First-in-man trial of ß3-adrenoceptor agonist treatment in chronic heart failure - impact on diastolic function.

Diastolic dysfunction (DD) in heart failure (HF) is associated with increased myocardial cytosolic calcium, and calcium-efflux via the sodium-calcium-exchanger depends on the sodium gradient. Beta-3-adrenoceptor (ß3-AR) agonists lower cytosolic sodium and have reversed organ congestion. Accordingly, ß3-AR agonists might improve diastolic function, which we aimed to assess. In a first-in-man, randomized, double-blinded trial, we assigned 70 patients with HF with reduced ejection fraction (HFrEF), NYHA II-III, and LVEF<40% to receive the ß3-AR agonist mirabegron (300 mg/day) or placebo for six months, in addition to recommended HF therapy. We performed echocardiography and cardiac computed tomography (CCT) and measured N-terminal pro-brain natriuretic peptide (NT-proBNP) at baseline and follow-up. DD was graded per multiple renowned algorithms. Baseline and follow-up data were available in 57 patients (59±11 years, 88% male, 49% ischemic heart disease). No clinically significant changes in diastolic measurements were found within or between groups by echocardiography (E/e' placebo: 13±7 to 13±5, p=0.21 vs mirabegron: 12±6 to 13±8, p=0.74, between group follow-up difference 0.2 [95% CI -3 to 4], p=0.89), or CCT (left atrial volume index: between group follow-up difference 9 ml/m2 [95% CI -3 to 19], p=0.15). DD gradings did not change within or between groups following two algorithms (p=0.72, p=0.75). NT-proBNP remained unchanged in both groups (p=0.74, p=0.64). In patients with HFrEF, no changes were identified in diastolic measurements, gradings or biomarker after ß3-AR stimulation compared to placebo. The findings add to previous literature questioning the role of impaired Na+-Ca2+ mediated calcium-export as a major culprit in DD. NCT01876433.

Hemodynamic Effects of Cyclic Guanosine Monophosphate-Dependent Signaling Through ß3 Adrenoceptor Stimulation in Patients With Advanced Heart Failure: A Randomized Invasive Clinical Trial.

BACKGROUND: ß3-AR (ß3-adrenergic receptor) stimulation improved systolic function in a sheep model of systolic heart failure (heart failure with reduced ejection fraction [HFrEF]). Exploratory findings in patients with New York Heart Association functional class II HFrEF treated with the ß3-AR-agonist mirabegron supported this observation. Here, we measured the hemodynamic response to mirabegron in patients with severe HFrEF. METHODS: In this randomized, double-blind, placebo-controlled trial we assigned patients with New York Heart Association functional class III-IV HFrEF, left ventricular ejection fraction <35% and increased NT-proBNP (N-terminal pro-B-type natriuretic peptide) levels to receive mirabegron (300 mg daily) or placebo orally for a week, as add on to recommended HF therapy. Invasive hemodynamic measurements during rest and submaximal exercise at baseline, 3 hours after first study dose and repeated after 1 week's treatment were obtained. Predefined parameters for analyses were changes in cardiac- and stroke volume index, pulmonary and systemic vascular resistance, heart rate, and blood pressure. RESULTS: We randomized 22 patients (age 66±11 years, 18 men, 16, New York Heart Association functional class III), left ventricular ejection fraction 20±7%, median NT-proBNP 1953 ng/L. No significant changes were seen after 3 hours, but after 1 week, there was a significantly larger increase in cardiac index in the mirabegron group compared with the placebo group (mean difference, 0.41 [CI, 0.07-0.75] L/min/BSA; P=0.039). Pulmonary vascular resistance decreased significantly more in the mirabegron group compared with the placebo group (-1.6 [CI, -0.4 to -2.8] Wood units; P=0.02). No significant differences were seen during exercise. There were no differences in changes in heart rate, systemic vascular resistance, blood pressure, or renal function between groups. Mirabegron was well-tolerated. CONCLUSIONS: Oral treatment with the ß3-AR-agonist mirabegron for 1 week increased cardiac index and decreased pulmonary vascular resistance in patients with moderate to severe HFrEF. Mirabegron may be useful in patients with worsening or terminal HF. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: 2016-002367-34.

Displacement of Native FXYD Protein From Na+/K+-ATPase With Novel FXYD Peptide Derivatives: Effects on Doxorubicin Cytotoxicity.

