Single Donor Infusion of S-Nitroso-Human-Serum-Albumin Attenuates Cardiac Isograft Fibrosis and Preserves Myocardial Micro-RNA-126-3p in a Murine Heterotopic Heart Transplant Model.

Objectives: Cold ischemia and subsequent reperfusion injury are non-immunologic cornerstones in the development of graft injury after heart transplantation. The nitric oxide donor S-nitroso-human-serum-albumin (S-NO-HSA) is known to attenuate myocardial ischemia-reperfusion (I/R)-injury. We assessed whether donor preservation with S-NO-HSA affects isograft injury and myocardial expression of GATA2 as well as miR-126-3p, which are considered protective against vascular and endothelial injury. Methods: Donor C57BL/6 mice received intravenous (0.1 µmol/kg/h) S-NO-HSA (n = 12), or 0.9% saline (control, n = 11) for 20 min. Donor hearts were stored in cold histidine-tryptophan-α-ketoglutarate-N solution for 12 h and underwent heterotopic, isogenic transplantation, except 5 hearts of each group, which were analysed immediately after preservation. Fibrosis was quantified and expression of GATA2 and miR-126-3p assessed by RT-qPCR after 60 days or immediately after preservation. Results: Fibrosis was significantly reduced in the S-NO-HSA group (6.47% ± 1.76 vs. 11.52% ± 2.16; p = 0.0023; 12 h-S-NO-HSA-hHTX vs. 12 h-control-hHTX). Expression of miR-126-3p was downregulated in all hearts after ischemia compared to native myocardium, but the effect was significantly attenuated when donors received S-NO-HSA (1 ± 0.27 vs. 0.33 ± 0.31; p = 0.0187; 12 h-S-NO-HSA-hHTX vs. 12 h-control-hHTX; normalized expression to U6 snRNA). Conclusion: Donor pre-treatment with S-NO-HSA lead to reduced fibrosis and preservation of myocardial miR-126-3p and GATA2 levels in murine cardiac isografts 60 days after transplantation.

Hepatic Energy Metabolism Underlying Differential Lipidomic Responses to High-Carbohydrate and High-Fat Diets in Male Wistar Rats.

BACKGROUND: Low-carbohydrate diets are suggested to exert metabolic benefits by reducing circulating triacylglycerol (TG) concentrations, possibly by enhancing mitochondrial activity. OBJECTIVE: We aimed to elucidate mechanisms by which dietary carbohydrate and fat differentially affect hepatic and circulating TG, and how these mechanisms relate to fatty acid composition. METHODS: Six-week-old, ∼300 g male Wistar rats were fed a high-carbohydrate, low-fat [HC; 61.3% of energy (E%) carbohydrate] or a low-carbohydrate, high-fat (HF; 63.5 E% fat) diet for 4 wk. Parameters of lipid metabolism and mitochondrial function were measured in plasma and liver, with fatty acid composition (GC), high-energy phosphates (HPLC), carnitine metabolites (HPLC-MS/MS), and hepatic gene expression (qPCR) as main outcomes. RESULTS: In HC-fed rats, plasma TG was double and hepatic TG 27% of that in HF-fed rats. The proportion of oleic acid (18:1n-9) was 60% higher after HF vs. HC feeding while the proportion of palmitoleic acid (16:1n-7) and vaccenic acid (18:1n-7), and estimated activities of stearoyl-CoA desaturase, SCD-16 (16:1n-7/16:0), and de novo lipogenesis (16:0/18:2n-6) were 1.5-7.5-fold in HC vs. HF-fed rats. Accordingly, hepatic expression of fatty acid synthase (Fasn) and acetyl-CoA carboxylase (Acaca/Acc) was strongly upregulated after HC feeding, accompanied with 8-fold higher FAS activity and doubled ACC activity. There were no differences in expression of liver-specific biomarkers of mitochondrial biogenesis and activity (Cytc, Tfam, Cpt1, Cpt2, Ucp2, Hmgcs2); concentrations of ATP, AMP, and energy charge; plasma carnitine/acylcarnitine metabolites; or peroxisomal fatty acid oxidation. CONCLUSIONS: In male Wistar rats, dietary carbohydrate was converted into specific fatty acids via hepatic lipogenesis, contributing to higher plasma TG and total fatty acids compared with high-fat feeding. In contrast, the high-fat, low-carbohydrate feeding increased hepatic fatty acid content, without affecting hepatic mitochondrial fatty acid oxidation.

