© 2000 American Heart Association, Inc.
Integrative Physiology |
Dephosphorylation and Intracellular Redistribution of Ventricular Connexin43 During Electrical Uncoupling Induced by Ischemia
From the Departments of Medicine, Surgery, Pediatrics and Pathology, and the Center for Cardiovascular Research, Washington University School of Medicine, St. Louis, Mo, and the Department of Physiology, University of Bern, Bern, Switzerland. Dr Beyer’s current address is Section of Pediatric Hematology/Oncology, University of Chicago Children’s Hospital, Chicago, Ill.
Correspondence to Jeffrey E. Saffitz, MD, PhD, Washington University School of Medicine, Department of Pathology, Box 8118, 660 S Euclid Ave, St. Louis, MO 63110. E-mail saffitz{at}pathology.wustl.edu
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Abstract |
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Abstract—Electrical uncoupling at gap junctions during acute myocardial ischemia contributes to conduction abnormalities and reentrant arrhythmias. Increased levels of intracellular Ca2+ and H+ and accumulation of amphipathic lipid metabolites during ischemia promote uncoupling, but other mechanisms may play a role. We tested the hypothesis that uncoupling induced by acute ischemia is associated with changes in phosphorylation of the major cardiac gap junction protein, connexin43 (Cx43). Adult rat hearts perfused on a Langendorff apparatus were subjected to ischemia or ischemia/reperfusion. Changes in coupling were monitored by measuring whole-tissue resistance. Changes in the amount and distribution of phosphorylated and nonphosphorylated isoforms of Cx43 were measured by immunoblotting and confocal immunofluorescence microscopy using isoform-specific antibodies. In control hearts, virtually all Cx43 identified immunohistochemically at apparent intercellular junctions was phosphorylated. During ischemia, however, Cx43 underwent progressive dephosphorylation with a time course similar to that of electrical uncoupling. The total amount of Cx43 did not change, but progressive reduction in total Cx43 immunofluorescent signal and concomitant accumulation of nonphosphorylated Cx43 signal occurred at sites of intercellular junctions. Functional recovery during reperfusion was associated with increased levels of phosphorylated Cx43. These observations suggest that uncoupling induced by ischemia is associated with dephosphorylation of Cx43, accumulation of nonphosphorylated Cx43 within gap junctions, and translocation of Cx43 from gap junctions into intracellular pools.
Key Words: connexin43 • gap junctions • ischemia • uncoupling • phosphorylation • arrhythmias
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Introduction |
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Alterations in electrical coupling of ventricular myocytes play an important role in arrhythmogenesis in acute and chronic ischemic heart disease.1 2 3 Mechanisms mediating uncoupling during ischemia are related to multiple pathophysiological processes including progressive increases in intracellular Ca2+ and H+ concentrations and accumulation of amphipathic lipid metabolites.4 5 6 7 8 Other mechanisms could also promote uncoupling, however, and contribute to the development of conduction abnormalities and arrhythmias. Insights into mechanisms responsible for uncoupling could suggest novel therapeutic strategies to limit lethal arrhythmias induced by ischemia.
Intercellular electrical coupling channels are composed of connexins, members of a family of proteins that form gap junctions.9 10 Studies of ventricular conduction in connexin43 (Cx43)–deficient mice have revealed that Cx43 is the principal ventricular electrical coupling protein.11 12 Like many of the connexins, Cx43 is a phosphoprotein.13 14 15 Changes in connexin phosphorylation can affect channel properties and connexin turnover dynamics.14 15 16 17 18 19 20 21 Because acute ischemia may activate or inhibit protein kinases and phosphatases,22 we performed the present study to test the hypothesis that electrical uncoupling induced by myocardial ischemia is mediated, at least in part, by alterations in phosphorylation of Cx43. We studied an isolated rat heart preparation in which the time course of uncoupling during ischemia was similar to that reported in rabbit23 and porcine24 hearts. We observed that in response to ischemia, Cx43 underwent marked dephosphorylation with a time course similar to that of uncoupling. Uncoupling was associated with diminished Cx43 immunoreactive signal and concomitant accumulation of nonphosphorylated Cx43 at sites of intercellular connections. Recovery of contractile function after reperfusion was associated with increased levels of phosphorylated Cx43. These observations suggest that uncoupling induced by ischemia may be related to dephosphorylation of Cx43 within gap junctions and translocation of Cx43 from gap junctions to intracellular sites.
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Materials and Methods |
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Animal Care
Animals were housed in barrier facilities under veterinary supervision. All protocols were approved by the Animal Studies Committee of Washington University School of Medicine.
