Glycogen Synthase 3β Inhibition Modulates Mitochondrial Pathways Resulting in Improved Myocardial Oxidative Stress and Collagen Production in Swine Model of Chronic Myocardial Ischemia and Metabolic Syndrome
Brittany A Potz, Laura A Scrimgeour, Sharif A Sabe, Neel R. Sodha, Frank W Sellke
Brown University, Providence, RI
Background: Glycogen synthase kinase 3β inhibition has been found to increase microvascular density and improve myocardial blood flow in a pig model of chronic myocardial ischemia in the setting of metabolic syndrome (MetS). Glycogen synthase kinase 3 beta (GSK-3β) activation is known to promote mitochondrial dysfunction leading to cellular apoptosis by inhibition of mcl-1 activity. Accumulation of the citric acid cycle intermediate succinate has been implicated in ischemic cardiomyocyte pathology. We hypothesized that GSK-3β inhibition would have a beneficial effect on oxidative stress in a pig model of chronic myocardial ischemia in the setting of metabolic syndrome.
Methods: Pigs were fed a high fat diet for 4 weeks, then underwent placement of an ameroid constrictor to their left circumflex artery. Three weeks later animals received either: no drug (DMSO placebo: CON) or a GSK-3β inhibitor (GSK-3βI). The diets and placebo/GSK-3βI were continued for an additional 5 weeks. The pigs were then euthanized and their myocardial tissue was harvested. Oxidative Stress was analyzed via Oxyblot Analysis (Milipore Billerica, MA). Protein expression of the harvested myocardial tissue samples were analyzed via western blotting. Collagen expression was analyzed via picrosirius staining.
Results: GSK-3β inhibition was associated with decreased expression of oxidative stress in the ischemic and non ischemic myocardial tissue compared to the control group. [Figure 1A] GSK-3β inhibition was also associated with decreased collagen expression in the ischemic and non ischemic myocardial tissue compared to the control group. [Figure 1B] Protein analysis identified decreased expression of Heat Shock Factor-1 (HSF-1) in the ischemic GSK-3β inhibited myocardial tissue compared to the control, which is consistent with decreased oxidative stress. There was also a decrease in expression of p-MCL-1 (ser 159), which is the inhibited form of the anti apoptotic BCL-2 family member protein, and a non significant decrease in expression of the pro apoptotic cleaved caspase 3/caspase 3 protein ratio (p=0.09) in the ischemic GSK-3β inhibited myocardial tissue compared to the control. GSK-3β inhibition was also associated with decreased expression of succinate dehydrogenase in the ischemic myocardial tissue compared to the control Finally, GSK-3β inhibition was associated with a decrease in matrix metalloproteinase 9 (MMP-9) expression in the GSK-3β inhibited group compared to the control. [Figure 1C]
Conclusion: In the setting of MetS, inhibition of GSK-3β is associated with decreased oxidative stress and collagen formation in the ischemic and nonischemic myocardial tissue. GSK-3β may be working by inhibiting mitochondrial induced myocardial apoptosis and decreasing succinate dehydrogenase and MMP-9 expression.
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