© 2000 American Heart Association, Inc.
Cellular Biology |
Sinoatrial Node Pacemaker Activity Requires Ca2+/Calmodulin-Dependent Protein Kinase II Activation
From the Laboratory of Cardiovascular Sciences (T.M.V., Y.-Y.Z., K.Y.B., D.Y., M.K., H.C., R.-P.X.), National Institute of Aging, Gerontology Research Center, Baltimore, Md, and National Laboratory of Biomembrane and Membrane Biotechnology (D.Y., H.C.), College of Life Sciences, Beijing University, Beijing, China.
Correspondence to Rui-Ping Xiao, MD, PhD, Laboratory of Cardiovascular Science, Gerontology Research Center, NIA, NIH, 5600 Nathan Shock Dr, Baltimore, MD 21224. E-mail xiaor{at}grc.nia.nih.gov \ © 2000 American Heart Association, Inc.
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Abstract |
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Abstract—Cardiac beating arises from the spontaneous rhythmic excitation of sinoatrial (SA) node cells. Here we report that SA node pacemaker activity is critically dependent on Ca2+/calmodulin-dependent protein kinase II (CaMKII). In freshly dissociated rabbit single SA node cells, inhibition of CaMKII by a specific peptide inhibitor, autocamtide-2 inhibitory peptide (AIP, 10 µmol/L), or by KN-93 (0.1 to 3.0 µmol/L), but not its inactive analog, KN-92, depressed the rate and amplitude of spontaneous action potentials (APs) in a dose-dependent manner. Strikingly, 10 µmol/L AIP and 3 µmol/L KN-93 completely arrested SA node cells, which indicates that basal CaMKII activation is obligatory to the genesis of pacemaker AP. To understand the ionic mechanisms of the CaMKII effects, we measured L-type Ca2+ current (ICa, L), which contributes both to AP upstroke and to pacemaker depolarization. KN-93 (1 µmol/L), but not its inactive analog, KN-92, decreased ICa, L amplitude from 12±2 to 6±1 pA/pF without altering the shape of the current-voltage relationship. Both AIP and KN-93 shifted the midpoint of the steady-state inactivation curve leftward and markedly slowed the recovery of ICa, L from inactivation. Similar results were observed using the fast Ca2+ chelator BAPTA, whereas the slow Ca2+ chelator EGTA had no significant effect, which suggests that CaMKII activity is preferentially regulated by local Ca2+ transients. Indeed, confocal immunocytochemical imaging showed that active CaMKII is highly localized beneath the surface membrane in the vicinity of L-type channels and that AIP and KN-93 significantly reduced CaMKII activity. Thus, we conclude that CaMKII plays a vital role in regulating cardiac pacemaker activity mainly via modulating ICa, L inactivation and reactivation, and local Ca2+ is critically involved in these processes.
Key Words: sinoatrial node • L-type Ca2+ channel • Ca2+/calmodulin-dependent kinase II • local Ca2+ signaling
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Introduction |
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The human heart faithfully supplies blood to the body by beating more than 3 billion times in a lifetime. The sinoatrial (SA) node possesses automaticity and serves as the primary physiological pacemaker of the heart. The pacemaker action potential (AP) is initiated in a small group of primary pacemaker cells located in the center of the SA node and then propagates through transitional, peripheral regions to the atrial tissue.1 2 A number of ionic currents are involved in the SA node pacemaker activity, including 2 delayed rectifier potassium currents (mainly the rapidly activated component, IKr), L- and T-type Ca2+ currents (ICa, L and ICa, T), hyperpolarization-activated cation current (If), and others.3 4 In primary SA node cells, ICa, L plays an obligatory role in the generation of rhythmic spontaneous APs, because ICa, L is an important source of inward current for the AP upstroke4 and diastolic depolarization.5 6
Ca2+/calmodulin-dependent protein kinase II (CaMKII), a ubiquitous and multifunctional enzyme, is widely involved in Ca2+-dependent cellular processes. Signals that increase [Ca2+]i activate CaMKII.7 This kinase also retrospectively targets an array of molecules that affect Ca2+ levels. In the heart, CaMKII regulates the sarcoplasmic reticulum (SR) Ca2+ cycling by phosphorylating the Ca2+ release channels and the SR Ca2+-ATPase regulator, phospholamban.8 9 In ventricular myocytes, activated CaMKII localizes closely to cardiac sarcolemmal membranes and mediates a frequency- and Ca2+-dependent facilitation of ICa, L, which counteracts the voltage- and Ca2+-dependent inactivation of the channel.10 11 12 Furthermore, autophosphorylated (active) CaMKII retains its catalytic activity even in the absence of an increase in [Ca2+]i13 14 15 ; this biochemical property enables CaMKII to prolong the action of a transient Ca2+ signal or to function as a "frequency detector" of repetitive Ca2+ signals, which makes it ideally suited for the regulation of rhythmic activities such as heart beats.
