Canagliflozin inhibits inflammasome activation in diabetic endothelial cells - Revealing a novel calcium-dependent anti-inflammatory effect of canagliflozin on human diabetic endothelial cells.

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-1ß, NLRP3, pro-caspase-1 and ASC. RESULTS: 10 µM CANA significantly reduced TNF-α related ROS generation, IL-1ß production and NLRP3 expression (P all <0.05), but NAC did not alter the inflammasome activation (P > 0.05). CANA and BAPTA both prevented intracellular Ca2+ increase in cells exposed to TNF-α (P both <0.05). Moreover, BAPTA and PD 98059 significantly reduced the TNF-α triggered IL-1ß production as well as NLRP3 and pro-caspase-1 expression (P all <0.05). CONCLUSION: CANA suppresses inflammasome activation by inhibition of (1) intracellular Ca2+ and (2) ERK1/2 phosphorylation, but not by ROS reduction.

NLRX1 Prevents M2 Macrophage Polarization and Excessive Renal Fibrosis in Chronic Obstructive Nephropathy.

BACKGROUND: Chronic kidney disease often leads to kidney dysfunction due to renal fibrosis, regardless of the initial cause of kidney damage. Macrophages are crucial players in the progression of renal fibrosis as they stimulate inflammation, activate fibroblasts, and contribute to extracellular matrix deposition, influenced by their metabolic state. Nucleotide-binding domain and LRR-containing protein X (NLRX1) is an innate immune receptor independent of inflammasomes and is found in mitochondria, and it plays a role in immune responses and cell metabolism. The specific impact of NLRX1 on macrophages and its involvement in renal fibrosis is not fully understood. METHODS: To explore the specific role of NLRX1 in macrophages, bone-marrow-derived macrophages (BMDMs) extracted from wild-type (WT) and NLRX1 knockout (KO) mice were stimulated with pro-inflammatory and pro-fibrotic factors to induce M1 and M2 polarization in vitro. The expression levels of macrophage polarization markers (Nos2, Mgl1, Arg1, and Mrc1), as well as the secretion of transforming growth factor ß (TGFß), were measured using RT-PCR and ELISA. Seahorse-based bioenergetics analysis was used to assess mitochondrial respiration in naïve and polarized BMDMs obtained from WT and NLRX1 KO mice. In vivo, WT and NLRX1 KO mice were subjected to unilateral ureter obstruction (UUO) surgery to induce renal fibrosis. Kidney injury, macrophage phenotypic profile, and fibrosis markers were assessed using RT-PCR. Histological staining (PASD and Sirius red) was used to quantify kidney injury and fibrosis. RESULTS: Compared to the WT group, an increased gene expression of M2 markers-including Mgl1 and Mrc1-and enhanced TGFß secretion were found in naïve BMDMs extracted from NLRX1 KO mice, indicating functional polarization towards the pro-fibrotic M2 subtype. NLRX1 KO naïve macrophages also showed a significantly enhanced oxygen consumption rate compared to WT cells and increased basal respiration and maximal respiration capacities that equal the level of M2-polarized macrophages. In vivo, we found that NLRX1 KO mice presented enhanced M2 polarization markers together with enhanced tubular injury and fibrosis demonstrated by augmented TGFß levels, fibronectin, and collagen accumulation. CONCLUSIONS: Our findings highlight the unique role of NLRX1 in regulating the metabolism and function of macrophages, ultimately protecting against excessive renal injury and fibrosis in UUO.

Empagliflozin reduces oxidative stress through inhibition of the novel inflammation/NHE/[Na+]c/ROS-pathway in human endothelial cells.

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.

Sodium Glucose Co-Transporter 2 Inhibitors Ameliorate Endothelium Barrier Dysfunction Induced by Cyclic Stretch through Inhibition of Reactive Oxygen Species.

