Short‐Chain Fatty Acid Butyrate Is an Inotropic Agent With Vasorelaxant and Cardioprotective Properties

Abstract

Background

The heart can metabolize the microbiota‐derived short‐chain fatty acid butyrate. Butyrate may have beneficial effects in heart failure, but the underlying mechanisms are unknown. We tested the hypothesis that butyrate elevates cardiac output by mechanisms involving direct stimulation of cardiac contractility and vasorelaxation in rats.

Methods and Results

We examined the effects of butyrate on (1) in vivo hemodynamics using parallel echocardiographic and invasive blood pressure measurements, (2) isolated perfused hearts in Langendorff systems under physiological conditions and after ischemia and reperfusion, and (3) isolated coronary arteries mounted in isometric wire myographs. We tested Na‐butyrate added to injection solutions or physiological buffers and compared its effects with equimolar doses of NaCl. Butyrate at plasma concentrations of 0.56 mM increased cardiac output by 48.8±14.9%, stroke volume by 38.5±12.1%, and left ventricular ejection fraction by 39.6±6.2%, and lowered systemic vascular resistance by 33.5±6.4% without affecting blood pressure or heart rate in vivo. In the range between 0.1 and 5 mM, butyrate increased left ventricular systolic pressure by up to 23.7±3.4% in isolated perfused hearts and by 9.4±2.9% following ischemia and reperfusion, while reducing myocardial infarct size by 81.7±16.9%. Butyrate relaxed isolated coronary septal arteries concentration dependently with an EC₅₀=0.57 mM (95% CI, 0.23–1.44).

Conclusions

We conclude that butyrate elevates cardiac output through mechanisms involving increased cardiac contractility and vasorelaxation. This effect of butyrate was not associated with adverse myocardial injury in damaged hearts exposed to ischemia and reperfusion.

Nonstandard Abbreviations and Acronyms

3‐OHB

3‐hydroxybutyate

KH

Krebs‐Henseleit

Research Perspective.

What Is New?

• Elevating the plasma concentration of the short‐chain fatty acid butyrate simultaneously increases cardiac output by 48% and ejection fraction by 39%, and lowers systemic vascular resistance by 33% in rats.

• In isolated hearts and arteries, butyrate increases cardiac contractility and induces vasorelaxation, respectively.

• Butyrate improves contractile function of the left ventricle and reduces myocardial injury after ischemia and reperfusion.

What Question Should Be Addressed Next?

• The hemodynamic effects of butyrate should be investigated in larger animal models and in clinical trials.

• The molecular mechanism responsible for simultaneous increased cardiac contractility, arterial vasodilation, and protection from ischemia‐induced myocardial injury should be elaborated.

Metabolic derangements of the cardiovascular system have emerged as part of the pathophysiology underlying heart failure. Exogenous administration of substances that improve cardiac metabolism may represent a new therapeutic approach. ¹ Recently, we showed that stimulation of the cardiovascular system with the ketone body 3‐hydroxybutyrate (3‐OHB) elevates cardiac output in healthy individuals and patients with heart failure without compromising cardiac energy supply and demand. ² This is particularly important for patients with heart failure, because the balance between tissue perfusion and oxidative demand is often disrupted.

The beneficial actions of 3‐OHB stem from its ability to increase cardiac contractility and induce simultaneous peripheral relaxation of resistance arteries. ³ However, achieving therapeutically meaningful plasma concentrations of 3‐OHB is challenging. Intravenous administration is confined to in‐hospital care, whereas ingestion of ketone esters is hindered by their unpleasant taste and gastrointestinal side effects. ⁴

Butyrate is a 4‐carbon short‐chain fatty acid produced by the intestinal microbiota during anaerobic fermentation of dietary fibers. ⁵ Local concentrations in the colon may be as high as 100 mM, whereas systemic concentrations usually remain within a low micromolar range. ⁷ Apart from fueling our intestinal mucosa, butyrate may have beneficial extraintestinal effects, such as lowering of blood pressure and inflammation and improving lipid metabolism. ⁸ , ⁹ Exogenous butyrate can be administered orally in the esterified and capsulated form of tributyrin, which is generally well tolerated and can elevate butyrate plasma levels to ≈0.1 mM. ¹⁰

Both butyrate and 3‐OHB serve as substrates for oxidative phosphorylation in the myocardium. ¹¹ , ¹² , ¹³ The failing heart upregulates key enzymes involved in the metabolism of 3‐OHB ¹⁴ , ¹⁵ and butyrate ¹¹ but favors butyrate over 3‐OHB for ATP generation. ¹¹ Butyrate dilates mesenteric and caudal arteries ¹⁶ , ¹⁷ and is associated with blood pressure reduction in both clinical and preclinical models of hypertension when supplied through probiotic stimulation of butyrate‐producing bacteria or butyrate‐enriched starch. ⁹ , ¹⁸ However, the specific cardiac effects and hemodynamic consequences of butyrate administration remain unknown.

