John E. Baker

Heat Shock Protein 90 Mediates the Balance of Nitric Oxide and Superoxide Anion from Endothelial Nitric-oxide Synthase

The balance of nitric oxide (·NO) and superoxide anion (O⨪2) plays an important role in vascular biology. The association of heat shock protein 90 (Hsp90) with endothelial nitric-oxide synthase (eNOS) is a critical step in the mechanisms by which eNOS generates ·NO. As eNOS is capable of generating both ·NO and O⨪2, we hypothesized that Hsp90 might also mediate eNOS-dependent O⨪2 production. To test this hypothesis, bovine coronary endothelial cells (BCEC) were pretreated with geldanamycin (GA, 10 μg/ml; 17.8 μm) and then stimulated with the calcium ionophore,A23187 (5 μm). GA significantly decreasedA23187-stimulated eNOS-dependent nitrite production (p < 0.001, n = 4) and significantly increased A23187-stimulated eNOS-dependent O⨪2production (p < 0.001, n = 8).A23187 increased phospho-eNOS(Ser-1179) levels by >1.6-fold over vehicle (V)-treated levels. Pretreatment with GA by itself or with A23187 increased phospho-eNOS levels. In unstimulated V-treated BCEC cultures low amounts of Hsp90 were found to associate with eNOS. Pretreatment with GA and/or A23187 increased the association of Hsp90 with eNOS. These data show that Hsp90 is essential for eNOS-dependent ·NO production and that inhibition of ATP-dependent conformational changes in Hsp90 uncouples eNOS activity and increases eNOS-dependent O⨪2production.

Chronic Hypoxia Activates Lung 15-Lipoxygenase, Which Catalyzes Production of 15-HETE and Enhances Constriction in Neonatal Rabbit Pulmonary Arteries

Abstract— Hypoxia causes localized pulmonary arterial (PA) constriction to divert blood flow to optimally ventilated regions of the lung. The biochemical mechanisms for this have remained elusive, especially during prolonged exposures to reduced Po2. We have evidence that subacute hypoxia activates 15-lipoxygenase (15-LO) in small PAs of neonatal rabbits maintained for 9 days in hypoxic environments (Fio2=0.12) compared with siblings raised under normoxia. PA microsomal products of 15-LO, 15-hydroxyeicosatetraenoic acid (HETE), 11,14,15-trihydroxyeicosatrienoic acid (THETA), and 11,12,15-THETA were identified by gas chromatography/mass spectrometry. Increased amounts of these products are synthesized in vivo and in vitro by the lungs of animal raised in hypoxic versus normoxic environments. 15-HETE formation is attenuated by lipoxygenase, but not cytochrome P450 or cyclooxygenase inhibitors. Activation of 15-LO is associated with translocation of the enzyme from the cytosol to membrane as seen by Western immunoblotting. Immunohistochemical analysis demonstrates that 15-LO expression is clearly localized in vascular cells in lungs from normoxic and hypoxic kits. 15-HETE causes concentration-dependent constriction of PA rings from animals exposed to hypoxic but not normoxic environments. In addition, lipoxygenase inhibitors reduce phenylephrine-induced constriction of PA rings. Therefore, subacute hypoxia increases expression of and activates 15-LO, and enhances sensitivity of pulmonary arteries to its product, 15-HETE. Because 15-HETE is a constrictor in this vascular bed, it may play an important role in hypoxia-induced pulmonary vasoconstriction in rabbit kits. Although a clear causal relationship remains to be demonstrated, these data suggest a previously unrecognized role for 15-LO in hypoxic vasoconstriction in neonatal mammals.

Erythropoietin protects the infant heart against ischemia–reperfusion injury by triggering multiple signaling pathways

