Ketone Ester Treatment Improves Cardiac Function and Reduces Pathologic Remodeling in Preclinical Models of Heart Failure

Abstract

Background: Accumulating evidence suggests that the failing heart reprograms fuel metabolism toward increased utilization of ketone bodies and that increasing cardiac ketone delivery ameliorates cardiac dysfunction. As an initial step toward development of ketone therapies, we investigated the effect of chronic oral ketone ester (KE) supplementation as a prevention or treatment strategy in rodent heart failure models.

Methods: Two independent rodent heart failure models were used for the studies: transverse aortic constriction/myocardial infarction (MI) in mice and post-MI remodeling in rats. Seventy-five mice underwent a prevention treatment strategy with a KE comprised of hexanoyl-hexyl-3-hydroxybutyrate KE (KE-1) diet, and 77 rats were treated in either a prevention or treatment regimen using a commercially available β-hydroxybutyrate-(R)-1,3-butanediol monoester (DeltaG; KE-2) diet.

Results: The KE-1 diet in mice elevated β-hydroxybutyrate levels during nocturnal feeding, whereas the KE-2 diet in rats induced ketonemia throughout a 24-hour period. The KE-1 diet preventive strategy attenuated development of left ventricular dysfunction and remodeling post-transverse aortic constriction/MI (left ventricular ejection fraction±SD, 36±8 in vehicle versus 45±11 in KE-1; P=0.016). The KE-2 diet therapeutic approach also attenuated left ventricular dysfunction and remodeling post-MI (left ventricular ejection fraction, 41±11 in MI-vehicle versus 61±7 in MI-KE-2; P<0.001). In addition, ventricular weight, cardiomyocyte cross-sectional area, and the expression of ANP (atrial natriuretic peptide) were significantly attenuated in the KE-2-treated MI group. However, treatment with KE-2 did not influence cardiac fibrosis post-MI. The myocardial expression of the ketone transporter and 2 ketolytic enzymes was significantly increased in rats fed KE-2 diet along with normalization of myocardial ATP levels to sham values.

Conclusions: Chronic oral supplementation with KE was effective in both prevention and treatment of heart failure in 2 preclinical animal models. In addition, our results indicate that treatment with KE reprogrammed the expression of genes involved in ketone body utilization and normalized myocardial ATP production following MI, consistent with provision of an auxiliary fuel. These findings provide rationale for the assessment of KEs as a treatment for patients with heart failure.

Keywords: esters; heart failure; ketone bodies; myocardial infarction; myocardium.

Conflict of interest statement

The University Medical Center Groningen, which employs Dr Yurista, Dr Silljé, K.T. Nijholt, Dr van Veldhuisen, Dr de Boer, and Dr Westenbrink, has received research grants and fees from Abbott, AstraZeneca, Bristol-Myers Squibb, Novartis, Novo Nordisk, and Roche. Dr de Boer received personal fees from Abbott, AstraZeneca, and Novartis. Drs Newman and Verdin are cofounders with equity interest in BHB Therapeutics, Inc. Dr Verdin is an advisor to KetoFuel, Inc. Dr Newman serves on the scientific advisory board of Virta Health, Inc, and Roche.

Figures

Figure 1.

Ketone ester diet increases nocturnal circulating β-hydroxybutyrate (βHB or BHB) in mice.A, Structure of the hexanoyl-hexyl-3-hydroxybutyrate ketone ester (KE-1) depicting a central βHB flanked by two 6-carbon moieties. B, Levels of blood βHB measured after 1 wk of feeding normal chow, 10% w/w KE-1 diet, or 15% w/w KE-1 diet (n=5–9). βHB measurements were taken during the day (09:00–13:00) and at night (00:00–02:00). Two-way ANOVA with Tukey multiple comparison test. C, Body weight normalized to starting weight measured after 1 wk of feeding normal chow, 10% w/w KE-1 diet, or 15% w/w KE-1 diet. Data are presented as mean±SEM. Comparisons between groups were performed using 1-way ANOVA with Tukey multiple comparison test.

Figure 2.

