A formação de aldeídos decorrente do estresse oxidativo é cardiotóxica e contribui para o aparecimento das doenças cardiovasculares. O aldeído 4-hidroxi-2-nonenal (4HNE) apresenta grande poder nocivo cardíaco. A enzima mitocondrial aldeído desidrogenase 2 (ALDH2) é a principal responsável pela remoção do 4HNE, sendo que a ativação farmacológica da ALDH2 previne danos cardíacos oriundos do processo de isquemia/reperfusão. Com o intuito de compreender o papel desta enzima na insuficiência cardíaca (IC) tivemos como objetivos: caracterizar o curso temporal de ativação da ALDH2 pós-infarto do miocárdio em ratos e estudar o efeito da ativação farmacológica sustentada da ALDH2 na IC. Nossos resultados apontam que na 1ª, 2ª e 4ª semanas pós-infarto a atividade da ALDH2 apresenta-se reduzida. Essa redução está associada à disfunção mitocondrial e cardíaca. Contudo, o tratamento com Alda-1 proporciona uma melhora dessas funções. Assim, concluímos que a ativação seletiva da ALDH2 reduz danos cardíacos, podendo ser considerada um novo alvo terapêutico no tratamento da IC. / Here we determined the benefits of chronic activation of ALDH2 on the progression of heart failure after the onset of symptoms using a post-myocardial infarction model. We showed that a six-week treatment of myocardial infarction-induced heart failure rats with Alda-1, a selective ALDH2 activator, enhanced contractile function, improved left ventricular compliance and increased diastolic dysfunction under basal conditions and after sudden pressure-overload stress. Moreover, sustained Alda-1 treatment showed no toxicity and promoted a cardiac anti-remodeling effect by suppressing myocardial hypertrophy and fibrosis. Moreover, ALDH2 activation-mediated cardioprotection was associated with improved mitochondrial function. Further Alda-1 treatment preserved mitochondrial function in vitro upon 4-HNE addition. Therefore, selective activation of mitochondrial ALDH2 reduces aldehydic load, preserves mitochondrial function and improves heart failure outcome, suggesting that ALDH2 activators have a potential therapeutic value for treating heart failure patients.

A formação de aldeídos decorrente do estresse oxidativo é cardiotóxica e contribui para o aparecimento das doenças cardiovasculares. O aldeído 4-hidroxi-2-nonenal (4HNE) apresenta grande poder nocivo cardíaco. A enzima mitocondrial aldeído desidrogenase 2 (ALDH2) é a principal responsável pela remoção do 4HNE, sendo que a ativação farmacológica da ALDH2 previne danos cardíacos oriundos do processo de isquemia/reperfusão. Com o intuito de compreender o papel desta enzima na insuficiência cardíaca (IC) tivemos como objetivos: caracterizar o curso temporal de ativação da ALDH2 pós-infarto do miocárdio em ratos e estudar o efeito da ativação farmacológica sustentada da ALDH2 na IC. Nossos resultados apontam que na 1ª, 2ª e 4ª semanas pós-infarto a atividade da ALDH2 apresenta-se reduzida. Essa redução está associada à disfunção mitocondrial e cardíaca. Contudo, o tratamento com Alda-1 proporciona uma melhora dessas funções. Assim, concluímos que a ativação seletiva da ALDH2 reduz danos cardíacos, podendo ser considerada um novo alvo terapêutico no tratamento da IC. / Here we determined the benefits of chronic activation of ALDH2 on the progression of heart failure after the onset of symptoms using a post-myocardial infarction model. We showed that a six-week treatment of myocardial infarction-induced heart failure rats with Alda-1, a selective ALDH2 activator, enhanced contractile function, improved left ventricular compliance and increased diastolic dysfunction under basal conditions and after sudden pressure-overload stress. Moreover, sustained Alda-1 treatment showed no toxicity and promoted a cardiac anti-remodeling effect by suppressing myocardial hypertrophy and fibrosis. Moreover, ALDH2 activation-mediated cardioprotection was associated with improved mitochondrial function. Further Alda-1 treatment preserved mitochondrial function in vitro upon 4-HNE addition. Therefore, selective activation of mitochondrial ALDH2 reduces aldehydic load, preserves mitochondrial function and improves heart failure outcome, suggesting that ALDH2 activators have a potential therapeutic value for treating heart failure patients.

