O 4-hidroxi-2-nonenal (4-HNE) é um dos principais produtos da peroxidação lipídica, processo exacerbado no quadro de estresse oxidativo. Em função de sua alta reatividade com biomoléculas, seu acúmulo tem sido relacionado ao estabalecimento e progressão de inúmeras doenças, incluindo as cardiovasculares. Recentemente, nosso grupo identificou a interação entre 4-HNE e a proteína Dicer em coração de ratos com insuficiência cardíaca (dados não publicados). Dicer é uma RNAse importante na biogênese de microRNAs (miRNA), com papel na regulação gênica póstranscricional, de modo que alterações em sua função poderiam afetar diversos processos celulares. Tanto a interação entre o aldeído e Dicer, quanto o efeito sobre a mesma não foram descritos na literatura. Nesse contexto, o presente estudo tem como objetivo avaliar o efeito do 4-HNE na atividade e a expressão da Dicer. Nossa hipótese é que o 4-HNE afete negativamente o perfil de atividade e expressão da Dicer. Para testar essa hipótese, utilizamos o modelo animal de disfunção cardíaca induzida cirurgicamente e avaliamos: a formação de adutos de 4-HNE-proteínas, atividade e expressão de Dicer, e os níveis de miRNAs cardíacos. Em cultura celular (H9C2, MEF e HEK293), por sua vez, avaliamos o efeito agudo de 4-HNE sobre as mesmas variáveis após sua adição no meio de cultura. E, por último, utilizando a proteína recombinante, analisamos o efeito direto do aldeído sobre a estabilidade e atividade da enzima in vitro. Como esperado, em ensaios com a proteína isolada, observamos que o 4-HNE interage diretamente com a RNAse Dicer, e a formação de conjugados Dicer-4-HNE é responsável pela inibição e perda de estabilidade da proteína de forma tempo- e concentração-dependentes. No modelo animal, demonstramos um prejuízo na atividade de Dicer no coração de animais com disfunção cardíaca induzida por infarto do miocárdio, sem alteração em sua expressão, acompanhado de diminuição dos níveis da maioria dos miomiRs analisados. Notavelmente, ambos os parâmetros, assim como os níveis de adutos de 4-HNE-proteínas, foram melhorados no grupo tratado com Alda-1, agonista alostérico da enzima ALDH2 (responsável pela remoção do 4-HNE). Dessa forma, sugerimos a existência de associação entre os níveis de 4-HNE, atividade de Dicer e alteração na expressão de miRNAs no quadro de disfunção cardíaca. Consistente com os dados observados in vivo, em modelos celulares, a exposição aguda ao 4-HNE demonstrou reduzir a atividade de Dicer e afetar a via de biossíntese de miRNAs. Porém, não observamos proteção por Alda nesse modelo. Conjuntamente, nossos dados sugerem que a atividade de Dicer é modulada por 4-HNE em quadros de estresse agudo e crônico de aldeídos. Contudo, mais estudos são necessários a fim de elucidar o mecanismo pelo qual essa modulação ocorre. Visto que o acúmulo de 4-HNE e a desregulação na biogênese de miRNAs tem sido associados ao desenvolvimento de patologias, o estudo da interação entre Dicer e o aldeído é importante na compreensão dessas doenças e planejamento de novas estratégias terapêuticas. / 4-hydroxy-2-nonenal (4-HNE) is a major by-product of lipid peroxidation, a process that is exacerbated under oxidative stress conditions. This aldehyde is a very reactive molecule associated with the establishment and progression of many diseases, including cardiovascular diseases. We recently found using proteomics that 4-HNE directly targets Dicer in failing hearts, a critical enzyme for miRNA biology (unpublished data). Neither the aldehyde-Dicer adduction, nor its effect on protein stability and activity has been previously reported. Therefore, this study aimed to fill this gap by further investigating 4-HNE-Dicer interaction and characterizing its effect on Dicer profile. We hypothesize that 4-HNE will make adducts with Dicer and compromise its function and levels. Using an animal model of cardiac dysfunction, we evaluated the following parameters: levels of 4-HNE adducted proteins, Dicer levels and activity, and the levels of heart specific miRNAs (myomiRs). The same variables were analyzed in distinct cellular models (H9C2, MEF, HEK293) after acute exposure to 4-HNE. Additionally, we synthetized recombinant Dicer, and protein function and stability were assessed in vitro. As expected, the experiments with recombinant protein revealed that 4-HNE directly interacts with Dicer, and the formation of 4-HNE-DICER adduct causes loss of Dicer cleavage activity and stability in a time- and concentration-dependent manner. Regarding the animal model, Dicer activity, but not protein levels, dropped in failing hearts, which was paralleled by a reduction of mature miRNA levels. Of interest, animals with cardiac dysfunction chronically treated with a small molecule activator of aldehyde dehydrogenase 2 (ALDH2), termed Alda-1, displayed an elevated cardiac Dicer activity and mature miRNA levels compared with vehicle-treated animals. ALDH2 is the mains enzyme responsible for 4-HNE clearance. In this context, this study points out a potential connection among 4-HNE levels, Dicer activity and myomiR levels in cardiac dysfunction. Consistent with our in vivo data, cells acutely exposed to 4-HNE showed an increase in 4-HNE-protein adducts followed by a reduction in Dicer activity and changes in miRNA biosynthesis. However, Alda showed no protective effect in the latter model. Taken together, our findings using animal and cellular models suggest that Dicer activity is impaired in chronic (cardiac dysfunction) and acute aldehyde stress conditions. However, the molecular mechanisms involved in this response are still unclear. As both 4-HNE accumulation and microRNAs have been linked to innumerous pathologies, clarifying the modulation of Dicer activity under such conditions will certainly contribute to a better understanding the diseases and future therapeutic strategies.

