Cardiovascular diseases (CVD) are the leading cause of death and absenteeism in the Western world, generating a considerable economic burden on treatment and prevention.Currently there are increasing experimental evidences indicating that chronic and acute overproduction of reactive oxygen species (ROS) and nitrogen species (RNS) mediate cardiac pathologies, together with other major diseases like cancer, neurodegenerative or immune disorders. Specifically for cardiac pathologies, ROS and RNS are involved in ischemia-reperfusion, heart failure, and hypertension. In this sense, two major factors have been suggested to induce damage and death of cardiomyocytes in response to ischemia-reperfusion (I/R): a) an increase in intracellular calcium concentration, and b) oxidative stress. Moreover, in response to cellular oxidative stress caused by displacement of the equilibrium state between pro-oxidant/anti-oxidant systems toward pro-oxidants, lipid peroxidation may happen. This process is characterized by a production of biologically active aldehydes, as final products, that promote disruption of biological membranes. Some of these aldehydes are considered toxic second messengers due to their ability to amplify the damage initially caused by ROS and RNS. Contrary to oxygen-derived free radicals, aldehydes are relatively stable and can diffuse inside or escape from the cell, but in both cases they attack targets that are far from where they were originated. Among these aldehydes, 4-hydroxy-2-nonenal (HNE) is considered of special interest, due to its toxic nature and high diffusivity. HNE is involved in the pathogenesis of several diseases as diabetes, chronic renal failure, cardiovascular and neurodegenerative diseases. HNE toxicity lies on its electrophilic nature, conferred by the conjugated system of a C2-C3 double bond and a carbonyl group (in C1), that provides a partial positive charge to carbon 3. This positive charge is further increased by the inductive effect of the hydroxyl group at carbon 4. Due to its three functional groups, which frequently act synergistically, HNE can undergo several reactions in the cell interacting with nucleophile targets, such as lipids, DNA and proteins. The interaction of HNE with proteins takes place by Michael addition or Schiff base formation with the amino acids Cys, His and Lys. In addition to its electrophilic nature, HNE toxicity could be explained as well by its high half-life and lipophilicity, making HNE reactive towards targets located far apart from its synthesis place.Importantly, several groups have shown that these protein-bound aldehydes can be detected in animal models of atherosclerosis and in human patients with high risk factors or clinical manifestations of atherosclerosis. These results suggest an implication of products like HNE in the genesis and development of CVD. In this sense, different evidences show that the formation of HNE-protein adducts is a key event in some of the effects associated with free radicals in the heart. These effects include: a) Disturbance of rhythm and myocardial contractility, b) modulation of signal transduction leading to adaptive responses or apoptosis, and c) alteration of mitochondrial energy metabolism.According to the background exposed above, it is clear that molecules produced during oxidative stress, including HNE, contribute to the pathogenesis of various cardiovascular diseases. Thus, antioxidant therapies may be effective in preventing or blocking the pathological effects caused by these species. The literature does not clearly establish the potential success of using antioxidants as prevention for CVD. Therefore, the development of a new class of antioxidants targeted to specific cellular compartments or to specific molecules, besides the development of sensitive and specific biomarkers that can be used clinically to assess oxidative stress phenotypes that underlie various vascular pathologies, will be a great tool against cardiovascular disease.The overall objectives in this thesis were: firstly, to clarify the cellular effect of 4-hydroxy-2-nonenal in neonatal rat cardiomyocytes (NRC); and secondly, to determine the possible protective role of antioxidant molecules isolated from medicinal plants, which could be potentially used in cardiovascular disease prevention. Our results show a lethal effect of HNE in NRC, being 4.38¿M the LC50 at 3h treatment, a concentration in the pathophysiological range. On the basis of the viability results, 5 and 10¿M were established as HNE doses on further experiments. Cell death induced by HNE is time-dependent, peaking at 3h, and its toxic effect is prevented by the antioxidant trolox, the ß-blocker carvedilol, thiol containing compounds (i.e., N-Acetyl-Cysteine (NAC) and ß-mercapto-propionyl-glycine (MPG)), and extremely small concentrations of Thymbra capitata essential oil. By one hand, a short exposure of NRC to HNE 5 and 10¿M (15min) increases intracellular concentration ([Ca2+]i), as well as Ca2+ dynamic loss. After 1h treatment mitochondrial alterations were observed, including a burst of ROS generation and loss of mitochondria membrane potential (¿¿m). On the other hand, the ATP and GSH intracellular pools decrease considerably after 3h treatment, being the loss of GSH levels completely prevented when HNE treatment is preceded by a pre-treatment with the GSH precursor NAC. MPG pre-treatment also protects against cell death, whereas the GSH pool was recovered in a lesser extent compared to NAC. To gain more insight in the cell death mechanism induced by HNE, we analyze, caspase-3 activity, DNA fragmentation, both classic apoptotic markers, and a necrotic marker like LDH release. Surprisingly, caspase-3 activity and DNA fragmentation appear as well as an increase in LDH release, which was evident after 3h