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

Abstract

Anthracyclines such as doxorubicin (DOX) 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, previous studies show anthracyclines perturb aspects of excitation-contraction coupling (ECC), though to our knowledge this has yet to be demonstrated in a large animal model. Furthermore, the extent to which altered ECC is dependent on anthracycline-induced reactive oxygen species (ROS) production / oxidative stress remains ambiguous. This is important given the key role of oxidative stress in other cardiotoxic processes. Therefore, we aimed to (1) characterise the effects of DOX on Ca handling and contractility in sheep ventricular myocytes and (2) quantify the dependence of any effect on oxidative stress.Sheep myocytes were enzymatically isolated in accordance with the Animals (Scientific Procedures) Act, UK, 1986. Cells were loaded with fura-2 (0.1 μM) then field stimulated at 0.5 Hz. Fura-2 was excited sequentially at 340 nm and 380 nm (200 Hz) and changes of cytoplasmic Ca inferred from the ratio of emitted light. Sarcomere length was measured simultaneously using a Myocam-S CCD camera and SarcLen acquisition module (Ion Optix, USA). Changes to sarcoplasmic reticulum (SR) Ca content were estimated by rapid application of caffeine (10 mM). Two-way ANOVA was used for statistical analysis.1 nM DOX reduced the amplitude of systolic Ca and shortening by 39 ± 3 % (n=49, p<0.001) and 42 ± 4 % (n=45, p<0.001) respectively. The amplitude of the caffeine-evoked Ca transient decreased by 33 ± 5 % (n=24, p<0.001). The rate of decay of the systolic Ca transient ( k sys ) represents the combined activity of SERCA and NCX and was reduced by 17 ± 3 % (n=46, p<0.001). The rate of decay of the caffeine-evoked Ca transient ( k caff ) represents NCX alone and was increased by 64 ± 26 % (n=23, p=0.002).Subtraction of k caff from k sys indicates SERCA activity ( k SERCA ), which was decreased by 34 ± 8 % (n=20, p<0.001). When the experiment was repeated in the presence of the antioxidant N-acetylcysteine (NAC), the effect of DOX on systolic Ca, systolic shortening and the caffeine-evoked Ca transient was attenuated by 52 % (n=29, p<0.001), 58 % (n=29, p<0.001) and 45 % (n=15, p=0.003) respectively. Furthermore, NAC prevented the effect of DOX on k sys , k caff and k SERCA .These data suggest DOX increases NCX activity and reduces SERCA activity which decreases SR Ca content thence systolic Ca and contractility. The effect of DOX on NCX and SERCA appears to be entirely ROS / oxidative stress dependent. However, other non-oxidative mechanisms likely contribute to net DOX-mediated negative cellular inotropy. Our next experiments will investigate this and the sources of anthracycline-induced oxidative stress. This work was funded by Kidscan childrens cancer research, UK. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.