The Failing Heart Relies on Ketone Bodies as a Fuel

Abstract

Background: Significant evidence indicates that the failing heart is energy starved. During the development of heart failure, the capacity of the heart to utilize fatty acids, the chief fuel, is diminished. Identification of alternate pathways for myocardial fuel oxidation could unveil novel strategies to treat heart failure.

Methods and results: Quantitative mitochondrial proteomics was used to identify energy metabolic derangements that occur during the development of cardiac hypertrophy and heart failure in well-defined mouse models. As expected, the amounts of proteins involved in fatty acid utilization were downregulated in myocardial samples from the failing heart. Conversely, expression of β-hydroxybutyrate dehydrogenase 1, a key enzyme in the ketone oxidation pathway, was increased in the heart failure samples. Studies of relative oxidation in an isolated heart preparation using ex vivo nuclear magnetic resonance combined with targeted quantitative myocardial metabolomic profiling using mass spectrometry revealed that the hypertrophied and failing heart shifts to oxidizing ketone bodies as a fuel source in the context of reduced capacity to oxidize fatty acids. Distinct myocardial metabolomic signatures of ketone oxidation were identified.

Conclusions: These results indicate that the hypertrophied and failing heart shifts to ketone bodies as a significant fuel source for oxidative ATP production. Specific metabolite biosignatures of in vivo cardiac ketone utilization were identified. Future studies aimed at determining whether this fuel shift is adaptive or maladaptive could unveil new therapeutic strategies for heart failure.

Keywords: fatty acids; heart failure; hypertrophy; metabolism; molecular biology.

Figure 1

Mitochondrial proteomic profiling in the hypertrophied and failing mouse heart. (A) Schematic of the experimental design for quantitative proteomic analysis using Stable Isotope Labeling by amino ACids (SILAC), in mitochondrial enriched fractions, from the ventricles of sham-operated, compensated hypertrophy (CH), and heart failure (HF) animals. (B) Venn diagram displaying the number of regulated proteins identified in the CH, HF or both groups using a cut-off of < −1.25 or > 1.5 fold change (FC) as compared to sham-operated controls (n=2 per group). (C) The graph denotes fold change in levels of proteins that meet the defined cut-offs; HF/sham (ordinate) and CH/sham (abscissa). The key denotes regulated proteins involved in two fuel utilization pathways of interest as described in the text: fatty acid β-oxidation (white) and ketone catabolism (black). Spearman’s correlation coefficient (r) was calculated to determine the relationship between the CH and HF protein changes.

Figure 2

Bdh1 expression is induced in the hypertrophied and failing mouse heart. (A) Bdh1 mRNA levels in cardiac ventricular tissue from mice 4 weeks after sham, TAC (CH), or TAC/MI (HF) surgeries. Expression is normalized to Rplp0 (36B4). Bars represent mean ±SEM values (n=9–11 per group). *p<0.05. (B) Representative immunoblot analyses performed using protein extracts prepared from mouse cardiac ventricular tissue homogenates 4 weeks post-sham, -CH, or -HF surgeries collected in the fed state (4h after feeding) or following a 24 hour fast. Antibodies used are shown on the left. Anti-VDAC was used as a mitochondrial protein loading control.

Figure 3

Increased βOHB oxidation in the hypertrophied heart. Left Panel: The fraction of ¹³C-enriched acetyl-CoA entering the TCA cycle from ¹³C-labeled βOHB (F_c, βOHB) is shown. Right panel: The fraction of carbon entering the TCA cycle via anaplerosis relative to that entering via citrate synthase (Y) is shown for CH and sham-operated controls. Data are shown as mean ±SEM (n=10, Sham and 11, CH). *p<0.05.

Figure 4

The myocardial metabolite profile of the failing heart is indicative of increased ketone utilization in the failing heart. (A) Schematic of the ketone metabolism pathway indicating relevant intermediary metabolite derivatives (dashed arrows). (B) Levels of ketone utilization pathway metabolite derivatives (C4OH-carnitine, succinate, C2-carnitine) in cardiac biventricular tissue from CH or HF mice and corresponding sham-operated controls 4 weeks post-surgery as measured previously using mass spectrometry-based quantitative metabolomics. Data are shown as mean ± SEM (n=6 per group). *p<0.05.

Figure 5

Myocardial metabolite profile on a ketogenic diet is similar to that observed for the HF mice on a standard chow diet. (A) Levels of R-C4OH-carnitine, S-C4OH-carnitine, and C2-carnitine in cardiac ventricular tissue from wild-type C57BL/6J mice fed a ketogenic (Keto) diet or standard (Std) chow diet for 4 weeks (n=5 per group). Values were determined using mass spectrometry. *p<0.05. (B) Total C4OH-carnitine levels in extracts prepared from neonatal rat ventricular myocytes (NRVMs) cultured in media +/− 8mM ketone, R-βOHB, in the presence of 1g/L glucose and 1mM carnitine, for 24h (n=3 per group, *p<0.05 by Student’s t-test). (C) Total plasma ketones (acetoacetate+βOHB), glucose, free fatty acids (FFA), and triglycerides in CH, HF and sham-operated control mice in the fed state (after a 4h morning fast; n=5–11 per group). Bars represent mean ± SEM for all panels.*p<0.05.