© 1999 American Heart Association, Inc.
Original Contribution |
Establishment of ß-Adrenergic Modulation of L-Type Ca2+ Current in the Early Stages of Cardiomyocyte Development
From the Division of Cardiovascular Medicine, Henry Ford Heart and Vascular Institute (V.A.M.), Detroit, Mich; Institut für Neurophysiologie der Universität zu Köln (G.J.J., B.K.F., J.H.), Cologne, Germany; and Institut für Pflanzengenetik und Kulturpflanzenforschung (A.M.W.), Gatersleben, Germany.
Correspondence to Jürgen Hescheler, Institut für Neurophysiologie, Robert-Koch str 39, 50931 Köln, Germany. E-mail jh{at}Physiologie.Uni-Koeln.de
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Abstract |
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Abstract—ß-Adrenergic modulation of the L-type Ca2+ current (ICaL) was characterized for different developmental stages in murine embryonic stem cell-derived cardiomyocytes using the whole-cell patch-clamp technique at 37°C. Cardiomyocytes first appeared in embryonic stem cell-derived embryoid bodies grown for 7 days (7d). ICaL was insensitive to isoproterenol, forskolin, and 8-bromo-cAMP in very early developmental stage (VEDS) cardiomyocytes (from 7+1d to 7+2d) but highly stimulated by these substances in late developmental stage (LDS) cardiomyocytes (from 7+9d to 7+12d), indicating that all signaling cascade components became functionally coupled during development. In early developmental stage (EDS) cells (from 7+3d to 7+5d), the stimulatory response to forskolin and 8-bromo-cAMP was relatively weak. The forskolin effect was strongly augmented by ATP-
-S. At this stage, basal
ICaL was stimulated by the nonselective
phosphodiesterase (PDE) inhibitor
isobutylmethylxanthine, by PDE
inhibitors selective for the PDE II, III, and IV isoforms,
as well as by the phosphatase inhibitor okadaic acid.
Stimulation of ICaL by the catalytic subunit
of the cAMP-dependent protein kinase A (PKA) was found to be similar
(about 3 times) throughout development and in adult mouse
ventricular cardiomyocytes, indicating that no
structural changes of the Ca2+ channel related to
phosphorylation occurred during development.
ICaL was stimulated by isoproterenol in the
presence of a PKA inhibitor and GTP--S in LDS but not
VEDS cardiomyocytes, suggesting the development of a
membrane-delimited stimulatory pathway mediated through the stimulatory
GTP binding protein, Gs. We conclude that uncoupling and/or
low expression of Gs protein accounted for the
ICaL insensitivity to ß-adrenergic
stimulation in VEDS cardiomyocytes. Furthermore, in EDS
cells at the 7+4d stage, the reduced ß-adrenergic response is due, at
least in part, to high intrinsic PDE and phosphatase activities.
Key Words: L-type Ca2+ channel • adenylyl cyclase • cAMP-dependent protein kinase A • phosphatase • phosphodiesterase
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An important process in regulating heart rate and excitation-contraction coupling by sympathetic nerves is the stimulation of the high-threshold (L-type) Ca2+ current (ICaL) by ß-adrenergic agonists. In adult mammalian cardiomyocytes, the signal transduction from receptor to the channel includes several steps of interaction of functionally coupled downstream components (Figure 1
). Binding
of an agonist to the ß-adrenoceptor activates a guanine
nucleotide-binding (G) protein, Gs,
which triggers the activation of adenylyl cyclase (AC) and, in turn,
increases the level of cytosolic cAMP.1 This is
followed by the phosphorylation of the
Ca2+ channel through the activation of
cAMP-dependent protein kinase A (PKA),2 3 leading to an
increase in channel open probability and channel
availability.4 However, a direct stimulation of
Ca2+ channels via Gs
protein was also suggested.5 Despite recent advances in
the understanding of the ß-adrenergic modulation of the
Ca2+ channel during embryonic6 and
postnatal development,7 the establishment of this
important signaling cascade during cardiomyocyte
development is still unresolved. Moreover, studies on isolated
cardiomyocytes highlighted the importance of the impaired
ß-adrenergic modulation in cardiac disorders, such as heart
hypertrophy and heart failure.8 9 The detailed
knowledge about cardiomyocytes of early developmental
stages has now become of particular interest, because recent studies
show that cardiomyocytes of failing hearts tend to
dedifferentiate toward an embryonic phenotype (see References
10 and 1110 11 ). On the other hand, dysfunctions in adult cells may stem
from activation of a dormant pathway developed earlier during cellular
differentiation.
