Proteome Alterations in Cardiac Fibroblasts: Insights from Experimental Myocardial Infarction and Clinical Ischaemic Cardiomyopathy

Abstract

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.

Keywords: TGF-β; cardiac remodelling; extracellular matrix; ischaemic heart disease; myocardial infarction; proteomics.

Figure 1

Proteomic characterisation of myocardial infarction experimental mice. Female C57BL6 mice underwent experimental myocardial infarction (MI) by permanent left coronary artery ligation. Mice were sacrificed on day 3, 1 week and 1 month post-surgery. (A) Heart tissue from n = 3 (day 3), n = 6 (week 1 and 1 month) mice were used to generate primary fibroblasts, which were treated with the pro-fibrotic cytokine TGF-b or DMSO (control) for 24 h before mass spectrometry analysis using dia-PASEF acquisition. (B) Principal component analysis of resulting mass spectrometry data reveals a unique proteomic profile in untreated (control) fibroblasts from MI mice at 1 month. (C) Welch’s t-test is applied to identify significant protein expression changes between MI and sham cardiac fibroblast cells on day 3, 1 week, and 1 month. (D,E) Two proteins (Neuroplastin and Trans-transcription factor1) are significantly elevated in MI mice at all time points. (F,G) In week 1 and 1 month, the average fold changes of extracellular matrix proteins (n = 59) are trending higher in MI cardiac fibroblasts. Bar charts show the mean ± standard error of the mean (SEM). Coloured dot plots (F) represent individual samples, and black dots (G) represent individual extracellular matrix (ECM) proteins. * p < 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, non-significant.

Figure 2

Pathway dysregulation in chronic myocardial infarction experimental model. (A,B) Gene Ontology analysis reveals biological processes (BP), cellular compartments (CC) and molecular functions (MF) that are more de-regulated by significantly down and upregulated genes 1 month post-myocardial infarction. (C) The top most enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (mouse) associated with protein changes at 1 month post-MI are also de-regulated at day 3 post-MI. ‡ = common up and downregulated genes in D3 and 1m datasets; ¥ = common up and downregulated genes at all time points.

Figure 3

Effect of TGF-β treatment on MI cardiac fibroblasts. (A) Welch’s t-test is applied to identify significant protein changes as a result of TGF-β treatment of MI cardiac fibroblasts at day 3, 1 week and 1 month time points. (B–D) Three of the commonly up- and downregulated proteins (Dclk1, Antrx1 and Prepl) at all time points in response to MI. Bar charts show the mean ± standard error of the mean (SEM). * p < 0.05; ns, non-significant.

Figure 4

MI sensitises cardiac fibroblasts to pro-fibrotic stimulation by TGF-β. (A,B) The extracellular matrix proteins Lox and Loxl3 are significantly elevated in MI cardiac fibroblasts after TGF-β treatment. (C) Lox and Loxl3 are significantly associated with a number of collagen proteins (red = negative correlation; green = positive correlation). (D) Lox and (E) Loxl3 are also elevated in human ISCM, compared to patients without clinical heart failure. Bar plots represent the mean ± standard error of the mean (SEM). * p < 0.05; ** p ≤ 0.01; ns, non-significant.

Figure 5

In silico validation of proteomic changes in cardiac fibroblasts. (A) Publicly available mass spectrometry data were accessed through ProteomeXchange for validation of observed protein expression changes (Shah et al. [9]). (B–G) Neuroplastin (Nptn), Lox and collagen type Vi alpha 1 (Col6A1) were verified as being significantly associated with MI. Bar plots represent the mean ± standard error of the mean (SEM). * p < 0.05; ** p ≤ 0.01; **** p ≤ 0.0001; ns, non-significant.