AJP - Heart Information on EB 2010
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 295: H2106-H2112, 2008. First published October 3, 2008; doi:10.1152/ajpheart.00456.2008
0363-6135/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
295/5/H2106    most recent
00456.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Totzeck, A.
Right arrow Articles by Schulz, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Totzeck, A.
Right arrow Articles by Schulz, R.

No impact of protein phosphatases on connexin 43 phosphorylation in ischemic preconditioning

Andreas Totzeck,1,* Kerstin Boengler,1,* Anita van de Sand,1 Ina Konietzka,1 Petra Gres,1 David Garcia-Dorado,2 Gerd Heusch,1 and Rainer Schulz1

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

Submitted 30 April 2008 ; accepted in final form 26 September 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cardiac connexin 43 (Cx43) is involved in infarct propagation, and the uncoupling of Cx43-formed channels reduces infarct size. Cx43-formed channels open upon Cx43 dephosphorylation, and ischemic preconditioning (IP) prevents the ischemia-induced Cx43 dephosphorylation. In addition to the sarcolemma, Cx43 is also present in the cardiomyocyte mitochondria. We now examined the interaction of Cx43 with protein phosphatases PP1{alpha}, PP2A{alpha}, and PP2B{alpha} and the role of such interaction for Cx43 phosphorylation in preconditioned myocardium. Infarct size (in %area at risk) in left ventricular anterior myocardium of Göttinger minipigs subjected to 90 min of low-flow ischemia and 120 min of reperfusion was 23.1 ± 2.7 [n = 7, nonpreconditioned (NIP) group] and was reduced by IP to 10.0 ± 3.2 (n = 6, P < 0.05). Mitochondrial and gap junctional Cx43 dephosphorylation increased after 85 min of ischemia in NIP myocardium, whereas Cx43 phosphorylation was preserved with IP. PP2A{alpha} and PP1{alpha}, but not PP2B{alpha}, were detected by Western blot analysis in the left ventricular myocardium. Cx43 coprecipitated with PP2A{alpha} but not with PP1{alpha}. Although the total PP2A{alpha} immunoreactivity (confocal laser scan) was increased to 154 ± 24% and 194 ± 13% of baseline (P < 0.05) after 85 min of ischemia in NIP and IP myocardium, respectively, the PP2A activities were similar between the groups. The amount of PP2A{alpha} coimmunoprecipitated with Cx43 remained unchanged. Only PP2A{alpha} coprecipitates with Cx43 in pig myocardium. This interaction is not affected by IP, suggesting that PP2A{alpha} is not involved in the prevention of the ischemia-induced Cx43 dephosphorylation by IP.

cardioprotection; ischemia-reperfusion

CONNEXINS ARE A FAMILY of transmembrane proteins consisting of four transmembrane, two extracellular, and three cytosolic domains (38). Cardiomyocytes predominantly express connexin 43 (Cx43), which is present both at the gap junctions and in the mitochondria (6). Gap junctional Cx43 polymerizes into hexamers and forms transmembrane channels termed hemichannels or connexons. Clusters of hemichannels in adjacent cells accumulate to form gap junctions (31, 33, 36, 38), which transmit depolarizing currents and thus coordinate the contraction between cells (20, 27). Cx43 is phosphorylated on serine, threonine, and tyrosine residues at the carboxy terminus (22) by multiple protein kinases (23, 24, 28, 35, 37), thus modulating cardiac hemichannel and gap junction channel permeability and conductance (22). The open probability of Cx43-formed sarcolemmal channels increases during Cx43 dephosphorylation, as occurring during ischemia (5, 21), and thus contributes to cell death and the propagation of irreversible cell injury (35). Indeed, the gap junction uncoupler heptanol when administered at the onset of reperfusion limits necrosis by reducing the progression of hypercontracture (14), and gap junctional Cx43 phosphorylation during ischemia is preserved by ischemic preconditioning (IP) (17, 19, 29, 34). The molecular mechanisms that maintain gap junctional Cx43 phosphorylation in preconditioned myocardium have not been identified. We propose that the interaction of Cx43 with protein kinases and protein phosphatases (PPs) regulates Cx43 phosphorylation. Whereas it has been clearly demonstrated that Cx43 is also located in the cardiomyocyte mitochondria and is important for the cardioprotection by IP (6, 15, 32), the phosphorylation status of mitochondrial Cx43 at baseline and after ischemia or IP has not been investigated in detail.

