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Correction for Strohm et al., Cardiovasc Res 56 (3) 492.
Cardiovascular Research 2002 55(3):602-618; doi:10.1016/S0008-6363(02)00453-4
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Transcription inhibitor actinomycin-D abolishes the cardioprotective effect of ischemic reconditioning

Claudia Strohma,,1, Miroslav Barancíkb,,1, Marie-Luise von Bruehla, Monika Strniskovab, Claudia Ullmanna, René Zimmermannc and Wolfgang Schapera,*

aDepartment of Experimental Cardiology, Max Planck Institute for Physiological and Clinical Research, Bad Nauheim, Germany
bInstitute for Heart Research, Slovak Academy of Sciences, Bratislava, Slovak Republic
cKerckhoff-Clinic, Vascular Genomics, Bad Nauheim, Germany

w.schaper{at}kerckhoff.mpg.de

* Corresponding author. Tel.: +49-6032-705-402; fax: +49-6032-705-419

Received 12 November 2001; accepted 1 May 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Our previous studies have suggested a role of mitogen-activated protein kinases (MAPKs) in cardioprotection in the porcine heart. To investigate, whether this could be due to modification of transcriptional events we studied the influence of actinomycin-D (act-D), a known RNA-synthesis inhibitor on (i) ischemic preconditioning, (ii) (IP)-mediated cardioprotection, (iii) transcription factors levels and MAPKs activation. Methods: The IP-design in our model included two cycles of 10' LAD occlusion (CO) and 10' reperfusion (RP), followed by 40' CO (index ischemia) and 60' RP. Act-D was infused intramyocardially (i.my.) or systemically (syst.) (0.05 or 0.12 mg/kg) during 15' before IP and during both RP cycles of the IP-protocol. The i.my. infusions occurred via four pairs of needles into the risk area (RA). Results: Systemic infusion of act-D (0.05 mg/kg) before index ischemia significantly increased the IS from 54.0±2.5 to 78.5±3.8%. IP significantly reduced the IS to 2.5±0.8%. Syst. of act-D completely abolished the IP-induced cardioprotection. At a dose of 0.12 mg/kg the IS was 88.6±1.7% of the risk area; at 0.05 mg/kg IS was 65.6±1.5%. Local infusion of act-D reduced the IP-induced cardioprotection in a concentration dependent manner. Syst. or i.my. infusion of DMSO in KHB did not influence the IP-induced cardioprotection. Western blot analysis with phospho-specific antibodies showed a significant increase in phosphorylation of cytosolic ERK1/2 and SAPK/JNKs at the end of IP procedure and act-D treatment inhibited IP-induced activation of these MAPKs. By Western blot analysis using phospho-specific antibodies against c-Jun, ATF-2, Elk-1 and c-Myc we found increased phosphorylation of all these transcription factors in the myocardial risk area at the end of IP protocol and both local and systemic infusion of act-D significantly (P<0.05) inhibited this increased phosphorylation. Unlike UO, act-D had no influence on the Akt-pathway but inhibited the increased expression of S100 protein induced by IP. Conclusions: We demonstrate in vivo that act-D, completely cancelled the IP-induced cardioprotection. The influence of act-D on cardioprotection, transcription factors, and activities of ERKs and JNKs indicates a possible transcriptional role of these MAPKs signal transduction pathways during ischemia and in IP.

KEYWORDS Gene expression; Infarction; Preconditioning; Protein kinases; Protein phosphorylation; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Cells of the cardiovascular system respond to a variety of stress stimuli. The stresses include osmotic shock, physical stretch or deformation, increased rates of contraction, oxidative stress, chemical stresses and ischemic stress. A number of these stimuli initiate a transmission of signals from the cell surface receptors to the cytoplasm and to the transcriptional machinery in the nucleus [1–4]. The activation of distinct protein kinases such as PKC or mitogen-activated protein kinases (MAPKs) plays an important role in transduction of these signals. MAPKs represent the family of at least four MAPK pathways—the ERKs (extracellular-signal regulated protein kinases), the SAPK/JNK (stress activated/c-Jun-N-terminal protein kinases), the p38-MAPK and the BMK (big mitogen kinase) [5–14]. In cardiac myocytes the MAPKs were found to be involved in cell growth, differentiation, transformation, regulation of contraction, ion transport, in apoptosis and probably gene expression and even more in some pathological states like hypertrophy or ischemia [11,14–16].

Short transient periods of ischemia render the myocardium more resistant to a subsequent prolonged coronary occlusion, resulting in a reduction of the infarct size (IS). This cardioprotective mechanism is known as ischemic preconditioning (IP) [1,17–20]. Moreover, several studies investigating the mechanism of cardioprotection afforded by ischemic preconditioning suggest the involvement of MAPKs in responses of myocardium to ischemia [21–26].

