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Am J Physiol Heart Circ Physiol 286: H2257-H2263, 2004. First published February 26, 2004; doi:10.1152/ajpheart.01043.2003
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Cav3.2 subunit underlies the functional T-type Ca2+ channel in murine hearts during the embryonic period

Noriko Niwa,1 Kenji Yasui,1 Tobias Opthof,2 Haruki Takemura,1 Atsuya Shimizu,1 Mitsuru Horiba,1 Jong-Kook Lee,1 Haruo Honjo,1 Kaichiro Kamiya,1 and Itsuo Kodama1

1Departments of Circulation and Humoral Regulation, Division of Regulation of Organ Function, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan; and 2Department of Medical Physiology, University Medical Center Utrecht, 3508 TA Utrecht, The Netherlands

Submitted 10 November 2003 ; accepted in final form 13 February 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
T-type Ca2+ channels are implicated in cardiac automaticity, cell growth, and cardiovascular remodeling. Two voltage-gated Ca2+ subtypes (Cav3.1 and Cav3.2) have been cloned for the pore-forming {alpha}1-subunit of the T-type Ca2+ channel in cardiac muscle, but their differential roles remain to be clarified. The aim of this study was to elucidate the relative contribution of the two subtypes in the normal development of mouse hearts. A whole cell patch clamp was used to record ionic currents from ventricular myocytes isolated from mice of early (E9.5) and late embryonic days (E18) and from adult 10-wk-old mice. Large T-type Ca2+ current (ICa,T) was observed at both E9.5 and E18, displaying similar voltage-dependence and kinetics of activation and inactivation. The current was inhibited by Ni2+ at relatively low concentrations (IC50 26–31 µM). ICa,T was undetectable in adult myocytes. Quantitative PCR analysis revealed that Cav3.2 mRNA is the predominant subtype encoding T-type Ca2+ channels at both E9.5 and E18. Cav3.1 mRNA increased from E9.5 to E18, but remained low compared with Cav3.2 mRNA during the whole embryonic period. In the adulthood, in contrast, Cav3.1 mRNA is greater than Cav3.2 mRNA. These results indicate that Cav3.2 underlies the functional T-type Ca2+ channels in the embryonic murine heart, and there is a subtype switching of transcripts from Cav3.2 to Cav3.1 in the perinatal period.

ion channel; gene expression; fetal development; {alpha}1-subunit; cardiac myocyte

TWO DISTINCT FAMILIES of voltage-gated Ca2+ channels (Cav) are identified in mammalian cardiac muscle: high-voltage-activated L-type Ca2+ channels and low-voltage-activated T-type Ca2+ channels (10). The L-type Ca2+ channels exist in large quantities in working myocardium and play an essential role in the excitation-contraction coupling by mediating plasma membrane Ca2+ influx required for Ca2+-induced Ca2+ release (1). In contrast, less information is available regarding the expression and function of T-type Ca2+ channels. T-type Ca2+ current (ICa,T) is observed in pacemaker cells from the sinoatrial node (8, 35) and Purkinje fibers (9). ICa,T is clearly described in embryonic (12) or neonatal cultured atrial and ventricular myocytes (17), whereas it is rarely observed in normal adult ventricular myocytes (19). ICa,T has been shown to reappear in ventricular myocytes under some pathological processes, such as ventricular hypertrophy (19, 24) and postmyocardial infarction (11). These facts suggest that T-type Ca2+ channels are associated with pacemaker activity, development, and postnatal growth of cardiac cells.

