In the failing heart, the extracellular matrix (ECM) undergoes structural remodelling with changes in the accumulation and organisation of ECM proteins. Ivabradine (IVA) an inhibitor of the pacemaker (If) current has been shown to have beneficial effects in the structure and function of the failing heart including the reversal of myocardial fibrosis. However, whether IVA alters the expression of specific ECM proteins during the progression of heart failure (HF) is unknown. In addition, changes in the fibroblast number and phenotype before and after IVA treatment have not been studied. HF was induced by permanent coronary artery ligation in rats anaesthetized with Isoflurane. Sham-operated animals (S) were used as controls. After 12 weeks, HF (HF-12) animals (ejection fraction<40%) were treated either with oral IVA (HF-IVA) (10mg/kg/day) or saline (HF-S) for a further 4 weeks. Values are presented as means±S.E.M. and compared by an ANOVA. Using immunofluorescence and confocal microscopy, sections were studied for specific ECM proteins and the percentage area fraction was quantified on 8-10 fields per section (3-4 hearts per group) using ImageJ software. IVA significantly reduced the levels of collagen I (S: 0.76±0.07%, n=69; HF-12: 1.12±0.08%, n=57; HF-S: 1.59±0.12%, n=70; HF-IVA: 0.68±0.06%, n=52; p<0.05), collagen III (S: 0.88±0.06%, n=78; HF-12: 1.26±0.09%, n=90; HF-S: 1.91±0.13%, n=74; HF-IVA: 0.75±0.65%, n=60; p<0.05) and elastin (S: 0.62±0.06%, n=52; HF-12: 1.16±0.06%, n=85; HF-S: 1.93±0.22%, n=68; HF-IVA: 0.52±0.09%, n=29; p<0.05) compared with HF-12. Vimentin, a marker for cardiac fibroblasts was measured using western blotting. IVA reduced the vimentin expression observed in HF-S to sham levels (in arbitrary units, S: 0.33±0.04, n=5; HF-S: 0.59±0.05, n=6; HF-IVA: 0.37±0.02, n=5; p<0.05). Since vimentin is also expressed in endothelial cells, von Willebrand factor (vWf) was measured. No change in vWf was observed suggesting that endothelial cell number was not affected. α-smooth muscle actin, a marker used to identify myofibroblasts but also present in smooth muscle cells, was also measured. IVA normalised the levels of α-smooth muscle actin to sham levels (S: 0.50±0.13, n=5; HF-S: 1.00±0.13, n=6; HF-IVA: 0.44±0.08, n=4; p<0.05). Expression of the hyperpolarisation-activated cyclic nucleotide-gated channel isoform 4 which encodes the If channels was up-regulated during HF but IVA down-regulated this overexpression (S: 0.31±0.05, n=5; HF-S: 0.57±0.05, n=6; HF-IVA: 0.38±0.05, n=5; p<0.05). Our results demonstrate that IVA reverses the accumulation of several components of the ECM and reduce fibroblast number and phenotype which may explain the changes observed in the ECM. Thus IVA appears to play a beneficial role in the structural integrity of the ECM which can be important in the reparative process of the failing heart.