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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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The effect of inhibiting PI3K and Akt upon reperfusion in IPC effluent-treated recipient hearts. When administering the PI3K-inhibitor WI and the Akt-inhibitor SH-6 at the onset of reperfusion in IPC effluent pre-ischemic (EffPre) or post-ischemic (EffRep and EffPost) treated hearts, the cardioprotective effect of the effluent was completely abolished. Ctr control; EffPre 10 min effluent administration prior to RI; EffRep 10 min effluent administration after RI; EffPost effluent administration 3× 30 s at start of reperfusion; WI 10 min Wortmannin (1 μM) at reperfusion; SH-6 10 min SH-6 (10 μM) at reperfusion. Bars represent mean ± SEM. N ≥ 6 in each group. *P < 0.05 versus Ctr
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Fig5: The effect of inhibiting PI3K and Akt upon reperfusion in IPC effluent-treated recipient hearts. When administering the PI3K-inhibitor WI and the Akt-inhibitor SH-6 at the onset of reperfusion in IPC effluent pre-ischemic (EffPre) or post-ischemic (EffRep and EffPost) treated hearts, the cardioprotective effect of the effluent was completely abolished. Ctr control; EffPre 10 min effluent administration prior to RI; EffRep 10 min effluent administration after RI; EffPost effluent administration 3× 30 s at start of reperfusion; WI 10 min Wortmannin (1 μM) at reperfusion; SH-6 10 min SH-6 (10 μM) at reperfusion. Bars represent mean ± SEM. N ≥ 6 in each group. *P < 0.05 versus Ctr

Mentions: Hausenloy et al. [9] have previously shown that Akt phosphorylation at reperfusion is essential for IPC-induced protection, since inhibition of this kinase during early reperfusion abrogated the IPC-mediated reduction in infarct size. Therefore, to elucidate whether IPC effluent exerts its cardioprotective effect through an PI3K/Akt-dependent signaling pathway activated at reperfusion, hearts were administered fresh IPC effluent for 10 min either prior to index ischemia (EffPre) or at reperfusion (EffRep or EffPost), and co-administered the PI3K-inhibitor WI or the Akt-inhibitor SH-6 for 10 min at reperfusion (see experimental protocol Fig. 1). The protective effect of IPC effluent was completely abolished by co-administering WI or SH-6 at reperfusion compared to the EffPre group (EffPre + WI: 51 ± 7%, EffPre + SH-6: 60 ± 4% vs. EffPre: 24 ± 6%, P < 0.05; Fig. 5) and the EffRep group (EffRep + WI: 56 ± 12%, EffRep + SH-6: 54 ± 8% vs. EffRep: 25 ± 4%, P < 0.05; Fig. 5) and EffPost group (EffPost + WI: 50 ± 11% vs. EffPost; 35 ± 3%, P < 0.05) (data not shown). SH-6 is a phosphatidylinositol (PI) analog that inhibits Akt without affecting the activity of its upstream kinase PDK-1 [16], and has also been shown to prevent phosphorylation of Akt at Ser473 [4, 32]. The inhibitors themselves had no effect on infarct size (WI: 54 ± 8%, SH-6: 52 ± 3% vs. Ctr: 54 ± 5%, ns). Furthermore, both WI and SH-6 significantly reduced Akt phosphorylation in IPC effluent-treated hearts (EffRep + WI: 0.7 ± 0.1 AU, EffRep + SH-6: 2.8 ± 0.2 AU vs. EffRep: 7.2 ± 0.2 AU, P < 0.05; Fig. 4c, d).Fig. 5


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)

The effect of inhibiting PI3K and Akt upon reperfusion in IPC effluent-treated recipient hearts. When administering the PI3K-inhibitor WI and the Akt-inhibitor SH-6 at the onset of reperfusion in IPC effluent pre-ischemic (EffPre) or post-ischemic (EffRep and EffPost) treated hearts, the cardioprotective effect of the effluent was completely abolished. Ctr control; EffPre 10 min effluent administration prior to RI; EffRep 10 min effluent administration after RI; EffPost effluent administration 3× 30 s at start of reperfusion; WI 10 min Wortmannin (1 μM) at reperfusion; SH-6 10 min SH-6 (10 μM) at reperfusion. Bars represent mean ± SEM. N ≥ 6 in each group. *P < 0.05 versus Ctr
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Fig5: The effect of inhibiting PI3K and Akt upon reperfusion in IPC effluent-treated recipient hearts. When administering the PI3K-inhibitor WI and the Akt-inhibitor SH-6 at the onset of reperfusion in IPC effluent pre-ischemic (EffPre) or post-ischemic (EffRep and EffPost) treated hearts, the cardioprotective effect of the effluent was completely abolished. Ctr control; EffPre 10 min effluent administration prior to RI; EffRep 10 min effluent administration after RI; EffPost effluent administration 3× 30 s at start of reperfusion; WI 10 min Wortmannin (1 μM) at reperfusion; SH-6 10 min SH-6 (10 μM) at reperfusion. Bars represent mean ± SEM. N ≥ 6 in each group. *P < 0.05 versus Ctr
Mentions: Hausenloy et al. [9] have previously shown that Akt phosphorylation at reperfusion is essential for IPC-induced protection, since inhibition of this kinase during early reperfusion abrogated the IPC-mediated reduction in infarct size. Therefore, to elucidate whether IPC effluent exerts its cardioprotective effect through an PI3K/Akt-dependent signaling pathway activated at reperfusion, hearts were administered fresh IPC effluent for 10 min either prior to index ischemia (EffPre) or at reperfusion (EffRep or EffPost), and co-administered the PI3K-inhibitor WI or the Akt-inhibitor SH-6 for 10 min at reperfusion (see experimental protocol Fig. 1). The protective effect of IPC effluent was completely abolished by co-administering WI or SH-6 at reperfusion compared to the EffPre group (EffPre + WI: 51 ± 7%, EffPre + SH-6: 60 ± 4% vs. EffPre: 24 ± 6%, P < 0.05; Fig. 5) and the EffRep group (EffRep + WI: 56 ± 12%, EffRep + SH-6: 54 ± 8% vs. EffRep: 25 ± 4%, P < 0.05; Fig. 5) and EffPost group (EffPost + WI: 50 ± 11% vs. EffPost; 35 ± 3%, P < 0.05) (data not shown). SH-6 is a phosphatidylinositol (PI) analog that inhibits Akt without affecting the activity of its upstream kinase PDK-1 [16], and has also been shown to prevent phosphorylation of Akt at Ser473 [4, 32]. The inhibitors themselves had no effect on infarct size (WI: 54 ± 8%, SH-6: 52 ± 3% vs. Ctr: 54 ± 5%, ns). Furthermore, both WI and SH-6 significantly reduced Akt phosphorylation in IPC effluent-treated hearts (EffRep + WI: 0.7 ± 0.1 AU, EffRep + SH-6: 2.8 ± 0.2 AU vs. EffRep: 7.2 ± 0.2 AU, P < 0.05; Fig. 4c, d).Fig. 5

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