During myocardial ischemia, connexin 43 (Cx43) is dephosphorylated in vitro, and the subsequent opening of gap junctions formed by two opposing Cx43 hexamers was suggested to propagate ischemia/reperfusion injury. Reduction of infarct size (IS) by ischemic preconditioning (IP) involves activation of protein kinase C (PKC) and p38 mitogen activated protein kinase (MAPK), both of which can phosphorylate Cx43. We now studied in anesthetized pigs whether IP impacts on Cx43 phosphorylation by measuring the density of non-phosphorylated and total Cx43 (confocal laser) during normoperfusion and 90-min ischemia in non-preconditioned and preconditioned hearts. Co-localization of PKCalpha, p38MAPKalpha, and p38MAPKbeta with Cx43 and the activity of p38MAPK were assessed. IP by 10 min ischemia and 15 min reperfusion reduced IS. Non-phosphorylated Cx43 remained unchanged during ischemia in preconditioned hearts, while it increased from 35+/-3 to 75+/-8 AU (P<0.05) in non-preconditioned hearts. Co-localization of PKCalpha, p38MAPKalpha, and p38MAPKbeta with Cx43 during ischemia increased only in preconditioned hearts. While the ischemia-induced increase in p38MAPKalpha activity was comparable in preconditioned and non-preconditioned hearts, p38MAPKbeta activity was increased only in preconditioned hearts. Blockade of p38MAPK by SB203580 attenuated the IS-reduction and the increased p38MAPK-Cx43 co-localization by IP. We conclude that IP increases co-localization of protein kinases with Cx43 and preserves phosphorylation of Cx43 during ischemia.

Abstract-Higher doses of inhaled NO exert effects beyond the pulmonary circulation. How such extrapulmonary effects can be reconciled with the presumed short half-life of NO in the blood is unclear. Whereas erythrocytes have been suggested to participate in NO transport, the exact role of plasma in NO delivery in humans is not clear. Therefore, we investigated potential routes of NO decomposition and transport in human plasma. NO consumption in plasma was accompanied by a concentration-dependent increase in nitrite and S-nitrosothiols (RSNOs), with no apparent saturation limit up to 200 mol/L. The supposedly rapid conversion of NO to biologically inactive metabolites in human blood formed the rationale for inhalation NO therapy, because the short half-life of NO should confine its effect to the pulmonary circulation. 4 However, recent evidence suggests that higher doses of inhaled NO may exert side effects beyond the pulmonary circulation. 5,6 Red blood cells (RBCs) are believed to be a major sink for NO by virtue of the rapid co-oxidation reaction of NO with oxyhemoglobin to form methemoglobin (metHb) and nitrate. Alternatively, NO may react with hemoglobin (Hb) to form either nitrosylhemoglobin (NOHb) or S-nitrosohemoglobin (SNOHb). 6,7 In addition to its reaction with RBCs, NO has to interact at some stage with plasma constituents, especially in view of the existence of an RBC-free zone close to the vessel wall. 8 A better knowledge of the fate of NO in plasma is an important prerequisite for a proper understanding of its physiology in blood and in the human circulation in general.Recently, we provided evidence that intra-arterially applied NO can be transported in a bioactive form over significant distances along the forearm circulation. 9 To date, no data have been reported on the systemic dilator effects of intravenously applied NO in the human peripheral vasculature, presumably because of its supposedly rapid clearance from blood. Our data challenge this current dogma. In the present study, we demonstrate that the intravenous infusion of NO solution results in the transport and delivery of NO as S-nitrosothiols (RSNOs), which are accompanied by systemic hemodynamic effects and vasodilation in conduit and resistance vessels.