SGLT-2i's exert direct anti-inflammatory and anti-oxidative effects on resting endothelial cells. However, endothelial cells are constantly exposed to mechanical forces such as cyclic stretch. Enhanced stretch increases the production of reactive oxygen species (ROS) and thereby impairs endothelial barrier function. We hypothesized that the SGLT-2i's empagliflozin (EMPA), dapagliflozin (DAPA) and canagliflozin (CANA) exert an anti-oxidative effect and alleviate cyclic stretch-induced endothelial permeability in human coronary artery endothelial cells (HCAECs). HCAECs were pre-incubated with one of the SGLT-2i's (1 µM EMPA, 1 µM DAPA and 3 µM CANA) for 2 h, followed by 10% stretch for 24 h. HCAECs exposed to 5% stretch were considered as control. Involvement of ROS was measured using N-acetyl-l-cysteine (NAC). The sodium-hydrogen exchanger 1 (NHE1) and NADPH oxidases (NOXs) were inhibited by cariporide, or GKT136901, respectively. Cell permeability and ROS were investigated by fluorescence intensity imaging. Cell permeability and ROS production were increased by 10% stretch; EMPA, DAPA and CANA decreased this effect significantly. Cariporide and GKT136901 inhibited stretch-induced ROS production but neither of them further reduced ROS production when combined with EMPA. SGLT-2i's improve the barrier dysfunction of HCAECs under enhanced stretch and this effect might be mediated through scavenging of ROS. Anti-oxidative effect of SGLT-2i's might be partially mediated by inhibition of NHE1 and NOXs.

Quantification of Myocardial Creatine and Triglyceride Content in the Human Heart: Precision and Accuracy of in vivo Proton Magnetic Resonance Spectroscopy.

BACKGROUND: Proton magnetic resonance spectroscopy (1 H-MRS) of the human heart is deemed to be a quantitative method to investigate myocardial metabolite content, but thorough validations of in vivo measurements against invasive techniques are lacking. PURPOSE: To determine measurement precision and accuracy for quantifications of myocardial total creatine and triglyceride content with localized 1 H-MRS. STUDY TYPE: Test-retest repeatability and measurement validation study. SUBJECTS: Sixteen volunteers and 22 patients scheduled for open-heart aortic valve replacement or septal myectomy. FIELD STRENGTH/SEQUENCE: Prospectively ECG-triggered respiratory-gated free-breathing single-voxel point-resolved spectroscopy (PRESS) sequence at 3 T. ASSESSMENT: Myocardial total creatine and triglyceride content were quantified relative to the total water content by fitting the 1 H-MR spectra. Precision was assessed with measurement repeatability. Accuracy was assessed by validating in vivo 1 H-MRS measurements against biochemical assays in myocardial tissue from the same subjects. STATISTICAL TESTS: Intrasession and intersession repeatability was assessed using Bland-Altman analyses. Agreement between 1 H-MRS measurements and biochemical assay was tested with regression analyses. RESULTS: The intersession repeatability coefficient for myocardial total creatine content was 41.8% with a mean value of 0.083% ± 0.020% of the total water signal, and 36.7% for myocardial triglyceride content with a mean value of 0.35% ± 0.13% of the total water signal. Ex vivo myocardial total creatine concentrations in tissue samples correlated with the in vivo myocardial total creatine content measured with 1 H-MRS: n = 22, r = 0.44; P < 0.05. Likewise, ex vivo myocardial triglyceride concentrations correlated with the in vivo myocardial triglyceride content: n = 20, r = 0.50; P < 0.05. DATA CONCLUSION: We validated the use of localized 1 H-MRS of the human heart at 3 T for quantitative assessments of in vivo myocardial tissue metabolite content by estimating the measurement precision and accuracy. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY STAGE: 2.

NLRX1 Deletion Increases Ischemia-Reperfusion Damage and Activates Glucose Metabolism in Mouse Heart.

Background: NOD-like receptors (NLR) are intracellular sensors of the innate immune system, with the NLRP3 being a pro-inflammatory member that modulates cardiac ischemia-reperfusion injury (IRI) and metabolism. No information is available on a possible role of anti-inflammatory NLRs on IRI and metabolism in the intact heart. Here we hypothesize that the constitutively expressed, anti-inflammatory mitochondrial NLRX1, affects IRI and metabolism of the isolated mouse heart. Methods: Isolated C57Bl/6J and NLRX1 knock-out (KO) mouse hearts were perfused with a physiological mixture of the essential substrates (lactate, glucose, pyruvate, fatty acid, glutamine) and insulin. For the IRI studies, hearts were subjected to either mild (20 min) or severe (35 min) ischemia and IRI was determined at 60 min reperfusion. Inflammatory mediators (IL-6, TNFα) and survival pathways (mito-HKII, p-Akt, p-AMPK, p-STAT3) were analyzed at 5 min of reperfusion. For the metabolism studies, hearts were perfused for 35 min with either 5.5 mM 13C-glucose or 0.4 mM 13C-palmitate under normoxic conditions, followed by LC-MS analysis and integrated, stepwise, mass-isotopomeric flux analysis (MIMOSA). Results: NLRX1 KO significantly increased IRI (infarct size from 63% to 73%, end-diastolic pressure from 59 mmHg to 75 mmHg, and rate-pressure-product recovery from 15% to 6%), following severe, but not mild, ischemia. The increased IRI in NLRX1 KO hearts was associated with depressed Akt signaling at early reperfusion; other survival pathways or inflammatory parameters were not affected. Metabolically, NLRX1 KO hearts displayed increased lactate production and glucose oxidation relative to fatty acid oxidation, associated with increased pyruvate dehydrogenase flux and 10% higher cardiac oxygen consumption. Conclusion: Deletion of the mitochondrially-located NOD-like sensor NLRX1 exacerbates severe cardiac IR injury, possibly through impaired Akt signaling, and increases cardiac glucose metabolism.

