Analysis of microRNA role in the development of left ventricular hypertrophy in the stroke-prone spontaneously hypertensive rat

MicroRNAs (miRs) are a group of short non-coding RNAs, on average 22 nucleotides in length, that form an important axis of post-transcriptional regulation of gene expression. They have been identified as major modulators of all biological processes including development, cell differentiation, growth and apoptosis as well as diseases such as cancer, diabetes and cardiovascular disease (CVD). In the developed world CVD remains the leading cause of morbidity and mortality, and a substantial burden on healthcare. Left ventricular hypertrophy (LVH) is defined as an increase in thickness of the myocardium and is an important risk factor in CVD. The stroke-prone spontaneously hypertensive rat (SHRSP) is an animal model of essential hypertension used in research of CVD together with a normotensive reference strain Wistar-Kyoto (WKY). The SHRSP animals exhibit an increase in the size of myocardium prior to the onset of hypertension and have established LVH at 16 weeks of age thus are a good model for investigating the genetics of this condition. The aim of this project was to identify signature expression patterns of novel and previously implicated microRNAs and to investigate their role in the development of LVH in the SHRSP. Furthermore, potential gene targets of candidate selected microRNAs were identified to investigate biological pathways involved in the disease process. MicroRNA microarray profiling was performed by Dr. McBride in the hearts of 5 week old SHRSP and WKY male rats using the LC Sciences (LCS) multispecies chip based on Sanger miRBase 11.0. The data were analysed (Drs. McBride and McClure) using Rank Product (RP) analysis method and evaluated in combination with the statistical analysis provided by LC Sciences (LCS). LCS data indicated 103 microRNAs differentially expressed at 5 weeks of age, 64 at 16 weeks of age, with 9 in common. The RP analysis identified 72 microRNAs differentially expressed between WKY and SHRSP at 5 weeks of age and 51 at 16 weeks of age, and 21 microRNAs were differentially regulated at both time points. Both methods identified a subset of 35 microRNAs in 5 week old hearts and 8 in 16 week old samples. TaqMan® microRNA assays were used to confirm these expression patterns. Based on these data and published literature candidate microRNAs – miR-195, miR-329 and miR-451 were selected for further experimental investigation. Expression of candidate microRNAs (miR-195, miR-329 and miR-451) in neonatal hearts of SHRSP and WKY rats was also investigated. It was found that all three candidate microRNAs were differentially expressed at this time point and there were significantly increased levels in the SHRSP compared to WKY. Cardiac cell line H9c2 AngII model of hypertrophy was used to investigate the effect of AngII on our candidate miRNA expression levels. A 96 hour stimulation of H9c2 cell with AngII resulted in a significant increase in cell size. Levels of miR-195 and miR-329 were not affected by addition of AngII; expression of miR-451 was significantly down-regulated immediately post stimulation, however levels were increased at the final assessment at 96 hours. Adenoviral vectors over-expressing miR-195, miR-329 and miR-451 were designed and generated. These vectors were used to investigate if overexpression of each individual miR could affect cell size in the selected in vitro model of cardiomyocyte hypertrophy. It was found that all candidate microRNAs reduced AngII mediated hypertrophic cell growth at higher doses. Identifying pathways and specific gene targets affected by changes in microRNA levels is of paramount importance. Availability of such data not only provides information about regulation of cardiac homeostasis, but also possible therapeutic approaches for treatment and prevention. Target prediction algorithms (DIANAmT, miRanda, miRDB, miRWalk, PICTAR5, PITA, RNA22, RNAhybrid and Targetscan) were used to identify potential gene targets for candidate microRNAs. To refine these lists to genes relevant to the experimental design Ingenuity Pathway analysis (IPA 9.0) software was used to overlay microRNA microarray data with results of heart mRNA gene expression data (M. McBride, personal communications) from the same cardiac tissue and to relate these to appropriate pathways and cellular functions. A list of 12 genes was generated: similar to CG4768-PA (RGD1309748), KN motif and ankyrin repeat domains 1 (Kank1), sterile alpha motif domain containing 4B (Samd4b), dual specificity phosphatase 10 (Dusp10), follistatin-like 3 (secreted glycoprotein) (Fstl3), jun D proto-oncogene (JunD), forkhead box M1 (Foxm1), SIN3 homolog A transcription regulator (yeast) (Sin3a), cyclin-dependent kinase 1 (Cdk1), kinesin family member 23 (Kif23), bone morphogenetic protein receptor type IA (Bmpr1a) and sestrin 1 (Sesn1). Expression of these candidate targets was assessed in heart tissues from neonates, 5 and 16 week old rats. Six out of ten of these targets were differentially expressed at one or more time points. To further investigate the proposed targeting of these genes by candidate microRNAs, expression levels were measured in each of the predicted targets in H9c2 cell transduced with miR over-expressing viruses. The expression patterns of Cdk1, Kif23, Kank1 and Sin3a were consistent with overexpression of the targeting microRNA, i.e. expression of each gene was down-regulated. In summary, data presented in this thesis elucidate the role of miR-195, miR-329 and miR-451 in the development of LVH in the SHRSP. Understanding the underlying cause for differential expression of these candidate microRNAs, confirming gene targets and identifying relevant pathways will improve the understanding of LVH at the molecular level. It will also help explain the pathophysiology of cardiovascular disease development in this rat model of human hypertension providing a basis for the development of novel therapeutic approaches to treat or prevent LVH.