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Am J Physiol Heart Circ Physiol 292: H1764-H1769, 2007. First published December 1, 2006; doi:10.1152/ajpheart.01071.2006 Free Article
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Loss of ischemic preconditioning's cardioprotection in aged mouse hearts is associated with reduced gap junctional and mitochondrial levels of connexin 43

Kerstin Boengler,1 Ina Konietzka,1 Astrid Buechert,1 Yvonne Heinen,1 David Garcia-Dorado,2 Gerd Heusch,1 and Rainer Schulz1

1Institut für Pathophysiologie, Universitätsklinikum Essen, Germany; and 2Servicio de Cardiologia, Hospital Universitari Vall D'Hebron, Barcelona, Spain

Submitted 30 September 2006 ; accepted in final form 22 November 2006


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
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Connexin 43 (Cx43) is localized at left ventricular (LV) gap junctions and in cardiomyocyte mitochondria. A genetically induced reduction of Cx43 as well as blockade of mitochondrial Cx43 import abolishes the infarct size (IS) reduction by ischemic preconditioning (IP). With progressing age, Cx43 content in ventricular and atrial tissue homogenates is reduced. We now investigated whether or not 1) the mitochondrial Cx43 content is reduced in aged mice hearts and 2) IS reduction by IP is lost in aged mice hearts in vivo. Confirming previous results, sarcolemmal Cx43 content was reduced in aged (>13 mo) compared with young (<3 mo) C57Bl/6 mice hearts, whereas the expression levels of protein kinase C {epsilon} and endothelial nitric oxide synthase remained unchanged. Also in mitochondria isolated from aged mice LV myocardium, Western blot analysis indicated a 40% decrease in Cx43 content compared with mitochondria isolated from young mice hearts. In young mice hearts, IP by one cycle of 10 min ischemia and 10 min reperfusion reduced IS (% of area at risk) following 30 min regional ischemia and 120 min reperfusion from 67.7 ± 3.3 (n = 17) to 34.2 ± 6.6 (n = 5, P < 0.05). In contrast, IP's cardioprotection was lost in aged mice hearts, since IS in nonpreconditioned (57.5 ± 4.0, n = 10) and preconditioned hearts (65.4 ± 6.3, n = 8, P = not significant) was not different. In conclusion, mitochondrial Cx43 content is decreased in aged mouse hearts. The reduced levels of Cx43 may contribute to the age-related loss of cardioprotection by IP.

left ventricle; infarct size; translocase of the outer membrane 20

BRIEF PERIODS OF ischemia-reperfusion delay the development of cell death from a subsequent prolonged episode of ischemia-reperfusion, a phenomenom known as ischemic preconditioning (IP; for review see Refs. 10 and 34). Connexin 43 (Cx43) is essential for cardioprotection (36). The cardioprotection by ischemic or pharmacological preconditioning with diazoxide is abolished both in in situ hearts and in isolated cardiomyocytes from heterozygous Cx43-deficient mice (17, 23, 37). Apart from its localization at the sarcolemma, Cx43 is also detected at the inner membrane of cardiomyocyte mitochondria (3, 32), i.e., in cell organelles that are central for triggering/mediating the cardioprotection by IP (21, 26, 28). Indeed, the reduction of mitochondrial Cx43 by geldanamycin, which inhibits heat shock protein 90 and thereby decreases the translocation of Cx43 to the mitochondria, abolishes the infarct size reduction by diazoxide otherwise seen in isolated rat hearts (32).

Reactive oxygen species (ROS), which are partially produced by uncoupling of oxidative phosphorylation, trigger IP's cardioprotection if present in small amounts (for review, see Ref. 27). ROS are also produced by diazoxide in isolated cardiomyocytes from wild type but not from heterozygous Cx43-deficient mice, thereby pointing to a role for mitochondrial Cx43 in the formation of ROS specifically in response to diazoxide (17).

A decrease of the myocardial Cx43 content has been described in failing hearts of rabbits (5) and patients (11), and in such diseased myocardium IP's cardioprotection is often abolished (13). Also during aging, a reduced level of Cx43 has been observed in the ventricles of hamsters (6) and in the sinoatrial node of guinea pigs (18); data on the mitochondrial Cx43 content in aged and/or diseased hearts are lacking. Whether or not IP is effective in the aged myocardium is discussed controversially (19). Depending on the species and the respective age of the animals at the time of the study, infarct size reduction by IP was present or absent.