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The aim of the present study was to investigate the establishment of the ß-adrenergic signaling cascade for early cardiomyocyte development. Because of the known difficulties to obtain cardiomyocytes of the very early developmental stages from the mammalian embryos (eg, before day 12 to 13 of gestation in mouse), we have used for the present study an in vitro model of cardiomyogenesis based on pluripotent mouse embryonic stem (ES) cells. The cardiomyogenesis observed in ES cell–derived embryoid bodies (EBs) is almost identical to mouse embryo12 during early stages of development. Moreover, late-stage ES cell–derived cardiomyocytes exhibit rod-shaped morphology, sarcomere formation, and cell-cell junctions similar to those observed in cardiac myocytes developing in vivo.13 The electrophysiological characteristics in late developmental stage ES cell–derived cardiomyocytes are identical to postnatal cardiomyocytes.14 15 16 The normal course of development of cardiomyocytes from ES cells has been recently corroborated by the finding that implanted ES cell–derived cardiomyocytes form stable functional grafts in the hearts of adult mice.17 18
The ES cell differentiation model provides the unique possibility to
examine the role of particular components involved in signal
transduction at different time points during
cardiomyogenesis.19 20 Early-stage ES cell–derived
cardiomyocytes express ICaL as
well as the delayed rectifying K+ current. These
currents were regulated by both ß-adrenergic and muscarinic signaling
pathways, suggesting the normal course of development for signaling
components.16 In preliminary experiments, we found a
strong developmental increase in the chronotropic response of EBs to
ß-adrenergic stimulation.14 Therefore, in the
present study, the modulation of ICaL
was used as a functional assay to test different components of the
ß-adrenergic signaling cascade during cardiomyocyte
development (Figure 1
).
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Materials and Methods |
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Cell Cultures, Differentiation Procedure, and Preparation of Single Cardiomyocytes
The ES cell line D321 was used throughout the present study. These cells were cultivated and differentiated into spontaneously beating cardiomyocytes as previously described.14 15 16 Briefly, ES cells were kept in an undifferentiated, pluripotent state using mitomycin C–inactivated feeder layer of primary cultures of mouse embryonic fibroblasts. They were cultivated on gelatin (0.1%)-coated plastic Petri dishes in DMEM supplemented with 15% FCS (selected batches), L-glutamine (2 mmol/L), and nonessential amino acids. The main steps of differentiation included the following: (1) the cultivation of a definite number of cells (400) in "hanging drops" as EBs for 2 days, (2) cultivation as suspension in bacteriological dishes for 5 days, (3) plating of 7 days' (7d) EBs on 24–well-microwell plates. About 10% of these 7d EBs already contained contracting cardiomyocytes. In EB outgrowths, cardiomyocytes appeared in the form of spontaneously contracting cell clusters. The clusters increased in size during differentiation. Single cardiomyocytes were isolated from the clusters by enzymatic dissociation with collagenase using a modified procedure of Isenberg and Klöckner22 described previously.16 Cardiomyocytes were isolated at 3 distinct developmental stages: (1) the very early developmental stage (VEDS), when first spontaneously contracting clusters of cardiomyocytes appeared (from 7+1d to 7+2d), (2) the early developmental stage (EDS) (from 7+3d to 7+5d), and (3) the late developmental stage (LDS) (from 7+9d to 7+12d). Ventricular cardiomyocytes were isolated from adult mice by collagenase treatment as previously reported.22
Stable Recording of ICaL by
Whole-Cell Patch-Clamp Technique
The whole-cell configuration of the patch-clamp
technique23 was used throughout the present study.
