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Sodium glucose cotransporter 2 inhibitors (SGLT2i) constitute a promising drug treatment for heart failure patients with either preserved or reduced ejection fraction. Whereas SGLT2i were originally developed to target SGLT2 in the kidney to facilitate glucosuria in diabetic patients, it is becoming increasingly clear that these drugs also have important effects outside of the kidney. In this review we summarize the literature on cardiac effects of SGLT2i, focussing on pro-inflammatory and oxidative stress processes, ion transport mechanisms controlling sodium and calcium homeostasis and metabolic/mitochondrial pathways. These mechanisms are particularly important as disturbances in these pathways result in endothelial dysfunction, diastolic dysfunction, cardiac stiffness, and cardiac arrhythmias that together contribute to heart failure. We review the findings that support the concept that SGLT2i directly and beneficially interfere with inflammation, oxidative stress, ionic homeostasis, and metabolism within the cardiac cell. However, given the very low levels of SGLT2 in cardiac cells, the evidence suggests that SGLT2-independent effects of this class of drugs likely occurs via off-target effects in the myocardium. Thus, while there is still much to be understood about the various factors which determine how SGLT2i affect cardiac cells, much of the research clearly demonstrates that direct cardiac effects of these SGLT2i exist, albeit mediated via SGLT2-independent pathways, and these pathways may play a role in explaining the beneficial effects of SGLT2 inhibitors in heart failure.

Background: Canagliflozin (CANA) shows anti-inflammatory and anti-oxidative effects on endothelial cells (ECs). In diabetes mellitus (DM), excessive reactive oxygen species (ROS) generation, increased intracellular calcium (Ca2+) and enhanced extracellular signal regulated kinase (ERK) 1/2 phosphorylation are crucial precursors for inflammasome activation. We hypothesized that: (1) CANA prevents the TNF-α triggered ROS generation in ECs from diabetic donors and in turn suppresses the inflammasome activation; and (2) the anti-inflammatory effect of CANA is mediated via intracellular Ca2+ and ERK1/2. Methods: Human coronary artery endothelial cells from donors with DM (D-HCAECs) were pre-incubated with either CANA or vehicle for 2 h before exposure to 50 ng/ml TNF-α for 2–48 h. NAC was applied to scavenge ROS, BAPTA-AM to chelate intracellular Ca2+, and PD 98059 to inhibit the activation of ERK1/2. Live cell imaging was performed at 6 h to measure ROS and intracellular Ca2+. At 48 h, ELISA and infra-red western blot were applied to detect IL-1β, NLRP3, pro-caspase-1 and ASC. Results: 10 µM CANA significantly reduced TNF-α related ROS generation, IL-1β production and NLRP3 expression (P all 0.05). CANA and BAPTA both prevented intracellular Ca2+ increase in cells exposed to TNF-α (P both

Cardioprotection encompasses a variety of strategies protecting the heart against myocardial injury that occurs during and after inadequate blood supply to the heart during myocardial infarction. While restoring reperfusion is crucial for salvaging myocardium from further damage, paradoxically, it itself accounts for additional cell death-a phenomenon named ischemia/reperfusion injury. Therefore, therapeutic strategies are necessary to render the heart protected against myocardial infarction. Ischemic pre- and postconditioning, by short periods of sublethal cardiac ischemia and reperfusion, are still the strongest mechanisms to achieve cardioprotection. However, it is highly impractical and far too invasive for clinical use. Fortunately, it can be mimicked pharmacologically, for example, by volatile anesthetics, noble gases, opioids, propofol, dexmedetomidine, and phosphodiesterase inhibitors. These substances are all routinely used in the clinical setting and seem promising candidates for successful translation of cardioprotection from experimental protocols to clinical trials. This review presents the fundamental mechanisms of conditioning strategies and provides an overview of the most recent and relevant findings on different concepts achieving cardioprotection in the experimental setting, specifically emphasizing pharmacological approaches in the perioperative context.

Sodium glucose co-transporter 2 inhibitors (SGLT-2is) improve cardiovascular outcomes in both diabetic and non-diabetic patients. Preclinical studies suggest that SGLT-2is directly affect endothelial function in a glucose-independent manner. The effects of SGLT-2is include decreased oxidative stress and inflammatory reactions in endothelial cells. Furthermore, SGLT2is restore endothelium-related vasodilation and regulate angiogenesis. The favourable cardiovascular effects of SGLT-2is could be mediated via a number of pathways: (1) inhibition of the overactive sodium-hydrogen exchanger; (2) decreased expression of nicotinamide adenine dinucleotide phosphate oxidases; (3) alleviation of mitochondrial injury; (4) suppression of inflammation-related signalling pathways (e.g., by affecting NF-κB); (5) modulation of glycolysis; and (6) recovery of impaired NO bioavailability. This review focuses on the most recent progress and existing gaps in preclinical investigations concerning the direct effects of SGLT-2is on endothelial dysfunction and the mechanisms underlying such effects.

