Baicalein Preconditioning Cardioprotection Involves Pro-Oxidant Signaling and Activation of Pyruvate Dehydrogenase.

Preconditioning has a powerful protective potential against myocardial ischemia-reperfusion injury (I/R). Our prior work demonstrated that baicalein, a flavonoid derived from the root of Scatellaria baicalensis Georgi (also known as Huangqin), confers this preconditioning protection. This study further explored the mechanisms of baicalein preconditioning (BC-PC) in mouse cardiomyocytes. Cells were treated with baicalein (10 µM) for a brief period of time (10 min) prior to simulated ischemia 90 min/reperfusion for 180 min. Baicalein triggered an induction of a small amount of mitochondrial reactive oxygen species (ROS) prior to the initiation of ischemia, assessed by 6-carboxy-2', 7'-dichlorodihydrofluorescein diacetate (6-carboxy-H2DCFDA). It also significantly increased cell viability measured by propidium iodide (PI) and lactate dehydrogenase and preserved mitochondrial membrane potential assessed by TMRM fluorescence intensity. Myxothiazol, a mitochondrial electron transport chain complex III inhibitor, partially blocked ROS generation induced by BC-PC and reduced cell viability. BC-PC increased phosphorylation of Akt (Thr308 and Ser473) and eNOS Ser1177, and nitric oxide (NO) production measured using 4,5-diaminofluorescein diacetate (DAF-2 DA, 1 µM). Akt inhibitor API-2 abolished Akt phosphorylation and reduced DAF-2 production and cell viability. In addition, BC-PC decreased phosphorylation of pyruvate dehydrogenase (PDH) reflecting upregulated PDH activity, and increased ATP production at 30 min during reperfusion. Taken together, baicalein preconditioning-induced cardioprotection involves pro-oxidant generation, activates survival signaling Akt/eNOS/NO, and improves metabolic recovery after I/R injury. Our work provides new perspectives on the effect of baicalein on cardiac preconditioning against I/R injury.

Therapeutic hypothermia cardioprotection via Akt- and nitric oxide-mediated attenuation of mitochondrial oxidants.

Therapeutic hypothermia (TH) is a promising cardioprotective treatment for cardiac arrest and acute myocardial infarction, but its cytoprotective mechanisms remain unknown. In this study, we developed a murine cardiomyocyte model of ischemia-reperfusion injury to better determine the mechanisms of TH cardioprotection. We hypothesized that TH manipulates Akt, a survival kinase that mediates mitochondrial protection by modulating reactive oxygen species (ROS) and nitric oxide (NO) generation. Cardiomyocytes, isolated from 1- to 2-day-old C57BL6/J mice, were exposed to 90 min simulated ischemia and 3 h reperfusion. For TH, cells were cooled to 32 degrees C during the last 20 min of ischemia and the first hour of reperfusion. Cell viability was evaluated by propidium iodide and lactate dehydrogenase release. ROS production was measured by 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate and mitochondrial membrane potential (DeltaPsim) by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazoly-carbocyanine iodide (JC-1). Phospho (p)-Akt (Thr308), p-Akt (Ser473), and phosphorylated heat shock protein 27 (p-HSP27) (Ser82) were analyzed by Western blot analysis. TH attenuated reperfusion ROS generation, increased NO, maintained DeltaPsim, and decreased cell death [19.3 + or - 3.3% (n = 11) vs. 44.7 + or - 2.7% (n = 10), P < 0.001]. TH also increased p-Akt during ischemia before reperfusion. TH protection and attenuation of ROS were blocked by the inhibition of Akt and NO synthase but not by a cGMP inhibitor. HSP27, a regulator of Akt, also exhibited increased phosphorylation (Ser82) during ischemia with TH. We conclude that TH cardioprotection is mediated by enhanced Akt/HSP27 phosphorylation and enhanced NO generation, resulting in the attenuation of ROS generation and the maintenance of DeltaPsim following ischemia-reperfusion.

Akt1 genetic deficiency limits hypothermia cardioprotection following murine cardiac arrest.

