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To commemorate the auspicious occasion of the 30th anniversary of IPC, leading pioneers in the field of cardioprotection gathered in Barcelona in May 2016 to review and discuss the history of IPC, its evolution to IPost and RIC, myocardial reperfusion injury as a therapeutic target, and future targets and strategies for cardioprotection. This article provides an overview of the major topics discussed at this special meeting and underscores the huge importance and impact, the discovery of IPC has made in the field of cardiovascular research. DJH was funded by the British Heart Foundation (Grant Number FS/10/039/28270), the Rosetrees Trust, and is supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre. HEB was funded by the Danish Council for Independent Research (11-108354), the Danish Council for Strategic Research (11-115818), the Novo-Nordisk Foundation and Trygfonden. PF was funded by the Hungarian Scientific Research Fund (OTKA K 109737 and ANN 107803). HACF is funded by a Startup Grant of the ``Excellence Cluster Cardio-Pulmonary System'' (ECCPS) from the German Research Foundation (DFG, Bonn, Germany) and the ``Peter und Traudl Engelhorn-Stiftung'' (Weilheim, Germany) and in part by the Russian Government Program for competitive growth of Kazan Federal University, Kazan (Russian Federation). GH was supported by the German Research Foundation (He 1320/18-3 and SFB 1116/B8). TM was funded by the Japan Society for the Promotion of Science (Grant-in-Aid for Scientific Research \#23591086, \#2646113). MRS was funded by Novo-Nordisk Foundation. DG-D is supported by the Spanish Institute of Health, Instituto de Salud Carlos III (Grants PIE 13/00027, RETICS-RIC, RD12/0042/0021, and PI14/01431). RS and PF were funded by the European Foundation for the Study of Diabetes (EFSD) New Horizons Collaborative Research Initiative from the European Association for the Study of Diabetes (EASD) and by the European Cooperation in Science and Technology (COST EU-ROS). PF was funded by the Hungarian Scientific Research Fund (OTKA K 109737 and ANN 107803). Sí

Hypoxia-reoxygenation induces loss of endothelial barrier function and oedema formation, which presents a major impediment for recovery of the organ. The integrity of the endothelial barrier is highly dependent on its contractile machinery and actin dynamics, which are precisely regulated by Rho GTPases. Perturbed activities of these Rho-GTPases under hypoxia-reoxygenation lead to derangement of the actin cytoskeleton and therefore may affect the integrity of the endothelial barrier. The aim of the present study was to analyse the role of these GTPases in regulating endothelial barrier function during hypoxia-reoxygenation in cultured porcine aortic endothelial cells and isolated perfused rat hearts. Hypoxia-reoxygenation induced an increase in albumin permeability of endothelial monolayers accompanied by an activation of the endothelial contractile machinery, derangement of the actin cytoskeleton and loss of VE-cadherin from cellular junctions. Inhibition of contractile activation with ML-7 partially protected against hypoxia-reoxygenation-induced hyperpermeability. Likewise, reoxygenation caused an increase in RhoA and a reduction in Rac1 activity accompanied by enhanced stress fibre formation and loss of peripheral actin. Inhibition of RhoA/rho kinase (Rock) signalling with RhoA or Rock inhibitors led to a complete depolymerisation and derangement of the actin cytoskeleton and worsened hypoxia-reoxygenation-induced hyperpermeability. Activation of Rac1 using a cAMP analogue, 8-CPT-O-Me-cAMP, which specifically activates Epac/Rap1 signalling, restored peripheral localisation of actin and VE-cadherin at cellular junctions and abrogated reoxygenation-induced hyperpermeability. Similar results were reproduced in isolated saline-perfused rat hearts. These data show that activation of Rac1 but not the inhibition of RhoA preserves endothelial integrity against reoxygenation-induced loss of barrier function.

Since adenine nucleotide translocase 1 (ANT1) overexpression improved cardiac function in rats with activated renin-angiotensin system (RAS) and angiotensin II is known to enhance transforming growth factor β (TGFβ) signaling in cardiomyocytes, we assumed that ANT1 might modulate the classical TGFβ/SMAD pathway. We therefore investigated whether the cardioprotective effect of ANT1 overexpression suppresses TGFβ(1)-induced apoptosis, whether mitochondrial permeability transition pore (MPTP) regulation is involved, and SMAD signaling pathway is affected.Ventricular cardiomyocytes isolated from wild-type (WT) and ANT1 transgenic rats were treated with the apoptosis-inducing agent TGFβ(1) (1 ng/ml). TGFβ(1) treatment of WT cells enhanced the number of apoptotic cells by 31.8 ± 11.7% (p0.01 vs. WT) measured by chromatin condensation. Apoptosis was blocked by 1μM cyclosporine A and by ANT1 overexpression. The protecting effect of ANT1 overexpression on TGFβ(1)-induced apoptosis was verified by reduced caspase 3/7 activity and increased Bcl-2 expression. In addition, TGFβ(1) decreased mitochondrial membrane potential as measured by JC-1 staining by 18.0 ± 3.7% in WT cardiomyocytes, but only by 7.2 ± 2.8% (p0.05 vs. WT) in ANT1 cardiomyocytes. Cyclosporine A also attenuated the decline in mitochondrial membrane potential under TGFβ(1) in WT cardiomyocytes. Determination of MPTP opening by Calcein assay in isolated cardiomyocytes and calcium retention assay in isolated mitochondria revealed a reduced open probability of MPTP after ANT1 overexpression. In addition to the effects of ANT1 on MPTP opening we investigated if ANT1 may interfere with the classical TGFβ signaling pathway. Interestingly, ANT1-transgenic cardiomyocytes expressed less TGFβ receptor II than WT cells. However, SMAD2 phosphorylation was already enhanced without TGFβ(1) stimulation in these cells. Although no additional increase in SMAD2 phosphorylation was detectable after TGFβ(1) treatment, SMAD signaling was still responsive to TGFβ(1) indicated by an upregulation of SMAD7, a TGFβ(1) target protein.Heart-specific overexpression of ANT1 leads to a reduced apoptotic response to TGFβ(1) by preservation of the mitochondrial membrane potential, resistance to MPTP opening and altered TGFβ signaling.

Aims Intermedin (IMD) is a novel member of the calcitonin gene-related peptide family, which acts via calcitonin receptor-like receptors (CLRs), mediating activation of cAMP signalling. The main objective of the present study was to analyse the molecular mechanisms of the differential effects of IMD on the macromolecule permeability of endothelial cells of different vascular beds. Methods and results Here we demonstrate that IMD increases permeability of rat coronary microvascular endothelial cells (RCECs) and reduces permeability of human umbilical vein endothelial cells (HUVECs) and rat aortic endothelial cells via CLRs and cAMP. Intermedin causes a derangement of the actin cytoskeleton accompanied by loss of vascular endothelial cadherin (VE-cadherin) in RCECs, while it causes a rearrangement of the actin cytoskeleton and VE-cadherin at cell–cell junctions in HUVECs. Intermedin inactivates the RhoA/Rho-kinase (Rock) pathway in both cell types; however, it inactivates Rac1 in RCECs but not in HUVECs. Inhibition and rescue experiments demonstrate that both RhoA and Rac1 are required for the RCEC barrier stability, while in HUVECs the inhibition of RhoA/Rock signalling does not interfere with basal permeability. Conclusion The opposite effects of IMD on permeability of RCECs and HUVECs are due to differential regulation of actin cytoskeleton dynamics via RhoA and Rac1. Moreover, Rac1 activity is regulated by the RhoA/Rock pathway in RCECs but not in HUVECs.

Recently, we showed that activated factor XIII (FXIIIa) has a direct influence on permeability (P) of cultured endothelial monolayer. Clinical investigation on children operated on for congenital heart disease has demonstrated a distinct correlation between decrease of endothelial barrier function (edema formation) and reduced FXIII activity. Our experiments investigated whether significant effects of FXIII could also be shown in a full-organ model. The effect of FXIII on endothelial barrier function was studied by determining the passage of trypan blue-labeled albumin in a model of cultured monolayers of porcine aortic endothelial cells and changes in myocardial water content of saline-perfused rat hearts in a Langendorff-circuit. The plasma FXIIIa (1 U/ml) reduced albumin permeability of aortic endothelial monolayers within 20 minutes from a basal value of 5.9±0.4×10−6 cm/second by 30±7% whereas the nonactivated plasma FXIII had no effect on permeability. Reduction of permeability to the same extent (by 34±9%) could also be obtained with a thrombin-activated recombinant factor XIII A subunit (rhu FXIII; 1 U/ml) in this culture model. The effect of factor XIII A* on permeability was dose dependent with maximum effect at 5 U/ml (52±11% reduction of permeability compared with control), and existed even if cells had hyperpermeability stress (KCN/2-DG). Activated rhuFXIII prevented the increase in myocardial water content in anoxic-reperfused rat hearts (448±24 vs 517±29 ml/100g dry weight). The data of the present study show that FXIII has a stabilizing effect at the site of endothelial cells not only in cultured monolayers but also in a model of the anoxic perfused rat heart.

