Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut 06269
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ABSTRACT |
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The
actin-binding proteins dystrophin and -actinin are members of a
family of actin-binding proteins that may link the cytoskeleton to
membrane proteins such as ion channels. Previous work demonstrated that
the activity of Ca2+ channels can be regulated by agents
that disrupt or stabilize the cytoskeleton. In the present study, we
employed immunohistochemical and electrophysiological techniques to
investigate the potential regulation of cardiac L-type Ca2+
channel activity by dystrophin and
-actinin in cardiac myocytes and
in heterologous cells. Both actin-binding proteins were found to
colocalize with the Ca2+ channel in mouse cardiac myocytes
and to modulate channel function. Inactivation of the Ca2+
channel in cardiac myocytes from mice lacking dystrophin
(mdx mice) was reduced compared with that in wild-type
myocytes, voltage dependence of activation was shifted by 5 mV to more
positive potentials, and stimulation by the
-adrenergic pathway and
the dihydropyridine agonist BAY K 8644 was increased. Furthermore, heterologous coexpression of the Ca2+ channel with muscle,
but not nonmuscle, forms of
-actinin was also found to reduce
inactivation. As might be predicted from a reduction of
Ca2+ channel inactivation, a prolonging of the mouse
electrocardiogram QT was observed in mdx mice. These results
suggest a combined role for dystrophin and
-actinin in regulating
the activity of the cardiac L-type Ca2+ channel and a
potential mechanism for cardiac dysfunction in Duchenne and Becker
muscular dystrophies.
muscular dystrophy; intracellular regulation
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INTRODUCTION |
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VOLTAGE-DEPENDENT CA2+ channels play an essential role in the regulation of many cellular processes, including gene transcription, muscle contraction, cell division, and exocytosis, by transducing a voltage signal into an elevation of intracellular Ca2+ (14). The cardiac voltage-gated L-type Ca2+ channel (CaV1.2) mediates the Ca2+ current that is responsible for the plateau phase of the cardiac action potential and for initiating contraction. Previous work suggests that the actin-based cytoskeleton contributes to the regulation of both voltage- and ligand-gated ion channels (16), and L-type Ca2+ channels in cardiac and smooth muscle have been reported as being regulated by actin filament organization (8, 18, 21, 26).
Two actin-binding proteins (ABPs), dystrophin and -actinin, are
known to link membrane-associated elements to the cytoskeleton (5). Dystrophin, a member of the spectrinlike superfamily
of actin-binding proteins, acts as a link between the actin
cytoskeleton, the plasmalemma, and the surrounding basal lamina.
Disruption of this link through deletion of the dystrophin gene results
in Duchenne and Becker muscular dystrophies (DMD, BMD),
characterized by progressive weakness and wasting of skeletal muscles,
nonprogressive cognitive impairment, and failure of the electrical
conduction system in the heart. In the mouse model of DMD, the
mdx mouse, the upregulation of a related protein, utrophin,
effectively prolongs the life span of these mice and decreases muscle
atrophy, but signs of skeletal and cardiac myopathy are still present
(11). Dystrophin and its associated proteins have been
implicated in playing a role in receptor/channel localization
(10, 22).
-Actinin functions to anchor parallel filaments of F-actin
throughout the cytoskeleton in all tissue types, as well as to anchor
antiparallel actin filaments in muscle tissue, forming the Z disk in
skeletal and cardiac muscle and the cytoplasmic-dense bodies in smooth
muscle. Recent data have suggested that
-actinin may also associate
with several types of ion channels. The voltage-gated K+
channel Kv1.5, expressed in the cardiovascular system and brain, binds
to
-actinin-2 and colocalizes at the membrane in transfected human
embryonic kidney (HEK) cells (23). This colocalization of
-actinin-2 with voltage-gated K+ channels was found to
modulate channel gating and current density (6, 23).
-Actinin has also been found to bind to the NR1 and NR2B subunits of
the N-methyl-D-aspartate (NMDA)-type glutamate receptor and
influence channel regulation (7). Krupp et al. (20) found that overexpressed muscle isoforms of
-actinin competed with calmodulin for binding to the NMDA receptor.
