Vitamin D deficiency induces cardiac hypertrophy and inflammation in epicardial adipose tissue in hypercholesterolemic swine

Abstract

Introduction: Vitamin D is a sectosteroid that functions through Vitamin D receptor (VDR), a transcription factor, which controls the transcription of many targets genes. Vitamin D deficiency has been linked with cardiovascular diseases, including heart failure and coronary artery disease. Suppressor of cytokine signaling (SOCS)3 regulates different biological processes such as inflammation and cellular differentiation and is an endogenous negative regulator of cardiac hypertrophy.

Objective: The purpose of this study was to test the hypothesis that vitamin D deficiency causes cardiomyocyte hypertrophy and increased proinflammatory profile in epicardial adipose tissue (EAT), and this correlates with decreased expression of SOCS3 in cardiomyocytes and EAT.

Methods: Eight female Yucatan miniswine were fed vitamin D-sufficient (900 IU/d) or vitamin D-deficient hypercholesterolemic diet. Lipid profile, metabolic panel, and serum 25(OH)D levels were regularly measured. After 12 months animals were euthanized and histological, immunohistochemical and qPCR studies were performed on myocardium and epicardial fat.

Results: Histological studies showed cardiac hypertrophy, as judged by cardiac myocyte cross sectional area, in the vitamin D-deficient group. Immunohistochemical and qPCR analyses showed significantly decreased mRNA and protein expression of VDR and SOCS3 in cardiomyocytes of vitamin D-deficient animals. EAT from vitamin D-deficient group had significantly higher expression of TNF-α, IL-6, MCP-1, and decreased adiponectin in association with increased inflammatory cellular infiltrate. Interestingly, EAT from vitamin D-deficient group had significantly decreased expression of SOCS3.

Conclusion: These data suggest that vitamin D deficiency induces hypertrophy in cardiomyocytes which is associated with decreased expression of VDR and SOCS3. Vitamin D deficiency is also associated with increased inflammatory markers in EAT. Activity of VDR in the body is controlled through regulation of vitamin D metabolites. Therefore, restoration of VDR function by supplementation of VDR ligands in vitamin D-deficient population might be helpful in reducing inflammation and cardiovascular risk.

Figure 1

The effects of vitamin D-deficient high cholesterol and vitamin D-sufficient high cholesterol diet on (A) serum 25-hydroxy vitamin D, (B) total cholesterol, (C) low density lipoprotein (LDL), and (D) high density lipoprotein (HDL) levels of the female Yucatan miniswine following 12 months of administration of the diet. Data are shown as mean ± SEM (N=8); *P <0.05, * *P< 0.01, * * *P< 0.001.

Figure 2

Histological analysis of left ventricular myocytes from vitamin D-deficient hypercholesterolemic and vitamin D-sufficient hypercholesterolemic swine. Paraffin embedded thin sections were cut, deparaffinized and histological analysis was performed using H&E, and Masson's trichrome stains. (A, B): Masson's trichrome staining of vitamin D-deficient hypercholesterolemic (A) and vitamin D-sufficient hypercholesterolemic swine (B); (C, D): H&E staining of vitamin D-deficient hypercholesterolemic (C) and vitamin D-sufficient hypercholesterolemic swine (D). Note the larger size of cardiomyocytes in vitamin D-deficient hypercholesterolemic group; E: Mean cross-sectional area of cardiomyocytes (±SEM) in vitamin D-deficient hypercholesterolemic and vitamin D-sufficient hypercholesterolemic swine was measured. Two-tailed unpaired Student t tests were performed to determine statistical relevance; significant P value is shown; *P <0.05, * *P< 0.01, * * *P< 0.001. Magnification 600×

