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 C (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-induccd 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 die I P-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 ERK 1/2 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 ischermia and in IP. (C) 2002 Elsevier Science BY All rights reserved.