AJP - Heart AJP: Lung Cellular and Molecular Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 286: H1963-H1969, 2004. First published January 8, 2004; doi:10.1152/ajpheart.01011.2003
0363-6135/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
286/5/H1963    most recent
01011.2003v1
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 ISI Web of Science
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 ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tipparaju, S. M.
Right arrow Articles by Wagner, M. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tipparaju, S. M.
Right arrow Articles by Wagner, M. B.

Developmental differences in L-type calcium current of human atrial myocytes

Srinivas M. Tipparaju, Rajiv Kumar, Yanggan Wang, Ronald W. Joyner, and Mary B. Wagner

Todd Franklin Cardiac Research Laboratory, Sibley Children's Heart Center, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322

Submitted 24 October 2003 ; accepted in final form 2 January 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We investigated differences in L-type Ca2+ current (ICa) between infant (INF, 1–12 mo old), young adult (YAD, 14–18 yr old), and older adult (AD) myocytes from biopsies of right atrial appendages. Basal ICa was smaller in INF myocytes (1.2 ± 0.1 pA/pF, n = 29, 6 ± 1 mo old, 11 patients) than in YAD (2.5 ± 0.2 pA/pF, n = 20, 16 ± 1 yr old, 5 patients) or AD (2.6 ± 0.3 pA/pF, n = 19, 66 ± 3 yr old, 9 patients) myocytes (P < 0.05). Maximal ICa produced by isoproterenol (Iso) was similar in INF, YAD, and AD cells: 8.4 ± 1.1, 9.6 ± 1.0, and 9.2 ± 1.3 pA/pF, respectively. Efficacy (Emax) was larger in INF (607 ± 50%) than for YAD (371 ± 29%) or AD (455 ± 12%) myocytes. Potency (EC50) was 8- to 10-fold higher in AD (0.82 ± 0.09 nM) or YAD (0.41 ± 0.14 nM) than in INF (7.6 ± 3.5 nM) myocytes. Protein levels were similar for Gi{alpha}2 but much greater for Gi{alpha}3 in INF than in AD or YAD atrial tissue. When Gi{alpha}3 activity was inhibited by inclusion of a Gi{alpha}3 COOH-terminal decapeptide in the pipette, basal ICa and the response to 10 nM Iso were increased in INF, but not in YAD, cells. We propose that basal ICa and the response to low-dose {beta}-adrenergic stimulation are inhibited in INF (but not YAD or AD) cells as a result of constitutive inhibitory effects of Gi{alpha}3.

{beta}-adrenergic receptors; human atrium; atrial function; isoproterenol

L-TYPE CALCIUM CURRENT (ICa) is a major determinant of the early plateau phase of the action potential and is the trigger (combined with Ca2+ release from the sarcoplasmic reticulum) for contraction in heart cells. The enhancement of ICa in cardiac cells by {beta}-adrenergic agonists is a mechanism for regulating heart rate and contractility by sympathetic nerve stimulation. During fetal and postnatal cardiac development, various changes occur in the cardiac contractile apparatus, sarcoplasmic reticulum, metabolic sensitivity, and hormonal regulation. However, little is known about the developmental changes in regulation of ionic currents and Ca2+ handling. The regulation of ICa is a complex interaction among many factors, including the voltage and time dependence of Ca2+ channels, intracellular release of Ca2+, circulating catecholamines, Ca2+ channel density, and effects of many pharmacological agents, as well as interactions between adenylyl cyclase, cyclic nucleotides, and G proteins. The basal levels of ICa, as well as the amplitude and time course of various K+ currents, in human infant atrial cells may be different from those in human adult atrial cells, and these differences may be important in the in vivo electrical and mechanical properties of the atrium. Differences in the waveform of the action potentials lead to differences in the refractory period and, thus, may be important in the genesis and maintenance of atrial arrhythmias.

Various animal studies on developmental changes in regulation of ICa were primarily focused on ventricular cells. We previously demonstrated (13, 19) lower ICa density (pA/pF) and less sensitivity to stimulation by isoproterenol (Iso) in newborn than in adult rabbit ventricular cells. We further showed (11) a tonic inhibition by inhibitory G proteins in newborn cells, which could account for their lower basal ICa amplitude and decreased potency for Iso stimulation. In other species, e.g., rat (7), newborn cells have been shown to have higher ICa than adult cells. In a previous study on ICa amplitude in human infant vs. adult atrial cells (21), no differences were found. In this study, the amplitude of ICa in infant hearts (1–12 mo old) was 1.0–1.2 pA/pF, while that in adult (47–79 yr old) atrial cells was somewhat larger (1.5–1.8 pA/pF) but not significantly different.

