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Am J Physiol Heart Circ Physiol 275: H577-H590, 1998;
0363-6135/98 $5.00
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Vol. 275, Issue 2, H577-H590, August 1998

Electrophysiological properties of neonatal rat ventricular myocytes with alpha 1-adrenergic-induced hypertrophy

John P. Gaughan, Colleen A. Hefner, and Steven R. Houser

Department of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

The electrophysiology of neonatal rat ventricular myocytes with and without hypertrophy has not been characterized. The alpha 1-adrenergic agonist phenylephrine induced hypertrophy in neonatal rat ventricular myocytes. After 48 h of exposure to 20 µM phenylephrine, cell surface area of hypertrophied myocytes was 44% larger than control. Action potential duration was significantly longer in hypertrophy than in control. There was an increase in L-type Ca2+ current in control after 48 h in culture, but current density was significantly less in hypertrophy (-4.7 ± 0.8 hypertrophy vs. -10.7 ± 1.2 control pA/pF, n = 22, P < 0.05). T-type Ca2+ current density was not different. The alpha -adrenergic antagonist prazosin blocked the hypertrophy and the chronic effect of phenylephrine on L-type Ca2+ current. Transient outward K+ current density was decreased 70% in hypertrophy and was blocked with 4-aminopyridine. No change in Na+ current density was observed. Staurosporine, a protein kinase C inhibitor, eliminated the hypertrophy and the effect on L-type Ca2+ current. These studies showed that phenylephrine-induced hypertrophy occurred via the alpha 1-adrenergic pathway and caused electrophysiological changes and effects on ion channel expression.

electrophysiological changes; ion channels

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

THE DENSITY AND TYPES of sarcolemmal ion channels found in cardiac myocytes change significantly during development and in hypertrophic heart disease and underlie the associated changes in action potential wave shape. The cellular signaling mechanisms that determine the number and types of ion channels expressed in cardiac myocytes are not well understood and are the subject of this investigation. Previous studies have shown that pressure-overload hypertrophy in vivo is accompanied by electrophysiological changes which are thought to be both proarrhythmic and contributory to the contractile dysfunction observed in hypertrophy and heart failure (4, 5, 27, 28, 39, 40). The density of transient outward K+ current is often decreased and may lead to the prolonged action potential duration of hypertrophied myocytes (7, 9, 39, 52). The specific factors that induce these changes have not been well defined.

Studies of the effects of activation of specific signaling pathways on the expression of functional proteins are most easily controlled when cultured cells are employed. Primary cultures of neonatal rat ventricular myocytes exposed to alpha -adrenergic agonists have been widely studied as a model of signaling pathways controlling cardiac cell growth and hypertrophy (48-50). Various other hormones (epinephrine, ANG II, endothelin, and thyroxine), growth factors (transforming growth factor-beta 1, fibroblast growth factor, and insulin-like growth factor I), and mechanical stimuli have also been shown to induce significant hypertrophy in this preparation (19, 20, 31, 42, 45).

Hypertrophy of cultured neonatal rat ventricular myocytes induced by alpha -adrenergic agonists is characterized by changes in gene expression (45). These changes include an increase in myosin light chain 2 and increased atrial natriuretic peptide gene expression (9, 10, 29, 36, 46). alpha 1-Adrenergic stimulation has been shown to cause hypertrophy through the activation of protein kinase C (PKC) and to induce the protooncogenes c-myc and c-fos (48, 51). The electrophysiological consequences of chronic alpha -adrenergic stimulation have not been defined. We studied the changes in electrophysiological properties of cultured neonatal rat ventricular myocytes with hypertrophy induced by the alpha 1-adrenergic agonist phenylephrine.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Tissue culture. Primary cultures of neonatal rat ventricular myocytes were prepared by the methods originally described by Simpson and Savion (50) with minor modifications. The hearts from 1- to 2-day-old Sprague-Dawley rats were removed, the ventricles were minced, and the myocytes were dissociated with trypsin (1.5 mg/ml, Difco, Pittsburgh, PA). Dispersed cells were preplated on 100-mm culture dishes (Falcon, Oxford, CA) for 30 min at 37°C in 1% CO2 to remove fibroblasts. Nonattached viable myocytes were collected, seeded onto glass coverslips, and placed into 35-mm culture dishes. Myocytes were plated at low density (500/mm2) to prevent confluent growth during the experimental period. Ventricular myocytes were incubated in Hanks' minimal essential medium (MEM; Sigma Chemical, St. Louis, MO) supplemented with 5% calf serum (Hyclone Laboratories, Logan, UT), penicillin, and vitamin B12 (Sigma) for 24 h and then replaced with serum-free Hanks' MEM supplemented with penicillin, vitamin B12, insulin, transferrin, BSA, and bromodeoxyuridine (all Sigma) for 24-72 h with or without 20 µM phenylephrine (Sigma). Measurements of myocyte two-dimensional area were made after 48 h of exposure to phenylephrine by using the Macintosh-based, public-domain NIH Image measurement program developed at the National Institutes of Health (available on Internet at http://rsb.info. nih.gov/nih-image/). Measurements using the NIH Image program were validated by measuring a two-dimensional rectangle of known dimensions formed by overlapping micrometer slides. Measurements were made only on nonoverlapping myocytes. After 24 h, cultures were supplemented with 1 mM 4-methylumbelliferyl-7-beta -D-xyloside (Sigma) to inhibit the formation of extracellular matrix and to facilitate patch-clamp seal formation (21).