We now analyzed 1) whether mitochondrial Cx43 levels are decreased in aged mouse myocardium and 2) whether or not IP is abolished in the aged mouse heart in vivo.


   MATERIALS AND METHODS
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 MATERIALS AND METHODS
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The present study was performed with approval by the Bioethical Committee of the district of Düsseldorf, Germany. It conforms to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (National Institutes of Health publication no. 85–23, revised 1996).

Young (<3 mo) and aged (>13 mo) C57/Bl6 mice were purchased from Charles River Laboratories (Kißlegg, Germany) or were retired breeders from our own animal facility.

Immunohistochemistry. Sections (4 µm) of paraffin-embedded tissue from the left ventricle of female C57/Bl6/J mice of different ages (<3 mo: n = 8, >13 mo: n = 13) were stained with antibodies against Cx43 (rabbit polyclonal anti-rat Cx43, dilution 1:100; Invitrogen, Carlsbad, CA) and the respective FITC-labeled secondary antibodies. Rhodamine-phalloidin was used to visualize actin. The samples were examined under the same settings by confocal laser scan microscopy (Pascal; Zeiss, Jena, Germany) at x630 magnification. Five optical fields per mouse were analyzed, and the Cx43 immunoreactivity at the gap junctions was quantified. Additionally, the area of the cardiomyocytes was measured in square micrometers. For negative control, the primary antibodies were omitted.

Isolation of mitochondria. The left ventricles of female C57/Bl6/J mice of different ages (<3 mo: n = 14, >13 mo: n = 10) were minced in isolation buffer (250 mM sucrose, 10 mM HEPES, 1 mM EGTA, and 0.5% BSA; pH 7.4). The buffer was changed several times to wash the tissue from blood. Tissue samples were homogenized (Ultra Turrax, 3 steps of ~5 s each) and then centrifuged at 700 g for 10 min. The supernatant was filtered through a nylon filter of ~250 µm pore size and then centrifuged at 10,780 g for 10 min. The sediment was resuspended in isolation buffer and centrifuged at 7,650 g for 10 min. Again, the sediment was resuspended in isolation buffer and then layered on top of a 30% Percoll solution in isolation buffer and centrifuged at 35,000 g for 30 min. The lower band reflecting the intact mitochondria was collected and washed two times in isolation buffer by centrifugation at 7,650 g for 5 min.

The purification procedure was validated as the enrichment in mitochondrial proteins as well as the elimination of other cellular constituents by means of Western blot analysis (3, 4).

Western blot analysis. The right ventricles (RV) of female C57/Bl6/J mice of different ages (<3 mo: n = 10, >13 mo: n = 18) or the mitochondria of the left ventricles were homogenized in 1x Cell Lysis buffer (Cell Signaling, Beverly, MA) containing 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerolphosphate, 1 mM Na3VO4 1, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1% Triton X-100, pH 7.5, supplemented with Complete Protease Inhibitors (Roche, Basel, Switzerland). The samples were centrifuged at 13,000 g for 10 min at 4°C. The supernatants were collected, and the protein concentrations were determined using the Dc protein assay (Bio-Rad, Hercules, CA). RV (20 µg) or mitochondrial proteins were electrophoretically separated on 10% SDS-PAGE and transferred to nitrocellulose membranes. After being blocked, the membranes were incubated with rabbit polyclonal anti-rat Cx43 (dilution 1:1,000; Invitrogen), goat polyclonal anti-human protein kinase c {epsilon} (PKC{epsilon}, dilution 1:500; Santa Cruz, Santa Cruz, CA), mouse monoclonal anti-human endothelial nitric oxide synthase (eNOS, dilution 1:300; BD-Transduction, San Jose, CA), rabbit polyclonal anti-human translocase of the outer membrane 20 (Tom20, dilution 1:500; Santa Cruz), or mouse monoclonal anti-rabbit Na+-K+-ATPase (dilution 1:500; Upstate, Lake Placid, NY) antibodies. Immunoreactive signals were detected by chemiluminescence (SuperSignal West Femto Maximum Sensitivity Substrate; Pierce, Rockford, IL) and quantified with the Scion Image software (Frederick, MD). The immunoreactive signals were normalized to Ponceau S staining. The Cx43 protein levels normalized to Ponceau S were not significantly different between the right and the left ventricles of <3 mo old C57/Bl6 mice {0.58 ± 0.12 arbitrary units (AU; in the RV) vs. 0.56 ± 0.20 AU [left ventricle (LV)], n = 5, P = not significant}.

