An extensive metabolomics workflow to discover cardiotoxin-induced molecular perturbations in microtissues

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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

32 Downloads (Pure)

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.

Original languageEnglish
Article number644
JournalMetabolites
Volume11
Issue number9
DOIs
Publication statusPublished - 21 Sep 2021

Bibliographical note

Funding Information:
This research was funded by UK Research and Innovation Biotechnology and Biological Sciences Research Council (BBSRC), grant number BB/S507064/1, and AstraZeneca, via iCASE PhD studentship to T.J.B. We thank Thermo Fisher Scientific for their support of this project, including the application of the Orbitrap ID-X Tribrid mass spectrometer, via the University of Birmingham Thermo Fisher Scientific Technology Alliance Partnership.

Keywords

  • Biomarkers
  • Cardiac microtissues
  • Cardiotoxicity
  • In vitro metabolomics
  • Mode of action
  • Sample harvesting
  • Sensitivity
  • Untargeted toxicokinetics
  • mode of action
  • sample harvesting
  • biomarkers
  • untargeted toxicokinetics
  • cardiotoxicity
  • in vitro metabolomics
  • sensitivity
  • cardiac microtissues

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry
  • Endocrinology, Diabetes and Metabolism

Fingerprint

Dive into the research topics of 'An extensive metabolomics workflow to discover cardiotoxin-induced molecular perturbations in microtissues'. Together they form a unique fingerprint.

Cite this