Sirt1 attenuates calcific aortic valve disease by inhibiting ferroptosis and enhancing mitochondrial function via the activation of the Nrf2/HO‑1/FTH1 axis

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  Figure 1. Downregulation of Sirt1 in calcified aortic valve tissues and osteogenic-induced aortic valve interstitial cells. (A) RT-qPCR analysis of Sirt1 expression levels in aortic valve tissues from the CAV and normal groups. (B) Western blot analysis of Sirt1 expression levels in aortic valve tissues from the CAV and Normal groups. (C and D) RT-qPCR and western blot analysis of Sirt1 expression levels in hAVICs under standard culture (control) and osteogenic medium (OM) stimulation. Data represent the mean ± SD from three independent experiments. Statistical significance was determined using an unpaired two-tailed Student's t-test. **P<0.01 vs. normal/control. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; CAV, calcific aortic valve; Sirt1, sirtuin 1; hAVICs, human aortic valve interstitial cells.

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  Figure 2. Overexpression of Sirt1 in vitro suppresses the calcification of osteogenically-induced aortic valve interstitial cells. (A) Western blot analysis of Sirt1 protein expression in each treatment group; (B) ALP staining illustrating ALP activity in each group; (C) Alizarin Red S staining reflecting calcified nodule formation in all groups; (D) western blot analysis of RUNX2 protein expression in nuclear and cytoplasmic fractions; (E) reverse transcription-quantitative polymerase chain reaction analysis of osteogenic markers RUNX2, OPN, osterix and ALP expression levels in all groups. Data represent the mean ± SD from three independent experiments. Comparisons were performed using one-way ANOVA. **P<0.01 vs. control; ##P<0.01 vs. OM + vector. Sirt1, sirtuin 1; ALP, alkaline phosphatase; RUNX2, RUNX family transcription factor 2; OPN, osteopontin; OM, osteogenic medium; oe, overexpression.

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  Figure 3. Overexpression of Sirt1 enhances mitochondrial function in osteogenically-induced aortic valve interstitial cells. (A and B) Fluorescent probes were used to detect (A) intracellular ROS and (B) mitochondrial ROS levels. (C) JC-1 staining was applied to assess mitochondrial membrane potential (JC-1). (D) Reverse transcription-quantitative polymerase chain reaction was used to measure the mtDNA content. (E) Intracellular ATP levels were detected using a luminescence-based ATP assay; (F-J) ELISA was performed to evaluate the expression of mitochondrial function-related proteins (PGC1α, MFN2, OPA1, FIS1 and DRP1). Data represent the mean ± SD from three independent experiments. Comparisons were performed using one-way ANOVA. **P<0.01 vs. control; ##P<0.01 vs. OM + vector. ROS, reactive oxygen species; mtDNA, mitochondrial DNA; ATP, adenosine triphosphate; PGC1α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; MFN2, mitofusin 2; OPA1, optic atrophy 1; FIS1, fission 1; DRP1, dynamin-related protein 1; Sirt1, sirtuin 1; OM, osteogenic medium; oe, overexpression.

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  Figure 4. Overexpression of Sirt1 inhibits ferroptosis in osteogenically-induced aortic valve interstitial cells. (A-D) Biochemical assays were used to quantify intracellular (A) MDA, (B), (C) Fe2+, and (D) total iron levels. (E and F) Western blot analysis was performed to evaluate the expression of ferroptosis-related proteins, including GPX4, ACSL4, SLC7A11 and FTH1. Data represent the mean ± SD from three independent experiments. Comparisons were performed using one-way ANOVA. **P<0.01 vs. control; ##P<0.01 vs. OM + vector. MDA, malondialdehyde; GSH, glutathione; Fe2+, ferrous ion; GPX4, glutathione peroxidase 4; ACSL4, acyl-CoA synthetase long-chain family member 4; SLC7A11, solute carrier family 7 member 11; FTH1, ferritin heavy chain 1; Sirt1, sirtuin 1; OM, osteogenic medium; oe, overexpression.

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  Figure 5. Overexpression of Sirt1 activates the Nrf2/HO-1 pathway in osteogenically-induced aortic valve interstitial cells. (A and B) Western blot analysis of KEAP1, HO-1 and NQO1 protein levels in cells from each group. (C and D) Cytoplasmic and nuclear fractions were isolated to evaluate Nrf2 protein localization using western blot analysis. Data represent the mean ± SD from three independent experiments. Comparisons were performed using one-way ANOVA. **P<0.01 vs. control; ##P<0.01 vs. OM + vector. KEAP1, Kelch-like ECH-associated protein 1; HO-1, heme oxygenase 1; NQO1, NAD(P)H quinone dehydrogenase 1; Sirt1, sirtuin 1; OM, osteogenic medium; oe, overexpression.

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  Figure 6. Inhibition of Nrf2 signaling or induction of ferroptosis reverses the inhibitory effect of Sirt1 overexpression on osteogenic differentiation and mineralization in hAVICs. (A) ALP and (B) Alizarin Red S staining were used to assess ALP activity and bone mineralization. Data represent the mean ± SD from three independent experiments. Comparisons were performed using one-way ANOVA. **P<0.01 vs. OM + vector; ##P<0.01 vs. OM + oe-Sirt1. ALP, alkaline phosphatase.

