AJP - Heart Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 290: H2267-H2276, 2006. First published December 9, 2005; doi:10.1152/ajpheart.01093.2005
0363-6135/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/6/H2267    most recent
01093.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Huang, J.
Right arrow Articles by Tibbits, G. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Huang, J.
Right arrow Articles by Tibbits, G. F.

L-type Ca2+ channel function and expression in neonatal rabbit ventricular myocytes

Jingbo Huang,*,1,2 Liqun Xu,*,1,2 Marion Thomas,1 Keith Whitaker,1,2 Leif Hove-Madsen,3 and Glen F. Tibbits1,2

1Cardiac Membrane Research Laboratory, Simon Fraser University, Burnaby, British Columbia; 2Cardiovascular Sciences, Child and Family Research Institute, Vancouver, British Columbia, Canada; and 3Cell Physiology Laboratory, Cardiology, Hospital de Santa Creus y Sant Pau, Barcelona, Spain

Submitted 17 October 2005 ; accepted in final form 30 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
L-type Ca2+ channel-mediated, Ca2+-induced Ca2+ release (CICR) is the dominant mode of excitation-contraction (E-C) coupling in the mature mammalian myocardium but is thought to be absent in the fetal and newborn mammalian myocardium. Furthermore, the characteristics and contributors of E-C coupling at the earliest developmental stages are poorly understood. In this study, we measured [3H](+)PN200-110 dihydropyridine binding capacity, functionality and expression of the L-type Ca2+ channel, and cytosolic [Ca2+] ([Ca2+]i) at various developmental stages (3, 6, 10, 20, and 56 days old) to characterize ontogenetic changes in E-C coupling. We found that 1) the whole cell L-type Ca2+ channel peak current (ICa) density increased slightly in parallel with cell growth, but the current-voltage relationship, the steady-state activation, and the maximum DHP binding and binding affinity did not exhibit significant developmental changes; 2) sarcoplasmic reticulum Ca2+ dependence of inactivation rates of L-type Ca2+ channel and peak of ICa density were only observed after 10 days of age, which temporally coincides with transverse (T)-tubule formation; 3) the relationship between [Ca2+]i and voltage changed from a linear relationship at the earliest developmental stages to a "bell-shaped" relationship at the later developmental stages, presumably corresponding to a switch from reverse-mode Na/Ca exchange-dependent to ICa-dependent E-C coupling; and 4) the expression of two different splice variants of CaV1.2, IVS3A and IVS3B, switched from predominantly IVS3A at the earliest stages to IVS3B at the later developmental stages. Our data suggest that whereas the density of functional dihydropyridine receptors (DHPRs) increases only slightly during ontogeny, the enhancement of functional coupling between DHPR and ryanodine receptor is dramatic between the second and third weeks after birth. Furthermore, we found that the differential expression of splice variants during development temporally correlated with the appearance of ICa-dependent E-C coupling and T-tubule formation.

neonate myocardium; contractility; excitation-contraction coupling; dihydropyridine receptor

CARDIAC EXCITATION-CONTRACTION (E-C) coupling is mediated by a transient increase in cytosolic calcium ([Ca2+]i). It is commonly accepted that the relatively small amount of Ca2+ that enters via the activation of L-type Ca2+ channels [dihydropyridine (DHP) receptors, DHPRs] triggers a larger amount of Ca2+ release from the sarcoplasmic reticulum (SR), in a process known as Ca2+-induced Ca2+ release (CICR) in adult mammalian myocytes (2, 6, 7, 19). L-type Ca2+ channel-mediated CICR depends on both transverse (T) tubules and SR structural and functional maturation (7, 21, 33). Therefore, before the T-SR system reaches its full maturation, immature hearts likely exhibit an alternative means of E-C coupling. There are several studies which demonstrate that E-C coupling in immature cardiomyocytes may not depend on the functional coupling of DHPR and ryanodine receptor (RyR) (13, 16, 25). However, the ages of the rabbits in those studies are fetal, neonatal (1–7 days old), and adult (1.5–3.0 kg). Substantial indirect evidence indicates that the first few weeks postpartum are crucial in myocardial development (10, 27). Our study is the first to collectively investigate postnatal development of Ca+ channel peak current (ICa) in 3- to 56-day-old rabbit ventricular myocytes using the perforated patch-clamp technique. Although the ICa current-voltage (I-V) relationship has been shown to be qualitatively similar in neonatal and mature heart (25, 40), the important relationship between [Ca2+]i and membrane potential (Em) has not been investigated thoroughly in neonates. It is known that the {alpha}1C-subunit of the cardiac L-type Ca2+ channel (CaV1.2) bears the most important known functional characteristics of the channels, including the channel gating properties and the Ca2+ channel antagonist receptors (35). Alternative splicing variants of the third transmembrane segment of the fourth motif (IVS3) of the CaV1.2 have been identified in different species (IVS3A and IVS3B) and tissue types and have shown developmental changes in the rat heart (5, 8, 29, 43). However, the role of this differential expression with ontogeny is not clear.

In the present study, a DHP binding assay, real time PCR of L-type Ca2+ channel splice variants measurement, the whole cell perforated patch-clamp technique, and Ca2+ transient measurements were used in rabbit ventricular myocytes at 3 (3d), 6 (6d), 10 (10d), 20 (20d), and 56 (56d) days of age. We have examined the postnatal changes of ICa and the time course of functional coupling of DHPR and RyR by characterizing the occurrence of SR Ca2+-dependent L-type Ca2+ channel inactivation. Our data suggest that whereas the density of functional DHPR increases slightly during ontogeny, the enhancement of functional coupling between DHPR and RyR is dramatic between the second and third weeks after birth. Furthermore, we observed developmental regulation of splice variants from a dominance of IVS3A at the earliest stages to IVS3B at the later developmental stages. On the one hand, this switch in CaV1.2 isoform expression was not associated with any obvious changes in the I-V plot parameters or activation kinetics of the channel. On the other hand, this differential expression pattern correlated temporally with the appearance and robustness of ICa-dependent E-C coupling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Ventricular homogenate preparation. Ventricular homogenates were prepared from the hearts of New Zealand White rabbits (of either sex) from five distinct age groups: 3d, 6d, 10d, 20d, and 56d postpartum by methods previously described (36).

