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Results: 12

1.
Figure 4

Figure 4. EET blocks fragmentation of nuclei by HR. From: Multiple anti-apoptotic targets of the PI3K-Akt survival pathway are activated by epoxyeicosatrienoic acids to protect cardiomyocytes from hypoxia/anoxia.

HL-1 cells (a) and neonatal myocytes (b) were subjected to HR in presence of vehicle or 14-15-EET (1 μM). Nuclei were stained with Hoechst reagent and imaged. There is visible condensation of chromatin (marked with arrows) in serum deprived cells that is absent in control cells and reduced in cells pretreated with EET after HR.

Anuradha Dhanasekaran, et al. Am J Physiol Heart Circ Physiol. ;294(2):H724-H735.
2.
Figure 2

Figure 2. EETs increase cell viability after HR. From: Multiple anti-apoptotic targets of the PI3K-Akt survival pathway are activated by epoxyeicosatrienoic acids to protect cardiomyocytes from hypoxia/anoxia.

HL-1 cells (a) and neonatal myocytes (b) were subjected to HR in presence of vehicle or EET. Cell viability was determined using the MTT assay (see Methods) and normalized to ‘percent control’. Bar graph showing the mean ± sem of percent decrease of viable cells over control (100%). * denotes p<0.01 for analyses compared to HR and # denotes p<0.01 as compared to control.

Anuradha Dhanasekaran, et al. Am J Physiol Heart Circ Physiol. ;294(2):H724-H735.
3.
Figure 10

Figure 10. EETs decrease caspase-9 activity. From: Multiple anti-apoptotic targets of the PI3K-Akt survival pathway are activated by epoxyeicosatrienoic acids to protect cardiomyocytes from hypoxia/anoxia.

HL-1 cells (a) and neonatal myocytes (b) were subjected to HR after pretreatment with vehicle or the 3 regioisomers of EET. Caspase-9 activity was measured as described in the ‘Materials and Methods’. * indicates a significant difference (p<0.05) compared to control (8h); # indicates a significant difference (p<0.05) compared to control (16h); & indicates a significant difference (p<0.05) compared to HR (8h); $ indicates a significant difference (p<0.05) compared to HR (16h). Controls were treated under normoxic conditions. Both cell types showed increased caspase-9 activity after HR, which was attenuated by 8,9-, 11,12-, or 14,15-EET by 16 h.

Anuradha Dhanasekaran, et al. Am J Physiol Heart Circ Physiol. ;294(2):H724-H735.
4.

Figure 8. EET increases intracellular p-Akt. From: Multiple anti-apoptotic targets of the PI3K-Akt survival pathway are activated by epoxyeicosatrienoic acids to protect cardiomyocytes from hypoxia/anoxia.

HL-1 cells (a) and neonatal myocytes (b) were subjected to HR after pretreatment with vehicle or 14,15 EET. Cells were treated as described in ‘Materials and Methods’ and stained with p-AKT antibody. Nuclei were counterstained with DAPI and the cells were viewed under fluorescent microscopy. Images were captured and overlaid as shown. Figures (c) and (d) represent the quantitation of green fluorescence, which indicates the increase in p-Akt levels. * p< 0.05 as compared to control and # p<0.05 as compared to HR.

Anuradha Dhanasekaran, et al. Am J Physiol Heart Circ Physiol. ;294(2):H724-H735.
5.
Figure 5

Figure 5. EETs decrease caspase-3 activity. From: Multiple anti-apoptotic targets of the PI3K-Akt survival pathway are activated by epoxyeicosatrienoic acids to protect cardiomyocytes from hypoxia/anoxia.

HL-1 cells (a) and neonatal myocytes (b) were subjected to HR after pretreatment with vehicle (marked HR) or one of the 3 regioisomers of EET as labeled. Caspase-3 activity was measured as described in the Materials and Methods. * indicates a significant difference (p<0.05) compared to control (8h); # indicates a significant difference (p<0.05) compared to control (16h); & indicates a significant difference (p<0.05) compared to HR (8h); $ indicates a significant difference (p<0.05) compared to HR (16h). Controls were treated under normoxic conditions. Both cell types showed increased caspase-3 activity after HR which was attenuated by 1 μM of 8,9-, 11,12-, or 14,15-EET.

Anuradha Dhanasekaran, et al. Am J Physiol Heart Circ Physiol. ;294(2):H724-H735.
6.
Figure 11

Figure 11. EET protects contractility of primary neonatal myocytes in culture. From: Multiple anti-apoptotic targets of the PI3K-Akt survival pathway are activated by epoxyeicosatrienoic acids to protect cardiomyocytes from hypoxia/anoxia.

Neonatal myocytes were subject to HR after pretreatment with vehicle or 14,15-EET (1 μM). The number of beating cells in a microscope field (a) and the frequency (beats/min) (b) were counted as described in Materials and Methods (a total of 3-5 batches of cells were examined for each treatment). Pretreatment with EET increased both total number and frequency of beating cells as compared to myocytes that were exposed to HR in the presence of vehicle.

Anuradha Dhanasekaran, et al. Am J Physiol Heart Circ Physiol. ;294(2):H724-H735.
7.
Figure 7

Figure 7. EETs increase PI3K activity. From: Multiple anti-apoptotic targets of the PI3K-Akt survival pathway are activated by epoxyeicosatrienoic acids to protect cardiomyocytes from hypoxia/anoxia.

