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Ischemia-induced apoptosis of endothelial cells may contribute to tissue injury, organ failure, and transplantation rejection. However, little is known about survival mechanisms capable to counteract endothelial apoptosis. This study investigated the potential role of an endogenous anti-apoptotic response elicited by transient hypoxia, capable to avert ongoing apoptosis in endothelial cells. Experiments were carried out in three different types of cultured endothelial cells (human umbilical vein, pig aorta, and from rat coronary microvasculature). As a pro-apoptotic challenge endothelial cells were cultured in serum-free medium and subjected to hypoxia for 2 h. We found that transient hypoxia reduced caspase 3 activation within 1 h of hypoxia. Accordingly, the number of apoptotic cells was reduced after 24 h of reoxygenation. This was true for all three cell types analyzed. Analysis of Akt and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathways revealed that hypoxia induced a transient activation of ERK 2 but not of Akt. ERK 2 phosphorylation preceded the phosphorylation of pro-apoptotic molecule Bad at Ser112, an inhibitory phosphorylation site specific for ERK. The protective effects of hypoxia regarding Bad phosphorylation, caspase 3 activation, and apoptosis were abolished by MEK 1/2 inhibitors, PD98059 or UO126, as well as by antisense oligonucleotides directed against ERK 1/2. Furthermore, inhibition of this pathway inhibited hypoxia-induced increase in mitochondrial membrane potential. The present study demonstrates that transient hypoxia induces a novel survival mechanism that protects endothelial cells against apoptosis. This endogenous process involves MEK/ERK-mediated inhibition of the pro-apoptotic molecule Bad and caspase 3. anti-apoptosis; Bad; extracellular signal-regulated kinases; phosphoinositide 3-kinase/Akt doi: 10.1152/ajpcell.00333.2009.

A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the space flight environment has never been accomplished because of significant technological and logistical hurdles. Moreover, the effects of space flight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared with identical ground control cultures. Global microarray and proteomic analyses revealed that 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground-based microgravity culture model. Space flight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during space flight missions and provide novel therapeutic options on Earth. microgravity | Space Shuttle | low shear modeled microgravity | rotating wall vessel | Salmonella

Opposite effect of cAMP signaling in endothelial barriers of different origin. Am J Physiol Cell Physiol 287: C1246-C1255, 2004; doi: 10.1152/ajpcell.00132.2004.--cAMP-mediated signaling mechanisms may destabilize or stabilize the endothelial barrier, depending on the origin of endothelial cells. Here, microvascular coronary [coronary endothelial cells (CEC)] and macrovascular aortic endothelial cell (AEC) monolayers with opposite responses to cAMP were analyzed. Macromolecule permeability, isometric force, activation state of contractile machinery [indicated by phosphorylation of regulatory myosin light chains (MLC), activity of MLC kinase, and MLC phosphatase], and dynamic changes of adhesion complex proteins (translocation of VE-cadherin and paxillin) were determined, cAMP signaling was stimulated by the adenosine receptor agonist 5'-N-(ethylcarboxamido)-adenosine (NECA), the [beta]-adrenoceptor agonist isoproterenol (Iso), or by the adenylyl cyclase activator forskolin (FSK). Permeability was increased in CEC and decreased in AEC on stimulation with NECA, Iso, or FSK. The effects could be inhibited by the PKA inhibitor Rp-8-CPT-cAMPS and imitated by the PKA activator Sp-cAMPS. Under cAMP/PKA-dependent stimulation, isometric force and MLC phosphorylation were reduced in monolayers of either cell type, due to an activation of MLC phosphatase. In CEC but not in AEC, FSK induced delocalization of VE-cadherin and paxillin from cellular adhesion complexes as indicated by cell fractionation and immunofluorescence microscopy. In conclusion, decline in contractile activation and isometric force contribute to cAMP/PKA-mediated stabilization of barrier function in AEC. In CEC, this stabilizing effect is overruled by cAMP-induced disintegration of cell adhesion structures. endothelial cell adhesion; endothelial permeability; isometric force; myosin light chain kinase; myosin light chain pbosphatase

