Ketone Catabolism Is Essential for Maintaining Normal Heart Function During Aging

Figures

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  Figure 1. Cardiac‐specific deletion of SCOT induces cardiac contractile dysfunction without overt hypertrophy in male mice. A, Immunoblots showing SCOT expression in the hearts of male mice at the indicated ages. B, Quantification of SCOT expression. n=5. C, Schematic representation of the OXCT1 gene‐targeting strategy. D, Immunoblots confirming cKO of SCOT in 10‐wk‐old male mice. E, Immunoblots showing SCOT and BDH1 expression in adult cardiomyocytes and nonmyocytes from 10‐wk‐old male mouse hearts. F, Schematic illustration of the experimental design. Both floxed and ⍺MHC‐Cre alone mice were used as controls in the 20‐wk and 30‐wk age groups. G, Representative M‐mode echocardiography images. Vertical scale bar, 5 mm; transverse scale bar, 1 or 0.5 s. H–J, Echocardiographic parameters of male mice at the indicated ages. n=5 (H); n=7 (f/f), 11 (cKO), and 5 (Cre) (I); n=10 (f/f), 7 (cKO), and 6 (Cre) (J). One‐way ANOVA followed by Tukey’s multiple comparison test. N represents biologically independent replicates. P values are indicated in the figure. Data are presented as mean±SEM. Source data are provided as a Source Data file. αMHC indicates α‐myosin heavy‐chain; BDH1, β‐hydroxybutyrate…

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  Figure 2. Cardiac‐specific deletion of SCOT induces cardiac dilatation without overt systemic effects in male mice. A–C, Male mice at 19 to 22 wks of age. n=7 (f/f), 10 (cKO), and 5 (Cre). D–F, Male mice at 28 to 30 wks of age. n=10 (f/f), 7 (cKO), and 6 (Cre). Necropsy data, including body weight, VW normalized to TL, lung weight/TL, liver weight/TL, and spleen weight/TL (A, D). Gross heart morphology of the indicated mice (B, E). Scale unit: mm. Serum β‐hydroxybutyrate levels under fed conditions (C, F). One‐way ANOVA followed by Tukey’s multiple comparison test. N represents biologically independent replicates. P values are indicated in the figure. Data are presented as mean±SEM. Source data are provided as a Source Data file. β‐OHB indicates β‐hydroxybutyrate; cKO, cardiac‐specific knockout; f/f, flox/flox; SCOT, succinyl‐CoA:3‐ketoacid CoA transferase; TL, tibia length; and VW, ventricular weight.

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  Figure 3. SCOT cardiac‐specific deletion induces heart failure with severe cardiac inflammation in male mice. A and B, Wheat germ agglutinin staining showing cardiomyocyte size, with quantitative analysis for 19‐ to 22‐wk‐old (A) and 28‐ to 30‐wk‐old (B) mice. Scale bar, 50 μm. C and D, Picric Acid Sirius Red staining, indicating cardiac fibrosis, with quantitative analysis for 19‐ to 22‐wk‐old (C) and 28‐ to 30‐wk‐old (D) mice. Scale bar, 100 μm. E and F, Gene expression analysis of markers associated with heart failure, cardiac remodeling, and inflammation in 19‐ to 22‐wk‐old (E) and 28‐ to 30‐wk‐old (F) mouse hearts. n=7 (f/f), 10 (cKO), and 5 (Cre) (A, C); n=9 (f/f), 7 (cKO), and 6 (Cre) (B, D); n=7 (f/f), 6 (cKO), and 5 (Cre) (E); n=10 (f/f), 7 (cKO), and 6 (Cre) (F). One‐way ANOVA followed by Tukey’s multiple comparison test. N represents biologically independent replicates. P values are shown in the figure. Data are presented as mean±SEM. Source data are provided as a Source Data file. cKO indicates cardiac‐specific knockout; f/f, flox/flox; and SCOT, succinyl‐CoA:3‐ketoacid CoA transferase.

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  Figure 4. Cardiac‐specific deletion of SCOT induces cardiac dysfunction in the absence of overt hypertrophy in female mice. A and B, Female mice were monitored up to 30 wks of age. Representative M‐mode echocardiography images (A). Vertical scale bar, 5 mm; transverse scale bar, 1 s. Schematic representation of the experimental design (B). C–E, Echocardiographic parameters of female mice at the indicated ages. n=5 (C); n=8 (f/f), 13 (cKO), and 5 (Cre) (D); n=9 (f/f), 8 (cKO), and 7 (Cre) (E). F–I, Necropsy data of female mice at 19–22 wks of age. n=8 (f/f), 13 (cKO), and 5 (Cre). J–L, Necropsy data of female mice at 28–30 wks of age. n=11 (f/f), 11 (cKO), and 7 (Cre). Necropsy data including body weight, ventricular weight (VW) normalized to tibia length (TL), lung weight/TL, liver weight/TL, and spleen weight/TL (F, J). Serum β‐hydroxybutyrate levels under fed conditions (G, K). Relative cardiomyocyte size, assessed by wheat germ agglutinin staining (H, L). Gross heart appearance of the indicated mice (I). Scale unit: mm. One‐way ANOVA followed by Tukey’s multiple comparison test. N represents biologically independent replicates. P values are shown in the figure. Data are presented as…

