Development and translation of thiometallate sulfide donors using a porcine model of coronary occlusion and reperfusion.

Sulfide-releasing compounds reduce reperfusion injury by decreasing mitochondria-derived reactive oxygen species production. We previously characterised ammonium tetrathiomolybdate (ATTM), a clinically used copper chelator, as a sulfide donor in rodents. Here we assessed translation to large mammals prior to clinical testing. In healthy pigs an intravenous ATTM dose escalation revealed a reproducible pharmacokinetic/pharmacodynamic (PK/PD) relationship with minimal adverse clinical or biochemical events. In a myocardial infarction (1-h occlusion of the left anterior descending coronary artery)-reperfusion model, intravenous ATTM or saline was commenced just prior to reperfusion. ATTM protected the heart (24-h histological examination) in a drug-exposure-dependent manner (r2 = 0.58, p < 0.05). Blood troponin T levels were significantly (p < 0.05) lower in ATTM-treated animals while myocardial glutathione peroxidase activity, an antioxidant selenoprotein, was elevated (p < 0.05). Overall, our study represents a significant advance in the development of sulfides as therapeutics and underlines the potential of ATTM as a novel adjunct therapy for reperfusion injury. Mechanistically, our study suggests that modulating selenoprotein activity could represent an additional mode of action of sulfide-releasing drugs.

A Targeted, Low-Throughput Compound Screen in a Drosophila Model of Neurofibromatosis Type 1 Identifies Simvastatin and BMS-204352 as Potential Therapies for Autism Spectrum Disorder (ASD).

Autism spectrum disorder (ASD) is a common neurodevelopmental condition for which there are no pharmacological therapies that effectively target its core symptomatology. Animal models of syndromic forms of ASD, such as neurofibromatosis type 1, may be of use in screening for such treatments. Drosophila larvae lacking Nf1 expression exhibit tactile hypersensitivity following mechanical stimulation, proposed to mirror the sensory sensitivity issues comprising part of the ASD diagnostic criteria. Such behavior is associated with synaptic dysfunction at the neuromuscular junction (NMJ). Both phenotypes may thus provide tractable outputs with which to screen for potential ASD therapies. In this study, we demonstrate that, while loss of Nf1 expression within the embryo is sufficient to impair NMJ synaptic transmission in the larva, constitutive Nf1 knock-down is required to induce tactile hypersensitivity, suggesting that a compound must be administered throughout development to rescue this behavior. With such a feeding regime, we identify two compounds from a targeted, low-throughput screen that significantly and consistently reduce, but do not fully rescue, tactile hypersensitivity in Nf1P1 larvae. These are the HMG CoA-reductase inhibitor simvastatin, and the BKCa channel activator BMS-204352. At the NMJ, both compounds induce a significant reduction in the enhanced spontaneous transmission frequency of Nf1P1 larvae, though again not to the level of vehicle-treated controls. However, both compounds fully rescue the increased quantal size of Nf1P1 mutants, with simvastatin also fully rescuing their reduced quantal content. Thus, the further study of both compounds as potential ASD interventions is warranted.

Loss of NF1 in Drosophila Larvae Causes Tactile Hypersensitivity and Impaired Synaptic Transmission at the Neuromuscular Junction.

Autism spectrum disorder (ASD) is a neurodevelopmental condition in which the mechanisms underlying its core symptomatology are largely unknown. Studying animal models of monogenic syndromes associated with ASD, such as neurofibromatosis type 1 (NF1), can offer insights into its etiology. Here, we show that loss of function of the Drosophila NF1 ortholog results in tactile hypersensitivity following brief mechanical stimulation in the larva (mixed sexes), paralleling the sensory abnormalities observed in individuals with ASD. Mutant larvae also exhibit synaptic transmission deficits at the glutamatergic neuromuscular junction (NMJ), with increased spontaneous but reduced evoked release. While the latter is homeostatically compensated for by a postsynaptic increase in input resistance, the former is consistent with neuronal hyperexcitability. Indeed, diminished expression of NF1 specifically within central cholinergic neurons induces both excessive neuronal firing and tactile hypersensitivity, suggesting the two may be linked. Furthermore, both impaired synaptic transmission and behavioral deficits are fully rescued via knock-down of Ras proteins. These findings validate NF1 -/- Drosophila as a tractable model of ASD with the potential to elucidate important pathophysiological mechanisms.SIGNIFICANCE STATEMENT Autism spectrum disorder (ASD) affects 1-2% of the overall population and can considerably impact an individual's quality of life. However, there are currently no treatments available for its core symptoms, largely because of a poor understanding of the underlying mechanisms involved. Examining how loss of function of the ASD-associated NF1 gene affects behavior and physiology in Drosophila may shed light on this. In this study, we identify a novel, ASD-relevant behavioral phenotype in NF1 -/- larvae, namely an enhanced response to mechanical stimulation, which is associated with Ras-dependent synaptic transmission deficits indicative of neuronal hyperexcitability. Such insights support the use of Drosophila neurofibromatosis type 1 (NF1) models in ASD research and may provide outputs for genetic or pharmacological screens in future studies.

