Main

In the heart, the L-type Ca2+ channel current (ICa,L) plays a crucial role in the excitation–contraction coupling in ventricles and spontaneous automaticity in the sinoatrial and atrioventricular nodes (1, 2). In the clinical setting, ICa,L provides an important therapeutic target for the treatment of various cardiovascular disorders and diseases. Currently, three classes of structurally different L-type Ca2+ channel antagonists, namely dihydropyridines, benzothiazepines, and phenylalkylamines, are clinically used to treat tachyarrhythmia and hypertension (2, 3). The phenylalkylamine verapamil is effective in terminating paroxysmal supraventricular tachycardia and some forms of ventricular tachycardia in adults (4, 5). However, the intravenous administration of verapamil has been regarded as contraindicated in neonates and infants (4, 6), because of a substantial risk of severe hypotension or bradycardia (7). In contrast, it is generally accepted that the dihydropyridines (nifedipine and isradipine) and benzothiazepine diltiazem can be safely used in neonates and infants in the clinical setting, without causing significant hypotension (8, 9, 10).

Previous studies have shown that the expression and function of Ca2+ handling proteins, involved in excitation–contraction coupling in the heart, undergo significant changes during postnatal development (11, 12). For example, current density of ICa,L exhibits a postnatal developmental increase in many mammalian species, including mouse and rabbit (13, 14, 15). In contrast, the expression of Na+/Ca2+ exchanger is highest in neonatal stages, and then gradually decreases during postnatal development in the hearts of rabbits and rats (16, 17). It is also known that, because the sarcoplasmic reticulum and transverse tubules are less developed in the neonatal stage than in adult stage (11, 18), the transsarcolemmal Ca2+ influx through ICa,L and/or the reverse mode Na+/Ca2+ exchanger plays a predominant role in providing the Ca2+ required for cardiomyocyte contraction (19, 20). Indeed, it has been demonstrated in rabbit that myocardial contractile force in immature hearts is more sensitive to the L-type Ca2+ channel antagonists (verapamil, nifedipine, and diltiazem) than that in the adult hearts (21, 22).

However, it remains unclear how phenylalkylamine verapamil exerts its more potent negative inotropic action in neonates and infants than dihydropyridines and benzothiazepines. The present study was undertaken to examine the possible postnatal developmental changes in the sensitivity of ICa,L to the inhibition by verapamil. Our results revealed that verapamil more potently inhibits ICa,L in ventricular myocytes of mice before the weaning age (~21 days after birth) than in mice after the weaning age.

Methods

Mice at Various Postnatal Ages

Mice on the day of birth were denoted as postnatal day 0 in age. We used six different age groups (day 0, day 7, day 14, day 21, day 28, and 10–15 weeks) of mice (C57BL/6J; Charles River, Yokohama, Japan) for the experiments investigating the blocking actions of verapamil, nifedipine, and diltiazem on ICa,L. Because mice are usually weaned ~21 days after birth (23), we may reasonably assume that mice younger than 21 days after birth correspond to neonatal and infant stages in humans. In contrast, mice at 28 days and 10–15 weeks after birth can be regarded as representing child and adult stages in humans, respectively (24). All protocols conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), and were approved by the institution’s Animal Care and Use Committee of Shiga University of Medical Science (approval number, 2014-8-2).