Sodium-glucose-cotransporter 2 inhibitors (SGLT2is) demonstrate large cardiovascular benefit in both diabetic and non-diabetic, acute and chronic heart failure patients. These inhibitors have on-target (SGLT2 inhibition in the kidney) and off-target effects that likely both contribute to the reported cardiovascular benefit. Here we review the literature on direct effects of SGLT2is on various cardiac cells and derive at an unifying working hypothesis. SGLT2is acutely and directly (1) inhibit cardiac sodium transporters and alter ion homeostasis, (2) reduce inflammation and oxidative stress, (3) influence metabolism, and (4) improve cardiac function. We postulate that cardiac benefit modulated by SGLT2i’s can be commonly attributed to their inhibition of sodium-loaders in the plasma membrane (NHE-1, Nav1.5, SGLT) affecting intracellular sodium-homeostasis (the sodium-interactome), thereby providing a unifying view on the various effects reported in separate studies. The SGLT2is effects are most apparent when cells or hearts are subjected to pathological conditions (reactive oxygen species, inflammation, acidosis, hypoxia, high saturated fatty acids, hypertension, hyperglycemia, and heart failure sympathetic stimulation) that are known to prime these plasmalemmal sodium-loaders. In conclusion, the cardiac sodium-interactome provides a unifying testable working hypothesis and a possible, at least partly, explanation to the clinical benefits of SGLT2is observed in the diseased patient.

Purpose Vascular inflammation and disturbed metabolism are observed in heart failure and type 2 diabetes mellitus. Glycolytic enzyme hexokinase II (HKII) is upregulated by inflammation. We hypothesized that SGLT2 inhibitors Canagliflozin (Cana), Empagliflozin (Empa) or Dapagliflozin (Dapa) reduces inflammation via HKII in endothelial cells, and that HKII-dependent inflammation is determined by ERK1/2, NF-κB. and/or AMPK activity in lipopolysaccharide (LPS)-stimulated human coronary artery endothelial cells (HCAECs). Methods HCAECs were pre-incubated with 3 μM or 10 μM Cana, 1 μM, 3 μM or 10 μM Empa or 0.5 μM, 3 μM or 10 μM Dapa (16 h) and subjected to 3 h LPS (1 μg/mL). HKII was silenced via siRNA transfection. Interleukin-6 (IL-6) release was measured by ELISA. Protein levels of HK I and II, ERK1/2, AMPK and NF-κB were detected using infra-red western blot. Results LPS increased IL-6 release and ERK1/2 phosphorylation; Cana prevented these pro-inflammatory responses (IL-6: pg/ml, control 46 ± 2, LPS 280 ± 154 p < 0.01 vs. control, LPS + Cana 96 ± 40, p < 0.05 vs. LPS). Cana reduced HKII expression (HKII/GAPDH, control 0.91 ± 0.16, Cana 0.71 ± 0.13 p < 0.05 vs. control, LPS 1.02 ± 0.25, LPS + Cana 0.82 ± 0.24 p < 0.05 vs. LPS). Empa and Dapa were without effect on IL-6 release and HKII expression in the model used. Knockdown of HKII by 37% resulted caused partial loss of Cana-mediated IL-6 reduction (pg/ml, control 35 ± 5, LPS 188 ± 115 p < 0.05 vs. control, LPS + Cana 124 ± 75) and ERK1/2 activation by LPS. In LPS-stimulated HCAECs, Cana, but not Empa or Dapa, activated AMPK. AMPK activator A769662 reduced IL-6 release. Conclusion Cana conveys anti-inflammatory actions in LPS-treated HCAECs through 1) reductions in HKII and ERK1/2 phosphorylation and 2) AMPK activation. These data suggest a novel anti-inflammatory mechanism of Cana through HKII.

Inflammation causing oxidative stress in endothelial cells contributes to heart failure development. Sodium/glucose cotransporter 2 inhibitors (SGLT2i's) were shown to reduce heart failure hospitalization and oxidative stress. However, how inflammation causes oxidative stress in endothelial cells, and how SGLT2i's can reduce this is unknown. Here we hypothesized that 1) TNF-α activates the Na

AimsSodium glucose cotransporter 2 (SGLT2) inhibitors have sodium–hydrogen exchanger (NHE) inhibition properties in isolated cardiomyocytes, but it is unknown whether these properties extend to the intact heart during ischaemia–reperfusion (IR) conditions. NHE inhibitors as Cariporide delay time to onset of contracture (TOC) during ischaemia and reduce IR injury. We hypothesized that, in the ex vivo heart, Empagliflozin (Empa) mimics Cariporide during IR by delaying TOC and reducing IR injury. To facilitate translation to in vivo conditions with insulin present, effects were examined in the absence and presence of insulin.Methods and resultsIsolated C57Bl/6NCrl mouse hearts were subjected to 25 min I and 120 min R without and with 50 mU/L insulin. Without insulin, Empa and Cari delayed TOC by 100 and 129 s, respectively, yet only Cariporide reduced IR injury [infarct size (mean ± SEM in %) from 51 ± 6 to 34 ± 5]. Empa did not delay TOC in the presence of the NHE1 inhibitor Eniporide. Insulin perfusion increased tissue glycogen content at baseline (from 2 ± 2 µmol to 42 ± 1 µmol glycosyl units/g heart dry weight), amplified G6P and lactate accumulation at end-ischaemia, thereby decreased mtHKII and exacerbated IR injury. Under these conditions, Empa (1 µM) and Cariporide (10 µM) were without effect on TOC and IR injury. Empa and Cariporide both inhibited NHE activity, in isolated cardiomyocytes, independent of insulin.ConclusionsIn the absence of insulin, Empa and Cariporide strongly delayed the time to onset of contracture during ischaemia. In the presence of insulin, both Empa and Cari were without effect on IR, possibly because of severe ischaemic acidification. Insulin exacerbates IR injury through increased glycogen depletion during ischaemia and consequently mtHKII dissociation. The data suggest that also in the ex vivo intact heart Empa exerts direct cardiac effects by inhibiting NHE during ischaemia, but not during reperfusion.