On the origin of nitrosylated hemoglobin in COVID-19: Endothelial NO capture or redox conversion of nitrite?: Experimental results and a cautionary note on challenges in translational research.

In blood, the majority of endothelial nitric oxide (NO) is scavenged by oxyhemoglobin, forming nitrate while a small part reacts with dissolved oxygen to nitrite; another fraction may bind to deoxyhemoglobin to generate nitrosylhemoglobin (HbNO) and/or react with a free cysteine to form a nitrosothiol. Circulating nitrite concentrations in healthy individuals are 200-700 nM, and can be even lower in patients with endothelial dysfunction. Those levels are similar to HbNO concentrations ([HbNO]) recently reported, whereby EPR-derived erythrocytic [HbNO] was lower in COVID-19 patients compared to uninfected subjects with similar cardiovascular risk load. We caution the values reported may not reflect true (patho)physiological concentrations but rather originate from complex chemical interactions of endogenous nitrite with hemoglobin and ascorbate/N-acetylcysteine. Using an orthogonal detection method, we find baseline [HbNO] to be in the single-digit nanomolar range; moreover, we find that these antioxidants, added to blood collection tubes to prevent degradation, artificially generate HbNO. Since circulating nitrite also varies with lifestyle, dietary habit and oral bacterial flora, [HbNO] may not reflect endothelial activity alone. Thus, its use as early marker of NO-dependent endothelial dysfunction to stratify COVID-19 patient risk may be premature. Moreover, oxidative stress not only impairs NO formation/bioavailability, but also shifts the chemical landscape into which NO is released, affecting its downstream metabolism. This compromises the endothelium's role as gatekeeper of tissue nutrient supply and modulator of blood cell function, challenging the body's ability to maintain redox balance. Further studies are warranted to clarify whether the nature of vascular dysfunction in COVID-19 is solely of endothelial nature or also includes altered erythrocyte function.

The Pharmacology and Therapeutic Utility of Sodium Hydroselenide.

Metabolically active gasotransmitters (nitric oxide, carbon monoxide and hydrogen sulfide) are important signalling molecules that show therapeutic utility in oxidative pathologies. The reduced form of selenium, hydrogen selenide (HSe-/H2Se), shares some characteristics with these molecules. The simple selenide salt, sodium hydroselenide (NaHSe) showed significant metabolic activity, dose-dependently decreasing ex vivo O2 consumption (rat soleus muscle, liver) and transiently inhibiting mitochondrial cytochrome C oxidase (liver, heart). Pharmacological manipulation of selenoprotein expression in HepG2 human hepatocytes revealed that the oxidation status of selenium impacts on protein expression; reduced selenide (NaHSe) increased, whereas (oxidized) sodium selenite decreased the abundance of two ubiquitous selenoproteins. An inhibitor of endogenous sulfide production (DL-propargylglycine; PAG) also reduced selenoprotein expression; this was reversed by exogenous NaHSe, but not sodium hydrosulfide (NaHS). NaHSe also conferred cytoprotection against an oxidative challenge (H2O2), and this was associated with an increase in mitochondrial membrane potential. Anesthetized Wistar rats receiving intravenous NaHSe exhibited significant bradycardia, metabolic acidosis and hyperlactataemia. In summary, NaHSe modulates metabolism by inhibition of cytochrome C oxidase. Modification of selenoprotein expression revealed the importance of oxidation status of selenium therapies, with implications for current clinical practice. The utility of NaHSe as a research tool and putative therapeutic is discussed.

