The signal transducer and activator of transcription 3 (STAT3) contributes to cardioprotection by ischemic pre- and postconditioning. Mitochondria are central elements of cardioprotective signaling, most likely by delaying mitochondrial permeability transition pore (MPTP) opening, and STAT3 has recently been identified in mitochondria. We now characterized the mitochondrial localization of STAT3 and its impact on respiration and MPTP opening. STAT3 was mainly present in the matrix of subsarcolemmal and interfibrillar cardiomyocyte mitochondria. STAT1, but not STAT5 was also detected in mitochondria under physiological conditions. ADP-stimulated respiration was reduced in mitochondria from mice with a cardiomyocyte-specific deletion of STAT3 (STAT3-KO) versus wildtypes and in rat mitochondria treated with the STAT3 inhibitor Stattic (STAT3 inhibitory compound, 6-Nitrobenzo[b]thiophene 1,1-dioxide). Mitochondria from STAT3-KO mice and Stattic-treated rat mitochondria tolerated less calcium until MPTP opening occurred. STAT3 co-immunoprecipitated with cyclophilin D, the target of the cardioprotective agent and MPTP inhibitor cyclosporine A (CsA). However, CsA reduced infarct size to a similar extent in wildtype and STAT3-KO mice in vivo. Thus, STAT3 possibly contributes to cardioprotection by stimulation of respiration and inhibition of MPTP opening.

Connexin-43 (Cx43), a predominant cardiac connexin, forms gap junctions (GJs) that facilitate electrical cell–cell coupling and unapposed/nonjunctional hemichannels that provide a pathway for the exchange of ions and metabolites between cytoplasm and extracellular milieu. Uncontrolled opening of hemichannels in the plasma membrane may be deleterious for the myocardium and blocking hemichannels may confer cardioprotection by preventing ionic imbalance, cell swelling and loss of critical metabolites. Currently, all known hemichannel inhibitors also block GJ channels, thereby disturbing electrical cell–cell communication. Here we aimed to characterize a nonapeptide, called Gap19, derived from the cytoplasmic loop (CL) of Cx43 as a hemichannel blocker and examined its effect on hemichannel currents in cardiomyocytes and its influence in cardiac outcome after ischemia/reperfusion. We report that Gap 19 inhibits Cx43 hemichannels without blocking GJ channels or Cx40/pannexin-1 hemichannels. Hemichannel inhibition is due to the binding of Gap19 to the C-terminus (CT) thereby preventing intramolecular CT–CL interactions. The peptide inhibited Cx43 hemichannel unitary currents in both HeLa cells exogenously expressing Cx43 and acutely isolated pig ventricular cardiomyocytes. Treatment with Gap19 prevented metabolic inhibition-enhanced hemichannel openings, protected cardiomyocytes against volume overload and cell death following ischemia/reperfusion in vitro and modestly decreased the infarct size after myocardial ischemia/reperfusion in mice in vivo. We conclude that preventing Cx43 hemichannel opening with Gap19 confers limited protective effects against myocardial ischemia/reperfusion injury.

