Effects and mechanisms of apolipoprotein A-V on the regulation of lipid accumulation in cardiomyocytes

Abstract

Background: Apolipoprotein (apo) A-V is a key regulator of triglyceride (TG) metabolism. We investigated effects of apoA-V on lipid metabolism in cardiomyocytes in this study.

Methods: We first examined whether apoA-V can be taken up by cardiomyocytes and whether low density lipoprotein receptor family members participate in this process. Next, triglyceride (TG) content and lipid droplet changes were detected at different concentrations of apoA-V in normal and lipid-accumulation cells in normal and obese animals. Finally, we tested the levels of fatty acids (FAs) taken up into cardiomyocytes and lipid secretion through [¹⁴C]-oleic acid.

Results: Our results show that heart tissue has apoA-V protein, and apoA-V is taken up by cardiomyocytes. When HL-1 cells were transfected with low density lipoprotein receptor (LDLR)-related protein 1(LRP1) siRNA, apoA-V intake decreased by 53% (P<0.05), while a 37% lipid accumulation in HL-1 cells remain unchanged. ApoA-V localized to the cytoplasm and was associated with lipid droplets in HL-1 cells. A 1200 and 1800 ng/mL apoA-V intervention decreased TG content by 28% and 45% in HL-1 cells, respectively and decreased TG content by 39% in mouse heart tissue (P<0.05). However, apoA-V had no effects on TG content in either normal HL-1 cells or mice. The levels of FAs taken up into cardiomyocytes decreased by 43% (P < 0.05), and the levels of TG and cholesterol ester secretion increased by 1.2-fold and 1.6-fold, respectively (P < 0.05).

Conclusion: ApoA-V is a novel regulator of lipid metabolism in cardiomyocytes.

Keywords: Apolipoprotein A-V; HL-1 cells; Lipid droplets; Triglyceride.

Fig. 1

Expression of apoA-V in both normal and obese mice. Normal and obese mice were sacrificed, and the heart tissue was rapidly excised. Western blot (A and B) and immunohistochemistry (C) results show that heart tissues from both groups contained apoA-V. Compared to normal mice, in the heart tissue of obese mice, the amount of apoA-V was increased. Liver tissues were used as a positive control. (C-a: immunohistochemistry image of heart tissue; C-b: immunohistochemistry images of liver tissue)

Fig. 2

Endocytosis of apoA-V in both normal and lipid-accumulated HL-1 cells. A Subcellular localization of apoA-V detected by fluorescence and confocal microscopy. ApoA-V was localized within HL-1 cells with (A-c) or without (A-b) lipid accumulation (scale bar: 25 μm; nuclei: blue; apoA-V: green; A-a: normal HL-1 cells without apoA-V). B The [¹²⁵I] radioactivity detection results of [¹²⁵I]-apoA-V in HL-1 cells. In normal HL-1 cells, ~ 20% of [¹²⁵I]-apoA-V uptake remained intracellular over a 24 h chase period, while 80% was degraded (B-a); however, in HL-1 cells with lipid accumulation, ~ 44% of [¹²⁵I]-apoA-V uptake remained intracellular, and 56% was degraded over a 24 h chase period (B-b). C Western blot analysis of the uptake of apoA-V by HL-1 cells. β-actin served as a loading control

Fig. 3

LDLR family members participate in endocytosis of apoA-V in HL-1 cells. A Determination of the optimum transfection concentration of LRP1 siRNA by Western blot analysis. B Radioactivity changes of [¹²⁵I]-apoA-V in HL-1 cells after transfection of LRP1 siRNA. Data are shown as the mean ± SE of values from three independent experiments. C Western blot analysis of the effect of LRP1 on the endocytosis of apoA-V by HL-1 cells. β-actin served as a loading control

Fig. 4

Association of apoA-V and lipid droplets in HL-1 cells. HL-1 cells were incubated with (b) or without (a) 0.5 mM oleic acid for 24 h. (scale bar 25 μm; lipid droplets: red; nuclei: blue; apoA-V: green)

Fig. 5

a Effect of apoA-V on the TG content in normal HL-1 cells. b Lipid droplet morphology visualized by fluorescence and confocal microscopy in normal HL-1 cells. c Effect of apoA-V on TG content in HL-1 cells with lipid accumulation. d Lipid droplet morphology visualized by fluorescence and confocal microscopy in normal HL-1 cells. Intervention with 1800 ng/ml of apoA-V significantly decreased the number of intracellular lipid droplets compared with the control group (without apoA-V intervention) but had no effect on the size of lipid droplets in HL-1 cells with lipid accumulation. (Confocal microscopy images were recorded at 1000× and 200× magnification, and scale bars are 25 and 75 μm, respectively; lipid droplets: red; nuclei: blue)

Fig. 6

A Immunohistochemistry images of heart tissue from normal mice with (A-c) or without (A-b) the apoA-V adenovirus transfection. Controls were treated with PBS buffer (A-a). B Lipid accumulation in the heart tissue with (B-c) or without (B-b) apoA-V adenovirus transfection was examined by Oil Red Staining. Controls were treated with PBS buffer (B-a) (40× magnification). C TG content in heart tissue of normal mice with or without the apoA-V adenovirus transfection was examined with a triglyceride quantification kit; data are shown as the mean ± SE. *p < 0.05 vs. control

Fig. 7

A Immunohistochemistry images of heart tissue from obese mice with (A-c) or without (A-b) apoA-V adenovirus transfection. Controls were treated with PBS buffer (A-a). B Western blot analysis of apoA-V levels in the heart tissue of control obese mice and those transfected with the apoA-V adenovirus. C Lipid accumulation in the heart tissue with (C)-c or without (C-b) apoA-V adenovirus transfection was examined by Oil Red Staining. Controls were treated with PBS buffer (C-a) (40× magnification). D TG content in the heart tissue of obese mice with or without apoA-V adenovirus transfection was examined with a Triglyceride Quantification Kit; data are shown as the mean ± SE. *p < 0.05 vs. control

Fig. 8

a mRNA expression levels involved in fatty acid and triglyceride metabolism in lipid-accumulated HL-1 cells with or without apoA-V treatment. b mRNA expression levels involved in fatty acid and triglyceride metabolism in the heart tissue of obese mice with or without the apoA-V transfection. c Western blot analysis of PPARα, PPARγ, MTP, and CD36 protein levels in HL-1 cells and mouse heart tissue

Fig. 9

a Measurement of fatty acid uptake in HL-1 cells with or without 1800 ng/mL apoA-V intervention. b Measurement of TG and cholesterol ester secretion from HL-1 cells with or without 1800 ng/mL apoA-V intervention, *p < 0.05 vs. control