Abstract
Calcium current was recorded from ventricular cardiomyocytes of rats at various stages of postnatal development using the whole cell patch-clamp technique. In cultured 3-day-old neonatal cells, the current carried by Ca2+ or Ba2+ (5 mM) was not completely inhibited by 2 μM nifedipine. A residual current was activated in the same voltage range as the L-type, nifedipine-sensitive Ca2+ current, but its steady-state inactivation was negatively shifted by 16 mV. This nifedipine-resistant calcium current was not further inhibited by other organic calcium current antagonists such as PN200-110, verapamil, and diltiazem nor by nickel, ω-conotoxin, or tetrodotoxin. It was completely blocked by cadmium and increased by isoproterenol and forskolin. This current was >20% of total calcium current in ventricular myocytes freshly isolated from neonatal rats, and it decreased during postnatal maturation, disappearing at the adult stage. This suggests that this current could be caused by an isoform of the L-type calcium channel expressed in a way that reflects the developmental stage of the rat heart.
- rats
- heart
- development
- Ca2+ channel isoform
postnatal cardiac development is characterized by a rapid withdrawal of myocytes from the cell cycle after birth (22), with growth assumed to take place via the enlargement of preexisting myocytes as a result of a large increase in protein synthesis (33). The myocyte phenotype shifts from a fetal phenotype to a more differentiated phenotype to attain the completely differentiated stage of adult cardiac cells with respect to metabolic processes (23), contractile protein functions (27), and excitation-contraction coupling mechanisms (35).
In this way, important developmental changes take place in relation to cellular electrophysiological properties during the postnatal growth period (39). For example, it has been reported that the activity of the rat cardiac Na+/Ca2+ exchanger (19, 31) and the density of the inward rectifier K+ current (24) are increased, whereas the activity of the Na+/H+ exchanger (15) is decreased during this period. Some discrepancies, however, have appeared in the literature in relation to developmental changes in the cardiac L-type calcium current (I Ca-L). The density of this current has been shown in different studies either to remain constant (12,35) or to decrease (6).
The major entry pathway of calcium into cardiac cells is assumed to be via voltage-activated calcium channels (VACC). The electrophysiological properties of these channels have been extensively characterized in terms of their voltage dependence, inactivation, single-channel conductance, and pharmacology, such that several types of channels (named L, N, P/Q, R, and T) have been identified (10, 20,25). Subsequent biochemical and molecular biology studies have established the structure of these channels as multi-subunit complexes composed of an ion-conducting α1-subunit protein associated with α2-, β-, γ-, and δ-regulatory subunits. Calcium currents are classically divided into two classes, low- and high-voltage-activated calcium channels (LVACC and HVACC), as a function of their voltage dependence.
In mammalian heart, two types of calcium currents have been reported, which show a cell-specific distribution. The T-type low-voltage-activated calcium current (I Ca-T) is expressed in the heart's conduction system as well as in pacemaker and atrial cells. It has also been shown in rat ventricular cardiomyocytes at the fetal and neonatal stages of development, but it disappears during postnatal maturation (40). This current has also been shown to be reexpressed in hypertrophied (3) and dedifferentiated adult ventricular cells in culture (9), suggesting a cell cycle-related expression of this calcium channel (13). The L-type high-voltage-activated calcium current has been uniformly observed in cardiac cells regardless of the stage of development. Its sensitivity to dihydropyridine (DHP) as a pharmacological hallmark was used to characterize its molecular structure and to clone the genes encoding for its multimeric protein components (1). In addition to these two types of calcium current, a DHP-resistant calcium current with some properties different from those of the T-type current was observed in 18-day-old rat fetal cardiomyocytes (32).
