Ketogenic diets inhibit mitochondrial biogenesis and induce cardiac fibrosis

Long-term KD exposure resulted in cardiac fibrosis

To survey the potential pathological effects of a KD on cardiac disease, we fed rats either a KD or normal diet and monitored changes in the rat heart (Supplementary Fig. 1a–c). After 16 weeks, besides a decrease in body weight (Supplementary Fig. 1d), fat mass (Supplementary Fig. 1e), and blood pressure (Supplementary Fig. 1f, g) in KD-fed rats, we observed increased heart rates and impaired cardiac function, as evaluated by echocardiography (Table 1). In KD-fed rats, the left ventricular posterior wall thickness (LVPWd) increased significantly compared with normal diet-fed rats, indicating a compensatory increase in cardiomyocytes, used to enhance cardiac contractility (Table 1). If the left heart function was decompensated, the blood accumulated in the pulmonary circulation. The right ventricular anterior wall thickness (RVAW) also increased, indicating increased pulmonary circulation resistance and corresponding thickening of the right ventricular myocardium. Furthermore, the left atrial diameter (LAD), left ventricular dimension in diastole (LVDd), and left ventricular dimension in systole (LVDs) increased in KD-fed rats, indicating that cardiac function was decompensated and entered a state of failure (Table 1). Taken together, the echocardiography results revealed impaired cardiac function in KD-fed rats.

Table 1 Echocardiographic data from rats fed a normal diet, ketogenic diet, or caloric restriction diet Full size table

Considering the fact that increased cardiac fibrosis is associated with cardiac hypertrophy, dilatation,24,25,26 and the onset of AF27 and that significantly increased ketone bodies were observed in the cardiac tissues of patients with AF,15 we next examined the cardiac fibrosis levels in rat atrial tissues. In accordance with impaired cardiac function, we observed the occurrence of fibrosis in the atrial tissues of KD-fed but not normal diet-fed rats, as analyzed by atrial tissue staining (Fig. 1a and Supplementary Fig. 2a). We also detected the expression levels of protein markers of fibrosis using both immunohistochemistry (IHC) and western blotting. Indeed, type I collagen, type III collagen, and α-smooth muscle actin (α-SMA) levels were increased in atrial tissues from KD-fed rats (Fig. 1b, c and Supplementary Fig. 2b, c). These results indicated that long-term KD feeding may cause cardiac fibrosis.

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. nsp > 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 Full size image

Because a KD usually provides fewer calories than a carbohydrate-rich diet, we employed caloric restriction (CR) in another group of rats as a control to determine whether KD induced cardiac fibrosis was caused by an insufficient supply of energy. We found CR did not induce cardiac fibrosis and cardiac function impairment in rats (Fig. 1a–c, Supplementary Figs. 1 and 2, and Table 1). Moreover, the KD caused increased levels of β-OHB and AcAc, the predominant forms of ketone bodies, in both plasma and heart tissues (Fig. 1d, e). In contrast, CR only induced a mild elevation of β-OHB and AcAc (Fig. 1d, e). These observations suggested that a considerable elevation in ketone bodies may account for the onset of cardiac fibrosis in rats.

Increased β-OHB resulted in cardiac fibrosis

To further elucidate whether and which ketone bodies induced fibrosis, we increased the levels of either β-OHB or AcAc in rats by intraperitoneal injection (Supplementary Fig. 3a–d) because β-OHB and AcAc were the predominant forms of ketone bodies elevated after KD feeding (Fig. 1d, e). During the 16-week injection period, water intake, daily calorie intake, body weight, fat mass, and blood pressure were not affected by either β-OHB or AcAc injection (Supplementary Fig. 3e–j). We found that increased levels of β-OHB, but not AcAc, resulted in impaired cardiac function, as evidenced by increased LAD, LVDs, LVDd, LVPWd, and RVAW in echocardiography analysis (Table 2). Furthermore, we observed the occurrence of cardiac fibrosis in β-OHB intraperitoneal injected rats, as evident by atrial staining (Fig. 1f and Supplementary Fig. 4a) and analysis of cardiac fibrosis molecular markers, namely type I collagen, type III collagen, and α-SMA (Fig. 1g, h and Supplementary Fig. 4b, c). We also observed that frequent deep fasting (Supplementary Fig. 5a, b) induced high levels of β-OHB (Supplementary Fig. 5c) and molecular phenotypes of cardiac fibrosis, including characteristic fiber staining results (Supplementary Fig. 5d), and expression of collagen and α-SMA (Supplementary Fig. 5e). Together, these findings supported the theory that increased levels of β-OHB resulted in cardiac fibrosis.