DHP binding assay. [3H](+)PN200-110 (PN) binding was performed as previously described (36) using ventricular homogenate preparations from four distinct age groups: 3d, 6d, 10d, and 20d (for technical reasons the 56d group was not included). All binding data were analyzed by iterative nonlinear regression and Scatchard analysis using GRAFIT (Erithacus Software) to determine the maximum DHP binding (Bmax) and binding affinity (Kd) values.

Real time PCR of L-type Ca2+ channel splice variants. Tissue (100–200 mg) was taken from the left ventricular homogenate preparations from five distinct age groups: 3d, 6d, 10d, 20d, and 56d. Polyadenylated RNA was immediately isolated from tissue samples using the MicroPoly(A) Pure mRNA purification kit (Ambion, Austin, TX) according to the manufacturer's protocol. Isolated mRNA was reverse transcribed using random primers and Superscript II Reverse Transcriptase (InVitrogen, Carlsbad, CA), ensuring that the final concentration of mRNA was 16.67 ng/µl. Primers and probes for use in real-time PCR reactions were designed using the Primer Express software (Table 1; PE Applied Biosystems, Foster City, CA). In each primer/probe combination, either the primer or the probe spanned the intron/exon boundary to eliminate genomic DNA contamination. PCR reactions were performed using an ABI Prism 7000 Sequence Detection System utilizing TaqMan technology (PE Applied Biosystems). PCR reactions were carried out using 12.5 µl of TaqMan Universal PCR master mix, 2.25 µl of both the forward and reverse primers (900 nM), 0.5 µl of probe (200 nM), and 2.4 µl (40 ng) of cDNA in a total reaction volume of 25 µl. The thermal protocol included an initial hold at 50°C for 2 min and a denaturing step at 95°C for 10 min. Thermal cycling followed, consisting of a denaturing step at 95°C for 15 s and an annealing/extension step at 60°C for 1 min, repeated for 42 cycles. Quantification of relative mRNA transcript levels was obtained by generating a standard curve expressing the threshold cycle values (Ct) as a function of the natural log of the initial cDNA concentration of four 10x serial dilutions. Experimental data points were obtained in triplicate, and mean Ct values were used to determine mRNA expression level from the standard curves. Both A and B variant values were normalized for the internal control beta-actin and arbitrarily expressed as the fraction of the average 6d IVS3B variant value.


View this table:
[in this window]
[in a new window]
  		Table 1. Primer and probe sequences used in real time PCR experiments

 
Isolation of ventricular myocytes. Ventricular myocytes were isolated from the hearts of New Zealand White rabbits (of either sex) from five distinct age groups: 3d, 6d, 10d, 20d, and 56d as described previously (11, 12, 31).

Whole cell perforated patch voltage clamp. Whole cell amphotericin-perforated voltage-clamp technique was used as described previously (11, 12). The internal pipette solution contained (in mM) 110 CsCl, 5 MgATP, 1 MgCl2, 20 tetraethyl ammonium, 5 Na2 phosphocreatine, and 10 HEPES, and pH was adjusted to 7.1 with CsOH. The standard external solution contained (in mM) 130 NaCl, 5 CsCl, 1 MgCl2, 2.0 CaCl2, 5 Na-pyruvate, 10 glucose, and 10 HEPES, and pH was adjusted to 7.4 with NaOH. The replacement of K+ with Cs+ and the addition of tetraethyl ammonium were used to eliminate K+ currents.

The voltage dependence of ICa (I-V relationship) was determined by depolarizing the cell to 12 different potentials (from –50 to +60 mV in increments of 10 mV) for 400 ms with a predepolarization step to –40 mV for 50 ms from a holding potential of –80 mV to inactivate Na+ and T-type Ca2+ channels. Peak ICa was determined as the difference between its peak inward current and baseline current at the end of depolarization. {tau}f and {tau}s, the fast and slow components of ICa inactivation time constants, respectively, were obtained by fitting the current decay with a second-order exponential equation (R2 > 0.99) using the least squares method (CLAMPFIT curve-fitting algorithm). The maximum ICa of steady-state activation curve was extrapolated from the linear portion of the I-V relationship. The data were well-fit with the Boltzmann equation (R2 > 0.95).

Measurement of Ca2+-dependent fluo-3 fluorescence. The [Ca2+]i was measured with the fluorescent Ca2+ indicator fluo-3-acetoxymethyl ester as described previously (11, 12). F0 was the difference in the background fluorescence determined in the absence and presence of a cell in the area of measurement. {Delta}F was the increment measured from baseline or the background fluorescence in the presence of a cell in the area of measurement.

Data and statistical analyses. Data are presented as means ± SE. Statistical significance of the results was tested using a one-way ANOVA (SPSS 11.0) or Student's t-test for paired or unpaired samples. Post hoc tests were taken using Tukey multiple comparisons. A P value of ≤0.05 was taken to be significant.

Materials. All reagents used were of the highest purity available and purchased from Sigma Chemicals (St. Louis, MO) unless specified otherwise. Radioisotopes were purchased from New England Nuclear (St. Laurent, QC, Canada).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Developmental changes in body weight and cell membrane surface area. As expected and shown in Table 2, body weight and total cell membrane capacitance (Cm), and therefore cell surface area, all increased significantly with age. Body weight and Cm increased ~20-fold and 4.5-fold, respectively, from 3d to 56d.


View this table:
[in this window]
[in a new window]
  		Table 2. Developmental changes in rabbit ventricular myocyte and ICa parameters

 
ICa and DHPR densities do not undergo significant developmental changes. Figure 1A shows representative ICa traces induced by depolarizations to –40, –10, +10, and +30 mV in both 3d and 56d groups. Inward ICa achieved a maximum value at +10 mV for both 3d and 56d. Figure 1B shows the ICa I-V relationship with ICa expressed as peak ICa (left), ICa density (right), and with the peak value taken as unity (bottom) as a function of age. I-V plots were qualitatively similar for all age groups. There were no significant voltage shifts in the peak current or the reversal potential. Furthermore, the steady-state activation curve of ICa did not show significant shifts as a function of age. Differences in the values of V1/2 (the Em producing half-maximum activation) and K (a measure of the steepness of the voltage dependence of the activation curve) did not reach a level of significance between any age groups (Table 2). Figure 1C shows Bmax (left) and Kd values (right) of DHP binding as a function of age derived from nonlinear regression analysis of individual binding curves. There were no significant differences between any of the age groups.