The pathogenesis of heart failure (HF) involves compensatory left ventricular hypertrophy. Reactive oxygen species (ROS) are elevated in HF and mediate myocardial hypertrophy. ROS also mediate formation of lipid peroxidation byproducts, yet little is known about their role in promoting hypertrophy. One lipid peroxidation byproduct, 4-hydroxy-trans-2-nonenal (HNE) is a reactive aldehyde that forms covalent adducts on proteins. HNE levels are also elevated in HF and may mediate hypertrophy via HNE-adduct formation. LKB1 - a tumor suppressor protein - regulates cellular growth through activation of the downstream kinase AMPK. Activation of AMPK suppresses functions that consume ATP and simultaneously activates processes to generate energy. The LKB1 protein is inhibited by oxidants, but whether this results in myocardial hypertrophy is unclear. I hypothesized that HNE can directly promote cardiac hypertrophy via the modification of LKB1. In HEK293T cells I observed that HNE adducts inhibit activity of LKB1 through direct oxidative modification. Mutation of LKB1 Lys-96 or Lys-97 resulted in less HNE-LKB1 adduct formation. Mutation of LKB1 Lys-97 prevented the inhibitory effect of HNE, suggesting that HNE-adduction at this residue is sufficient to inhibit LKB1. In cardiomyocytes HNE inhibited both LKB1 and AMPK, increased phosphorylation of mTOR, p70S6K, and S6K, and increased protein synthesis. HNE also activated Erk1/2, which contributed to S6K activation but was not required for cellular growth. Hypertrophic S6K activation was dependent on mTOR. Mice fed a high-fat high-sucrose (HFHS) diet have myocardial hypertrophy that can be prevented by antioxidants. Hearts of HFHS mice have HNE-LKB1 adducts, inhibited LKB1 activity, yet no change in AMPK activation. Mice lacking aldehyde dehydrogenase 2 (ALDH2), an enzyme involved in HNE detoxification, have increased myocardial hypertrophy when fed HFHS diet yet have increased LKB1 activity. In summary HNE directly causes hypertrophy in cardiomyocytes. This occurs through inhibition of LKB1 and in part through Erk1/2 activation. In HFHS-fed mice HNE-LKB1 adduct formation is associated with decreased LKB1 activity. Impairing detoxification of reactive aldehydes in the ALDH2-KO mice is sufficient to increase myocardial hypertrophy, but this appears to be independent of LKB1. This study demonstrates a novel mechanism of cardiac hypertrophy caused by reactive aldehydes.

