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

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.

Effects of ketone body 3-hydroxybutyrate on cardiac and mitochondrial function during donation after circulatory death heart transplantation.

Normothermic regional perfusion (NRP) allows assessment of therapeutic interventions prior to donation after circulatory death transplantation. Sodium-3-hydroxybutyrate (3-OHB) increases cardiac output in heart failure patients and diminishes ischemia-reperfusion injury, presumably by improving mitochondrial metabolism. We investigated effects of 3-OHB on cardiac and mitochondrial function in transplanted hearts and in cardiac organoids. Donor pigs (n = 14) underwent circulatory death followed by NRP. Following static cold storage, hearts were transplanted into recipient pigs. 3-OHB or Ringer's acetate infusions were initiated during NRP and after transplantation. We evaluated hemodynamics and mitochondrial function. 3-OHB mediated effects on contractility, relaxation, calcium, and conduction were tested in cardiac organoids from human pluripotent stem cells. Following NRP, 3-OHB increased cardiac output (P < 0.0001) by increasing stroke volume (P = 0.006), dP/dt (P = 0.02) and reducing arterial elastance (P = 0.02). Following transplantation, infusion of 3-OHB maintained mitochondrial respiration (P = 0.009) but caused inotropy-resistant vasoplegia that prevented weaning. In cardiac organoids, 3-OHB increased contraction amplitude (P = 0.002) and shortened contraction duration (P = 0.013) without affecting calcium handling or conduction velocity. 3-OHB had beneficial cardiac effects and may have a potential to secure cardiac function during heart transplantation. Further studies are needed to optimize administration practice in donors and recipients and to validate the effect on mitochondrial function.

The Role of Plasma Extracellular Vesicles in Remote Ischemic Conditioning and Exercise-Induced Ischemic Tolerance.

Ischemic conditioning and exercise have been suggested for protecting against brain ischemia-reperfusion injury. However, the endogenous protective mechanisms stimulated by these interventions remain unclear. Here, in a comprehensive translational study, we investigated the protective role of extracellular vesicles (EVs) released after remote ischemic conditioning (RIC), blood flow restricted resistance exercise (BFRRE), or high-load resistance exercise (HLRE). Blood samples were collected from human participants before and at serial time points after intervention. RIC and BFRRE plasma EVs released early after stimulation improved viability of endothelial cells subjected to oxygen-glucose deprivation. Furthermore, post-RIC EVs accumulated in the ischemic area of a stroke mouse model, and a mean decrease in infarct volume was observed for post-RIC EVs, although not reaching statistical significance. Thus, circulating EVs induced by RIC and BFRRE can mediate protection, but the in vivo and translational effects of conditioned EVs require further experimental verification.

Remodeling after myocardial infarction and effects of heart failure treatment investigated by hyperpolarized [1-13 C]pyruvate magnetic resonance spectroscopy.

PURPOSE: Hyperpolarized [1-13 C]pyruvate MRS can measure cardiac metabolism in vivo. We investigated whether [1-13 C]pyruvate MRS could predict left ventricular remodeling following myocardial infarction (MI), long-term left ventricular effects of heart failure medication, and could identify responders to treatment. METHODS: Thirty-five rats were scanned with hyperpolarized [1-13 C]pyruvate MRS 3 days after MI or sham surgery. The animals were re-examined after 30 days of therapy with ß-blockers and ACE-inhibitors (active group, n = 12), placebo treatment (placebo group, n = 13) or no treatment (sham group, n = 10). Furthermore, heart tissue mitochondrial respiratory capacity was assessed by high-resolution respirometry. Metabolic results were compared between groups, over time and correlated to functional MR data at each time point. RESULTS: At 30 ± 0.5 days post MI, left ventricular ejection fraction (LVEF) differed between groups (sham, 77% ± 1%; placebo, 52% ± 3%; active, 63% ± 2%, P < .001). Cardiac metabolism, measured by both hyperpolarized [1-13 C]pyruvate MRS and respirometry, neither differed between groups nor between baseline and follow-up. Three days post MI, low bicarbonate + CO2 /pyruvate ratio was associated with low LVEF. At follow-up, in the active group, a poor recovery of LVEF was associated with high bicarbonate + CO2 /pyruvate ratio, as measured by hyperpolarized MRS. CONCLUSION: In a rat model of moderate heart failure, medical treatment improved function, but did not on average influence [1-13 C]pyruvate flux as measured by MRS; however, responders to heart failure medication had reduced capacity for carbohydrate metabolism.

