickinson) and analyzed using CellQuest (Becton Dickinson) or Win MDI software (Scripps Institute). Cells transfected with a plasmid expressing LacZ were used as nonfluorescent controls. Green fluorescent protein (GFP)-positive cells were measured as those whose fluorescence intensity exceeded the fluorescence of 99.9% of the control cells (488/530 nm excitation/emission).

Ventricular Myocyte Isolation
Guinea pig left ventricular myocytes were isolated using Langendorff perfusion, as previously described.²¹ After digestion, cells were stored at room temperature in a high potassium solution (mmol/L: K-glutamate 120, KCl 25, MgCl₂ 1, glucose 10, HEPES 10, and EGTA 1; pH 7.4) for 30 minutes. Myocytes were then placed on laminin-coated (20 µL/mL culture medium; Becton Dickinson Labware) cover slips in 6-well plates in medium 199 (Cellgro, Mediatech) supplemented with 2% FBS (Life Technologies) and maintained at 37°C in a 5% CO₂ humidified incubator for 1 hour.

Cell Fusion
Control (CHO) or test (CHO-Kv4.3) cells were grown to 70% confluency in 75 cm² flasks. Before fusion, CHO cells were transiently transfected with GFP, or for the stable CHO cells in which the GFP signal was faint, loaded with calcein-AM (2 µL/mL growth medium; 1 mmol/L stock solution in dimethyl sulfoxide; Molecular Probes) to increase the cytosolic fluorescent marker. After staining, cells were trypsinized, centrifuged, and resuspended in 6 mL medium 199 supplemented with leukoagglutinin 40 µg/mL (Sigma Chemical Co). The myocyte growth medium was exchanged with this CHO cell suspension at 0.5 mL/well. One hour after coplating, myocytes and CHO cells were fused with prewarmed (37°C) polyethylene glycol 1500 40% (PEG) (Boehringer Mannheim) in H₂O. After 2 to 4 minutes of exposure to PEG, cells were rehydrated with high potassium solution (same solution that was used after myocyte isolation) for 5 to 10 minutes and then superfused with physiological saline solution (see below) for patch-clamp experiments.

Electrophysiology
Experiments were carried out using standard microelectrode whole-cell patch-clamp techniques²² with an Axopatch 1D amplifier (Axon instruments) while sampling at 10 kHz and filtering at 2 kHz. All experiments were performed at a temperature of 37°C.

Cells were superfused with a physiological saline solution containing (mmol/L) NaCl 138, KCl 5, CaCl₂ 2, glucose 10, MgCl₂ 0.5, and HEPES 10; pH 7.4. The micropipette electrode solution was composed of (mmol/L): K-glutamate 130, KCl 9, NaCl 8, MgCl₂ 0.5, HEPES 10, EGTA 2, and Mg-ATP 5; pH 7.2. Microelectrodes had tip resistances of 1 to 5 M when filled with the internal recording solution.

Voltage-clamp experiments were performed with an interepisode interval of 2.5 seconds. Action potentials were initiated by short depolarizing current pulses (2 to 3 ms, 500 to 800 pA) at a cycle length of 2 seconds. Action potential duration was measured as the time from the overshoot to 50% or 90% repolarization (APD₅₀, APD₉₀).

Membrane capacitance, quantified from 10-mV depolarizing test pulses from a holding potential of -80 mV, was not different between myocytes fused with CHO-Kv4.3 cells (99.1±10.5 pF; n=19), myocytes fused with nontransfected CHO cells (84.5±9.2 pF; n=5), and nonfused myocytes (86.2±9.8 pA/pF; n=6). Data were corrected for the measured liquid junction potential (-18 mV).²³ A xenon arc lamp was used to view calcein fluorescence or GFP at 488/530 nm (excitation/emission).

Confocal Imaging
Images were taken on a laser confocal microscope (PCM 2000, Nikon Inc) with a x60 water immersion objective lens. GFP was imaged with an argon laser at 488-nm excitation/520±15-nm emission; R-phycoerythrin–conjugated CD8 antibody (Sigma Chemical Co) was visualized with a helium-neon laser at 543-nm excitation/605±16-nm emission.

Statistical Analysis
Pooled data are presented as mean±SEM when appropriate. Regression analysis was used to test for a relationship between I_to1 density and repolarization velocity, plateau height, and action potential duration. Comparisons between groups were made using unpaired Student t tests. Values of P<0.05 were considered significant.