In this study, we investigate the hemodynamic effects of exogenous butyrate administration in rats. We hypothesize that butyrate elevates cardiac output through increased cardiac contractility and vasorelaxation of resistance arteries. Furthermore, we explore whether butyrate affects myocardial viability following ischemia and reperfusion.

Methods

The authors declare that all supporting data of the present study are available in the article. Original data underlying this article will be shared upon reasonable request to the corresponding author. Unless otherwise specified, chemicals were obtained from Sigma‐Aldrich (Germany). The experimental protocol conformed to the Danish law for animal research and the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. The experimental protocols were approved by the Danish Animal Experiments Inspectorate (2018‐15‐0201‐01475, 2018‐15‐0201‐01446, and 2021‐15‐0201‐00986). We followed the Animal Research: Reporting of In Vivo Experiments 2.0 guidelines. ¹⁹ , ²⁰ A full description of the intended experimental design and analysis has not been published in a preclinical registry. Experiments on rats were performed between December 2022 and August 2023.

Animals

Male Sprague‐Dawley rats (n=122) (9–12 weeks old) from Taconic Biosciences (Denmark) were housed at Aarhus University and acclimatized for 7 days before experiments under a 12‐hour light/dark cycle with ad libitum access to food and water.

Design

We investigated the effect of butyrate by comparing Na‐butyrate added to injection solutions or physiological buffers with NaCl at equimolar concentrations in 4 series of experiments. Schematic illustrations of the experimental series are provided in Figures S1–S4.

In Vivo Hemodynamics

We investigated hemodynamic effects of Na‐butyrate as detailed below. We compared echocardiographic variables and invasive blood pressure measurements between intraperitoneal injections with Na‐butyrate (n=8) or NaCl (n=6) at increasing doses (10‐minute intervals; Figure S1).

Ex Vivo Isolated Hearts

We investigated butyrate‐mediated effects on left ventricular contractile function in isolated perfused hearts under constant pre‐ and afterload in Langendorff systems. Following stabilization, we tested effects on cardiac variables of adding Na‐butyrate or NaCl at 0.1 mM (n=9–10), 0.5 mM (n=8–10), or 5 mM (n=9–10) to a standard Krebs‐Henseleit (KH) buffer (Figure S2).

Ex Vivo Isolated Hearts Following Ischemia and Reperfusion

We explored whether butyrate‐induced inotropic stimulation affects the vulnerability of hearts to ischemia and reperfusion injury, as might be expected from conventional inotropic agents. ²¹ , ²² As elaborated below, following ischemia and reperfusion with KH buffer, we tested the effects of adding Na‐butyrate or NaCl at 0.5 mM (n=8) and 5 mM (n=6–8) to the perfusion buffer for the remaining reperfusion period. We measured changes in cardiac parameters and the final infarct size (Figure S3).

Ex Vivo Isolated Coronary Arteries

To elucidate the direct effect of butyrate on resistance arteries, we tested vascular responses in coronary septal arteries mounted in wire myographs. Concentration‐response curves were obtained by cumulative stepwise additions (0.1–30 mM) of either Na‐butyrate or NaCl (n=7) for 10‐minute durations at each concentration (Figure S4).

Echocardiography and Blood Pressure Measurements

Rats were anesthetized in an induction chamber with 8% sevoflurane, intubated, and mechanically ventilated with a rodent ventilator (Ugo Basile 7025, Italy). The rats were immobilized in a supine position on a heating pad with integrated ECG electrodes, and anesthesia was maintained with 3% to 3.5% sevoflurane (in balanced air, 0.5 L/min). Ventilation was adjusted to target an end‐tidal CO₂ fraction of 4% using a capnograph (Type 340; Harvard Apparatus, Holliston, MA). A rectal thermal probe was inserted and the core temperature kept at 37 °C. A single bolus of 50 IU heparin was injected intramuscularly. The right carotid artery was exposed through dissection, and a solid‐state pressure catheter (SPR‐869; Millar Instruments, TX) was inserted. The left femoral artery was cannulated and a sheet inserted for repeated blood sampling. B‐mode ultrasound images in short‐ and long‐axis view were acquired with a 21‐MHz dedicated rat probe connected to a Vevo 2100 high‐frequency ultrasound system (Fujifilm VisualSonics, Canada). Ultrasound images, blood pressure, and heart rate data were acquired following 10 minutes of stabilization (baseline) and after subsequent intraperitoneal injections of 0.033, 0.165, and 0.661 g/kg bodyweight of Na‐butyrate (Thermo Fisher Scientific, MA) or equimolar NaCl, separated by 10‐minute intervals. Na‐butyrate and NaCl were dissolved in Milli‐Q water to 300 mM and adjusted to pH 7.40 at 37 °C. No specific inclusion/exclusion criteria were set for this experimental series; 2 animals died from bleeding during cannulation.