Preventive treatment with ketone ester diet attenuates pathological cardiac remodeling and left ventricular dysfunction in a mouse model of heart failure.A, Schematic showing the experimental timeline. Male C57Bl/6NJ mice aged 7 wk were randomized to diet of either standard chow or 10% hexanoyl-hexyl-3-hydroxybutyrate ketone ester (KE-1) diet. One week later, mice received either sham surgery or transverse aortic constriction (TAC)/myocardial infarction (MI) surgery. B, Ratio of biventricular weight (BV) to tibia length (T). C, Left ventricular (LV) end-systolic volume (ESV) and end-diastolic volume (EDV) 4 wk after TAC/MI surgery as determined by echocardiography. D, LV ejection fraction (LVEF) 4 wk post-TAC/MI surgery (n=11–16). Data are presented as mean±SEM. Comparisons between groups were performed using 2-way ANOVA with Tukey multiple comparison test.

Figure 3.

Ketone ester supplementation induces sustained ketonemia in rats.A, Experimental design of early and late β-hydroxybutyrate (βHB or BHB)–butanediol monoester (KE-2) diet supplementation regimens in rats with heart failure (HF) after myocardial infarction (MI). B, Plasma βHB concentrations measured after 10 d of KE-2 supplementation during the day (09:00–10:30) and at night (22:00–22:30; n=8–26). C, Body weight at the end of the study (n=8–20). Data are presented as mean±SEM. Comparisons between groups were performed using 2-way ANOVA with Tukey multiple comparison test. Echo indicates echocardiography.

Figure 4.

Ketone ester treatment improves pathological cardiac remodeling and left ventricular (LV) dysfunction in a rat model of heart failure.A, Representative LV sections stained with Masson trichrome. B, Quantification of infarct size from Masson trichrome stained section (n=7–22). C, Ratio of biventricular weight (BV) to tibia length (T; n=7–21). D, LV internal dimensions in diastole (LVIDd) as determined by echocardiography (n=7–22). E, LV ejection fraction (LVEF; n=7–22). Comparisons between groups were performed using 1-way ANOVA with Tukey multiple comparison test. F, Longitudinal change in LVEF between 2 wk post-myocardial infarction (MI) and termination in the βHB-butanediol monoester (KE-2)-late (KE-2-L) compared with the chow control group, tested using Wilcoxon signed-rank test. Data are presented as mean±SEM. KE-2-E indicates KE-2-early.

Figure 5.

Ketone ester treatment attenuates cardiac hypertrophy independent of fibrotic response.A, Representative left ventricular (LV) sections stained with wheat germ agglutinin (WGA) and Masson trichrome to assess cardiomyocyte hypertrophy and fibrosis, respectively. B, Quantification of cardiomyocyte cross-sectional area from WGA-stained section (n=7–22). C, Quantification of fibrosis in noninfarcted LV from Masson trichrome–stained section (n=7–22). The graph denotes fold change over sham-chow control group. D and E, Measurement of mRNA levels to assess molecular markers for remodeling and fibrosis, respectively, normalized to 36B4 (n=7–22). The graph denotes fold change over sham-chow group. Data are presented as mean±SEM. Comparisons between groups were performed using 1-way ANOVA with Tukey multiple comparison test. ANP indicates atrial natriuretic peptide; COL1A1, collagen type I alpha 1; KE-2-E, βHB-butanediol monoester-early; KE-2-L, βHB-butanediol monoester-late; and MI, myocardial infarction.

Figure 6.

Ketone ester treatment enhances cardiac energy. Levels of mRNA encoding MCT1 (monocarboxylate transporter 1; n=9–21; A) and BDH1 (3-hydroxybutyrate dehydrogenase 1; n=9–21; B) in left ventricular (LV) free wall remote from the infarction (as determined by quantitative polymerase chain reaction) shown as arbitrary units normalized to the value of sham-chow control (+1.0). C, Top, Representative immunoblot analyses of succinyl CoA:3-oxo acid CoA transferase (SCOT) and GAPDH from protein extracts derived from the noninfarcted LV free wall. Bottom, Average SCOT protein expression normalized to GADPH and presented as fold change over the sham-chow group (n=5–6). D, ATP levels in the LV tissue (n=9–21). Data are presented as mean±SEM. Comparisons between groups were performed using Kruskal-Wallis test with Dunn multiple comparisons test. MI indicates myocardial infarction.