Cyclic Hypoxia Conditioning Alters the Content of Myoblast-Derived Extracellular Vesicles and Enhances Their Cell-Protective Functions.

Remote ischemic conditioning (RIC) is a procedure that can attenuate ischemic-reperfusion injury by conducting brief cycles of ischemia and reperfusion in the arm or leg. Extracellular vesicles (EVs) circulating in the bloodstream can release their content into recipient cells to confer protective function on ischemia-reperfusion injured (IRI) organs. Skeletal muscle cells are potential candidates to release EVs as a protective signal during RIC. In this study, we used C2C12 cells as a model system and performed cyclic hypoxia-reoxygenation (HR) to mimic RIC. EVs were collected and subjected to small RNA profiling and proteomics. HR induced a distinct shift in the miRNA profile and protein content in EVs. HR EV treatment restored cell viability, dampened inflammation, and enhanced tube formation in in vitro assays. In vivo, HR EVs showed increased accumulation in the ischemic brain compared to EVs secreted from normoxic culture (N EVs) in a mouse undergoing transient middle cerebral artery occlusion (tMCAO). We conclude that HR conditioning changes the miRNA and protein profile in EVs released by C2C12 cells and enhances the protective signal in the EVs to recipient cells in vitro.

Cardioprotective effects of empagliflozin after ischemia and reperfusion in rats.

The Sodium Glucose Co-Transporter-2 inhibitor, empagliflozin (EMPA), reduces mortality and hospitalisation for heart failure following myocardial infarction irrespective of diabetes status. While the findings suggest an inherent cardioprotective capacity, the mechanism remains unknown. We studied infarct size (IS) ex-vivo in isolated hearts exposed to global IR injury and in-vivo in rats subjected to regional myocardial ischemia reperfusion (IR) injury, in whom we followed left ventricular dysfunction for 28 days. We compared rats that were given EMPA orally for 7 days before, EMPA 1.5 h before IR injury and at onset of reperfusion and continued orally during the follow-up period. We used echocardiography, high resolution respirometry, microdialysis and plasma levels of ß-hydroxybutyrate to assess myocardial performance, mitochondrial respiration and intermediary metabolism, respectively. Pretreatment with EMPA for 7 days reduced IS in-vivo (65 ± 7% vs. 46 ± 8%, p < 0.0001 while administration 1.5 h before IR, at onset of reperfusion or ex-vivo did not. EMPA alleviated LV dysfunction irrespective of the reduction in IS. EMPA improved mitochondrial respiration and modulated myocardial interstitial metabolism while the concentration of ß-hydroxybutyric acid was only transiently increased without any association with IS reduction. EMPA reduces infarct size and yields cardioprotection in non-diabetic rats with ischemic LV dysfunction by an indirect, delayed intrinsic mechanism that also improves systolic function beyond infarct size reduction. The mechanism involves enhanced mitochondrial respiratory capacity and modulated myocardial metabolism but not hyperketonemia.

Influence of strain, age, origin, and anesthesia on the cardioprotective efficacy by local and remote ischemic conditioning in an ex vivo rat model.