Recent advances in molecular and genetic characterization have identified three different subtypes of gene encoding {alpha}1-subunits of T-type Ca2+ channels: Cav3.1, Cav3.2, and Cav3.3 (2, 15, 25). The functional expression of these cDNAs in heterologous systems generated currents analogous to native ICa,T in terms of voltage dependence and kinetics of activation and inactivation, but they are not identical (13, 21). Whereas Cav3.3 was mostly detected in the brain (15), Cav3.1 and Cav3.2 mRNAs were also detected in human, rat, and mouse hearts (2, 25). The subtype distribution is also development stage dependent (22), but the issue remains controversial. Cribbs et al. (3) have shown in mouse embryonic hearts that only Cav3.1 underlies functional T-type Ca2+ channels in midgestational (E14) fetal myocardium. In rat hearts at middle-to-late embryonic (E16–21) and perinatal periods, substantial participation of both Cav3.1 and Cav3.2 to the functional T-type Ca2+ channels has been reported (7). To our knowledge, no data are available for the subtype-specificity of functional T-type Ca2+ channels at earlier embryonic stages of mammalian hearts.

In the present study, we investigated developmental changes of subtype expression for the functional T-type Ca2+ channels in mouse hearts from the earliest embryonic stage at which the heart starts to beat regularly (E9.5) to adulthood. Our observations in cell electrophysiology and mRNA quantification have revealed that Cav3.2 underlies the functional T-type Ca2+ channels in the embryonic murine heart, and there is a subtype switching of transcripts from Cav3.2 to Cav3.1 in the perinatal period.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Animals. ICR mice at three developmental stages were employed for the present study: an early and a late embryonic day (E9.5 and E18) and 10-wk-old female adult. All animal procedures were approved by the Animal Care and Use Committee, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.

Dissection of ventricles from E9.5 mouse embryos. Pregnant (9.5 days postcoitum) mice were euthanized by cervical dislocation, the uteri were isolated, and whole embryos were exposed. The ventricles were separated carefully from tubular hearts with the use of a sharp needle under a stereoscopic microscope. During the dissection procedure, the tissues were kept in Hanks' balanced salt solution (Sigma; St. Louis, MO).

Electrophysiological recordings. Single myocytes were prepared from ventricles of E9.5, E18, and adult mice by methods previously described (18, 34). Whole cell Ca2+ currents were recorded from the myocytes using the patch-clamp technique. All experiments were carried out at 32–35°C. To isolate Ca2+ currents, the myocytes were perfused with Na+- and K+-free external solution containing (in mM) 140 TEA-Cl, 1 MgCl2, 5 HEPES, 10 glucose, 5 CaCl2, 5 4-aminopyridine, and 0.005 tetrodotoxin (pH 7.4 with CsOH). The internal pipette solution contained (in mM) 60 CsOH, 80 CsCl, 40 aspartate, 5 HEPES, 10 EGTA, 5 MgATP, 5 Na2-creatine phosphate, and 0.65 CaCl2 (pH 7.2 with CsOH). The recording pipettes had tip resistances ranging from 3.8 to 4.5 M{Omega} when filled with the internal solution. Liquid junction potential of the internal pipette solution was minimal (2.0 ± 0.1 mV; n = 6). Therefore, we did not correct the membrane potential (Vm) by the liquid junction potential. NiCl2, which blocks Cav3.2-based T-type Ca2+ current at lower concentrations than Cav3.1-based T-type Ca2+ current (16), was diluted in the external solution from a stock solution (1 M) to the appropriate concentrations.

Current recordings were made with an Axopatch-200B amplifier (Axon Instruments). Series resistance was compensated for ~75%. Command voltage pulse generation and data acquisition were performed with a Digidata 1200 and pCLAMP8 software (Axon Instruments). Cell capacitance was measured by applying a ramp voltage pulse of 0.5 V/s at a potential ranging between –50 and +70 mV. The average cell capacitance was 25.5 ± 1.6 pF for E9.5 myocytes (n = 10), 28.9 ± 1.8 pF for E18 myocytes (n = 9), and 154.3 ± 10.7 pF for adult myocytes (n = 11).