Transcription activator protein 1 mediates [alpha]- but not [beta]-adrenergic hypertrophic growth responses in adult cardiomyocytes. Am J Physiol Heart Circ Physiol 286: H2369-H2375, 2004. First published February 5, 2004; 10.1152/ajpheart.00741.2003.--In some models of cardiac hypertrophy, activation of activator protein 1 (AP-1) correlates with growth. However, AP-1 is also activated by stimuli not involved in cardiac growth. This raises the following questions: does AP-1 plays a causal role for cardiomyocyte growth, and is this role model or stimulus dependent? We used a single model to address these questions, i.e., ventricular cardiomyocytes of adult rats, and two growth stimuli, i.e., [alpha]-and [beta]-adrenoceptor agonists [10 [micro]M phenylephrine (PE) and 1 [micro]M isoprenaline (Iso), respectively]. After 1 h of stimulation with PE, mRNA expression of c-Fos and c-Jun was upregulated to 185 [+ or -] 32 and 132 [+ or -] 13% of control. Fos and Jun proteins formed the AP-1 complex. PE stimulated DNA binding activity of AP-1 to 165 [+ or -] 22% of control within 2 h and increased protein synthesis to 161 [+ or -] 27% of control and cross-sectional area to 126 [+ or -] 4% of control. Inhibition of AP-1 binding activity by cAMP response element (CRE) decoy oligonucleotides abolished both of these growth responses. Iso stimulated AP-1 binding activity to 203 [+ or -] 19% of control within 2 h and stimulated protein synthesis to 145 [+ or -] 17% of control. However, the growth effect of Iso was not abolished by CRE decoys: Iso increased protein synthesis to 158 [+ or -] 17% of control in the presence of CRE. In conclusion, AP-1 is a causal mediator of the [alpha]-adrenergic, but not the [beta]-adrenergic, growth response of cardiomyocytes. hypertrophy; adrenoceptors; immediate early genes; transcription factors

Klingenberg, D., D. Gunduz, F. Hartel, K. Bindewald, M. Schafer, H. M. Piper, and T. Noll. MEK/MAPK as a signaling element in ATP control of endothelial myosin light chain. Am J Physiol Cell Physiol 286: C807-C812, 2004; 10.1152/ajpcell.00002.2003.--Phosphorylation of endothelial myosin light chains (MLC) is a key mechanism in control of endothelial contractile machinery. Extracellular ATP influences endothelial MLC phosphorylation by either activation of [Ca.sup.2+] -dependent MLC kinase or [Ca.sup.2+]-independent MLC phosphatase. Here, the role of the MEK/MAPK pathway in this signaling was investigated in porcine aortic endothelial cells. Phosphorylation of ERK2 and phosphorylation of MLC were analyzed in cultured aortic endothelial cells. ATP (10 [micro]M) increased ERK2 phosphorylation from basal 17 [+ or -] 3 to 53 [+ or -] 4%, an effect suppressed in the presence of the MEK inhibitors PD-98059 (20 [micro]M) or U0126 (10 [micro]M). Phosphorylation of ERK2 was not dependent on the ATP-induced cytosolic [Ca.sup.2+] rise, because it was unaltered when this was suppressed by the [Ca.sup.2+] chelator BAPTA (10 [micro]M) or xestospongin C (3 [micro]M), an inhibitor of the inositol 1,4,5 trisphosphate-sensitive [Ca.sup.2+] release mechanism of the endoplasmic reticulum. Phosphorylation of ERK2 was neither induced by the adenosine analog 5'-(N-ethylcarboxamido)adenosine (l [micro]M) nor inhibited in the presence of the adenosine receptor antagonist 8-phenyltheophylline (10 [micro]M). ATP increased MLC kinase activity, and this was blocked in presence of PD-98059. ATP also increased MLC phosphatase activity, which was not inhibited by PD-98059. The MEK/MAPK pathway is a [Ca.sup.2+] -independent part of ATP signaling toward MLC kinase but not of ATP signaling toward MLC phosphatase. mitogen-activated protein kinase; contractile machinery; myosin light chain kinase; myosin light chain phosphatase

Research was conducted to examine the cardioprotective effects of KB-R7943 (KB) in isolated rat cardiomyocytes exposed to 80 minutes of simulated ischemia and 15 minutes of reoxygenation. The objective was to investigate, on the cellular level, the effects ofKB on cytosolic ion homeostatis during ATP depletion. Results indicate that the treatment of KB during anoxia effectively inhibits CA2+ overload and attenuates reoxygenation-induced injury of cardiomyocytes.