Given the important role of ICa, L in initiating SA node pacemaker activity and the role of CaMKII in modulating ICa, L, we hypothesize that CaMKII may be critically involved in the regulation of SA node spontaneous excitations. Here we examined the effects of CaMKII on SA node spontaneous excitation and underlying ionic mechanisms, particularly modulation of ICa, L. The present results demonstrate that inhibition of CaMKII with a specific peptide inhibitor or a synthetic inhibitor, KN-93, can completely arrest SA node cells largely as a result of depressed ICa, L amplitude, reduced window current, and slowed recovery of L-type Ca2+ channels from inactivation. This finding shows, for the first time, a pivotal role of CaMKII in regulating cardiac pacemaker activity.
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Materials and Methods |
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SA Node Cell Isolation
Single SA node cells were isolated according to the modified method of Ito and Ono.16 Albino rabbits weighing 1.8 to 2.2 kg were deeply anesthetized with sodium pentobarbital (50 to 90 mg/kg). The heart was rapidly excised, and the SA node region was excised and cut into small strips (0.5 to 1.0 mm wide) perpendicular to the crista terminalis. The strips were first incubated in Ca2+-free Tyrode solution containing (in mmol/L) NaCl 140, KCl 5.4, MgCl2 0.5, NaH2PO4 0.33, HEPES 5, and glucose 5.5 (pH 6.9) at 33°C and then in the Ca2+-free Tyrode solution containing elastase type IIA (0.6 mg/mL; Sigma), collagenase B (0.6 mg/mL; Boehringer Mannheim), and 0.1% BSA for 30 minutes at 33°C. Thereafter, the strips were washed and dispersed in a modified Kraftbruhe solution by gentle pipetting and were stored at 4°C.
Electrophysiological Recordings
Perforated- or ruptured-patch-clamp techniques were
used to record spontaneous APs or ICa,
L, respectively, with Axopatch-1D patch-clamp amplifier
(Axon Instruments). The bath temperature was maintained at 34±0.5°C.
For perforated-patch experiments, amphotericin B (400 µg/mL, Sigma)
or ß-escin (40 to 50
µmol/L,17 Sigma)
was added to the pipette solution. All potentials were corrected by
subtracting the pipette-to-bath liquid junction potential, which was 13
mV for the perforated patch recordings of APs, as calculated using the
Clampex 7 software package (Axon Instruments).
For ICa, L recordings, depolarizing voltage-clamp pulses (300 ms) were applied from a holding potential of -50 mV; 10 to 30 µmol/L tetrodotoxin and 4 mmol/L 4-aminopyridine were added to block interfering currents. Activation-voltage relationships of ICa, L were estimated from normalized conductance-voltage curves.11 18 Steady-state inactivation-voltage relationships of ICa, L were measured using a 2-pulse protocol in which a variable-amplitude prepulse of 2000 ms was followed by a 200-ms test pulse to 0 mV from a holding potential of -70 mV. To measure the time required for recovery of Ca2+ channels from voltage-dependent inactivation, the cell was depolarized to 0 mV for 300 ms to inactivate Ca2+ channels, then repolarized to -50 mV for various durations to allow channel recovery (20 to 2300 ms), and finally depolarized to 0 mV for 300 ms. The percentage of ICa, L restored in the test pulse was used to calculate the channel recovery.
Immunocytochemical Staining of CaMKII
The intracellular distribution of total and
autophosphorylated (active) CaMKII was visualized in isolated SA node
cells by confocal fluorescence
microscopy.10 Cells
were incubated overnight at 4°C with the monoclonal CaMKII antibodies
(Affinity Bioreagents Inc), followed by an incubation for 4 hours with
a Texas Red–conjugated anti-mouse antibody (Vector Laboratories Inc).