In the present study, we provide evidence for the presence in neonatal rat ventricular cardiomyocytes of a component of calcium current, resistant to nifedipine, that has some properties different from those of the T-type and L-type calcium currents. This current, which has been characterized in neonatal ventricular cells in culture, can also be found in freshly dissociated cells at the neonatal stage but then progressively diminishes to the point of being absent in adult cells. We suggest here that this current could be caused by a calcium channel isoform, which is expressed as a function of postnatal development and then as a function of the cell cycle.
METHODS
Neonatal ventricular cardiomyocyte culture.
Single-cell cultures were prepared according to previously described methods (28). Briefly, hearts from 1- to 2-day-old Wistar rats killed by cervical dislocation followed by decapitation were excised and placed in a filter-sterilized Ca2+- and Mg2+-free spinner medium. Ventricles were isolated, cut into small pieces, and incubated at 37°C in a spinner solution containing 0.1% trypsin (Seromed). The dissociated cells were preplated for 90 min in a culture medium containing Ham's F-10 (GIBCO Life Technologies) supplemented with 10% FCS (Boehringer Mannheim), 10% heat-inactivated horse serum (GIBCO-BRL), and 1% antibiotics (100 U/ml penicillin G and 50 U/ml streptomycin; Sigma). This preplating step removes most of the rapidly adhering nonmyocyte cells (2). The suspended cells were subsequently collected and counted and then plated and incubated at a density of 300 cells/mm2 in 35-mm dishes. After 1 day in culture, the growth medium was renewed, with the exception of the FCS, which was omitted.
Freshly isolated neonatal and adult ventricular cardiomyocytes.
Neonatal rat ventricles were isolated and dissected as described for the neonatal cell cultures. Small pieces of ventricle were incubated at 37°C for 30 min in spinner solution containing 0.05% collagenase (type II, Worthington) under gentle agitation. The cell suspension was collected and centrifuged (100 g for 5 min), and the resulting pellet was rinsed three times in a Kraftbrühe (KB) solution, gradually resuspended in a normal Tyrode solution, and kept at room temperature until used.
Adult ventricular cardiomyocytes were dissociated as previously described (8). Adult male Wistar rats were injected intraperitoneally with 1,000 IU heparin and anesthetized with ether. The heart was quickly removed via a thoracotomy and transferred to an ice-cold Tyrode solution. The aorta was cannulated, and the heart was mounted on a Langendorff apparatus and successively perfused (at 37°C) with the following oxygenated solutions: 5 min with Tyrode solution, 4 min with a nominally Ca2+-free Tyrode solution, and ∼20 min with the same solution supplemented with 0.05% collagenase (type II, Worthington), 0.06 mM CaCl2 and 0.1% BSA. When the heart was flaccid, it was rinsed with KB solution for 2 min. The ventricles were dissected out, cut into small pieces, and gently stirred in KB solution. Isolated cells were filtered, maintained for 1 h in the KB solution, and gradually resuspended in Tyrode solution.
Electrophysiological measurements.
Voltage-clamp experiments were performed at room temperature (20–22°C), using the whole cell patch-clamp technique (14). Micropipettes (2–4 MΩ) were pulled on a two-stage patch pipette puller (PP83, Narishige Scientific Instrument) connected to the head stage of a patch-clamp amplifier (RK 300, Biologic) and driven by a PC-compatible microcomputer via an A/D-D/A conversion board (Labmaster TM-40, Scientific Solutions). Membrane voltage clamping, data acquisition, and analysis were performed using pCLAMP software (version 6, Axon Instruments, Foster City, CA).
Solutions and procedures.
The spinner medium for ventricular neonatal cell dissociation contained (in mM) 116 NaCl, 5.3 KCl, 8 NaH2PO4, 22.6 NaHCO3, 10 HEPES, and 5.6 d-glucose, pH 7.4 with NaOH. Thirty minutes before voltage-clamp experiments, cultured cells were rinsed twice and maintained in Tyrode solution. Calcium currents were recorded in a nominally Na+- and K+-free external solution containing (in mM) 130N-methyl-d-glucamine (NMG), 20 tetraethylammonium-Cl (TEA-Cl), 5.0 CaCl2 or BaCl2, 2 MgCl2, 10 HEPES, and 10d-glucose, pH 7.4 with HCl. The pipette solution, from which potassium ions were also omitted, contained (in mM) 110 CsCl, 20 TEA-Cl, 5 MgATP, 0.3 NaGTP, 2 Na2ATP, 5 sodium phosphocreatine, and 5 HEPES, pH 7.2 with Tris.
