Fig 1: Myocardial ketone oxidation is impaired in heart failure with preserved ejection fraction (HFpEF). (A) HFpEF protocol and empagliflozin (EMPA) and ketone supplement (KS) protocol (i), body weight changes over the HFpEF protocol (ii), blood glucose changes over the 2‐h course of intraperitoneal glucose‐tolerance test (GTT), which were performed by injection of glucose (2 g kg−1 in saline) after 16‐h fasting (iii), fasting blood ketone levels assessed by sampling blood from mice tail and read with FreeStyle precision ß‐Ketone test strips paired with its analyser (n = 14–17) (iv), cardiac tissue levels of βOHB assessed with a colorimetric assay kit from Abcam (ab83390) (n = 6) (v), transthoracic echocardiographic analysis of LV ejection fraction (%EF) (n = 11–17) (vi), isovolumic relaxation time (IVRT) (n = 11–17) (vii) and LV mass (LV mass) (n = 11–17) (viii). (B) βOHB/ketone oxidation rates (n = 5–8) (i), glucose oxidation rates (n = 4–6) (ii), glycolysis (n = 4–8) (iii) and palmitate/fatty acid oxidation rates (n = 4–5) (iv) in isolated working hearts perfused with appropriately radiolabelled 5‐mM glucose, 0.8‐mM palmitate, 3% bovine serum albumin (BSA), 100 μU/mL insulin and either 0.6‐ or 1‐mM βOHB. (C) ATP production rates were calculated based on the oxidation rates from glucose (31 moles of ATP from glucose oxidation), fatty acid (104 moles of ATP from palmitate oxidation) and βOHB (21.25 moles of ATP from βOHB oxidation) (i), %ATP production is calculated by matching the ATP production rates attributed to each of the four metabolic pathways normalized to the sum of ATP production rates (ii). (D) Quantification of western blotting showing expression of enzymes involved in myocardial ketone oxidation (ACAT1 [i], BDH1 [ii] and SCOT [iii]), and representative blots (n = 6–8) (iv). Data are mean ± SEM. Statistics were performed in GraphPad Prism 10. Significant differences were determined by using a one‐way or two‐way analysis of variance (ANOVA) followed by a Tukey's multiple comparisons post hoc test. For Panel C, *P < 0.05 compared with control group under the same condition, #P < 0.05 compared with HFpEF under the same condition. For all other panels, *P < 0.05 compared with corresponding group with bracket. Mouse studies were approved by the University of Alberta Animal Care and Use Committee and comply with Animal Research: Reporting of In Vivo Experiments (ARRIVE) and National Institutes of Health guidelines. Abbreviations: ACAT1: acetyl‐CoA Acetyltransferase 1; ATP, adenosine triphosphate; βOHB, β‐hydroxybutyrate; BDH1, β‐hydroxybutyrate dehydrogenase 1; EMPA, empagliflozin; EF, ejection fraction; HFD, high‐fat diet; HFpEF, heart failure with preserved ejection fraction; IVRT, isovolumetric relaxation time; KS, ketone supplement; L‐NAME, L‐NG‐nitroarginine methyl ester, N(G)‐nitro‐L‐arginine methyl ester; LV, left ventricle; SCOT, succinyl‐CoA:3‐ketoacid CoA transferase; Supp, supplement.
Fig 2: Glucose specific regulation of ketone body utilizing genes and protein by cardiac specific glucose transporter 4 (GLUT4)‐induced glucose delivery.Gene expression from qPCR analysis showing expression of Bdh1 (A) and Oxct1 (B) in mice 4 weeks after GLUT4 induction in the heart (mG4H). Western analysis of whole cell extracts from ventricular heart tissue for BDH1 and SCOT. C, Upper panel: Immunoblot illustrating BDH1 levels in ventricular tissue of mG4H mice compared with Control (Con) mice. Lower panel: Densitometric analysis of immunoblots shown in upper panel. D, Upper panel: Immunoblot illustrating SCOT levels in ventricular tissue of mG4H mice compared with Con mice. mRNA expression of Hmgcs2 (E), and ketone transport genes, Slc16a1 and Slc16a7 (F) in ventricular tissue. G, Tissue metabolomics data of cardiac β‐hydroxybutyrate (β‐OHB) levels. n≥6; Means±SEM; BDH1 indicates β‐hydroxybutyrate dehydrogenase; MG4H, transgenic inducible cardiac‐restricted expression of glucose transporter 4; qPCR, quantitative polymerase chain reaction; SCOT, succinyl‐CoA:3‐oxoacid CoA transferase. **P<0.01; vs. Con.