Immunostaining of an autophosphorylated form of CaMKII was repeated
after 30-minute pretreatment of cells with either of the CaMKII
inhibitors KN-93 or autocamtide-2 inhibitory peptide (AIP).
Immunostaining was then detected with a laser scanning confocal
microscope (LSM-410, Zeiss).
Statistical Analysis
Data are given as mean±SEM. The difference of mean
values were analyzed by Student t test; ANOVA was used
to compare groups of data, and P<0.05 was considered
statistically significant.
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
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Results |
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Inhibition of CaMKII Suppresses Spontaneous AP in SA Node Cells
We studied spontaneous excitation of freshly isolated, spindle-shaped SA node cells using the perforated patch-clamp technique (Figure 1
). Under control conditions at 34°C, parameters of
AP were as follows: amplitude, 84.2±2.9 mV; duration at 50%
repolarization, 102.2±5.7 ms; upstroke velocity (dV/dt), 8.5±1.0 V/s;
and cycle length, 357.1±17.0 ms (n=24). The diastolic depolarization
rate was 57.6±3.8 mV/s, and the maximum diastolic potential (MDP) was
-65.5±1.4 mV (n=24). These values are highly comparable with those
reported
previously.4
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Figure 1
shows effects of a highly specific peptide
inhibitor of CaMKII (AIP, membrane-permeable form, 10 µmol/L) on SA
node pacemaking. This peptide, which corresponds to an
"autoinhibitory" domain of
CaMKII,19 decreased
AP amplitude and absolute value of MDP, reduced the rate of spontaneous
excitations of the SA node cells, and in 3 out of 4 experiments
completely arrested SA node cells
(Figure 1). These effects were achieved within 20 to 30
minutes and were largely reversible on washout
(Figure 1A). Further evidence that SA node pacemaking depends
on CaMKII was obtained by superfusing cells with a specific CaMKII
inhibitor, KN-93.20
Figures 2A through 2C show representative continuous
recordings of APs in the absence (left panels) and presence (right
panels) of KN-93, at different concentrations. KN-93 at a low
concentration (0.3 µmol/L) reduced the rate of spontaneous
excitations (P<0.05) and absolute value of MDP
(Figure 2A); these effects were reversible on washout of the
inhibitor (data not shown). In the presence of 1 µmol/L KN-93, the
negative chronotropic effect was more pronounced; excitations became
irregular, and AP amplitudes became unstable. The disturbed pacemaker
activity was associated with a 
10-mV reduction of MDP
(Figure 2B). More strikingly, application of a higher
concentration of KN-93 (3 µmol/L) completely abolished spontaneous
excitations
(Figure 2C). In contrast, KN-92 (1 µmol/L, n=6), an
inactive KN-93 analogue, had no significant effect on any examined AP
parameter
(Figure 2D), confirming the specificity of the CaMKII
inhibitor.
Figures 3A through 3D summarize the average effects of KN-93
on AP parameters that were similar to those induced by AIP
(Figure 1B). The CaMKII inhibitor induced a robust change in
all AP parameters in a dose-dependent manner. Spontaneous excitations
were abolished in 2 of 6 cells and in 4 of 4 cells by KN-93 at
concentrations of 1 and 3 µmol/L, respectively. Taken together, our
data indicate that CaMKII plays an essential role in SA node pacemaking
and that basal CaMKII activation is indispensable for the SA node cell
to generate APs.
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) of MDP. D, Maximum velocity of AP upstroke (dV/dt).
*P<0.05, 
P<0.01 vs control values
(n=4 to 6).
Effects of CaMKII on
ICa, L
To delineate the ionic mechanisms underlying
CaMKII-mediated modulation of SA node pacemaker activity, we measured
ICa, L in the presence or
absence of the CaMKII inhibitors, using a whole-cell patch-clamp
configuration. KN-93 (1 µmol/L) induced a 50% decrease in
ICa, L amplitude (from 12±2 to
6±1 pA/pF at 0 mV, P<0.01) without altering the
current-voltage relationship
(Figure 4A). The time-dependent inactivation of
ICa, L fitted by a
double-exponential function was not significantly altered by KN-93
(
f=6.9±0.27 ms and
s=37.6±1.4 ms [n=19] in control cells;
f=7.7±0.31 ms and
s=33.9±1.4 [n=10] in KN-93–treated
cells). The inactive analog KN-92 (1 µmol/L) had no significant
effect on ICa, L amplitude
(13±1 pA/pF at 0 mV [n=3, P>0.05 versus control])
or other parameters examined (data not shown).
