Therapeutic modulation of miRNA-320a to prevent diabetic cardiomyopathy
Recent findings reveal that more than 70% of diabetic individuals are prone to developing some form of heart disease during their life span. Population-based studies have shown that the risk of heart failure is augmented 2 to 3 fold by diabetes signifying that diabetes itself is an independent risk factor for cardiovascular disease. The occurrence of diabetes substantially accelerates the development of heart failure in patients with myocardial infarction, hypertension, or atrial fibrillation. The impact of diabetes on the development of vascular disease has been established earlier whereas, non-ischemic heart failure associated with diabetes termed as diabetic cardiomyopathy has received less attention compared to coronary and cerebrovascular events. Diabetic cardiomyopathy is a chronic complication in individuals with diabetes which is characterized by ventricular dilation and hypertrophy, systolic and diastolic dysfunction, eventually resulting in heart failure. Despite being well characterized, the fundamental mechanisms leading to diabetic cardiomyopathy are still elusive. Recent studies identified the involvement of small non-coding small RNA molecules such as microRNAs (miRNAs) playing a key role in the etiology of diabetic cardiomyopathy. Therefore, miRNAs associated with diabetic cardiomyopathy represents a new class of targets for the development of mechanistic therapeutics, which may yield marked benefits compared to other therapeutic approaches. Indeed, a few miRNAs are currently under active clinical investigation, with many expressing cautious optimism that miRNAs based therapies will succeed in the coming years. MiRNA plays its role by regulating cellular and metabolic processes by modulating specific targets. Hence, the selection of miRNA candidates is a major challenge to obtain the desired therapeutic effect by its modulation. In this thesis, I focused specifically on miRNA-320a which projects a distinct effect on apoptosis in diabetic conditions by targeting the insulin growth factor-1 gene. Previous studies demonstrated that insulin growth factor-1 plays a major role in the apoptotic signaling pathway which allows me to select this miRNA candidate for my modulation study. To date, no study has been specifically designed to validate the onset of miRNA-320a expression and correlation with cardiac dysfunction in diabetic conditions. Therefore, the overarching aim of this thesis was to investigate the expression of miRNA-320a in diabetic human hearts, type-2 diabetic db/db mouse heart, and high glucose-cultured human ventricular cardiomyocytes, along with the effectiveness of miRNA-320a modulation by CRISPR/Cas9 and Locked Nucleic Acid-anti-miRNA in high glucose-cultured human ventricular cardiomyocytes and type-2 diabetic db/db mouse heart respectively. We investigated the expression of miRNA-320a in human right atrial appendage tissue collected from non-diabetic with no ischemic heart disease, non-diabetic with ischemic heart disease, and diabetic with ischemic heart disease patients and evaluated the expression of target protein insulin growth factor-1 and apoptotic markers like B-cell lymphoma 2 and cleaved caspase-3. By subjecting diabetic vs non-diabetic mice age-matched from 8 weeks up to 32 weeks, we investigated the onset of miRNA-320a expression and target protein with apoptotic markers. Eventually, we extended my study to assess the expression of miRNA-320a, target protein, and level of apoptosis in high and normal glucose-cultured human ventricular cardiomyocytes. In the next step, we investigate the effect of cell survival after the knockdown of miRNA-320a genomic DNA by CRISPR/Cas9. Eventually, we evaluated the cell survival in the whole heart of type-2 diabetic db/db mouse after the miRNA-320a knockdown by Locked Nucleic Acid-anti-miRNA. Finally, the cardiac functional improvement was measured after miRNA-320a knockdown in type-2 diabetic db/db mice. In conclusion, this thesis provides evidence that miRNA-320a is a late responding miRNA that has no effect on initiating apoptosis and cardiac dysfunction in the diabetic heart. Though it may not initiate apoptosis and cardiac dysfunction, but we found this miRNA aggravates the process of apoptosis and cardiac dysfunction. Whereas, effective modulation of miRNA-320a can prevent cell death and partially improve cardiac dysfunction in the diabetic heart.
Advisor: Katare, Rajesh; Chatterjee, Aniruddha
Degree Name: Doctor of Philosophy
Degree Discipline: Physiology
Publisher: University of Otago
Keywords: New Zealand; Department; Nilanjan; microRNA-320
Research Type: Thesis