Empagliflozin and Dapagliflozin Reduce ROS Generation and Restore NO Bioavailability in Tumor Necrosis Factor α-Stimulated Human Coronary Arterial Endothelial Cells.

BACKGROUND/AIMS: Heart failure is characterized by chronic low-grade vascular inflammation, which in itself can lead to endothelial dysfunction. Clinical trials showed reductions in heart failure-related hospitalizations of type 2 diabetic patients using sodium glucose co-transporter 2 inhibitors (SGLT2i's). Whether and how SGLT2i's directly affect the endothelium under inflammatory conditions is not completely understood. The aim of the study was to investigate whether the SGLT2i Empagliflozin (EMPA) and Dapagliflozin (DAPA) reduce tumor necrosis factor α (TNFα) induced endothelial inflammation in vitro. METHODS: Human coronary arterial endothelial cells (HCAECs) and human umbilical vein endothelial cells (HUVECs) were (pre-)incubated with 1 µM EMPA or DAPA and subsequently exposed to 10 ng/ml TNFα. ROS and NO were measured using live cell imaging. Target proteins were either determined by infrared western blotting or fluorescence activated cell sorting (FACS). The connection between Cav-1 and eNOS was determined by co-immunoprecipitation. RESULTS: Nitric oxide (NO) bioavailability was reduced by TNFα and both EMPA and DAPA restored NO levels in TNFα-stimulated HCAECs. Intracellular ROS was increased by TNFα, and this increase was completely abolished by EMPA and DAPA in HCAECs by means of live cell imaging. eNOS signaling was significantly disturbed after 24 h when cells were exposed to TNFα for 24h, yet the presence of both SGLT2is did not prevent this disruption. TNFα-induced enhanced permeability at t=24h was unaffected in HUVECs by EMPA. Similarly, adhesion molecule expression (VCAM-1 and ICAM-1) was elevated after 4h TNFα (1.5-5.5 fold increase of VCAM-1 and 4-12 fold increase of ICAM-1) but were unaffected by EMPA and DAPA in both cell types. Although we detected expression of SGLT2 protein levels, the fact that we could not silence this expression by means of siRNA and the mRNA levels of SGLT2 were not detectable in HCAECs, suggests aspecificity or our SGLT2 antibody and absence of SGLT2 in our cells. CONCLUSION: These data suggest that EMPA and DAPA rather restore NO bioavailability by inhibiting ROS generation than by affecting eNOS expression or signaling, barrier function and adhesion molecules expression in TNFα-induced endothelial cells. Furthermore, the observed effects cannot be ascribed to the inhibition of SGLT2 in endothelial cells.

Divergent Effects of Hypertonic Fluid Resuscitation on Renal Pathophysiological and Structural Parameters in Rat Model of Lower Body Ischemia/Reperfusion-Induced Sterile Inflammation.

