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Experimental and clinical studies demonstrated that postconditioning confers protection against myocardial ischemia/reperfusion injury. However the underlying cellular mechanisms responsible for the beneficial effect of postconditioning are still poorly understood. The aim of the present study was to examine the role of cytosolic and mitochondrial Ca(2+)-handling. For this purpose adult rat cardiomyocytes were subjected to simulated in vitro ischemia (glucose-free hypoxia at pH6.4) followed by simulated reperfusion with a normoxic buffer (pH7.4; 2.5 mmol/L glucose). Postconditioning, i.e., 2 repetitive cycles of normoxic (5s) and hypoxic (2.5 min) superfusion, was applied during the first 5 min of reoxygenation. Mitochondrial membrane potential (ΔΨm), cytosolic and mitochondrial Ca(2+) concentrations, cytosolic pH and necrosis were analysed applying JC-1, fura-2, fura-2/manganese, BCECF and propidium iodide, respectively. Mitochondrial permeability transition pore (MPTP) opening was detected by calcein release. Hypoxic treatment led to a reduction of ΔΨm, an increase in cytosolic and mitochondrial Ca(2+) concentration, and acidification of cardiomyocytes. During the first minutes of reoxygenation, ΔΨm transiently recovered, but irreversibly collapsed after 7 min of reoxygenation, which was accompanied by MPTP opening. Simultaneously, mitochondrial Ca(2+) increased during reperfusion and cardiomyocytes developed spontaneous cytosolic Ca(2+) oscillations and severe contracture followed by necrosis after 25 min of reoxygenation. In postconditioned cells, the collapse in ΔΨm as well as the leak of calcein, the increase in mitochondrial Ca(2+), cytosolic Ca(2+) oscillations, contracture and necrosis were significantly reduced. Furthermore postconditioning delayed cardiomyocyte pH recovery. Postconditioning by hypoxia/reoxygenation was as protective as treatment with cyclosporine A. Combining cyclosporine A and postconditioning had no additive effect. The data of the present study demonstrate that postconditioning by hypoxia/reoxygenation prevents reperfusion injury by limiting mitochondrial Ca(2+) load and thus opening of the MPTP in isolated cardiomyocytes. These effects seem to be supported by postconditioning-induced delay in pH recovery and suppression of Ca(2+) oscillations.

Uncontrolled release of Ca(2+) from the sarcoplasmic reticulum (SR) contributes to the reperfusion-induced cardiomyocyte injury, e.g. hypercontracture and necrosis. To find out the underlying cellular mechanisms of this phenomenon, we investigated whether the opening of mitochondrial permeability transition pores (MPTP), resulting in ATP depletion and reactive oxygen species (ROS) formation, may be involved. For this purpose, isolated cardiac myocytes from adult rats were subjected to simulated ischemia and reperfusion. MPTP opening was detected by calcein release and by monitoring the ΔΨ(m). Fura-2 was used to monitor cytosolic [Ca(2+)](i) or mitochondrial calcium [Ca(2+)](m), after quenching the cytosolic compartment with MnCl(2). Mitochondrial ROS [ROS](m) production was detected with MitoSOX Red and mag-fura-2 was used to monitor Mg(2+) concentration, which reflects changes in cellular ATP. Necrosis was determined by propidium iodide staining. Reperfusion led to a calcein release from mitochondria, ΔΨ(m) collapse and disturbance of ATP recovery. Simultaneously, Ca(2+) oscillations occurred, [Ca(2+)](m) and [ROS](m) increased, cells developed hypercontracture and underwent necrosis. Inhibition of the SR-driven Ca(2+) cycling with thapsigargine or ryanodine prevented mitochondrial dysfunction, ROS formation and MPTP opening. Suppression of the mitochondrial Ca(2+) uptake (Ru360) or MPTP (cyclosporine A) significantly attenuated Ca(2+) cycling, hypercontracture and necrosis. ROS scavengers (2-mercaptopropionyl glycine or N-acetylcysteine) had no effect on these parameters, but reduced [ROS](m). In conclusion, MPTP opening occurs early during reperfusion and is due to the Ca(2+) oscillations originating primarily from the SR and supported by MPTP. The interplay between Ca(2+) cycling and MPTP promotes the reperfusion-induced cardiomyocyte hypercontracture and necrosis. Mitochondrial ROS formation is a result rather than a cause of MPTP opening.

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.

