Ketone body 3-hydroxybutyrate elevates cardiac output through peripheral vasorelaxation and enhanced cardiac contractility

We tested hemodynamic effects of 3-OHB in vivo and related them to actions of 3-OHB directly on isolated hearts and blood vessels ex vivo. As detailed below, we investigated effects of 3-OHB on (a) in vivo hemodynamic variables using echocardiography and invasive blood pressure measurements, (b) isolated perfused hearts mounted in Langendorff systems, and (c) isolated arteries (coronary, cerebral, femoral, mesenteric, renal) and veins (femoral, brachial, mesenteric) mounted in isometric wire myographs. We compared effects of racemic mixtures or individual enantiomers of Na-3-OHB to equimolar doses or concentrations of NaCl. To test concentrations of 3-OHB exceeding the pathophysiological range (i.e., >20 mM) and estimate EC₅₀ values ex vivo, we substituted Na-3-OHB for NaCl to maintain the osmolarity in the physiological range.

We performed transthoracic echocardiography on lightly sedated (3% sevoflurane) rats immobilized in supine position on a heating pad with integrated ECG electrodes. Rectal temperature was measured with a thermal probe and kept constant at 37 °C. Using a 21 MHz rat probe connected to a Vevo 2100 high-frequency ultrasound system (FUJIFILM VisualSonics, Canada), we acquired B-mode images in both short and long axis views 10 minutes after induction of anesthesia (baseline) and again 20 minutes after intraperitoneal administration of racemic Na-3-OHB (1.32 g/kg body weight) or an equimolar dose of NaCl. Na-3-OHB and NaCl were both dissolved at 300 mM in Milli-Q water with pH adjusted to 7.40 at 37 °C. Images were analyzed using Vevo Lab software (FUJIFILM VisualSonics), and left ventricular (LV) volumes calculated using the bullet method [33, 53]: LV volume = 5/6 × LV area × LV length. We report changes in echocardiography parameters from baseline for Na-3-OHB after subtraction of the response to equimolar NaCl.

In the same rats evaluated by echocardiography, we one day later measured systemic arterial blood pressure. To prevent coagulation during instrumentation, we administered 50 IU heparin intramuscularly. We placed a solid-state pressure catheter (SPR-869, Millar Instruments, USA) in the right carotid artery of intubated rats that were mechanically ventilated with 3.5% sevoflurane. Rectal temperature was maintained at 37 °C using a heating pad. We measured blood pressure and heart rate 15-20 minutes after intraperitoneal administration of racemic Na-3-OHB (1.32 g/kg body weight) and report differences relative to equimolar NaCl. Each rat received the same compound (Na-3-OHB or NaCl) during echocardiography and blood pressure measurements. We determined dP/dt_max from the arterial blood pressure traces using the blood pressure module of the LabChart 8 software (ADInstruments, New Zealand).

Tail vein blood samples were drawn immediately after echocardiography and blood pressure measurements. We quantified the combined concentration of D- and L-3-OHB by hydrophilic interaction liquid chromatography tandem mass spectrometry, as previously described [56].

To gauge the baseline blood level of 3-OHB before injection of exogenous 3-OHB, we analyzed a drop of capillary tail blood using the Freestyle Precision Neo point-of-care device (Abbott, USA).

The physiological saline solution (PSS) used for myograph experiments consisted of (in mM [13]): 119 NaCl, 22 NaHCO₃, 10 HEPES, 1.2 MgSO₄, 2.82 KCl, 5.5 glucose, 1.18 KH₂PO₄, 0.03 EDTA, 1.6 CaCl₂. The Krebs-Henseleit (KH) solution used for Langendorff experiments consisted of (in mM): 118.5 NaCl, 22 NaHCO₃, 1.2 MgSO₄, 4.7 KCl, 11 glucose, 1.2 KH₂PO₄, 2.4 CaCl₂. Solutions with elevated K⁺ were produced by equimolar substitution for Na⁺. For ex vivo experiments, NaCl and the racemic mixture and stereospecific enantiomers of Na-3-OHB were dissolved in PSS or KH buffer heated to 37 °C and aerated with gas mixtures of 5% CO₂/balance O₂ (hearts) or 5% CO₂/balance air (blood vessels) before pH was adjusted to 7.40 using NaOH or HCl.