The seven mammalian FXYD proteins associate closely with α/ß heterodimers of Na+/K+-ATPase. Most of them protect the ß1 subunit against glutathionylation, an oxidative modification that destabilizes the heterodimer and inhibits Na+/K+-ATPase activity. A specific cysteine (Cys) residue of FXYD proteins is critical for such protection. One of the FXYD proteins, FXYD3, confers treatment resistance when overexpressed in cancer cells. We developed two FXYD3 peptide derivatives. FXYD3-pep CKCK retained the Cys residue that can undergo glutathionylation and that is critical for protecting the ß1 subunit against glutathionylation. FXYD3-pep SKSK had all Cys residues mutated to Serine (Ser). The chemotherapeutic doxorubicin induces oxidative stress, and suppression of FXYD3 with siRNA in pancreatic- and breast cancer cells that strongly express FXYD3 increased doxorubicin-induced cytotoxicity. Exposing cells to FXYD3-pep SKSK decreased co-immunoprecipitation of FXYD3 with the α1 Na+/K+-ATPase subunit. FXYD3-pep SKSK reproduced the increase in doxorubicin-induced cytotoxicity seen after FXYD3 siRNA transfection in pancreatic- and breast cancer cells that overexpressed FXYD3, while FXYD3-pep CKCK boosted the native protein's protection against doxorubicin. Neither peptide affected doxorubicin's cytotoxicity on cells with no or low FXYD3 expression. Fluorescently labeled FXYD3-pep SKSK was detected in a perinuclear distribution in the cells overexpressing FXYD3, and plasmalemmal Na+/K+-ATPase turnover could not be implicated in the increased sensitivity to doxorubicin that FXYD3-pep SKSK caused. FXYD peptide derivatives allow rapid elimination or amplification of native FXYD protein function. Here, their effects implicate the Cys residue that is critical for countering ß1 subunit glutathionylation in the augmentation of cytotoxicity with siRNA-induced downregulation of FXYD3.

S-glutathionylation of the Na+-K+ Pump: A Novel Redox Mechanism in Preeclampsia.

CONTEXT: Reduced Na+-K+ pump activity is widely reported in preeclampsia and may be caused by a reversible oxidative modification that is a novel pathological feature of preeclampsia. OBJECTIVE: This work aims to determine whether ß 1 subunit (GSS-ß 1) protein glutathionylation of the Na+-K + pump occurs in preeclampsia. METHODS: The GSS-ß1 of the Na+-K+ pump and its subunit expression in human placentas were compared between women with healthy pregnancies and women with preeclampsia. Human placental samples of pregnant women with preeclampsia (n = 11, mean gestational age 36.5 weeks) were used to examine the GSS-ß 1 of the Na+-K+ pump, compared to healthy pregnancies (n = 11, mean gestational age 39 weeks).The potential pathogenetic role of GSS-ß 1-mediated Na+-K+ pump dysfunction in preeclampsia was investigated. RESULTS: Protein expression of the ß 1 subunit was unchanged in placentas from women with preeclampsia vs those with normotensive pregnancies. Preeclamptic placentas had a significantly increased GSS-ß 1 of the Na+-K+ pump compared to those from healthy pregnancies, and this was linked to a decrease in α 1/ß 1 subunit coimmunoprecipitation. The cytosolic p47phox nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase subunit and its coimmunoprecipitation with the α 1 Na+-K+ pump subunit was increased in preeclamptic placentas, thus implicating NADPH oxidase-dependent pump inhibition. CONCLUSIONS: The high level of ß 1 pump subunit glutathionylation provides new insights into the mechanism of Na+-K+ pump dysfunction in preeclampsia.

Targeting Cardiac Myocyte Na+-K+ Pump Function With ß3 Adrenergic Agonist in Rabbit Model of Severe Congestive Heart Failure.