Isolation of Ventricular Myocytes

Ventricular myocytes were enzymatically dissociated from the hearts of mice in all age groups (0, 7, 14, 21, 28 days, and 10–15 weeks) using a similar Langendorff-perfusion method (15, 25, 26). Mice were killed by an intraperitoneal injection of sodium pentobarbital overdose (300 mg/kg) with heparin (8,000 U/kg). In all age groups, the heart was rapidly removed and perfused at 37 °C for 3 min in a Langendorff mode with cell isolation buffer (CIB), supplemented with 0.4 mM EGTA. The CIB contained (in mM) 130 NaCl, 5.4 KCl, 0.5 MgCl2, 0.33 NaH2PO4, 22 glucose, 25 HEPES (pH adjusted to 7.4 with NaOH), and 50 U/ml bovine insulin. The hearts of day 0 and day 7 mice were then perfused for 3–5 min with CIB supplemented with 1 mg/ml collagenase (type 2; Worthington Biochemical Corporation, Lakewood, NJ), 0.03 mg/ml trypsin (Sigma, St. Louis, MO), 0.03 mg/ml protease (Sigma), and 0.15 mM CaCl2. The heart was then removed from the Langendorff apparatus, and the ventricles were gently agitated to disperse the myocytes in CIB, supplemented with 1.2 mM CaCl2 and 2 mg/ml bovine serum albumin (BSA, Sigma). The hearts of day 14, day 21, day 28, and 10–15-week mice were Langendorff-perfused for 6–8 min with CIB supplemented with 1 mg/ml collagenase, 0.06 mg/ml trypsin, 0.06 mg/ml protease, and 0.3 mM CaCl2. The hearts were removed from the Langendorff apparatus, chopped into small pieces, and further incubated at 37 °C for 10 min in CIB supplemented with 1 mg/ml collagenase, 0.03 mg/ml trypsin, 0.03 mg/ml protease, 0.7 mM CaCl2, and 2 mg/ml BSA. The supernatant was centrifuged (14g for 3 min), and the myocyte pellet was resuspended in CIB supplemented with 1.2 mM CaCl2 and 2 mg/ml BSA. The myocytes were further incubated for 10 min, centrifuged (14g for 3 min), and resuspended in normal Tyrode solution supplemented with 2 mg/ml BSA. The normal Tyrode solution contained (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.33 NaH2PO4, 5.5 glucose, and 5.0 HEPES (pH adjusted to 7.4 with NaOH). It should be noted that chunk method has been commonly used to isolate ventricular myocytes having spherical shape from neonatal mice, possibly because of the technical difficulty in introducing the Langendorff-perfusion method. The present investigation adopted the Langendorff-perfusion method, which enables the harvesting of spindle- or rod-shaped healthy ventricular myocytes from neonatal mice (15).

Whole-cell patch-clamp experiments

The whole-cell patch-clamp technique (27) was used to record ICa,L in mouse ventricular myocytes of all age groups, with an EPC-8 patch-clamp amplifier (HEKA, Lambrecht, Germany). The patch electrodes, fabricated from glass capillaries using a horizontal microelectrode puller (P-97; Sutter Instrument, Novato, CA), had a resistance of 2.0–4.0 MΩ, when filled with a Cs+-rich pipette solution that contained (in mM) 90 cesium aspartate, 30 CsCl, 20 tetraethylammonium chloride, 2 MgCl2, 5 ATP (magnesium salt; Sigma), 0.1 GTP (dilithium salt; Roche Diagnostics, GmbH, Mannheim, Germany), 5 phosphocreatine (disodium salt; Sigma), 5 EGTA, and 5 HEPES (pH adjusted to 7.2 with CsOH). Ventricular myocytes were transferred to a recording chamber (0.5 ml in volume) mounted on the stage of an inverted microscope, and continuously superfused at 36–37 °C with normal Tyrode solution. After the establishment of whole-cell patch-clamp mode, ventricular myocytes were superfused with a Cs+-Tyrode solution containing (in mM) 140 NaCl, 5.4 CsCl, 1.8 CaCl2, 0.5 MgCl2, 5.5 glucose, and 5 HEPES (pH adjusted to 7.4 with NaOH). ICa,L was activated by depolarizing voltage-clamp steps applied from a holding potential of –40 mV to test potentials of –30 mV through +40 mV in 10 mV steps, before and during exposure to verapamil (hydrochloride, 10−8 to 10−6 M; Sigma), nifedipine (10−9 to 10−7 M; Sigma), or diltiazem (hydrochloride, 10−8 to 10−6 M; Sigma). In experiments examining the blocking effects of these L-type Ca2+ channel antagonists, only one concentration of the drug was tested in each ventricular myocyte. The percentage reduction in the amplitude of ICa,L was measured at 0 mV by the presence of each test concentration of the drug, and was fitted with a Hill equation: R=100/(1+(IC50/[drug])nH), where R is the percent reduction of ICa,L, IC50 is the concentration of drug causing a half-maximal inhibition, and nH is a Hill coefficient. The conductance of ICa,L (gCa,L) was calculated by dividing the current amplitude at each test potential by the driving force for Ca2+ influx through ICa,L and was then normalized with the maximum value at +10 or +20 mV (gCa,L,max) (15, 28). The normalized conductance (gCa,L) was fitted with a Boltzmann equation: gCa,L=1/(1+exp((V1/2–Vt)/k)), where V1/2 is the voltage at half-maximal activation, Vt is test potential, and k is the slope factor. Frequency-dependent block of ICa,L by verapamil, nifedipine, or diltiazem was evaluated in ventricular myocytes of day 7 and 10–15-week mice by repetitively (0.2 or 2 Hz) applying depolarizing steps to 0 mV from a holding potential of –40 mV in the presence of verapamil, nifedipine, or diltiazem. The concentration of verapamil, nifedipine, or diltiazem was set to near the IC50 values for the inhibition by each antagonists; verapamil (50 nM for day 7 and 150 nM for 10–15-week ventricular myocytes), nifedipine (10 nM for both day 7 and 10–15-week ventricular myocytes), and diltiazem (150 nM for both day 7 and 10–15-week ventricular myocytes). Cell membrane capacitance (Cm) was measured from the capacitive transients in response to voltage-clamp steps (±5 mV) applied from a holding potential of –40 mV, using the follow equation; Cm=τC I0/ΔVm (1−I∞/I0) (15, 29), where τC is the time constant of the capacitive transient, I0 is the initial peak current amplitude, I∞ is the steady-state current value, ΔVm is the amplitude of voltage step (5 mV).

