Dihydrotanshinone I preconditions myocardium against ischemic injury via PKM2 glutathionylation sensitive to ROS

Ischemic preconditioning (IPC) is a potential intervention known to protect the heart against ischemia/reperfusion injury, but its role in the no-reflow phenomenon that follows reperfusion is unclear. Dihydrotanshinone I (DT) is a natural compound and this study illustrates its role in cardiac ischemic injury from the aspect of IPC. Pretreatment with DT induced modest ROS production and protected cardiomyocytes against oxygen and glucose deprivation (OGD), but the protection was prevented by a ROS scavenger. In addition, DT administration protected the heart against isoprenaline challenge. Mechanistically, PKM2 reacted to transient ROS via oxidization at Cys423/Cys424, leading to glutathionylation and nuclear translocation in dimer form. In the nucleus, PKM2 served as a co-factor to promote HIF-1α-dependent gene induction, contributing to adaptive responses. In mice subjected to permanent coronary ligation, cardiac-specific knockdown of Pkm2 blocked DT-mediated preconditioning protection, which was rescued by overexpression of wild-type Pkm2, rather than Cys423/424-mutated Pkm2. In conclusion, PKM2 is sensitive to oxidation, and subsequent glutathionylation promotes its nuclear translocation. Although IPC has been viewed as a protective means against reperfusion injury, our study reveals its potential role in protection of the heart from no-reflow ischemia.

Angiotensin II inhibits Na+/K+ATPase activity in pulmonary artery smooth muscle cells via glutathionylation and with the involvement of a 15.6 kDa inhibitor protein.

The role of angiotensin II in regulating Na+/K+-ATPase activity has been investigated in bovine pulmonary artery smooth muscle cells (BPASMCs). Our study reveals that angiotensin II inhibits the Na+/K+ATPase activity via glutathionylation of the pump with the involvement of an increase in NADPH oxidase-derived O2.-. Additionally, angiotensin II treatment to the cells increases the inhibitory potency of the 15.6 kDa inhibitor towards the Na+/K+ATPase activity.

Investigação de potenciais biomarcadores redox - um enfoque em aldeídos e seus produtos / Potential redox biomarkers investigation - focus on aldehydes and their products

As espécies reativas são associadas a processos toxicológicos e fisiopatológicos, agindo como importantes mediadores, por exemplo, na sinalização celular. Diversas classes de compostos têm sido utilizadas como possíveis biomarcadores de estresse redox, destacando-se os aldeídos α,ß-insaturados, capazes de alquilar biomoléculas como o DNA. Para evitar efeitos deletérios, estes aldeídos são detoxificados por glutationilação e posterior metabolização a derivados mercaptúricos. Contudo, avaliar o estado redox em sistemas biológicos ainda é tarefa bastante complexa, sendo a dificuldade em quantificar de forma prática e acurada os efeitos de sinalização e/ou dano molecular o maior problema dos estudos redox. Assim, o objetivo deste trabalho foi desenvolver métodos acurados e sensíveis de análise de potenciais biomarcadores de estresse redox, isto é: nucleosídeos modificados, aldeídos endógenos e exógenos, glutationa e produtos de glutationilação, e avaliá-los em sistemas modelos, celular e animal, e em humanos. A avaliação dos níveis urinários de três nucleosídeos modificados por metodologia de HPLC-MS/MS desenvolvida pelo grupo em moradores da cidade de São Paulo - região com poluição atmosférica - demonstrou aumento significativo de 1,N2-propanodGuo comparado aos moradores de região não poluída. Ademais, comprova-se pela primeira vez que células deficientes em reparo de ligações cruzadas apresentam níveis basais elevados de 1,N2-propanodGuo, em duas linhagens independentes, colocando este aduto como potencial mediador de carcinogênese em pacientes portadores de Anemia de Fanconi. Utilizando cérebro de ratos SOD1G93A (modelo de Esclerose Lateral Amiotrófica - ELA), verificou-se aumento de 50% nos níveis de 1,N2-propanodGuo e de 100% nos de 1,N6-εdAdo em fase sintomática, sugerindo influência do conteúdo lipídico cerebral, levando a comprometimento do metabolismo neuronal e morte celular. O perfil de aldeídos determinado em cérebro de ratos SOD1G93A demonstrou aumento de trans-hexa-2-enal e trans,trans-hexa-2,4-dienal em fase assintomática e de trans,trans-deca-2,4-dienal em fase sintomática, não sendo observada nenhuma alteração na medula. Conhecer estas variações permite direcionar estudos de modificações em biomoléculas, além de a metodologia per se corroborar com as áreas de análises lipidômicas. Técnicas distintas e o preparo de amostras refletiram nos níveis de glutationa reduzida (GSH) e oxidada (GSSG) relatados. A técnica de espectrometria de massas mostrou-se mais precisa que a detecção eletroquímica; e a alquilação do grupo tiol minimizou interferências de matriz. Por análise de HPLC-UV/Vis-ESI-MS/MS, a quantificação de trans-4-hidroxi-2-nonenal (HNE) e crotonaldeido conjugados com GSH demonstrou não haver alterações em cérebro e medula de ratos SOD1G93A. Contudo, há formação esteroespecífica dos adutos de HNE in vivo. Ressalta-se que a metodologia desenvolvida é extremamente sensível e específica e permite análise simultânea de GSH, GSSG, cisteína, cistina e dos adutos supracitados, servindo para análise de outros adutos de glutationilação de aldeídos que possam ser importantes em doenças associadas a estresse redox