L-type Ca2+ current (ICa,L) and ICa,T were separated by application of 200-ms voltage steps in 10-mV increments, with a pulse interval of 5 s, to different test potentials from holding potentials (HP) of –100 and –50 mV. Isolation of ICa,T was performed by subtracting the currents obtained from the same test potential taken at the different HPs. Current amplitude was determined as the difference between the peak inward current and the steady-state current recorded at the end of the test pulse. The current amplitude was divided by cell capacitance to obtain current density.

In experiments to study the activation and inactivation properties of ICa,T, the current was recorded from HP of –100 mV in the presence of nisoldipine (3 µM) to eliminate ICa,L. The voltage-dependence of activation was estimated from conductance (G)-voltage relationship obtained by the equation G = Ipeak/(Vm – Erev), where Ipeak is the peak ICa,T and Erev is the extrapolated reversal potential of ICa,T. Conductance values were then normalized to the maximal conductance and fitted to the following Boltzmann equation: G/Gmax = 1/{1 + exp[–(Vm – V0.5)/k]}, where Gmax is the maximal Ca2+ conductance, V0.5 is the potential at which the conductance is half-maximally activated, and k is the slope factor.

The voltage dependence of steady-state inactivation for ICa,T was determined with the use of a double-pulse protocol. A conditioning pulse for 1 s to various voltages ranging from –90 to –50 mV was followed by a test pulse to –40 mV for 200 ms to elicit ICa,T. Data were normalized by dividing the test current by the maximal current elicited and fitted according to the Boltzmann equation.

The recovery of ICa,T from inactivation was studied by applying two test pulses to –40 mV for 50 ms from a HP of –100 mV with increasing intervals from 10 to 3,000 ms every 5 s. The fractional recovery was calculated as the ratio of the current during the test pulse to the maximum current during the conditioning pulse.

PCR analysis. Total RNA of cardiac ventricles was extracted from E9.5 mouse embryos using a RNeasy Mini Kit (Qiagen; Hilden, Germany) and from E18 embryos and adult mice by the acid guanidinium-phenol-chloroform method. Single-strand cDNA was synthesized from DNase I (Boehringer; Ingelheim, Germany)-treated RNA and incubated with oligo (dT) primer and Superscript II RT (GIBCO-BRL; Gaithersburg, MD). The enzyme was then heat inactivated, and RNase H (GIBCO-BRL) was added to degrade residual RNA.

Two types PCR were carried out: conventional PCR and real-time PCR. GAPDH mRNA was used as an internal control. Primers and Taqman probes for Cav3.1, Cav3.2, and GAPDH were designed by using Primer Express (Perkin Elmer Applied Biosystems) (Table 1). Conventional PCR was first performed using AmpliTaq Gold (Roche Molecular Systems). After 30 cycles of PCR, amplified fragments were resolved on a 2% agarose gel. PCR products for Cav3.1, Cav3.2, and GAPDH genes were subcloned by TA cloning (pGEM-T Easy; Promega) and were verified by sequencing.


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  		Table 1. Sequence of oligonucleotides used as PCR primers and sequence-specific probes

 
To quantify mRNA expression of Cav3.1, Cav3.2, and GAPDH, we applied a real-time fluorogenic 5'-nuclease PCR assay, ABI Prism 7700 (Perkin Elmer Applied Biosystems). The Taqman probe contains reporter dye at the 5' end and quencher dye at the 3' end. During the PCR reaction, the 5' nuclease activity of AmpliTaq Gold cleaves the Taqman probe to separate the reporter dye and the quencher dye, which results in increased fluorescence of the reporter dye. Accumulation of PCR products was detected directly by monitoring the increase in fluorescence. The fluorescence signals were analyzed and converted into a relative number of copies of target molecules. Standard curves for the quantification were constructed by amplification of successive template quantity of cDNA (from 1 x 103 to 1 x 107). The threshold cycles were plotted against the logarithm of the initial molecule number of cDNA standards with linear relationships. Slope factors of Cav3.1, Cav3.2, and GAPDH were –3.275, –3.447, and –3.425 cycles/log decade, respectively. In a real-time PCR experiment, 300 ng cDNA of the sample was added to each tube.