We have previously demonstrated that Cx43 is involved in the signal transduction cascade of IP, potentially by modifying ROS formation/release (17). To attenuate IP's cardioprotection, Cx43 (at the level of the mitochondria) has to be reduced before the preconditioning cycle of ischemia-reperfusion. Therefore, the mitochondrial Cx43 content was analyzed before the sustained ischemia.

In vivo mouse model. C57Bl6/J mice of different ages were anesthetized with pentobarbital sodium (80 mg/kg ip). The temperature of the animals was kept stable between 36.6 and 37.4°C using heating pads, and the electrocardiogram was monitored continuously. After intubation, the animals were ventilated with a stroke rate of 130/min and a tidal volume of 1 ml. The left anterior descending coronary artery of C57Bl6/J mice of different ages was occluded for 30 min and reperfused for 120 min (young: n = 19, 12 males, 7 females; aged: n = 10, 5 males, 5 females). IP was induced by a cycle of 10 min ischemia and 10 min reperfusion before 30 min sustained ischemia and 120 min reperfusion (young: n = 6, 5 males, 1 female; aged: n = 9, 7 males, 2 females). Because it has been demonstrated that the infarct size is not affected by reperfusion times between 2 and 24 h (12, 16), in vivo experiments were performed with 120 min reperfusion. The area at risk (AAR) was measured by Evans blue, and the infarct size was determined by 2,3,5-triphenyl tetrazolium chloride staining.

Statistics. Data are reported as mean values ± SE. Immunohistochemical and Western blot data were compared by unpaired Student's t-test. Infarct size data were compared using two-way ANOVA and post hoc tests (Fisher least-significant difference). A P < 0.05 was considered to indicate a significant difference.


   RESULTS
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The area of cardiomyocytes and the Cx43 protein content at the gap junctions of left ventricular (LV) tissue sections of mice of different ages were analyzed by immunohistochemistry and confocal laser scan microscopy (Fig. 1A). The measurement of the cardiomyocyte area indicated that the cardiomyocytes became hypertrophic with aging (young mice: 377.2 ± 11.6 µm2, n = 8 vs. aged mice: 573.9 ± 25.1 µm2, n = 13, P < 0.05; Fig. 1B). However, both the gap junctional Cx43 level, which was not normalized to the cell area (young: 59.21 ± 0.57 AU, n = 8 vs. aged: 37.63 ± 1.91 AU, n = 13, P < 0.05; Fig. 1D) and the gap junctional Cx43 level normalized to the cardiomyocyte area (young: 2.0 ± 0.07 AU, n = 8, vs. aged: 1.14 ± 0.03 AU, n = 13, P < 0.05; Fig. 1C), were reduced in aged compared with young mouse myocardium.


Figure 1
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  		Fig. 1. Connexin 43 (Cx43) content at the gap junctions in the left ventricular (LV) myocardium of mice of different ages. A: LV tissue sections of mice of different age (<3 mo and >13 mo) were stained with antibodies against Cx43 (green) and analyzed by confocal laser scan microscopy. Actin was labeled with phalloidin (red). B: area of cardiomyocytes in LV tissue sections was measured in young (n = 8) and aged (n = 13) C57/Bl6/J mice. *P < 0.05. C: Cx43 immunoreactivity at the gap junctions of one cell was normalized to the area of the respective cardiomyocyte in tissue sections from young (n = 8) and aged (n = 13) mice. *P < 0.05. D: quantification of the Cx43 immunoreactivity at the gap junctions in LV mouse myocardium of different ages (young: n = 8, aged: n = 13) without normalization to the cardiomyocyte area. *P < 0.05.