The topical application of factor XIII (Fibrogammin HS) is a new concept in the treatment of ulcerative leg disease. In addition to the mode of application, the utilization of a fibrin-stabilizing effect in ulcer treatment is a completely different strategy, since many other medications used for local wound treatment contain a fibrinolytic component. Of the 29 inpatients treated topically during the last 3 years, 17, 12 female and 5 male averaging 60.8±13.6 years, suffered ulcerative leg disease due to a postthrombotic syndrome. The average period since first clinical manifestation of venous ulcer was 3.5±2.1 years. Thirteen patients (76.4%) with chronic ulceration showed such a distinct improvement of topical site after an average period of 3.02±1.05 weeks with topically applied factor XIII (Fibrogammin HS) combined with compression bandaging that they were discharged for further ambulant treatment. Apart from a wound surface reduction and a clinical improvement of granulation, we observed a marked reduction of bleeding and secretion tendency within the ulcus area, especially in patients with severe venous insufficiency. Our hypothesis was that high doses of topically applied factor XIII could improve barrier function of endothelial cells in the wound area. For experimental investigation, porcine aortic endothelial cells were cultured on filter membranes to confluent monolayers. To monitor the barrier function, permeability of the monolayer was continuously measured as flux of trypan blue-labeled albumin. Confluent endothelial monolayers exposed to factor XIII (Fibrogammin HS) showed a significant (n=8,p

Activation of cAMP signalling abrogates thrombin-induced hyperpermeability. One of the mechanisms underlying this protective effect is the inactivation of endothelial contractile machinery, one of the major determinants of endothelial barrier function, mainly via the activation of myosin light chain phosphatase (MLCP). To date, the mechanisms of cAMP-mediated MLCP activation are only partially understood. Here the contribution of two cAMP effectors, PKA and Epac, in the regulation of endothelial contractile machinery and barrier function was studied.Endothelial contractile machinery and barrier function were analysed in cultured human umbilical vein endothelial cells (HUVEC). The cAMP analogues 8-CPT-cAMP and 6-Bnz-cAMP were used to activate Epac and PKA, respectively, and forskolin (FSK) was used to activate adenylyl cyclase. The cells were challenged by thrombin to inhibit MLCP via the RhoA/Rock pathway. Activation of either PKA or Epac partially blocked thrombin-induced hyperpermeability. Simultaneous activation of PKA and Epac had additive effects that were comparable to that of FSK. Activation of PKA but not Epac inhibited thrombin-induced phosphorylation of MLC and the MLCP regulatory subunit MYPT1, partly via inhibition of the RhoA/Rock pathway. FSK activated the MLCP catalytic subunit PP1 via dephosphorylation and dissociation of the PP1 inhibitory protein CPI-17. FSK blunted thrombin-induced CPI-17 phosphorylation, CPI-17/PP1 complex formation, and PP1 inactivation. Down-regulation of CPI-17 attenuated thrombin-induced hyperpermeability and abolished the antagonistic effect of the PKA activator, whereas the Epac activator retained its antagonistic effect.cAMP/PKA regulates the endothelial barrier via inhibition of the contractile machinery, mainly by the activation of MLCP via inhibition of CPI-17 and RhoA/Rock. The permeability-lowering effect of the cAMP/Epac pathway is independent of CPI-17.

1.1 Definitions ‘Reperfusion injury’ has been for some time an imprecise and controversial term referring to a variety of alterations occurring at the time of restoration of blood flow, from functional impairment to cell death. The term ‘lethal reperfusion injury’ specifically refers to cell death associated with transient ischaemia that can be prevented by interventions applied at the time of reperfusion.1 Therefore, it is the component of cell death occurring as a consequence of reperfusion. There are aspects of lethal reperfusion injury upon which there is general agreement based on solid information. ### 1.2 Existence and relevance The existence of lethal myocardial reperfusion injury has been demonstrated by an overwhelming body of evidence obtained mainly in laboratory experiments, but also, and increasingly, in humans.2 Different interventions have consistently been shown to limit myocardial necrosis when applied at the time of reperfusion in a variety of models and preparations, and it is unquestionable that, at least under certain conditions, lethal myocardial reperfusion injury occurs. It is also clear from those studies that lethal reperfusion injury can be responsible for a significant proportion (one-third or more) of cell death caused by transient global or regional myocardial ischaemia. ### 1.3 Initial minutes after reperfusion Many studies have shown a reduction of the protection afforded by several pharmacological interventions and postconditioning protocols when they are first applied only a few minutes later than reperfusion onset.3 Prolonging intracellular acidosis for only 3 min at the onset of reperfusion has a dramatic protective effect.4,5 These observations demonstrate that at least part of lethal reperfusion injury takes place very early after restoration of flow, and they are consistent with the fact that most changes associated with cell death, such as calcium oscillations, calpain activation, hypercontracture, oxidative stress, or mitochondrial permeability transition,6 occur during the very first minutes of reflow. ### 1.4 Necrosis The rapid time course …

Aims Platelet-derived growth factor BB (PDGF-BB) has been assigned a critical role in vascular growth and recruitment of perivascular mural cells. The purpose of the present study is to investigate the signalling events underlying the stimulation of vasculogenesis of mouse embryonic stem (ES) cells by PDGF-BB. Methods and results PDGF-BB increased vascular sprouting and branching of capillary-like structures in embryoid bodies as evaluated by computer-assisted analysis of CD31-positive cell structures. It also activated extracellular-regulated kinase 1,2 (ERK1,2) and c-Jun N-terminal kinase but not p38 mitogen-activated protein kinase or PI 3-kinase. Microfluorometric analysis of fluo-4 fluorescence revealed that treatment with PDGF-BB raised intracellular Ca2+ levels in differentiating ES cells expressing the PDGF receptor β, an effect that was abolished in the presence of the intracellular Ca2+ chelator BAPTA. Furthermore, PDGF-BB raised reactive oxygen species (ROS) levels in embryoid bodies as evaluated using the redox-sensitive dye H2DCF-DA. ROS generation was blunted in the presence of the NADPH oxidase inhibitors diphenylen iodonium (DPI) and apocynin as well as in the presence of BAPTA, suggesting that ROS generation is regulated by intracellular Ca2+ transients. The stimulation of vasculogenesis of ES cells upon treatment with PDGF-BB was significantly inhibited by the ERK1,2 inhibitor U0126, the NADPH oxidase inhibitors DPI, apocynin, 4-(2-aminoethyl)benzenesulfonylfluoride and VAS2870, the free radical scavengers vitamin E, and N-(2-mercaptopropionyl)glycin as well as by BAPTA. Conclusion Our data demonstrate that the pro-vasculogenic effects of PDGF-BB are mediated by Ca2+-induced ROS generation, resulting in the activation of an ERK1,2-mediated signal transduction cascade.

Rapid interventional restoration of coronary blood flow is the most effective therapy to limit infarct size in today}s cardiology. The early phase of reperfusion represents, however, a window of therapeutic opportunities largely unused in the clinic. Experimentally it has been clearly shown that the modalities of reperfusion have a substantial impact on infarct size, since reperfusion itself can damage the myocardium (reperfusion injury). The major cause for acute injury of the cardiomyocytes in reperfusion is their hypercontracture. Cytosolic Ca 2+ overload and malfunction of cell organelles, i. e. sarcoplasmic reticulum and mitochondria, determine the pathophysiology of reperfusion injury. The underlying mechanisms can be influenced in the first minutes of reperfusion by activation of protective signalling pathways (reperfusion therapy). First clinical studies confirm the efficacy of acute reperfusion therapy.

Bicarbonate transport (BT) has been previously shown to participate in apoptosis induced by various stress factors. However, the precise role of BT in ischaemia-induced apoptosis is still unknown. To investigate this subject, rat coronary endothelial cells (EC) were exposed to simulated ischaemia (glucose free anoxia at Ph 6.4) for 2 hrs and cells undergoing apoptosis were visualized by nuclear staining or by determination of cas-pase- 3 activity. To inhibit BT, EC were either treated with the inhibitor of BT 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS, 300 μmol/l) or exposed to ischaemia in bicarbonate free, 4-(2-hydroxyethyl)-I-piperazi-neethanesulphonic acid (HEPES)-buffered medium. Simulated ischaemia in bicarbonate-buffered medium (Bic) increased caspase-3 activity and the number of apoptotic cell (23.7 + 1.4%versus 5.1 + 1.2% in control). Omission of bicarbonate during ischaemia further significantly increased caspase-3 activity and the number of apoptotic cells (36.7 1.7%). Similar proapoptotic effect was produced by DIDS treatment during ischaemia in Bic, whereas DIDS had no effect when applied in bicarbonate-free, HEPES-buffered medium (Hep). Inhibition of BT was without influence on cytosolic acidification during ischaemia and slightly reduced cytosolic Ca2+ accumulation. Initial characterization of the underlying mechanism leading to apoptosis induced by BT inhibition revealed activation of the mitochondrial pathway of apoptosis, i.e., increase of cytochrome C release, depolarization of mitochondria and translocation of Bax protein to mitochondria. In contrast, no activation of death receptor-dependent pathway (caspase-8 cleavage) and endoplasmic reticulum- dependent pathway (caspase-12 cleavage) was detected. In conclusion, BT plays an important role in ischaemia-induced apoptosis of coronary EC by suppression of mitochondria-dependent apoptotic pathway.