Purpose Sonlicromanol is a phase IIB clinical stage compound developed for treatment of mitochondrial diseases. Its active component, KH176m, functions as an antioxidant, directly scavenging reactive oxygen species (ROS), and redox activator, boosting the peroxiredoxin-thioredoxin system. Here, we examined KH176m’s potential to protect against acute cardiac ischemia-reperfusion injury (IRI), compare it with the classic antioxidant N-(2-mercaptopropionyl)-glycine (MPG), and determine whether protection depends on duration (severity) of ischemia. Methods Isolated C56Bl/6N mouse hearts were Langendorff-perfused and subjected to short (20 min) or long (30 min) ischemia, followed by reperfusion. During perfusion, hearts were treated with saline, 10 μM KH176m, or 1 mM MPG. Cardiac function, cell death (necrosis), and mitochondrial damage (cytochrome c (CytC) release) were evaluated. In additional series, the effect of KH176m treatment on the irreversible oxidative stress marker 4-hydroxy-2-nonenal (4-HNE), formed during ischemia only, was determined at 30-min reperfusion. Results During baseline conditions, both drugs reduced cardiac performance, with opposing effects on vascular resistance (increased with KH176m, decreased with MPG). For short ischemia, KH176m robustly reduced all cell death parameters: LDH release (0.2 ± 0.2 vs 0.8 ± 0.5 U/min/GWW), infarct size (15 ± 8 vs 31 ± 20%), and CytC release (168.0 ± 151.9 vs 790.8 ± 453.6 ng/min/GWW). Protection by KH176m was associated with decreased cardiac 4-HNE. MPG only reduced CytC release. Following long ischemia, IRI was doubled, and KH176m and MPG now only reduced LDH release. The reduced protection against long ischemia was associated with the inability to reduce cardiac 4-HNE. Conclusion Protection against cardiac IRI by the antioxidant KH176m is critically dependent on duration of ischemia. The data suggest that with longer ischemia, the capacity of KH176m to reduce cardiac oxidative stress is rate-limiting, irreversible ischemic oxidative damage maximally accumulates, and antioxidant protection is strongly diminished.

The aim of pharmacological conditioning is to protect the heart against myocardial ischemia-reperfusion (I/R) injury and its consequences. There is extensive literature that reports a multitude of different cardioprotective signaling molecules and mechanisms in diverse experimental protocols. Several pharmacological agents have been evaluated in terms of myocardial I/R injury. While results from experimental studies are immensely encouraging, translation into the clinical setting remains unsatisfactory. This narrative review wants to focus on two aspects: (1) give a comprehensive update on new developments of pharmacological conditioning in the experimental setting concentrating on recent literature of the last two years and (2) briefly summarize clinical evidence of these cardioprotective substances in the perioperative setting highlighting their clinical implications. By directly opposing each pharmacological agent regarding its recent experimental knowledge and most important available clinical data, a clear overview is given demonstrating the remaining gap between basic research and clinical practice. Finally, future perspectives are given on how we might overcome the limited translatability in the field of pharmacological conditioning.

Background and objectives Red-blood-cell (RBC) transfusion is associated with lung injury, which is further exacerbated by mechanical ventilation. Manufacturing methods of blood products differ globally and may play a role in the induction of pulmonary cell activation through alteration of the immunomodulatory property of the products. Here, the effect of different manufacturing methods on pulmonary cell activation was investigated in an in vitro model of mechanical ventilation. Materials and methods Pulmonary type II cells were incubated with supernatant from fresh and old RBC products obtained via whole blood filtration (WBF), red cell filtration (RCF), apheresis-derived (AD) or whole blood-derived (WBD) methods. Lung cells were subjected to 25% stretch for 24 h. Controls were non-stretched or non-incubated cells. Results Fresh but not old AD products and WBF products induce lung cell production of pro-inflammatory cytokines and chemokines, which was not observed with WBD or RCF products. Effects were associated with an increased amount of platelet-derived vesicles and an increased thrombin-generating capacity. Mechanical stretching of lung cells induced more severe cell injury compared to un-stretched controls, including alterations in the cytoskeleton, which was further augmented by incubation with AD products. In all read-out parameters, RCF products seemed to induce less injury compared to the other products. Conclusions Our findings show that manufacturing methods of RBC products impact pulmonary cell activation, which may be mediated by the generation of vesicles in the product. We suggest RBC manufacturing method may be an important factor in understanding the association between RBC transfusion and lung injury.

Caveolins are involved in anaesthetic-induced cardioprotection. Actin filaments are located in close connection to Caveolins in the plasma membrane. We hypothesised that helium might affect the cytoskeleton and induce secretion of Caveolin. HCAEC, HUVEC and Cav-1 siRNA transfected HUVEC were exposed for 20 minutes to either helium (5% CO2, 25% O2, 70% He) or control gas (5% CO2, 25% O2, 70% N2). Cells and supernatants were collected for infrared Western blot analysis, immunofluorescence staining, nanoparticle tracking analysis and permeability measurements. Helium treatment increased the cortical localisation of F-actin fibers in HUVEC. After 6 hours, helium decreased cellular Caveolin-1 (Cav-1) levels and increased Cav-1 levels in the supernatant. Cell permeability was decreased 6 and 12 hours after helium treatment, and increased levels of Vascular Endothelial - Cadherin (VE-Cadherin) and Connexin 43 (Cx43) were observed. Transfection with Cav-1 siRNA abolished the effects of helium treatment on VE-Cadherin, Cx43 levels and permeability. Supernatant obtained after helium treatment reduced cellular permeability in remote HUVEC, indicating that increased levels of Cav-1 are responsible for the observed alterations. These findings suggest that Cav-1 is secreted after helium exposure in vitro, altering the cytoskeleton and increasing VE-Cadherin and Cx43 expression resulting in decreased permeability in HUVEC.