The serine/threonine phosphatase protein family was originally classified into two types, PP1 and PP2 (18) (subclassified into PP2A, PP2B, and PP2C), based on their reaction with certain inhibitors and their requirement for cations. Today, serine/threonine phosphatases are classified according to their encoding gene families phospho-protein-phosphatase (PPP), PP Mg-dependent, and transcription factor II F-dependent carboxyterminal domain phosphatase 1, defined by amino acid sequences and crystal structures (10, 16, 40, 41). The PPP family can be further divided into subfamilies PPP1-7 based on their sequences. The major cellular phosphatases PP1, PP2A, and PP2B are also known as PPP1-3 (PP1 = PPP1; PP2A = PPP2; and PP2B = PPP3) and are the most thoroughly studied. Up until now, more than 50 mammalian regulatory subunits of PP1C have been identified (11). PP1, PP2A, and PP2B have been implicated in Cx43 dephosphorylation (16), especially in the context of heart failure (1). We hypothesize that IP maintains Cx43 phosphorylation by inhibiting phosphatases. In the present study, we therefore analyzed 1) the phosphorylation of gap junctional and mitochondrial Cx43, 2) the protein levels and activities of PP in a pig model of IP, 3) the interactions between Cx43 and PP1, PP2A, or PP2B, and 4) the dependence of infarct size reduction on the interactions between Cx43 and PP in IP.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The present study was performed with the approval of 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 National Institutes of Health (NIH Publication No. 85-23, Revised 1996). Göttinger minipigs (20–40 kg, n = 13) were initially sedated with 1 g (25 mg/kg) ketamine hydrochloride intramuscularly and then anesthetized with thiopental sodium (Trapanal, 500 mg, 12.5 mg/kg iv). Anesthesia was maintained with enflurane (1–1.5% oxygen-nitrous oxide mixture, 40%/60%). Arterial blood gases were measured throughout the study (Radiometer, Copenhagen, Denmark) and maintained within the normal range. A left lateral thoracotomy was performed; the left anterior descending artery was cannulated and perfused from an extracorporal circuit. Following surgical instrumentation, the minipigs underwent 90 min of low-flow ischemia without [n = 7; nonpreconditioned (NIP)] or with (n = 6, IP) a preceding preconditioning cycle of 10 min of ischemia and 15 min of reperfusion. Regional myocardial blood flow was assessed at baseline and at 5 min of ischemia using radiolabeled microspheres. Drill biopsies (8–12 mg) were acquired from the anterior myocardium at baseline and after 85 min of ischemia and snap frozen in liquid nitrogen. Transmural tissue samples (5 to 6 g) were collected from the anterior and posterior myocardial wall at the end of the 90-min ischemic period. Reperfusion was initiated for 120 min to allow for the quantification of infarct size by 2,3,5-triphenyltetrazolium chloride staining. The amount of infarcted tissue is expressed as a percentage of the left ventricular (LV) area at risk, as determined by the microspheres technique (30). A scheme of the experimental protocols is shown in Fig. 1.


Figure 1
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 1. Experimental protocols. Pig myocardium was subjected to 90 min ischemia without (NIP) and with ischemic preconditioning (IP) by 10 min ischemia and 15 min reperfusion. Drill biopsies for immunohistochemistry and Western blot analysis of nonphosphorylated and total connexin 43 (Cx43) were taken from the anterior ventricular wall (AW) at baseline and after 85 min ischemia. Larger tissue samples (5 to 6 g) from the AW and posterior ventricular wall (PW) of NIP and IP pig myocardium after 90 min ischemia were collected for immunoprecipitation, protein phosphatase (PP)2A activity assay, and mitochondrial isolation (tissue sampling; IP, n = 4–6; and NIP, n = 4–6). In a second set of animals, pigs were subjected to 90 min ischemia and 120 min reperfusion without and with IP to assess regional myocardial blood flow (RMBF) at baseline (BL) and at 5 min ischemia and to measure infarct size by 2,3,5-triphenyltetrazolium chloride (TTC) staining (IP n = 6, and NIP n = 7).

 
Mitochondria isolation. Mitochondria were isolated from the anterior and posterior wall of NIP and IP pig myocardium after 85 min of ischemia (n = 6) by differential centrifugation and Percoll-gradient ultracentrifugation as described previously (6). The purity of the mitochondrial preparations was controlled by Western blot analysis using antibodies for other cellular compartments.

Immunohistochemistry. Paraffin-embedded tissue samples of the anterior myocardial wall were cut into 4-µm sections, dewaxed, and rehydrated with graded alcohols and rinsed in phosphate-buffered saline. The sections were pretreated with 0.01 M citrate buffer for antigen retrieval, incubated with rabbit anti-total Cx43 and mouse anti-nonphosphorylated Cx43, or with rat anti-PP2A{alpha} antibodies diluted in antibody diluent S3022 (Dako Cytomation, Copenhagen, Denmark) for 1 h at 37°C and subsequently with the respective fluorochrome-labeled secondary antibodies. For the negative control, primary antibodies were omitted. The samples were covered with Vectashield (H-1000, Vector, Burlingame, CA) and examined by confocal laser scan microscopy at x630 magnification (LSM Pascal 5, Zeiss, Jena, Germany). For the analysis of PP2A{alpha}, the density of FITC immunofluorescence was detected in the total area (0.0214 mm2 each) and expressed as arbitrary units (AU). To eliminate background staining, the AU values from the negative control tissue samples were subtracted. The mean values of the AU in the baseline biopsies were set to 100%, and the values of the tissue samples obtained after 85 min of ischemia were calculated as the percentage of the respective baseline.

Phosphatase treatment. Mitochondria isolated from the control posterior wall were lysed in a buffer containing 1% Triton X-100, 0.4% SDS, 1 mM EDTA, and 10 mM Tris·HCl (pH 7.4), supplemented with 1x protease inhibitors (Complete, Roche, Basel, Switzerland). Mitochondrial proteins (50 µg) were incubated for 30 min at 37°C with different amounts of antarctic phosphatase (NEB, Ipswich, MA) in 1x phosphatase reaction buffer. The reaction was stopped by adding SDS sample buffer and incubating the samples at 95°C for 5 min (n = 5). Proteins were characterized by Western blot analysis.