We have observed a differential regulation of MAPK cascades during ischemia and reperfusion [27]. Moreover, our previous studies with protein kinase stimulators and inhibitors have indicated different roles of the various protein kinases in IP in pig myocardium [28]. Several lines of evidence pointed to the positive role of ERK cascade during ischemia and by cardioprotection. The FGFs and IGFs, also known as potent stimulatory factors for ERKs cascade, protected the pig myocardium against necrotic damage after ischemia [29,30]. Moreover, short periods of ischemia and reperfusion (IP) that protected against sustained ischemia induced also significant activation of ERKs [25]. The inhibition of this ERKs activation using specific inhibitors, PD98059 and UO126 reversed the protective effects of IP. However, the precise role and target structures (mechanism of action) of distinct protein kinase pathways is not resolved yet. A common feature of all MAPKs is their ability to phosphorylate the transactivation domain of numerous transcription factors and thereby modulate their transcriptional activities [31–35]. Distinct MAPK-pathways were found to influence the activation of transcription factors such as ATF-2, Elk-1, c-Fos, c-Jun, MEF-2 and c-Myc [36–38]. Studies involving IP have indicated also changes in expression of several stress-related genes and proteins. We have also previously shown that brief coronary occlusions lead to upregulation of proto-oncogenes c-jun and c-fos in pig myocardium [39,40]. The protein synthesis can be regulated at the transcriptional or translational levels. It was found that some protein synthesis inhibitors acting at the translational level, such as anisomycin, influence both responses of myocardium to ischemia and MAPK activities [21,24,41]. In the present study we used actinomycin-D (act-D), an inhibitor acting at the transcription level. We investigated the effects of both systemic and local myocardial infusions of act-D before and during the IP protocol on the IP-induced cardioprotection. Moreover, also the effect of systemic infusion of act-D before index ischemia was determined. In another part of in vivo experiments we tested the effect of actinomycin-D infusion on IP-mediated cardioprotection in the presence of UO126, an inhibitor of ERK pathway. Moreover, we investigated also the effects of parallel application of both act-D and UO126 before index ischemia on infarct size.

To elucidate the possible mechanisms of act-D action the levels and phosphorylation of several nuclear transcription factors as well as levels and activation of MAPKs and Akt cascades were determined. Moreover, the effects of both act-D and UO126 on the level of the regulatory calcium binding protein, S100, were compared.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
The experimental protocol of this study was approved by the Bioethical Committee of the District of Darmstadt, Germany. Furthermore, all animals in this study were handled in accordance with the guiding principles in care and use of animals as approved by the American Physiology Society and the investigation conformed with the Guide for care and use of laboratory animals, published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.1 Chemicals
Piritramide was purchased from Janssen Pharmaceutica. Ketamin 10% was received from Medistar GmbH. Heparin (Liquemin® N 25000) was obtained from Roche. Actimomycin-D and UO126 were ordered from Alexis biochemicals. {alpha}-Chloralose, triphenyl-tetrazolium-chloride (TTC) and other biochemicals were from Sigma. The fluorescent zinc–cadmium sulfide microspheres (diameter 2–15 µm) were purchased from Duke Scientific Corp. The antibodies for detection of MAPKinases and transcription factors were from Santa Cruz Biotechnology. The antibodies for detection of phosphorylated forms of MAPKinases, Akt kinase and transcription factors were from Cellular Signaling. The antibody for detection of S100 was purchased from Biozol. Nitrocellulose membranes, molecular mass markers, the horseradish peroxidase-linked goat anti-rabbit and anti-mouse immunoglobulins, the enhanced chemiluminiscence (ECL) reagents, autoradiography films and [{gamma}-P32]-ATP were from Amersham, Pharmacia Biotech.

2.2 Animal preparation
The experiments were carried out in 44 anesthetized, ventilated, open chested, male castrated pigs (35.4±5.6 kg BW). A loose reversible ligature was placed halfway around the left anterior descending artery (LAD) and was subsequently tightened to occlude the vessels. In pigs subjected to intramyocardial microinfusion, eight 26 gauge needles connected by tubing with a peristaltic pump were placed in pairs along the LAD into the myocardium perpendicular to the epicardial surface. act-D or UO126 were dissolved in DMSO and finally diluted in Krebs–Henseleit-buffer (KHB; final concentration of DMSO was 0.1%). The infusion of KHB with DMSO served as a negative control.