 
With the use of Western blot analysis, the Cx43 protein levels in total ventricular protein extracts of mice were determined and normalized to Ponceau S staining. Compared with young mice (0.16 ± 0.03 AU, n = 10), the amount of Cx43 was decreased in old mice (0.06 ± 0.01 AU, n = 18, P < 0.05). However, the protein levels of both eNOS and PKC{epsilon} were not altered with progressing age (Fig. 2).


Figure 2
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  		Fig. 2. Cx43, but not endothelial nitric oxide synthase (eNOS) or protein kinase C {epsilon} (PKC{epsilon}) protein levels, are reduced with increasing age in the mouse ventricle. A: eNOS, PKC{epsilon}, and Cx43 protein levels in total right ventricular protein extracts from mice of <3 mo and >13 mo of age were determined by Western blot analysis. Ponceau S staining demonstrates equal protein loading. B: bar graphs representing eNOS, PKC{epsilon}, and Cx43 immunoreactivities in right ventricular protein extracts from mice of different ages (<3 mo: eNOS and PKC{epsilon}: n = 7, Cx43: n = 10; >13 mo: eNOS and PKC{epsilon}: n = 17, Cx43: n = 18). *P < 0.05.

 
Isolated mitochondria were not contaminated with plasma membrane proteins, as shown by the absence of Na+-K+-ATPase immunoreactivity in the mitochondrial preparations using Western blot analysis (Fig. 3A). The Cx43 level, which was normalized to Ponceau S, decreased from 0.17 ± 0.02 AU (n = 14) in mitochondria from young mice to 0.11 ± 0.02 AU (n = 10, P < 0.05) in mitochondria from >13-mo-old mice (Fig. 3C). The mitochondrial protein content of Tom20, the presequence receptor of the mitochondrial import machinery, was reduced from 0.19 ± 0.01 AU in mitochondria from young mice (n = 14) to 0.15 ± 0.02 AU in mitochondria isolated from aged mice (n = 10, P < 0.05; Fig. 3B).


Figure 3
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  		Fig. 3. Cx43 and translocase of the outer membrane 20 (Tom20) protein level in mitochondria isolated from LV mycardium from young and aged mice. A: Western blot analysis was performed on mitochondrial protein extracts from young (<3 mo) and aged (>13 mo) mice and on total right ventricular proteins (<3 mo) for Na+-K+-ATPase and Cx43. Ponceau S staining demonstrates equal protein loading. B: Western blot analysis was performed for Tom20 on mitochondrial protein extracts from young and aged mice and on total right ventricular proteins. Ponceau S staining demonstrates equal protein loading. C: quantification of the Cx43 and Tom20 immunoreactivity in mitochondria of young (<3 mo, n = 14) and aged (>13 mo, n = 10) mice normalized to Ponceau S staining. *P < 0.05 vs. <3 mo.

 
Young and aged mice underwent 30 min of regional ischemia and 120 min of reperfusion without or with IP. During coronary occlusion, electrocardiogram recordings demonstrated an elevation of the S-T segment (data not shown). Two young mice in the ischemia group as well as one young and one old mouse in the IP group died prematurely and were excluded from the study. There were no differences in mortality induced by ischemia-reperfusion between the groups. The AAR (% of the LV) was 38.7 ± 3.0% in the young (n = 17) and 33.3 ± 3.9% in the old (n = 10) ischemia group and was 37.6 ± 5.5% in the young (n = 5) and 39.1 ± 3.4% in the old (n = 8) IP group. There was no significant difference in the AAR between the groups. The infarct size in the young mice was 67.7 ± 3.3% of the AAR. IP by a preceding cycle of 10 min ischemia and 10 min reperfusion reduced the infarct size to 34.2 ± 6.6% of the AAR (P < 0.05). In the old mice hearts subjected to 30 min of regional ischemia and 120 min of reperfusion, the infarct size was 57.5 ± 4.0% of the AAR. In contrast to the young mice, IP's cardioprotection was lost in old mice hearts (infarct size 65.4 ± 6.3% of the AAR; Fig. 4).