Table 2 Echocardiographic data from rats injected with saline, AcAc, or β-OHB Full size table

We next investigated how β-OHB induced cardiac fibrosis. In cultured human cardiomyocytes (HCM), rat cardiomyoblasts (H9C2), and mouse cardiac muscle cells (HL-1), increased levels of β-OHB led to elevated rates of apoptosis; however, β-OHB did not induce significant increases in apoptosis in fibroblasts, such as mouse embryonic fibroblasts (MEFs) and 3T3 cells (Fig. 2a). In primary cells isolated from neonatal mice, β-OHB treatment also led to an increase in apoptosis in primary mouse cardiomyocytes, but not in primary mouse cardiac fibroblast (Fig. 2b). In accordance with increased apoptosis, elevated β-OHB led to increases in the levels of cleaved caspase 3 in HCM and H9C2 cells, but not in MEFs (Fig. 2c). Moreover, in rat atrial tissue, we confirmed that high β-OHB levels, induced by either KD feeding or β-OHB injection, led to increased rates of apoptosis, as evidenced by in situ TUNEL assays (Fig. 2d) and detection of the levels of cleaved caspase 3 in rat atrial tissue (Fig. 2e). These results suggested that a high concentration of β-OHB caused cell-type-specific cardiomyocyte apoptosis and may underlie β-OHB induced cardiac fibrosis, which is known to be caused by cardiomyocyte apoptosis.28,29

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 Full size image

We next evaluated the mechanisms underlying cardiomyocyte apoptosis induced by β-OHB. First, given that β-OHB constitutes an important alternative energy source that can be catalyzed into acetyl-CoA for processing through the citrate cycle,30 we examined whether β-OHB affected energy metabolism through enhancing the ketolysis pathway. By measuring the expression of proteins within the ketolysis pathway in H9C2, HL-1, and HCM cells treated with or without β-OHB, we observed decreased expression of β-hydroxybutyrate dehydrogenase 1 (BDH1) and succinyl-CoA:3-ketoacid CoA transferase (SCOT) in β-OHB-treated cells (Fig. 2f), and atrial tissues from KD-fed and β-OHB-injected rats (Fig. 2g), suggesting that increased input of ketone bodies did not enhance ketolysis. We next evaluated the glycolytic and citrate metabolic flux in cultured cell lines and primary cardiomyocytes. Decreased levels of citrate cycle metabolites and increased levels of the glycolytic metabolites pyruvate and lactate were observed in β-OHB-treated cells (Fig. 2h). Together with our results showing that decreased levels of citrate cycle metabolites and increased levels of the glycolytic metabolites were also observed in atrial tissues from rats with high β-OHB levels (Fig. 2i), these findings indicated that high β-OHB did not enhance mitochondrial metabolism. Instead, increased β-OHB may lead to reduced mitochondrion numbers because the levels of some mitochondrial enzymes decreased (Fig. 2f, g).

β-OHB decreased mitochondrion levels in cardiomyocyte

Next, we evaluated the number of mitochondria in response to β-OHB treatment. Our results showed that β-OHB treatment decreased MitoTracker Green staining levels (Fig. 3a) and decreased the ratio of mitochondrial (mt) DNA to nucleic DNA (Fig. 3b) in HCM, H9C2, and HL-1 cells, in a concentration-dependent manner. In primary mouse cardiomyocytes isolated from neonatal mice, β-OHB treatment also led to a decrease in mitochondrion mass, as determined by measuring the ratio of mtDNA to nucleic DNA (Fig. 3c). In accordance with the observations in cultured cells, in rats fed a KD or injected intraperitoneally with β-OHB, significant decreases in cardiac mitochondrion mass were observed compared with those in untreated animals, as determined by measuring the ratio of mtDNA to nucleic DNA (Fig. 3d, e). Moreover, the mtDNA damage levels were not altered significantly in β-OHB-treated cells (Supplementary Fig. 6a) and cardiomyocytes from rats fed a KD or injected intraperitoneally with β-OHB (Supplementary Fig. 6b, c). These results indicated that β-OHB decreased mitochondrial numbers in cultured cells and rat heart tissue.