The pathogenesis of acute kidney injury (AKI) is characterized by the deterioration of tissue perfusion and oxygenation and enhanced inflammation. The purpose of this study was to investigate whether or not the hemodynamic and inflammatory effects of hypertonic saline (HS) protect the kidney by promoting renal microcirculatory oxygenation and possible deleterious effects of HS due to its high sodium content on renal functional and structural injury following ischemia/reperfusion. Mechanically ventilated and anesthetized rats were randomly divided into four groups (n = 6 per group): a sham-operated control group; a group subjected to renal ischemia for 45 min by supra-aortic occlusion followed by 2 h of reperfusion (I/R); and I/R group treated with a continuous i.v. infusion (5 mL/kg/h) of either % 0.9 NaCl (IR+NS) or %10 NaCl (I/R+HS) after releasing the clamp. Systemic and renal hemodynamic, renal cortical (CµPO2), and medullar microcirculatory pO2 (MµPO2) are measured by the oxygen-dependent quenching of the phosphorescence lifetime technique. Renal functional, inflammatory, and tissues damage parameters were also assessed. HS, but not NS, treatment restored I/R-induced reduced mean arterial pressure, CµPO2, renal oxygen deliver (DO2ren), and consumption (VO2ren). HS caused a decrease in tubular sodium reabsorption (TNa) that correlated with an elevation of fractional sodium excretion (EFNa) and urine output. HS had an anti-inflammatory effect by reducing the levels TNF-α, IL-6, and hyaluronic acid in the renal tissue samples as compared with the I/R and I/R+NS groups (P < 0.05). HS treatment was also associated with mild acidosis and an increased renal tubular damage. Despite HS resuscitation improving the systemic hemodynamics, microcirculatory oxygenation, and renal oxygen consumption as well as inflammation, it should be limited or strictly controlled for long-term use because of provoking widespread renal structural damage.

A randomized trial of remote ischemic preconditioning and control treatment for cardioprotection in sevoflurane-anesthetized CABG patients.

BACKGROUND: Remote ischemic preconditioning (RIPC) efficacy is debated. Possibly, because propofol, which has a RIPC-inhibiting action, is used in most RIPC trials. It has been suggested that clinical efficacy is, however, present with volatile anesthesia in the absence of propofol, although this is based on one phase 1 trial only. Therefore, in the present study we further explore the relation between RIPC and cardioprotection with perioperative anesthesia restricted to sevoflurane and fentanyl, in CABG patients without concomitant procedures. METHODS: In a single-center study, we aimed to randomize 46 patients to either RIPC (3x5 min inflation of a blood pressure cuff around the arm) or control treatment (deflated cuff around the arm). Blood samples were obtained before and after RIPC to evaluate potential RIPC-induced mediators (Interleukin (IL)-6, IL-10, Tumor Necrosis Factor-α, Macrophage Inhibitory Factor). An atrial tissue sample was obtained at cannulation of the appendix of the right atrium for determination of mitochondrial bound hexokinase II (mtHKII) and other survival proteins (Akt and AMP-activated protein kinase α). In blood samples taken before and 6, 12 and 24 h after surgery cardiac troponin T (cTnT) and C-reactive protein (CRP) were determined. Surgery was strictly performed under sevoflurane anesthesia (no propofol). RESULTS: We actually randomized 16 patients to control treatment and 13 patients to RIPC. The mean 24 h area under the curve (AUC) cTnT was 11.44 (standard deviation 4.66) in the control group and 10.90 (standard deviation 4.73) in the RIPC group (mean difference 0.54, 95% CI -3.06 to 4.13; p = 0.76). The mean 24 h AUC CRP was 1319 (standard deviation 92) in the control group and 1273 (standard deviation 141) in the RIPC group (mean difference 46.2, 95% CI -288 to 380; p = 0.78). RIPC was without effect on survival proteins in atrial tissue samples obtained before surgery (mitochondrial hexokinase, Akt and AMPK) and inflammatory mediators obtained before and immediately after RIPC (IL-6, IL-10, TNF-α, macrophage migration inhibitory factor). CONCLUSION: Many factors can interfere with the outcome of RIPC. Trying to correct for this led to strict inclusion criteria, which, in combination with a decreased institutional frequency of CABG without concomitant procedures and a change in institutional anesthetic regimen away from volatile anesthetics towards total intravenous anesthesia, caused slow inclusion and halting of this trial after 3 years, before target inclusion could be reached. Therefore this study is underpowered to prove its primary goal that RIPC reduced AUC cTnT by < 25%. Nevertheless, we have shown that the effect of RIPC on 24 h AUC cTnT, in cardiac surgery with anesthesia during surgery restricted to sevoflurane/fentanyl (no propofol), was between a decrease of 27% and an increase of 36%. These findings are not in line with previous studies in this field. TRIAL REGISTRATION: The Netherlands Trial Register: NTR2915 ; Registered 25 Mei 2011.

Reduced acute myocardial ischemia-reperfusion injury in IL-6-deficient mice employing a closed-chest model.