Rats were euthanized by exsanguination during deep CO₂ or sevoflurane anesthesia. Tissues were immediately excised and transferred to ice-cold PSS. Segments of coronary septal arteries, middle cerebral arteries, mesenteric arteries and veins, renal interlobar arteries, caudal femoral arteries and veins, and profound brachial veins were dissected under a stereomicroscope and mounted in PSS-filled wire myograph chambers (DMT 610M and 620M, Denmark) for isometric evaluation [26]. The myograph chambers were bubbled with 5% CO₂/balance air and heated to 37 °C. Arteries were normalized to 90% of the internal diameter corresponding to a transmural pressure of 100 mmHg [41], whereas veins were normalized to an internal diameter corresponding to a transmural pressure of 20 mmHg [61]. All blood vessels were exposed to a warm-up procedure consisting of five 1-minute long contractions elicited by 60 mM extracellular K⁺. Then, we induced an initial maximal contraction by stimulation with 120 mM extracellular K⁺ and 0.1 μM thromboxane analogue U46619. Vasorelaxation was tested in blood vessels pre-contracted by U46619 to a stable tension equivalent to ~60% of the initial maximal contraction. The response was averaged in the interval of peak vasorelaxation, from 5 to 10 minutes after application of the test compound, and reported relative to the pre-contraction level. Multiple concentrations of Na-3-OHB and NaCl were tested on each blood vessel in alternating order between experiments to control for potential effects of time. We report changes from the pre-contraction level for Na-3-OHB after subtraction of the response to equimolar NaCl. In order to reach a vasomotor response plateau needed for EC₅₀ estimation, we performed on coronary septal arteries ex vivo an experimental series where Na-3-OHB was applied in concentrations exceeding the pathophysiological range (i.e., >20 mM). In this series of experiments, we substituted Na-3-OHB for NaCl thereby maintaining osmolarity at physiological level, and we report the vasorelaxations relative to time control experiments. We excluded blood vessels that required more than 1 µM U46619 to reach the desired pre-contraction level. Vasocontractions are illustrated and reported relative to the initial maximal contraction.

Isolated perfused rat hearts were prepared as previously described [53]. In brief, rats were anesthetized by a subcutaneous injection of Hypnorm-Dormicum (fentanyl citrate, 0.158 mg/kg body weight; fluanisone, 0.5 mg/kg body weight; midazolam, 0.5 mg/kg body weight) and mechanically ventilated through a tracheostomy. A bolus of 500 IU heparin for anticoagulation was administered through the femoral vein. Then, the ascending aorta was cannulated via a thoracoabdominal incision and retrogradely perfused at a constant pressure of 80 mmHg with 37 °C KH buffer continuously aerated with 5% CO₂/balance O₂. Following transfer to the Langendorff system, a balloon was inserted in the left ventricle through the left atrial appendage and pressurized to 5–8 mmHg to simulate preload. After the hearts had stabilized in KH buffer for 45 minutes, we tested the influence of adding 3 or 10 mM racemic Na-3-OHB or NaCl. We report changes from baseline (measured 10 or 20 minutes after buffer change) for Na-3-OHB after subtraction of the response to equimolar NaCl. We excluded hearts that during stabilization did not fulfill the following inclusion criteria adjusted for rat size [14]: Coronary flow: 10–35 mL/min, arrhythmias: <10 ectopic beats and no sustained ventricular tachycardia or fibrillation, heart rate: 150–400 min^–1, left ventricular systolic pressure: >120 mmHg.

Normally distributed, continuous data are expressed as mean±SEM. P values smaller than 0.05 were considered statistically significant. The n-values represent the number of rats (i.e., biological replicates) unless otherwise specified. Sample sizes were chosen based on previous experience involving similar methods and tissues. Investigators were not blinded for intervention. Effects of time were eliminated by alternating the order of interventions or performing control and intervention experiments on separate preparations in parallel. We applied two-tailed Student’s t-tests to compare a single variable between two groups and used logarithmic transformation or Welch’s correction if variances were unequal between the groups. We evaluated effects of two variables on a third variable using two-way ANOVA followed by Šídák's multiple comparisons tests or, in case of missing values, by mixed-effects analysis. Concentration-response relationships were fitted to sigmoidal functions and the derived parameters were compared using extra sum-of-squares F-tests. Data were processed in Microsoft Excel 2016, and statistical analyses performed using GraphPad Prism 10.0.2 software (CA, USA).

For the echocardiographic evaluation, intraperitoneal injection of racemic Na-3-OHB increased plasma 3-OHB concentrations by 1.9±0.3 mM compared to an equimolar dose of NaCl (Fig. 1A). Relative to the osmotic control, 3-OHB raised cardiac output by 28.3±7.8% due to an increase in stroke volume of 22.4±6.0% with no change in heart rate (Fig. 1B). As illustrated in Fig. 1B, the larger stroke volume in response to 3-OHB was associated with an increase in end-diastolic volume of 10.0±4.5% and a similar—yet not significant—relative decrease in end-systolic volume. Together, these effects raised the left ventricular ejection fraction by 13.3±4.6% (Fig. 1B).

For the invasive blood pressure measurements, intraperitoneal injection of racemic Na-3-OHB increased plasma 3-OHB concentrations by 3.2±0.4 mM compared to equimolar NaCl injection (Fig. 1C). The recordings revealed no significant effect of 3-OHB on mean arterial blood pressure, pulse pressure, systolic or diastolic blood pressure (Fig. 1D). However, consistent with higher cardiac contractility—suggested by the elevated left ventricular ejection fraction (Fig. 1B)—3-OHB increased the maximal rate of rise of the arterial blood pressure (dP/dt_max) by 31.9±12.9% (Fig. 1D).

