CARDIOMYOCYTE RESISTANCE TO DOXORUBICIN TOXICITY MAY BE ACHIEVED BY ADENOSINE SIGNALING Asher Shainberg, Avishag Forkus, Liaman Mamedova & Vladimir Shneyvays. Faculty of Life Sciences, Rar-!lan University, Ramat Cian, Israel

llse of anthracycline antibtotic doxorublcin (DOX), also called adriamycin, is limited because of its dosc- dependent cardiotoxicity, leading to irreversible degenerative cardiomyopathy. Adenosine (ADO) is a well-known regulator of a variety of physiological functions in the heart. The A, ADO receptor (A,R) is the most recently identified subtype. Stimulation of A1 adenosine receptors with the highly selective agonists leads to protection cultured cardiomyocytes treated with DOX. However, the mechanism of tolerance acquisition to doxorubicin detrimental side effects remains unclear. In this study regulating pathways, leading to cells resistance to DOX were studied by: I. Evaluation of the ATP-sensitive potassium (KA.,p) channels. 2. Investigation of redox states of mitochondria and ATP production. 3. Evaluation of free and intramitochondrial calcium through adenosine A3 receptor regulation. Activation of A3-subtype but not Al-subtype receptors attenuated doxorubicin cardiotoxicity. Since DOX is a drug with tremendous therapeutic anticancer potential, understanding the mechanisms leading to resistance of heart cells to DOX, will facilitate development of more effective treatments for cancers.

ASCORBIC ACID INHIBITS ISCHEMIC PRECON- DITIONINGIN PIGS. Andreas Skyschally, Rainer Schulz, Petra Gres, Matthias Thielmann & Gerd Heusch. Depanment of Pathophysiology, University of Essen Medical School, Germany

The involvement of free oxyradlcals In lschemlc preconditlonlng in rats and rabbits has been established. Data from larger mammals which are closer to man are lacking We have therefore tested the impact of the natural radical scavenger ascorbic acid on ischemic preconditionmg in pigs In 32 anestheuzed pigs the LAD coronary artery was perfused from an extracorporal circuit. Subendocardial blood flow (radioactive microspheres) and myocardial infarct size (TX-staining) were determined. lschemic preconditiontng (IP) by IO min ischemia followed by 15 min reperfusion reduced infarct size after 90 mm severe ischemia and 120 min reperfusion (l/R). The pretreatment with ascorbic acid (A; i.v., 30 min before I or IP. resp.; 2g bolus; maintenance dose 25mg/mm up to the end of I) had no effect on infarct size per se (A+I/R). but abolished the infarct size reduction by ischemrc preconditioning (A+IP+I/R)

I Infarct size 1 Subendocardial blood flow Group 1 [% area at risk; 1 __ [rnl/min/g] l/R (n=9) 1 ‘26.9 f 3.9 1 0.048 * 0.007 lP+uR (11=‘)1 6.4 * 2.4 - A+r/R (n=5) 24.0 ct 7.7 Aetl’+IIR (n=9) 19.1 f 5.4 Mean&EM: * ~~0.05 vs. I/R;

0054 t 0.011 0.053 * 0.014 0 046 + 0.010

Conclusion: Scavenging free oxyradicals with ascorbic acid prevents the beneficial effect of ischemic preconditionmg on infarct size in pigs. Therefore, free oxyradicals are also involved in the signal transduction of ischemic preconditioning In pigs. Their role as triggers or mediators remams lo be established.

lschemlc preconditioning attenuates the dephosphorylatlon of connrxln 43 In pigs in sllu Andrees Skyschally. Ina Konietzka, Petra Gres, Rainer Schulz. Gerd Heusch. Dept of Pathophysiology. University of Essen. Germany

Objectives While almost all connexln 43 (Cx43) is phosphorylated in isolated normoperfused rat hearts, it becomes progressively dephosphorylated dunng lschemla Such dephosphorylation corresponds to electrical uncoupling (Circ Res 87: 656-662, 2000). The effects of ischemic preconditioning (IP) on the phosphorylation status of Cx43 in hearts in vivo is unknown. We therefore analyzed myocardial biopsies from 4 control pigs, undergoing 90 min ischemia and 120 min reperfusion. and 4 pigs preconditioned by a single cycle of 10 min &hernia and 15 min reperfusion preceding the sustained ischemia. Biopsies were taken durmg normoperfusion, at 5 min and 85 min of the sustained ischemia. stained for total Cx43 and non-phosphorylaled Cx43 using commercially available antibodies. and analyzed using confocal laser microscoov. The sianal ratio of non-ohosohorvhtad Cx43 lo total Cx43’ it the i&ercellular juncti’ons ‘was- caIculated (analyzed area: control group 1373*330flm’ [mean&D]; IP group 1567671flm*; n.s.).- Subendocardial blood flow (microspheres) and systemic hemodynamics did not differ belween groups, infarct size (?X- staining) was reduced from 20.2&3% 10 7.7*9.0% in preconditioned hearts. The ratio of non-phosphorylated Cx43 lo total Cx43 did not change from baseline (0.30&03) to 5 min ischemia (0.3m.O3), but increased to 1 Olto.51 at 85 mln ischemia (~~0.05) in control hearts. Following IP, the increase in the non-phosphotylated Cx43 was completely abolished. 0.3(ko.O1 at baseline vs. 03061.03 after 85 min ischemla. Total Cx43 remained unchanged during &hernia and did not differ between groups. Conclusion: lschemic precondltionlng attenuates the ischemiainduced dephosphorylatlon of connexln 43 in pig hearts in vivo.

CHANGES IN CATECHOlAMINES AND CGRP LEVELS IN HEART Df ~OZOTOCIN-DUC RATS lana Slavlkovb, Jltka Kuncod, Jkka &&rovi. Dapt of physiology, Fat of Med, Charka Unlv, f+kai%, Czech Republic.

The present study examined long-term changes In the efferent sympathebc adrenerglc and afferent sensory innervation in the time course of developing diabetes mellitus in the rat. Model of Type I diabetes was induced by a single, intravenous streptozotocin (SE’) inJection (65 mg/kg) to 60-day old animals. Rats which had glycaemia 3” day after the ST2 administration higher than 20 mmol/l were selected for the studies. Vehicle-treated animals served as controls. The hearts were isolated after 4, 8, and 16 weeks duration of the study and catecholamines noradrenaline (NA), adrenaline (A), and dopamine (DA) concentrations were determined separately In all heart compartments by radioimmunoassay. Also levels of calcitonin gene-related peptide (CGRP), a neurobansmitter of sensory motor Innervation, were analysed usmg the same experimental protocol. The hearts from 4-week diabetic rats did not show any significant changes In the levels of all the investigated neurotransmitters. After 8 weeks, however, concenbations of NA and A decreased significantly in both atria and ventricles. Also DA levels lowered significantly in both atria, while in ventricles they remained at control values. In contrast, CGRP concentrations in ventricles from b-week ST2 rats increased twofold in comparison with controls, whereas levels in atria remained approximately constant. After 16 weeks, however, ST2 diabetes CGRP concentrations significantly increased In all heart compartments. The results indicate, that the sympathetic neuropathy in the untreated SR diabetes differs at the onset and Intensih/ of Impairment between the heart compartments. An Increased level of CGRP in long-term diabetes may reflect its positive trophic effect during progressjon of this disease.

Supported by GA CR 305/01/0263