Cardiomyocyte death secondary to transient ischemia occurs mainly during the first minutes of reperfusion, in the form of contraction band necrosis involving sarcolemmal rupture. Cardiomyocyte hypercontracture caused by re-energisation and pH recovery in the presence of impaired cytosolic Ca(2+) control as well as calpain-mediated cytoskeletal fragility play prominent roles in this type of cell death. Hypercontracture can propagate to adjacent cells through gap junctions. More recently, opening of the mitochondrial permeability transition pore has been shown to participate in reperfusion-induced necrosis, although its precise relation with hypercontracture has not been established. Experimental studies have convincingly demonstrated that infarct size can be markedly reduced by therapeutic interventions applied at the time of reperfusion, including contractile blockers, inhibitors of Na(+)/Ca(2+) exchange, gap junction blockers, or particulate guanylyl cyclase agonists. However, in most cases drugs for use in humans have not been developed and tested for these targets, while the effect of existing drugs with potential cardioprotective effect is not well established or understood. Research effort should be addressed to elucidate the unsolved issues of the molecular mechanisms of reperfusion-induced cell death, to identify and validate new targets and to develop appropriate drugs. The potential benefits of limiting infarct size in patients with acute myocardial infarction receiving reperfusion therapy are enormous.

Background: Parathyroid hormone-related protein (PTHrP) is an endothelial-derived vasoactive peptide. This study investigated whether bioactive PTHrP is locally released in a pressure-dependent way. Methods: A PTHrP antibody directed against the midregional part of PTHrP was used to analyze PTHrP in plasma samples. The biological activity of this PTHrP-like peptide was investigated in vitro. Plasma values were determined in samples from the left pulmonary artery and the arteria femoralis, taken under basal conditions and after the application of oxygen or iloprost to lower the pulmonary pressure. Twenty young patients (mean age 6.5 years), who were catheterized for an analysis of the reactivity of the pulmonary bed, were investigated. Endothelial function was investigated by acetylcholine responsiveness. Results: The antibody recognized a 30-kDa protein with in vitro PTHrP-like activity. In 11 patients (responders) with intact endothelial function, the PTHrP values determined in the left pulmonary artery were higher than those in the arteria femoralis. The local increase in the PTHrP concentration was reduced when either oxygen or iloprost lowered the pressure. Nine patients with endothelial dysfunction did not show any concentration gradients at any time (nonresponders). Conclusions: The local concentration of bioactive PTHrP is increased in patients with pulmonary hypertension and normal endothelial function.

Objective : The transcription factor AP-1 is a mediator of hypertrophic growth and apoptosis in cardiomyocytes. This puts AP-1 in the center of two important processes found in the failing heart and implies that variations (i) in the AP-1 composition itself or (ii) in additional, interacting transcription factors are responsible for the diverse actions of AP-1. To test this hypothesis, we performed studies on isolated ventricular cardiomyocytes of rat under hypertrophy- or apoptosis-inducing conditions. Methods and results : The NO donor SNAP (100 μM), which is a pro-apoptotic stimulus in cardiomyocytes, activated AP-1 within 2 h. c-Jun, JunB and FosB are identified as the main components of this AP-1 complex. This complex formation is identical to the composition of AP-1 found under hypertrophic growth stimulation by phenylephrine (PE, 10 μM). Analysis of other transcription factors able to interact with AP-1 revealed activation of SMAD activity only during stimulation with SNAP to 131 ± 9.6% ( p

Quercetin is one of the dietary-derived flavonoids that are held responsible for the beneficial effects of red wine drinking in coronary artery disease known as the "French paradox". We examined whether quercetin modulates endothelial function by influencing Ca2+-activated K+ channels with large conductance (BK(Ca)) in cultured human endothelial cells. Membrane potential and intracellular Ca2+ concentrations of cultured human endothelial cells derived from umbilical cord veins (HUVEC) were measured using the fluorescence dyes DiBAC, and FURA-2, respectively. NO production was examined using a cGMP radioimmunoassay. HUVEC proliferation was analyzed by cell counts and thymidine incorporation. A dose-dependent hyperpolarization of HUVEC was recorded when quercetin was added (5-100 micromol/L). The maximum effect (50 micromol/L) was significantly reduced by the addition of the highly selective BK(Ca) inhibitor iberiotoxin (100 nmol/L), but not by blockers of other Ca2+-activated K+ channels (n = 30; p < 0.05). This BK(Ca)-induced hyperpolarization caused a transmembrane Ca2+ influx, because the quercetin-induced increase of intracellular Ca2+ was blocked by iberiotoxin, or by applying 2-aminoethoxydiphenylborate (100 micromol/L)--an inhibitor of capacitative Ca2+ entry (n = 30; p < 0.05). Quercetin-induced cGMP levels were significantly reduced by the eNOS-inhibitor l-NMMA (300 micromol/L), and by iberiotoxin (n = 10; p < 0.05). Endothelial proliferation was significantly reduced by 56 % when cells were incubated with quercetin (n = 12; p < 0.05). This effect was due to the increased NO production, because it was reversed when the cells were treated with a combination of quercetin and l-NMMA. In conclusion quercetin improves endothelial dysfunction by increasing NO synthesis involving BK(Ca)-dependent membrane hyperpolarization-induced capacitative Ca 2+ entry. Increased NO production is responsible for the quercetin-dependent inhibition of endothelial proliferation.

Measuring the diffusion and impact of scientific papers is a difficult task with many potential applications. In recent years, download monitoring has emerged as a new tool that can help serve this purpose. Currently, virtually all of the most prominent scientific journals have electronic versions that may be accessed over the Internet, and while journals that are exclusively electronic still have a very limited impact no matter how it is measured, electronic access has become an increasingly important method of diffusion of scientific papers as compared to classical, mail-distributed printed issues. Monitoring of downloads permits an estimation of the number of readers attracted by an individual article, something that was not possible when diffusion was based only on hard-copy mailing. It is clear that simply reading an article is different from finding new and useful information in it that may contribute to subsequent scientific production, which is what the ISI impact factor aims to measure. It is also clear that download monitoring ignores the readers who obtain their articles as printed copies only. Nevertheless, download monitoring has the advantage of providing immediate information, something that any index based on citations cannot provide. We may take it now as an “interest factor” for our electronically published manuscripts. Unfortunately, this parameter can easily be spoiled, e.g., if authors become aware of download monitoring and start downloading their own articles. We have analyzed downloads of articles published in Cardiovascular Research between June, 2003, and June, 2004. The number of times any individual article was downloaded during the 90 days following its online publication was counted, and articles were ranked according to this number. Analysis of this ranking provides a clear indication about the preferences of our readership. It must be stressed that, in principle, readers decide to download an article before having seen … *Corresponding author. Tel.: +49 641 99 47 242; fax: +49 641 99 47 209.

Background: Vascular endothelial growth factor (VEGF) induces proliferation of endothelial cells (EC) in vitro and angiogenesis in vivo. Furthermore, a role of VEGF in K+ channel, nitric oxide (NO) and Ca2+ signaling was reported. We examined whether the K+ channel blocker margatoxin (MTX) influences VEGF-induced signaling in human EC. Methods: Fluorescence imaging was used to analyze changes in the membrane potential (DiBAC), intracellular Ca2+ (FURA-2) and NO (DAF) levels in cultured human EC derived from human umbilical vein EC (HUVEC). Proliferation of HUVEC was examined by cell counts (CC) and [3H]-thymidine incorporation (TI).Results: VEGF (5–50 ng/ml) caused a dose-dependent hyperpolarization of EC, with a maximum at 30 ng/ml (n = 30, p < 0.05). This effect was completely blocked by MTX (5 µmol/l). VEGF caused an increase in transmembrane Ca2+ influx (n = 30, p < 0.05) that was sensitive to MTX and the blocker of transmembrane Ca2+ entry 2-aminoethoxydiphenyl borate (APB, 100 µmol/l). VEGF-induced NO production was significantly reduced by MTX, APB and a reduction in extracellular Ca2+ (n = 30, p < 0.05). HUVEC proliferation, examined by CC and TI, was significantly increased by VEGF and inhibited by MTX (CC: –58%, TI –121%); APB (CC –99%, TI –187%); N-monomethyl-L-arginine (300 µmol/l: CC: –86%, TI –164%). Conclusions: VEGF caused an MTX-sensitive hyperpolarization which results in an increased transmembrane Ca2+ entry that is responsible for the effects on endothelial proliferation and NO production.