Figure 4
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  		Fig. 4. Cardioprotection by ischemic preconditioning in young and aged mice. Infarct size as percent of the area at risk (AAR) in young (<3 mo) and aged (>13 mo) mice after 30 min ischemia and 120 min reperfusion (Isch, <3 mo: n = 17, >13 mo: n = 10) or after a preconditioning cycle of 10 min ischemia and 10 min reperfusion followed by 30 min ischemia and 120 min reperfusion (IP, <3 mo: n = 5, >13 mo: n = 8). Results of the individual animals and mean values ± SE are reported. bullet, Male mice; {circ}, female mice. *P < 0.05 vs. Isch <3 mo.

 

   DISCUSSION
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The data reported in our study show that IP's cardioprotection is lost in aged mouse heart in vivo. In the aged compared with young ventricular tissue, the Cx43 protein level was reduced both at the sarcolemma and in mitochondria.

Several genes are differentially expressed in the aged myocardium (40), among them genes encoding proteins involved in the signal transduction of cardioprotection (38). Because IP is dependent on the Cx43 content, we determined the protein level of Cx43 by Western blot analysis in total RV protein extracts from mice of different ages. In the heart, the eNOS content and activity remains stable with progressing age (39); therefore, the eNOS protein level was analyzed for control. Our results confirmed an unaltered eNOS protein content in mouse myocardium between <3 mo and >13 mo of age.

PKC{epsilon} is involved in the signal transduction cascade of IP (30), and Cx43 represents one target of PKC{epsilon} (9). A decrease of the PKC{epsilon} level in the aged rat heart has been described in the soluble fraction only, whereas its content is unaltered in the particulate fraction (20). In the present study, where PKC{epsilon} expression was determined in total extracts only (which does not allow one to distinguish between subcellular localizations of the protein), no differential expression of PKC{epsilon} was measured with age.

A decreased Cx43 level has been described in aged endothelial cells and human dental pulp (25, 42). The amount of Cx43 is reduced with increasing age in the sinoatrial node of guinea pigs (18), and furthermore a decrease of Cx43 has been described in ventricles from adult (13–18 wk) to old (>45 wk) hamsters (6). However, in rabbit hearts from 6 to 32 mo of age, i.e., in a model in which IP's cardioprotection is effective (31), a redistribution of Cx43 from the gap junctions to the lateral cardiomyocyte sarcolemma rather than a reduction of the protein level has been observed (8).

In our study, young mice were compared with >13-mo-old mice, and we confirmed the decrease of the Cx43 level with progressing age at ventricular gap junctions as well as in total RV protein extracts.

The mitochondrial Cx43 content was also affected by age. Our data show that the Cx43 protein level in mitochondria is reduced with increasing age. Thus it is not likely that a Cx43 redistribution between the sarcolemma and the mitochondria occurs with increasing age.

When studying the response of the heart to ischemia-reperfusion, the aged myocardium shows a reduced tolerance to ischemic injury (1). In C57/Bl6 mice, the intrinsic myocardial tolerance to ischemia is decreased with 12 mo of age (41), and an increase in the infarct size induced by ischemia/reperfusion has been described in aged (22–24 mo) C57/Bl6 mice (2). However, also a reduction in infarct size 24 h after coronary ligation has been observed in aged (14 mo) compared with young mice (14). The reason for these different findings are unclear at present.

Whether IP's cardioprotection is impaired in aged myocardium has been studied in animal models and in the human heart. However, the results on the effectiveness of IP in the aged myocardium remain controversial (for review, see Ref. 19). Focusing on in vivo studies, IP's cardioprotection was preserved in 4-yr-old rabbits and 5.7- to 8-yr-old sheep (31), whereas IP's cardioprotection was impaired in the senescent rat heart (24 mo old; see Ref. 43). Our study is the first in which IP's cardioprotection was analyzed in the aged mouse heart in situ, and, in contrast to young hearts, IP is ineffective at an age >13 mo. Whether or not the different results relate to species differences (mice and rats vs. rabbits and sheep) or the age (relative to the maximal life span of the respective species, which is ~2 yr for mice, 3 yr for rats, 9 yr for rabbits, and 15 yr for sheep) at the time of investigation remains unanswered.

