Ragnar Huhn

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.

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.

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.

Cyclosporine A administered during reperfusion fails to restore cardioprotection in prediabetic Zucker obese rats in vivo.

BACKGROUND AND AIMS Hyperglycaemia blocks sevoflurane-induced postconditioning, and cardioprotection in hyperglycaemic myocardium can be restored by inhibition of the mitochondrial permeability transition pore (mPTP). We investigated whether sevoflurane-induced postconditioning is also blocked in the prediabetic heart and if so, whether cardioprotection could be restored by inhibiting mPTP. METHODS AND RESULTS Zucker lean (ZL) and Zucker obese (ZO) rats were assigned to one of seven groups. Animals underwent 25 min of ischaemia and 120 min of reperfusion. Control (ZL-/ZO Con) animals were not further treated. postconditioning groups (ZL-/ZO Sevo-post) received sevoflurane for 5 min starting 1min prior to the onset of reperfusion. The mPTP inhibitor cyclosporine A (CsA) was administered intravenously in a concentration of 5 (ZO CsA and ZO CsA+Sevo-post) or 10 mg/kg (ZO CsA10+Sevo-post) 5 min before the onset of reperfusion. At the end of reperfusion, infarct sizes were measured by TTC staining. Blood samples were collected to measure plasma levels of insulin, cholesterol and triglycerides. Sevoflurane postconditioning reduced infarct size in ZL rats to 35±12% (p<0.05 vs. ZL Con: 60±6%). In ZO rats sevoflurane postconditioning was abolished (ZO Sevo-post: 59±12%, n.s. vs. ZO Con: 58±6%). 5 mg and 10 mg CsA could not restore cardioprotection (ZO CsA+Sevo-post: 59±7%, ZO CsA10+Sevo-post: 57±14%; n.s. vs. ZO Con). In ZO rats insulin, cholesterol and triglyceride levels were significant higher than in ZL rats (all p<0.05). CONCLUSION Inhibition of mPTP with CsA failed to restore cardioprotection in the prediabetic but normoglycaemic heart of Zucker obese rats in vivo.

Helium-induced late preconditioning in the rat heart in vivo

BACKGROUND A recent study showed that the noble gas helium induces early myocardial preconditioning. Cyclooxygenase-2 (COX-2) has been shown to be an important mediator in the signal transduction of late preconditioning. In the present study, we investigated whether helium induces late preconditioning in a concentration-dependent, time-dependent, or in both manner and whether COX-2 activity, mitochondrial function, or both are involved. METHODS The study was performed in male Wistar rats and consisted of two parts. In part 1, late preconditioning was achieved by administration of 70%, 50%, 30%, and 10% helium for 15 min 24 h before ischaemia/reperfusion (I/R). Based on the findings of part 1, in additional experiments 30% helium was administered subsequently three and two days before I/R. Furthermore, additional rats were pretreated with the COX-2 inhibitor NS-398 (5 mg kg(-1)) with and without 30% helium. Additional experiments were performed for mitochondrial analysis. RESULTS Helium concentrations of 70%, 50%, and 30% but not 10% reduced infarct size [He-LPC 70: 37(13)%, He-LPC 50: 34(16)%, He-LPC 30: 40(9)%; each P<0.05 vs CONTROL 55(8)%, He-LPC 10: 53(4)%; P>0.05 vs CONTROL]. Repeated administration of helium did not further enhance cardioprotection. NS-398 completely abolished cardioprotection by 30% helium [He-LPC 30+NS-398: 57(9)%; P<0.05 vs He-LPC 30] but had itself no effect on infarct size [NS-398: 55(9)%; P>0.05 vs CONTROL]. There were no differences in mitochondrial function after helium preconditioning. CONCLUSIONS Helium induces late preconditioning. Cardioprotection is already maximal with administration of one cycle of 30% helium and is abolished by functional blockade of COX-2 activity.

Cardioprotection by remote ischemic preconditioning exhibits a signaling pattern different from local ischemic preconditioning