Electrophysiology. Suction-type patch pipettes were prepared from borosilicate glass (1B150F, World Precision Instruments, Sarasota, FL) with a two-stage pipette puller (model BB-CH, Mecanex, Geneva, Switzerland). The pipette tip was lightly heat polished before use, and the tip resistance was 4-8 MOmega . For studies of T- and L-type Ca2+ currents, Ba2+ was used as the charge carrier and pipettes were filled with Na+- and K+-free solution composed of (in mM) 100 cesium chloride, 20 tetraethylammonium chloride, 10 EGTA, 10 HEPES, 5 potassium ATP, and 0.2 lithium GTP (pH 7.3 with cesium hydroxide). The bath solution was composed of (in mM) 140 tetraethylammonium chloride, 2 magnesium chloride, 5.4 barium chloride, 10 dextrose, and 10 HEPES (pH 7.4 with tetraethylammonium hydroxide) (15). Separation of T- and L- type Ca2+ currents was by subtraction of currents obtained from holding potentials of -90 and -50 mV, respectively (15). For the recording of K+ currents, the pipette solution was composed of (in mM) 140 potassium chloride, 1 magnesium chloride, 5 sodium ATP, 5 HEPES, and 10 EGTA (pH 7.2 with potassium hydroxide). The bath solution was composed of (in mM) 150 sodium chloride, 10 dextrose, 5.4 potassium chloride, 1.2 magnesium chloride, 2 calcium chloride, 2 sodium pyruvate, and 5 HEPES (pH 7.4 with sodium hydroxide). Cadmium chloride (0.5 mM) was added to block Ca2+ channels. For recording Na+ currents, the pipette solution was composed of (in mM) 125 cesium chloride, 1 magnesium chloride, 10 EGTA, 5 sodium ATP, and 10 HEPES (pH 7.2 with cesium hydroxide). The bath solution was composed of (in mM) 100 tetraethylammonium chloride, 40 sodium chloride, 10 glucose, 1 magnesium chloride, 5 cesium chloride, 0.1 calcium chloride, 1 nickel chloride, and 10 HEPES (pH 7.4 with cesium hydroxide). The physiological pipette solution used for the recording of action potentials was composed of (in mM) 140 potassium chloride, 1 magnesium chloride, 5 HEPES, and 5 sodium ATP (pH 7.2 with potassium hydroxide).

The pipette was attached to the input stage of a patch-clamp amplifier (Axoclamp II, Axon Instruments, Foster City, CA) as described in detail previously (27, 28). Whole cell currents and voltages were measured by the discontinuous switch-clamp method (14). A sampling rate of 10 kHz ensured critical damping (maximal stability) of the discontinuous voltage clamp, assuming an average cell capacitance of 30 pF and an average gain of 0.1 nA/mV. The data were sampled using a 12-bit analog-to-digital converter (Labmaster TL-1, Scientific Solutions, Foster City, CA). Data acquisition and analysis were controlled by pCLAMP software (Axon Instruments) utilizing a DOS-based personal computer. All experiments were carried out at 37°C except for experiments on Na+ currents, which were carried out at 25°C.

All experiments were performed on single myocytes with no obvious contacts with neighboring cells. After seal formation and membrane rupture, a single 10-mV hyperpolarizing step (from -50 mV) was applied. The resulting capacitative current transient was integrated and divided by the voltage step (10 mV) to determine the capacitative surface area of the cell. This measurement (in pF) was used to index the membrane currents to calculate membrane current density. Adequate voltage control was assessed by visual inspection of actual voltage traces recorded simultaneously with current traces. The adequacy of the voltage clamp for recording Na+ currents was assessed by using only cells in which the capacitance transient had settled within 1.5 ms. The peaks of the inward Na+ currents occurred 2-5 ms after the initiation of the voltage step (see Fig. 9). Because of the extremely small size of these cells (20-30 pF), rundown of membrane currents precluded extensive study of individual cells. For this reason, after membrane rupture, study was limited to a single protocol carried out on each cell within 5 min (rundown data are presented in RESULTS; see below).

Cells attached to coverslips were placed in a Lucite perfusion chamber (~20 × 10 × 10 mm) for voltage-clamp experiments or studied directly in culture dishes for action potentials. They were mounted on the heated stage of an inverted microscope (Nikon Diaphot-TMD). The experimental chamber was perfused with various ion-containing "bath" solutions for voltage-clamp experiments (see above for composition of solutions) at a rate of 1-2 ml/min. Before the start of a voltage-clamp experiment, cells were superfused with the bath solution for 2-5 min.