Immunoprecipitation and Western blot analysis. Myocardial and mitochondrial protein extracts were prepared by a homogenization of nitrogen-frozen myocardial tissue in Cell lysis buffer (Cell Signaling, Beverly, MA) containing (in mM) 20 Tris, 150 NaCl, 1 EDTA, 1 EGTA, 2.5 sodium pyrophosphate, 1 β-glycerolphosphate, 1 Na3VO4, 1 PMSF, 1 µg/ml leupeptin, and 1% Triton X-100 (pH 7.5), supplemented with Complete Protease Inhibitors. The samples were sonicated for 20 s and 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). Western blot analysis for the total and/or nonphosphorylated Cx43 or protein phosphatases was performed on 25 µg of myocardial or mitochondrial protein extracts as described previously (6).

Protein extracts (25 µg) of pig LV myocardium and cerebrum, tissue known to express high amounts of all PPs, were subjected to Western blot analysis for PP1{alpha}, PP2A{alpha}, or PP2B{alpha}.

For immunoprecipitation, 300 µg of pig myocardial proteins were incubated with rabbit anti-Cx43, mouse anti-PP1{alpha}, sheep anti-PP-2A{alpha} antibodies, or with anti-rabbit, anti-mouse, or anti-sheep horseradish peroxidase-conjugated IgGs for 1 h at 4°C. Protein A/G plus Agarose (Santa Cruz) was added to each sample followed by an overnight incubation at 4°C. The Protein A/G plus Agarose beads were washed three times with 500 µl 1x Cell lysis buffer-Complete Protease Inhibitors. After the sample buffer was added, the samples were boiled, and the supernatants were subjected to Western blot analysis. The immunoprecipitations to characterize the interactions between Cx43 and PP were performed at least three times independently. To quantify the coprecipitation of PP2A with Cx43, Cx43 was immunoprecipitated from the proteins extracted from the anterior and posterior wall of NIP (n = 5) and IP (n = 4) hearts.

Myocardial and mitochondrial proteins as well as immunoprecipitated proteins and supernatants of immunoprecipitations were electrophoretically separated on 10% polyacrylamide gels and transferred to nitrocellulose membranes, which were stained with Ponceau S. After an incubation with primary and secondary antibodies, immunoreactive bands were detected using the SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL) or LumiGLO Chemiluminescent Substrate (Cell Signaling). Immunoreactivities for nonphosphorylated Cx43 were normalized to Ponceau S staining and were quantified with Scion Image software (Scion, Frederick, MD).

PP2A activity. PP2A activity was measured using the Serine/Threonine Phosphatase Assay System (Promega, Madison, WI). Tissue samples (~100–300 mg) obtained after 85 min of ischemia from the anterior and posterior wall of NIP (n = 4) and IP (n = 4) pig myocardium were homogenized in extraction buffer containing 0.1 mM EDTA, 0.1 mM EGTA, 50 mM Tris·HCl, 1 mM dithiothreitol, 0.5% Triton X-100, and 10% glycerol (pH 7), supplemented with 1x protease inhibitors. The homogenate was centrifuged at 100,000 g for 1 h at 4°C, and the soluble fraction was passed through a Sephadex G-25 spin column to reduce the free phosphate level in the samples. The protein concentration of the flow through was determined with the DC protein assay (Bio-Rad). The phosphatase assay, which detects the amount of free phosphate generated mainly by PP2 phosphatases from chemically synthesized phosphopeptides, was performed according to the manufacturer's instruction using 5 µg protein/well in PP2A reaction buffer. PP2A-specific activities were defined as the activity that was inhibited by 200 nM okadaic acid. Free phosphate was used to generate a standard curve.

Antibodies. The following antibodies were used: rabbit polyclonal anti-rat total Cx43 (directed against the carboxy terminus of the protein; detects all molecular weights of Cx43; 1 µg antibodies/120 µg pig protein extracts for immunoprecipitation; dilution 1:500 for Western blot analysis and 1:100 for immunohistochemistry; catalog no. 71-0700; Invitrogen, Carlsbad, CA), mouse monoclonal anti-rat nonphosphorylated Cx43 (recognizes Cx43 only when Ser368, a protein kinase C phosphorylation site, is nonphosphorylated; detects predominantly Cx43 migrating at 43 kDa; dilution 1:200 for Western blot analysis and dilution 1:100 for immunohistochemistry; catalog no. 13-8300; Invitrogen), mouse monoclonal anti-rabbit PP1{alpha} (1 µg antibodies/20 µg pig protein extracts for immunoprecipitation; dilution 1:200 for Western blot analysis; catalog no. P7607; Sigma, St. Louis, MO), rabbit polyclonal anti-mouse/human serine/threonine PP1{alpha} (dilution 1:200 for Western blot analysis; catalog no. P100P; Exalpha Biologicals, Watertown, MA), rabbit polyclonal anti-mouse/human serine/threonine PP2A{alpha} (dilution 1:200 for Western blot analysis; catalog no. P135P; Exalpha Biologicals), sheep polyclonal anti-mouse/human serine/threonine PP2A{alpha} (1 µg antibodies/20 µg pig protein extracts for immunoprecipitation; catalog no. P140P; Exalpha Biologicals), rat monoclonal anti-human PP2A{alpha} (dilution 1:200 for Western blot analysis and 1:10 for immunohistochemistry; catalog no. P6498, Sigma), rabbit polyclonal anti-mouse/human serine/threonine PP2B{alpha} (dilution 1:200 for Western blot analysis; catalog no. P195P; Exalpha Biologicals), sheep anti-mouse/human serine/threonine PP2B{alpha} (dilution 1:200 for Western blot analysis; catalog no. P245P; Exalpha Biologicals), and mouse monoclonal anti-human PP2B{alpha} (dilution 1:200 for Western blot analysis; catalog no. H00005530-M03; Abnova, Tapei, Taiwan).

