Ketogenic diets inhibit mitochondrial biogenesis and induce cardiac fibrosis - PubMed (original) (raw)

Ketogenic diets inhibit mitochondrial biogenesis and induce cardiac fibrosis

Sha Xu et al. Signal Transduct Target Ther. 2021.

Abstract

In addition to their use in relieving the symptoms of various diseases, ketogenic diets (KDs) have also been adopted by healthy individuals to prevent being overweight. Herein, we reported that prolonged KD exposure induced cardiac fibrosis. In rats, KD or frequent deep fasting decreased mitochondrial biogenesis, reduced cell respiration, and increased cardiomyocyte apoptosis and cardiac fibrosis. Mechanistically, increased levels of the ketone body β-hydroxybutyrate (β-OHB), an HDAC2 inhibitor, promoted histone acetylation of the Sirt7 promoter and activated Sirt7 transcription. This in turn inhibited the transcription of mitochondrial ribosome-encoding genes and mitochondrial biogenesis, leading to cardiomyocyte apoptosis and cardiac fibrosis. Exogenous β-OHB administration mimicked the effects of a KD in rats. Notably, increased β-OHB levels and SIRT7 expression, decreased mitochondrial biogenesis, and increased cardiac fibrosis were detected in human atrial fibrillation heart tissues. Our results highlighted the unknown detrimental effects of KDs and provided insights into strategies for preventing cardiac fibrosis in patients for whom KDs are medically necessary.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1

Increased β-OHB induced cardiac fibrosis in animal models. a, b H&E, Sirius Red, and Masson trichrome staining (a) results in atrial tissues from rats fed a normal diet, KD, or CR diet, and immunohistochemical (IHC) analysis of α-SMA, collagen I, and collagen III in atrial tissues from rats according to diet (b). Representative results (top) and summary of quantitative analysis are shown. c Western blotting analysis of α-SMA, collagen I, and collagen III in atrial tissues of rats fed the different diets. d, e Ketone body concentrations in the plasma (d) and atrial tissues (e) of rats fed the different diets. f, g H&E, Sirius Red, and Masson staining results of atrial tissues from rats intraperitoneally injected with saline, AcAc, or β-OHB (f) and IHC analysis of α-SMA, collagen I, and collagen III in atrial tissues from rats according to treatment (g). Representative results (top) and summary of quantitative analysis are shown. h Western blotting analysis of α-SMA, collagen I, and collagen III in atrial tissues of rats according to treatment. Data shown are means ± standard errors. ns_p_ > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 versus the corresponding control group according to Student’s t tests. Some full-length blots are presented in the Supplementary Fig. 13

Fig. 2

Increased β-OHB promoted cardiomyocyte apoptosis by decreasing mitochondrial metabolism. a Cell apoptosis associated with various treatments in different cultured cell lines. Trichostatin A (TSA) is a general histone deacetylase inhibitor; n = 5. b Cell apoptosis associated with various treatments in primary cells isolated from neonatal mice; n = 5. c Western blotting analysis of caspase 3 in different cell lines with various treatments. d TUNEL analysis of atrial tissues from rats with different treatments; n = 3. e Western blotting analysis of caspase 3 in atrial tissues from rats with various treatments. f Western blotting analysis of BDH1 and SCOT in different cell lines treated with or without β-OHB. g Western blotting analysis of BDH1 and SCOT in atrial tissues from rats with various treatments. h Metabolite concentrations in cell lines and primary cardiomyocyte treated with or without β-OHB; n = 5. i Metabolite concentrations in atrial tissues from rats; n = 5. Some full-length blots are presented in the Supplementary Fig. 13

Fig. 3

Increased β-OHB decreased mitochondrion levels. a, b Mitochondrial mass, as determined by MitoTracker Green staining (a) and mitochondrial DNA quantification (b) in cells with different concentrations of β-OHB; n = 3. c Mitochondrial mass determined by mitochondrial DNA quantification in mouse primary cardiomyocytes; n = 3. d, e Mitochondrial mass determined by mitochondrial DNA quantification in atrial tissues from rats with different treatments; n = 6/group. f, g Oxygen consumption rates (OCRs) in H9C2 (f) and HL-1 (g) cells treated with β-OHB or trichostatin A (TSA). h, i OCRs in primary cardiomyocytes isolated from the atrial tissues of rats with various treatments. j, k Apoptosis in cell lines and primary mouse cardiomyocytes treated with β-OHB and oxyrase; n = 3. Data shown are means ± standard errors. ns_p_ > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 versus the corresponding control group according to Student’s t tests