It has been demonstrated that 10 min ischemia followed by 2 h reperfusion induce an infarct size of 3.5% of the AAR (12). The preconditioning protocol used in the present study (preconditioning episode of 10 min ischemia and 10 min reperfusion), however, was sufficient to reduce infarct size in young mice, whereas IP's cardioprotection was lost in aged mice. The infarct size between young and aged mice after ischemia-reperfusion was not significantly different; therefore, the ~50% reduction of Cx43 with progressing age does not seem to play a role in the irreversible tissue damage induced by ischemia-reperfusion alone. However, when gap junctional communication was almost completely inhibited by the use of gap junction uncouplers such as heptanol, 18-glycyrrhetinic acid, or palmitoleic acid, a limitation of ischemia-reperfusion injury has been observed (33). When interpreting these results, it has to be taken into account that the large safety factor of gap junction-mediated intercellular communication predicts that a 50% reduction in the number of channels should have no detectable effect on electrical or chemical cell coupling.

A reduction of the Cx43 protein level may influence the susceptibility of the heart toward arrhythmias. Whereas under physiological conditions a decrease of Cx43 to ~60% of control values was not sufficient to induce tachyarrhythmias (7), the analysis of heterozygous Cx43-deficient mice demonstrated the occurrence of tachyarrhythmias during 60 min of ischemia (22). In contrast, the in situ analysis of Cx43+/– mice in our own laboratory did not show differences in electrocardiographic recordings compared with wild-type hearts (37).

Because IP's cardioprotection is not dependent on the presence of gap junctions (23) and is not mediated by effects of cell-cell electrical coupling (29), it is unlikely that the loss of gap junctional Cx43 during aging is involved in the loss of cardioprotection by IP in the aged mice. Rather, the reduced content of mitochondrial Cx43, which is implicated in the trigger phase of preconditioning (17), may be related to the abolition of IP's cardioprotection in the >13-mo-old mice. It has been demonstrated in a previous study that the cardioprotection by pharmacological preconditioning with diazoxide is abolished when the mitochondrial Cx43 level is decreased below a certain threshold level (32). The application of geldanamycin before sustained ischemia induces such a critical reduction of mitochondrial Cx43 and thereby abolishes pharmacological preconditioning with diazoxide. Therefore, in experiments in which gap junctional Cx43 is not altered but mitochondrial Cx43 is decreased below a certain threshold level, the cardioprotection is lost.

IP induced an about threefold increase of the mitochondrial Cx43 in preconditioned pigs after 85 min low-flow ischemia compared with nonpreconditioned animals (3). At the same time point, the sarcolemmal Cx43 content was not different between preconditioned and nonpreconditioned pigs (35). This finding makes is unlikely that the increased mitochondrial Cx43 content is the result of a translocation of the protein from the plasma membrane to the mitochondria. In isolated rat hearts, an increase of mitochondrial Cx43 was already detected after two short cycles of 5 min ischemia and reperfusion. The time frame of 20 min is too short to explain the enhancement of mitochondrial Cx43 level by de novo synthesis of the protein only. Therefore, the increased mitochondrial Cx43 level induced by IP is presumably the result of an increased import, a decreased degradation of the protein, or both.

Because Cx43 interacts with the presequence receptor Tom20 of the TOM (translocase of the outer membrane) complex of the mitochondrial import machinery (32) and the Tom20 protein level controls the import of proteins in the mitochondria (15, 24), we analyzed the Tom20 protein content in mitochondria isolated from young and aged mice and found the Tom20 protein level significantly reduced in aged mitochondria. Therefore, it is possible that the reduced Tom20 protein content is involved, at least in part, in impairing Cx43 translocation in the mitochondria in the aged cell organelles.

Taken together, our data demonstrate that the protein level of Cx43 at the sarcolemmal gap junctions as well as at the mitochondria is significantly reduced in aged compared with young mouse myocardium. In the aged mouse heart in vivo, IP is not effective. Because Cx43 is important for IP's cardioprotection, a reduced Cx43 content with increasing age, specifically at the level of mitochondria, may contribute to the loss of cardioprotection by ischemic preconditioning.


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R. Schulz was the recipient of a grant from the Deutsche Forschungsgemeinschaft (Schu 843/7-1).


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Schulz, Institut für Pathophysiologie, Zentrum für Innere Medizin, Universitätsklinikum Essen, Hufelandstr. 55, 45122 Essen, Germany (e-mail: rainer_schulz{at}uni-essen.de)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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