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  Figure 5. Systemic effects of a ketogenic diet in SCOT cardiac‐specific knockout mice. A, Schematic representation of the experimental design. B, Body weight change in SCOT cKO and control (WT: flox/flox or αMHC‐Cre alone) mice fed a ketogenic or control diet for 12 d, starting at 9–10 wks of age. C, Casual plasma glucose levels. D, Serum β‐OHB levels under fed conditions. n=6 (WT; n=3 flox/flox and n=3 ⍺MHC‐Cre) and 5 (cKO) in male mice; n=8 (WT; n=4 flox/flox and n=4 ⍺MHC‐Cre) and 6 (cKO) in the control diet group and n=5 (WT; n=3 flox/flox and n=2 ⍺MHC‐Cre) in the ketogenic diet group for female mice. Two‐way ANOVA followed by Tukey’s multiple comparison test. N represents biologically independent replicates. P values are indicated in the figure. Data are presented as mean±SEM. Source data are provided as a Source Data file. αMHC indicates α‐myosin heavy‐chain; β‐OHB, β‐hydroxybutyrate; BW, body weight; cKO, cardiac‐specific knockout; SCOT, succinyl‐CoA:3‐ketoacid CoA transferase; and WT, wild type.

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  Figure 6. A ketogenic diet mitigates cardiomyopathy in cardiac‐specific SCOT knockout male mice. A, Schematic representation of the experimental design. Data were collected after 4 wks of ketogenic or control diet feeding. B, Body weight under fed conditions. C and D, Representative M‐mode echocardiography images (C) and echocardiographic parameters (D). Vertical scale bar, 5 mm; transverse scale bar, 0.5 s. E, Necropsy data, including VW normalized to TL, lung weight/TL, liver weight/TL, and spleen weight/TL. F, Wheat germ agglutinin staining showing cardiomyocyte size of the indicated mice after 4 wks of ketogenic diet, with quantitative analysis. Scale bar, 50 μm. G, Gene expression analysis of markers associated with heart failure, cardiac remodeling, and inflammation in the hearts of the indicated mice after 4 wks of ketogenic diet. H, β‐OHB levels under fed conditions in the indicated mice after 4 wks of ketogenic diet. n=6 (f/f), 8 (cKO), 4 (Cre), and 7 (cKO on a control diet). One‐way ANOVA followed by Tukey’s multiple comparison test. N represents biologically independent replicates. P values are shown in the figure. Data are presented as mean±SEM. Source data are provided as a…

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  Figure 7. A ketogenic diet attenuates cardiomyopathy in cardiac‐specific SCOT knockout female mice. A, Schematic representation of the experimental design. Data were collected after 4 wks of ketogenic or control diet feeding. B, Body weight under fed conditions. C and D, Representative M‐mode echocardiography images (C) and echocardiographic parameters (D). Vertical scale bar, 5 mm; transverse scale bar, 0.5 s. E, Necropsy data, including VW normalized to TL, lung weight/TL, liver weight/TL, and spleen weight/TL. F, Wheat germ agglutinin staining showing cardiomyocyte size, with quantitative analysis. Scale bar, 50 μm. G, Serum β‐OHB levels under fed conditions. n=5. One‐way ANOVA followed by Tukey’s multiple comparison test. N represents biologically independent replicates. P values are shown in the figure. Data are presented as mean±SEM. Source data are provided as a Source Data file. αMHC indicates α‐myosin heavy‐chain; β‐OHB, β‐hydroxybutyrate; cKO, cardiac‐specific knockout; Con, control diet; f/f, flox/flox; HR, heart rate; IVSd, interventricular septal at end diastole; KD, ketogenic diet; LVDd, left ventricular diameter at end diastole; LVDs, left ventricular diameter at end…

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  Figure 8. SCOT overexpression tends to enhance mitochondrial maximal respiratory capacity in H9C2 cells. A, Immunoblots showing adenovirus‐mediated FLAG‐tagged SCOT overexpression in H9C2 cells. B and C, Mitochondrial function was assessed using Seahorse experiments. Representative OCR plots (B) in H9C2 cells transfected with FLAG‐SCOT or control LacZ for 48 h in the absence of 100 μM β‐hydroxybutyrate. Histograms showing basal respiration, ATP‐linked respiration, maximal respiratory capacity, and nonmitochondrial respiration (C). Representative profile n=4 wells per cohort (B) and n=6 independent experiments, each conducted using separately prepared cell cultures per cohort (C). D, Schematic representation of SCOT downregulation‐mediated cardiac dysfunction during aging. Unpaired t test. P values are indicated in the figure. Data are presented as mean±SEM. Source data are provided as a Source Data file. Ad‐LacZ indicates adenovirus harboring beta‐galactosidase; FCCP, carbonylcyanidep‐triflouromethoxyphenylhydrazone; OCR, oxygen consumption rate; and SCOT, succinyl‐CoA:3‐ketoacid CoA transferase.