Objective: The duration of extracellular signal-regulated kinase (ERK) activation and the ERK-dependency of hypertrophic growth differ between stimulation of α-adrenoceptors or angiotensin II receptors. As both receptor systems activate different protein kinase C (PKC) isoforms, we hypothesized that PKC isoforms contribute to the specific effect of α-adrenoceptor stimulation. Methods: Isolated adult ventricular cardiomyocytes from rats were used. Different PKC isoforms were inhibited either pharmacologically by six different PKC inhibitors or specifically downregulated by antisense oligonucleotides. ERK activation was determined by phosphorylation relative to total ERK. The rate of protein synthesis was determined by 14C-phenylalanine incorporation. Results: The hypertrophic response of phenylephrine was inhibited in a concentration-dependent fashion by three different inhibitors of Ca2+-independent PKC isoforms (Go6983, rottlerin, Go6850), but not by three distinct PKC inhibitors directed preferentially against Ca2+-dependent PKC isoforms (Ro32-0432, HBDDE, Go6976). Antisense oligonucleotides directed against PKC-α, -δ, or -e downregulated their specific isoforms. Their corresponding sense oligonucleotides did not affect PKC isoform expression. The phenylephrine-induced increase in protein synthesis was blocked by antisense oligonucleotides directed against PKC-δ or PKC-e but not PKC-α, confirming the pharmacological experiments. Inhibition of Ca2+-dependent PKC isoforms by HBDDE or Go6976 converted a transient activation of ERK by phenylephrine into a sustained response. Under these conditions, phenylephrine increased protein synthesis in an ERK-dependent way. Conclusion: Inhibition of Ca2+-dependent PKC isoforms converts the ERK-independent effect of phenylephrine on protein synthesis into an ERK-dependent induction of protein synthesis. We conclude that co-activation of Ca2+-dependent PKC isoforms by phenylephrine contributes to the specific effect on adult ventricular cardiomyocytes from rat.

During the past decade, the understanding has grown that control of the conditions of reperfusion is critical for salvaging ischemic-reperfused myocardium. The first few minutes of reperfusion constitute a critical phase, as here lethal tissue injury in addition to that already developed during ischemia may be initiated. The identification of the mechanisms of reperfusion-induced cell death opens a new window of opportunity for cardioprotection in the clinic. Development of cardiomyocyte hypercontracture is a predominant feature of reperfusion injury. We and others have shown that control of hypercontracture in reperfusion reduces the extent of tissue injury. On the cellular level, it was shown that reperfusion-induced hypercontracture might either originate from a rigor-type mechanism, when energy recovery proceeds very slowly, or from Ca2+ overload, when energy recovery is rapid but cytosolic Ca2+ load is high. These two mechanisms can be influenced by various interventions that either connect with cytosolic Ca2+ control or myofibrillar Ca2+ sensitivity or with mitochondrial energy production. These experimental approaches will hopefully lead to novel strategies for clinical cardioprotection during the early phase of reperfusion.

Recent findings on the induction of anti-apoptotic gene expression in ischemic/reperfused hearts encouraged us to investigate whether ischemic/reperfused hearts may be protected against apoptosis induction. To analyze this hypothesis we performed studies on isolated perfused hearts of rat. For apoptosis induction, hearts were perfused with the NO donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP, 10 microM) for 30 minutes. Four hours thereafter apoptosis was detected by DNA laddering and TUNEL assay. Under normoperfusion SNAP induced 5.5 +/- 1.4 TUNEL-positive myocytes per tissue section (vs. 1.8 +/- 0.5 in controls). But when hearts were subjected to 20 minutes of no flow ischemia, which was sufficient for energy depletion of the hearts without inducing severe necrotic or apoptotic cell death, reperfusion in the presence of SNAP did not induce apoptosis. To analyze if this mode of protection is a property of the cardiomyocytes, we performed corresponding experiments on ventricular cardiomyocytes of rat. Again, under normoxic conditions SNAP (100 (microM) increased the number of TUNEL-positive cells to 12.6 +/- 4.9 % (vs. 5.4 +/- 0.7 % in controls). But when SNAP was added after 3 h of simulated ischemia, which was sufficient for energy depletion of the cells without inducing apoptotic cell death, the number of apoptotic cells did not increase. The ischemia-induced protection of hearts and cardiomyocytes goes along with an increased expression of several anti-apoptotic genes, mainly of the bcl-2 family. This indicates that ischemic conditions induce an anti-apoptotic gene program in cardiomyocytes, which may also be responsible for the observed anti-apoptotic actions in the intact ischemic/reperfused myocardium.

Objective: There is recent evidence that Ca2+ influx via reverse mode Na+/Ca2+ exchange (NCX) at the time of reperfusion can contribute to cardiomyocyte hypercontracture. However, forward NCX is essential for normalization of [Ca2+]i during reperfusion, and its inhibition may be detrimental. This study investigates the effect of NCX inhibition with KB-R7943 at the time of reperfusion on cell viability. Methods: The effect of several concentrations of KB-R7943 added at reperfusion was studied in Fura-2 loaded quiescent cardiomyocytes submitted to 40 min of simulated ischemia (NaCN 2 mM, pH 6.4), and in rat hearts submitted to 60 min of ischemia. [Ca2+]i and cell length were monitored in myocytes, and functional recovery and LDH release in isolated hearts. From these experiments an optimal concentration of KB-R7943 was identified and tested in pigs submitted to 48 min of coronary occlusion and 2 h of reperfusion. Results: In myocytes, KB-R7943 at concentrations up to 15 μM reduced [Ca2+]i rise and the probability of hypercontracture during re-energization ( P

The influence of the sodium-proton-exchanger-1 (NHE-1) inhibitor HOE694 on alpha- or beta-adrenoceptor mediated stimulation of protein synthesis was investigated in cultured ventricular cardiomyocytes from adult rat pre-treated with fetal calf serum to induce hypertrophic responsiveness to beta-adrenoceptor stimulation. Stimulation of alpha-adrenoceptors with phenylephrine (10 microM) in bicarbonate-free medium caused cellular alkalization (Delta pH(i): + 0.17 +/- 0.02, n = 5, P0.05). HOE694, an NHE-1 inhibitor, completely abolished this effect. [14C]phenylalanine incorporation into cellular protein mass increased in the presence of phenylephrine by 23+/-8%, and this effect was also abolished in the presence of HOE694. HOE694 (1 mciroM) neither influenced basal protein synthesis nor interfered with alpha-adrenoceptor mediated activation of ERK2. Phorbol myristate acetate, a direct stimulator of protein kinase C, mimicked the effect of alpha-adrenoceptor stimulation in regard to protein synthesis, but did not lead to cellular alkalization. Protein synthesis increased in the presence of isoprenaline, a beta-adrenoceptor agonist also. Again, HOE694 attenuated the stimulation of protein synthesis although isoprenaline did not cause cellular alkalization. In conclusion, the growth response to different hypertrophic stimuli, namely alpha- or beta-adrenoceptor stimulation, is attenuated in the presence of the NHE-1 inhibitor HOE694 and this inhibition is independent from cellular alkalization.

Objective: The hypertrophic response of adult rat cardiomyocytes to norepinephrine via α-adrenoceptor stimulation is limited by an inhibitory cross-talk of simultaneously β-adrenoceptor stimulation. On the other hand, neuropeptide Y (NPY), known to be co-secreted with norepinephrine from intramural nerve endings of the heart, exerts an anti-β-adrenergic effect. Therefore, it should be expected that NPY enhances the hypertrophic response to norepinephrine. This hypothesis was addressed in the present study. Methods: Isolated adult ventricular cardiomyocytes from rats were used. As parameters of hypertrophic growth we investigated cell volume, cross-sectional area, protein mass. Protein and RNA synthesis were determined by incorporation of [14C]phenylalanine or [14C]uridine, respectively. Results: Norepinephrine (1 μmol/l) did not significantly increase protein or RNA synthesis. In co-presence of NPY (100 nmol/l), however, norepinephrine increased protein synthesis by 44% and RNA synthesis by 18%. Under the same conditions, NPY enhanced the effect of norepinephrine on cell volume from +6.4 to +18.2%, its effect on cross-sectional area from +16 to +23%, and increased the protein/DNA ratio from 32.5 to 35.6 mg/mg. In parallel, norepinephrine caused a translocation of PKC-α and PKC-δ into the particular fractions and this effect of norepinephrine was also enhanced by co-presence of NPY. In contrast, NPY did not enhance ERK-activation caused by norepinephrine. Conclusion: Our study indicates the anti-β-adrenergic effect of NPY is sufficient to modulate the hypertrophic response of adult ventricular cardiomyocytes to norepinephrine. The results suggest that the hypertrophic effect of norepinephrine via α-adrenoceptor stimulation can be modulated by co-release of NPY from intramural nerve endings.

