Poster T17 - Identification of cardioprotective strategies to prevent anthracycline-induced cardiotoxicity using an engineered human cardiac microtissue model

Introduction: Advances in anticancer therapy continue to improve patient outcomes, yet treatment-induced cardiotoxicities remain a major challenge to preserving the long-term health of cancer survivors. The anthracycline doxorubicin is used in combination with newer therapeutics to treat both solid tumors and hematologic malignancies in up to 70% of cancer patients, but the cumulative dose is directly correlated to increased risk of heart failure. While limits are placed on total exposure to doxorubicin, reducing the efficacy of treatment, approximately 5% of doxorubicin-treated patients will still develop heart failure. Current clinical practice relies on monitoring cardiovascular health during and after treatment and the initiation of conventional heart failure regimens (i.e., statins, ACE inhibitors, and beta-blockers) after the discovery of reduced cardiac function. However, these treatments are not curative and delays in detection or treatment result in substantially worse outcomes. Thus, therapeutic strategies for preserving cardiac function during anthracycline treatment are needed to maintain the longevity and quality of life of cancer survivors. The ACE inhibitor enalapril, an existing heart failure drug, has been proposed as one potential cardioprotective compound, but its efficacy as a concurrent therapy remains incomplete. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a readily accessible cell type that have been shown to recapitulate multiple aspects of human cardiac physiology and pathophysiology in vitro, including doxorubicin-induced cardiotoxicity. Leveraging our existing hiPSC-CM-based three-dimensional human cardiac microtissue model, we examined doxorubicin toxicity and enalapril cardioprotection via multiple functional cardiac endpoints to develop a novel framework by which to proactively reduced anthracycline-induced cardiotoxicity.

Materials and

Methods: Using a method previously established in our lab, self-assembling, engineered cardiac microtissues were formed by combining metabolically purified human induced-pluripotent stem cell-derived ventricular cardiomyocytes (hiPSC-vCMs) and primary cardiac fibroblasts in round-bottom agarose microwells in a 95:5 ratio. After one week in culture, microtissues were treated with either doxorubicin alone or a DMSO vehicle (negative control) for an additional three days prior to downstream analysis. For cardioprotection experiments, microtissues were treated with a combination of 1 or 3 µM doxorubicin and 5 or 10 µM enalapril over the same three-day period. Doxorubicin toxicity was first established by measuring concentration-dependent changes in viability via the metabolic MTT assay, functional contractile changes via optical tracking, and metabolism via Seahorse Assay. Enalapril cardioprotection was examined by measuring cell death via fluorescent LIVE/DEAD staining and contractility assessment via optical tracking. Because the generation of reactive oxygen species (ROS) is a one mechanism of doxorubicin-induced cardiotoxicity, mitochondrial ROS production was measured via the fluorescence intensity of MitoSOX.

Results, Conclusions, and Discussions: Doxorubicin cardiotoxicity was confirmed in our engineered cardiac tissue model due to concentration-dependent changes in cell viability (Figure 1A), contraction (Figure 1B), and metabolism (Figure 1C) after three days of doxorubicin exposure. We observed significant reductions in cell viability and contractility compared to vehicle controls at higher doses (>1 µM) of doxorubicin and at 0.1 µM in metabolism. Higher concentrations (1 and 3 µM) were used for subsequent cardioprotection experiments. Accordingly, quantification of fluorescent LIVE/DEAD staining revealed significant increases in cell death in doxorubicin-treated microtissues compared to vehicle controls at both 1 and 3 µM (Figure 1D). When microtissues were co-treated with enalapril, dead stain intensity was reduced, suggesting that enalapril reduces doxorubicin-induced cell death. These observations were mirrored by our functional contractility measurements (Figure 1E). Microtissues treated with doxorubicin alone had significantly reduced function compared to vehicle controls while co-treatment with 10 µM enalapril ameliorated deficits in contractility (displacement). We further hypothesized that co-treatment with 10 µM enalapril would cause a reduction in ROS generation, resulting in the observed cardioprotection. Interestingly, however, MitoSOX intensity did not differ between doxorubicin-only and enalapril co-treated microtissues (Figure 1F), suggesting that enalapril cardioprotection may be independent of ROS generation. Together, our results demonstrate that co-treatment with the ACE inhibitor enalapril may offer a viable strategy with which to prevent the initiation of the anthracycline-induced cardiotoxicity that often leads to heart failure in cancer survivors. Ongoing studies will aim to identify the specific mechanism by which enalapril prevents cardiotoxicity, informing future drug and therapy development to advance anticancer therapies.

Acknowledgements (Optional):