Abstract-Extracellular ATP liberated during hypoxia and inflammation can either signal directly on purinergic receptors or can activate adenosine receptors following phosphohydrolysis to adenosine. Given the association of polymorphonuclear leukocytes (PMNs) with adenine-nucleotide/nucleoside signaling in the inflammatory milieu, we hypothesized that PMNs are a source of extracellular ATP. Initial studies using high-performance liquid chromatography and luminometric ATP detection assays revealed that PMNs release ATP through activation-dependent pathways.In vitro models of endothelial barrier function and neutrophil/endothelial adhesion indicated that PMN-derived ATP signals through endothelial adenosine receptors, thereby promoting endothelial barrier function and attenuating PMN/endothelial adhesion. Metabolism of extracellular ATP to adenosine required PMNs, and studies addressing these metabolic steps revealed that PMN express surface ecto-apyrase (CD39). In fact, studies with PMNs derived from cd39 Ϫ/Ϫ mice showed significantly increased levels of extracellular ATP and lack of ATP dissipation from their supernatants. After excluding lytic ATP release, we used pharmacological strategies to reveal a potential mechanism involved in PMN-dependent ATP release (eg, verapamil, dipyridamole, brefeldin A, 18-␣-glycyrrhetinic acid, connexin-mimetic peptides). These studies showed that PMN ATP release occurs through connexin 43 (Cx43) hemichannels in a protein/phosphatase-A-dependent manner. Findings in human PMNs were confirmed in PMNs derived from induced Cx43 Ϫ/Ϫ mice, whereby activated PMNs release less than 15% of ATP relative to littermate controls, whereas Cx43 heterozygote PMNs were intermediate in their capacity for ATP release (PϽ0.01). Taken together, our results identify a previously unappreciated role for Cx43 in activated PMN ATP release, therein contributing to the innate metabolic control of the inflammatory milieu. (Circ Res. 2006;99:1100-1108.) Key Words: nucleotide Ⅲ nucleoside Ⅲ adenosine Ⅲ endothelia Ⅲ inflammation Ⅲ ATP Ⅲ connexin Ⅲ inflammation Ⅲ hypoxia P ast studies have revealed a central role of extracellular nucleotide phosphohydrolysis and nucleoside signaling in innate immune responses during conditions of limited oxygen availability (hypoxia) or during acute inflammation. For example, metabolic enzymes and vascular nucleotide levels are consistently increased during hypoxia. 1,2 The contribution of individual nucleotides (ATP, ADP, AMP) to these innate responses remain unclear. Polymorphonuclear granulocytes (PMNs) function as a first line of cellular response during acute inflammatory episodes. 3 Previous reports have suggested that PMNs may release ATP during conditions of inflammation or hypoxia. 1 Such extracellular ATP can either signal directly to vascular ATP receptors 4 or may function as a metabolite following conversion via ecto-apyrase (CD39, conversion of ATP to AMP) and ecto-5Ј-nucleotidase (CD73, conversion of AMP to adenosine).In the present study, we aimed to identify mol...

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Not only the prevalence, but also the mortality due to ischaemic cardiovascular disease is higher in older than in young humans, and the demographic shift towards an ageing population will further increase the prevalence of age-related cardiovascular disease. In order to develop strategies aimed to limit reversible and irreversible myocardial damage in older patients, there is a need to better understand age-induced alterations in protein expression and cell signalling. Cardioprotective phenomena such as ischaemic and pharmacological pre and postconditioning attenuate ischaemia/reperfusion injury in young hearts. Whether or not pre and postconditioning are still effective in aged organs, animals, or patients, i.e. under conditions where such cardioprotection is most relevant, is still a matter of debate; most studies suggest a loss of protection in aged hearts. The present review discusses changes in protein expression and cell signalling important to ischaemia/reperfusion injury with myocardial ageing. The efficacy of cardioprotective manoeuvres, e.g. ischaemic pre and postconditioning in aged organs and animals will be discussed, and the development of strategies aimed to antagonize the age-induced loss of protection will be addressed.

Abstract-The cardioprotection by ischemic preconditioning is lost in aged wild-type and in STAT3 (signal transducer and activator of transcription 3)-deficient mice. The aim of the present study was to analyze whether or not ischemic postconditioning (iPoco) was effective in aged mice hearts and whether iPoco was dependent on STAT3. Young (3 months) and aged (Ͼ13 months) C57Bl6/J mice underwent 30 minutes of ischemia and 2 hours of reperfusion without or with iPoco (3 cycles of 10 seconds of ischemia/10 seconds of reperfusion [3ϫ10] or 5 cycles of 5 seconds of ischemia/5 seconds of reperfusion [5ϫ5] at the beginning of reperfusion). In young mice, both iPoco3ϫ10 and iPoco5ϫ5 reduced infarct size (IS), whereas in aged mice, only iPoco5ϫ5 was effective in reducing IS. In young mice, iPoco3ϫ10 increased the phosphorylated over total STAT3 (phosphorylated STAT3/STAT3) ratio at 10 minutes of reperfusion in the postconditioned anterior wall compared with the control posterior wall. In aged mice hearts, total STAT3 and phosphorylated STAT3/STAT3 in the anterior wall at reperfusion were reduced compared with young mice hearts. In young mice hearts subjected to iPoco3ϫ10 but pretreated with the JAK-2 inhibitor AG-490, phosphorylated STAT3/STAT3 was reduced in the anterior wall compared with untreated young mice hearts, and IS reduction by iPoco3ϫ10 was abolished. Furthermore, in young mice with a cardiomyocyte-restricted deletion of STAT3, iPoco3ϫ10 failed to reduce IS, whereas iPoco5ϫ5 reduced IS. Thus, cardioprotection by iPoco is dependent on the postconditioning protocol in aged and STAT3-deficient hearts. The reduced levels of STAT3 with increasing age may contribute to the age-related loss of iPoco. Key Words: signal transducer and activator of transcription 3 Ⅲ ischemic postconditioning Ⅲ aging T ransient periods of ischemia/reperfusion reduce the irreversible tissue injury by a subsequent prolonged episode of ischemia/reperfusion, a phenomenon known as ischemic preconditioning (IP). Previous studies have demonstrated that IP is not effective in C57Bl6/J mice older than 13 months. 1 STAT3 (signal transducer and activator of transcription 3) transduces stress signals from the plasma membrane to the nucleus, leading to changes in gene transcription. 2 Besides its role in postpartum cardiomyopathy, cardiomyocyte hypertrophy, and apoptosis, STAT3 is involved in ischemia/reperfusion injury. 3-7 IP induces an increase in the STAT3 protein content in the nucleus. 8 Furthermore, the phosphorylation of STAT3 and the binding to the STAT target site are enhanced after IP. 8,9 The importance of STAT3 for cardioprotection by IP has been clearly demonstrated in mice with a cardiacspecific deletion of STAT3, in which the IP-induced infarct size (IS) reduction was abolished. 10 In ischemic postconditioning (iPoco), the IS is reduced by short cycles of ischemia/reperfusion immediately following a sustained ischemic insult. The mechanisms and signal transduction cascade of iPoco, its age dependence, and especially the role of STAT3...