Inflammation causing oxidative stress in endothelial cells contributes to heart failure development. Sodium/glucose cotransporter 2 inhibitors (SGLT2i's) were shown to reduce heart failure hospitalization and oxidative stress. However, how inflammation causes oxidative stress in endothelial cells, and how SGLT2i's can reduce this is unknown. Here we hypothesized that 1) TNF-α activates the Na+/H+ exchanger (NHE) and raises cytoplasmatic Na+ ([Na+]c), 2) increased [Na+]c causes reactive oxygen species (ROS) production, and 3) empagliflozin (EMPA) reduces inflammation-induced ROS through NHE inhibition and lowering of [Na+]c in human endothelial cells. Human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAECs) were incubated with vehicle (V), 10 ng/ml TNF-α, 1 µM EMPA or the NHE inhibitor Cariporide (CARI, 10 µM) and NHE activity, intracellular [Na+]c and ROS were analyzed. TNF-α enhanced NHE activity in HCAECs and HUVECs by 92% (p < 0.01) and 51% (p < 0.05), respectively, and increased [Na+]c from 8.2 ± 1.6 to 11.2 ± 0.1 mM (p < 0.05) in HCAECs. Increasing [Na+]c by ouabain elevated ROS generation in both HCAECs and HUVECs. EMPA inhibited NHE activity in HCAECs and in HUVECs. EMPA concomitantly lowered [Na+]c in both cell types. In both cell types, TNF α-induced ROS was lowered by EMPA or CARI, with no further ROS lowering by EMPA in the presence of CARI, indicating EMPA attenuated ROS through NHE inhibition. In conclusion, inflammation induces oxidative stress in human endothelial cells through NHE activation causing elevations in [Na+]c, a process that is inhibited by EMPA through NHE inhibition.

Aims/hypothesis Sodium-glucose cotransporter 2 (SGLT2) inhibitors (SGLT2i) constitute a novel class of glucose-lowering (type 2) kidney-targeted agents. We recently reported that the SGLT2i empagliflozin (EMPA) reduced cardiac cytosolic Na+ ([Na+](c)) and cytosolic Ca2+ ([Ca2+](c)) concentrations through inhibition of Na+/H+ exchanger (NHE). Here, we examine (1) whether the SGLT2i dapagliflozin (DAPA) and canagliflozin (CANA) also inhibit NHE and reduce [Na+](c); (2) a structural model for the interaction of SGLT2i to NHE; (3) to what extent SGLT2i affect the haemodynamic and metabolic performance of isolated hearts of healthy mice. Methods Cardiac NHE activity and [Na+](c) in mouse cardiomyocytes were measured in the presence of clinically relevant concentrations of EMPA (1 mu mol/l), DAPA (1 mu mol/l), CANA (3 mu mol/l) or vehicle. NHE docking simulation studies were applied to explore potential binding sites for SGTL2i. Constant-flow Langendorff-perfused mouse hearts were subjected to SGLT2i for 30 min, and cardiovascular function, O-2 consumption and energetics (phosphocreatine (PCr)/ATP) were determined. Results EMPA, DAPA and CANA inhibited NHE activity (measured through low pH recovery after NH4+ pulse: EMPA 6.69 +/- 0.09, DAPA 6.77 +/- 0.12 and CANA 6.80 +/- 0.18 vs vehicle 7.09 +/- 0.09; p

BackgroundThe noble gas helium induces cardio- and neuroprotection by pre- and post-conditioning. We investigated the effects of helium pre- and post-conditioning on the brain and heart in a rat resuscitation model. MethodsAfter approval by the Animal Care Committee, 96 Wistar rats underwent cardiac arrest for 6min induced by ventricular fibrillation. Animals received 70% helium and 30% oxygen for 5min before cardiac arrest and for 30min after restoration of spontaneous circulation (ROSC). Control animals received 70% nitrogen and 30% oxygen. Hearts and brains were excised after 2, 4h or 7days. Neurological degeneration was evaluated using TUNEL and Nissl staining in the hippocampal CA-1 sector. Cognitive function after 7days was detected with the tape removal test. Molecular targets were measured by infrared western blot. Data are shown as median [Interquartile range]. ResultsHelium treatment resulted in significantly less apoptosis (TUNEL positive cells/100 pixel 73.5 [60.3-78.6] vs.78.2 [70.4-92.9] P=0.023). Changes in Caveolin-3 expression in the membrane fraction and Hexokinase-II in the mitochondrial fraction were observed in the heart. Caveolin-1 expression of treated animals significantly differed from control animals in the membrane fraction of the heart and brain after ROSC. ConclusionTreatment with helium reduced apoptosis in our resuscitation model. Differential expression levels of Caveolin-1, Caveolin-3 and Hexokinase II in the heart were found after helium pre- and post-conditioning. No beneficial effects were seen on neurofunctional outcome

