Mechanisms of anthracycline-induced dysfunction of ca2+ handling proteins in the heart
Creators: name: Hanna, Amy
Publisher: name: The Australian National University
Year: 2014
Subjects: subject: Heart Diseases
subject: Anthracyclines
subject: Doxorubicin
subject: Ryanodine Receptors
subject: Calcium-binding proteins

description: Anthracyclines, such as doxorubicin and daunorubicin, are powerful chemotherapy agents whose use is limited due to the onset of potentially fatal cardiac side effects which include arrhythmogenesis and heart failure. Several proteins important in intracellular Ca2+ signalling have been identified as drug binding targets, including the ryanodine receptor Ca2+ release channel (RyR2), the Ca2+ binding protein calsequestrin (CSQ2) and the Sarco/Endoplasmic Reticulum Ca-ATPase (SERCA2A). The drug metabolites are believed to be important in the devastating cardiac effects of anthracyclines but their actions have been poorly characterized. Previous work showed that daunorubicin modulates RyR2 and that its effects were attributable to ligand binding and thiol oxidation. 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. Anthracyclines caused biphasic modulation of RyR2 where there was an increase in channel activity followed by an inhibitory phase. RyR2 channel activation, but not inhibition, could be reversed by drug washout, typical of a ligand binding effect. This was supported by affinity chromatography experiments showing that doxorubicin and doxorubicinol bind to RyR2. Conversely, the irreversible nature of the inhibitory effect suggested a non-ligand binding effect. Treatment with anthracyclines reduced the number of thiols on RyR2, indicative of a drug-induced thiol-modification such as oxidation. Together, these results support the earlier hypothesis that activation of RyR2 by anthracyclines is due to ligand binding, while the inhibitory effect is due to direct thiol oxidation. In addition to modulating RyR2, doxorubicinol was found to alter other aspects of SR Ca2+ handling. For the first time, the effect of doxorubicinol on the luminal Ca2+ sensitivity of RyR2 channels has been assessed. Doxorubicinol abolished the response of RyR2 to changes in luminal Ca2+. Additional experiments revealed that the abolition of luminal Ca2+ sensing was due to an interaction between doxorubicinol and CSQ2. Furthermore, in SR vesicles, a decrease in the Ca2+ uptake rate showed that doxorubicinol inhibits the function of SERCA2A. This effect could be prevented by pre-treatment with the thiol protective agent dithiothreitol, indicating that doxorubicinol's inhibition of SERCA2A was due to thiol oxidation. Hence doxorubicinol causes substantial dysfunction of SR Ca2+ handling proteins, affecting both Ca2+ release and Ca2+ uptake pathways. To determine the effects of doxorubicinol in an intact cell, cardiomyocytes were isolated from adult mouse hearts and loaded with the Ca2+ indicator Fluo-4. Pre-treatment with doxorubicinol reduced cytoplasmic Ca2+ transients, depleted SR load and inhibited SERCA2A and the Na+- Ca2+ exchanger. Furthermore, doxorubicinol-treated myocytes exhibited more spontaneous Ca2+ release events and had a higher resting Ca2+ concentration. These effects resulted in an overall impairment in contractile function. This project provides novel insight into cellular mechanisms of anthracyclines and is the most thorough characterization of the effects of these drugs on cardiomyocyte Ca2+ handling to date. The results suggest that by targeting multiple Ca2+ handling proteins, anthracyclines severely disturb cardiomyocyte Ca2+ homeostasis and that these effects may have an important role in the onset of anthracycline-mediated arrhythmia and heart failure.