Figure 1
View larger version (16K):
[in this window]
[in a new window]
  		Fig. 1. Ca2+ current (ICa) current-voltage (I-V) plot and dihydropyridine (DHP) binding during development. A: representative ICa traces induced by depolarizations of –40, –10, +10, and +30 mV (as indicated) in both 3 days (3d) and 56 days (56d), respectively. B: "bell-shaped" voltage dependence of ICa as a function of age, expressed as three different parameters: peak of ICa (left), peak of ICa density (right), and ICa normalized by peak of ICa (bottom). There were no significant voltage shifts in the I-V relationships among all age groups. C: maximal binding (Bmax) and affinity binding (Kd) values of DHP binding as a function of age. There were no significant differences between any of the age groups. n of A and B is equal to that shown in Table 2; n of C equals 9, 13, 16, and 9 for 3d, 6d, 10d and 20d, respectively.

 
Voltage dependence and kinetics of [Ca2+]i change from NCX to ICa dependency with age. Figure 2A shows representative Ca2+ transient traces induced by 12 different depolarizations (when using the same protocol as in Fig. 1) both with and without 15 µM of the L-type Ca2+ channel blocker nifedipine (Nif) for both 3d and 56d groups. This dose of Nif was able to block ICa (~98%) in all age groups (data not shown). Figure 2B shows the voltage dependence of peak [Ca2+]i as a function of age (3d and 56d) in CON, Nif, and Nif sensitive, the latter of which was obtained by subtraction of the Ca2+ transient with Nif from the CON. Ca2+ transients increased linearly as a function of Em in 3d in both CON and Nif solutions. In contrast, the Ca2+ transient magnitude reached a peak at +10 mV and then decreased with further depolarization (up to +50 mV) in 56d in CON solution. In the 56d group, Nif significantly reduced the magnitude of the Ca2+ transients at all potentials, and the relationship between Em and [Ca2+]i became linear over the range of +10 to +50 mV. The Nif-resistant Ca2+ transients were abolished in both 3d and 56d by the addition of 10 µM KB-R 7943 (KB-R), which blocks reverse-mode NCX (data not shown). The slopes of the regression line ({Delta}F/F0/mV) of the Ca2+ transients in the presence of Nif were 0.009 and 0.180, respectively for 56d and 3d. Furthermore, the regression line in 56d was shifted to more positive potentials compared with that in 3d. However, a "bell-shaped" voltage dependence of peak ICa density was observed for both 3d and 56d groups in CON solution (Fig. 2C), in contrast to that observed for the voltage dependence of peak [Ca2+]i.


Figure 2
View larger version (18K):
[in this window]
[in a new window]
  		Fig. 2. Voltage dependence of intracellular Ca2+ concentration ([Ca2+]i) as a function of age. A: representative Ca2+ transient traces induced by the same depolarizations as those employed in Fig. 1 in both control solution (CON) and the presence of 15 µM nifedipine (Nif) in 3d and 56d, respectively. Ca2+ transients increased in magnitude monotonically over more positive depolarizations in 3d. In contrast, Ca2+ transients reached a peak at a depolarization of +10 mV and then decreased in response to more positive depolarizations (up to +50 mV). B: peak of CON, Nif, and Nif-sensitive Ca2+ transients as a function of voltage for 3d and 56d, respectively. A linear [Ca2+]i-V relationship was observed for both Nif and CON Ca2+ transients in 3d. However, a "bell shape" was present in CON Ca2+ transient and replaced by a linear relationship in Nif Ca2+ transients in 56d. Slopes of regression line ({Delta}F/F0/mV) of the Nif Ca2+ transients were 0.009 and 0.18, for 56d and 3d, respectively. The "bell-shaped" [Ca2+]i-V corresponding to I-V relationship was observed in the Nif-sensitive group in 56d. C: qualitatively similar "bell-shaped" voltage dependence of peak ICa density were observed in both 3d and 56d under control conditions. n = 8.

 
Ca2+ transients in response to +10 and +60 mV depolarizations were used as reporters on the relative role of ICa- and NCX-dependent E-C coupling in the age groups 3d and 56d. In these studies, the time-to-peak (TTP) of the Ca2+ transients generated in response to these two depolarizations was used to characterize E-C coupling changes with ontogeny.

Figure 3A shows representative Ca2+ transients at depolarizations to +10 and +60 mV for both 3d and 56d in the CON group. TTP of the Ca2+ transient was measured from the starting point of upstroke to the peak [Ca2+]i. When depolarized to +10 mV, the peak of the Ca2+ transient occurred significantly earlier in 56d (TTP ~100 ms) compared with that in 3d (TTP ~400 ms). However, Ca2+ transient peaks were observed at about the same time for both 3d and 56d (TTP ~400 ms) when depolarized to +60 mV. Noticeably, a smaller and more rapid Ca2+ transient rise (indicated as a shoulder) was observed at +10 mV in 3d. The TPP of the shoulder (~100 ms) was similar to that observed at +10 mV in 56d. Both the shoulder of the Ca2+ transient in 3d and the peak of Ca2+ transients in 56d at +10 mV were abolished in the presence of Nif; in contrast, the peak of Ca2+ transients at +10 mV in 3d and at +60 mV in both 3d and 56d were unaffected in the presence of Nif but were abolished by the addition of KB-R (Fig. 2). Figure 3B shows TTP as a function of age in response to depolarization of both +10 and +60 mV. TTP in response to depolarization to +10 mV significantly decreased with age, whereas those at +60 mV did not.


Figure 3
View larger version (14K):
[in this window]
[in a new window]
  		Fig. 3. Time to peak of Ca2+ transient (TTP) as a function of age and voltage. A: representative Ca2+ transient traces at depolarizations of +10 (solid lines) and +60 mV (shaded lines) for both 3d and 56d, respectively. In 56d, there was a rapid upstroke of the Ca2+ transient at a depolarization of +10 mV compared with that of +60 mV. A smaller, but more rapid, Ca2+ transient rise (indicated as "shoulder") compared with the peak observed at a depolarization of +10 mV in 3d. B: TTP as a function of age for depolarizations of +10 (solid bars) and +60 (shaded bars) mV, respectively. There were significant differences between either 3d or 10d vs. either 20d or 56d (***P < 0.005). Difference in TTP observed at a depolarization of + 10 mV was not observed when depolarized to +60 mV. n = 12.

