Circulation Research. 2000;86:723-728
(Circulation Research. 2000;86:723.)
© 2000 American Heart Association, Inc.
Connexin Expression and Turnover
Implications for Cardiac Excitability
Jeffrey E. Saffitz,
James G. Laing,
Kathryn A. Yamada
From the Departments of Pathology and Medicine and the Center for
Cardiovascular Research, Washington University, St. Louis, Mo.
Correspondence to Jeffrey E. Saffitz, Department of Pathology, Box 8118, Washington University School of Medicine, 660 S Euclid Ave, St. Louis, MO 63110. E-mail saffitz{at}pathbox.wustl.edu
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Abstract
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AbstractElectrical activation of
the heart requires current
transfer from one cell to another via gap
junctions, arrays
of densely packed intercellular channels. The extent
to which
cardiac myocytes are coupled is determined by multiple
mechanisms,
including tissue-specific patterns of expression of diverse
gap
junction channel proteins (connexins), and regulatory pathways
that
control connexin synthesis, intracellular trafficking,
assembly into
channels, and degradation. Many connexins, including
those expressed in
the heart, have been found to turn over rapidly.
Recent studies in the
intact adult heart suggest that connexin43,
the principal cardiac
connexin, is surprisingly short-lived
(half-life

1.3 hours). Both
the proteasome and the lysosome
participate in connexin43
degradation. Other ion channel proteins,
such as those forming selected
voltage-gated K
+ channels, may
also exhibit rapid turnover
kinetics. Regulation of connexin
degradation may be an important
mechanism for adjusting intercellular
coupling in the heart under
normal and pathophysiological conditions.
Key Words: gap junctions connexins proteolysis proteasome lysosome
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Introduction
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Gap junctions couple cardiac myocytes electrically and
metabolically
by facilitating the intercellular exchange of
ions, signaling
molecules (<1 kDa in size), and other molecular
information.
1 At least 4 different members of the connexin
family of gap
junction channel proteins are expressed in the heart:
connexin
(Cx) 43, Cx45, Cx40, and Cx37.
2 3 4 5 6 These proteins
are
expressed in different amounts and combinations in different
cell
types and regions of the heart. Each protein forms channels
with
distinct biophysical properties, including unitary conductances
and
voltage sensitivities.
7 8 9 10 A particular pattern of
connexin
expression may, therefore, confer specific conduction
properties
on a given cardiac tissue. Results of recent studies have
implicated
other potential determinants of intercellular coupling,
including
mechanisms regulating connexin synthesis, assembly, and
degradation.
This review focuses on connexin turnover kinetics and
explores
the hypothesis that regulation of connexin degradation may
modulate
cell-cell coupling of cardiac myocytes and thereby affect
impulse
propagation, especially in response to
pathophysiological stimuli.
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Rapid Turnover of Connexins
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Electron microscopic images of the intercalated disk create
the
impression that gap junctions are static structures. In
fact, the
opposite is true. Many connexins, including those
expressed in the
heart (eg, Cx43, Cx45, and Cx37) and those
expressed abundantly in the
liver and elsewhere (eg, Cx32 and
Cx26), have half-lives of 1 to 5
hours.
11 12 13 14 15 16 17 Although not all connexins are short-lived
(Cx46 in the lens
has an apparent half-life of >1 day,
18
which may reflect
the unusual metabolic conditions in the
lens), dynamic turnover
of connexins in the heart may be an important
mechanism for
modulating intercellular coupling and impulse
propagation.
The first evidence that gap junction proteins turn over rapidly in
cardiac myocytes came from studies by Laird et al,14 who
showed that newly synthesized Cx43 disappeared with a half-life of
2
hours in cultured neonatal rat ventricular myocytes. The
discovery of the rapid turnover of Cx43 raised questions about the
intracellular pathways responsible for degradation of gap junction
proteins. Insights into this issue were first revealed in studies of
Cx43 turnover in E36 Chinese hamster ovary cells and ts20 cells, a
mutant line of E36 cells, which carry a temperature-sensitive mutation
in the E1 ubiquitin-activating protein and, therefore, fail to degrade
proteins dependent on ubiquitin pathways when exposed to elevated
temperatures.19 Metabolic labeling and
pulse-chase studies in control E36 cells revealed a Cx43 half-life of
2 hours, whereas Cx43 accumulated in ts20 cells when the
ubiquitin-conjugation system was inactivated at the
restrictive temperature.19
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Connexin Degradation by Proteasomal and Lysosomal Pathways
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The major organelles of intracellular proteolysis are the
proteasome
and the lysosome.