2.3 Experimental groups
The experimental design for this study is presented in Fig. 1.


Figure 1
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  		Fig. 1 Experimental design. This study consisted of seven experimental groups. In group I (control group; n = 9) the index ischemia was achieved by a period of 40 min ischemia (LAD occlusion) followed by 60 min reperfusion. In group II the hearts were subjected to a preceding preconditioning protocol of two cycles of 10 min ischemia followed by 10 min reperfusion to the following index ischemia (n = 6). In group III actinomycin-D (0.05 mg/kg of BW) or KHB/DMSO (DMSO 0.1%) were infused systemic intravenously 60 min before index ischemia (n = 7). In group IV both actinomycin-D (0.05 mg/kg of BW) and UO126 (0.25 mg/kg BW) were infused parallel systemic intravenously 60 min before index ischemia (n = 6). In group V actinomycin-D (0.05 and 0.12 mg/kg of BW) was infused systemically for 40 min before and during both reperfusion periods of IP procedure (n = 12). In group VI actinomycin-D (0.05 and 0.12 mg/kg of BW) and UO126 (0.25 mg/kg BW) were infused parallel systemically for 40 min before and during both reperfusion periods of IP procedure (n = 6). The experimental design of group VII was similar to that of group V except for a local microinfusion of actinomycin-D (12.5, 25 and 50 µM) or KHB/DMSO (n = 11). For the in vitro biochemical assays left ventricular biopsies of the infused myocardium were taken in group II, IV and V before and after IP period. The drill biopsies weighed about 80 mg, were 4 mm long and reached from epi- to mid-myocardium. Biopsies were taken after the actinomycin-D treatment from the area of infusion and from control tissue (KHB/DMSO-infusion, non-risk area (NRA) of the left ventricle; and risk-area (RA) of the left ventricle without microinfusion. The application of actinomycin-D occurred in group V via three pairs of needles. Through the fourth pair of needles the solvent (DMSO in KHB 0.1%) was infused (negative control).

 
2.4 Determination of infarct size (IS)
We expressed infarct size (IS) as the infarct area (IA) relative to the risk area (RA), based on the weights of the heart slices that we used for planimetry. We used a combined staining method using zinc cadmium fluorescent microspheres (diameter 2–15 µm) and 1% triphenyl-tetrazolium-chloride (TTC) as described before [22,25].

2.5 Preparation of cellular (soluble and nuclear) fractions
After homogenization and denaturation of the ventricular biopsies the probes were applied to SDS–PAGE and used for Western blot analysis as previously described [22,25].

2.6 Immunoblot analysis
Soluble or nuclear fractions were subjected to SDS–PAGE and proteins after separation were transferred onto nitrocellulose membranes. Anti-ERK, anti-JNKs, anti-p38-MAPK (all from Santa Cruz), anti-phospho-ERK, antiphospho-JNK, anti-phospho-p38-MAPK, anti-phospho-Akt kinase, anti-Elk-1, anti-phospho-Elk-1, anti-c-Jun, anti-phospho-c-Jun, anti-ATF-2, anti-phospho-ATF-2, anti-c-Myc, anti-phospho-c-Myc (all from Cell Signaling) and anti-S100 (BioPrime) antibodies were used for primary immunodetection. As the secondary antibodies were used peroxidase labeled anti-mouse or anti-rabbit immunoglobulins. Bound antibodies were detected by the ECL Western blot detection method (Pharmacia Biotech).

2.7 Molecular probes and Northern blot analysis
The following molecular probes were used for Northern blot analysis of gene expression: mouse c-jun (a gift of Dr. R. Bravo), human ATF-2 (IMAGp998D16283; RZPD, Berlin, Germany), human Elk-1 (IMAGp998E173968; RZPD, Berlin, Germany), human c-fos (ATCC41042, Rockville, USA), human c-myc (PCR-product; a gift of Dr. H.S. Sharma) and a murine 18S ribosomal RNA cDNA probe, a gift from Dr. I. Oberbäumer (Rostock, Germany). For Northern blot analysis, cDNA probes were randomly labelled to a specific activity of approximately 108 cpm/µg using the Prime-It labelling kit (Amersham Pharmacia Biotech) and 40 µCi of [{alpha}-P32]-dCTP (3000 Ci/mmol). Total RNA from frozen heart tissue biopsies was isolated according to the method of Chomczynski and Sacchi [42]. A 15 µg amount of total RNA from NRA and RA of KHB/DMSO and act-D (0.22 mg/kg) treated tissue were size-fractionated on 1% agarose gel containing 0.66 M formaldehyde. Blotting and hybridization were performed as described previously [42]. After washing to a final stringency of 0.2xSSC/0.1% SDS at 60 °C the filters were exposed at –80 °C to Kodak X-OMAT AR films using intensifying screens up to 5 days. Filters were sequentially hybridized with all (different) probes and finally rehybridized with an 18S cDNA probe for control purpose.

2.8 Statistical analysis
The one ‘factorial analysis of variance’ with subsequent multiple comparisons by Bonferroni were performed to compare the infarct size of different groups; P<0.05 was accepted as significant. For Western blot assays the act-D treated tissue biopsy materials were compared with control (untreated: RA- and NRA-tissue) and KHB-treated tissue (negative control). The differences were evaluated by the Student's t test. The accepted level of significance was P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Hemodynamic data
The hemodynamic parameters remained unchanged during local microinfusion of the test compounds (act-D) or solvent (KHB/DMSO) and also during systemic application of both act-D and UO126. Compared to the control values no significant change was observed in any measured parameters. In addition, no ventricular premature beats were detected during the infusion.

3.2 The effect of systemic application of actinomycin-D before sustained (index) ischemia on infarct size in pig myocardium
The systemic infusion of act-D for 60 min before index ischemia (group III) significantly increased infarct size compared to index ischemia (group I). The application of act-D at the concentration of 0.05 mg/kg of BW increased the infarct size from 54.0±2.5 to 78.5±3.8% (Fig. 2). The effect of systemic application of UO126 (0.25 mg/kg BW) was published in our previous data and resulted also in an increased infarct size (69.8±3.8%) [25].


