Signaling responses after exposure to 5 alpha-dihydrotestosterone or 17 beta-estradiol in norepinephrine-induced hypertrophy of neonatal rat ventricular myocytes

Abstract

Androgens appear to enhance, whereas estrogens mitigate, cardiac hypertrophy. However, signaling pathways in cells for short (3 min) and longer term (48 h) treatment with 17beta-estradiol (E2) or 5 alpha-dihydrotestosterone (DHT) are understudied. We compared the effect of adrenergic stimulation by norepinephrine (NE; 1 microM) alone or in combination with DHT (10 nM) or E2 (10 nM) treatment in neonatal rat ventricular myocytes (NRVMs) by cell area, protein synthesis, sarcomeric structure, gene expression, phosphorylation of extracellular signal-regulated (ERK), and focal adhesion kinases (FAK), and phospho-FAK nuclear localization. NE alone elicited the expected hypertrophy and strong sarcomeric organization, and DHT alone gave a similar but more modest response, whereas E2 did not alter cell size. Effects of NE dominated when used with either E2 or DHT with all combinations. Both sex hormones alone rapidly activated FAK but not ERK. Long-term or brief exposure to E2 attenuated NE-induced FAK phosphorylation, whereas DHT had no effect. Neither hormone altered NE-elicited ERK activation. Longer term exposure to E2 alone reduced FAK phosphorylation and reduced nuclear phospho-FAK, whereas its elevation was seen in the presence of NE with both sex hormones. The mitigating effects of E2 on the NE-elicited increase in cell size and the hypertrophic effect of DHT in NRVMs are in accordance with results observed in whole animal models. This is the first report of rapid, nongenomic sex hormone signaling via FAK activation and altered FAK trafficking to the nucleus in heart cells.

Fig. 1.

Effect of sex hormones on norepinephrine (NE)-induced cardiac hypertrophy. Cells were treated with sex hormones alone, NE alone, or NE with sex hormones for 48 h (protocol 1). E2, 17β-estradiol; DHT, 5α-dihydrotestosterone; NVRMs, neonatal rat ventricular myocytes. A: schematic illustration of protocol 1. B: light microscopy showing cells at ×40 magnification. PE, phenylephrine. C: 2-dimensional cell surface area as a percentage of control. Bars represents means ± SE of determinations from 3 separate experiments. D: NRVMs were treated with E2, DHT, and NE in the presence of 0.5 μCi of [³H]leucine for 48 h. Cells were harvested, and ³H incorporation was determined by liquid scintillation counting. Bars represent mean (±SE) disintegrations per minute (DPM) (n = 3). Except for E2, all treatments are significantly different from control *P < 0.05, significantly different from control (no treatment). #P < 0.05, significantly different from NE. ^@P < 0.05, significant difference between E2 and DHT treatment in either the presence or absence of NE.

Fig. 2.

Effect of sex hormones and/or NE treatment on NRVM sarcomeric organization. Cells were treated with sex hormones alone, NE alone, or NE with sex hormones for 48 h (protocol 1). Confocal micrographs of myocytes after incubation with E2, DHT, and/or NE for 48 h. Actin is visualized with FITC-conjugated phalloidin stain (green), and the nucleus is visualized by 4′,6′-diamidino-2-phenylindole staining (blue).

Fig. 3.

Effect of sex hormones on NE-elicited alterations in expression of marker hypertrophic genes. NRVMs were treated with sex hormones alone, NE alone, or NE with sex hormones for 48 h (protocol 1) and then harvested to determine levels of brain natriuretic peptide (BNP), atrial natriuretic peptide (ANP), skeletal actin, and β-myosin heavy chain (β-MHC) mRNA by real-time RT-PCR. Bars represent mean (±SE) mRNA as a percentage of control value from 4 separate experiments. *P < 0.05, significantly different from control. #P < 0.05, significantly different from NE alone.

Fig. 4.

Effect of long-term (48 h) sex hormone exposure on rapid (2 min) NE-elicited alterations in ERK and focal adhesion kinase (FAK) phosphorylation. NRVMs were incubated with either E2 or DHT for 48 h and then stimulated with NE (or diluent, control) for 2 min (protocol 2). A: schematic illustration of protocol 2. B: typical Western blots. C and D: mean (±SE) phosphorylated total ERK (pERK; C) and phosphorylated FAK (pFAK; D) normalized to GADPH and expressed as a percentage of control from 3 separate experiments. *P < 0.05, significantly different from control. #P < 0.05, significantly different from NE alone. @P < 0.05, significant difference between sex hormone treatments.

Fig. 5.

Effect of short-term sex hormone exposure on NE-elicited ERK and FAK phosphorylation. One minute after exposure to sex hormones, NRVMs were stimulated with NE or diluent for 2 min (protocol 3). A: schematic illustration of protocol 3. B: typical Western blots. C and D: mean (±SE) phosphorylated total ERK (C) or FAK (D) normalized to GADPH and expressed as a percentage of control from 3 separate experiments. *P < 0.05, significantly different from control. #P < 0.05, significantly different from NE alone.

Fig. 6.

Effect of sex hormone and NE treatment on FAK phosphorylation and localization. Distribution of phosphorylated FAK was examined in NRVMs treated with sex hormones alone, NE alone, or NE with sex hormones for 48 h (protocol 1). A: schematic illustration of protocol 1. B: distribution of phosphorylated FAK after 48 h of treatment with NE alone and sex hormone in the presence or absence of NE. Images are confocal micrographs of immunostained NRVMs. Cell nuclei are stained blue, phosphorylated FAK (Y397) is immunostained red, and actin is stained green.