Membrane capacitance (C
m) was estimated from the capacitative transient elicited by a 10-mV depolarizing step (10-ms duration) from a holding potential (HP) of −90 mV and calculated according to the equation
where τc is the time constant of the membrane capacitance, I
0 is the initial current value,I
∞ is the amplitude of the steady-state current and ΔV
m is the amplitude of the voltage step (change in membrane potential). No capacitance or series resistance correction was used.
Calcium currents were generated using a double-pulse protocol to obtain current-voltage (I-V) curves and steady-state activation and inactivation curves. From a HP of −80 mV, 1-s depolarizing pulses to different membrane potentials (10-mV increments, i.e., the conditioning pulse) were followed by a 5-ms return to −80 mV and then by a 1-s test pulse to 0 mV, at which the maximum L-type calcium current (I Ca-L) was obtained.
The peak current amplitudes for I-V curves and steady-state activation curves were measured from the conditioning pulses. Activation curves were estimated according to the relation (17)
where G
Ca is the peak conductance,I
Ca is the peak of calcium current for the test potential V
m, and V
rev is the apparent reversal potential for Ca2+. Steady-state inactivation curves were plotted from the normalized current recorded during test pulses.
The data for activation and inactivation curves were fitted with a simple Boltzmann function
where I/I
max is the relative current, V
0.5 is the half-maximum voltage of activation or inactivation, and k is the slope factor.
The time course of decay of calcium currents was analyzed by using a biexponential fitting of current traces obtained at 0 mV according to the equation
where I
Ca(f) andI
Ca(s) represent the amplitudes and τf and τs the time constants of the fast and slow components of calcium current decay, respectively.
Chemicals and drugs.
Nifedipine, PN200-110, diltiazem, and verapamil (all purchased from Sigma) were each dissolved in DMSO to form 10−3 M stock solutions, ω-conotoxin GVIA (Sigma) was dissolved in distilled water to form a 10−5 M stock solution, and TTX (Latoxan) was dissolved in dilute acetic acid for to form 10−3 M stock solution. When used, these drugs were added from the stock solutions to the test solution at the desired final concentration.
Statistical analysis.
Data are expressed as means ± SE. Statistical analysis of data was performed using ANOVA followed by a Newman-Keuls test. Values of P < 0.05 were considered statistically significant.
RESULTS
Evidence for a nifedipine-resistant calcium current in neonatal rat ventricular cardiomyocytes in culture.
Figure 1 A shows calcium currents elicited in cultured 3-day-old neonatal rat ventricular cardiomyocytes in response to depolarizations increasing in 10-mV steps from a HP of −80 mV. I-V curves (Fig.1 B) plotted from these current traces show that the whole cell calcium currents obtained in a Na+- and K+- free external medium containing 5 mM Ca2+exhibited I Ca-L properties with potential values of −40, 0, and +50 mV for activation threshold, peak, and reversal of the current, respectively. The application of nifedipine (2 μM), aI Ca-L blocker, did not however, completely block this calcium current (Fig. 1 A). That is, the peak of the residual current at 0 mV (Fig. 1 B) represented 27.4 ± 1.9% of the total calcium current density (−2.0 ± 0.4 vs. −7.3 ± 0.7 pA/pF, n = 17 cells). Under these experimental conditions, the density of theI Ca-L blocked by nifedipine (measured by subtracting residual calcium current from total calcium current) was −5.3 ± 0.5 pA/pF (n = 17 cells).