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To define the specific mode of CaMKII-mediated modulation of
ICa, L , we examined the
voltage-dependent activation and steady-state inactivation of
ICa, L. As shown in
Figure 4B, the threshold for
ICa, L activation was -40
mV, and the saturation occurred at +10 mV in the SA node cells.
Inhibition of CaMKII (KN-93, 1 µmol/L) had virtually no effect on the
slope factor (7.3±0.2 mV in controls versus 7.4±0.4 mV with KN-93),
the midpoint voltage
(Vh=-16.1±1.3 mV in control
versus -16.1±1.5 mV with KN-93), or the overall shape of the
activation curve. In contrast, inhibition of CaMKII had a profound
effect on the steady-state inactivation of the channel, as manifested
by a parallel shift of the inactivation curve by 11.2±1.1 mV
(P<0.01) toward more negative potentials
(Figure 4B). In the presence of KN-93, the midpoint
inactivation voltage was -45.2±1.3 mV, and the slope factor for
inactivation was 6.1±0.2 mV, which indicates that a substantial number
of L-type channels must be inactivated at the MDPs (-54.4±3.2 mV,
n=4) in the presence of CaMKII inhibitor
(Figure 4B). Similarly, AIP (100 µmol/L in the pipette
solution, >30 minutes) shifted the steady-state inactivation curve of
ICa, L leftward by 5.3±0.3 mV
(P<0.01) without altering the activation curve
(Figure 5A). Thus, CaMKII activity is required to alleviate
the steady-state inactivation of L-type Ca2+
channels, maintaining the channel availability in the pacemaker
cells.
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In cells undergoing rhythmical excitations, L-type channel
recovery from inactivation is another important determinant of channel
availability. To gain further insight into the CaMKII-mediated
modulation of pacemaker activity, we measured the time course for
recovery of ICa, L from
inactivation using a dual-pulse protocol (see Materials and Methods).
The results are summarized in
Figure 5B. At a holding potential of -50 mV, the recovery
of ICa, L was fitted by 2
exponentials, with a fast component being predominant
(1=71 ms,
A1=70%;
2=612 ms,
A2=30%). The role of CaMKII in
L-type channel reactivation was confirmed by the fact that AIP markedly
slowed the recovery of Ca2+ channels from
inactivation (1=174 ms,
A1=50%;
2=1085 ms,
A2=50%)
(Figure 5B). In the presence of KN-93 (1 µmol/L, n=6), both
time constants were also markedly prolonged
(1=120 ms; 2=2000
ms), with the slow component being dominant
(A1=26%;
A2=74%). KN-92 (1 µmol/L,
n=6), the inactive analogue of KN-93, had no significant effect on the
recovery kinetics of the channel. These results reveal another mode of
CaMKII action, ie, ensuring the reactivation of L-type channels during
each excitation cycle. The markedly slowed recovery of the channel from
inactivation contributes, at least in part, to KN-93–induced reduction
of ICa, L amplitude depicted in
Figure 4A. The effects of AIP and KN-93 on
ICa, L indicate that some basal
CaMKII activity exists in SA node cells dialyzed with 10 mmol/L EGTA,
as is the case in rat ventricular
myocytes.10
The results described above suggest that the
inhibitory effects of CaMKII inhibitors on SA node pacemaker activity
are largely mediated by suppressing ICa,
L activation and reactivation. Next, we directly tested
this idea using an L-type Ca2+ channel
blocker, nifedipine (n=4). A representative example is shown in
Figure 6. Similar to the CaMKII inhibitor, nifedipine (0.2
µmol/L) depressed all of the parameters of AP, including AP
frequency, amplitude, rate of AP upstroke, and reduced MDP
(Figure 6B). After a 5-minute perfusion, nifedipine
completely arrested the pacemaker cell
(Figure 6C). Thus, the effects of CaMKII inhibition on the
pacemaker activity can be largely mimicked by a selective blockade of
ICa, L.