Western Blotting and Immunocytochemistry

A full description of western blotting and immunocytochemistry, including primary antibodies used for these experiments, is available in Methods in online Supplementary Information.

Statistical Analysis

Data are presented as mean±SD. Statistical significance was evaluated using either Student’s unpaired t-test or an analysis of variance, followed by a post hoc Dunnett or Tukey test, as appropriate. A value of P<0.05 was considered to be statistically significant.

Results

Electrophysiological Properties of ICa,L in Ventricular Myocytes Isolated from Different Age Groups of Mouse Heart

We first examined the cell membrane capacitance (Cm) of ventricular myocytes isolated from the hearts of six different age groups (0, 7, 14, 21, 28 days, and 10–15 weeks) of mice. As shown in Figure 1, Cm, which is proportional to the cell surface area, increased from 20.7±8.3 pF (n=44) in day 0 ventricular myocytes to 142.6±10.7 pF (n=75) in 10–15-week ventricular myocytes. These values of Cm are qualitatively similar to the previous observations in mouse ventricular myocytes and indicate that the ventricular cell size gradually increases with postnatal growth of the heart (15, 30).

Figure 1
figure 1

Postnatal developmental increases in ventricular cell size. Cell membrane capacitance (Cm) of ventricular myocytes obtained from six different age groups from day 0 to 10–15 weeks. Data represent mean±SD (number of cells).

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We then investigated the conductance properties of ICa,L in mouse ventricular myocytes of all age groups. As described in Figure 2a, ICa,L exhibited its maximum amplitude at a test potential of –10 or 0 mV in both postnatal day 0 and 10–15-week ventricular myocytes. However, the maximum amplitudes of ICa,L, measured at a test potential of –10 or 0 mV, gradually increased during postnatal development from 4.8±0.9 pA/pF (n=8) in day 0 to 11.0±2.2 pA/pF (n=8) in 10–15-week ventricular myocytes (Figure 2c), consistent with the previous study (15). The conductance of ICa,L at each test potential was then calculated by dividing the peak amplitude of ICa,L by the driving force for Ca2+ influx through ICa,L. The smooth curves through the data points represent the least-squares fit of the Boltzmann equation (Figure 2d), yielding V1/2 and k. There were no significant differences in the values of V1/2 (Figure 2e) or k (Figure 2f) among all age groups, suggesting that the voltage-dependence of ICa,L activation does not differ during the postnatal development of mice from day 0 through 10–15 weeks. Previous studies have also shown that there are no significant developmental changes in the voltage-dependence of ICa,L activation in ventricular myocytes from neonatal to adult stages in mouse and rabbit (13, 14, 15).