BACKGROUND: Abnormally high cytosolic Na+ concentrations in advanced heart failure impair myocardial contractility. Stimulation of the membrane Na+-K+ pump should lower Na+ concentrations, and the ß3 adrenoceptor (ß3 AR) mediates pump stimulation in myocytes. We examined if ß3 AR-selective agonists given in vivo increase myocyte Na+-K+ pump activity and reverse organ congestion in severe heart failure (HF). METHODS: Indices for HF were lung-, heart-, and liver: body weight ratios and ascites after circumflex coronary artery ligation in rabbits. Na+-K+ pump current, Ip, was measured in voltage-clamped myocytes from noninfarct myocardium. Rabbits were treated with the ß3 AR agonists CL316,243 or ASP9531, starting 2 weeks after coronary ligation. RESULTS: Coronary ligation caused ascites in most rabbits, significantly increased lung-, heart-, and liver: body weight ratios, and decreased Ip relative to that for 10 sham-operated rabbits. Treatment with CL316,243 for 3 days significantly reduced lung-, heart-, and liver: body weight ratios and prevalence of ascites in 8 rabbits with HF relative to indices for 13 untreated rabbits with HF. It also increased Ip significantly to levels of myocytes from sham-operated rabbits. Treatment with ASP9531 for 14 days significantly reduced indices of organ congestion in 6 rabbits with HF relative to indices of 6 untreated rabbits, and it eliminated ascites. ß3 AR agonists did not significantly change heart rates or blood pressures. CONCLUSIONS: Parallel ß3 AR agonists-induced reversal of Na+-K+ pump inhibition and indices of congestion suggest pump inhibition is a useful target for treatment with ß3 AR agonists in congestive HF.

The first-in-man randomized trial of a beta3 adrenoceptor agonist in chronic heart failure: the BEAT-HF trial.

AIMS: The third isotype of beta adrenergic receptors (ß3 ARs) has distinctly different effects on cardiomyocytes compared with ß1 and ß2 ARs. Stimulation of ß3 ARs may reduce cardiomyocyte Na+ overload and reduce oxidative stress in heart failure (HF). We examined if treatment with the ß3 AR agonist mirabegron increases LVEF in patients with HF. METHODS AND RESULTS: In a double-blind trial we randomly assigned 70 patients with NYHA class II-III HF and LVEF <40% at screening-echocardiography to receive mirabegron or placebo for 6 months as add-on to optimized standard therapy. The primary endpoint was an increase in LVEF after 6 months as measured by computed tomography (CT). Changes in LVEF after 6 months between treatment groups were not significantly different (0.4%, -3.5 to 3.8%, P = 0.82). In an exploratory analysis, based on an expectation that the pathophysiological substrate targeted with treatment is dependent on the baseline LVEF, patients with LVEF <40% by CT given mirabegron had a significant increase in LVEF while no increase was seen in patients given placebo. The changes were significantly different between groups (5.5%, 0.6-10.4%, P < 0.03). Additionally, there was interaction between baseline LVEF and change in LVEF in the entire group of patients treated with mirabegron (R2 = 0.40, ß = -0.63, P < 0.001), but not in the placebo group (R2 = 0.00, ß = -0.01, P = 0.95). Treatment was generally well tolerated. Three patients in each group had fatal or life-threatening events. CONCLUSIONS: The primary endpoint was not reached. Exploratory analysis indicated that ß3 AR stimulation by mirabegron increased LVEF in patients with severe HF. Treatment appeared safe. Additional studies in severe HF are needed. TRIAL REGISTRATION: NCT01876433.

Glutathionylation-Dependence of Na(+)-K(+)-Pump Currents Can Mimic Reduced Subsarcolemmal Na(+) Diffusion.

The existence of a subsarcolemmal space with restricted diffusion for Na(+) in cardiac myocytes has been inferred from a transient peak electrogenic Na(+)-K(+) pump current beyond steady state on reexposure of myocytes to K(+) after a period of exposure to K(+)-free extracellular solution. The transient peak current is attributed to enhanced electrogenic pumping of Na(+) that accumulated in the diffusion-restricted space during pump inhibition in K(+)-free extracellular solution. However, there are no known physical barriers that account for such restricted Na(+) diffusion, and we examined if changes of activity of the Na(+)-K(+) pump itself cause the transient peak current. Reexposure to K(+) reproduced a transient current beyond steady state in voltage-clamped ventricular myocytes as reported by others. Persistence of it when the Na(+) concentration in patch pipette solutions perfusing the intracellular compartment was high and elimination of it with K(+)-free pipette solution could not be reconciled with restricted subsarcolemmal Na(+) diffusion. The pattern of the transient current early after pump activation was dependent on transmembrane Na(+)- and K(+) concentration gradients suggesting the currents were related to the conformational poise imposed on the pump. We examined if the currents might be accounted for by changes in glutathionylation of the ß1 Na(+)-K(+) pump subunit, a reversible oxidative modification that inhibits the pump. Susceptibility of the ß1 subunit to glutathionylation depends on the conformational poise of the Na(+)-K(+) pump, and glutathionylation with the pump stabilized in conformations equivalent to those expected to be imposed on voltage-clamped myocytes supported this hypothesis. So did elimination of the transient K(+)-induced peak Na(+)-K(+) pump current when we included glutaredoxin 1 in patch pipette solutions to reverse glutathionylation. We conclude that transient K(+)-induced peak Na(+)-K(+) pump current reflects the effect of conformation-dependent ß1 pump subunit glutathionylation, not restricted subsarcolemmal diffusion of Na(+).

