Recent work has shown pre-incubation with nanomolar concentrations of anthracyclines inhibits caffeine- 'induced calcium release from canine cardiac sarcoplasmic reticulum (SR) vesicles (Olson, et al., Tox. Appl. Pharm., 2000, 169, 168-176) This inhibition is important to understand because it may contribute to the clinical cardiotoxicity of anthracyclines. The inhibitory effects are hypothesized to be a result of a direct interaction between anthracyclines and the SR calcium binding protein, calsequestrin, which results in a drug-dependent disruption of proper calsequestrin function. This proposal is designed to probe the mechanism of inhibition, with particular attention to the roles of calsequestrin and the ryanodine receptor calcium release channel. Comparison of the anthracyclines daunorubicin, daunorubicinol and 5-iminodaunorubicin, which vary in their ability to inhibit calcium release, will allow the importance of various structural moieties for inhibition to be explored. The proposed research consists of four specific aims: 1) to study calcium-dependent conformational changes in calsequestrin in the presence of anthracyclines using fluorescence spectroscopy; 2) to investigate whether anthracyclines alter the aggregation behavior of calsequestrin using flow-field flow fractionation and multi-angle light scattering detection (flow-FFF/MALS); 3) to probe the ability of anthracyclines to inhibit calcium binding to calsequestrin using Ca-45 competitive binding assays; 4) to determine the conformation of the cardiac ryanodine receptor (RyR2) in isolated SR vesicles after preincubation with anthracyclines; ryanodine binding assays will be used. The proposed experiments will determine the ability of anthracyclines to interfere with normal calsequestrin function and will define the contribution of RyR2 channel gating to inhibition of SR calcium release. The results will provide insight into the details of molecular mechanisms involved in disruption of proper cardiac calcium handling by anthracychnes and will thus inform the design of improved anthracyclines with decreased cardiotoxicity.
| Charlier Jr, Henry A; Olson, Richard D; Thornock, Carissa M et al. (2005) Investigations of calsequestrin as a target for anthracyclines: comparison of functional effects of daunorubicin, daunorubicinol, and trifluoperazine. Mol Pharmacol 67:1505-12 |