Abstract The immediate protective effect of erythropoietin (EPO) against ischemia in heart suggests a role beyond hematopoiesis and the treatment of anemia. We determined the role of JAK/STAT and Ras/Rac/MAPK in the protective effect of EPO against ischemia–reperfusion injury in infant rabbit heart. EPO (1.0 U/ml) administered 15 minutes prior to 30–minutes global ischemia and 35 minutes reperfusion resulted in increased recovery of postischemic ventricular developed pressure in rabbit hearts. EPO exerted its immediate cardioprotective effect via activation of multiple signaling pathways by: 1) phosphorylation and activation of JAK1/2, STAT3 and STAT5A but not of STAT1α and STAT5B, 2) phosphorylation and activation of PI3 kinase and its downstream kinases Akt and Rac, 3) activation of PKCε, Raf, MEK1/2, p42/44 MAPK and p38 MAPK. Pretreatment with Wortmannin abolished EPO–induced Akt activation and phosphorylation. Pretreatment with Chelerythrine followed by EPO treatment resulted in partial inhibition of Raf activation, and abolished PKCε and p38 MAPK activation without any effect on Akt, MEK1/2 and p42/44 MAPK. PD98059 abolished MEK1/2 and p42/44 MAPK activation with no effect on Akt, Raf and p38 MAPK activation. SB203580 inhibited only p38 MAPK activation by EPO. We can conclude EPO increases immediate cardioprotection through the activation of multiple signal transduction pathways.

Intestinal microbiota determine severity of myocardial infarction in rats

Signals from the intestinal microbiota are important for normal host physiology; alteration of the microbiota (dysbiosis) is associated with multiple disease states. We determined the effect of antibiotic‐induced intestinal dysbiosis on circulating cytokine levels and severity of ischemia/reperfusion injury in the heart. Treatment of Dahl S rats with a minimally absorbed antibiotic vancomycin, in the drinking water, decreased circulating leptin levels by 38%, resulted in smaller myocardial infarcts (27% reduction), and improved recovery of postischemic mechanical function (35%) as compared with untreated controls. Vancomycin altered the abundance of intestinal bacteria and fungi, measured by 16S and 18S ribosomal DNA quantity. Pretreatment with leptin (0.12 μg/kg i.v.) 24 h before ischemia/reperfusion abolished cardioprotection produced by vancomycin treatment. Dahl S rats fed the commercially available probiotic product Goodbelly, which contains the leptin‐suppressing bacteria Lactobacillus plantarum 299v, also resulted in decreased circulating leptin levels by 41%, smaller myocardial infarcts (29% reduction), and greater recovery of postischemic mechanical function (23%). Pretreatment with leptin (0.12 μg/kg i.v.) abolished cardioprotection produced by Goodbelly. This proof‐of‐concept study is the first to identify a mechanistic link between changes in intestinal microbiota and myocardial infarction and demonstrates that a probiotic supplement can reduce myocardial infarct size.—Lam, V., Su, J., Koprowski, S., Hsu, A., Tweddell, J. S., Rafiee, P., Gross, G. J., Salzman, N. H., Baker, J. E. Intestinal microbiota determine severity of myocardial infarction in rats. FASEB J. 26, 1727‐1735 (2012). www.fasebj.org

Chronic Hypoxia Increases Endothelial Nitric Oxide Synthase Generation of Nitric Oxide by Increasing Heat Shock Protein 90 Association and Serine Phosphorylation

Abstract— Chronic hypoxia increases endothelial nitric oxide synthase (eNOS) production of nitric oxide (·NO) and cardioprotection in neonatal rabbit hearts. However, the mechanism by which this occurs remains unclear. Recent studies suggest that heat shock protein 90 (hsp90) alters eNOS function. In the present study, we examined the role of hsp90 in eNOS-dependent cardioprotection in neonatal rabbit hearts. Chronic hypoxia increased recovery of postischemic left ventricular developed pressure (LVDP). Geldanamycin (GA), which inhibits hsp90 and increases oxidative stress, decreased functional recovery in normoxic and hypoxic hearts. To determine if a loss in ·NO, afforded by GA, decreased recovery, GA-treated hearts were perfused with S-nitrosoglutathione (GSNO) as a source of ·NO. GSNO increased recovery of postischemic LVDP in GA-treated normoxic and hypoxic hearts to baseline levels. Although chronic hypoxia decreased phosphorylated eNOS (S1177) levels by ≈4- to 5-fold and total Akt and phosphorylated Akt by 4- and 5-fold, it also increased hsp90 association with eNOS by more than 3-fold. Using hydroethidine (HEt), a fluorescent probe for superoxide, we found that hypoxic hearts contained less ethidine (Et) staining than normoxic hearts. Normoxic hearts generated 3 times more superoxide by an N&ohgr;-nitro-l-arginine methyl ester (L-NAME)-inhibitable mechanism than hypoxic hearts. Taken together, these data indicate that the association of hsp90 with eNOS is important for increasing ·NO production and limiting eNOS-dependent superoxide anion generation. Such changes in eNOS function appear to play a critical role in protecting the myocardium against ischemic injury.