We examined colocalization and regulation of the Ca2+
channel by the actin-binding proteins -actinin and dystrophin using
immunofluorescence and whole cell patch-clamp techniques. Within adult
mouse cardiac tissue, the Ca2+ channel was found to
colocalize with dystrophin at both the Z and M lines, whereas
-actinin was found to colocalize only over regions of the Z line.
This colocalization with dystrophin and
-actinin indicates the
potential for channel regulation by these proteins. In cardiac myocytes
from mdx mice, which lack dystrophin, inactivation was
reduced by a positive shift in the voltage dependence of activation, a
likely allosteric change in channel conformation that made it more
sensitive to stimulation by the
-adrenergic agonist
l-isoproterenol and the dihydropyridine agonist
l-BAY K 8644. This change in channel kinetics may have led
to a slowing of ventricular repolarization, observed as an increase in
the QT interval in mdx mice. Coexpression of the
Ca2+ channel with muscle isoforms of
-actinin in
heterologous cells resulted in a similar slowing of channel
inactivation. Together, these data suggest a regulatory role for the
ABPs
-actinin and dystrophin, acting on the Ca2+ channel
by either direct or indirect interactions.
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MATERIALS AND METHODS |
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Animals. Mice used for immunofluorescence studies and electrocardiograms were 2- to 3-mo-old C57BL/10SnJ (wild type) and C57BL/10ScSn-Dmdmdx (mdx), both from Jackson Laboratories (Bar Harbor, ME), weighing 20-30 g. Cardiac myocytes were prepared from mice between 1 and 4 days old. The animals were killed in accordance with the guidelines of the Institutional Animal Care and Use Committee at the University of Connecticut.
Immunofluorescence analysis.
Wild-type mice were injected with heparin (5000 U/kg ip), anesthetized
30 min later with xylazine-ketamine (7 mg/kg xylazine, 80 mg/kg
ketamine ip), and perfused with 10 ml of PBS and then with 10 ml of 4%
formaldehyde in PBS. Hearts and brains were removed and postfixed in
4% PBS-buffered formaldehyde overnight at 4°C. After being washed in
PBS for 30 min (3 times), the tissues were successively sunk in 30%
sucrose in PBS for 4 h and 30% sucrose in PBS-OCT mounting medium
(1:1; Tissue-Tek 4583; Sakura Finetek) overnight at 4°C.
Tissue was then immediately frozen in OCT mounting medium, and
10-µm-thick sections were cut on a cryostat (model HM 500 OM; Carl
Zeiss, Thornwood, NY). Sections were washed and hydrated twice for 10 min in PBS, once for 30 min in PBS containing 2% BSA, and once for 15 min in PBS containing 2% BSA or 3% goat serum and 0.3% Triton X100.
Sections were incubated in PBS containing 2% BSA or 3% goat serum
overnight at 4°C. Sections were labeled in incubation buffer
containing 0.3% Triton X-100 with rabbit polyclonal
anti-Ca2+ channel 1C antibody and/or mouse
monoclonal anti-
-actinin antibody overnight at 4°C. Sections were
first labeled with polyclonal antibody against L-type Ca2+
channel
1-subunit, followed by a monoclonal antibody
against
-actinin, monoclonal antibody against dystrophin, or
fluorescent phalloidin to label F-actin. An average of four to eight
cryosectioned tissues from different animals were transferred to
gelatin-coated glass slides and processed for immunolabeling.
Antibodies used in this study included 1) affinity-purified
rabbit polyclonal antibody against the Ca2+ channel
1-subunit (13); secondary antibody for this
was goat anti-rabbit conjugated with Texas red (Vector Laboratories);
2) mouse monoclonal antibody against the dystrophin rod
domain (Novocastra Laboratories); secondary antibody was goat
anti-mouse conjugated with FITC (Sigma-Aldrich, St. Louis, MO); and
3) mouse monoclonal antibody against sarcomeric
-actinin;
secondary antibody was goat anti-mouse conjugated with FITC
(Sigma-Aldrich). F-actin was labeled with fluorescent phalloidin.
Labeling of all of these structures was specific, because it was not
obtained with nonimmune rabbit antibodies or any secondary antibody
that failed to bind rabbit IgG. Cells were imaged using fluorescent microscopy.
Cell preparation and electrophysiology.
