Nina C. Weber

The noble gas xenon induces pharmacological preconditioning in the rat heart in vivo via induction of PKC-ɛ and p38 MAPK

1 Xenon is an anesthetic with minimal hemodynamic side effects, making it an ideal agent for cardiocompromised patients. We investigated if xenon induces pharmacological preconditioning (PC) of the rat heart and elucidated the underlying molecular mechanisms. 2 For infarct size measurements, anesthetized rats were subjected to 25 min of coronary artery occlusion followed by 120 min of reperfusion. Rats received either the anesthetic gas xenon, the volatile anesthetic isoflurane or as positive control ischemic preconditioning (IPC) during three 5‐min periods before 25‐min ischemia. Control animals remained untreated for 45 min. To investigate the involvement of protein kinase C (PKC) and p38 mitogen‐activated protein kinase (MAPK), rats were pretreated with the PKC inhibitor calphostin C (0.1 mg kg−1) or the p38 MAPK inhibitor SB203580 (1 mg kg−1). Additional hearts were excised for Western blot and immunohistochemistry. 3 Infarct size was reduced from 50.9±16.7% in controls to 28.1±10.3% in xenon, 28.6±9.9% in isoflurane and to 28.5±5.4% in IPC hearts. Both, calphostin C and SB203580, abolished the observed cardioprotection after xenon and isoflurane administration but not after IPC. Immunofluorescence staining and Western blot assay revealed an increased phosphorylation and translocation of PKC‐ɛ in xenon treated hearts. This effect could be blocked by calphostin C but not by SB203580. Moreover, the phosphorylation of p38 MAPK was induced by xenon and this effect was blocked by calphostin C. 4 In summary, we demonstrate that xenon induces cardioprotection by PC and that activation of PKC‐ɛ and its downstream target p38 MAPK are central molecular mechanisms involved. Thus, the results of the present study may contribute to elucidate the beneficial cardioprotective effects of this anesthetic gas.

Molecular Mechanisms Transducing the Anesthetic, Analgesic, and Organ-protective Actions of Xenon

The anesthetic properties of xenon have been known for more than 50 yr, and the safety and efficacy of xenon inhalational anesthesia has been demonstrated in several recent clinical studies. In addition, xenon demonstrates many favorable pharmacodynamic and pharmacokinetic properties, which could be used in certain niche clinical settings such as cardiopulmonary bypass. This inert gas is capable of interacting with a variety of molecular targets, and some of them are also modulated in anesthesia-relevant brain regions. Besides these anesthetic and analgesic effects, xenon has been shown to exert substantial organoprotective properties, especially in the brain and the heart. Several experimental studies have demonstrated a reduction in cerebral and myocardial infarction after xenon application. Whether this translates to a clinical benefit must be determined because preservation of myocardial and cerebral function may outweigh the significant cost of xenon administration. Clinical trials to assess the impact of xenon in settings with a high probability of injury such as cardiopulmonary bypass and neonatal asphyxia should be designed and underpinned with investigation of the molecular targets that transduce these effects.

Remote Ischemic Conditioning to Protect against Ischemia-Reperfusion Injury: A Systematic Review and Meta-Analysis

Background Remote ischemic conditioning is gaining interest as potential method to induce resistance against ischemia reperfusion injury in a variety of clinical settings. We performed a systematic review and meta-analysis to investigate whether remote ischemic conditioning reduces mortality, major adverse cardiovascular events, length of stay in hospital and in the intensive care unit and biomarker release in patients who suffer from or are at risk for ischemia reperfusion injury. Methods and Results Medline, EMBASE and Cochrane databases were searched for randomized clinical trials comparing remote ischemic conditioning, regardless of timing, with no conditioning. Two investigators independently selected suitable trials, assessed trial quality and extracted data. 23 studies in patients undergoing cardiac surgery (15 studies), percutaneous coronary intervention (four studies) and vascular surgery (four studies), comprising in total 1878 patients, were included in this review. Compared to no conditioning, remote ischemic conditioning did not reduce mortality (odds ratio 1.22 [95% confidence interval 0.48, 3.07]) or major adverse cardiovascular events (0.65 [0.38, 1.14]). However, the incidence of myocardial infarction was reduced with remote ischemic conditioning (0.50 [0.31, 0.82]), as was peak troponin release (standardized mean difference −0.28 [−0.47, −0.09]). Conclusion There is no evidence that remote ischemic conditioning reduces mortality associated with ischemic events; nor does it reduce major adverse cardiovascular events. However, remote ischemic conditioning did reduce the incidence of peri-procedural myocardial infarctions, as well as the release of troponin.