Ultrasound images were analyzed with Vevo Lab software (Fujifilm VisualSonics) using the bullet method to calculate left ventricular volumes: volume=5/6 × area × length. ²³ , ²⁴ We show changes in hemodynamic parameters induced by butyrate from baseline after subtracting the response to equimolar NaCl for cardiac output, stroke volume, left ventricular end‐systolic volume, left ventricular end‐diastolic volume, ejection fraction, mean arterial pressure, heart rate, systolic blood pressure, diastolic blood pressure, and systemic vascular resistance.

Quantification of Butyrate in Plasma

Blood samples were drawn from a femoral artery sheet immediately after each echocardiography and blood pressure measurement.

Concentrations of butyrate were quantified by liquid chromatography tandem mass spectrometry after extraction from plasma with a method using butyrate reference compound for calibration and stable isotope labeled internal standard. We added plasma (25 μL) to 25 μL solvent A (water:methanol 98:2 with 0.013% acetic acid), 25 μL internal standard solution (1 mM ¹³C₄‐butyrate (TRC, Canada)), and finally 250 μL of methanol:acetonitrile (1:1). The extract was vortex‐mixed and centrifuged for 5 minutes at 10 000g, and 20 μL of the resulting supernatant was diluted with 180 μL solvent A. Pure calibrant samples (7 including blank) were prepared from butyrate reference compound using the plasma sample preparation method at nominal concentrations equivalent to 0, 0.04, 0.1, 0.5, 2, 5, and 10 mM.

Sample solution (7 μL) was injected into a Waters ultraperformance liquid chromatography system (Waters, MA), equipped with an ultra‐performance liquid chromatography HSS T3 column (Waters Premier T3 1.8 μm, 2.1 × 100 mM) maintained at 30 °C. The flow rate was 0.3 mL/min, and the initial condition was 100% mobile phase A (water:methanol 98:2 with 0.013% acetic acid) that was changed through a linear gradient to 8% mobile phase B (methanol with 0.013% acetic acid) over 3 minutes. From 3 to 6 minutes, the gradient was changed to 60% B, and to 90% B at 8 minutes, returned to 100% A at 8.5 minutes, and allowed to equilibrate for 2 minutes. The compounds were analyzed with a Waters Xevo TQS mass spectrometer using electrospray ionization in the negative mode with a capillary voltage of 2.5 kV, with source and desolvation temperatures of 150 °C and 500 °C, respectively. Butyrate did not undergo fragmentation during electrospray and collision‐induced dissociation, so the analytes were monitored by their parent ion transitions with the following parameters: butyrate (m/z: 87 → 87, collision energy 2 eV), ¹³C₄‐butyrate (m/z: 91 → 91). Calibration curves were constructed by linear regression of the peak area ratio (analyte/internal standard) versus the nominal concentrations of the calibrator samples with a weighting factor of 1/X using targetlynx in masslynx vers 4.2 (Waters).

Solutions for Ex Vivo Experiments

For myograph experiments a physiological saline solution was used (in millimolars: 119 NaCl, 22 NaHCO₃, 10 HEPES, 1.2 MgSO₄, 2.82 KCl, 5.5 glucose, 1.18 KH₂PO₄, 0.03 EDTA, 1.6 CaCl₂). ²⁵ A standard KH solution was used for Langendorff experiments (in millimolars: 118.5 NaCl, 22 NaHCO₃, 1.2 MgSO₄, 4.7 KCl, 11 glucose, 1.2 KH₂PO₄, 2.4 CaCl₂). ²⁶ We adjusted the pH of all solutions to 7.40 °C at 37 °C using HCl or NaOH during continuous bubbling with 5% CO₂.