SCH 79797, a selective PAR1 antagonist, limits myocardial ischemia/reperfusion injury in rat hearts

Myocardial ischemia/reperfusion (I/R) injury is partly mediated by thrombin. In support, the functional inhibition of thrombin has been shown to decrease infarct size after I/R. Several cellular responses to thrombin are mediated by a G-protein coupled protease-activated receptor 1 (PAR1).However, the role of PAR1 in myocardial I/R injury has not been well characterized. Therefore, we hypothesized that PAR1 inhibition will reduce the amount of myocardial I/R injury. After we detected the presence of PAR1 mRNA and protein in the rat heart by RT-PCR and immunoblot analysis,we assessed the potential protective role of SCH 79797, a selective PAR1 antagonist, in two rat models of myocardial I/R injury. SCH 79797 treatment immediately before or during ischemia reduced myocardial necrosis following I/R in the intact rat heart. This response was dose-dependent with the optimal dose being 25 μg/kg IV. Likewise, SCH 79797 treatment before ischemia in the isolated heart model reduced infarct size and increased ventricular recovery following I/R in the isolated heart model with an optimal concentration of 1 μM. This reduction was abolished by a PAR1 selective agonist. SCH 79797-induced resistance to myocardial ischemia was abolished by wortmannin, an inhibitor of PI3 kinase; L-NMA, a NOS inhibitor; and glibenclamide, a nonselective KATP channel blocker. PAR1 activating peptide,wortmannin, L-NMA and glibenclamide alone had no effect on functional recovery or infarct size. A single treatment of SCH 79797 administered prior to or during ischemia confers immediate cardioprotection suggesting a potential therapeutic role of PAR1 antagonist in the treatment of injury resulting from myocardial ischemia and reperfusion.

Increased tolerance of the chronically hypoxic immature heart to ischemia. Contribution of the KATP channel.

BACKGROUND Hypoxia from birth in immature rabbits increases the tolerance of isolated hearts to ischemia compared with age-matched normoxic rabbits. We determined whether this increased tolerance to ischemia was due to an alteration in the ATP-sensitive potassium (KATP) channel and whether increased KATP channel activation was associated with increases in intracellular lactate. METHODS AND RESULTS Isolated immature rabbit hearts (7 to 10 days old) were perfused with bicarbonate buffer at 39 degrees C in the Langendorff mode at a constant pressure. Saline-filled latex balloons were placed in the left and right ventricles for measurement of developed pressure. A KATP channel agonist (bimakalim) or a KATP channel antagonist (glibenclamide) was added 15 minutes before a global ischemic period of 18 minutes, followed by 35 minutes of reperfusion. Rabbits raised from birth in hypoxic conditions (FIO2 = 0.12) displayed significantly enhanced recovery of developed pressure. The right ventricle was more tolerant of ischemia than the left ventricle in normoxic and hypoxic hearts. Bimakalim (1 mumol/L) increased the recovery of left ventricular developed pressure in normoxic hearts to values not different from those of hypoxic controls (43 +/- 3% to 67 +/- 5%) and slightly increased developed pressure in hypoxic hearts (67 +/- 5% to 72 +/- 5%). Glibenclamide (3 mumol/L) abolished the cardioprotective effect of hypoxia (67 +/- 5% to 43 +/- 5%). Constant-flow studies indicated that the effects of bimakalim and glibenclamide were independent of their actions on coronary flow. Ventricular lactate and lactate dehydrogenase concentrations were elevated in hypoxic hearts compared with normoxic control hearts. CONCLUSIONS Increased tolerance to ischemia exhibited by chronically hypoxic rabbit hearts is associated with increased activation of the KATP channel. This increased KATP activity may be the result of increased intracellular concentrations of lactate.

Activation of protein kinases in chronically hypoxic infant human and rabbit hearts: role in cardioprotection.