Figure 2
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  		Fig. 2 Heart slices after double exposure. The slices demonstrate the effect of the systemic infusion of actinomycin-D (act-D) before index ischemia (group III) on infarct size in pig myocardium. Heart slice of a control group I (A) and act-D-treated group III animal (B). The area at risk (RA; area surrounded by yellow spots) is unstained by fluorescent microspheres. Viable myocardium of the area at risk is stained by the TTC reaction (ib=ischemic border zone); infarcted area appears unstained (area surrounded by blue spots). The non-risk area (NRA; area surrounded by green spots) is stained by orange fluorescent microspheres. (C) Average of infarct size (IS) per group I, III and IV; graphical depiction. IS-determination=infarcted area/risk area (IA/RA). Effect of the single systemic infusion of act-D and parallel systemic infusion of both act-D and UO126 on infarct size in pig myocardium. Values are expressed as percentage of the area at risk of infarction. Group I—control group, index ischemia; group III—systemic infusion of 0.05 mg/kg BW act-D; group IV—parallel systemic infusion of both act-D (0.05 mg/kg BW) and UO126 (0.25 mg/kg BW). Each bar represents the mean±S.E.M.

 
3.3 The effect of parallel systemic application of actinomycin-D and UO126 before sustained (index) ischemia on infarct size in pig myocardium
The systemic infusion of both act-D and UO126 for 60 min before index ischemia (group IV) significantly increased infarct size compared to index ischemia (group I). Moreover, the parallel application of both substances increased the infarct size even more than observed after infusion of act-D only. The application of act-D at the concentration of 0.05 mg/kg of BW increased the infarct size from 54.0±2.5 to 78.5±3.8% the parallel application of both act-D and UO126 (0.25 mg/kg BW) increased the infarct size to 84.4±0.7% (Fig. 2C).

3.4 The effect of systemic application of actinomycin-D before and during ischemic preconditioning on infarct size in pig myocardium
The ischemic preconditioning procedure (group II) significantly reduced the infarct size from 54.0±2.5% (control index ischemia, group I) to 2.5±0.75%. The systemic infusion of act-D for 40 min before and during both reperfusion phases of IP protocol (group V) completely abolished the IP-induced cardioprotection (Fig. 3). Using the dose of 0.12 mg act-D per kg BW the IS was 88.6±1.7%; with 0.05 mg IS was 65.6±1.5%. The infusion of DMSO in KHB (negative control) did not influence the IP-induced cardioprotection.


Figure 3
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  		Fig. 3 Panel (A) shows a slice of an animal, that underwent the IP protocol (group II), which resulted in this visible reduction of the infarct size. Panel (B) shows a heart slice of a group IV animal, that was treated with actinomycin-D systemically (0.012 mg/kg BW), the IS reduction of the IP protocol was completely abolished. (C). The diagram shows the effect of the systemic infusion of 0.05 and 0.12 mg/kg BW actinomycin-D (group V) and parallel systemic infusion of both act-D (0.05 mg/kg BW) and UO126 (7.5 mg/animal) (group VI) before and during reperfusion phases of ischemic preconditioning (IP) procedure on the cardioprotection induced by IP (group II). Values are expressed as percentage of the area at risk of infarction. Group I, control group-index ischemia. Each bar represents the mean±S.E.M.

 
The local and systemic infusion of the ERK inhibitor UO126 reduced the IP-induced cardioprotection in a concentration-dependent fashion. The inhibition results at the highest concentrations in a complete cancellation of the effect (IS: 68.7±2%) [25].

3.5 The effect of parallel systemic application of actinomycin-D and UO126 before and during ischemic preconditioning on infarct size in pig myocardium
The parallel systemic infusion of both act-D and UO126 for 40 min before and during both reperfusion phases of IP protocol (group VI) completely abolished the IP-induced cardioprotection. Moreover, the parallel application of both substances increased the infarct size even more than observed after infusion of act-D only. The application of act-D (0.05 mg/kg of BW) increased the infarct size from 2.5±0.75 to 65.6±1.5%, the parallel application of both act-D (0.05 mg/kg of BW) and UO126 (0.25 mg/kg BW) increased the infarct size to 76.1±1.1% (Fig. 3C).

3.6 The effect of actinomycin-D microinfusion before and during ischemic preconditioning on infarct size
The local microinfusion of act-D for 40 min before and during both reperfusion phases of IP protocol (group VII) significantly reduced the IP-induced cardioprotection (with regard to infarct size, Fig. 4). In the areas receiving 50 µM act-D infusion significant wedge-shaped infarcts were observable (IS: 39.7±4.8%), and with decreased concentrations of act-D the IS decreased (25 µM: 26.4±1.8%; 12.5 µM: 15.6±2.3). The infarct size after microinfusion of 25 and 50 µM act-D was significantly higher compared to IP (IS: 2.5±0.1%) (Fig. 4A). Thus, the effects of act-D on IS were concentration dependent (Fig. 4B). The local microinfusion of DMSO in KHB (negative control) did not influence the IP-induced cardioprotection.


