Gender-based differences found in cardiovascular diseases raise the possibility that estrogen may have direct effects on cardiac tissue. Therefore we investigated whether cardiac myocytes and fibroblasts express functional estrogen receptors. Immunofluorescence demonstrated estrogen receptor protein expression in both female and male rat cardiac myocytes and fibroblasts. Nuclear translocation of the estrogen receptor protein was observed after stimulation of cardiomyocytes with 17ß-estradiol (E 2 ). Cells transfected with an estrogen-responsive reporter plasmid showed that treatment with E 2 induced a significant increase in reporter activity. Furthermore, E 2 induced a significant increase in expression of the estrogen receptors a and ß, progesterone receptor and connexin 43 in cardiac myocytes. Cardiac myocytes and fibroblasts contain functional estrogen receptors and estrogen regulates expression of specific cardiac genes. These data suggest that gender-based differences in cardiac diseases may in part be due to direct effects of estrogen on the heart.

The role of cardiac oestrogen receptor expression and local oestrogen synthesis in the pathogenesis of cardiovascular disease is poorly understood. Therefore we studied the effects of the oestrogen precursors androstendione and testosterone on the expression of cyp450 aromatase, oestrogen receptor alpha and beta, and inducible NO synthase (iNOS) in neonatal rat cardiac myocytes. Here, we show that cyp450 aromatase is expressed in cardiac myocytes and incubation of cardiac myocytes with oestrogen precursors leads to sexual dimorphic transactivation of an oestrogen-responsive reporter plasmid. Furthermore, incubation with oestrogen precursors stimulated expression of oestrogen receptor alpha and beta, and iNOS in a gender-specific fashion. These data suggest that local oestrogen biosynthesis of the heart is effective to activate oestrogen receptor alpha and beta, and downstream target genes in a gender-based fashion and may therefore contribute to the beneficial effects of oestrogen in the pathogenesis of cardiovascular disease.

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We studied the effects of angiotensin converting enzyme (ACE) inhibitors on angiotensin II (Ang II) induced growth related signalling pathways in neonatal rat cardiac fibroblasts. In BrdU proliferation assays, Ang II (10(-9)-10(-7) M) stimulated cardiac fibroblast growth in a dose-dependent fashion (maximum at 10(-7) M, 5.22 +/- 0.01-fold, n = 9). 2-2-(1-(ethoxycarbonyl)-3-phenylpropyl)[amino-oxopropyl]-6,7-dimethoxy- 1,2,3,4-tetrahydroisoquinoline-3 carboxylic acid (moexiprilat) led to a dose-dependent inhibition of the Ang II induced cardiac fibroblast growth. A less pronounced effect on cellular proliferation was seen with the ACE inhibitor enalaprilat. To elucidate the mechanisms involved in this direct antiproliferative effect of ACE inhibitors in cardiac fibroblasts, we studied the activation of mitogen-activated protein kinases [MAPKs: extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38-MAPK] and JAK/STAT janus kinases/signal transducer and activator of transcription) signal transduction pathways. Ang II (10(-7) M) caused an increase in MAPKs activity with an increased phosphorylation of ERK1/2 (1.7-fold) and p38-MAPK (3.6-fold). This effect was completely inhibited by moexiprilat (10(-7) M) and enalaprilat (10(-7) M). Stimulation with Ang II (10(-7) M) also led to an increased phosphorylation of STAT3, which is one of the key effector proteins in the JAK/STAT signalling pathway. This effect was also completely inhibited by moexiprilat (10(-7) M) and enalaprilat (10(-7) M). These data show that the ACE inhibitors moexiprilat and enalaprilat inhibit Ang II induced proliferation of cardiac fibroblasts according to their relative potency of ACE inhibition in vitro. This novel effect of ACE inhibitors is accompanied by blocking the Ang II induced activation of several intracellular signal transduction pathways (ERK1/2, p38-MAPK and STAT3).

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