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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 20, 2003 as doi:10.1096/fj.02-0975fje. |
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Institut für Pathophysiologie, Zentrum für Innere Medizin, Universitätsklinikum Essen, 45122 Essen, Germany
2Correspondence: Institut für Pathophysiologie, Zentrum für Innere Medizin, Universitätsklinikum Essen, Hufelandstrasse 55, 45122 Essen, Germany. E-mail: rainer_schulz{at}uni-essen.de
SPECIFIC AIMS
The present study investigated whether nonphosphorylated and total Cx43 are altered during normoperfusion and subsequent 90 min ischemia in non-preconditioned and preconditioned hearts in vivo. We assessed the colocalization of PKC
, PKC
, p38MAPK, and p38MAPKß with Cx43 at the level of cell–cell contacts and measured the activity of p38MAPK. In a subset of experiments, infarct size and p38MAPK colocalization with Cx43 were studied in hearts in which an intracoronary infusion of the p38MAPK inhibitor SB203580 was started before the preconditioning ischemia and maintained until the end of the sustained ischemia.
PRINCIPAL FINDINGS
1. Infarct size
As expected, infarct size was reduced from 25.5 ± 4.2 to 10.3 ± 2.3% of the area at risk (P<0.05) by ischemic preconditioning, but remained at 20.0±3.9% in preconditioned hearts receiving SB203580. Heart rate, left ventricular pressure, area at risk, and ischemic myocardial blood flow were similar in non-preconditioned and preconditioned hearts in the absence or presence of SB203580.
2. Cx43
The density of total Cx43 and nonphosphorylated Cx43 at the intercalated disks did not differ between non-preconditioned and preconditioned hearts at baseline (see representative example in Fig. 1
). At 5 min of sustained ischemia, all parameters remained unchanged in non-preconditioned and preconditioned hearts (Fig. 1
and Fig. 2
). However, prolongation of ischemia to 85–90 min induced marked changes in the density of total and nonphosphorylated Cx43 in the non-preconditioned hearts, i.e., total Cx43 decreased whereas nonphosphorylated Cx43 increased (Figs. 1
and 2)
. In contrast, in preconditioned hearts total Cx43 and nonphosphorylated Cx43 remained stable (Figs. 1
and 2)
. At 30 min reperfusion, the density of nonphosphorylated Cx43 returned toward baseline values in non-preconditioned hearts (Figs. 1
and 2)
.
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3. Colocalization of PKC and PKC with Cx43
The density of PKC (91±7 vs. 90±6 AU) and PKC (106±6 vs. 113±5 AU) at the intercalated disks did not differ between non-preconditioned and preconditioned hearts at baseline. At 5 min of the sustained ischemia, no significant change in the colocalization of Cx43 with either protein kinase was observed in non-preconditioned and preconditioned hearts. With prolongation of ischemia to 85–90 min, colocalization of PKC (85±9 AU) with Cx43 at the intercalated disks remained unchanged in non-preconditioned hearts. At 30 min reperfusion, the colocalization of PKC (87±10 AU) with Cx43 at the intercalated disks remained unchanged in non-preconditioned hearts. In contrast, the colocalization of PKC with Cx43 was increased in preconditioned hearts at 85–90 min ischemia (103±3 AU) (P<0.05 vs. baseline and non-preconditioned hearts) and at 30 min reperfusion (105±7 AU). PKC colocalization with Cx43 remained unaltered in non-preconditioned and preconditioned hearts.
4. PKC and PKC protein contents
At 5 min and 85 min ischemia, expression of PKC in the cytosolic fraction tended to be increased in non-preconditioned and preconditioned hearts. Expression of PKC in the membrane fraction was increased at 5 min ischemia in non-preconditioned and preconditioned hearts, without any significant difference between groups. The increased expression of PKC at the membrane was preserved even when ischemia was prolonged to 85–90 min. No significant change in PKC within the cytosolic or the membrane fraction was detected in either group.
5. Colocalization of p38MAPK and p38MAPKß with Cx43
The density of p38MAPK (94±2 vs. 89±2 AU) and p38MAPKß (74±4 vs. 75±4 AU) at the intercalated disks did not differ between non-preconditioned and preconditioned hearts at baseline. At 5 min of the sustained ischemia, no significant change in the colocalization of Cx43 with either kinase was observed in non-preconditioned and preconditioned hearts. With prolongation of ischemia to 85–90 min, colocalization of p38MAPK (95±1 AU) and p38MAPKß (76±5 AU) with Cx43 at the intercalated disks remained unchanged in non-preconditioned hearts. The colocalization p38MAPK (101±2 AU) and p38MAPKß (89±4 AU) with Cx43 was increased in preconditioned hearts (all P<0.05 vs. baseline and non-preconditioned hearts). At 30 min reperfusion, the colocalization of p38MAPK (95±4 AU) and p38MAPKß (85±3 AU) with Cx43 at the intercalated disks was not significantly different from baseline in non-preconditioned hearts, but colocalization of p38MAPK (114±6 AU) and p38MAPKß (101±5 AU) with Cx43 remained elevated in preconditioned hearts. Intracoronary infusion of SB203580 significantly attenuated colocalization of p38 MAPK and p38MAPKß with Cx43 at 85 min ischemia. Neither the infarct size reduction nor the increased colocalization achieved by ischemic preconditioning was completely blocked following SB203580. Moreover, a significant correlation between the increased colocalization of p38 MAPKß with connexin 43 at 85 min ischemia and reduction in infarct size was observed in preconditioned hearts in the absence or presence of SB203580 (y=0.71x-68.88, r=0.73, P<0.05). Such close correlation was not detected for the increased colocalization of p38MAPK with connexin 43 at 85 min ischemia and the reduction in infarct size (y=0.49x-41.96, r=0.44).