More than 100 million animals are used for research purposes around the world annually, most of them in Europe and the USA. Rats, mice, and other rodents make up 85–90% of all research animals. 1–1.5% correspond to dogs and cats and less than 1% is represented by non-human primates. Despite a significant decrease in the numbers of animals used in biomedical research and testing in the last two decades, in part due to more restrictive policies and development of alternative methods ( in vitro tests, availability of human cells and tissues, computational models, and others), the magnitude of these numbers, together with an increasing social sensitivity towards animal pain and suffering, makes the debate about the use of animals in experimental research an important and indisputable ethical issue among scientists, scientific journals, regulatory committees, and local, national, and international agencies. The use of animals in research has historically represented an irresolvable moral dilemma between the value of the expected benefits and the suffering, pain, confinement, and death to which it subjects sensitive living beings. Scientists using animals have to work on the assumption that the resulting increase in knowledge or improvement in human health outweighs all these associated costs, which in some cases has been arguable. The Cartesian view that prevailed up to the second half of the last century tried to circumvent this ethical conflict by depriving the animals of any sensitive and intellectual capacity. This view of animal status was extended throughout Europe for centuries due to the important impact the French philosopher Descartes (1596–1650) had on our concept about animal consciousness. Non-human living beings were considered to lack cognitive abilities and could not feel pain or distress but only automatic reactions in response to external stimuli. This doctrine had a pernicious effect on animal welfare and gave rise …

Parathyroid hormone-related peptide (PTHrP) is expressed throughout the cardiovascular system including coronary endothelial cells. Factors involved in the regulation of cardiac PTHrP expression have not been examined before. This study investigates the influence of transforming growth factor (TGF)- β 1 on ventricular PTHrP expression. Coronary endothelial cells were isolated from ventricles of adult rats and PTHrP protein expression in these cultures was analysed by immunoblotting. TGF- β 1 caused a concentration-dependent reduction in PTHrP protein within 24 h. In transgenic mice over-expressing TGF- β 1 ventricular PTHrP protein expression and release was reduced compared to non-transgenic littermates. Similar concerns hold for PTHrP mRNA content (RT-PCR). Since ventricular TGF- β 1 expression increases under pathophysiological conditions like arterial hypertension, ventricular PTHrP expression was further determined in aging spontaneously hypertensive (SHR-SP) and normotensive rats. TGF- β 1 expression was increased in SHR-SP and ventricular PTHrP mRNA expression was downregulated at the age of 10 months. PTHrP expression did not recover in elder SHR-SP in which TGF- β 1 expression was normalized again. Finally, we investigated ventricular PTHrP expression in rats after banding of the ascending aorta which generates a pressure induced hypertrophy without an induction of TGF- β 1 expression. In ventricles from these animals, PTHrP expression was transiently increased and normalized at day 3. In conclusion, PTHrP expression was reduced under all conditions in which coronary endothelial cells were exposed to TGF- β 1 . PTHrP expression does not correlate with cardiac hypertrophy. Since coronary endothelial cells represent the majority of PTHrP producing cells in the ventricle its downregulation by TGF- β 1 seems to be relevant for the paracrine effects of PTHrP.

Adult ventricular cardiomyocytes show an unusual structure-function relationship for cyclic AMP-dependent effects of PTHrP. We investigated whether PTHrP(1 – 16), void of biological activity on classical PTHrP target cells, is able to mimic the positive contractile effect of PTHrP(1 – 34), a fully biological agonist on cardiomyocytes. Adult ventricular cardiomyocytes were paced at a constant frequency of 0.5 Hz and cell contraction was monitored using a cell-edge-detection system. Twitch amplitudes, expressed as per cent cell shortening of the diastolic cell length, and rate constants for maximal contraction and relaxation velocity were analysed. PTHrP(1 – 16) (1 μmol l−1) mimicked the contractile effects of PTHrP(1 – 34) (1 μmol l−1). It increased the twitch amplitude from 5.33±0.72 to 8.95±1.10 (% dl l−1) without changing the kinetic of contraction. PTH(1 – 34) (10 μmol l−1) affected the positive contractile effect of PTHrP(1 – 34), but not that of PTHrP(1 – 16). RpcAMPS (10 μmol l−1) inhibited the positive contractile effect of PTHrP(1 – 34), but not that of PTHrP(1 – 16). The positive contractile effect of PTHrP(1 – 16) was antagonized by the ETA receptor antagonist BQ123. Sarafotoxin 6b and PTHrP(1 – 16), but not PTHrP(1 – 34), replaced 3H-BQ123 from cardiac binding sites. We conclude that N-terminal PTHrP peptides void of a PTH/PTHrP-receptor binding domain are able to bind to, and activate cardiac ETA receptors. British Journal of Pharmacology (2001) 132, 427–432; doi:10.1038/sj.bjp.0703830

The study investigated whether beta-adrenoceptor antagonists augment the hypertrophic response of cardiomyocytes evoked by norepinephrine.In adult ventricular cardiomyocytes, stimulation of alpha- but not beta-adrenoceptors induces myocardial hypertrophy. Natural catecholamines, like norepinephrine, stimulate simultaneously alpha- and beta-adrenoceptors. We investigated whether beta-adrenoceptor stimulation interferes with the hypertrophic response caused by alpha-adrenoceptor stimulation.Adult ventricular cardiomyocytes isolated from rats were used as an experimental model. Hypertrophic parameters under investigation were stimulation of phenylalanine incorporation and protein mass, stimulation of 14C-uridine incorporation and RNA mass, and increases in cell shape.Norepinephrine (0.01 to 10 micromol/liter) increased concentration-dependent phenylalanine incorporation; pEC50 value was 5.9 +/- 0.1 (n = 8). The alpha1-adrenoceptor antagonist prazosin (0.1 micromol/liter) suppressed norepinephrine-induced increase in rate of protein synthesis. Conversely, propranolol (1 micromol/liter) and the beta1-adrenoceptor selective antagonists CPG 20712A (300 nmol/liter) or atenolol (1 micromol/liter) augmented increases in phenylalanine incorporation caused by norepinephrine. Addition of the beta2-adrenoceptor antagonist ICI 118,551 (55 nmol/liter) did not influence the hypertrophic effect of norepinephrine. Atenolol augmented the norepinephrine-induced increases of all hypertrophic parameters investigated (i.e., protein mass, uridine incorporation, RNA mass, cell volume, and cross-sectional area). In the presence of norepinephrine, inhibition of beta1-adrenoceptors increased the amount of protein kinase C-alpha and -delta isoforms translocated into the particulate fraction. The effect of pharmacological inhibition of beta1-adrenoceptors could be mimicked by Rp-cAMPS (adenosine-3', 5'-cyclic phosphorothiolate-Rp). The inhibitory effect of beta1-adrenoceptor stimulation on the alpha-adrenoceptor-mediated effect persisted in cardiomyocytes isolated from hypertrophic hearts of rats submitted to aortic banding.In isolated ventricular cardiomyocytes from rats, beta1-adrenoceptor stimulation attenuates the hypertrophic response evoked by alpha1-adrenoceptor stimulation.

We investigated whether selective β1-adrenoceptor stimulation causes hypertrophic growth on isolated ventricular cardiomyocytes from adult rat. As parameters for the induction of hypertrophic growth, the increases of [14C]phenylalanine incorporation, protein and RNA mass, and cell size were determined. Isoproterenol (Iso, 10 μM) alone had no growth effect. In the presence of the β2-adrenoceptor antagonist ICI-118551 (ICI, 10 μM), Iso caused an increase in [14C]phenylalanine incorporation, protein and RNA mass, cell volume, and cross-sectional area. We showed for phenylalanine incorporation that the growth effect of Iso+ICI could be antagonized by β1-adrenoceptor blockade with atenolol (10 μM) or metoprolol (10 μM), indicating that it was caused by selective β1-adrenoceptor stimulation. The growth response to Iso+ICI was accompanied by an increase in ornithine decarboxylase (ODC) activity and expression. Inhibition of ODC by the ODC antagonist difluoromethylornithine (1 mM) attenuated this hypertrophic response, indicating that ODC induction is causally involved. The growth response to Iso+ICI was found to be cAMP independent but was sensitive to genistein (100 μM) or rapamycin (0.1 μM). The reaction was enhanced in the presence of pertussis toxin (10 μM). We conclude that selective β1-adrenoceptor stimulation causes hypertrophic growth of ventricular cardiomyocytes by a mechanism that is independent of cAMP but dependent on a tyrosine kinase and ODC.

