Abstract P5-18-12: Cardioprotective effects of walnuts against aromatase inhibitor: Pilot study in a zebrafish model
Authors: Hilakivi-Clarke Leena , Da Silva Joas Lucas , Blaes Anne , Hoeppner Luke 
Published: March 2022

BACKGROUND: Breast cancer patients on aromatase inhibitor (AI) are at a significantly higher risk of heart failure and cardiovascular disease (CVD) compared with patients on tamoxifen, and trend toward increased risks of myocardial infarction and ischemic stroke. In a clinical setting, AI has been shown to induce endothelial dysfunction that is a predictor of CVD. Further, patients on extended AI therapy exhibit significantly higher number of cardiovascular events when compared with patients who took AI for no longer than 5 years. AI effects on cardiovascular health may reflect the expression of functional estrogen receptors (ERs) in vascular endothelial and smooth muscle cells and cardiomyocytes, especially the membrane bound G-protein coupled ER (GPER). Activation of nuclear ER causes enhanced vasorelaxation and re-endovascularization, whilst GPER has been identified as a mediator of estrogen-induced vasodilation, either acting through nitric oxide (NO) or independent of it. GPER activation also protects against cholesterol-induced endothelial changes and endothelial inflammation. Since walnuts and walnut oil have many cardioprotective properties, we investigated if walnuts might prevent adverse effects of AI on a zebrafish model. Walnuts are rich in plant-derived n-3 PUFA alpha-linolenic acid (ALA).METHODS: Zebrafish embryos are used in cardiotoxicity studies (reduction of heartbeat) to model various human diseases. It has been shown that at 72 hours post fertilization (hpf) the primary and sprout vessels in the zebrafish embryo heart become active, and the blood vessel structure closely resembles of that in the human body. In our study, zebrafish embryos were treated with 4 mL of corn oil, soybean oil, docosahexaenoic acid (DHA) added to soybean oil, walnut oil or walnut powder added to walnut oil when they were 24 hpf. Oils were administered to yolk sac. 10-500 mM letrozole was added to water when fish were 48 hpf, and finally heart beat was measured from 72 hpf embryos. For the heart beat measurements, zebrafish embryos were anesthetized, and recorded to calculate the heart rate. After zebrafish was euthanized, RNA and protein were collected to assess changes in Esr2a, Esr2b, Gper1, Nos1, Nos2a, and Nos2b (shares properties with Nos3/eNOS). RESULTS: Administration of 500 mM letrozole significantly (p&lt;0.001) reduced the heartbeat in zebrafish, indicating that it impaired cardiac function. None of the oils significantly altered the heartbeat, compared with controls. However, both walnut oil (p&lt;0.006) and walnut powder enriched walnut oil (p&lt;0.001) increased heartbeat in letrozole treated zebrafish, compared with zebrafish only given letrozole. Thus, walnuts prevented letrozole induced reduction in heartbeat. RT-qPCR analysis indicated that letrozole significantly reduced Gper (p&lt;0.001) levels. Walnut oil prevented this change. Walnut oil also caused a significant reduction in Nos1 (p=0.014) levels during letrozole treatment. The effect of walnut powder on gene expression has not yet been studied.CONCLUSIONS: Although preliminary, our data suggest that walnuts might be cardioprotective during letrozole treatment. The protective effect may be mediated by prevention of letrozole-induced inhibition of GPER which acts as a vasodilator in the vasculature and cardiomyocytes. Future studies are planned to investigate if feeding adult zebrafish walnut powder and/or walnut oil has cardioprotective effects against chronic letrozole administration, whether GPER is the mediator of these effects, and if GPER acts through activating pathways such as EGFR, ERK, PI3K/Akt and cAMP/PKA. Further, the possibility that walnuts might either adversely or beneficially affect AIs’ effects on breast cancer will be studied.

Citation Format: Leena Hilakivi-Clarke, Joas Lucas Da Silva, Anne Blaes, Luke Hoeppner. Cardioprotective effects of walnuts against aromatase inhibitor: Pilot study in a zebrafish model [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-18-12.