This configuration allowed the cell to be dialyzed with various
compounds, testing the development of different components of the
ß-adrenergic signaling cascade. Stimulation effects of
ICaL were measured when
ICaL rundown was minimized. Fast rundown of
Ca2+ channel current has been previously reported
in embryonic mouse cardiomyocytes when
Ba2+ was used as a charge
carrier.6 This was also true for
cardiomyocytes differentiated from ES cells (authors'
recent unpublished observation). However, we found that
ICaL rundown was smaller when
Ca2+ was used as a charge carrier. Furthermore,
the intracellular ATP concentration was raised to 5 mmol/L to
minimize ICaL rundown.24
Under these conditions, the decrease in
ICaL amplitude was small particularly
during the first 10 minutes after establishment of the whole-cell
configuration using 1.8 mmol/L Ca2+ as a
charge carrier.
As previously reported,16 the size of
cardiomyocytes increases during development. For the
developmental stages characterized in the present study, VEDS, EDS,
and LDS, the average membrane capacities were 19.5±1.4 pF (n=16),
29.2±1.7 (n=39), and 35.9±2.4 pF (n=58), respectively. Because cell
constituents may be washed out more rapidly in smaller cells and lead
to possible artifacts, we ensured that this was insignificant for our
investigations on ICaL stimulation. Indeed,
the isoproterenol effect on ICaL measured
at 5 and 20 minutes after establishment of the whole-cell configuration
was identical (difference <10%, n=8; data not shown). Furthermore,
although cell dialysis depends even more on pipette size, we detected
no correlation between ICaL stimulation and
pipette size (pipette resistances from 0.8 to 4.5 M
). Our stable
recordings of ICaL allowed us to
accurately measure and compare effects of
ICaL stimulation at
physiological levels of
[Ca2+]o.
Measurements of ICaL
Stimulation
ICaL was measured under
voltage-clamp conditions by L/M EPC-7 patch-clamp amplifier (List
Electronic) or an Axopatch 200A (Axon Instruments) amplifier. Cells
were constantly superfused using a gravitational perfusion system, the
perfusion rate being 
2 mL/min. The chamber volume was 0.5 mL. The
temperature of the bath as well as of the perfusion solutions was kept
constant at 37°C. The pipette solution contained (in mmol/L)
CsCl 120, MgCl2 3, MgATP 5, EGTA 10, and HEPES 5
(pH 7.4; CsOH). The extracellular solution was of the following
composition (in mmol/L): NaCl 120, KCl 5,
CaCl2 1.8, TEA-Cl 20, MgCl2
1, and HEPES 10 (pH 7.4; TEA-OH). For some experiments on
ICaL stimulation, the concentration of
[Ca2+]o was raised to
3.6 mmol/L CaCl2 to increase the amplitude
of ICaL. The internal and external
solutions contained Cs+ and
tetraethylammonium, respectively, to
effectively block K+ current. The
ICaL peak was measured repetitively at a
test potential of 0 mV for 300 or 20 ms from a holding potential of
-40 or -50 mV. For the recording of the time course of peak
ICaL, depolarizing voltage pulses were
applied at a frequency of 0.2 Hz. To evaluate the degree of
ICaL stimulation, the maximum
ICaL density was taken before and after
drug application. The stimulation of ICaL
is reported in terms of the percentage of the increase of
ICaL density. The average stimulation
effect at a given developmental stage was calculated by averaging
individual cell responses. If not stated otherwise, only those cells
with an increase of ICaL density of >10%
after drug application were considered responding cells and included
into the statistics. The membrane capacity was measured by applying a
voltage ramp16 or by using appropriate software
(MFK). Current densities are expressed as the
ICaL peak value per capacity. The results
are presented as mean±SEM for n cells. The
statistical significance of mean values was determined by Student
t test for unpaired data. If not stated otherwise, the 2
pools of data were considered to be significantly different at
P<0.01.
