Doxazosin induces apoptosis of cells expressing hERG K+ channels

Abstract

The antihypertensive drug doxazosin has been associated with an increased risk for congestive heart failure and cardiomyocyte apoptosis. Human ether-a-go-go-related gene (hERG) K(+) channels, previously shown to be blocked by doxazosin at therapeutically relevant concentrations, represent plasma membrane receptors for the antihypertensive drug. To elucidate the molecular basis for doxazosin-associated pro-apoptotic effects, cell death was studied in human embryonic kidney cells using three independent apoptosis assays. Doxazosin specifically induced apoptosis in hERG-expressing HEK cells, while untransfected control groups were insensitive to treatment with the antihypertensive agent. An unexpected biological mechanism has emerged: binding of doxazosin to its novel membrane receptor, hERG, triggers apoptosis, possibly representing a broader pathophysiological mechanism in drug-induced heart failure.

Fig. 1

Doxazosin induces apoptosis in hERG-positive cells. A Fluorescence microphotographs corresponding to TUNEL assays of controls and of cells treated with 30 μM doxazosin (24 h). Green nuclear fluorescence reflects endonucleolytic DNA degradation and induction of apoptosis. Four independent experiments with similar results have been performed. Scale bar, 50 μm. B Mean (± S.E.M.) apoptosis rates (n = 4 independent experiments) in HEK cells assessed by TUNEL staining and cell counting. In the graph, TUNEL-positive cells are expressed as a percentage of the total number of cells (n = 4 independent assays; * indicates p < 0.05 versus untreated cells). Multiple comparisons were performed using one-way ANOVA followed by Bonferroni post hoc testing. C Morphologic changes induced by doxazosin in HEK-hERG cells. Doxazosin-associated apoptotic nuclei show fluorescence and fragmentation (arrow). Nuclei were visualized by staining with Hoechst 33342 dye in untreated cells and after application of 30 μM doxazosin (42 h). Representative fluorescence microphotographs of one typical experiment are shown. Three independent experiments with similar results have been performed. Scale bar, 15 μm .

Fig. 2

A Flow cytometric analysis illustrating time-dependence of pro-apoptotic effects of doxazosin. Cells were treated with 30 μM doxazosin and analyzed for subdiploid DNA content as hallmark for apoptosis using the assay described by Nicoletti et al. (1991). One representative FACS result is shown. B Mean values (± S.E.M) obtained from three experiments (* indicates p < 0.05 versus drug-free conditions at 0 h). Statistical significance was calculated using Student's t tests. C Attenuation of doxazosin block by pore mutations demonstrates direct doxazosin binding to hERG channels. Original current traces are shown, illustrating the effects of doxazosin on wild type or mutant Y652A and F656C currents (1 and 10 μM doxazosin, respectively). D Concentration-response relationships for doxazosin blockade of hERG constructs transiently expressed in HEK cells. Calculated IC₅₀ values are indicated (mean ± S.E.M.). E Pore mutations with attenuated doxazosin binding affinity show reduced apoptosis levels. Mean (± S.E.M.) apoptosis rates (n = 3 independent experiments) in transiently transfected HEK cells after treatment with 30 μM doxazosin (72 h), assessed by FACS analyses as in panel A. Multiple comparisons were performed using one-way ANOVA followed by Bonferroni post hoc testing (* indicates p < 0.05 versus WT+doxazosin). WT, hERG wild type; Y, hERG Y652A; F, hERG F656C.