An investigation of the relationship of the progression of hyperpermeability of endothelial monolayers during the initial state of energy to the delocalization of vinculin from adhesion complexes was conducted. The vinculin localization and phosphorylation, protein kinase C activity and albumen permeability were examined in cultured coronary endothelial monolayers from rats. Results indicated that vinculin phosphorylation is involved in the development of hyperpermeability during energy depletion in coronary endothelial monolayers.

Researchers have investigated new mechanisms to prevent hypercontracture produced by reoxygenation in isolated cardiomyocytes, using isolated ventricular cardiomyocytes from adult rats as the experimental model. In particular, attention was given to the impact of atrial natriuretic peptide (ANP) and the related peptide urodilatin. It was established that the temporary presence of the stimulators of particulate guanylate cyclase, ANP or urodilatin, just before and during the initial phase of reoxygenation can protect against reoxygenation-induced hypercontracture.

Monolayers of cultured porcine endothelial cells were analyzed after the administration of guanosine 3',5'- cyclic monophosphate (cGMP) analog to determine its effects on endothelial cell membrane permeability. Administration of 8-bromoguanosine 3',5'-cyclic monophosphate increased membrane permeability in confluent cultured porcine endothelial cell monolayers. Furthermore, cGMP analog-induced membrane permeability was mediated by elevations in intracellular free Ca2+ which was inhibited in the absence of exogenous Ca2+.

An experiment using cultures of coronary endothelial cells from Wistar rats has provided evidence for the functional role of neuropeptide Y (NPY) in these cells. Measurements of albumin across the monolayers indicated that NPY reduces macromolecule permeability of coronary endothelial monolayers. The effect of NPY was abolished by pertussis toxin. It was shown that NPY reduced permeability by reducing cAMP contents of the cells.

Increased osmotic fragility of isolated adult rat cardiomyocytes can be attenuated at the onset of reoxygenation by interventions of nitric oxide donors or a lipophilic antilipid peroxidant. Reoxygenation causes enzyme loss if combined with osmotic stress. The protective effects of nitric oxide donors and antilipid peroxidant diphenylphenylenediamine are independent of influence on reoxygenation-induced hypercontracture. Their effect is also independent of any guanosine 3',5'-cyclic monophosphate mediation.

Adenosine cyclic monophosphate (cAMP) and guanosine cyclic monophosphate (cGMP) have opposing effects on cell permeability. cAMP increases the uptake of substrate such as albumin, whose movement can be followed through labelling with fluorescein isothiocyanate. Inhibitors of cAMP reduced albumin uptake. Meanwhile, cGMP and its precursor guanylyl cyclase raised albumin uptake. These effects may be mediated by protein kinase G.

The use of cultured, isolated adult ventricular cardiomyocytes in studies of myocardial protein metabolism was introduced in the 1980s. Such studies have led to better knowledge of myocardial hypertrophy-induced abnormalities in cardiomyocyte protein synthesis. Thus, it has been discovered that alpha-1-adrenoceptor stimulation, neuropeptide Y and mechanical stretch play a role in the regulation of protein synthesis in cardiomyocytes.

The prevention of Na(super +)/H(super +) exchange process and cytosolic acidosis in reoxygenated heart cells has a protective effect on the structure and function of heart cells which provides their protection against hypercontracture. The inhibition of a rapid increase in cytosolic pH and a reduction of Na(super +) influx through Na(super +)/H(super +) exchange process are the main reasons for such an effect. Isolated cardiomyocytes from the ventricular tissue of adult rats have been used in the experiment.

A contractile mechanism causes rapid initiation of hyperpermeability in the coronary endothelial cells of adult rats. These cells received 5mM potassium cyanide (KCN) and 5mM 2-deoxy-D-glucose (DG) as metabolic inhibitors. Analysis covered albumin transcendothelial flux, cellular adinosine triphosphate (ATP) content and cytosolic Ca2+ concentration. A 28% loss of ATP reserves caused a 41% raise in albumin flux. Rapid energy depletion attenuated hyperpermeability.