Isolated Perfused Hearts

Rat hearts were isolated and perfused ex vivo as previously described. ²⁴ , ²⁷ Rats were anesthetized by a mixture of Hypnorm and Dormicum (containing in milligrams per kilogram body weight: fluanisone, 0.5; fentanyl citrate, 0.158, midazolam, 0.5). Through a tracheostomy, rats were mechanically ventilated (Ugo Basile 7025, Italy), and a bolus of 500 IU heparin was administered through the femoral vein for anticoagulation. A thoracotomy and laparotomy were performed, and the ascending aorta was cannulated under continuous retrograde perfusion with an aerated (5% CO₂/95% O₂) and pressurized (80 mm Hg) KH buffer. Perfused hearts were excised and transferred to an isolated perfused heart system (IH‐SR type844/1; HSE, Germany) and submerged in an organ bath with a constant temperature of 37 °C. A balloon (size 7; HSE) connected to a pressure transducer was inserted in the left ventricle through the left atrial appendage and adjusted to 5 to 8 mm Hg to simulate preload. Following stabilization in KH buffer for 45 minutes (baseline), we examined the effects of adding 0.1, 0.5, and 5 mM Na‐butyrate or NaCl on left ventricular systolic pressure, heart rate, and coronary flow rate.

A subset of hearts underwent 40 minutes of stabilization in KH buffer, 20 minutes of global ischemia, 30 minutes of reperfusion with KH (baseline), followed by the addition of 0.5 and 5 mM Na‐butyrate or NaCl to the perfusion buffer for the remaining reperfusion period. We excluded hearts that during stabilization did not fulfill the following prespecified inclusion criteria adjusted for rat size ²⁶ : coronary flow: 10 to 35 mL/min, arrhythmias: <10 ectopic beats and no sustained ventricular tachycardia or fibrillation, heart rate: 150 to 400 min⁻¹, left ventricular systolic pressure: >120 mm Hg (experimental series 2: n=2, experimental series 3: n=4).

Data were digitally converted (DT9804; Data Translation, MA) and analyzed using Notochord Hem Software (Notochord Systems, France).

Infarct Size

We measured infarct sizes in isolated perfused hearts by a conventional method ²⁶ ; hearts were demounted and frozen at −80 °C, and heart slices (2 mm) were incubated for 3 minutes with 1% 2, 3, 5‐triphenyltetrazoliumchloride. After 24 hours storage in 4% formaldehyde (VWR International, Belgium), the area at risk and infarct size were evaluated with ImageJ 2 software (National Institutes of Health) and adjusted to the wet weight of the individual slices. The final infarct sizes are expressed as the ratio infarct size/area at risk, where area at risk corresponds to the entire left ventricle.

Isometric Wire Myography

Rats were euthanized by exsanguination in deep CO₂ anesthesia. Hearts were immediately excised and transferred to ice‐cold physiological saline solution. Coronary septal arteries were isolated by dissection under a stereo microscope. Small segments of blood vessels were mounted in physiological saline solution‐filled myograph chambers (DMT 610M, Denmark) using 40‐μm stainless steel wires. Myograph chambers were heated to 37 °C and continuously bubbled with 5% CO₂/balance air. Arteries were normalized to 90% of the internal diameter corresponding to a transmural pressure of 100 mm Hg. ²⁸ All arteries were exposed to the same warm‐up procedure: 5 contractions elicited by 60 mM K⁺ (1 minute per contraction) followed by an initial maximal contraction elicited by stimulation with 120 mM K⁺ and 0.1 μM U46619 (thromboxane analogue). Vascular responses to Na‐butyrate or NaCl were tested in U46619 precontracted vessels that developed a stable tension equivalent to ~60% of the initial maximal response. Concentration‐response curves were obtained by cumulative stepwise additions (10‐minute intervals) of either Na‐butyrate or NaCl (0.1–30 mM), and we report vasorelaxations relative to time control experiments with equimolar NaCl. The response to each concentration level was averaged over the last 30 seconds of the 10‐minute exposure. We report relative changes from the precontraction level and excluded blood vessels (n=1) that required >1 μM U46619 to reach the predefined level of precontraction.

Statistical Analysis

Normally distributed continuous data are expressed as mean±SEM unless otherwise specified, and P values <0.05 were considered statistically significant. Sample sizes were chosen based on previous experience with similar methods. To eliminate potential time effects, we performed experiments with parallel intervention and control solutions. Animals were randomly allocated to receive either butyrate or control solutions. Investigators were not blinded to interventions but were blinded during analyses. Single variables were compared between 2 groups using a 2‐tailed Student t‐test. The effects of 2 categorical variables, time and randomization group, on a dependent variable (end point) were evaluated using a mixed‐effects model for repeated measures. This model included time and randomization group as fixed effects, with rats serving as the unit of random effect to account for the intrasubject correlation of repeated measurements over time. Although the model assumed a symmetrical variance structure, it did not presuppose sphericity (equal variability of differences). Consequently, the Geissner‐Greenhouse correction was applied to adjust for potential violations of the sphericity assumption. Bonferroni's correction was applied for multiple comparisons. We fitted a sigmoidal nonlinear regression to the log[butyrate] and vasoactive response relationships to obtain an EC₅₀ value of the dose–response relationship. Data were analyzed using GraphPad Prism 10.1.1 software (Boston, MA).

