title: Identifying early genetic determinants of adverse cardiac remodelling in complex genetic models of human cardiovascular disease
Creators: name: Trivett, Cara
Publisher: name: University of Glasgow
Year: 2024

description: Globally, cardiovascular diseases (CVD) are the leading cause of morbidity and mortality. High blood pressure, or hypertension, is the primary modifiable risk factor contributing to the development of CVD. An independent risk factor of cardiac and all cause mortality is an enlargement of the heart, specifically the left ventricle (LV), clinically defined as left ventricle hypertrophy (LVH). Accounting for known biological and environmental factors, a large part of variability in LV mass is unexplained, and is thought to come under genetic control. Studies of heritability estimate structural measures of the heart are highly heritable, ranging between 15–60%. Translation of genetic variation into mechanisms underlying progression of human disease can be methodologically difficult, and thus far understanding of the genetic architecture of LV mass is not well-developed. Genetically determined differences are required to alter molecular phenotypes to result in measurable differences in cardiac structure. Unravelling connections between genetic variation and pathways of pathology are therefore key to defining genetic contribution to LVM index (LVMI). The rat is an effective small rodent model to recapitulate human disease phenotypes without surgical or dietary intervention. Genetic models of hypertension include the Stroke Prone Spontaneously Hypertensive Rat (SHRSP) and its closest genetic comparator, Wistar Kyoto (WKY).

To determine potential genetic factors underlying the development of LV hypertrophy, a linkage study in WKY and SHRSP rats identified a region of chromosome 14 independently associated with LVMI. Following this, the chromosome 14 region associated with LVMI was the target of congenic strain generation, where a segment of chromosome 14 was introgressed from donor WKY and SHRSP strains into the opposing strain (SP.WKYGla14a and WKY.SPGla14a respectively). Initial phenotyping, in male animals, show cardiac phenotypes of chromosome 14 congenic strains diverged from their background strain, with measurable differences detectable from as early as 5-weeks. The WKY.SPGla14a developed increased LVMI without an excessively increased blood pressure, whilst the SP.WKYGla14a developed increased blood pressure without significant remodelling of the LV. Following the development and establishment of the chromosome 14 congenic strains on both normotensive and hypertensive backgrounds, work undertaken in this thesis comprised a series of investigations in-vivo, ex-vivo, in-vitro, and in-silico to determine potential key genetic factors and molecular effectors predicting and resulting in excessively increased LVMI in rat models of human hypertension.

Short-read DNA sequences, from WKY and SHRSP colonies housed in Glasgow, were aligned to the recently published SHRSP/BbbUtx reference quality genome. High quality variant calling analysis identified a number of key sites of variance between SHRSP and WKY strains, which could be contributing to differences in cardiac gene expression and LV structure. These include localised clusters of high impact variants within previously identified QTLs for cardiac mass on chromosomes 1 &amp; 3. Prior to this work, secreted phosphoprotein 1 (Spp1 ) was identified as a positional and functional candidate gene governing increased LVMI associated with the SHRSP chromosome 14 region. Variants within the Spp1 gene and upstream of its transcriptional start site were fine mapped between the WKY and SHRSP strains for future investigations determining causal variants underlying observed differences in expression of Spp1 in cardiac tissues.

Expanding on initial phenotyping, in-vivo studies were conducted to determine the effect of introgression of SHRSP ‘risk’ haplotypes into the ‘protected’ WKY background (and vice versa), in both female and male congenic strains. Phenotype assessment in neonate, 5-, and 16-week animals included transthoracic ultrasound echocardiography (5- &amp; 16-week) and tail cuff plethysmography (16-weeks). Male and female SHRSP animals developed a significantly increased LVMI compared to sex- and age-matched WKYs by 5-weeks of age. This was accompanied by a persistent upregulation of Spp1 mRNA in the LV, which was more pronounced in females at 16-weeks of age. The chromosome 14 congenic strains did not develop a cardiac phenotype which differed significantly from their respective parental strain. Despite this, Spp1 mRNA expression was increased in the LV of strains harbouring SHRSP genome at the chromosome 14 locus. The increase in Spp1 expression in SHRSP and WKY.SPGla14a hearts persisted across all measured time-points; gestational day 18.5, 1–3 day neonate, 5-, and 16- weeks.

To better understand biological pathways altered by genetic variation in parental and congenic strains, short read RNA sequencing was performed in the gestational day 18.5 heart. Although prior to the development of divergent cardiac phenotypes, introgression of the SHRSP genome significantly and dramatically altered gene expression and transcript usage during cardiac development. Alignment of the RNA short read sequences to the SHRSP/BbbUtx, revealed variance within genes that also displayed evidence of significant differential expression and transcript usage. The hearts of SHRSP and WKY.SPGla14a strains are potentially primed for future dysfunction through dysregulation of genes to increase mitochondrial dysfunction and decrease oxidative phosphorylation. In addition, key receptors and signalling factors involved in the maintenance and production of the extracellular matrix were dysregulated in SHRSP and WKY.SPGla14a hearts during development.

Molecular investigations focused on Spp1, an extracellular matrix protein and a known biomarker of established heart disease. Adapting a H9c2 cell model used in Angiotensin-II (Ang-II) studies, transfection of H9c2 cells with vectors containing Spp1 mRNA increased cell size 48-hours post-transfection compared to pcDNA controls. The H9c2 cell model was further expanded to investigate the role of small extracellular vesicles (sEV). These membrane-bound vesicles (&lt;200nm) are produced by cardiac and other cells of the heart for cell-to-cell communication and signal transduction, carrying nucleotide species and/or protein messengers to mediate physiological and pathological processes. sEVs derived from cells transfected with Spp1 were applied to naive H9c2 cells resulting in an equal increase in cell size to direct transfection of Spp1. This increase in cell size following incubation with sEV was blocked when cells were treated with 60µM of EV uptake inhibitor, dynasore.

Finally, characterisation of a CRISPR/Cas9 Spp1 knock-out on the SHRSP strain suggested alternative transcripts of Spp1 are produced in the rat cardiac transcriptome. The alternative transcript identified resembled human short forms of SPP1 known as osteopontin-c. Alternative Spp1 transcripts produced by the knock-out removes the exon containing point mutations and may rescue osteopontin protein expression in these animals. In the H9c2 model, overexpression of Spp1 transcripts produced by the knock-out have equal function to canonical Spp1.

Taken together, these data have implicated a role for sEV in the pathway of Spp1 overexpression that results in H9c2 cell size to increase. Genomic and transcriptomic studies support a role of Spp1 dysregulation underpinning differences in cardiac phenotypes associated with the SHRSP chromosome 14 region. These studies have generated a basis to further validate and translate findings in described experimental models. The wealth of genomic data provided by large consortia such as GTEx, the GWAS catalogue, and the UK BioBank represent rich resources to combine and correlate findings from genetic rat models to targets in human disease.