OBJECTIVE AND DESIGN: We examined the role of IL-6 in the temporal development of cardiac ischemia-reperfusion injury employing a closed-chest I/R model. MATERIALS/METHODS: Infarction, local and systemic inflammation, neutrophil infiltration, coagulation and ST elevation/resolution were compared between wild-type (WT) and IL-6-deficient (IL-6(-/-)) mice after 1 h ischemia and 0, ½, 3, and 24 h reperfusion. RESULTS: IL-6 deficiency reduced infarct size at 3 h reperfusion (28.8 ± 4.5 % WT vs 17.6 ± 2.5 % IL-6(-/-)), which reduction persisted and remained similar at 24 h reperfusion (25.1 ± 3.0 % WT vs 14.6 ± 4.4 % IL-6(-/-)). Serum Amyloid A was reduced at 24 h reperfusion only (57.5 ± 4.9 WT vs 24.8 ± 5.6 ug/ml IL-6(-/-) mice). Cardiac cytokines (IL-6, IL-1ß and TNFα) peaked at 3 h reperfusion, but IL-1ß and TNFα levels were unaffected by IL-6 deficiency. Significant neutrophil influx was only detected at 24 h reperfusion and was similar for WT and IL-6(-/-). Tissue factor peaked at 24 h reperfusion, whereas fibrin/fibrinogen peaked at 3 h reperfusion and was completely resolved at 24 h reperfusion; both coagulation factors were unaltered by IL-6 deficiency. Prolonged ST elevation was observed during ischemia that completely resolved for both genotypes at early reperfusion. CONCLUSIONS: The data suggest that, in the absence of major surgical intervention, IL-6 contributes to the development of infarct size in the early phase of reperfusion; this contribution did not depend on neutrophil influx, IL-1ß and TNFα, tissue factor and fibrin.

Reducing mitochondrial bound hexokinase II mediates transition from non-injurious into injurious ischemia/reperfusion of the intact heart.

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.

In vivo mouse myocardial (31)P MRS using three-dimensional image-selected in vivo spectroscopy (3D ISIS): technical considerations and biochemical validations.

(31)P MRS provides a unique non-invasive window into myocardial energy homeostasis. Mouse models of cardiac disease are widely used in preclinical studies, but the application of (31)P MRS in the in vivo mouse heart has been limited. The small-sized, fast-beating mouse heart imposes challenges regarding localized signal acquisition devoid of contamination with signal originating from surrounding tissues. Here, we report the implementation and validation of three-dimensional image-selected in vivo spectroscopy (3D ISIS) for localized (31)P MRS of the in vivo mouse heart at 9.4 T. Cardiac (31)P MR spectra were acquired in vivo in healthy mice (n = 9) and in transverse aortic constricted (TAC) mice (n = 8) using respiratory-gated, cardiac-triggered 3D ISIS. Localization and potential signal contamination were assessed with (31)P MRS experiments in the anterior myocardial wall, liver, skeletal muscle and blood. For healthy hearts, results were validated against ex vivo biochemical assays. Effects of isoflurane anesthesia were assessed by measuring in vivo hemodynamics and blood gases. The myocardial energy status, assessed via the phosphocreatine (PCr) to adenosine 5'-triphosphate (ATP) ratio, was approximately 25% lower in TAC mice compared with controls (0.76 ± 0.13 versus 1.00 ± 0.15; P < 0.01). Localization with one-dimensional (1D) ISIS resulted in two-fold higher PCr/ATP ratios than measured with 3D ISIS, because of the high PCr levels of chest skeletal muscle that contaminate the 1D ISIS measurements. Ex vivo determinations of the myocardial PCr/ATP ratio (0.94 ± 0.24; n = 8) confirmed the in vivo observations in control mice. Heart rate (497 ± 76 beats/min), mean arterial pressure (90 ± 3.3 mmHg) and blood oxygen saturation (96.2 ± 0.6%) during the experimental conditions of in vivo (31)P MRS were within the normal physiological range. Our results show that respiratory-gated, cardiac-triggered 3D ISIS allows for non-invasive assessments of in vivo mouse myocardial energy homeostasis with (31)P MRS under physiological conditions.

Effect of metformin pretreatment on myocardial injury during coronary artery bypass surgery in patients without diabetes (MetCAB): a double-blind, randomised controlled trial.