Effects of nifedipine (2 μM) on calcium currents in cultured 3-day-old neonatal rat ventricular cardiomyocytes superfused with 5 mM Ca2+. A: superimposed traces of calcium current elicited by depolarization increments from a holding potential (HP) of −80 mV before (○, total calcium current) and after exposure to nifedipine (2 μM). Traces of L-type calcium current inhibited by nifedipine (●) were obtained by subtraction of nifedipine-resistant current traces (▴) from the total current. B: averaged current-voltage relationships for total (○), L-type (●), and residual (▴) calcium current components. Values are means ± SE (n = 17 cells).
When experiments were carried out with 5 mM Ba2+ in the external medium instead of Ca2+, the total inward current was increased in amplitude (Fig. 2) and displayed a slower time course of inactivation (Fig. 2 A). After the application of nifedipine (2 μM), a residual calcium current was revealed with the same characteristics as those observed with 5 mM Ca2+ in the external medium, particularly in relation to the time course of inactivation of the current (Fig.2 A). The density of this current (−4.3 ± 0.6 pA/pF,n = 21 cells) was higher than that measured with Ca2+ but represented a similar ratio (32 ± 2.8%,n = 21 cells) of the total current density (−12.8 ± 0.9 pA/pF, n = 21 cells).
Effects of nifedipine (2 μM) on barium current amplitude in cultured 3-day-old neonatal rat ventricular cardiomyocytes superfused with 5 mM Ba2+. A: superimposed traces of barium current elicited by depolarization increments from a HP of −80 mV before (○) and after the application of nifedipine (2 μM). Traces of L-type currents inhibited by nifedipine (●) were obtained by subtraction of nifedipine-resistant current traces (▴) from the total current (○). B: averaged current-voltage relationships for total (○), L-type (●), and residual (▴) calcium current components. Values are means ± SE (n = 21 cells).
Dose-response curves generated to examine the potency of nifedipine (Fig. 3) show that, in the range of 0.001 to 50 μM, nifedipine failed to completely block the total barium current, irrespective of the value of the holding potential. The peak density of the residual current measured in the presence of 50 μM nifedipine represented 20.5 ± 2.1% (n = 20 cells) and 10.0 ± 1.2% of the total current measured for HPs of −80 and −50 mV, respectively. These results clearly confirm the presence of a nifedipine-resistant calcium current in newborn rat ventricular cardiomyocytes in culture. This current component was observed in all cultured cells up to 3 days of age as well as in those cultured for up to 6 days.
Dose-response curves for the inhibition by nifedipine of calcium currents elicited at 0 mV in cultured 3-day-old cardiomyocytes. Mean dose-response curves were obtained under conditions in which 5 mM external Ba2+ was used. Currents were elicited from holding potentials (HPs) of −80 (⧫) and −50 (◊) mV by means of protocol indicated ininset. Values are means ± SE for number of cells indicated over points on curve. [nifedipine], Nifedipine concentration.
Characterization of nifedipine-resistant calcium current.