Statistics. Data are reported as means ± SE. Hemodynamics and regional myocardial blood flow at baseline and 5 min ischemia were compared using two-way ANOVA for repeated measures and the Fisher least significant difference method. Data on infarct size and nonphosphorylated mitochondrial Cx43 were compared by unpaired Student's t-test. Western blot analysis for nonphosphorylated Cx43 in total myocardial extracts, immunohistochemistry, and immunoprecipitation of Cx43 with coprecipitated PP2A{alpha} were compared using two-way ANOVA and Fisher least significant difference. A P < 0.05 was considered to indicate a significant difference.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The hemodynamics throughout the protocol of ischemia-reperfusion did not differ among pigs without or with IP. At baseline, the coronary blood flow was 55.0 ± 6.1 and 49.5 ± 5.0 ml/min in NIP and IP hearts, respectively. During ischemia, the coronary blood flow was reduced to 6.4 ± 0.3 and 7.3 ± 1.1 ml/min in nonpreconditioned and preconditioned hearts, respectively [P = not significant (NS)]. The regional myocardial blood flow at baseline and at 5 min ischemia (0.04 ± 0.01 vs. 0.05 ± 0.01 ml·min–1·g–1) did not differ between nonpreconditioned and preconditioned hearts. The area at risk was 50.9 ± 2.1% and 51.5 ± 0.6% of the LV in nonpreconditioned and preconditioned hearts, respectively (P = NS). The infarct size was reduced from 23.1 ± 2.7% in NIP to 10.0 ± 3.2% of the area at risk in IP hearts.

The phosphorylation status of Cx43 in the LV anterior wall of pigs undergoing ischemia without or with preconditioning was analyzed by immunohistochemistry and confocal laser scan microscopy (Fig. 2). Ischemia induced a dephosphorylation of Cx43, and this dephosphorylation was prevented by IP. Western blot analysis demonstrated that the immunoreactivity of nonphosphorylated Cx43 in the anterior wall increased from 0.14 ± 0.02 at baseline to 0.62 ± 0.15 AU after 85 min ischemia in the NIP group (n = 4, P < 0.05; Fig. 3). In preconditioned pig myocardium, the immunoreactivity after 85 min ischemia was 0.27 ± 0.15 AU and was significantly lower than in nonpreconditioned myocardium (n = 4).


Figure 2
View larger version (104K):
[in this window]
[in a new window]

  		Fig. 2. Paraffin-embedded samples of the AW of IP and NIP porcine myocardial tissue at BL and after 85 min ischemia (I85) were stained with antibodies against total Cx43 (red) and Cx43 nonphosphorylated at Ser368 (green) and examined by confocal laser scan microscopy. Overlay pixels are shown in yellow.

 

Figure 3
View larger version (29K):
[in this window]
[in a new window]

  		Fig. 3. Cx43 phosphorylation in total myocardial protein extracts. A: Western blot analysis was performed for total Cx43 (top) and Cx43 nonphosphorylated at Ser368 [DP-Ser368 Cx43, in arbitrary units (AU), bottom] on left ventricular proteins isolated from the AW at BL and after I85 of NIP and IP pig myocardium. Western blot analysis for total Cx43 includes the phosphorylated (P) and dephosphorylated (deP) forms of the protein. B: ratio of DP-Ser368 Cx43 to Ponceau S staining at BL and after I85 in NIP and IP pig myocardium (n = 4) . *P < 0.05 vs. BL NIP; #P < 0.05 vs. I85 IP.

 
When comparing the mitochondria isolated from the anterior wall after 85 min of ischemia and from the control posterior wall of NIP and IP pig myocardium, nonphosphorylated Cx43 was detected only in the anterior wall mitochondria (Fig. 4). Phosphatase treatment of mitochondrial proteins resulted in a dose-dependent increase in nonphosphorylated Cx43, as shown by Western blot analysis. The quantification of the amount of nonphosphorylated Cx43 revealed that only 16.2 ± 10.2% of Cx43 in the posterior wall mitochondria was nonphosphorylated compared with the mitochondrial proteins treated with 0.83 U/µl phosphatase, which were set as 100% (n = 5). There was a trend toward an increase in the level of nonphosphorylated Cx43 in the anterior wall mitochondria isolated from NIP compared with IP pig myocardium; however, the difference was not statistically significant.


Figure 4
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 4. Cx43 phosphorylation in mitochondrial protein extracts. A: Western blot analysis was performed for DP-Ser368 Cx43 on mitochondria isolated from the AW and control PW of pig myocardium subjected to I85 of NIP and IP (exposition time, 15 min). B: PW mitochondria were treated with the indicated concentrations of antarctic phosphatase (AP) and were analyzed for the level of total Cx43 (including the P and deP forms of the protein) and DP-Ser368 Cx43 by Western blot analysis (exposition time, 1 min). C: the immunoreactivity for DP-Ser368 Cx43 normalized to Ponceau S staining in mitochondria isolated after I85 from the AW of NIP and IP pig myocardium (n = 6).