Figure 4
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  		Fig. 4 Heart slices after double exposure. Experiment with intramyocardial act-D and KHB/DMSO microinfusion (=group V). Effect of the local microinfusion of 50, 25 and 12.5 µM actinomycin-D (group V) before and during reperfusion phases of ischemic preconditioning (IP) procedure on the cardioprotection induced by IP (group II). Heart slices showing the effect of intramyocardial microinfusion of (A) 50 µM, (B) 25 µM and (C) 12.5 µM actinomycin-D. Intramyocardial infusion of KHB/DMSO served as a negative control (D). The needles for intramyocardial microinfusion were placed into the subsequently ischemic part of the left ventricle. After triphenyl-tetrazolium-chloride (TTC) staining the unstained areas around the microinfusion needles represent infarcted myocardium, the remaining viable myocardium is stained red. (E) Graph showing the effect of actinomycin-D on infarct size. Values are expressed as percentage of the area at risk of infarction. Group I, control group-index ischemia. Each bar represents the mean±S.E.M. (F) Dose response curve of infarcted area after intramyocardial infusion of actinomycin-D. Data were obtained after the experimental design of group V (semilogarithmic presentation).

 
All the tested compounds we have also checked by microinfusion into non-ischemic myocardium. Here we had not observed any evidence of cell necrosis as a consequence of the local microinfusion of the inhibitors, even at the highest concentrations. Also the local infusion of the solvent alone was not associated with any infarction.

3.7 Effect of systemic actinomycin-D infusion before and during ischemic preconditioning on levels and activation of MAPKs
We also checked, whether systemic actinomycin-D treatment directly affected the MAPKs in the cytosolic fraction.

The ERK1/2 phosphorylation was significantly reduced in actinomycin-D treated animals. The activation of distinct MAPKs was investigated using phospho-specific antibodies. Regarding ERK1/2 we saw the expected increase within 5 and 10 min of coronary occlusion and in particular during reperfusion of the first IP cycle and after completion of IP. Western blot analysis with a phospho-specific ERKs (Thr202/Tyr204) antibody showed a significant increase in phosphorylation of cytosolic ERK1/2 during and at the end of IP procedure compared to control tissue (NRA). The act-D treatment inhibited this IP-induced increase in ERK1/2 phosphorylation (Fig. 5A/C).


Figure 5
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  		Fig. 5 Effect of IP-procedure and systemic infusion of actinomycin-D on phosphorylation state of ERKs. (A) Record after Western blot analysis with a phospho-specific ERK (Thr202/Tyr204) antibody. The arrows on the right indicate the position of ERK-1: p44 extracellular-signal regulated kinase and ERK-2: p42 extracellular-signal regulated kinase. (B) Record after Western blot analysis showing the effect of IP-procedure and systemic infusion of actinomycin-D on protein levels of ERKs. RA: risk area; NRA: control non-risk area; KHB: KHB/DMSO treated tissue; act-D: actinomycin-D treated tissue. (C) Quantification of ERK phosphorylation. Data were obtained from Western blot assays and are expressed as a percentage of value of the corresponding non-preconditioned myocardium (NRA). Each bar represents the mean±S.E.M. * P<0.05 vs. KHB treated myocardium.

 
By Western blot analysis we observed no changes of levels or cellular distribution of the total, non-phosphorylated ERKs after IP with or without actinomycin-D or KHB/DMSO treatment (Fig. 5B).

Using specific anti-phospho-JNKS antibody directed against Thr183/Tyr185 we observed a significant increase in phosphorylation of both JNKs during IP with the maximal stimulation reached after the completion of the IP protocol. The presence of act-D significantly blocked the increased phosphorylation (Fig. 6A/C).


Figure 6
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  		Fig. 6 (A) Western-blot assay of myocardial tissue (cytosolic fractions) after IP and act-D-treatment using a specific antibody against phospho-SAPK/JNKs (Thr183/Tyr185). The arrows on the right indicate the position of the phosphorylated (P-JNK1/2) form of JNK-55 and JNK-46. (B) Record after Western blot analysis showing the effect of IP-procedure and systemic infusion of actinomycin-D on protein levels of NKs. RA: risk area; NRA: control non-risk area; KHB: KHB/DMSO treated tissue; act-D: actinomycin-D treated tissue. (C) Quantification of JNKs phosphorylation. Data were derived from Western blot assays and are expressed as a percentage of value for control non-preconditioned myocardium (NRA). Each bar represents the mean±S.E.M. * P<0.05 vs. KHB treated myocardium. CO—coronary occlusion; RP—reperfusion; NRA—non-risk control area; KHB—KHB/DMSO treated tissue from area at risk; act-D—actinomycin-D treated tissue from the area at risk; 5', 10'=fifth or tenth min of the ischemic or reperfusion episode.