Measurements of ICa from isolated adult human atrial cells at room temperature have been reported with a variety of "basal" ICa amplitudes: from 1.2 ± 0.1 to 12 ± 4 pA/pF (5, 10, 16, 17, 20, 21, 24, 26). Similarly, the maximum effect of Iso to stimulate ICa in adult human atrial cells has also been reported with considerable variability: from 150 to 853% (5, 16, 20, 24). In the present study, we have isolated human atrial cells from older adult, young adult, and infant patients to determine whether the basal ICa and the sensitivity to Iso in infant cells is less than that in adult cells and, if so, whether these changes occur early or late in life.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients. Biopsies from the right atrial appendage were obtained at the time of initiation of cardiopulmonary bypass from older adult (AD, 51–79 yr old, n = 11), young adult (YAD, 14–18 yr old, n = 6), and infant (INF, 3–10 mo old, n = 19) patients. The percentages of the patients that were female were 45% of AD, 17% of YAD, and 42% of INF. All the patients were in normal sinus rhythm at the time of surgery, with no documented history of previous atrial fibrillation or atrial dilation. The characteristics of AD, YAD, and INF patients are given in Table 1, as well as whether the biopsy was used to study ICa and/or for Western blot analysis. The study protocols for using adult and infant atrial biopsies were approved by the Emory University Human Investigation Committee and by Children's Healthcare of Atlanta.


View this table:
[in this window]
[in a new window]
  		Table 1. Patient characteristics

 

Preparation of isolated cells. As in our previous work (29), atrial tissue obtained at the time of surgery was placed in oxygenated Ca2+-free Krebs-Ringer (KR) solution and transported to the laboratory within 5 min after excision and cut into small chunks. Tissue chunks were stirred in oxygenated Ca2+-free KR solution at 34–36°C, and the solution was changed three times. After 15 min, the solution was replaced with Ca2+-free KR solution + collagenase (1 mg/ml, type I; Sigma), protease (0.4 mg/ml, type XXIV; Sigma), and BSA (1 mg/ml) for 40 min. Tissue was transferred to Ca2+-free KR solution + collagenase (1 mg/ml, type I; Sigma). Tissue chunks were triturated, and the supernatant was tested every 15 min under the microscope until cells started to appear. Cells were then separated by centrifugation and resuspended in storage solution containing 10 mg/ml BSA and kept in a refrigerator until they were used.

Recording whole cell ICa. The cells were placed in a chamber that was continuously perfused with normal test solution at room temperature, as in our previous work with rabbit ventricular cells (19). Whole cell voltage-clamp experiments were performed with pipette resistances of 1.0–2.0 M{Omega} with compensation for capacitance and series resistance but without leak correction. The cell was depolarized every 10 s from a holding potential (Vh) of –45 mV to a test potential of +10 mV for 360 ms. In each age group, we also performed experiments using a Vh of –80 mV with a prepulse of 200 ms to –45 mV to inhibit Na+ current and found nearly identical values of ICa without and with Iso compared with using a Vh of –45 mV. The potential of the pipette was set to zero within the bath solution, and no compensation was made for differences in tip potential during exposure to the cytoplasmic solution. We express all the current data as current density (pA/pF) by normalizing the current for each cell to its capacitance. Cells that showed rundown were excluded from analysis.

Solutions and drugs. Ca2+-free KR solution contained (in mM) 35 NaCl, 4.75 KCl, 1.2 KH2PO4, 16 Na2HPO4, 134 sucrose, 25 NaHCO3, 10 glucose, and 10 HEPES, with pH adjusted to 7.4 with NaOH. Storage solution contained (in mM) 100 K-glutamate, 25 KCl, 10 KH2PO4, 0.5 EGTA, 1 MgSO4, 20 taurine, 10 glucose, and 5 HEPES, with pH adjusted to 7.2 with KOH. ICa test solution consisted of (in mM) 130 NaCl, 1.8 CaCl2, 20 CsCl, 0.53 MgCl2, 5 HEPES, and 5 glucose, with pH adjusted to 7.4 with NaOH. Pipette solution consisted of (in mM) 110 CsOH, 90 aspartic acid, 20 CsCl, 10 tetraethylammonium chloride, 5 HEPES, 10 EGTA, 5 MgATP, 5 Na2 creatine phosphate, 0.4 GTP-Tris salt, and 0.1 leupeptin, with pH adjusted to 7.2 with CsOH. Modifications to these solutions are described below for specific protocols. The "EC" peptide as used previously by Aridor et al. (3) and in our previous study (11) to inhibit Gi{alpha}3 is a synthetic decapeptide corresponding to the COOH terminus of Gi{alpha}3 and was produced in the Emory University Microchemistry Facility.

Determination of G protein levels by Western blotting. We analyzed amounts of Gi{alpha}2 or Gi{alpha}3 in total homogenates from INF, YAD, and AD atrial biopsies as we previously described (13, 18). We used polyclonal antibodies for Gi{alpha}2 (Santa Cruz Biotechnology) at 1:500 dilution and for Gi{alpha}3 (Calbiochem) at 1:1,000 dilution. For detection of bands, we used an enhanced chemiluminescence detection kit (ECL plus, Amersham) with Lumigen PS-3 substrate. To compare relative amounts of specific proteins in multiple lanes, we analyzed immunostained bands with a two-dimensional gel imaging system and LabWorks image acquisition and analysis software (UVP, Upland, CA) with data presented as arbitrary optical density units.

