Abstract
Type 2 diabetes is a metabolic disorder characterised by high glucose levels and insulin resistance. Molecular alterations in the diabetic heart have been shown to accelerate apoptotic cell death. MicroRNAs (miRs) are a class of non-coding RNAs that can regulate physiological and pathological processes by controlling the expression of genes. In doing this, miRs have been demonstrated to affect multiple pathological pathways in the diabetic heart. MiR-532 was demonstrated to regulate apoptotic cell death in cardiomyocytes by targeting the apoptosis repressor with caspase recruitment domain (ARC). Additionally, other studies have demonstrated an association between ARC and the casein kinase 2 alpha 1 (CK2A1) subunit; thereby indicating a possible association between CK2A1 and miR-532.
Preliminary analysis indicated a significant upregulation in miR-532 in high glucose-induced mouse atrial cardiomyocytes and human diabetic right atrial appendage (RAA) samples. Therefore, my project aimed to investigate the effect of therapeutic inhibition of miR532 in high glucose-treated human ventricular cardiomyocytes (AC-16 cells) and in ex vivo cultured human RAA tissues. The first objective of my project was to optimise the culture and transfection protocol for the AC-16 cell line. The expression of miR-532 was significantly upregulated in AC-16 cardiomyocytes following 48 hours of high glucose exposure. This increase was associated with a significant decrease in the expression of target proteins ARC and CK2A1. Therapeutic knockdown of miR-532 under high glucose conditions restored the expression of target proteins and significantly reduced high glucose-induced cardiomyocyte apoptosis. The second objective of this project was to optimise the ex vivo culture and transfection protocol for RAA tissues. To my knowledge, this is the first time that human RAA tissues have been cultured ex vivo tested for their transfection efficiency. Caspase assays confirmed the sustained viability of ex vivo cultured tissues for at least 96 hours in culture. However, while the therapeutic knockdown of miR-532 in the ex vivo cultured tissues was successful, miR-532 failed to replicate the beneficial effects on apoptosis and target protein expression that was observed in vitro cardiomyocyte culture. Being a novel protocol, further optimisation is required for the miR transfection of the ex vivo cultured tissues.
Collectively my data demonstrates miR-532 plays a key role in high glucose-induced apoptosis in cardiomyocytes by regulating the anti-apoptotic ARC and casein kinase 2 catalytic subunit CK2A1. Additionally, this project was able to provide preliminary data on the novel model for the ex vivo culture and transfection of human RAA tissues.