 
The analysis of the expression of protein phosphatases in posterior wall pig myocardium and cerebrum showed that PP1{alpha}, PP2A{alpha}, and PP2B{alpha} were most abundant in pig cerebrum (Fig. 5A). Whereas PP1{alpha} and PP2A{alpha} were also detected in myocardial protein extracts, almost no PP2B{alpha} was found.


Figure 5
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 5. A: Western blot analysis was performed for PP1{alpha}, PP2A{alpha}, PP2B{alpha}, and GAPDH in protein extracts of the PW of the pig left ventricle and of the cerebrum (CB). Immunoprecipitation (precip) was performed for total Cx43 (including the P and deP forms of the protein) and PP1{alpha} (B), PP2A{alpha} (C), and horseradish peroxidase-conjugated IgG in pig myocardial protein extracts (n = 3 for each antibody tested). Western blot analysis for Cx43, PP1{alpha}, and PP2A{alpha} was performed on the precipitated proteins and on pig left ventricular proteins.

 
Immunoprecipitation of Cx43 from control LV protein extracts resulted in a coprecipitation of PP2A{alpha}. Conversely, with the use of the anti-PP2A{alpha} antibody for immunoprecipitation, a coprecipitation of Cx43 was detected (Fig. 5, B and C). No coprecipitation of PP1{alpha} with Cx43 was observed (n = 3 for each antibody tested), even though different antibodies were used for immunoprecipitations. The precipitation of IgGs, used as negative control, did not result in any Cx43 or PP immunoreactivity.

With the use of confocal laser scan microscopy, PP2A{alpha} immunoreactivity in the whole myocardial area increased to 154 ± 24% of baseline at 85 min ischemia in nonpreconditioned hearts (n = 5, P < 0.05) and to 194 ± 13% of baseline in preconditioned hearts (n = 4, P < 0.05 vs. baseline). The PP2A activity (in pmol phosphate/µg protein) in posterior wall tissue samples was 59.8 ± 5.2 in IP and 70.1 ± 6.3 in NIP myocardium, and it was 62.4 ± 8.2 and 47.4 ± 6.2 in anterior wall tissue samples of IP and NIP myocardium, respectively. The PP2A activities in the anterior wall pig myocardium were similar between groups.

Total Cx43 was precipitated from the anterior and the control posterior wall of NIP and IP hearts after 85 min of ischemia. In the myocardium from nonpreconditioned hearts, the ratio of dephosphorylated Cx43 (migrating at lower molecular weight) over total Cx43 was 0.06 ± 0.01 AU in the posterior control wall and 0.15 ± 0.01 AU in the anterior wall, indicating a significant decrease in phosphorylation during ischemia (P < 0.05). In preconditioned myocardium, no change of the dephosphorylated Cx43-to-total Cx43 ratio of the anterior wall (0.11 ± 0.02 AU) compared with the posterior wall (0.08 ± 0.03 AU) was seen at 85 min low-flow ischemia. Western blot analysis was performed for PP2A{alpha} coimmunoprecipitated with total Cx43. However, the amount of PP2A{alpha} coprecipitated with total Cx43 was similar between the two experimental groups (PP2A{alpha}-to-total Cx43 ratio was 0.20 ± 0.05 in the posterior and 0.19 ± 0.05 AU in the anterior wall of NIP hearts and 0.19 ± 0.05 in the posterior vs. 0.21 ± 0.03 AU in the anterior wall of IP hearts, respectively, P = NS; Fig. 6). The immunoprecipitation of Cx43 from the LV anterior and posterior wall of NIP and IP pig hearts did not result in a coprecipitation of PP1{alpha} (not shown).


Figure 6
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 6. A: Cx43 was immunoprecipitated from protein extracts of the preconditioned AW and control PW of NIP (n = 5) and IP (n = 4) pig myocardium. Western blot analysis was performed for total Cx43, including P and deP Cx43, and PP2A{alpha}. For control, a supernatant of a Cx43 precipitation from the PW of a NIP pig heart is shown. B: the ratios of coprecipitated PP2A{alpha} to total Cx43 in PW and AW of IP and NIP animals.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we analyzed the serine/threonine phosphatases PP1, PP2A, and PP2B, which have been detected in myocardial tissue of species other than the pig in previous studies (9).

Whereas PP1{alpha} and PP2A{alpha} were also detected in pig myocardium, PP2B{alpha} was almost absent. Since three different antibodies against PP2B{alpha} were tested and all antibodies detected PP2B{alpha} in pig cerebrum protein extracts, the lack of PP2B{alpha} immunoreactivity cannot be explained by the lack of cross-reactivity of the antibodies with porcine proteins. The modification of PP expression and/or the activity affects Cx43 content and phosphorylation. The overexpression of active PP2B in mouse cardiomyocytes induced a reduction of the Cx43 protein level, an increased Cx43-dephosphorylation, and a redistribution of Cx43 away from the intercalated discs (8). In HeLa cells, an overexpression of PP1β dephosphorylated connexin (7), and in a rabbit model of heart failure, where a downregulation and dephosphorylation of Cx43 were observed, the interaction between Cx43 and PP2A was enhanced (1).

