Download Adrenergic regulation of conduction velocity in cultures of immature

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Interuniversity Cardiology
Institute of the Netherlands
Adrenergic regulation of conduction velocity in
cultures of immature cardiomyocytes
T. P. de Boer, H.V.M. van Rijen, M.A.G. van der Heyden, J.M.T. de Bakker, T.A.B. van Veen
During cardiac maturation, increased exposure of the
heart to circulating catecholamines correlates with
increased conduction velocity and growth of the heart. We
used an in vitro approach to study the underlying
mechanisms of adrenergic stimulation induced changes in
conduction velocity. By combining functional measurements and molecular techniques, we were able to
demonstrate that the increased conduction velocity after
β-adrenergic stimulation is probably not caused by
changes in intercellular coupling. Instead, RT-PCR
experiments and action potential measurements have
shown an increased excitability that may well explain the
observed increase in conduction velocity. Apart from
being relevant to cardiac maturation, our findings are
relevant in the context of stem cells and cardiac repair. Preconditioning of stem cell derived cardiomyocytes may
help to enhance electrical maturation of de novo generated
cardiomyocytes and consequently reduce their proarrhythmogenic potential. (Neth Heart J 2008;16:106-9.)
Keywords: action potential, catecholamines, cardiomyocytes,
gap junction, impulse propagation, ion channel
T.P. de Boer
H.V.M. van Rijen
M.A.G. van der Heyden
T.A.B. van Veen
Department of Medical Physiology, Division of Heart & Lungs,
University Medical Center Utrecht, the Netherlands
J.M.T. de Bakker
Interuniversity Cardiology Institute of the Netherlands, Utrecht and
Heart Failure Research Center, Academic Medical Center, Amsterdam,
the Netherlands
Correspondence to: T.A.B. van Veen
Department of Medical Physiology, Division of Heart & Lungs,
University Medical Center Utrecht, PO Box 85500, 3508 GA Utrecht,
the Netherlands
E-mail: [email protected]
106
he autonomic nervous system equips the heart with a
delicate regulatory mode to adapt rhythm and contractility
to continuously changing physiological demands. Whereas
the intrinsically predominant parasympathetic side of the
system tends to temper rhythm and contractility, activation
of the sympathetic side enables a rapid increase in rhythm and
contractility in order to force cardiac output under conditions
of physical exercise, fear or stress. The mechanism through
which the heart is able to increase its rhythm is well known
and relies on a hyperexcitability of the autorhythmic cells in
the sino-atrial node which in turn is triggered by an increased
calcium influx through phosphorylation of voltage-gated
calcium channels. More generally, adrenergic stimuli are
important determinants of cardiac function throughout life.
In immature stages of the heart, development of cardiac
innervation coincides with increasing levels of circulating
catecholamines and an increase in expression of β-adrenoreceptors.1 After birth, cardiomyocyte hyperplasia decreases
and is followed by hypertrophic growth and maturation of
cardiomyocytes.2 In this process, also the initially slow impulse
conduction becomes faster.3 In contrast to the effect of
adrenergic stimulation in the context of cardiac rhythm, the
effect on conduction velocity is less clear.
Impulse propagation throughout the heart is determined
by several factors. Cell-to-cell propagation is facilitated by
specialised membrane proteins that form conducting channels
(gap junctions) which connect cells electrically and are
encoded by the genes connexin 40, 43 and 45.4-8 These gene
products constitute hemichannels that can connect to a hemichannel expressed by an adjoining cell, thus forming an
intercellular junction that allows exchange of ions and small
molecules. Efficiency of intercellular coupling by gap junctions
is determined by the number of channels expressed, open
probability of the expressed channel, conductance of the
channel when in its open state and the specific characteristics
of the involved isoform.9
A second, important factor in impulse propagation is the
intrinsic excitability of the cardiomyocytes, which is de-
T
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A
B
C
Figure 1. A: Geometrically defined preparation used in this study (left panel). Notice that cardiomyocytes adhered in the star-shaped pattern
only. Typical extracellular potentials recorded from the embedded electrodes are depicted in the right panel. B: Conduction velocity was
increased in ISO-stimulated preparations, but not in control preparations after 24 hours. C: Action potentials were recorded and upstroke
velocity was determined, demonstrating a faster upstroke after ISO treatment. *p<0.05. Figure adapted with permission from De Boer et al.10
termined by the expression of ion channels. In mature hearts,
sodium channel expression enables a fast upstroke of the action
potential. In immature hearts, action potential upstroke is
slower because depolarisation of cardiomyocytes highly
depends on calcium channels that have relatively slow kinetics
when compared with the less abundant fast sodium channels.
Since cardiomyocytes with rapid upstrokes are faster in exciting
a neighbouring cardiomyocyte, upstroke velocity is an important determinant of conduction velocity.
A third factor that affects impulse propagation is tissue
architecture, which in our cell culture model involves cardiomyocyte size and shape. These parameters affect conduction
velocity by altering cytoplasmic resistance.