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 …

Aims Apoptosis of vascular smooth muscle cells (VSMC) in advanced atherosclerotic plaques is an important cause of plaque instability. Oxysterols have been suggested as important inducers of apoptosis in VSMC, but the precise mechanism is still poorly understood. Here we aimed to analyse the role of the soluble adenylyl cyclase (sAC). Methods and results VSMC derived from rat aorta were treated with either 25-hydroxycholesterol or 7-ketocholesterol for 24 h. Apoptosis was detected by TUNEL staining and caspases cleavage. Oxysterols treatment led to the activation of the mitochondrial pathway of apoptosis (cytochrome c release and caspase-9 cleavage) and mitochondrial ROS formation, which were suppressed by the pharmacological inhibition or knockdown of sAC. Scavenging ROS with N -acetyl-l-cysteine prevented oxysterol-induced apoptosis. Analyses of the downstream pathway suggest that protein kinase A (PKA)-dependent phosphorylation and the mitochondrial translocation of the pro-apoptotic protein Bax is a key link between sAC and oxysterol-induced ROS formation and apoptosis. To distinguish between intra-mitochondrial and extra-mitochondrial/cytosolic sAC pools, sAC was overexpressed in mitochondria or in the cytosol. sAC expression in the cytosol, but not in mitochondria, significantly promoted apoptosis and ROS formation during oxysterol treatment. Conclusion These results suggest that the sAC/PKA axis plays a key role in the oxysterol-induced apoptosis of VSMC by controlling mitochondrial Bax translocation and ROS formation and that cytosolic sAC, rather than the mitochondrial pool, is involved in the apoptotic mechanism.

Background Calcium (Ca2+) is a cofactor of multiple cellular processes. The mechanisms that lead to elevated cytosolic Ca2+ concentration are unclear. Objective To illuminate how bloody cerebrospinal fluid (bCSF) from patients with intraventricular hemorrhage causes cell death of cultured human astrocytes. Methods Cultured astrocytes were incubated with bCSF. In control experiments, native CSF was used. Cytosolic Ca2+ concentration was measured by fura-2 fluorescence. Apoptosis and necrosis were evaluated by staining with Hoechst-3342 and propidium iodide. Results Incubation of astrocytes with bCSF provoked a steep Ca2+ concentration peak that was followed by a slow Ca2+ rise during the observation period of 50 minutes. Necrosis, but not apoptosis, was induced. Blockade of ATP-sensitive P2 receptors with suramin inhibited the bCSF-induced initial Ca2+ peak and necrosis. Blockade of P1 receptors with 8-phenyltheophylline or of N-methyl-D-aspartate receptors with D(-)-2-amino-5-phosphopentanoic acid had no significant effect. Preincubation with xestospongin D, a blocker of inositol 1,4,5-trisphosphate receptors, prevented the initial Ca2+ rise and reduced the rate of necrosis. Preemptying of the endoplasmic reticulum with thapsigargin protected astrocytes from the bCSF-induced Ca2+ peak. Inhibition of mitochondrial permeability transition pores opening with cyclosporin A reduced the rate of astrocytic necrosis significantly, although it did not influence the initial Ca peak. Conclusion bCSF elicits a steep, transient Ca rise when administered to human astrocytes by activation of ATP-sensitive P2 receptors and subsequent inositol 1,4,5-trisphosphate-dependent Ca release from endoplasmic reticulum. This massive Ca overload leads to subsequent mitochondrial permeability transition pores opening and necrosis of the cells.

Aims Cardiac progenitor cells (CPCs) have been shown to promote cardiac regeneration in vivo. Understanding the function of CPCs is essential for further implementation of these cells in the treatment of cardiac diseases. The present study tested the hypothesis that adult CPC exert paracrine effects that lead to an improvement in the functional characteristics of cardiomyocytes. This study also investigated whether aging (we included patients aged between 4 months and 81 years) has any effect on the paracrine mechanisms of CPC. Methods and results The supernatant of CPC generated both from human and rat hearts—so called ‘conditioned cardiosphere medium’ improved the contractile behaviour of isolated adult cardiomyocytes in a concentration-dependent manner after incubation for 24 h and increased the SERCA/NCX ratio. The observed positive effects on contractile behaviour were independent of the CPC donors' age. Conditioned cardiosphere media also normalized angiotensin II-induced contractile dysfunction. Cytokines released by CPC into the media were detected by cytokine arrays. Conclusion The observed diversity of cytokines released by CPC needs to be further elucidated in detail. Nevertheless, CPC are a promising therapeutic approach in the field of cardiac disease. The methods described allow investigation of the underlying paracrine mechanisms in a standardized in vitro situation.

