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Remote postconditioning by humoral factors in effluent from ischemic preconditioned rat hearts is mediated via PI3K/Akt-dependent cell-survival signaling at reperfusion.

Breivik L, Helgeland E, Aarnes EK, Mrdalj J, Jonassen AK - Basic Res. Cardiol. (2010)

Bottom Line: Transferring coronary effluent collected during IPC treatment to un-preconditioned recipient hearts protects from lethal ischemic insults.Effluent administration for 10 min at immediate reperfusion (reperfusion therapy) or as a mimetic of pharmacological postconditioning (remote postconditioning, RIPost) significantly reduced infarct size compared to control (P < 0.05).Fractionation of coronary IPC effluent revealed that cytoprotective humoral mediator(s) released during the conditioning phase were of hydrophobic nature as all hydrophobic fractions with molecules under 30 kDa significantly reduced infarct size versus the control and hydrophilic fraction-treated hearts (P < 0.05).

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedicine, Faculty of Medicine and Dentistry, University of Bergen, Bergen, Norway. lars.breivik@biomed.uib.no

ABSTRACT
Short non-lethal ischemic episodes administered to hearts prior to (ischemic preconditioning, IPC) or directly after (ischemic postconditioning, IPost) ischemic events facilitate myocardial protection. Transferring coronary effluent collected during IPC treatment to un-preconditioned recipient hearts protects from lethal ischemic insults. We propose that coronary IPC effluent contains hydrophobic cytoprotective mediators acting via PI3K/Akt-dependent pro-survival signaling at ischemic reperfusion. Ex vivo rat hearts were subjected to 30 min of regional ischemia and 120 min of reperfusion. IPC effluent administered for 10 min prior to index ischemia attenuated infarct size by ≥55% versus control hearts (P < 0.05). Effluent administration for 10 min at immediate reperfusion (reperfusion therapy) or as a mimetic of pharmacological postconditioning (remote postconditioning, RIPost) significantly reduced infarct size compared to control (P < 0.05). The IPC effluent significantly increased Akt phosphorylation in un-preconditioned hearts when administered before ischemia or at reperfusion, while pharmacological inhibition of PI3K/Akt-signaling at reperfusion completely abrogated the cardioprotection offered by effluent administration. Fractionation of coronary IPC effluent revealed that cytoprotective humoral mediator(s) released during the conditioning phase were of hydrophobic nature as all hydrophobic fractions with molecules under 30 kDa significantly reduced infarct size versus the control and hydrophilic fraction-treated hearts (P < 0.05). The total hydrophobic effluent fraction significantly reduced infarct size independently of temporal administration (before ischemia, at reperfusion or as remote postconditioning). In conclusion, the IPC effluent retains strong cardioprotective properties, containing hydrophobic mediator(s) < 30 kDa offering cytoprotection via PI3K/Akt-dependent signaling at ischemic reperfusion.

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Phosphorylation status of myocardial Akt in recipient hearts exposed to treatment with IPC effluent. a Representative immunoblots of Akt phosphorylation (Ser473) showing the effects of treatment with IPC effluent (EffPre) in recipient hearts (tissue harvested at end of treatment), as compared to hearts exposed to the standard IPC protocol (IPC) and baseline hearts (CB). c Representative immunoblots of Akt phosphorylation (Ser473) in pre-ischemic IPC effluent-treated hearts that was also reperfused for 15 min (EffPre+Rep) and reperfusion treatment with IPC effluent (EffRep) in recipient hearts as compared to ischemic reperfused control hearts (CtrRep) (tissues harvested at 15 min of reperfusion). Administering the PI3K-inhibitor WI (1 μM) and the Akt-inhibitor SH-6 (10 μM) for 10 min at reperfusion abrogated Akt phosphorylation in IPC effluent-treated hearts. Total Akt and GADPH indicates equal loading. b, d Densitometric analysis of total and phosphorylated Akt immunoblots expressed in arbitrary units (AU). p-Akt expressed as a ratio of total Akt with CB = 1. Bars represent mean ± SEM. N ≥ 3 in each group. *P < 0.05 versus CB, †P < 0.05 versus IPC, §P < 0.05 versus EffPre+Rep, ¤P < 0.05 versus EffRep, #P < 0.05 versus CtrRep
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Fig4: Phosphorylation status of myocardial Akt in recipient hearts exposed to treatment with IPC effluent. a Representative immunoblots of Akt phosphorylation (Ser473) showing the effects of treatment with IPC effluent (EffPre) in recipient hearts (tissue harvested at end of treatment), as compared to hearts exposed to the standard IPC protocol (IPC) and baseline hearts (CB). c Representative immunoblots of Akt phosphorylation (Ser473) in pre-ischemic IPC effluent-treated hearts that was also reperfused for 15 min (EffPre+Rep) and reperfusion treatment with IPC effluent (EffRep) in recipient hearts as compared to ischemic reperfused control hearts (CtrRep) (tissues harvested at 15 min of reperfusion). Administering the PI3K-inhibitor WI (1 μM) and the Akt-inhibitor SH-6 (10 μM) for 10 min at reperfusion abrogated Akt phosphorylation in IPC effluent-treated hearts. Total Akt and GADPH indicates equal loading. b, d Densitometric analysis of total and phosphorylated Akt immunoblots expressed in arbitrary units (AU). p-Akt expressed as a ratio of total Akt with CB = 1. Bars represent mean ± SEM. N ≥ 3 in each group. *P < 0.05 versus CB, †P < 0.05 versus IPC, §P < 0.05 versus EffPre+Rep, ¤P < 0.05 versus EffRep, #P < 0.05 versus CtrRep