Recently, several investigators suggested that the expression level of GAPDH mRNA could alter under cell proliferating condition. To test the suitability of GAPDH as an internal control, we investigated whether there is the existence of developmental change in GAPDH mRNA expression. We observed no significant changes in the expression GAPDH mRNA among these stages [459 x 104 ± 68 x 104 at E9.5 (n = 6), 425 x 104 ± 26 x 104 at E18 (n = 6), and 345 x 104 ± 67 x 104 at 10 wk old (n = 7); P = 0.38].

Statistics. Data analysis, statistics, and curve fitting were performed with pCLAMP8 software (Axon Instruments), Origin software (Microcal Software), and Microsoft Excel computer software. Values are presented as means ± SE and were analyzed by the unpaired t-test, ANOVA, and analysis of covariance. For multiple comparisons, Scheffé's F-test was followed. Differences were considered significant at P < 0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
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Two components of Ca2+ current in embryonic ventricular myocytes. Two components of Ca2+ current were recorded from ventricular myocytes with different HPs. Figure 1 shows representative current traces recorded from an E9.5 myocyte. Depolarizing pulses to various voltages (–40 to +30 mV) from a HP of –100 mV caused greater inward currents than those from a HP of –50 mV. Subtraction of the currents has revealed the presence of low-voltage-activated ICa,T in addition to high-voltage-activated ICa,L.



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  		Fig. 1. Ca2+ current traces in mouse ventricular myocytes at early embryonic days (E9.5). Currents were recorded in Na+- and K+-free solution by a whole cell patch-clamp method. Depolarization protocols are illustrated at the top. Depolarizing steps to various voltages from two different holding potentials (HP) of –100 mV (left traces) and of –50 mV (middle traces) elicited Ca2+ current. Right traces show T-type Ca2+ current (ICa,T) obtained by subtracting currents elicited at the two HPs.

 
Figure 2 summarizes the current-voltage relationships (I-V curves) of Ca2+ current recorded from ventricular myocytes at E9.5, E18, and adult, respectively. Both in E9.5 (Fig. 2A) and E18 (Fig. 2B) myocytes, the difference current (ICa,T) was activated negative to –40 mV and peaked at around –20 mV. The peak ICa,T density of E18 myocytes tended to be larger at more depolarized membrane potential than that of E9.5 myocytes, but the differences did not reach a statistical significance. In adult myocytes (Fig. 2C), the two I-V curves from HP of –100 and –50 mV were almost superimposed, giving rise to minimal difference current at potentials more positive than –20 mV; the difference current is not the result of ICa,T, but may reflect a partial voltage-dependent inactivation of ICa,L, because its apparent I-V curve was proportional to the current recorded with a HP of –50 mV.



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  		Fig. 2. Current-voltage relationships (I-V curve) obtained from myocyte at E9.5 (n = 6; A), late embryonic day (E18) (n = 5; B), and 10-wk adulthood (n = 5, C). I-V curve obtained from HP of –100 mV ({bullet}) is mixture of L-type Ca2+ current (ICa,L) and ICa,T, and I-V curve obtained from HP of –50 mV ({blacktriangledown}) is ICa,L. Difference of those two I-V curves ({circ}) indicates ICa,T.

 
Activation and inactivation properties of ICa,T. The current properties of ICa,T in embryonic myocytes were studied after application of nisoldipine (3 µM) to eliminate ICa,L. It was confirmed in our different series of experiments that ICa,L in either embryonic or adult ventricular myocytes is abolished completely by 3 µM nisoldipine (data not shown). In both E9.5 and E18 myocytes, ICa,T was activated and inactivated rapidly during depolarization to –40 mV (for 200 ms) from a HP of –100 mV. The activation and inactivation time courses were fitted by single-exponential functions (Fig. 3). There were no significant differences in the activation ({tau}act) and inactivation time constants ({tau}inac) between the two embryonic stages.