TsA-201 cells (large T antigen-transformed human embryonic kidney
cells, HEK-293) were maintained at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and 100 units of penicillin and streptomycin (GIBCO-Life Technologies) in a
humidified atmosphere containing 5% carbon dioxide. Calcium phosphate
coprecipitation method was used for transfections (17). All cultures were transiently transfected with the following
Ca2+ channel subunits: rabbit 1C
(32),
2A (27), and rat
2
(30). Cotransfected
-actinin
isoforms were human skeletal muscle
-actinin-2 (1, 33),
chicken smooth muscle
-actinin, chicken nonmuscle
-actinin
containing only the spectrin domain, or full-length chicken nonmuscle
-actinin (20, 31). CD8 reporter plasmid was used for
identification of transfected cells (EBO-pCD-Leu2, American Type
Culture Collection). Before patch clamping, cell cultures were
incubated with CD8 antibody-coated beads (M-450 Dynabeads, Dynal
Biotech) for 1 min.
Electrocardiograms. Wild-type and mdx mice, 4-8 wk old, were anesthetized with xylazine-ketamine (7 mg/kg xylazine, 80 mg/kg ketamine ip). Two fine needle electrodes were inserted subcutaneously on the shoulder at the junction between the chest and the left and right forelimbs (lead I). Electrodes were connected to an electrocardiogram (ECG) amplifier (World Precision Instruments) and digitized at 5-200 Hz. An average of 4-10 successive cycles were used to determine heart rate, QT, PR, QRS, and ST intervals, and amplitude of S, T, and R waves. Mean heart rate was calculated from the R-R intervals of the ECG. Heart rate-corrected QT values (QTc) were obtained as described (25). All measurements and recordings were made using the HEKA EPC-9 system (HEKA Electronik). Data are presented as means ± SE.
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RESULTS |
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Colocalization of actin-binding proteins -actinin and dystrophin
with the Ca2+ channel.
To determine whether the Ca2+ channel colocalizes with
actin or ABPs, we employed immunofluorescent labeling of the
Ca2+ channel, actin,
-actinin, and dystrophin in
cryosections of cardiac and neural tissues. The Ca2+
channel was at highest density over Z lines (Fig.
1, A,
D, and G), which confirms previous reports on the
distribution of the L-type Ca2+ channel in the cardiac
myocyte (9). Z and M lines could be distinguished from
each other because structures over Z lines are usually wider than those
over M lines. Labeling of actin (Fig. 1E), which is absent
in the H zone of sarcomeres, also helped to distinguish the striated
banding pattern.
-Actinin (Fig. 1B), a major protein
forming Z disks, was distributed over the Z line of costameres as
expected. Figure 1C shows colocalization of
-actinin and
the Ca2+ channel over Z lines. However, the absence of
cortical
-actinin near the M line suggests that the Ca2+
channel may be associated with another binding protein or isoform of
-actinin that links it to the cortical actin cytoskeleton. As shown
in Fig. 1H, immunofluorescent labeling of dystrophin revealed a pattern that is consistent with previous findings of dystrophin localization over Z and M lines in striated muscle (29). Dystrophin colocalized intensely with the
Ca2+ channel over Z lines (Fig. 1I). Labeling of
dystrophin over the M line was similar to that found for the
Ca2+ channel (compare G and H, Fig.
1).
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Effect of dystrophin deletion on Ca2+ channel inactivation and voltage dependence. Cardiac myocytes isolated from mdx mice could not be distinguished by gross morphology from those isolated from wild-type mice. Cells were of similar size as estimated from cell capacitance (wild type: 30 ± 3 pF, n = 33; mdx: 28 ± 2 pF, n = 29) and showed no signs of hypo- or hypertrophy. Ca2+ channel expression and peak activity were also similar, as determined from current density (wild type: 22 ± 2 pA/pF; mdx: 19 ± 2 pA/pF, n = 34). The absence of dystrophin did not appear to affect the ability of the channel to localize within the membrane and did not result in any visible cell changes.
