Comprehending Cardiac Dysfunction by Oxidative Stress: Untargeted Metabolomics of In Vitro Samples

Alan Gonçalves Amaral; Isabela Aparecida Moretto; Flávia da Silva Zandonadi; Hans Rolando Zamora-Obando; Isabela Rocha; Alessandra Sussulini; André Alexandre de Thomaz; Regina Vincenzi Oliveira; Aline Mara Dos Santos; Ana Valéria Colnaghi Simionato

doi:10.3389/fchem.2022.836478

Comprehending Cardiac Dysfunction by Oxidative Stress: Untargeted Metabolomics of In Vitro Samples

Front Chem. 2022 Apr 8:10:836478. doi: 10.3389/fchem.2022.836478. eCollection 2022.

Affiliations

¹ Laboratory of Analysis of Biomolecules Tiselius, Department of Analytical Chemistry, Institute of Chemistry, State University of Campinas, Campinas, Brazil.
² Institute of Biology, Department of Structural and Functional Biology, State University of Campinas, Campinas, Brazil.
³ Laboratory of Bioanalytics and Integrated Omics, Department of Analytical Chemistry, Institute of Chemistry, State University of Campinas, Campinas, Brazil.
⁴ National Institute of Science and Technology for Bioanalytics-INCTBio, Institute of Chemistry, State University of Campinas, Campinas, Brazil.
⁵ Institute of Physics "Gleb Wataghin", State University of Campinas, Campinas, Brazil.
⁶ Department of Chemistry, Federal University of São Carlos, São Carlos, Brazil.

Abstract

Cardiovascular diseases (CVDs) are noncommunicable diseases known for their complex etiology and high mortality rate. Oxidative stress (OS), a condition in which the release of free radical exceeds endogenous antioxidant capacity, is pivotal in CVC, such as myocardial infarction, ischemia/reperfusion, and heart failure. Due to the lack of information about the implications of OS on cardiovascular conditions, several methodologies have been applied to investigate the causes and consequences, and to find new ways of diagnosis and treatment as well. In the present study, cardiac dysfunction was evaluated by analyzing cells' alterations with untargeted metabolomics, after simulation of an oxidative stress condition using hydrogen peroxide (H₂O₂) in H9c2 myocytes. Optimizations of H₂O₂ concentration, cell exposure, and cell recovery times were performed through MTT assays. Intracellular metabolites were analyzed right after the oxidative stress (oxidative stress group) and after 48 h of cell recovery (recovery group) by ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) in positive and negative ESI ionization mode. Significant alterations were found in pathways such as "alanine, aspartate and glutamate metabolism", "glycolysis", and "glutathione metabolism", mostly with increased metabolites (upregulated). Furthermore, our results indicated that the LC-MS method is effective for studying metabolism in cardiomyocytes and generated excellent fit (R²Y > 0.987) and predictability (Q² > 0.84) values.

Keywords: LC-MS; cardiovascular diseases; cell culture; metabolomics; oxidative stress.