Figure 2
figure 2

Conductance property of ICa,L in mouse ventricular myocytes. (a) Superimposed current traces of ICa,L recorded during depolarizing steps applied from a holding potential of −40 mV to test potentials of –30 to +40 mV, from day 0 (upper panel) and 10–15-week (lower panel) ventricular myocytes. (b) Mean current–voltage relationships for peak ICa,L, obtained from day 0 (upper panel, n=5) and 10–15-week (lower panel, n=5) ventricular myocytes. (c) Postnatal increase in the maximal amplitude of ICa,L from day 0 to 10–15-week ventricular myocytes. (d) Mean normalized conductance–voltage relationships for ICa,L in ventricular myocytes of all postnatal stages. Data points were fitted with the Boltzmann equation. V1/2 (e) and k (f) obtained from the Boltzmann fit shown in d. The number of cells is indicated in parentheses. Note that there are no significant differences in V1/2 and k among all age groups.

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Postnatal Developmental Changes in the Sensitivity of ICa,L to the Inhibition by Verapamil

We next investigated the pharmacological sensitivity of ICa,L in ventricular myocytes at various developmental stages to three structurally different L-type Ca2+ channel antagonists; namely phenylalkylamine verapamil, dihydropyridine nifedipine, and benzodiazepine diltiazem. Figure 3a shows the superimposed current traces of ICa,L recorded at test potentials of –30 through +40 mV in days 0, 7, 14, 21, 28, and 10–15-week ventricular myocytes before and during exposure to 100 nM verapamil. Under control conditions, the amplitude of ICa,L was maximum at test potentials of –10 or 0 mV in all of these ventricular myocytes (Figure 3b). Verapamil (100 nM) was found to reduce ICa,L in all developmental stages, which appeared, however, more potent in younger stages than in older ones.

Figure 3
figure 3

ICa,L recorded from the ventricular myocytes of various developmental stages in the absence and presence of 100 nM verapamil. (a) Superimposed current traces of ICa,L recorded during 200-ms depolarizing steps to test potentials of –30 to +40 mV, before and 2–3 min after exposure to 100 nM verapamil. (b) Current–voltage relationships for ICa,L measured before (empty symbols) and after (filled symbols) the exposure to 100 nM verapamil.

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To quantitatively analyze the blocking effect of verapamil on ICa,L, we measured the percentage inhibition of ICa,L by various concentrations (10−8 to 10−6 M) of verapamil at a test potential of 0 mV in ventricular myocytes of all developmental stages. The potency of verapamil in blocking ICa,L was examined by constructing the concentration–response relationships for the percent inhibition of ICa,L by verapamil in days 0, 7, 14, 21, 28, and 10–15-week ventricular myocytes (Figure 4a). As summarized in Figure 4d, IC50 value for the inhibition of ICa,L in days 0, 7, 14, 21 ventricular myocytes was smaller than that in day 28 and 10–15-week ventricular myocytes, showing that verapamil inhibits ICa,L more potently in ventricular myocytes at younger stages than at older stages. However, there were no significant changes in IC50 value for the blocking action of nifedipine on ICa,L among ventricular myocytes at any developmental stages (Figure 4b). Similarly, IC50 value for diltiazem did not differ among ventricular myocytes at any developmental stage (Figure 4c).

Figure 4
figure 4

Blocking potency of verapamil, nifedipine, and diltiazem on ICa,L in ventricular myocytes of all developmental stages. (a–c) The concentration–response relationships for the inhibition of ICa,L by verapamil (a), nifedipine (b), and diltiazem (c). The percent inhibition of ICa,L by verapamil, nifedipine, and diltiazem, measured at 0 mV, was plotted against their concentrations. (d–f) IC50 for the block of ICa,L by verapamil (d), nifedipine (e), and diltiazem (f). Note that the blocking potency of verapamil on ICa,L was stronger in day 0, day 7, day 14, and day 21 ventricular myocytes than in day 28 and 10–15-week ventricular myocytes. *P<0.05 compared with 10–15-week ventricular myocytes. N.S., not significant.

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Frequency-Dependent Blocking Property of Verapamil on ICa,L

Verapamil, nifedipine, and diltiazem have been shown to exhibit differential frequency-dependent block of ICa,L in heart cells (31, 32, 33). We then investigated the frequency-dependent blocking property of verapamil, nifedipine, or diltiazem on ICa,L in ventricular myocytes of day 7 and 10–15-week mice. For this purpose, verapamil was applied to the ventricular myocytes of day 7 and 10–15-week mice at concentrations near to their respective IC50 values to block ICa,L (50 nM and 150 nM for day 7 and 10–15-week ventricular myocytes, respectively). Ventricular myocytes were repetitively depolarized from a holding potential of –40 mV to a test potential of 0 mV in the presence of verapamil at a frequency of 0.2 and 2 Hz. As demonstrated in Figure 5a, the amplitude of ICa,L at the 15th step applied at 0.2 Hz was moderately reduced, compared with that at the 1st step in both day 7 and 10–15-week ventricular myocytes. In contrast, the amplitude of ICa,L at the 15th step applied at 2 Hz was markedly reduced, compared with the 1st step in both day 7 and 10–15-week ventricular myocytes (Figure 5b).