Results

Animals

Of the total 122 animals housed at Aarhus University, 107 were included in the final analysis. Specific reasons for exclusion are provided in a Consolidated Standards of Reporting Trials diagram (Figure S5).

Effects of Butyrate on Hemodynamic Variables

Figure 1A illustrates the experimental setup of the in vivo series. Evaluating hemodynamic changes with echocardiography and invasive blood pressure measurements, we observed no significant differences in baseline parameters between the butyrate and control group (Table S1).

Figure 1. In vivo hemodynamic changes after injections with butyrate.

A, Schematic illustration of the experimental setup. B, Plasma concentrations of butyrate during echocardiography and blood pressure measurements after IP injection with 0.033 (1. Injection), 0.165 (2. Injection), and 0.661 (3. Injection) g/kg body weight Na‐butyrate or NaCl (n=5–7). Values below the method specific detection limit of 0.03 mM are reported as 0. C through E, Cardiac and hemodynamic changes induced by increasing butyrate plasma concentrations following injections with 1: 0.033, 2: 0.165, 3: 0.661 g butyrate/kg body weight. C, Changes in cardiac variables (n=6–8). D, Changes in MAP, HR, SBP, and DBP (n=6–8). E, Changes in SVR (n=6–8). Bars represent relative differences between Na‐butyrate and equimolar NaCl, and absolute differences are reported below the bars for selected comparisons. Bars represent mean±SEM. Data in panels C through E were compared using a mixed‐effects model for repeated measures with Bonferroni's multiple comparisons test. The schematic illustration was created with BioRender.com. CO indicates cardiac output; DBP, diastolic blood pressure; EF, ejection fraction; HR, heart rate; LVEDV, left ventricular end‐diastolic volume; LVESV, left ventricular end‐systolic volume; MAP, mean arterial pressure; SBP, systolic blood pressure; SV, stroke volume; and SVR, systemic vascular resistance.

Intraperitoneal injections of Na‐butyrate in increasing doses from 0.033, 0.165, to 0.661 g/kg bodyweight raised plasma levels of butyrate by 0.03±0.01, 0.11±0.03, and 0.56±0.06 mM (Figure 1B). Echocardiographic evaluation revealed an increase in cardiac output by up to 48.8±14.9% relative to the osmotic NaCl control (Figure 1C). The increment was caused by an increase in stroke volume by 38.5±12.1%, because heart rate did not change significantly (Figure 1D). The change in stroke volume was associated with a decrease in end‐systolic volume by 50.1±6.7%, whereas end‐diastolic volume remained unaffected (Figure 1C). Accordingly, butyrate elevated left ventricular ejection fraction by 39.6±6.2% (Figure 1C).

Invasive blood pressure measurements revealed no significant change in mean arterial pressure resulting from any butyrate dose when compared with equimolar NaCl (Figure 1D). Because arterial pressure, cardiac output, and heart rate were measured simultaneously, we estimated a corresponding reduction in systemic vascular resistance of 33.5±6.4% (Figure 1E) elicited by the highest butyrate concentration. In addition to the relative values reported here, absolute values are provided in Table S2.

Effects of Butyrate on Cardiac Contractility in Isolated Hearts

Figure 2A shows the average trace of left ventricular systolic pressure during the experimental protocol with 5 mM Na‐butyrate or equimolar NaCl. At baseline we observed no significant differences between groups of isolated perfused hearts (Table S3).

Figure 2. Effects of butyrate at different concentrations on isolated perfused hearts.

A, Average trace showing changes in left ventricular systolic pressure after adding 5 mM Na‐butyrate or 5 mM NaCl (n=9–10) to a standard Krebs‐Henseleit buffer. B, Relative changes in LVSP, HR, and CFR after addition of 0.1, 0.5, and 5 mM Na‐butyrate (n=8–10). Data from the last time point in (A) are also represented in (B). Bars represent relative differences between Na‐butyrate and equimolar NaCl. Data are mean±SEM. Data in (B) were compared by unpaired 2‐tailed Student t‐test. CFR indicates coronary flow rate; HR, heart rate; and LVSP, left ventricular systolic pressure.