Investigation of the Establishment of ß-Adrenergic Signal
Transduction During Development
The functional coupling of different components of the
ß-adrenergic signaling cascade was studied during
cardiomyocyte development. For this purpose,
ICaL peak amplitude was measured using
different agents. Key molecules involved in signal transduction and
specific molecular tools testing their functions are shown
schematically in Figure 1. The functional expression of
ß-adrenergic receptors was tested by extracellular application of
isoproterenol (1 µmol/L). The function of AC was tested by its
specific activator, forskolin (1 µmol/L). Maximum
stimulation of ICaL by cAMP was evaluated
by cell dialysis with the cAMP analog 8-bromo-cAMP (8-Br-cAMP; 400
µmol/L). The function of phosphodiesterases (PDEs) was tested by
using the nonselective inhibitor
isobutylmethylxanthine (IBMX) as well as the
selective inhibitors EHNA
(Erythro-9-[2-hydroxy-3-nonyl]adenine), milrinone, and rolipram for
the various PDE isoforms. The receptor-mediated
phosphorylation was investigated by coapplication of
forskolin (1 µmol/L) and the thiophosphorylating compound
ATP--S (2 mmol/L via patch pipette). The effect of
Ca2+ channel phosphorylation was
tested by cell dialysis with the catalytic subunit of PKA (7
µmol/L). ATP--S (1 mmol/L) was added into the pipette
solution in addition to the catalytic subunit of PKA to make channel
phosphorylation irreversible. To test the direct
stimulation of ICaL via
Gs protein, isoproterenol was applied
extracellularly when cells were dialyzed with 10 µmol/L of PKA
inhibitor (PKI) and 1 mmol/L GTP--S. Okadaic acid
(10 µmol/L) was used as a phosphatase type 1 and type 2A
inhibitor.
Source of Substances
Forskolin was purchased from Serva, the catalytic subunit of PKA
from Promega, and PKI 5-24 (lot LK-102) from Calbiochem. Cell
cultivation medium, FCS, and all other substances for cell cultures
were purchased from Gibco BRL. All the other substances were purchased
from Sigma Chemical Co.
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Results |
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Isoproterenol, Forskolin, and 8-Br-cAMP Stimulated ICaL in LDS but not VEDS Cardiomyocytes
The effect of isoproterenol on ICaL was different in VEDS and LDS cardiomyocytes. Although ICaL was insensitive to isoproterenol in VEDS cardiomyocytes (n=28), ICaL was stimulated by isoproterenol in all (n=24) LDS cardiomyocytes assayed (see examples in Figure 2
), suggesting the establishment of a
functional ß-adrenergic cascade during development. Peak current
amplitudes changed insignificantly (remaining within 10% of initial
value) during at least 5 minutes after isoproterenol application in
VEDS cells (in 25 of 28 cells) within the entire range of tested
voltages from -30 to 40 mV (Figure 2A). In those 3 responding
cells, the average increase in ICaL density
was very small (16±5%). In contrast, in LDS cells,
ICaL stimulation amounted to 70±8% (n=24,
measured at 0 mV). The stimulation of ICaL
was observed in the whole range of test potentials applied, from -30
to 40 mV (Figure 2B).
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) and after isoproterenol (Isoprot)
application (
). Original ICaL traces used
for the peak current determinations are shown on the right. The
membrane voltage levels are indicated at the respective traces.
Cm indicates membrane capacity.
The different sensitivity of the cells to isoproterenol may be a result
of the low expression of ß-adrenoceptors, as has been previously
reported for cardiomyocytes from the fetal murine
heart.25 If the low expression of ß-adrenoceptors was
the only reason for the unresponsiveness of VEDS cells to
isoproterenol, ICaL would still be
stimulated by either forskolin, an activator of AC or
8-Br-cAMP. We also noticed significant changes of
ICaL sensitivity to both forskolin (Figure 3A and 3B) and intracellular application
of 8-Br-cAMP (400 µmol/L) during development (Figure 3C and 3D). Whereas in VEDS cells forskolin or 8-Br-cAMP did not influence
ICaL, all LDS cardiomyocytes
were highly responsive to both drugs. ICaL
density increase amounted to 82±12% (n=16) and 156±32% (n=12) in
LDS cells treated with forskolin and 8-Br-cAMP, respectively (see
Table for absolute values of
ICaL densities). This indicated that the
full functional coupling of the ß-adrenergic signaling cascade was
established only in LDS cardiomyocytes. Conversely, the
lack of response to isoproterenol, forskolin, and 8-Br-cAMP in VEDS
cardiomyocytes (see Figures 2 and 3 and
Table) is consistent with a signaling defect at a number
of levels in these cells.
