O conteúdo de zinco (Zn) reativo cerebral é importante para o equilíbrio da sinaptofisiologia neural. A prova disto é que um aumento nos seus níveis, após evento hipóxico-isquêmico, resulta em neurotoxicidade, o que tem estimulado o tratamento desta disfunção cerebral com quelantes de Zn, tal como o dietilditiocarabamato (DEDTC). No caso do DEDTC, o uso deste composto sobre esta disfunção deve ser analisado com cuidado, pois ele também apresenta muitos efeitos colaterais sobre o sistema nervoso central. Desta forma, para atender este propósito, é necessário antes obter uma concentração de DEDTC com menores efeitos colaterais. Por esta razão, no presente trabalho, nós decidimos usar um modelo vertebrado mais simples, tal como o peixe-zebra, o qual permitiria a triagem, em larga escala, dos efeitos de DEDTC sobre o Zn reativo. Entretanto, jamais foi mostrada a presença de Zn reativo no cérebro de peixe-zebra. Com isto, através de marcações histológicas, nós conseguimos mostrar pela primeira vez a distribuição citoarquitectônica de Zn reativo em neurônios glutamatérgicos, bem como o número desses neurônios contendo Zn no cérebro de peixe-zebra. Isto nos permitiu avaliar o efeito de diferentes concentrações de DEDTC sobre o conteúdo de Zn cerebral do peixe-zebra, o qual foi intensamente quelado por elevadas quantidades do composto, induzindo comportamentos tipo-crise. Neste mesmo estudo nós obtemos também uma concentração de DEDTC com poucos efeitos colaterais que poderia exercer neuroproteção sobre o aumento de Zn reativo induzido pela hipóxia-isquemia. Assim, após a padronização de um modelo de hipóxia em peixe-zebra, que demonstra danos relacionados à isquemia, nós testamos se essa concentração de DEDTC poderia ser neuroprotetora sobre este modelo. Contudo, DEDTC apresentou efeitos pró-oxidantes, embora ele tenha atenuado o elevado conteúdo de Zn reativo induzido pela hipóxia. Portanto, mesmo que o DEDTC tenha falhado, este modelo, agora, está apto para a triagem de outros fármacos com potencial ação sobre o alterado conteúdo de Zn reativo que ocorre em eventos hipóxicos-isquêmicos. / The content of brain reactive zinc (Zn) is important for the synaptophysiology in the central nervous system (CNS). This is evidenced in hypoxic-ischemic events, when an increase in their levels results in neurotoxicity. Consequently, this has stimulated the treatment of cerebral ischemia with Zn chelators, such as diethyldithiocarbamate (DEDTC). In the case of DEDTC, the use of this compound in this dysfunction should be examined carefully, because it also has many side effects on the (CNS). Thus, to meet this, it is necessary first to obtain a concentration of DEDTC with negligible side effects. Here, we decided to use a simpler vertebrate model, such as zebrafish, which would allow large-scale screening of DEDTC effects on reactive Zn. However, the presence of reactive Zn has never been shown in zebrafish brain. Then, using histological markers, we were able to show for the first time the cytoarchitectonic distribution of reactive Zn in glutamatergic neurons as well as the number of these neurons containing Zn in the zebrafish brain. This allowed us to evaluate the effect of different DEDTC concentrations on the brain content of Zn in zebrafish. As a result, high levels of the compound did strongly chelate the metal, inducing seizure-like behaviors. In this study we also obtained a DEDTC concentration with few side effects that could exert neuroprotection on the increased reactive Zn induced by hypoxia-ischemia. Then, after the standardization of an ischemic-sensitive model of hypoxia in zebrafish, we tested if this DEDTC concentration could be neuroprotective on this model. Nevertheless, DEDTC showed pro-oxidant effects, though it had mitigated the elevated content of reactive Zn induced hypoxia. Therefore, despite the DEDTC have failed as neuroprotective drug, this model enables the screening of other chemical agents with potential action on the increased content of reactive Zn that occurs in hypoxic-ischemic events.

