title: Investigating the role of Osteopontin in cardiac hypertrophy
Creators: name: Asirvatham, Amrita Lucette
Publisher: name: University of Glasgow
Year: 2022

description: Cardiovascular disease (CVD) remains to be the leading cause of morbidity and mortality in the developed world. Cardiac hypertrophy is an adaptive response to pressure or volume stress, mutations of sarcomeric (or other) proteins, or loss of contractile mass from prior infarction. Hypertrophic growth accompanies many forms of heart disease, including ischemic disease, hypertension, heart failure, and valvular disease. The stroke-prone spontaneously hypertensive rat (SHRSP) is an animal model of essential hypertension and is used in research of CVD together with a normotensive reference strain Wistar-Kyoto (WKY). The SHRSP animals exhibit a significant increase in the left ventricular mass index (LVMI) compared to the WKY strain and this significant phenotypic difference is apparent at 5 weeks of age, prior to the onset of hypertension in this model. Osteopontin or secreted phosphoprotein 1 (Spp1) plays an important role in cardiac remodeling in CVDs, including coronary heart disease and heart failure. Patients and animal models with acute and chronic CVD have increased Spp1 expression in the heart predominantly within cardiomyocytes resulting in cardiac hypertrophy. The aim of this project was to determine that early overexpression of Spp1 leads to a susceptibility of cardiac phenotypes in the SHRSP strain. Multiple single nucleotide polymorphisms (SNPs) exist within the Spp1 promoter region between the SHRSP and WKY rat strains. To determine whether the overexpression of Spp1 begins during gestational development, leading to increased levels of cardiac hypertrophy and fibrosis, we used gestational day 18 rat hearts from the SHRSP and WKY strains. We aimed to quantify and compare the expression levels of Spp1 by conducting quantitative real-time polymerase chain reaction (qRTPCR). We found a significant increase in Spp1 expression during gestational development in the SHRSP strain compared to the WKY strain even before the onset of the disease. We also generated promoter constructs for the SHRSP and WKY strains and compare the transcriptional activity of segments of the Spp1 promoter by using dual luciferase assay. When these constructs were transfected into H9c2, we observed a trend towards increased luciferase activity in the SHRSP constructs when compared to the WKY constructs. However, when this experiment was repeated using HeLa cells our results were inconsistent. Microarray analysis was conducted on 5 weeks vs neonatal SHRSP vs WKY.SPGla14a strains (chromosome 14 congenic strain) to capture gene expression changes responsible for the disease phenotype. Using Ingenuity Pathway Analysis (IPA), 101 genes were identified that were differentially expressed and found in common between the SHRSP and WKY.SPGla14a strains. From these 101 genes, a Tox analysis was conducted to identify genes downstream that potentially contribute towards cardiac hypertrophy and are connected to Spp1. Desmin (DES), Matrix metalloproteinase-14 (MMP14), Cathepsin-D (CTSD), Solute carrier family 25 member 11 (SLC25A11), and Myomesin-1 (MYOM1) were the 5 genes identified downstream that are connected to Spp1 and the enlargement of the heart. Following this, we aimed to establish and characterize the minimally invasive transverse aortic constriction (TAC) pressure overload model of left ventricular hypertrophy (LVH) in the WKY, WKY.SPGla14a congenic strain, SHRSP Spp1 WT, and SHRSP Spp1em1 (Spp1 knock out) rats. We assessed the impact of TAC surgery on body weight and blood pressure, assessed differences in LVMI and assessed cardiac phenotype parameters such as relative wall thickness (RWT), stroke volume (SV) fractional shortening (FS), ejection fraction (EF) and cardiac output (CO) in the SHAM and TAC operated rats. We also aimed to examine strain differences in response to TAC-induced pressure overload in these strains. Our results showed that we were able to successfully establish the TAC model in our WKY and chromosome 14 congenic strain by showing significantly increased LVH in these strains 8 weeks post-TAC surgery. We however were unable to show an exaggerated hypertrophic response in the hearts of the SHRSP Spp1 WT and SHRSP Spp1em1 rats with no evidence of progression to heart failure. Finally, to investigate the functional response of Spp1 overexpression we used the cardiac cell line H9c2 as a model in our experiments to determine cell size. H9c2 cells were transfected with Spp1 and pcDNA control to measure any changes in cell size post-transfection. We observed a significant increase in cell size in cells transfected with Spp1 when compared to the control. Furthermore, we aimed to assess the functional role of EVs isolated from Spp1 transfected H9c2 and HeLa conditioned media on H9c2 cells. Over-expression of Spp1 in H9c2 cells results in a significant increase in cell size, most likely contributing to cardiac hypertrophy in models of cardiovascular disease. We also investigate the role of extracellular vesicles (EVs) in Spp1 trafficking released from Spp1 transfected H9c2 cells. EVs were isolated from H9c2 cells transfected with Spp1 and then overlayed onto a fresh batch of H9c2 cells to observe any changes in cell size. By doing this we observed a significant increase in cell size indicating the potential role EVs play in the mediation of Spp1 trafficking. This study also implicates the role of EVs in cardiac disease. Following this, mass spectrometry analysis was conducted on EVs isolated from transfected H9c2 cells to characterize protein content. The results showed high quantities of foetal bovine serum (FBS) proteins and cross-contamination of keratin from the surrounding environment while the samples were being prepared. This was rectified by optimizing the protocol and preparing EV samples inside a sterilized tissue culture hood. However, despite there being cross-contaminants and high quantities of FBS, we were able to identify proteins of interest that were found in common between the control-EV and Spp1-EV samples, desmoplakin or DSP gene and zinc transporter ZIP8 synthesized by the SLC39A8 gene. RNA sequencing (RNAseq) was conducted on RNA isolated from control and Spp1 transfected EVs to determine whether Spp1 was transferred into EVs posttransfection and to identify any significantly differentially expressed protein and long non-coding RNAs that may be involved in processes affecting cell size. Initial test samples revealed Spp1 to be ranked 8th among the genes identified in the Spp1 transfected EV-RNA sample with a coverage of 1178 reads per kilobase per million (RPKM). The control group showed Spp1 to be ranked 774th with a coverage of 2.84 RPKM. This indicates that Spp1 was successfully transferred from transfected H9c2 cells into the EVs with a higher coverage compared to the control pcDNA EV sample. Further analysis using a new set of EV-RNA samples showed Spp1 expression was significantly increased, with a fold change of 5598 in the Spp1 EVRNA sample when compared to the control. Analysis of the two data sets suggests that Spp1 was successfully transferred in the EVs. In summary, the data presented in this thesis elucidate Spp1 as a key modulator for LVH and cardiac fibrosis and how early changes in gene expression initiate cardiac phenotypes in the SHRSP strain even before the onset of the disease.