Mentions: IPC is known to activate the pro-survival kinase Akt directly after the IPC protocol [34]. Interestingly, administration of IPC effluent prior to index ischemia caused a significant increase in Akt phosphorylation compared to hearts exposed to IPC treatment only (EffPre: 5.2 ± 0.2 AU vs. IPC: 2.0 ± 0.2 AU, P < 0.05; Fig. 4a, b), indicating an increased potential of the IPC effluent to activate the pro-survival Akt-signaling as compared to the IPC stimulus itself. Furthermore, administration of IPC effluent at reperfusion elevated the Akt-phosphorylation compared to I/R control hearts (EffRep: 7.2 ± 0.2 AU vs. CtrRep: 2.0 ± 0.5 AU, P < 0.05; Fig. 4c, d). Treatment with pre-ischemic IPC effluent and analysis of Akt phosphorylation at reperfusion (EffPre+Rep) also increased phosphorylation status of Akt as compared to the I/R control group (EffPre+Rep: 4.2 ± 0.6 AU vs. CtrRep: 2.0 ± 0.5 AU, P < 0.05; Fig. 4c, d).Fig. 4


Remote postconditioning by humoral factors in effluent from ischemic preconditioned rat hearts is mediated via PI3K/Akt-dependent cell-survival signaling at reperfusion.

Breivik L, Helgeland E, Aarnes EK, Mrdalj J, Jonassen AK - Basic Res. Cardiol. (2010)

Phosphorylation status of myocardial Akt in recipient hearts exposed to treatment with IPC effluent. a Representative immunoblots of Akt phosphorylation (Ser473) showing the effects of treatment with IPC effluent (EffPre) in recipient hearts (tissue harvested at end of treatment), as compared to hearts exposed to the standard IPC protocol (IPC) and baseline hearts (CB). c Representative immunoblots of Akt phosphorylation (Ser473) in pre-ischemic IPC effluent-treated hearts that was also reperfused for 15 min (EffPre+Rep) and reperfusion treatment with IPC effluent (EffRep) in recipient hearts as compared to ischemic reperfused control hearts (CtrRep) (tissues harvested at 15 min of reperfusion). Administering the PI3K-inhibitor WI (1 μM) and the Akt-inhibitor SH-6 (10 μM) for 10 min at reperfusion abrogated Akt phosphorylation in IPC effluent-treated hearts. Total Akt and GADPH indicates equal loading. b, d Densitometric analysis of total and phosphorylated Akt immunoblots expressed in arbitrary units (AU). p-Akt expressed as a ratio of total Akt with CB = 1. Bars represent mean ± SEM. N ≥ 3 in each group. *P < 0.05 versus CB, †P < 0.05 versus IPC, §P < 0.05 versus EffPre+Rep, ¤P < 0.05 versus EffRep, #P < 0.05 versus CtrRep
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Fig4: Phosphorylation status of myocardial Akt in recipient hearts exposed to treatment with IPC effluent. a Representative immunoblots of Akt phosphorylation (Ser473) showing the effects of treatment with IPC effluent (EffPre) in recipient hearts (tissue harvested at end of treatment), as compared to hearts exposed to the standard IPC protocol (IPC) and baseline hearts (CB). c Representative immunoblots of Akt phosphorylation (Ser473) in pre-ischemic IPC effluent-treated hearts that was also reperfused for 15 min (EffPre+Rep) and reperfusion treatment with IPC effluent (EffRep) in recipient hearts as compared to ischemic reperfused control hearts (CtrRep) (tissues harvested at 15 min of reperfusion). Administering the PI3K-inhibitor WI (1 μM) and the Akt-inhibitor SH-6 (10 μM) for 10 min at reperfusion abrogated Akt phosphorylation in IPC effluent-treated hearts. Total Akt and GADPH indicates equal loading. b, d Densitometric analysis of total and phosphorylated Akt immunoblots expressed in arbitrary units (AU). p-Akt expressed as a ratio of total Akt with CB = 1. Bars represent mean ± SEM. N ≥ 3 in each group. *P < 0.05 versus CB, †P < 0.05 versus IPC, §P < 0.05 versus EffPre+Rep, ¤P < 0.05 versus EffRep, #P < 0.05 versus CtrRep
Mentions: IPC is known to activate the pro-survival kinase Akt directly after the IPC protocol [34]. Interestingly, administration of IPC effluent prior to index ischemia caused a significant increase in Akt phosphorylation compared to hearts exposed to IPC treatment only (EffPre: 5.2 ± 0.2 AU vs. IPC: 2.0 ± 0.2 AU, P < 0.05; Fig. 4a, b), indicating an increased potential of the IPC effluent to activate the pro-survival Akt-signaling as compared to the IPC stimulus itself. Furthermore, administration of IPC effluent at reperfusion elevated the Akt-phosphorylation compared to I/R control hearts (EffRep: 7.2 ± 0.2 AU vs. CtrRep: 2.0 ± 0.5 AU, P < 0.05; Fig. 4c, d). Treatment with pre-ischemic IPC effluent and analysis of Akt phosphorylation at reperfusion (EffPre+Rep) also increased phosphorylation status of Akt as compared to the I/R control group (EffPre+Rep: 4.2 ± 0.6 AU vs. CtrRep: 2.0 ± 0.5 AU, P < 0.05; Fig. 4c, d).Fig. 4

