Background: Doxorubicin (Dox), an anticancer drug, is known to induce cardiac toxicity by causing mitochondrial dysfunction. Although CaMKII and its phosphorylation targets, Drp1 to control mitochondrial fission and MCU to control mitochondrial Ca²+ uptake, regulate mitochondrial homeostasis, the involvement of these molecules in the Dox-induced mitochondrial dysfunction remains unclear.
Method: To study the effects of Dox on mitochondrial homeostasis, we evaluated mitochondrial membrane potential (MMP), mitophagy, and mitochondrial Ca²+ content ([Ca²+]m) in H9C2 cells with the following fluorescent dyes, JC-1, Mtphagy, and Rhod2-AM, respectively. To examine the activating effect of Dox on CaMKII, we evaluated the phosphorylation levels of CaMKII by western blotting. To test the involvement of CaMKII, Drp1, and MCU in the Dox-induced mitochondrial dysfunction, the specific inhibitors, KN-93, Mdivi-1, and Ru360, respectively, were used.
Result: Dox treatment dose-dependently reduced MMP and increased the number of cells with mitophagy and [Ca²+]m (p<0.05 in all). Dox treatment significantly increased the phosphorylation levels of CaMKII (p<0.05). The inhibition of CaMKII suppressed the effects of Dox on the MMP and the mitophagy (p<0.05), but not on [Ca2+]m. Contrarily, the inhibition of Drp1 and MCU failed to suppress the decrease in MMP by Dox. Similarly, the inhibition of Drp1 did not reverse the increase in mitophagy by Dox, nor did the inhibition of MCU suppress the elevation of [Ca²+]m by Dox.
Conclusion: These results suggested that activated CaMKII, but not Drp1 and MCU, is involved in the impairment of MMP leading to Dox-induced mitochondrial dysfunction and that the excessive fission by Drp1 and the increased uptake [Ca²+]m by MCU are not the mechanism for the Dox-induced MMP reduction.