 
The degree of p38-MAPK phosphorylation during and after completion of the IP protocol was investigated with a specific anti-phospho-p38-MAPK antibody (Thr180/Tyr182). At the tenth minute of the first ischemic period of IP we observed an increase in p38-MAPK phosphorylation and the amount of phosphorylated p38-MAPK declined during the course of IP procedure (data not shown [22]). In contrast to ERKs and JNKs, the act-D treatment did not significantly influence the phosphorylation of p38-MAPK and similar levels of phospho-p38-MAPK were observed in act-D treated as well as in KHB/DMSO treated myocardium, during and after IP. We found no changes in levels or in cellular distribution of p38-MAPK and SAPK/JNK on completion of the IP protocol with or without actinomycin-D or KHB/DMSO treatment (Fig. 6B).

3.8 Effect of systemic actinomycin-D or UO126 infusion before and during ischemic preconditioning on the activation of Akt kinase
The activation of the Akt signaling pathway was investigated using phospho-Akt (Ser473) antibody at the end of the IP procedure and also after infusion of either act-D (0.22 mg/kg) or UO126 (7.5 mg/animal). We found that IP increased phosphorylation of Akt and the content of phosphorylated Akt in cytosolic fractions increased after completion of the IP protocol about 2.2-fold (P<0.001); (Fig. 7). This IP-mediated activation of Akt kinase was inhibited by the application of UO126 but not by Act-D (Fig. 7).


Figure 7
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  		Fig. 7 Effect of IP-procedure and systemic infusion of actinomycin-D and UO126 on phosphorylation of Akt kinase. (A) Western-blot assay of myocardial tissue (cytosolic fractions) after IP, act-D or UO126 treatment was performed using a specific antibody against phospho-Akt (Ser473). (B) Con: NRA—non-risk control area at the end of IP; RA— risk area at the end of IP procedure; act-D—actinomycin-D treated tissue from the area at risk after IP; UO—UO126 treated Akt-phosphorylation. Data were derived from Western blot assays and are expressed as a percentage of value for control non-preconditioned myocardium (NRA). Each bar represents the mean±S.E.M. * P<0.001 vs. KHB treated myocardium (con). IP—ischemic preconditioning; NRA—non-risk control area; KHB—KHB/DMSO treated tissue from area at risk(c/con); act-D—actinomycin-D treated tissue from the area at risk.

 
3.9 Effect of actinomycin-D infusion before and during IP protocol on the protein levels and phosphorylation of several transcription factors
Actinomycin-D is known as a transcription inhibitor. To determine the effect of act-D at the transcription level we investigated the levels and the in vivo phosphorylation (activation) of several transcription factors at the end of IP. By Western blot analysis using specific antibodies we did not observe changes in abundance (total protein levels) of c-Jun, ATF-2, Elk-1 and c-Myc after IP procedure and after act-D treatment, whether it was risk-area or non-risk-area tissue, with or without actinomycin-D treatment. Fig. 8F shows the results for c-Jun, Elk-1 and c-Myc. Similar results were obtained also for ATF-2 (data not shown).


Figure 8
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  		Fig. 8 Effect of IP-procedure and systemic infusion of actinomycin-D on the phosphorylation state of several transcription factors. (A) the content of ATF-2, (B) c-Jun, (C) Elk-1 and (D) c-Myc. RA—tissue after IP procedure from the area at risk; NRA—tissue after IP procedure from the non-risk area; KHB— KHB/DMSO infusion; act-D—actinomycin-D infusion; 5', 10'=fifth or tenth minute of the ischemic or reperfusion episode. (E) Quantification of transcription factors phosphorylation during and after IP. The data are expressed as a percentage of the mean obtained from corresponding preconditioned control tissue (KHB infusion). Each bar represents the mean±S.E.M. (F) Effect of IP-procedure and systemic infusion of actinomycin-D on the protein levels of several transcription factors. It shows the content of c-Jun, Elk-1 and c-Myc. RA—tissue after IP procedure from the area at risk; NRA—tissue after IP procedure from the non-risk area; KHB—KHB/DMSO infusion; act-D—actinomycin-D infusion.

 
The investigation of phosphorylation of these transcription factors revealed their increased phosphorylation in the myocardial risk area at the end of IP protocol (Fig. 8A–D). The local or systemic infusion of act-D significantly inhibited this IP-mediated phosphorylation of transcription factors (Fig. 8E). The quantification of the phosphorylation of the transcription factors showed a 15.4-fold decrease for the phospho-ATF-2, a 5.3-fold decrease for phospho-c-Myc, a 3.2-fold decrease for phospho-Elk-1 and a 1.9-fold decrease for phospho-c-Jun after systemic infusion of act-D (Fig. 8E).

3.10 Effect of actinomycin-D infusion during IP protocol on transcription factors expression
mRNA levels for c-jun, ATF-2, Elk-1, c-fos and c-myc were determined by Northern blot analysis. In control myocardium without IP only slightly expressed transcripts for c-jun (ca. 2.7 kb), c-fos (ca. 2.1 kb) and c-myc (ca. 2.3 kb) were found. After IP procedure the mRNA levels of transcription factor c-jun, c-fos and c-myc increased in the risk area of KHB/DMSO treated myocardium. However, the changes in m-RNA levels were not connected with similar changes at the protein levels (Fig. 8F). Act-D treatment inhibited the IP-mediated mRNAs expressions (Fig. 9A–C) but not the protein levels. No transcripts of ATF-2 and Elk-1 were detected by means of Northern blot hybridization in control or preconditioned myocardium.