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  		Fig. 3. Activation and inactivation time courses of ICa,T. ICa,T were elicited by the depolarization pulse to –40 mV from a HP of –100 mV. Data are expressed as mean ± SE. {tau}act, Time constant of activation; {tau}inac, time constant of inactivation.

 
The voltage dependence of activation and that of steady-state inactivation of ICa,T were also studied in the presence of nisoldipine (Fig. 4A). The activation curves of individual cells were fitted to a Boltzmann equation, and the parameters obtained were averaged. For ICa,T recorded from E9.5 myocytes, the V0.5 was at –40.9 ± 1.1 mV and k was 5.3 ± 0.3 mV (n = 9). For ICa,T recorded from E18 myocytes, V0.5 was –38.4 ± 1.1 mV and k was 5.0 ± 0.5 mV (n = 6). The steady-state inactivation curves analyzed similarly give rise to V0.5 at –63.4 ± 0.9 mV with a k of –4.2 ± 0.5 mV for E9.5 myocytes (n = 7) and V0.5 at –60.2 ± 1.3 mV with a k of –4.7 ± 0.2 mV for E18 myocytes (n = 7). There were no significant differences in V0.5 and k values between the two embryonic stages for both activation and inactivation, and the activation and steady-state inactivation curves fitted to averaged data of myocytes at E9.5 and E18 (Fig. 4A) were almost superimposed.



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  		Fig. 4. A: voltage dependence of activation and steady-state inactivation of ICa,T. Voltage-clamp experiments to measure ICa,T were carried out in the presence of nisoldipine (3 µM) according to the procedure described in METHODS. Data points plotted are means ± SE for E9.5 myocytes ({bullet} and {blacktriangleup}, n = 9) and for E18 myocytes ({circ} and {triangleup}, n = 5). The solid lines represent the best fit with the Boltzmann equation for the respective averaged data. B: time dependence of recovery from inactivation of ICa,T. A two-pulse protocol with various interpulse intervals ({Delta}t) was used to estimate the recovery kinetics (inset). Relative peak current for the test pulse [(Itest) normalized to the maximum current (Imax) for the conditioning pulse] was plotted against {Delta}t. Data points are means ± SE for E9.5 myocytes ({bullet}, n = 6) and for E18 myocytes ({circ}, n = 5). The solid lines represent the best fit with a double exponential function for the respective averaged data.

 
Figure 4B illustrates time-dependent ICa,T recovery from inactivation in the embryonic myocytes (E9.5 and E18). The fractional recovery expressed as the ratio of the current during the test pulse to the maximum current during the conditioning pulse was plotted as a function of the interpulse duration. The relations were best fitted by a double exponential function. The curve fits provided time constants {tau}fast and {tau}slow and amplitudes Afast and Aslow for fast and slowly recovering current fractions, respectively. The recovery kinetics at E9.5 ({tau}fast, 37.2 ± 6.9 ms; Afast, 0.45 ± 0.08; {tau}slow, 276 ± 21 ms; and Aslow, 0.54 ± 0.07; n = 6) were similar to those at E18 ({tau}fast, 26.9 ± 5.0 ms; Afast, 0.50 ± 0.03; {tau}slow, 300 ± 23 ms; and Aslow, 0.41 ± 0.05; n = 8).

