Anthracyclines are the critical component in a majority of pediatric chemotherapy regimens due to their broad anticancer efficacy. Unfortunately, the vast majority of long-term childhood cancer survivors will develop a chronic health condition caused by their successful treatments and severe cardiac disease is a common life-threatening outcome that is unequivocally linked to previous anthracycline exposure. The intricacies of how anthracyclines such as doxorubicin, damage the heart and initiate a disease process that progresses over multiple decades is not fully understood. One area left largely unstudied is the role of the cardiac fibroblast, a key cell type in cardiac maturation and injury response. In this study, we demonstrate the effect of doxorubicin on cardiac fibroblast function in the presence and absence of the critical DNA damage response protein p53. In wildtype cardiac fibroblasts, doxorubicin-induced damage correlated with decreased proliferation and migration, cell cycle arrest, and a dilated cardiomyopathy gene expression profile. Interestingly, these doxorubicin-induced changes were completely or partially restored in p53-/cardiac fibroblasts. Moreover, in wildtype cardiac fibroblasts, doxorubicin produced DNA damage and mitochondrial dysfunction, both of which are well-characterized cell stress responses induced by cytotoxic chemotherapy and varied forms of heart injury. A 3-fold increase in p53 (p = 0.004) prevented the completion of mitophagy (p = 0.032) through sequestration of Parkin. Interactions between p53 and Parkin increased in doxorubicin-treated cardiac fibroblasts (p = 0.0003). Finally, Parkin was unable to localize to the mitochondria in wildtype cardiac fibroblasts, but mitochondrial localization was restored in p53-/cardiac fibroblasts. These findings strongly suggest that cardiac fibroblasts are an important myocardial cell type that merits further study in the context of doxorubicin treatment. A more robust knowledge of the role cardiac fibroblasts play in the development of doxorubicin-induced cardiotoxicity will lead to novel clinical strategies that will improve the quality of life of cancer survivors.

Cardiac non-myocytes comprise a diverse and crucial cell population in the heart that plays dynamic roles in cardiac wound healing and growth. Non-myocytes broadly fall into four cell types: endothelium, fibroblasts, leukocytes, and pericytes. Here we characterize the diversity of the non-myocytes in vivo and in vitro using mass cytometry. By leveraging single-cell RNA sequencing we inform the design of a mass cytometry panel. To aid in annotation of the mass cytometry datasets, we utilize data integration with a neural network. We introduce approximately 460,000~ single cell proteomes of non-myocytes as well as 5,000~ CD31 negative single cell transcriptomes. Using our data, as well as previously reported datasets, we characterize cardiac non-myocytes with high depth in six mice, characterizing novel surface markers (CD9, CD200, Notch3, and FolR2). Further, we find that extended cell culture promotes the proliferation of CD45+CD11b+FolR2+IAIE-myeloid cells in addition to fibroblasts.

Childhood cancer survivors (CCSs) face lifelong side effects related to their treatment with chemotherapy. Anthracycline agents, such as doxorubicin (DOX), are important in the treatment of childhood cancers but are associated with cardiotoxicity. Cardiac toxicities represent a significant source of chronic disability that cancer survivors face; despite this, the chronic cardiotoxicity phenotype and how it relates to acute toxicity remains poorly defined., To address this critical knowledge gap, we studied the acute effect of DOX on murine cardiac non-myocytes in vivo. Determination of the acute cellular effects of DOX on non-myocytes, a cell pool with finite replicative capacity, provides a basis for understanding the pathogenesis of the chronic heart disease that CCSs face. To investigate the acute cellular effects of DOX, we present scRNAseq data from homeostatic cardiac non-myocytes and compare it to pre-existing datasets as well as a novel CyTOF datasets. SCANPY, a python-based single cell analysis, was used to assess the heterogeneity of cells detected in scRNAseq and CyTOF. To further assist in CyTOF data annotation, joint analyses of scRNAseq and CyTOF data using an artificial neural network known as sparse autoencoder for clustering, imputation, and embedding (SAUCIE) are performed. Lastly, the panel is tested on a mouse model of acute DOX exposure at two time points (24 and 72 hours) after the last dose of doxorubicin and examined with joint clustering. In sum, we report the first ever CyTOF study of cardiac non-myocytes and characterize the effect of acute DOX exposure with scRNAseq and CyTOF.