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ICaL Stimulation in EDS
Cardiomyocytes
To determine the critical period for the development of the
functional ß-adrenergic signaling cascade, we tested on a
"day-by-day" basis the effects of 8-Br-cAMP, isoproterenol, and
forskolin on ICaL (Figure 4). Most (71%, n=7) of 7+3d myocytes
responded to 8-Br-cAMP exposure with an average increase of
ICaL density of 18.6±3.2%. Cells of 7+4d
stage (79%, n=14) responded with a significantly larger increase of
ICaL density (68.2±12.1%). In 7+5d cells,
ICaL stimulation amounted to 84±21.8%
(n=7, 100% response rate). An important finding was that all cells
responded to 8-Br-cAMP after 7+5d, suggesting that significant
developmental changes occur during this short period from day 7+3d to
day 7+4d in the EDS cells. However, during this transitory period, the
stimulation of ICaL by isoproterenol still
remained relatively small, 38.7±5.1% (n=20, 45% response rate, see
Figure 4A and Table).
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A possible interpretation of these findings in EDS cells was a reduced
expression of functional ß-adrenoceptors and/or a defective coupling
to AC. We tested therefore different components of the signal
transduction cascade in EDS cells to identify the mechanism(s)
responsible for the observed changes. ICaL
was stimulated by forskolin in EDS cells (from 7+3d to 7+4d), but the
effect was small in comparison to LDS cells and could not be observed
in all cells tested (Figure 4B). The 7+3d cells responded (67%,
n=9) with a 33.3±10.5% ICaL density
increase. The stimulatory effect of forskolin on
ICaL was still relatively small (45±11.4%
ICaL density increase) in 7+4d cells (n=8,
100% of cells responding), whereas 7+5d cardiomyocytes
(n=17, all cells responding) displayed significantly greater
stimulation, ie, an 84.5±9.9% increase of
ICaL density. The forskolin effect was
strongly augmented by coapplication of forskolin plus ATP--S (Figure 5), particularly at the 7+4d stage. The
7+3d cardiomyocytes (n=19, 100% of cells responding)
showed a 68.2±15.4% stimulation of ICaL
density, and the 7+4d cells (n=14, 14 cells responding) responded with
a 131±30% stimulation. In contrast, in LDS cells, coapplication of
forskolin plus ATP--S resulted in a current density increase
(103±26%, n=3) that was similar to the stimulation produced by
forskolin alone (Figure 4B).
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Effect of ATP--S and Okadaic Acid on
ICaL in EDS and LDS Cells
Because coapplication of forskolin plus ATP--S in EDS cells
caused a strongly increased stimulation of
ICaL density compared with forskolin alone,
an involvement of phosphatases was suspected. Therefore, the
stimulatory effect of intracellular ATP--S (2 mmol/L)
application via the patch pipette was tested in EDS and LDS cells
(Figure 6A, 6B, and 6D).
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ATP--S resulted in a large increase of the basal
ICaL density (70.3±13%, n=5) in EDS
cells, whereas it augmented ICaL slightly
in LDS cells (9.7±15.6%, n=4, averaging all cells examined). A high
intrinsic phosphatase activity in EDS cells was further confirmed by
examining the effect of okadaic acid, an inhibitor of type
1 and type 2A phosphatases on ICaL density
at this developmental stage. Infusion of okadaic acid (10
µmol/L) via the patch pipette resulted in an increase of
ICaL density by 22±5% (n=5) (Figure 6C and 6D).
