Restoration of transient outward current by norepinephrine in cultured canine cardiac myocytes

Department of Physiology, Cornell University, Ithaca, New York 14853-6401

	ABSTRACT

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The mechanism for the reduction of the transient outward K⁺ current (I_to) in diseased myocardium is unknown. To identify potential mechanisms, the reduction of I_to and its subsequent restoration by norepinephrine (NE) were studied in cultured canine epicardial myocytes. After myocytes were cultured for 9 days (day 9), I_to density was decreased compared with density on the day of isolation (day 0) (3.2 ± 0.4 vs. 10.4 ± 0.4 pA/pF; mean ± SE). The time constant of current decay (_decay) was increased, the time course of recovery from inactivation was prolonged, and the half-inactivation voltage (V_1/2) was shifted to less negative potentials. Exposure of myocytes on day 8 to 1 µM NE or isoproterenol (Iso) for 1 h had no acute effect on I_to but restored I_to density to 7.6 ± 1.2 or 9.7 ± 2.3 pA/pF, respectively, on day 9. Recovery from inactivation and _decay remained slowed, and V_1/2 remained shifted to less negative potentials. The effects of NE and Iso were blocked by actinomycin D and were not mimicked by phenylephrine or phorbol ester. A-23187 (1 µM) also restored I_to. Thus -adrenergic agonists restored normal I_to density, but not normal I_to kinetics, in cultured epicardial myocytes, possibly via increased intracellular Ca²⁺ concentration.

potassium currents; sympathetic nervous system

	INTRODUCTION

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THE DENSITY of the 4-aminopyridine (4-AP)-sensitive transient outward K⁺ current (I_to) typically is low in the neonatal heart and in hypertrophic and myopathic adult hearts (9, 13, 14, 17, 21, 23-25, 31). Although the mechanism for the reduction of I_to density in developing or diseased myocardium has not been established, several lines of evidence suggest that the expression of I_to in the developing heart is modulated by a trophic effect of the sympathetic nervous system and that the reduction of I_to in diseased myocardium is caused by the loss of that effect (9, 12, 13, 26). However, studies of the mechanisms responsible for the reduction of I_to in disease states such as Chagas' disease (12, 23), X-linked muscular dystrophy (24), and inherited sudden death (9) have been hampered by the cost of maintaining colonies of affected animals and by variability between animals in the expression of their disease. To circumvent these limitations, we have in the present study exploited the observation by Schackow et al. (28) that I_to density decreases in adult feline ventricular myocytes with time in primary culture. Our expectation was that, by identifying the mechanism for the reduction of I_to in cultured myocytes, we would gain insights into potential mechanisms for the reduction of I_to in diseased myocardium.

The initial objective of our study was to determine whether the density of I_to is reduced in cultured adult canine ventricular myocytes as it is in cultured feline myocytes (28). Once it became apparent that I_to density was reduced progressively with time in culture, we tested whether I_to could be restored by exposure to norepinephrine (NE) and whether such a restorative effect was mediated by - or -adrenergic receptors. We also tested whether the restorative effect of NE on I_to was mediated by increased intracellular Ca²⁺ concentration.

	METHODS

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Epicardial myocyte isolation. Adult beagle dogs (n = 24) of either sex were obtained from a colony of inbred dogs maintained by Cornell University. The dogs were anesthetized with Fatal-Plus (390 mg/ml pentobarbital sodium, 0.2 mg/4.5 kg iv; Vortech Pharmaceuticals, Dearborn, MI). Hearts were removed rapidly via a left thoracotomy and placed in cold, oxygenated (95% O₂-5% CO₂) Tyrode solution containing (in mM) 0.7 MgCl₂, 0.9 NaH₂PO₄, 2.0 CaCl₂, 124 NaCl, 24 NaHCO₃, 4 KCl, and 5.5 glucose; pH 7.4.

The circumflex coronary artery or a branch of the left anterior descending coronary artery was cannulated and a portion of the left ventricle excised. The tissue initially was perfused with Tyrode solution at 37°C. After 10-15 min, the perfusion was switched to a Ca²⁺-free solution containing (in mM) 118 NaCl, 4.8 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 0.68 glutamine, 11 glucose, 25 NaHCO₃, 5 pyruvate, 2 mannitol, and 10 taurine; pH 7.3. At ~3-5 min, collagenase (0.4 mg/ml; type II, Worthington Biochemical) and BSA (0.5 mg/ml; Sigma) were added and perfusion was continued for another 10-12 min. Digested tissue was sliced away from the subepicardial area, placed in 10 ml of enzyme solution, and swirled. The supernatant was collected, and 10 ml of fresh Ca²⁺-free solution with 0.4 mg/ml collagenase and 0.5 mg/ml BSA was added to the slurry and gently bubbled in a water bath maintained at 37°C. Supernatant was collected for six subsequent washes. After 5 min of settling, the final pellet was washed in 10 ml of incubation buffer containing (in mM) 118 NaCl, 4.8 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 0.68 glutamine, 11 glucose, 20 NaHCO₃, 5 HEPES, 5 pyruvate, 10 taurine, and 0.5 CaCl₂ plus 2% BSA. After another 30 min of settling, the pellet was washed a second time with incubation buffer now containing 1 mM CaCl₂ and was allowed to equilibrate at room temperature for 60 min.