	Results

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CHO Cell Line Stably Expressing Kv4.3
A CHO cell line stably expressing Kv4.3 was designed to facilitate the introduction of transient outward K⁺ currents into myocytes by cell fusion. Transfected CHO cells were selected by their fluorescence. As shown in Figure 1A, mean fluorescence measured by FACS analysis of the stable CHO-Kv4.3 clone (clone A) used in the present experiments clearly exceeded the background autofluorescence of nontransfected CHO cells. To prove that cells from this clone stably expressed Kv4.3, transient outward currents were elicited at +40 mV from a holding potential of -100 mV. Figure 1B shows a representative fully inactivating outward current that was similarly observed in every cell tested for this clone (n=12). Mean peak current density at +40 mV, obtained by subtracting the prepulse-inactivated current (0 mV prepulse) from the fully primed current (-100 mV prepulse), was 139.9±16.6 pA/pF (range 37.9 pA/pF to 241.9 pA/pF; mean cell capacitance 13.7±0.8 pF; n=12).

View larger version (30K): [in this window] [in a new window]   Figure 1. CHO cell clones stably expressing Kv4.3. Mean fluorescence measured by FACS analysis of 4 different clones stably expressing Kv4.3 (Kv4.3A through Kv4.3D) clearly exceeded background autofluorescence of noninfected CHO cells (A). In CHO-Kv4.3 cells of clone A (clone used in these experiments), depolarization to +40 mV from a holding potential of -100 mV elicited a fully inactivating transient outward current (B).

Cell Fusion of Myocytes With Noninfected CHO Cells Has No Effect on Action Potential Morphology
To test the role of I_to1 on repolarization of cardiomyocytes, freshly isolated guinea pig myocytes were chosen for cell fusion experiments, because guinea pig myocytes physiologically lack transient outward K⁺ currents. Before attempting to modify the electrophysiology of guinea pig cells, we needed to confirm that myocytes could be fused with CHO cells without altering the basic electrophysiology. Cell fusion between guinea pig myocytes and CHO cells was verified by the obvious transfer of cytosolic GFP or calcein from CHO cells to the cardiomyocytes. Figure 2 depicts a confocal image of a typical heterokaryon of a guinea pig myocyte and a CHO cell. Fused myocytes were readily distinguishable from the background autofluorescence of nonfused cells. Cell fusion had no obvious effects on myocyte morphology, except at the site of fusion itself. To test for nonspecific effects of cell fusion on the electrophysiology of guinea pig myocytes, action potentials were recorded in nonfused cells that had not undergone PEG exposure and compared with recordings obtained in myocytes fused with nontransfected CHO cells. Cell fusion did not produce any appreciable effects on the waveform or duration of action potentials. Mean resting membrane potentials (-87.0±1.0 mV versus -88.3±3.0 mV), overshoot (39.0±2.4 mV versus 38.2±2.2 mV), and action potential durations measured at 50% (181.8±12.7 ms versus 155.8±26.4 ms) and 90% repolarization (214.3±9.2 ms versus 188.5±25.9 ms) did not differ significantly in myocytes that had not undergone PEG exposure (n=6) and myocytes fused with nonexpressing CHO cells (n=5), respectively.

View larger version (177K): [in this window] [in a new window]   Figure 2. Confocal fluorescence image (green plane) of typical heterokaryons of guinea pig myocytes and CHO cells (transmitted light image shown in blue plane). Cell fusion between the guinea pig myocyte and CHO cells (arrow) was verified by the obvious transfer of cytosolic GFP from CHO cells to the cardiomyocyte. Fused myocytes were readily distinguishable from the background autofluorescence of nonfused cells. Cell fusion had no obvious effects on myocyte morphology, except at the site of fusion itself.

Effect of Myocyte/CHO-Kv4.3 Cell Fusion on Action Potential Plateau and Duration
Myocytes fused with CHO-Kv4.3 cells exhibited robust transient outward currents. The density of I_to1 introduced into guinea pig myocytes by cell fusion ranged from 1.9 pA/pF to 44.2 pA/pF at +40 mV. Figures 3C, 3E, and 3G show transient outward currents recorded in three different myocytes fused with CHO-Kv4.3 cells that exhibited different I_to1 amplitudes. Mean peak current density was 16.5±2.6 pA/pF at +40 mV (n=19). In myocytes fused with nontransfected CHO cells, no transient outward currents were recorded (n=5) (Figure 3A).