Functional Expression of PDEs in EDS Cells
Because 8-Br-cAMP stimulated ICaL
significantly stronger (about 2 times) than forskolin in 7+4d cells, an
involvement of PDEs in cAMP degradation was suspected. Therefore,
effects of nonselective and selective PDE inhibitors on
ICaL density were examined (Figure 7). IBMX, a nonselective PDE
inhibitor, had a strong stimulatory effect on
ICaL density (61±7.5%) in 7+3d and 7+4d
cardiomyocytes in all cells tested (n=21). The PDE II
inhibitor EHNA increased ICaL
density by 30.2±7.4% in 89% of cells (n=9). The PDE III blocker
milrinone stimulated ICaL density by
44.2±5.3% in 64% of the tested cells (n=17). Furthermore, the
cAMP-dependent PDE IV inhibitor rolipram increased
ICaL density by 41±9.3% in 69% of cells
tested (n=12).
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ICaL Was Stimulated by Cell Dialysis
With the Catalytic Subunit of PKA in Both VEDS and LDS Cells
The fact that ICaL in VEDS cells was
not stimulated by forskolin or 8-Br-cAMP indicated that uncoupling of
signal transduction at this very early developmental stage could also
occur downstream to the ß-adrenoceptor or AC, possibly as a result of
unresponsiveness of the Ca2+ channel molecule
itself. However, ICaL was strongly
stimulated in all VEDS cells tested (n=6) on intracellular application
of the catalytic subunit of PKA via the patch pipette. After
establishment of the whole-cell configuration,
ICaL density gradually increased. The
increase of ICaL density attained
saturation (increase by 213±25%, n=6) within 5 minutes of cell
dialysis. A similar stimulatory effect induced by the catalytic subunit
of PKA was observed in LDS cardiomyocytes (increase by
208±28%, n=10) as well as in ventricular
cardiomyocytes isolated from adult mice (increase by
202±15%, n=3). This indicates that during all developmental stages,
the channel itself or a closely associated protein can be effectively
phosphorylated, leading to an increase of
ICaL amplitude. The final
ICaL density was higher in LDS
cardiomyocytes than in VEDS cells (see Table),
reflecting an increase of ICaL density of
1.7 times during development (see also Reference 1616 ).
ICaL Stimulation by a Membrane-Delimited
Pathway in LDS but not VEDS Cardiomyocytes
In adult cardiomyocytes, a direct pathway for
ICaL stimulation independent of
cAMP-dependent phosphorylation is believed to be
mediated via direct interaction of Gs with
Ca2+ channels.26 Therefore, the next set of
experiments was designed to test whether ES cell–derived
cardiomyocytes also developed this alternative pathway. To
discriminate between these 2 pathways, the cAMP-dependent
phosphorylation was prevented by cell dialysis with the
peptide inhibitor 5-24, a known potent blocker of PKA
activity.27 In addition, GTP--S (1 mmol/L) was
included into the pipette to fully activate all available
Gs (see also Reference 2828 ).
Under these conditions, the amplitude of
ICaL varied insignificantly with time. The
reduction of ICaL remained within 10%
during the first 10 minutes of cell dialysis, similar to control cells
(see Figure 8A). The stability of the
ICaL amplitude was important for the
evaluation of the ß-adrenergic stimulation. To demonstrate that PKI
completely blocked the PKA activation, we tested whether inclusion of
PKI plus GTP--S in the patch pipette prevented forskolin (1
µmol/L)-mediated stimulation of ICaL in
LDS cells. Indeed, ICaL density did not
change on forskolin application in 6 cells 10 minutes after rupture of
the cell membrane (see example in Figure 8B). Accordingly, the
isoproterenol effect was assayed 10 minutes after starting cell
dialysis with PKI plus GTP--S. ICaL was
stimulated by isoproterenol (Figure 8C) in all LDS
cardiomyocytes (n=8), suggesting the functional activity of
the alternative pathway for ICaL
regulation. The stimulating effect of isoproterenol on
ICaL under these conditions was
31±7%.