Lysophosphatidic Acid Induces Aerobic Glycolysis, Lipogenesis, and Increased Amino Acid Uptake in BV-2 Microglia.

Lysophosphatidic acid (LPA) species are a family of bioactive lipids that transmit signals via six cognate G protein-coupled receptors, which are required for brain development and function of the nervous system. LPA affects the function of all cell types in the brain and can display beneficial or detrimental effects on microglia function. During earlier studies we reported that LPA treatment of microglia induces polarization towards a neurotoxic phenotype. In the present study we investigated whether these alterations are accompanied by the induction of a specific immunometabolic phenotype in LPA-treated BV-2 microglia. In response to LPA (1 µM) we observed slightly decreased mitochondrial respiration, increased lactate secretion and reduced ATP/ADP ratios indicating a switch towards aerobic glycolysis. Pathway analyses demonstrated induction of the Akt-mTOR-Hif1α axis under normoxic conditions. LPA treatment resulted in dephosphorylation of AMP-activated kinase, de-repression of acetyl-CoA-carboxylase and increased fatty acid content in the phospholipid and triacylglycerol fraction of BV-2 microglia lipid extracts, indicating de novo lipogenesis. LPA led to increased intracellular amino acid content at one or more time points. Finally, we observed LPA-dependent generation of reactive oxygen species (ROS), phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2), upregulated protein expression of the Nrf2 target regulatory subunit of glutamate-cysteine ligase and increased glutathione synthesis. Our observations suggest that LPA, as a bioactive lipid, induces subtle alterations of the immunometabolic program in BV-2 microglia.

Empagliflozin protects heart from inflammation and energy depletion via AMPK activation.

AIMS: Sodium-glucose co-transporter 2 (SGLT2) were originally developed as kidney-targeting anti-diabetic drugs. However, due to their beneficial cardiac off-target effects (as SGLT2 is not expressed in the heart), these antagonists currently receive intense clinical interest in the context of heart failure (HF) in patients with or without diabetes mellitus (DM). Since the mechanisms by which these beneficial effects are mediated are still unclear yet, inflammation that is present in DM and HF has been proposed as a potential pharmacological intervention strategy. Therefore, we tested the hypothesis that the SGLT2 inhibitor, empagliflozin, displays anti-inflammatory potential along with its glucose-lowering property. METHODS AND RESULTS: Lipopolysaccharide (LPS) was used to induce inflammation in vitro and in vivo. In cardiomyocytes and macrophages empagliflozin attenuated LPS-induced TNFα and iNOS expression. Analysis of intracellular signalling pathways suggested that empagliflozin activates AMP kinase (AMPK) in both cell types with or without LPS-treatment. Moreover, the SGLT2 inhibitor increased the expression of anti-inflammatory M2 marker proteins in LPS-treated macrophages. Additionally, empagliflozin-mediated AMPK activation prevented LPS-induced ATP/ADP depletion. In vivo administration of LPS in mice impaired cardiac contractility and aortic endothelial relaxation in response to acetylcholine, whereby co-administration of empagliflozin preserved cardiovascular function. These findings were accompanied by improved cardiac AMPK phosphorylation and ATP/ADP, reduced cardiac iNOS, plasma TNFα and creatine kinase MB levels. CONCLUSION: Our data identify a novel cardio protective mechanism of SGLT2 inhibitor, empagliflozin, suggesting that AMPK activation-mediated energy repletion and reduced inflammation contribute to the observed cardiovascular benefits of the drug in HF.

Increased hepatic mitochondrial FA oxidation reduces plasma and liver TG levels and is associated with regulation of UCPs and APOC-III in rats.

