Mechanisms of Anthracycline-Induced Dysfunction of Calcium Handling Proteins in the Heart

Abstract

Anthracyclines are powerful chemotherapy agents, whose use is limited due to the onset of potentially fatal cardiotoxicity which includes arrhythmogenesis and heart failure. Several proteins important in intracellular Ca2+ signalling have been identified as drug binding targets, including the cardiac ryanodine receptor Ca2+ release channel (RyR2), the Ca2+ binding protein calsequestrin (CSQ2) and the Sarco/Endoplasmic Reticulum Ca-ATPase (SERCA2a). The effects of the drug metabolites have been poorly characterized but are believed to be important in the devastating cardiac effects of these drugs.The functional effect of doxorubicin and its metabolite, doxorubicinol on RyR2 was assessed by adding clinically relevant drug concentrations to single RyR2 channels in lipid bilayers. Both drugs caused biphasic modulation of RyR2 activity where there was an early increase in channel activity followed by a later, inhibitory phase. RyR2 channel activation, but not inhibition, could be reversed by drug washout, typical of a ligand binding effect. Conversely, the irreversible nature of the inhibitory effect suggested a non-ligand binding effect. Treatment with doxorubicin/doxorubicinol reduced the number of thiols on RyR2, indicative of drug-induced thiol oxidation. Additionally, doxorubicinol abolished the response of RyR2 to changes in luminal Ca2+. Further experiments revealed that the loss of luminal Ca2+ sensing was due to an interaction between doxOL and CSQ2. Finally, we found that doxorubicinol inhibits SERCA2a Ca2+ uptake into SR vesicles and that this was prevented by pretreatment with DTT. These results provide novel insight into the cellular mechanisms of anthracyclines. We suggest that by targeting multiple Ca2+ handling proteins in cardiac muscle, anthracyclines severely disturb cardiomyocyte Ca2+ homeostasis and that these effects have an important role in the onset of anthracycline-mediated arrhythmia and heart failure.