Figure 9
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  		Fig. 9 A representative Northern blot demonstrating the changes of the mRNA levels of c-jun (Top), c-fos (Center) and c-myc (Bottom) in KHB/DMSO and actinomycin-D (act-D) treated myocardium after IP. Lanes 1 and 2: non-risk area (NRA), left ventricle, after KHB/DMSO treatment. Lanes 3 and 4: NRA, right ventricle, after KHB/DMSO treatment. Lanes 5–9: risk area (RA), left ventricle, after KHB/DMSO treatment. Lanes 10 and 11: NRA, left ventricle, after act-D treatment. Lanes 12 and 13: NRA, left ventricle, after act-D treatment. Lanes 14–18: risk area, left ventricle, after act-D treatment.

 
3.11 Effects of actinomycin-D or UO126 infusions before and during IP on the levels of S100 protein
Using a specific antibody we found increased immunoreactivity for S100 at the end of the IP procedure in the area of risk compared to control (from 100±7 to 249±10%). Infusion of both actinomycin-D and UO126 significantly reversed the IP-mediated increase in S100 immunoreactivity (Fig. 10A/B). UO126 decreased the immunoreactivity from 249±10 (IP) to 110±9%. With act-D the decrease was even more pronounced to 77±7% (P<0.001).


Figure 10
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  		Fig. 10 Effect of IP-procedure and systemic infusion of actinomycin-D and UO126 on protein levels of S100 calcium binding protein. (A) Western-blot assay of myocardial tissue (cytosolic fractions) after IP and act-D or UO126 treatment was performed using a specific antibody against S100A1. (B) Quantification of S100 levels. The data are expressed as a percentage of the mean obtained from corresponding control tissue. Each bar represents the mean±S.E.M. 0.001 vs. NRA (con)—non-risk control area; RA—risk area at the end of IP procedure; act-D (RA)—actinomycin-D treated tissue from the area at risk; UO (RA)—UO126 treated tissue from the area at risk.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
We have previously shown that inhibition of the ERK branch of the MAPKs abolishes the cardioprotective effects of ischemic preconditioning [25]. This MAPK cascade is known to translate extracellular signals and regulate gene expression. Therefore, the protective effect of IP might be at least partially mediated by transcriptional activity. To test this hypothesis we used the transcription inhibitor actinomycin-D in our in vivo porcine myocardial ischemia model.

Our results show that actinomycin-D

1. completely inhibits the protective effects of IP in a dose-dependent manner,
2. increases infarct size after index ischemia without exerting a cardiotoxic effect in non-ischemic myocardium,
3. does not influence the protein levels of MAPKs but inhibits the IP-mediated activation of the MAPKinases ERK:1/2 and JNK:1/2,
4. does not influence the protein levels but inhibits the IP-mediated phosphorylation of the transcription factors ATF-2, c-Jun, Elk-1 and c-Myc.
5. inhibits the mRNA expression of the transcription factor c-jun, c-fos and c-myc
6. declines the IP-mediated increase in the level of S100 protein, opposed to Akt-phosphorylation

Actinomycin-D is a transcription inhibitor that binds to DNA, blocks the movement of RNA polymerase and prevents RNA synthesis [43–48]. The inhibitory effect of act-D was also shown for a number of cytosolic enzymes, especially for tyrosine kinases and adaptor-proteins including SH2 and SH3 domains [49,50].

In our current study we demonstrate an effect of IP on the transcriptional and phosphorylation level of certain protooncogenes. The experimental design may not have allowed enough time for the observed transcriptional effects of IP or act-D to result in different levels of the respective proteins, despite their short turnover time.

Several studies investigated the role of protein synthesis in IP-mediated cardioprotection using inhibitors of transcription or translation. Neither the inhibition of translation by cycloheximide, nor the inhibition of transcription by actinomycin-D blocked IP in an open-chest model of anesthetized rabbits, when administered before coronary occlusion [51]. However, Rowland et al. [52] demonstrated that IP was abolished by pretreatment with cycloheximide but not by actinomycin-D. The authors concluded that IP is regulated at the level of translation rather than gene transcription. A recent study confirmed that cycloheximide, but not actinomycin-D, reverses the infarct-size reducing effect of an IP-protocol [53]. These studies support an important role for translational events in IP. On the other hand, Singh et al. [54] showed that both, actinomycin-D and cycloheximide, markedly attenuate the IP-induced delayed cardioprotection in anaesthetized rat hearts. Most of the studies mentioned above are at variance with our observations. One possible explanation is the use of different animal models and experimental protocols. Lepran et al. [55] administered actinomycin-D or cycloheximide 24 h and 1 or 4 h before inducing coronary occlusion in conscious rats, which offered marked protection against postinfarction ventricular fibrillation and sudden death. When actinomycin-D was given 4 h prior to coronary ligation, the outcome of myocardial infarction was not influenced. These results suggest that protein factors may be involved in the early events of myocardial infarction. In contrast to this and other studies [51,52] we infused actinomycin-D not only before the first coronary occlusion but also during both reperfusion phases of the IP protocol. This may reflect the importance of reperfusion episodes for the protective effect of IP.