Addition of butyrate increased left ventricular systolic pressure (Figure 2B). Relative to the NaCl control, the addition of 0.1 mM butyrate had no significant effect on left ventricular systolic pressure. In contrast, 0.5 mM butyrate increased left ventricular systolic pressure by 14.1±4.1%, and 5 mM butyrate by 23.7±3.4% (Figure 2B). None of the tested doses of butyrate led to significant changes in heart rate (Figure 2B). These data suggest that increased cardiac contractility contributes to the observed hemodynamic response, beginning at concentrations from around 0.5 mM.

Effects of Butyrate on Reperfusion Injury and Cardiac Contractility in the Injured Heart

Figure 3A shows the average trace of left ventricular systolic pressure during the experimental protocol with 0.5 mM Na‐butyrate or NaCl. We considered the baseline to be 30 minutes after reperfusion, when peak pressures had reached a plateau (Figure 3A). At this time point, we observed no differences in left ventricular pressures between groups (Table S3). Relative to baseline, butyrate increased left ventricular systolic pressure immediately after addition, and compared with the osmotic control, butyrate kept left ventricular systolic pressure elevated by 9.4±2.9% until the end of the reperfusion period (Figure 3B). Despite the observed increase in left ventricular systolic pressure, butyrate administration was associated with a reduced mean infarct size of 57.1±27.9% for 0.5 mM butyrate and 81.7±16.9% for 5 mM butyrate, relative to the osmotic control (Figure 3C and 3D).

Figure 3. Effects of butyrate on isolated perfused hearts following global ischemia and reperfusion.

A, Average trace of left ventricular systolic pressure with addition of 0.5 mM Na‐butyrate (n=8) or NaCl (n=8). B, Relative difference in left ventricular systolic pressure at the end of reperfusion with 0.5 mM (n=8) and 5 mM Na‐butyrate or NaCl (n=6–8). C and D, Differences in infarct size relative to area at risk in isolated perfused hearts following global ischemia and reperfusion with 0.5 mM (n=8) and 5 mM (n=6–8) Na‐butyrate or NaCl with representative images of heart slices at the end of each graph. Bars represent mean±SEM. Data in (B through D) were compared by unpaired 2‐tailed Student t test. LVSP indicates left ventricular systolic pressure.

Butyrate and Coronary Artery Tone

Butyrate decreased systemic vascular resistance (Figure 1E) and increased coronary flow rate in hearts mounted in Langendorff systems (20.9±7.1% at 5 mM) (Figure 2B). Figure 4A illustrates a representative trace showing changes in relative active tension in isolated coronary arteries during cumulative, stepwise additions of Na‐butyrate or NaCl. In accordance, butyrate induced concentration‐dependent vasorelaxation in precontracted arteries, with an EC₅₀ of 0.57 mM (95% CI, 0.23–1.44) (Figure 4B). Complete relaxation occurred in response to butyrate concentrations >10 mM (Figure 4B). There were no differences at baseline in the initial maximal contraction or in lumen diameter between segments of vessels exposed to Na‐butyrate or NaCl (Table S4).

Figure 4. Effects of butyrate on isolated coronary resistance arteries precontracted with U46619.

A, Representative trace of changes in relative active tension in coronary arteries during cumulative, stepwise additions of NaCl or Na‐butyrate. B, Concentration‐dependent vasorelaxant response of coronary arteries to butyrate (n=7). The vasorelaxant response was calculated relative to time control experiments with equimolar NaCl. Symbols and bars represent mean±SEM. Data in (B) were fitted a sigmoidal nonlinear regression.

Discussion

In this study, we demonstrate that the short‐chain fatty acid butyrate substantially elevates cardiac output without significant effects on blood pressure or heart rate. We assessed the underlying cardiovascular mechanism and identified 2 targets: (1) enhancement of cardiac contractility, resulting in increased left ventricular ejection fraction and (2) vasorelaxation of resistance arteries, resulting in reduced systemic vascular resistance. Finally, we demonstrate that butyrate did not exert a deleterious effect despite its positive inotropic action but rather also had beneficial effects in an injured heart exposed to ischemia–reperfusion.

