Kerstin Boengler

Cardioprotection Nitric Oxide, Protein Kinases, and Mitochondria

Over the past 2 to 3 decades, several phenomena have been identified that provide powerful protection against myocardial infarction and other sequelae of ischemia/reperfusion1: myocardial hibernation that is related to stunning,2 ischemic preconditioning,3 delayed or second-window ischemic preconditioning,4 ischemic postconditioning,5 and their pharmacological recruitment. Stunning and hibernation share contractile function as an end point. In stunning, reduced postischemic contractile function is viewed as reversible injury, whereas in hibernation dysfunction is viewed as an adaptive response. Ischemic preconditioning is characterized by infarct size reduction as its most robust end point but shares with hibernation the underlying idea of a regulated protective response. These phenomena have been confirmed in patients with coronary artery disease.2,6–8 Nitric oxide (NO) and mitochondria also are important in patients with coronary artery disease. Nitroglycerin induces delayed protection against periinterventional ischemic ECG alterations, contractile dysfunction, and pain sensation,7 and cyclosporin A, which inhibits opening of the mitochondrial permeability transition pore (MPTP), attenuates reperfusion injury in patients with acute myocardial infarction.9 Articles pp 1961 and 1970 The “new ischemic syndromes” have intrigued scientists and clinicians trying to understand their underlying pathophysiology and to recruit their cardioprotective potential. This initial enthusiasm has not materialized into translational medicine.10 Why is that? There has been a long but ultimately fruitless debate as to whether the phenotype of hibernating myocardium is the result of ongoing ischemia with reduced baseline coronary blood flow or repetitive cycles of ischemia/reperfusion and consequent stunning without reduced baseline flow. Clearly, perfusion-contraction matching cannot be maintained for >12 hours. Conversely, repetitive cycles of ischemia/reperfusion with subsequent stunning finally result in hibernating myocardium with reduced contractile function and baseline blood flow.2 Thus, the cumulative effect of stunning is a progression to hibernation. True stunning, if seen at all, is …

ATP Release From Activated Neutrophils Occurs via Connexin 43 and Modulates Adenosine-Dependent Endothelial Cell Function

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−/− 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-α-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−/− 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 (P<0.01). 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.

Ischemic Postconditioning in Pigs: No Causal Role for RISK Activation

Ischemic postconditioning (IPoC) reduces infarct size following ischemia/reperfusion. Whether or not phosphorylation of RISK (reperfusion injury salvage kinases) (AKT, ERK1/2, P70S6K, GSK3&bgr;) is causal for protection by IPoC is controversial. We therefore studied the impact of RISK on IPoC in anesthetized pigs subjected to 90 minutes of left anterior descending coronary artery hypoperfusion and 120 minutes of reperfusion. In protocol 1, IPoC, by 6 cycles of 20/20 seconds of reperfusion/reocclusion (n=13), was compared with immediate full reperfusion (IFR) (n=15). In protocol 2, IPoC (n=4) or IFR (n=4) was performed with pharmacological RISK blockade by IC coinfusion of Wortmannin and U0126. Infarct size was determined by TTC staining, and the expression of phosphorylated RISK proteins by Western blot analysis in biopsies. In protocol 1, infarct size was 20±3% (percentage of area at risk; mean±SEM) with IPoC and 33±4% (P<0.05) with IFR. RISK phosphorylation increased with reperfusion but was not different between IPoC and IFR. In protocol 2, Wortmannin and U0126 blocked the increases in RISK phosphorylation during reperfusion, but infarct size was still smaller with IPoC (15±7%) than with IFR (35±6%; P <0.05).

Loss of cardioprotection with ageing

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.

The myocardial JAK/STAT pathway: from protection to failure.

Proteins of the interleukin-6 (IL-6) family bind to receptors in the plasma membrane. Subsequent signal transduction involves activation of the janus kinase (JAK) and signal transducer and activator of transcription (STAT) proteins. STAT proteins are translocated into the nucleus, where they bind to the promoter region of target genes and are thereby involved in regulating the transcription of target genes. In the first part, the present review focusses on the role of STAT3 in ischemia/reperfusion injury and in cardioprotection by ischemic pre- and postconditioning. In the heart, ischemia induces an increase in IL-6 cytokines, which is associated with activation of STAT3. Genetic modification of the myocardial STAT3 protein content shows a protective role of STAT3 on infarct size after ischemia/reperfusion injury. The cardioprotection by both early and late ischemic preconditioning as well as by ischemic postconditioning involves an activation of STAT3 and is dependent on STAT3 protein level. Whereas the infarct-sparing effect of late preconditioning is clearly mediated by an increase in transcription-mediated protein synthesis, early preconditioning is independent of gene transcription, suggesting a role of STAT3 independent of transcriptional regulation. Possibly, STAT3 plays a role in modifying mitochondrial function, organelles central for the cardioprotection by pre- and postconditioning. In the second part, the role of STAT3 in physiological stresses such as aging and pregnancy, as well as in pathophysiological situations such as myocardial infarction and heart failure is summarized. Furthermore, the requirements for the use of STAT3 as a target for treatment strategies of cardiovascular diseases is discussed.

Cardioprotection by Ischemic Postconditioning Is Lost in Aged and STAT3-Deficient Mice

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 (>13 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 [3×10] or 5 cycles of 5 seconds of ischemia/5 seconds of reperfusion [5×5] at the beginning of reperfusion). In young mice, both iPoco3×10 and iPoco5×5 reduced infarct size (IS), whereas in aged mice, only iPoco5×5 was effective in reducing IS. In young mice, iPoco3×10 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 iPoco3×10 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 iPoco3×10 was abolished. Furthermore, in young mice with a cardiomyocyte-restricted deletion of STAT3, iPoco3×10 failed to reduce IS, whereas iPoco5×5 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.