Statistics. Values are means ± SE. Comparison of measured parameters was done using ANOVA. Student-Newman-Keuls post hoc test was used to compare variations between groups. P < 0.05 was defined as significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Basal and Iso-stimulated ICa in AD, INF, and YAD human atrial cells. Figure 1 compares typical current traces for INF, YAD, and AD atrial cells in the control solution and, for the same cells, with 100 nM Iso. Figure 1A shows data from an INF (9 mo old) atrial cell [capacitance (Cm) = 27 pF] with a control ICa density of 1.5 pA/pF, which increased to 8.3 pA/pF with 100 nM Iso. Figure 1B shows data from a YAD (19-yr-old) atrial cell (Cm = 67 pF) with a control ICa of 2.4 pA/pF, which increased to 9.9 pA/pF with 100 nM Iso. Figure 1C shows data from an AD (75-yr-old) atrial cell (Cm = 51 pF) with a control ICa of 2.3 pA/pF, which increased to 10.5 pA/pF with 100 nM Iso. The voltage step is to +10 mV from a Vh of –45 mV.



View larger version (10K):
[in this window]
[in a new window]
  		Fig. 1. Recordings of L-type Ca2+ current (ICa) from an infant (INF, A), young adult (YAD, B), and adult (AD, C) atrial cell in control solution and after addition of 100 nM isoproterenol (Iso).

 

Because we recorded ICa from all the cells as a voltage step to +10 mV, we needed to show that this voltage step was appropriate for determining the maximum ICa in control solution and in Iso. Figure 2A shows current-voltage relations for ICa recorded from some of the INF, YAD, and AD cells in control solution and in 100 nM Iso produced by a sequence of voltage steps (each from Vh = –45 mV) to +40 mV, with the peak ICa plotted against the test potential. In control solution, the maximum current was obtained at +10 or +15 mV in each age group; in the Iso solution the curves shift leftward, as expected, and the peak occurred at +5 or +10 mV. We used a test potential of +10 mV for the measurements of peak ICa density reported below.



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 2. A: current-voltage relations for peak ICa density for test pulses in 5-mV steps from –30 to +40 (from a holding potential of –45 mV) in control solution and after addition of 100 nM Iso. B: basal ICa values and Iso (1, 10, and 100 nM)-stimulated ICa for INF, YAD, and AD atrial cells. INF basal ICa values are significantly less than YAD or AD ICa values. INF ICa values are also less than AD or YAD ICa values for 1 and 10 nM Iso. There are no significant differences among INF, YAD, or AD cells for 100 nM Iso-stimulated ICa. AD and YAD values of ICa are not significantly different from each other under basal conditions or for any concentration of Iso. *Significantly different than YAD and AD.

 

To determine whether there are developmental changes in the basal ICa and in the response of ICa to Iso, we recorded ICa from a total of 29 INF (from 11 INF patients), 19 AD (from 9 AD patients), and 20 YAD (from 5 YAD patients) human atrial cells. For all these cells, we also recorded ICa after exposure to one or more concentrations of Iso and compared the data among the three age groups. Figure 2B summarizes the basal ICa and the ICa produced by application of 1, 10, or 100 nM Iso in INF, YAD, and AD atrial cells. The average basal ICa recorded from INF atrial cells was significantly lower (1.2 ± 0.1 pA/pF) than that recorded from AD (2.6 ± 0.3 pA/pF) and YAD (2.5 ± 0.2 pA/pF) atrial cells. At 1 and 10 nM Iso, ICa in INF cells was also significantly less than in AD and YAD cells. The maximum current achieved in response to 100 nM Iso in INF (8.4 ± 1.1 pA/pF, n = 5), YAD (9.6 ± 1.0 pA/pF, n = 5), and AD (9.2+1.3 pA/pF, n = 6) human atrial cells was not significantly different. There were no significant differences in ICa between AD and YAD cells in control or at any concentration of Iso. As expected, the capacitance values were lower in INF (24 ± 2 pF) than in YAD (69 ± 5 pF) or AD (71 ± 5 pF) cells, with no significant differences for Cm between AD and YAD cells.

The concentration-dependent effect of Iso on ICa for INF, YAD, and AD human atrial cells is summarized in Fig. 3, plotted as the percent increase in ICa as a function of Iso concentration. The data were fit using the logistic equation to determine Emax and EC50 for the different age groups. For INF atrial cells (Fig. 3A), the calculated Emax was 607 ± 50%, while EC50 was 7.6 ± 3.5 nM. For YAD cells (Fig. 3B), EC50 was 0.41 ± 0.05 nM, with Emax of 371 ± 29%. For AD atrial cells (Fig. 3C), EC50 was 0.82 ± 0.09 nM, with Emax of 455 ± 12. The AD and YAD cells are similar, in that they have a high potency for Iso (EC50 < 1 nM), with Emax of ~400%. INF cells are distinctly different from AD and YAD cells, in that they have a lower potency (higher EC50 by a factor of ~10), a broader dose-response curve, and a higher Emax in terms of percent change by Iso (although not a higher level of ICa produced by Iso; Fig. 2).



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 3. Dose-response curves for percent change in ICa for INF, YAD, and AD atrial cells in response to Iso. Calculated values for EC50 (potency) and Emax (efficacy) are shown for INF (A), YAD (B), and AD (C) cells. *Significantly increased compared with control.

