Pulmonary vasoconstrictor influence of endothelin in exercising swine depends critically on phosphodiesterase 5 activity.

Both phosphodiesterase 5 (PDE5) inhibition and endothelin (ET) receptor blockade have been shown to induce pulmonary vasodilation. However, little is known about the effect of combined blockade of these two vasoconstrictor pathways. Since nitric oxide (NO) exerts its pulmonary vasodilator influence via production of cyclic guanosine monophosphate (cGMP) as well as through inhibition of ET, we hypothesized that interaction between the respective signaling pathways precludes an additive vasodilator effect. We tested this hypothesis in chronically instrumented swine exercising on a treadmill by comparing the vasodilator effect of the PDE5 inhibitor EMD360527, the ETA/ETB antagonist tezosentan, and combined EMD360527 and tezosentan. In the systemic circulation, vasodilation by tezosentan and EMD360527 was additive, both at rest and during exercise, resulting in a 17 ± 2% drop in blood pressure. In the pulmonary circulation, both EMD360527 and tezosentan produced vasodilation. However, tezosentan produced no additional pulmonary vasodilation in the presence of EMD360527, either at rest or during exercise. Moreover, in isolated preconstricted porcine pulmonary small arteries (∼300 µm) EMD360527 (1 nM-10 µM) induced dose-dependent vasodilation, whereas tezosentan (1 nM-10 µM) failed to elicit vasodilation irrespective of the presence of EMD360527. However, both PDE5 inhibition and 8Br-cGMP, but not 8Br-cAMP, blunted pulmonary small artery contraction to ET and its precursor Big ET in vitro. In conclusion, in healthy swine, either at rest or during exercise, PDE5 inhibition and the associated increase in cGMP produce pulmonary vasodilation that is mediated in part through inhibition of the ET pathway, thereby precluding an additional vasodilator effect of ETA/ETB receptor blockade in the presence of PDE5 inhibition.

Anammox growth on pretreated municipal wastewater.

Autotrophic nitrogen removal from municipal wastewater enables development of energy autarkic wastewater treatment plants. In this study we report the evaluation of the anammox process in a granular sludge fluidized bed lab-scale reactor continuously fed with the actual effluent of the A-stage of the WWTP of Dokhaven, Rotterdam. The reactor was anoxic, and nitrite was dosed continuously to support anammox activity only. The system was operated for more than ten months at temperatures between 20 and 10 °C. COD was also consumed during the process, but heterotrophs could not outcompete anammox bacteria. Volumetric N-removal rates obtained were comparable or higher than those of conventional N-removal systems, with values higher than 0.4 g-N L(-1) d(-1) when operated at 10 °C. The biomass specific N-removal rate at 10 °C was on average 50±7 mg-N g-VSS(-1) d(-1) during the last month of operations, almost two times higher than previously reported activities at this temperature. FISH analysis revealed that the dominant anammox species was Candidatus Brocadia Fulgida throughout the experimentation. Evidence for growth of anammox bacteria at mainstream conditions was demonstrated for the entire temperature range tested (10-20 °C), and new granules were shown to be actively formed and efficiently retained in the system.

Cardiomyocytes derived from pluripotent stem cells recapitulate electrophysiological characteristics of an overlap syndrome of cardiac sodium channel disease.

BACKGROUND: Pluripotent stem cells (PSCs) offer a new paradigm for modeling genetic cardiac diseases, but it is unclear whether mouse and human PSCs can truly model both gain- and loss-of-function genetic disorders affecting the Na(+) current (I(Na)) because of the immaturity of the PSC-derived cardiomyocytes. To address this issue, we generated multiple PSC lines containing a Na(+) channel mutation causing a cardiac Na(+) channel overlap syndrome. METHOD AND RESULTS: Induced PSC (iPSC) lines were generated from mice carrying the Scn5a(1798insD/+) (Scn5a-het) mutation. These mouse iPSCs, along with wild-type mouse iPSCs, were compared with the targeted mouse embryonic stem cell line used to generate the mutant mice and with the wild-type mouse embryonic stem cell line. Patch-clamp experiments showed that the Scn5a-het cardiomyocytes had a significant decrease in I(Na) density and a larger persistent I(Na) compared with Scn5a-wt cardiomyocytes. Action potential measurements showed a reduced upstroke velocity and longer action potential duration in Scn5a-het myocytes. These characteristics recapitulated findings from primary cardiomyocytes isolated directly from adult Scn5a-het mice. Finally, iPSCs were generated from a patient with the equivalent SCN5A(1795insD/+) mutation. Patch-clamp measurements on the derivative cardiomyocytes revealed changes similar to those in the mouse PSC-derived cardiomyocytes. CONCLUSION: Here, we demonstrate that both embryonic stem cell- and iPSC-derived cardiomyocytes can recapitulate the characteristics of a combined gain- and loss-of-function Na(+) channel mutation and that the electrophysiological immaturity of PSC-derived cardiomyocytes does not preclude their use as an accurate model for cardiac Na(+) channel disease.

