Investigating links between factors involved in adverse cardiac remodelling and expression of the therapeutic target Runx1

Abstract

Cardiovascular disease (CVD) is a major cause of morbidity and mortality, responsible for approximately 26 million people suffering worldwide and costing the healthcare system heavily. CVD includes hypertension, congestive heart failure, and ischaemic heart disease and involves exertion of continuous stress on the myocardium to meet physiological demands of body tissues. It gradually drives structural and functional changes in the myocardium, making it less viable and susceptible to inflammation (adverse cardiac remodelling). Adverse cardiac remodelling occurs in response to various mechanical, neurohormonal, genetic factors and physiological stress ultimately impairing cardiac contractile function. Current therapeutic interventions used to prevent or reverse adverse cardiac remodelling remain inadequate. More research is needed to identify novel therapeutic targets and understand the pathophysiological mechanisms behind adverse cardiac remodelling. This thesis investigates the role and modulation of the runt-related transcription factor-1 (RUNX1) in the pathophysiology of CVD and adverse cardiac remodelling. RUNX1 belongs to the RUNX family transcription factor and is linked to CVD and cardiac remodelling. This gene has been intensively studied and linked to cancer and haematological disorders, but its role in CVD is less well known, making it an ideal research candidate. Several studies demonstrate increased expression of RUNX1 in cardiovascular pathology, including chronic dilated cardiomyopathy, myocardial infarction, diabetic cardiomyopathy, and pressure overload. This research aimed to investigate the extent to which factors involved in adverse cardiac remodelling (in particular Angiotensin and TGFβ) are responsible for changing expression of RUNX1 and its other isoforms (RUNX2 and RUNX3). Neonatal and adult rat cardiomyocytes (NRCMs and ARCMs, respectively) were utilised to study the effect of factors that modulate adverse cardiac remodelling on expression of RUNX1. NRCMs were isolated from rat pups immediately after birth (1-5 day), while ARCMs were harvested from adult rats at three months of age. Following treatment with Angiotensin II (Ang II) at 200, 500 and 1000 nM, the Runx1 mRNA level increased in cardiomyocytes after 24 h in NRCMs, whereas no increase was detected in ARCMs. On the contrary, no changes were detected for Runx2 and Runx3 mRNA expression. ANP and BNP (markers of cardiac hypertrophy) also increased following administration of Ang II at 500nM in NRCMs. Confocal ii

microscopy was used to confirm the NRCM hypertrophy, and Ang II increased the size of the cytoskeleton at 200 and 500 nM. Separate in vivo experiments in mice, showed a significant increase in both Runx1 and Runx2, but not Runx3 in response to Ang II. These findings suggest a novel link between Ang II and Runx1 both in vitro and in vivo. Another factor discovered to regulate RUNX1 was transforming growth factor beta 1 (TGFβ1). RUNX1 mRNA and protein expression were upregulated in both NRCMs and ARCMs. Runx1 expression was normalised by pre-incubating the cardiomyocytes with an inhibitor of the ALK5 pathway of TGFβ1, suggesting the involvement of Smad2/3 signalling in upregulation of Runx1. These findings suggest a mechanistic and potentially therapeutic inhibitor of RUNX1. Experimental approaches were also used to target Runx1-mediated cardiac remodelling using either small interfering RNA (siRNA) to downregulate Runx1 expression or the pharmacological small molecule Ro5-3335 to inhibit RUNX1 activity. A reduction of Runx1 mRNA expression using siRunx1 revealed a positive correlation between a selection of genes known to be modulated by adverse cardiac remodelling. Correlation analysis of in vitro and in vivo experiments demonstrates that Runx1 is positively correlated with Runx2 and Runx3 and may also be positively or negatively correlated with Yap1 in both NRCMs and adult LV tissue. However, no significant effect of Runx1 knockdown was observed on proliferation of NRCMs. Collectively, these findings provide new insight linking factors involved in adverse cardiac remodelling with expression of Runx1 emphasising the importance of Runx1 in the progression of CVD.