Experimental analysis of the ability of nitric oxide donor 3-morpholinosydnonimine (SIN-1) to protect cardiomyocytes from reoxygeneration-induced hypercontracture reveals that the temporary existence of high levels of 3-morpholinoiminoacetonitrile (SIN-1C) or SIN-1 is effective in protecting the cardiomyocytes from hypercontracture. The temporary presence of SIN-1 and SIN-1C during the last and first 15 mins of anoxia and reoxygenation, respectively, results in 96% and 72% inhibition of hypercontracture. Methylene blue reduces the effect of SIN-1 to the same as that of SIN-1C, while SIN-1C combines with sodium nitroprusside to yield the same degree of protection as SIN-1.

Isolated ventricular cardiomyocytes from rat heart are used to examine the function of Na(+) in reoxygenated myocardial cells for recovery from chronic anoxic Ca(2+) overload. External Na(+) and an active sarcolemmal Na(+) pump are required for complete recovery of cytosolic Ca(2+) regulation. Extrusion of most of the accumulated Ca(2+) occurs through the sarcolemmal Na(+)/Ca(2+) exchange process.

The disintegration of F-actin filaments in energy-depleted endothelial cells was examined. The cellular contents and intracellular localization of F-actin were determined by histochemical microscopy and sodium dodecyl sulfate- polyacrylamide gel electrophoresis. Results indicate that fragmentation and not depolymerization is responsible for the disintegration of F-actin filaments during a long-lasting reversible period of energy depletion and cystolic Ca2+ overload.

Schafer, M., D. Bahde, B. Bosche, Y. Ladilov, C. Schafer, H. M. Piper, and T. Noll. Modulation of early [[Ca.sup.2+]].sub.i] rise in metabolically inhibited endothelial cells by xestospongin C. Am J Physiol Heart Circ Physiol 280: H1002-H1010, 2001.--When energy metabolism is disrupted, endothelial cells lose [Ca.sup.2+] from endoplasmic reticulum (ER) and the cytosolic [Ca.sup.2+] concentration ([[Ca.sup.2+]].sub.i]) increases. The importance of glycolytic energy production and the mechanism of [Ca.sup.2+] loss from the ER were analyzed. Endothelial cells from porcine aorta in culture and in situ were used as models. 2-Deoxy-D-glucose (2-DG, 10 mM), an inhibitor of glycolysis, caused an increase in [[Ca.sup.2+]].sub.i] (measured with fura 2) within 1 min when total cellular ATP contents were not yet affected. Stimulation of oxidative energy production with pyruvate (5 mM) did not attenuate this 2-DG-induced rise of [[Ca.sup.2+]].sub.i], while this maneuver preserved cellular ATP contents. The inhibitor of ER-[Ca.sup.2+]-ATPase, thapsigargin (10 nM), augmented the 2-DG-induced rise of [[Ca.sup.2+]].sub.i]. Xestospongin C (3 [micro]M), an inhibitor of D-myo-inositol 3-phosphate [Ins(3)P]-sensitive ER-[Ca.sup.2+] release, abolished the rise. The results demonstrate that the ER of endothelial cells is very sensitive to glycolytic metabolic inhibition. When this occurs, the ER [Ca.sup.2+] store is discharged by opening of the Ins(3)P-sensitive release channel. Xestospongin C can effectively suppress the early [[Ca.sup.2+]].sub.i] rise in metabolically inhibited endothelial cells. D-myo-inositol 3-phosphate-sensitive [Ca.sup.2+] release; ischemia; hypoxia; cytosolic [Ca.sup.2+] concentration

Research was conducted to examine the role of (Ca2+)(sub i) changes in endothelial cell preparations in which ATP reduces permeability. A comparison of the influence of ATP with those of ionomycin, a Ca2+ ionophore that causes a rise in (Ca2+)(sub i) in a receptor-independent manner, was also carried out. Results suggest that ATP reduces macromolecule permeability through a phospholipase C-mediated mechanism that is independent of the concomitant effects of ATP on cytosolic Ca2+, protein kinase C or cyclic nucleotides.