Desflurane Preconditioning Induces Time-dependent Activation of Protein Kinase C Epsilon and Extracellular Signal-regulated Kinase 1 and 2 in the Rat Heart In Vivo

Background:Activation of protein kinase C epsilon (PKC-&egr;) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) are important for cardioprotection by preconditioning. The present study investigated the time dependency of PKC-&egr; and ERK1/2 activation during desflurane-induced preconditioning in the rat heart. Methods:Anesthetized rats were subjected to regional myocardial ischemia and reperfusion, and infarct size was measured by triphenyltetrazoliumchloride staining (percentage of area at risk). In three groups, desflurane-induced preconditioning was induced by two 5-min periods of desflurane inhalation (1 minimal alveolar concentration), interspersed with two 10-min periods of washout. Three groups did not undergo desflurane-induced preconditioning. The rats received 0.9% saline, the PKC blocker calphostin C, or the ERK1/2 inhibitor PD98059 with or without desflurane preconditioning (each group, n = 7). Additional hearts were excised at four different time points with or without PKC or ERK1/2 blockade: without further treatment, after the first or the second period of desflurane-induced preconditioning, or at the end of the last washout phase (each time point, n = 4). Phosphorylated cytosolic PKC-&egr; and ERK1/2, and membrane translocation of PKC-&egr; were determined by Western blot analysis (average light intensity). Results:Desflurane significantly reduced infarct size from 57.2 ± 4.7% in controls to 35.2 ± 16.7% (desflurane-induced preconditioning, mean ± SD, P < 0.05). Both calphostin C and PD98059 abolished this effect (58.8 ± 13.2% and 64.2 ± 15.4% respectively, both P < 0.05 versus desflurane-induced preconditioning). Cytosolic phosphorylated PKC-&egr; reached its maximum after the second desflurane-induced preconditioning and returned to baseline after the last washout period. Both calphostin C and PD98059 inhibited PKC-&egr; activation. ERK1/2 phosphorylation reached its maximum after the first desflurane-induced preconditioning and returned to baseline after the last washout period. Calphostin C had no effect on ERK1/2 phosphorylation. Conclusions:Both, PKC and ERK1/2 mediate desflurane-induced preconditioning. PKC-&egr; and ERK1/2 are both activated in a time dependent manner during desflurane-induced preconditioning, but ERK1/2 activation during desflurane-induced preconditioning is not PKC dependent. Moreover, ERK1/2 blockade abolished PKC-&egr; activation, suggesting ERK-dependent activation of PKC-&egr; during desflurane-induced preconditioning.

Hyperglycaemia blocks sevoflurane-induced postconditioning in the rat heart in vivo: cardioprotection can be restored by blocking the mitochondrial permeability transition pore

BACKGROUND Recent studies showed that hyperglycaemia (HG) blocks anaesthetic-induced preconditioning. The influence of HG on anaesthetic-induced postconditioning (post) has not yet been determined. We investigated whether sevoflurane (Sevo)-induced postconditioning is blocked by HG and whether the blockade could be reversed by inhibiting the mitochondrial permeability transition pore (mPTP) with cyclosporine A (CsA). METHODS Chloralose-anaesthetized rats (n=7-11 per group) were subjected to 25 min coronary artery occlusion followed by 120 min reperfusion. Postconditioning was achieved by administration of 1 or 2 MAC sevoflurane for the first 5 min of early reperfusion. HG was induced by infusion of glucose 50% (G 50) for 35 min, starting 5 min before ischaemia up to 5 min of reperfusion. CsA (5 or 10 mg kg(-1)) was administered i.v. 5 min before the onset of reperfusion. At the end of the experiments, hearts were excised for infarct size measurements. RESULTS Infarct size (% of area at risk) was reduced from 51.4 (5.0)% [mean (sd)] in controls to 32.7 (12.8)% after sevoflurane postconditioning (Sevo-post) (P<0.05). This infarct size reduction was completely abolished by HG [51.1 (13.2)%, P<0.05 vs Sevo-post], but was restored by administration of sevoflurane with CsA [35.2 (5.2)%, P<0.05 vs HG+Sevo-post]. Increased concentrations of sevoflurane or CsA alone could not restore cardioprotection in a state of HG [Sevo-post2, 54.1 (12.6)%, P>0.05 vs HG+Sevo-post; CsA10, 58.8 (11.3)%, P>0.05 vs HG+CsA]. CONCLUSIONS Sevoflurane-induced postconditioning is blocked by HG. Inhibition of the mPTP with CsA is able to reverse this loss of cardioprotection.

