ANALYSIS OF LIPID AND PROTEIN OXIDATION STATUS IN HEART FAILURE PATIENTS

Increasing body of evidence supports that oxidative stress is involved systolic and diastolic myocardial dysfunction in HF. Indeed, it has been well established an association between cardiac remodeling (hypertrophy, apoptosis or contractile dysfunction) and oxidative stress. Hence, the identification, quantification and fully characterization of oxidized biomolecules present in the plasma of HF patients can contribute to understand the mechanisms responsible for disease development but also as biological markers. However, the classic biomarkers of oxidative stress identified in HF do not provide thorough information but report about a general oxidative stress status. For a better understanding of the pathology of HF it is needed to unveil not only protein and lipid targets of oxidative damage but also the residues undergoing the modification within the protein and the structure of the oxidation products. Analysis of all of these oxPTMs and lipid peroxidation products by mass spectrometry is extremely challenging. Indeed, oxPTMs tend to occur randomly on a number of susceptible residues and proteins, making it extremely difficult to define all protein species. Along this project, plasma samples from HF patients belonging to different NYHA groups (from class I to class IV) have been employed to identify oxidized biomolecules as potential biomarkers of HF, and eventually subjected to several plasma prefractionation strategies combined with high-throughput untargeted and targeted mass spectrometry techniques. Consequently, part of this project has been focused on the study of oxidative modifications in human serum albumin (HSA), the most abundant protein in the circulatory system, associated with HF. HSA is involved in a wide range of biological functions and, due to its long half-life and high concentration in plasma, HSA is highly sensitive to undergo oxPTMs that may lead to its functional loss, thus contributing to the progression of HF. Taking advantage of the high abundance of HSA, we relatively quantified for the first time plasma levels of cysteinylated HSA (cys-HSA) and also levels of early glycated HSA (GA), and we observed a significant increase in HF patients with respect to the healthy subjects. A positive correlation between the levels of both isoforms and HF severity was highlighted whereas the abundance of total HSA showed a tendency to decrease when raising the severity degree of HF. For a deeper characterization of GA, we elucidated for the first time the early glycation pattern of HSA associated to HF by means of the newest generation of Tribrid MS instruments. Lys233 and lys525 were observed as the two most abundant glycated amino acids (79% and 13% respectively). Considering that further modifications of GA, such as rearrangement, oxidation, polymerization, and cleavage give rise to irreversible conjugates, called advanced glycation end products (AGEs), we also aimed to unveil the plasma advanced glycation pattern associated to HF. In this case, lys20, arg98, and lys402 emerged as the three most abundant carboxymethylated residues. Therefore, the results suggested that on one hand lys233 and lys525, and on the other lys20, arg98 and lys402, might represent the main potential therapeutic targets to reduce GA and AGE-HSA levels in HF patients, respectively. Besides the study of oxidized isoforms of HSA, we also tried to identify other less abundant advanced glycation and lipoxidation end products (AGEs and ALEs) in plasma by means of an enrichment strategy based on the ability of RAGE receptor to bind AGEs and ALEs. Due to the relevance of HSA in circulation, we have also evaluated the potential causal role of GA in the etiopathogenesis of HF. Hence, we pursued to evaluate the biological effects of GA on cardiac myocytes. Results highlighted that GA modulates in vitro the cardiomyocyte expression of inflammatory cytokines such as IL-6 or TNF-α. Furthermore, we reported that GA induces oxidative stress and oxidative modifications of a multitude of cellular proteins within cardiac myocytes. In addition, GA modulates the secretion of several cardiac proteins involved in response to stress biological processes. On the other hand, lipids play a crucial role in physiological processes and phospholipid disruption may participate in cardiovascular disease events, therefore the phospholipidome profiling has emerged as a powerful tool to explore novel biomarkers and mechanisms in several pathologies. Hence, we have also studied for the first time the plasma phospholipidome of HF patients. In this pilot study, we have applied both untargeted and targeted (MRM) strategies together with fractionating methods to separate the phospholipid content from the rest of plasma components, in order to decrease the heterogeneity and complexity of this biological matrix. The results suggested that HF plasma phospholipid signatures were gender-specific. In the case of females, several PE species were more abundant in HF than in the control group. On the contrary, male HF patients mainly showed a higher abundance of LPC species when compared to the male control group. Additionally, lipidomic approaches (LC-MS/MS and PRM) have been carried out to define for the first time a circulating oxidative lipid pattern associated with HF. In this case, we have focused on long- and short-chain products of lipid peroxidation since their identification and characterization in the pathology of HF were still lacking. Promising results have been pinpointed so far: we have detected 9 lipid peroxidation products (full-chain oxidized lipids together with fragmented species). In conclusion, the results gathered along this Ph.D. project provide evidence at the molecular level of the mechanisms associated to oxidative stress and oxidative damage underlying the development and progression of HF, thus contributing to a better understanding of the pathology. Indeed, the elucidation of specific modified residues within key circulating proteins such as HSA, or the characterization of long- and fragmented-chain lipid peroxidation products present in HF plasma samples reported in this thesis may pave the way to new therapies to reduce and treat HF. We have also contributed to expand the awareness of the biological effects of GA in the cardiac context. Additionally, this project has pointed out a gender-dependent adaptation of the plasma phospholipidome occurring in the pathology of HF, highlighting that gender is a major and often underestimated factor that should be carefully considered when screening lipidomes or proteomes of pathological processes. Finally, we have demonstrated that either proteomic or lipidomic technologies can provide deep biological insight into human health and disease.