Background—Many infants who undergo heart surgery have a congenital cyanotic defect in which the heart is chronically perfused with hypoxic blood. However, the signaling pathways by which infant hearts adapt to chronic hypoxia and resist subsequent surgical ischemia is unknown. Method and Results—We determined the activation and translocation of protein kinase C (PKC) isoforms and mitogen activated protein kinases (MAP kinases) in 15 infants with cyanotic (Sao2<85%) or acyanotic (Sao2>95%) heart defects undergoing surgical repair and in 80 rabbits raised from birth in a hypoxic (Sao2<85%) or normoxic (Sao2>95%) environment. Tissues from infant human and rabbit hearts were processed for Western and in vitro kinase analysis. In human infants with cyanotic heart defects, PKC&egr;, p38 MAP kinase, and JUN kinase but not p42/44 MAP kinase were activated and translocated from the cytosolic to the particulate fraction compared with acyanotic heart defects. In rabbit infants there was a parallel response for PKC&egr;, p38 MAP kinase, and JUN kinase similar to humans. In infant rabbit hearts inhibition of PKC&egr; with chelerythrine, p38 MAP kinase, with SB203580 and JUN kinase with curcumin abolished the cardioprotective effects of chronic hypoxia but had no effects on normoxic hearts. Conclusions—Infant human and rabbit hearts adapt to chronic hypoxia through activation of PKC&egr;, p38 MAP kinase, and JUN kinase signal transduction pathways. These pathways may be responsible for cardioprotection in the chronically hypoxic infant rabbit heart.

Preconditioning in Immature Rabbit Hearts Role of KATP Channels

BACKGROUND The protective effects of ischemic preconditioning have been shown to occur in adult hearts of all species studied. We determined whether immature hearts normoxic or chronically hypoxic from birth could be preconditioned, the time window or memory of the cardioprotective effect, and the involvement of the KATP channel. METHODS AND RESULTS Isolated immature rabbit hearts (7 to 10 days old) were subjected to 0, 1, or 3 cycles of preconditioning consisting of 5 minutes of global ischemia plus 10 minutes of reperfusion. This was followed by 30 minutes of global ischemia and 35 minutes of reperfusion. Normoxic hearts (FIO2=0.21) subjected to 1 cycle of preconditioning recovered 70+/-7% of left ventricular developed pressure compared with 43+/-8% recovery in nonpreconditioned controls. Three cycles of preconditioning did not result in additional recovery (63+/-8%). Hearts from rabbits raised from birth in hypoxic conditions (FIO2=0.12) and subjected to 1 and 3 preconditioning cycles did not show increased recovery (68+/-8% and 65+/-5%) compared with nonpreconditioned hypoxic controls (63+/-9%), although the recovery was greater in chronically hypoxic hearts than in age-matched normoxic controls. Increasing the recovery period after the preconditioning stimulus from 10 to 30 minutes resulted in a loss of cardioprotection. Pretreatment of normoxic hearts for 30 minutes with the KATP channel blocker 5-hydroxydecanoate (300 micromol/L) completely abolished preconditioning (70+/-7% to 35+/-9%) but had no effect on nonpreconditioned hearts (40+/-8%). CONCLUSIONS Immature hearts normoxic from birth can be preconditioned, whereas immature hearts hypoxic from birth cannot. Preconditioning in normoxic immature hearts is associated with activation of the KATP channel.

Chronic myocardial hypoxia increases nitric oxide synthase and decreases caveolin-3.

Nitric oxide synthase (NOS) is believed to play an important role in protecting the myocardium against ischemia. Chronic hypoxia from birth increases NOS activity in the myocardium resulting in enhanced nitric oxide production and increased resistance to ischemia. We examined the effects of chronic hypoxia on NOS gene and protein expression and on NOS protein association with caveolin-3. Rabbits were raised from birth in a normoxic (F(I)O(2) = 0.21) or a hypoxic (F(I)O(2) = 0.12) environment for 9 d, and then the hearts were isolated. Ribonuclease protection assays revealed that chronic hypoxia did not alter NOS transcript levels for NOS1, NOS2, or NOS3. The most abundant transcript was NOS3. Western analysis revealed NOS3 was the only isoform detected. Immunoblots of NOS3 immunoprecipitates showed that chronic hypoxia increases NOS3 protein by 2.0 +/- 0.4-fold and decreases the amount of caveolin-3 that can be coprecipitated with NOS3 by 5.5 +/- 0.9-fold. Immunoblots of normoxic and hypoxic hearts showed that chronic hypoxia decreases the amount of caveolin-3 in heart homogenates by 2. 2 +/- 0.5-fold. These data suggest that a decrease in caveolin-3 plays a role in the mechanisms by which chronic hypoxia increases NOS3 activity in the myocardium.