For recording of action potentials, a continuous current-clamp mode was used. To initiate an action potential, a square current pulse (5-ms duration) was applied at various frequencies (0.5, 1.0, 1.5, and 2.0 Hz). The stimulus simultaneously activated the recording of the action potential via a personal computer for storage and later measurement. For the recording of action potentials, cells were studied in serum-free culture medium (with 2 mM Ca2+) maintained at 37°C for 4 days with or without 20 µM phenylephrine. Before action potentials were recorded, phenylephrine was removed from the culture medium to preclude the acute effects of phenylephrine. Myocytes chosen for study of action potentials exhibited prominent nuclei and were observed to beat spontaneously in the culture media.

To record Ba2+ currents through Ca2+ channels, the holding potential was initially set at -50 mV to inactivate T-type Ca2+ channels (15), and a series of 11 depolarizing voltage steps was applied in increasing 10-mV increments for 200 ms each to activate L-type Ca2+ channels. A series of 15 voltage steps in increasing 10-mV increments was then applied for 200 ms each from a holding potential of -90 mV to elicit T-type and L-type Ba2+ currents through Ca2+ channels (6). Values for T-type currents were calculated by subtracting the peak current values obtained at each test voltage with the -50-mV protocol from the peak current values obtained with the -90-mV protocol. This procedure effectively eliminated T-type current from measurements of L-type current (Fig. 8). An interval of 3 s was used between voltage steps. Ba2+ was used as the charge carrier to minimize Ca2+-induced current inactivation common with L-type Ca2+ currents (32).

For the recording of Na+ currents, a holding potential of -90 mV was used, followed by 30-ms test pulses up to +40 mV in increments of 10 mV. To achieve adequate voltage control for Na+ currents, the external Na+ concentration was reduced to 40 mM, and the experiments were carried out at room temperature (25°C). Nevertheless, Na+ currents as large as 1 nA were recorded. The peak Na+ current was measured as the difference between the inward peak and the baseline at the end of the pulse. For the recording of transient outward K+ currents, Na+-containing bath solution was used. A holding potential of -80 mV was followed by a 10-ms prepulse to -40 mV (to inactivate Na+ current) and then stepped to -30 through +70 mV (in 10-mV increments).

The peak inward Ba2+ current for each "voltage step" was determined with pCLAMP software and measured from the baseline to the peak current that occurred during the 200-ms test pulse. Outward K+ currents were measured as the difference between the peak current obtained at each voltage step and the steady-state current at the end of the test pulse.

Data analysis. Each voltage-clamp experiment was analyzed by using a repeated-measures ANOVA for a two-factor factorial experiment with repeated measures on the second factor, test voltage, at either 11 or 15 levels.

The statistical analysis was based on an additive linear model and employed a least-squares ANOVA. Measurements of action potentials were analyzed using Student's t-test for independent samples. All data are presented as means ± SE. Differences were considered significant at P <=  0.05.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Significant changes in cell morphology occurred in neonatal ventricular myocytes after exposure to 20 µM phenylephrine for 48 h in culture. The size was seen to increase to a diameter of 20-30 µm from 10 µm during this period, and the two-dimensional cell area was 44% greater in hypertrophied myocytes than in control myocytes (889 ± 88 µm2 hypertrophy, n = 30 vs. 619 ± 36 µm2 control, n = 46, means ± SE), similar to previously reported values (50). Figure 1 shows that myocytes exposed to phenylephrine were hypertrophied with prominent nuclei and cytoplasmic extensions. The application of propranolol (10-6 M) along with phenylephrine had no effect on the hypertrophy, whereas the application of prazosin (10-5 M) blocked the hypertrophic response. A PKC inhibitor, staurosporine (1 nM), when applied concurrently with the phenylephrine, also blocked the hypertrophic response. Average cell area was not significantly different from control myocytes after application of the staurosporine (483 ± 55 µm2 staurosporine, n = 23 vs. 619 ± 36 µm2 control, n = 46). Cell capacitance measured after 48 h was not significantly different between control and hypertrophied myocytes (26.1 ± 1.1 pF, n = 122 control vs. 30.1 ± 1.1 pF, n = 118 hypertrophy, means ± SE). After 72-96 h in culture, both control and hypertrophied myocytes were observed to beat spontaneously and sporadically in the culture medium as previously reported by Simpson (47).


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  		Fig. 1.   Control (A) and hypertrophied (B) neonatal rat ventricular myocytes (72-h cultures). After 24 h in serum-supplemented culture, myocytes were placed in serum-free culture medium with or without 20 µM phenylephrine for an additional 48 h. Control myocytes were ~10 µm in diameter, and myocytes exposed to phenylephrine were 2-3 times larger.