HL-1 cells (a) and neonatal myocytes (b) were subjected to HR after pretreatment with vehicle or EET in the absence or presence of wortmannin (WT, 0.2-1 μM). Reoxygenation was performed for 1 hour. PI3K assays were conducted as mentioned in ‘Materials and Methods’. The values were determined by plotting the OD on the standard curve obtained from the known values (standards). * p< 0.001 as compared to control or HR-treated group.

Anuradha Dhanasekaran, et al. Am J Physiol Heart Circ Physiol. ;294(2):H724-H735.
8.
Figure 12

Figure 12. Schematic representation of a pathway for protection of cardiomyocytes by EET via the PI3K/Akt pro-survival pathway. From: Multiple anti-apoptotic targets of the PI3K-Akt survival pathway are activated by epoxyeicosatrienoic acids to protect cardiomyocytes from hypoxia/anoxia.

We have reported that pretreatment with EET increases (i) PI3K activity (ii) phosphorylation of Akt (iii) phosphorylation of BAD (iv) intracellular XIAP and (v) inhibition of activity of caspases-9. Each of these events decreases activity of caspase 3, a late effector, to protect the cardiomyocyte from apoptosis by HR. These targets are known to align in a signaling cascade as illustrated in the schematic. It should be noted that the results do not implicate absence of other pro-survival or mitotic pathways that may also be targeted by EETs.

Anuradha Dhanasekaran, et al. Am J Physiol Heart Circ Physiol. ;294(2):H724-H735.
9.
Figure 6

Figure 6. EET decreases intracellular cleaved caspase 3. From: Multiple anti-apoptotic targets of the PI3K-Akt survival pathway are activated by epoxyeicosatrienoic acids to protect cardiomyocytes from hypoxia/anoxia.

HL-1 cells (a) and neonatal myocytes (b) were subjected to HR after pretreatment with vehicle or 14,15-EET (1 μM). After treatment cells were processed as described in ‘Materials and Methods’ and treated with a caspase-3 antibody which recognizes only the cleaved caspase and not the pro-caspase. The cells were then stained with corresponding FITC-tagged secondary antibody and analyzed by fluorescent microscopy. Note increased caspase 3 activity in HR as compared to HR+14,15-EET or control (normoxia).

Anuradha Dhanasekaran, et al. Am J Physiol Heart Circ Physiol. ;294(2):H724-H735.
10.
Figure 9

Figure 9. Western analysis demonstrating effect of EETs on Akt, BAD and XIAP. From: Multiple anti-apoptotic targets of the PI3K-Akt survival pathway are activated by epoxyeicosatrienoic acids to protect cardiomyocytes from hypoxia/anoxia.

HL-1 cells and neonatal myocytes were grown and subjected to HR with or without pretreatment with EETs. The cells were lysed in the presence of phosphatase inhibitors and the lysates were developed by western analysis with antibody to (a) p–Akt and Akt; samples labeled WT were pretreated with wortmannin (0.2-1 μM) for 30 min (b) p-BAD, BAD and XIAP in neonatal myocytes (left panel) and HL-1 cells (right panel). Samples treated with EET showed increase in phosphorylation of Akt (p-Akt) and BAD (p-BAD) and higher levels of XIAP in both cell types, while Akt and BAD remain unaltered and demonstrate equal protein loading in all lanes in (a) and (b) respectively.

Anuradha Dhanasekaran, et al. Am J Physiol Heart Circ Physiol. ;294(2):H724-H735.
11.
Figure 3

Figure 3. EETs decrease annexin V binding after HR. From: Multiple anti-apoptotic targets of the PI3K-Akt survival pathway are activated by epoxyeicosatrienoic acids to protect cardiomyocytes from hypoxia/anoxia.

HL-1 cells (a-d) and neonatal myocytes (e-h) were subjected to HR in presence of vehicle or EET. Cells were incubated with binding buffer containing annexin V as described in Materials and Methods. Graphs (a-h) represent typical histograms comparing fluorescent intensity of control cells (exposed to normoxia) with those exposed to HR in the presence of vehicle (labeled HR) or EET (traced in bold lines). Note an increase in fluorescence after HR which shifts towards control in samples pretreated with EET. (I) Bar graph showing the mean ± sem of percent increase of annexin binding over control (100%). * denotes p<0.05 compared to control; ** denotes p<0.05 compared to HR-treated cells.

Anuradha Dhanasekaran, et al. Am J Physiol Heart Circ Physiol. ;294(2):H724-H735.
12.
Figure 1

Figure 1. EET stimulates phosphorylation of the prosurvival protein Akt. From: Multiple anti-apoptotic targets of the PI3K-Akt survival pathway are activated by epoxyeicosatrienoic acids to protect cardiomyocytes from hypoxia/anoxia.

(a) neonatal myocytes were exposed to increasing concentrations of 14,15-EET for 30 minutes as indicated in the figure. Detection of pAkt and Akt were carried out by western blots. Increasing concentrations of 14,15-EET up to 1 μM enhanced phosphorylation of Akt while 10 μM was less effective (upper panel figure 1a). The lower panel depicts corresponding levels of Akt in the cells (loading controls). (b) Four independent experiments were repeated as in (a) and the densitometric intensities of the pAkt and Akt bands were measured, to calculate pAkt/Akt ratios in each sample. These were normalized against the vehicle in each experiment. * denotes p<0.01 as compared to vehicle.

Anuradha Dhanasekaran, et al. Am J Physiol Heart Circ Physiol. ;294(2):H724-H735.

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