Our previous studies have indicated that chronic treatment with 1-[(2-dimethylamino) ethylamino]-4-(hydroxymethyl)-7-[(4-methylphenyl) sulfonyl oxy]-9H-
xanthene-9-one (XNT), an
angiotensin-converting enzyme 2 (ACE2) activator, reverses
hypertension-induced cardiac and renal
fibrosis in spontaneously hypertensive rats (SHRs). Furthermore, XNT prevented pulmonary vascular remodelling and
right ventricular hypertrophy and
fibrosis in a rat model of
monocrotaline-induced
pulmonary hypertension. The aim of this study was to determine the mechanisms underlying the protective effects of XNT against cardiac
fibrosis.
Hydroxyproline assay was used to measure cardiac
collagen content in control and XNT-treated (200 ng kg(-1) min(-1) for 28 days) SHRs. Cardiac ACE2 activity and
protein levels were determined using the fluorogenic
peptide assay and Western blot analysis, respectively.
Extracellular signal-regulated kinases (ERKs; p44 and p42) and
angiotensin II type 1 (AT(1)) receptor levels were quantified by Western blotting. Cardiac
ACE2 protein levels were ∼15% lower in SHRs compared with Wistar-Kyoto control animals (ACE2/
glyceraldehyde 3-phosphate dehydrogenase ratio: Wistar-Kyoto, 1.00 ± 0.02 versus SHR, 0.87 ± 0.01). However, treatment of SHRs with XNT completely restored the decreased cardiac ACE2 levels. Also, chronic infusion of XNT significantly increased cardiac ACE2 activity in SHRs. This increase in ACE2 activity was associated with decreased cardiac
collagen content. Furthermore, the antifibrotic effect of XNT correlated with increased cardiac
angiotensin-(1-7) immunostaining, though no change in cardiac AT(1)
protein levels was observed. The beneficial effects of XNT were also accompanied by a reduction in ERK phosphorylation (phospho-ERK/total ERK ratio: Wistar-Kyoto, 1.00 ± 0.04; control SHR, 1.46 ± 0.25; treated SHR, 0.86 ± 0.02). Our observations demonstrate that XNT activates cardiac ACE2 and inhibits
fibrosis. These effects are associated with increases in
angiotensin-(1-7) and inhibition of cardiac ERK signalling.