Figure 5
figure 5

Frequency-dependent block of ICa,L by verapamil, nifedipine, or diltiazem in ventricular myocytes of day 7 and 10–15-week mice. Depolarizing voltage-clamp steps were applied to a test potential of 0 mV from a holding potential of −40 mV at a frequency of 0.2 or 2 Hz in the presence of verapamil, nifedipine, or diltiazem. The concentration of verapamil, nifedipine, or diltiazem was set to near the IC50 value for inhibition by verapamil (50 nM for day 7 or 150 nM for 10–15-week ventricular myocytes), nifedipine (10 nM for both day 7 and 10–15-week ventricular myocytes), or diltiazem (150 nM for both day 7 and 10–15-week ventricular myocytes). (a, b) Superimposed current traces of ICa,L at the 1st and 15th depolarizing steps applied at a frequency of 0.2 Hz (a) or 2 Hz (b) in the presence of verapamil in day 7 (left panel) and 10–15-week (right panel) ventricular myocytes. (c–e) The peak amplitude of ICa,L at each depolarizing step applied at 0.2 and 2 Hz was normalized with reference to the value at the first step (In/I1) and plotted against the step number in the presence of verapamil (c), nifedipine (d), or diltiazem (e) in day 7 (left panel) and 10–15-week (right panel) ventricular myocytes. (f) The relative amplitude of ICa,L at the 15th step to that at the 1st step at a frequency of 0.2 and 2 Hz in the presence of verapamil, nifedipine, or diltiazem in day 7 (left panel) and 10–15-week (right panel) ventricular myocytes. *P<0.05. N.S., not significant.

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Figure 5c summarizes the changes in the peak amplitude of ICa,L during the trains of 15 depolarizing steps applied at 0.2 and 2 Hz in the presence of verapamil in day 7 (left panel) and 10–15-week (right panel) ventricular myocytes. Using the same protocol, we measured the changes in the peak amplitude of ICa,L in the presence of nifedipine (10 nM, Figure 5d) or diltiazem (150 nM, Figure 5e) in day 7 (left panel) and 10–15-week (right panel) ventricular myocytes. To assess the frequency-dependent block by verapamil, nifedipine, or diltiazem, the peak amplitude of ICa,L at the 15th step (I15) was normalized to that at the 1st step (I1), and this ratio in current amplitudes (I15/I1) was compared at frequencies of 0.2 and 2 Hz. In both day 7 and 10–15-week ventricular myocytes, the ratio of I15/I1 obtained at 2 Hz was significantly smaller than that at 0.2 Hz in verapamil and diltiazem (Figure 5f), suggesting that a frequency-dependent block of ICa,L was observed for these two antagonists in both day 7 and 10–15-week ventricular myocytes. However, there was no significant change in I15/I1 obtained at 0.2 or 2 Hz in nifedipine (Figure 5f), suggesting that a frequency-dependent block of ICa,L was scarcely observed for this antagonist in both day 7 and 10–15-week ventricular myocytes.

Expression and Cellular Localization of CaV1.2 and CaV1.3 Channel Proteins in Day 7 and 10–15-Week Ventricular Myocytes