Há décadas os campos magnéticos (CMs) são alvo de investigação científica. Entretanto, o grande corpo de evidências, está relacionado a campos eletromagnéticos e não a campos magnéticos contínuos. Nosso interesse é direcionado para a modulação das respostas comportamentais e motoras, na preservação de neurônios pós-lesão isquêmica e na possibilidade de uma interferência funcional com drogas que modifiquem a neurotransmissão encefálica. Utilizamos 130 Gerbilos, alocados em 13 grupos experimentais. Grupos específicos foram submetidos a isquemia encefálica global bilateral e a implantação de um capacete magnético com potencia de 3200G. Quatro dias após os procedimentos cirúrgicos os animais foram avaliados no monitor de atividades e no Rotarod, após receberem uma injeção de Apomorfina (APO) (2,5mg /kg) ou Racloprida (RAC) (0,9ml/kg). Valor de p significativo <0,05. No monitor de atividades, os animais do grupo isquemia atravessaram o maior número de sensores horizontais (F12,117: 9,39) e verticais (F12,117: 10,60) do que todos os outros grupos. Animais isquêmicos injetados com APO e tratados com campo magnético atravessam um número menor de sensores do que os grupos isquêmicos. Os isquêmicos injetados com APO e estimulados com o polo norte dispararam menos sensores do que os isquêmicos injetados com APO. Animais injetados com RAC com ou sem estimulação magnética disparam menos sensores do que os animais isquêmicos e isquêmicos tratados com polo norte e sul. No teste do Rotarod, o grupo isquêmico apresentou o menor tempo de permanência no teste do que os demais grupos. Ainda, animais isquêmicos tratados com APO e RAC e estimulação magnética pelo polo norte apresentam maior tempo de permanência em relação aos grupos isquemia e isquemia injetado com Apo e RAC (F12,117: 11,29). Nossos dados confirmam a possibilidade de interação dos polos magnéticos e os mecanismos de ação das drogas utilizadas no experimento. / For decades, magnetic fields (CMs) have been the subject of scientific research. However, the great body of evidence is related to electromagnetic fields and not to continuous magnetic fields. Our interest is directed to the modulation of behavioral and motor responses, the preservation of neurons after brain ischemic injury and the possibility of functional interference with drugs that modify brain neurotransmission. We used 130 gerbils, allocated in 13 experimental groups. Specific groups were submitted to bilateral global brain ischemia and the implantation of a magnetic helmet with power of 3200G. Four days after the surgical procedures, the animals were evaluated on the activity monitor and Rotarod after receiving an injection of Apomorphine (APO) (2.5 mg / kg) or Raclopride (RAC) (0.9 ml / kg). Significant p value was set as <0.05. In the activity monitor, the animals in the ischemia group crossed fired the largest number of horizontal sensors (F12,117: 9,39) and vertical sensors (F12,117: 10,60) than all the other groups. Ischemic animals injected with APO and magnetic field fired a smaller number of sensors than the ischemic groups and injected with APO. Animals stimulated with the north pole fired fewer sensors than ischemic animals injected with APO. RAC injected animals, with or without magnetic stimulation fired fewer sensors than ischemic animals or ischemic with north and south magnetic stimulation. In the Rotarod test, the ischemic group had the shortest permanence time in the test than the other groups. Still, ischemic animals treated with APO and RAC and North Pole magnetic stimulation present a longer permanence time in comparison to the ischemia group and ischemia with APO and RAC. Ischemic animals injected with APO and magnetic field pass through a smaller number of sensors than the ischemic groups and injected with APO. In the Rotarod test, the ischemic group had the shortest residence time in the test than the other groups. Still, ischemic animals treated with APO and north pole present a longer residence time in relation to the ischemia groups with APO and RAC (F12,117: 11,29). Our data confirm the possibility of interaction of the magnetic poles and the action mechanisms of action of the drugs used in the experiment.

