Transcription inhibitor actinomycin-D abolishes the cardioprotective effect of ischemic reconditioning.
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.
Strohm
Claudia
C
Department of Experimental Cardiology, Max Planck Institute for Physiological and Clinical Research, Bad Nauheim, Germany.
Barancík
Miroslav
M
von Bruehl
Marie
M
Strniskova
Monika
M
Ullmann
Claudia
C
Zimmermann
René
R
Schaper
Wolfgang
W
D051698:Activating Transcription Factor 2; D000704:Analysis of Variance; D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics; D004268:DNA-Binding Proteins; D003609:Dactinomycin / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015151:Immunoblotting; D019157:Ischemic Preconditioning, Myocardial / Q000379:methods*; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D009203:Myocardial Infarction / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D009206:Myocardium / Q000378:metabolism / Q000473:pathology; D009570:Nitriles / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger / Q000032:analysis; D013552:Swine; D014157:Transcription Factors / Q000235:genetics; D050807:ets-Domain Protein Elk-1
D051698:Activating Transcription Factor 2; D002070:Butadienes; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D009570:Nitriles; D019384:Nucleic Acid Synthesis Inhibitors; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D014157:Transcription Factors; C113580:U 0126; D050807:ets-Domain Protein Elk-1; D003609:Dactinomycin; D020928:Mitogen-Activated Protein Kinases