After opening in response to a membrane depolarization, most voltage-gated ion channels turn themselves off in a process known as inactivation. This process occurs despite continued membrane depolarization and prevents the cell from becoming overloaded with Ca2+. Inactivation occurs by multiple mechanisms, two of which are known for Ca2+ channels: a slow, voltage-dependent mechanism and a faster, Ca2+-dependent mechanism (12). Alterations in the cytoskeleton are known to influence both types of inactivation (16). To study primarily the voltage-dependent component, Ba2+ was used as the charge carrier because it does not substitute for Ca2+ as effectively in the ion-dependent inactivation mechanism (12). When we examined the time course of channel inactivation in mdx mice, we found that there was a significant slowing. Figure 2 compares the time course of inactivation in mdx and wild-type cardiac myocytes during 1-s depolarizations (
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Influence of dystrophin on -adrenergic and dihydropyridine
modulation of the Ca2+ channel.
Adrenergic agonists stimulate cAMP production and activate
cAMP-dependent protein kinase (PKA) in cardiac myocytes, leading to
increased phosphorylation of the Ca2+ channel, enhancement
of channel current, and an increase in the force of contraction
(24). PKA is localized near the L-type Ca2+
channel by the binding of its regulatory subunit to proteins known as
A-kinase anchoring proteins (AKAPs) (16). Because of the
importance of PKA anchoring near the channel, disruption of submembrane
structure could result in aberrant regulation of the channel. We
undertook experiments on
-adrenergic modulation of the L-type
Ca2+ channel in cardiac myocytes with the hypothesis that
the absence of dystrophin in the mdx mouse might disrupt the
interaction between the Ca2+ channel and PKA and that the
positive shift in channel voltage dependence and slowing of
inactivation could be explained by a reduction in tonic channel
phosphorylation by PKA.
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Alterations in the mdx mouse ECG.
In the heart, a slowing of Ca2+ channel inactivation as
observed in mdx myocytes might be predicted to prolong
ventricular depolarization (prolong QT interval) and could also lead to
cardiac arrhythmia. Dystrophin-deficient mice (mdx) were
found to have heart rates similar to those of wild-type mice (Fig.
6A, 237 beats/min compared with 248 beats/min). No significant difference was found in PR and QRS
intervals or among amplitudes of S, T, or R waves. However, QT (112 ms
compared with 79 ms, P < 0.003) and QTc intervals (70 ms compared with 50 ms, P < 0.003) were significantly
prolonged in mdx mice (Fig. 6, B and
C). Figure 6, D-I, shows characteristic ECG
records for wild-type and mdx mice. We found notched QRS
complexes in ~20% of ECGs from mdx mice (Fig.
6H) but not in any ECGs from wild-type mice. QRS notches
were also observed in patients with DMD (15). Abnormal
regulation of the Ca2+ channel or other ion channels in
mdx cardiac myocytes may be manifest at the organ level as a
prolonging of the QT interval.
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Regulation of Ca2+ channel
voltage-dependent inactivation by muscle isoforms of -actinin.
We next examined whether
-actinin could modulate the activity of the
Ca2+ channel in heterologous cells. Channel subunits
(
1C,
2A, and
2
) were
coexpressed with muscle and nonmuscle isoforms of
-actinin in the
TsA-201 cell line, and channel activity was compared with that in
control cells expressing only Ca2+ channel subunits.
Current density, a measure of active channel density in the membrane,
showed a trend (although not statistically significant) toward smaller
currents in the presence of muscle isoforms (control: 21 ± 3 pA/pF, n = 7; smooth muscle: 15 ± 3, n = 6; skeletal muscle: 16 ± 3, n = 5) and larger currents in the presence of nonmuscle isoforms
(nonmuscle: 30 ± 6 pA/pF, n = 5; spectrin repeat:
28 ± 5 pA/pF, n = 10).
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DISCUSSION |
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In the current study, we found that alterations in the expression
of either -actinin or dystrophin lead to changes in Ca2+
channel voltage dependence and kinetics of inactivation. These actin-binding proteins colocalize with the Ca2+ channel at
the Z line of cardiac myocytes and the Ca2+ channel and
-actinin colocalize in the brain. At the cardiac myocyte M line
where
-actinin is absent, we found that dystrophin colocalizes with
the channel. Dystrophin-associated proteins such as syntrophin interact
with other ion channels and therefore may also be involved in
localization and regulation of the Ca2+ channel in these
regions. Our results are in accord with those of Gao et al.