Figure 4 shows the voltage-dependent availability of the nifedipine-sensitive I Ca-Land the nifedipine-resistant calcium current (I Ca-NR) elicited in 3-day-old cultured cardiomyocytes. In the presence of Ca2+ as the charge carrier (Fig. 4 A), activation curves for the two experimental conditions could be superimposed. Activation parameters were not significantly different from each other [V 0.5 = −11.1 ± 0.3 and −12.9 ± 0.5 mV and k = −7.2 ± 0.2 and −6.8 ± 0.4 (n = 17 cells) for I Ca-L andI Ca-NR, respectively]. However, inactivation curves obtained using the double-pulse protocol described inmethods showed that the curve forI Ca-NR was shifted to more negative potentials (V 0.5 = −39.3 ± 0.5 mV,k = 6.7 ± 0.5; n = 17 cells) compared with that measured for I Ca-L(V 0.5 = −23.3 ± 0.2 mV,k = 5.4 ± 0.2; n = 17 cells). In these conditions, the window current obtained forI Ca-NR was significantly reduced compared with that observed for I Ca-L, which has been shown to be more prominent at the neonatal than at the adult stage (6,12). Similar results describing V 0.5 andk for activation of I Ca-L andI Ca-NR were obtained when Ca2+ was replaced by Ba2+ [V 0.5 = −17.5 ± 0.1 and −18.4 ± 0.1 mV and k = −5.1 ± 0.1 and −4.9 ± 0.1 for L-type barium current (I Ba-L) and nifedipine-resistant barium current (I Ba-NR), respectively (n = 21 cells); Fig. 4 B]. Inactivation parameters similar to those described above were also found after the replacement of external Ca2+ with Ba2+[V 0.5 = −23.9 ± 0.8 and −38.4 ± 0.6 mV and k = 7.5 ± 0.7 and 6.8 ± 0.5 (n = 21 cells) for I Ba-L andI Ba-NR, respectively]. For concentrations of nifedipine higher than 1 μM, which blocked L-type calcium current, Table 1 shows that steady-state inactivation parameters for the residual current were not significantly changed. This suggests that the shift observed in the voltage-dependent inactivation is not caused by nifedipine.
Availability of calcium currents in cultured 3-day-old neonatal rat ventricular cardiomyocytes. Mean steady-state activation and inactivation curves were obtained (by means of the voltage-clamp protocol indicated in the inset) for L-type current (●) and nifedipine-resistant (NR) current (▴) under conditions in which 5 mM external Ca2+ (A; n = 17 cells) or 5 mM external Ba2+ (B; n = 21 cells) was used. Data are means ± SE.I/I max, relative current; ΔV m, voltage step amplitude.
Steady-state inactivation parameters of Ba2+ current obtained in cultured 3-day-old neonatal rat ventricular cardiomyocytes in absence or presence of nifedipine
It can be clearly seen in Fig. 5 that L-type calcium current increases in amplitude and exhibits slower inactivation time courses when Ca2+ was replaced by Ba2+. Figure 5 B shows that when barium was substituted for calcium (see also Fig. 2 A), the nifedipine-resistant barium current was increased in amplitude, in a proportion similar to that observed for L-type current (Fig.5 A) but without significant changes of its inactivation kinetics. A comparison of current decay time courses (Table2) shows that L-type calcium current was best fitted with two time constants when 5 mM Ca2+ was present in the extracellular medium [10.0 ± 0.9 ms for the fast component (τf) and 93.0 ± 4.2 ms for the slow component (τs) n = 16 cells], whereas only one time constant was required when Ba2+ was used in the extracellular medium (182.1 ± 8.7 ms; n = 16 cells). Furthermore, the nifedipine-resistant current was best fitted with two time constants under conditions when Ca2+ was used in the extracellular medium [12.2 ± 0.9 and 94.3 ± 8.9 ms for τf and τs, respectively;n = 16 cells] as well when Ba2+ was used (20.3 ± 1.0 and 114.0 ± 10.0 ms for τf and τs, respectively; n = 16 cells).
Effects of substituting barium for calcium on L-type and NR currents elicited at 0 mV in cultured 3-day-old neonatal rat ventricular cardiomyocytes. Traces of L-type current (A) and NR current (B) elicited at 0 mV in the presence of 5 mM external Ca2+ (○) or Ba2+(●) are shown. Traces of L-type calcium current inhibited by nifedipine were obtained by subtraction of NR current traces from the total current obtained by means of the voltage-clamp protocol indicated in the inset.