View larger version (19K): [in this window] [in a new window]   Figure 3. Transient outward currents and action potentials of myocytes fused with CHO-Kv4.3 cells and nontransfected CHO cells (Ito1=0 pA/pF). In myocytes fused with nontransfected CHO cells, no Ito1 could be recorded (A). Introduction of Ito1 into guinea pig myocytes (C, E, and G) substantially changed the action potential waveform (D, F, and H) compared with myocytes fused with nontransfected CHO cells (B). These changes became more pronounced with increasing densities of transient outward currents, resulting in a suppression of the plateau potential and abbreviation of the overall action potential duration. Introduction of very large Ito1 densities (G) caused a spike-like action potential (H).

I_to1 in fused guinea pig myocytes had similar current properties as native I_to1 in myocytes of other mammalian species and humans.^{5 24 25 26 27 28} I_to1 activated at -40 to -50 mV, with a half-activation of -2.5±1.1 mV (n=4). Half-maximal inactivation was -55.7±0.4 mV with a slope factor of 8.1±0.3 mV (n=8) (Figure 4A). This value is very similar to rat myocytes (-55 mV) in the absence of extracellular divalent cations²⁸ and to dogs and humans (-34 to -37 mV) after correction for the voltage shift (20 mV) due to the use of [Cd²⁺]_o by these authors.^{2 24 25} Using monoexponential fits, we determined the mean time constant of inactivation to be 11.6±4.4 ms (n=7); 90% recovery (at –100 mV) of I_to1 from inactivation (verified in two fused myocytes) was 30 ms (Figure 4B).

View larger version (13K): [in this window] [in a new window]   Figure 4. Voltage dependence of activation and inactivation (A) and recovery from inactivation (B) of Ito1 in fused guinea pig myocytes. A, Inactivation curve (left) was calculated by fitting a Boltzmann distribution to normalized peak current amplitudes of 8 fused myocytes. Half-maximal inactivation was -55.7±0.4 mV. Activation curve (right) obtained by a Boltzmann fit of normalized peak current conductances yielded a half-activation of -2.5±1.1 mV (n=4). B, Recovery of Ito1 at -100 mV from an inactivating pulse to +60 mV in a fused guinea pig myocyte is plotted as a function of the interpulse-interval. A monoexponential fit of data points yielded a time constant of 11.6 ms and 90% recovery of inactivation after 30 ms.

Action potential recordings from heterokaryons demonstrated that the resting membrane potential in myocytes fused with CHO-Kv4.3 cells (-89.9±1.2 mV; n=19) and nontransfected CHO cells (-88.3±3.0 mV; n=5) was not different. Figures 3D, 3F, and 3H demonstrate the effect of I_to1 on repolarization of guinea pig cardiomyocytes. The introduction of I_to1 substantially changed the action potential waveform compared with myocytes fused with control CHO cells (Figure 3B). The modification of the action potential became more pronounced as the amount of I_to1 increased. Extremely large I_to1 densities resulted in a spike-like configuration of the action potential reminiscent of that recorded in normal rat ventriculocytes (Figure 3H).

I_to1 accelerated the initial repolarization velocity of fused guinea pig myocytes. The mean repolarization velocity measured 2 and 3 ms after the overshoot in myocytes fused with CHO-Kv4.3 cells was -8.5±1.2 mV/ms and -5.3±0.6 mV/ms, respectively (n=19), compared with -5.5±0.8 mV/ms and -3.3±0.3 mV/ms (n=5), in myocytes fused with nontransfected CHO cells (P=0.03 and P=0.02, respectively). Figure 5A plots the repolarization velocity measured 3 ms after the overshoot as a function of the density of I_to1 recorded at +40 mV. Initial repolarization velocity became progressively faster with increasing I_to1 density (r=-0.68; P=0.0003).

View larger version (12K): [in this window] [in a new window]   Figure 5. Effect of Ito1 current size on early repolarization velocity and height of the action potential plateau in myocytes fused with CHO-Kv4.3 cells and nontransfected CHO cells (Ito1=0 pA/pF). A, Repolarization velocity measured at 3 ms after the overshoot is plotted versus Ito1 density at +40 mV. Early repolarization became progressively faster with increasing Ito1 density. B, Introduction of Ito1 into guinea pig myocytes depressed the action potential plateau. The suppression of the voltage of the early plateau phase measured at d2V/dt2=0 (at the transition from early repolarization to final repolarization) correlated well with the introduced Ito1 density.

