Anthraquinone-sensitized Ca²⁺ release channel from rat cardiac sarcoplasmic reticulum: Possible receptor-mediated mechanism of doxorubicin cardiomyopathy

Rat cardiac membrane vesicles enriched in biochemical markers of the junctional region of sarcoplasmic reticulum (SR) and exhibiting ruthenium red-sensitive rapid Ca²⁺ release have been prepared. Doxorubicin and seven congeners are shown to enhance the binding of [³H]ryanodine to the ryanodine receptor with a strong structural requirement. Doxorubicin enhances the binding of [³H]ryanodine to SR membranes and soluble receptor preparations and induces Ca²⁺ release from SR vesicles in a highly Ca²⁺-dependent manner, suggesting that anthraquinones promote the open state of the junctional Ca²⁺ release channel by increasing the affinity of the Ca²⁺ activator site for Ca²⁺. Doxorubicin reduces the K_d of [³H]ryanodine binding solely by enhancing the rate of association. Caffeine competes for the same site with anthraquinones, because the caffeine-activated binding of [³H]ryanodine is inhibited by doxorubicin and vice versa. The acute effect of doxorubicin on the cardiac Ca²⁺ release channel is fully reversible; however, long term treatment (up to 24 hr) with doxorubicin increases the sensitivity of the preparation to subsequent acute challenge with doxorubicin. The thiol-reductive agent dithiothreitol enhances, whereas the reactive disulfide 4,4′-dithiodipyridine reduces, the doxorubicin-enhanced binding of [³H]ryanodine. These results demonstrate that the acute and chronic cardiotoxicity of anthraquinones may be accounted for by a receptor-mediated mechanism. Our findings suggest that the chronic effects observed with the clinical use of anthraquinones may be the result of a receptor-mediated shift in the redox equilibrium of allosteric thiols at the ryanodine receptor complex, which in turn leads to long term sensitization of the Ca²⁺ release channel.