Protein kinase C⑀ (PKC⑀) plays a central role in ischemic preconditioning (IP) in mice and rabbits, and activated PKC⑀ colocalizes with and phosphorylates connexin43 (Cx43) in rats and humans. Whether or not Cx43 contributes to the mechanism(s) of IP in vivo is yet unknown. Therefore, wild-type (n ϭ 8) and heterozygous Cx43-deficient mice (n ϭ 8) were subjected to 30 min occlusion and 120 min reperfusion of the left anterior descending coronary artery. IP was induced by one cycle of 5 min occlusion and 10 min reperfusion (n ϭ 8/8 mice) before the sustained occlusion. Infarct size was reduced by IP in wild-type mice [11.3 Ϯ 3.4% vs. 23.7 Ϯ 7.2% of the left ventricle (LV), P Ͻ 0.05] but not in Cx43-deficient mice (26.0 Ϯ 6.0% vs. 25.1 Ϯ 3.8% of LV). Also, three cycles of 5 min occlusion and 10 min reperfusion (n ϭ 5) did not induce protection in Cx43-deficient mice (27.6 Ϯ 5.5 % of LV). Thus Cx43 contributes to the protection of IP in mice in vivo. mouse heart in situ; infarct size; gap junctions ISCHEMIC PRECONDITIONING (IP) by brief episodes of ischemia-reperfusion protects the myocardium from the damage induced by a subsequent more prolonged ischemia. Several triggers and mediators of IP have been identified, whereas the final end effector is still unknown (15). Protein kinase C⑀ (PKC⑀) is an established mediator of IP in mice and rabbits, whereas other PKC isoforms may be more important in other species (15). PKC⑀ is involved in signaling complexes with at least 36 proteins (1, 9, 18), among them connexin43 (Cx43) (10), an integral protein of myocardial gap junctions. Activated PKC⑀ colocalizes with Cx43 and contributes to phosphorylation of Cx43 in rats (3) and humans (2), which might then modulate gap junction transmission characteristics and intercellular communication. Indeed, in isolated mouse hearts, uncoupling of gap junctions using heptanol abolished infarct size reduction by IP (8). The data on the effect of gap junction uncoupling on infarct size per se are controversial. Pretreatment with the gap junction uncoupler heptanol had no effect on infarct size in isolated rabbit hearts (6), but heptanol given during early ischemia decreased infarct size in isolated rabbit hearts (16). Also, in pigs in vivo heptanol given during early reperfusion decreased infarct size (4). Data on the importance of Cx43 for IP in vivo, however, are lacking. We therefore studied whether or not Cx43 is involved in the cardioprotection by IP using heterozygous Cx43-deficient mice. EXPERIMENTAL PROCEDURESThe experimental protocols were approved by the bioethics committee of the district of Dü sseldorf, Germany. Mice were handled according to the guidelines of the American Physiological Society.We used the in situ mouse heart model developed by Guo et al. (5). Briefly, male and female C57BL/6J wild-type and heterozygous Cx43-deficient mice (B6.129-Gja1 tm1Kdr , JAX mice; Bar Harbor, ME) (12) (weight: 30.5 Ϯ 4.5 g, age: 14-20 wk) were anesthetized with pentobarbital sodium (80 mg/kg ip) and atropine sulfate (0.04 mg/kg ip). Ele...

Background-The frequency and importance of microembolization in patients with acute coronary syndromes and during coronary interventions have recently been appreciated. Experimental microembolization induces immediate ischemic dysfunction, which recovers within minutes. Subsequently, progressive contractile dysfunction develops over several hours and is not associated with reduced regional myocardial blood flow (perfusion-contraction mismatch) but rather with a local inflammatory reaction. We have now studied the effect of antiinflammatory glucocorticoid treatment on this progressive contractile dysfunction. Methods and Results-Microembolization was induced by injecting microspheres (42-m diameter) into the left circumflex coronary artery. Anesthetized dogs were followed up for 8 hours and received placebo (nϭ7) or methylprednisolone 30 mg/kg IV either 30 minutes before (nϭ7) or 30 minutes after (nϭ5) microembolization. In addition, chronically instrumented dogs received either placebo (nϭ4) or methylprednisolone (nϭ4) 30 minutes after microembolization and were followed up for 1 week. In acute placebo dogs, posterior systolic wall thickening was decreased from 20.0Ϯ2.1% (meanϮSEM) at baseline to 5.8Ϯ0.6% at 8 hours after microembolization. Methylprednisolone prevented the progressive myocardial dysfunction. Increased leukocyte infiltration in the embolized myocardium was prevented only when methylprednisolone was given before microembolization. In chronic placebo dogs, progressive dysfunction recovered from 5.0Ϯ0.7% at 4 to 6 hours after microembolization back to baseline (19.1Ϯ1.6%) within 5 days. Again, methylprednisolone prevented the progressive myocardial dysfunction. Conclusions-Methylprednisolone, even when given after microembolization, prevents progressive contractile dysfunction.

In animals and patients with severe heart failure (HF), the serum tumor necrosis factor-alpha (TNF-alpha) concentration is increased. It is, however, still controversial whether or not such increased serum TNF-alpha originates from the heart itself or is of peripheral origin secondary to gastrointestinal congestion and increased endotoxin concentration. We therefore now examined TNF-alpha in serum, myocardium, and liver of sham-operated and HF rabbits. In nine rabbits in which HF was induced by left ventricular (LV) pacing at 400 beats/min for 3 wk, LV end-diastolic diameter was increased and systolic shortening fraction (9.4 +/- 1.0 vs. 28.5 +/- 1.3%, echocardiography, P < 0.05) was reduced. Serum TNF-alpha was higher in HF than in sham-operated rabbits (240 +/- 24 vs. 150 +/- 22 U/ml, WEHI-cell assay, P < 0.05). In the heart, TNF-alpha was located mainly in the vascular endothelium (immunohistochemistry), and TNF-alpha protein (920 +/- 160 vs. 900 +/- 95 U/g) did not differ between groups. In the liver of HF rabbits, hepatocytes expressed TNF-alpha, and TNF-alpha protein was increased compared with sham-operated rabbits (2,390 +/- 310 vs. 1,220 +/- 135 U/g, P < 0.05) and correlated to the number of hepatic leukocytes (r = 0.85) and serum TNF-alpha (r = 0.69). The intestinal endotoxin concentration was 24.5 +/- 1.2 vs. 17.0 +/- 3.1 endotoxin units/g wet wt (P < 0.05) in HF compared with sham-operated rabbits. In this HF model, serum but not myocardial TNF-alpha is increased. The increased serum TNF-alpha originates from peripheral sources.