Hepatic mitochondrial function, APOC-III, and LPL are potential targets for triglyceride (TG)-lowering drugs. After 3 weeks of dietary treatment with the compound 2-(tridec-12-yn-1-ylthio)acetic acid (1-triple TTA), the hepatic mitochondrial FA oxidation increased more than 5-fold in male Wistar rats. Gene expression analysis in liver showed significant downregulation of APOC-III and upregulation of LPL and the VLDL receptor. This led to lower hepatic (53%) and plasma (73%) TG levels. Concomitantly, liver-specific biomarkers related to mitochondrial biogenesis and function (mitochondrial DNA, citrate synthase activity, and cytochrome c and TFAM gene expression) were elevated. Interestingly, 1-triple TTA lowered plasma acetylcarnitine levels, whereas the concentration of ß-hydroxybutyrate was increased. The hepatic energy state was reduced in 1-triple TTA-treated rats, as reflected by increased AMP/ATP and decreased ATP/ADP ratios, whereas the energy state remained unchanged in muscle and heart. The 1-triple TTA administration induced gene expression of uncoupling protein (UCP)2 and UCP3 in liver. In conclusion, the 1-triple TTA-mediated clearance of blood TG may result from lowered APOC-III production, increased hepatic LPL gene expression, mitochondrial FA oxidation, and (re)uptake of VLDL facilitating drainage of FAs to the liver for ß-oxidation and production of ketone bodies as extrahepatic fuel. The possibility that UCP2 and UCP3 mediate a moderate degree of mitochondrial uncoupling should be considered.

Blood transfusions may impair endothelium-dependent vasodilatation during coronary artery bypass surgery.

OBJECTIVE: The hemolytic product free-hemoglobin (fHb) reduces nitric oxide (NO) bioavailability. The present study aims to establish whether administration of different blood transfusions result in increased circulating fHb levels and NO consumption with effects on arterial NO-dependent blood flow in patients undergoing CABG surgery. METHODS: Ninety-five consecutive patients undergoing elective CABG surgery were prospectively divided in four groups based on blood transfusion requirements during surgery: stored blood cells (SBC, n. 21), intraoperative autologous salvaged blood (ASB, n. 25), SBC and ASB (n.22), no transfusion (control, n. 27). Blood samples were collected before and after intervention to analyse plasma levels of fHb and NO consumption. Endothelium-dependent relaxation was assessed in left internal mammary artery (LIMA) rings harvested before chest closure. Peripheral artery tonometry was assessed after intervention. RESULTS: Transfusions with SBC increased plasma fHb (p<0.05). Transfusions of ASB resulted in higher plasma fHb compared to SBC (p<0.01). fHb concentrations directly correlated with NO consumption (r=0.65, p<0.001). Maximal endothelium-dependent relaxation in LIMA was significantly attenuated in SBC and ASB patients compared to control (15.2±3.1% vs 21.1±2.5% vs 43±5.0% respectively; p<0.01). Significant correlations were identified between the aortic pressure wave velocity, plasma fHb concentration and NO consumption (p<0.01). CONCLUSIONS: Intraoperative blood transfusions and particularly autologous salvaged blood impair endothelium-dependent relaxation through NO scavenging by fHb. These findings obtained in vitro and in vivo provide new insights into the adverse relation between blood transfusions and patient outcome.

Real-time visualization of distinct nitric oxide generation of nitric oxide synthase isoforms in single cells.

The members of the nitric oxide synthase (NOS) family, eNOS, nNOS and iNOS, are well-characterized enzymes. However, due to the lack of suitable direct NO sensors, little is known about the kinetic properties of cellular NO generation by the different nitric oxide synthase isoenzymes. Very recently, we developed a novel class of fluorescent protein-based NO-probes, the geNOps, which allow real-time measurement of cellular NO generation and fluctuation. By applying these genetic NO biosensors to nNOS-, eNOS- and iNOS-expressing HEK293 cells we were able to characterize the respective NO dynamics in single cells that exhibited identical Ca2+ signaling as comparable activator of nNOS and eNOS. Our data demonstrate that upon Ca2+ mobilization nNOS-derived NO signals occur instantly and strictly follow the Ca2+ elevation while NO release by eNOS occurs gradually and sustained. To detect high NO levels in cells expressing iNOS, a new ratiometric probe based on two fluorescent proteins was developed. This novel geNOp variant allows the measurement of the high NO levels in cells expressing iNOS. Moreover, we used this probe to study the L-arginine-dependency of NO generation by iNOS on the level of single cells. Our experiments highlight that the geNOps technology is suitable to detect obvious differences in the kinetics, amplitude and substrate-dependence of cellular NO signals-derived from all three nitric oxide synthase isoforms.

IP3-mediated STIM1 oligomerization requires intact mitochondrial Ca2+ uptake.