All isolation solutions were filter sterilized and contained penicillin-streptomycin (100 U/ml penicillin-100 µg/ml streptomycin). The perfusion apparatus also was sterilized, and perfusion and subsequent bubblings were conducted in a laminar flow hood.

Epicardial myocyte culture. Once isolated, the myocytes were plated at a density of ~7 × 10³ cells/cm² on plastic culture dishes coated with laminin (10 µg/ml; GIBCO). The culture medium consisted of Eagle's minimum essential medium (GIBCO) with the following additions: nonessential amino acids (GIBCO), vitamins (2X; GIBCO); 10 µg/ml insulin (Sigma), 10 µg/ml transferrin (GIBCO); 5% fetal bovine serum, and 100 U/ml penicillin-100 µg/ml streptomycin. Cells were incubated at 37°C in 5% CO₂ in a humidified environment. For the first 7 days in culture, cytosine -D-arabinofuranoside (10 µM; Sigma) was added to the medium to prevent fibroblast proliferation. Culture medium was changed every 3 days. Culture medium was removed before drug treatment, and the cells were maintained in serum-free medium from the time of drug exposure until I_to was measured 24 h later. On the day of electrophysiological recordings, the myocytes were washed three times with sterile PBS and covered with a trypsin-EDTA solution (0.05%, Sigma) until cells were free floating.

Voltage-clamp technique. Current was measured in the whole cell configuration (11) with the use of an Axopatch-1D amplifier (Axon Instruments, Burlingame, CA) interfaced with a personal computer (Dell System 320LX), as described in detail previously (23-25). Data acquisition and analysis were performed using a commercial program (pCLAMP, v. 5.5.1, Axon Instruments). Current records were filtered at 5 kHz and sampled at a frequency of 10 kHz. After data analysis had been performed, the current traces were imported into AcqKnowledge 3.2 (BIOPAC Systems), where they were smoothed using a moving average of three samples. Smoothed traces were used for Figs. 1, 6, and 7 to increase clarity, particularly that of the low-amplitude traces.

I_to was measured in a bath solution containing (in mM) 125 N-methylglucamine, 4 KCl, 10 HEPES, 1 MgCl₂, 1 CaCl₂, 5 glucose, 0.2 CdCl₂, and 0.03 tetrodotoxin (Sigma); pH 7.3. The pipette solution contained (in mM) 125 potassium aspartate, 20 KCl, 11 EGTA, 5 ATP (Mg salt), 1 CaCl₂, 1 MgCl₂, and 5 HEPES; pH 7.3. Seals were made in serum-free medium at 22-25°C. Activation of I_to was determined using 300-ms duration test pulses from 70 to +60 mV from a holding potential of 80 mV in 10-mV steps. The amplitude of I_to was measured as the difference between peak outward current and minimum current during the depolarizing pulse after the peak. Measurements were made before and 5 min after exposure to 4-AP (2 mM), and peak I_to was defined as the 4-AP-sensitive current.

The decay of I_to was analyzed using a single exponential fit of the current recorded after clamp steps from 10 to +40 mV. The voltage dependence of steady-state inactivation was determined with the use of a double-pulse protocol, in which a 500-ms conditioning pulse, ranging from 80 to +40 mV, was followed by a 300-ms test pulse to +40 mV. The results were fit to a Boltzmann function, I/I_max = 1/{1 + exp [(V_1/2  V_m)/k]}, where V_m is the membrane potential, V_1/2 is the membrane potential at which half-inactivation occurs, and k is the slope factor when V_m = V_1/2. Recovery from inactivation was examined with the use of a double-pulse protocol, in which the time interval between two 300-ms duration pulses to +40 mV from a holding potential of 80 mV was varied between 5 and 750 ms. Cell capacitance was measured by integrating the area beneath the capacitive transient elicited by 10-mV depolarizing steps and dividing that area by the change in voltage.

