PMC

(A) Schematic diagram of experimental protocols. Effect of hypoxia–reoxygenation, in the presence or absence of isoflurane (ISO), on cell survival measured by lactate dehydrogenase (LDH) activity in co-culture (CM+EC). (B) Effect of the addition of N^G-nitro-l-arginine methyl ester (l-NAME) at different time points during the experimental protocol.

Histograms depicting nitric oxide (NO) production in endothelial cells (EC), cardiomyocytes (CM) and co-cultures (EC and CM cultured together; CC) in the presence (ISO) or absence (CON) of isoflurane during hypoxia (panel A) and reoxygenation (panel B). NO-levels in pmol per mg protein, expressed as % decrease or increase from baseline. Data are mean ± SEM (n = 6/group); *P<0.05 vs. CON EC (hypoxia), **P<0.05 vs. ISO EC (hypoxia), ^†P<0.05 vs. CON co-culture (hypoxia), ^‡P<0.05 vs. ISO EC (hypoxia), ^§P<0.05 vs. CON EC (reoxygenation), ^#P<0.05 vs. ISO EC (reoxygenation).

(A) Representative Western blot illustrating temporal expression of hypoxiainducible factor (HIF1α) in isoflurane treated endothelial cells (EC). HIF1α protein amount is expressed as fold change of control. (B) Expression of HIF1α in cardiomyocytes (CM) and EC treated with isoflurane (ISO). HIF1α protein amount is expressed as fold change to respective untreated control (CON). Data are mean ± SEM (n = 6/group); *P<0.05 vs. baseline, 30 or 120 min of ISO exposure, ^†P<0.05 vs. CON EC.

(A) Histograms depicting lactate dehydrogenase (LDH) activity in cardiomyocytes (CM) and endothelial cells (EC) undergoing hypoxia–reoxygenation (H/R) or normoxia in the presence (H/R + ISO) or absence (H/R) of isoflurane. Panel (B) summarizes the effects of H/R in the presence (ISO) or absence (CON) of isoflurane on co-culture (CM + EC) with or without the addition of NG-nitro-l-arginine methyl ester (l-NAME). LDH activity is expressed as fold change over the respective normoxic-group. Data are mean ± SEM (n = 8/group); *P<0.05 vs. respective normoxia, ^†P<0.05 vs. CON, ^‡P<0.05 vs. ISO.

(A) Immunofluorescence depicting expression and subcellular localization of hypoxia-inducible factor (HIF1α) in endothelial cells (EC) in the presence and absence of isoflurane and pretreated with the Mitogen-activated Protein/Extracellular Signal-regulated Kinase inhibitor UO126 (green = HIF1α, blue = DAPI nuclear staining). (B) Representative Western blot studies showing cytosolic and nuclear HIF1α protein expression in EC with and without isoflurane (ISO) and in the presence of UO126 (ISO + UO126). HIF1α protein amount is expressed as fold change of respective untreated control (CON). Data are mean ± SEM (n = 6/group); *P<0.05 vs. cytosolic CON, **P<0.05 vs. cytosolic ISO, ^§P<0.05 vs. nuclear CON, ^†P<0.05 vs. nuclear ISO.

(A) Photoexcitation-generated oxidative stress induces mitochondrial permeability transition pore (mPTP) opening as observed by the rapid dissipation of tetramethylrhodamine ethyl ester (TMRE) fluorescence in isoflurane (ISO) stimulated cardiomyocytes (CM + ISO) and co-culture (EC and CM cultured together; CC + ISO). (B) The time by which TMRE fluorescence intensity decreased by half between initial and residual fluorescence intensity was considered as the “arbitrary mPTP opening time” (arrows) and was compared among experimental groups (a.u. = arbitrary units). Panel (C) shows a histogram representing the arbitrary mPTP opening times. CysA = cyclosporine A (n = 8/group). Data are mean ± SEM; *P<0.05 vs. CM, ^#P<0.05 vs. CM + ISO, ^§P<0.05 vs. CC.

Panel (A) representative Western blot depicting HIF1α protein in uninfected EC (CON), EC with lentiviral vector carrying a scrambled sequence (SCR) and EC infected with lentivirus containing shRNA for HIF1α. HIF1α immunoreactive bands are expressed as fold change over CON (n=6/group). Panel (B) summarizes lactate dehydrogenase (LDH) activity in co-cultures (CM + EC) with uninfected endothelial cells (EC, control), EC infected with lentivirus expressing scrambled shRNA (scrambled shRNA) and lentiviral infection of EC with shRNA for hypoxia-inducible factor (HIF1α shRNA). Co-cultures were subjected to hypoxia and reoxygenation (H/R) in the presence or absence of isoflurane (ISO). LDH activity is expressed as fold change over the normoxic-group (n = 6/group). Panel (C) representative Western blots showing eNOS and phosphorylated eNOS expression in uninfected EC, EC with lentiviral vector carrying a scrambled sequence (SCR), EC in the presence of N^G-nitro-l-arginine methyl ester (l-NAME) and EC infected with lentivirus containing shRNA for HIF1α (shRNA), subjected to H/R in the presence or absence of ISO (n = 6/group). Data are mean ± SEM; *P<0.05 vs. normoxia, ^†P<0.05 vs. H/R (control), ^‡P<0.05 vs. H/R (scrambled shRNA), ^§P<0.05 vs. H/R + ISO (scrambled shRNA), ^#P<0.05 vs. CON, ^¶P<0.05 vs. SCR, ^&P<0.05 vs. respective control (CON), ^$P<0.05 vs. EC CON, ^¢P<0.05 vs. EC (SCR) CON, ^¥P<0.05 vs. EC ISO, ^£P<0.05 vs. EC (SCR) ISO.