20 21 Pathways
responsible for degradation
of proteins can be elucidated by incubating
cells with specific
and selective inhibitors of the
proteasome (peptide aldehydes
such as acetyl-leucyl-leucyl-norleucinal
[ALLN] or carboxybenzoyl-leucyl-leucyl-leucinal
[MG132]
22 or the fungal metabolite
lactacystin
23 ) or the lysosome (lysosomotropic
amines
such as primaquine or chloroquine, weak bases such as ammonium
chloride,
24 or cathepsin inhibitors such as
leupeptin or E-64
25 ). By using
this approach, treatment of
BWEM cells (a smooth muscle-like
cell line originally derived from
embryonic rat heart) with
inhibitors of either pathway
resulted in accumulation of Cx43,
26 indicating that both
the lysosome and proteasome participate
in Cx43 degradation.
Confocal microscopy of BWEM cells treated
with the proteasomal
inhibitor lactacystin revealed accumulation
of Cx43 signal
at or near appositional membranes, whereas intracellular
vesicular
staining was more abundant in cells treated with the
lysosomal
inhibitor E-64.
26 Thus, proteasomal
degradation may
involve initial targeting of Cx43 at the junctional
membrane,
causing protein to accumulate there when proteasomal
proteolysis
is inhibited. Conversely, internalization of Cx43 leading
to
its accumulation in intracellular vesicles may occur when lysosomal
proteolysis
is inhibited.
To further test the hypothesis that rapid degradation of Cx43 involves
protein present in cell-surface gap junctions, studies have been
performed in BWEM cells treated with brefeldin A (BFA),26
a compound that disrupts delivery of newly synthesized proteins to the
cell surface. Cells exposed to BFA exhibited dramatic loss of Cx43
membrane staining that coincided with a decrease in the total cellular
content of Cx43 assessed by
immunoblotting.26 BFA-induced loss of Cx43
was prevented by coincubating cells with proteasomal or lysosomal
inhibitors.26 However, when cells were treated
simultaneously with BFA and a proteasomal
inhibitor (either MG132 or lactacystin), plasma membrane
staining of Cx43 was comparable to that in untreated
cells.26 These results provide further evidence that Cx43
already assembled within gap junctional plaques undergoes proteasomal
degradation.
Intracellular sites and pathways of connexin degradation vary in
different cell types, and results observed in established cell lines
may not be applicable to cardiac myocytes. Accordingly, additional
studies have been performed using proteolysis inhibitors in
primary cultures of neonatal rat ventricular cardiac
myocytes. As observed in previous studies of BWEM cells,26
Cx43 accumulated in myocytes incubated with either lysosomal or
proteasomal inhibitors,27 indicating that both
pathways degrade Cx43 in cardiac myocytes. In contrast to what was
observed in established cell lines, however, lysosomal inhibition
caused Cx43 to accumulate in appositional membranes rather than in
intracellular vesicles.27 In addition, electron microscopy
revealed increased length of gap junction profiles in cells treated
with either chloroquine or lactacystin.27 Thus, both
proteasomal and lysosomal pathways are involved in the degradation of
Cx43 that resides within the intercalated disk in cardiac myocytes.
An important consideration in analysis of connexin turnover in
cultured cardiac myocytes concerns the effects of cell isolation.
Freshly disaggregated adult myocytes contain intact gap junctions on
their surfaces,28 29 indicating the presence of junctional
membranes of former neighbors. These gap junctions become internalized
and disappear rapidly.28 29 Because disaggregation
followed by active reestablishment of cell junctions in culture could
increase connexin synthesis and degradation rates compared with those
in the intact heart, studies of connexin turnover dynamics in intact
myocardium are of particular interest.
Metabolic labeling and pulse-chase studies have been
performed in isolated, perfused adult rat hearts.17
Radioactive Cx43 disappeared in a monoexponential
fashion with a calculated half-life of
1.3 hours,17 a
rate similar to that seen in cultured myocytes.14 16 In
contrast, the amount of radioactivity in actin did not change
measurably, consistent with its reported half-life of
11
days. Thus, Cx43 turns over as rapidly in the intact adult heart as in
cultured myocytes.