BACKGROUND: Local ischemic preconditioning (IPC) and remote ischemic conditioning (RIC) induced by brief periods of ischemia and reperfusion protect against ischemia-reperfusion injury. METHODS: We studied the sensitivity to IR-injury and the influence of strain, age, supplier, and anesthesia upon the efficacy of IPC and RIC in 7- and 16-weeks-old Sprague-Dawley and Wistar rats from three different suppliers. The influence of sedation with a hypnorm and midazolam mixture (rodent mixture) and pentobarbiturate was compared. RESULTS: IPC attenuated infarct size in both 7-weeks-old Sprague-Dawley (48.4 ± 17.7% vs. 20.3 ± 6.9, p < 0.001) and 7-weeks-old Wistar (55.6 ± 10.9% vs. 26.8 ± 5.0%, p < 0.001) rats. Infarct size was larger in 16-weeks-old Sprague-Dawley rats, however, IPC still lowered infarct size (78.8 ± 9.2% vs. 58.3 ± 12.3%, p < 0.01). RIC reduced infarct sizes in 7-weeks-old Sprague-Dawley (75.3 ± 11.8% vs. 58.6 ± 8.9%, p < 0.05), but not in 7-weeks-old Wistar rats (31.7 ± 17.6% and 24.0 ± 12.6%, p = 0.2). In 16-weeks-old Sprague-Dawley rats, RIC did not induce protection (76.4 ± 5.5% and 73.2 ± 14.7%, p = 0.6). However, RIC induced protection in 16-weeks-old Wistar rats (45.2 ± 8.5% vs. 14.7 ± 10.8%, p < 0.001). RIC did not reduce infarct size in 7-weeks-old Sprague-Dawley rats from Charles River (62.0 ± 13.5% and 69.4 ± 10.4% p = 0.3) or 16-weeks-old Wistar rats from Janvier (50.7 ± 11.3 and 49.2 ± 16.2, p = 0.8). There was no difference between sedation with rodent mixture or pentobarbiturate. CONCLUSION: The cardioprotective effect of IPC is consistent across rat strains independent of age, strain, and supplier. RIC seems to be less reproducible, but still yields protection across different rat strains. However, age, animal supplier, and anesthetics may modulate the sensitivity of IR-injury and the response to RIC.

Myocardial salvage by succinate dehydrogenase inhibition in ischemia-reperfusion injury depends on diabetes stage in rats.

Inhibition of succinate dehydrogenase (SDH) by Dimethyl Malonate (DiMal) reduces cardiac ischemia-reperfusion (IR) injury. We investigated the cardioprotective effect of DiMal in a rat model during advancing type 2 diabetes. Zucker Diabetic Fatty rats and lean controls were investigated corresponding to prediabetes, onset and mature diabetes. Hearts were mounted in an isolated perfused model, and subjected to IR for investigation of infarct size (IS) and mitochondrial respiratory control ratio (RCR). DiMal was administered for 10 min before ischemia. Compared with age-matched non-diabetic rats, prediabetic rats had larger IS (49 ± 4% vs. 36 ± 2%, p = 0.007), rats with onset diabetes smaller IS (51 ± 3% vs. 62 ± 3%, p = 0.05) and rats with mature diabetes had larger IS (79 ± 3% vs. 69 ± 2%, p = 0.06). At the prediabetic stage DiMal did not alter IS. At onset of diabetes DiMal 0.6 mM increased IS in diabetic but not in non-diabetic control rats (72 ± 4% vs. 51 ± 3%, p = 0.003). At mature diabetes DiMal 0.1 and 0.6 mM reduced IS (68 ± 3% vs. 79 ± 3% and 64 ± 5% vs. 79 ± 3%, p = 0.1 and p = 0.01), respectively. DiMal 0.1 mM alone reduced IS in age-matched non-diabetic animals (55 ± 3% vs. 69 ± 2% p = 0.01). RCR was reduced at mature diabetes but not modulated by DiMal. Modulation of SDH activity results in variable infarct size reduction depending on presence and the stage of diabetes. Modulation of SDH activity may be an unpredictable cardioprotective approach.

Cardioprotection by remote ischemic conditioning is transferable by plasma and mediated by extracellular vesicles.