Study groups. The groups of myocytes studied were as follows: 1) day 0 myocytes, from which recordings were made on the day of isolation; 2) day 9 myocytes, from which recordings were made on day 9 of culture; and 3) drug treatment myocytes, which were exposed to drug treatment for 1 h on day 8 of culture and from which recordings were made on day 9. Drug treatments included NE (1 µM), actinomycin D (1 µg/ml), isoproterenol (Iso; 1 µM), phenylephrine (PE; 1 µM), phorbol 12-myristate 13-acetate (PMA; 0.1 mM), and A-23187 (1 µM).

Data are reported as means ± SE. Statistically significant differences between groups were evaluated initially using ANOVA (StatView, Abacus Concepts, Berkeley, CA), followed by a Scheffé's F-test. P < 0.05 was considered statistically significant.

	RESULTS

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Time-dependent reduction of I_to. I_to density was reduced from 10.4 ± 0.4 pA/pF on day 0 (n = 29) to 7.8 ± 1.6 pA/pF on day 5 (n = 12) to 3.2 ± 0.4 pA/pF on day 9 (n = 32) of culture. Little or no current was recorded from 6 myocytes after 14 days in culture. Given these results, we elected to determine the effects of NE and other interventions on I_to after 9 days in culture. At this time, I_to density was significantly reduced, but I_to could still be measured and current kinetics determined reliably.

Restoration of I_to by NE. I_to density was significantly reduced in day 9 cells compared with day 0 cells (Figs. 1 and 2). Exposure of myocytes to NE (1 µM) on day 8 of culture had no significant effect on I_to within 2 h of exposure (not shown) but increased I_to density 24 h after exposure (Figs. 1 and 2). The restoration of I_to did not occur in the presence of the transcriptional inhibitor actinomycin D (I_to density = 3.7 ± 1.3 pA/pF; n = 5).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Transient outward K+ current (Ito) activation in day 0 (A), day 9 (B), and norepinephrine (NE; 1 µM)-treated myocytes (C). Currents were elicited by voltage-clamp steps from a holding potential of 80 mV to 30 to +60 mV.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Normalized current-voltage relationships for Ito in day 0 (n = 29), day 9 (n = 32), and NE (1 µM)-treated (n = 13) myocytes. Ito was recorded during depolarizing steps of 300 ms duration from a holding potential of 80 mV to test potentials between 20 and +60 mV.

The kinetics of I_to also were altered with time in culture. The time constant of current decay (_decay) at +40 mV was significantly increased in day 9 cells compared with day 0 cells (48.6 ± 2.8 vs. 29.3 ± 0.9 ms) (Fig. 3), and the voltage dependence of steady-state inactivation was shifted to more positive potentials (V_1/2 = 20.3 ± 1.9 vs. 30.5 ± 0.8 mV) (Fig. 3). In addition, the rapid time constant of recovery from inactivation (₁) was slightly, but significantly, increased in day 9 cells compared with day 0 cells, and the slow time constant of recovery from inactivation (₂) was markedly prolonged (Fig. 4).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   A: time constant of current decay (decay) determined with a single exponential fit of Ito at +40 mV for Ito in day 0 (n = 29), day 9 (n = 32), and NE (1 µM)-treated (n = 13) myocytes. Values are means ± SE. B: steady-state inactivation determined using a double-pulse protocol consisting of a conditioning pulse ranging from 80 to 40 mV, followed by a test pulse to +40 mV. Results are fitted to a Boltzmann function I/Imax.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Fast (1; A) and slow (2; B) time constants of recovery of Ito from inactivation for Ito in day 0 (n = 25), day 9 (n = 28), and NE (1 µM)-treated (n = 10) myocytes as determined using a double-pulse protocol, in which the time interval between two 300-ms pulses to +40 mV from a holding potential of 80 mV was varied between 50 and 1,000 ms. Values are means ± SE.

Although NE restored I_to density, it did not restore _decay, which remained prolonged (Fig. 3), or alter the voltage dependence of steady-state inactivation (V_1/2 = 19.3 ± 2.2 mV) (Fig. 3). The rapid and slow time constants of recovery from inactivation also were not altered by NE exposure (Fig. 4).