 
SR Ca2+-dependent modulation of ICa amplitude and kinetics increases with maturation. To investigate the developmental changes of SR Ca2+-dependent modulation of ICa or the "back talk" between the DHPR and RyR, ICa amplitude and its inactivation kinetics were measured with SR Ca2+ load under both steady-state and Ca2+-depleted states. To do this, the SR Ca2+ was first cleared by a rapid application of 10 mM caffeine. A train of 20 repetitive depolarizations at 0.12 Hz to +10 mV for 400 ms (including a predepolarization step to –40 mV for 50 ms from a holding potential of –80 mV) was then applied. The 20th and 1st depolarizations were considered representative of the steady state and the SR Ca2+-depleted state, respectively. Figure 4A shows representative traces of ICa and the corresponding Ca2+ transients triggered by the 1st and 20th depolarizations in a 3d and a 56d myocyte. Ca2+ transient peaks were smaller for both the 3d (~35%) and the 56d (~25%) myocytes in the 1st compared with those observed in response to the 20th depolarization. However, ICa was smaller in the 20th compared with that in the 1st depolarization in 56d, consistent with SR Ca2+ dependence of ICa, which was not observed in 3d. As shown in Fig. 4B, the SR Ca2+ dependence of ICa was observed after 10d. Although the peak of ICa in both the 1st and 20th depolarizations increased significantly with age (Table 2), its density (normalized by cell capacitance, pF) exhibited a much smaller increase with ontogeny (Fig. 4B). A significant increase in peak ICa density from 3d–6d to 20d–56d was observed compared with that of the 1st depolarization but not with that of the 20th. The peak ICa density in response to the 20th depolarization was smaller than that in response to the 1st depolarization but only in the 10d, 20d, and 56d groups. Figure 4C shows the ICa inactivation time constants as a function of age for both the 1st and 20th depolarizations. The 20th {tau}f and {tau}s in 56d were significantly faster than that in other age groups. In 56d myocytes, inactivation of the steady-state ICa was significantly faster than that in response to the 1st depolarization, suggestive of SR Ca2+-dependent inactivation of ICa. In 20d, only {tau}f showed a SR Ca2+ dependence. SR Ca2+ dependence on the inactivation kinetics of ICa was not observed in any other age group.


Figure 4
View larger version (17K):
[in this window]
[in a new window]
  		Fig. 4. Inactivation time constants ({tau}) and peak ICa density as a function of age. A: representative traces of ICa and its corresponding Ca2+ transient triggered by the 1st (shaded line) and 20th (solid line) depolarizations (from –40 to +10 mV for 400 ms at 0.12 Hz) for 3d and 56d, respectively. {tau}f and {tau}s are indicated by arrows. Sarcoplasmic reticulum (SR) Ca2+ dependence of ICa inactivation constants was observed in 56d but not in 3d. B: ICa peak density (normalized by cell capacitance, pF) as a function of age. The 1st ICa (shaded bars) in either 20d (*P < 0.05) or 56d (**P < 0.01) was significantly greater than that in either 3d or 6d; however, the magnitude of ICa density for 20th ICa (solid bars) was not significant between the groups. SR Ca2+ dependence of ICa was observed among 10d ({dagger}P < 0.05), 20d, and 56d groups ({dagger}{dagger}P < 0.01). C: ICa inactivation time constants for depolarizations of 1st (shaded bars and shaded hatched bars for {tau}f and {tau}s, respectively) and the 20th (solid bars and hatched bars for {tau}f and {tau}s, respectively) as a function of age. The 20th {tau}f and {tau}s in 56d were significantly faster compared with other groups (**P < 0.01 and *P < 0.05, respectively). SR Ca2+-dependent {tau}f was observed in both 20d and 56d ({dagger}P < 0.05), and SR Ca2+-dependent {tau}s was observed only in 56d ({dagger}P < 0.05). n values are shown in Table 2.

 
L-type Ca2+ channel splice variant expression changes with ontogeny. Because developmentally regulated specific splice variant expression of CaV1.2 during ontogeny has been reported in the murine heart, the possibility of their existence in the rabbit heart as well as the correlation between the splice variant expression and functionality of CaV1.2 during the development were examined.

Two different splice variants of the IVS3 region of CaV1.2 subunit of the L-type Ca2+ channel were found in the rabbit ventricle, and they exhibited a high degree of identity with those found previously (5) in the rat heart. The sequences of the two splice variants were as follows:

Formula
where U1 represents the last five amino acids of the first exon upstream of the splice variant exon; D1 represents the first four amino acids of the first exon downstream of the splice variant exon and the symbols : and . represent amino acid identity and a conservative substitution, respectively.

Relative splice variant expression levels as a function of age are shown in Fig. 5. The IVS3A variant transcript levels remained approximately constant throughout neonatal development up to 56 days of age. In contrast, the IVS3B variant significantly increased with age. In both 3d and 6d, the IVS3A variant was dominant, with an expression level 1.8-fold greater than IVS3B. The period around 10d appears to represent a transition in isoform dominance because relatively equal amounts of the IVS3A and IVS3B variants were expressed in this developmental stage. The expression of IVS3B variant was approximately twofold greater than the IVS3A variant in 56d.


Figure 5
View larger version (9K):
[in this window]
[in a new window]
  		Fig. 5. Expression of IVS3A and IVS3B variants as a function of age. IVS3A variant transcript levels remained approximately constant throughout neonatal development up to 56 days of age. In contrast, IVS3B variant transcript levels significantly increased with age. In 56d group, IVS3B variant level was statistically higher than that of any other age group (*P < 0.05). In addition, IVS3B variant expression in 20d was significantly higher than that of the 6d (*P < 0.05). Ratio of IVS3A over IVS3B (A/B, open bars) shows a significant decrease with age (56d vs. either 3d or 6d, {dagger}{dagger}{dagger}P < 0.001). n: 11, 12, 11, 10, and 11 for 3d, 6d, 10d, 20d, and 56d, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Functionality of L-type Ca2+ channels during development. The peak of the absolute ICa increased with age (Table 2) and was 6.5- and 5.2-fold greater in 56d compared with that in 3d in a SR Ca2+-depleted state and steady state, respectively. However, the increase in ICa density (normalized by Cm) with ontogeny was substantially smaller in magnitude (Fig. 1B and Fig. 4B) because Cm predictably increased with maturation (4.5-fold greater in 56 than in 3d). Therefore, the increase in peak ICa during development is mainly attributed to the increase in total cell surface area due to cellular hypertrophy and T-tubule appearance. In rat ventricular myocytes, two groups have found that the peak of ICa density was fairly constant from 1 day to 150 days of age (15, 38). However, in rabbit ventricular myocytes, various results have been observed. A developmental increase of peak ICa density from 1.3- to 3-fold has been reported (13, 25, 40). Our results (1.3-fold) are similar to those determined by Osaka and Joyner (25) and Wetzel et al. (40) (1.7- and 1.3-fold, respectively). The ICa peak density (3.3–3.5 pA/pF) in 3d–6d observed in our study is slightly higher than the 2.6 pA/pF reported by Osaka and Joyner (26) but lower than the 4.3–5.6 pA/pF reported in 1- to 5-day-old rabbit ventricular myocytes reported by other investigators (13, 25). The differences are likely due to variations in species, cell preparations, experimental techniques, and conditions used.