Introduction of I_to1 into guinea pig myocytes caused a depression (hyperpolarization) of the action potential plateau. In most fused myocytes, the whole plateau phase was suppressed; in only one fused myocyte was a small notch and dome obtained (Figure 3F). To assess the plateau height of the action potential, we measured the voltage during the plateau phase at d²V/dt²=0, ie, at the transition from phase 1 to phase 2 repolarization, as previously reported for human ventricular subendocardial myocytes.²⁴ For the fused myocyte that exhibited the small notch and dome, the maximum plateau voltage after the notch at phase 1 was obtained. As shown in Figure 5B, the suppression of the voltage level of the early plateau phase correlated well with the introduced I_to1 density (r=-0.90; P<0.0001).

Perhaps most surprisingly, I_to1 also decreased the overall action potential duration. Figure 6 shows that the reduction of the action potential duration both at 50% (APD₅₀; r=-0.76; P<0.0001) and 90% repolarization (APD₉₀; r=-0.65; P=0.0006) became more pronounced with progressively larger introduced transient outward currents. I_to1 density was not correlated to the maintained outward current measured at the end of a 500-ms depolarization pulse to +20 mV (r=0.43) or +40 mV (r=0.39). The mean (I_to1 related) maintained outward components at +20 mV and +40 mV, obtained by subtraction of the fully activated current and the prepulse (0 mV) inactivated current, were -0.3±0.1 pA/pF and -0.2±0.1 pA/pF (n=8), respectively. These data indicate that the Kv4.3-encoded I_to1 channels inactivated completely during the 500-ms pulse. The effects cannot be attributed simply to a component of maintained outward current.

View larger version (12K): [in this window] [in a new window]   Figure 6. Effect of Ito1 current size on the action potential duration in myocytes fused with CHO-Kv4.3 cells and nontransfected CHO cells (Ito1=0 pA/pF). Action potential duration at 50% (APD50) (A) and 90% repolarization (APD90) (B) are plotted versus Ito1 current density at +40 mV. Reduction of APD50 and APD90 correlated well with Ito1 density evaluated by regression analysis.

Confocal Imaging of Myocytes Fused With CHO Cells Expressing GFP and CD8
To test whether cell fusion of guinea pig myocytes with CHO cells led only to cytosolic exchange between fused cells or also to intermixing of cell membrane proteins, myocytes were fused with CHO cells transiently transfected with GFP and the cell surface antigen CD8. CD8 was visualized with R-phycoerythrin–conjugated CD8 antibodies. Figure 7 shows that surface membrane proteins from the CHO cell spread into the myocyte surface membrane (cell fixing 2 hours after cell fusion). The cytosolic green fluorescence of the myocyte verifies cell fusion between the myocyte and CHO cell. Red staining of the surface membrane and of t-tubular structures of the fused myocyte indicates a redistribution of CD8 from the CHO cell membrane with the membranes of the myocyte.

View larger version (89K): [in this window] [in a new window]   Figure 7. Confocal fluorescence image of a typical heterokaryon of a guinea pig myocyte and CHO cell expressing GFP and CD8. CHO cells were transiently transfected with GFP and CD8. After cell fusion, cells were fixed and CD8 was visualized with R-phycoerythrin–conjugated monoclonal CD8 antibodies. The cytosolic green fluorescence of the myocyte indicates cell fusion with the CHO cell. Red staining of the surface membrane and of t-tubular structures of the myocyte verifies that CD8 from the CHO cell membrane mixed with the membranes of the myocyte (cells were fixed 2 hours after cell fusion).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study demonstrates that I_to1 introduced into freshly isolated guinea pig myocytes by cell fusion substantially changes the early repolarization phase and abbreviates action potential duration. The role of I_to1 in the action potential has been previously assessed indirectly by comparison of I_to1 densities and action potentials throughout the layers of the myocardial wall (ie, humans,^{24 25} dogs,^{29 30 31} cats,³² rats,³³ and rabbits³⁴ ) or in disease states such as hypertrophy and heart failure.^{1 2 3} To further elucidate the role of I_to1, I_to1 has been blocked pharmacologically, knocked out by dominant-negative constructs, or artificially introduced into heart failure cells with brief repolarizing current pulses.^{2 6 33 35} However, K⁺ channel blockers such as 4-aminopyridine also exhibit nonspecific effects,³³ current pulses may lack physiological current kinetics, and previous knockout studies were limited by changes of gene expression during cell culture⁶ or adaptive upregulation of other outward currents in transgenic mice.³⁵ Using cell fusion to introduce Kv4.3 in freshly isolated guinea pig cardiomyocytes to probe the role of I_to1 on repolarization, we obviated culture-related alterations of the action potential and nonspecific drug effects in the present study.