 

Comparison between effects of Iso and cAMP in AD and INF human atrial cells. It is not always the case that Iso can maximize ICa in cardiac cells. To examine the maximal stimulation of ICa by activation of the PKA pathway, we applied 100 µM cAMP by intracellular dialysis to AD and INF human atrial cells. We previously showed that intracellular application of 100 µM cAMP increased ICa maximally in adult rabbit atrial cells (28) as well as in newborn and adult rabbit ventricular cells (19) but that the maximum effects of Iso were significantly less than those of intracellularly applied cAMP in newborn rabbit ventricular cells. For the human AD atrial cells, there was no significant difference between maximal ICa densities obtained with externally applied Iso (9.2±1.3 pA/pF, n = 6) and internally applied 100 µM cAMP (11.5 ± 1.8 pA/pF, n = 3). For INF human atrial cells, the maximal value of ICa achieved with intracellular application of cAMP was 9.4 ± 0.7 pA/pF (n = 8), which was not significantly different from the maximal ICa obtained with Iso (8.4 ± 1.1 pA/pF, n = 5). Thus it appears that Iso can maximize ICa in AD and INF human atrial cells, although with a higher EC50 for INF cells, which is distinctively different from the results we obtained with the newborn and adult rabbit ventricular cells (19).

Levels of Gi protein isoforms in AD, YAD, and INF atrial tissue. The data reported above showing decreased basal ICa and a decreased potency for Iso stimulation for INF cells vs. YAD or AD cells represent developmental differences similar to those we showed previously for newborn vs. adult rabbit ventricular cells. In the rabbit cells, we also showed that levels of Gi proteins were significantly greater in newborn than in adult ventricular cells and that a tonic inhibitory effect from these G proteins was part of the mechanism for the developmental differences in ICa amplitude and Iso sensitivity. To determine whether there are developmental differences in Gi proteins in human atrial cells, we obtained proteins from three AD, three YAD, and four INF atrial biopsies for comparisons by Western blotting.

Figure 4 shows Western blots of total homogenate protein prepared from atrial biopsies obtained from INF and AD patients. Protein levels for Gi{alpha}2 (Fig. 4, top) are similar for the two age groups, whereas protein levels for Gi{alpha}3 (Fig. 4, bottom) are clearly higher for the INF patients than for the AD patients. Densitometric analysis of INF and AD bands (optical density in arbitrary units) showed that Gi{alpha}2 in INF patients (252 ± 17, n = 3) was not significantly different from that in AD patients (211 ± 64, n = 3). However, Gi{alpha}3 in INF tissue (298 ± 37, n = 3) was 13 times greater (P < 0.05) than the mean level of Gi{alpha}3 in AD tissue (22 ± 10, n = 3). The Western blots in Fig. 5 show relative amounts of Gi{alpha}2 and Gi{alpha}3 in INF vs. YAD tissue. Densitometric analysis of INF and YAD bands (optical density in arbitrary units) showed that Gi{alpha}2 in INF patients was not significantly different from that in YAD patients: 104 ± 6 vs. 85 ± 5 (n = 3). However, Gi{alpha}3 was six times greater in INF tissue than in YAD tissue: 61 ± 16 vs. 11 ± 6 (n = 3, P < 0.05). These results show similar levels of Gi{alpha}2 in INF and YAD atrial tissue but much lower levels of Gi{alpha}3 in YAD than in INF tissue, the same relation we showed for AD tissue. In our previous work with rabbit ventricular cells, Gi{alpha}2 and Gi{alpha}3 were significantly greater in newborn than in adult cells (13); this finding differs from the results obtained in the present study for human atrial cells.



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 4. Western blots in which lanes labeled INF and AD each contained 40 µg of total homogenate protein prepared from atrial biopsies obtained from 3 INF (patients 6, 9, and 12 in Table 1) and 3 AD (patients 4, 7, and 11 in Table 1) patients, respectively.

 


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 5. Western blots in which lanes labeled INF and YAD each contained 40 µg of total homogenate protein prepared from atrial biopsies obtained from 3 INF (patients 6, 9, and 15 in Table 1) and 3 YAD (patients 1, 3, and 4 in Table 1) patients, respectively.

 

Effects of a decapeptide corresponding to the COOH terminus of Gi{alpha}3. To determine whether Gi proteins, specifically Gi{alpha}3, are playing a tonic inhibitory role in basal ICa levels in INF, but not in YAD, atrial cells, we carried out experiments to determine whether the addition of a decapeptide corresponding to the COOH terminus of Gi{alpha}3 (EC peptide) to the pipette solution would increase basal ICa in INF, but not in YAD, atrial cells. Additionally, we determined whether EC peptide increased the sensitivity of the response of INF atrial cells to Iso. In these experiments, we included EC peptide in the pipette solution; thus we cannot record a stable basal ICa without the effect, if any, of EC peptide. Thus we compared cells for which EC peptide was in the pipette solution with other cells (from the same age group) for which EC peptide was not in the pipette. Figure 6, left, shows current traces from an INF atrial cell with 500 µg/ml EC peptide in the pipette. For nine INF cells, we recorded basal ICa with the EC peptide in the peptide and also applied one or more concentrations of Iso. The basal ICa with EC peptide in the pipette (2.2 ± 0.3 pA/pF, n = 9) was significantly greater than the basal ICa without EC peptide in the pipette (1.2 ± 0.1 pA/pF, n = 29). In addition, the ICa for 10 nM Iso with EC peptide in the pipette (8.1 ± 1.0 pA/pF, n = 5) was significantly greater than the ICa for 10 nM Iso without EC peptide in the pipette (4.7 ± 0.4 pA/pF, n = 14). However, the current with 100 nM Iso (with EC peptide) was 7.5 ± 0.1 pA/pF, which was not significantly different from the current for INF atrial cells with 100 nM Iso (without EC peptide): 8.4 ± 1.0 pA/pF. Thus the presence of EC peptide increased the basal ICa and the ICa at intermediate levels of Iso but not the maximum obtainable ICa for these INF cells.