With the use of mass spectrometry, the serine phosphorylation sites of Cx43 have been systematically studied (4). In these experiments in isolated rat hearts, the number of phosphorylated Cx43 epitopes decreased with ischemia; however, both a dephosphorylation (e.g., Ser306 or Ser368) and a phosphorylation of specific Cx43 serine residues (e.g., Ser330) occurred.

Within cardiomyocytes, Cx43 is not only present at gap junctions but is also localized at the inner membrane of the mitochondria (6). In the present study, we analyzed the phosphorylation status of both gap junctional and mitochondrial Cx43. Ischemia-induced dephosphorylation of gap junctional Cx43 was prevented by IP (17, 19, 29). This effect was also confirmed in our pig model of low-flow ischemia by immunohistochemistry (34). Performing a Western blot analysis on the total myocardial proteins does not allow us to distinguish between gap junctional and mitochondrial Cx43. However, since mitochondrial Cx43 represents only about 4% of total Cx43 (32), Western blot analysis for Cx43 predominantly detects the gap junctional form of the protein. Our Western blot analysis data confirmed that IP inhibits the ischemia-induced dephosphorylation of Cx43.

Studying the phosphorylation status of mitochondrial Cx43 isolated from the posterior wall, we detected immunoreactivity for nonphosphorylated Cx43 only in phosphatase-treated protein extracts. Our data therefore indicate that under physiological conditions, the mitochondrial Cx43 is highly phosphorylated. The quantification of the amount of nonphosphorylated Cx43 within the mitochondria demonstrated that only about 16% of the protein is in a nonphosphorylated state. This is in agreement with previous data showing that in mitochondria isolated from the rat LV, more than 80% of Cx43 is phosphorylated (6). However, in mitochondria isolated from the myocardium subjected to 85 min of ischemia without and with IP, immunoreactivity for nonphosphorylated Cx43 was detected. Whereas there was a trend toward a prevention of an ischemia-induced increase in Cx43 dephosphorylation by IP, the differences were not statistically significant.

Mitochondrial Cx43 is important for the generation of reactive oxygen species (ROS), important trigger molecules for the cardioprotection by IP (26), since the ROS production induced by the cardioprotective agent diazoxide is attenuated in heterozygous Cx43-deficient cardiomyocytes (15). A dephosphorylation of mitochondrial Cx43 could affect its ability to contribute to ROS formation and thus to cardioprotection.

The pharmacological inhibition of PP may increase the phosphorylation status of target proteins involved in the cardioprotection of IP. Indeed, the PP2A-selective protein phosphatase inhibitor fostriecin protected from infarction even when administered after the onset of ischemia, although IP had no effect on the activities of PP1 or PP2A (39). Furthermore, fostriecin at a PP2A-selective concentration, mimicked preconditioning in both ischemic rabbit and pig cardiomyocytes (3), and both fostriecin and calyculin A, an inhibitor of both PP1 and PP2A, protected rabbit cardiomyocytes when given in late ischemia (2). Okadaic acid, another inhibitor of both PP1 and PP2A, also reduced ischemia-induced cellular death in young and aged rat cardiomyocytes (13). The inhibition of PP can reduce ischemia-reperfusion injury, and the inhibition of PP1 also preserves Cx43 phosphorylation during ischemia in rats (12, 21). Whereas the coprecipitation of PP1 with Cx43 has been demonstrated in rabbit myocardium (1), no coprecipitation of PP1{alpha} and Cx43 was found in either protein extracts derived from control or ischemic pig myocardial tissue in the present study, suggesting that the association between Cx43 and PP1 is species dependent. However, we were able to demonstrate an interaction of PP2A{alpha} with Cx43 in pig myocardium, in control myocardium as well as in myocardium subjected to ischemia without or with IP. Although the immunoprecipitation experiments were performed on protein extracts derived from tissue in which a significant increase in the amount of PP2{alpha} was detected by immunohistochemistry, the amount of PP2A{alpha} coprecipitating with Cx43 was similar between groups. Thus, whereas PP2A appears to be associated with Cx43, its impact on the Cx43 phosphorylation status can be neglected. Our data confirm the results of a previous study in which the blockade of PP2A by fostriecin did not affect ischemia-induced dephosphorylation of Cx43 in rat myocardium (21). Similar results were obtained in astrocytes (25). Furthermore, our data show that the activity of PP2A was not affected by IP, thereby supporting previous results obtained in rabbit myocardium (39). Thus the prevention of ischemia-induced dephosphorylation of Cx43 by IP is not a direct consequence of the interaction with PP2{alpha} or of an altered activity of the phosphatase.

In conclusion, our data reveal the coprecipitation of only PP2A{alpha} and Cx43 in porcine LV myocardium. Whereas the total amount of PP2A{alpha} was increased by ischemia in both nonpreconditioned and preconditioned hearts, there was no increase of PP2A{alpha}, which coprecipitated with Cx43. Therefore, PP2A{alpha} is not directly responsible for the prevention of the ischemia-induced dephosphorylation of Cx43 by IP.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Interne Forschungsförderung Essen research program of the University of Duisburg-Essen and the German Research Foundation (DFG Schu 843/7-1).


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Schulz, Institut für Pathophysiologie, Universitätsklinikum Essen, Hufelandstrabe 55, 45122, Essen, Germany (e-mail: rainer.schulz{at}uk-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.