treatment, but not after 1h30min. Although LDH release is a necrotic cell death marker, we suggest that HNE toxicity could take place through apoptosis although the membrane integrity is not kept intact until the end of the process. Moreover, this observation could be explained by the fact that cultured cells at the end stage of apoptosis can undergo a procedure known as secondary necrosis due to the absence of phagocytic cells in the culture able to engulf the cell debris (also called apoptotic bodies). In addition, immunocytochemistry with anti-tubulin (marker for microtubules) and staining with Rhodamine-Phalloidine (marker of actin fibers) showed a loss of general cytoskeletal integrity after three hours treatment, whereas after 1h30min treatment the cytoskeleton organization was not affected. The cytoskeleton alterations are irreversible and could be related with the loss of membrane integrity, described after 3h treatment. Regarding antioxidant compounds a dose dependent dual action was observed for Thymbra capitata essential oil extracts. While 0.002 % of essential oil has a strong antioxidant effect (preventing cell death, ROS generation and loss of ¿¿) higher concentrations are highly toxic. The second part of this work was to determine the mechanism responsible of Ca2+ overload. For this purpose, we used sarcoplasmic reticulum (SR) vesicles (from rabbit skeletal muscle), a well established in vitro model for the study of calcium transport in membranes. We tested initially, Ca2+-ATPase (SERCA1a), the most abundant protein in SR vesicles, as the target for HNE action on Ca2+ homeostasis. However, high doses (100-500¿M) of HNE were required to obtain a significantly inhibition of ATPase activity. The inhibition of ATPase activity was time, dose, pH (the loss in the ability of SERCA1a to hydrolyze ATP is faster under alkaline conditions), and temperature dependent. Moreover, Ca2+ uptake was inhibited in addition to ATP hydrolysis. Ca2+ binding was not affected by HNE, whereas the MgATP binding to the catalytic site decreases considerably. HNE vs. MgATP competition was measured using both a direct ATP binding technique and changes in the intrinsic fluorescence of the protein induced by MgATP. As a consequence of reduced ATP binding by HNE treatment, the phosphoenzyme (EP) formation was inhibited too. In order to study HNE direct interactions with Lys residues, we carried out competition assays, using two specific markers: FITC, which label Lys 515, and PLP, to label Lys 492. The results revealed direct interaction of HNE with Lys515 but not Lys492. Since ATP binding was inhibited in a lesser extent than ATP hydrolysis, we suggest that the loss of ATPase activity after reaction with HNE is partially due to a reduction in the ability of SERCA1a to bind MgATP. The quantification of ¿SH groups with DTNB, in addition to MALDI-TOF assays, showed the formation of HNE-Cys adducts upon HNE treatment, which could contribute to the SERCA1a functional loss. Even though HNE causes a loss in SERCA1a functionality, the high HNE concentrations required would not explain the cellular toxicity of HNE. Nonetheless, it is of special interest that treatment of SR with HNE increased the passive permeability of this membrane to Ca2+ to a very large extent (higher than the ionophore A23187). This effect is especially fast at low HNE concentrations (in the 10-20¿M range). The previous result is coherent with the increase on ATPase activity in absence of ionophore or detergent at low HNE concentrations. After discarding the lipids as HNE target and therefore responsible for this increased permeability; we hypothesized that other proteins present in SR membranes, different to SERCA, could be HNE targets and responsible of the calcium leak. Immunodetection with an antibody against HNE-protein conjugates did not detect SERCA1a-HNE adducts when the treatment was carried out at low HNE concentration (20¿M). Strikingly, a 150-170kDa band appeared in the PVDF membrane. After an extensive study of vesicles proteome, we hypothesized that this band could fit with two proteins responsible for regulating the Ryanodine-Receptor (RyR) activity: Histidine Rich Calcium Binding Protein (HRC) and Sarcalumenin (SAR). The phosphorylation of these proteins by cellular kinases perturbs ryanodine binding to its receptor, and therefore, it might impair the Ca2+ channel opening. These proteins are potential candidates as HNE targets at low concentrations, as their reaction with HNE may indeed be expected to open leakage pathways in the SR membranes via the nearby RyR. This prompts us to analyze the effect of HNE on different SR fractions (Light SR enriched in longitudinal SR and Heavy SR enriched in junctional SR). The results obtained point to HRC as HNE main target, because HNE-protein 150-170kDa adducts are present in a larger amount in the Heavy SR population, where HRC is mainly located, whereas SAR is placed in longitudinal tubes. Collectively, these findings suggest that HNE modulates intracellular redox status (loss of GSH pool and modification of the redox state of proteins through interaction with ¿SH groups), affecting Ca2+ homeostasis. The Ca2+ overload and the redox alterations, as consequence of HNE treatment, may be responsible of triggering mitochondrial dysfunction (ROS production and ¿¿m) related with the onset of apoptotic cell death (caspase-3 activity and DNA fragmentation). Future studies will need to explore the role of cardiac HRC and SAR in HNE induced Ca2+ homeostasis alterations, as well as deeper analysis of 150-170 kDa HNE-Protein adducts, in order to demonstrate HRC and/or SAR involvement on calcium leak in SR vesicle. To determine properly the mechanism of cell death, further analysis should be done to establish the mechanism responsible of caspase-3 activation.