Action potential duration. The effects of chronic exposure to phenylephrine (72 h) on the wave shape of the myocyte action potential were studied. One hour before action potentials were recorded, cells were washed with fresh culture medium (without phenylephrine) to preclude the acute effects of phenylephrine. Only nonoverlapping myocytes, similar to those used in voltage-clamp experiments, were chosen for study. Action potential duration measured at 25, 50, and 75% repolarization (APD25, APD50, and APD75) was significantly prolonged in the hypertrophied cells (see Fig. 2). Differences between control and hypertrophied cells are presented in Table 1. The APD25 was prolonged 45%, APD50 42%, and APD75 61% (P < 0.05). There were no significant differences either in resting membrane potential (-68.3 ± 1.3 mV control vs. -65.7 ± 1.1 mV hypertrophy) or overshoot (29.3 ± 2.2 mV control vs. 33.1 ± 1.5 mV hypertrophy). There was noticeable change in the shape of the early repolarization phase of the action potential between control and hypertrophied myocytes. The differences in action potential characteristics between control and hypertrophied myocytes were present at all rates of stimulation between 0.5 and 2 Hz (Fig. 1C). Rate-dependent shortening of the action potential duration was apparent in control and hypertrophied myocytes The action potential duration, resting membrane potential, and overshoot for control ventricular myocytes were similar to values from published studies of cultured neonatal rat ventricular myocytes (11-13, 26, 53). The present experiments show that hypertrophied myocytes induced by phenylephrine in culture have prolonged action potentials. The ionic basis of these action potential changes were studied with whole cell voltage-clamp techniques (see below).


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  		Fig. 2.   Representative action potentials from control (A) and hypertrophied (B) neonatal rat ventricular myocytes. Hypertrophied myocytes had been exposed to 20 µM phenylephrine for 72 h but were studied in absence of phenylephrine. Controls were cultured in defined medium for an equal period of time. Myocytes were stimulated at 0.5-2 Hz. Lines show 0-mV potential. C: rate-dependent shortening of action potential in control and hypertrophied myocytes. APD90, action potential duration at 90% repolarization.

                              
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  		Table 1.   Characteristics of action potentials from control and hypertrophied neonatal rat ventricular myocytes

Ba2+ current through L-type Ca2+ channels: acute effects of phenylephrine. Ba2+ current through L-type Ca2+ channels was measured in 24-h cultures of neonatal rat myocytes exposed to 20 µM phenylephrine for 5-30 min to study acute effects. Representative current tracings are shown in Fig. 3. The peak inward current was significantly larger in myocytes exposed to phenylephrine. This effect was apparent as early as 5 min after exposure. Maximal increases were approximately the same at 5 and 30 min. Average peak inward Ba2+ current density through L-type Ca2+ channels was 247% larger in myocytes exposed to phenylephrine when the test potential was stepped to -10 mV from a holding potential of -50 mV. Figure 3 shows that Ba2+ current through L-type Ca2+ channels was significantly increased by acute exposure to 20 µM phenylephrine and that the effect was eliminated by the beta -adrenergic receptor blocker propranolol (10-6 M) but not by the alpha -adrenergic receptor blocker prazosin (10-5 M). These results indicate that the acute effect of phenylephrine was via activation of the beta -adrenergic receptor.


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  		Fig. 3.   Acute effects of phenylephrine (20 µM) on Ba2+ currents of 24-h cultures of neonatal rat ventricular myocytes are shown. Voltage-clamp experiments were from a holding potential of -90 mV and stepped to test voltages indicated for 200 ms each at 0.33 Hz. Control myocyte (A) had a similar capacitance (20 pF) to myocyte exposed to 20 µM phenylephrine (B) for 5 min. T-type Ba2+ currents can be seen in voltage step to -30 mV. Maximal L-type Ba2+ current (IBa,L ) occurred at voltage step to -10 mV. C: acute exposure to phenylephrine (Phenyl) caused a large increase in Ba2+ current density. D: this effect was blocked by beta -adrenergic blocker, propranolol, but not by alpha -adrenergic blocker, prazosin. * Significant difference, P <=  0.05.

Ba2+ current through L-type Ca2+ channels: chronic effects of phenylephrine. Twenty-four-hour cultures of neonatal rat ventricular myocytes were incubated in serum-free Hanks' MEM with 20 µM phenylephrine for an additional 48 h. Experiments were then conducted in the absence of phenylephrine. Voltage-clamp experiments were carried out on 23 time-matched control and 22 phenylephrine-treated, hypertrophied myocytes. Representative tracings of inward Ba2+ currents from these myocytes are shown in Fig. 4. The peak Ba2+ current density was significantly smaller in the myocytes exposed to phenylephrine. On average, the peak current density was at least 50% smaller in hypertrophied myocytes than in time-matched controls between -10 and +20 mV (see Fig. 4). Similar results were obtained with Ca2+ as the charge carrier (see Fig. 4, insets). It is important to note that, in control cells, the peak Ba2+ current density approximately doubled during the 48-h culture period, from -4 to -8 pA/pF. Therefore exposure to phenylephrine eliminated the increase in current density with time in culture. This increase in Ba2+ current density in control cells with time in culture agrees with results of Nalivaiko et al. (37).