Furthermore, qualitatively the "bell-shaped" I-V relationship and activation parameters (V1/2 and K) of ICa as well as DHP binding did not change with age (Fig. 1), which is consistent with other reports in different species (13, 14, 25, 40). These findings suggest that there were no significant developmental changes in the functionality of ICa.

Voltage dependence and kinetics of [Ca2+]i during development. In contrast to the well-conserved "bell-shaped" ICa I-V relationship observed during development, there is a dearth of knowledge on the relationship between either the Ca2+ transient or cell contraction with voltage during development. There are a number of findings in adult cardiomyocytes that support the "bell-shaped" contraction-voltage relationship (2, 19). However, some investigators have reported that both contractions or Ca2+ transients do not decline as much at positive potentials as does ICa under certain experimental conditions, particularly with an increased intracellular [Na+] (17, 18, 24). The present study supports the "bell-shaped" relationship in 56d and further demonstrated a linear relationship in 3d between the Ca2+ transient and voltage (Fig. 2).

Furthermore, the peak of the Nif-sensitive [Ca2+]i transient appeared earlier than that of the KB-R-sensitive transient (Fig. 3). Therefore, the kinetics of the Ca2+ transients (TTP) correlated with different modes of E-C coupling, with ICa-dependent transients exhibiting a more rapid rise (~110 ms), and reverse-mode NCX-dependent transients were associated with a slower rise (~400 ms). The shortened TTP of the ICa-dependent Ca2+ transient is likely related to its greater and faster Ca2+ influx rate and synchronized ICa-mediated CICR due to well-developed T tubules in the mature cells. One would predict, therefore, that the ICa-dependent Ca2+ transient would not be reduced by a shortening of the action potential duration (APD) to ~200 ms (30). The prolonged TTP of reverse-mode NCX-dependent Ca2+ transient is likely to be associated with its slower Ca2+ influx rate and consequently its slower termination. Recently, it has been reported that a more profound impact of APD on Ca2+ transients was observed in neonate cardiomyocytes compared with that in adult cardiomyocytes (9). Sipido et al. (34) reported different TTPs in adult guinea pig ventricular myocytes, 120 ms and 225 ms for depolarizations of 225 ms for nisoldipine-sensitive and nisoldipine-resistant Ca2+ transients, respectively, which are in accordance with our results (110 and 400 ms for a duration of 400-ms depolarization for Nif-sensitive and Nif-resistant, respectively). The present study gives the first evidence that the kinetic changes of the Ca2+ transients reflect developmental changes in E-C coupling, from reverse-mode NCX-dependent to ICa-dependent mechanisms.

"Forward talk" between DHPR and RyR during development. The Ca2+ transient in 56d was sensitive to Nif (Fig. 2) as well as SR Ca2+ (Fig. 4A), indicating the presence of ICa-induced Ca2+ release or "forward talk" between DHPR and RyR. In contrast, both our study and that of others (41) have shown that Nif has very little reduction in the peak of the Ca2+ transient or cell contraction in the neonatal heart. However, the apparent lack of ICa-mediated CICR in the neonate is difficult to reconcile with the following observations: 1) ICa peak density under steady-state conditions was not significantly different from the adult; 2) there is likely a higher surface-to-volume ratio present in the neonate myocyte compared with that of the adult (10); and 3) the SR Ca2+ load (normalized to surface area) in the neonate cardiomyocytes is at least comparable to that in adults (11, 12). There are several possible explanations for this discrepancy. One is that the contribution of ICa to the rise in [Ca2+]i is underestimated by the determination of the peak Ca2+ transient or maximum contraction amplitude. As shown in Fig. 3A, the Nif-sensitive "shoulder" observed at +10 mV in 3d was ~28% of the peak Ca2+ transient; however, the Nif-sensitive Ca2+ transient peak was <10% of the peak Ca2+ transient. The other and arguably the more important postulate is that the DHPR and RyR are not functionally coupled in the earliest developmental stages. We hypothesize, therefore, that the role of ICa in E-C coupling is greatly diminished in neonate compared with that in adult due to the lack of functional DHPR-RyR coupling.

"Back talk" between DHPR and RyR during development. The reduction in the rate of ICa inactivation observed in the SR Ca2+-depleted state produced by either caffeine or RyR has been reported by other investigators in adult dog, rat, and mouse cardiac myocytes (20, 33, 37). SR Ca2+-dependent developmental time course changes in ICa inactivation kinetics have been reported in rat ventricular myocytes (15, 38). In rabbit ventricular myocytes, Wetzel et al. (40) found the time courses of inactivation of ICa were not significantly different from day 21 of gestation to adult ({tau}f were 16 and 17 ms and {tau}s were 81, 70 ms for adult and 2–5d, respectively); in contrast, Osaka and Joyner (25) have shown developmental changes in ICa inactivation. The values obtained in the present study ({tau}f of 13 and 19 ms and {tau}s of 60 and 95 ms for 56d and 3d, respectively) are close to those reported by Osaka and Joyner but are much greater than those reported in rat ventricular myocytes ({tau}f of 5.9–6.6 and 4.5–10.7 ms and {tau}s of 17.0–21, 18.0–27.1 ms for adult and 1–7d, respectively) and guinea pig ventricular myocytes ({tau}f of 7.6 and 5.2 ms and {tau}s of 7.6, 5.2 ms for adult and 1–5d, respectively) (14). Besides the species differences, the dissimilar techniques and experiment conditions undoubtedly contributed to the differences in the time courses of ICa inactivation as well.