Perfusion of Isolated Hearts
Hearts excised from adult male Sprague-Dawley rats were
transferred to a Langendorff apparatus and perfused via an
aortic cannula as previously described.20 After a
10-minute stabilization interval of normoxic perfusion, hearts were
made ischemic by cessation of perfusion for 0 to 40 minutes. In
some studies, hearts were subjected to global ischemia for 20
minutes and then reperfused with oxygenated buffer for 30
minutes.
Measurement of Whole-Tissue Resistance
The time course of electrical uncoupling induced by
ischemia was characterized in 5 additional hearts by measuring
changes in whole-tissue resistance,
rt.3 23 24 25 This method
has been validated in rabbit papillary muscle by cable
analysis.3 Briefly,
polytetrafluoroethylene (Teflon)–coated
silver wire electrodes (0.045-inch coated diameter) were placed on the
anterior surface of the heart in an orientation roughly parallel to the
long axis of the epicardial ventricular fibers. Electrical
recordings were made at a point 
50 µm below the
epicardial surface where the Teflon insulation had been removed from
each wire. The outer 2 electrodes, each separated from its adjacent
inner electrode by a distance of 1.0 mm, were connected to a
current source, and the inner 2 electrodes, separated from each other
by a distance of 1.5 mm, were connected to a voltage amplifier. A
subthreshold current of 20-ms duration was delivered across the outer 2
electrodes, and the voltage drop across the inner 2 electrodes was
recorded. Tissue resistance, rt, is a
measure of the resistance of the extracellular
(ro) and intracellular
(ri) spaces arranged in parallel
(1/rt=1/ro+1/ri).3
Data were normalized to control measurements obtained during normoxic
perfusion in each heart to permit comparisons between hearts. Once the
electrodes had been placed, hearts were perfused with
oxygenated buffer, and baseline
rt values were obtained during a
preischemic perfusion period of 10 minutes. Hearts were
then subjected to global ischemia, and
rt was measured every 2 minutes. During
ischemia, rt showed the
characteristic time course defined by an immediate early rise (first
phase, vascular collapse), a subsequent slow rise (second phase, rise
in extracellular resistance), and a marked final rise (third phase,
cell-to-cell uncoupling).3 The onset of uncoupling
was determined in each experiment by the transition from the second to
the third phase (see Figure 1
, top).
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Antibodies
A rabbit polyclonal antibody (Zymed) directed against epitopes
in the C terminus of rat Cx43 was used in
immunoblotting and
immunofluorescence studies as described
previously.20 26 27 We also used a mouse monoclonal
antibody (Zymed) shown by Nagy et al28 to bind selectively
to nonphosphorylated Cx43. As detailed below, we have
confirmed this finding and used this antibody to characterize the
amount and distribution of nonphosphorylated Cx43 in
rat hearts subjected to ischemia.
Preparation and Quantification of Immunoblots
Hearts were removed from the perfusion apparatus,
trimmed of atria and great vessels, and immediately freeze-clamped.
Pulverized samples were prepared and analyzed by quantitative
immunoblotting as described
previously.12 20
Immunofluorescence and Confocal
Microscopy
Hearts were removed from the perfusion apparatus and
fixed in 10% neutral buffered formalin in preparation for paraffin
embedding and confocal immunohistochemical analysis as
previously described.26 27 The amount of high-intensity
Cx43 signal in discrete spots at intercellular junctions was measured
as described previously26 27 and expressed as a proportion
of total tissue area.
Statistical Analysis
Data are expressed as mean±SD except where indicated.
Differences between sample populations in immunoblot
studies were determined with 1-way ANOVA using Tukey post hoc testing.
Differences in confocal immunofluorescence signal
intensities were determined with Kruskal-Wallis 1-way ANOVA using
Dunnett post hoc testing. A value of P<0.05 was considered
to be statistically significant.