Objective: TGF-β stimulation of cardiac myocytes induces a hypertrophic responsiveness to β-adrenoceptor stimulation. This study investigates whether this β-adrenoceptor mediated effect depends on induction of ornithine decarboxylase (ODC). Methods: Isolated adult ventricular cardiomyocytes from rats were used as an experimental model. Cells were either cultured in 20% (v/v) FCS to activate autocrine released TGF-β or used without pre-treatment. The hypertrophic response was characterized by an increased 14C-phenylalanine incorporation, RNA and protein mass or by an increased expression of atrionatriurectic factor and ODC. The results on cell cultures were compared to those achieved by isoprenaline perfused mice hearts from transgenic mice overexpressing TGF-β1. Results: ODC activity and expression increased within 2 h in TGF-β1 pre-treated cells under isoprenaline. In the presence of ODC inhibitors (α-methylornithine or difluoromethylornithine) this increase remained absent and the increases in 14C-phenylalanine incorporation, protein and RNA mass under isoprenaline were abolished. In cells not exposed to TGF-β no induction of ODC was observed. Isoprenaline also induced ODC in isolated perfused ventricles from transgenic mice overexpressing TGF-β1, but not in ventricles from their nontransgenic counterparts. Conclusions: This study shows first, a pivotal role for ODC induction in the hypertrophic response of cardiomyocytes to β-adrenoceptor stimulation and second, that ODC induction in vivo and in vitro requires pre-treatment of cardiomyocytes with TGF-β. It is concluded that TGF-β induces a hypertrophic responsiveness to β-adrenoceptor stimulation that is characterized by ODC induction.

The effect of factor XIII on endothelial barrier function was studied in a model of cultured monolayers of porcine aortic endothelial cells and saline-perfused rat hearts. The thrombin-activated plasma factor XIII (1 U/ml) reduced albumin permeability of endothelial monolayers within 20 min by 30 ± 7% (basal value of 5.9 ± 0.4 × 10−6 cm/s), whereas the nonactivated plasma factor XIII had no effect. Reduction of permeability to the same extent, i.e., by 34 ± 9% could be obtained with the thrombin-activated A subunit of factor XIII (1 U/ml), whereas the iodoacetamide-inactivated A subunit as well as the B subunit had no effect on permeability. Endothelial monolayers exposed to the activated factor XIII A exhibited immunoreactive deposition of itself at interfaces of adjacent cells; however, these were not found on exposure to nonactivated factor XIII A or factor XIII B. Hyperpermeability induced by metabolic inhibition (1 mM potassium cyanide plus 1 mM 2-deoxy-d-glucose) was prevented in the presence of the activated factor XIII A. Likewise, the increase in myocardial water content in ischemic-reperfused rat hearts was prevented in its presence. This study shows that activated factor XIII reduces endothelial permeability. It can prevent the loss of endothelial barrier function under conditions of energy depletion. Its effect seems related to a modification of the paracellular passageways in endothelial monolayers.

The present study investigated the role of early response kinase (ERK) and phosphatidylinositol 3 (PI 3)-kinase in ventricular cardiomyocytes from adult rat for the hypertrophic response to alpha-adrenoceptor stimulation. Parameters of the hypertrophic response were stimulation of protein synthesis and induction of creatine kinase BB. The alpha-adrenoceptor agonist phenylephrine (10 micromol/l) activated ERK2 and PI 3-kinase. The protein kinase C inhibitor bisindolylmaleimide (5 micromol/l) and the mitogen-activated protein kinase kinase inhibitor PD-98059 (10 micromol/l) but not the tyrosine kinase inhibitor genistein (100 micromol/l) blocked ERK2 activation. Inhibition of ERK2 activation abolished induction of creatine kinase BB by phenylephrine but not the increase in protein synthesis. The PI 3-kinase inhibitor wortmannin (100 nmol/l) blocked protein synthesis under alpha-adrenoceptor stimulation but did not interfere with ERK2 activation. Inhibition of the ERK2 pathway with PD-98059 did not affect PI 3-kinase activation. We conclude that ERK2- and PI 3-kinase-dependent pathways represent two mutually exclusive ways of signaling that lead to different aspects of the hypertrophic response to alpha-adrenoceptor stimulation.

The Editorial Team at Cardiovascular Research ( CVR ) is constantly trying to evaluate which research topics are the most interesting to its readers. Two ways we do this is to look at which articles are most often downloaded from the Journal's website on the Internet and which are cited most frequently in the literature. Now, at the end of 2008, we are in a position to look back at the year 2007 to see how well the papers from last year performed and which were most popular. In addition, this will give us a look ahead at our next impact factor for 2008. Table 1 shows the 20 most-cited articles from 2007. It is not surprising that review articles were the most cited, with all of the top 10 being reviews. … *Corresponding author. Tel: +49 641 99 47242; fax: +49 641 99 47209. E-mail address : CVR{at}physiologie.med.uni-giessen.de

Over the past several years, the editors of leading international cardiovascular journals have met to form the HEART Group and to discuss areas of growing, common interest. Recently, the HEART Group has developed a document that addresses general ethical principles in the conduct of the scientific process with which all of the editors concur. Published essentially simultaneously in all of the participating journals, including this journal, this document presents the ethical tenets accepted by all of the undersigned editors that will (continue to) guide their decisions in the editorial process. These are the general principles on which the HEART Group is based and by which we, as a group, abide; however, please note that individual journal members and their respective societies may have their own rules and regulations that supersede the guidelines of the HEART Group. Acta Cardiologica Hugo Ector, MD, PhD Editor-in-Chief Patrizio Lancellotti, MD Editor-in-Chief American Journal of Cardiology William C. Roberts, MD Editor-in-Chief American Journal of Geriatric Cardiology Nanette K. Wenger, MD Editor-in-Chief Annals of Noninvasive Electrocardiology Arthur J. Moss, MD Editor-in-Chief Canadian Journal of Cardiology Eldon R. Smith, MD Editor-in-Chief Cardiology Jeffrey S. Borer, MD Editor-in-Chief Cardiosource Review Journal Kim A. Eagle, MD Editor-in-Chief Cardiovascular Drug Reviews Jane Freedman, MD Incoming Editor-in-Chief Henry Krum, PhD Incoming Editor-in-Chief Chim Lang, MD Incoming Editor-in-Chief Cardiovascular Drugs and Therapy Willem J. Remme, MD, PhD Editor-in-Chief Cardiovascular Research Hans Michael Piper, MD, PhD Editor-in-Chief Catheterization and Cardiovascular Interventions Christopher J. White, MD Editor-in-Chief Circulation Joseph Loscalzo, MD, PhD Editor-in-Chief Circulation Research Eduardo Marban, MD, PhD Editor-in-Chief Coronary Artery Disease Burton E. Sobel, MD Editor Current Opinion in Cardiology Robert Roberts, MD Editor Current Problems in Cardiology Shahbudin H. Rahimtoola, MD Editor Europace A. John Camm, MD Editor-in-Chief European Heart Journal Frans Van de Werf, MD Editor-in-Chief European Journal of Heart Failure Karl Swedberg, MD, PhD Editor-in-Chief Heart Adam D. Timmis, MD Editor Heart & Lung: The Journal of Acute and Critical Care Kathleen S. Stone, PhD, RN Editor-in-Chief Heart Rhythm Douglas P. Zipes, MD Editor-in-Chief International Journal of Interventional Cardioangiology David G. Iosseliani, MD Editor-in-Chief Journal of Cardiovascular Computed Tomography Allen J. Taylor, MD Editor-in-Chief Journal of Cardiovascular Pharmacology Michael R. Rosen, MD Editor Journal of Interventional Cardiology Cindy L. Grines, MD Editor-in-Chief Journal of the American College of Cardiology Anthony N. DeMaria, MD Editor-in-Chief JACC: Cardiovascular Imaging Jagat Narula, MD, PhD Editor-in-Chief JACC: Cardiovascular Interventions Spencer B. King III, MD Editor-in-Chief Journal of Electrocardiology Galen S. Wagner, MD Editor-in-Chief Journal of Interventional Cardiac Electrophysiology Sanjeev Saksena, MD Editor-in-Chief Journal of the American Society of Echocardiography Alan S. Pearlman, MD Editor-in-Chief Journal of Heart Valve Disease Endre Bodnar, MD, PhD Editor-in-Chief Robert W. Emery, MD Incoming Editor-in-Chief Journal of Thoracic and Cardiovascular Surgery Lawrence H. Cohn, MD Editor-in-Chief Netherlands Heart Journal Ernst E. van der Wall, MD Editor-in-Chief Pediatric Cardiology Ra-id Abdulla, MD Editor-in-Chief Progress in Cardiovascular Diseases Michael Lesch, MD Editor Revista Espanola de Cadiologia Fernando Alfonso, MD, PhD Editor-in-Chief Scandinavian Cardiovascular Journal Rolf Ekroth, MD Chief Editor

