INTRODUCTION

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IT IS WELL KNOWN that the -adrenergic system plays an important role in the regulation of cardiac excitation and contraction in mature mammals. The modulation of L-type Ca²⁺-channel currents in cardiomyocytes by -adrenergic receptors is the main process in the regulation of heart rate and excitation-contraction coupling. Although it is well established that -adrenergic agonists, by activating the G_s protein, stimulate cardiac Ca²⁺-channel currents via dual pathways, cAMP dependent and cAMP independent (24, 25), the -adrenergic modulation of Ca²⁺ channels in the embryonic heart is still ambiguous.

There is a considerable discrepancy in the literature as to the role of -adrenergic receptors in embryonic hearts at an early stage (within a couple of days after the initiation of beating). An et al. (1) reported that in the embryonic mouse heart, L-type Ca²⁺ channels are responsive to isoproterenol (Iso) in late-stage [17-20 days postcoitum (dpc)] cardiomyocytes but not in early-stage (11-13 dpc) cardiomyocytes. In rat cardiomyocytes, Iso has little effect on Ca²⁺-channel currents at fetal days 15 and 18 but has a marked stimulatory effect from fetal day 20 (14). However, biochemical studies proved the expression of -adrenergic receptors in 13-day-old fetal mouse heart or 12-day-old fetal rat heart (4, 5, 22). On the other hand, Robkin et al. (18, 19) reported that the chronotropic -adrenergic response of the rat heart appears at fetal day 10.5 or 11 in the explanted whole embryo, concomitant with the onset of heart beating (a rat embryo at day 11 of gestation corresponds to a mouse embryo at day 10 in terms of the stage of development). Similarly, Hall (8) found chronotropic -adrenergic response of isolated embryonic rat hearts at 10.5-13.5 dpc.

To clarify the functional role of -adrenergic receptors in the early-stage embryonic mouse heart, we examined the effect of Iso on the heart beating rate and electrophysiological properties of ventricular myocytes (action potential configuration and L-type Ca²⁺-channel currents) at 9.5 dpc. For comparison, we also examined the effect of Iso on L-type Ca²⁺-channel currents in 18-dpc ventricular myocytes. Our results indicate that activation of -adrenergic receptors does modulate heart beating and L-type Ca²⁺ channels in the 9.5-dpc embryonic mouse heart, although the modulatory potency is appreciably less than in the 18-dpc embryonic heart.

	MATERIALS AND METHODS

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Dissection of 9.5-dpc mouse embryos and ventricles. Pregnant (9.5 dpc) mice were killed by cervical dislocation, the uteri were isolated, and the whole embryos were exposed. Ventricles were cut from the exposed embryos. During the dissection procedures, the tissues were kept in Hanks' balanced salt solution (GIBCO).

Isolation of 9.5-dpc cardiomyocytes. The dissected ventricles were incubated in Ca²⁺-free saline with 0.3 mg/ml collagenase type II (Worthington Biochemical) containing (in mM) 116 NaCl, 20 HEPES, 1.0 NaH₂PO₄, 5.5 glucose, 5.0 KCl, and 0.8 MgSO₄ (pH 7.35) for 20 min at 37°C and then rinsed with the same buffer solution without enzyme. The ventricles were mechanically dissociated by trituration. The cell suspensions were centrifuged at 1,000 rpm for 5 min, and the isolated cells were plated on collagen-coated glass coverslips and incubated in Dulbecco's modified Eagle medium (GIBCO) with 10% fetal bovine serum (GIBCO) and 10 µg/ml gentamicin at 37°C in a humidified CO₂ incubator.

Isolation of 18-dpc cardiomyocytes. Ventricles were dissected from 18-dpc embryonic hearts and cut into pieces. The ventricular pieces were incubated in Dulbecco's phosphate-buffered saline containing 0.16 mg/ml collagenase (Yakult) with stirring at 37°C for 12 min, and cell suspension was collected; this was repeated four times. The collected suspension was centrifuged at 1,000 rpm for 5 min, and the isolated cells were cultured in the same way as 9.5-dpc cardiomyocytes.

Observation of heart beating. Embryos or ventricles (9.5 dpc) were transferred to a chamber perfused with normal Tyrode solution containing (in mM) 143 NaCl, 5.4 KCl, 1.8 CaCl₂, 0.5 MgCl₂, 5.5 glucose, and 5 HEPES (pH 7.4) maintained at 37°C. The heart rate was measured by the naked eye under an inverted microscope (Nikon).

