Cardiovascular Research 2002 55(3):602-618; doi:10.1016/S0008-6363(02)00453-4
© 2002 by European Society of Cardiology
Copyright © 2002, European Society of Cardiology
Transcription inhibitor actinomycin-D abolishes the cardioprotective effect of ischemic reconditioning
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
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 Both authors contributed equally to this study.
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