Muscle cells, and in particular cardiomyocytes, consume a large amount of chemical energy to produce mechanical work. Myofilament contraction is regulated and synchronized within the cell by rapid, transient changes in the cytosolic Ca2+ concentration. Most of this calcium is released from the sarcoplasmic reticulum (SR), which has a large surface for Ca2+ transport, in close contact with the myofilaments. In between the myofilaments, cardiomyocytes are packed with mitochondria. These generate the energy needed for myofilament contraction, but serve also as an additional store for Ca2+. Thus, SR and mitochondria interact physiologically to control cellular Ca2+ homeostasis. It is partly through the Ca2+ loading of the mitochondria that their energy production is matched with the contractile energy demand. Mitochondria are now recognized as an essential effector structure determining cell death and survival. This is not only because of their central role in cellular energy metabolism, but also as key players in survival and death signalling. Alterations in the function of the SR are now known to be of primary importance for the development of hypertrophy and heart failure and in the genesis of life-threatening arrhythmias. Therefore, interaction between SR and mitochondria not only determines cardiomyocyte function under physiological conditions, but also is relevant for the development of cardiomyocyte dysfunction or death under pathophysiological conditions. The present Spotlight Issue of Cardiovascular Research aims to update our understanding of these important and rapidly evolving aspects of cardiac physiology and pathophysiology through a series of invited reviews and original contributions. In this issue, Orchard and Brette review the structure and function of t-tubules, their contribution to Ca2+ transients in cooperation with the SR, and the pathophysiological importance of their remodelling in … *Corresponding author. Tel: +34 93 489 4038; fax: +34 93 489 4032. E-mail address : dgdorado{at}ir.vhebron.net

Currently, restoring blood flow in an acutely occluded vessel represents the most effective, long-term clinical therapy for acute myocardial infarction. Experimental research over the past years has demonstrated that the first minutes of reperfusion represent a window of opportunity for additional therapy that is yet unused in the clinical setting. Experimentally, it has been shown that a considerable part of myocardial injury developing after an episode of prolonged ischaemia and subsequent reperfusion is due to causes emerging during the period of reperfusion.1–6 This type of injury has been termed ‘reperfusion injury’. For a long time it had been difficult to prove the existence of this type of injury; this could only be done by identifying interventions applied just at the time of reperfusion that would reduce the otherwise resulting myocardial injury. Early studies in this area had shown that interventions during reperfusion aimed at controlling cytosolic [Ca2+] or the activation of the contractile machinery in cardiomyocytes could reduce cardiac cell necrosis.7–10 More recently, a protective protocol called ‘postconditioning’11 has been discovered that can be used in the clinical setting. In this protocol, very brief, repetitive bouts of ischaemia are deliberately applied during the first minutes of reperfusion. In ground-breaking work, Ovize and co-workers12,13 showed that postconditiong can reduce the signs of tissue injury, such as the release of myocardial enzymes, in patients receiving acute coronary revascularisation. In a rapidly growing number of manuscripts, details of the protective signalling of postconditioning and pharmacological interventions designed to exploit its protective potential have been identified. It has become apparent that activation of specific protein kinases can provide and mediate cardiac protection during the reperfusion period. These have been termed ‘reperfusion injury salvage kinases’ (RISK).14–16 …

The atrial natriuretic peptide (ANP)-C receptor is generally believed to clear ANP; however, the ANP-C receptor may serve to reduce cAMP by inhibiting adenylate cyclase. ANP decreases endothelial permeability in coronary endothelial cell monolayers. We tested the hypothesis that part of this effect might be mediated by the ANP-C receptor. We used an endothelial cell monolayer from rat coronary endothelium and measured albumin flux. We applied either ANP or a ring-deleted ANP (C-ANP), which only stimulates the ANP-C receptor. ANP and C-ANP both decreased permeability from 100 pM to 100 nM by 60 and 30%, respectively. ANP increased endothelial cGMP contents 5.5-fold, whereas C-ANP had no effect. ANP reduced endothelial cAMP contents by 75%, which was only partly blocked by pertussis toxin. C-ANP also reduced cAMP; however, this effect was completely blocked by pertussis toxin. Protein kinase G inhibition blocked the ANP-mediated decrease in permeability by 50%. In contrast, pretreatment with pertussis toxin, in the face of protein kinase G inhibition, blocked the effect completely. C-ANP decreased permeability by half the amount of ANP. This C-ANP effect was completely blocked by pertussis toxin but not by protein kinase G inhibition. Isoproterenol (10 μM) increased permeability by almost 50%, which was completely blocked by ANP but only partially blocked by C-ANP. The C-ANP effect was blocked completely by pertussis toxin. Isoproterenol increased cAMP threefold, which was abolished by ANP. C-ANP reduced the isoproterenol-induced increase in cAMP by 50%. Isoproterenol had no effect on cGMP. We conclude that agonist binding to the ANP-C receptor inhibits cAMP production via a Giprotein-coupled signaling system. This inhibition may contribute to the decreased endothelial permeability evoked by ANP in this system.

Cardiovascular cells (cardiomyocytes and smooth muscle cells) are target cells for parathyroid hormone (PTH) and the structurally related peptide parathyroid hormone-related peptide (PTH-rP). PTH activates protein kinase C (PKC) of cardiomyocytes via a PKC activating domain previously identified on chondrocytes. Activation of PKC leads to hypertrophic growth and re-expression of fetal type proteins in cardiomyocytes. This hypertrophic effect of PTH might contribute to left ventricular hypertrophy in hemodialysis patients with secondary hyperparathyroidism. PTH-rP is expressed in cardiovascular cells (endothelial cells and smooth muscle cells). It does not mimic the above described actions of PTH but exerts effects of its own on cardiomyocytes. These effects involve activation of protein kinase A, via a N-terminal domain distinct from that identified on PTH, and activation of PKC, via a C-terminally located domain distinct from that found on PTH. On smooth muscle cells PTH and PTH-rP reduce the influence of extracellular calcium, through cAMP-dependent mechanisms. These inhibitory effects on voltage-dependent L-type calcium channels of smooth muscle cells cause vasorelaxation. Present studies concerning cardiovascular actions of either PTH and PTH-rP suggest that increased plasma levels of PTH and PTH-rP influence cardiomyocyte and smooth muscle cell physiology. It can be assumed that PTH-rP acts as a paracrine or autocrine modulator in heart and vessels.

After many years of debate during which the preponderance of opinion was skeptical about the experimental and clinical existence of reperfusion injury (with attitudes ranging between polite condescendence and overt denial), it can be said that the controversy has been now largely resolved, and that the death of cardiomyocytes during reperfusion is now generally admitted as a fact. Perhaps this agreement is still more firm in the experimental literature than in clinical cardiology. The discovery of the phenomenon of postconditioning [1] has undoubtedly contributed to this change of opinion. The first reports of postconditioning the human heart by Laskey et al. and Ovize et al. certainly support the concept of reperfusion injury as a clinical entity that can be measured even in a small group of patients [2,3]. The report by Zhao et al. [1] has aroused a renewed interest in the mechanisms of cell death during ischemia–reperfusion, and of endogenous protection against it [4]. Postconditioning is as powerful as preconditioning and has the potential of being clinically applicable in the most common situation of unexpected coronary occlusion and acute myocardial infarction [3]. Yet, the mechanisms by which postconditioning limits reperfusion-induced cell death and the relationship between the mechanisms in pre- and postconditioning are far from being completely understood. The present Spotlight Issue presents a series of review articles with the aim of providing an up-to-date overview of the state of the art in the rapidly moving field of research on endogenous protection against myocardial cell death secondary to ischemia–reperfusion. The reason for the resurrection of this field of research is that it is necessary to gain an adequate … * Corresponding author. Tel.: +34 93 489 4038; fax: +34 93 489 4032. Email address: dgdorado{at}vhebron.net

The newly developed concept of postconditioning as a way of reducing reperfusion injury has been receiving much attention in the literature, both in basic science and clinical circles. Cardiovascular Research will be hosting a Spotlight Issue entitled “ Cardiac Protection by Pre- and Postconditioning ” in May of this year. This editorial is thought to serve as an introduction to the upcoming issue. One of the major advances of all times in cardiovascular therapy has been the development and generalization of reperfusion treatment in patients with acute coronary occlusion and evolving myocardial infarction. The experimental basis for that therapy was provided in the early 1980s by the group of Jennings and Reimer at Duke University with the demonstration that early reperfusion can salvage ischemic myocardium that otherwise would die, and that the effectiveness of reperfusion depends on the severity and duration of prior ischemia [1]. Despite its overall beneficial effect, experimental myocardial reperfusion was associated with prominent arrhythmias, enzyme release, and other adverse phenomena that were grouped under the imprecise term “reperfusion injury”. Reperfusion injury attracted the attention of many basic scientists, but the success of large clinical trials demonstrating the beneficial effect of reperfusion in patients pushed research on reperfusion injury into the background [2]. A second origin of the concept of reperfusion injury came from the work of Hearse et al. in London who showed in the mid-1970s that reperfusion of an isolated ischemic heart could provoke myocardial contracture and massive myocytolysis [3]. Since they attributed this phenomenon to tissue reoxygenation and the potentially detrimental role of oxygen, they termed this phenomenon the “oxygen paradox” [3,4]. In …

