Abstract
Despite decades of effort from researchers, clinicians, and public health teams, cardiovascular disease (CVD) remains the leading cause of global mortality and morbidity. Myocardial ischaemia (inadequate blood flow and thus oxygen delivery to the myocardium) is a pathological hallmark of numerous CVDs including ischaemic heart disease, acute myocardial infarction and chronic heart failure. While current treatments have contributed to reduced mortality rates, novel therapeutics targeting underlying pathophysiological processes are being urgently sought. Over the past two decades, the role of non-coding RNA molecules termed microRNAs (miRNAs) in the progression of ischaemic cardiomyopathies has come to light. Notably, a subset of miRNAs are thought to be directly regulated by cellular hypoxia, termed ‘hypoxamiRs’. Dysregulation of these hypoxamiRs is thought to contribute to pathological cardiac remodelling by increasing apoptosis, fibrosis, hypertrophy, and by reducing the formation of new vasculature within the ischaemic heart. While the modulation of hypoxamiRs is a promising therapeutic strategy, several gaps in current knowledge exist.
Our knowledge of hypoxamiR regulation within the ischaemic heart has primarily been derived from in vitro studies of individual cardiac cell types, employing various hypoxic stimuli. This has made it challenging to form conclusions about both cell-specific and temporal patterns of hypoxamiR regulation. Furthermore, cardiac cell types don’t function in isolation, but rather as co-ordinators and effectors of a tissue-level response. As such, more complex in vitro models are required which better replicate features of the in vivo myocardium. These models may provide an appropriate platform for both the study of integrated cellular responses, and for the evaluation of therapeutic miRNA modulation within the ischaemic heart. This thesis aimed to first produce a 3D cardiac spheroid model, before assessing its suitability for the investigation of both the dysregulation and therapeutic modulation of hypoxamiRs.
Cardiac spheroids were successfully formed by the combination of cardiomyocytes, endothelial cells and patient-derived cardiac fibroblasts in ultra-low attachment plates. The produced cardiac spheroid model was found to maintain its morphology over an extended culture duration, with evidence of successful incorporation of all cell types and hypoxamiRs of interest. Cardiac spheroids demonstrated features of increased similarity to the in vivo myocardium, with the formation of extensive microvascular-like networks and the increased expression of sarcomeric gene transcripts. Successful model characterisation permitted the downstream investigation of both hypoxamiR regulation and therapeutic modulation.
Four key cardiac hypoxamiRs were selected for investigation in both 2D cell lines and the cardiac spheroid model – miR-21-5p, miR-30c-5p, miR-34a-5p and miR-126-3p. These hypoxamiRs were selected to investigate a range of processes involved in pathological cardiac remodelling. Divergent patterns of hypoxamiR expression were observed between the major cardiac cell types. Most notably, miR-30c-5p and miR-34a-5p demonstrated increased expression in cardiomyocytes and cardiac fibroblasts, with decreased expression in endothelial cells under a matched hypoxic stress. This evidence is critical for informing future therapeutic studies, providing an argument for the cell-specific modulation of hypoxamiRs in the wake of myocardial ischaemia. Interestingly, no differences in hypoxamiR expression were identified in the cardiac spheroid model, with plausible explanations including increased hypoxic resistance and the summation of opposing cell-specific directions of dysregulation.
The therapeutic modulation of hypoxamiRs miR-34a-5p and miR-126-3p was attempted in cardiac spheroids using lipofection. Results from this study highlighted the successful intracellular uptake of miRNA therapeutics, however with evidence of disruption of the extracellular matrix and the induction of pro-inflammatory signalling. The uptake of miRNA therapeutics as assessed by confocal microscopy appeared limited to the peripheral cell layers of cardiac spheroids, with 3D culture likely posing significant physical barriers to lipofection. Despite these uptake challenges, several promising results worthy of further investigation were identified. Most notably, preliminary evidence was provided to support a reduction in pro-fibrotic signalling following the delivery of a miR-34a-5p inhibitor and miR-126-3p mimic versus modulation of either hypoxamiR alone, with biological plausibility. Evidence further supported modulation of IL6/JAK/STAT3 and interferon-alpha signalling by miR-34a-5p and miR-126-3p modulation respectively, relationships which have been rarely explored in a cardiac context.
To our knowledge, this study is the first to evaluate both intracellular hypoxamiR expression and the therapeutic modulation of hypoxamiRs within a 3D in vitro cardiac model. Collectively, this study has improved our understanding of the nuances of hypoxamiR regulation within the ischaemic myocardium, and the utility of 3D cardiac models as platforms for the investigation of both hypoxamiR expression and their therapeutic modulation.