As the increase in circulating catecholamines in the
developing neonatal heart is associated with an increase in
conduction velocity, it is not unlikely that adrenergic stimuli
simultaneously modulate the different determinants of conduction velocity. Elucidating this process in an intact heart
during development is highly complex as tissue geometry,
excitability and intercellular communication undergo rapid
changes. In order to address this issue, we used a reductionistic
approach by subjecting neonatal rat cardiomyocytes, which
were cultured on geometrically defined substrates, to
adrenergic stimuli. Extracellular electrodes embedded in the
substrate allowed us to measure conduction velocity using
the same preparations (figure 1A), before and after 24-hour
stimulation with isoproterenol (ISO, β-adrenergic agonist)
or phenylephrine (PE, α-adrenergic agonist). In addition we
determined action potential waveform, cell capacitance and
expression levels of cardiac connexins and ion channels.
Results
Measurement of conduction velocity demonstrated that under
control conditions, preparations measured at t=0 h and
t=24 h revealed similar conduction velocities (30.9±1.9 and
32.4±4.4 cm/s respectively, n=7, p<0.70). Stimulation with
Netherlands Heart Journal, Volume 16, Number 3, March 2008
the β-adrenergic agonist ISO, (100 nM) for 24 hours resulted
in a significantly increased conduction velocity, from 28.0±2.0
to 34.8±2.2 cm/s at t=0 h and t=24 h, respectively (n=5,
p<0.002, see figure 1B). After 24 hours of stimulation with
the α-adrenergic agonist PE (10 µM), no increase in conduction velocity was detected (29.3±5.8 at t = 0 h vs. 26.3±1.8
cm/s at t=24 h, n=4, p<0.57).
Proceeding experiments in which we used micro-electrodes
to measure action potential waveform demonstrated that the
increased conduction velocity after β-adrenergic stimulation
was accompanied by an increased upstroke velocity of the
action potential (figure 1C). Control preparations had an
average upstroke velocity of 33.9±3.6 V/s (n=12), which
increased significantly to 52.6±7.7 V/s (n=11) after 24 hours
of stimulation with ISO.
An important prerequisite to validate the use of our simplified model to determine the molecular mechanism underlying
the observed alterations is that tissue geometry remains
preserved. Since the substrates on which cells are cultured are
defined and fibrosis is not present, changes in geometry would
be due to changes in cell size only. Analysis of cell size by
immunohistochemistry did not, however, indicate that cell
size was affected by adrenergic stimuli. To verify this in an
alternative way and to be able to quantify cell size, we used
voltage clamp techniques to measure cell capacitance; a
reflection of membrane surface area and thus cell size. These
measurements indeed confirmed that no differences were
observed between control cells (16.7±1.9 pF, n=6) and ISOtreated cells (18.6±2.0 pF, n=5).
To identify which molecular substrate had been responsible for the increased CV upon β-adrenergic stimulation,
expression of cardiac gap junction proteins was first evaluated.
RT-PCR revealed no differences in the expression of Cx40,
Cx43 and Cx45 (figure 2B). On the protein level, no signals
were detected for Cx40 and Cx45 in both control and
stimulated preparations, which was probably caused by the very
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low expression levels. We did detect expression of Cx43, the
main ventricular connexin isoform, which was visualised on
a Western blot in a typical three-banded pattern (n=4, figure
2A). In this pattern, one unphosphorylated (P0) and two
phosphorylated bands (P1 and P2) were found. No changes
in total protein expression were detected as the total density
of the three bands was similar under control and ISO conditions. We did, however, observe an increase in phosphorylation of Cx43, as evidenced by increased density of the P2
band. This shift in phosphorylation was absent in preparations
pretreated with atenolol, a specific β1-antagonist. The two
phosphorylated forms of Cx43 are commonly regarded to
constitute functional channels in the sarcolemma while
dephosphorylation of the protein is related to degradation
which is increased under pathophysiological conditions.
During myocardial ischaemia, dephosphorylation of Cx43
results in intercellular uncoupling, increased degradation of
Cx43, heterogeneity of impulse propagation and development
of arrhythmogeneity. Western blots additionally showed no
differences in expression of structural proteins such as α-actinin
and desmin, which underlines the absence of alterations in
cell size and shape (data not shown).
To evaluate potential effects on expression of ion channels,
we performed semi-quantitative RT-PCR analysis (n=4,
figure 2B). After 24 hours of stimulation with ISO we
observed an increase in expression of both the L-type calcium
channel (α1C) and to a lesser extent the cardiac sodium
channel (SNC5A). Equal loading in those experiments was
confirmed by equal expression of GAPDH, while specificity
was confirmed by the absence of PCR product without reverse
transcriptase (-RT).
To determine whether increased conduction velocity
could be attributed specifically to β-adrenergic stimulation,
similar experiments were performed with preparations
stimulated with the α-adrenergic agonist phenylephrine (10
µM). No changes were detected in Cx43 protein expression,
and mRNA levels of Cx40, Cx43, Cx45, SCN5A and α1C
were similar in control and phenylephrine-treated preparations. Together, these molecular findings are in line with
the absence of changed conduction velocity after α-adrenergic
stimulation.