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The existence of a distinct G protein–dependent pathway for ICaL regulation was also investigated in VEDS cells. As shown in the present study, ICaL in VEDS cells was unresponsive to isoproterenol. The reason for this could be a relatively weak G protein activity because of low intracellular GTP.
To rule out this possibility, additional experiments were performed
with VEDS cells, dialyzed with PKI and GTP--S, using the identical
experimental protocol as for LDS cardiomyocytes. None of
VEDS cells (n=6) responded to isoproterenol (Figure 8D),
suggesting the lack of this pathway during very early stages of cardiac
development.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, using the stimulation of ICaL as a functional assay, we have characterized important changes in the functional expression of components of the ß-adrenergic signaling cascade occurring during early stages of cardiomyogenesis. Although VEDS cardiomyocytes were insensitive to ß-adrenergic stimulation, LDS cardiomyocytes responded to isoproterenol with a large increase of ICaL density (by
70%), similarly as reported for mammalian
cardiomyocytes during postnatal development
(100%7 ). Our data are in line with reports that
embryonic mouse and rat cardiomyocytes are unresponsive to
ß-adrenergic stimulation.6 29 In addition, the
membrane-delimited pathway via direct Ca2+
channel activation by Gs previously reported for
adult cardiomyocytes26 30 was absent in VEDS
cells. In the present study, we took advantage of the ES cell–derived cardiomyogenesis to isolate cardiomyocytes at very early developmental stages, when the first contractions occurred.16 Optical recordings of contractions of rat embryos indicated that the paired cardiac primordia are just fused at the time of the first spontaneous contractions at the late period of the 3-somite stage.31 In contrast to the ES cell differentiation system, access to the mammalian heart at such an early developmental stage is complicated.31 For the VEDS and EDS cardiomyocytes, we developed an experimental protocol of relatively stable recordings of ICaL at physiological Ca2+ concentrations and temperature (37°C). The latter is particularly important with respect to the high temperature sensitivity of the regulatory enzyme cascade examined in the present study. This experimental approach allowed the characterization of the mechanisms responsible for changes in the ß-adrenergic modulation of ICaL that occurred during development.
Changes of ICaL Modulation During
Development
The lack of ß-adrenergic modulation in VEDS
cardiomyocytes suggests that one or more components of the
signaling cascade were missing or functionally inactive. To identify
these components, we assayed the functional activity of different
elements in the ß-adrenergic enzymatic cascade. A key finding was
that ICaL was strongly stimulated by cell
dialysis with the catalytic subunit of PKA in both VEDS and LDS
cardiomyocytes. In contrast to a previous
report6 in which fast current rundown was observed
using Ba2+ as a charge carrier, we could
accurately measure the stimulating effect of the catalytic subunit of
PKA on ICaL in VEDS
cardiomyocytes. The catalytic subunit of PKA produced a
maximal ICaL stimulation. Importantly,
ICaL stimulation by the catalytic subunit
of PKA was found to be similar (3 times) in VEDS, LDS, and
ventricular cardiomyocytes isolated from the
adult mouse heart. These data provide strong evidence for similar
structural properties of the Ca2+ channel or a
closely related protein during development at least in regard to
phosphorylation sites. Because
ICaL was strongly stimulated by the
catalytic subunit of PKA but relatively insensitive to either forskolin
or 8-Br-cAMP, one explanation for the lack of ß-adrenergic modulation
in VEDS cardiomyocytes could be a low expression of the PKA
holoenzyme, as suggested by An et al.6
The EDS cells proved to be particularly interesting, because forskolin
and 8-Br-cAMP already had a stimulatory effect on
ICaL density, but the majority of cells
still were not stimulated by isoproterenol. Furthermore, we detected a
strong stimulation of ICaL by the combined
application of forskolin plus the thiophosphorylating agent ATP--S.