Bicarbonato é uma importante espécie química para os seres vivos, sendo o principal tampão celular, alem de apresentar uma negligenciada atividade redox. Isquemia é um evento no qual existe inibição do aporte de nutrientes e oxigênio, sendo a reperfusão o retorno do fluxo de nutrientes e oxigênio, que é acompanhada por alta produção de radicais livres e morte celular. Nessa tese estudamos o efeito da presença de bicarbonato durante a isquemia-reperfusão. Em nosso modelo nós mantivemos o pH constante e modulamos a quantidade de bicarbonato enquanto células, órgãos e animais foram submetidos a isquemia-reperfusão. Utilizamos condições sem a presença de bicarbonato, a concentração basal sanguínea e uma concentração mais alta simulando o acúmulo de bicarbonato em condições isquêmicas. Nesses diversos modelos mostramos que a presença de bicarbonato aumenta o dano provocado por isquemia-reperfusão e provoca um aumento do acúmulo de proteínas oxidadas. A presença do bicarbonato não modifica a respiração, produção de espécies reativas de oxigênio, ou a morfologia mitocondrial, também não detectamos mudança na atividade do proteassoma e nos indicadores de autofagia geral. Entretanto detectamos um acúmulo de marcadores autofágicos na fração mitocondrial indicando inibição da mitofagia. Essa inibição foi confirmada ao detectarmos o acúmulo de uma proteína degradada especificamente por mitofagia enquanto não houve mudança em outra degradada pelo proteassoma. Além disso, ao inibirmos farmacologicamente a autofagia, reproduzimos o fenótipo causado pelo bicarbonato mesmo na sua ausência. Em conclusão, a presença de bicarbonato é deletéria em condições de isquemia/reperfusão devido a inibição da mitofagia / Bicarbonate is an important molecule in all living being, acting as the main cellular buffer. However, its biological and redox activity has been mostly neglected to date. Ischemia is an event in which an inhibition of nutrient availablity and oxygen flow occurs, while reperfusion is the return of nutrients and oxygen, accompanied of a burst of reactive oxygen species production and cell death. Here, we studied the effects of bicarbonate during cardiac ischemia-reperfusion. In our model, we kept the pH stable and changed the concentration of the bicarbonate. We then subjected cells, organs and animals to ischemia-reperfusion under conditions where there was no presence, basal blood concentration or a higher concentration of bicarbonate. In these diverse models, we found that the presence of bicarbonate increased damage after a ischemia-reperfusion, and promoted the accumulation of oxidized proteins. Bicarbonate did not change respiration, production of reactive oxygen species or the morphology of the mitochondria. There were also no changes in proteasome activity and in global autophagy markers, although there was an accumulation of mitophagy markers. We also found that mitophagy was responsible for the increased damage observed, since pharmacological inhibiting of autophagy abolished the increased damage caused by the presence of bicarbonate. In conclusion the presence of bicarbonate is deleterious in ischemia-reperfusion due mitophagy inhibition

Em todo o mundo, milhões de pacientes são atendidos em emergências por apresentarem dor torácica de início aguda, mas apenas uma parcela deve-se a síndrome coronariana aguda (SCA). Em situações como essa é de extrema importância distinguir quando a dor torácica é devido à isquemia do miocárdio, pois esta é de alto risco e o início do tratamento deve ser imediato. Novos biomarcadores são necessários para auxiliar no diagnóstico e conduta clínica a ser tomada diante de situações de emergência como esta. Recentemente a quantificação de troponinas através de ensaios ultrassensíveis tem sido amplamente utilizado para diagnósticos e prognóstico de isquemia cardíaca, porém esses ensaios não tiveram seus valores de referências estabelecidos e validados para diversas situações clínicas. O presente estudo identificou a troponina I cardíaca nitrada como um novo biomarcador para isquemia cardíaca. Através de experimentos de imunoluorecência, foi possível colocalizar a marcação de troponina I cardíaca e nitrotirosina em modelos celulares e murinos de isquemia cardíaca, sugerindo assim que a troponina I cardíaca é nitrada. A partir do soro de modelos porcinos de isquemia, foi realizado o enriquecimento de proteínas nitradas por imunoprecipitação seguido da identificação da troponina I cardíaca por western blot. Dessa maneira foi possível identificar a troponina I cardíaca nitrada no soro poucos minutos após o evento x isquêmico, a qual permaneceu circulante por até 24 horas. Nessas mesmas amostras outros