Aims Ischaemia–reperfusion provokes barrier failure of the coronary microvasculature, impeding functional recovery of the heart during reperfusion. The aim of the present study was to investigate whether the stimulation of cGMP signalling by activation of soluble guanylyl cyclase (sGC) can reduce reperfusion-induced endothelial intercellular gap formation and to determine whether this is due to an influence on endothelial cytosolic Ca2+ homeostasis during reperfusion. Methods and results Experiments were performed with cultured coronary endothelial monolayers and isolated saline-perfused rat hearts. HMR1766 (1 µmol/L) or DEAnonoate (0.5 µmol/L) were used to activate sGC. After exposure to simulated ischaemic conditions, reperfusion of endothelial cells led to a pronounced increase in cytosolic calcium levels and intercellular gaps. Stimulation of cGMP signalling during reperfusion increased Ca2+ sequestration in the endoplasmic reticulum (ER) and attenuated the reperfusion-induced increase in cytosolic [Ca2+]. Phosphorylation of phospholamban was also increased, indicating a de-inhibition of the ER Ca2+ pump (SERCA). Reperfusion-induced intercellular gap formation was reduced. Reduction of myosin light chain phosphorylation indicated inactivation of the endothelial contractile machinery. Effects on cytsolic Ca2+ and gaps were abrogated by inhibition of cGMP-dependent protein kinase (PKG) with KT5823. In reperfused hearts, stimulation of cGMP signalling led to decreased oedema development. Conclusion sGC/PKG activation during reperfusion reduces reperfusion-induced endothelial intercellular gap formation by attenuation of cytosolic calcium overload and reduction of contractile activation in endothelial cells. This mechanism protects the heart against reperfusion-induced oedema.

Objective: Myocardial ischemia has been shown to induce apoptosis of endothelial cells (EC). However, the mechanism of this endothelial injury is still poorly understood. To analyse the signaling pathway of ischemia-induced EC apoptosis was the aim of the present study. Methods: The primary culture of rat coronary EC was exposed to simulated ischemia (glucose-free anoxia at pHo 6.4). Apoptosis was defined by staining of nuclei with Hoechst-33342 and TUNEL. Cytosolic Ca2+ and pH were measured with Fura-2 and BCECF, respectively. Results: Apoptosis (29.2±1.7% of cells) induced by exposure to simulated ischemia for 2 h was accompanied by cytosolic Ca2+ overload (1090±52 nmol/l) and acidosis (pHi=6.52±0.13). Simulated ischemia had no significant effect on caspase-8 cleavage, but induced cleavage of caspase-3 and caspase-12 and led to a slight release of cytochrome C . Prevention of cytosolic acidosis (anoxia at pHo 7.4) had no effect on cytochrome C release, but significantly reduced apoptosis, attenuated cytosolic Ca2+ overload, and prevented cleavage of caspase-12. A similar effect was achieved by inhibition of Ca2+ release channels in the endoplasmic reticulum with ryanodine and xestospongin C. Knock-down of caspase-12 with small interfering RNA suppressed caspase-3 activation and reduced apoptotic cell number by about 70%. Conclusion: Acidosis, rather than anoxia, is an important trigger of apoptosis in EC under simulated ischemia. The main pathway of the simulated ischemia-induced apoptosis consists of the Ca2+ leak from the ER followed by activation of caspase-12 and caspase-3.

Prolonged myocardial ischemia leads to the loss of viable tissue in the heart, even if the ischemic episode is eventually ended by reperfusion. As we now know today, the extent of infarct is only partially due to the ischemic injury. Another part of tissue loss is due to death signals generated at the time of and by the specific circumstances of reperfusion, a phenomenon termed “reperfusion injury” [1,2]. The existence of reperfusion injury has been revealed by successful attempts to interfere with such causes at the time of reperfusion and limit, thereby, the extent of final tissue loss. Reperfusion injury is initiated within the first minutes of reperfusion. This is why these minutes represent a valuable “window of opportunity” for myocardial protection. One of the most recent strategies to interfere within that time window is postconditioning, i.e. the repetitive application of brief bouts of ischemia within the first minutes of reperfusion [3]. This protocol is so promising because it has brought reperfusion protection to the clinical arena and reperfusion injury to the broad attention of clinical cardiologists. The nature of the death signals activated during the first minutes of reperfusion is still a matter of debate. Opinions are also divided on whether the ensuing cell loss is due to necrosis or apoptosis. Cell damage in reperfused myocardium exhibits a peculiar histological appearance. It is characterized by hypercontracted sarcomeres (“contraction bands”) and frequent sarcolemmal ruptures that gave rise to the term “contraction band necrosis” [4,5]. The histological picture has led to a hypothesis on the pathogenesis of acute reperfusion injury. According to this hypothesis, it is an uncontrolled activation of the contractile machinery in myocardial cells upon reperfusion that produces the final, decisive blow to cardiomyocytes already weakened by the ischemic history and ends their fate by disruption …