It has been shown that SR Ca2+-dependent inactivation of ICa or "back talk" is effective within a microdomain because it has been shown that the application of the Ca2+ buffers, EGTA, or BAPTA used at concentrations that abolished cell contraction had no apparent effect on its fast and slow inactivation kinetics (33). From these and other data it has been calculated that the L-type Ca2+ channel inactivation sites and RyRs are within 10–20 nm of each other, a spatial proximity that is fundamental for the functional coupling of DHPR and RyR (3, 33). The "back talk" became apparent after 10d, which temporally correlates with the formation of T tubules and the appearance of "forward talk" (Figs. 2 and 4). The lack of "forward talk" and "back talk" at the earliest developmental stages (Fig. 4C) suggests that DHPR and RyR are not within the same restricted microdomain, which is consistent with other reports (16, 22). Our previous work has demonstrated that there is a much greater amount of Ca2+ stored in the SR in the earliest developmental stages (~3-fold greater in 3d compared with that in 56) (11, 12) than previously thought. Recently, it has been demonstrated that RyR isolated from neonate hearts have gating properties in planar lipid bilayers similar to those from adults (28). Therefore, the lack of DHPR and RyR functional coupling in the neonatal heart is not due to the lack of SR Ca2+ and/or a higher threshold for RyR opening but is likely due to the DHPR and RyR not being within 10–20 nm of each other. We have investigated the developmental changes of the colocalization between the two proteins using double-labeling immunofluorescence and confocal laser scanning microscopy (31, 32). We found that even as early as 3d there was a certain degree of colocalization of DHPRs and RyRs (43% in 3d compared with 79% in 20d after deconvolution). However, it should be noted that even with deconvolution of the confocal images, the microscope optics limit the resolution of colocalization at a voxel size of approximately 100 x 100 x 250 nm (x, y, z). Therefore, colocalization of DHPR and RyR within the same voxel does not ensure functional coupling between these proteins. The [Ca2+]i-dependent inactivation of ICa might be a better indicator of physical proximity between the DHPRs and RyRs compared with the double immunolabeling. Furthermore, the time course of the appearance of the adult-dominant splice variant (IVS3B) (Fig. 5) fits well with the appearance of DHPR and RyR functional coupling in the present study and the advent of T tubules (Figs. 2 and 4). However, because of the high degree of identity between the two splice variants and the lack of unique epitopes, we were not able to generate splice variant-specific antibodies. Thus it was not possible to determine whether the IVS3B variant is preferentially expressed in the T tubules, although this remains an intriguing hypothesis.

L-type Ca2+ channel splice variants and age-dependent differential expression. Diebold et al. (5) found that the neonate (1d) rat heart expressed both IVS3A and IVS3B splice variants of CaV1.2, whereas the adult rat heart expressed only the IVS3B variant (5); interestingly, adult rats have been shown to revert back to a neonatal (IVS3A dominant) phenotype following myocardial infarction (8). In the adult human heart, the IVS3A variant is dominant compared with IVS3B variant; however, the IVS3B variant was demonstrated to be dominant in failing human heart tissue (42). In the present study, isoform switching occurred predominately ~10d, from a preponderance of IVS3A at the earliest stages to IVS3B at the later developmental stages (Fig. 5).

Although the presence of these variants in various species and tissue types has been known for some time, there is still confusion surrounding their role in developmental processes. With respect to the L-type calcium channel current, the following parameters were not different as a function of developmental stage in the present study: Bmax and Kd of DHP binding, voltage at peak current, the general shape of the I-V plot, and the steady-state voltage-dependent activation kinetics (Fig. 1). In addition, electrophysiological data collected on these two variants demonstrated Ba2+ currents with similar voltage ranges of activation and inactivation (44). For these reasons it is unlikely that this region of the channel directly modulates the electrophysiological properties; however, the splice variants could confer other properties (e.g., differences in protein-protein interactions), which may not be reflected in the electrophysiological differences but could determine, for example, molecular localization within the cell. For instance, the presence of a potential caveolin-binding motif as defined by Couet et al. (4) in the IVS3 region (amino acids 1308–1316) may allow for interaction with caveolin 3, a protein associated with caveolae and membrane trafficking processes. Furthermore, we speculate that the different isoforms might be associated with distinct spatial distributions of L-type Ca2+ channels or IVS3A might be favorably expressed on the sarcolemma and IVS3B might be expressed preferentially in the T tubules. Therefore, the isoform switching from adult form to neonatal form in response to pathological conditions imply that the IVS3 variant might result in a downregulation of ICa-mediated E-C coupling. These possibilities, however, need to be explored further.

In conclusion, our results show although the density of functional DHPRs increased slightly during ontogeny, the enhancement of functional coupling between DHPR and RyR is dramatic between the second and third weeks after birth. Furthermore, the time course of isoform switching was correlated with the appearance of ICa-mediated E-C coupling and T-tubule formation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The generous support of the Canadian Institutes of Health Research and the Heart and Stroke Foundation of BC and Yukon to GFT is gratefully acknowledged. J. Huang is the beneficiary of a Canada Research Scholarship from the Canadian Institutes of Health Research. L. Hove-Madsen is the holder of a Ramon y Cajal grant from the Spanish Ministry of Science and Technology, and G. F. Tibbits is the recipient of a Tier I Canada Research Chair.

Present address of L. Xu: Ottawa Heart Institute, Ottawa, ON, Canada. Present address of M. Thomas: University of Oslo, Oslo, Norway.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. F. Tibbits, Cardiac Membrane Research Laboratory, Simon Fraser Univ., Burnaby, BC, V5A 1S6 Canada (e-mail: tibbits{at}sfu.ca)

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.