Perfusion of isolated adult rat hearts with either proteasomal or
lysosomal inhibitors produced a marked increase in C43
immunofluorescence at intercalated disks in the
known distribution of gap junctions.17 The intracellular
pattern of vesicular Cx43 staining reported in previous studies of
established cell lines treated with lysosomal
inhibitors26 was not, however, observed in
ventricular myocytes of intact rat hearts perfused with
ammonium chloride.17 Furthermore, immunoblots
prepared from ventricular homogenates revealed
that nonphosphorylated Cx43 accumulated in
ALLN-treated hearts, whereas inhibition of lysosomal proteolysis with
leupeptin or ammonium chloride causes marked accumulation of
phosphorylated Cx43.17 It is not known how
changes in phosphorylation state contribute to protein
stability or whether dephosphorylated Cx43 may be a
target for degradation. It is also not known whether protein that
accumulates in response to inhibition of lysosomal or proteasomal
degradation is present in functional channels that could alter
intercellular coupling. These questions have important implications for
potential therapeutic strategies designed to interfere with connexin
degradation and thereby reduce the incidence of arrhythmias
dependent on impaired coupling.
All of the cardiac connexins are subject to
phosphorylation14 15 16 30 31 involving, in
most cases, serine residues. Phosphorylation of Cx43
has been characterized most extensively. Progressive
phosphorylation of newly synthesized Cx43 occurs over
time and is associated with its assembly into channels and transport to
the cell surface.15 Phosphorylated
isoforms of Cx43, identified by their altered mobility on
SDS-polyacrylamide gels, are resistant to extraction in
1% Triton X-100, suggesting that they are associated with the cortical
cytoskeleton at the plasma membrane.15 Cx43 can be
phosphorylated by protein kinase C,
mitogen-activated protein kinase, and the src protein
kinase.32 33 34 Actions of these kinases usually lead to
diminished single-channel or whole-cell conductances or diminished dye
coupling, but the responsible mechanisms are poorly
defined.35 36 A recent study showed that src-dependent
phosphorylation of Cx43, but not Cx45, occurs in
cardiomyopathic hamster hearts.37 The
exact sites and the biological consequences of
phosphorylation of the cardiac connexins have not been
elucidated.
Increasing evidence has focused attention on the role of
phosphorylation in the regulation of connexin
stability. Results of recent studies suggest that
phosphorylation of specific serine residues on Cx43 and
Cx45 alters connexin degradation38 39 but by complex,
disparate mechanisms. For example, phosphorylation of
Cx43 on ser255 by p34cdc2 kinase in Rat1 cells in
the G2/M phase of the cell cycle promotes
endocytosis and degradation of Cx43.38 In contrast,
phosphorylation of serine residues in the carboxyl
terminal of Cx45 dramatically stabilizes the protein in transfected
HeLa cells.39 Thus, phosphorylation of
serine residues may alter connexin stability and/or target the protein
for degradation.
It might be argued that previous studies involving
metabolic labeling and pulse-chase strategies have not
formally ruled out the possibility that rapid turnover of connexins
occurs selectively in an intracellular pool of protein, whereas some or
all of the protein in gap junctions is sufficiently long-lived that it
does not become labeled during relatively brief pulse intervals.
However, results of recent studies in which Cx43 has been visualized in
living cells previously transfected to express Cx43 tagged on its
carboxyl terminal with green fluorescent protein (Cx43-GFP)
have provided dramatic evidence of the dynamic nature of Cx43 in
cell-surface gap junctions.40 Time-lapse movies have shown
continuous transport of apparently newly synthesized Cx43-GFP to the
plasma membrane of MDCK cells where discrete patches of
fluorescent signal (presumed gap junctional plaques) were seen
to oscillate and occasionally to coalesce. Cx43-GFP was removed from
the plasma membrane by budding and internalization and usually formed
distinct endocytic vesicles of two different sizes. The smaller of
these vesicles appeared to deliver Cx43-GFP back to the cell
surface.40 Although the fate of Cx43-GFP in living cells
has not been followed for more than 40 minutes (due to technical
limitations), future studies may be able to directly visualize turnover
of Cx43 in gap junction plaques.
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Stress- or Injury-Induced Acceleration of Cx43 Degradation
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Electron microscopy has revealed intracellular membrane-bound
vesicular
structures containing morphologically identifiable gap
junctions
referred to as annular gap junctions. Annular gap junctions
occur
in many cell types including cardiac
myocytes.