Silencing overexpression of FXYD3 protein in breast cancer cells amplifies effects of doxorubicin and γ-radiation on Na(+)/K(+)-ATPase and cell survival.

FXYD3, also known as mammary tumor protein 8, is overexpressed in several common cancers, including in many breast cancers. We examined if such overexpression might protect Na(+)/K(+)-ATPase and cancer cells against the high levels of oxidative stress characteristic of many tumors and often induced by cancer treatments. We measured FXYD3 expression, Na(+)/K(+)-ATPase activity and glutathionylation of the ß1 subunit of Na(+)/K(+)-ATPase, a reversible oxidative modification that inhibits the ATPase, in MCF-7 and MDA-MB-468 cells. Expression of FXYD3 was suppressed by transfection with FXYD3 siRNA. A colorimetric end-point assay was used to estimate cell viability. Apoptosis was estimated by caspase 3/7 (DEVDase) activation using a Caspase fluorogenic substrate kit. Expression of FXYD3 in MCF-7 breast cancer cells was ~eightfold and ~twofold higher than in non-cancer MCF-10A cells and MDA-MB-468 cancer cells, respectively. A ~50 % reduction in FXYD3 expression increased glutathionylation of the ß1 Na(+)/K(+)-ATPase subunit and reduced Na(+)/K(+)-ATPase activity by ~50 %, consistent with the role of FXYD3 to facilitate reversal of glutathionylation of the ß1 subunit of Na(+)/K(+)-ATPase and glutathionylation-induced inhibition of Na(+)/K(+)-ATPase. Treatment of MCF-7 and MDA-MB- 468 cells with doxorubicin or γ-radiation decreased cell viability and induced apoptosis. The treatments upregulated FXYD3 expression in MCF-7 but not in MDA-MB-468 cells and suppression of FXYD3 in MCF-7 but not in MDA-MB-468 cells amplified effects of treatments on Na(+)/K(+)-ATPase activity and treatment-induced cell death and apoptosis. Overexpression of FXYD3 may be a marker of resistance to cancer treatments and a potentially important therapeutic target.

Routine aspiration thrombectomy improves the diagnosis and management of embolic myocardial infarction.

OBJECTIVES: Assess the effect of aspiration thrombectomy on diagnosis and management of embolic acute myocardial infarction. BACKGROUND: Discrimination of embolic acute myocardial infarction from atherosclerotic plaque rupture/erosion prompts oral anticoagulation treatment of source of embolus, as well as avoiding unnecessary stenting and dual antiplatelet therapy. However, detection is difficult without aspiration. METHODS: We compared rates of diagnosis of embolic infarction for 2.5 years prior to (pre-RAT) and 2.5 years post routine aspiration thrombectomy (post-RAT). Baseline demographics, outcomes, and treatment strategies were also compared between the embolic infarction and atherosclerotic infarction. RESULTS: Diagnosed embolic infarction rose from 1.2% in the pre-RAT era to 2.8% in the post-RAT period (P < 0.05). In addition, more successful removal of thrombus by aspiration led to less stenting (20% vs. 55% P < 0.05) in the post-RAT period thus avoiding the hazards of "triple therapy." Embolic infarction was more frequently associated with atrial fibrillation (55% vs. 8%), had higher mortality (17% vs. 4%), and had higher rates of embolic stroke (13% vs. 0.3%) when compared with atherosclerotic MI (all P < 0.05). CONCLUSIONS: Routine aspiration thrombectomy more readily identifies embolic infarction allowing more specific therapy and avoidance of stenting and triple anticoagulant therapy.