Therapeutic hypothermia (TH) cardioprotection has recently been associated with increased Akt signaling in a rat model of cardiac arrest. However, it is not known whether Akt is required for this beneficial effect of TH. We used a mouse model of cardiac arrest demonstrating TH cardioprotection to study the response of mice deficient in an Akt1 allele. We hypothesized that Akt1 mediates TH cardioprotection and that decreases in Akt1 content would diminish such protection. Adult C57BL/6 wild-type (WT) mice underwent an 8-min cardiac arrest. After 6 min, the mice were randomized to normothermia (WT(NT), 37 degrees C) or TH (WT(TH), 30 degrees C). Following cardiopulmonary resuscitation and the return of spontaneous circulation (ROSC), the animals were hemodynamically monitored for 240 min (R240). At R240, cardiac tissue Akt content and phosphorylation were assayed. Studies were repeated in Akt1 heterozygous (Akt1(+/-)) mice. As a result, baseline characteristics and ROSC rates were equivalent across groups. At R240, WT(TH) mice exhibited lower heart rate, larger stroke volume, and higher cardiac output than WT(NT) animals (P < 0.05). Cardioprotection in WT(TH) at R240 was associated with increased cardiac Akt phosphorylation at Ser473 and Thr308 compared with that in WT(NT) (P < 0.05). TH-associated alterations in Akt phosphorylation, stroke volume, heart rate, and cardiac output were abrogated in Akt1(+/-) animals. In conclusion, TH improves post-ROSC cardiac function and increases Akt phosphorylation in WT, but not Akt1(+/-), mice. The Akt1 isoform appears necessary for TH-mediated cardioprotection.

Grape seed proanthocyanidins protect cardiomyocytes from ischemia and reperfusion injury via Akt-NOS signaling.

Ischemia/reperfusion (I/R) injury in cardiomyocytes is related to excess reactive oxygen species (ROS) generation and can be modulated by nitric oxide (NO). We have previously shown that grape seed proanthocyanidin extract (GSPE), a naturally occurring antioxidant, decreased ROS and may potentially stimulate NO production. In this study, we investigated whether GSPE administration at reperfusion was associated with cardioprotection and enhanced NO production in a cardiomyocyte I/R model. GSPE attenuated I/R-induced cell death [18.0 +/- 1.8% (GSPE, 50 microg/ml) vs. 42.3 +/- 3.0% (I/R control), P < 0.001], restored contractility (6/6 vs. 0/6, respectively), and increased NO release. The NO synthase (NOS) inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME, 200 microM) significantly reduced GSPE-induced NO release and its associated cardioprotection [32.7 +/- 2.7% (GSPE + L-NAME) vs. 18.0 +/- 1.8% (GSPE alone), P < 0.01]. To determine whether GSPE induced NO production was mediated by the Akt-eNOS pathway, we utilized the Akt inhibitor API-2. API-2 (10 microM) abrogated GSPE-induced protection [44.3% +/- 2.2% (GSPE + API-2) vs. 27.0% +/- 4.3% (GSPE alone), P < 0.01], attenuated the enhanced phosphorylation of Akt at Ser473 in GSPE-treated cells and attenuated GSPE-induced NO increases. Simultaneously blocking NOS activation (L-NAME) and Akt (API-2) resulted in decreased NO levels similar to using each inhibitor independently. These data suggest that in the context of GSPE stimulation, Akt may help activate eNOS, leading to protective levels of NO. GSPE offers an alternative approach to therapeutic cardioprotection against I/R injury and may offer unique opportunities to improve cardiovascular health by enhancing NO production and increasing Akt-eNOS signaling.

Altering CO2 during reperfusion of ischemic cardiomyocytes modifies mitochondrial oxidant injury.