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  Figure 7. Inhibition of Nrf2 signaling or induction of ferroptosis reverses the molecular protective effects of Sirt1 overexpression in hAVICs. (A) Reverse transcription-quantitative polymerase chain reaction was used to evaluate the expression of the osteogenic markers, RUNX2, OPN, osterix and ALP; (B) Mitochondrial function-related proteins PGC1α, MFN2, OPA1, FIS1 and DRP1 were measured using ELISA; (C) Western blot analysis of ferroptosis-related proteins (GPX4, ACSL4, SLC7A11 and FTH1). Data represent the mean ± SD from three independent experiments. Comparisons were performed using one-way ANOVA. **P<0.01 vs. OM + vector; ##P<0.01 vs. OM + oe-Sirt1. ALP, alkaline phosphatase; RUNX2, RUNX family transcription factor 2; OPN, osteopontin; PGC1α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; MFN2, mitofusin 2; OPA1, optic atrophy 1; FIS1, fission 1; DRP1, dynamin-related protein 1; GPX4, glutathione peroxidase 4; ACSL4, acyl-CoA synthetase long-chain family member 4; SLC7A11, solute carrier family 7 member 11; FTH1, ferritin heavy chain 1. ML385, Nrf2 inhibitor; erastin, ferroptosis inducer; Sirt1, sirtuin 1; OM, osteogenic medium; oe, overexpression.

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  Figure 8. HO-1 and FTH1 contribute to the Sirt1-mediated inhibition of osteogenic differentiation and calcification in hAVICs. (A) Alizarin Red S staining and (B) ALP staining were performed to assess mineralization and osteogenic activity in each group; (C) RT-qPCR analysis of Sirt1, HO-1 and FTH1 mRNA expression levels in the different groups; (D) RT-qPCR analysis of osteogenic markers RUNX2, OPN, osterix and ALP expression levels. Data represent the mean ± SD from three independent experiments. Comparisons were performed using one-way ANOVA. **P<0.01 vs. OM + vector + shNC; #P<0.05 and ##P<0.01 vs. OM + oe-Sirt1+ shNC. RT-qPCR, reverse transcription-quantitative PCR; OM, osteogenic medium; Sirt1, sirtuin 1; HO-1, heme oxygenase 1; FTH1, ferritin heavy chain 1; RUNX2, RUNX family transcription factor 2; OPN, osteopontin; ALP, alkaline phosphatase.

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  Figure 9. Activation of Sirt1 in vivo attenuates calcification-associated pathological remodeling in Apoe−/− mice. (A) H&E staining for examining the pathological damage of aortic valve tissues from all groups; (B) reverse transcription-quantitative PCR analysis of osteogenic marker (RUNX2, OPN, osterix and ALP) expression levels in aortic valve tissues; (C) western blot analysis of KEAP1, HO-1 and NQO1 protein levels in aortic valve tissues; (D) western blot analysis of Nrf2 protein levels in cytoplasmic and nuclear fractions. For animal experiments, each individual mouse was considered as one biological replicate (n=10 per group). Comparisons were performed using one-way ANOVA. **P<0.01 vs. normal; ##P<0.01 vs. calcified + SRT2104. H&E, hematoxylin and eosin; RUNX2, RUNX family transcription factor 2; OPN, osteopontin; ALP, alkaline phosphatase; KEAP1, Kelch-like ECH-associated protein 1; HO-1, heme oxygenase 1; NQO1, NAD (P) H quinone dehydrogenase 1. SRT2104, a pharmacological sirtuin 1 activator.

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  Figure 10. HO-1 and FTH1 contribute to the protective effects of Sirt1 activation against aortic valve calcification in vivo. Representative (A) H&E staining and (B) Alizarin Red S staining of mouse aortic valve sections illustrating leaflet morphology and pathological remodeling in different experimental groups. (C) Reverse transcription-quantitative PCR analysis of the expression levels of the osteogenic markers, RUNX2, OPN, osterix and ALP. Data represent the mean ± SD. For animal experiments, each individual mouse was considered as one biological replicate (n=10 per group). Comparisons were performed using one-way ANOVA. **P<0.01 vs. normal; ##P<0.01 vs. calcified; &&P<0.01 vs. calcified + SRT2104. Normal, Apoe−/− mice mice on a standard diet; calcified, Apoe−/− mice fed a Western diet; Calcified + SRT2104, Apoe−/− mice treated with SRT2104; AAV-shHO-1, adeno-associated virus-mediated HO-1 knockdown; AAV-shFTH1, adeno-associated virus-mediated FTH1 knockdown; RUNX2, RUNX family transcription factor 2; OPN, osteopontin; ALP, alkaline phosphatase; KEAP1, Kelch-like ECH-associated protein 1; Nrf2, nuclear factor erythroid 2-related factor 2; HO-1, heme oxygenase 1; NQO1, NAD(P)H quinone…