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Intracellular Localization of CaMKII
To determine intracellular distribution of
autophosphorylated (active) as well as total CaMKII in the SA node
cell, we used a site-specific antibody against CaMKII phosphorylated at
Thr286 and another antibody that recognizes CaMKII regardless of its
phosphorylation state. Confocal immunofluorescence imaging showed that
active CaMKII was concentrated beneath the surface membrane
(Figure 7B), whereas total CaMKII was present uniformly in
the SA node cell
(Figure 7A).
Figure 7C shows the negative control image obtained in the
absence of any primary antibody; the nonspecific staining was
negligible. The restricted localization of active CaMKII to the surface
membrane is consistent with the idea that CaMKII targets sarcolemmal
membrane–delimited substrates, particularly L-type
Ca2+ channels, and that CaMKII activity is
likely regulated by local Ca2+ gradients in
the submembrane microdomains (see below). Pretreatment of cells with
agents blocking CaMKII activity, KN-93 or AIP, significantly decreased
the amount of the active form of CaMKII
(Figure 7D), which substantiates the idea that both agents
acted through inhibition of CaMKII.
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Role of Local Ca2+
in CaMKII-Mediated Modulation of Pacemaker Activity
Theoretically, Ca2+ influx
through L-type channels as well as ICa,
L-triggered Ca2+ release from
the abutting SR may generate a local increase in
[Ca2+], the activator of CaMKII. We
therefore hypothesized that CaMKII is preferentially activated by local
high Ca2+ transients in the microdomain of
the surface membrane and that the active CaMKII, in
turn,mediates a positive feedback regulation of L-type
channels, which contributes to the pacemaker activity. To test this
hypothesis, we examined susceptibility of pacemaker APs and
ICa, L to
Ca2+ buffers with different kinetics, EGTA
and BAPTA. Because the kinetics of BAPTA are 100-fold faster than
those of EGTA,21
BAPTA is much more efficient in buffering local
Ca2+ transients, even though both can
effectively suppress global Ca2+ transients.
Confocal imaging verified that global Ca2+
transients, as measured by the Ca2+
indicator fluo-4, nearly vanished after an exposure of the SA node
cells to either BAPTA-AM (5 µmol/L) or EGTA-AM (30 µmol/L) for 10
minutes at 34°C (data not shown). However, only BAPTA significantly
reduced the rate and amplitude of spontaneous AP by 54% and 14%,
respectively, and reduced MDP by 8 mV
(Figures 8B through 8D). It is noteworthy that BAPTA also led
to an irregular beating pattern that was characterized by missing beats
(Figure 8A). In contrast, the slow
Ca2+ buffer, EGTA, only slightly reduced the
rate of spontaneous excitations and did not change the amplitude of AP
as well as MDP
(Figure 8). In voltage-clamped cells, BAPTA hampered the
ICa, L recovery from
inactivation in a manner similar to that of the CaMKII inhibitor KN-93.
Specifically, when EGTA (10 mmol/L) in the pipette solution was
substituted by BAPTA (10 mmol/L), the ICa,
L recovery time constants were increased to
1=120 and 2=3660
ms, respectively. Taken together, these results indicate that in SA
node cells, the spontaneous AP and ICa,
L are far more sensitive to BAPTA than to EGTA, which
supports the idea that local Ca2+ transients
are critically involved in CaMKII-dependent pacemaker
activity.