BACKGROUND: Remote ischemic conditioning (RIC) by brief periods of limb ischemia and reperfusion protects against ischemia-reperfusion injury. We studied the cardioprotective role of extracellular vesicles (EV)s released into the circulation after RIC and EV accumulation in injured myocardium. METHODS: We used plasma from healthy human volunteers before and after RIC (pre-PLA and post-PLA) to evaluate the transferability of RIC. Pre- and post-RIC plasma samples were separated into an EV enriched fraction (pre-EV + and post-EV +) and an EV poor fraction (pre-EV- and post-EV-) by size exclusion chromatography. Small non-coding RNAs from pre-EV + and post-EV + were purified and profiled by NanoString Technology. Infarct size was compared in Sprague-Dawley rat hearts perfused with isolated plasma and fractions in a Langendorff model. In addition, fluorescently labeled EVs were used to assess homing in an in vivo rat model. (ClinicalTrials.gov, number: NCT03380663) RESULTS: Post-PLA reduced infarct size by 15% points compared with Pre-PLA (55 ± 4% (n = 7) vs 70 ± 6% (n = 8), p = 0.03). Post-EV + reduced infarct size by 16% points compared with pre-EV + (53 ± 15% (n = 13) vs 68 ± 12% (n = 14), p = 0.03). Post-EV- did not affect infarct size compared to pre-EV- (64 ± 3% (n = 15) and 68 ± 10% (n = 16), p > 0.99). Three miRNAs (miR-16-5p, miR-144-3p and miR-451a) that target the mTOR pathway were significantly up-regulated in the post-EV + group. Labelled EVs accumulated more intensely in the infarct area than in sham hearts. CONCLUSION: Cardioprotection by RIC can be mediated by circulating EVs that accumulate in injured myocardium. The underlying mechanism involves modulation of EV miRNA that may promote cell survival during reperfusion.

Differences in intrinsic aerobic capacity alters sensitivity to ischemia-reperfusion injury but not cardioprotective capacity by ischemic preconditioning in rats.

INTRODUCTION: Aerobic capacity is a strong predictor of cardiovascular mortality. Whether aerobic capacity influences myocardial ischemia and reperfusion (IR) injury is unknown. PURPOSE: To investigate the impact of intrinsic differences in aerobic capacity and the cardioprotective potential on IR injury. METHODS: We studied hearts from rats developed by selective breeding for high (HCR) or low (LCR) capacity for treadmill running. The rats were randomized to: (1) control, (2) local ischemic preconditioning (IPC) or (3) remote ischemic preconditioning (RIC) followed by 30 minutes of ischemia and 120 minutes of reperfusion in an isolated perfused heart model. The primary endpoint was infarct size. Secondary endpoints included uptake of labelled glucose, content of selected mitochondrial proteins in skeletal and cardiac muscle, and activation of AMP-activated kinase (AMPK). RESULTS: At baseline, running distance was 203±7 m in LCR vs 1905±51 m in HCR rats (p<0.01). Infarct size was significantly lower in LCR than in HCR controls (49±5% vs 68±5%, p = 0.04). IPC reduced infarct size by 47% in LCR (p<0.01) and by 31% in HCR rats (p = 0.01). RIC did not modulate infarct size (LCR: 52±5, p>0.99; HCR: 69±6%, p>0.99, respectively). Phosphorylaion of AMPK did not differ between LCR and HCR controls. IPC did not modulate cardiac phosphorylation of AMPK. Glucose uptake during reperfusion was similar in LCR and HCR rats. IPC increased glucose uptake during reperfusion in LCR animals (p = 0.02). Mitochondrial protein content in skeletal muscle was lower in LCR than in HCR (0.77±0.10 arbitrary units (AU) vs 1.09±0.07 AU, p = 0.02), but not in cardiac muscle. CONCLUSION: Aerobic capacity is associated with altered myocardial sensitivity to IR injury, but the cardioprotective effect of IPC is not. Glucose uptake, AMPK activation immediately prior to ischemia and basal mitochondrial protein content in the heart seem to be of minor importance as underlying mechanisms for the cardioprotective effects.

