Kimberly R. Wojcik

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 µg/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 Nω‐nitro‐L‐arginine methyl ester (L‐NAME, 200 µM) 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 µM) 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. J. Cell. Biochem. 107: 697–705, 2009.

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.

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 &mgr;M) 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 &mgr;M) 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 Profiles of Airway Epithelium After Repair of Mechanical Injury in Guinea Pigs

Glycosylated structures on the cell surface have a role in cell adhesion, migration, and proliferation. Repair of the airway epithelium after injury requires each of these processes, but the expression of cell surface glycosylation of airway epithelial cells after injury is not known. We examined cell surface glycosylation using lectin-binding profiles of normal and repairing epithelia in Hartley guinea pigs from 0 to 14 days after mechanical injury. The epithelium regenerated completely over 7 days. In normal trachea, galactose- or galactosamine-specific lectins (14 of 20 tested) labelled epithelial cells, but fucose, mannose, and other sugar-specific lectins (15 tested) did not. GSA-2, a glucosamine-specific lectin, labelled epithelial cells weakly in uninjured tracheas, but intense labelling was noted in basal and non-ciliated columnar cells adjacent to the injury site over 3 h to 14 days after injury. Labelling of these cells peaked at 12 h and 5 days after injury respectively. Similar patterns were seen with lectins AlloA and HAA but not with CPA during repair. The binding of the lectin DSA to proteins collected from primary cultures of airway epithelial cells decreased substantially after treatment for 24 h with either transforming growth factor-β or interleukin-1β, but that of the CPA lectin did not. We demonstrate changes in glycosylation profiles of airway epithelial cells coordinate with repair after mechanical injury. These changes may be useful to study mechanisms by which repair is regulated.

Blockade of caspase-2 Activity inhibits ischemia/ Reperfusion-induced Mitochondrial Reactive Oxygen Burst and cell Death in cardiomyocytes

We previously showed that initiator caspases-2 and -8 are prominently activated in ischemia/reperfusion (I/R)-induced injury in cardiomyocytes, but while blockade of caspase-2 activity enhanced cell survival, blockade of caspase-8 activity did not protect cardiomyocytes. Because apoptotic death in these cells is characterized by a burst of reactive oxygen species (ROS) at reperfusion and their survival by inhibition of this burst, we examined the effects of blocking caspase-2 and caspase-8 activities on ROS production. Caspase-2 inhibition blocked the reperfusion-induced ROS burst, while inhibition of caspase-8 did not. We also examined effects of caspase inhibition on mitochondrial membrane potential (∆ψ m) and mitochondrial function and found that blocking caspase-2, but not caspase-8, allowed recovery of ∆ψ m and mitochondrial functionality. Furthermore, knockdown of caspase-2 by small-interfering (si)RNA confirmed caspase-2 participation in cytochrome c release, which correlates with loss of ∆ψm and cell death in these cardiomyocytes.