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 6. Left: current traces from an INF atrial cell with 500 µg/ml EC peptide in the pipette. Basal ICa is 2.5 pA/pF and the current increases to 5.0 pA/pF with 0.3 nM Iso and then further increases to 10.1 pA/pF with addition of 10 nM Iso. Right: dose-response relation of ICa for 0.3, 1, 10, and 100 nM Iso in INF atrial cells with EC peptide in the pipette. In INF cells, Emax for Iso with EC peptide in the pipette (303 + 34%) is considerably reduced compared with that for Iso without EC peptide in the pipette (607 ± 50%, see Fig. 3). EC50 for Iso with EC peptide in the pipette (1.1 ± 0.6 nM) is also considerably reduced compared with that for Iso without EC peptide in the pipette (7.6 ± 3.5 nM, see Fig. 3). *Significantly increased compared with control.

 

The Iso dose response for INF atrial cells with EC peptide in the pipette is shown in Fig. 6, right. For INF cells, the Emax for Iso with EC peptide in the pipette was considerably reduced compared with the Emax for Iso without EC peptide in the pipette. Similarly, the EC50 for Iso with EC peptide in the pipette was also considerably reduced compared with the EC50 for Iso without EC peptide in the pipette. For INF cells, the presence of EC peptide in the pipette made the values of EC50 and Emax close to those obtained for YAD or AD cells without EC peptide in the pipette.

To determine whether the effects of EC peptide on basal ICa were present exclusively for INF cells, we also recorded from six cells from YAD patients with the same EC peptide in the pipette and obtained values of basal ICa (2.7 ± 0.6 pA/pF) and ICa with 10 nM Iso (9.0 ± 1.0 pA/pF, n = 4) that were not significantly different from the values for YAD atrial cells without EC peptide in the pipette (2.5 ± 0.2 and 10.0 ± 1.4 pA/pF for basal ICa and ICa with 10 nM Iso, respectively); these data show that EC peptide did not affect the basal ICa or the maximum Iso effect on ICa for YAD cells.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our data indicate significant differences in the amplitude and regulation of ICa of infant vs. young or older adult human atrial cells as determined by square-wave voltage-clamp waveforms at room temperature. Specifically, the basal ICa and the potency for Iso stimulation are lower for infant than for young and older adult cells. However, the magnitude of the maximal ICa obtained with direct dialysis of cAMP into the cells or with maximal levels of Iso was not different for infant vs. adult cells.

Our studies on amplitude and regulation of ICa were done at room temperature to maximize the stability of the preparations. Previous studies on the amplitude of ICa in human atrial cells have predominantly been done on adult cells. At room temperature, basal values for adult cells of 1.2 ± 0.1 pA/pF (16), 1.5–1.8 pA/pF (21), 1.7 ± 0.1 pA/pF (5), 1.8 ± 0.3 pA/pF (10), 1.96 ± 0.12 pA/pF (24), 2.2 ± 0.3 pA/pF (17), 3.2 ± 0.2 pA/pF (26), and 12 ± 4 pA/pF (20) have been reported. Our value of 2.6 ± 0.3 pA/pF for adult human atrial cells falls within this range. Other investigators have recorded 2.47 ± 0.23 pA/pF (9) for ICa from infants from 3 days to 4 yr of age. In the study by Roca et al. (21), the amplitude of ICa from infant atrial cells (1–12 mo old) was 1.0–1.2 pA/pF, which is not largely different from our results for infants of the same age range (1.2 ± 0.1 pA/pF), whereas their amplitude for adult human atrial cells (47–79 yr old, 1.5–1.8 pA/pF) is less than the 2.6 ± 0.3 pA/pF we obtained and may explain why we demonstrated a significant difference between infant and adult cells and they did not.

Prior studies on the amplitude of ICa of adult human atrial cells at physiological temperature have shown, as expected, higher amplitudes of ICa, with reported values of 6.76 ± 1.14 pA/pF (30), 9.3 ± 1.0 pA/pF (27), 10.4 pA/pF (1), and 10.8 ± 1.1 pA/pF (14). We recently reported (29) a value of 4.1 ± 0.4 pA/pF for peak ICa of infant human atrial cells at physiological temperature. These data suggest that the approximately two-fold difference in basal current between infant and adult human atrial cells persists at physiological temperature, but comparable experiments on regulation of ICa by Iso or by intracellular cAMP at physiological temperature have not been reported.

