Defense Date
2024
Document Type
Thesis
Degree Name
Master of Science
Department
Physiology and Biophysics
First Advisor
Anindita Das, PhD
Abstract
Background: Cardiovascular disease (CVD) is the leading cause of death worldwide. The risk of developing CVD is increased two-to-four-fold in patients with diabetes, and, following a myocardial infarction, patients with diabetes often have worse outcomes. Dysregulation of individual or clusters of microRNAs is linked to the pathogenesis of heart diseases and its risk factors, such as diabetes, hypertension, atherosclerosis, myocardial ischemia/reperfusion (I/R) injury, and heart failure. Our study identified that the microRNA-17-92 cluster (miR-17-92) is suppressed in diabetic hearts with CVD. The purpose of the present study is to define how miR-17-92 attenuates injurious signaling in the post ischemic heart under diabetic condition and identify targets of the cluster to find potential therapeutic modalities.
Methods: AC16 human cardiomyocytes were transfected with miR-20a-mimic (one of the seven miRs encoded by the miR-17-92 cluster) and incubated with high glucose (25 mM). After 24 hours, the cells were subjected to 4 hours of simulated ischemia (SI) and 24 hours of reoxygenation (RO) with high glucose. Expression level of miR-20a-mimic and target proteins was assessed by Real-Time PCR and Western blot. Cell death was measured by Trypan blue staining. Cardiomyocytes were isolated from cardiac-specific tamoxifen-inducible miR-17-92 knock-in (KI) and knock-out (KO) mice and incubated with high glucose (25 mM). After one hour, the cells were subjected to SI for 40 minutes followed by a RO period of one hour with high glucose. Expression of miR-20a was assessed by Real-Time PCR. Cell death was measured by trypan blue staining and apoptosis was measured by TUNEL assay.
Results: Overexpression of miR-20 protects AC-16 against SI/RO injury under normal and hyperglycemic conditions. miR-20 reduced sphingolipid biosynthesis enzymes, ceramide synthase 6 (CerS6) and sphingomyelin synthase 2 (SMS2) as well as phosphorylation of YAP. Post SI/RO, cell death and apoptosis are higher in cardiomyocytes from miR-17-92 KO mice compared to wild type mice, while cell death is suppressed in cardiomyocytes from miR-17-92 KI mice under hyperglycemic conditions.
Conclusion: The study demonstrated that overexpression of miR-17-92 protects against ischemia/ reperfusion (I/R) injury by suppressing biosynthesis of sphingolipids, via targeting CerS6 and SMS2 in cardiomyocytes, as well as by targeting YAP, the central modulator of the Hippo-YAP pathway. These pathways promote production of molecules that majorly contribute to lipotoxicity in the cell and stimulate pro-apoptotic cascades and inflammatory responses. These findings provide a promising direction for modulation of I/R injury and potential therapeutic intervention for diabetic patients with myocardial infarction.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
8-8-2024