I_to1 accelerated the early repolarization velocity and suppressed the voltage level of the plateau phase in guinea pig myocytes. Both effects became more pronounced as I_to1 density increased. Therefore, these results support the conclusion that I_to1 exerts significant effects on early repolarization and plays an important role in setting the voltage of the plateau phase. Previously, reduction of I_to1 density in tachycardia-induced heart failure of dogs has also been related to observed changes in the plateau height, ie, an elevation of the plateau voltage in failing canine ventricular myocytes compared with nonfailing control cells.² In human subendocardial myocytes that exhibit small I_to1 sizes, the plateau voltage tended to be higher than in subepicardial cells, which have significantly larger I_to1 densities.²⁴

Except for one myocyte, cell fusion did not lead to a typical notch-and-dome configuration of the action potential. The variable prominence of a notch in phase 1 repolarization has been related to I_to1 size in subepicardial cells compared with subendocardial cells in humans,^{24 25} dogs,^{29 30 31} cats,³² and rabbits³⁴ as well as in hypertrophy and heart failure.^{1 2 3} Human subendocardial myocytes exhibiting small I_to1 densities lack a notch but demonstrate a monotonic repolarization similar to our recordings of guinea pig myocytes with small introduced I_to1 densities.²⁴ Very large transient outward K⁺ currents like those in rat subepicardial cells lead to rapid repolarization and spike-like action potentials.³³ Introduction of comparable I_to1 densities into guinea pig cardiomyocytes in the present study led to similar spike-like action potentials. In myocytes with intermediate I_to1 densities, action potential shape is variable in different species. Human subepicardial and dog cells exhibit a notch-and-dome waveform, whereas rat subendocardial cells with I_to1 densities in a similar range exhibit no notch but a continuous repolarization as observed in most of our fused guinea pig myocytes.^{24 25 29 30 31 33} These findings suggest that the configuration of phase 1 repolarization and the presence or absence of a notch-and-dome morphology are importantly influenced by the balance of other repolarizing and depolarizing currents.

The introduction of I_to1 into guinea pig myocytes shortened the action potential duration in a current-density dependent manner. Although the rapid inactivation of Kv4.3 makes a direct contribution to the late phase of repolarization unlikely, the observed changes in the action potential duration may be due to indirect effects on other currents secondary to changes in the early plateau potential.^{24 36}

Cell fusion has been previously used in embryonic cardiomyocytes and skeletal muscle cells.^{9 14 16 17} In the present study, we demonstrated that heterologous cell fusion is also possible with adult cardiomyocytes without any appreciable effect on cell morphology or viability. Because the space constant of myocardial tissue is much larger (10x) than the length of an isolated myocyte,³⁷ punctate delivery of current into a myocyte will electrically affect the entire cell. Thus, the observed changes of the action potential waveform of guinea pig myocytes fused to CHO-Kv4.3 cells would be readily explained even if the Kv4.3 channels remained localized in the CHO cell membrane. Nevertheless, fusion of myocytes with CHO cells transiently expressing GFP and CD8 demonstrated that there is not only prompt cytosolic exchange but also eventual redistribution of membrane proteins between fused cells. Thus, this technique enables further applications to introduce premade proteins into cardiomyocytes and to study their interaction with the endogenous myocyte membrane.

Recordings in the present study were performed with a pipette solution containing EGTA. Thus, similar to most previous studies that claim an effect of I_to1 on the action potential duration,^{1 2 24 30 34} abbreviation of action potentials by I_to1 introduction into guinea pig myocytes was observed in the presence of [Ca²⁺]_i buffering in the present study. However, it still must be determined whether I_to1 modulates the overall action potential duration when [Ca²⁺]_i cycling is intact.^{38 39}

	Acknowledgments

 
This study was supported by a grant from Tanabe Seiyaku Co Ltd (to E.M.), the Deutsche Forschungsgemeinschaft (to U.C.H.), and National Institutes of Health grant R01 HL61711 (to B.O'R.). General laboratory support was provided by a National Institutes of Health grant (R37 HL-36957).

	Footnotes

 
This manuscript was sent to Michael R. Rosen, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

¹ Both authors contributed equally to this study.

Received December 21, 1998; accepted February 18, 1999.

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