Cytochrome P-450 2C9 exerts a vasoconstrictor influence on coronary resistance vessels in swine at rest and during exercise.

A significant endothelium-dependent vasodilation persists after inhibition of nitric oxide synthase (NOS) and cyclooxygenase (COX) in the coronary vasculature, which has been linked to the activation of cytochrome P-450 (CYP) epoxygenases expressed in endothelial cells and subsequent generation of vasodilator epoxyeicosatrienoic acids. Here, we investigated the contribution of CYP 2C9 metabolites to regulation of porcine coronary vasomotor tone in vivo and in vitro. Twenty-six swine were chronically instrumented. Inhibition of CYP 2C9 with sulfaphenazole (5 mg/kg iv) alone had no effect on bradykinin-induced endothelium-dependent coronary vasodilation in vivo but slightly attenuated bradykinin-induced vasodilation in the presence of combined NOS/COX blockade with N(ω)-nitro-L-arginine (20 mg/kg iv) and indomethacin (10 mg/kg iv). Sulfaphenazole had minimal effects on coronary resistance vessel tone at rest or during exercise. Surprisingly, in the presence of combined NOS/COX blockade, a significant coronary vasodilator response to sulfaphenzole became apparent, both at rest and during exercise. Subsequently, we investigated in isolated porcine coronary small arteries (∼250 µm) the possible involvement of reactive oxygen species (ROS) in the paradoxical vasoconstrictor influence of CYP 2C9 activity. The vasodilation by bradykinin in vitro in the presence of NOS/COX blockade was markedly potentiated by sulfaphenazole under control conditions but not in the presence of the ROS scavenger N-(2-mercaptoproprionyl)-glycine. In conclusion, CYP 2C9 can produce both vasoconstrictor and vasodilator metabolites. Production of these metabolites is enhanced by combined NOS/COX blockade and is critically dependent on the experimental conditions. Thus production of vasoconstrictors slightly outweighed the production of vasodilators at rest and during exercise. Pharmacological stimulation with bradykinin resulted in vasodilator CYP 2C9 metabolite production when administered in vivo, whereas vasoconstrictor CYP 2C9 metabolites, most likely ROS, were dominant when administered in vitro.

HCN4 current during human sinoatrial node-like action potentials.

BACKGROUND: Despite the many studies carried out over the past 40 years, the contribution of the HCN4 encoded hyperpolarization-activated 'funny' current (If) to pacemaker activity in the mammalian sinoatrial node (SAN), and the human SAN in particular, is still controversial and not fully established. OBJECTIVE: To study the contribution of If to diastolic depolarization of human SAN cells and its dependence on heart rate, cAMP levels, and atrial load. METHODS: HCN4 channels were expressed in human cardiac myocyte progenitor cells (CMPCs) and HCN4 currents assessed using perforated patch-clamp in traditional voltage clamp mode and during action potential clamp with human SAN-like action potential waveforms with 500-1500 ms cycle length, in absence or presence of forskolin to mimic ß-adrenergic stimulation and a -15 mV command potential offset to mimic atrial load. RESULTS: Forskolin significantly increased the fully-activated HCN4 current density at -140 mV by 14% and shifted the steady-state activation curve by +7.4 mV without affecting its slope. In addition, forskolin significantly accelerated current activation but slowed deactivation. The HCN4 current did not completely deactivate before the subsequent diastolic depolarization during action potential clamp. The amplitude of HCN4 current increased with increasing cycle length, was significantly larger in the presence of forskolin at all cycle lengths, and was significantly increased upon the negative offset to the command potential. CONCLUSIONS: If is active during a human SAN action potential waveform and its amplitude is modulated by heart rate, ß-adrenergic stimulation, and diastolic voltage range, such that If is under delicate control.

Both beta1- and beta2-adrenoceptors contribute to feedforward coronary resistance vessel dilation during exercise.