28 29 41 42 They
are especially abundant in
recently disaggregated adult myocytes.
28 29 Their presence
suggests that arrays of channels at the cell
surface can be
internalized and possibly degraded by lysosomes.
It is
currently not known whether degradation of gap junctions
may contribute
to abnormal electrical remodeling in the heart
and arrhythmogenesis in
the setting of hypertrophy, ischemia,
or
infarction.
Recent studies in which cultured neonatal cardiac myocytes have been
subjected to heat stress bear on the relation between myocyte injury
and Cx43 degradation.27 Exposure of cultured myocytes to
heat stress (43.5°C for 30 minutes) resulted in dramatic loss of Cx43
protein content (assessed by immunoblotting or
immunohistochemistry). Heat stress also resulted in accumulation of the
heat shock protein HSP70. Degradation of Cx43 was prevented during an
interval of heat stress by simultaneously incubating cells
with proteasomal or lysosomal inhibitors, suggesting that
both pathways participate in heat-induced proteolysis of Cx43.
Furthermore, when heat-stressed cells were allowed to reaccumulate Cx43
during a 3-hour recovery interval, a subsequent interval of heat shock
failed to cause degradation of Cx43. These results suggest that a
factor induced during an initial interval of heat stress (possibly
HSP70) may have protected against subsequent Cx43 degradation in
response to additional injury.27
The wider implications of these results with respect to changes in gap
junction structure and function during ischemic preconditioning
have not been explored in detail. Enhanced degradation and turnover of
connexins could reduce cell-cell coupling, slow conduction, and promote
reentrant arrhythmias. Differential targeting of Cx43 to
ubiquitin-dependent or -independent proteolysis pathways could provide
mechanisms by which cardiac myocytes regulate "normal" and
"pathophysiologic" degradation responses. Furthermore, disparate
ubiquitin-dependent pathways may result in either protein degradation
or stabilization. Phosphorylation at PEST motifs
(regions rich in pro, glu, ser, and thr sequences) has been identified
as a general mechanism for targeting proteins for rapid
degradation.43 44 Conversely,
phosphorylation of c-Mos, c-Fos, and c-Jun by
mitogen-activated protein kinases suppresses their
ubiquitination and degradation.45 Little is known about
the specific signaling pathways and enzymes responsible for targeting
connexins and other proteins for degradation in the heart, much less
the wide range of physiological and
pathophysiological conditions in which these
reactions occur.
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Degradation of Other Channel Proteins
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The discovery that connexins turn over rapidly raises questions
about
whether other proteins involved in cardiac excitability and
conduction
are similarly regulated. This issue has not been studied
systematically.
Of the voltage-gated channels studied thus far, Kv1.5
proteins
are among those reported to have the shortest half-lives (

4
hours)
based on disappearance of protein in cells treated with the
protein
synthesis inhibitor cycloheximide.
46
Levitan and Takimoto
47 have suggested that rapid
upregulation of voltage-gated K
+ channels and
concomitant regulation of changes in excitability
may be mediated by
rapid turnover of Kv channel proteins. Voltage-gated
Na
+ and Ca
2+ channels have
been reported to have half-lives of 15
to 26 hours.
48 49
Thus, turnover of K
+ channel and gap junction
proteins
but apparently not Na
+ or
Ca
2+ channel proteins may influence
minute-to-minute
changes in excitability. Other membrane-bound
receptors have
been reported to turn over with half-lives of 6 to 8
hours (A
1 adenosine
receptors,
50 IP
3
receptors
51 52 ), days (fetal acetylcholine
receptors
53 ), or weeks (adult acetylcholine
receptors,
53 sarcoplasmic
reticulum
Ca
2+-ATPase pump
52 ).