Ni2+ sensitivity. Ni2+ applied to myocytes acts as a blocker of ICa,T. As previously reported from heterologous expression systems, the Ni2+ sensitivity of Cav3.2- and Cav3.1-related current strongly differs (IC50 = 12 and >150 µM, respectively) (16). We therefore examined the effects of Ni2+ at concentrations ranging from 1 to 1,000 µM on ICa,T recorded from embryonic (E9.5 and E18) ventricular myocytes. Representative records are shown in Fig. 5A. ICa,T was measured at a test pulse to –40 mV from a HP of –100 mV in the presence of nisoldipine. Bath application of 30 µM Ni2+ resulted in a prompt reduction of ICa,T amplitude to approximately half of control at both E9.5 and E18, indicating relatively high sensitivity of the current to Ni2+. Figure 5B shows the average dose-response curves obtained from E9.5 (n = 10) and E18 (n = 7) myocytes. The two curves were almost superimposed and IC50 were 31 ± 4 µM at E9.5 and 26 ± 5 µM at E18.



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  		Fig. 5. Effect of Ni2+ on ICa,T of ventricular myocytes at E9.5 and E18. A: representative ICa,T elicited during test pulses to –40 mV in control condition and in the presence of 30 µM Ni2+. Both ICa,T of E9.5 (top traces) and E18 (bottom traces) were effectively blocked by relatively low concentration of Ni2+. B: dose-response curves for the block by Ni2+ of ICa,T of E9.5 ({bullet}, n = 10) and E18 ({circ}, n = 5). Inhibition of ICa,T were normalized to control and then plotted as a function of drug concentration.

 
Quantification of mRNA expression of T-type Ca2+ channel {alpha}1-subunit. Conventional RT-PCR revealed that Cav3.1 mRNA was hardly detectable in E9.5 but present in E18 and in the adult ventricle. On the other hand, Cav3.2 mRNA was prominent in E9.5, reduced in E18, and minimal in the adult ventricle (Fig. 6A). The expression of these transcripts was quantified by a real-time PCR assay. Figure 6B shows the data normalized to GAPDH mRNA (1 x 105 molecules). At E9.5, Cav3.2 mRNA was expressed abundantly (7,177 ± 105; n = 7), whereas Cav3.1 mRNA expression was minimal (40 ± 3; n = 7). At E18, Cav3.1 mRNA was increased (1,226 ± 121; n = 7), whereas Cav3.2 mRNA was decreased (4,041 ± 629; n = 7), but it still remained to be dominant subtype. In adult ventricular tissue, a substantial level of Cav3.1 mRNA expression (741 ± 148, n = 7) was detected, whereas Cav3.2 mRNA expression was minimal (57 ± 19, n = 7).



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  		Fig. 6. Expression of mRNA encoding T-type channels in mouse cardiac ventricles. A: identification of Cav3.1 mRNA (top bands) and Cav3.2 mRNA (middle bands) in mouse cardiac ventricle by RT-PCR assay. GAPDH mRNA (bottom bands) was used as an internal control. Left lane represents mRNA expression at E9.5, middle lane represents mRNA expression at E18, and right lane represents mRNA expression at 10-wk adulthood. B: developmental change of Cav3.1 mRNA (open bars) and Cav3.2 mRNA (solid bars) expression. Quantitative analysis was performed by a real-time PCR assay. mRNA levels are normalized to the amount of GAPDH mRNA. Left bars show the level of mRNA expression at E9.5 (n = 7), middle bars show the level of mRNA expression at E18 (n = 7), and right bars show the level of mRNA expression at 10-wk adulthood (n = 7).

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
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In this study, we have described ICa,T in ventricular myocytes isolated from murine embryonic hearts from the onset of the heart beat at E9.5 until briefly before birth (E18). The current, which is inhibited by relatively low concentration of Ni2+ (IC50 26–31 µM), can be recorded in association with the temporal expression of Cav3.2 mRNA. Interestingly, Cav3.2 mRNA is downregulated by ~50% during the second half of embryonic development. In 10-wk-old mice, ICa,T is virtually absent, and Cav3.2 mRNA has almost disappeared. During the second half of embryonic development, Cav3.1 mRNA is upregulated by ~300-fold, although the other subtype (Cav3.2) mRNA remains predominant. The reverse is observed in adult myocytes: Cav3.1 mRNA is predominant, although its level of expression is lower than the embryonic period.