biomarcadores de isquemia como CKMB, Troponina I e Troponina T ultrassensível foram dosados e nenhum marcador de elevou após a isquemia cardíaca seguida de reperfusão. A troponina I cardíaca nitrada foi caracterizada por espectrometria de massas. Esse proteína é um potencial marcador circulante sensível para o diagnóstico e prognóstico precoce de isquemia cardíaca com ou sem necrose do miocárdio / Worldwide, millions of patients are treated in emergencies because they had acute-onset chest pain, but only a portion is due to coronary syndrome. In situations like this is extremely important to distinguish when the chest pain is due to myocardial ischemia, as this is high risk and initiation of treatment should be immediate. New biomarkers are needed to assist clinical decision-making in ACS. Recently, the quantification of ultra-sensitive tests for troponins has been widely used for diagnosis and prognosis of myocardial ischemia, however the reference values was not well validated and established for different subjects groups. The present study identified the nitrated cardiac troponin I as a novel biomarker of cardiac ischemia. We performed immunofluorescence colocalization marking of cardiac troponin I and nitrotyrosine in cell and rat model of cardiac ischemia, suggesting that cardiac troponin I is a nitrated protein. From serum of porcine models cardiac ischemia was made enrichment of nitrated proteins by immunoprecipitation with anti-nitrotyrosine followed by detection of cardiac troponin I by western blot. It was possible to identify the cardiac troponin I in serum nitrated few minutes after the ischemic event, which remains current for up to 24 hours. In these samples, other markers of cardiac ischemia such as CK-MB, troponin I and ultra-sensitive troponin T did not increase after ischemia followed by reperfusion. Nitrated cardiac troponin I was characterized by MS/MS. The xii nitrated cardiac troponin I is a potential circulating marker sensitive for the diagnosis and prognosis for early cardiac ischemia with or without myocardial necrosis

Activation of sympathetic nervous system is a key component of myocardial remodeling that generally occurs following ischemia/reperfusion (I/R) injury and myocardial infarction. It induces cardiac myocyte apoptosis and myocardial fibrosis, leading to myocardial dysfunction. Intracellular ubiquitin (UB) regulates protein turnover by the UB-proteosome pathway. The biological functions of extracellular UB in the heart remain largely unexplored. Previously, our lab has shown that β-adrenergic receptor (β-AR) stimulation increases extracellular UB levels, and extracellular UB inhibits β-AR-stimulated apoptosis in adult rat ventricular myocytes (ARVMs). This study explores the role of extracellular UB in myocyte apoptosis, fibroblast phenotype and function, and myocardial remodeling following β-AR stimulation and I/R injury. First, left ventricular (LV) structural and functional remodeling was studied 7 days after chronic β-AR-stimulation in the presence or absence of UB infusion. Echocardiographic analyses showed UB infusion decreases β-AR-stimulated increases in percent fractional shortening and ejection fraction. It decreased cardiac myocyte apoptosis and myocardial fibrosis. UB activated Akt, and inhibition of Akt inhibited β-AR-stimulated increases in matrix metalloproteinase-2 expression. Second, using cardiac fibroblasts, we provide evidence that extracellular UB interacts with the cell surface and co-immunoprecipitates with CXCR4. UB treatment increased expression of α-smooth muscle actin (myofibroblast marker), and induced rearrangement of actin into stress fibers. It inhibited lamellopodia and filopodia formation, and cell migration into the wound. Third, using isolated mouse heart and I/R injury as a model, we provide evidence that UB treatment decreases I/R-mediated increases in infarct size. UB treatment improved functional recovery of the heart as measured by increased % LV developed pressure. Activation of proapoptotic proteins, p-STAT-1 and caspase-9, was significantly lower in UB I/R hearts versus I/R alone. In ARVMs, UB treatment decreased simulated I/R-induced apoptosis. It activated Akt (anti-apoptotic kinase) and inhibited activation of GSK-3β (pro-apoptotic kinase). It decreased I/R-induced oxidative stress and protected anoxia-induced mitochondrial polarization. In fibroblast and ARVMs, CXCR4 antagonism negated the effects of UB, while mutated UBs (unable to interact with CXCR4) had no effect. Thus, extracellular UB, most likely acting via CXCR4, modulates myocardial remodeling with effects on heart function, fibroblast phenotype and function and myocyte apoptosis.