Figure 3

Expression of VDR in cardiomyocytes of hypercholesterolemic swine following vitamin D deficiency. A: Total RNA was isolated from cardiac tissue and expression of VDR was detected by quantitative PCR. The fold change in VDR expression between samples was calculated by Fold change= 2 –ΔΔCt method. The results were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (B, C): Immunofluorescence of cardiac VDR expression in vitamin D-deficient hypercholesterolemic (B) and vitamin D-sufficient hypercholesterolemic (C) swine. Sections were stained using mouse anti- VDR antibody and goat anti-mouse cy3 as secondary antibody. DAPI was used to stain the nuclei. DAPI overlay is shown with mouse anti- VDR and goat anti-mouse cy3 as secondary antibody; D: Mean fluorescent intensity of staining obtained with anti-VDR antibody was measured in arbitrary units using NIH Image J software. Two-tailed unpaired student t tests were performed to determine statistical relevance; significant P values are shown. Data are shown as mean ± SEM (N=8); *P <0.05, * *P< 0.01, * * *P< 0.001. Magnifications 200×.

Figure 4

Expression of SOCS3 in cardiomyocytes of hypercholesterolemic swine following vitamin D deficiency. A: Total RNA was isolated from cardiac tissue and expression of SOCS3 was detected by quantitative PCR. The fold change in SOCS3 expression between samples was calculated by Fold change= 2 –ΔΔCt method. The results were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data shown are mean ± SEM (N=8). *P <0.05, * *P< 0.01, * * *P< 0.001. (C, D): Immunohistochemical expression of cardiac SOCS3 in vitamin D-deficient hypercholesterolemic (C) and vitamin D-sufficient hypercholesterolemic (D) swine. Sections were stained with DAB as chromogen and counterstained using hematoxylin. Decreased SOCS3 expression was observed in vitamin D-deficient hypercholesterolemic group. Negative control is shown (B). Magnification 600×

Figure 5

Vitamin D- deficiency mediated increased inflammatory infiltrates and decreased SOCS3 expression in EAT of hypercholesterolemic swine. (A, B): H&E staining of paraffin sections of EAT from both vitamin D-deficient hypercholesterolemic (A) and vitamin D-sufficient hypercholesterolemic swine (B). Arrows indicate inflammatory cells. EAT of vitamin D-deficient hypercholesterolemic swine showed significantly increased inflammatory cellular infiltrate. Magnification (200-600×); (C, D): Immunofluorescence of SOCS3 expression in vitamin D-deficient hypercholesterolemic (C) and vitamin D-sufficient hypercholesterolemic (D) swine EAT. Sections were stained using rabbit anti- SOCS3 antibody and goat anti-rabbit cy3 as secondary antibody. DAPI was used to stain the nuclei. DAPI overlay is shown with rabbit anti-SOCS3 and goat anti-rabbit cy3 as secondary antibody. Decreased expression of SOCS3 was observed in vitamin D-deficient hypercholesterolemic group. Magnification (200×)

Figure 6

Immunohistochemical detection of adipokines in EAT. Sections were stained with DAB as chromogen and counterstained using hematoxylin. (A, B): Expression of TNF-α in EAT of vitamin D-deficient hypercholesterolemic (A) and vitamin D-sufficient hypercholesterolemic swine (B). Increased TNF-α expression (arrows) was observed in vitamin D-deficient hypercholesterolemic group. (C, D): Expression of MCP-1 in EAT of vitamin D-deficient hypercholesterolemic (C) and vitamin D-sufficient hypercholesterolemic swine (D). Increased MCP-1 expression (arrows) was observed in vitamin D-deficient hypercholesterolemic group. Magnification (200 ×- 600×).

Figure 7

Immunohistochemical detection of adipokines in EAT. Sections were stained with DAB as chromogen and counterstained using hematoxylin. (A, B): Expression of IL-6 in EAT of vitamin D-deficient hypercholesterolemic (A) and vitamin D-sufficient hypercholesterolemic swine (B). Increased IL-6 expression (arrows) was observed in vitamin D-deficient hypercholesterolemic group. (C, D): Expression of adiponectin in EAT of vitamin D-deficient hypercholesterolemic (C) and vitamin D-sufficient hypercholesterolemic swine (D). Adiponectin expression (arrows) was higher in vitamin D-sufficient hypercholesterolemic group. Magnification (200 ×- 600×).