It has been demonstrated in the mouse heart that although CaV1.2 channel primarily contributes to ICa,L in adult ventricular myocytes, CaV1.3 channel is mainly responsible for ICa,L in the early embryonic ventricular myocytes (34). It is, however, still unknown whether CaV1.3 channel is expressed in mouse ventricular myocytes at early postnatal stages. We therefore examined the expression of CaV1.2 and CaV1.3 channel proteins in day 7 and 10–15-week ventricular myocytes, using Western blotting and immunocytochemistry. CaV1.2 channel protein was detected by Western blotting in both day 7 and 10–15-week ventricular myocytes (Figure 6a). On the other hand, CaV1.3 channel protein was detected in day 7 ventricular myocytes, but was not clearly observed in 10–15-week ventricular myocytes (Figure 6b). Immunocytochemistry confirmed that CaV1.2 channel was expressed in both day 7 and 10–15-week ventricular myocytes (Figure 6c). A merged image of the immunofluorescent signals of CaV1.2 channel and α-actinin in both day 7 and 10–15-week ventricular myocytes indicated that this channel was orderly expressed along with the sarcomeres. In contrast, the immunofluorescent signals of CaV1.3 channel were not detected in 10–15-week ventricular myocytes. In day 7 ventricular myocytes, the immunofluorescent signals of CaV1.3 channel were only detected in the perinuclear area (Figure 6d). A similar perinuclear localization of CaV1.3 channel has been reported in the neonatal rat ventricular myocytes (35).

Figure 6
figure 6

Expression and cellular localization of CaV1.2 and CaV1.3 channel proteins in day 7 and 10–15-week mouse ventricles. (a, b) Western blotting of CaV1.2 (a) and CaV1.3 (b) channel expression in day 7 and 10–15-week ventricular tissues. CaV1.2 and CaV1.3 channel antibodies preincubated with control peptide antigen in day 7 mouse ventricular tissue (absorption test). GAPDH was used as an internal control. (c, d) Confocal laser scanning microscopy of double immunostaining for α-actinin and CaV1.2 (c), or CaV1.3 (d) channel, DAPI staining for nuclei and differential interference contrast (DIC) images of day 7 and 10–15-week ventricular myocytes. Bar=25 μm.

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Discussion

Previous studies have shown that excitation–contraction coupling in the heart undergoes significant postnatal developmental changes in experimental animals (11, 12). Because of the poor development of sarcoplasmic reticulum and transverse tubules, the transsarcolemmal Ca2+ influx appears to be a more important source of Ca2+ to trigger the contraction of cardiac myocytes than Ca2+ release from the sarcoplasmic reticulum (19, 20). Indeed, the blockade of ICa,L by verapamil and diltiazem has been reported to produce more potent negative inotropic action in the neonatal rabbit hearts than in the adult rabbit hearts (22). However, the precise mechanism mediating the more marked inhibitory effect of verapamil on cardiac contractile function in the immature heart than that of dihydropyridine and benzothiazepine classes of L-type Ca2+ channel antagonists has yet to be fully elucidated.

The present experiments using a mouse heart model revealed that, although the sensitivity of ICa,L to inhibition by nifedipine or diltiazem are similar at all developmental stages, the sensitivity of ICa,L to verapamil is higher in mice of ≤21 days of age than in those of ≥28 days of age (Figure 4). Thus, this study showed for the first time that postnatal developmental changes in the sensitivity of ICa,L are present in verapamil, but not in nifedipine or diltiazem. In the clinical setting, the maximum concentration of free verapamil in the serum has been reported to be approximately 80 nM (36), where a substantial difference in the degree of ICa,L inhibition was observed between the neonatal and adult ventricular myocytes (~60% and 40% inhibition in day 0 and 10–15-week mice, respectively; Figure 4a). This difference might be involved in the differential sensitivity of ventricular contractile function to verapamil between neonates and adults.

Four pore-forming α subunits of ICa,L channel have been identified, namely, CaV1.1 (α1S), CaV1.2 (α1C), CaV1.3 (α1D), and CaV1.4 (α1F) (1, 2). Among these, CaV1.2 and CaV1.3 channels underlie ICa,L in mammalian ventricles (34). It has been shown in mouse ventricular myocytes that CaV1.3 channel is functionally expressed at the early embryonic stages; however, it is gradually downregulated during the second-half of the embryonic stage, leading to a negligibly low expression level at 18 days post coitum (34). In contrast, the expression of CaV1.2 channel is gradually increased throughout the embryonic stages (34, 37), and hence CaV1.2 channel becomes the predominant isoform at 18 days post coitum, which continues thereafter (34). In the present study, Western blotting and immunocytochemistry revealed that CaV1.2 channel is expressed in sarcomeres in both day 7 and 10–15-week ventricular myocytes (Figure 6a), supporting the view that the CaV1.2 channel is functionally expressed in the ventricular myocytes of both ages.