The different plasma concentrations of 3-OHB reached during echocardiography (Fig. 1A) and arterial pressure catheter instrumentation (Fig. 1C) complicate quantitative interpretations. Nevertheless, based on rats with matched recordings of cardiac output and mean arterial blood pressure, we estimate that 3-OHB lowers systemic vascular resistance by 30.6±11.2% (Fig. 1E).

To identify the cardiovascular sites of action for 3-OHB, we next evaluated contractile function in isolated perfused hearts mounted in Langendorff systems with constant pre- and afterload (Fig. 2). We compared effects of racemic Na-3-OHB to equimolar NaCl administered to the cardiac perfusate. Relative to the osmotic control, addition of 3–10 mM 3-OHB increased left ventricular systolic pressure by up to 27.0±7.5 mmHg (Fig. 2A, B) and left ventricular developed pressure by up to 26.3±7.5 mmHg (Fig. 2B) within 10 minutes, whereas heart rate was unaffected (Fig. 2B). The effects after 20 minutes were very similar (Supplementary Fig. S1). These findings further support that 3-OHB increases cardiac contractility.

We also evaluated effects of 3-OHB on coronary perfusion in isolated perfused hearts mounted in Langendorff systems. Compared to the osmotic control, 10 mM 3-OHB increased coronary flow rate by 20.2±9.5% (Fig. 2B).

To determine whether the raised coronary perfusion in response to 3-OHB is explained by direct vasorelaxation or is secondary to metabolic regulation elicited through effects on cardiomyocytes, we next investigated the function of isolated coronary arteries. The arteries were mounted in wire myographs and pre-contracted with the thromboxane analog U46619 (Fig. 3). 3-OHB caused concentration-dependent vasorelaxation of coronary septal arteries beginning at concentrations above 1 mM and with an EC₅₀ value of 12.4 mM (Fig. 3A, B). The vasorelaxant influence of 3-OHB ex vivo peaked after approximately 10 minutes and then reached a plateau that was stable for at least 30 minutes (Fig. 3A).

Racemic 3-OHB consists of two enantiomers, but D-3-OHB dominates endogenous production. To evaluate whether the observations made with racemic 3-OHB are relevant also to conditions with increased endogenous levels—for instance, fasting, ketogenic diets or metabolic disease—we individually tested the two enantiomers. The coronary septal arteries relaxed at similar rate and with similar magnitude when exposed separately to the two enantiomers D-3-OHB (Fig. 3C) and L-3-OHB (Fig. 3D).

In the physiologically relevant concentration range between 2 and 4 mM, the coronary vasorelaxation to 3-OHB (Fig. 3B) was 20–25% of that to 5 µM of the endothelium-dependent vasorelaxant acetylcholine (Fig. 3E). When we elevated 3-OHB concentrations to 30–100 mM—exceeding the pathophysiologically and therapeutically relevant range—vasorelaxation of the coronary arteries was almost complete (Fig. 3B) and similar in magnitude to that induced by 5 µM acetylcholine (Fig. 3E).

We next explored whether the effect of 3-OHB involves changes in endothelium-dependent vasoactive mechanisms. After denudation of the endothelial cell layer—achieved by passing air bubbles through the lumen—the coronary arteries, as expected, did not relax in response to 5 µM acetylcholine (Fig. 3E). However, the endothelial denudation did not restrict the relaxation to 10 mM 3-OHB (Fig. 3F). We also observed no influence on the 3-OHB-induced vasorelaxation when coronary arteries were pre-incubated with 3 µM indomethacin (Fig. 3G), supporting that a cyclooxygenase-dependent prostanoid is not involved.

Finally, we tested whether incubation with 3-OHB influences the sensitivity of septal coronary arteries to a vasoconstrictor agent. Indeed, 10 mM 3-OHB substantially rightward-shifted the concentration-dependency of the vasoconstrictor response to U46619 (Fig. 3H).

Taken together, we conclude that 3-OHB causes endothelium-independent vasorelaxation through a direct action on coronary arteries at concentrations that are both physiologically and pathophysiologically relevant.

We next expanded our evaluation of 3-OHB-induced vasorelaxation to vascular beds other than coronary arteries (Fig. 4A, B), including both arteries and veins. Supplementary Figs. S2 and S3 show average tension traces for each separate concentration of Na-3-OHB and NaCl tested.

3-OHB relaxed caudal femoral, middle cerebral, and mesenteric arteries in a concentration-dependent manner, beginning at 6–10 mM (Fig. 4C–H). In contrast, the tone of renal interlobar arteries was only minimally reduced in response to 3-OHB even at a concentration of 10 mM (Fig. 4I). 3-OHB also relaxed caudal femoral, profound brachial, and mesenteric veins in a concentration-dependent manner (Fig. 5A–D). Whereas the profound brachial veins relaxed even in response to 1 mM 3-OHB (Fig. 5C), the caudal femoral and mesenteric veins responded significantly only when 3-OHB concentrations were elevated to 6 mM or higher (Fig. 5B, D).