Action potential recording. Action potentials (AP) were recorded from spontaneously beating myocytes, cultured for ~20 h, in a normal Tyrode solution. Pipettes (15-20 M) filled with an internal solution containing (in mM) 60 KOH, 80 KCl, 40 aspartate, 5 HEPES, 10 EGTA, 5 MgATP, 5 Na₂-phosphocreatine, and 0.65 CaCl₂ (pH 7.2) were sealed to the myocytes, and whole cell recording was initiated by suction with negative pressure. Electrical signals were fed into an Axopatch-1D (Axon Instruments), filtered at 2 kHz, and digitized at a sampling rate of 5 kHz.

Measurement of Ca²⁺-channel currents. Whole cell voltage-clamp recordings were performed from cardiac myocytes 20-28 h after plating. To isolate Ca²⁺-channel currents, the cells were perfused with a Na⁺- and K⁺-free solution containing (in mM) 2 BaCl₂, 50 tetraethylammonium chloride, 100 Tris · Cl, 0.5 MgCl₂, 3 4-aminopyridine, 5 HEPES, and 5.5 glucose (pH 7.4). Pipettes were pulled to resistances of 3.5-5.0 M when filled with an internal solution containing (in mM) 80 CsCl, 60 CsOH, 40 aspartate, 5 HEPES, 10 EGTA, 5 MgATP, 5 Na₂-phosphocreatine, and 0.65 CaCl₂ (pH 7.2). Data were recorded with an Axopatch-1D (Axon Instruments), filtered at 2 kHz, digitized at 5 kHz, and stored on a microcomputer disk for subsequent off-line analysis. Experiments were performed at 33°C.

Chemicals. l-Isoproterenol, forskolin, and dl-propranolol were purchased from Sigma (St. Louis, MO).

Data analysis. Data were analyzed using the pCLAMP program (Axon Instruments). Current density was calculated using the measured membrane capacitance. The cell capacitance was determined by applying a ramp voltage pulse of ±0.5 V/s at a potential ranging between 50 mV and +70 mV. Data are expressed as means ± SE. The mean percentage of change was the average value of individual percentage. Statistical analysis was performed using Student's t-test, and values of P < 0.05 were considered to indicate a significant difference.

	RESULTS

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Effect of Iso on beating rates of 9.5-dpc whole hearts, ventricles, and ventricular myocytes. Table 1 compares the effects of Iso on beating rates of the whole hearts in vivo and those of the dissected ventricles and firing rates of AP recorded from isolated ventricular myocytes. There was no significant difference in the control beating rates between whole hearts and dissected ventricles. The spontaneous firing rate of isolated ventricular myocytes was slightly higher (by 13-15%) than the beating rates of whole hearts and of dissected ventricles. The increase in beating rate after Iso (3 µM) application in the whole hearts (by 17.0 ± 4.9%, n = 8) was similar to that in the dissected ventricles (by 16.5 ± 6.5%, n = 10). The corresponding value in ventricular myocytes (29.4 ± 3.8%, n = 8) was slightly larger than those in the whole hearts and dissected ventricles.

Effects of Iso on AP in ventricular myocytes. Figure 1 shows examples of AP before, during, and after the application of Iso (3 µM). AP fired spontaneously (101/min), with a slow diastolic depolarization. The maximal upstroke velocity (V_max) under the control condition amounted to 70.4 V/s (Fig. 1A). During the application of Iso (3 µM), the AP firing rate increased to 138/min (Fig. 1B). This positive chronotropic effect was associated with an increase in the slope of slow diastolic depolarization and a prolongation of AP duration. The AP duration measured at 50 mV (APD₅₀) was prolonged by 30 ms. However, V_max was decreased to 56.2 V/s. These AP changes partially recovered after the withdrawal of Iso (Fig. 1C).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Effects of isoproterenol (Iso, 3 µM) on spontaneous action potentials in 9.5-day postcoitum (dpc) embryonic mouse ventricular myocyte. Representative action potentials (AP) were recorded in current-clamp mode. A, B, and C show AP before, during, and after application of Iso, respectively. Iso reversibly increased AP frequency, slope of slow diastolic potential (pacemaker potential), and prolonged AP duration. Horizontal lines indicate 0 mV potential.

