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

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.

A Strategy for Uncovering the Serum Metabolome by Direct-Infusion High-Resolution Mass Spectrometry

“…In metabolomics analysis, nanoelectrospray ionization (nESI) DI-HRMS provides similar discrimination capabilities to conventional liquid chromatography-mass spectrometry (LC-MS) while greatly reducing the total analysis time [ 3 , 4 ]. DI-HRMS approach has been widely applied in various metabolomics studies [ 5 , 6 , 7 ], such as urinary metabolic profiling in human epidemiological investigations [ 8 ], metabolite analysis for the diagnosis of inborn errors of metabolism [ 9 , 10 , 11 ], and serum metabolomics analysis in diabetes [ 12 ].…”