During exercise, beta-feedforward coronary vasodilation has been shown to contribute to the matching of myocardial oxygen supply with the demand of the myocardium. Since both beta(1)- and beta(2)-adrenoceptors are present in the coronary microvasculature, we investigated the relative contribution of these subtypes to beta-feedforward coronary vasodilation during exercise as well as to infusion of the beta(1)-agonist norepinephrine and the beta(1)- and beta(2)-agonist isoproterenol. Chronically instrumented swine were studied at rest and during graded treadmill exercise (1-5 km/h) under control conditions and after beta(1)-blockade with metoprolol (0.5 mg/kg iv) and beta(1)/beta(2)-blockade with propranolol (0.5 mg/kg iv). The selectivity and degree of beta-blockade of metoprolol and propranolol were confirmed using isoproterenol infusion (0.05-0.4 microg. kg(-1).min(-1)) under resting conditions. Isoproterenol-induced coronary vasodilation was mediated through the beta(2)-adrenoceptor, whereas norepinephrine-induced coronary vasodilation was principally mediated through the beta(1)-adrenoceptor. Exercise resulted in a significant increase in left ventricular norepinephrine release and epinephrine uptake. beta(1)-Adrenoceptor blockade with metoprolol had very little effect under resting conditions. However, during exercise, metoprolol attenuated the increase in myocardial oxygen supply in excess of the reduction in myocardial oxygen demand, as evidenced by a progressive decrease in coronary venous Po(2). Consequently, metoprolol caused a clockwise rotation of the relationship between myocardial oxygen consumption and coronary venous Po(2). Additional beta(2)-adrenoceptor blockade with propranolol further inhibited myocardial oxygen supply during exercise, resulting in a further clockwise rotation of the relationship between myocardial oxygen consumption and coronary venous Po(2). In conclusion, both beta(1)- and beta(2)-adrenoceptors contribute to the beta-feedforward coronary resistance vessel dilation during exercise.

Integrated control of pulmonary vascular tone by endothelin and angiotensin II in exercising swine depends on gender.

The lungs are now recognized as an active metabolic organ that is a major determinant of the plasma concentrations of the vasoconstrictors endothelin (ET) and ANG II. Several studies have suggested a complex interaction between ET and ANG II in the systemic and coronary vascular beds that is different at rest and during exercise. To date, the interaction between these vasoconstrictor peptides has barely been investigated in relation to the pulmonary vascular bed. Consequently, we investigated the integrated control of pulmonary vasomotor tone by ET and ANG II in 24 chronically instrumented swine (15 female and 9 male) at rest and during graded treadmill exercise. In the systemic circulation, ANG II type 1 (AT(1)) receptor blockade with irbesartan and mixed ET(A)/ET(B) blockade with tezosentan each produced vasodilation. The systemic vasodilator effect of ET(A)/ET(B) blockade was enhanced after AT(1) blockade in female swine, whereas a trend toward an increase was observed in male swine. In the pulmonary circulation, AT(1) receptor blockade had no effect on pulmonary vascular tone in male swine, whereas it resulted in an unexpected increase in pulmonary vasomotor tone in female swine. ET(A)/ET(B) receptor blockade did not result in a decrease in pulmonary vasomotor tone at rest but produced a decrease in vasomotor tone during exercise in both genders. This pulmonary vasodilation by ET(A)/ET(B) receptor blockade was enhanced after prior AT(1) blockade in female swine but not in male swine. In conclusion, in both the systemic and pulmonary circulation of female swine, ANG II inhibits the vasoconstrictor influence of ET. This interaction is gender specific. The observation that plasma ET levels were not altered by AT(1) blockade in either gender suggests that the interaction between these vasoconstrictors occurs locally in the vasculature.

Towards mainstream anammox: lessons learned from pilot-scale research at WWTP Dokhaven.

The aim of this research was to study the biological feasibility of the Partial Nitritation/Anammox (PN/A) technology to remove nitrogen from municipal mainstream wastewaters. During stable process operations at summer temperatures (23.2 ± 1.3°C), the total nitrogen removal rate was 0.223 ± 0.029 kg N (m3 d)-1 while at winter temperatures (13.4 ± 1.1°C) the total nitrogen removal rate was 0.097 ± 0.016 kg N (m3 d)-1. Nitrite-oxidizing bacteria (NOB) suppression was successfully achieved at the complete temperature range of municipal mainstream wastewater. Despite the presence of NOB as observed in activity tests, their activity could be successfully suppressed due to a relative low dissolved oxygen concentration. An overcapacity of ammonia-oxidizing bacteria and anammox activity was always present. Long-term stability is a focus point for future research, especially in relation to the stability of the biological oxygen demand removing step, preceding the PN/A reactor.