Membrane accessibility of glutathione.

Regulation of the ion pumping activity of the Na+,K+-ATPase is crucial to the survival of animal cells. Recent evidence has suggested that the activity of the enzyme could be controlled by glutathionylation of cysteine residue 45 of the ß-subunit. Crystal structures so far available indicate that this cysteine is in a transmembrane domain of the protein. Here we have analysed via fluorescence and NMR spectroscopy as well as molecular dynamics simulations whether glutathione is able to penetrate into the interior of a lipid membrane. No evidence for any penetration of glutathione into the membrane was found. Therefore, the most likely mechanism whereby the cysteine residue could become glutathionylated is via a loosening of the α-ß subunit association, creating a hydrophilic passageway between them to allow access of glutathione to the cysteine residue. By such a mechanism, glutathionylation of the protein would be expected to anchor the modified cysteine residue in a hydrophilic environment, inhibiting further motion of the ß-subunit during the enzyme's catalytic cycle and suppressing enzymatic activity, as has been experimentally observed. The results obtained, therefore, suggest a possible structural mechanism of how the Na+,K+-ATPase could be regulated by glutathione.

Stimulation of the cardiac myocyte Na+-K+ pump due to reversal of its constitutive oxidative inhibition.

Protein kinase C can activate NADPH oxidase and induce glutathionylation of the ß1-Na(+)-K(+) pump subunit, inhibiting activity of the catalytic α-subunit. To examine if signaling of nitric oxide-induced soluble guanylyl cyclase (sGC)/cGMP/protein kinase G can cause Na(+)-K(+) pump stimulation by counteracting PKC/NADPH oxidase-dependent inhibition, cardiac myocytes were exposed to ANG II to activate NADPH oxidase and inhibit Na(+)-K(+) pump current (Ip). Coexposure to 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) to stimulate sGC prevented the decrease of Ip. Prevention of the decrease was abolished by inhibition of protein phosphatases (PP) 2A but not by inhibition of PP1, and it was reproduced by an activator of PP2A. Consistent with a reciprocal relationship between ß1-Na(+)-K(+) pump subunit glutathionylation and pump activity, YC-1 decreased ANG II-induced ß1-subunit glutathionylation. The decrease induced by YC-1 was abolished by a PP2A inhibitor. YC-1 decreased phosphorylation of the cytosolic p47(phox) NADPH oxidase subunit and its coimmunoprecipitation with the membranous p22(phox) subunit, and it decreased O2 (·-)-sensitive dihydroethidium fluorescence of myocytes. Addition of recombinant PP2A to myocyte lysate decreased phosphorylation of p47(phox) indicating the subunit could be a substrate for PP2A. The effects of YC-1 to decrease coimmunoprecipitation of p22(phox) and p47(phox) NADPH oxidase subunits and decrease ß1-Na(+)-K(+) pump subunit glutathionylation were reproduced by activation of nitric oxide-dependent receptor signaling. We conclude that sGC activation in cardiac myocytes causes a PP2A-dependent decrease in NADPH oxidase activity and a decrease in ß1 pump subunit glutathionylation. This could account for pump stimulation with neurohormonal oxidative stress expected in vivo.

ß3-Adrenoceptor activation relieves oxidative inhibition of the cardiac Na+-K+ pump in hyperglycemia induced by insulin receptor blockade.

