Suppression of collagen production in norepinephrine stimulated cardiac fibroblasts culture: differential effect of alpha and beta-adrenoreceptor antagonism.

OBJECTIVES: Prolonged sympathetic activation is damaging to the heart and experimental norepinephrine (NE) infusion induces the deposition of myocardial collagen. This study investigated the effects of NE on collagen and transforming growth factor-beta1 (TGF-beta1) gene expression in rat cardiac fibroblasts (CF) culture, and compared the anti-fibrotic effect of alpha and beta (both selective and non-selective adrenergic receptor antagonists) receptors.

METHODS AND RESULTS: Rat CF were cultured in the presence of NE (0.01 to 100 muM) for 24 hours. Procollagen types I and III as well as TGF-beta1 gene expressions were measured by real-time quantitative PCR method. Collagen protein level was measured by Sirius red-based colorimetric method and Western blot analysis. The optimal dose of NE on fibrogenesis was 0.1 muM. Incubation for 24 hours increased procollagen I, III and TGF-beta1 gene expression by 1.35 +/- 0.23, 1.26 +/- 0.16 and 1.35 +/- 0.21 fold, respectively (all p < 0.05). The collagen protein was increased by both Sirius-red assay (0.120 +/- 0.03 vs 0.093 +/- 0.04 microg/total microg of protein, p < 0.05) and Western blot analysis (1.29 +/- 0.26 fold, p < 0.05), when compared with the control group. Addition of carvedilol (a non-selective beta-blocker with alpha-blockage activity) inhibited the effect of NE on procollagen I (0.64 +/- 0.17 vs 1.28 +/- 0.08 fold, p < 0.01), procollagen III gene (0.47 +/- 0.16 vs 1.45 +/- 0.29 fold, p < 0.01) and collagen protein expressions (0.99 +/- 0.12 vs 1.26 +/- 0.31 fold, p < 0.05, respectively). Doxazosin (an alpha-blocker) also inhibited the effect of NE on procollagen I (0.88 +/- 0.30 vs 1.28 +/- 0.08 fold, p < 0.01), procollagen III gene (0.64 +/- 0.13 vs 1.45 +/- 0.29 fold, p < 0.01) and collagen protein expression (0.90 +/- 0.11 vs 1.26 +/- 0.31 fold, p < 0.01 respectively). Such inhibitory effects were not seen in metoprolol (a beta1-selective blocker) and propranolol (a non-selective beta blocker). Furthermore, all the 4 drugs were unable to inhibit the NE induced TGF-beta1 gene over-expression.

CONCLUSIONS: In conclusion, NE increased collagen gene and protein expressions in CF culture. This effect is likely mediated through alpha-receptor as they were normalized by pretreatment with carvedilol and doxazosin, but not beta-blockers such as propranolol and metoprolol. Also, TGF-beta1 doesn't seem to play a role in carvedilol inhibition of NE induced fibrogenesis.