Figure 1.
OA treatment attenuates oxidative stress in H9c2 cells.
Quantification of oxidative stress (DCFDA staining) in H9c2 cells in response to simulated chronic hyperglycemia (33 mM glucose) vs. control (5.5 mM glucose) ± treatment with 20 µM or 50 µM OA for 6 and 24 hr, respectively. (A) and (C) Fluorescence microscopy; (B) and (D) Flow cytometry. Values are expressed as mean ± SEM (n = 9). *p<0.05, **p<0.01, ***p<0.001 vs. respective controls.
Figure 2.
Decreased apoptotic cell death in H9c2 cells treated with OA (caspase activity).
Evaluation of caspase activity in H9c2 cells in response to simulated chronic hyperglycemia vs. control ± treatment with 20 µM and 50 µM OA, respectively, for 24 hr. Values are expressed as mean ± SEM (n = 9). ***p<0.001 vs. respective controls.
Figure 3.
Diminished apoptosis in OA-treated H9c2 cells (flow cytometry).
Flow cytometric analysis using the Annexin V/FITC apoptosis assay kit to evaluate the effects of 100 µM OA treatment under control and high glucose culturing conditions (24 hr). (A), (B), (C) and (D) Representative FACS analyses of four individual experiments corresponding to control and high glucose ± OA treatment, respectively. (E) and (F) Quantification of OA treatment after 6 and 24 hr, respectively. Values are normalized to the control and expressed as mean ± SEM (n = 4). ***p<0.001 vs. controls and ##, ### p<0.01, p<0.001 vs. high glucose exposure without OA treatment.
Figure 4.
OA treatment does not affect pre-ischemic cardiac function.
Isolated rat hearts were perfused under simulated hyperglycemic conditions (33 mM glucose) vs. controls (11 mM glucose) ±100 µM OA treatment. We initially perfused for 60 min, whereafter OA was added for a further 20 min. Subsequently, the buffer initially used was returned and hearts perfused for an additional 20 min. (A) Left ventricular developed pressure at baseline (11 mM) and (B) high glucose conditions (33 mM). Rate pressure product (RPP) at baseline (C) and (D) high glucose levels. Values are expressed as mean ± SEM (n = 9).
Figure 5.
OA treatment blunts high glucose-induced cardiac dysfunction following ischemia and reperfusion.
Isolated rat hearts were perfused under simulated hyperglycemic conditions (33 mM glucose) vs. controls (11 mM glucose) and subjected to 20 min of global ischemia, followed by 60 min of reperfusion. For OA treatments groups, 100 µM OA was added during the first 20 min of reperfusion. (A) Left ventricular developed pressure (% recovery) at baseline glucose levels (11 mM), and (B) with high glucose (33 mM). Rate pressure product (RPP) at baseline glucose levels (C), and (D) under high glucose conditions. Values are expressed as mean ± SEM (n = 9).*p<0.05, ***p<0.001 vs. respective controls.
Figure 6.
OA administration decreases infarct size under high glucose perfusion conditions.
Isolated rat hearts were perfused under high glucose conditions vs. controls and subjected to regional ischemia. For OA treated groups, 100 µM OA was added during the first 20 min of the two hr reperfusion period. Evans blue dye and TTC staining enabled visualization of viable tissue (blue), infarcted area (white) and the area at risk (red). Values are expressed as mean ± SEM (n = 6). *p<0.05, ***p<0.001 vs. respective controls.
Figure 7.
OA treatment decreases infarct size following coronary artery ligation in streptozotocin-diabetic rats.
Wistar rats were injected with STZ and followed for a 1-week period. Subsequently, 0.45 mg/kg OA was injected via the penile vein within the first two min of reperfusion. Evans blue dye and TTC staining enabled visualization of viable tissue (blue), infarcted area (white) and the area at risk (red). Values are expressed as mean ± SEM (n = 6). *p<0.05, **p<0.01 vs. respective controls.
Table 1.
Body weight and blood glucose levels after 1 week of STZ injection.
Table 2.
Effects of OA on in vivo heart rate, ST height, systolic and diastolic blood pressures during early reperfusion.
Figure 8.
Long-term OA treatment improves cardiac function in STZ-diabetic rats.
Sprague-Dawley rats were injected with STZ and followed for a 2-week period ± daily OA treatment. Subsequently, isolated hearts from STZ-diabetic and matched controls were perfused and action potentials recorded via a force transducer. Values are expressed as mean ± SEM (n = 6). **p<0.05 vs. non-diabetic control and ***p<0.01 vs. respective controls.
