Yoshitaka Inamura

Sevoflurane enhances ethanol-induced cardiac preconditioning through modulation of protein kinase C, mitochondrial KATP channels, and nitric oxide synthase, in guinea pig hearts.

BACKGROUND:Volatile anesthetics and regular ethanol consumption induce cardioprotection mimicking ischemic preconditioning. We investigated whether sevoflurane enhances ethanol preconditioning and whether inhibition of protein kinase C (PKC) and mitochondrial KATP channels attenuated this enhanced cardioprotection. The effects of regular ethanol consumption on expression of inducible (iNOS) and endothelial (eNOS) nitric oxide synthase were determined. METHODS:Isolated perfused guinea pig hearts underwent 30-min global ischemia and 120-min reperfusion (Control: CTL). The ethanol group (EtOH) received 2.5% ethanol in their drinking water for 6 wk. Anesthetic preconditioning was elicited by 10-min exposure to sevoflurane (1 minimum alveolar anesthetic concentration; 2%) in ethanol (EtOH + SEVO) or nonethanol (SEVO) hearts. PKC and mitochondrial KATP channels were inhibited with chelerythrine and 5-hydroxydecanoate pretreatment, respectively. Contractile recovery was assessed by monitoring of left ventricular developed and end-diastolic pressures. Infarct size was determined by triphenyltetrazolium chloride staining. Expression of iNOS and eNOS were determined by Western blot analysis. RESULTS:After ischemia-reperfusion, hearts from the EtOH, sevoflurane (SEVO), and EtOH + SEVO groups had higher left ventricular developed pressure and lower left ventricular end-diastolic pressure compared with CTL. Infarct size was reduced in EtOH and SEVO hearts compared with CTL (27% and 23% vs 45%, respectively, P < 0.001). Sevoflurane further reduced infarct size in EtOH hearts (27% vs 15%, P < 0.001). Chelerythrine and 5-hydroxydecanoate abolished cardioprotection in both SEVO and EtOH cardioprotected hearts. iNOS expression was reduced and eNOS expression was increased in EtOH hearts. CONCLUSIONS:Sevoflurane enhances cardiac preconditioning induced by regular EtOH consumption. This effect is mediated in part by modulation of PKC and mitochondrial KATP channels, and possibly by altered modulation of NOS expression.

Aprotinin Abolishes Sevoflurane Postconditioning by Inhibiting Nitric Oxide Production and Phosphorylation of Protein Kinase C-δ and Glycogen Synthase Kinase 3β

Background:It remains controversial whether aprotinin use during cardiac surgery is cardioprotective or detrimental. In contrast, volatile anesthetics may offer cardioprotection perioperatively. Increased nitric oxide, protein kinase C activation, and glycogen synthase kinase 3β inhibition play a role in sevoflurane-induced cardioprotection. The authors investigated whether aprotinin affects sevoflurane postconditioning. Methods:Isolated guinea pig hearts underwent 30 min of global ischemia and 120 min of reperfusion (control [CTL]). Postconditioning was elicited with sevoflurane (2%) for 2 min at reperfusion onset (POST). Aprotinin (250 kallikrein inhibitor units/ml) was administered for 5 min at reperfusion onset (POST + APRO and CTL + APRO). In additional experiments, both sevoflurane and aprotinin were given before ischemia and throughout the reperfusion period (SEVO + APRO (throughout)) to mimic clinical conditions. Left ventricular developed and end-diastolic pressures and infarct size were measured. Western blot analysis determined phosphorylated protein kinase C-&dgr;, protein kinase C-&dgr;, Akt, and glycogen synthase kinase 3β expression. Nitric oxide production during reperfusion was measured by nitric oxide sensor. Results:After ischemia–reperfusion, POST had significantly higher left ventricular developed (56 ± 11 vs. 26 ± 8 mmHg [mean ± SD]) and lower end-diastolic pressures (20 ± 9 vs. 47 ± 15 mmHg) and reduced infarct size (15 ± 3% vs. 41 ± 10%) versus CTL. Aprotinin abolished these improvements. Expressions of phospho-Akt (activated), phospho–protein kinase C-&dgr; (activated), and phospho–glycogen synthase kinase 3β (inhibited) were significantly increased in POST. Aprotinin attenuated these increased expressions. Nitric oxide production after reperfusion was higher in POST than in CTL, but not in POST + APRO. Conclusions:Aprotinin abolishes sevoflurane postconditioning, associated with inhibited phosphorylation of Akt, protein kinase C-&dgr;, and glycogen synthase kinase 3β and reduced nitric oxide production.