Free radicais and oxidant species are associated with toxicological and pathophysiological processes. It has been demonstrated that production of reactive oxygen species may be involved in cell signaling and regulation. Several biomarkers of redox processes have been used, including adducts formed through the reaction of α,ß-unsaturated aldehydes with biomolecules such as DNA. In order to avoid these deleterious effects, aldehydes are detoxified through glutathionylation and further metabolized to mercapturic derivatives. However, assessing the redox status in biological systems is still a very complex task, and the difficulty in practical and accurate quantification of signaling effects and/or molecular damage is a major problem in redox studies. The objective of this work was to develop accurate and sensitive methods for analysis of potential biomarkers of redox stress, i.e., modified nucleosides, endogenous and exogenous aldehydes, glutathione and glutathionylation products, and their evaluation in cell, animal model and humans. Evaluation of urinary levels of 1,N2-propano-2'-deoxyguanosine (1,N2-propanodGuo), 1,N2-etheno-2'-deoxyguanosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine in residents of São Paulo City - polluted region - showed a significant increase (p<0.05) in 1,N2-propanodGuo levels compared to residents of an unpolluted region by a HPLC-MS/MS methodology developed by the group. Moreover, it was proven, for the first time, that repair deficient cells have basal levels of 1,N2-propanodGuo higher than proficient cells in two independent strains, placing 1,N2-propanodGuo as a potential mediator of carcinogenesis in Fanconi Anemia patients. In an Amyotrophic Lateral Sclerosis (ALS) animal model (SOD1G93A rat) , a 50% increase in the levels of 1,N2-propanodGuo and 100% in the 1,N6-etheno-2'-deoxyadenosine in brain tissue in the symptomatic phase was observed, suggesting that the high brain lipid content may play a role, leading to impairment of cell metabolism and neuronal cell death. There is an increase of trans-hex-2-enal and trans,trans-hexa-2,4-dienal in asymptomatic SOD1G93A rats brain and of trans,trans-deca-2,4-dienal in symptomatic ones. However, no alteration was observed in spinal cord. Our approach contributes to a better understanding of the aldehyde status in vivo and allows us to predict biomolecule modifications. The developed methodology can contribute to lipidomic studies. The use of different techniques and sample preparation reflected in the reported levels of reduced (GSH) and oxidized glutathione (GSSG). The mass spectrometry technique proved to be more accurate than the electrochemical one, and the use of thiol alkylating agent minimizes matrix interference. No changes were observed in the levels of the GSH conjugates of trans-4-hydroxy-2-nonenal (HNE) and crotonaldehyde in brain and spinal cord of SOD1G93A rats quantified by HPLC-UV/Vis-ESI-MS/MS compared to controls. However, it was observed stereospecific HNE adducts formation in vivo. Note that this methodology is extremely sensitive and specific and allows simultaneous analysis of GSH, GSSG, Cys, cystine and the aforementioned adducts, serving for analysis of other aldehyde-glutathionylation adducts that may be important in pathologies associated with stress redox