Bottom Line: Transferring coronary effluent collected during IPC treatment to un-preconditioned recipient hearts protects from lethal ischemic insults.Effluent administration for 10 min at immediate reperfusion (reperfusion therapy) or as a mimetic of pharmacological postconditioning (remote postconditioning, RIPost) significantly reduced infarct size compared to control (P < 0.05).Fractionation of coronary IPC effluent revealed that cytoprotective humoral mediator(s) released during the conditioning phase were of hydrophobic nature as all hydrophobic fractions with molecules under 30 kDa significantly reduced infarct size versus the control and hydrophilic fraction-treated hearts (P < 0.05).

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedicine, Faculty of Medicine and Dentistry, University of Bergen, Bergen, Norway. lars.breivik@biomed.uib.no

ABSTRACT
Short non-lethal ischemic episodes administered to hearts prior to (ischemic preconditioning, IPC) or directly after (ischemic postconditioning, IPost) ischemic events facilitate myocardial protection. Transferring coronary effluent collected during IPC treatment to un-preconditioned recipient hearts protects from lethal ischemic insults. We propose that coronary IPC effluent contains hydrophobic cytoprotective mediators acting via PI3K/Akt-dependent pro-survival signaling at ischemic reperfusion. Ex vivo rat hearts were subjected to 30 min of regional ischemia and 120 min of reperfusion. IPC effluent administered for 10 min prior to index ischemia attenuated infarct size by ≥55% versus control hearts (P < 0.05). Effluent administration for 10 min at immediate reperfusion (reperfusion therapy) or as a mimetic of pharmacological postconditioning (remote postconditioning, RIPost) significantly reduced infarct size compared to control (P < 0.05). The IPC effluent significantly increased Akt phosphorylation in un-preconditioned hearts when administered before ischemia or at reperfusion, while pharmacological inhibition of PI3K/Akt-signaling at reperfusion completely abrogated the cardioprotection offered by effluent administration. Fractionation of coronary IPC effluent revealed that cytoprotective humoral mediator(s) released during the conditioning phase were of hydrophobic nature as all hydrophobic fractions with molecules under 30 kDa significantly reduced infarct size versus the control and hydrophilic fraction-treated hearts (P < 0.05). The total hydrophobic effluent fraction significantly reduced infarct size independently of temporal administration (before ischemia, at reperfusion or as remote postconditioning). In conclusion, the IPC effluent retains strong cardioprotective properties, containing hydrophobic mediator(s) < 30 kDa offering cytoprotection via PI3K/Akt-dependent signaling at ischemic reperfusion.

Show MeSH
Related in: MedlinePlus