We chose to explore the hemodynamic effects of butyrate and its potential for treatment of heart failure to follow‐up on the encouraging hemodynamic effects of the ketone body, 3‐OHB, in heart failure. ² , ³ Butyrate is structurally related to 3‐OHB, and both are substrates for oxidative metabolism in cardiomyocytes. ¹¹ However, therapeutically efficient plasma concentrations of 3‐OHB are challenging to obtain in patients, because administration is confined to in‐hospital care, and ingestion of ketone esters is hindered by their lack of palatability and gastrointestinal side effects. ⁴ Also, we were encouraged by a few recent studies that indicated a potential of butyrate treatment in patients with heart failure. Mollar et al recently demonstrated that intestinal dysbiosis in patients with heart failure admitted for decompensation was associated with low levels of circulating butyrate, suggesting that butyrate might play a role in the pathology of heart failure. ²⁹ Butyrate is more capably oxidized than ketone bodies in failing hearts. ¹¹ Butyrate seems to be cardioprotective by reducing infarct size in experimental models of myocardial infarction. ³⁰ , ³¹ , ³²

Our findings demonstrate that butyrate elevates cardiac output due to a combination of a moderate positive inotropic effect and a significant decrease in systemic vascular resistance. Butyrate did not induce an increase end‐diastolic volume, reflecting an unchanged preload. Despite a significant reduction in systemic vascular resistance of 33%, mean arterial pressure and heart rate did not change significantly. Of note, the largest hemodynamic changes were observed at a plasma concentration of butyrate of approximately 0.6 mM, although the hemodynamic response was present at much lower concentrations. In our experiments on isolated perfused hearts, characterized by constant pre‐ and afterload, butyrate induced a significant increase in left ventricular peak amplitude of pressure, suggesting an increase in cardiac contractility, ³³ which applies for both the intact and injured myocardium. This effect was achieved with 0.5 mM butyrate in the perfusate, directly relatable to our in vivo findings. Previous findings from experiments on isolated hearts suggest that butyrate reinstates contractile reserve after ischemia, reported as an elevation in the derivate of pressure relative to preischemic values. ³⁴ Although this may be attributed to direct inotropic stimulation, it may also be explained by reduced myocardial damage, as demonstrated here and elsewhere. ³⁰ , ³¹ , ³²

We explored the potential of inotropic stimulation of hearts subjected to ischemia and reperfusion injury because conventional inotropic agents have been associated with increased myocardial injury. ²¹ , ²² , ³⁵ Following ischemia and reperfusion, butyrate demonstrated the ability to increase left ventricular systolic pressure by ≈10%, suggesting a restored capacity for inotropic stimulation within the damaged heart. Our model using left ventricular systolic pressure reflects a global measure of left ventricular function. Therefore, our approach does not allow us to decipher whether the improved cardiac function is caused by the infarct size reduction per se or whether it relates to increased contractility of remaining healthy myocardium. Our aim was to clarify whether the inotropic effect of butyrate was associated with a deleterious effect on the injured heart, and we extended previous associations between butyrate and cardioprotection ³⁰ , ³¹ by demonstrating a reduction in final infarct size, with 5 mM butyrate yielding a remarkable 81% reduction. A reduction of this magnitude separately improves left ventricular function to an extent that may not allow detection of further stimulatory improvement of undamaged myocardium. With larger infarct size and a 57% infarct size reduction by 0.5 mM butyrate, the improved cardiac function reflects that butyrate induced contractile mobilization of the remaining intact myocardium in the damaged heart distinct from the infarct sparring effect such as our findings in the intact heart.

In contrast to conventional inotropes, such as dobutamine, which increase cardiac energy demand, ³⁶ ketone body infusion in patients with heart failure and healthy volunteers has been shown to improve cardiac output without compromising myocardial external efficiency. ² Although we did not investigate myocardial energy metabolism in the present study, this finding may extend to butyrate, particularly given the preference for butyrate over ketone bodies for oxidative metabolization in the failing heart. ¹¹ Moreover, butyrate restores intramyocardial ATP levels without deteriorating cardiac work in a murine model of metabolic heart disease. ³⁷ Detailed investigations of the effect of butyrate on cardiac energy demand warrants further exploration.

Conventional inotropic agents are used for in‐hospital treatment of acute decompensated heart failure to restore hemodynamic properties and tissue perfusion. ³⁸ However, their usage is limited to short durations due to the propensity of inotropes, especially those with adrenergic stimulation, to induce myocardial ischemia and arrhythmias associated with heightened mortality. ³⁹ Consequently, the therapeutic strategy of patients with acute decompensated heart failure involves coadministration of inotropes and veno‐/vasodilators to reduce venous return and mitigate load‐induced strain on the left ventricle. ³⁹ Consistent with our observation that butyrate decreased systemic vascular resistance significantly in vivo, butyrate relaxed coronary arteries mounted in wire myographs in a concentration‐dependent manner. The corresponding EC₅₀ value of 0.57 mM highlights the substantial vasodilatory potential within a therapeutically relevant range. Although our current focus was on coronary arteries, previous studies on mesenteric and caudal arteries suggest that butyrate‐induced vasorelaxation is present in other vascular beds. ¹⁶ , ¹⁷ In addition to the direct vascular effects, several studies report that butyrate lowers systolic blood pressure in patients with arterial hypertension ⁹ and in experimental models of hypertension. ⁹ , ⁴⁰ , ⁴¹ , ⁴² The mechanism behind the direct vascular response to both 3‐OHB and butyrate is not well understood but might involve an endothelium‐independent pathway because both substances induce vasorelaxation in isolated arteries without functional endothelium, ³ , ¹⁶ whereas various different targets are proposed to be involved in butyrate‐mediated blood pressure reduction, ranging from neuronal modulation to specific molecular pathways. ⁸

