Multiple Interference of Anthracyclines with Mitochondrial Creatine Kinases: Preferential Damage of the Cardiac Isoenzyme and Its Implications for Drug Cardiotoxicity MALGORZATA TOKARSKA-SCHLATTNER, THEO WALLIMANN, and UWE SCHLATTNER Institute of Cell Biology, Swiss Federal Institute of Technology (ETH), Ho ¨ nggerberg, Zu ¨ rich, Switzerland Received July 10, 2001; accepted December 5, 2001 This article is available online at http://molpharm.aspetjournals.org ABSTRACT Anthracyclines are among the most efficient drugs of cancer chemotherapy, but their use is limited by a significant risk of cardiotoxicity, which is still far from being understood. This study investigates whether impairment of mitochondrial creat- ine kinase (MtCK), a key enzyme in cellular energy metabolism, could be involved in anthracycline cardiotoxicity. We have an- alyzed the effects of three anthracyclines, doxorubicin, dauno- rubicin, and idarubicin, on two MtCK isoenzymes, sarcomeric/ cardiac sMtCK and ubiquitous uMtCK, from human and chicken. Using surface plasmon resonance, gel filtration, and enzyme assays, we have quantified properties that are of basic importance for MtCK functioning in vivo: membrane binding, octameric state, and enzymatic activity. Anthracyclines signif- icantly impaired all three properties with differences in dose-, time-, and drug-dependence. Membrane binding and enzy- matic activity were already affected at low anthracycline con- centrations (5–100 M), indicating high clinical relevance. Ef- fects on membrane binding were immediate, probably because of competitive binding of the drug to cardiolipin. In contrast, dissociation of MtCK octamers into dimers, enzymatic inacti- vation and cross-linking occurred only after hours to days. Different protection assays suggest that the deleterious effects were caused by oxidative damage, mainly affecting the highly susceptible MtCK cysteines, followed by generation of free oxygen radicals at higher drug concentrations. Enzymatic in- activation occurred mainly at the active site and involved Cys278, as indicated by experiments with protective agents and sMtCK mutant C278G. All anthracycline effects were sig- nificantly more pronounced for sMtCK than for uMtCK. These in vitro results suggest that sMtCK damage may play a role in anthracycline cardiotoxicity. Anthracyclines are among the most efficient drugs of can- cer chemotherapy, but a significant risk of cardiotoxicity limits their use (Olson and Mushlin, 1990; Singal et al., 1997). The molecular mechanisms of anthracycline cardiotox- icity are still far from being clear. Cardiac injury has been related to the impairment of mitochondrial functions, such as respiratory rate, and generation of high-energy phosphates. Numerous mechanisms for inactivation of the cardiac mito- chondrial respiratory chain by anthracyclines have been pro- posed, such as generation of free radicals, interaction with mitochondrial DNA, disruption of cardiac gene expression, alteration of calcium exchange, lipid peroxidation inducing disturbance of mitochondrial membranes, and cardiomyocyte apoptosis (Singal et al., 1997; Minotti et al., 1999; Arola et al., 2000; Horenstein et al., 2000). This study focuses on an alternative mechanism that could lead to heart energy depletion: the impairment of different key properties of mitochondrial creatine kinase (MtCK). Cells and tissues with high or fluctuating energy demands, like heart and brain, use creatine and isoenzymes of creatine kinase (CK) to cope with high ATP requirements (Wallimann et al., 1992). CK, catalyzing the reversible transphosphory- lation between ATP and phosphocreatine (PCr), is able to stock the “high energy” of ATP in form of PCr and, vice versa, to use PCr to replenish cellular ATP pools. The interplay between dimeric cytosolic and mainly octameric mitochon- drial CK isoenzymes, referred to as CK/PCr-circuit, repre- sents an “energy buffer” and also provides an “energy shut- tle” bridging sites of energy generation with sites of energy consumption (Wallimann et al., 1992). Two MtCK isoen- This work was supported by grants from the Schweizerische Herzstiftung, the Wolfermann-Na ¨ geli-Stiftung; and the Novartis Stiftung (to T.W.); the Sandoz Family Office (to M.T.-S.); and the Schweizer Krebsliga and the Zen- tralschweizer Krebsliga (SKL) (to U.S. and T.W.). Some preliminary results were reported in abstract form: Tokarska-Schlat- tner M, Wallimann T, and Schlattner U (2000) Surface plasmon resonance studies (BiaCore) of protein-membrane interactions using mitochondrial cre- atine kinase. Biophys J 78(Suppl):410A, and Tokarska-Schlattner M, Walli- mann T and Schlattner U (2000) In vitro effect of the anthracycline drugs on MtCK isoform functions. Eur J Cell Biol 79(Suppl 52):178. ABBREVIATIONS: MtCK, mitochondrial creatine kinase; CK, creatine kinase; PCr, phosphocreatine; sMtCK, sarcomeric MtCK; uMtCK, ubiqui- tous MtCK; -ME, -mercaptoethanol; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; SPR, surface plasmon resonance; CL, cardiolipin; PC, phosphatidylcholine; TSAC, transition state analog complex; SOD, superoxide dismutase; ROS, reactive oxygen species. 0026-895X/02/6103-516 –523$3.00 MOLECULAR PHARMACOLOGY Vol. 61, No. 3 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1212/967809 Mol Pharmacol 61:516–523, 2002 Printed in U.S.A. 516 at ASPET Journals on February 14, 2018 molpharm.aspetjournals.org Downloaded from