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  		Fig. 4.   Effects of 48-h exposure to phenylephrine on Ba2+ currents through L-type Ca2+ channels. Representative tracings of inward Ba2+ currents from a control myocyte (cell capacitance = 23.3 pF; A), a hypertrophied myocyte exposed to phenylephrine (cell capacitance = 21.0 pF; B), and a myocyte exposed to phenylephrine and staurosporine (cell capacitance = 34.3 pF; C). Insets: currents from similar cells in which Ca2+ was used as charge carrier. Voltage-clamp experiments were from a holding potential of -90 mV and stepped to test voltages indicated for 200 ms at 0.33 Hz. Control myocytes (A) had significantly larger Ba2+ currents than those exposed to 20 µM phenylephrine (B) for 48 h. Staurosporine (1 nM) effectively blocked hypertrophy and effect on L-type current (C). Current-voltage relationship (D) shows L-type Ba2+ current densities were significantly smaller in hypertrophied myocytes exposed to phenylephrine. Peak current densities occurred in voltage step to -10 mV. * Significant difference, P <=  0.05.

Rundown of Ba2+ currents in control and hypertrophied cells was a concern during all experiments. For this reason, after membrane rupture, study was limited to a single protocol on each cell. Figure 5 shows that significant rundown of Ba2+ currents did not occur during 2 min of study either in control or hypertrophied myocytes. Another concern was that because capacitance measurements were similar for control and hypertrophied myocytes, we may not have observed the average effect on the hypertrophied myocytes. To address this issue, we analyzed L-type Ba2+ current density as a function of cell capacitance (Fig. 6). This analysis showed that L-type Ba2+ current density was smaller in hypertrophied myocytes than in control cells at all cell capacitances and was a result of exposure to phenylephrine.


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  		Fig. 5.   Representative current tracings of Ba2+ currents through L-type Ca2+ channels in a control (A) and a hypertrophied myocyte exposed to phenylephrine (B) after 72 h in culture. A and B: left panels, a series of voltage steps from a holding potential of -50 mV 1 min after membrane rupture; right panels, identical protocol carried out 2 min after membrane rupture, indicating a lack of significant rundown of L-type current; bottom panels, current-voltage relationships for test cells.


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  		Fig. 6.   L-type Ba2+ current (IBaL) density of control and hypertrophied myocytes as a function of cell capacitance (Cm). After 48 h of exposure to phenylephrine, hypertrophied myocytes had decreased L-type Ca2+ current density at all levels of cell capacitance compared with control myocytes.

The involvement of specific adrenergic (alpha  and beta ) signaling mechanisms on myocyte hypertrophy and Ca2+ current was studied. Although the beta -adrenergic blocker propranolol eliminated the acute effect of phenylephrine on Ba2+ current density, it had no significant effect on the hypertrophy that developed or on the associated decrease in Ba2+ current density that occurred with chronic exposure to phenylephrine (Fig. 7). However, when the alpha -adrenergic blocker prazosin (10-5 M) was included in the culture medium (with phenylephrine), the hypertrophy did not occur and Ba2+ current density was not significantly different from control (Fig. 7). Staurosporine (1 nM), an inhibitor of PKC, when included in the culture medium, prevented the hypertrophy and the effect on L-type current (Fig. 4). These data strongly suggest that the hypertrophy and the reduction in Ba2+ current density through L-type Ca2+ channels were mediated via the alpha -adrenergic receptor signaling pathway and may have involved the activation of PKC.


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  		Fig. 7.   Effects of alpha  (prazosin)- and beta  (propranolol)-blockers on chronic effects of phenylephrine exposure: current-voltage relationship of peak L-type Ba2+ current density of myocytes exposed to 20 µM phenylephrine for 48 h with or without propranolol (10-6 M; A) and with or without prazosin (10-5 M; B). One hour before analysis, fresh culture medium without test agents was substituted to preclude any acute effects of test agents. Voltage-clamp experiments were from a holding potential of -50 mV and steps were to test voltages indicated for 200 ms each at 0.33 Hz. Propranolol had no effect on hypertrophy or on effect (reduced current density) of phenylephrine on L-type Ba2+ current density. Prazosin blocked hypertrophy and phenylephrine-induced decrease of L-type Ba2+ current density. * Significant difference, P <=  0.05.

Ba2+ current through T-type Ca2+ channels: acute and chronic effects of phenylephrine. All myocytes studied demonstrated robust T-type currents. T- and L-type current were effectively separated by holding potential as described in METHODS and in previous studies (15) (Fig. 8). To further confirm that this approach could separate the T- and the L-type Ba2+ current, we applied 10 µM nitrendipine, which virtually eliminated the L-type current, and 50 µM nickel chloride, which eliminated the T-type current (Fig. 8A). These experiments confirmed that from -50 mV the inward current was almost entirely L-type current, whereas from -90 mV both L- and T-type currents were elicited. In myocytes in culture for 24 h, peak T-type current density was about -2 pA/pF (Fig. 8B), similar to the value reported previously for neonatal rat ventricular myocyte cultures (15). Acute exposure (5-30 min) of these myocytes to phenylephrine had no significant effect on T-type current density (Fig. 8B).