BACKGROUND: During coronary artery bypass graft (CABG) surgery, ischaemia and reperfusion damage myocardial tissue, and increased postoperative plasma troponin concentration is associated with a worse outcome. We investigated whether metformin pretreatment limits cardiac injury, assessed by troponin concentrations, during CABG surgery in patients without diabetes. METHODS: We did a placebo-controlled, double-blind, single-centre study in an academic hospital in Nijmegen (Netherlands) in adult patients without diabetes undergoing an elective on-pump CABG procedure. We randomly assigned patients (1:1) in blocks of ten via a computer-generated randomisation sequence to either metformin hydrochloride (500 mg three times per day) or placebo (three times per day) for 3 days before surgery. The last dose was given roughly 3 h before surgery. Patients, investigators, trial staff, and the statistician were all masked to treatment allocation. The primary endpoint was the plasma concentration of high-sensitive troponin I at 6, 12, and 24 h postreperfusion after surgery, analysed in the per-protocol population with a mixed-model analysis using all these timepoints. Secondary endpoints included the occurrence of clinically relevant arrhythmias within 24 hours after reperfusion, the need for inotropic support, time to detubation, duration of stay in the intensive-care unit, and postoperative use of insulin. This study is registered with ClinicalTrials.gov, number NCT01438723. FINDINGS: Between Nov 8, 2011, and Nov 22, 2013, we randomly assigned 111 patients to treatment (57 to metformin and 54 to placebo). Five patients dropped out from the metformin group, and six from the placebo group. 52 patients in the metformin group and 48 patients in the placebo group were included in the per-protocol analysis. Geometric mean high-sensitivity troponin I increased from 0 µg/L to 3·67 µg/L (95% CI 3·06-4·41) with metformin and to 3·32 µg/L (2·75-4·01) with placebo at 6 h after reperfusion; 2·84 µg/L (2·37-3·41) and 2·45 µg/L (2·02-2·96), respectively, at 12 h; and to 1·77 µg/L (1·47-2·12) and 1·60 µg/L (1·32-1·94) at 24 h. The concentrations did not differ significantly between the groups (difference 12·3% for all timepoints [95% CI -12·4 to 44·1] p=0·35). Occurrence of arrhythmias did not differ between groups (three [5·8%] of 52 patients who received metformin vs three [6·3%] of 48 patients who received placebo; p=1·00). There was no difference between groups in the need for inotropic support, time to detubation, duration of stay in the intensive-care unit, or postoperative use of insulin. No patients died within 30 days after surgery. Occurrence of gastrointestinal discomfort (mostly diarrhoea) was significantly higher with metformin than with placebo (11 [21·2%] of 52 vs two [4·2%] of 48 patients; p=0·01). INTERPRETATION: Short-term metformin pretreatment, although safe, does not seem to be an effective strategy to reduce periprocedural myocardial injury in patients without diabetes undergoing CABG surgery. FUNDING: Netherlands Organisation for Health Research and Development and Netherlands Heart Foundation.

Optimizing anesthetic regimen for surgery in mice through minimization of hemodynamic, metabolic, and inflammatory perturbations.

The role of anesthetics in animal research models is crucial, yet often ignored, and is almost never the primary focus of examination. Here, we investigated the impact of anesthetic regimens on different parameters of hemodynamics (blood pressure (BP) and heart rate (HR)), metabolism (glucose, insulin, and free fatty acids (FFA)), and inflammation (IL-6 and TNF-α) in two frequently used mouse strains (C57BL/6 and FVB). All animals were at a similar surgical plane of anesthesia, mechanically ventilated, and monitored for 60 min. The following anesthetic regimens were studied: (1) fentanyl-ketamine-midazolam (FKM), (2) fentanyl-midazolam-haldol (FMH), (3) pentobarbital (P), (4) fentanyl-fluanisone-midazolam (FFM), (5) fentanyl-midazolam-acepromazine (FMA), (6) ketamine-medetomidine-atropine (KMA), (7) isoflurane (ISO), and (8) propofol-fentanyl-midazolam (PFM). Metabolic and inflammatory parameters were compared with those obtained from non-anesthetized animals. Hemodynamics: BP >80 mm Hg were only obtained with KMA, whereas hypotension (BP <60 mm Hg) was observed with FKM and P. HR >500 beats/min was observed with ISO and PFM, whereas HR <400 beats/min was induced with KMA, FMH (BL/6), P (BL/6), and FKM (FVB). Metabolism: Glucose and insulin were most disturbed by KMA and ISO and mildly disturbed by FMA, whereas FFM, PFM, and P did not have any effect. FFA increased largely by FMA, with ISO and FKM having no effects. Inflammation: Cytokines were increased least with ISO/FFM/FMA, whereas FKM and KMA induced the largest increases in cytokines. When aiming at achieving surgical anesthesia without large disturbances in hemodynamic, metabolic, and inflammatory profiles, FFM, ISO, or PFM may be the most neutral anesthetic regimens in mice.