On the other hand, although the expression of CaV1.3 channel in day 7 ventricular myocytes was detected by Western blotting (Figure 6b), the CaV1.3 channel was found to be mainly localized in the perinuclear region, and was scarcely detected in the plasma membrane by immunocytochemistry (Figure 6d). It therefore seems likely that the functional expression of CaV1.3 channel is minimal in day 7 mouse ventricular myocytes. Consistent with a previous study using mouse ventricular myocytes (34), the expression of CaV1.3 channel was not detected in 10–15-week ventricular myocytes by either Western blotting or immunocytochemistry (Figure 6b). Taken together, it seems reasonable and plausible to assume that ICa,L recorded from mouse ventricular myocytes in various postnatal stages in the present study was primarily based on CaV1.2 channel rather than CaV1.3 channel.

Previous studies have shown that CaV1.3 channel activates at a more negative membrane potential than CaV1.2 channel, and exhibits less sensitivity to dihydropyridines than CaV1.2 channel (38, 39). The present study confirms that there were no significant differences in both the voltage dependence of ICa,L activation (Figure 2d–f) or the sensitivity of ICa,L to nifedipine (Figure 4b), among all developmental stages. These findings also appear to be consistent with the view that CaV1.2 channel primarily contributes to the functional ICa,L in the mouse ventricle after birth.

The precise mechanisms underlying the postnatal developmental changes in the sensitivity of ICa,L to inhibition by verapamil remain unclear. ICa,L channel is composed of the α1, α2δ, and β subunits in the heart (1, 2). Interestingly, patch-clamp experiments using heterologous expression systems have shown that α1C/β3 channel exhibits higher sensitivity to verapamil but a similar sensitivity to the dihydropyridine isradipine, compared with α1C channel (40), suggesting that the subunit composition of ICa,L channel substantially affects the sensitivity to inhibition by L-type Ca2+ channel antagonists (40, 41). However, because the β3 subunit is only expressed in the heart at the early embryonic stages, and not after birth (42), this possibility may not account for the postnatal developmental changes in the sensitivity of ICa,L to verapamil. Future studies are thus required to elucidate the molecular basis for the postnatal developmental changes in the sensitivity of ICa,L to inhibition by verapamil.

Three classes of L-type Ca2+ channel antagonists are known to exhibit different degrees of frequency-dependent block of ICa,L. Phenylalkylamines, such as verapamil and D600 (methoxy-verapamil), exhibit a marked frequency-dependent block of ICa,L (31, 32, 33), which is assumed to arise from the preferential block of the channel in the open-state (43, 44). In contrast, nifedipine shows tonic block of ICa,L without an appreciable frequency-dependent block of ICa,L (32). Diltiazem shows both tonic and frequency-dependent block of ICa,L (31, 32). In the present study, verapamil and diltiazem (Figure 5c) but not nifedipine (Figure 5d) showed a frequency-dependent blocking action on ICa,L in ventricular myocytes of both day 7 and 10–15-week mice. Given that the heart rate in neonates and infants is considerably higher than that in adults in humans, it is expected that inhibitory action of verapamil and diltiazem on ICa,L becomes more potent in neonates and infants than in adults. Although diltiazem can be safely used in infants in the clinical setting (10), verapamil produces a serious depression in the cardiac function of neonates and infants (4, 6, 7). Thus, it seems likely that the frequency-dependent blocking action on ICa,L could not be a critical factor contraindicating its use in neonates and infants. It is thus expected that the higher sensitivity of ICa,L to verapamil in neonates and infants (in comparison to adults) contributes to the serious depressant effects on the ventricular contractile function.

Limitations of the Study

We may assume that day 0 mice correspond to neonates in humans; days 7, 14, and 21 mice correspond to infants; day 28 mice correspond to children, and 10–15-week mice correspond to adults, based on the weaning age of postnatal day 21 in mice (23). However, the precise correlation in age-dependent development of the heart between mice and human has yet to be fully elucidated.

Conclusion

Our electrophysiological studies using a mouse heart model, have detected, for the first time, a higher sensitivity of ventricular ICa,L to verapamil in neonatal and infant stages than in child and adult stages. This developmental change in the sensitivity of ICa,L to verapamil is at least partly responsible for the verapamil-induced negative inotropic action observed in neonates and infants in the clinical setting.