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Results |
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Changes in rt During Acute Global Ischemia
Figure 1
shows changes in
rt induced by ischemia. As shown
previously with cable analysis in rabbit
hearts,3 changes in rt in
no-flow ischemia occurred in characteristic phases. A rapid
initial phase was associated with vascular collapse after induction of
ischemia. Between 4 and 14 minutes,
rt increased slowly, presumably because of
osmotic water shifts from the extracellular to the intracellular
compartment as previously described.3 After 14
minutes, a sustained rise in rt
attributable to uncoupling occurred and reached a plateau 22 minutes
later. The onset of uncoupling was distinct in each individual
experiment (Figure 1, top) but became somewhat blurred by
superimposition of 5 curves with slightly different time courses
(Figure 1, bottom). The mean time of onset of uncoupling in 5
hearts was 15.2±3.3 minutes. The observed pattern was virtually
identical to results of previous studies.3 23 24 The
plateau in rt seen 22 minutes after the
onset of uncoupling indicated that complete uncoupling had
occurred.29 30
Characterization of Cx43 Phosphorylation Isoforms
by Immunoblot Analysis
The polyclonal anti-Cx43 antibody detected major bands at 44 and
46 kDa and a faint band at 41 kDa in a blot prepared from control rat
ventricular homogenate (Figure 2), consistent with previous
reports that most of the Cx43 in the heart is
phosphorylated.20 28 31 When the
homogenate was preincubated with alkaline phosphatase, only
a single intense band at 41 kDa was observed. These results indicate
that the higher molecular weight bands comprise
phosphorylated isoforms of Cx43 and the rabbit
polyclonal antibody recognizes both phosphorylated and
nonphosphorylated isoforms on polyacrylamide
gels. The mouse monoclonal anti-Cx43 antibody barely recognized a faint
band at 41 kDa (Figure 2); it did not react with the more
prominent, higher molecular weight bands seen with the polyclonal
antibody. When the homogenate was preincubated with
alkaline phosphatase, this antibody recognized an intense band at 41
kDa. These results confirm that the mouse monoclonal antibody
selectively binds nonphosphorylated Cx43.
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Changes in Cx43 Phosphorylation During Acute
Ischemia and Ischemia/Reperfusion
Isolated perfused rat hearts were subjected to global
ischemia for 15, 30, or 40 minutes. The 15-minute time point
corresponded to the average time of onset of cell-to-cell uncoupling,
and the 30- and 40-minute points were times of more advanced and full
uncoupling, respectively (see Figure 1). Figure 3A shows a representative
immunoblot prepared with the polyclonal antibody. Acute
ischemia was associated with marked loss of
phosphorylated Cx43 (bands at 44 to 46 kDa) and a
corresponding increase in nonphosphorylated Cx43
(41-kDa signal). Loss of phosphorylated Cx43 was
apparent after 15 minutes of ischemia when uncoupling had just
begun and became more marked after 30 or 40 minutes of
ischemia. Densitometric analysis of
immunoblots from 4 hearts at each time point (Figure 3B) revealed no change in the total amount of Cx43 signal
(phosphorylated plus nonphosphorylated
isoforms). Thus, a 40-minute interval of global ischemia is
associated with progressive dephosphorylation of Cx43
but no net loss of total Cx43 protein content from
ventricular myocardium.
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To further characterize the temporal relationship between uncoupling
and Cx43 dephosphorylation, we prepared
immunoblots from 5 hearts that had undergone
ischemia for 7 minutes. At this time, all hearts had ceased
contractile activity but electrical uncoupling had not yet begun, as
shown in Figure 1 and indicated by the mean time of onset of
uncoupling (15.2±3.3 minutes; n=5). As shown in Figure 4A, there was little change in the
relative proportions of phosphorylated and
nonphosphorylated Cx43 in 3
representative hearts made globally ischemic
for 7 minutes.
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To determine whether reperfusion after ischemia led to
reaccumulation of phosphorylated Cx43, we studied
hearts subjected to 20 minutes of ischemia followed by
reperfusion for 30 minutes. We selected 20 minutes of ischemia
because by this time, electrical uncoupling and Cx43
dephosphorylation had begun (see Figures 1 and 3). Furthermore, some hearts resumed contractile activity during
a subsequent 30-minute interval of reperfusion, whereas others failed
to resume contractions and had presumably become irreversibly injured.