Dysregulated nitric oxide (NO)- and superoxide (O2 (·-))-dependent signaling contributes to the pathobiology of diabetes-induced cardiovascular complications. We examined if stimulation of ß3-adrenergic receptors (ß3-ARs), coupled to endothelial NO synthase (eNOS) activation, relieves oxidative inhibition of eNOS and the Na(+)-K(+) pump induced by hyperglycemia. Hyperglycemia was established in male New Zealand White rabbits by infusion of the insulin receptor antagonist S961 for 7 days. Hyperglycemia increased tissue and blood indexes of oxidative stress. It induced glutathionylation of the Na(+)-K(+) pump ß1-subunit in cardiac myocytes, an oxidative modification causing pump inhibition, and reduced the electrogenic pump current in voltage-clamped myocytes. Hyperglycemia also increased glutathionylation of eNOS, which causes its uncoupling, and increased coimmunoprecipitation of cytosolic p47(phox) and membranous p22(phox) NADPH oxidase subunits, consistent with NADPH oxidase activation. Blocking translocation of p47(phox) to p22(phox) with the gp91ds-tat peptide in cardiac myocytes ex vivo abolished the hyperglycemia-induced increase in glutathionylation of the Na(+)-K(+) pump ß1-subunit and decrease in pump current. In vivo treatment with the ß3-AR agonist CL316243 for 3 days eliminated the increase in indexes of oxidative stress, decreased coimmunoprecipitation of p22(phox) with p47(phox), abolished the hyperglycemia-induced increase in glutathionylation of eNOS and the Na(+)-K(+) pump ß1-subunit, and abolished the decrease in pump current. CL316243 also increased coimmunoprecipitation of glutaredoxin-1 with the Na(+)-K(+) pump ß1-subunit, which may reflect facilitation of deglutathionylation. In vivo ß3-AR activation relieves oxidative inhibition of key cardiac myocyte proteins in hyperglycemia and may be effective in targeting the deleterious cardiac effects of diabetes.

Outcomes by day and night for patients bypassing the emergency department presenting with ST-segment elevation myocardial infarction identified with a pre-hospital electrocardiogram.

BACKGROUND: Pre-hospital ECG and emergency department (ED) bypass direct to the catheter laboratory may optimize reperfusion times for patients with ST-segment elevation myocardial infarction. Questions remain over feasibility and safety during off hours. AIMS: To determine if presenting time of day is associated with differences in in-hospital and 30-day mortality and key reperfusion times. METHODS/RESULTS: Seven hundred and twenty consecutive patients with STEMI triaged directly from the field to the catheter laboratory between June 2004-May 2013. Vital status was reported as of August 2013. The mean age was 65 ± 14 years, and 75.1% were male. Overall mortality (in-hospital/30 days) did not significantly differ for patients (3.4% in hours and 3.1% off hours; P = N/S). Symptom onset-to-arrival to the heart attack was non-significantly lower (100 minutes off hours (IQR 78-174) versus 110 minutes in hours (IQR 75-199), P = N/S). Call-to-balloon time was not significantly affected by the time of presentation: 150 min in hours (IQR 111-239) versus 154 minutes during off hours (IQR 115-225) P = N/S. Overall door-to-balloon time was 36 minutes (IQR 25-51), 34 minutes in hours (IQR 24-49) versus 40 minutes off hours (IQR 29-55) P = N/S. The overall false positive activation rate was only 13.1%, (in hours 12.2% vs. off hours 14.6%, respectively, P = N/S). CONCLUSIONS: In a unit with an established field triage system facilitating ED bypass, reperfusion times and mortality are not significantly influenced by whether the patient presents during standard working hours or outside of these hours.

Identification of electric-field-dependent steps in the Na(+),K(+)-pump cycle.

The charge-transporting activity of the Na(+),K(+)-ATPase depends on its surrounding electric field. To isolate which steps of the enzyme's reaction cycle involve charge movement, we have investigated the response of the voltage-sensitive fluorescent probe RH421 to interaction of the protein with BTEA (benzyltriethylammonium), which binds from the extracellular medium to the Na(+),K(+)-ATPase's transport sites in competition with Na(+) and K(+), but is not occluded within the protein. We find that only the occludable ions Na(+), K(+), Rb(+), and Cs(+) cause a drop in RH421 fluorescence. We conclude that RH421 detects intramembrane electric field strength changes arising from charge transport associated with conformational changes occluding the transported ions within the protein, not the electric fields of the bound ions themselves. This appears at first to conflict with electrophysiological studies suggesting extracellular Na(+) or K(+) binding in a high field access channel is a major electrogenic reaction of the Na(+),K(+)-ATPase. All results can be explained consistently if ion occlusion involves local deformations in the lipid membrane surrounding the protein occurring simultaneously with conformational changes necessary for ion occlusion. The most likely origin of the RH421 fluorescence response is a change in membrane dipole potential caused by membrane deformation.

Crystal structure and magnetism of Fe2(OH)[B2O4(OH)].