Optogenetic Stimulation of Vagal Efferent Activity Preserves Left Ventricular Function in Experimental Heart Failure.

Large clinical trials designed to test the efficacy of vagus nerve stimulation (VNS) in patients with heart failure did not demonstrate benefits with respect to the primary endpoints. The nonselective nature of VNS may account for the failure to translate promising results of preclinical and earlier clinical studies. This study showed that optogenetic stimulation of vagal pre-ganglionic neurons transduced to express light-sensitive channels preserved left ventricular function and exercise capacity in a rat model of myocardial infarction-induced heart failure. These data suggested that stimulation of vagal efferent activity is critically important to deliver the therapeutic benefit of VNS in heart failure.

Neuroprotective effects of ammonium tetrathiomolybdate, a slow-release sulfide donor, in a rodent model of regional stroke.

BACKGROUND: Several therapeutic strategies to rescue the brain from ischemic injury have improved outcomes after stroke; however, there is no treatment as yet for reperfusion injury, the secondary damage caused by necessary revascularization. Recently we characterized ammonium tetrathiomolybdate (ATTM), a drug used as a copper chelator over many decades in humans, as a new class of sulfide donor that shows efficacy in preclinical injury models. We hypothesized that ATTM could confer neuroprotection in a relevant rodent model of regional stroke. METHODS AND RESULTS: Brain ischemia was induced by transient (90-min) middle cerebral artery occlusion (tMCAO) in anesthetized Wistar rats. To mimic a clinical scenario, ATTM (or saline) was administered intravenously just prior to reperfusion. At 24 h or 7 days post-reperfusion, rats were assessed using functional (rotarod test, spontaneous locomotor activity), histological (infarct size), and molecular (anti-oxidant enzyme capacity, oxidative damage, and inflammation) outcome measurements. ATTM-treated animals showed improved functional activity at both 24 h and 7-days post-reperfusion, in parallel with a significant reduction in infarct size. These effects were additionally associated with increased brain antioxidant enzyme capacity, decreased oxidative damage, and a late (7-day) effect on pro-inflammatory cytokine levels and nitric oxide products. CONCLUSION: ATTM confers significant neuroprotection that, along with its known safety profile in humans, provides encouragement for its development as a novel adjunct therapy for revascularization following stroke.

Nimodipine Reduces Dysfunction and Demyelination in Models of Multiple Sclerosis.

OBJECTIVE: Treatment of relapses in multiple sclerosis (MS) has not advanced beyond steroid use, which reduces acute loss of function, but has little effect on residual disability. Acute loss of function in an MS model (experimental autoimmune encephalomyelitis [EAE]) is partly due to central nervous system (CNS) hypoxia, and function can promptly improve upon breathing oxygen. Here, we investigate the cause of the hypoxia and whether it is due to a deficit in oxygen supply arising from impaired vascular perfusion. We also explore whether the CNS-selective vasodilating agent, nimodipine, may provide a therapy to restore function, and protect from demyelination in 2 MS models. METHODS: A variety of methods have been used to measure basic cardiovascular physiology, spinal oxygenation, mitochondrial function, and tissue perfusion in EAE. RESULTS: We report that the tissue hypoxia in EAE is associated with a profound hypoperfusion of the inflamed spinal cord. Treatment with nimodipine restores spinal oxygenation and can rapidly improve function. Nimodipine therapy also reduces demyelination in both EAE and a model of the early MS lesion. INTERPRETATION: Loss of function in EAE, and demyelination in EAE, and the model of the early MS lesion, seem to be due, at least in part, to tissue hypoxia due to local spinal hypoperfusion. Therapy to improve blood flow not only protects neurological function but also reduces demyelination. We conclude that nimodipine could be repurposed to offer substantial clinical benefit in MS. ANN NEUROL 2020 ANN NEUROL 2020;88:123-136.

Chemistry, pharmacology, and cellular uptake mechanisms of thiometallate sulfide donors.