This ischemia/reperfusion protocol was, therefore, well suited
to investigating the relationship between functional recovery after
ischemia and phosphorylation of Cx43. We
monitored the return of contractile activity rather than a decrease in
rt as an indicator of functional recovery
because in preliminary studies we sometimes observed a rapid, dramatic
fall in rt toward the baseline value after
reperfusion even when no contractile function had returned. This rapid
decrease in rt is apparently related to
disruption of myocyte membranes and/or to marked extracellular edema
after reperfusion, each of which interferes with maintenance of
separate extracellular and intracellular resistive components in the
electrical bidomain.3
Figures 4B and 4C show immunoblots from
hearts that had undergone 20 minutes of ischemia or 20 minutes
of ischemia followed by 30 minutes of reperfusion. As shown in
a blot prepared with the polyclonal anti-Cx43 antibody (Figure 4B), considerable dephosphorylation of Cx43
occurred after 20 minutes of ischemia (lanes 2 and 3) compared
with a control heart (lane 1). Figure 4B also shows results from
4 hearts that had undergone 20 minutes of ischemia followed by
30 minutes of reperfusion. Two of these hearts failed to recover
contractile activity during reperfusion and had presumably become
irreversibly injured. There was no apparent increase in
phosphorylated Cx43 in these 2 hearts (Figure 4B, lanes 5 and 6). Indeed, there appeared to be further
dephosphorylation. In contrast, there was a noticeable
increase in the amount of phosphorylated Cx43 in 2
other hearts that resumed vigorous contractile activity during
reperfusion (Figure 4B, lanes 4 and 7). When the blot shown in
Figure 4B was stripped and reprobed with the monoclonal
antibody, the relative differences in the content of
nonphosphorylated Cx43 were readily apparent (Figure 4C). Little nonphosphorylated Cx43 was seen in
the control (nonischemic) heart, whereas accumulation of
nonphosphorylated Cx43 was apparent after 20 minutes of
ischemia. A marked difference was apparent in the level of
nonphosphorylated Cx43 in reperfused hearts that had
recovered contractile function and those that were irreversibly
injured. Taken together, these results suggest that significant
dephosphorylation of Cx43 does not occur until the
tissue uncouples, whereas reversal of uncoupling with reperfusion,
reflected by a recovery of contractile function, is associated with
reaccumulation of phosphorylated Cx43.
Changes in the Distribution of Cx43 Isoforms During Acute
Ischemia
Figure 5 shows
representative confocal images from a control heart and
hearts subjected to global ischemia. Figure 6 shows quantitative digital image
analysis of the proportion of tissue area occupied by discrete
spots of intense immunofluorescent signal in 5 fields per heart
from 4 hearts at each time point. In sections stained with the
polyclonal antibody, intense immunofluorescent signal was seen
in control hearts at discrete sites of intercellular apposition. In
contrast, virtually no signal was seen in control
myocardium stained with the monoclonal antibody. This
observation confirms previous findings suggesting that most if not all
of the Cx43 in gap junctions in normal myocardium is
phosphorylated.20 28 31 With
ischemia, progressive loss of immunoreactive signal at apparent
gap junctions occurred in sections stained with the polyclonal antibody
with a corresponding increase in signal in sections stained with the
monoclonal antibody. Because the relative titers and binding affinities
of the 2 anti-Cx43 antibodies used in these studies are not known, it
is not possible to directly compare the relative amounts of
phosphorylated and nonphosphorylated
Cx43 in gap junctions at any point during ischemia.
Nevertheless, the results in Figures 5 and 6 clearly
indicate that during uncoupling, nonphosphorylated Cx43
accumulated in sites of intercellular apposition (presumed gap
junctions) while at the same time the amount of total Cx43
(phosphorylated and nonphosphorylated)
in gap junctions was progressively reduced. The concentration of
intracellular Cx43 was sufficiently low that it was not detected by the
image-processing method used to analyze signal in intercellular
junctions. However, because the total tissue content of Cx43 did not
change during ischemia (Figure 3B), the
immunofluorescence data indicate that Cx43
translocates from gap junctions to intracellular site(s) during
uncoupling.
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Qualitative immunofluorescence analysis was
performed on representative hearts subjected to 20
minutes of ischemia followed by 30 minutes of reperfusion. As
shown in Figure 7, the amount of total
Cx43 signal was dramatically reduced and the amount of
nonphosphorylated Cx43 signal was greatly increased in
hearts that failed to recover contractile activity during reperfusion,
consistent with the immunoblot results shown in
Figure 4. In contrast, total Cx43 signal increased and
nonphosphorylated Cx43 signal was noticeably reduced in
hearts that did recover. Thus, functional recovery (and, presumably,
electrical recoupling) is associated with reaccumulation of
phosphorylated Cx43 and an increase in the amount of
Cx43 immunoreactivity at intercellular junctions.