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  		Fig. 8.   Separation of Ba2+ currents through L- and T-type Ca2+ channels (A; left, control). A holding potential (Vh) of -90 mV elicited both T- and L-type currents. T-type current could be clearly seen in step to -30 mV and both T- and L-type were apparent in step to -10 mV. A holding potential of -50 mV elicited only L-type currents (A, bottom). Addition of 10 µM nitrendipine effectively blocked L-type current leaving T-type current, which peaked at -10 mV (A, middle). Addition of 50 µM NiCl2 effectively blocked T-type current leaving L-type current, which likewise peaked at -10 mV (A, right). Bottom: current-voltage relationship for T-type Ba2+ current (IBaT) density after 24 h in culture (B) and 72 h in culture (C). Phenylephrine had no significant effect on T-type current density when applied acutely (B) or chronically (C) to cultured myocytes.

In hypertrophied myocytes (exposed to 20 µM phenylephrine for 48 h), the T-type current density was not significantly different from that of 24-h cultures (-3 pA/pF; Fig. 8C) or from time-matched controls. The voltage dependence of steady-state inactivation for the T-type Ba2+ currents was not significantly different between control (n = 4) and hypertrophied (n = 4) myocytes (data not shown). These results are similar to the observations of Nalivaiko et al. (37), who recorded a slight increase in T-type Ca2+ current density after 72 h in culture. Our results showed that T-type channel expression was sufficient to maintain T-type Ca2+ current density after exposure to phenylephrine.

Effect of phenylephrine-induced hypertrophy on transient outward K+ current. Transient outward K+ current (IKto1) in hypertrophied cells was measured and compared with nonhypertrophied, time-matched controls (representative current tracings are shown in Fig. 9). These experiments showed increasingly large transient outward currents between 0 and +70 mV in control myocytes. Peak IKto1 density was 76% smaller in hypertrophied myocytes than in controls (see Fig. 9). Control cells had IKto1 densities similar to those reported for acutely isolated neonatal cells of similar age, 4-5 pA/pF, at a test potential of +50 mV (53). The recovery of IKto1 was investigated, since it probably played an important role in the rate of repolarization and wave shape of the action potential (see above). Figure 10 shows the time to recovery for IKto1 at different pulse intervals. The current required ~300 ms for complete recovery, indicating full recovery of the current between pulses during action potential studies at stimulation frequencies of 0.5-2.0 Hz. This process was not easily studied in hypertrophied myocytes because the current magnitude was so small in these cells. Application of 4-aminopyridine (4-AP, 2 mM), a blocker of Ca2+-independent IKto1, when applied to control cells (in bath solution), reduced the transient outward current 63%, providing evidence that the transient outward current was carried by K+. The failure of 4-AP to block the sustained, time-independent outward current (data not shown) suggested that the nature of this current was different. It has been shown that, in rabbit atrial myocytes, the sustained outward current (IKto2), resistant to blockade by 4-AP, is a Cl- current (54). This idea was not pursued in the present study. Staurosporine (1 nM), a PKC inhibitor, when included in the culture medium along with phenylephrine, eliminated the effect of phenylephrine on IKto1 (10.0 ± 1.1 pA/pF, mean ± SE, at test voltage of +70 mV, n = 2). The acute application of 20 µM phenylephrine had no significant effect on the IKto1 (n = 5, data not shown).


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  		Fig. 9.   Effect of chronic (72 h) phenylephrine exposure on transient outward K+ current (IKto1). Representative tracings (A-C) and current-voltage relationship (D) of control (A), control plus 4-aminopyridine (4-AP, 2 mM; B), and myocytes exposed to phenylephrine (C). Experimental protocol is described in METHODS. 4-AP (2 mM) reduced IKto1 in control cells (B). IKto1 current density was significantly smaller in hypertrophied myocytes exposed to phenylephrine (C) but was not different from controls after exposure to 4-AP. Current-voltage relationship (D) shows that exposure to phenylephrine caused significant decreases in K+ current density. * Significant difference, P <=  0.05.


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  		Fig. 10.   Recovery from inactivation of IKto1. Recovery was determined by a 2-pulse protocol consisting of a 300-ms prepulse from a holding potential of -70 mV to a test potential of +80 mV followed by an identical test pulse after a varying recovery period of 10-450 ms. Plot shows average recovery of 4 control cells (data are means ± SE) to be essentially complete within 300 ms. Data were fitted to a single-parameter exponential function, test current-to-maximal current ratio (Itest/Imax) = 1 - e-t/tau (tau  = 66.7 ms, 95% confidence limits, 53.1, 80.3 ms).

