Heart extracellular matrix gene expression profile in the vitamin D receptor knockout mice

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Abstract

1α,25-Dihydroxyvitamin D3 [1,25D] deficiency and vitamin D receptor [VDR] genotypes are risk factors for several diseases and disorders including heart diseases. Extracellular matrix (ECM) remodeling mediated by matrix metalloproteinases [MMPs] contributes to progressive left ventricular remodeling, dilation, and heart failure. In the present study, we used high-density oligonucleotide microarray to examine gene expression profile in wild type [WT] and vitamin D receptor knockout mice (VDR KO) which was further validated by RT-PCR. Microarray analysis revealed tissue inhibitors of metalloproteinases [TIMP-1 and TIMP-3] were significantly under expressed in VDR KO mice as compared to WT mice which was further validated by RT-PCR. Zymography and RT-PCR showed that MMP-2 and MMP-9 were up regulated in VDR KO mice. In addition, cross-sectional diameter and longitudinal width of the VDR KO heart myofibrils showed highly significant cellular hypertrophy. Trichrome staining showed marked increase in fibrotic lesions in the VDR KO mice. Heart weight to body weight ratio showed ∼41% increase in VDR KO mice when compared to WT mice. This data supports a role for 1,25D in heart ECM metabolism and suggests that MMPs and TIMPs expression may be modulated by vitamin D.

Introduction

The biologically active form of vitamin D3, 1,25D has a broad range of physiological effects [1], [2]. This hormone functions through the vitamin D receptor (VDR), a member of the nuclear hormone receptor family, to regulate transcription of target genes. In addition to modulating rates of gene expression, 1,25D also mediates non-genomic and more rapid effects which are absent in cells isolated from VDR-null mice [3], [4].

Previous studies from our and other labs have shown that 1,25D receptor exists in the rat and human heart [5], [6] and vitamin D3 deficiency increases myocardial contractility and fibrosis, and regulates proliferation and hypertrophy in rat cardiomyocytes [7]. Activation of MMP is known to contribute to the degradation of extracellular matrix (ECM), myocardial wall thinning, and left ventricular (LV) dilation [8]. Dysregulated MMP/TIMP production has been demonstrated in the failing heart [9]. It has also been reported that collagen structure and functional properties are altered in cardiomyopathies [10].

In many respects, 1,25D signaling is ideally suited to genomic analysis because VDR is a direct regulator of gene transcription with a well-characterized binding site. In the present study, using microarray analysis and RT-PCR technique, we studied gene regulation in WT and VDR KO mice. Our results provide insights into the range of molecular genetic events underlying the broad physiological actions of 1,25D, including its effects on MMP regulation and collagen formation in heart.

Section snippets

Animal maintenance

All the studies were approved by the institutional animal care committee at University of Michigan. A breeding colony of VDR HET mice (+/−) was established from three mice generously provided by Dr. Marie Demay (Harvard Medical School, Boston, MA). All VDR KO (−/−) and WT mice (+/+) were fed a diet containing 2% calcium, 1.25% phosphate and 20% lactose with 2.2 IU vitamin D3/g (Teklad Diet #TD.96348, Madison, WI). At 12 months, animals were weighed, heparnized (1500 U/kg, i.p.) then after 20 min

Increased heart/body weight ratio in VDR KO mice

The values for the heart to body weight ratio (mg/g) for VDR KO and WT determined at the age of 12 months were mean 6.9 ± 1.03 and 4.9 ± 0.64, respectively (P < 0.05).

VDR KO mice develop cardiac hypertrophy and fibrosis

Fig. 1 shows heart tissue using H&E staining which revealed increased cell size. Trichrome staining showed (blue stain) increased collagen deposition mostly in ECM suggesting cardiac fibrosis in VDR KO mice.

Effect of VDR on mRNA expression of ECM regulatory systems

In the present study, using gene microarray analysis we observed that there were low levels of TIMP-1 and TIMP-3 gene expression in

Discussion

Using the VDR KO mouse, we showed that the 1,25D pathway plays an important role in suppressing MMP expression in cardiac tissue. MMPs are a family of zinc containing metalloproteinases that play an important role in ECM degradation, synthesis and remodeling. Previous studies have shown an increase in MMP-2 and MMP-9 gelatinolytic activity, extensive collagen deposition, and denaturation and ECM remodeling in mice with heart failure [11]. Several studies suggest that a positive correlation

Acknowledgement

This work was supported by the National Institutes of Health Grant #5 RO1-HL074894-02.

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