Dilated cardiomyopathy (DCM) is the most common cause of heart failure, with a complex aetiology involving multiple cell types. We aimed to detect cell-specific transcriptomic alterations in DCM through analysis that leveraged recent advancements in single-cell analytical tools. Single-cell RNA sequencing (scRNA-seq) data from human DCM cardiac tissue were subjected to an updated bioinformatic workflow in which unsupervised clustering was paired with reference label transfer to more comprehensively annotate the dataset. Differential gene expression was detected primarily in the cardiac fibroblast population. Bulk RNA sequencing was performed on an independent cohort of human cardiac tissue and compared with scRNA-seq gene alterations to generate a stratified list of higher-confidence, fibroblast-specific expression candidates for further validation. Concordant gene dysregulation was confirmed in TGFβ-induced fibroblasts. Functional assessment of gene candidates showed that AEBP1 may play a significant role in fibroblast activation. This unbiased approach enabled improved resolution of cardiac cell-type-specific transcriptomic alterations in DCM.

Aims: Dynamic alterations in cardiac DNA methylation have been implicated in the development of heart failure (HF) with evidence of ischaemic heart disease (IHD); however, there is limited research into cell specific, DNA methylation sensitive genes that are affected by dysregulated DNA methylation patterns. In this study, we aimed to identify DNA methylation sensitive genes in the ischaemic heart and elucidate their role in cardiac fibrosis. Methods: A multi-omics integrative analysis was carried out on RNA sequencing and methylation sequencing on HF with IHD (n = 9) versus non-failing (n = 9) left ventricular tissue, which identified Integrin beta-like 1 (ITGBL1) as a gene of interest. Expression of Itgbl1 was assessed in three animal models of HF; an ischaemia-reperfusion pig model, a myocardial infarction mouse model and an angiotensin-II infused mouse model. Single nuclei RNA sequencing was carried out on heart tissue from angiotensin-II infused mice to establish the expression profile of Itgbl1 across cardiac cell populations. Subsequent in vitro analyses were conducted to elucidate a role for ITGBL1 in human cardiac fibroblasts. DNA pyrosequencing was applied to assess ITGBL1 CpG methylation status in genomic DNA from human cardiac tissue and stimulated cardiac fibroblasts. Results: ITGBL1 was >2-fold up-regulated (FDR adj P = 0.03) and >10-fold hypomethylated (FDR adj P = 0.01) in human HF with IHD left ventricular tissue compared with non-failing controls. Expression of Itgbl1 was up-regulated in three isolated animal models of HF and showed conserved correlation between increased Itgbl1 and diastolic dysfunction. Single nuclei RNA sequencing highlighted that Itgbl1 is primarily expressed in cardiac fibroblasts, while functional studies elucidated a role for ITGBL1 in cardiac fibroblast migration, evident in 50% reduced 24 h fibroblast wound closure occurring subsequent to siRNA-targeted ITGBL1 knockdown. Lastly, evidence provided from DNA pyrosequencing supports the theory that differential expression of ITGBL1 is caused by DNA hypomethylation. Conclusions: ITGBL1 is a gene that is mainly expressed in fibroblasts, plays an important role in cardiac fibroblast migration, and whose expression is significantly increased in the failing heart. The mechanism by which increased ITGBL1 occurs is through DNA hypomethylation.

Ischaemic heart disease (IHD) is a chronic condition that can cause pathological cardiac remodelling and heart failure (HF). In this study, we sought to determine how cardiac fibroblasts were altered post-experimental myocardial infarction (MI). Female C57BL6 mice underwent experimental MI by permanent left coronary artery ligation. Cardiac fibroblasts were isolated from extracted heart tissue of experimental MI mice and subsequently treated with the pro-fibrotic cytokine, TGF-β, for 24 h and analysed using high throughput LC-MS/MS analysis. Findings were validated using mass spectrometry data generated from human left ventricular tissue analysis, which were collected from patients with ischaemic cardiomyopathy (ISCM) and age/sex-matched patients without clinical HF (NF). Proteomic analysis revealed significant protein expression changes in mouse cardiac fibroblasts after MI. These changes were most pronounced at 1 month post-MI, compared to earlier time points (3 days and 1 week). TGF-β treatment profoundly affected fibroblast cells extracted from MI mice, indicating a heightened sensitivity to pro-fibrotic factors after myocardial injury. Extracellular matrix (ECM) proteins significantly altered in MI fibroblasts following TGF-β treatment were significantly associated with cardiac remodelling. Notably, Lox was significantly changed in both isolated fibroblasts treated with TGF-β from experiment MI mice and human ISCM. Isolated cardiac fibroblasts from MI mice are more susceptible to developing pathogenic traits following TGF-β treatment than isolated fibroblasts from normal heart tissue. ECM proteins associated with these enhanced fibroblast activities and functions are evident. These altered proteins may play a functional role in MI-associated cardiac dysfunction.