Deterioration of the anammox process at decreasing temperatures and long SRTs.

The implementation of autotrophic nitrogen removal in the mainstream of a municipal wastewater treatment plant is currently pursued. Among the crucial unknown factors are the kinetic properties of anaerobic ammonium oxidising (anammox) bacteria at low temperatures. In this study we investigated the adaptation of a fast-growing anammox culture to a lower temperature. In a membrane bioreactor a highly enriched anammox community was obtained at 30°C, 25°C and 20°C. This culture was exposed to long- and short-term temperature changes. In short-term experiments the decrease in biomass-specific activity due to decrease in temperature can be described by an activation energy of 64 ± 28 kJ mol-1. Prolonged cultivation (months) implies that cultivation at low temperatures resulted in deterioration of biomass-specific activity (EaLT 239 kJ mol-1). The growth rate and specific anammox activity in the system decreased from 0.33 d-1 and 4.47 g NO2-N g VSS-1 d-1 at 30°C to 0.0011 d-1 and 0.037 g NO2-N g VSS-1 d-1 at 20°C. The reason for the deterioration of the system was related to the required long SRT in the system. The long SRT leads to an increase of non-active and non-anammox cells in the reactor, thereby decreasing the biomass-specific activity.

Anti-arrhythmic potential of the late sodium current inhibitor GS-458967 in murine Scn5a-1798insD+/- and human SCN5A-1795insD+/- iPSC-derived cardiomyocytes.

AIMS: Selective inhibition of cardiac late sodium current (INaL) is an emerging target in the treatment of ventricular arrhythmias. We investigated the electrophysiological effects of GS-458967 (GS967), a potent, selective inhibitor of INaL, in an overlap syndrome model of both gain and loss of sodium channel function, comprising cardiomyocytes derived from both human SCN5A-1795insD+/- induced pluripotent stem cells (hiPSC-CMs) and mice carrying the homologous mutation Scn5a-1798insD+/-. METHODS AND RESULTS: On patch-clamp analysis, GS967 (300 nmol/l) reduced INaL and action potential (AP) duration in isolated ventricular myocytes from wild type and Scn5a-1798insD+/- mice, as well as in SCN5A-1795insD+/- hiPSC-CMs. GS967 did not affect the amplitude of peak INa, but slowed its recovery, and caused a negative shift in voltage-dependence of INa inactivation. GS967 reduced AP upstroke velocity in Scn5a-1798insD+/- myocytes and SCN5A-1795insD+/- hiPSC-CMs. However, the same concentration of GS967 did not affect conduction velocity in Scn5a-1798insD+/- mouse isolated hearts, as assessed by epicardial mapping. GS967 decreased the amplitude of delayed after depolarizations and prevented triggered activity in mouse Scn5a-1798insD+/- cardiomyocytes. CONCLUSION: The INaL inhibitor GS967 decreases repolarization abnormalities and has anti-arrhythmic effects in the absence of deleterious effects on cardiac conduction. Thus, selective inhibition of INaL constitutes a promising pharmacological treatment of cardiac channelopathies associated with enhanced INaL. Our findings furthermore implement hiPSC-CMs as a valuable tool for assessment of novel pharmacological approaches in inherited sodium channelopathies.

Effects of the residual ammonium concentration on NOB repression during partial nitritation with granular sludge.

Partial nitritation was stably achieved in a bench-scale airlift reactor (1.5L) containing granular sludge. Continuous operation at 20 °C treating low-strength synthetic wastewater (50 mg N-NH4+/L and no COD) achieved nitrogen loading rates of 0.8 g N-NH4+/(L·d) during partial nitritation. The switch between nitrite-oxidizing bacteria (NOB) repression and NOB proliferation was observed when ammonium concentrations in the reactor were below 2-5 mg N-NH4+/L for DO concentrations lower than 4 mg O2/L at 20 °C. Nitrospira spp. were detected to be the dominant NOB population during the entire reactor operation, whereas Nitrobacter spp. were found to be increasing in numbers over time. Stratification of the granule structure, with ammonia-oxidizing bacteria (AOB) occupying the outer shell, was found to be highly important in the repression of NOB in the long term. The pH gradient in the granule, containing a pH difference of ca. 0.4 between the granule surface and the granule centre, creates a decreasing gradient of ammonia towards the centre of the granule. Higher residual ammonium concentration enhances the ammonium oxidation rate of those cells located further away from the granule surface, where the competition for oxygen between AOB and NOB is more important, and it contributes to the stratification of both populations in the biofilm.

