Mass Spectrometry-Based Redox and Protein Profiling of Failing Human Hearts

Abstract

Oxidative stress contributes to detrimental functional decline of the myocardium, leading to the impairment of the antioxidative defense, dysregulation of redox signaling, and protein damage. In order to precisely dissect the changes of the myocardial redox state correlated with oxidative stress and heart failure, we subjected left-ventricular tissue specimens collected from control or failing human hearts to comprehensive mass spectrometry-based redox and quantitative proteomics, as well as glutathione status analyses. As a result, we report that failing hearts have lower glutathione to glutathione disulfide ratios and increased oxidation of a number of different proteins, including constituents of the contractile machinery as well as glycolytic enzymes. Furthermore, quantitative proteomics of failing hearts revealed a higher abundance of proteins responsible for extracellular matrix remodeling and reduced abundance of several ion transporters, corroborating contractile impairment. Similar effects were recapitulated by an in vitro cell culture model under a controlled oxygen atmosphere. Together, this study provides to our knowledge the most comprehensive report integrating analyses of protein abundance and global and peptide-level redox state in end-stage failing human hearts as well as oxygen-dependent redox and global proteome profiles of cultured human cardiomyocytes.

Reversible thiol oxidation increases mitochondrial electron transport complex enzyme activity but not respiration in cardiomyocytes from patients with end-stage heart failure

“…A key feature of and contributing factor to myocardial dysfunction in HFrEF is excessive accumulation of reactive oxygen species (ROS). Markers of an oxidized shift in the cellular redox tone have been observed in ventricular biopsies from patients with HFrEF [14,25,33]. The mitochondria are a prominent source of increased ROS production and, by proximity, a target of redox reactions.…”

“…While there are reports of decreased mitochondrial content that contribute to energetic insufficiency [5], Sheeran et al [25] argue that mitochondrial dysfunction stems from oxidized post-translational modifications that impair enzyme and organelle function. They report increased markers of oxidation on subunits of complexes I, IV, and V of the ETS that correspond to decreased enzyme activity [25], while redox proteomics in ventricular biopsies also observed increased oxidation of subunits associated with complexes I and II [14]. Specifically, five subunits in Complex I showed increased markers of oxidation (carbonylation and nitrotyrosine) and all subunits contain Fe-S clusters that facilitate the transfer of electrons.…”

“…Two cysteines on the 75 kDa subunit of complex I (NDUSFI) (Cys 531 and Cys 704 ) are particularly sensitive to redox reactions that can impair enzyme function [37][38][39]. While specific cysteines were not investigated, the NDUSFI subunit experienced significant oxidative shifts in myocardial biopsies from patients with HFrEF compared to controls [14]. It follows that redox reactions on these residues may relate to impaired function that can be targeted with a reducing agent.…”

“…Mitochondria are considered a major source of increased oxidants in the failing heart and, by proximity, are also a primary target for redox reactions [12,13]. Redox proteomics in left ventricle biopsies from patients with end-stage HFrEF reveal increases in reversibly oxidized cysteine residues in proteins of complexes I and II of the electron transport system (ETS) in cardiac mitochondria [14]. Reversible oxidation of thiol moieties causes mitochondrial dysfunction in cardiomyocytes from mice fed a high fat, high sucrose diet [15] and from rats treated with doxorubicin [16].…”

Mitochondrial Bioenergetics and Dynamism in the Failing Heart

“…Cardiac metabolism maintains a dynamic state of equilibrium for efficient energy transfer and is a highly concerted plethora of chemical reactions leading to the conversion of ATP both to sustain cell function and allow contraction, growth, repair, and regeneration. The metabolic alterations in the failing myocardium have been explored; from many points of view [ 23 ], HF is considered a return to fetal stages due to the shift from FAO-based metabolism (which mainly manifests as a 35% decrease in the ATP concentration and changes in substrate utilization) to glycolysis (the main active pathway during the fetal period) accompanied by progressive degeneration of the myocardium [ 24 , 25 ] ( Figure 1 ). Accordingly, cardiac metabolism in HF was recognized as a field of active research for well over a century [ 26 ] and will be discussed in the first part of this review.…”