Developmental change of molecular identity. There is a considerable controversy among investigators as to the molecular identity of the T-type Ca2+ channel in embryonic hearts. Cribbs et al. (3) have demonstrated in developing mouse hearts that only Cav3.1d mRNA (a splice variant of Cav3.1) but no other subtypes or splice variants are expressed in embryonic (E12.5–E14) ventricular myocardium. They also showed positive immunostaining for Cav3.1 protein (localized to the cell membrane) in the embryonic myocytes but negative for Cav3.2 protein. The Ni2+ sensitivity of ICa,T recorded from the myocytes was relatively low (blocked by 38% with 100 µM Ni2+). On the basis of these observations, they concluded that Cav3.1 contributes to the functional ICa,T in midgestational fetal myocardium.

Our data on the dominance of Cav3.2 in the murine embryonic heart are in conflict with those of Cribbs et al. (3). We were able to detect Cav3.1 mRNA at E18 (3), but we observed much higher Cav3.2 mRNA levels, and ICa,T was more sensitive to Ni2+. Given the facts, we propose that Cav3.2-associated ICa,T is predominant at least in the period directly after the onset of the heartbeat. The discrepancy could be attributed to different methods employed for ICa,T recording (e.g., temperature and external solution) and for mRNA quantification (e.g., PCR assays, primers, and probes). We have investigated a broader age range than has been done previously. This experimental design has an advantage to shed light on the ongoing switch in Cav3 gene expression that occurs over somewhat prolonged period of development. It is interesting to note that as the isoform switch occurs at the mRNA level, the amount of ICa,T is decreasing. Cav3.1d is a splice variant in the III-IV linker (alternate splice of exon 25) of Cav3.1. Because the primers and probes employed in the present study correspond to a domain in the COOH-terminal (exon 35–36), mRNAs of Cav3.1 and Cav3.1d should have been amplified similarly.

It was shown recently by Xu et al. (32) in mouse early embryonic hearts (E9.5) that expression of Cav3.1 mRNA is more abundant than that of Cav3.2 mRNA in tissue samples. In isolated cardiomyocytes at E9.5, unlike in the total heart, they showed a small but substantial mRNA expression only for Cav3.2 (Cav3.1 mRNA was below the level of detection). On the basis of these observations, Xu et al. (32) speculated that Cav3.1 at this embryonic stage could be expressed in other cell types than cardiomyocytes.

In rat hearts, both Cav3.1 and Cav3.2 transcripts of comparable amounts were shown to be expressed in middle-to-late embryonic stages (E16–21). In a study by Larsen et al. (14) Cav3.2 mRNA decreased rapidly after birth and became undetectable at 5 wk, whereas Cav3.1 mRNA levels remained high in the postnatal periods. Ferron et al. (7) demonstrated that ICa,T is expressed in fetal rat ventricular myocytes, but decreased soon after birth to an undetectable level. They also showed progressive decrease of Ni2+ sensitivity of ICa,T from the midembryonic stage (IC50 = 49 µM at E16) to the neonatal stage (IC50 = 291 µM at 1 day after birth). Ferron et al. (7) therefore suggested that the relative participation of each subunit varies during developmental stages: with a major contribution of Cav3.2 at E16, whereas Cav3.1 in newborn myocytes. These observations are concordant with our data indicating the dominance of Cav3.2 subunit in the mouse embryonic heart and subtype switching of transcripts from Cav3.2 to Cav3.1 after birth. Progressive decline of Cav3.2 transcripts during development from early gestation to adult stages in the heart has also been demonstrated in human hearts (26).