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Effects of Iso and forskolin on L-type Ca²⁺-channel currents in ventricular myocytes. Whole cell voltage-clamp recordings were performed on dissociated ventricular myocytes at 9.5 dpc to examine the effect of Iso on L-type Ca²⁺-channel currents. To minimize rundown of Ca²⁺-channel currents and to obtain higher conductance, Ba²⁺ was used as a charge carrier of Ca²⁺-channel currents instead of Ca²⁺. The inward currents, evoked by 200-ms depolarizing pulses from a holding potential of 50 mV to 0 mV, were identified as an L-type Ca²⁺-channel current (I_Ba) because of their complete blockade by 1 µM nisoldipine (data not shown). Figure 2, A and B, shows an example: I_Ba was augmented by 28.6% after Iso (3 µM) application and recovered to control level after the addition of its antagonist, propranolol (3 µM).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Effects of Iso and forskolin (For) on Ca2+-channel currents measured with Ba2+ as charge carrier (IBa) in 9.5-dpc embryonic mouse ventricular myocytes. A: peak IBa amplitude is plotted against time. Iso (3 µM) increased IBa, and propranolol (Pro, 3 µM) reversed Iso effect. IBa was elicited by a 200-ms voltage step to 0 mV from a holding potential of 50 mV. B: selected current traces are illustrated at points denoted on time course in A; a and b were recorded in absence and presence of Iso, respectively, and c in presence of Iso and Pro (3 µM). C: typical current traces showing effect of For (10 µM) on IBa in 9.5-dpc embryonic mouse ventricular myocyte. Pulse protocol is same as shown in B. Control and Forskolin, IBa recorded in absence and presence of 10 µM For, respectively.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Effect of Iso on current-voltage (I-V) relationship of IBa in 9.5-dpc embryonic mouse ventricular myocytes. Peak I-V curve was obtained before () and during () application of Iso (3 µM). IBa was expressed as current density (pA/pF) calculated using membrane capacitance. Data points plotted are means ± SE (n = 10). Threshold was about 30 mV, and maximal activation of IBa occurred at 0 mV.

	DISCUSSION

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Initiation of heartbeat and Iso. During mouse embryogenesis, the primitive heart normally begins to contract irregularly at 8.5 dpc, whereas at 9 dpc of development the primitive atria and ventricles beat regularly and powerfully (10). In this study, an Iso-induced increase in the heart beating rate was observed at 9.5 dpc.

AP and Iso. Couch et al. (6) reported developmental changes in AP of ventricular muscles of prenatal rats (10.5-20.5 dpc). However, the AP recorded from these rat hearts had limited magnitudes of both resting potential (positive to 50 mV in average) and V_max (<25 V/s), suggesting that the tip of a Woodbury-Brady type of floating electrode may not be able to be impaled completely into the intracellular space of such immature and downsized ventricular muscle cells. AP of single ventricular myocytes recorded through a suction pipette electrode in the present study were comparable to those of myocytes in prenatal or neonatal rat hearts in terms of MDP, APA, and V_max (7, 11). In addition, Iso significantly reduced the V_max of AP (see Table 2). This is consistent with an inhibition of Na⁺-channel currents by Iso in adult mammalian ventricular myocytes, which has been linked to the G_s protein pathway (17, 20, 21). The fact that the slow diastolic depolarization was enhanced by Iso suggests the possible modulatory effect of Iso on pacemaker currents. Because the I-V curve shows that L-type Ca²⁺-channel currents at such negative potentials are almost inactive, other pacemaker currents may be involved in the response to Iso.

Stimulation of L-type Ca²⁺-channel currents by Iso. Our main finding in this report is that Iso, a -adrenergic agonist, enhanced I_Ba in 9.5-dpc mouse cardiomyocytes. The effect of Iso on I_Ba was mediated by -adrenergic receptors because of its reversible blockade by propranolol.

Physiological significance. In mature mammalian cardiomyocytes, excitation-contraction coupling is mediated mainly by Ca²⁺ release from the sarcoplasmic reticulum (SR), which is triggered by Ca²⁺ entry through sarcolemmal L-type Ca²⁺ channels (2, 9, 16). However, during early embryonic stages of development, cardiac contraction is more dependent on transsarcolemmal Ca²⁺ influx through Ca²⁺ channels because of immature Ca²⁺ regulatory properties of SR (3, 12). The expression of functional L-type Ca²⁺ channels in 9.5-dpc mouse embryos is in agreement with the fact that the primitive mouse heart begins to beat rhythmically at 9.0 dpc (10).