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Doxorubicin directly binds to the cardiac-type ryanodine receptor
Kazuhiko Saeki1, Ichiro Obi, Noriko Ogiku
1Discovery Research Laboratory, Tanabe Seiyaku Co, Saitama, Japan. kazu@tanabe.co.jp
Abstract:
The clinical use of doxorubicin, an antineoplasmic agent, is limited by its extensive cardiotoxicity which is mediated by the mobilization of intracellular Ca2+ from SR. In order to elucidate the mechanism of Ca2+ release, we analyzed the binding sites of doxorubicin on rabbit cardiac SR (sarcoplasmic reticulum). One of the binding sites was identified as cardiac-type ryanodine receptor (RyR2) which was purified by immunoprecipitation from solubilized cardiac SR in the presence of DTT. Ligand blot analysis revealed the direct binding of doxorubicin to RyR2. The binding of doxorubicin to RyR2 was specific and displaced by caffeine. Both doxorubicin and caffeine enhanced [3H]-ryanodine binding to RyR2 in a Ca2+ dependent manner. These results suggest that there is a doxorubicin binding site on RyR2.
Insights
Doxorubicin, an antineoplastic drug, causes cardiotoxicity by affecting intracellular calcium release. This study identifies a direct binding site for doxorubicin on the cardiac ryanodine receptor (RyR2), a key protein in calcium handling.
Area of Science:
- Cardiovascular Pharmacology
- Molecular Cardiology
- Cancer Therapeutics
Background:
- Doxorubicin is a vital antineoplastic agent.
- Its clinical utility is significantly limited by dose-dependent cardiotoxicity.
- This cardiotoxicity is associated with intracellular calcium (Ca2+) dysregulation, specifically release from the sarcoplasmic reticulum (SR).
Purpose of the Study:
- To investigate the molecular mechanisms underlying doxorubicin-induced cardiotoxicity.
- To identify specific binding sites of doxorubicin within cardiac sarcoplasmic reticulum (SR).
Main Methods:
- Purification of cardiac-type ryanodine receptor (RyR2) from rabbit cardiac SR using immunoprecipitation.
- Ligand blot analysis to assess direct doxorubicin binding to purified RyR2.
- Assessment of doxorubicin and caffeine's effect on [3H]-ryanodine binding to RyR2 in a Ca2+-dependent manner.
Main Results:
- Doxorubicin was found to bind directly and specifically to the cardiac ryanodine receptor (RyR2).
- Caffeine, a known modulator of RyR2, displaced doxorubicin binding.
- Both doxorubicin and caffeine enhanced Ca2+-dependent [3H]-ryanodine binding to RyR2.
Conclusions:
- The cardiac ryanodine receptor (RyR2) represents a direct binding site for doxorubicin in cardiac cells.
- This interaction suggests RyR2 as a potential mediator of doxorubicin-induced cardiotoxicity through modulation of Ca2+ release.
- Understanding this interaction may lead to strategies for mitigating doxorubicin cardiotoxicity.