Cardioprotective effect of succinate dehydrogenase inhibition in rat hearts and human myocardium with and without diabetes mellitus.

Ischemia reperfusion (IR) injury may be attenuated through succinate dehydrogenase (SDH) inhibition by dimethyl malonate (DiMAL). Whether SDH inhibition yields protection in diabetic individuals and translates into human cardiac tissue remain unknown. In isolated perfused hearts from 24 weeks old male Zucker diabetic fatty (ZDF) and age matched non-diabetic control rats and atrial trabeculae from patients with and without diabetes, we compared infarct size, contractile force recovery and mitochondrial function. The cardioprotective effect of a 10 minutes DiMAL administration prior to global ischemia and ischemic preconditioning (IPC) was evaluated. In non-diabetic hearts exposed to IR, DiMAL 0.1 mM reduced infarct size compared to IR (55 ± 7% vs. 69 ± 6%, p < 0.05). Mitochondrial respiration was reduced by DiMAL 0.6 mM compared to sham and DiMAL 0.1 mM (p < 0.05). In diabetic hearts an increased concentration of DiMAL (0.6 mM) was required for protection compared to IR (64 ± 13% vs. 79 ± 8%, p < 0.05). Mitochondrial function remained unchanged. In trabeculae from humans without diabetes, IPC and DiMAL improved contractile force recovery compared to IR (43 ± 12% and 43 ± 13% vs. 23 ± 13%, p < 0.05) but in patients with diabetes only IPC provided protection compared to IR (51 ± 15% vs. 21 ± 8%, p < 0.05). Neither IPC nor DiMAL modulated mitochondrial respiration in patients. Cardioprotection by SDH inhibition is possible in human tissue, but depends on diabetes status. The narrow therapeutic range and discrepancy in respiration between experimental and human studies may limit clinical translation.

Cardiovascular Effects of Treatment With the Ketone Body 3-Hydroxybutyrate in Chronic Heart Failure Patients.

BACKGROUND: Myocardial utilization of 3-hydroxybutyrate (3-OHB) is increased in patients with heart failure and reduced ejection fraction (HFrEF). However, the cardiovascular effects of increased circulating plasma-3-OHB levels in these patients are unknown. Consequently, the authors' aim was to modulate circulating 3-OHB levels in HFrEF patients and evaluate: (1) changes in cardiac output (CO); (2) a potential dose-response relationship between 3-OHB levels and CO; (3) the impact on myocardial external energy efficiency (MEE) and oxygen consumption (MVO2); and (4) whether the cardiovascular response differed between HFrEF patients and age-matched volunteers. METHODS: Study 1: 16 chronic HFrEF patients (left ventricular ejection fraction: 37±3%) were randomized in a crossover design to 3-hour of 3-OHB or placebo infusion. Patients were monitored invasively with a Swan-Ganz catheter and with echocardiography. Study 2: In a dose-response study, 8 HFrEF patients were examined at increasing 3-OHB infusion rates. Study 3 to 4: 10 HFrEF patients and 10 age-matched volunteers were randomized in a crossover design to 3-hour 3-OHB or placebo infusion. MEE and MVO2 were evaluated using 11C-acetate positron emission tomography. RESULTS: 3-OHB infusion increased circulating levels of plasma 3-OHB from 0.4±0.3 to 3.3±0.4 mM ( P<0.001). CO rose by 2.0±0.2 L/min ( P<0.001) because of an increase in stroke volume of 20±2 mL ( P<0.001) and heart rate of 7±2 beats per minute (bpm) ( P<0.001). Left ventricular ejection fraction increased 8±1% ( P<0.001) numerically. There was a dose-response relationship with a significant CO increase of 0.3 L/min already at plasma-3-OHB levels of 0.7 mM ( P<0.001). 3-OHB increased MVO2 without altering MEE. The response to 3-OHB infusion in terms of MEE and CO did not differ between HFrEF patents and age-matched volunteers. CONCLUSIONS: 3-OHB has beneficial hemodynamic effects in HFrEF patients without impairing MEE. These beneficial effects are detectable in the physiological concentration range of circulating 3-OHB levels. The hemodynamic effects of 3-OHB were observed in both HFrEF patients and age-matched volunteers. 3-OHB may potentially constitute a novel treatment principle in HFrEF patients.