Regulators of G-protein signaling (RGS) proteins are a family of proteins that act as GTPase accelerating proteins (GAPs) through their interaction with GΑ subunits, including GΑo, GΑi, and GΑq but not GΑs. This increased rate of hydrolysis of GTP to GDP temporally regulates G-protein coupled receptor (GPCR) signaling. A member of this family, RGS4, has been implicated in several neurological disorders including Parkinson's Disease (PD). A hallmark of PD is the induction of oxidative stress within dopaminergic neurons. In this thesis, we evaluate the role of oxidative stress, including lipid peroxidation products with 4-hydroxy-2-nonenal (4HNE) as a model, in regulating RGS4 activity within neurons. Utilizing transfected RGS4, we evaluated whether RGS4 is readily modified by physiologically relevant concentrations of 4HNE by immuonoprecipitation of RGS4 from 4HNE treated cells. Further examination of recombinant RGS4 by mass spectrometry, revealed that RGS4 is readily modified at several cysteine residues by 4HNE, including C148. Modification at this residue has been shown to be a critical site for allosteric regulation of RGS4. This is confirmed through a malachite green based phosphate generation assay we developed to observe the GAP activity of RGS4 on its native binding partner GΑi. This malachite green based assay was then adapted for high throughput screening. The assay was successfully miniaturized to a 1536-well format. In a screen of 2300 compounds, 4 were identified as hits. The development of this simple and cheap assay can be adapted for usage with a variety of RGS proteins with little work to interrogate other pathways and identify novel RGS modulators. Finally, expansive study of PD has linked oxidative stress to the pathology of both diseases. What has not been discerned is the potential relationship between oxidative stress and the induction of RGS4. In support of the hypothesis, we evaluated the potential relationship between oxidative stress and RGS4 expression. This was accomplished by evaluating two striatal neuron like cell lines, SH-SY5Y and HCN-1A. After treatment with hydrogen peroxide, both cell lines showed increased RGS4 in response to oxidative stress. This response is not however related to mRNA expression, indicating this change is most likely an adjustment of proteasomal regulation of RGS4. This phenomenon may explain the rapid onset of Parkinsonian motor symptoms in reserpine treated animal models of PD, as excess dopamine in the cytoplasm may be rapidly metabolized in reactive products.

The modification of proteins by xenobiotic and endogenous electrophilic species produced in cells undergoing oxidative stress contributes to cellular toxicity and disease processes. Many xenobiotics are themselves reactive electrophiles; however non-reactive compounds may become reactive towards proteins and DNA following metabolism. Identifying actual sites of adduction on target proteins is critical for determining the structural and functional consequences associated with the modification. 1,4-benzoquinone (BQ) is a reactive quinone and environmental toxicant, formed from oxidative metabolism of benzene, an aromatic hydrocarbon found in gasoline and other fuels. Although environmental and occupational exposure to benzene is associated with the development of aplastic anemia and leukemia, the mechanism of toxicity remains elusive. Due to the electrophilic nature of BQ, it reacts with glutathione to form quinol-thioether (QT) conjugates that retain the ability to redox cycle between the reduced (HQ) and oxidized (BQ) forms. BQ and its QT metabolites are reactive, and can produce cellular necrosis through oxidative stress and protein modification. One further consequence of oxidative stress is the elevation of cellular membrane lipid peroxidation, resulting in the formation of reactive lipid-aldehydes such as 4-hydroxynonenal (4HNE). Adduction of critical amino acid residues in target bone marrow proteins by 4HNE and QTs following exposure to benzene could contribute to its hematotoxic effects. This dissertation builds upon the foundation of proteins targeted by electrophilic adduction by outlining techniques to pinpoint the specific amino acids targeted and furthermore predict the functional releavance of adduction. For the first time, protein targets of reactive endogenous lipid aldehydes are reported in the bone marrow of chemically treated rats. Furthermore, novel sites of adduction by aldehydes and benzene-glutathione conjugates are reported within functional regions of topoisomerase II. Inhibition of bone marrow DNA topoisomerase II by benzene metabolites is implicated as a potential mechanism of benzene-induced hematotoxicity and acute-myeloid leukemia. The strong inhibitory effect of these compounds on topoisomerase II activity suggests that their presence in the bone marrow may play a role in benzene-induced myelotoxicity.