Physiological and pathological implications. T-type Ca2+ channels coexist with L-type Ca2+ channels in the heart. The density of T-type Ca2+ channels is 0.1–0.3/µm2 in adult guinea pig ventricular myocytes (4). Peak inward ICa,T is <10% of ICa,L in guinea pig ventricular myocytes (31, 36). It is, however, much higher in canine Purkinje fibers (9) or the rabbit sinus node (6). The T-type Ca2+ channel seems absent in the rabbit atrium, rabbit ventricle, rat ventricle, and human atrium (20). ICa,T may thus play a functional role only in the specialized conduction system of normal matured hearts. In our study, ICa,T was also virtually absent in adult ventricular myocytes.

Because ICa,T is prominent in embryonic hearts, it may play a role in sustaining ventricular automaticity together with hyperpolarization-activated inward current (If), which we described in embryonic murine ventricular myocytes previously (33). The "pacemaker current" (If) disappears by ~80% during the second half of embryonic development, which is concomitant with the loss of ventricular automaticity. Figure 2, however, shows that there is no significant difference in ICa,T density between E9.5 and E18, despite the changes in Cav3.1 mRNA and Cav3.2 mRNA. It seems, therefore, that the amount of ICa,T in the absence of substantial If is insufficient to maintain regular ventricular automaticity in the embryonic heart.

Several studies have reported an upregulation of ICa,T in adult ventricular myocytes after experimentally induced hypertrophy (19, 24) or myocardial infarction (11). Information available for the molecular identity of such reexpressed ICa,T is still limited. In rat model of myocardial infarction, an increase of ICa,T was associated with an upregulation of Cav3.1 mRNA (11). On the other hand, ICa,T recorded from ventricular myocytes of rat with pressure overload-induced hypertrophy (19) and that of a genetically determined cardiomyopathic hamster (30) showed relatively high Ni2+ sensitivity (IC50 = 40–50 µM), suggesting a substantial contribution of Cav3.2 subunit to the reexpressed ICa,T. An increase of ICa,T in those diseased hearts could be arrhythmogenic through an increase of action potential duration, an induction of early and delayed afterdepolarization (27), or an enhancement of electrical and structural remodeling (5, 28).

Limitations. In our data, ICa,T recorded from E9.5 and E18 showed similar electrophysiological properties in terms of current density, kinetics of activation/inactivation, voltage dependence of activation, steady-state inactivation, and recovery kinetics from inactivation. This does not parallel the mRNA expression; Cav3.2 mRNA amount decreased, whereas the Cav3.1 mRNA amount increased from the early to the late embryonic period. An electrophysiological signature to discriminate the cloned Cav3.1 and Cav3.2 channels is the recovery kinetics from inactivation: Cav3.1 recovers more rapidly than Cav3.2 (13, 21). If participation of Cav3.1 to the functional T-type Ca2+ channels are increased from E9.5 to E18 in response to altered expression of mRNA amounts, it would cause a faster recovery from inactivation of ICa,T in E18 myocytes. In adult myocytes, no ICa,T was recorded despite substantial amount Cav3.1mRNA. These discrepancies may be interpreted in part by developmental changes of posttranscriptional and/or posttranslational regulations. Quantification of Cav3.1 and Cav3.2 protein amounts in different developmental stages will be required to get an insight into these regulations. We were unable to quantify the protein amounts, because there are no specific and reliable antibodies commercially available for the two subunits. Another possibility is a regulation of functional T-type Ca2+ channel by auxiliary subunits, known to modulate ionic current like ICa,L (23). Further studies investigating these issues should be very useful to increase our understanding on expression of ICa,T in developing mammalian hearts.


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 GRANTS
REFERENCES
 
This study was supported by a Grant-In-Aid for Scientific Research 14570654 from the Ministry of Education, Science, Sports, and Culture.


   ACKNOWLEDGMENTS
 
We thank Mayumi Hojo for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Yasui, Dept. of Circulation, Div. of Regulation of Organ Function, Research Institute of Environmental Medicine, Nagoya Univ., Nagoya 464-8601, Japan (E-mail: kenji{at}riem.nagoya-u.ac.jp).

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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