Background Remote ischemic preconditioning (RIPC) is less effective in patients with diabetes, and the molecular mechanisms behind this lack of protection are not fully understood. In this study we investigated the effects of remote ischemic preconditioning on the intracellular signal cascade (known as the RISK pathway), e.g. ERK1/2, STAT3 and STAT5 in patients with and without diabetes undergoing coronary artery bypass graft (CABG) surgery. Methods Twenty-six patients with and 24 patients without diabetes mellitus undergoing CABG surgery under a total-intravenous, propofol based anesthetics regime, were randomized to either remote ischemic preconditioning induced by 3x 5 minutes of forearm ischemia or untreated controls. In each patient, two atrial tissue samples were collected: the first before, and the second after preconditioning, but before initiation of cardiopulmonary bypass. Tissue samples were homogenized and the ratio of total-to-phosphorylated ERK1 and 2, STAT 3 and STAT5 were determined by Western Blotting. Myocardial injury after CABG was assessed by analyzing postoperative troponin release. Results In the patients without diabetes, no effect of RIPC on any of the molecular markers was observed. In addition, we found no reduction in postoperative peak troponin release after RIPC. In patients with diabetes, ERK1 and ERK2 phosphorylation was increased in the control group, while in the RIPC treated patients only ERK2 was significantly increased. STAT3 phosphorylation was increased in both, the control and RIPC group, while there was no effect on STAT5 phosphorylation. Again, there was no effect on postoperative troponin release. Discussion We found no effect of RIPC on ERK 1, ERK2, STAT 3 or STAT 5 in either patients with or without diabetes. Additionally, there was no reduction in troponin T release by RIPC. The major limitation of our trial is the small sample size. Trial registration The trial was prospectively registered in the Dutch trial register as NTR1931.

Purpose Remote ischemic preconditioning protects peripheral organs against prolonged ischemia/reperfusion injury via circulating protective factors. Preconditioning with helium protected healthy volunteers against postischemic endothelial dysfunction. We investigated whether plasma from helium-treated volunteers can protect human umbilical vein endothelial cells (HUVECs) against hypoxia in vitro through release of circulating of factors. Methods Healthy male volunteers inhaled heliox (79% helium, 21% oxygen) or air for 30 min. Plasma was collected at baseline, directly after inhalation, 6 h and 24 h after start of the experiment. HUVECs were incubated with either 5% or 10% of the plasma for 1 or 2 h and subjected to enzymatically induced hypoxia. Cell damage was measured by LDH content. Furthermore, caveolin 1 (Cav-1), hypoxia-inducible factor (HIF1α), extracellular signal-regulated kinase (ERK)1/2, signal transducer and activator of transcription (STAT3) and endothelial nitric oxide synthase (eNOS) were determined. Results Prehypoxic exposure to 10% plasma obtained 6 h after helium inhalation decreased hypoxia-induced cell damage in HUVEC. Cav-1 knockdown in HUVEC abolished this effect. Conclusions Plasma of healthy volunteers breathing helium protects HUVEC against hypoxic cell damage, possibly involving circulating Cav-1. Electronic supplementary material The online version of this article (10.1007/s10557-019-06880-2) contains supplementary material, which is available to authorized users.

In patients with diabetes, metabolic disorders disturb the physiological balance of coagulation and fibrinolysis, leading to a prothrombotic state characterized by platelet hypersensitivity, coagulation disorders and hypofibrinolysis. Hyperglycemia and insulin resistance cause changes in platelet number and activation, as well as qualitative and/or quantitative modifications of coagulatory and fibrinolytic factors, resulting in the formation of fibrinolysis-resistant clots in patients with diabetes. Other coexisting factors like hypoglycemia, obesity and dyslipidemia also contribute to coagulation disorders in patients with diabetes. Management of the prothrombotic state includes antiplatelet and anticoagulation therapies for diabetes patients with either a history of cardiovascular disease or prone to a higher risk of thrombus generation, but current guidelines lack recommendations on the optimal antithrombotic treatment for these patients. Metabolic optimizations like glucose control, lipid-lowering, and weight loss also improve coagulation disorders of diabetes patients. Intriguing, glucose-lowering drugs, especially cardiovascular beneficial agents, such as glucagon-like peptide-1 receptor agonists and sodium glucose co-transporter inhibitors, have been shown to exert direct anticoagulation effects in patients with diabetes. This review focuses on the most recent progress in the development and management of diabetes related prothrombotic state.

Ischemia/reperfusion (I/R) of the heart becomes injurious when duration of the ischemic insult exceeds a certain threshold (approximately ≥20 min). Mitochondrial bound hexokinase II (mtHKII) protects against I/R injury, with the amount of mtHKII correlating with injury. Here, we examine whether mtHKII can induce the transition from non-injurious to injurious I/R, by detaching HKII from mitochondria during a non-injurious I/R interval. Additionally, we examine possible underlying mechanisms (increased reactive oxygen species (ROS), increased oxygen consumption (MVO2) and decreased cardiac energetics) associated with this transition. Langendorff perfused rat hearts were treated for 20 min with saline, TAT-only or 200 nM TAT-HKII, a peptide that translocates HKII from mitochondria. Then, hearts were exposed to non-injurious 15-min ischemia, followed by 30-min reperfusion. I/R injury was determined by necrosis (LDH release) and cardiac mechanical recovery. ROS were measured by DHE fluorescence. Changes in cardiac respiratory activity (cardiac MVO2 and efficiency and mitochondrial oxygen tension (mitoPO2) using protoporphyrin IX) and cardiac energetics (ATP, PCr, ∆GATP) were determined following peptide treatment. When exposed to 15-min ischemia, control hearts had no necrosis and 85% recovery of function. Conversely, TAT-HKII treatment resulted in significant LDH release and reduced cardiac recovery (25%), indicating injurious I/R. This was associated with increased ROS during ischemia and reperfusion. TAT-HKII treatment reduced MVO2 and improved energetics (increased PCr) before ischemia, without affecting MVO2/RPP ratio or mitoPO2. In conclusion, a reduction in mtHKII turns non-injurious I/R into injurious I/R. Loss of mtHKII was associated with increased ROS during ischemia and reperfusion, but not with increased MVO2 or decreased cardiac energetics before damage occurs.