Ataxia telangiectasia (AT), caused by mutations in the gene encoding ataxia telangiectasia mutated kinase (ATM), is a rare autosomal recessive disorder. AT individuals exhibit neuronal degeneration and are predisposed to cancer. Carriers of this disorder are predisposed to cancer and ischemic heart disease. Heart disease, mostly due to myocardial infarction (MI), is a leading cause of death in the US. Following MI, release of catecholamines in the heart stimulates β- adrenergic receptors (β-AR). Our lab has shown that β-AR stimulation increases ATM expression in the heart and myocytes, and ATM plays an important role in β-AR-stimulated myocardial remodeling with effects on function, fibrosis and apoptosis. Using wild-type (WT) and ATM heterozygous knockout (hKO) mice, this study investigated the role of ATM in the inflammatory, proliferative and maturation phases of infarct healing post-MI. During the inflammatory phase, 1 and 3 days post-MI, a deficiency of ATM resulted in decreased left ventricular dilation as measured by echocardiography. It decreased the number of neutrophils and macrophages in the heart 1 day post-MI. Myocardial fibrosis, expression of alpha-smooth muscle actin (α-sma) and apoptosis were higher in the infarct region of ATM deficient hearts. Akt activation (anti-apoptotic) was lower, while Bax expression (pro-apoptotic) was higher in the infarct region of ATM deficient hearts. During the proliferative phase, 7 days post-MI, ATM deficiency attenuated cardiac dysfunction as measured by echocardiography. ATM deficient hearts exhibited increased fibrosis and expression of α-sma in the infarct region with increased myocyte apoptosis in the border area. During the maturation phase, 14 and 28 days post-MI, ATM deficiency resulted in exaggerated cardiac function. It associated with increased fibrosis, expression of α-sma and decreased cardiac cell apoptosis in the infarct region 28 days post-MI. Myocyte hypertrophy was greater in the non-infarct region during ATM deficiency. ATM deficiency decreased expression of p16 (marker of cell senescence) and activation of proapoptotic protein, GSK-3β. Thus, ATM modulates the remodeling processes of the heart including function, fibrosis, apoptosis and hypertrophy post-MI. ATM (1) delays the inflammatory response post-MI, (2) decreases dilative remodeling during inflammatory and proliferative phases and (3) exaggerates dysfunction during the maturation phase.

β-Adrenergic receptor (β-AR) stimulation increases extracellular levels of ubiquitin (UB) in myocytes, and exogenous UB decreases β-AR-stimulated myocyte apoptosis and myocardial fibrosis. Here, we hypothesized that exogenous UB modulates the inflammatory response, thereby playing a protective role in cardiac remodeling after ischemia-reperfusion (I/R) injury. C57BL/6 mice infused with vehicle or UB (1 μg·g−1·h−1) were subjected to myocardial I/R injury. Functional and biochemical parameters of the heart were examined 3 days post-I/R. Heart weight-to-body weight ratios were similarly increased in I/R and UB + I/R groups. The area at risk and infarct size were significantly lower in UB + I/R versus I/R groups. Measurement of heart function using echocardiography revealed that I/R decreases percent fractional shortening and percent ejection fraction. However, the decrease in fractional shortening and ejection fraction was significantly lower in the UB + I/R group. The UB + I/R group displayed a significant decrease in inflammatory infiltrates, neutrophils, and macrophages versus the I/R group. Neutrophil activity was significantly lower in the UB + I/R group. Analysis of the concentration of a panel of 23 cytokines/chemokines in the serum using a Bio-Plex assay revealed a significantly lower concentration of IL-12 subunit p40 in the UB + I/R versus I/R group. The concentration of monocyte chemotactic protein-1 was lower, whereas the concentration of macrophage inflammatory protein-1α was significantly higher, in the UB+I/R group versus the sham group. Expression of matrix metalloproteinase (MMP)-2 and activity of MMP-9 were higher in the UB + I/R group versus the I/R group. Levels of ubiquitinated proteins and tissue inhibitor of metalloproteinase 2 expression were increased to a similar extent in both I/R groups. Thus, exogenous UB plays a protective role in myocardial remodeling post-I/R with effects on cardiac function, area at risk/infarct size, the inflammatory response, levels of serum cytokines/chemokines, and MMP expression and activity.

Previous studies demonstrated that intravenous administration of human umbilical cord blood (HUCB) cells could improve behavioral and neurological recovery of stroked animals following middle cerebral artery occlusion (MCAO). In addition, HUCB cell recipients had less of an inflammatory response with less leukocyte infiltration. In these studies we explored how HUCB cells change the inflammatory response of neurons, astrocytes, and microglia to hypoxia/ischemia. Initiation of the inflammatory response occurs with the expression of chemokines. We determined that monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein 1alph (MIP-1a), which are upregulated in the brain early after a stroke, induce migration of HUCB cells to the site of injury. Neutralizing these chemokines with antibodies prevented migration in an in vitro migration assay. We next explored the interaction of the whole HUCB mononuclear cell fraction, as well as subpopulations from within the mononuclear fraction (T cell alone, B cell alone, and monocytes/macrophage alone) with cultures of enriched neurons, astrocytes or microglia exposed to hypoxia in an oxygen, glucose deprivation paradigm. We showed that HUCB cells increased the cell viability of neurons and astrocytes, while decreasing cell viability of microglia. There was also a change in the cytokine secretion profile from the cells exposed to HUCB cells under hypoxic conditions. These results suggested that chemokines, MCP-1 and MIP-1a increased in stroked brain, and they played an important role in recruitment of HUCB into the CNS after intravenous administration. Once inside the brain, HUCB could suppress the immune response by promoting microglial death and modulating the function of astrocytes. In addition, HUCB cells provide neuron protection against the injury caused by stroke. However, it is unlikely to contribute the effect of HUCB to a single population of HUCB.