Inhibition of mitochondrial permeability transition pore opening: the holy grail of cardioprotection

Cardioprotection is a fairly vague term which refers to the reduction of damage from myocardial ischemia/reperfusion by several endogenous phenomena, such as hibernation [23, 37], ischemic preconditioning [32, 41], ischemic postconditioning [20, 43], and remote conditioning [19, 22] as well as by pharmacological interventions. With the recognition of the postconditioning phenomenon [43], myocardial reperfusion injury has been appreciated as a reality [42] from which protection is clinically feasible [36, 38, 39]. The signal transduction of cardioprotection is under intense investigation, with the ultimate aim to identify targets for pharmacological recruitment of cardioprotection [21]; three major pathways have been characterized—the cGMP/PKG-pathway [11], the reperfusion injury salvage kinase (RISK)-pathway [18] and the survivor activating factor enhancement (SAFE)-pathway [31]. These three major pathways are not mutually exclusive, but potentially cooperative, and they are recruited by the above cardioprotective phenomena to a different extent. The cardioprotective signaling pathways are thought to converge on the mitochondria [21], and various mitochondrial proteins without or with channel structure have been identified as central elements in cardioprotection. Several signaling pathways of cardioprotection converge to inhibit glycogen synthase kinase-3b which in its phosphorylated state increases the threshold for mitochondrial permeability transition pore (MPTP) opening [25, 26]. The MPTP is a large-conductance megachannel which is traditionally thought to be formed by an arrangement of the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane, the adenine nucleotide transporter (ANT) in the inner membrane and cyclophilin D in the matrix [30]. The MPTP is either not present or mostly closed under physiological conditions, but opens in response to high concentrations of calcium. The calciuminduced opening of MPTP is favoured by high concentrations of inorganic phosphate, reactive oxygen species, and nitric oxide and a reduction of the inner membrane potential—all conditions which occur during myocardial ischemia and reperfusion; acidosis and magnesium ions inhibit/delay MPTP opening [8, 13, 29, 30, 40, 44]. In addition, proand anti-apoptotic members of the Bcl-family interact with the MPTP [3]. Formation and opening of the MPTP results in depolarization of the inner membrane potential and matrix swelling and ultimately in rupture of the outer membrane. Proteins, among them cytochrome C, are then released from the intermembrane space into the cytosol and activate caspase cascades to initiate cellular fragmentation and ultimately cell death. In 1993, both the groups of Crompton [12] and Halestrap [16] demonstrated that MPTP opened upon reperfusion following myocardial ischemia and that cyclosporine A protected from reperfusion injury. Cyclosporine A inhibits binding of cyclophilin D to ANT, thereby MPTP opening and ultimately cell death; ablation of the Ppif gene which encodes for cyclophilin D [4, 33] and cyclosporine A [4] therefore reduce infarct size resulting from myocardial ischemia and reperfusion. Yellon et al. first proposed that inhibition of MPTP opening could be the effector of ischemic preconditioning [17], and Ovize et al. shortly thereafter proposed MPTP inhibition as the effector of ischemic postconditioning [2]; G. Heusch (&) K. Boengler R. Schulz Institute for Pathophysiology, University of Essen Medical School, Hufelandstr. 55, 45122 Essen, Germany e-mail: gerd.heusch@uk-essen.de

Translocation of Connexin 43 to the Inner Mitochondrial Membrane of Cardiomyocytes Through the Heat Shock Protein 90–Dependent TOM Pathway and Its Importance for Cardioprotection

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.

Impairment of Diazoxide-Induced Formation of Reactive Oxygen Species and Loss of Cardioprotection in Connexin 43 Deficient Mice

Protection by ischemic preconditioning is lost in cardiomyocytes and hearts of heterozygous connexin 43 deficient (Cx43+/−) mice. Because connexin 43 (Cx43) is localized in cardiomyocyte mitochondria and mitochondrial Cx43 content is increased with ischemic preconditioning, we now tried to identify a functional defect at the level of the mitochondria in Cx43+/− mice by use of diazoxide and menadione. Diazoxide stimulates the mitochondrial formation of reactive oxygen species (ROS) and menadione generates superoxide at multiple intracellular sites; both substances elicit cardioprotection through increased ROS formation. ROS formation in response to the potassium ionophore valinomycin was also measured for comparison. Menadione (2 &mgr;mol/L) and valinomycin (10 nmol/L) induced similar ROS formation in wild-type (WT) and Cx43+/− cardiomyocytes. In contrast, diazoxide (200 &mgr;mol/L) increased ROS formation by 43±10% versus vehicle in WT, but only by 18±4% in Cx43+/− cardiomyoctes (P<0.05). Two hour–simulated ischemia and oxygenated, hypo-osmolar reperfusion reduced viability as compared with normoxia (WT: 7±1% versus 39±2%, Cx43+/−: 8±1% versus 40±3%, P<0.01). Although menadione protected WT and Cx43+/− cardiomyocytes, diazoxide increased viability (17±2%, P<0.01) in WT, but not in Cx43+/− (9±1%). Menadione (37 &mgr;g/kg i.v.) before 30 minutes coronary occlusion and 2 hour reperfusion reduced infarct size in WT and Cx43+/− mice (24±4% versus 24±5%). In contrast, diazoxide (5 mg/kg i.v.) reduced infarct size in WT (35±4% versus 55±3% of area at risk, P<0.01), but not in Cx43+/− mice (56±2% versus 54±3%). Cardiomyocytes of Cx43+/− mice have a specific functional deficit in ROS formation in response to diazoxide and accordingly less protection.

Selective inhibition of Cx43 hemichannels by Gap19 and its impact on myocardial ischemia/reperfusion injury

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.