* J. Huang and L. Xu contributed equally to this study. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Bers DM and Peskoff A. Diffusion around a cardiac calcium channel and the role of surface bound calcium. Biophys J 59: 703–721, 1991.[Web of Science][Medline]
  2. Beuckelmann DJ and Wier WG. Mechanism of release of calcium from sarcoplasmic reticulum of guinea-pig cardiac cells. J Physiol 405: 233–255, 1988.[Abstract/Free Full Text]
  3. Carl SL, Felix K, Caswell AH, Brandt NR, Ball WJ Jr, Vaghy PL, Meissner G, and Ferguson DG. Immunolocalization of sarcolemmal dihydropyridine receptor and sarcoplasmic reticular triadin and ryanodine receptor in rabbit ventricle and atrium. J Cell Biol 129: 672–682, 1995.[Medline]
  4. Couet J, Li S, Okamoto T, Ikezu T, and Lisanti MP. Identification of peptide and protein ligands for the caveolin-scaffolding domain. Implications for the interaction of caveolin with caveolae-associated proteins. J Biol Chem 272: 6525–6533, 1997.[Abstract/Free Full Text]
  5. Diebold RJ, Koch WJ, Ellinor PT, Wang JJ, Muthuchamy M, Wieczorek DF, and Schwartz A. Mutually exclusive exon splicing of the cardiac calcium channel alpha 1 subunit gene generates developmentally regulated isoforms in the rat heart. Proc Natl Acad Sci USA 89: 1497–1501, 1992.[Abstract/Free Full Text]
  6. DuBell WH and Houser SR. Voltage and beat dependence of Ca2+ transient in feline ventricular myocytes. Am J Physiol Heart Circ Physiol 257: H746–H759, 1989.[Abstract/Free Full Text]
  7. Fabiato A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol Cell Physiol 245: C1–C14, 1983.[Abstract/Free Full Text]
  8. Gidh-Jain M, Huang B, Jain P, Battula V, and el-Sherif N. Reemergence of the fetal pattern of L-type calcium channel gene expression in non infarcted myocardium during left ventricular remodeling. Biochem Biophys Res Commun 216: 892–897, 1995.[CrossRef][Web of Science][Medline]
  9. Go A, Srivastava S, Collis L, Coetzee WA, and Artman M. Negative inotropic effect of nifedipine in the immature rabbit heart is due to shortening of the action potential. Pediatr Res 57: 399–403, 2005.[CrossRef][Web of Science][Medline]
  10. Hoerter J, Mazet F, and Vassort G. Perinatal growth of the rabbit cardiac cell: possible implications for the mechanism of relaxation. J Mol Cell Cardiol 13: 725–740, 1981.[CrossRef][Web of Science][Medline]
  11. Huang J, Hove-Madsen L, and Tibbits GF. Na+/Ca2+ exchange activity in neonatal rabbit ventricular myocytes. Am J Physiol Cell Physiol 288: C195–C203, 2005.[Abstract/Free Full Text]
  12. Huang J, van Breemen C, Hove-Madsen L, and Tibbits GF. Store-operated Ca2+ entry modulates SR Ca2+ loading in neonatal rabbit cardiac ventricular myocytes (Abstract). Am J Physiol Cell Physiol 86: 384a, 2004.
  13. Huynh TV, Chen F, Wetzel GT, Friedman WF, and Klitzner TS. Developmental changes in membrane Ca2+ and K+ currents in fetal, neonatal, and adult rabbit ventricular myocytes. Circ Res 70: 508–515, 1992.[Abstract/Free Full Text]
  14. Kato Y, Masumiya H, Agata N, Tanaka H, and Shigenobu K. Developmental changes in action potential and membrane currents in fetal, neonatal and adult guinea-pig ventricular myocytes. J Mol Cell Cardiol 28: 1515–1522, 1996.[CrossRef][Web of Science][Medline]
  15. Katsube Y, Yokoshiki H, Nguyen L, Yamamoto M, and Sperelakis N. L-type Ca2+ currents in ventricular myocytes from neonatal and adult rats. Can J Physiol Pharmacol 76: 873–881, 1998.[CrossRef][Web of Science][Medline]
  16. Klitzner TS. Maturational changes in excitation-contraction coupling in mammalian myocardium. J Am Coll Cardiol 17: 218–225, 1991.[Abstract]
  17. Levi AJ, Li J, Litwin SE, and Spitzer KW. Effect of internal sodium and cellular calcium load on voltage-dependence of the Indo-1 transient in guinea-pig ventricular myocytes. Cardiovasc Res 32: 534–550, 1996.[CrossRef][Web of Science][Medline]
  18. Litwin SE, Li J, and Bridge JH. Na+-Ca2+ exchange and the trigger for sarcoplasmic reticulum Ca2+ release: studies in adult rabbit ventricular myocytes. Biophys J 75: 359–371, 1998.[Web of Science][Medline]
  19. London B and Krueger JW. Contraction in voltage-clamped, internally perfused single heart cells. J Gen Physiol 88: 475–505, 1986.[Abstract/Free Full Text]
  20. Masaki H, Sako H, Kadambi VJ, Sato Y, Kranias EG, and Yatani A. Overexpression of phospholamban alters inactivation kinetics of L-type Ca2+ channel currents in mouse atrial myocytes. J Mol Cell Cardiol 30: 317–325, 1998.[CrossRef][Web of Science][Medline]
  21. Morad M. Signaling of Ca2+ release and contraction in cardiac myocytes. Adv Exp Med Biol 382: 89–96, 1995.[Medline]
  22. Nakanishi T, Okuda H, Kamata K, Abe K, Sekiguchi M, and Takao A. Development of myocardial contractile system in the fetal rabbit. Pediatr Res 22: 201–207, 1987.[Web of Science][Medline]
  23. Nascimento JH. Electrophysiological properties of L-type Ca2+ currents in isolated adult mouse ventricular myocytes. Braz J Med Biol Res 29: 1397–1405, 1996.[Web of Science][Medline]
  24. Nuss HB and Houser SR. Sodium-calcium exchange-mediated contractions in feline ventricular myocytes. Am J Physiol Heart Circ Physiol 263: H1161–H1169, 1992.[Abstract/Free Full Text]
  25. Osaka T and Joyner RW. Developmental changes in calcium currents of rabbit ventricular cells. Circ Res 68: 788–796, 1991.[Abstract/Free Full Text]
  26. Osaka T and Joyner RW. Developmental changes in the beta-adrenergic modulation of calcium currents in rabbit ventricular cells. Circ Res 70: 104–115, 1992.[Abstract/Free Full Text]
  27. Page E and Buecker JL. Development of dyadic junctional complexes between sarcoplasmic reticulum and plasmalemma in rabbit left ventricular myocardial cells. Morphometric analysis. Circ Res 48: 519–522, 1981.[Abstract/Free Full Text]
