An Extensive Metabolomics Workflow to Discover Cardiotoxin-Induced Molecular Perturbations in Microtissues.

Tara J Bowen,Andrew R Hall,Amy Pointon,Gavin R Lloyd,Ralf J M Weber,Mark R Viant,Amanda Wilson

doi:10.3390/metabo11090644

Tara J Bowen, Andrew R Hall + Show 5 more

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https://doi.org/10.3390/metabo11090644

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Abstract

Discovering modes of action and predictive biomarkers of drug-induced structural cardiotoxicity offers the potential to improve cardiac safety assessment of lead compounds and enhance preclinical to clinical translation during drug development. Cardiac microtissues are a promising, physiologically relevant, in vitro model, each composed of ca. 500 cells. While untargeted metabolomics is capable of generating hypotheses on toxicological modes of action and discovering metabolic biomarkers, applying this technology to low-biomass microtissues in suspension is experimentally challenging. Thus, we first evaluated a filtration-based approach for harvesting microtissues and assessed the sensitivity and reproducibility of nanoelectrospray direct infusion mass spectrometry (nESI-DIMS) measurements of intracellular extracts, revealing samples consisting of 28 pooled microtissues, harvested by filtration, are suitable for profiling the intracellular metabolome and lipidome. Subsequently, an extensive workflow combining nESI-DIMS untargeted metabolomics and lipidomics of intracellular extracts with ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) analysis of spent culture medium, to profile the metabolic footprint and quantify drug exposure concentrations, was implemented. Using the synthetic drug and model cardiotoxin sunitinib, time-resolved metabolic and lipid perturbations in cardiac microtissues were investigated, providing valuable data for generating hypotheses on toxicological modes of action and identifying putative biomarkers such as disruption of purine metabolism and perturbation of polyunsaturated fatty acid levels.

Highlights

Drug-induced cardiotoxicity is a major cause of attrition during preclinical and clinical drug development and post-approval withdrawal of medicines [1,2,3,4]
Comparing the time scales for the two sampling approaches, the filtration method required ca. 60 s from collecting the microtissues from their culture plate through to the quenching of metabolism, while the centrifugation method required over 10 min, opening the potential for significant and unwanted metabolic changes
Measurements of the metabolome and lipidome revealed that somewhat more consistent intracellular metabolic and lipidomic signatures can be obtained when the microtissues are sampled rapidly via filtration (Figure 1). This finding is further evidenced by the lower median relative standard deviation; an established metric for describing the variance of metabolic features [34], measured across filtered, vs. centrifuged, biological replicates (Table 1)

Summary

Introduction

Drug-induced cardiotoxicity is a major cause of attrition during preclinical and clinical drug development and post-approval withdrawal of medicines [1,2,3,4]. The recent development and use of more complex in vitro models encompassing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in 3D co-cultures alongside fibroblasts and endothelial cells, which together represent the majority of non-cardiomyocytes in the heart, has enhanced the specificity and sensitivity of assays which aim to predict the cardiac safety risk of screened compounds [12,17]. These spontaneously beating 3D co-cultures, composed of approximately 500 cells, are termed cardiac microtissues [17]

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