Mechanisms of xenon- and isoflurane-induced preconditioning – a potential link to the cytoskeleton via the MAPKAPK-2/HSP27 pathway

We previously demonstrated that the anesthetic gas xenon exerts cardioprotection by preconditioning in vivo via activation of protein kinase C (PKC)‐ɛ and p38 mitogen‐activated protein kinase (MAPK). P38 MAPK interacts with the actin cytoskeleton via the MAPK‐activated protein kinase‐2 (MAPKAPK‐2) and heat‐shock protein 27 (HSP27). The present study further elucidated the underlying molecular mechanism of xenon‐induced preconditioning (Xe‐PC) by focusing on a potential link of xenon to the cytoskeleton. Anesthetized rats received either xenon (Xe‐PC, n=6) or the volatile anesthetic isoflurane (Iso‐PC, n=6) during three 5‐min periods interspersed with two 5‐min and one final 10‐min washout period. Control rats (n=6) remained untreated for 45 min. Additional rats were either pretreated with the PKC inhibitor Calphostin C (0.1 mg kg−1) or with the p38 MAPK inhibitor SB203580 (1 mg kg−1) with and without anesthetic preconditioning (each, n=6). Hearts were excised for immunohistochemistry of F‐actin fibers and phosphorylated HSP27. Phosphorylation of MAPKAPK‐2 and HSP27 were assessed by Western blot. HSP27 and actin colocalization were investigated by co‐immunoprecipitation. Xe‐PC induced phosphorylation of MAPKAPK‐2 (control 1.0±0.2 vs Xe‐PC 1.6±0.1, P<0.05) and HSP27 (control 5.0±0.5 vs Xe‐PC 9.8±1.0, P<0.001). Both effects were blocked by Calphostin C and SB203580. Xe‐PC enhanced translocation of HSP27 to the particulate fraction and increased F‐actin polymerization. F‐actin and pHSP27 were colocalized after Xe‐PC. Xe‐PC activates MAPKAPK‐2 and HSP27 downstream of PKC and p38 MAPK. These data link Xe‐PC to the cytoskeleton, revealing new insights into the mechanisms of Xe‐PC in vivo.

Impact of preconditioning protocol on anesthetic-induced cardioprotection in patients having coronary artery bypass surgery

OBJECTIVE Anesthetic preconditioning may contribute to the cardioprotective effects of sevoflurane in patients having coronary artery bypass surgery. We investigated whether 2 different sevoflurane administration protocols can induce preconditioning in patients having coronary artery bypass. METHODS Thirty patients were randomly allocated to 1 of 3 groups. All patients received a total intravenous anesthesia with sufentanil (0.3 microg(-1) x kg x h(-1)) and propofol as target controlled infusion (2.5 microg/mL). The control group had no further intervention; 10 minutes prior to establishing the extracorporeal circulation, patients of the sevoflurane-I group received 1 minimum alveolar concentration of sevoflurane for 5 minutes. Patients of the sevoflurane-II group received (2 times) 5 minutes of sevoflurane, interspersed by 5-minute washout 10 minutes prior to extracorporeal circulation. Troponin I was measured as marker of cardiac cellular damage. RESULTS Peak levels of troponin I release were observed at 4 hours after cardiopulmonary bypass and were not affected by 1 cycle of sevoflurane administration (controls: 14 +/- 3 ng/mL vs sevoflurane-I group, 14 +/- 3 ng/mL). Two periods of sevoflurane preconditioning significantly reduced cellular damage compared with controls (peak troponin I level sevoflurane-II group, 7 +/- 2 ng/mL). CONCLUSION These data show that sevoflurane-induced preconditioning is reproducible in patients having coronary artery bypass but depends on the preconditioning protocol used.

Helium-induced Preconditioning in Young and Old Rat Heart Impact of Mitochondrial Ca 2 -sensitive Potassium Channel Activation