Sensitivity of the human atrial cell ICa to Iso has been reported with wide variability. In room-temperature studies of adult human atrial cells, Ouadid et al. (20) reported a maximal stimulation by Iso of 853% with an EC50 of ~100 nM, Maier et al. (16) reported a 271% increase in ICa with 10 nM Iso, Skasa et al. (24) reported a 350% increase with 100 nM Iso, and Boixel et al. (4) reported a 150% increase with 1 µM Iso. At physiological temperature, Li and Nattel (14) reported a 138% increase, while van Wagoner et al. (27) reported a 280% increase in response to 1 µM Iso. At room temperature, for infants from 3 days to 4 yr of age, Hatem et al. (9) reported a 172% increase with 1 µM Iso. No prior study has reported dose-response relations for Iso for infant and adult human atrial cells with the same dissociation technique and protocol. Our data show that, in terms of current density (pA/pF), INF atrial cells have a lower basal value than YAD or AD cells but that the maximal current densities obtained with Iso or intracellular dialysis with cAMP are the same for INF, YAD, and AD cells.

Various biochemical studies have been done to compare atrial tissue from children and adults with regard to different components of the regulatory pathways for ICa. Brodde et al. (6) analyzed specimens of right atrial appendage from children (3.7 ± 1 yr old) vs. adults (37.9 ± 2.3 yr old) vs. older adults (66.1 ± 1.5 yr old). Neither the {beta}-adrenoceptor number (fmol iodocyanopindolol/mg protein) nor the {beta}1-to-{beta}2 ratio were different among the three groups. However, adenylyl cyclase basal and stimulated activity (pmol cAMP·mg protein–1· min–1) were higher in the children than in the adults or older adults. Although Gs{alpha} levels were not different for the three age groups, Gi{alpha} levels (with use of an antibody that detects Gi{alpha}1, Gi{alpha}2, and transducin) at 37 and 46 kDa were slightly higher in the older adults. However, specific comparisons of Gi{alpha}1, Gi{alpha}2, and Gi{alpha}3 were not reported. We previously showed (13) developmental differences of rabbit ventricular cells primarily in Gi{alpha}2 and Gi{alpha}3. These Gi{alpha} changes also led to a greater sensitivity to carbachol and adenosine for inhibiting ICa, which had been enhanced by Iso in newborn compared with adult rabbit ventricular cells (13). Suto et al. (25) showed a similar increased neonatal sensitivity to adenosine in inhibiting Iso-stimulated ICa in rabbit atrial cells. The varying age ranges of the "children" groups of these biochemical studies make it difficult to extrapolate to our studies on infant (<1 yr old) vs. young or older adult atrial cells. Our Western blotting studies show that infant atrial tissue has similar levels of Gi{alpha}2 but much greater levels of Gi{alpha}3 than young or older adult human atrial tissue. These results suggest that one mechanism of lower basal ICa and lower potency for Iso in infant cells than in adult cells may be constitutive activity of Gi{alpha}3, producing a tonic inhibition of adenylyl cyclase activity in the infant cells. Our results using EC peptide in the pipette solution for INF or YAD cells showed that inclusion of EC peptide in the pipette increased the basal ICa and the 10 nM Iso-stimulated ICa in the INF cells but not the 100 nM Iso-stimulated ICa in the INF cells. However, EC peptide had no effect on the basal ICa or the 10 nM Iso-stimulated ICa in the YAD cells, which is consistent with our proposal of a tonic inhibition via Gi{alpha}3 in INF, but not in YAD or AD, cells. EC peptide decreased Emax and EC50 of INF cells close to the levels of AD and YAD cells without EC peptide.

Constitutive activity of a wide variety of G protein-coupled receptors is widely recognized (for review see Ref. 22) and has been shown specifically (23) for human adenosine A1 receptors transfected into Chinese hamster ovary cells as a decreased GTP{gamma}S binding in response to the application of inverse agonists. G protein {alpha}-subunits link to specific receptors by specific amino acid sequences at their COOH-terminal end. Freissmuth et al. (8) reported preferential coupling of Gi{alpha}3 to adenosine A1 receptors in a reconstituted system. They demonstrated that adenosine A1 receptors had a 10-fold higher affinity for recombinant Gi{alpha}3 synthesized in Escherichia coli (rGi{alpha}3) than for rGi{alpha}1 or rGi{alpha}2 and suggested that the specific coupling between adenosine A1 receptors and Gi{alpha}3 is likely to govern transmembrane signaling pathways in vivo. Aridor et al. (3) reported that the synthetic decapeptide (EC) that corresponds to the COOH-terminal end sequence of Gi{alpha}3 specifically blocked the mastparan- and compound 48/80-induced histamine exocytosis from the mast cell. Aridor et al. (3) also showed that the inclusion of decapeptides corresponding to the COOH terminus of Gi{alpha}1 or Gi{alpha}2 had much smaller effects and that the inclusion of an irrelevant peptide of similar size had no effect. In our previous work on neonatal vs. adult rabbit ventricular cells (11), we showed that interruption of constitutive activity of Gi{alpha}3 by the intracellular application of EC peptide increased basal ICa, enhanced the Iso response, and blocked the inhibitory effects of adenosine for neonatal cells.