6. p38MAPK or p38MAPKß activity
The activity of p38MAPK was increased at 5 min and more so at 85 min ischemia in non-preconditioned hearts (166±79% and 290±79% of baseline, respectively, both P<0.05) and preconditioned hearts (167±58% and 401±160% of baseline, respectively, both P<0.05). Whereas p38MAPKß activity tended to be increased in non-preconditioned and preconditioned hearts at 5 min ischemia (218±62% vs. 162±73%, respectively), at 85–90 min ischemia it remained increased only in preconditioned hearts (273±45% vs. 93±44% in non-preconditioned hearts, P<0.05 vs. baseline and between groups).
CONCLUSIONS AND SIGNIFICANCE
The present study demonstrates for the first time in vivo that dephosphorylation of Cx43 occurs during sustained ischemia. Such dephosphorylation of Cx43 is abolished by ischemic preconditioning. Colocalization of certain protein kinases with Cx43 at the intercalated disks is increased by ischemic preconditioning. Finally, the present study demonstrates for the first time a differential activation pattern of p38MAPK and p38MAPKß activity during sustained ischemia in non-preconditioned vs. preconditioned hearts.
In isolated buffer-perfused rat hearts, Cx43 is dephosphorylated during sustained ischemia. This result was extended in the present study to the in vivo pig heart. Dephosphorylation of Cx43 increases the permeability of gap junctions and hemichannels, thereby contributing to the propagation of ischemia/reperfusion injury. In support of this idea, ischemic preconditioning reduced infarct size and completely abolished the dephosphorylation of Cx43 in the present study.
Functional gap junctions and/or hemichannels are necessary for the signal transduction of ischemic preconditioning, since uncoupling of gap junctions and/or hemichannels by pretreatment with heptanol or the reduction in the number of functional gap junctions and/or hemichannels in heterozygous deficiency of Cx43 abolished ischemic preconditioning’s protection in mice hearts. Such an ambivalent role (i.e., involvement not only in ischemia/reperfusion damage but also in ischemic preconditioning’s protection) has been demonstrated for free radicals: free radicals per se contribute to irreversible tissue injury during sustained ischemia but are involved in the signal transduction cascade of ischemic preconditioning’s protection.
PKC is an established mediator of ischemic preconditioning in mice and rabbits and is involved in signaling complexes with at least 36 proteins in mice, among them Cx43. Indeed, activated PKC colocalizes with Cx43 and contributes to phosphorylation of Cx43 in rats. In larger mammals, PKC appears to be more important for ischemic preconditioning’s protection; although in the present study PKC and PKC were expressed in the membrane fraction at baseline, only PKC expression was significantly increased by ischemia/reperfusion. PKC contributes to phosphorylation of Cx43 at its cytoplasmic domain Ser368 in rats in vitro. Since the antibody directed against the nonphosphorylated Cx43 recognizes Ser368, it appears likely that the increased colocalization of PKC with Cx43 at the intercalated disks in preconditioned hearts contributes to the preservation of Cx43 phosphorylation. In humans, activated PKC and PKC colocalize with Cx43. Whether PKC is required for such Cx43 phosphorylation in pigs is unknown.
p38MAPK is phosphorylated within minutes during global or regional no-flow ischemia in isolated rat hearts as well as in rat, dog, and pig hearts in vivo. With prolongation of ischemia, however, phosphorylation of p38MAPK may be reduced toward preischemic values but is once again increased upon reperfusion. p38MAPK and p38MAPKß isoforms are expressed within the heart and appear to mediate opposing biological functions. The present study demonstrates that p38MAPK and p38MAPKß activities are increased at 5 min ischemia in non-preconditioned and preconditioned hearts. However, whereas the activity of p38MAPK remains elevated with prolongation of ischemia in non-preconditioned and preconditioned hearts, p38MAPKß activity remains increased in preconditioned hearts but is once again decreased toward baseline values in non-preconditioned hearts. Apart from differences in p38MAPK activity, p38MAPK and p38MAPKß isoforms colocalize with Cx43 and such colocalization is increased during sustained ischemia only in preconditioned hearts. Such increased colocalization of p38MAPK and p38MAPKß with Cx43 is prevented by SB203580 as is the infarct size reduction by ischemic preconditioning. Increased Cx43 phosphorylation after angiotensin II stimulation is mediated by p38MAPK in neonatal rat ventricular cardiomyocytes, since it was abolished by the p38MAPK antagonist SB202190.
Phosphorylation of Cx43 will decrease the permeability and conductance of gap junctions and hemichannels. Whether modulation of the permeability and conductance of gap junctions (and thus cell–cell communication) or hemichannels (and thus volume regulation) or both, is responsible for the cardioprotection obtained by ischemic preconditioning is unknown and requires further in vitro studies.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0975fje; doi: 10.1096/fj.02-0975fje 
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