OBJECTIVE: Acute changes in tissue CO2 and pH during reperfusion of the ischemic heart may affect ischemia/reperfusion injury. We tested whether gradual vs. acute decreases in CO2 after cardiomyocyte ischemia affect reperfusion oxidants and injury. DESIGN: Comparative laboratory investigation. SETTING: Institutional laboratory. SUBJECTS: Embryonic chick cardiomyocytes. INTERVENTIONS: Microscope fields of approximately 500 chick cardiomyocytes were monitored throughout 1 hr of simulated ischemia (PO2 of 3-5 torr, PCO2 of 144 torr, pH 6.8), followed by 3 hrs of reperfusion (PO2 of 149 torr, PCO2 of 36 torr, pH 7.4), and compared with cells reperfused with relative hypercarbia (PCO2 of 71 torr, pH 6.8) or hypocarbia (PCO2 of 7 torr, pH 7.9). MEASUREMENTS AND MAIN RESULTS: The measured outcomes included cell viability (via propidium iodide) and oxidant generation (reactive oxygen species via 2',7'-dichlorofluorescin oxidation and nitric oxide [NO] via 4,5-diaminofluorescein diacetate oxidation). Compared with normocarbic reperfusion, hypercarbia significantly reduced cell death from 54.8% +/- 4.0% to 26.3% +/- 2.8% (p < .001), significantly decreased reperfusion reactive oxygen species (p < .05), and increased NO at a later phase of reperfusion (p < .01). The NO synthase inhibitor N-nitro-L-arginine methyl ester (200 microM) reversed this oxidant attenuation (p < .05), NO increase (p < .05), and the cardioprotection conferred by hypercarbic reperfusion (increasing death to 54.3% +/- 6.0% [p < .05]). Conversely, hypocarbic reperfusion increased cell death to 80.4% +/- 4.5% (p < .01). It also increased reactive oxygen species by almost two-fold (p = .052), without affecting the NO level thereafter. Increased reactive oxygen species was attenuated by the mitochondrial complex III inhibitor stigmatellin (20 nM) when given at reperfusion (p < .05). Cell death also decreased from 85.9% +/- 4.5% to 52.2% +/- 6.5% (p < .01). The nicotinamide adenine dinucleotide phosphate oxidase inhibitor apocynin (300 microM) had no effect on reperfusion reactive oxygen species. CONCLUSIONS: Altering CO2 content during reperfusion can significantly affect myocardial postresuscitation injury, in part by modifying mitochondrial oxidants and NO synthase-induced NO production.

Glycosylation and annexin II cell surface translocation mediate airway epithelial wound repair.

Glycosylation of cell surface proteins can regulate multiple cellular functions. We hypothesized that glycosylation and expression of glycoproteins after epithelial injury is important in mediating repair. We report the use of an in vitro culture model of human airway epithelial cells (1HAEo(-)) to identify mediators of epithelial repair. We characterized carbohydrate moieties associated with repair by their interaction with the lectin from Cicer arietinum, chickpea agglutinin (CPA). Using CPA, we identified changes in cell surface glycosylation during wound repair. Following mechanical wounding of confluent monolayers of 1HAEo(-) cells, CPA staining increases on the cell surface of groups of cells in proximity to the wound edge. Blocking the CPA carbohydrate ligand inhibited wound repair highlighting the role of the CPA carbohydrate ligand in epithelial repair. Annexin II (AII), a calcium-dependent, membrane-associated protein, was identified as a protein associated with the CPA ligand. By membrane protein biotinylation and immunodetection, we have shown that following mechanical wounding, the presentation of AII on the cell surface increases coordinate with repair. Cell surface AII accumulates in proximity to the wound. Furthermore, translocation of AII to the cell surface is N-glycosylation dependent. We are the first to demonstrate that following injury, N-glycosylation events and AII presentation on the cell surface of airway epithelial cells are important mediators in repair.

Airway epithelial wound repair: role of carbohydrate sialyl Lewisx.

Epithelial repair is a complex cellular and molecular process, the details of which are still not clearly understood. Plasma membrane glycoconjugates can modulate cell function by altering the function of protein and lipids. Sialyl Lewisx (sLex), a fucose-containing tetrasaccharide, decorates membrane-bound and secreted proteins and mediates cell-cell interaction. In the present study we investigated the role of sLex in airway epithelial repair. Using immunohistochemistry, we showed an increased expression of sLex in areas of damaged bronchial epithelium compared with intact regions. Confluent monolayers of airway epithelial cells were mechanically wounded and allowed to close. Wounded monolayers were photographed for wound closure kinetics, fixed for immunocytochemical studies, or subjected to RNA extraction. Examining the expression of different alpha1,3-fucosyltransferases (FucT), enzymes that mediate the final step in the synthesis of sLex, we found that FucT-IV was the common gene expressed in all cell lines and primary airway epithelial cells. We demonstrated an increased expression of sLex over time after mechanical injury. Blocking of sLex with an inhibitory antibody completely prevented epithelial repair. Our data suggest an essential functional role for sLex in epithelial repair. Further studies are necessary to explore the exact mechanism for sLex in mediating cell-cell interaction in bronchial epithelial cells to facilitate epithelial migration and repair.