Induction of autophagy restores the loss of sevoflurane cardiac preconditioning seen with prolonged ischemic insult

Sevoflurane preconditioning against myocardial ischemia-reperfusion injury is lost if the ischemic insult is too long. Emerging evidence suggests that induction of autophagy may also confer cardioprotection against ischemia-reperfusion injury. We examined whether induction of autophagy prolongs sevoflurane preconditioning protection during a longer ischemic insult. Isolated guinea pigs hearts were subjected to 30 or 45 min ischemia, followed by 120 min reperfusion (control). Anesthetic preconditioning was elicited with 2% sevoflurane for 10 min prior to ischemia (SEVO-30, SEVO-45). Chloramphenicol (autophagy upregulator, 300 µM) was administered starting 20 min before ischemia and throughout reperfusion in SEVO-45 (SEVO-45+CAP). To inhibit autophagy, 3-methyladenine (10 μM) was administered during sevoflurane administration in SEVO-45+CAP. Infarct size was determined by triphenyltetrazolium chloride stain. Tissue samples were obtained before ischemia to determine autophagy-related protein (microtubule-associated protein light chain I and II: LC3-I, II), Akt and glycogen synthase kinase 3β (GSK3β) expression using Western blot analysis. The effect of autophagy on calcium-induced mitochondrial permeability transition pore (MPTP) opening in isolated calcein-loaded mitochondria was assessed. Electron microscopy was used to detect autophagosomes. Infarct size was significantly reduced in SEVO-30, but not in SEVO-45. Chloramphenicol restored sevoflurane preconditioning lost by 45 min ischemia. There were more abundant autophagozomes and LC3-II expression was significantly increased in SEVO-45+CAP. Induction of autophagy before ischemia enhanced GSK3β phosphorylation and inhibition of calcium-induced MPTP opening. These effects were abolished by 3-methyladenine. Pre-ischemic induction of autophagy restores sevoflurane preconditioning lost by longer ischemic insult. This effect is associated with enhanced inhibition of MPTP by autophagy.

Acute memory phase of sevoflurane preconditioning is associated with sustained translocation of protein kinase C-α and ε, but not δ, in isolated guinea pig hearts

Background and objective Anaesthetic preconditioning (APC) exerts cardioprotective effects by reducing infarct size and improving recovery of contractile function after ischaemia–reperfusion. The interval between brief exposure to volatile anaesthetic and sustained ischaemia, the acute memory phase, is dependent on intracellular signalling mediating this cardioprotection. Intramyocyte translocation of protein kinase C (PKC) is known to be a key mediator in APC. We examined the relationship between the time frame of the acute memory phase of sevoflurane preconditioning and intramyocyte translocation of PKC-α, δ and ϵ to the particulate fraction. Methods Isolated perfused guinea pig hearts were subjected to 30 min ischaemia and 120 min reperfusion. APC was elicited with one minimum alveolar concentration sevoflurane for 10 min. Washout times of 10, 30, 60 and 90 min were studied. Contractile recovery was assessed by monitoring left ventricular developed pressures. Infarct size was determined by triphenyltetrazolium chloride staining. Translocation of PKC was examined by western blot analysis. Results After ischaemia–reperfusion, left ventricular developed pressure recovered to a greater degree with APC compared with control for washout times of 10 and 30 min, but not 60 and 90 min. Similarly, infarct size was reduced for washout times of 10 and 30 min, but not 60 and 90 min. Sustained translocation of PKC-α and ϵ, but not δ, was associated with the time frame of the acute memory phase. Conclusion The acute memory phase of sevoflurane preconditioning is limited to less than 60 min. Sustained translocation of PKC-α and ϵ, but not δ, correlates with this acute memory phase of sevoflurane preconditioning.