There are important differences between the results we demonstrated previously comparing newborn with adult rabbit ventricular cells (11–13, 15, 19) and the present work comparing infant with young or older adult human atrial cells. Newborn rabbit ventricular cells had decreased basal ICa, increased EC50, and decreased Emax for Iso stimulation compared with adult cells and also had higher levels of Gi{alpha}2 and Gi{alpha}3 than adult cells. Infant human cells have decreased basal ICa and increased EC50 for Iso stimulation but increased Emax for Iso stimulation and increased levels of Gi{alpha}3, but not Gi{alpha}2, compared with young adult or older adult cells. In addition, for newborn, but not adult, rabbit ventricular cells, Iso was not able to increase ICa to the maximum levels achieved with forskolin or intracellular cAMP. For human infant and adult atrial cells, the maximum current obtained with Iso was comparable to that obtained with intracellular cAMP, suggesting that the tonic inhibition of ICa in infant human atrial cells was perhaps weaker than that observed in newborn rabbit cells, because it could be overcome by higher levels of Iso. This is consistent with our finding that the INF human atrial cells had higher levels of Gi{alpha}3, but not Gi{alpha}2, than YAD or AD atrial cells. There are other significant differences for newborn rabbit ventricular cells compared with adult rabbit ventricular cells that we have not evaluated for human atrial cells, including increased sensitivity to inhibitors of phosphatases or phosphodiesterases and increased inhibitory potency of adenosine and carbachol (2, 11, 15).


   ACKNOWLEDGMENTS
 
GRANTS

This work was partially supported by National Heart, Lung, and Blood Institute Grants HL-56787 (R. Kumar) and HL-22475 (R. W. Joyner), a Scientist Development Grant from the American Heart Association, a Biomedical Engineering Research Grant from the Whitaker Foundation (M. B. Wagner), The Sibley Children's Heart Center, and the Emory Egleston Children's Research Center.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. B. Wagner, Todd Franklin Cardiac Research Laboratory, The Sibley Children's Heart Center, Dept. of Pediatrics, Emory Univ. School of Medicine, 2040 Ridgewood Dr. NE, Atlanta, GA 30322 (E-mail: mwagner{at}cellbio.emory.edu).