To determine whether the induction of I_to by NE in the cultured myocytes was mediated by an ₁-adrenergic receptor pathway, as in chagasic myocytes (12), myocyte cultures were treated with the ₁-adrenergic agonist PE (1 µM) or the protein kinase C activator PMA (0.1 mM). As shown by the normalized current-voltage relationships in Fig. 5, neither PE nor PMA exposure increased I_to density. Given these results, we then tested whether the induction of I_to by NE was mediated by -adrenergic receptors. Exposure to 1 µM Iso restored I_to density to day 0 levels (Fig. 6). However, after exposure to Iso, the time course of recovery from inactivation remained slowed (see Fig. 8).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Normalized current-voltage relationships for day 0 (n = 29), day 9 (n = 32), phenylephrine (PE; 1 µM)-treated (n = 5), and phorbol 12-myristate 13-acetate (PMA; 0.1 mM)-treated (n = 5) myocytes.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   A: representative Ito recording from a cultured myocyte exposed to isoproterenol (Iso; 1 µM). B: normalized current-voltage relationships for day 0 (n = 29), day 9 (n = 32), NE (1 mM)-treated (n = 13), and Iso (1 mM)-treated (n = 10) myocytes.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   A: representative Ito traces of a cultured myocyte exposed to the calcium ionophore A-23187 (1 µM). B: normalized current-voltage relationships for day 0 (n = 29), day 9 (n= 32), Iso (1 µM)-treated (n = 10), and A-23187 (1 µM)-treated (n = 10) myocytes.

Restoration of I_to secondary to activation of -adrenergic receptors could be mediated by a number of effects, including elevation of intracellular Ca²⁺. To test whether elevated intracellular Ca²⁺ could restore I_to, myocyte cultures were exposed to the calcium ionophore A-23187 (1 µM) for 1 h on day 8, and I_to was measured on day 9. A-23187 increased I_to density (Fig. 7) but, like Iso, did not restore normal kinetics of recovery from inactivation (Fig. 8).

View larger version (27K): [in this window] [in a new window]   Fig. 8.   1 (A) and 2 (B) of recovery of Ito from inactivation for Ito in day 0 (n = 25), day 9 (n = 28), Iso (1 µM; n = 7)- and A-23187 (1 µM)-treated (n = 9) myocytes. Values are means ± SE.

	DISCUSSION

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I_to in cultured adult ventricular myocytes. The results of this study indicate that after 9 days in primary culture, peak I_to density was significantly decreased, whereas _decay was increased and the time course of recovery from inactivation was prolonged. In addition, V_1/2 was shifted to less negative potentials. These results are similar to those reported previously by Schackow et al. (28), who studied alterations of I_to in cultured adult feline ventricular myocytes. In those studies, a 50% reduction in I_to density occurred after 7-9 days in culture, with virtual disappearance of the current within 2 wk. The mechanism for the reduction of I_to was not identified but apparently did not involve decreased I_to availability or the expression of a noninactivating I_to. In addition, the reduction of I_to could not be attributed to the cell culture procedure per se or to the presence of serum in the cell culture medium.

Developmental regulation of I_to. I_to is known to be developmentally regulated in canine ventricular myocardium (13, 25). Maturation of I_to is particularly marked between 10 and 20 wk of age (13, 25), a time of parallel maturation of the sympathetic nervous system (32). Consequently, it has been proposed that the developmental changes in I_to may be regulated by functional sympathetic innervation (13). Previous studies (33, 34) have provided evidence for a trophic effect of sympathetic innervation on the development of L-type Ca²⁺ channels (22) and on the maturation of Na⁺ current (33, 34). However, there is little direct evidence to support a role for the sympathetic nervous system in the regulation of I_to development.

Given that the density of I_to is lower in neonatal ventricular myocytes than in adult myocytes (13, 25), the reduction of I_to in cultured adult ventricular myocytes may represent a return to a "fetal program" of gene expression (15, 16). If the normal maturation of I_to is facilitated by, or even dependent on, sympathetic innervation of developing myocytes, it might be expected that the reduction of I_to in cultured adult ventricular myocytes could be reversed by exposure to sympathetic neurotransmitters. Our observation that the reduction of I_to in cultured myocytes was reversed by brief exposure to NE and that this effect was prevented by actinomycin D supports this idea.

Sympathetic regulation of I_to in diseased myocardium. If I_to gene expression is regulated by the sympathetic nervous system, NE would be expected to restore I_to in cells in which I_to has been reduced by a disease process that destroys sympathetic nerves or otherwise interferes with neuronal release of NE. Recently, we have shown that I_to is reduced during the acute stage of Chagas' disease in dogs but returns to normal in a more chronic stage of the disease (23). The acute stage of the disease is associated with marked parasitemia (23) and degeneration of sympathetic nerve terminals (18), whereas, in the chronic stage, the parasitemia abates (23) and sympathetic nerve terminals reappear (18). Exposure of acutely infected chagasic myocytes to NE restores I_to 20-24 h after the initiation of NE exposure, suggesting that the reduction of I_to during the acute stage of the disease is caused by the loss of a trophic effect of sympathetic innervation. The effects of NE required binding to an ₁-adrenergic receptor and activation of protein kinase C, with the latter effect most likely involving a pertussis toxin-insensitive G protein and activation of phospholipase C (12).