BACKGROUND Red blood cell (RBC) transfusion is associated with organ failure. The mechanism remains unknown, but may include adherence of blood cells to the microvasculature. We hypothesized that RBC-derived extracellular vesicles (EVs) interact with monocytes to activate endothelial cells. STUDY DESIGN AND METHODS Human umbilical vein endothelial cells were incubated with supernatant from fresh and stored RBC units either containing EVs or depleted from EVs, with or without the addition of immune cells. We measured expression of adhesion markers by flow cytometry and markers of coagulation and inflammation in the culture medium. We studied phagocytosis of EVs by monocytes by using confocal microscopy and flow cytometry. RESULTS Incubation of endothelial cells with monocytes alone did not induce up regulation of adhesion markers. The addition of both monocytes and supernatant from RBCs containing EVs resulted in up regulation of endothelial expression of intercellular adhesion molecule 1 and E-selectin when compared to baseline. Up regulation was absent when stimulated with RBC supernatant depleted from EVs. EVs are phagocytosed by monocytes, which was partly abrogated after coincubation with two different complement receptor 3 (CR3)-blocking antibodies. Addition of RBC-derived EVs also increased levels of von Willebrand factor (VWF). There were no differences between groups related to storage time. CONCLUSION EVs from RBC transfusion bags activate monocytes with subsequent up regulation of endothelial cell adhesion markers. EVs are phagocytosed by monocytes through CR3. Furthermore, these EVs proved to be a source of VWF. These effects are unrelated to storage time. Thereby, EVs from RBC transfusion bags induce a proinflammatory and procoagulant endothelial cell response.

The noble gas helium (He) induces cardioprotection in vivo through unknown molecular mechanisms. He can interact with and modify cellular membranes. Caveolae are cholesterol and sphingolipid-enriched invaginations of the plasma-membrane-containing caveolin (Cav) proteins that are critical in protection of the heart. Mice (C57BL/6J) inhaled either He gas or adjusted room air. Functional measurements were performed in the isolated Langendorff perfused heart at 24 h post He inhalation. Electron paramagnetic resonance spectrometry (EPR) of samples was carried out at 24 h post He inhalation. Immunoblotting was used to detect Cav-1/3 expression in whole-heart tissue, exosomes isolated from platelet free plasma (PFP) and membrane fractions. Additionally, transmission electron microscopy analysis of cardiac tissue and serum function and metabolomic analysis were performed. In contrast to cardioprotection observed in in vivo models, the isolated Langendorff perfused heart revealed no protection after He inhalation. However, levels of Cav-1/3 were reduced 24 h after He inhalation in whole-heart tissue, and Cav-3 was increased in exosomes from PFP. Addition of serum to muscle cells in culture or na&iuml, ve ventricular tissue increased mitochondrial metabolism without increasing reactive oxygen species generation. Primary and lipid metabolites determined potential changes in ceramide by He exposure. In addition to direct effects on myocardium, He likely induces the release of secreted membrane factors enriched in caveolae. Our results suggest a critical role for such circulating factors in He-induced organ protection.

Caveolae, lipid enriched invaginations of the plasma membrane, are epicentres of cellular signal transduction. The structural proteins of caveolae, caveolins, regulate effector pathways in anaesthetic-induced cardioprotection, including the RISK pathway. Helium (He) postconditioning (HePoc) is known to mimic anaesthetic conditioning and to prevent damage from myocardial infarction. We hypothesize that HePoc regulates caveolin-1 and caveolin-3 (Cav-1 and Cav-3) expression in the rat heart and activates the RISK pathway. Male Wistar rats (n=8, each group) were subjected to 25min of cardiac ischaemia followed by reperfusion (I/R) for 5, 15 or 30min (I/R 5/15/30). The HePoc groups underwent I/R with 70% helium ventilation during reperfusion (IR+He 5/15/30min). Sham animals received surgical treatment without I/R. After each protocol blood and hearts were retrieved. Tissue was obtained from the area-at-risk (AAR) and non-area-at-risk (NAAR) and processed for western blot analyses and reverse-transcription-real-time-polymerase-chain-reaction (RT-qPCR). Protein analyses revealed increased amounts of Cav-1 and Cav-3 in the membrane of I/R+He15 (AAR: Cav-1, P

Introduction: There are few reports of in vivo muscle strength measurements in animal models of ICU‐acquired weakness (ICU‐AW). In this study we investigated whether the Escherichia coli (E. coli) septic peritonitis mouse model may serve as an ICU‐AW model using in vivo strength measurements and myosin/actin assays, and whether development of ICU‐AW is age‐dependent in this model. Methods: Young and old mice were injected intraperitoneally with E. coli and treated with ceftriaxone. Forelimb grip strength was measured at multiple time points, and the myosin/actin ratio in muscle was determined. Results: E. coli administration was not associated with grip strength decrease, neither in young nor in old mice. In old mice, the myosin/actin ratio was lower in E. coli mice at t = 48 h and higher at t = 72 h compared with controls. Conclusions: This E. coli septic peritonitis mouse model did not induce decreased grip strength. In its current form, it seems unsuitable as a model for ICU‐AW. Muscle Nerve 53: 127–133, 2016

Research data from the past decade indicate that noble gases like xenon and helium exert profound cardioprotection when applied before, during or afte r organ ischemia. Of all noble gases, especially helium, has gained interest in the past years because it does not have an anesthetic “side effect” like xenon, allowing application of this specific gas in numerous clinical ischemia/reperfusion situations. Because helium has several unique characteristics and no hemodynamic side effects, helium could be administered in severely ill patients. Investigations in animals as well as in humans have proven that this noble gas is not completely inert and can induce several biological effects. Though the underlying molecular mechanisms of helium-induced cardiac protection are still not yet fully understood, recently different signaling pathways have been elucidated.