Effect of phenylephrine-induced hypertrophy on Na+ current. Na+ currents were recorded from hypertrophied (n = 4) and time-matched control (n = 4) myocytes. Rapidly activating currents with properties sufficient to explain the rapid upstroke of the action potential were observed in these cells. Specifically, the peak inward Na+ current occurred 2-5 ms after the initiation of the voltage step (Fig. 11). Although the Na+ current was as large as 1 nA (in reduced extracellular Na+), there was no difference in Na+ current density between control and hypertrophied myocytes (see Fig. 11), suggesting that exposure to phenylephrine had no significant effect on Na+ current. The apparent reversal potential was consistent with our experimental conditions.


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  		Fig. 11.   Effects of chronic phenylephrine exposure on Na+ current (INa) density. Voltage-clamp experiments were from a holding potential of -90 mV and stepped to various positive test voltages for 30 ms each. Representative tracings of a control (A) and a hypertrophied myocyte exposed to phenylephrine (B) are shown. C: current-voltage relationship for 4 control and 4 hypertrophied myocytes. Na+ current density and voltage dependence of current were not significantly different in control and hypertrophied myocytes.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The objective of the present study was to investigate factors responsible for determining the electrophysiological phenotype of cardiac myocytes. We began this research because the wave shape of the cardiac action potential changes with development and disease and the bases of these changes are not well known. The action potential duration is relatively long in the fetal and neonatal period and shortens with development in the rat (11, 12). If a sustained pressure overload is imposed on the adult ventricle, there is hypertrophy with a lengthening of the action potential duration (9) resembling that of fetal myocytes. The lengthening of the action potential may be part of a generalized return to a fetal phenotype that is seen in adult myocardium with pressure overload-induced hypertrophy (2, 3, 23, 25).

Studies suggest that significant changes in T- and L-type Ca2+ currents and transient outward, inward, and delayed rectifier K+ currents may all play a role in action potential changes (27, 28, 40, 41).

Cellular signaling pathways that regulate the expression of cardiac myocyte ion channels have not been well studied. Cultured neonatal rat ventricular myocytes, an in vitro system, were chosen for studies of factors that regulate ion channels (47, 48, 50). These studies have shown that when neonatal rat ventricular myocytes are exposed to phenylephrine, the alpha 1-adrenergic receptor pathway is activated, and this leads to hypertrophy and activation of the fetal gene program (18). An important limitation of the study is that two-dimensional measurement of hypertrophy may overestimate the phenylephrine-induced increase in cell volume. The major findings of our study were as follows: 1) acute exposure (i.e., 5-30 min) to 20 µM phenylephrine caused a significant increase in Ba2+ currents through L- but not T-type Ca2+ channels, and this effect was blocked by the beta -adrenergic antagonist propranolol but not by the alpha -adrenergic blocker prazosin; 2) chronic exposure (48-72 h) to phenylephrine caused hypertrophy and an increase in the duration of the action potential; and 3) chronic exposure (48-72 h) to phenylephrine caused a decrease in Ba2+ current density through L-type Ca2+ channels, no change in T-type current density or Na+ current density, and a decrease in the density of the IKto1. The hypertrophy and the accompanying changes in ionic currents were eliminated by the nonselective alpha -adrenergic blocker prazosin and by the PKC inhibitor staurosporine but not by the beta -adrenergic blocker propranolol. These results strongly support the involvement of the alpha -adrenergic signaling pathway(s) and the subsequent activation of PKC in the changes in electrophysiological properties of these myocytes.

Electrophysiological properties of hypertrophied myocytes. Significant changes in cell morphology occurred in neonatal ventricular myocytes after exposure to 20 µM phenylephrine for 48 h in culture. The two-dimensional cell area was 44% greater in hypertrophied myocytes than in control myocytes, similar to previously reported values (50). It is not clear why we did not find an increase in cell capacitance in hypertrophied cells. One explanation is that there is a change (or disruption) in the complex arrangement of cellular membranes that contributes to the electrical capacitance of the cell. Keung and Aronson (23) have reported similar findings in hypertrophied adult rat ventricular myocardium in which the total capacitance was observed to decrease despite the "increased total area per cell." They suggest that this finding in hypertrophied myocardium may be due to a lack of effective charging of the T-tubular system. Another possibility is that the surface membrane is less folded in hypertrophied myocytes. This issue requires further study.

Prolongation of the action potential is one of the hallmarks of adult ventricular myocytes with hypertrophy induced by hemodynamic overload and, in particular, pressure overload (2, 3, 9, 23, 24, 41). This electrophysiological change is thought to be proarrhythmic and may contribute to the slow relaxation in hypertrophied and/or failing myocytes. A number of laboratories have examined the changes in membrane currents that underlie this prolongation. These studies have shown that there are changes in both Ca2+ (27) and K+ (28) currents in hypertrophied myocytes. The changes in L-type Ca2+ current appear to be dependent on the magnitude of hypertrophy with a significant reduction only in severe hypertrophy (16, 39). T-type Ca2+ currents are not found in high density in normal adult ventricular myocytes but are reexpressed in pressure overload hypertrophy (40). The current that appears to change to the greatest extent in hypertrophy is the IKto1, which is significantly reduced (9, 28). The composite changes in these currents are thought to produce the new electrophysiological phenotype of hypertrophied myocytes. The results of the present experiments show that activation of the alpha -adrenergic signaling pathway in cultured neonatal myocytes induces an electrophysiological phenotype characterized by a prolongation of the action potential and disturbed ion channel expression.

