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. 2008 Dec;52(6):1106-12.
doi: 10.1161/HYPERTENSIONAHA.108.119602. Epub 2008 Oct 20.

Expression of the Vitamin D Receptor Is Increased in the Hypertrophic Heart

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Free PMC article

Expression of the Vitamin D Receptor Is Increased in the Hypertrophic Heart

Songcang Chen et al. Hypertension. .
Free PMC article

Abstract

The liganded vitamin D receptor (VDR) is thought to play an important role in controlling cardiac function. Specifically, this system has been implicated as playing an antihypertrophic role in the heart. Despite this, studies of VDR in the heart have been limited in number and scope. In the present study, we used a combination of real-time polymerase chain reaction, Western blot analysis, immunofluorescence, and transient transfection analysis to document the presence of functional VDR in both the myocytes and fibroblasts of the heart, as well as in the intact ventricular myocardium. We also demonstrated the presence of 1-alpha-hydroxylase and 24-hydroxylase in the heart, 2 enzymes involved in the synthesis and metabolism of 1,25 dihydroxyvitamin D. VDR is shown to interact directly with the human B-type natriuretic peptide gene promoter, a surrogate marker of the transcriptional response to hypertrophy. Of note, induction of myocyte hypertrophy either in vitro or in vivo leads to an increase in VDR mRNA and protein levels. Collectively, these findings suggest that the key components required for a functional 1,25 dihydroxyvitamin D-dependent signaling system are present in the heart and that this putatively antihypertrophic system is amplified in the setting of cardiac hypertrophy.

Figures

Figure 1
Figure 1
ET increases VDR protein and mRNA expression in cardiac myocytes and fibroblasts. ET stimulates VDR protein expression in a dose-dependent fashion in cardiac myocytes (n=3–5) (A) and fibroblasts (n=4) (B). (C) VDR immunoreactivity was blocked by VDR competing peptide (CP). The protein from cardiac myocytes (CM), fibroblasts (F), heart tissue (H), and inner medullary collecting duct (IMCD) cells were transferred onto membranes and incubated with VDR antibody alone or with the antibody-competing peptide mixture. (D) ET (10−7 mol/L) increases VDR mRNA levels in cardiac myocytes and fibroblasts. Quiescent cells were treated with ET for 14 hours and total RNA was collected (n=3). *P<0.05, **P<0.001 vs. control.
Figure 2
Figure 2
Immunocytochemistry of VDR in cardiac cells. VDR was visualized by immunofluorescence in cardiac myocytes (100×, oil immersion) and in cardiac fibroblasts (60×). (A) Cardiac myocytes were double stained with a polyclonal anti-VDR antibody, visualized with Cy3 (red) conjugated anti-rabbit antibody, and sarcomere-staining, polyclonal anti-α-actinin (green), visualized by AG488 conjugated anti-mouse antibody. VDR co-localized to the nucleus, stained with DAPI (blue). (B) Cardiac fibroblasts were double stained with anti- VDR, visualized with Cy3 conjugated anti-rabbit antibody, and anti-vimentin, visualized with AG488 conjugated anti-mouse antibody. VDR co-localized to the nucleus stained with DAPI (n=3).
Figure 3
Figure 3
VD3 inhibits fibroblast proliferation and procollagen I synthesis. (A) VD3 reduces [3H]-thymidine incorporation in cultured ventricular fibroblasts. Values are normalized to controls in each experiment (n=4). (B) VD3 inhibits procollagen I synthesis in cultured ventricular fibroblasts. Cells were treated with VD3 for 48 hours in SS media. Procollagen I protein was detected by Western blot (n=4). Values are normalized to control in each experiment. *P<.05 vs. control.
Figure 4
Figure 4
VD3, 25-hydroxyvitamin D, paracalcitol and active hectorol inhibit -1595 BNP promoter activity in cardiomyocytes. (A) -1595 BNP-Luc and Renilla-Luc were cotransfected with VDR/RXR expression vectors into the myocytes. Twenty-four hours following transfection, cells were treated with different doses of VD3, paracalcitol, active hectorol or vehicle for 48 hours and ET for 24 hours prior to collecting cells (n=3). BNP luciferase activity was normalized for Renilla luciferase activity. (B) Ventricular myocytes were transfected with -1595 BNP-Luc, Renilla-Luc and VDR/RXR expression vectors for 24 hours and then treated with different doses of 25-hydroxyvitamin D for 48 hours (n = 5). (C) 1-α-hydroxylase expressed in cardiomyocytes as assessed by Western blot and real time PCR (n=3). *P<0.05, **P<0.01 vs. Control; +P<0.01 vs. ET alone.
Figure 5
Figure 5
Liganded VDR inhibits hBNP promoter activity through direct interaction. (A) -198 hBNP-Luc and Renilla-Luc were co-transfected with or without VDR/RXR expression vectors into cardiac myocytes. Twenty four hours post-transfection, cells were treated with vehicle or VD3 for 48 hours. BNP/Renilla activities were measured and pooled data (n=3) are shown. *P<0.05, **P<0.01 vs. Control. (B) Myocytes were transfected with -198 hBNP luciferase or pcDNA3 plasmid. Cells were treated with VD3 followed by ET. DNA immunoprecipitation assay was carried out as described in Methods (n=3).
Figure 6
Figure 6
Isoproterenol (ISO) induces cardiac hypertrophy and stimulates VDR expression in the Wistar rat. (A) Rats were infused with ISO or vehicle for 7 days. Animals were euthanized and body weight (BW), left ventricular weight (LVW), and tibial length (LT) were measured (n=7). (B) Left ventricular samples from control and ISO-infused rats were homogenized in extraction buffer and total RNA was isolated. ANP mRNA/GAPDH mRNA levels were measured by real-time PCR (n=5). (C) VDR expression assessed by Western blot and real time PCR. Representative immunoblot and pooled data (n=7) for VDR protein and mRNA measurements are presented. (D) 24-hydroxylase and 1-α-hydroxylase expression assessed by Western blot (n=7). **P<0.01 vs. control.

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