The perioperative period is characterized by profound changes in the body's homoeostatic processes. This review seeks to address whether epigenetic mechanisms may influence an individual's reaction to surgery and anaesthesia. Evidence from animal and human studies suggests that epigenetic mechanisms can explain many facets of susceptibility to acute and chronic pain, making them potential therapeutic targets. Modern pain management is still based upon opiates, and both the developmental expression of opioid receptors and opioid-induced hyperalgesia have been linked to epigenetic mechanisms. In general, opiates seem to increase global DNA methylation levels. This is in contrast to local anaesthetics, which have been ascribed a global demethylating effect. Even though no direct investigations have been carried out, the potential influence of epigenetics on the inflammatory response that follows surgery seems a promising area for research. There is a considerable body of evidence that supports the involvement of epigenetics in the complex process of wound healing. Epigenetics is an important emerging research topic in perioperative medicine, with a huge potential to positively influence patient outcome.

Linked Articles This article is part of a themed section on Conditioning the Heart – Pathways to Translation. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-8

Several noble gases, although classified as inert substances, exert a tissue-protective effect in different experimental models when applied before organ ischaemia as an early or late preconditioning stimulus, after ischaemia as a post-conditioning stimulus or when given in combination before, during and/or after ischaemia. A wide range of organs can be protected by these inert substances, in particular cardiac and neuronal tissue. In this review we summarize the data on noble gas-induced cardioprotection, focusing on the underlying protective mechanisms. We will also look at translatability of experimental data to the clinical situation.

Aims/hypothesis Sodium–glucose cotransporter 2 (SGLT2) inhibitors (SGLT2i) constitute a novel class of glucose-lowering (type 2) kidney-targeted agents. We recently reported that the SGLT2i empagliflozin (EMPA) reduced cardiac cytosolic Na+ ([Na+]c) and cytosolic Ca2+ ([Ca2+]c) concentrations through inhibition of Na+/H+ exchanger (NHE). Here, we examine (1) whether the SGLT2i dapagliflozin (DAPA) and canagliflozin (CANA) also inhibit NHE and reduce [Na+]c; (2) a structural model for the interaction of SGLT2i to NHE; (3) to what extent SGLT2i affect the haemodynamic and metabolic performance of isolated hearts of healthy mice. Methods Cardiac NHE activity and [Na+]c in mouse cardiomyocytes were measured in the presence of clinically relevant concentrations of EMPA (1 μmol/l), DAPA (1 μmol/l), CANA (3 μmol/l) or vehicle. NHE docking simulation studies were applied to explore potential binding sites for SGTL2i. Constant-flow Langendorff-perfused mouse hearts were subjected to SGLT2i for 30 min, and cardiovascular function, O2 consumption and energetics (phosphocreatine (PCr)/ATP) were determined. Results EMPA, DAPA and CANA inhibited NHE activity (measured through low pH recovery after NH4 + pulse: EMPA 6.69 ± 0.09, DAPA 6.77 ± 0.12 and CANA 6.80 ± 0.18 vs vehicle 7.09 ± 0.09; p

Both the absence of cyclophilin D (CypD) and the presence of mitochondrial bound hexokinase II (mtHKII) protect the heart against ischemia/reperfusion (I/R) injury. It is unknown whether CypD determines the amount of mtHKII in the heart. We examined whether CypD affects mtHK in normoxic, ischemic and preconditioned isolated mouse hearts. Wild type (WT) and CypD−/− mouse hearts were perfused with glucose only and subjected to 25 min ischemia and reperfusion. At baseline, cytosolic and mtHK was similar between hearts. CypD ablation protected against I/R injury and increased ischemic preconditioning (IPC) effects, without affecting end-ischemic mtHK. When hearts were perfused with glucose, glutamine, pyruvate and lactate, the preparation was more stable and CypD ablation−resulted in more protection that was associated with increased mtHK activity, leaving little room for additional protection by IPC. In conclusion, in glucose only-perfused hearts, deletion of CypD is not associated with end-ischemic mitochondrial-HK binding. In contrast, in the physiologically more relevant multiple-substrate perfusion model, deletion of CypD is associated with an increased mtHK activity, possibly explaining the increased protection against I/R injury.

Helium, a noble gas, has been used safely in humans. In animal models of regional myocardial ischemia/reperfusion (I/R) it was shown that helium conditioning reduces infarct size. Currently, it is not known how helium exerts its cytoprotective effects and which cell death/survival pathways are affected. The objective of this study, therefore, was to investigate the cell protective effects of helium postconditioning by PCR array analysis of genes involved in necrosis, apoptosis and autophagy. Male rats were subjected to 25 min of ischemia and 5, 15 or 30 min of reperfusion. Semiquantitative histological analysis revealed that 15 min of helium postconditioning reduced the extent of I/R-induced cell damage. This effect was not observed after 5 and 30 min of helium postconditioning. Analysis of the differential expression of genes showed that 15 min of helium postconditioning mainly caused upregulation of genes involved in autophagy and inhibition of apoptosis versus I/R alone. The results suggest that the cytoprotective effects of helium inhalation may be caused by a switch from pro-cell-death signaling to activation of cell survival mechanisms, which appears to affect a wide range of pathways.