Effects of substituting Ba2+ for Ca2+ on L-type and nifedipine-resistant current decay in cultured 3-day-old neonatal rat ventricular cardiomyocytes
Figure 6 shows the effects of several Ca2+ current inhibitors on the nifedipine inward barium current. The current was inhibited only slightly by PN200-110 (2 μM; Fig. 6 A), which is known to be a more potent antagonist than nifedipine (11). It was not further inhibited by verapamil (2 μM) or diltiazem (2 μM) (data not shown). Moreover, under these conditions, I Ba-L was not completely blocked as a result of reciprocal allosteric interactions between the respective domains of inhibitors (10). Figure 6 B shows that I Ba-NR was not further inhibited by nickel (30 μM), but it could be completely blocked by 500 μM cadmium (Fig.6 C). Finally, I Ba-NR was insensitive to tetrodotoxin (10 μM) and ω-conotoxin GVIA (1 μM) (data not shown).
Effects of calcium current inhibitors on the NR barium current (I Ba-NR). Effects of PN200-110 (2 μM; ▵, A), nickel (30 μM; □,B), and cadmium (500 μM; ■, C) on nifedipine-resistant current carried by Ba2+(▴) obtained by means of the voltage-clamp protocol indicated in the inset.
Figure 7 shows that isoproterenol (1 μM) increased both I Ba-NR andI Ba-L to a similar extent [54.3 ± 5.1% and 47.5 ± 4.9% for I Ba-L andI Ba-NR, respectively; n = 10 cells]. An additional increase in both currents was observed in the presence of 1 μM forskolin [24.9 ± 3.2% and 32.5 ± 3.4% for I Ba-L andI Ba-NR, respectively; n = 10 cells], suggesting that the channel responsible forI Ba-NR could be phosphorylated similarly to the L-type calcium channel.
Effects of isoproterenol and forskolin on L-type and NR currents carried by Ba2+. Current traces of L-type barium current (I Ba-L, A) andI Ba-NR (B) measured at 0 mV under control conditions (5 mM Ba2+, ○), in the presence of 1 μM isoproterenol (●), or in the presence of isoproterenol + 1 μM forskolin (■) are shown. Traces of I Ba-L inhibited by nifedipine were obtained by subtraction of NR current traces from total current traces.
Presence of ICa-NR in ventricular cardiomyocytes as a function of development.
To test whether the I Ca-NR described above could be caused by the conditions under which cardiomyocytes were cultured, the effects of nifedipine were studied on calcium currents (with Ba2+ as the charge carrier) recorded from freshly dissociated ventricular cells obtained from 4-, 10-, and 15-day-old immature rats and from young adult (2–3 mo) rats.
Figure 8 A confirms that cell membrane capacitance increased as a function of age in young rats, as previously reported (12, 24). When normalized to the mean values of C m presented in Fig. 8 A, the density of I Ba-L increased slightly but not significantly (as previously shown; see Refs. 12, 35) during the developmental phase [−6.2 ± 1.1 (n = 11 cells), −6.5 ± 1.2 (n = 11 cells), −7.1 ± 1.0 (n = 13 cells), and −8.9 ± 1.5 (n = 17) pA/pF for 4-, 10-, and 15-day-old rats and adult rats, respectively; Fig. 8 B]. In contrast, although IBa-NR was clearly expressed at the neonatal stage (Fig.8 B), its density significantly decreased during postnatal development [−1.9 ± 0.5 (n = 11 cells), −1.5 ± 0.4 (n = 11 cells), and −0.8 ± 0.3 (n = 13 cells) pA/pF in cardiomyocytes from 4-, 10-, and 15-day-old rats, respectively]. In adult rat hearts,I Ba-NR was observed in <10% of the isolated cells tested, with its mean density being only −0.2 ± 0.07 pA/pF (n = 7 cells).
Changes in L-type and NR currents density during postnatal development of cardiomyocytes. A: representative histogram of age-dependent increase in cell membrane capacitance (C m). B: representative histograms ofI Ba-L (top) andI Ba-NR density (bottom) at 0 mV. Data are means ± SE for number of cells indicated over bars. † P < 0.05; ‡ P < 0.01 compared with values obtained in cardiomyocytes from 4-day-old rats.