* A. Totzeck and K. Boengler contributed equally to this study. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ai X, Pogwizd SM. Connexin 43 downregulation and dephosphorylation in nonischemic heart failure is associated with enhanced colocalized protein phosphatase type 2A. Circ Res 96: 54–63, 2005.[Abstract/Free Full Text]
  2. Armstrong SC, Gao W, Lane JR, Ganote CE. Protein phosphatase inhibitors calyculin A and fostriecin protect rabbit cardiomyocytes in late ischemia. J Mol Cell Cardiol 30: 61–73, 1998.[CrossRef][Web of Science][Medline]
  3. Armstrong SC, Hoover DB, Shivell LC, Ganote CE. Preconditioning of isolated rabbit cardiomyocytes: no evident separation of induction, memory and protection. J Mol Cell Cardiol 29: 2285–2298, 1997.[CrossRef][Web of Science][Medline]
  4. Axelsen LN, Stahlhut M, Mohammed S, Larsen BD, Nielsen MS, Holstein-Rathlou NH, Andersen S, Jensen ON, Hennan JK, Kjolbye AL. Identification of ischemia-regulated phosphorylation sites in connexin43: A possible target for the antiarrhythmic peptide analogue rotigaptide (ZP123). J Mol Cell Cardiol 40: 790–798, 2006.[CrossRef][Web of Science][Medline]
  5. Beardslee MA, Lerner DL, Tadros PN, Laing JG, Beyer EC, Yamada KA, Kleber AG, Schuessler RB, Saffitz JE. Dephosphorylation and intracellular redistribution of ventricular connexin43 during electrical uncoupling induced by ischemia. Circ Res 87: 656–662, 2000.[Abstract/Free Full Text]
  6. Boengler K, Dodoni G, Ruiz-Meana M, Cabestrero A, Rodriguez-Sinovas A, Garcia-Dorado D, Gres P, Di Lisa F, Heusch G, Schulz R. Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic preconditioning. Cardiovasc Res 67: 234–244, 2005.[Abstract/Free Full Text]
  7. Chen HH, Baty CJ, Maeda T, Brooks S, Baker LC, Ueyama T, Gursoy E, Saba S, Salama G, London B, Stewart AF. Transcription enhancer factor-1-related factor-transgenic mice develop cardiac conduction defects associated with altered connexin phosphorylation. Circulation 110: 2980–2987, 2004.[Abstract/Free Full Text]
  8. Chu G, Carr AN, Young KB, Lester JW, Yatani A, Sanbe A, Colbert MC, Schwartz SM, Frank KF, Lampe PD, Robbins J, Molkentin JD, Kranias EG. Enhanced myocyte contractility and Ca2+ handling in a calcineurin transgenic model of heart failure. Cardiovasc Res 54: 105–116, 2002.[Abstract/Free Full Text]
  9. Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem 58: 453–508, 1989.[CrossRef][Web of Science][Medline]
  10. Cohen PT. Novel protein serine/threonine phosphatases: variety is the spice of life. Trends Biochem Sci 22: 245–251, 1997.[CrossRef][Web of Science][Medline]
  11. Cohen PT. Protein phosphatase 1—targeted in many directions. J Cell Sci 115: 241–256, 2002.[Abstract/Free Full Text]
  12. Duthe F, Plaisance I, Sarrouilhe D, Herve JC. Endogenous protein phosphatase 1 runs down gap junctional communication of rat ventricular myocytes. Am J Physiol Cell Physiol 281: C1648–C1656, 2001.[Abstract/Free Full Text]
  13. Fenton RA, Dickson EW, Dobson JG Jr. Inhibition of phosphatase activity enhances preconditioning and limits cell death in the ischemic/reperfused aged rat heart. Life Sci 77: 3375–3388, 2005.[CrossRef][Web of Science][Medline]
  14. Garcia-Dorado D, Inserte J, Ruiz-Meana M, Gonzalez MA, Solares J, Julia M, Barrabés JA, Soler-Soler J. Gap junction uncoupler heptanol prevents cell-to-cell progression of hypercontracture and limits necrosis during myocardial reperfusion. Circulation 96: 3579–3586, 1997.[Abstract/Free Full Text]
  15. Heinzel FR, Luo Y, Li X, Boengler K, Buechert A, García-Dorado D, Di Lisa F, Schulz R, Heusch G. Impairment of diazoxide-induced formation of reactive oxygen species and loss of cardioprotection in connexin 43 deficient mice. Circ Res 97: 583–586, 2005.[Abstract/Free Full Text]
  16. Herve JC, Sarrouilhe D. Protein phosphatase modulation of the intercellular junctional communication: importance in cardiac myocytes. Prog Biophys Mol Biol 90: 225–248, 2006.[CrossRef][Web of Science][Medline]
  17. Hund TJ, Lerner DL, Yamada KA, Schuessler RB, Saffitz JE. Protein kinase Cepsilon mediates salutary effects on electrical coupling induced by ischemic preconditioning. Heart Rhythm 4: 1183–1193, 2007.[CrossRef][Web of Science][Medline]
  18. Ingebritsen TS, Cohen P. Protein phosphatases: properties and role in cellular regulation. Science 221: 331–338, 1983.[Abstract/Free Full Text]
  19. Jain SK, Schuessler RB, Saffitz JE. Mechanisms of delayed electrical uncoupling induced by ischemic preconditioning. Circ Res 92: 1138–1144, 2003.[Abstract/Free Full Text]