These data demonstrate that Cx43 is localized at cardiomyocyte mitochondria and that IP enhances such mitochondrial localization.

Abstract-We have previously shown that connexin 43 (Cx43) is present in mitochondria, that its genetic depletion abolishes the protection of ischemia-and diazoxide-induced preconditioning, and that it is involved in reactive oxygen species (ROS) formation in response to diazoxide. Here we investigated the intramitochondrial localization of Cx43, the mechanism of Cx43 translocation to mitochondria and the effect of inhibiting translocation on the protection of preconditioning. Confocal microscopy of mitochondria devoid of the outer membrane and Western blotting on fractionated mitochondria showed that Cx43 is located at the inner mitochondrial membrane, and coimmunoprecipitation of Cx43 with Tom20 (Translocase of the outer membrane 20) and with heat shock protein 90 (Hsp90) indicated that it interacts with the regular mitochondrial protein import machinery. In isolated rat hearts, geldanamycin, a blocker of Hsp90-dependent translocation of proteins to the inner mitochondrial membrane through the TOM pathway, rapidly (15 minutes) reduced mitochondrial Cx43 content by approximately one-third in the absence or presence of diazoxide. Geldanamycin alone had no effect on infarct size, but it ablated the protection against infarction afforded by diazoxide. Geldanamycin abolished the 2-fold increase in mitochondrial Cx43 induced by 2 preconditioning cycles of ischemia/reperfusion, but this effect was not associated with reduced protection. These results demonstrate that Cx43 is transported to the inner mitochondrial membrane through translocation via the TOM complex and that a normal mitochondrial Cx43 content is important for the diazoxide-related pathway of preconditioning. Key Words: mitochondria Ⅲ heat shock protein Ⅲ geldanamycin Ⅲ connexin 43 Ⅲ TOM (Translocase of the Outer Membrane) complex C ardiomyocyte death during acute coronary syndromes determines survival and quality of life of patients with coronary artery disease. 1 In the majority of these patients, cardiomyocyte death is the consequence of transient, prolonged ischemia, and there is strong evidence that a substantial part of cell death occurs at the time of reperfusion. 2,3 Preconditioning, a state of increased resistance against cell death induced by ischemia-reperfusion, is elicited by brief ischemia/reperfusion episodes or by certain pharmacological stimuli and has received particular attention. 4 A wealth of information has been collected on the molecular mechanisms involved in preconditioning, but many aspects of the signaling pathways and of the end effectors of the protection remain unknown. 4,5 An intriguing and unresolved aspect is the involvement of connexin 43 (Cx43), the protein forming gap junctions connecting adjacent ventricular cardiomyocytes, 6,7 in the genesis of preconditioning. 8,9 The protection of preconditioning is abolished in Cx43-deficient mice 10 but also in isolated cardiomyocytes from Cx43-deficient hearts, 11 indicating that it cannot be explained by effects of preconditioning on gap junction-mediated cell-to-cell c...