Acute effects of phenylephrine on Ba2+ currents through Ca2+ channels. In the present study, we found that acute exposure to 20 µM phenylephrine caused a significant increase in the Ba2+ current density through L-type Ca2+ channels. We used different myocytes for control and test measurements because Ba2+ current "runs down" rapidly in these small cells. Perforated-patch techniques did not appreciably prevent this rundown. The magnitude of the observed acute phenylephrine effect was similar to that reported previously (18, 33, 34). We found that the acute effect could be eliminated by blocking the beta -adrenergic receptors but not the alpha -adrenergic receptors. Liu et al. (33, 34) found that alpha -adrenergic blockers were effective in their preparation. The reasons for these differences are not clear at present. One possible explanation is that we used a higher concentration of phenylephrine (20 µM) than did Liu et al. (10 µM), and this could be responsible for the greater effect on the beta -adrenergic pathway. Another possibility is that the adrenergic receptors were not fully differentiated in rats of this age. However, this does not explain why we were unable to observe acute alpha -adrenergic effects on the Ca2+ current such as those previously reported (33, 34). Maki et al. (35) reported that a transient increase in mRNA levels of the dihydropyridine receptor in cultured neonatal myocytes was a beta -adrenergic effect. It is interesting to note that we previously observed that phenylephrine increases L-type Ca2+ current density in normal adult feline ventricular myocytes (17) via beta -adrenergic but not alpha -adrenergic pathways. These studies show that high concentrations of phenylephrine can activate beta - as well as alpha -adrenergic receptors.

Chronic effects of phenylephrine on Ca2+ and K+ currents. The present results demonstrate that chronic exposure to phenylephrine caused prolongation of the action potential and that changes in the expression of Ca2+ and K+ current densities were responsible. These effects were shown to occur via the alpha -adrenergic receptor and may have involved the activation of PKC, since the hypertrophy and the effect on L-type Ca2+ current was blocked by staurosporine, a PKC inhibitor. Chronic exposure to phenylephrine has been shown to reduce dihydropyridine receptor mRNA levels and Ca2+ current density in cultured myocytes (35). A number of studies have shown that phenylephrine can activate PKC and may play a central role in the reexpression of fetal genes in cardiac myocyte hypertrophy (1, 18, 22, 30, 34, 36, 43, 46, 48, 49).

The reduction in L-type current density that we observed in cultured neonatal myocytes exposed to phenylephrine is larger than that observed in mild-to-moderate hypertrophy in vivo (8, 27, 44) but is similar to that in severe hypertrophy and heart failure (39).

T-type Ca2+ currents are found in higher density in neonatal than in adult ventricular myocytes (15, 40). In pressure overload-induced hypertrophy the density of T-type Ca2+ channels is increased (40). In the present studies, T-type Ca2+ current density remained constant as myocytes enlarged in response to phenylephrine, consistent with the hypothesis that T-type Ca2+ channel expression is associated with myocyte growth (15). They also show that chronic alpha -adrenergic stimulation has different effects on T- and L-type Ca2+ channel expression. Future studies should address whether alpha 1-adrenergic activation in adult myocytes can produce reexpression of T-type Ca2+ channels. An important limitation of this study is that ionic disturbances which occurred with our in vitro model using neonatal rat myocytes may be a function of the model and species.

The membrane current with the greatest quantitative change in neonatal rat ventricular myocytes chronically exposed to phenylephrine was IKto1 (Fig. 9). In the rat, this current has been shown to increase in density during development (12) and decrease in adult ventricular myocytes with hypertrophy induced by pressure overload (7, 9, 52). These studies support an important role for transient outward current in developmental and hypertrophic changes in the action potential duration. The present study suggests that activation of the alpha 1-adrenergic pathway causes reduced expression of this channel.

No evidence of a delayed rectifier K+ current could be demonstrated in our experiments. This result is similar to that observed by others (13). Therefore the effect of the alpha 1-adrenergic signaling pathway on the expression of this channel could not be determined.

In summary, the present study shows that chronic exposure of neonatal rat ventricular myocytes to alpha -adrenergic stimulation induces hypertrophy and changes in action potential wave shape. Our findings suggest that activation of the alpha 1-adrenergic signaling pathway has complex influences on the expression of many different cardiac myocyte ion channels and that activation of PKC is required for these effects.

    FOOTNOTES

Address for reprint requests: J. P. Gaughan, Dept. of Physiology, Temple University School of Medicine, 3420 North Broad St., Philadelphia, PA 19140.

Received 11 August 1997; accepted in final form 17 April 1998.

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Top
Abstract
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Methods
Results
Discussion
References

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