Figure 9.
OA treatment does not affect pre-ischemic superoxide dismutase and caspase activities.
Isolated rat hearts were perfused under simulated hyperglycemic conditions (33 mM glucose) vs. controls (11 mM glucose) ±100 µM OA treatment. We initially perfused for 60 min, whereafter OA was added for a further 20 min. Subsequently, the buffer initially used was returned and hearts perfused for an additional 20 min. (A) Superoxide dismutase activity (% inhibition) in response to high glucose vs. control ± OA treatment; (B) Caspase activity. Values are expressed as mean ± SEM (n = 6).
Figure 10.
Anti-oxidant effects of OA in hearts subjected to ischemia-reperfusion under high glucose perfusion conditions.
Isolated rat hearts were perfused under high glucose conditions vs. controls and subjected to ischemia-reperfusion. For OA treatments groups, 100 µM OA was added during the first 20 min of reperfusion. (A) Superoxide levels under high glucose conditions vs. control ± OA treatment; (B) Superoxide dismutase activity (% inhibition) in response to high glucose vs. control ± OA treatment; (C) Time course for SOD activity following ischemic insult. Values are expressed as mean ± SEM (n = 9). *p<0.05, **p<0.01, ***p<0.001 vs. respective controls.
Figure 11.
OA treatment decreases carbonylation levels in hearts subjected to ischemia-reperfusion under high glucose conditions.
Isolated rat hearts were perfused under high glucose conditions vs. controls and subjected to ischemia-reperfusion. For OA treatments groups, 100 µM OA was added during the first 20 min of reperfusion. (A) Degree of carbonylation under high glucose conditions vs. control; (B) OA treatment at baseline (11 mM glucose) and (C) under high glucose conditions (33 mM). Values are expressed as mean ± SEM (n = 9). ***p<0.001 vs. respective controls.
Figure 12.
Anti-apoptotic effects of OA in hearts subjected to ischemia-reperfusion under high glucose conditions.
Isolated rat hearts were perfused under high glucose conditions vs. controls and subjected to ischemia-reperfusion. For OA treatments groups, 100 µM OA was added during the first 20 min of reperfusion. (A) Caspase activity; (B) p-BAD/BAD peptide levels; (C) Caspase-3 peptide levels; (D) Time course of myocardial apoptosis following the ischemic insult. Values are expressed as mean ± SEM (n = 9). *p<0.05, **p<0.01, ***p<0.001 vs. respective controls.
Figure 13.
OA treatment attenuates O-GlcNAcylation in hearts subjected to ischemia-reperfusion under high glucose conditions.
Isolated rat hearts were perfused under high glucose conditions vs. controls and subjected to ischemia-reperfusion ± OA treatment during reperfusion. Western blot analysis for overall O-GlcNAcylation is shown with β–actin as loading control. Densitometric analysis for O-GlcNAcylation is displayed below gel image (normalized to corresponding β–actin values). Values are expressed as mean ± SEM (n = 6). *p<0.05, **p<0.01 vs. respective controls.
Figure 14.
Increased proteasomal activity in hearts subjected to ischemia-reperfusion under high glucose conditions.
Isolated rat hearts were perfused under high glucose conditions vs. controls and subjected to ischemia-reperfusion. A) Trypsin-like proteasomal, (B) chymotrypsin-like, and (C) caspase-like activities after 60 min of reperfusion under high glucose conditions (33 mM) vs. control (11 mM). Values are expressed as mean ± SEM (n = 9). *p<0.05, **p<0.01 vs. respective controls.
Figure 15.
OA attenuates high glucose-induced proteasomal activity following ischemia-reperfusion.
Isolated rat hearts were perfused under high glucose conditions vs. controls and subjected to ischemia-reperfusion. For OA treatments groups, 100 µM OA was added during the first 20 min of reperfusion. (A) Trypsin-like proteasomal, (B) chymotrypsin-like, and (C) caspase-like activities after 60 min of reperfusion at baseline (11 mM glucose). (D) Trypsin-like proteasomal, (E) chymotrypsin-like, and (F) caspase-like activities after 60 min of reperfusion under high glucose conditions (33 mM glucose). Values are expressed as mean ± SEM (n = 9). *p<0.05 vs. respective controls.