Fig. 4

β-OHB impaired mitochondrial biogenesis by activating SIRT7. a Western blotting analysis of proteins involved in mitochondrial biogenesis and autophagy in different cell lines treated with or without β-OHB. b Analysis of mRNA expression of genes involved in the mitochondrial biogenesis pathway in different cell lines treated with or without β-OHB; n = 3. c Sirt7 mRNA levels in different cell lines treated with different concentrations of β-OHB. d SIRT7 protein levels in different cell lines treated with different concentrations of β-OHB. e Gfm2 and Mrpl24 mRNA levels in SIRT7-knockdown H9C2 and control cells treated with or without β-OHB; n = 3. f Gfm2 and Mrpl24 mRNA levels in SIRT7-overexpressing H9C2 cells and control cells treated with or without β-OHB; n = 3. g Sirt7, Gfm2, and Mrpl24 mRNA levels in atrial tissues from rats with different treatments. h SIRT7 protein levels in rat atrial tissues according to different treatments. i, j Mitochondrial mass determined by mitochondrial DNA quantification in H9C2 cells with different treatments; n = 3. k Double immunofluorescence staining of SIRT7 and SOD2 in rat atrial tissues. Right panel shows the quantitative results of three replicates. Data shown are means ± standard errors. ns_p_ > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 versus the control group according to Student’s t tests. Some full-length blots are presented in the Supplementary Fig. 13

Fig. 5

β-OHB activated Sirt7 transcription by inhibiting HDAC2. a Sirt7 mRNA levels in Hdac2_-knockdown or control H9C2 cells treated with or without β-OHB; n = 3. The lower panel shows the knockdown efficiency. b Sirt7 mRNA levels in H9C2 cells overexpressing different histone deacetylases (HDACs); n = 3. The lower panel shows the knockdown efficiency. c ChIP followed by PCR showing HDAC2 and H3K9Ac occupancy at the Sirt7 promoter. d ChIP followed by qRT-PCR showing the binding affinities of H3K9Ac and H3K14Ac to the Sirt7 promoter in cells treated with or without β-OHB; n = 3. e ChIP followed by qRT-PCR showing the binding affinities of H3K9Ac and H3K14Ac to the Sirt7 promoter in rats with different treatments; n = 3. f In vitro assay showing the effects of β-OHB on inhibition of HDAC2 activity (IC50: 2.4 mM). g Total histone acetylation levels in H9C2 cells treated with different concentrations of β-OHB. h H3K9Ac and H3K14Ac levels in H9C2 and HCM cells treated with different concentrations of β-OHB. i CACNA1H and CACNA2D2 levels in different cells treated with or without β-OHB. j CACNA1H and CACNA2D2 levels in atrial tissues from rats fed a normal diet or KD. Data shown are means ± standard errors. ns_p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 versus the corresponding control group according to Student’s t tests. Some full-length blots are presented in the Supplementary Fig. 13

Fig. 6

Increased β-OHB was associated with increased risk of atrial fibrillation (AF). a Concentrations of β-OHB and AcAc in atrial tissues from patients with sinus rhythm (SR) and AF. b The left panel shows representative western blotting analysis of SIRT7, CACNA1H, CACNA2D2, GFM2, MRPL24, collagen I, collagen III, and α-SMA in atrial tissues from patients with SR and AF. The right panel shows the quantitative results. c IHC analysis of SIRT7 in atrial tissues from patients with SR and AF. The right panel shows the quantitative results. d Mitochondrial mass determined by mtDNA quantification in atrial tissues from patients with SR and AF. e Correlation between β-OHB concentration and mitochondrial mass in atrial tissues from patients with SR and AF. f Schematic illustration showing how KD increases the risk of cardiac fibrosis. Data shown are means ± standard errors. ns_p_ > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 versus the corresponding control group according to Student’s t tests. Some full-length blots are presented in the Supplementary Fig. 13

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Ketogenic diets inhibit mitochondrial biogenesis and induce cardiac fibrosis - PubMed (original) (raw)