  20. Jalife J, Morley GE, Vaidya D. Connexins and impulse propagation in the mouse heart. J Cardiovasc Electrophysiol 10: 1649–1663, 1999.[Web of Science][Medline]
  21. Jeyaraman M, Tanguy S, Fandrich RR, Lukas A, Kardami E. Ischemia-induced dephosphorylation of cardiomyocyte connexin-43 is reduced by okadaic acid and calyculin A but not fostriecin. Mol Cell Biochem 242: 129–134, 2003.[CrossRef][Web of Science][Medline]
  22. Kwak BR, Jongsma HJ. Regulation of cardiac gap junction channel permeability and conductance by several phosphorylating conditions. Mol Cell Biochem 157: 93–99, 1996.[CrossRef][Web of Science][Medline]
  23. Laird DW. Connexin phosphorylation as a regulatory event linked to gap junction internalization and degradation. Biochim Biophys Acta 1711: 172–182, 2005.[Medline]
  24. Lampe PD, Lau AF. Regulation of gap junctions by phosphorylation of connexins. Arch Biochem Biophys 384: 205–215, 2000.[CrossRef][Web of Science][Medline]
  25. Li WE, Nagy JI. Connexin43 phosphorylation state and intercellular communication in cultured astrocytes following hypoxia and protein phosphatase inhibition. Eur J Neurosci 12: 2644–2650, 2000.[CrossRef][Web of Science][Medline]
  26. Liu Y, Yang XM, Iliodromitis EK, Kremastinos DT, Dost T, Cohen MV, Downey JM. Redox signaling at reperfusion is required for protection from ischemic preconditioning but not from a direct PKC activator. Basic Res Cardiol 103: 54–59, 2008.[CrossRef][Web of Science][Medline]
  27. Lo CW. Role of gap junctions in cardiac conduction and development: insights from the connexin knockout mice. Circ Res 87: 346–348, 2000.[Free Full Text]
  28. Moreno AP, Lau AF. Gap junction channel gating modulated through protein phosphorylation. Prog Biophys Mol Biol 94: 107–119, 2007.[CrossRef][Web of Science][Medline]
  29. Papp R, Gonczi M, Kovacs M, Seprenyi G, Vegh A. Gap junctional uncoupling plays a trigger role in the antiarrhythmic effect of ischaemic preconditioning. Cardiovasc Res 74: 396–405, 2007.[Abstract/Free Full Text]
  30. Post H, Schulz R, Behrends M, Gres P, Umschlag C, Heusch G. No involvement of endogenous nitric oxide in classical ischemic preconditioning in swine. J Mol Cell Cardiol 32: 725–733, 2000.[CrossRef][Web of Science][Medline]
  31. Revel JP, Karnovsky MJ. Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. J Cell Biol 33: C7–C12, 1967.[Free Full Text]
  32. Rodriguez-Sinovas A, Boengler K, Cabestrero A, Gres P, Morente M, Ruiz-Meana M, Konietzka I, Miró E, Totzeck A, Heusch G, Schulz R, Garcia-Dorado D. Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection. Circ Res 99: 93–101, 2006.[Abstract/Free Full Text]
  33. Saez JC, Berthoud VM, Branes MC, Martinez AD, Beyer EC. Plasma membrane channels formed by connexins: their regulation and functions. Physiol Rev 83: 1359–1400, 2003.[Abstract/Free Full Text]
  34. Schulz R, Gres P, Skyschally A, Duschin A, Belosjorow S, Konietzka I, Heusch G. Ischemic preconditioning preserves connexin 43 phosphorylation during sustained ischemia in pig hearts in vivo. FASEB J 17: 1355–1357, 2003.[Abstract/Free Full Text]
  35. Schulz R, Heusch G. Connexin 43 and ischemic preconditioning. Cardiovasc Res 62: 335–344, 2004.[Abstract/Free Full Text]
  36. Sohl G, Willecke K. Gap junctions and the connexin protein family. Cardiovasc Res 62: 228–232, 2004.[Abstract/Free Full Text]
  37. Solan JL, Lampe PD. Connexin phosphorylation as a regulatory event linked to gap junction channel assembly. Biochim Biophys Acta 1711: 154–163, 2005.[Medline]
  38. Sosinsky GE, Nicholson BJ. Structural organization of gap junction channels. Biochim Biophys Acta 1711: 99–125, 2005.[Medline]
  39. Weinbrenner C, Baines CP, Liu GS, Armstrong SC, Ganote CE, Walsh AH, Honkanen RE, Cohen MV, Downey JM. Fostriecin, an inhibitor of protein phosphatase 2A, limits myocardial infarct size even when administered after onset of ischemia. Circulation 98: 899–905, 1998.[Abstract/Free Full Text]
  40. Yeo M, Lin PS. Functional characterization of small CTD phosphatases. Methods Mol Biol 365: 335–346, 2007.[Medline]
  41. Yeo M, Lin PS, Dahmus ME, Gill GN. A novel RNA polymerase II C-terminal domain phosphatase that preferentially dephosphorylates serine 5. J Biol Chem 278: 26078–26085, 2003.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
295/5/H2106    most recent
00456.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Totzeck, A.
Right arrow Articles by Schulz, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Totzeck, A.
Right arrow Articles by Schulz, R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.