Clinical translation of myocardial conditioning.

Rapid admission and acute interventional treatment combined with modern antithrombotic pharmacologic therapy have improved outcomes in patients with ST elevation myocardial infarction. The next major target to further advance outcomes needs to address ischemia-reperfusion injury, which may contribute significantly to the final infarct size and hence mortality and postinfarction heart failure. Mechanical conditioning strategies including local and remote ischemic pre-, per-, and postconditioning have demonstrated consistent cardioprotective capacities in experimental models of acute ischemia-reperfusion injury. Their translation to the clinical scenario has been challenging. At present, the most promising mechanical protection strategy of the heart seems to be remote ischemic conditioning, which increases myocardial salvage beyond acute reperfusion therapy. An additional aspect that has gained recent focus is the potential of extended conditioning strategies to improve physical rehabilitation not only after an acute ischemia-reperfusion event such as acute myocardial infarction and cardiac surgery but also in patients with heart failure. Experimental and preliminary clinical evidence suggests that remote ischemic conditioning may modify cardiac remodeling and additionally enhance skeletal muscle strength therapy to prevent muscle waste, known as an inherent component of a postoperative period and in heart failure. Blood flow restriction exercise and enhanced external counterpulsation may represent cardioprotective corollaries. Combined with exercise, remote ischemic conditioning or, alternatively, blood flow restriction exercise may be of aid in optimizing physical rehabilitation in populations that are not able to perform exercise practice at intensity levels required to promote optimal outcomes.

Effect of paroxetine on left ventricular remodeling in an in vivo rat model of myocardial infarction.

Left ventricular (LV) remodeling following a myocardial infarction (MI) involves formation of reactive oxygen species (ROS). Paroxetine, a selective serotonin reuptake inhibitor, has an antioxidant effect in the vascular wall. We investigated whether paroxetine reduces myocardial ROS formation and LV remodeling following a MI. In a total of 32 Wistar rats, MI was induced by a 30-min ligation of the left anterior descending artery followed by 7- or 28-day reperfusion. During the 28 days of reperfusion, LV remodeling was evaluated by magnetic resonance imaging (MRI) and echocardiography (n = 20). After 28 days of reperfusion, the susceptibility to ventricular tachycardia was evaluated prior to sacrifice and histological assessment of myocyte cross-sectional area, fibrosis, and presence of myofibroblasts. Myocardial ROS formation was measured with dihydroethidium after 7 days of reperfusion in separate groups (n = 12). Diastolic LV volume, evaluated by MRI (417 ± 60 vs. 511 ± 64 µL, p < 0.05), and echocardiography (515 ± 80 vs. 596 ± 83 µL, p < 0.05) as well as diastolic LV internal diameter evaluated with echocardiography (7.2 ± 0.6 vs. 8.1 ± 0.7 mm, p < 0.05) were lower in the paroxetine group than in controls. Furthermore, myocyte cross-sectional area was reduced in the paroxetine group compared with controls (277 ± 26 vs. 354 ± 23 mm3, p < 0.05) and ROS formation was reduced in the remote myocardium (0.415 ± 0.19 normalized to controls, p < 0.05). However, no differences in the presence of fibrosis or myofibroblasts were observed. Finally, paroxetine reduced the susceptibility to ventricular tachycardia (induced in 2/11 vs. 6/8 rats, p < 0.05). Paroxetine treatment following MI decreases LV remodeling and susceptibility to arrhythmias, probably by reducing ROS formation.