Cardioprotective mechanisms of PPAR gamma in diabetic heart
Type of Degreedissertation
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Patients with diabetes exhibit significantly altered renin-angiotensin system (RAS). Recently it has been determined that hyperglycemic conditions induce an increase in angiotensin II (AT II) expression; specifically in cardiomyocytes. Altered RAS has been shown to be associated with an increase in oxidative stress and cardiac dysfunction leading to the development of cardiac hypertrophy. The transient outward potassium current (Ito) in cardiac myocytes is mainly mediated by members of the Kv subfamily of voltage gated potassium channels and has been shown to be altered in cellular localization and expression during the development of cardiac hypertrophy. However it is not clear as to how AT II affects the pore forming complex at the cell membrane and thus directly affects the Ito current. In the current study, we explored the protective effect of PPAR gamma ligands on cardiomyocyte Ito by preventing NADPH Oxidase activation and the ensuing ROS formation. Furthermore, short term PPAR gamma activation in diabetic leptin deficient db/db mice displayed improvements in the membrane association of the molecular components of Ito as well as prolonged QT interval. These findings demonstrate that PPAR gamma agonist have the potential to attenuate cardiomyocyte dysfunction associated with diabetes. Even though we and others have demonstrated that the use of PPAR gamma agonists exerts a profound protection against diabetes mediated cardiovascular pathologies, the use of these drugs in a clinical setting is restricted because of its detrimental side effects with long term use. Therefore, development of new therapeutic agents which can modulate the PPAR gamma activity without the adverse effects can be beneficial to mitigate diabetes induced cardiovascular diseases. The objective of the second study reported in this dissertation is to develop novel dual PPAR gamma/delta agonists without the deleterious side effects associated with full PPAR gamma agonists. Docking simulations of 23 novel compounds within the ligand binding domain of PPAR gamma/ delta were performed using AutoDock Vina which consistently reproduced experimental binding poses from known PPAR agonists. Comparisons were made and described with other docking programs AutoDock and Surflex-Dock (from SYBYL-X). Biological evaluation of compounds was accomplished by transcriptional promoter activity assays, quantitative PCR gene analysis for known PPAR gamma/ delta targets as well as in vitro assays for lipid accumulation and mitochondrial biogenesis verses known PPAR agonists. We found one (compound 9) out of the 23 compounds evaluated, to be the most potent and selective dual PPAR gamma/ delta agonist which did not display the deleterious side effects associated with full PPAR gamma agonists. Additionally, we have observed that PPAR gamma agonist treatment can offer a protection against I/R injury in the diabetic heart/ The major form of fatality due to diabetes is development of congestive heart failure triggered by myocardial infarction (MI). The impaired insulin signaling in the diabetic heart leads to myocardial energy dysregulation that compromises the cardioprotective mechanism against ischemic injury. We observed that db/db mice (leptin deficient, type 2 diabetic mice) have increased infarction size compared to wild type mice after ischemia/reperfusion (I/R) injury by TTC stain. We also found that activity of Hypoxia inducible factor-1 (HIF-1 alpha), a master transcription factor involved in the cardioprotective response to ischemia, is impaired in db/db hearts. HIF-1 alpha is known to transcriptionally regulate genes involved in myocardial energetics. We recently found that HIF-1 alpha transcriptionally regulates the mitochondrial protein frataxin in cardiomyocytes as determined by luciferase assays. In vitro studies indicate that hypoxic conditions increase frataxin protein expression in cardiomyocytes as determined by western analysis. Frataxin plays an important role in the Fe-S cluster biogenesis required to maintain the activity of aconitase, succinate dehydrogenase and complexes in the mitochondria. Interestingly, we observed decreased expression of frataxin in the ischemic diabetic heart. Therefore, we postulate that attenuated HIF-1 alpha –frataxin signaling in ischemic db/db hearts leads to abnormally enlarged infarction size in response to I/R. The decline in HIF-1 alpha activity in response to hypoxia was further validated in cardiomyocytes cultured in high glucose media. The significance of frataxin against hypoxic injury was confirmed by utilizing over-expressed frataxin cardiomyocytes via MTT, ATP and aconitase activity assays. Currently we are attempting to identify the hypoxia response element (HRE) in frataxin promoter to further validate the transcriptional activity of HIF-1 alpha. In addition, we are completing the I/R surgeries on HIF-1 alpha KO mice to address the cardioprotective nature of HIF-1 alpha-frataxin signaling against MI.