Mitochondria contribute to cell signaling by controlling store-operated Ca(2+) entry (SOCE). SOCE is activated by Ca(2+) release from the endoplasmic reticulum (ER), whereupon stromal interacting molecule 1 (STIM1) forms oligomers, redistributes to ER-plasma-membrane junctions and opens plasma membrane Ca(2+) channels. The mechanisms by which mitochondria interfere with the complex process of SOCE are insufficiently clarified. In this study, we used an shRNA approach to investigate the direct involvement of mitochondrial Ca(2+) buffering in SOCE. We demonstrate that knockdown of either of two proteins that are essential for mitochondrial Ca(2+) uptake, the mitochondrial calcium uniporter (MCU) or uncoupling protein 2 (UCP2), results in decelerated STIM1 oligomerization and impaired SOCE following cell stimulation with an inositol-1,4,5-trisphosphate (IP3)-generating agonist. Upon artificially augmented cytosolic Ca(2+) buffering or ER Ca(2+) depletion by sarcoplasmic or endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitors, STIM1 oligomerization did not rely on intact mitochondrial Ca(2+) uptake. However, MCU-dependent mitochondrial sequestration of Ca(2+) entering through the SOCE pathway was essential to prevent slow deactivation of SOCE. Our findings show a stimulus-specific contribution of mitochondrial Ca(2+) uptake to the SOCE machinery, likely through a role in shaping cytosolic Ca(2+) micro-domains.

Functional impairment of endothelial cells by the antimycotic amphotericin B.

We set out to determine the membrane potential (Vm) of the endothelial cell line EA.hy926 and its sensitivity to the antimycotic amphotericin B (AmB), a commonly used antifungal component in cell culture media. We measured the endothelial Vm under various experimental conditions by patch clamp technique and found that Vm of AmB-treated cells is (-12.1 ± 9.3) mV, while in AmB-untreated (control) cells it is (-57.1 ± 4.1) mV. In AmB-free extracellular solutions, Vm recovered toward control levels and this gain in Vm rapidly dissipated upon re-addition of AmB, demonstrating a rapid and reversible effect of AmB on endothelial Vm. The consequences of AmB dependent alterations in endothelial transmembrane potential were tested at the levels of Ca(2+) signaling, of nucleotide concentrations, and energy metabolism. In AmB-treated cells we found substantially reduced Ca(2+) entry (to about 60% of that in control cells) in response to histamine induced endoplasmic reticulum (ER) Ca(2+) depletion, and diminished the ATP-to-ADP ratio (by >30%). Our data demonstrate a marked and experimentally relevant dependence of basic functional parameters of cultured endothelial cells on the presence of the ionophoric antimycotic AmB. The profound and reversible effects of the widely used culture media component AmB need careful consideration when interpreting experimental data obtained under respective culture conditions.

I(f) blocking potency of ivabradine is preserved under elevated endotoxin levels in human atrial myocytes.

Lower heart rate is associated with better survival in patients with multiple organ dysfunction syndrome (MODS), a disease mostly caused by sepsis. The benefits of heart rate reduction by ivabradine during MODS are currently being investigated in the MODIfY clinical trial. Ivabradine is a selective inhibitor of the pacemaker current If and since If is impaired by lipopolysaccharide (LPS, endotoxin), a trigger of sepsis, we aimed to explore If blocking potency of ivabradine under elevated endotoxin levels in human atrial cardiomyocytes. Treatment of myocytes with S-LPS (containing the lipid A moiety, a core oligosaccharide and an O-polysaccharide chain) but not R595 (an O-chain lacking LPS-form) caused If inhibition under acute and chronic septic conditions. The specific interaction of S-LPS but not R595 to pacemaker channels HCN2 and HCN4 proves the necessity of O-chain for S-LPS-HCN interaction. The efficacy of ivabradine to block If was reduced under septic conditions, an observation that correlated with lower intracellular ivabradine concentrations in S-LPS- but not R595-treated cardiomyocytes. Computational analysis using a sinoatrial pacemaker cell model revealed that despite a reduction of If under septic conditions, ivabradine further decelerated pacemaking activity. This novel finding, i.e. If inhibition by ivabradine under elevated endotoxin levels in vitro, may provide a molecular understanding for the efficacy of this drug on heart rate reduction under septic conditions in vivo, e.g. the MODIfY clinical trial.

