Investigating the complexities of microrna-target interactions in cardiac biology

Abstract

One in three Australians die of cardiovascular disease and despite advances in treatment it remains the leading cause of death in Australia. MiRNAs play critical roles in the heart and specific miRNAs can promote or prevent cardiac hypertrophy, sparking interest to use miRNAs as therapeutic targets. However, cells can produce both miRNAs and their mRNA targets in multiple processing variants increasing the complexity of miRNA-mediated control. It is therefore critical to elucidate the role of these variants in cardiac disease. The aim of this thesis was to profile miRNA and mRNA 3' UTRs in cardiac hypertrophy and to interrogate this data for evidence of processing variation. We established a murine model of left ventricular hypertrophy using transverse aortic constriction as confirmed using haemodynamic, physiological and biochemical measurements. Cardiomyocyctes were isolated from pre-hypertrophic, hypertrophic and control hearts and RNA extracted for next-generation sequencing of miRNAs and mRNA 3' ends. We identified 17 miRNAs and 776 mRNAs that changed in level during the hypertrophy, with the majority being upregulated. Overall, more mRNAs changed expression in the pre-hypertrophic cardiomyocytes, while the miRNAs were altered mostly in the hypertrophic samples. This is the first study to document the expression of miRNAs and mRNAs simultaneously in purified cardiomyocytes and their change in hypertrophy. Analysing the datasets for processing diversity revealed that nearly all miRNAs in cardiomyocytes showed some degree of processing diversity including expression from both hairpin arms, 5' and 3' isomiRs, or the presence of non-templated additions. There was little directional change in miRNA processing during hypertrophy but two examples were identified where the bias for particular variants changed significantly. This is the first documentation of variable miRNA processing in purified cardiomyocytes. Such variants provide a mechanism to change the mRNA targeting properties of miRNAs from a given locus without changing the genomic sequence. In support of this, in vitro luciferase assays demonstrated that the different 5' isomiRs of the cardiac miR-133a-3p have distinct targeting preferences. Analysis of the 3' end sequencing data demonstrated that mRNAs expressed in cardiomyocytes had on average 2.35 distinct 3' UTRs with an average length of 3.7 kb. The proportion of short versus long 3' UTRs was altered for 424 mRNAs, with 272 3' UTRs getting shorter and 152 getting longer, in the pre-hypertrophic cardiomyocytes. This is the first study to provide an in-depth mapping of mRNA 3' UTR diversity in purified cardiomyocytes and document changes to the proportion of these variants in cardiac hypertrophy. Alterations in the length of 3' UTRs can lead to inclusion of or exclusion of miRNA target sites on mRNAs and thus change their sensitivity to regulation by miRNAs. Overall, the datasets reported here provide global information on expression changes to both miRNA sequence and mRNA 3' UTR lengths forming the basis for an improved systems level understanding of miRNA-regulation during cardiac hypertrophy. The realisation that cardiac miRNAs and their targets exist as currently under-appreciated variants with potentially complex effects on target specificities has important implications for the role of miRNAs in cardiac disease.