Targeting hexokinase II to mitochondria to modulate energy metabolism and reduce ischaemia-reperfusion injury in heart.

Mitochondrially bound hexokinase II (mtHKII) has long been known to confer cancer cells with their resilience against cell death. More recently, mtHKII has emerged as a powerful protector against cardiac cell death. mtHKII protects against ischaemia-reperfusion (IR) injury in skeletal muscle and heart, attenuates cardiac hypertrophy and remodelling, and is one of the major end-effectors through which ischaemic preconditioning protects against myocardial IR injury. Mechanisms of mtHKII cardioprotection against reperfusion injury entail the maintenance of regulated outer mitochondrial membrane (OMM) permeability during ischaemia and reperfusion resulting in stabilization of mitochondrial membrane potential, the prevention of OMM breakage and cytochrome C release, and reduced reactive oxygen species production. Increasing mtHK may also have important metabolic consequences, such as improvement of glucose-induced insulin release, prevention of acidosis through enhanced coupling of glycolysis and glucose oxidation, and inhibition of fatty acid oxidation. Deficiencies in expression and distorted cellular signalling of HKII may contribute to the altered sensitivity of diabetes to cardiac ischaemic diseases. The interaction of HKII with the mitochondrion constitutes a powerful endogenous molecular mechanism to protect against cell death in almost all cell types examined (neurons, tumours, kidney, lung, skeletal muscle, heart). The challenge now is to harness mtHKII in the treatment of infarction, stroke, elective surgery and transplantation. Remote ischaemic preconditioning, metformin administration and miR-155/miR-144 manipulations are potential means of doing just that.

The mechanisms and physiological relevance of glycocalyx degradation in hepatic ischemia/reperfusion injury.

SIGNIFICANCE: Hepatic ischemia/reperfusion (I/R) injury is an inevitable side effect of major liver surgery that can culminate in liver failure. The bulk of I/R-induced liver injury results from an overproduction of reactive oxygen and nitrogen species (ROS/RNS), which inflict both parenchymal and microcirculatory damage. A structure that is particularly prone to oxidative attack and modification is the glycocalyx (GCX), a meshwork of proteoglycans and glycosaminoglycans (GAGs) that covers the lumenal endothelial surface and safeguards microvascular homeostasis. ROS/RNS-mediated degradation of the GCX may exacerbate I/R injury by, for example, inducing vasoconstriction, facilitating leukocyte adherence, and directly activating innate immune cells. RECENT ADVANCES: Preliminary experiments revealed that hepatic sinusoids contain a functional GCX that is damaged during murine hepatic I/R and major liver surgery in patients. There are three ROS that mediate GCX degradation: hydroxyl radicals, carbonate radical anions, and hypochlorous acid (HOCl). HOCl converts GAGs in the GCX to GAG chloramides that become site-specific targets for oxidizing and reducing species and are more efficiently fragmented than the parent molecules. In addition to ROS/RNS, the GAG-degrading enzyme heparanase acts at the endothelial surface to shed the GCX. CRITICAL ISSUES: The GCX seems to be degraded during major liver surgery, but the underlying cause remains ill-defined. FUTURE DIRECTIONS: The relative contribution of the different ROS and RNS intermediates to GCX degradation in vivo, the immunogenic potential of the shed GCX fragments, and the role of heparanase in liver I/R injury all warrant further investigation.

Deletion of the innate immune NLRP3 receptor abolishes cardiac ischemic preconditioning and is associated with decreased Il-6/STAT3 signaling.

