The Novel Iron Chelator, 2-Pyridylcarboxaldehyde 2-Thiophenecarboxyl Hydrazone, Reduces Catecholamine-Mediated Myocardial Toxicity Pr˘emyslMlade ˘nka, †,‡ Danuta S. Kalinowski, ‡,§ Pavlı ´na Has ˇkova ´, ‡,| Zuzana Bobrovova ´, † Radomı ´r Hrdina, † Toma ´s ˇS ˇ imu ˚nek, | Petr Nachtigal, ⊥ Vladimı ´r Semecky ´, ⊥ Jaroslava Va ´vrova ´, # Magdale ´na Holec ˇkova ´, # Vladimir Palicka, # Yvona Mazurova ´, ∇ Patric J. Jansson, § and Des R. Richardson* ,§ Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Kra ´loVe ´, Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kra ´loVe ´, Department of Biological and Medical Science, Faculty of Pharmacy in Hradec Kra ´loVe ´, Institute of Clinical Biochemistry and Diagnostics, Faculty of Medicine in Hradec Kra ´loVe ´, Department of Histology and Embryology, Faculty of Medicine in Hradec Kra ´loVe ´, Charles UniVersity in Prague, Czech Republic, and Iron Metabolism and Chelation Program, Bosch Institute and Department of Pathology, UniVersity of Sydney, Sydney, New South Wales, 2006 Australia ReceiVed September 3, 2008 Iron (Fe) chelators are used clinically for the treatment of Fe overload disease. Iron also plays a role in the pathology of many other conditions, and these potentially include the cardiotoxicity induced by catecholamines such as isoprenaline (ISO). The current study examined the potential of Fe chelators to prevent ISO cardiotoxicity. This was done as like other catecholamines, ISO contains the classical catechol moiety that binds Fe and may form redox-active and cytotoxic Fe complexes. Studies in vitro used the cardiomyocyte cell line, H9c2, which was treated with ISO in the presence or absence of the chelator, desferrioxamine (DFO), or the lipophilic ligand, 2-pyridylcarboxaldehyde 2-thiophenecarboxyl hydrazone (PCTH). Both of these chelators were not cardiotoxic and significantly reduced ISO cardiotoxicity in vitro. However, PCTH was far more effective than DFO, with the latter showing activity only at a high, clinically unachievable concentration. Further studies in vitro showed that interaction of ISO with Fe(II)/ (III) did not increase cytotoxic radical generation, suggesting that this mechanism was not involved. Studies in vivo were initiated using rats pretreated intravenously with DFO or PCTH before subcutaneous administration of ISO (100 mg/kg). DFO at a clinically used dose (50 mg/kg) failed to reduce catecholamine cardiotoxicity, while PCTH at an equimolar dose totally prevented catecholamine-induced mortality and reduced cardiotoxicity. This study demonstrates that PCTH reduced ISO-induced cardiotoxicity in vitro and in vivo, demonstrating that Fe plays a role, in part, in the pathology observed. Introduction Iron (Fe) chelators are well-known therapeutic agents adminis- tered to prevent complications associated with transfusional Fe overload in diseases such as -thalassemia major (1-6). Iron chelators that are specifically designed for the treatment of Fe overload disease act by avidly binding Fe, preventing it from participating in deleterious Fenton chemistry that results in the generation of reactive oxygen species (ROS) 1 and oxidative cell damage (4, 7). Endogenous catecholamines such as epinephrine (Figure 1) and norepinephrine are known to bind Fe(III) through their catechol groups, forming 3:1 ligand:Fe complexes (8-10) (Figure 1). Catecholamines are elevated in cardiovascular disease and may trigger acute myocardial infarction (AMI) (11, 12). In addition, sufficient doses of the synthetic catecholamine and nonselective -adrenergic agonist, isoprenaline (ISO; Figure 1), * To whom correspondence should be addressed. Tel: +61-2-9036-6548. Fax: +61-2-9036-6549. † Department of Pharmacology and Toxicology, Charles University in Prague. ‡ These authors contributed equally to this manuscript. § University of Sydney. | Department of Biochemical Sciences, Charles University in Prague. ⊥ Department of Biological and Medical Science, Charles University in Prague. # Institute of Clinical Biochemistry and Diagnostics, Charles University in Prague. ∇ Department of Histology and Embryology, Charles University in Prague. 1 Abbreviations: AMI, acute myocardial infarction; cTnT, cardiac troponin T; DCF, 2′,7′-dichlorofluorescein; DFO, desferrioxamine; Dp44mT, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone; H 2 DCF, 2′,7′-dichlo- rodihydrofluorescein; H 2 DCF-DA, 2′,7′-dichlorodihydrofluorescein-diac- etate; IBE, iron-binding equivalent; ISO, isoprenaline; NR, neutral red; PCIH, 2-pyridylcarboxaldehyde isonicotinoyl hydrazone; PCTH, 2-pyridyl- carboxaldehyde 2-thiophenecarboxyl hydrazone; PG, propylene glycol; PIH, pyridoxal isonicotinoyl hydrazone; PI, propidium iodide; ROS, reactive oxygen species; Tf, transferrin. Figure 1. Chemical structures of epinephrine, ISO, the 3:1 ISO:Fe(III) complex, DFO, and PCTH. Chem. Res. Toxicol. 2009, 22, 208–217 208 10.1021/tx800331j CCC: $40.75  2009 American Chemical Society Published on Web 12/12/2008