The urea decomposition product cyanate promotes endothelial dysfunction.

The dramatic cardiovascular mortality of patients with chronic kidney disease is attributable in a significant proportion to endothelial dysfunction. Cyanate, a reactive species in equilibrium with urea, is formed in excess in chronic kidney disease. Cyanate is thought to have a causal role in promoting cardiovascular disease, but the underlying mechanisms remain unclear. Immunohistochemical analysis performed in the present study revealed that carbamylated epitopes associate mainly with endothelial cells in human atherosclerotic lesions. Cyanate treatment of human coronary artery endothelial cells reduced expression of endothelial nitric oxide synthase, and increased tissue factor and plasminogen activator inhibitor-1 expression. In mice, administration of cyanate, promoting protein carbamylation at levels observed in uremic patients, attenuated arterial vasorelaxation of aortic rings in response to acetylcholine without affecting the sodium nitroprusside-induced relaxation. Total endothelial nitric oxide synthase and nitric oxide production were significantly reduced in aortic tissue of cyanate-treated mice. This coincided with a marked increase of tissue factor and plasminogen activator inhibitor-1 protein levels in aortas of cyanate-treated mice. Thus, cyanate compromises endothelial functionality in vitro and in vivo. This may contribute to the dramatic cardiovascular risk of patients suffering from chronic kidney disease.

Inhibition of autophagy rescues palmitic acid-induced necroptosis of endothelial cells.

Accumulation of palmitic acid (PA) in cells from nonadipose tissues is known to induce lipotoxicity resulting in cellular dysfunction and death. The exact molecular pathways of PA-induced cell death are still mysterious. Here, we show that PA triggers autophagy, which did not counteract but in contrast promoted endothelial cell death. The PA-induced cell death was predominantly necrotic as indicated by annexin V and propidium iodide (PI) staining, absence of caspase activity, low levels of DNA hypoploidy, and an early ATP depletion. In addition PA induced a strong elevation of mRNA levels of ubiquitin carboxyl-terminal hydrolase (CYLD), a known mediator of necroptosis. Moreover, siRNA-mediated knockdown of CYLD significantly antagonized PA-induced necrosis of endothelial cells. In contrast, inhibition and knockdown of receptor interacting protein kinase 1 (RIPK1) had no effect on PA-induced necrosis, indicating the induction of a CYLD-dependent but RIPK1-independent cell death pathway. PA was recognized as a strong and early inducer of autophagy. The inhibition of autophagy by both pharmacological inhibitors and genetic knockdown of the autophagy-specific genes, vacuolar protein sorting 34 (VPS34), and autophagy-related protein 7 (ATG7), could rescue the PA-induced death of endothelial cells. Moreover, the initiation of autophagy and cell death by PA was reduced in endothelial cells loaded with the Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-(acetoxymethyl) ester (BAPTA-AM), indicating that Ca(2+) triggers the fatal signaling of PA. In summary, we introduce an unexpected mechanism of lipotoxicity in endothelial cells and provide several novel strategies to counteract the lipotoxic signaling of PA.

Energy status of pig donor organs after ischemia is independent of donor type.

BACKGROUND: Literature is controversial whether organs from living donors have a better graft function than brain dead (BD) and non-heart-beating donor organs. Success of transplantation has been correlated with high-energy phosphate (HEP) contents of the graft. METHODS: HEP contents in heart, liver, kidney, and pancreas from living, BD, and donation after cardiac death in a pig model (n=6 per donor type) were evaluated systematically. BD was induced under general anesthesia by inflating a balloon in the epidural space. Ten hours after confirmation, organs were retrieved. Cardiac arrest was induced by 9V direct current. After 10min of ventricular fibrillation without cardiac output, mechanical and medical reanimation was performed for 30min before organ retrieval. In living donors, organs were explanted immediately. Freeze-clamped biopsies were taken before perfusion with Celsior solution (heart) or University of Wisconsin solution (abdominal organs) in BD and living donors or with Histidine-Tryptophan-Ketoglutaric solution (all organs) in non-heart-beating donors, after perfusion, and after cold ischemia (4h for heart, 6h for liver and pancreas, and 12h for kidney). HEPs (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, and phosphocreatine), xanthine, and hypoxanthine were measured by high-performance liquid chromatography. Energy charge and adenosine triphosphate-to-adenosine diphosphate ratio were calculated. RESULTS: After ischemia, organs from different donor types showed no difference in energy status. In all organs, a decrease of HEP and an increase in hypoxanthine contents were observed during perfusion and ischemia, irrespective of the donor type. CONCLUSION: Organs from BD or non-heart-beating donors do not differ from living donor organs in their energy status after average tolerable ischemia.