Abstract: Objectives: Extracellular ATP stabilizes the endothelial barrier and inactivates the contractile machinery of endothelial cells. This inactivation relies on dephosphorylation of the regulatory myosin light chain (MLC) due to an activation of the MLC phosphatase (MLCP). To date, activation and function of MLCP in endothelial cells are only partially understood. Methods: Here, the mechanism of extracellular ATP-mediated activation of MLCP was analyzed in human endothelial cells from umbilical veins. Cells were transfected with the endogenous protein phosphatase 1 (PP1)-specific inhibitor-2 (I-2). Results: Overexpression of I-2 led to inhibition of PP1 activity and abrogation of the ATP-induced dephosphorylation of MLC. This indicates that the PP1 catalytic subunit is the principal phosphatase catalyzing the MLC dephosphorylation induced by extracellular ATP. As demonstrated by immunoprecipitation analysis, extracellular ATP recruits the PP1δ catalytic subunit and the myosin phosphatase targeting subunit (MYPT1) to form a complex. ATP stimulated dephosphorylation of MYPT1 at the inhibitory phosphorylation sites threonine 850 and 696. However, extracellular ATP failed to stimulate MYPT1 dephosphorylation in I-2-overexpressing cells. Conclusions: The present study shows for the first time that, in endothelial cells, extracellular ATP causes activation of MLCP through recruitment of PP1δ and MYPT1 into a MLCP holoenzyme complex and PP1-mediated reduction of the inhibitory phosphorylation of MYPT1. [Copyright &y& Elsevier]

Abstract: Objective: Reperfusion injury of the myocardium is characterised by development of cardiomyocyte hypercontracture. Previous studies have shown that cGMP-mediated stimuli protect against reperfusion injury, but the cellular mechanism is still unknown. Methods: To simulate ischemia/reperfusion, adult rat cardiomyocytes were incubated anoxically (pHo 6.4) and then reoxygenated (pHo 7.4). Cytosolic calcium [Ca2+] i (fura-2 ratio), pH i (BCECF ratio), cell length, and phospholamban phosphorylation were analysed. Under simulated ischemia cardiomyocytes develop [Ca2+] i overload. When reoxygenated they rapidly undergo hypercontracture, triggered by oscillations of [Ca2+] i . We investigated whether cGMP-mediated stimuli can modulate [Ca2+] i or pH i recovery and whether this contributes to their protective effect. Membrane-permeable cGMP analogues, 8-bromo-cGMP (1 mmol/L) or 8-pCPT-cGMP (10 μmmol/L), or a receptor-mediated activator of particulate guanylyl cyclase, urodilatin (1 μmol/L), were applied. Results: The investigated stimuli protect against reoxygenation-induced hypercontracture (cell length as percent of end-ischemic length; control: 68±1.6; 8-bromo-cGMP: 88±1.5*; 8-pCPT-cGMP: 84±2.9*; urodilatin: 87±1.1*; n=24; *p<0.05). Recovery from [Ca2+] i overload after 2 min reoxygenation [fura-2 ratio (a.u.); control: 1.43±0.15; 8-bromo-cGMP: 1.86±0.15*; 8-pCPT-cGMP: 1.92±0.19*; urodilatin: 1.93±0.24*; n=25; *p<0.05] was accelerated, and the frequency of [Ca2+] i oscillations (min−1) was significantly reduced (control: 49±5.0 min−1; 8-bromo-cGMP: 18±3.5* min−1; 8-pCPT-cGMP: 18±4.5* min−1; urodilatin: 16±4.1* min−1; n=24; *p<0.05). cGMP-mediated stimuli increased sarcoplasmic Ca2+ sequestration (caffeine-releasable Ca2+ pool: 2–3 fold increase vs. control). Inhibition of sarcoplasmic Ca2+-ATPase (SERCA) by thapsigargin (150 nmol/L) or of protein kinase G with KT-5823 (1 μmol/L) abolished the effect of these stimuli on [Ca2+] i recovery. The investigated stimuli significantly enhanced phospholamban phosphorylation. Conclusions: We conclude that cGMP-dependent signals activate SERCA via a protein kinase G-dependent phosphorylation of phospholamban. The increase in SERCA activity seems to reduce peak [Ca2+] i and [Ca2+] i oscillation during reoxygenation and to attenuate the excessive activation of the contractile machinery that otherwise leads to the development of hypercontracture. [Copyright &y& Elsevier]