BACKGROUND AND PURPOSE: A clinical need exists for targeted, safe, and effective sulfide donors. We recently reported that ammonium tetrathiomolybdate (ATTM) belongs to a new class of sulfide-releasing drugs. Here, we investigated the cellular uptake mechanisms of this drug class compared to sodium hydrosulfide (NaHS) and the effects of a thiometallate tungsten congener of ATTM, ammonium tetrathiotungstate (ATTT). EXPERIMENTAL APPROACH: In vitro H2 S release was determined by headspace gas sampling of vials containing dissolved thiometallates. Thiometallate and NaHS bioactivity was assessed by spectrophotometry-derived sulfhaemoglobin formation. Cellular uptake dependence on the anion exchange protein (AE)-1 was investigated in human red blood cells. ATTM/glutathione interactions were assessed by LC-MS/MS. Rodent pharmacokinetic and pharmacodynamic studies focused on haemodynamics and inhibition of aerobic respiration. KEY RESULTS: ATTM and ATTT both exhibit temperature-, pH-, and thiol-dependence of sulfide release. ATTM/glutathione interactions revealed the generation of inorganic and organic persulfides and polysulfides. ATTM showed greater ex vivo and in vivo bioactivity over ATTT, notwithstanding similar pharmacokinetic profiles. Cellular uptake mechanisms of the two drug classes are distinct; thiometallates show dependence on AE-1, while hydrosulfide itself was unaffected by inhibition of this pathway. CONCLUSIONS AND IMPLICATIONS: The cellular uptake of thiometallates relies upon a plasma membrane ion channel. This advances our pharmacological knowledge of this drug class, and further supports their utility as cell-targeted sulfide donor therapies. Our results indicate that, as a more stable form, ATTT is better suited as a copper chelator. ATTM, a superior sulfide donor, may additionally participate in intracellular redox recycling. LINKED ARTICLES: This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc.

Selenium and hydrogen selenide: essential micronutrient and the fourth gasotransmitter?

Selenium (Se) is an essential micronutrient required by organisms of diverse lineage. Dietary Se is converted to hydrogen selenide either enzymatically or by endogenous antioxidant proteins. This convergent biochemical step crucially underlies the subsequent biological activity of Se and argues for inclusion of hydrogen selenide as the fourth endogenous gasotransmitter alongside nitric oxide, carbon monoxide and hydrogen sulfide.Endogenously generated hydrogen selenide is incorporated into numerous 'selenoprotein' oxidoreductase enzymes, essential for maintaining redox-status homeostasis in health and disease. Direct effects of endogenous hydrogen selenide on cellular and molecular targets are currently unknown. Given exogenously, hydrogen selenide acts as a modulator of metabolism via transient inhibition of mitochondrial cytochrome C oxidase. Here we provide an overview of Se biology, its impact on several physiological systems (immune, endocrine, cardiovascular and metabolic) and its utility as a supplement in acute and critical illness states. We further explore the evidence base supporting its role as the fourth gasotransmitter and propose a strategic case towards generation of novel selenomimetic therapeutics.

In vivo validation of a miniaturized electrochemical oxygen sensor for measuring intestinal oxygen tension.

Recent advances in the fields of electronics and microfabrication techniques have led to the development of implantable medical devices for use within the field of precision medicine. Monitoring visceral surface tissue O2 tension (PTo2) by means of an implantable sensor is potentially useful in many clinical situations, including the perioperative management of patients undergoing intestinal resection and anastomosis. This concept could provide a means by which treatment could be tailored to individual patients. This study describes the in vivo validation of a novel, miniaturized electrochemical O2 sensor to provide real-time data on intestinal PTo2. A single O2 sensor was placed onto the serosal surface of the small intestine of anesthetized rats that were exposed to ischemic (superior mesenteric artery occlusion) and hypoxemic (alterations in inspired fractional O2 concentrations) insults. Control experiments demonstrated that the sensors can function and remain stable in an in vivo environment. Intestinal PTo2 decreased following superior mesenteric artery occlusion and with reductions in inspired O2 concentrations. These results were reversible after reinstating blood flow or by increasing inspired O2 concentrations. We have successfully developed an anesthetized rat intestinal ischemic and hypoxic model for validation of a miniaturized O2 sensor to provide real-time measurement of intestinal PTo2. Our results support further validation of the sensors in physiological conditions using a large animal model to provide evidence of their use in clinical applications where monitoring visceral surface tissue O2 tension is important.NEW & NOTEWORTHY This is the first report of real-time continuous measurements of intestinal oxygen tension made using a microfabricated O2 sensor. Using a developed rodent model, we have validated this sensor's ability to accurately measure dynamic and reversible changes in intestinal oxygenation that occur through ischemic and hypoxemic insults. Continuous monitoring of local intestinal oxygenation could have value in the postoperative monitoring of patients having undergone intestinal surgery.

