The Protective Effect of 1,25(OH)2D3 on Myocardial Function is Mediated via Sirtuin 3-Regulated Fatty Acid Metabolism

Figures

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  FIGURE 1. General graphical outline of the experimental design.

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  FIGURE 2. Effect of vitamin D on energy metabolism in mice fed a high-fat diet. (A) Body weight changes in control or KO mice receiving CCE or a high-fat diet for 14 weeks (*p < 0.05 or **p < 0.01). (B) Food intake, (C) water intake, and (D) body temperature in each group were monitored for 5 days, and no changes were observed in all groups (p > 0.05). (E) O2 consumption and (F) respiratory exchange rate indicated a lower metabolic rate in the CCE + HF group than in the HF group. Serum lipid levels analysis: (G) free fatty acids, (H) serum calcium, and (I) serum potassium, with relative changes shown for all groups (*p < 0.05 vs. Ctrl, #p < 0.05 vs. HF). Values are expressed as the mean ± SEM. Ctrl, control group. CCE, Fed cholecalciferol cholesterol emulsion [a precursor of 1, 25(OH)2D3] group. HF, Fed high fat die group. CCE + HF, Fed cholecalciferol cholesterol emulsion [a precursor of 1, 25(OH)2D3] and high fat die group. KO, Cyp27b1–/– mice, KO + HF, Cyp27b1–/– mice fed with high fat die group.

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  FIGURE 3. Effect of vitamin D on lipid metabolism and cardiac function in vivo. (A) Western blot of lipid metabolism-associated proteins. (B) The left ventricular pressure-volume loop analysis revealed that heart contractility of Cyp27b1–/– mice was lower than the baseline. (C) Heart weight normalized to mouse body weight in animals fed a high-fat diet with or without CCE. (D) Invasive blood pressure measured via the carotid artery was lower in the CCE + HF group than the HF group (*p < 0.05, **p < 0.01, ***p < 0.001). (E) The curves of ECG and myocardial contractility were recorded for each group. Ctrl, control group. CCE, Fed cholecalciferol cholesterol emulsion [a precursor of 1, 25(OH)2D3] group. HF, Fed high fat die group. CCE + HF, Fed cholecalciferol cholesterol emulsion [a precursor of 1, 25(OH)2D3] and high fat die group. KO, Cyp27b1–/– mice. KO + HF, Cyp27b1–/– mice fed with high fat die group.

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  FIGURE 4. Vitamin D increased the expression of SIRT3 and mitochondrial complexes in high-fat diet-fed mice. (A,B) RT-qPCR analysis of sirtuins, VDR, and 1α(OH)ase revealed that the expression levels of Sirt3, VDR, and 1α(OH)ase were decreased in the KO group and increased following CCE treatment. Values are expressed as the mean ± SEM, *p < 0.05, **p ≤ 0.01, and ***p ≤ 0.001. (C) Western blotting analysis of SIRT3, LCAD, and VDR protein levels. (D) Electron microscopy images indicated mitochondrial arrangement and morphological characteristics (12,000×). Aberrant mitochondrial structures are indicated by a black arrow. (E) Western blotting indicated that CCE upregulated mitochondrial complex I, III, and IV protein expression. Serum levels of (F) MDA and (G) SOD with relative changes shown for the CCE + HF group compared with the HF group (#p < 0.05 vs. Ctrl, *p < 0.05 vs. HF). Ctrl, control group. CCE, Fed cholecalciferol cholesterol emulsion [a precursor of 1, 25(OH)2D3] group. HF, Fed high fat die group. CCE + HF, Fed cholecalciferol cholesterol emulsion [a precursor of 1, 25(OH)2D3] and high fat die group. KO, Cyp27b1–/– mice. KO + HF, Cyp27b1–/– mice fed with high fat die group.

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  FIGURE 5. VD/VDR regulated lipid metabolism and mitochondrial respiratory metabolism via SIRT3 to protect HL-1 cells against oxidative damage. (A,B) representative Oil Red O staining in cells treated with 1,25(OH)2D3, oleic acid (OA), and AGK7 (SIRT3 inhibitor) (original magnification: 400×). *p < 0.05, ##p < 0.01. (C) Protein levels of SIRT3, LCAD, VDR, complexes I, III, and IV in HL-1 cells treated with 1,25(OH)2D3, OA, and AGK7 were analyzed via western blot. Analysis of (D) MDA and (E) SOD with relative changes shown for the 1,25(OH)2D3 + OA group compared with the OA group (*p < 0.05 vs. OA). (F,G) Western blot analysis of SIRT3, LCAD, complexes I, III and IV in cells with and/or without 1,25(OH)2D3, knockout of Sirt3 via CRISPR/Cas9, and OA, indicated that VD/VDR did not regulate fatty acid metabolism in the absence SIRT3.OA, oleic acid. PX458-Sirt3, knockout of Sirt3 via CRISPR/Cas9. AGK7, SIRT3 inhibitor.

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  FIGURE 6. VD/VDR regulated energy substrate homeostasis in HL-1 cells via SIRT3. (A) Analysis of oxygen consumption rate (OCR) in the mitochondria of HL-1 cells treated with 1,25(OH)2D3 and OA. (B,D) The analysis of OCR in HL-1 cells treated with 1,25(OH)2D3, OA, and AGK7, indicating that VD treatment had no effect in the absence of SIRT3 *p < 0.05, **p < 0.01, ##p ≤ 0.01. (C) ATP turnover was determined based on the OCR prior to oligomycin minus the OCR after oligomycin, indicating that mitochondrial respiration was decreased, and ATP was increased in the 1,25(OH)2D3 + OA-treated cells compared with the OA-treated cells. (E,F) The analysis of extracellular acidification rate (ECAR) in HL-1 cells treated with 1,25(OH)2D3, OA, and AGK7. Ctrl, control group. OA, oleic acid. AGK7, SIRT3 inhibitor.

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  FIGURE 7. The binding of VDR to the VDRE in the promoter region of Sirt3 increased Sirt3 transcriptional activity. (A) Luciferase assay analysis indicated that 1,25(OH)2D3 increased Sirt3 transcriptional activity, *p < 0.05. (B,C) PCR and RT-qPCR analysis of chromatin immunoprecipitation confirmed that VD promoted the binding of VDR to the VDRE in the Sirt3 promoter, *p < 0.05.

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  FIGURE 8. Schematic model for the role of vitamin D in the regulation of cardiac fatty acid metabolism. The VDR activated by CCE and calcitriol upregulated the expression of Sirt3 through transcriptional regulation. SIRT3 inhibits the expression of LCAD and stimulates that of mitochondrial complexes through the use of common substrate NAD+, resulting in decreased fatty acid β-oxidation. VDR does not affect the inflow (Fat/CD36) and outflow (MTTP) of fatty acids, and ultimately suppresses CPT-1β to reduce the uptake of fatty acids in the mitochondria. In addition, to balance energy substrate use, VDR upregulates GLUT4, thus, promoting glucose metabolism. These changes lead to a balance of glucose and fatty acid metabolism in myocardial cells, which is beneficial to myocardial function.