In some models of cardiac hypertrophy, activation of activator protein 1 (AP-1) correlates with growth. However, AP-1 is also activated by stimuli not involved in cardiac growth. This raises the following questions: does AP-1 plays a causal role for cardiomyocyte growth, and is this role model or stimulus dependent? We used a single model to address these questions, i.e., ventricular cardiomyocytes of adult rats, and two growth stimuli, i.e., α- and β-adrenoceptor agonists [10 µM phenylephrine (PE) and 1 µM isoprenaline (Iso), respectively]. After 1 h of stimulation with PE, mRNA expression of c-Fos and c-Jun was upregulated to 185 ± 32 and 132 ± 13% of control. Fos and Jun proteins formed the AP-1 complex. PE stimulated DNA binding activity of AP-1 to 165 ± 22% of control within 2 h and increased protein synthesis to 161 ± 27% of control and cross-sectional area to 126 ± 4% of control. Inhibition of AP-1 binding activity by cAMP response element (CRE) decoy oligonucleotides abolished both of these growth responses. Iso stimulated AP-1 binding activity to 203 ± 19% of control within 2 h and stimulated protein synthesis to 145 ± 17% of control. However, the growth effect of Iso was not abolished by CRE decoys: Iso increased protein synthesis to 158 ± 17% of control in the presence of CRE. In conclusion, AP-1 is a causal mediator of the α-adrenergic, but not the β-adrenergic, growth response of cardiomyocytes. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

Objective: Barrier function of coronary endothelium becomes disturbed by ischemia–reperfusion. We investigated the mechanism of reperfusion-induced endothelial gap formation in monolayers of cultured endothelial cells (CEC) of the rat, exposed to simulated ischemia (40 min anoxia, pHo 6.4) and reperfusion (30 min reoxygenation, pHo 7.4). Methods: Cytosolic Ca2+ (fura-2) and intercellular gap formation (planimetrical analysis) were determined. Reoxygenation conditions were varied: (a) continuing perfusion at pHo 6.4, (b) with or without glucose (2.5 mM), (c) in presence of NaCN (2 mM), (d) with Ca2+ (10 mM) or BAPTA/AM (25 μM), (e) in the presence of myosin light chain kinase inhibitors ML-7 (5 μM) or wortmannin (1 μM). Results: During anoxia, CEC developed cytosolic Ca2+ overload which was not reversed during 30 min reoxygenation. Intercellular gap formation started during anoxia, but was increased during reoxygenation. Reoxygenation-related gap formation was largest in presence of glucose, lower when glucose was withdrawn or NaCN was added. Presence of ML-7 or wortmannin also reduced gap formation during reoxygenation. Conclusions: Reoxygenation induces gap formation. This is dependent on (i) Ca2+ overload during reoxygenation, (ii) energy production and (iii) activation of myosin light chain kinase. Together these results indicate that activation of the endothelial contractile machinery is the underlying cause. [Copyright &y& Elsevier]

Objective: We have recently shown that spontaneous Ca2+ oscillations elicit irreversible hypercontracture of cardiomyocytes during reoxygenation. The aim of this study was to investigate whether influx of exterior Ca2+ through the reverse mode of the Na+/Ca2+ exchanger (NCE) contributes to the development of these oscillations and, therefore, to reoxygenation-induced hypercontracture. Methods: Isolated cardiomyocytes and hearts from rats were used as models. Cardiomyocytes were exposed to 60 min simulated ischemia (pHo 6.4) and 10 min reoxygenation (pHo 7.4). During reoxygenation cardiomyocytes were superfused with medium containing 1 mmol/l Ca2+ (control), with nominally Ca2+-free medium or with medium containing 10 μmol/l KB-R 7943 (KB), a selective inhibitor of the reverse mode of the NCE. Results: In reoxygenated cardiomyocytes rapid Ca2+ oscillations occurred which were reduced under Ca2+-free conditions or in presence of KB. Hypercontracture was also significantly reduced under Ca2+-free conditions or in presence of KB. After 30 min of normoxic perfusion isolated rat hearts were subjected to 60 min global ischemia and reperfusion. KB (10 μmol/l) was present during the first 10 min of reperfusion. LVEDP, LVdevP and lactate dehydrogenase (LDH) release were measured. Presence of KB reduced post-ischemic LVEDP and improved left ventricular function (LVdevP). In KB treated hearts the reperfusion induced release of LDH was markedly reduced from 81.1±9.9 (control) to 49.3±8.8 U/60 min/g dry weight. Conclusion: Our study shows that inhibition of the reverse mode of the NCE, during reperfusion only, protects cardiomyocytes and whole hearts against reperfusion injury. [ABSTRACT FROM PUBLISHER]

Presents information on a study which investigated whether inhibition of the sodium/hydrogen ions exchanger during ischemia is protective due to reduction of cytosolic calcium ions accumulation or enhanced acidosis in cardiomyocytes. Methodology of the study; Results and discussion on the study.

