The effects of anthracyclines on calcium handling and contractility in sheep ventricular myocytes; role of oxidative stress

Anthracyclines such as doxorubicin (DOX) and daunorubicin (DAUN) are effective chemotherapeutics and contribute to improved cancer survival rates in children and adults. However, anthracyclines exhibit acute and chronic cardiotoxicity which can produce heart failure in cancer survivors. While the cellular basis remains unclear, limited previous studies show DOX perturbs certain aspects of excitation-contraction coupling and increases production of reactive oxygen species (ROS). Fewer studies have investigated the effects of DAUN and the effects of either anthracycline on excitation-contraction coupling (ECC) in a large animal model has yet to be demonstrated. Furthermore, the extent to which altered ECC is dependent on anthracycline-induced ROS production remains ambiguous. This is compounded by the fact that no studies have investigated whether elevated ROS production produces oxidative stress in cardiac myocytes. To address these gaps in our understanding, we performed the first integrative investigation of the effects of DOX and DAUN in sheep ventricular myocytes. We also measured the effect of DOX and DAUN on oxidative stress in these cells and further elucidated the underlying sources of ROS. Furthermore, we investigated the dependence of perturbed ECC on ROS thence oxidative stress elevations.Sheep ventricular myocytes were enzymatically isolated in accordance with the Animals (Scientific Procedures) Act, UK, 1986 and used for all experiments. Intracellular calcium and contractility dynamics were measured using epi-fluorescent photometry and video sarcomere detection simultaneously. Cells were field stimulated at 0.5 Hz then acutely exposed to 1 nM DOX or DAUN. Rapid application of 10 mM caffeine was used to measure SR Ca content. Oxidative stress was measured using CellROX red. Fluorescent images were captured using the cytation imaging system and cell fluorescence determined using ImageJ software. DOX reduced the activity of SERCA and increased the activity of NCX resulting in a reduction in SR Ca content. DAUN also reduced SR Ca content however due to an interaction with caffeine the mechanism could not be fully elucidated. The decrease in SR Ca content accounted for a decrease in systolic Ca which underpinned a decrease in systolic shortening. Both DOX and DAUN increased myofilament sensitivity to Ca, potentially offsetting the effect on contractility. DOX increased oxidative stress in a concentration and time-dependent manner. DAUN also increased oxidative stress, but only at relatively high concentrations (10 mM). Removal of oxidative stress by n-acetylcysteine (NAC) attenuated the effects of DOX on the majority of Ca handling and contractility parameters. For example, the effect of DOX on SR Ca content and Ca transient amplitude were reduced by approximately 50 %. Inhibition of the ROS producing enzymes NADPH oxidase (NOX) and xanthine oxidase (XO) reduced DOX-mediated oxidative stress by ~50 % and ~20 % respectively and attenuated the effects on ECC.In cells from sheep with heart failure, DOX reduced SR Ca content thence systolic Ca and contractility but had no effect on SERCA and NCX. These findings suggest that in a large animal model, DOX and DAUN decrease SR Ca content leading to a reduction in systolic Ca thence contractility. In the case of DOX, decreased SERCA and increased NCX activity likely contribute to the decrease of SR Ca content. However, that this isn’t the case in heart failure suggests a role for other mechanisms. These findings are also the first to show that DOX and DAUN increase intracellular oxidative stress in the heart and that NOX and XO are key enzymatic sources of ROS. Furthermore, these findings show this increase in oxidative stress is pathologically important as it accounts for approximately half of the effects of DOX on ECC. Collectively, these findings further elucidate the effects of anthracyclines on ECC and make important contributions to the understanding of the cellular basis of anthracycline-mediated cardiotoxicity. Furthermore, the dependence on and sources of oxidative stress reveal clinically relevant therapeutic targets