Role of calcium desensitization in the treatment of myocardial dysfunction after deep hypothermic circulatory arrest.

INTRODUCTION: Rewarming from deep hypothermic circulatory arrest (DHCA) produces calcium desensitization by troponin I (cTnI) phosphorylation which results in myocardial dysfunction. This study investigated the acute overall hemodynamic and metabolic effects of epinephrine and levosimendan, a calcium sensitizer, on myocardial function after rewarming from DHCA. METHODS: Forty male Wistar rats (400 to 500 g) underwent cardiopulmonary bypass (CPB) through central cannulation and were cooled to a core temperature of 13°C to 15°C within 30 minutes. After DHCA (20 minutes) and CPB-assisted rewarming (60 minutes) rats were randomly assigned to 60 minute intravenous infusion with levosimendan (0.2 µg/kg/min; n = 15), epinephrine (0.1 µg/kg/min; n = 15) or saline (control; n = 10). Systolic and diastolic functions were evaluated at different preloads with a conductance catheter. RESULTS: The slope of left ventricular end-systolic pressure volume relationship (Ees) and preload recruitable stroke work (PRSW) recovered significantly better with levosimendan compared to epinephrine (Ees: 85 ± 9% vs 51 ± 11%, P<0.003 and PRSW: 78 ± 5% vs 48 ± 8%, P<0.005; baseline: 100%). Levosimendan but not epinephrine reduced left ventricular stiffness shown by the end-diastolic pressure-volume relationship and improved ventricular relaxation (Tau). Levosimendan preserved ATP myocardial content as well as energy charge and reduced plasma lactate concentrations. In normothermia experiments epinephrine in contrast to Levosimendan increased cTnI phosphorylation 3.5-fold. After rewarming from DHCA, cTnI phosphorylation increased 4.5-fold in the saline and epinephrine group compared to normothermia but remained unchanged with levosimendan. CONCLUSIONS: Levosimendan due to prevention of calcium desensitization by cTnI phosphorylation is more effective than epinephrine for treatment of myocardial dysfunction after rewarming from DHCA.

Normothermic blood polarizing versus depolarizing cardioplegia in a porcine model of cardiopulmonary bypass.

OBJECTIVES: We have previously demonstrated beneficial cardiac protection with hypothermic polarizing cardioplegia compared to a hyperkalemic depolarizing cardioplegia. In this study, a porcine model of cardiopulmonary bypass was used to compare the protective effects of normothermic blood-based polarizing and depolarizing cardioplegia during cardiac arrest. METHODS: Thirteen pigs were randomized to receive either normothermic polarizing (n = 8) or depolarizing (n = 5) blood-based cardioplegia. After initiation of cardiopulmonary bypass, normothermic arrest (34°C, 60 min) was followed by 60 min of on-pump and 90 min of off-pump reperfusion. Primary outcome was myocardial injury measured as arterial myocardial creatine kinase concentration. Secondary outcome was haemodynamic function and the energy state of the hearts. RESULTS: During reperfusion, release of myocardial creatine kinase was comparable between groups (P = 0.36). In addition, most haemodynamic parameters showed comparable results between groups, but stroke volume (P = 0.03) was significantly lower in the polarizing group. Adenosine triphosphate levels were significantly (18.41 ± 3.86 vs 22.97 ± 2.73 nmol/mg; P = 0.03) lower in polarizing hearts, and the requirement for noradrenaline administration (P = 0.002) and temporary pacing (6 vs 0; P = 0.02) during reperfusion were significantly higher in polarizing hearts. CONCLUSIONS: Under normothermic conditions, polarizing blood cardioplegia was associated with similar myocardial injury to depolarizing blood cardioplegia. Reduced haemodynamic and metabolic outcome and a higher need for temporary pacing with polarized arrest may be associated with the blood-based dilution of this solution.

Evidence for a Physiological Role of T-Type Ca Channels in Ventricular Cardiomyocytes of Adult Mice.