Fig 3: Glucose specific regulation of ketone body utilizing protein activity and cardiac metabolic rates. A, Catalytic activity of BDH1 in mitochondrial fractions prepared from the hearts of mG4H mice. B, SCOT enzymatic activity was measured in the CoA transferase–enriched fractions from the heart. C, After 2 weeks of transgene induction, hearts from mG4H mice were isolated and perfused ex vivo in presence of D‐[5‐3H]‐glucose for glycolysis (C) and [U‐14C]‐βOHB for oxidation (D). Values are expressed as means±SEM (n=5–9; sample size range varies dependent on the parameter investigated); *P<0.05; ***P<0.001; vs. Con. BDH1 indicates β‐hydroxybutyrate dehydrogenase; MG4H, transgenic inducible cardiac‐restricted expression of glucose transporter 4; SCOT, succinyl‐CoA:3‐oxoacid CoA transferase.
Fig 4: Regulation of ketone body utilizing genes, protein, and activity by high‐fat diet (HFD)‐induced obesity.qPCR analysis showing expression of Bdh1 (A) and Oxct1 (B) in mice after 12 weeks of HFD or control diet (Con) feeding. mRNA expression of Hmgcs2 (C), and ketone transport genes, Slc16a1 and Slc16a7 (D) in ventricular tissue. Western analysis of whole cell extracts prepared from left ventricular heart tissue for β‐hydroxybutyrate dehydrogenase (BDH1) and succinyl‐CoA:3‐oxoacid CoA transferase (SCOT). E, Upper panel: Immunoblot illustrating BDH1 levels in ventricular tissue of HFD mice compared with Con mice. Lower panel: Densitometric analysis of immunoblots shown in upper panel. F, Upper panel: Immunoblot illustrating SCOT levels in ventricular tissue of HFD mice compared with Con mice. Lower panel: Densitometric analysis of immunoblots shown in upper panel. Cardiac BDH1 (G) and SCOT (H) enzyme activities in Con and HFD mice. n=6; Means±SEM. qPCR indicates quantitative polymerase chain reaction. **P<0.01; ***P<0.001; vs. Con.
Fig 5: Inoculation of B. adolescentis and B. virosa improves post-MI cardiac function in germ-free mice.a Shotgun metagenomics analysis revealed the enrichment of ketogenesis in STEMIT1 samples. Abundance of genes encoding ketogenic enzymes in Ctrl and STEMI samples is presented in the dot plot. b Butyrate production capabilities of B. adolescentis, B. virosa and S. parasanguinis. c β-hydroxybutyrate production capabilities of B. adolescentis, B. virosa and S. parasanguinis. d Experimental design of the myocardial infarction (MI) model in gnotobiotic mice inoculated with B. adolescentis, B. virosa and S. parasanguinis (upper panel). The survival curve of gnotobioic mice subjected to MI for 21 days (lower panel). e Changes in bacterial load in gnotobiotic mice determined with 16S rRNA qPCR. f Bacterial load of B. adolescentis, B. virosa and S. parasanguinis in gnotobiotic mice. g Echocardiographic analysis of the left ventricular ejection fraction (EF, %) and fraction shortening (FS, %) in gnotobiotic mice on MI day 21. h Post-MI cardiac function analysis in gnotobiotic mice, evaluating ESPVR, EDPVR, PRSW and dP/dt max (vs. EDV) using cardiac catheterization. i Representative histology of cardiac infarct size (left panel) and statistics (right panel) in gnotobiotic mice on MI day 21. Cardiac tissues were stained with picrosirius red to label fibrosis. j Colorimetric analysis of the plasma level of β-hydroxybutyrate in gnotobiotic mice on MI day 21. k Representative images of the gut in gnotobiotic mice (left panel), intestinal length (upper right panel) and the length of colon (lower right panel) on MI day 21. The number of biologically independent samples and mice are indicated in each chart. Data were analyzed with Kruskal–Wallis test followed by FDR correction. Data are represented as mean ± SEM. ACAT Acetyl-CoA acetyltransferase, HMGCS 3-hydroxy-3-methylglutaryl-CoA synthase, HMGCL 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) 2 lyase, OXCT succinyl-CoA:3-oxoacid CoA transferase (OXCT), ADC acetoacetate decarboxylase, BDH beta-hydroxybutyrate dehydrogenase, Bifidobacterium adolescentis (B. adolescentis); Butyricimonas virosa (B. virosa); Streptococcus parasanguinis (S. parasanguinis).
Supplier Page from Abcam for beta Hydroxybutyrate (beta HB) Assay Kit (Colorimetric)