Optimising organ perfusion in the high-risk surgical and critical care patient: a narrative review.

Maintenance or prompt restoration of an oxygen supply sufficient to facilitate adequate cellular metabolism is fundamental in maintaining organ function. This is particularly relevant when metabolic needs change markedly, for example in response to major surgery or critical illness. The consequences of inadequate tissue oxygenation include wound and anastomotic breakdown, organ dysfunction, and death. However, our ability to identify those at risk and to promptly recognise and correct tissue hypoperfusion is limited. Reliance is placed upon surrogate markers of tissue oxygenation such as arterial blood pressure and serum lactate that are insensitive to early organ compromise. Advances in oxygen sensing technology will facilitate monitoring in various organ beds and allow more precise titration of therapies to physiologically relevant endpoints. Clinical trials will be needed to evaluate any impact on outcomes, however accurate on-line monitoring of the adequacy of tissue oxygenation offers the promise of a paradigm shift in resuscitation and perioperative practice. This narrative review examines current evidence for goal-directed therapy in the optimisation of organ perfusion in high-risk surgical and critically ill patients, and offers arguments to support the potential utility of tissue oxygen monitoring.

Protein recycling and limb muscle recovery after critical illness in slow- and fast-twitch limb muscle.

An impaired capacity of muscle to regenerate after critical illness results in long-term functional disability. We previously described in a long-term rat peritonitis model that gastrocnemius displays near-normal histology whereas soleus demonstrates a necrotizing phenotype. We thus investigated the link between the necrotizing phenotype of critical illness myopathy and proteasome activity in these two limb muscles. We studied male Wistar rats that underwent an intraperitoneal injection of the fungal cell wall constituent zymosan or n-saline as a sham-treated control. Rats (n = 74) were killed at 2, 7, and 14 days postintervention with gastrocnemius and soleus muscle removed and studied ex vivo. Zymosan-treated animals displayed an initial reduction of body weight but a persistent decrease in mass of both lower hindlimb muscles. Zymosan increased chymotrypsin- and trypsin-like proteasome activities in gastrocnemius at days 2 and 7 but in soleus at day 2 only. Activated caspases-3 and -9, polyubiquitin proteins, and 14-kDa fragments of myofibrillar actin (proteasome substrates) remained persistently increased from day 2 to day 14 in soleus but not in gastrocnemius. These results suggest that a relative proteasome deficiency in soleus is associated with a necrotizing phenotype during long-term critical illness. Rescuing proteasome clearance may offer a potential therapeutic option to prevent long-term functional disability in critically ill patients.

Ammonium tetrathiomolybdate following ischemia/reperfusion injury: Chemistry, pharmacology, and impact of a new class of sulfide donor in preclinical injury models.

BACKGROUND: Early revascularization of ischemic organs is key to improving outcomes, yet consequent reperfusion injury may be harmful. Reperfusion injury is largely attributed to excess mitochondrial production of reactive oxygen species (ROS). Sulfide inhibits mitochondria and reduces ROS production. Ammonium tetrathiomolybdate (ATTM), a copper chelator, releases sulfide in a controlled and novel manner, and may offer potential therapeutic utility. METHODS AND FINDINGS: In vitro, ATTM releases sulfide in a time-, pH-, temperature-, and thiol-dependent manner. Controlled sulfide release from ATTM reduces metabolism (measured as oxygen consumption) both in vivo in awake rats and ex vivo in skeletal muscle tissue, with a superior safety profile compared to standard sulfide generators. Given intravenously at reperfusion/resuscitation to rats, ATTM significantly reduced infarct size following either myocardial or cerebral ischemia, and conferred survival benefit following severe hemorrhage. Mechanistic studies (in vitro anoxia/reoxygenation) demonstrated a mitochondrial site of action (decreased MitoSOX fluorescence), where the majority of damaging ROS is produced. CONCLUSIONS: The inorganic thiometallate ATTM represents a new class of sulfide-releasing drugs. Our findings provide impetus for further investigation of this compound as a novel adjunct therapy for reperfusion injury.

