Elucidating the Role of Two Inotropic Agents in Cardiac Hypertrophy: the Ceramides and Apelins

Abstract

The steady increase in prevalence of cardiovascular diseases (CVDs) globally, necessitatesthe urgent identification of novel druggable targets to reduce the development and progression of CVD. The inotropic agents, the ceramides and apelins, represent two potential druggable targets that influence cardiovascular function. In this dissertation, we examined the impact of silencing the ceramide synthases (CERS) responsible for the production of the putative protective very long-chain (VLC) ceramides 22:0 and 24:0 (CERS2) and detrimental long-chain (LC) 16:0 ceramide (CERS5/6) on cardiomyocyte health and induced hypertrophy. In this work, we demonstrate that the VLC and LC ceramides play contrasting roles in cardiomyocyte health. The silencing of CERS2 in healthy cardiomyocytes leads to pathway changes indicative of a decline in cardiomyocyte health, while CERS2 knockdown (KD) in cardiomyocytes subjected to hypertrophic conditions displayed an exacerbated response. Interestingly, transcriptional changes in healthy cardiomyocytes with silenced CERS5/6 resulted in pathway changes suggesting improved cardiomyocyte health, while the hypertrophic response in cardiomyocytes with CERS5/6 KD appeared tempered compared to controls. These findings suggest that VLC ceramides may be protective against CVD progression, while LC ceramides may contribute to the progression of CVD. Additionally, we probed how the different components of the apelinergic system (AS) affect both healthy cardiomyocytes and cellular hypertrophy progression. Here we show that both the apelin receptor (APJ) and apelin are necessary to elicit a protective response against cardiac hypertrophy, as well as identify many hypertrophic response changes due to the addition of the various AS components. To conclude this project, we present the development of a potential technique to enable the high-throughput screening of compounds against GPCR targets, such as APJ. We discuss the utilization of virus-like particles (VLPs) overexpressing APJ and demonstrate the ability to enrich the binding of positive control apelin, in both simple and complex mixtures containing a mock screening library with over 2500 peptides. The work in this thesis provides mechanistic insight into the impact the ceramides and apelins have on the progression of hypertrophy and proposes a unique technique that can be utilized to more efficiently generate future therapeutics targeting APJ and other GPCRs.