Single-nucleus analysis of exercise-driven transcriptomic and epigenetic restoration in the ageing heart

Ageing is a fundamental and inevitable biological process marked by a progressive decline in cellular and tissue function, leading to increased susceptibility to chronic diseases. In the context of cardiac health, ageing significantly increases the risk of cardiovascular disease, which is the leading cause of death in developed nations; therefore, understanding the molecular drivers of ageing and discovering mechanisms to mitigate their impact is critically important. Ageing is associated with the stiffening of the heart due to cardiac fibrosis, a reduced regenerative capacity, and increased inflammation. These changes contribute to reduced cardiac output and a heightened risk of heart failure, arrhythmias, and vascular stiffening. Regular exercise has been shown to have a cardioprotective effect on heart health, lowering the risk of cardiovascular disease. However, the molecular mechanism by which exercise can protect the heart from ageing remains an open question. Exercise has been shown to improve cardiomyocyte survival following injury, reduce fibrotic remodeling, and shift immune cell states towards anti-inflammatory phenotypes. To explore these changes, the transcriptome provides a foundational measure of cellular activity, with changes in gene expression ultimately resulting in functional changes. In parallel, the epigenome is increasingly recognised as a key dynamic regulator of transcriptional output, with changes in chromatin accessibility at transcription factor binding sites influencing enhancer activity and downstream gene expression. Recent advances in single-cell sequencing technologies allow for the resolution of distinct cell types within complex tissues, enabling the detection of both shared and cell-type-specific responses to ageing and exercise across multiple modalities. In this thesis, I utilise single-nucleus RNA-seq and single-nucleus ATAC-seq data from murine hearts across young and aged mice, with and without extended exercise training (6 months), to characterise the transcriptomic and epigenetic changes induced by ageing and exercise. I found that ageing was associated with altered RNA splicing, activation of fibrotic pathways, and reduced expression of genes involved in cardiac contractility and development. In contrast, exercise training opposed these age-associated patterns, restoring youthful gene expression profiles across a range of cell types. By profiling transcription factor binding sites, I found that exercise was associated with more youthful binding of AP-1 and bHLH-PAS transcription factors, with links to altered gene expression. Finally, an in-depth exploration of cardiac macrophages found that exercise drives an increase in the proportion of the anti-inflammatory M2 subpopulation as well as the expansion of a macrophage-fibroblast hybrid cell state that regulates the extracellular matrix. These findings provide a cell-type-resolved exploration of cardiac ageing and identify specific molecular mechanisms through which exercise acts to mitigate detrimental effects.