Improvements in medical care over recent decades have increased the number of premature and low birth weight infants that survive hypoxic-ischemic (H-I) insults. Because there is a rising incidence in diseases associated with these events, it is critical to develop effective therapies to treat the various resulting neuropathies. Extracellular matrix constitutes the majority of brain parenchyma. Lecticans and matrix-degrading proteases including ADAMTSs (a disintegrin and metalloproteinase with thrombospondin repeats) and matrix metalloproteinases (MMPs) exert effects on cell viability and may be associated with either protective or destructive processes after H-I. Both ADAMTSs (Cross et al. 2006; Tian et al. 2007) and MMPs (del Zoppo et al. 2007; Gu et al. 2005; Rosenberg et al. 2001) have been associated with pathological states in brain, yet the relative contributions of lecticans, ADAMTSs and MMPs to inflammation and cell death remain unknown. In the present study, the first series of experiments were conducted to characterize cellular damage and neuroinflammation in the postnatal day 7 rat after exposure to H-I, and to determine if cell death and inflammation were associated with alterations in lectican expression. Data showed that reduced brevican expression occurred 4 days after H-I in lesioned hippocampus. Additionally, reduced versican expression in white matter was concomitant with pre-OL cell death at this endpoint. In contrast, both lecticans were elevated at later endpoints (14, 21 days) that were associated with increased neuroinflammation and cavitary infarction. These data suggest that lectican loss is associated with cell death at the early endpoint, whereas increased lectican deposition over time likely leads to glial scar formation and a reduced capacity for neuroplasticity. Two subsequent series of experiments were conducted to determine the relative contributions of matrix-degrading proteases to injury, and whether proteolytic activity was associated with neuroinflammatory events. The first objective was to determine whether treatment with AG3340, a selective inhibitor of gelatin-degrading MMPs, or the anti-inflammatory compound minocycline, could provide neuroprotection when administered at a delayed time point after insult, and to compare the efficacy of AG3340 with that of the well-known anti-inflammatory compound minocycline. Data showed that both compounds effectively dampened the recruitment of microglia/macrophages to the lesion site when administered 24 hrs after H-I. These effects were associated with reduced neurodegeneration, indicating that these compounds neuroprotect at a clinically relevant time point. The final series of experiments tested whether these compounds could neuroprotect in an ex vivo model of oxygen glucose deprivation (OGD) that lacks peripheral immune cell involvement, thus providing insight into the relative contributions of resident microglia and gelatinase activity to the inflammatory sequelae. Results showed that both compounds blocked the OGD-induced increase in gelatinase activity and were neuroprotective in the absence of peripheral immune cells. Taken together, these data indicate that resident microglia contribute to H-I injury through gelatinase activation. Thus, the present study demonstrates that gelatin-degrading MMPs are important targets to consider when developing therapies to combat neonatal H-I injury.

Hydrogen sulfide (H2S) reduces ischemia reperfusion (IR) injury by stimulating adenosine triphosphate (ATP) sensitive potassium channels (KATP) [1-5]. Demonstrating H2S stimulation is unique to KATP, as other inwardly rectifying potassium (Kir) channels demonstrate inhibition or are unaffected [6]. We recently showed that H2S inhibits Kir2 and Kir3 by decreasing channel sensitivity to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2) [6]. Here, we test the hypothesis that H2S regulation of Kir6.2, a pore-forming subunit of the KATP channel, is also dependent on PIP2. Using whole-cell patch-clamp we show that H2S increases the activity of Kir6.2 channels expressed in HEK-293 cells. To study the mechanism, we modulated PIP2 levels by expressing a light- activated phosphatase, or by including high levels of a water-soluble PIP2 analog in the patch pipette. The results suggest that H2S augmentation of Kir6.2 channel activity is increased when PIP2 levels are elevated.