In diabetes hyperglycemia and lack of insulin signaling are key factors in the induction of diabetic sensory neuropathy. The combination of these factors in diabetes may enhance oxidative stress and trigger distal nerve damage in the peripheral nervous system. The link between elevated reactive oxygen species (ROS) levels and nerve degeneration is not clear. We tested the hypothesis that elevation of 4-hydroxy-2-nonenal (4-HNE) induced by oxidative stress in diabetes impairs mitochondrial activity and axonal regeneration in dorsal root ganglion (DRG) neurons. Also, we investigated the association between mitochondrial dysfunction and altered mitochondrial proteome in the axons of streptozotocin–induced diabetic rats. Research design and methods. Cultured adult rat DRG sensory neurons were treated exogenously with 4-HNE, and cell survival, axonal morphology, and level of axon outgrowth assessed. Western blot and fluorescence imaging were used to determine changes in the levels of adducts of 4-HNE and abnormalities in the mitochondria. Proteomic analysis using stable isotope labeling with amino acids in cell culture (SILAC) determined expression of proteins in the mitochondria. Results. 4-HNE impaired axonal regeneration, mitochondrial activity and induced aberrant axonal structures along the axons, which mimicked axon pathology observed in nerve isolated from diabetic rats and replicated aspects of neurodegeneration observed in human diabetic neuropathy. Proteins associated with mitochondrial dysfunction, oxidative phosphorylation and biosynthesis were down regulated in diabetic samples. The axons of diabetic neurons exhibited oxidative stress and depolarized mitochondria. CNTF and resveratrol reversed abnormalities in the mitochondrial membrane potential induced by diabetes and treatment of neurons with 4-HNE. CONCLUSIONS. Elevation of 4-HNE levels in diabetes was associated with impaired mitochondrial function and might be an important link between increased ROS levels and nerve degeneration in diabetic neuropathy. Abnormal mitochondrial function correlated with a down-regulation of mitochondrial proteins, with components of the respiratory chain targeted in lumbar DRG in diabetes. The reduced activity of the respiratory chain was associated with diminished superoxide generation within the mitochondrial matrix and did not contribute to oxidative stress in axons of diabetic neurons.

In diabetes hyperglycemia and lack of insulin signaling are key factors in the induction of diabetic sensory neuropathy. The combination of these factors in diabetes may enhance oxidative stress and trigger distal nerve damage in the peripheral nervous system. The link between elevated reactive oxygen species (ROS) levels and nerve degeneration is not clear. We tested the hypothesis that elevation of 4-hydroxy-2-nonenal (4-HNE) induced by oxidative stress in diabetes impairs mitochondrial activity and axonal regeneration in dorsal root ganglion (DRG) neurons. Also, we investigated the association between mitochondrial dysfunction and altered mitochondrial proteome in the axons of streptozotocin–induced diabetic rats. Research design and methods. Cultured adult rat DRG sensory neurons were treated exogenously with 4-HNE, and cell survival, axonal morphology, and level of axon outgrowth assessed. Western blot and fluorescence imaging were used to determine changes in the levels of adducts of 4-HNE and abnormalities in the mitochondria. Proteomic analysis using stable isotope labeling with amino acids in cell culture (SILAC) determined expression of proteins in the mitochondria. Results. 4-HNE impaired axonal regeneration, mitochondrial activity and induced aberrant axonal structures along the axons, which mimicked axon pathology observed in nerve isolated from diabetic rats and replicated aspects of neurodegeneration observed in human diabetic neuropathy. Proteins associated with mitochondrial dysfunction, oxidative phosphorylation and biosynthesis were down regulated in diabetic samples. The axons of diabetic neurons exhibited oxidative stress and depolarized mitochondria. CNTF and resveratrol reversed abnormalities in the mitochondrial membrane potential induced by diabetes and treatment of neurons with 4-HNE. CONCLUSIONS. Elevation of 4-HNE levels in diabetes was associated with impaired mitochondrial function and might be an important link between increased ROS levels and nerve degeneration in diabetic neuropathy. Abnormal mitochondrial function correlated with a down-regulation of mitochondrial proteins, with components of the respiratory chain targeted in lumbar DRG in diabetes. The reduced activity of the respiratory chain was associated with diminished superoxide generation within the mitochondrial matrix and did not contribute to oxidative stress in axons of diabetic neurons.

No description available.

No description available.

No description available.