The consequence of actinomycin-D infusion was inhibition of ERKs and SAPK/JNKs during and at the end of IP procedure. This suggest that one possible explanation of actinomycin-D effects could be that actinomycin-D acts via inhibition of the MAPKinase cascades dependent genes. Modulation of the activities of MAPKs can positively or negatively influence the response of myocardial tissue to ischemic damage and several studies demonstrated an important role of MAPKs in myocardial protection [21–23]. Numerous transcription factors are possible target structures for MAPKs in vivo. These transcription factors are phosphorylated at their transactivation domains and this phosphorylation mediates the upregulation of gene expression [36–38]. The relationship between MAPKs and transcription factor-phosphorylation was confirmed by several studies. The activation of p38-MAPK was found to be coincident with an increased phosphorylation of ATF-2 and c-Jun in adult rat hearts [56]. Increased phosphorylation of ATF-2 and c-Jun as well as upregulation of c-Jun protein were observed after activation of MAPKs by pro-inflammatory cytokines in neonatal rat ventricular cardiomyocytes [36]. In a recent study, we observed an inhibitory effect of ERK inhibitors on cardioprotection [25] and this was associated with an inhibition of Elk-1 phosphorylation in pig myocardium. The observed effects of actinomycin-D on mRNA expression of the transcription factor and the IP-mediated phosphorylation of several transcription factors as well as effects on ERKs and SAPK/JNKs activities implicate a possible involvement of these MAPKs in the regulation of transcriptional processes during IP. However, actinomycin-D induced changes in phosphorylation state of investigated transcription factors but did not alter the protein expression of these proteins. This indicates that also other effects of actinomycin-D, unrelated to protein synthesis events, should be considered. One possibility is that inhibition of MAPKs by actinomycin-D may lead to an alteration of the functional state of some other cytosolic or membrane-bound proteins involved in cardioprotection. It is worth speculating that actinomycin-D might reduce the levels of an unidentified phosphatase inhibitor. The subsequent increase in activity of phosphatases like MKP1/3 might explain the effects of actinomycin-D on the phosphorylation state of ERK and JNK and therefore certain transcription factors [57–59]. Addition of actinomycin-D abrogated the IL-1β-induced rise in COX-2 protein, COX activity and PGE2 release in human pulmonary A549 cells [60]. In mouse peritoneal macrophages actinomycin-D was also found to decrease the release of arachidonic acid after stimulation of the ERK and JNK pathways with PMA and okadaic acid. These effects were similar to the consequences of a specific inhibition of the ERK pathway by UO126 [61]. We also observed comparable effects of both actinomycin-D and UO126 on reversal of IP-induced changes on the content of the S100A1 calcium binding protein. This protein belongs to the family of S100 proteins that translate the Ca2+ signal into tissue-specific responses [62]. It was shown that this protein may be an early diagnostic marker for ischemic coronary diseases and is also important determinant of contractile dysfunction in cardiomyocytes [63–65].

The effects of single actinomycin-D infusions (present study) with regard to the reversal of IP-mediated cardioprotection and the inhibition of ERKs activation were comparable with effects observed previously for UO126 [25]. Interesting is the finding that the parallel application of both actinomycin-D and UO126 revealed a higher degree of myocardial damage (with regard to infarct size) when compared to infusions of actinomycin-D only. This can suggest some additive mechanisms or differences in the action of act-D and UO126 that showed different effects on Akt kinase pathway. UO126 but not Act-D inhibited the IP-mediated activation of Akt kinase. UO126 is an inhibitor of ERK pathway and its effect on activation of Akt kinase could reflect some connection between these two signaling pathways (ERK and Akt). This cross-talk was demonstrated in recent studies showing cross-regulation of ERK pathway by the PI3K-Akt pathway [66,67].

In summary, in this study we have demonstrated that an inhibitor of RNA synthesis, actinomycin-D, abolishes dose dependently the cardiac protective effect of IP in an in vivo pig model. Moreover, application of actinomycin-D increases infarct size without direct cardiotoxic effects on the non-ischemic myocardium. Actinomycin-D inhibits the activation of ERKs and JNKs. The inhibitory action of actinomycin-D on IP is associated with a suppression of IP-mediated mRNA expression and phosphorylation of ERKs and JNKs-dependent transcription factors. Thus, our results demonstrate the involvement of ERK and JNK cascades in IP.

Time for primary review 22 days.


   Acknowledgements
 
The authors gratefully acknowledge Armin Helisch and Swen Wolfram for the help during the preparation of the manuscript and suggestions about the figures. This study and the research fellowship for M.B. was supported in part by the Kuhl-Stiftung (Stifterverband), Essen; Germany.


   Notes
 
1 Both authors contributed equally to this study. Back


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
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