T-type Ca channels are strongly expressed and important in the developing heart. In the adult heart, these channels play a significant role in pacemaker tissues, but there is uncertainty about their presence and physiological relevance in the working myocardium. Here, we show that the T-type Ca channel isoforms Cav3.1 and Cav3.2 are expressed at a protein level in ventricular cardiomyocytes from healthy adult C57/BL6 mice. Myocytes isolated from adult wild-type and Cav3.2 KO mice showed considerable whole cell T-type Ca currents under beta-adrenergic stimulation with isoprenaline. We further show that the detectability of basal T-type Ca currents in murine wild-type cardiomyocytes depends on the applied experimental conditions. Together, these findings reveal the presence of functional T-type Ca channels in the membrane of ventricular myocytes. In addition, electrically evoked Ca release from the sarcoplasmic reticulum was significantly impaired in Cav3.2 KO compared to wild-type cardiomyocytes. Our work implies a physiological role of T-type Ca channels in the healthy adult murine ventricular working myocardium.

The Antiplatelet Action of S-Nitroso Human Serum Albumin in Whole Blood.

Nitric oxide donors (NO-donors) have been shown to have therapeutic potential (e.g., ischemia/reperfusion injury). However, due to their release rate/antiplatelet properties, they may cause bleeding in patients. We therefore studied the antiplatelet effects of the two different NO-donors, i.e., S-NO-Human Serum Albumin (S-NO-HSA) and Diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate) in whole blood (WB) samples. WB samples were spiked with S-NO-HSA or DEA-NONOate (100 µmol/L or 200 µmol/L), and the NO release rate (nitrite/nitrate levels via HPLC) and antiplatelet efficacy (impedance aggregometry, platelet function analyzer, Cone-and-platelet analyzer, thrombelastometry) were assessed. S-NO-HSA had a significantly lower NO release compared to equimolar concentrations of DEA-NONOate. Virtually no antiplatelet action of S-NO-HSA was observed in WB samples, whereas DEA-NONOate significantly attenuated platelet function in WB. Impedance aggregometry measurements revealed that Amplitudes (slope: -0.04022 ± 0.01045 ohm/µmol/L, p = 0.008) and Lag times (slope: 0.6389 ± 0.2075 s/µmol/L, p = 0.0051) were dose-dependently decreased and prolonged by DEA-NONOate. Closure times (Cone-and-platelet analyzer) were dose-dependently prolonged (slope: 0.3738 ± 0.1403 s/µmol/L, p = 0.0174 with collagen/ADP coating; slope: -0.5340 ± 0.1473 s/µmol/L, p = 0.0019 with collagen/epinephrine coating) by DEA-NONOate. These results in WB further support the pharmacological potential of S-NO-HSA as an NO-donor due to its ability to presumably prevent bleeding events even at high concentrations up to 200 µmol/L.

Remote Ischemic Perconditioning Ameliorates Myocardial Ischemia and Reperfusion-Induced Coronary Endothelial Dysfunction and Aortic Stiffness in Rats.

BACKGROUND: Vascular stiffness and endothelial dysfunction are accelerated by acute myocardial infarction (AMI) and subsequently increase the risk for recurrent coronary events. AIM: To explore whether remote ischemic perconditioning (RIPerc) protects against coronary and aorta endothelial dysfunction as well as aortic stiffness following AMI. METHODS: Male OFA-1 rats were subjected to 30 min of occlusion of the left anterior descending artery (LAD) followed by reperfusion either 3 or 28 days with or without RIPerc. Three groups: (1) sham operated (Sham, without LAD occlusion); (2) myocardial ischemia and reperfusion (MIR) and (3) MIR + RIPerc group with 3 cycles of 5 minutes of IR on hindlimb performed during myocardial ischemia were used. Assessment of vascular reactivity in isolated septal coronary arteries (non-occluded) and aortic rings as well as aortic stiffness was assessed by wire myography either 3 or 28 days after AMI, respectively. Markers of pro-inflammatory cytokines, adhesion molecules were assessed by RT-qPCR and ELISA. RESULTS: MIR promotes impaired endothelial-dependent relaxation in septal coronary artery segments, increased aortic stiffness and adverse left ventricular remodeling. These changes were markedly attenuated in rats treated with RIPerc and associated with a significant decline in P-selectin, IL-6 and TNF-α expression either in infarcted or non-infarcted myocardial tissue samples. CONCLUSIONS: Our study for the first time demonstrated that RIPerc alleviates MIR-induced coronary artery endothelial dysfunction in non-occluded artery segments and attenuates aortic stiffness in rats. The vascular protective effects of RIPerc are associated with ameliorated inflammation and might therefore be caused by reduced inflammatory signaling.