The reduction of I_to in the hearts of German shepherd dogs with inherited ventricular arrhythmias also may be linked to abnormal sympathetic innervation. I_to is reduced in left ventricular, but not right ventricular, epicardial and Purkinje myocytes isolated from the hearts of affected dogs (9). Sympathetic innervation, as assessed with the use of [¹²³I]metaiodobenzylguanidine scintigraphy and immunohistochemical localization of tyrosine hydroxylase, is reduced in the left ventricle, but not in the right ventricle, of these dogs (5). In an initial attempt to determine whether the association between reduced I_to and denervation was merely coincidental, we tested whether chronic NE exposure restored I_to in left ventricular epicardial myocytes isolated from affected dogs (26). Our results indicate that NE increases I_to density, suggesting that the reduction of I_to in affected myocytes may reflect the lack of a trophic effect of NE on expression of I_to.

I_to also is known to be reduced in other forms of cardiac disease, such as subacute myocardial infarction (17), myocardial hypertrophy (31), hypertrophic cardiomyopathy (21), pacing-induced heart failure (14), and X-linked muscular dystrophy (24). It remains to be determined whether the loss of I_to in failing, hypertrophied, or infarcted hearts is related to the abnormalities of sympathetic function that frequently accompany such disease states.

Demonstration that the development of I_to is regulated by sympathetic innervation would require that NE increase the expression of the gene encoding the I_to channel protein. Several candidates for the I_to channel protein exist, including Kv1.4, Kv4.2, and Kv4.3. In rat and ferret myocardium, it appears that Kv4.2 is the I_to channel protein, given that expression of this protein in oocytes produces a current with electrophysiological properties similar to those of native I_to (2) and that messages for both the Kv4.2 (4, 6) and the Kv4.2 protein (1) are abundant in ventricular myocardium. However, recent studies have indicated that Kv4.2 mRNA levels are low in adult canine ventricular myocardium; I_to in this tissue appears to be most similar to Kv4.3 (7). When expressed in oocytes, the Kv1.4 protein also is associated with a transient K⁺ current, but the recovery kinetics of this current are significantly slower than those of native I_to (27). Further studies are needed in cultured canine myocytes to determine whether NE exposure induces the native I_to -subunit but fails to induce a regulatory component of the channel (e.g., a -subunit) that is responsible for normal kinetics or whether NE may be inducing the expression of a different transient K⁺ channel, such as Kv1.4.

Mechanism for the trophic effect of NE on I_to. The mechanism for the induction of I_to by NE and isoproterenol remains to be determined. -Adrenergic stimulation has not been shown to directly stimulate mitogen-activated protein kinase (MAPK) pathways involved in activation of transcription factors. However, -adrenergic stimulation results in an elevation of intracellular Ca²⁺ via the activation of voltage-gated Ca²⁺ channels. Ca²⁺ has been shown to be involved in signal transduction pathways such as the MAPK pathways (3, 8) or calcium binding pathways (10, 29, 30), ultimately influencing gene expression through the induction of transcription factors. In this regard, Mori et al.(19) have shown that the induction of Kv1.5 by cAMP derivatives may be mediated by a cis response element-cAMP response element binding protein binding site on the 5' flanking region of the Kv1.5 gene.

Another unresolved issue relates to the observation that the effects of NE on I_to in chagasic myocytes were mediated by -adrenergic receptors (12), whereas the effects of NE in the cultured myocytes were mediated by -adrenergic receptors. In general, the electrophysiological and inotropic effects of -adrenergic stimulation predominate over those of -adrenergic stimulation in canine ventricle. In Chagas' disease, however, the generation of anti--receptor antibodies may blunt the response to -adrenergic agonists (20). The reason for the failure of the cultured myocytes to respond to -adrenergic agonists, at least as far as the induction of I_to is concerned, presently is not clear. It seems possible that -adrenergic receptors or elements of their associated signaling cascade may be altered by the culture procedure. Consequently, regulation of I_to in cultured cells may differ in important respects from that in the intact heart.

	ACKNOWLEDGEMENTS

We thank M. Lisa Lee and Dr. Steven C. Barr for assistance with the cell culture procedures and Dr. Mark S. Roberson for helpful discussions.

	FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests: Robert F. Gilmour, Jr., Dept. of Physiology, T8 012B VRT, Cornell Univ., Ithaca, NY 14853-6401.

Received 15 April 1998; accepted in final form 30 July 1998.

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