Background Resupply of oxygen to the myocardium after extended periods of ischemia or hypoxia can rapidly aggravate the already existing injury by provoking hypercontracture of cardiomyocytes (acute reperfusion injury). Previous studies indicated that halothane can protect ischemic-reperfused myocardium. The aim of the present study was to analyze on the cellular level the mechanism by which halothane may protect against reoxygenation-induced hypercontracture. Methods and Results To simulate ischemia-reperfusion, isolated adult rat cardiomyocytes were incubated at pH 6.4 under anoxia and reoxygenated at pH 7.4 in the presence or absence of 0.4 mmol/L halothane. Reoxygenation was started when intracellular Ca 2+ (measured with fura 2) had increased to ≥10 −5 mol/L and pH i (BCECF) had decreased to 6.5. Development of hypercontracture was determined microscopically. In the control group, reoxygenation provoked oscillations of cytosolic Ca 2+ (72±9 per minute at fourth minute of reoxygenation) accompanied by development of hypercontracture (to 65±3% of end-ischemic cell length). When halothane was added on reoxygenation, Ca 2+ oscillations were markedly reduced (4±2 per minute, P P i during reoxygenation. Similar effects on Ca 2+ oscillations and hypercontracture were observed when ryanodine (3 μmol/L), an inhibitor of the sarcoplasmic reticulum Ca 2+ release, or cyclopiazonic acid (10 μmol/L), an inhibitor of the sarcoplasmic reticulum Ca 2+ pump, were applied instead of halothane. Conclusions Halothane protects cardiomyocytes against reoxygenation-induced hypercontracture by preventing oscillations of intracellular Ca 2+ during the early phase of reoxygenation.

We investigated whether parathyroid hormone-related peptide (PTH-rP), recently found expressed in the heart, exerts growth and contractile effects on adult cardiomyocytes from rat hearts. Synthetic PTH-rP peptides were used covering either a protein kinase C (PKC)-activating domain [PTH-rP(107-111)], or an adenylate cyclase activating domain [PTH-rP(1-34) and PTH-rP(7-34)]. PTH-rP(107-111) (1 micro M) increased creatine kinase BB activity (CK-BB), a CK isoform re-expressed during cardiac hypertrophy, within 24 h by 62+/-12%. This induction was abolished in the presence of the mitogen-activated-protein (MAP)-kinase-kinase inhibitor PD 98059. PTH-rP(107-111) activated p42-MAP-kinase within 15 min, increased protein synthesis (19+/- 4%), total protein mass (19+/-5%), cell volume (45+/-7%), and cross-sectional area (38+/-9%) of cardiomyocytes. Activation of p42-MAP-kinase and increase in protein synthesis were abolished in presence of bisindolylmaleimide, a PKC inhibitor. PTH- rP(107-111) did not directly influence contractile activity but reduced the contractile response to isoprenaline. In contrast, PTH-rP(1-34) and PTH-rP(7-34) induced spontaneous contractile activity in 3-day-old cultures. This induction was abolished in presence of Rp-cAMPS, a protein kinase A inhibitor, indicating an involvement of cAMP in this response. PTH-rP(1-34) also increased the cellular accumulation of cAMP. It is concluded that PTH-rP exert direct effects on adult cardiomyocytes by activating either PKC via a functional domain covered by amino acids 107-111 or by activation of cAMP-dependent protein kinase via a functional domain covered by amino acids 7-34. Since these parts of PTH-rP have either no homology [PTH-rP(107-111)] or only a limited structural similarity [PTH-rP(7-34)] to parathyroid hormone, these activities of PTH-rP have to be clearly distinguished from those described for parathyroid hormone.

Abstract Whether organic nitrates are bioactivated to NO in cardiac muscle cells and may thus directly affect cardiac contractile function has remained an open question. Therefore, we determined the effects of the organic nitrates glyceryl trinitrate (100 μmol/L), pentaerythritol tetranitrate (10 μmol/L), and isosorbide-5-mononitrate on electrically stimulated contractile response (CR) and cAMP and cGMP content of isolated adult rat ventricular cardiomyocytes compared with different concentrations of the spontaneous NO donors S -nitroso- N -acetyl-d,l-penicillamine (SNAP) and 2,2-diethyl-1-hydroxy-1-nitroso-hydrazine (DEA/NO). A high concentration of spontaneous NO donors (100 μmol/L) caused a large increase in cGMP content that was accompanied by a decrease in CR to 73.8±6.7% (SNAP) and 80.9±6.1% (DEA/NO) of the control values. Inhibition of cGMP-dependent protein kinase by 10 μmol/L KT 5822 converted this effect into a pronounced improvement of CR (163.5±14.0%). By contrast, the organic nitrates caused a small but significant increase in cGMP, which was accompanied by an increase in cAMP and CR identical to that induced by 10 nmol/L isoprenaline (141.6±6.4%). A similar effect was observed with a low concentration (1 μmol/L) of SNAP and DEA/NO. All increases in CR induced by nitrates were abolished after inhibition of cAMP-dependent protein kinase by Rp-cAMPS (10 μmol/L). The positive contractile effect of isoprenaline was enhanced by 1 μmol/L SNAP. This effect was also demonstrated in isolated rat papillary muscles. These results indicate that in cardiac muscle (1) organic nitrates are bioactivated to NO; (2) this results in a moderate increase in cGMP, which causes an improved CR by increasing cAMP and activating cAMP-dependent protein kinase; and (3) a large increase in cGMP, produced by high doses of NO donors, reduces CR because of the activation of cGMP-dependent protein kinase.

Hypoxia–reoxygenation induces loss of endothelial barrier function and oedema formation, which presents a major impediment for recovery of the organ. The integrity of the endothelial barrier is highly dependent on its contractile machinery and actin dynamics, which are precisely regulated by Rho GTPases. Perturbed activities of these Rho-GTPases under hypoxia–reoxygenation lead to derangement of the actin cytoskeleton and therefore may affect the integrity of the endothelial barrier. The aim of the present study was to analyse the role of these GTPases in regulating endothelial barrier function during hypoxia–reoxygenation in cultured porcine aortic endothelial cells and isolated perfused rat hearts. Hypoxia–reoxygenation induced an increase in albumin permeability of endothelial monolayers accompanied by an activation of the endothelial contractile machinery, derangement of the actin cytoskeleton and loss of VE-cadherin from cellular junctions. Inhibition of contractile activation with ML-7 partially protected against hypoxia–reoxygenation-induced hyperpermeability. Likewise, reoxygenation caused an increase in RhoA and a reduction in Rac1 activity accompanied by enhanced stress fibre formation and loss of peripheral actin. Inhibition of RhoA/rho kinase (Rock) signalling with RhoA or Rock inhibitors led to a complete depolymerisation and derangement of the actin cytoskeleton and worsened hypoxia–reoxygenation-induced hyperpermeability. Activation of Rac1 using a cAMP analogue, 8-CPT-O′-Me-cAMP, which specifically activates Epac/Rap1 signalling, restored peripheral localisation of actin and VE-cadherin at cellular junctions and abrogated reoxygenation-induced hyperpermeability. Similar results were reproduced in isolated saline-perfused rat hearts. These data show that activation of Rac1 but not the inhibition of RhoA preserves endothelial integrity against reoxygenation-induced loss of barrier function.

Reperfusion may induce additional cell death in patients with acute myocardial infarction receiving primary angioplasty or thrombolysis. Altered intracellular Ca(2+) handling was initially considered an essential mechanism of reperfusion-induced cardiomyocyte death. However, more recent studies have demonstrated the importance of Ca(2+)-independent mechanisms that converge on mitochondrial permeability transition (MPT) and are shared by cardiomyocytes and other cell types. This article analyses the importance of Ca(2+)-dependent cell death in light of these new observations. Altered Ca(2+) handling includes increased cytosolic Ca(2+) levels, leading to activation of calpain-mediated proteolysis and sarcoplasmic reticulum-driven oscillations; this can induce hypercontracture, but also MPT due to the privileged Ca(2+) transfer between sarcoplasmic reticulum and mitochondria through cytosolic Ca(2+) microdomains. In the opposite direction, permeability transition can worsen altered Ca(2+) handling and favour hypercontracture. Ca(2+) appears to play an important role in cell death during the initial minutes of reperfusion, particularly after brief periods of ischaemia. Developing effective and safe treatments to prevent Ca(2+)-mediated cardiomyocyte death in patients with transient ischaemia, by targeting Ca(2+) influx, intracellular Ca(2+) handling, or Ca(2+)-induced cell death effectors, is an unmet challenge with important therapeutic implications and large potential clinical impact.