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. nsp > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 versus the corresponding control group according to Student’s t tests Full size image

Consistent with this observation, mitochondrial respiration was significantly reduced after β-OHB treatment in both H9C2 and HL-1 cells (Fig. 3f, g). We also observed decreased mitochondrial respiration in primary cardiomyocytes isolated from KD-fed or β-OHB-injected rats (Fig. 3h, i). Furthermore, oxyrase-induced hypoxia caused apoptosis, and saturated the proapoptotic effects of β-OHB in H9C2 and HL-1 cells (Fig. 3j) and primary mouse cardiomyocytes (Fig. 3k). These results were consistent with the hypothesis that a decrease in mitochondrion numbers may lead to cardiomyocyte apoptosis, which has been shown to be induced by hypoxia.31,32,33,34,35 In addition, we observed that β-OHB slightly decreased the mitochondrial mass in MEFs and primary mouse cardiac fibroblasts (Supplementary Fig. 7a, b), and did not exhibit strong inhibitory effects on mitochondrial respiration (Supplementary Fig. 7c, d). Accordingly, the apoptosis levels of MEFs and primary cardiac fibroblasts were not altered in response to hypoxia and β-OHB treatments (Supplementary Fig. 7e, f). These observations suggested that β-OHB caused cell-type-specific cardiomyocyte apoptosis through inhibiting mitochondrial respiration, consistent with previous findings demonstrating that cardiomyocytes are more sensitive to mitochondrial inhibition than fibroblasts.36,37,38 Taken together, these results supported the hypothesis that high levels of β-OHB may induce cardiomyocyte apoptosis by inhibiting mitochondria-mediated oxygen utilization.

β-OHB impaired mitochondrial biogenesis by activating SIRT7

By monitoring protein markers of mitochondrial biogenesis and degradation, we found that β-OHB decreased protein levels of mitochondrial ribosomal protein L16 (MRPL16), mitochondrial ribosomal protein L24 (MRPL24), and G elongation factor mitochondrial 2 (GFM2), markers of mitochondrial biogenesis (Fig. 4a).39 β-OHB also decreased GFM2, MRPL16, and MRPL24 mRNA levels (Fig. 4b). Moreover, the expression of PTEN-induced kinase 1 (PINK1) and parkin (PRKN), along with the relative ratio of LC3-I to LC3-II, markers of mitophagy, were not affected by β-OHB treatment, suggesting that β-OHB did not promote mitochondrial autophagy (Fig. 4a). These results collectively suggested that β-OHB inhibited cell respiration via the suppression of mitochondrial biogenesis.

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. nsp > 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 Full size image

In an attempt to identify genes that were transcriptionally regulated by β-OHB, we evaluated the gene expression profiles of H9C2 cells with or without β-OHB treatment. The results showed that the transcription of Sirt4 and Sirt7 was activated by β-OHB (Supplementary Fig. 8a). Because other markers of mitochondrial biogenesis, such as Polrmt, Tfb1m, and Tfam, were not reduced after β-OHB treatment and because SIRT7 specifically regulates MRPL16, MRPL24, and GFM2,40 we hypothesized that β-OHB blocked mitochondrial activity via SIRT7-mediated transcriptional regulation. In cultured H9C2, HL-1, and HCM cells, we confirmed that β-OHB increased SIRT7 expression at both the mRNA and protein levels in a concentration-dependent manner (Fig. 4c, d). Knockdown of SIRT7 increased the transcription of Gfm2 and Mrpl24, and attenuated the inhibitory effects of β-OHB treatment on Gfm2 and Mrpl24 transcription (Fig. 4e). Conversely, overexpression of SIRT7 resulted in decreased Gfm2 and Mrpl24 transcription, and saturated the effects of β-OHB treatment (Fig. 4f). Moreover, rats with high levels of β-OHB, including KD-fed and β-OHB-injected rats, exhibited increased cardiac SIRT7 mRNA and protein expression (Fig. 4g, h), as well as decreased Gfm2 and Mrpl24 mRNA levels (Fig. 4g). Furthermore, overexpression of SIRT7 decreased mitochondrion levels, whereas knockdown of SIRT7 slightly increased mitochondrion levels in cultured cells (Fig. 4i, j). Either overexpressing or knocking down SIRT7 in cells attenuated the inhibitory effects of β-OHB treatment (Fig. 4i, j). Double immunofluorescence staining of atrial tissues from β-OHB-injected or normal rats revealed that β-OHB promoted SIRT7 expression and reduced mitochondrial numbers, as indicated by measuring the level of superoxide dismutase 2 (SOD2); this further indicated that β-OHB impaired mitochondrial biogenesis by activating SIRT7 (Fig. 4k). Furthermore, although high β-OHB induced SIRT4 overexpression, forced SIRT4 overexpression had no effect on Gfm2 or Mrpl24 expression (Supplementary Fig. 8b). These results collectively confirmed that β-OHB inhibited mitochondrial biogenesis by promoting SIRT7 overexpression.