TECRL, a new life-threatening inherited arrhythmia gene associated with overlapping clinical features of both LQTS and CPVT.

Genetic causes of many familial arrhythmia syndromes remain elusive. In this study, whole-exome sequencing (WES) was carried out on patients from three different families that presented with life-threatening arrhythmias and high risk of sudden cardiac death (SCD). Two French Canadian probands carried identical homozygous rare variant in TECRL gene (p.Arg196Gln), which encodes the trans-2,3-enoyl-CoA reductase-like protein. Both patients had cardiac arrest, stress-induced atrial and ventricular tachycardia, and QT prolongation on adrenergic stimulation. A third patient from a consanguineous Sudanese family diagnosed with catecholaminergic polymorphic ventricular tachycardia (CPVT) had a homozygous splice site mutation (c.331+1G>A) in TECRL Analysis of intracellular calcium ([Ca2+]i) dynamics in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated from this individual (TECRLHom-hiPSCs), his heterozygous but clinically asymptomatic father (TECRLHet-hiPSCs), and a healthy individual (CTRL-hiPSCs) from the same Sudanese family, revealed smaller [Ca2+]i transient amplitudes as well as elevated diastolic [Ca2+]i in TECRLHom-hiPSC-CMs compared with CTRL-hiPSC-CMs. The [Ca2+]i transient also rose markedly slower and contained lower sarcoplasmic reticulum (SR) calcium stores, evidenced by the decreased magnitude of caffeine-induced [Ca2+]i transients. In addition, the decay phase of the [Ca2+]i transient was slower in TECRLHom-hiPSC-CMs due to decreased SERCA and NCX activities. Furthermore, TECRLHom-hiPSC-CMs showed prolonged action potentials (APs) compared with CTRL-hiPSC-CMs. TECRL knockdown in control human embryonic stem cell-derived CMs (hESC-CMs) also resulted in significantly longer APs. Moreover, stimulation by noradrenaline (NA) significantly increased the propensity for triggered activity based on delayed afterdepolarizations (DADs) in TECRLHom-hiPSC-CMs and treatment with flecainide, a class Ic antiarrhythmic drug, significantly reduced the triggered activity in these cells. In summary, we report that mutations in TECRL are associated with inherited arrhythmias characterized by clinical features of both LQTS and CPVT Patient-specific hiPSC-CMs recapitulated salient features of the clinical phenotype and provide a platform for drug screening evidenced by initial identification of flecainide as a potential therapeutic. These findings have implications for diagnosis and treatment of inherited cardiac arrhythmias.

Induced pluripotent stem cell derived cardiomyocytes as models for cardiac arrhythmias.

Cardiac arrhythmias are a major cause of morbidity and mortality. In younger patients, the majority of sudden cardiac deaths have an underlying Mendelian genetic cause. Over the last 15 years, enormous progress has been made in identifying the distinct clinical phenotypes and in studying the basic cellular and genetic mechanisms associated with the primary Mendelian (monogenic) arrhythmia syndromes. Investigation of the electrophysiological consequences of an ion channel mutation is ideally done in the native cardiomyocyte (CM) environment. However, the majority of such studies so far have relied on heterologous expression systems in which single ion channel genes are expressed in non-cardiac cells. In some cases, transgenic mouse models have been generated, but these also have significant shortcomings, primarily related to species differences. The discovery that somatic cells can be reprogrammed to pluripotency as induced pluripotent stem cells (iPSC) has generated much interest since it presents an opportunity to generate patient- and disease-specific cell lines from which normal and diseased human CMs can be obtained These genetically diverse human model systems can be studied in vitro and used to decipher mechanisms of disease and identify strategies and reagents for new therapies. Here, we review the present state of the art with respect to cardiac disease models already generated using IPSC technology and which have been (partially) characterized. Human iPSC (hiPSC) models have been described for the cardiac arrhythmia syndromes, including LQT1, LQT2, LQT3-Brugada Syndrome, LQT8/Timothy syndrome and catecholaminergic polymorphic ventricular tachycardia (CPVT). In most cases, the hiPSC-derived cardiomyoctes recapitulate the disease phenotype and have already provided opportunities for novel insight into cardiac pathophysiology. It is expected that the lines will be useful in the development of pharmacological agents for the management of these disorders.