  28. Perez CG, Copello JA, Li Y, Karko KL, Gomez LN, Ramos-Franco J, Fill M, Escobar AL, and Mejia-Alvarez R. Ryanodine receptor function in newborn rat heart. Am J Physiol Heart Circ Physiol 288: H2527–H2540, 2005.[Abstract/Free Full Text]
  29. Perez-Reyes E, Wei XY, Castellano A, and Birnbaumer L. Molecular diversity of L-type calcium channels. Evidence for alternative splicing of the transcripts of three non-allelic genes. J Biol Chem 265: 20430–20436, 1990.[Abstract/Free Full Text]
  30. Puglisi JL and Bers DM. LabHEART: an interactive computer model of rabbit ventricular myocyte ion channels and Ca2+ transport. Am J Physiol Cell Physiol 281: C2049–C2060, 2001.[Abstract/Free Full Text]
  31. Sedarat F, Lin E, Moore ED, and Tibbits GF. Deconvolution of confocal images of dihydropyridine and ryanodine receptors in developing cardiomyocytes. J Appl Physiol 97: 1098–1103, 2004.[Abstract/Free Full Text]
  32. Sedarat F, Xu L, Moore ED, and Tibbits GF. Colocalization of dihydropyridine and ryanodine receptors in neonate rabbit heart using confocal microscopy. Am J Physiol Heart Circ Physiol 279: H202–H209, 2000.[Abstract/Free Full Text]
  33. Sham JS, Cleemann L, and Morad M. Functional coupling of Ca2+ channels and ryanodine receptors in cardiac myocytes. Proc Natl Acad Sci USA 92: 121–125, 1995.[Abstract/Free Full Text]
  34. Sipido KR, Maes M, and Van de Werf F. Low efficiency of Ca2+ entry through the Na+-Ca2+ exchanger as trigger for Ca2+ release from the sarcoplasmic reticulum. A comparison between L-type Ca2+ current and reverse-mode Na+-Ca2+ exchange. Circ Res 81: 1034–1044, 1997.[Abstract/Free Full Text]
  35. Tanabe T, Adams BA, Numa S, and Beam KG. Repeat I of the dihydropyridine receptor is critical in determining calcium channel activation kinetics. Nature 352: 800–803, 1991.[CrossRef][Medline]
  36. Thomas MJ, Hamman BN, and Tibbits GF. Dihydropyridine and ryanodine binding in ventricles from rat, trout, dogfish and hagfish. J Exp Biol 199: 1999–2009, 1996.[Abstract]
  37. Tseng GN. Calcium current restitution in mammalian ventricular myocytes is modulated by intracellular calcium. Circ Res 63: 468–482, 1988.[Abstract/Free Full Text]
  38. Vornanen M. Contribution of sarcolemmal calcium current to total cellular calcium in postnatally developing rat heart. Cardiovasc Res 32: 400–410, 1996.[Abstract/Free Full Text]
  39. Wetzel GT, Chen F, Friedman WF, and Klitzner TS. Calcium current measurements in acutely isolated neonatal cardiac myocytes. Pediatr Res 30: 83–88, 1991.[Web of Science][Medline]
  40. Wetzel GT, Chen F, and Klitzner TS. Ca2+ channel kinetics in acutely isolated fetal, neonatal, and adult rabbit cardiac myocytes. Circ Res 72: 1065–1074, 1993.[Abstract/Free Full Text]
  41. Wetzel GT, Chen F, and Klitzner TS. Na+/Ca2+ exchange and cell contraction in isolated neonatal and adult rabbit cardiac myocytes. Am J Physiol Heart Circ Physiol 268: H1723–H1733, 1995.[Abstract/Free Full Text]
  42. Yang Y, Chen X, Margulies K, Jeevanandam V, Pollack P, Bailey BA, and Houser SR. L-type Ca2+ channel alpha 1c subunit isoform switching in failing human ventricular myocardium. J Mol Cell Cardiol 32: 973–984, 2000.[CrossRef][Web of Science][Medline]
  43. Yuan W, Ginsburg KS, and Bers DM. Comparison of sarcolemmal calcium channel current in rabbit and rat ventricular myocytes. J Physiol 493: 733–746, 1996.[Abstract/Free Full Text]
  44. Zuhlke RD, Bouron A, Soldatov NM, and Reuter H. Ca2+ channel sensitivity towards the blocker isradipine is affected by alternative splicing of the human alpha1C subunit gene. FEBS Lett 427: 220–224, 1998.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
G. Ding, R. F. Wiegerinck, M. Shen, A. Cojoc, C. M. Zeidenweber, and M. B. Wagner
Dopamine increases L-type calcium current more in newborn than adult rabbit cardiomyocytes via D1 and {beta}2 receptors
Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H2327 - H2335.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
J. Huang, L. Hove-Madsen, and G. F. Tibbits
Ontogeny of Ca2+-induced Ca2+ release in rabbit ventricular myocytes
Am J Physiol Cell Physiol, February 1, 2008; 294(2): C516 - C525.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
J. Huang, L. Hove-Madsen, and G. F. Tibbits
SR Ca2+ refilling upon depletion and SR Ca2+ uptake rates during development in rabbit ventricular myocytes
Am J Physiol Cell Physiol, December 1, 2007; 293(6): C1906 - C1915.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
L. P. Collis, S. Srivastava, W. A. Coetzee, and M. Artman
beta2-Adrenergic receptor agonists stimulate L-type calcium current independent of PKA in newborn rabbit ventricular myocytes
Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H2826 - H2835.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
T. Namiki, R. W. Joyner, and M. B. Wagner
Developmental changes in time course of recovery from inactivation in L-type calcium currents of rabbit ventricular myocytes
Am J Physiol Heart Circ Physiol, January 1, 2007; 292(1): H295 - H303.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
J. Huang, C. van Breemen, K.-H. Kuo, L. Hove-Madsen, and G. F. Tibbits
Store-operated Ca2+ entry modulates sarcoplasmic reticulum Ca2+ loading in neonatal rabbit cardiac ventricular myocytes
Am J Physiol Cell Physiol, June 1, 2006; 290(6): C1572 - C1582.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/6/H2267    most recent
01093.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Huang, J.
Right arrow Articles by Tibbits, G. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Huang, J.
Right arrow Articles by Tibbits, G. F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.