Objective: Nitric oxide (NO) has been shown to induce apoptosis in cardiomyocytes under normoxic conditions. The ability of NO to induce apoptosis after ischemia–reperfusion, a situation of increased NO release in vivo, has not been investigated. The present study was undertaken to characterize the pathway of induction of apoptosis by NO and the influence of ischemia on this pathway in cardiomyocytes. Methods: The study was performed on isolated adult cardiomyocytes of the rat. Ischemia was simulated by anoxia in a glucose free medium, pH 6.4. Induction of apoptosis was detected (1) by annexinV-fluorescein isothiocyanate (annexinV-FITC) binding to cells under exclusion of propidium iodide and (2) by laddering of genomic DNA. Results: Incubation of cardiomyocytes with the NO-donor (±)-S-nitroso-N-acetylpenicillamine (SNAP, 100 μM) induced apoptosis in 14.1±1.9% of the cells and necrosis in 24.4±4.6%. The induction of apoptosis but not necrosis could be blocked by inhibition of soluble guanylyl cyclase or of protein kinase G. Apoptosis induction was mimicked by incubation of cardiomyocytes with 8-pCPT-cGMP (100 μM, 9.6±0.6% apoptotic cells) or YC-1 (75 μM, 14.6±2.8% apoptotic cells), a direct activator of soluble guanylyl cyclase. After 3 h of anoxia, cardiomyocytes were transiently protected against apoptosis induced by NO, but not by 8-pCPT-cGMP or YC-1 (8.9±0.7% or 13.4±2.4% apoptotic cells). A correlation of the apoptotic response to SNAP or YC-1 with an increased activity of soluble guanylyl cyclase, determined by measurements of intracellular cGMP contents, was found. Conclusions: NO induces apoptosis in a cGMP dependent manner in isolated adult cardiomyocytes whereas induction of necrosis seems cGMP-independent. After simulated in vitro ischemia the activation of soluble guanylyl cyclase by NO is transiently inhibited resulting in a transient anti-apoptotic protection. [ABSTRACT FROM PUBLISHER]

Objectives: Apoptosis is one feature of myocardial damage after ischemia–reperfusion, but the causes for its induction are unclear. The present study was undertaken to investigate whether apoptosis in cardiomyocytes is directly initiated by their sub-lethal injury that results from ischemia–reperfusion. Methods: Ischemia was simulated on isolated ventricular cardiomyocytes of adult rats by anoxia in a glucose free medium, pH 6.4. Induction of apoptosis was detected by (1) DNA laddering of genomic DNA, (2) TUNEL positive cells (terminal deoxynucleotidyl transferase-mediated-UTP nick end labelling) and (3) annexinV–fluorescein isothiocyanate (annexinV–FITC) binding to cells under exclusion of propidium iodide. Necrotic cells were identified by (1) staining with both annexinV–FITC and propidium iodide, (2) unspecific DNA degradation and (3) enzyme release. Results: Simulated ischemia caused a >75% loss of high-energy phosphates within 2 h, which was reversible upon reoxygenation at pH 7.4. Even after 18 h of simulated ischemia, creatine phosphate contents recovered to 55.2±7.3% of control within 1 h. Apoptosis could be induced by UV irradiation (80 J/m2), H2O2 and the NO-donor N2-acetyl-S-nitroso-d,l-penicillinaminamide. In contrast to this, simulated ischemia and reoxygenation could not induce apoptosis in the cells, but with prolonged ischemia more cells became necrotic. After 18 hours of simulated ischemia and 4 h of reoxygenation 41.2±10.2% myocytes were necrotic (vs. 6.3±4.4% of control) and only 1.7±0.5% (vs. 8.7±4.6% of control) were apoptotic. The percentage of necrotic cells correlated with an increase in lactate dehydrogenase release from 9.9±0.6% (of total activity) of normoxic controls to 37.9±5.1% after 18 h of simulated ischemia and 12 h of reoxygenation. Conclusions: Simulated ischemia-reoxygenation causes necrosis of isolated cardiomyocytes but is not sufficient for induction of apoptosis. [ABSTRACT FROM PUBLISHER]