Remote ischemic preconditioning (RIPC) and local ischemic preconditioning (IPC) protect the myocardium from subsequent ischemia/reperfusion (I/R) injury. In this study, the protective effects of early RIPC, IPC, and the combination of both (RIPC-IPC) were characterized. Furthermore, the hypothesis was tested that protein kinase C (PKC) and mitogen-activated protein kinases (MAPKs), important mediators of IPC, are activated in RIPC. Infarct size, serum troponin T, and creatine kinase levels were assessed after 4 × 5-min noninvasive RIPC, local IPC, or a combination of both and 35 min of regional ischemia and 120 min of reperfusion. Protein kinase C ϵ and the MAPKs extracellular signal-regulated MAPK (ERK), c-jun N-terminal kinase (JNK), and p38 MAPK were analyzed by Western blot analysis and activity assays in the myocardium and skeletal muscle immediately after the preconditioning protocol. Remote ischemic preconditioning, IPC, and RIPC-IPC significantly reduced myocardial infarct size (RIPC-I/R: 54% ± 15%; IPC-I/R: 33% ± 15%; RIPC-IPC-I/R: 33% ± 15%; P < 0.05 vs. I/R [76% ± 14%]) and troponin T release (RIPC-I/R: 15.4 ± 6.4 ng/mL; IPC-I/R: 10.9 ± 7.0 ng/mL; RIPC-IPC-I/R: 9.8 ± 5.6 ng/mL; P < 0.05 vs. I/R [27.1 ± 12.0 ng/mL]) after myocardial I/R. Ischemic preconditioning led to an activation of PKCϵ and ERK 1/2, whereas RIPC did not lead to a translocation of PKCϵ to the mitochondria or phosphorylation of the MAPKs ERK 1/2, JNK 1/2, and p38 MAPK. Remote ischemic preconditioning did not induce translocation of PKCϵ to the mitochondria or phosphorylation of MAPKs in the preconditioned muscle tissue. Remote ischemic preconditioning, IPC, and RIPC-IPC exert early protection against myocardial I/R injury. Remote ischemic preconditioning and local IPC exhibit different activation dynamics of signal transducers in the myocardium. The studied PKC-MAPK pathway is likely not involved in the protective effects of RIPC.ABBREVIATIONS-AAR-area at risk; CK-creatine kinase; ERK-extracellular signal-regulated MAPK; IPC-ischemic preconditioning; I/R-ischemia and reperfusion; JNK-c-jun N-terminal kinase; MAPK-mitogen-activated protein kinase; PKC-protein kinase C; RIPC-remote ischemic preconditioning

Sevoflurane-induced Preconditioning Impact of Protocol and Aprotinin Administration on Infarct Size and Endothelial Nitric-Oxide Synthase Phosphorylation in the Rat Heart In Vivo

Background:Sevoflurane induces preconditioning (SevoPC). The effect of aprotinin and the involvement of endothelial nitric-oxide synthase (NOS) on SevoPC are unknown. We investigated (1) whether SevoPC is strengthened by multiple preconditioning cycles, (2) whether SevoPC is blocked by aprotinin, and (3) whether endothelial NOS plays a crucial role in SevoPC. Methods:Anesthetized male Wistar rats were randomized to 15 groups (each n = 6) and underwent 25-min regional myocardial ischemia and 2-h reperfusion. Controls were not treated further. Preconditioning groups inhaled 1 minimum alveolar concentration of sevoflurane for 5 min (SEVO-I), twice for 5 min each (SEVO-II), three times for 5 min each (SEVO-III), or six times for 5 min each (SEVO-VI). Aprotinin was administered with and without sevoflurane. Involvement of endothelial NOS was determined with the nonspecific NOS blocker N-nitro-l-arginine-methyl-ester, the specific neuronal NOS blocker 7-nitroindazole, and the specific inducible NOS blocker aminoguanidine. Results:SevoPC reduced infarct size in all protocols (SEVO-I, 42 ± 6%; SEVO-II, 33 ± 4%; SEVO-III, 11 ± 5%; SEVO-VI, 16 ± 4%; all P < 0.001 vs. control, 67 ± 3%) and was least after three and six cycles of sevoflurane (P < 0.001 vs. SEVO-II and -I, respectively). Aprotinin alone had no effect on infarct size but blocked SevoPC. N-nitro-l-arginine-methyl-ester abolished SevoPC (67 ± 4%; P < 0.05 vs. SEVO-III). Aminoguanidine and 7-nitroindazole blocked SevoPC only partially (25 ± 6 and 31 ± 6%, respectively; P < 0.05 vs. SEVO-III and control). SevoPC induced endothelial NOS phosphorylation, which was abrogated by aprotinin. Conclusion:SevoPC is strengthened by multiple preconditioning cycles, and phosphorylation of endothelial NOS is a crucial step in mediating SevoPC. These effects are abolished by aprotinin.

Age-related loss of cardiac preconditioning: impact of protein kinase A.