This indicated an involvement of phosphatases in the modulation of
ICaL at this developmental stage. Our
hypothesis was confirmed by the experimental observation that ATP--S
led to a pronounced stimulation of basal
ICaL in EDS cells, whereas at the LDS cell
stage only a small stimulation was seen, similarly as reported for
guinea-pig ventricular
cardiomyocytes.32 These results were
further corroborated by experiments using the phosphatase (type 1 and
type 2A inhibitor) okadaic acid.33 EDS cells
dialyzed with okadaic acid displayed an increase of
ICaL density, suggesting that there is an
intrinsic AC activity as well as the functional expression of PKA
leading to the phosphorylation of Ca2+
channels at rest besides an intrinsic phosphatase activity. Because of
the intrinsic AC activity, we tested whether cAMP degradation was
controlled by intrinsic PDE activity. Indeed, application of the
nonselective PDE inhibitor IBMX caused a strong increase of
ICaL density. The use of selective PDE
inhibitors confirmed the functional expression of the
cGMP-dependent type II and III and the cAMP-dependent type IV PDE
isoforms. Thus, EDS cells are characterized by high intrinsic AC
activity, which is counterbalanced by high intrinsic activity of PDEs
and phosphatases.
The strong ICa stimulation by
ß-adrenergic agonists in the presence of ATP--S has been
previously demonstrated in adult
cardiomyocytes.34 Interestingly,
forskolin plus ATP--S led to a much stronger stimulation compared
with forskolin alone in 7+4d cells, whereas in 7+3d cells this effect
was significantly less pronounced. This suggests that at 7+3d, not all
ß-adrenergic signaling components are fully functional, and,
therefore, the role of phosphatases is less important. One day later,
however, nearly full coupling is established, and a strong functional
role of phosphatases in the dephosphorylation of
stimulated ICaL, even more pronounced than
in LDS cells, is observed.
We also tested for the establishment of the membrane-delimited pathway for ICaL regulation via direct interaction of Gs and the Ca2+ channel. This modulatory pathway was found only in LDS but not VEDS cardiomyocytes. One of the possible reasons for the lack of both the membrane-delimited and the cAMP-mediated functional coupling of ß-adrenoceptors may be a low level of the Gs protein.35 This is in line with the observation that in the mouse embryonic heart, ß-adrenoceptors appear before a detectable heart rate response to isoproterenol.25 Also, important developmental changes in the coupling between ß-adrenoceptors and G proteins have been recently reported for fetal rat heart.36
Possible Physiological Significance
Functional abnormalities of the cardiac sympathetic nervous
system during development were suggested to be involved in the genesis
of cardiac arrhythmias, particularly in sudden infant death
syndrome.37 Depressed function of
ß-adrenoceptors,38 deficient production of
cAMP,39 and altered expression of G
proteins40 are characteristic for
cardiomyocytes of failing hearts. Furthermore, an altered
coupling of G proteins was suggested to account for decreased
stimulation of ICaL by ß-adrenergic
agonists in hypertrophied8 and
diabetic41 hearts. The ß-adrenergic receptor system in
heart failure is markedly desensitized due to, at least in part,
altered expression of ß-adrenergic receptor kinase acting in concert
with an inhibitor protein, ß-arrestin.42 In
addition, cardiomyocytes from failing hearts have an
altered program of gene expression. Many studies demonstrate that
cardiac hypertrophy as well as heart failure is associated
with reexpression of an ensemble of genes characteristic of the
embryonic heart.10 11 These findings clearly show that
detailed knowledge about the establishment of the ß-adrenergic
signaling cascade during development is important for a better
understanding of abnormalities in signaling detected in heart disease.
Because cardiac disease states are characterized by hyporesponsiveness
to ß-adrenergic agonists, it is tempting to speculate that high PDE
as well as phosphatase activities may contribute, similar to our
findings in EDS cells, to this functional disorder.
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Acknowledgments |
|---|
This research was supported by Deutsche Forschungsgemeinschaft (DFG, SFB 366 YW1/YE1), Ministry of Science and Research of Sachsen-Anhalt, and ZEBET, Bundesgesundheitsamt, of FRG. The skillful technical assistance of Oda Weiss, Sabine Sommerfeld, Marianne Faulhaber, and Birgit Hops is gratefully acknowledged.
Received August 6, 1998; accepted October 22, 1998.
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References |
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