Sevoflurane Confers Additive Cardioprotection to Ethanol Preconditioning Associated With Enhanced Phosphorylation of Glycogen Synthase Kinase-3β and Inhibition of Mitochondrial Permeability Transition Pore Opening

OBJECTIVE The purposes of this study were to investigate whether sevoflurane (SEVO) enhances moderate-dose ethanol (EtOH) preconditioning and whether this additional cardioprotection is associated with glycogen synthase kinase-3β (GSK-3β), protein kinase B (Akt), mammalian target of rapamycin (mTOR), 70-kDa ribosomal s6 kinase-1 (p70s6K), and/or mitochondrial permeability transition pore (MPTP) opening. DESIGN In vitro study using an isolated heart Langendorff preparation. SETTING University research laboratory. PARTICIPANTS Male guinea pigs (n = 170). INTERVENTIONS Isolated perfused guinea pig hearts underwent 30-minute ischemia and 120-minute reperfusion (control). The EtOH group received 5% EtOH in the drinking water for 8 weeks. Anesthetic preconditioning was elicited by a 10-minute exposure to 2% SEVO in EtOH (EtOH + SEVO group) or non-EtOH (SEVO group) hearts. The inhibition of GSK-3β phosphorylation and mTOR was achieved with LY294002 and rapamycin, respectively. GSK-3β, Akt, mTOR, and p70s6K expressions were determined by western blot. Calcium-induced MPTP opening was assessed in isolated calcein-loaded mitochondria. MEASUREMENTS AND MAIN RESULTS After ischemia-reperfusion, the EtOH, SEVO, and EtOH + SEVO groups had higher left ventricular developed pressure recovery and lower end-diastolic pressure versus the control group. Infarct size was smaller in the EtOH and SEVO groups versus control and even smaller in the EtOH + SEVO group. Phosphorylation of GSK-3β and Akt, but not mTOR and p70s6K, was increased in the EtOH and SEVO groups. Phosphorylation of GSK-3β, but not mTOR and p70s6K, was further increased in the EtOH + SEVO group. The EtOH and SEVO groups exhibited a smaller calcium-induced MPTP opening, and the EtOH + SEVO presented an even smaller MPTP opening. CONCLUSIONS SEVO and chronic EtOH preconditioning offer additive cardioprotection. This effect is associated with an increased GSK-3β phosphorylation and an inhibition of MPTP opening.

Regular dipyridamole therapy produces sustained protection against cardiac ischemia–reperfusion injury: Is it time to revisit PARIS?

BACKGROUND Increased activated Akt and eNOS expression coincide with this persistent cardioprotection. Emergent coronary reperfusion therapies are rarely carried out before considerable myocardial injury has occurred. Moreover, reperfusion after prolonged ischemia produces paradoxical ischemia-reperfusion injury, attenuating the efficacy of reperfusion therapies. This has provided impetus for identifying chronic therapies to protect against ischemia-reperfusion injury in those at risk. We previously found that regular dipyridamole therapy produces a chronic preconditioning-like effect mediated through adenosine A1 receptors. METHODS To determine how long this chronic preconditioning effect of dipyridamole remains present after discontinuing therapy, guinea pigs received 4 mg/kg/day in their water for 6 weeks. Ischemia-reperfusion was performed at 0, 2, 3, and 4 days after dipyridamole discontinuation (0 day, 2 days, 3 days and 4 days; n=8 per group). Left ventricular developed pressure (LVDP), end-diastolic pressure (LVEDP), coronary flow (CF), infarct size, and western blot analyses for Akt and endothelial nitric oxide synthase (eNOS) were studied. RESULTS After ischemia-reperfusion, 0 day, 2 days and 3 days, but not 4 days, had significantly higher LVDP and lower LVEDP compared to control. Myocardial infarct size was significantly reduced at 0 day, 2 days and 3 days, but not 4 days, compared to control. Western blot analyses demonstrated upregulation of phospho-Akt and phospho-eNOS expression at 0 day, 2 days, and 3 days, but not 4 days. CONCLUSIONS A chronic preconditioning-like cardioprotection by regular dipyridamole treatment persists for 3 days after discontinuing therapy. Increased activated Akt and eNOS expression may play a role in this persistent cardioprotection.