Plasma concentrations of butyrate in humans typically remain below 1 μM, ⁷ , ⁴³ , ⁴⁴ even following dietary enrichment with fermentable fibers. ⁴⁴ Based on our findings, butyrate does not naturally reach sufficient plasma concentrations to yield a noticeable hemodynamic response. However, exogenous administration of butyrate in the form of tributyrin increases plasma levels up to 0.5 mM, ¹⁰ and ingestion of butyrate‐enriched starch reduces systolic blood pressure in humans at plasma butyrate of 0.03 mM. ⁹ Our ex vivo findings demonstrate an effective response of butyrate, beginning in the range from 0.1 to 0.5 mM for both isolated hearts and isolated coronary arteries. At 0.5 mM we observe substantial relaxation of coronary arteries and a 15% increase in cardiac contractility. For comparison, we recently quantified the cardiac and vascular effects of 3‐OHB administration. ³ Isolated hearts and blood vessels that showed similar responses to 3‐OHB, only at much higher concentrations, were exemplified by an EC₅₀ value for coronary artery relaxation of 12.6 mM. However, hemodynamic changes are associated with 3‐OHB concentrations as low as 0.7 mM in humans. ² Despite potential interspecies differences in pharmacokinetics and pharmacodynamics, both 3‐OHB and butyrate appear to elicit hemodynamic responses in vivo at lower concentrations than observed ex vivo. Despite the induction of an almost 50% relaxation of isolated coronary arteries at 0.5 mM, a significant increase in coronary flow rate was observed only at 5 mM butyrate in isolated perfused hearts. Furthermore, cardiac output was elevated at 0.03 mM, a concentration much lower than those effective in our ex vivo models. Although the exact reasons for these disparities are not clear, autoregulation of coronary flow rate, responding to myocardial metabolic demand, may influence isolated perfused hearts but not isolated coronary arteries, where the surrounding tissue is removed. In addition, the absence of the whole‐body environment excludes any potential indirect actions of butyrate. Most importantly, a neurohormonal influence on the hemodynamic response in vivo is eliminated in the in vitro setting. ⁴²

Both 3‐OHB and butyrate are considered substrates for the monocarboxylate transporters for cellular uptake and transcellular transfer. ⁴⁵ Both substrates can enter the cardiomyocytes to yield acetyl‐CoA for oxidative metabolism. ¹¹ , ³⁷ When offered to the heart, butyrate contributes to oxidative metabolism and is consumed to a larger extent than ketone bodies in both healthy and failing hearts. ¹¹ In patients with heart failure, the microbiome responsible for butyrate production from dietary fibers is inhibited, and the enzymatic apparatus for its metabolization is altered. ¹¹ , ⁴⁶ Notably, fiber enrichment‐induced changes in the gut microbiota have been observed to protect against heart failure development in murine models. ⁴⁷ Nevertheless, the efficacious concentrations reported here are probably only achievable with butyrate supplements such as tributyrin‐ or butyrate‐enriched diets and not possible through dietary fiber intake alone, presenting challenges in terms of direct translatability. ⁹ , ⁴⁴

Our findings suggest that butyrate possesses the potential to enhance cardiac output (Figure 1) through the concurrent augmentation of cardiac contractility (Figure 2) and vasodilation (Figure 4). Moreover, its cardioprotective advantage per se (Figure 3) makes it an appealing candidate to investigate as a potential therapeutic intervention in heart failure.

Conclusions

The short‐chain fatty acid butyrate elevates cardiac output by increasing cardiac contractility and reducing artery tone. The combined action of butyrate was not associated with adverse myocardial injury in damaged hearts exposed to ischemia and reperfusion but rather reduced myocardial damage.

Sources of Funding

This project was funded by grants from the Novo Nordisk Foundation (NNF19OC0058543 to H.E.B and NNF21OC0071822 to E.B.) and the Independent Research Fund Denmark (0134‐00294B to H.E.B).

Disclosures

None.

Supporting information

Data S1