Background: The noble gas helium induces cardiac preconditioning. Whether activation of mitochondrial K+ channels is involved in helium preconditioning (He-PC) is unknown. The authors investigated whether He-PC (1) is mediated by activation of Ca2+-sensitive potassium channels, (2) results in mitochondrial uncoupling, and (3) is age dependent. Methods: Anesthetized Wistar rats were randomly assigned to six groups (n = 10 each). Young (2–3 months) control (Con) and aged (22–24 months) control animals (Age Con) were not treated further. Preconditioning groups (He-PC and Age He-PC) inhaled 70% helium for 3 × 5 min. The Ca2+-sensitive potassium channel blocker iberiotoxin was administered in young animals, with and without helium (He-PC+Ibtx and Ibtx). Animals underwent 25 min of regional myocardial ischemia and 120 min of reperfusion. In additional experiments, cardiac mitochondria were isolated, and the respiratory control index was calculated (state 3/state 4). Results: Helium reduced infarct size in young rats from 61 ± 7% to 36 ± 14% (P < 0.05 vs. Con). Infarct size reduction was abolished by iberiotoxin (60 ± 11%; P < 0.05 vs. He-PC), whereas iberiotoxin alone had no effect (59 ± 8%; not significant vs. Con). In aged animals, helium had no effect on infarct size (Age Con: 59 ± 7% vs. Age He-PC: 58 ± 8%; not significant). Helium reduced respiratory control index in young animals (2.76 ± 0.05 to 2.43 ± 0.15; P < 0.05) but not in aged animals (Age Con: 2.87 ± 0.17 vs. Age He-PC: 2.87 ± 0.07; not significant). Iberiotoxin abrogated the helium effect on respiratory control index (2.73 ± 0.15; P < 0.05 vs. He-PC) but had no effect itself on mitochondrial respiration (2.75 ± 0.05; not significant vs. Con). Conclusion: Helium causes mitochondrial uncoupling and induces preconditioning in young rats via Ca2+-sensitive potassium channel activation. However, these effects are lost in aged rats.

Morphine induces late cardioprotection in rat hearts in vivo: the involvement of opioid receptors and nuclear transcription factor kappaB.

&dgr;1-opioid receptor agonists can induce cardioprotection by early and late preconditioning (LPC). Morphine (MO) is commonly used for pain treatment during acute coronary syndromes. We investigated whether MO can induce myocardial protection by LPC and whether a nuclear transcription factor &kgr;B (NF-&kgr;B)-dependent intracellular signaling pathway is involved. Rats were subjected to 25 min of regional ischemia and 2 h of reperfusion 24 h after treatment with saline (NaCl; 0.9% 5 mL), lipopolysaccharide of Escherichia coli (LPS; 1 mg/kg), or MO (3 mg/kg). LPS is a trigger of LPC and served as positive control. Naloxone (NAL) was used to investigate the role of opioid receptors in LPC and was given before NaCl, LPS, or MO application (trigger phase) or before ischemia-reperfusion (mediator phase). Infarct size (percentage area at risk) was 59% ± 9%, 51% ± 6%, or 53% ± 10% in the NaCl, NAL-NaCl, and NaCl-NAL groups, respectively. Pretreatment with MO reduced infarct size to 20% ± 6% after 24 h (MO-24h), and this effect was abolished by NAL in the trigger (NAL-MO, 53% ± 14%) and in the mediator (MO-NAL, 60% ± 8%) phases. Pretreatment with LPS reduced infarct size to 23% ± 8%. NAL administration in the trigger phase had no effect on infarct size (NAL-LPS 30% ± 16%), whereas NAL during the mediator phase of LPC abolished the LPS-induced cardioprotection (LPS-NAL 54% ± 8%). The role of NF-&kgr;B in morphine-induced LPC was investigated by Western blot and electrophoretic mobility shift assay. Morphine and LPS treatment increased phosphorylation of the inhibitory protein &kgr;B, leading to an increased activity of NF-&kgr;B. Thus, MO induces LPC similarly to LPS and it is likely that this cardioprotection is mediated at least in part by activation of NF-&kgr;B. Opioid receptors are involved as mediators in both MO- and LPS-induced LPC but as triggers only in MO-induced LPC.

Hypoxia-inducible factor 1 and related gene products in anaesthetic-induced preconditioning

Volatile anaesthetics induce early and late preconditioning in several organs, including the heart. This phenomenon is of particular interest in the clinical setting to reduce infarct size and to elicit adaptive functions of the heart. One possible mechanism of anaesthetic-induced preconditioning is the activation of the transcription factor hypoxia-inducible factor 1α (HIF-1α) and its target gene responses. It was shown that pharmacological activation of the hypoxia-inducible factor 1α pathway is organ protective, and recent studies demonstrated that isoflurane and xenon lead to hypoxia-inducible factor 1α upregulation, which is related to the preconditioning effect of the inhalational anaesthetics. A better understanding of the molecular mechanisms that mediate cardioprotection by volatile anaesthetics might help to introduce specific applications of these substances for organ-protective purposes in patients with cardiovascular diseases. Abbreviations siRNA: small interfering RNA; VEGF: vascular endothelial growth factor.