S-nitroso human serum albumin as a nitric oxide donor in drug-eluting vascular grafts: Biofunctionality and preclinical evaluation.

Currently available synthetic small diameter vascular grafts reveal low patency rates due to thrombosis and intimal hyperplasia. Biofunctionalized grafts releasing nitric oxide (NO) in situ may overcome these limitations. In this study, a drug-eluting vascular graft was designed by blending polycaprolactone (PCL) with S-nitroso-human-serum-albumin (S-NO-HSA), a nitric oxide donor with prolonged half-life. PCL-S-NO-HSA grafts and patches were fabricated via electrospinning. The fabrication process was optimized. Patches were characterized in vitro for their morphology, drug release, biomechanics, inflammatory effects, cell proliferation, and expression of adhesion molecules. The selected optimized formulation (8%PCL-S-NO-HSA) had superior mechanical/morphological properties with high protein content revealing extended NO release (for 28 days). 8%PCL-S-NO-HSA patches significantly promoted endothelial cell proliferation while limiting smooth muscle cell proliferation. Expression of adhesion molecules (ICAM-1, VCAM-1) and pro-inflammatory macrophage/cytokine markers (CD80, IL-1α, TNF-α) was significantly reduced. 8%PCL-S-NO-HSA patches had superior immunomodulatory properties by up-regulating anti-inflammatory cytokines (IL-10) and M2 macrophage marker (CD163) at final time points. Grafts were further evaluated in a small rodent model as aortic implants up to 12 weeks. Grafts were assessed by magnetic resonance imaging angiography (MRI) in vivo and after retrieval by histology. All grafts remained 100 % patent with no signs of thrombosis or calcification. 8%PCL-S-NO-HSA vascular grafts supported rapid endothelialization, whereas smooth muscle cell proliferation was hampered in earlier phases. This study indicates that 8%PCL-S-NO-HSA grafts effectively support long-term in situ release of bioactive NO. The beneficial effects observed can be promising features for long-term success of small diameter vascular grafts. STATEMENT OF SIGNIFICANCE: Despite extensive research in the field of small diameter vascular graft replacement, there is still no appropriate substitute to autografts yet. Various limitations are associated with currently available synthetic vascular grafts such as thrombogenicity and intimal hyperplasia. Therefore, developing new generations of such conduits has become a major focus of research. One of the most significant signaling molecules that are involved in homeostasis of the vascular system is nitric oxide. The new designed nitric-oxide eluting vascular grafts described in this study induce rapid surface endothelialization and late migration of SMCs into the graft wall. These beneficial effects have potential to improve current limitations of small diameter vascular grafts.

Lysophosphatidic acid receptor activation affects the C13NJ microglia cell line proteome leading to alterations in glycolysis, motility, and cytoskeletal architecture.

Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to injury and microglia migration towards and homing at damaged tissue plays a key role in CNS regeneration. Lysophosphatidic acid (LPA) is involved in signaling events evoking microglia responses through cognate G protein-coupled receptors. Here we show that human immortalized C13NJ microglia express LPA receptor subtypes LPA(1), LPA(2), and LPA(3) on mRNA and protein level. LPA activation of C13NJ cells induced Rho and extracellular signal-regulated kinase activation and enhanced cellular ATP production. In addition, LPA induced process retraction, cell spreading, led to pronounced changes of the actin cytoskeleton and reduced cell motility, which could be reversed by inhibition of Rho activity. To get an indication about LPA-induced global alterations in protein expression patterns a 2-D DIGE/LC-ESI-MS proteomic approach was applied. On the proteome level the most prominent changes in response to LPA were observed for glycolytic enzymes and proteins regulating cell motility and/or cytoskeletal dynamics. The present findings suggest that naturally occurring LPA is a potent regulator of microglia biology. This might be of particular relevance in the pathophysiological context of neurodegenerative disorders where LPA concentrations can be significantly elevated in the CNS.