β-OHB inhibited histone deacetylase 2 and activated Sirt7 transcription

We next investigated how β-OHB regulated the transcription of Sirt7. We hypothesized that histone acetylation may play a role, as β-OHB is an established inhibitor of histone deacetylases (HDACs), include HDAC1, HDAC3, HDAC4, and HDAC6.12 Knockdown of HDACs individually in β-OHB-treated H9C2 cells revealed that only Hdac2 knockdown increased Sirt7 expression and abrogated the ability of β-OHB to activate Sirt7 transcription (Fig. 5a and Supplementary Fig. 9). In turn, overexpression of HDAC2, but not other HDACs, inhibited Sirt7 transcription in H9C2 cells (Fig. 5b), indicating that HDAC2 was the HDAC that regulated Sirt7 transcription. Accordingly, using chromatin immunoprecipitation (ChIP), we found that HDAC2 bound to the core promoter region of Sirt7 and localized to the same region as acetylated H3K9 (Fig. 5c). Moreover, the acetylation levels of H3K9 and H3K14 in the promoter of Sirt7 were increased in β-OHB-treated cultured H9C2 cells (Fig. 5d). In addition, we observed increased acetylation levels of H3K9 and H3K14 in the promoter of Sirt7 in the heart tissues of KD-fed and β-OHB-injected rats, when compared with those in the corresponding control rats (Fig. 5e). These results suggested that Sirt7 transcriptional activation was related to alterations in acetylation, which were regulated by the β-OHB/HDAC2 pathway.

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 (IC 50 : 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. nsp > 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 Full size image

Although β-OHB was previously shown to be negatively correlated with cellular HDAC2 activity,41 direct evidence has not yet been obtained to validate whether β-OHB inhibits HDAC2 deacetylase activity. Therefore, we conducted an in vitro deacetylation assay and found that β-OHB had significant inhibitory effects on HDAC2, with an IC 50 of 2.4 mM in vitro (Fig. 5f), suggesting that KD-induced elevation of β-OHB in the heart decreased HDAC2 activity by more than half. In cultured cells, we confirmed that β-OHB treatment resulted in increased levels of total histone acetylation (Fig. 5g), particularly of H3K9 and H3K14, downstream targets of HDAC2 (Fig. 5h). Trichostatin A, a general HDAC inhibitor, caused phenotypes similar to those of β-OHB, including activation of Sirt7 transcription (Supplementary Fig. 10a), downregulation of Gfm2 and Mrpl24 expression (Supplementary Fig. 10a), inhibition of mitochondrial biogenesis (Supplementary Fig. 10b), inhibition of mitochondrial respiration (Fig. 3f, g), and promotion of apoptosis in cardiomyocytes (Fig. 2a–c). In addition, we confirmed that the expression of voltage-dependent T-type calcium channel subunit alpha-1H (CACNA1H) and voltage-dependent T-type calcium channel subunit alpha-2 delta-2 (CACNA2D2), which are known to be inhibited by HDAC2,42 were increased in β-OHB-treated cells (Fig. 5i) and heart tissues from KD-fed rats (Fig. 5j). Taken together, these results indicated that β-OHB activated Sirt7 transcription by inhibiting HDAC2.

Increased β-OHB and its consequences were observed in patients with AF

In order to confirm the causal role of β-OHB in cardiac fibrosis, we examined cardiac fibrosis, β-OHB levels, and changes in β-OHB-associated markers in patients with AF. First, we found that β-OHB levels in cardiac tissues were 3.3-fold higher in patients with AF than in those with sinus rhythm (SR; Fig. 6a). Second, CACNA1H and CACNA2D2 expression was higher in cardiac tissues from patients with AF, indicating that HDAC2 was inhibited in the cardiac tissues of these patients (Fig. 6b and Supplementary Fig. 11a). Third, increased levels of SIRT7 were observed in cardiac tissues from patients with AF compared with those in patients with SR, according to both western blotting (Fig. 6b and Supplementary Fig. 11a) and IHC (Fig. 6c and Supplementary Fig. 11b). Fourth, markers of fibrosis, including type I collagen, type III collagen, and α-SMA, were higher in patients with AF (Fig. 6b and Supplementary Fig. 11a). Lastly, the number of mitochondria was significantly lower in cardiac tissues from patients with AF than in those from patients with SR, as indicated by the ratio of mtDNA to nucleic DNA (Fig. 6d). These findings, together with the observation that the cardiac β-OHB concentration was negatively correlated with the number of mitochondria (Fig. 6e), confirmed that elevations in β-OHB were associated with cardiac fibrosis and an increased risk of AF.