Discussion
In this study, experiments with geometrically defined cultures
of immature cardiomyocytes demonstrated that an increased
conduction velocity that is induced by β-, but not α-adrenergic
stimulation, can most likely be attributed to changes in
intrinsic excitability of the cardiomyocytes.
The first important determinant of conduction velocity,
gap junctional coupling, was not significantly altered by βadrenergic stimulation, as demonstrated by similar expression
levels of the predominantly expressed isoform Cx43. Since
Western blots indicate an increased phosphorylation upon
stimulation with ISO and previous investigations indicated
that phosphorylation of the protein increases the open
probability of the channels,9 we cannot exclude that this effect
could have contributed to the increased conduction velocity
that we measured. Nonetheless, several studies using knockout
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A
B
Figure 2. A: Western blot analysis of Cx43 expression and
phosphorylation in control preparations and preparations
stimulated with ISO, demonstrating similar total expression, but
increased phosphorylation after ISO. B: Semi-quantitative RTPCR experiments showed unchanged expression of the cardiac
connexins after 24 h ISO. In contrast, α1C and to a lesser extent
SCN5A showed increased mRNA expression. Figure adapted with
permission from De Boer et al.10
strategies have indicated that changes in gap junctional
coupling have to be very robust in order to affect conduction
velocity.11,12 Conduction velocity in hearts of transgenic mice
with a 50% reduction of Cx43 protein was comparable with
control mice, which indicates that the safety factor of conduction with respect to the expression of gap junctions is
rather large.
Since the geometry of our preparations is defined, and no
changes were detected in cell size either, the observed
increased conduction velocity is probably related to increased
intrinsic excitability of the cardiomyocytes. Indeed, we found
both functional (increased upstroke velocity) and molecular
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(increased expression of α1C and SCN5A mRNA) evidence
supporting this concept. Although we did not determine
whether β-adrenergic stimulation functionally increased
current density of calcium and sodium currents in our preparations, other researchers have demonstrated increased
calcium current density in neonatal cardiomyocytes after
stimulation with ISO.13,14 Experiments on genetically modified
mouse models in which the molecular substrate underlying
excitation was modulated revealed that apparently the safety
factor for conduction is relatively sensitive to changes in
excitability when compared with changes in intercellular
coupling. In that respect, moderate changes in expression of
calcium and sodium channels might well have functional
consequences. A 50% reduction in sodium channel expression
reduces conduction velocity by about 18%, whereas a 50%
reduction in connexin43 expression does not affect conduction velocity.
Of course, extrapolation of our results to adrenergic
regulation of conduction in adult hearts is limited by the use
of immature cardiomyocytes in our model. Not only the
expression levels and distribution of gap junction proteins
and ion channels might differ between the two stages of
maturation, also the fact that in immature cardiomyocytes
upstroke of the action potential is facilitated by gating of both
calcium and sodium channels while in mature hearts gating
of sodium channels is the principal determinant. However, our
model makes it possible to study changes observed during
cardiac maturation, and the finding that β-adrenergic
stimulation increases conduction velocity is interesting for
related topics as well. The potential use of stem cell derived
cardiomyocytes in cardiac repair strategies will require cardiomyocytes that are electrically and mechanically compatible
with the recipient (mature) myocardium in order to support
its compromised performance and to avoid an increased
propensity to develop cardiac arrhythmias. Most sources of
in vitro generated cardiomyocytes have electrical characteristics
comparable with the cardiomyocytes used in this study.
Automaticity, sensitivity to adrenergic stimulation, action
potentials with a clear phase 4 depolarisation, and gating of
calcium channels as the primarily responsible determinant for
the upstroke of the action potential are clear alignments. With
their highly reproducible isolation and culture procedures, in
vitro studies like this can provide useful information on the
mechanisms that regulate electrophysiological maturation.
Insight into this regulation might make it possible to direct
in vitro generated cardiomyocytes into a requested phenotype
suitable to use in transplantation strategies in order to repair
different forms of cardiac tissue (e.g. nodal, ventricular) or in
testing of cardiovascular drugs with a specific mode of activity.
Netherlands Heart Journal, Volume 16, Number 3, March 2008
Acknowledgements
S.J.A. Tasseron and S.C.M. van Amersfoorth (Department of
Experimental Cardiology, AMC, Amsterdam) are kindly
acknowledged for their contribution in coating the specific
preparations and for isolation and culture of the cardiomyocytes on these preparations. This study was supported by
the Netherlands Heart Foundation (grant 2003B07304,
TdB), the Technology Foundation (STW programme DPTE,
grant #MKG5942, MAGvdH and grant UGT.6746,
TABvV) and the Netherlands Organisation for Scientific
Research (NWO, grant 916.36.012, TABvV). ■
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