Objective Experimental and clinical studies have shown that administration of insulin during reperfusion is cardioprotective, but the underlying mechanisms are still unknown. In this study, we investigated in isolated rat cardiomyocytes subjected to hypoxia and reoxygenation whether administration of insulin during reoxygenation reduces reoxygenation-induced hypercontracture, a hallmark of acute reperfusion injury. The effects of insulin on potential pro-survival kinases, i.e., PI 3-kinase, NO synthase (eNOS), and cGMP-dependent protein kinase (PKG), and on cytosolic Ca2+ control in reoxygenated cardiomyocytes were investigated. Results Administration of insulin (10mU/L) during reoxygenation protected cardiomyocytes against hypercontracture development (cell length as % of end-hypoxic length: control 61.6±3.2; insulin 76.3±2.9*; n =26; * p

Objective: Ischemia–reperfusion provokes barrier failure of the coronary microvasculature, leading to myocardial edema development that jeopardizes functional recovery of the heart during reperfusion. Here, we tested whether adenosine 5′-triphosphate (ATP), either exogenously applied or spontaneously released during reperfusion, protects the endothelial barrier against an imminent reperfusion injury and whether interventions preventing ATP breakdown augment this protective ATP effect. Methods Cultured microvascular coronary endothelial monolayers and isolated-perfused hearts of rat were used. Results: After ischemic conditions were induced, reperfusion of endothelial monolayers activated the endothelial contractile machinery and caused intercellular gap formation. It also led to the release of ATP. When its breakdown was inhibited by 6- N , N -diethyl-β,γ-dibromomethylene-d-ATP (ARL 67156; 100 μM), a selective ectonucleotidase inhibitor, contractile activation and gap formation were significantly reduced. Reperfusion in the presence of exogenously added ATP (10 μM) plus ARL caused an additional reduction of both aforementioned effects. In contrast, elevation of ATP degradation by apyrase (1 U/ml), a soluble ectonucleotidase, or addition of adenosine (10 μM) provoked an increase in gap formation during reperfusion that could be completely inhibited by 8-phenyltheophylline (8-PT; 10 μM), an adenosine receptor antagonist. In Langendorff-perfused rat hearts, the reperfusion-induced increase in water content was significantly reduced by ARL plus ATP. Under conditions favouring ATP degradation, an increase in myocardial edema was observed that could be blocked by 8-PT. Conclusion ATP, either released from cells or exogenously applied, protects against reperfusion-induced failure of the coronary endothelial barrier. Inhibition of ATP degradation enhances the stabilizing effect of ATP on barrier function.

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

The hypothesis tested was that reoxygenation-induced contracture of myocardial cells, a form of reperfusion injury, can be due to a rigor-type mechanism. Isolated adult cardiomyocytes were exposed to 30- or 60-min anoxia (pH 6.4) and reoxygenation (pH 7.4). In cardiomyocytes, cytosolic Ca(2+) and cell length, and in isolated rat hearts left ventricular end-diastolic pressure (LVEDP) were measured. During reoxygenation, cardiomyocytes developed contracture. When energy recovery was slowed down, less Ca(2+) overload was required for contracture: (1) after 30-min anoxia Ca(20) (cytosolic Ca(2+) concentration in cells with 20% cell length reduction) was 1.42 +/- 0.11 micromol/l; (2) after 30-min anoxia with partial mitochondrial inhibition during reoxygenation (NaCN, 0.1 mmol/l) Ca(20) was reduced to 0.69 +/- 0.05 micromol/l; (3) after 60-min anoxia Ca(20) was reduced to 0.78 +/- 0.05 micromol/l and (4) when energy recovery was accelerated (succinate, 0.2 mmol/l), Ca(20) rose to 1.35 +/- 0.05 micromol/l. In isolated hearts, the reperfusion-induced rise in LVEDP was modulated by the same interventions. Slow recovery of energy production favors reoxygenation-induced contracture in cardiomyocytes and hearts. This shows that rigor contracture contributes to reoxygenation-induced cell injury.

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.