OBJECTIVE: Recent studies indicate that the innate immune system is not only triggered by exogenous pathogens and pollutants, but also by endogenous danger signals released during ischemia and necrosis. As triggers for the innate immune NLRP3 inflammasome protein complex appear to overlap with those for cardiac ischemia-reperfusion (I/R) and ischemic preconditioning (IPC), we explored the possibility that the NLRP3 inflammasome is involved in IPC and acute I/R injury of the heart. PRINCIPAL FINDINGS: Baseline cardiac performance and acute I/R injury were investigated in isolated, Langendorff-perfused hearts from wild-type (WT), ASC(-/-) and NLRP3(-/-) mice. Deletion of NLRP3 inflammasome components ASC(-/-) or NLRP3(-/-) did not affect baseline performance. The deletions exacerbated I/R-induced mechanical dysfunction, but were without effect on I/R-induced cell death. When subjected to IPC, WT and ASC(-/-) hearts were protected against I/R injury (improved function and less cell death). However, IPC did not protect NLRP3(-/-) hearts against I/R injury. NLRP3(-/-) hearts had significantly decreased cardiac IL-6 levels with a trend towards lower IL-1ß levels at end reperfusion, suggesting abrogation of IPC through diminished IL-6 and/or IL-1ß signaling. Subsequent experiments showed that neutralising IL-6 using an antibody against IL-6 abrogated IPC in WT hearts. However, inhibition of the IL-1r receptor with the IL-1 receptor inhibitor Anakinra (100 mg/L) did not abrogate IPC in WT hearts. Analysis of survival kinases after IPC demonstrated decreased STAT3 expression in NLRP3(-/-) hearts when compared to WT hearts. CONCLUSIONS: The data suggest that the innate immune NLRP3 protein, in an NLRP3-inflammasome-independent fashion, is an integral component of IPC in the isolated heart, possibly through an IL-6/STAT3 dependent mechanism.

Blood pressure influences end-stage renal disease of Cd151 knockout mice.

Podocytes of the kidney adhere tightly to the underlying glomerular basement membrane (GBM) in order to maintain a functional filtration barrier. The clinical importance of podocyte binding to the GBM via an integrin-laminin-actin axis has been illustrated in models with altered function of α3ß1 integrin, integrin-linked kinase, laminin-521, and α-actinin 4. Here we expanded on the podocyte-GBM binding model by showing that the main podocyte adhesion receptor, integrin α3ß1, interacts with the tetraspanin CD151 in situ in humans. Deletion of Cd151 in mouse glomerular epithelial cells led to reduced adhesive strength to laminin by redistributing α3ß1 at the cell-matrix interface. Moreover, in vivo podocyte-specific deletion of Cd151 led to glomerular nephropathy. Although global Cd151-null B6 mice were not susceptible to renal disease, as has been shown previously, increasing blood and transcapillary filtration pressure induced nephropathy in these mice. Importantly, blocking the angiotensin-converting enzyme in renal disease-susceptible global Cd151-null FVB mice prolonged their median life span. Together, these results establish CD151 as a crucial modifier of integrin-mediated adhesion of podocytes to the GBM and show that blood pressure is an important factor in the initiation and progression of Cd151 knockout-induced nephropathy.

Ischemic preconditioning, insulin, and morphine all cause hexokinase redistribution.

Association of hexokinase (HK) with mitochondria preserves mitochondrial integrity and is an important mechanism by which cancer cells are protected against hypoxic conditions. Maintenance of mitochondrial integrity also figures prominently as a major characteristic of many cardioprotective manipulations. In this study, we provide evidence that cardioprotective interventions may promote HK redistribution from the cytosol to the mitochondria in the heart. Isolated Langendorff-perfused rat hearts (n = 6/group) were subjected to normoxic perfusion (control, Con), three 5-min ischemia-reperfusion periods (ischemic preconditioning, IPC), 1 U/l insulin (Ins), or 1 microM morphine (Mor). Hearts were immediately homogenized and centrifuged to obtain whole cell, cytosolic, and mitochondrial fractions. HK, lactate dehydrogenase (LDH), and citrate synthase (CS) enzyme activities were determined. No change in LDH or CS present in the cytosol fraction relative to whole cell activity was observed with any of the cardioprotective interventions. By contrast, HK present in the cytosol fraction relative to whole cell activity decreased significantly (P < 0.05) with all cardioprotective interventions, from 0.58 +/- 0.03 (Con) to 0.46 +/- 0.04 (IPC), 0.41 +/- 0.01 (Ins), and 0.45 +/- 0.02 (Mor). In addition, HK relative to CS activity in the mitochondrial fraction increased significantly with cardioprotection, from 0.15 +/- 0.001 (Con) to 0.21 +/- 0.002 (IPC), 0.18 +/- 0.003 (Ins), and 0.21 +/- 0.005 (Mor). Our novel data suggest that well-known cardioprotective interventions share a common end-effector mechanism of cytosolic HK translocation. Association of HK with mitochondria may promote inhibition of the mitochondrial permeability transition pore and thereby reduce cell death and apoptosis.