Dose-response curves describing the effects of nifedipine on current amplitude (Fig. 9) show that ∼14% of the barium current was not blocked by 50 μM nifedipine in cardiomyocytes from 4-day-old rats, whereas it was completely abolished in cells from adult rats. Superimposition of the curves revealed that the blocking action of nifedipine was more efficient as a function of the postnatal age of the animals used, with IC50 values of 0.091, 0.087, 0.050, and 0.022 μM measured for cardiomyocytes obtained from 4-, 10-, and 15-day-old immature rats and adult rats, respectively.
Dose-response curves for the inhibition by nifedipine of calcium currents elicited at 0 mV during postnatal development of cardiomyocytes. Mean dose-response curves showing the effects of nifedipine on calcium current elicited at 0 mV from a HP of −80 mV in freshly dissociated ventricular cells from 4 (▾)-, 10 (□)-, and 15 (■)-day-old immature rats and from adult rat hearts (●). ○, Curve obtained in neonatal cultured cells (Fig. 3). Data are given as means ± SE for n = 8–25 cells.
DISCUSSION
Our data show that a component of the slow inward calcium current, not inhibited by nifedipine or any other major calcium channel antagonist, was observed in neonatal rat ventricular cardiomyocytes in culture. This current was also expressed in ventricular cardiomyocytes obtained from newborn rats and progressively disappeared during postnatal development. The main voltage-dependent and pharmacological properties of this current are quite similar to those of the DHP-resistant calcium current found in rat fetal cardiomyocytes (32). Moreover, properties of this nifedipine-resistant current seem also quite comparable to those of the nifedipine-insensitive calcium current recently found in the guinea pig mesenteric artery (26).
The first evidence for the presence of I Ca-NR in cultured neonatal rat ventricular cardiomyocytes could be seen on the application of 2 μM nifedipine to the extracellular medium. Although this concentration of nifedipine is usually able to completely block the inward L-type calcium current in cardiac cells, we observed that 28% of the calcium current was not inhibited in cultured 3-day-old cardiomyocytes. Moreover, 20% of the current was still present when the concentration of nifedipine was increased to 50 μM (Fig. 3).
In agreement with the voltage-dependent inhibitory effect of nifedipine on L-type Ca2+ current (16), the total calcium current measured here was more effectively inhibited when cells were voltage-clamped at a HP of −50 mV. However, even under these conditions, 10% of the current was not inhibited in the presence of 50 μM nifedipine (Fig. 4). In addition, the nifedipine-resistant current was not further blocked by PN200-110 (2 μM; Fig. 6 A), an L-type calcium current inhibitor known to be more potent than nifedipine, nor was it blocked by other specific inhibitors (phenylalkylamines and benzothiazepines) of the L-type calcium current.
An analysis of I-V curves showed thatI Ca-NR was activated in a voltage range similar to that seen for I Ca-L, whereas its steady-state inactivation curve was shifted toward more negative potentials compared with those of I Ca-L (Fig. 4). From this result, the hypothesis that this current component could be a T-type calcium current, as previously observed in ventricular cardiomyocytes obtained from 2-day-old rats (12), can be ruled out. Evidence to support this conclusion can be found in the fact thatI Ba-NR was not affected by 30 μM nickel (Fig.6 B) and its density was increased when Ba2+ was substituted for Ca2+ as the major charge carrier (Fig.5 B). Furthermore, the lack of an effect of ω-conotoxin GVIA and TTX allows us to exclude the hypothesis of an involvement in this current of N-type calcium channels or of the passage of Ca2+ through TTX-sensitive sodium channels. According to the study of Morita et al. (26), this nifedipine-resistant current presents properties close to those of the R-type calcium current. However, these authors have shown by a molecular approach with RT-PCR that the α1E-subunit mRNA was not detected under their conditions.