The structure and magnetism of Fe2(OH)[B2O4(OH)] are reported. Powder x-ray diffraction reveals a characteristic structure containing two crystallographically independent zigzag-ladder chains of magnetic Fe(2+) ions. Magnetization measurements reveal a phase transition at 85 K, below which a weak spontaneous magnetization (≈ 0.15 µB/Fe) appears. Below 85 K, magnetization increases with decreasing temperature down to 70 K, below which it decreases and approaches a constant value at low temperature. The Mössbauer spectrum at room temperature is composed of two paramagnetic doublets corresponding to the two crystallographic Fe(2+) sites. Below 85 K, each doublet undergoes further splitting because of the magnetic hyperfine fields. The temperature dependence of the hyperfine field is qualitatively different for the two distinguishable Fe(2+) sites. This is responsible for the anomalous temperature dependence of the magnetization.

Glutathionylation mediates angiotensin II-induced eNOS uncoupling, amplifying NADPH oxidase-dependent endothelial dysfunction.

BACKGROUND: Glutathionylation of endothelial nitric oxide synthase (eNOS) "uncouples" the enzyme, switching its function from nitric oxide (NO) to O2(•-) generation. We examined whether this reversible redox modification plays a role in angiotensin II (Ang II)-induced endothelial dysfunction. METHODS AND RESULTS: Ang II increased eNOS glutathionylation in cultured human umbilical vein endothelial cells (HUVECs), rabbit aorta, and human arteries in vitro. This was associated with decreased NO bioavailability and eNOS activity as well as increased O2(•-) generation. Ang II-induced decrease in eNOS activity was mediated by glutathionylation, as shown by restoration of function by glutaredoxin-1. Moreover, Ang II-induced increase in O2(•-) and decrease in NO were abolished in HUVECs transiently transfected, with mutant eNOS rendered resistant to glutathionylation. Ang II effects were nicotinamide adenine dinucleotide phosphate (NADPH) oxidase dependent because preincubation with gp 91ds-tat, an inhibitor of NADPH oxidase, abolished the increase in eNOS glutathionylation and loss of eNOS activity. Functional significance of glutathionylation in intact vessels was supported by Ang II-induced impairment of endothelium-dependent vasorelaxation that was abolished by the disulfide reducing agent, dithiothreitol. Furthermore, attenuation of Ang II signaling in vivo by administration of an angiotensin converting enzyme (ACE) inhibitor reduced eNOS glutathionylation, increased NO, diminished O2(•-), improved endothelium-dependent vasorelaxation and reduced blood pressure. CONCLUSIONS: Uncoupling of eNOS by glutathionylation is a key mediator of Ang II-induced endothelial dysfunction, and its reversal is a mechanism for cardiovascular protection by ACE inhibition. We suggest that Ang II-induced O2(•-) generation in endothelial cells, although dependent on NADPH oxidase, is amplified by glutathionylation-dependent eNOS uncoupling.

Extracellular allosteric Na(+) binding to the Na(+),K(+)-ATPase in cardiac myocytes.

Whole-cell patch-clamp measurements of the current, Ip, produced by the Na(+),K(+)-ATPase across the plasma membrane of rabbit cardiac myocytes show an increase in Ip over the extracellular Na(+) concentration range 0-50 mM. This is not predicted by the classical Albers-Post scheme of the Na(+),K(+)-ATPase mechanism, where extracellular Na(+) should act as a competitive inhibitor of extracellular K(+) binding, which is necessary for the stimulation of enzyme dephosphorylation and the pumping of K(+) ions into the cytoplasm. The increase in Ip is consistent with Na(+) binding to an extracellular allosteric site, independent of the ion transport sites, and an increase in turnover via an acceleration of the rate-determining release of K(+) to the cytoplasm, E2(K(+))2 → E1 + 2K(+). At normal physiological concentrations of extracellular Na(+) of 140 mM, it is to be expected that binding of Na(+) to the allosteric site would be nearly saturated. Its purpose would seem to be simply to optimize the enzyme's ion pumping rate under its normal physiological conditions. Based on published crystal structures, a possible location of the allosteric site is within a cleft between the α- and ß-subunits of the enzyme.

Quantitative calculation of the role of the Na(+),K(+)-ATPase in thermogenesis.