Helium induces preconditioning (He-PC) by mitochondrial calcium-sensitive potassium (mK(Ca)) channel-activation, but this effect is lost in the aged myocardium. Both, the upstream signalling pathway of He-PC and the underlying mechanisms for an age-related loss of preconditioning are unknown. A possible candidate as upstream regulator of mK(Ca) channels is protein kinase A (PKA). We investigated whether 1) regulation of PKA is involved in He-PC and 2) regulation of PKA is age-dependent. Young (2-3 months) and aged (22-24 months) Wistar rats were randomised to eight groups (each n=8). All animals underwent 25 min regional myocardial ischemia and 120 min reperfusion. Control (Con, Age Con) animals were not further treated. Young rats inhaled 70% helium for 3×5 min (He-PC). The PKA-blocker H-89 (10 μg/kg) was administered with and without helium (He-PC+H-89, H-89). Furthermore, we tested the effect of direct activation of mK(Ca) channels with NS1619. The adenylyl cyclase activator forskolin (For) was administered in young (300 μg/kg) and aged animals (300 and 1000 μg/kg). He-PC reduced infarct size from 60±4% (Con) to 37±10% (p<0.05). Infarct size reduction was completely abolished by H-89 (58±5%; p<0.05), but H-89 alone had no effect (57±2%). NS1619 reduced infarct size in the same concentration in both, young and aged rats (35±6%; p<0.05 vs. Con and 34±8%; p<0.05 vs. Age Con). Forskolin in a concentration of 300 μg/kg reduced infarct size in young (37±6%; p<0.05) but not in aged rats (48±13%; n.s.). In contrast, 1000 μg/kg Forskolin reduced infarct size also in aged rats (28±3%; p<0.05). He-PC is mediated by activation of PKA. Alterations in PKA regulation might be an underlying mechanism for the age-dependent loss of preconditioning.

Effects of remote ischemic preconditioning and myocardial ischemia on microRNA-1 expression in the rat heart in vivo.

ABSTRACT Remote ischemic preconditioning (RIPC) is an easily applicable method for protecting the heart against a subsequent ischemia and reperfusion (I/R) injury. However, the exact molecular mechanisms underlying RIPC are unknown. We examined the involvement of microRNAs (miRNAs) and in particular the expression of miRNA-1 (miR-1) in RIPC and myocardial ischemia. Remote ischemic preconditioning was conducted by four cycles of 5-min bilateral hind-limb ischemia in male Wistar rats. Cardiac ischemia was induced by ligation of the left anterior descending coronary artery for 35 min followed by 2 or 6 h of reperfusion. MicroRNA expression was analyzed by Taqman miRNA arrays and quantitative polymerase chain reaction assays. Luciferase assays were performed to validate the miR-1 target gene brain-derived neurotrophic factor (BDNF). Brain-derived neurotrophic factor mRNA and protein levels were analyzed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Remote ischemic preconditioning led to a differential expression of miRNAs. The most abundant cardiac miRNA, miR-1, was downregulated by RIPC without following ischemia as well as after I/R and RIPC followed by I/R after 2 h of reperfusion. After 6 h of reperfusion, RIPC led to an upregulation of miR-1, whereas ischemia had no effect on miR-1 expression. Luciferase assays confirmed the interaction of miR-1 with BDNF, a protein that has been shown to exert cardioprotective effects. Brain-derived neurotrophic factor protein levels in rat hearts measured by enzyme-linked immunosorbent assay were not significantly altered after 2 or 6 h of reperfusion in all intervention groups. Remote ischemic preconditioning leads to changes in the expression levels of the most abundant cardiac miRNA, miR-1. MicroRNA 1 levels did not correlate with protein levels of BDNF, a known miR-1 target, in vivo. Further studies are needed to explore the biological significance of changes in miR-1 expression levels and the potential interaction with BDNF in RIPC-induced cardioprotection.

Ischaemic and morphine-induced post-conditioning: impact of mK(Ca) channels

BACKGROUND Mitochondrial calcium-sensitive potassium (mK(Ca)) channels are involved in cardiac preconditioning. In the present study, we investigated whether also ischaemic-, morphine-induced post-conditioning, or both is mediated by the activation of mK(Ca) channels in the rat heart in vitro. METHODS Animals were treated in compliance with institutional and national guidelines. Male Wistar rats were randomly assigned to one of seven groups (each n = 7). Control animals were not further treated. Post-conditioning was induced either by 3 × 30 s of ischaemia/reperfusion (I-PostC) or by administration of morphine (M-PostC, 1 µM) for 15 min at the onset of reperfusion. The mK(Ca)-channel inhibitor paxilline (1 µM) was given with and without post-conditioning interventions (M-PostC+Pax, I-PostC+Pax, and Pax). As a positive control, we determined whether direct activation of mK(Ca) channels with NS1619 (10 µM) induced cardiac post-conditioning (NS1619). Isolated hearts underwent 35 min ischaemia followed by 120 min reperfusion. At the end of reperfusion, infarct sizes were measured by triphenyltetrazolium chloride staining. RESULTS In the control group, infarct size was 53 (5)% of the area at risk. Morphine- and ischaemic post-conditioning reduced infarct size in the same range [M-PostC: 37 (4)%, I-PostC: 35 (5)%; each P<0.05 vs control]. The mK(Ca)-channel inhibitor paxilline completely blocked post-conditioning [M-PostC+Pax: 47 (7)%, I-PostC+Pax: 51 (3)%; each P<0.05 vs M-PostC and I-PostC, respectively]. Paxilline itself had no effect on infarct size (NS vs control). NS1619 reduced infarct size to 33 (4)% (P < 0.05 vs control). CONCLUSIONS Ischaemic- and morphine-induced post-conditioning is mediated by the activation of mK(Ca) channels.