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Discussion |
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CaMKII Activation Is Essential to Cardiac Pacemaker Activity
The spontaneous excitation of SA node pacemaker cells in the mammalian heart is under tight neuronal and hormonal control. For instance, ß-adrenergic stimulation by the adrenal hormone epinephrine or the sympathetic neurotransmitter norepinephrine elicits a positive chronotropic effect through a cAMP/protein kinase A (PKA) signaling pathway.4 22 Conversely, parasympathetic stimulation exerts a potent negative chronotropic effect.23 In the present study, we demonstrated that in SA node cells basal CaMKII activation is essential for pacemaker activity. This conclusion is based on the following lines of evidence: (1) SA node cell pacemaker activity is depressed by the CaMKII inhibitor in a dose-dependent manner (Figures 1 through 3
); (2) the abolition of spontaneous
excitations by a high concentration of KN-93 (3 µmol/L) or inhibitory
peptide AIP (10 µmol/L) suggests that a minimal level of CaMKII
activation is obligatory to the genesis of the spontaneous excitations;
and (3) an intermediate inhibition of CaMKII by KN-93 (0.3 to 1
µmol/L) or BAPTA (5 µmol/L) disrupts the rhythm and stability of
pacemaker APs, which indicates that a certain amount of CaMKII
activation is required to ensure optimal pacemaker
function. Compared with the well-characterized nervous and hormonal regulation, the CaMKII-mediated regulation of pacemaker activity is unique in several important aspects. First, CaMKII-dependent modulation is intrinsic to the SA node cells, operating tonically to maintain the excitability of the pacemaker cells. Second, in addition to its strong modulatory effects, CaMKII also plays an important permissive role in cardiac pacemaking, as demonstrated in the present study. In this regard, the effect of PKA is mostly modulatory because inhibition of basal PKA activity by 2 µmol/L H-89,24 which fully prevents the positive chronotropic effect induced by the ß-adrenergic agonist isoproterenol, fails to abolish SA node pacemaker activity (data not shown). Third, CaMKII regulation of the pacemaker activity is a positive feedback by nature because CaMKII, which augments ICa, L, is activated by local Ca2+ transients produced directly by ICa, L or indirectly by ICa, L-induced Ca2+ release from the SR. Finally, the CaMKII-mediated modulation could be self-adaptive. For example, an increase in heart rate would, on one hand, reduce the pacemaker ICa, L because of enhanced voltage-dependent inactivation and insufficient time for the channel recovery from inactivation. On the other hand, on the basis of its "memory" properties, CaMKII would serve as the "frequency detector" to integrate the local Ca2+ signals; the faster the heart beats, the more frequent the local Ca2+ transients, and thus the greater the CaMKII activity. The enhanced CaMKII activity alleviates steady-state inactivation and promotes channel recovery from inactivation, maintaining pacemaker activity at a higher set point. Thus, although negative feedback regulation, eg, ICa, L inactivation, stabilizes an established pacemaker frequency, the self-adaptive CaMKII activity permits the heart rate to change over a wider dynamic range.
Ionic Mechanism of CaMKII Action
The most prominent effects of CaMKII inhibition were
the decrease of AP parameters (frequency, amplitude, and upstroke rate)
or even an abolition of the spontaneous excitations
(Figures 1 through 3). A decrease of MDP after CaMKII
inhibition per se could not explain the changes in AP parameters,
because in control cells membrane depolarization ( mV) induced an
increase rather than a decrease in the rate of spontaneous excitations
(data not shown). In KN-93–treated cells, AP parameters were only
partially restored when MDP was restored by injection of a
hyperpolarizing current (data not shown). To unravel the ionic
mechanism underlying the modulatory effects of CaMKII on SA node
pacemaker activity, we examined the possible involvement of
ICa, L and found that
suppression of CaMKII activity by KN-93 reduces
ICa, L amplitude by 50%
(Figure 4A), which is similar to previous observations in
ventricular
myocytes.25 26
The recovery of ICa, L from
inactivation is also markedly slowed by AIP
(Figure 5B), KN-93, or the fast
Ca2+ buffer BAPTA. This is consistent with
the leftward shift of the steady-state inactivation curve caused by the
CaMKII inhibitors, which contributes to the decrease in L-type channel
availability, particularly at the depolarized MDP.
In SA node cells, the leftward shift of steady-state
inactivation curve of ICa, L is
not associated with any shift of the voltage-dependent activation of
the current, resulting in a markedly reduced "window" current (the
overlap area of the steady-state inactivation and activation curves;
Figures 4B and 5A). It has been shown that in rabbit SA node
cells, the L-type window current contributes to the pacemaker
potential.27 Thus,
the decrease in the window current could, in part, explain the
inhibitory effects of the CaMKII inhibitors on spontaneous excitations
of SA node cells.