By taking into account the I-V curves obtained here and their dependence on the charge carrier used (Figs. 1, 2, and5) as well as the susceptibility of the channels to phosphorylation processes (Fig. 7), the nifedipine-resistant current could be suitably compared with the I Ca-L. However, differences in the sensitivity of I Ca-NR to specific inhibitors of I Ca-L, and altered steady-state inactivation and activation decay characteristics, clearly support the notion ofI Ca-NR as another component of the calcium current and possibly as an isotype of I Ca-L.
Cloning and expression studies have outlined the molecular determinants of Ca2+ channel properties and function (34,37). Properties such as cation permeation and drug sensitivity would be attributed to the α1-subunit constituting the ion-conducting pore of the channel, whereas the current amplitude and the gating of the channel would be mainly regulated by other α2-, δ-, β-, and γ-subunits. Concerning the cardiac L-type calcium channel, recent studies have shown that DHP sensitivity (38) as well as inactivation properties (30) could be mainly attributed to the α1-subunit. Therefore, if the present nifedipine-resistant calcium channel is an isoform of the L-type channel, it can be speculated that it results from the expression of an alternative splicing of the α1C-subunit. However, because it has also been shown that auxiliary subunits could regulate gating properties, particularly the β-subunit (4), possible expression of isoforms of these subunits cannot be excluded. To test these hypotheses, single-channel studies and a molecular approach will be necessary.
The strong possibility that the present nifedipine-resistant calcium current arises from the same molecular entity as the DHP-resistant calcium current found in rat fetal cardiomyocytes (32) argues for its developmentally regulated expression, as has been shown for the T-type LVACC (12, 18, 36, 39). To support such an hypothesis, Diebold et al. (7) showed that the gene for the α1-subunit of the cardiac calcium channel in rat heart generates developmentally regulated isoforms. In this way, variant isoforms of the IVS3 motif were expressed in fetal and newborn rat heart, whereas only a single isoform was predominant in adult rat heart. Secondly, the postnatal decrease in the expression ofI Ba-NR reported here (Fig. 8) is well correlated with a reduction of the 5-bromo-2′-deoxyuridine labeling index (used to evaluate the cell proliferative capacity) during the postnatal development of rat ventricles (5).
It has been demonstrated (13) that expression of theI Ca-T at the neonatal stage of development decreases in parallel with the rapid withdrawal of ventricular cardiomyocytes from the cell cycle soon after birth, suggesting a cell cycle-related form of channel expression. This was also proposed in vascular smooth muscle cells (21), in whichI Ca-T has been shown to increase during cell proliferation in culture and to decrease once cells reach confluence, suggesting that this current is important in cell proliferation (29). Such a physiological role ofI Ca-NR in cell signaling can also be proposed. However, present data have shown that its density was ∼30% of the total calcium current in cultured 3-day-old neonatal cells and >20% in ventricular cardiomyocytes from 4-day-old rats. A physiological role similar to that of the I Ca-L, as proposed for vascular smooth muscle cells (26), cannot then be excluded but requires further investigation. In addition, investigations on the presence of this current in other cardiac cells such as atrial cells or in ventricular cells from other species as a function of developmental process would provide further informations about its role.
The expression of this current component at the neonatal stage of development but not in adult ventricular cells provides further evidence for a developmental shift in the expression of calcium channels in cardiac muscle.
Acknowledgments
We thank Dr. M. F. Patterson for critically reading the manuscript and for correcting the language.
Footnotes
This work was financially supported by grants from the CNRS (UMR 6558), the University of Poitiers, and the Fondation Langlois.
Address for reprint requests and other correspondence: D. Potreau, Univ. of Poitiers, UMR 6558 LBSC, Faculty of Sciences, 40 Ave. du Recteur Pineau, 86022 Poitiers cedex, France (E-mail:daniel.potreau{at}univ-poitiers.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2000 the American Physiological Society
REFERENCES
StumbleUpon
More in this TOC Section
Related Articles
Cited By...