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.
The primary culture of rat coronary EC was exposed to simulated ischemia (glucose-free anoxia at pH(o) 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.
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 pH(o) 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%.
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.
The influence of the sodium‐proton‐exchanger‐1 (NHE‐1) inhibitor HOE694 on α‐ or β‐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 β‐adrenoceptor stimulation. Stimulation of α‐adrenoceptors with phenylephrine (10 μM) in bicarbonate‐free medium caused cellular alkalization (ΔpHi: +0.17±0.02, n‐5, P<0.05). HOE694, an NHE‐1 inhibitor, completely abolished this effect. 14Cphenylalanine 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 μM) neither influenced basal protein synthesis nor interfered with α‐adrenoceptor mediated activation of ERK2. Phorbol myristate acetate, a direct stimulator of protein kinase C, mimicked the effect of α‐adrenoceptor stimulation in regard to protein synthesis, but did not lead to cellular alkalization. Protein synthesis increased in the presence of isoprenaline, a β‐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 α‐ or β‐adrenoceptor stimulation, is attenuated in the presence of the NHE‐1 inhibitor HOE694 and this inhibition is independent from cellular alkalization.
There is recent evidence that Ca(2+) influx via reverse mode Na(+)/Ca(2+) exchange (NCX) at the time of reperfusion can contribute to cardiomyocyte hypercontracture. However, forward NCX is essential ...for normalization of Ca(2+)(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.
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. Ca(2+)(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.
In myocytes, KB-R7943 at concentrations up to 15 microM reduced Ca(2+)(i) rise and the probability of hypercontracture during re-energization (P<0.01). Nevertheless, in rat hearts, the effects of KB-R7943 applied during reperfusion after 60 min of ischemia depended on concentration and timing of administration. During the first 5 min of reperfusion, KB-R7943 (0.3-30 microM) induced a dose-dependent reduction in LDH release (half-response concentration 0.29 microM). Beyond 6 min of re-flow, KB-R7943 had no effect on LDH release, except at concentrations > or = 15 microM, which increased LDH. KB-R7943 at 5 microM given during the first 10 min of reflow reduced contractile dysfunction (P=0.011), LDH release (P=0.019) and contraction band necrosis (P=0.014) during reperfusion. Intracoronary administration of this concentration during the first 10 min of reperfusion reduced infarct size by 34% (P=0.033) in pigs submitted to 48 min of coronary occlusion.
These results are consistent with the hypothesis that during initial reperfusion NCX activity results in net reverse mode operation contributing to Ca(2+) overload, hypercontracture and cell death, and that NCX inhibition during this phase is beneficial. Beyond this phase, NCX inhibition may impair forward mode-dependent Ca(2+) extrusion and be detrimental. These findings may help in the design of therapeutic strategies against lethal reperfusion injury, with NCX as the target.
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.
In ischemic-reperfused myocardium, necrosis of cardiomyocytes may develop not only due to the ischemic conditions but also the specific circumstances of reperfusion. The existence of reperfusion ...injury becomes apparent when modifications of the conditions of reperfusion can prevent cell death otherwise occurring. Three prime causes of rapidly developing reperfusion injury are here discussed, ie, reenergization of cells at increased cytosolic Ca2+ contents, rapid normalization of tissue pH, and rapid normalization of tissue osmolality. All three causes lead to severe mechanical stress of cardiomyocytes which can cause their rapid deterioration. Propagation of cell injury among adjacent cells can cause a spreading of necrosis throughout myocardial tissue. The understanding of these initial causes of rapidly developing lethal reperfusion injury leads to new concepts for specific protection of reperfused myocardium.
ABSTRACT
Release of nitric oxide (NO) during inflammation can induce apoptosis in the heart. Here we analyzed the involvement of members of the mitogen‐activated protein kinase (MAPK) family and ...their downstream target, the transcription factor AP‐1, in induction of apoptosis by NO in isolated adult cardiomyocytes of rat. The NO‐donor (±)‐S‐nitroso‐N‐acetylpenicillamine (100 μM SNAP)‐induced apoptosis in 10.5 ± 0.7% of cardiomyocytes and activated the transcription activator protein AP‐1 by 333.6 ± 122.3%. Intracellular scavenging of AP‐1 with decoy‐oligonucleotides blocked NO‐induced apoptosis to control levels (3.8 ± 0.5% apoptotic cells). Activation of AP‐1 with a c‐Jun amino‐terminal kinase (JNK) activator (Ro318220, 10 μM) provoked apoptosis in 18.7 ± 1.2% cardiomyocytes, which was again blocked by intracellular scavenging of AP‐1. NO activated JNK by 87.0 ± 27.3% and extracellular signal‐regulated kinase (ERK) by 35 ± 3%. Inhibition of ERK by the mitogen‐activated protein kinase kinase (MEK1) inhibitor PD98059 (10 μM) abolished AP‐1 activation and apoptosis induction with SNAP. Evidence that p38 MAPK plays a role in NO‐induced apoptosis was not found. These results clearly demonstrate the involvement of the transcription factor AP‐1 in NO‐induced apoptosis in cardiomyocytes. The activation of AP‐1 is mediated by the two MAP kinases JNK and ERK.
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 ET
A
receptor antagonist BQ123.
Sarafotoxin 6b and PTHrP(1 – 16), but not PTHrP(1 – 34), replaced
3
H‐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 ET
A
receptors.
British Journal of Pharmacology
(2001)
132
, 427–432; doi:
10.1038/sj.bjp.0703830
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