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Discussion |
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The results of this study show that during the process of electrical uncoupling induced by ischemia, the principal cardiac gap-junction channel protein, Cx43, undergoes marked dephosphorylation. In previous studies, perfusion of isolated rat hearts with oxygenated buffer for 4 hours caused no change in Cx43 phosphorylation.20 Thus, dephosphorylation of Cx43 in the present studies was not related to the isolated perfused heart preparation. Although measurement of rt provides only a qualitative index of cell-to-cell coupling, the different phases of uncoupling during ischemia can be clearly discerned. With this approach, uncoupling and Cx43 dephosphorylation were first detected at about the same time after the onset of ischemia and progressed with a similar time course. Reperfusion after an interval of ischemia sufficient to cause both uncoupling and Cx43 dephosphorylation led to at least partial restoration of the control level of phosphorylated Cx43 in hearts that recovered contractile activity, whereas further dephosphorylation of Cx43 occurred in hearts that failed to recover. Immunohistochemical analysis using isoform-selective antibodies revealed that under physiological conditions, nearly all Cx43 at sites of intercellular apposition was phosphorylated. With uncoupling, however, there was progressive loss of Cx43 signal at sites of intercellular apposition concomitant with a marked increase in nonphosphorylated Cx43. In light of immunoblot data showing that the total amount of Cx43 remained constant throughout the ischemic interval, these observations suggest that uncoupling resulted in progressive dephosphorylation of Cx43, accumulation of the nonphosphorylated isoform within gap junctions, and translocation of Cx43 from gap junctions into an intracellular pool.
Although abundant evidence indicates that phosphorylated and nonphosphorylated isoforms of Cx43 migrate on SDS-polyacrylamide gels at 44 to 46 and 41 kDa, respectively, specific phospho–amino acid residues and the diversity of phosphorylated isoforms of Cx43 in the heart have not been defined precisely. Shifts in the migration of Cx43 bands and changes in the intensity of immunoreactive signal detected by antibodies used in the present study indicated that marked alterations in the amount and distribution of phosphorylated Cx43 occurred during ischemia, but no conclusions can be reached about specific biochemical changes in Cx43. It is possible that the monoclonal antibody could have detected some phosphorylated isoforms of Cx43 that migrated with apparent molecular weights at or near 41 kDa.32
Multiple mechanisms likely contribute to uncoupling during
ischemia, including increased levels of intracellular
Ca2+,4 5 8 progressive intracellular
acidosis,4,5,30 accumulation of lipid metabolites
that might act as uncoupling agents, 6,7 and
other potential mechanisms. Our results provide new evidence that
rapid, reversible Cx43 dephosphorylation could also
contribute to myocardial uncoupling and, thereby, play a role in
arrhythmogenesis during acute ischemia. One potential mechanism
of ischemia-induced accumulation of
dephosphorylated Cx43 is decreased intracellular ATP
concentration and decreased thermodynamic driving force for
phosphorylation (the free-energy change of ATP
hydrolysis, 
ATP). Interestingly, the decrease in ATP during
ischemia is biphasic with a moderate immediate decrease and a
marked secondary decrease that coincides with cell-to-cell
uncoupling.33 Although it remains unknown whether
uncoupling is preceded and caused by Cx43
dephosphorylation, whether
dephosphorylation occurs in channels that have already
uncoupled, or whether Cx43 dephosphorylation is the
result of increased phosphatase and/or decreased kinase activity, it is
likely that a reduction in ATP shifts the thermodynamic equilibrium
toward dephosphorylation. In any case,
dephosphorylation could serve as an initial step in
translocation of Cx43 from gap junctions into the cytoplasm during
ischemia.
The biological consequences of specific changes in Cx43 phosphorylation are not understood in detail. Dramatic decreases in gap junctional communication, and concomitant loss of phosphorylated Cx43 and accumulation of nonphosphorylated Cx43 have been reported in epithelial cells treated with 18ß-glycyrrhetinic acid34 and glial cells exposed to oleamide35 but, as in the present study, the relationship between changes in phosphorylation at specific residues and uncoupling was not defined. Although phosphorylation of serine residues in the carboxyl-terminal intracellular domain of Cx43 appears to be the major post-translation modification in Cx43 migrating on gels at 44 to 46 kDa,14 31 and generalized loss of serine phosphorylation reflected by a shift to 41 kDa is associated with uncoupling,34 35 phosphorylation of tyrosine residues can occur in Cx43 and is also associated with uncoupling.15 17 36 It is not known whether tyrosine phosphorylation of Cx43 occurred during uncoupling induced by ischemia in the present studies or whether the monoclonal antibody detected Cx43 isoforms containing phosphotyrosine. Future studies will be required to define the precise pathophysiological relationship between changes in phosphorylation at specific amino acid residues of Cx43 and uncoupling and the potential effects of modulating Cx43 phosphorylation on the development of arrhythmias during ischemia.
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Acknowledgments |
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This work was supported by NIH Grant HL50598 and the Swiss National Science Foundation. We thank Karen Green and Evelyn Kanter for expert technical assistance and Dr Kenneth Schechtman for assistance with statistical analyses.
Received July 26, 2000; revision received August 14, 2000; accepted August 18, 2000.
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