Background The noble gas helium induces pre- and postconditioning in animals and humans. Volatile anesthetics induce cardioprotection in humans undergoing coronary artery bypass graft (CABG) surgery. We hypothesized that helium induces pre- and postconditioning in CABG-patients, affecting signaling molecules protein kinase C-epsilon (PKC-ε), p38 mitogen activated protein kinase (p38 MAPK), extracellular signal-regulated kinase 1/2 (ERK-1/2) and heat shock protein 27 (HSP-27) within cardiac tissue, and reducing postoperative troponin levels. Methods After ethical approval and informed consent, 125 elective patients undergoing CABG surgery were randomised into this prospective, placebo controlled, investigator blinded, parallel arm single-centre study. Helium preconditioning (3 × 5 min of 70 % helium and 30 % oxygen) was applied before aortic cross clamping; postconditioning (15 min of helium) was applied before release of the aortic cross clamp. Signaling molecules were measured in right atrial appendix specimens. Troponin-T was measured at 4, 12, 24 and 48 h postoperatively. Results Baseline characteristics of all groups were similar. Helium preconditioning did not significantly alter the primary outcome (molecular levels of kinases PKC-ε and HSP-27, ratio of activated p38 MAPK or ERK ½). Postoperative troponin T was 11 arbitrary units [5, 31; area-under-the-curve (interquartile range)] for controls, and no statistically significant changes were observed after helium preconditioning [He-pre: 11 (6, 18)], helium postconditioning [He-post: 11 (8, 15)], helium pre- and postconditioning [He-PP: 14 (6, 20)] and after sevoflurane preconditioning [APC: 12 (8, 24), p = 0.13]. No adverse effects related to study treatment were observed in this study. Conclusions No effect was observed of helium preconditioning, postconditioning or the combination thereof on activation of p38 MAPK, ERK 1/2 or levels of HSP27 and PKC-ε in the human heart. Helium pre- and postconditioning did not affect postoperative troponin release in patients undergoing CABG surgery. Clinical trial number Dutch trial register (http://www.trialregister.nl/) number NTR1226 Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-1045-z) contains supplementary material, which is available to authorized users.

Helium induces preconditioning (He-PC) by mitochondrial calcium-sensitive potassium (mK(Ca)) channel-activation, but this effect is lost in the aged myocardium. Both, the upstream signalling pathway of He-PC and the underlying mechanisms for an age-related loss of preconditioning are unknown. A possible candidate as upstream regulator of mK(Ca) channels is protein kinase A (PKA). We investigated whether 1) regulation of PKA is involved in He-PC and 2) regulation of PKA is age-dependent. Young (2-3 months) and aged (22-24 months) Wistar rats were randomised to eight groups (each n =8). All animals underwent 25 min regional myocardial ischemia and 120 min reperfusion. Control (Con, Age Con) animals were not further treated. Young rats inhaled 70% helium for 3 x 5min (He-PC). The PKA-blocker H-89 (10 mu g/kg) was administered with and without helium (He-PC + H-89, H-89). Furthermore, we tested the effect of direct activation of mK(Ca) channels with NS1619. The adenylyl cyclase activator forskolin (For) was administered in young (300 mu g/kg) and aged animals (300 and 1000 mu g/kg). He-PC reduced infarct size from 60 +/- 4% (Con) to 37 +/- 10% (p

Background Sevoflurane induces preconditioning (SevoPC). The effect of aprotinin and the involvement of endothelial nitric-oxide synthase (NOS) on SevoPC are unknown. We investigated (1) whether SevoPC is strengthened by multiple preconditioning cycles, (2) whether SevoPC is blocked by aprotinin, and (3) whether endothelial NOS plays a crucial role in SevoPC. Methods Anesthetized male Wistar rats were randomized to 15 groups (each n = 6) and underwent 25-min regional myocardial ischemia and 2-h reperfusion. Controls were not treated further. Preconditioning groups inhaled 1 minimum alveolar concentration of sevoflurane for 5 min (SEVO-I), twice for 5 min each (SEVO-II), three times for 5 min each (SEVO-III), or six times for 5 min each (SEVO-VI). Aprotinin was administered with and without sevoflurane. Involvement of endothelial NOS was determined with the nonspecific NOS blocker N-nitro-l-arginine-methyl-ester, the specific neuronal NOS blocker 7-nitroindazole, and the specific inducible NOS blocker aminoguanidine. Results SevoPC reduced infarct size in all protocols (SEVO-I, 42 ± 6%; SEVO-II, 33 ± 4%; SEVO-III, 11 ± 5%; SEVO-VI, 16 ± 4%; all P < 0.001 vs. control, 67 ± 3%) and was least after three and six cycles of sevoflurane (P < 0.001 vs. SEVO-II and -I, respectively). Aprotinin alone had no effect on infarct size but blocked SevoPC. N-nitro-l-arginine-methyl-ester abolished SevoPC (67 ± 4%; P < 0.05 vs. SEVO-III). Aminoguanidine and 7-nitroindazole blocked SevoPC only partially (25 ± 6 and 31 ± 6%, respectively; P < 0.05 vs. SEVO-III and control). SevoPC induced endothelial NOS phosphorylation, which was abrogated by aprotinin. Conclusion SevoPC is strengthened by multiple preconditioning cycles, and phosphorylation of endothelial NOS is a crucial step in mediating SevoPC. These effects are abolished by aprotinin.