The Na(+),K(+)-ATPase is accepted as an important source of heat generation (thermogenesis) in animals. Based on information gained on the kinetics of the enzyme's partial reactions we consider via computer simulation whether modifications to the function of the combined Na(+),K(+)-ATPase/plasma membrane complex system could lead to an increased body temperature, either through the course of evolution or during an individual's lifespan. The enzyme's kinetics must be considered because it is the rate of heat generation which determines body temperature, not simply the amount of heat per enzymatic cycle. The results obtained indicate that a decrease in thermodynamic efficiency of the Na(+),K(+)-ATPase, which could come about by Na(+) substituting for K(+) on the enzyme's extracellular face, could not account for increased thermogenesis. The only feasible mechanisms are an increase in the enzyme's expression level or an increase in its ion pumping activity. The major source of Na(+),K(+)-ATPase-related thermogenesis (72% of heat production) is found to derive from passive Na(+) diffusion into the cell, which counterbalances outward Na(+) pumping to maintain a constant Na(+) concentration gradient across the membrane. A simultaneous increase in both Na(+),K(+)-ATPase activity and the membrane's passive Na(+) permeability could promote a higher body temperature.

Redox-dependent regulation of the Na⁺-K⁺ pump: new twists to an old target for treatment of heart failure.

By the time it was appreciated that the positive inotropic effect of cardiac glycosides is due to inhibition of the membrane Na(+)-K(+) pump, glycosides had been used for treatment of heart failure on an empiric basis for ~200 years. The subsequent documentation of their lack of clinical efficacy and possible harmful effect largely coincided with the discovery that a raised Na(+) concentration in cardiac myocytes plays an important role in the electromechanical phenotype of heart failure syndromes. Consistent with this, efficacious pharmacological treatments for heart failure have been found to stimulate the Na(+)-K(+) pump, effectively the only export route for intracellular Na(+) in the heart failure. A paradigm has emerged that implicates pump inhibition in the raised Na(+) levels in heart failure. It invokes protein kinase-dependent activation of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) and glutathionylation, a reversible oxidative modification, of the Na(+)-K(+) pump molecular complex that inhibits its activity. Since treatments of proven efficacy reverse the oxidative Na(+)-K(+) pump inhibition, the pump retains its status as a key pharmacological target in heart failure. Its role as a target is well integrated with the paradigms of neurohormonal abnormalities, raised myocardial oxidative stress and energy deficiency implicated in the pathophysiology of the failing heart. We propose that targeting oxidative inhibition of the pump is useful for the exploration of future treatment strategies. This article is part of a Special Issue entitled "Na(+)Regulation in Cardiac Myocytes".

Protein kinase-dependent oxidative regulation of the cardiac Na+-K+ pump: evidence from in vivo and in vitro modulation of cell signalling.

The widely reported stimulation of the cardiac Na(+)-K(+) pump by protein kinase A (PKA) should oppose other effects of PKA to increase contractility of the normal heart. It should also reduce harmful raised myocyte Na(+) levels in heart failure, yet blockade of the ß1 adrenergic receptor (AR), coupled to PKA signalling, is beneficial. We treated rabbits with the ß1 AR antagonist metoprolol to modulate PKA activity and studied cardiac myocytes ex vivo. Metoprolol increased electrogenic pump current (Ip) in voltage clamped myocytes and reduced glutathionylation of the ß1 pump subunit, an oxidative modification causally related to pump inhibition. Activation of adenylyl cyclase with forskolin to enhance cAMP synthesis or inclusion of the catalytic subunit of PKA in patch pipette solutions abolished the increase in Ip in voltage clamped myocytes induced by treatment with metoprolol, supporting cAMP/PKA-mediated pump inhibition. Metoprolol reduced myocardial PKA and protein kinase C (PKC) activities, reduced coimmunoprecipitation of cytosolic p47(phox) and membranous p22(phox) NADPH oxidase subunits and reduced myocardial O2(•-)-sensitive dihydroethidium fluorescence. Treatment also enhanced coimmunoprecipitation of the ß1 pump subunit with glutaredoxin 1 that catalyses de-glutathionylation. Since angiotensin II induces PKC-dependent activation of NADPH oxidase, we examined the effects of angiotensin-converting enzyme inhibition with captopril. This treatment had no effect on PKA activity but reduced the activity of PKC, reduced ß1 subunit glutathionylation and increased Ip. The PKA-induced Na(+)-K(+) pump inhibition we report should act with other mechanisms that enhance contractility of the normal heart but accentuate the harmful effects of raised cytosolic Na(+) in the failing heart. This scheme is consistent with the efficacy of ß1 AR blockade in the treatment of heart failure.