Because ICa, L
constitutes one of the main ionic currents responsible for excitations
of SA node cells, the suppression of ICa,
L provides a straightforward explanation for inhibitory
effects of the CaMKII inhibitor on AP. However, the paradox is that a
reduction of the inward current was accompanied by a depolarization,
instead of a hyperpolarization, of the membrane potential. One possible
explanation is that the depression or abolition of AP may
secondarily reduce or preclude K+
conductance activated by an AP (eg, delayed rectifier
K+
current4 16 )
such that the net result is a reduction in the outward currents and
thereby membrane depolarization. This interpretation is supported by a
computer simulation of the SA node pacemaker using the OXSOFT HEART
model.28 Direct
evidence is given in
Figure 6, which shows that the L-type channel antagonist
nifedipine similarly reduces MDP, consistent with the previous
reports.4 29
Taking these data together, we conclude that modulation of the voltage-
and time-dependent properties of L-type channel inactivation by CaMKII
is the primary mechanism underlying the CaMKII-mediated regulation of
SA node pacemaker activity.
In addition to ICa, L, several other ionic currents are involved in SA node pacemaker activity, including IKr, If, sustained current, T-type Ca2+ current, and the muscarinic K+ current (IKACh). However, inhibition of If causes only minor changes in AP parameters.30 Recent studies in rabbit ventricular myocytes have demonstrated that CaMKII inhibition by peptide AC3-I has no significant effect on IKr,26 which suggests that IKr and If are not critically involved in CaMKII-dependent regulation of spontaneous excitations. Further studies are required to determine possible contributions of the other pacemaker currents to CaMKII-dependent regulation of SA node pacemaker activity.
Regulation of CaMKII Activation by Local
Ca2+ Signaling
CaMKII activity is regulated in a
Ca2+- and calmodulin-dependent manner. To
delineate the contribution of local versus global
Ca2+ transients in the temporal and spatial
control of CaMKII activation, we directly visualized the intracellular
distribution of the active versus the total CaMKII and found a uniform
distribution of the total CaMKII but a highly localized distribution of
the active CaMKII to the subsarcolemmal microdomain. This spatial
pattern of active CaMKII in SA node cells fits nicely with the
identified functional role of CaMKII in regulating the sarcolemmal
Ca2+ channels and supports the idea of a
local control of CaMKII activation by local
Ca2+ transients. Indeed, a fast
Ca2+ buffer, BAPTA, significantly suppressed
the spontaneous excitations of SA node cells and slowed
ICa, L recovery from
inactivation, which mimics the effects of direct inhibition of CaMKII.
In contrast, the slow Ca2+ buffer EGTA has
no significant effect on either AP or ICa,
L in these SA node cells, probably because it cannot
effectively buffer the local
Ca2+.21
These results are in agreement with our previous observation in rat
ventricular myocytes that BAPTA, but not EGTA, abolishes
CaMKII-dependent ICa, L
facilitation during repetitive
depolarizations.10
Thus, subsarcolemmal Ca2+ transients play a
critical role in the local activation of CaMKII, which, in turn,
mediates a positive feedback regulation of
ICa, L in SA node
cells.
In summary, whereas previous studies focused on the role of ß-adrenergic and muscarinic stimulation in modulation of the heart rate, the present study demonstrates that SA node pacemaker activity is subject to an intrinsic regulation by CaMKII. The CaMKII-mediated regulation is unique as compared with the well-established hormonal or neuronal control because of its inherent positive feedback and self-adaptive properties. In addition, CaMKII may afford an important integrating mechanism for distinct Ca2+31 and other signals32 to regulate heart rate. For example, ß-adrenergic receptor stimulation may cross-talk with the CaMKII signaling pathway by enhancing ICa, L and SR Ca2+ cycling or by PKA-mediated, Ca2+-independent phosphorylation of CaMKII at Thr286.33 Thus, in SA node cells under physiological conditions, CaMKII plays both permissive and modulatory roles in cardiac pacemaker activity via modulating L-type Ca2+ channels.
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Acknowledgments |
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This work was supported by the NIH intramural research programs (R.-P.X., H.C.); the NIH, National Academy of Sciences, and National Research Council associateship programs (T.M.V.); and a Chinese National Science Fund Award for Outstanding Investigators Overseas (H.C.). We are deeply grateful to Drs Edward G. Lakatta, Shi-Qiang Wang, and Ira R. Josephson for helpful discussions and critical review of the manuscript and to Dr Harold A. Spurgeon and Bruce Ziman for help and technical support.
Received August 9, 2000; revision received September 1, 2000; accepted September 1, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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