Intracellular pathways responsible for degrading voltage-gated ion
channel proteins have not been elucidated in detail. Of the various
integral plasma membrane proteins in animal cells found to undergo
ubiquitination, the only ion channel is the epithelial
Na+ channel (ENaC), a short-lived protein with a
half-life of
1 hour.54 Mutations in the ENaC PPxY
motif, which interacts with WW domains of the ubiquitin-protein ligase
Nedd4, disrupt ubiquitin-mediated degradation of ENaC and result in
increased ENaC activity and Liddles syndrome.55 Other
channels, receptors, and transporters may be degraded by
ubiquitin-mediated pathways. A proteolytic cleavage product of the
skeletal muscle ryanodine receptor bears homology to S5a, a proteasome
subunit that targets polyubiquitinated proteins to the 26S
proteasome.56 Agonist stimulation may accelerate protein
turnover. For example, the muscarinic agonist, carbachol, increases the
rate of degradation of the IP3 type I receptor by
a Ca2+-dependent mechanism.51
Diminished Ca2+ entry may initiate acetylcholine
receptor loss at the neuromuscular junction.57 Blockade of
acetylcholine receptors by
-bungarotoxin results in a shift in the
half-life of the protein from 14 days to 1 day.58 A number
of growth factor receptors have also been found to undergo
ubiquitin-mediated lysosomal degradation in response to agonist
stimulation.24
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Summary of Intracellular Pathways of Cx43 Degradation
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The Figure

shows a proposed model of the
degradation of gap
junction proteins based on current knowledge. The
connexin polypeptide
is synthesized on endoplasmic reticulum
(ER)associated
ribosomes and inserted into the ER membrane. Some
unoligomerized
connexin polypeptides may be degraded in the ER in the
same
manner as other misfolded or improperly oligomerized secretory
and
membrane proteins. Excess, defective, or unused protein
could undergo
ubiquitination and be exported from the ER to
the cytosol where it
becomes degraded by the proteasome. Most
newly synthesized connexin
polypeptides in the ER are subsequently
oligomerized to form
hemichannels either in the ER or the Golgi.
The hemichannels are
transported to the sarcolemma where they
may dock with hemichannels of
neighboring cells to form complete
gap junction channels. Individual
channels then aggregate to
form gap junctional plaques. Degradation of
gap junctional plaques
requires actions of both the proteasome and the
lysosome, which
may occur in parallel or sequentially. Gap
junction channels
may be internalized within intracellular vesicles
either as
channel arrays (annular gap junctions) or as channels
disrupted
from the plaques by partial proteasomal
proteolysis.
59 Either
form may undergo lysosomal
degradation.

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Figure 1. Proposed model of intracellular pathways of connexin
degradation. Refer to text for explanation and references.
|
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Implications for Cardiac Excitability
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Because gap junctions play a fundamental role in cell-cell
communication
and impulse propagation, it seems likely that cardiac
myocytes
have evolved multiple regulatory mechanisms to control the
level
of intercellular communication in the heart. The fact that Cx43
turns
over so rapidly in the intact heart suggests that one mechanism
controlling
coupling is rapid adjustment in the number of channels
between
cells. Undoubtedly, other mechanisms controlling connexin
synthesis,
assembly into channels, and single-channel properties,
including
unitary conductances and open-probability times, also play
crucial
roles in regulating the extent to which cells are coupled to
one
another, especially under pathophysiological
conditions. Many
of these regulatory mechanisms may be mediated by
changes in
connexin phosphorylation. Cardiac myocytes
may uncouple rapidly
and completely, as occurs in the setting of acute,
severe ischemia.
In the face of sublethal injury, however,
cardiac myocytes may
uncouple partially as part of a more generalized
cellular response
to injury involving complex structural and functional
adaptations.
Alterations in intercellular coupling in viable but
structurally
remodeled myocardium undoubtedly change the
conduction properties
of the tissue and, in some settings, may create
anatomic substrates
of arrhythmias dependent on derangements in
conduction. Alterations
in connexin expression and spatial remodeling
of gap junctions
in regions bordering healing infarcts have been
strongly implicated
in the development of slow,
heterogeneous conduction and conduction
block critical in
reentrant arrhythmogenesis.
60 Downregulation
of Cx43
expression and loss of the largest gap junctions have
been described in
hibernating myocardium in patients with chronic
heart
failure,
61 although the contributions of these changes
to
electrical and contractile dysfunction have not been elucidated.
The
extent to which regulation of connexin turnover dynamics
is used by
cells to control levels of intercellular coupling
is not known, nor is
it clear whether alterations in connexin
expression in diseased
myocardium are mediated by changes in
the delicate balance
between connexin synthesis and connexin
degradation. More detailed
understanding of mechanisms underlying
cellular responses to injury
will not only provide novel insights
into fundamental disease processes
that promote arrhythmogenesis
but may also lead to novel therapies to
prevent lethal rhythm
disturbances in patients with heart
disease.
Received January 28, 2000;
accepted February 10, 2000.
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