Doxazosin Stimulates Galectin-3 Expression and Collagen Synthesis in HL-1 Cardiomyocytes Independent of Protein Kinase C Pathway

Abstract

Doxazosin, a drug commonly prescribed for hypertension and prostate disease, increases heart failure risk. However, the underlying mechanism remains unclear. Galectin-3 is an important mediator that plays a pathogenic role in cardiac hypertrophy and heart failure. In the present study, we investigated whether doxazosin could stimulate galectin-3 expression and collagen synthesis in cultured HL-1 cardiomyocytes. We found that doxazosin dose-dependently induced galectin-3 protein expression, with a statistically significant increase in expression with a dose as low as 0.01 μM. Doxazosin upregulated collagen I and α-smooth muscle actin (α-SMA) protein levels and also induced apoptotic protein caspase-3 in HL-1 cardiomyocytes. Although we previously reported that activation of protein kinase C (PKC) stimulates galectin-3 expression, blocking the PKC pathway with the PKC inhibitor chelerythrine did not prevent doxazosin-induced galectin-3 and collagen expression. Consistently, doxazosin treatment did not alter total and phosphorylated PKC. These results suggest that doxazosin-stimulated galectin-3 is independent of PKC pathway. To determine if the α1-adrenergic pathway is involved, we pretreated the cells with the irreversible α-adrenergic receptor blocker phenoxybenzamine and found that doxazosin-stimulated galectin-3 and collagen expression was similar to controls, suggesting that doxazosin acts independently of α1-adrenergic receptor blockade. Collectively, we show a novel effect of doxazosin on cardiomycytes by stimulating heart fibrosis factor galectin-3 expression. The mechanism of action of doxazosin is not mediated through either activation of the PKC pathway or antagonism of α1-adrenergic receptors.

Keywords: adrenergic receptor; cardiac fibrosis; collagen; protein kinase C.

FIGURE 1

Effect of doxazosin on galectin-3 expression. (A) HL-1 cardiomyocytes were treated with doxazosin at doses of 0, 0.01, 0.1, and 10 μM for 12 h. (B) HL-1 cells were incubated with 0.1 μM doxazosin (Doxa) or 0.1 μM prazosin (Prazo) for 12 h. Cells were collected and galectin-3 expression was analyzed by Western blot. Experiments were performed in triplicate and data is shown as the mean ± SD (n = 3, ^∗∗p < 0.01 compared to control).

FIGURE 2

Effect of doxazosin on collagen I and α-SMA expression. HL-1 cardiomyocytes were treated with doxazosin at increasing doses for 12 h. Collagen Iα1 and α-SMA expression was analyzed by Western blot and data is shown as the mean ± SD (n = 3, ^∗∗p < 0.01 compared to control).

FIGURE 3

Protein kinase C (PKC) is not involved in doxazosin-stimulated collagen and galectin-3 expression. HL-1 cardiomyocytes were pre-treated with 2 μM chelerythrine (Chel) for 30 min then treated with 1 μM doxazosin for 12 h. Galectin-3, collagen I, and PKC were assessed by Western blot with appropriate antibodies.

FIGURE 4

Effect of doxazosin on PKC protein expression. HL-1 cardiomyocytes were treated with 1 μM doxazosin for 12 h, total PKC-α protein and phosphorylated PKC-α at Thr497, Thr638 and Thr641 were analyzed by immunoblotting with appropriate antibodies. Experiments were performed in triplicate and data is shown as the mean ± SD. Statistics show no significance of doxazosin treated groups compared to control.

FIGURE 5

Doxazosin induces cell apoptosis in HL-1 cardiomyocytes. HL-1 cells were treated with doxazosin at increasing concentrations for 12 h, and the apoptotic protein caspase-3 was examined by Western blot. Caspase-3 signals were quantified and is shown as the mean ± SD (^∗p < 0.05, ^∗∗p < 0.01 compared to control, n = 3).

FIGURE 6

Effect of α1-adrenergic receptor blocker on doxazosin-stimulated galectin-3 and collagen expression. HL-1 cells were pre-treated with phenoxybenzamine (Phen) for 4 h then treated with doxazosin (Doxa) for 12 h. Galectin-3 and collagen I were analyzed by Western blot. Signals were quantified as the mean ± SD (^∗∗p < 0.01 compared to control without Doxa treatment, n = 3).