(9), who noted an association between the L-type Ca2+ channel and
-actinin in rabbit cardiac myocytes at
the Z line.
The absence of dystrophin in mdx myocytes was found to slow
inactivation of the L-type Ca2+ channel by shifting the
voltage dependence of the channel to more positive potentials, and
coexpression of muscle forms of -actinin with the Ca2+
channel in heterologous cells reduced Ca2+ channel
inactivation. One interpretation of these results is that
-actinin
may replace dystrophin when it is missing. Coming from the same family,
these proteins bear many homologous regions and therefore may both be
able to bind to the Ca2+ channel. The positive shift in
channel voltage dependence in mdx cardiac myocytes was not
apparent in these heterologous expression experiments, suggesting that
dystrophin and
-actinin modulate the Ca2+ channel by
different molecular mechanisms. One possible explanation of the
differing results is that
-actinin might modulate channel inactivation, whereas dystrophin might regulate channel voltage dependence. Disruption of dystrophin in mdx myocytes would
then cause changes in both properties by altering the channel's
interaction with
-actinin, whereas only inactivation is reduced in
heterologous cells because dystrophin expression is unchanged.
The difference between the two isoforms of -actinin (muscle and
nonmuscle) lies primarily in the putative Ca2+-binding
regions known as EF-hand domains, located near the carboxy terminus.
The nonmuscle isoform has two EF-hand domains that are believed to be
Ca2+ sensitive, whereas the muscle isoform may have only
one EF-hand domain that may not be capable of binding Ca2+.
Cotransfection of these isoforms with the NMDA-type glutamate receptor
gave similar results (20), whereas inactivation of the
receptor was slowed by the muscle isoform alone.
An unexpected result in these experiments was that channel modulation
by the -adrenergic pathway was enhanced in mdx compared with wild-type cardiac myocytes. Similarly, BAY K 8644 increased Ca2+ channel activity in mdx cardiac myocytes
more than in wild-type myocytes, suggesting that the channel itself may
be allosterically modified such that it is more sensitive to
upregulation. Ca2+ channel voltage dependence and
inactivation (properties of the pore-forming
1-subunit)
have been found to be modified by interactions with channel auxiliary
subunits, G proteins, and intracellular synaptic proteins, making it
likely that any interaction the channel has with the cytoskeleton
would have similar consequences. Dystrophin itself or one of the
proteins in the dystrophin-associated protein complex (DPC) is likely a
partner. This could include an interaction with auxiliary subunits such
as the
-subunit, or a direct allosteric effect on channel gating.
These results have implications for the mechanism of cardiac and central nervous system dysfunction in BMD and DMD. Progressive weakness and wasting of skeletal muscles, nonprogressive cognitive impairment, and failure of the electrical conduction system in the heart are associated with defects or deficiencies in dystrophin (2-4, 34). Increased sensitivity of the channel to sympathetic stimulation may cause cardiac tissue to become more susceptible to damage from Ca2+ loading. In addition, cytoskeletal disruption appears to alter Ca2+ channel kinetics, producing a late Ca2+ current that might prolong ventricular depolarization. The significant prolongation of the QT interval in the mdx ECG suggests that Ca2+ channel abnormalities play a role in the potential for arrhythmias in these muscular dystrophies. The importance of Ca2+ channels in the development and functioning of the brain may help to explain some of the differences observed in the central nervous system (4, 28).
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ACKNOWLEDGEMENTS |
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We thank the LoTurco laboratory for help with immunolabeling and Chu Ngo for preparing cultures, cryosectioning, and other technical assistance.
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FOOTNOTES |
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This work was supported by an American Heart Association Scientist Development Grant (B. D. Johnson) and by start-up funds from the University of Connecticut Research Foundation.
Address for reprint requests and other correspondence: B. D. Johnson, Dept. of Pharmacology and Therapeutics, Univ. of British Columbia, 3650 Wesbrook Mall, Vancouver, Canada BC V6S 2L2 (E-mail: barrydjohnson{at}telus.net).
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.
First published February 13, 2002;10.1152/ajpcell.00435.2001
Received 10 September 2001; accepted in final form 11 February 2002.
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