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
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ajiro Y, Hagiwara N, Katsube Y, Sperelakis N, and Kasanuki H. Levosimendan increases L-type Ca2+ current via phosphodiesterase-3 inhibition in human cardiac myocytes. Eur J Pharmacol 435: 27–33, 2002.[CrossRef][ISI][Medline]
  2. Akita T, Joyner RW, Lu C, Kumar R, and Hartzell HC. Developmental changes in modulation on calcium currents of rabbit ventricular cells by phosphodiesterase inhibitors. Circulation 90: 469–478, 1994.[Abstract/Free Full Text]
  3. Aridor M, Rajmilevich G, Beaven MA, and Sagi-Eisenberg R. Activation of exocytosis by the heterotrimeric G protein Gi3. Science 262: 1569–1572, 1993.[Abstract/Free Full Text]
  4. Boixel C, Dinanian S, Lang-Lazdunski L, Mercadier JJ, and Hatem SN. Characterization of effects of endothelin-1 on the L-type Ca2 + current in human atrial myocytes. Am J Physiol Heart Circ Physiol 281: H764–H773, 2001.[Abstract/Free Full Text]
  5. Boixel C, Tessier S, Pansard Y, Lang-Lazdunski L, Mercadier JJ, and Hatem SN. Tyrosine kinase and protein kinase C regulate L-type Ca2+ current cooperatively in human atrial myocytes. Am J Physiol Heart Circ Physiol 278: H670–H676, 2000.[Abstract/Free Full Text]
  6. Brodde OE, Zerkowski HR, Schranz D, Broede-Sitz A, Michel-Reher M, Schafer-Beisenbusch E, Piotrowski JA, and Oelert H. Age-dependent changes in the {beta}-adrenoceptor-G-protein(s)-adenylyl cyclase system in human right atrium. J Cardiovasc Pharmacol 26: 20–26, 1995.[CrossRef][ISI][Medline]
  7. Cohen NM and Lederer WJ. Changes in the calcium current of rat heart ventricular myocytes during development. J Physiol 406: 115–146, 1988.[Abstract/Free Full Text]
  8. Freissmuth M, Schutz W, and Linder ME. Interactions of the bovine brain A1-adenosine receptor with recombinant G protein {alpha}-subunits: selectivity for rGi{alpha}3. J Biol Chem 266: 17778–17783, 1991.[Abstract/Free Full Text]
  9. Hatem SN, Sweeten T, Vetter V, and Morad M. Evidence for presence of Ca2+ channel-gated Ca2+ stores in neonatal human atrial myocytes. Am J Physiol Heart Circ Physiol 268: H1195–H1201, 1995.[Abstract/Free Full Text]
  10. Huneke R, Jungling E, Skasa M, Rossaint R, and Luckhoff A. Effects of the anesthetic gases xenon, halothane, and isoflurane on calcium and potassium currents in human atrial cardiomyocytes. Anesthesiology 95: 999–1006, 2001.[ISI][Medline]
  11. Kumar R, Akita T, and Joyner RW. Adenosine and carbachol are not equivalent in their effects on L-type calcium current in rabbit ventricular cells. J Mol Cell Cardiol 28: 403–415, 1996.[CrossRef][ISI][Medline]
  12. Kumar R and Joyner RW. Expression of protein phosphatases during postnatal development of rabbit heart. Mol Cell Biochem 245: 91–98, 2003.[CrossRef][ISI][Medline]
  13. Kumar R, Joyner RW, Hartzell HC, Ellingsen D, Rishi F, Eaton DC, Lu C, and Akita T. Postnatal changes in the G-proteins, cyclic nucleotides and adenylyl cyclase activity in rabbit heart cells. J Mol Cell Cardiol 26: 1537–1550, 1994.[CrossRef][ISI][Medline]
  14. Li GR and Nattel S. Properties of human atrial ICa at physiological temperatures and relevance to action potential. Am J Physiol Heart Circ Physiol 272: H227–H235, 1997.[Abstract/Free Full Text]
  15. Lu C, Kumar R, Akita T, and Joyner RW. Developmental changes in the actions of phosphatase inhibitors on calcium current of rabbit heart cells. Pflügers Arch 427: 389–398, 1994.[CrossRef][ISI][Medline]
  16. Maier S, Aulbach F, Simm A, Lange V, Langenfeld H, Behre H, Kersting U, Walter U, and Kirstein M. Stimulation of L-type Ca2+ current in human atrial myocytes by insulin. Cardiovasc Res 44: 390–397, 1999.[Abstract/Free Full Text]
  17. Mewes T and Ravens U. L-type calcium currents of human myocytes from ventricle of non-failing and failing hearts and from atrium. J Mol Cell Cardiol 26: 1307–1320, 1994.[CrossRef][ISI][Medline]
  18. Mishra M, Wagner MB, Wang Y, Joyner RW, and Kumar R. Expression of cGMP-dependent protein kinase in human atrium. J Mol Cell Cardiol 33: 1467–1476, 2001.[CrossRef][ISI][Medline]
  19. Osaka T, Joyner RW, and Kumar R. Postnatal decrease in muscarinic cholinergic influence on Ca2+ currents of rabbit ventricular cells. Am J Physiol Heart Circ Physiol 264: H1916–H1925, 1993.[Abstract/Free Full Text]
  20. Ouadid H, Albat B, and Nargeot J. Calcium currents in diseased human cardiac cells. J Cardiovasc Pharmacol 25: 282–291, 1995.[ISI][Medline]
  21. Roca TP, Pigott JD, Clarkson CW, and Crumb WJ Jr. L-type calcium current in pediatric and adult human atrial myocytes: evidence for developmental changes in channel inactivation. Pediatr Res 40: 462–468, 1996.[ISI][Medline]
  22. Seifert R and Wenzel-Seifert K. Constitutive activity of G-protein-coupled receptors: cause of disease and common property of wild-type receptors. Naunyn Schmiedebergs Arch Pharmacol 366: 381–416, 2002.[CrossRef][ISI][Medline]
  23. Shryock JC, Ozeck MJ, and Belardinelli L. Inverse agonists and neutral antagonists of recombinant human A1 adenosine receptors stably expressed in Chinese hamster ovary cells. Mol Pharmacol 53: 886–893, 1998.[Abstract/Free Full Text]
  24. Skasa M, Jungling E, Picht E, Schondube F, and Luckhoff A. L-type calcium currents in atrial myocytes from patients with persistent and non-persistent atrial fibrillation. Basic Res Cardiol 96: 151–159, 2001.[CrossRef][ISI][Medline]
  25. Suto F, Habuchi Y, Yamamoto T, Tanaka H, and Hamaoka K. Increased sensitivity of neonate atrial myocytes to adenosine A1 receptor stimulation in regulation of the L-type Ca2+ current. Eur J Pharmacol 409: 213–221, 2000.[CrossRef][ISI][Medline]
  26. Vandecasteele G, Verde I, Rucker-Martin C, Donzeau-Gouge P, and Fischmeister R. Cyclic GMP regulation of the L-type Ca2+ channel current in human atrial myocytes. J Physiol 533: 329–340, 2001.[Abstract/Free Full Text]
  27. Van Wagoner DR, Pond AL, Lamorgese M, Rossie SS, McCarthy PM, and Nerbonne JM. Atrial L-type Ca2+ currents and human atrial fibrillation. Circ Res 85: 428–436, 1999.[Abstract/Free Full Text]
  28. Wang Y, Wagner MB, Joyner RW, and Kumar R. cGMP-dependent protein kinase mediates stimulation of L-type calcium current by cGMP in rabbit atrial cells. Cardiovasc Res 48: 310–322, 2000.[Abstract/Free Full Text]
  29. Wang Y, Xu H, Kumar R, Tipparaju SM, Wagner MB, and Joyner RW. Differences in transient outward current properties between neonatal and adult human atrial myocytes. J Mol Cell Cardiol 35: 1083–1092, 2003.[CrossRef][ISI][Medline]
  30. Workman AJ, Kane KA, Russell JA, Norrie J, and Rankin AC. Chronic {beta}-adrenoceptor blockade and human atrial cell electrophysiology: evidence of pharmacological remodelling. Cardiovasc Res 58: 518–525, 2003.[Abstract/Free Full Text]



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]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
286/5/H1963    most recent
01011.2003v1
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 ISI Web of Science
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 ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tipparaju, S. M.
Right arrow Articles by Wagner, M. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tipparaju, S. M.
Right arrow Articles by Wagner, M. B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.