Ventilation strategy has a major influence on remote ischaemic preconditioning in mice.

Whether oxygen should be administered acutely during ST-segment elevation myocardial infarction is debated. Despite this controversy, the possible influence of supplementary oxygen on animal models of ischaemia-reperfusion injury or cardioprotection is rarely considered. We used an in vivo mouse model of ischaemia and reperfusion to investigate the effect of ventilation with room air versus 100% oxygen. The coronary artery of anaesthetized mice was occluded for 40 min. followed by 2-hrs reperfusion. Infarct size was measured by tetrazolium staining and expressed as a percentage of area at risk, determined using Evan's blue. Unexpectedly, infarct size in mice ventilated with 100% oxygen was significantly smaller than in those ventilated with room air (33 ± 5% versus 46 ± 3%; n = 6; P < 0.01). We tested a standard protocol of 3 × 5 min. cycles of remote ischaemic preconditioning (RIPC) and found this was unable to protect mice ventilated with 100% oxygen. RIPC protocols using 2.5- or 10-min. occlusion were similarly ineffective in mice ventilated with oxygen. Similar disparate results were obtained with direct cardiac ischaemic preconditioning. In contrast, pharmacological protection using bradykinin administered at reperfusion was effective even in mice ventilated with 100% oxygen, reducing infarct size from 33 ± 5% to 21 ± 3% (n = 4-6; P < 0.01). Laser speckle contrast imaging of blood flow and direct pO2 measurements were made in the hindlimb, but these measurements did not correlate with protection. In conclusion, ventilation protocol can have a major influence on infarct size and ischaemic preconditioning protocols in mice.

Improved Survival in a Long-Term Rat Model of Sepsis Is Associated With Reduced Mitochondrial Calcium Uptake Despite Increased Energetic Demand.

OBJECTIVES: To investigate the relationship between prognosis, changes in mitochondrial calcium uptake, and bioenergetic status in the heart during sepsis. DESIGN: In vivo and ex vivo controlled experimental studies. SETTING: University research laboratory. SUBJECTS: Male adult Wistar rats. INTERVENTIONS: Sepsis was induced by intraperitoneal injection of fecal slurry. Sham-operated animals served as controls. Confocal microscopy was used to study functional and bioenergetic parameters in cardiomyocytes isolated after 24-hour sepsis. Electron microscopy was used to characterize structural changes in mitochondria and sarcoplasmic reticulum. The functional response to dobutamine was assessed in vivo by echocardiography. MEASUREMENTS AND MAIN RESULTS: Peak aortic blood flow velocity measured at 24 hours was a good discriminator for 72-hour survival (area under the receiver operator characteristic, 0.84 ± 0.1; p = 0.03) and was used in ex vivo experiments at 24 hours to identify septic animals with good prognosis. Measurements from animals with good prognostic showed 1) a smaller increase in mitochondrial calcium content and in nicotinamide adenine dinucleotide fluorescence following pacing and 2) increased distance between mitochondria and sarcoplasmic reticulum on electron microscopy, and 3) nicotinamide adenine dinucleotide redox potential and adenosine triphosphate/adenosine diphosphate failed to reach a new steady state following pacing, suggesting impaired matching of energy supply and demand. In vivo, good prognosis animals had a blunted response to dobutamine with respect to stroke volume and kinetic energy. CONCLUSIONS: In situations of higher energetic demand decreased mitochondrial calcium uptake may constitute an adaptive cellular response that confers a survival advantage in response to sepsis at a cost of decreased oxidative capacity.

[Pyr1]Apelin-13(1-12) Is a Biologically Active ACE2 Metabolite of the Endogenous Cardiovascular Peptide [Pyr1]Apelin-13.

Aims: Apelin is a predicted substrate for ACE2, a novel therapeutic target. Our aim was to demonstrate the endogenous presence of the putative ACE2 product [Pyr1]apelin-13(1-12) in human cardiovascular tissues and to confirm it retains significant biological activity for the apelin receptor in vitro and in vivo. The minimum active apelin fragment was also investigated. Methods and Results: [Pyr1]apelin-13 incubated with recombinant human ACE2 resulted in de novo generation of [Pyr1]apelin-13(1-12) identified by mass spectrometry. Endogenous [Pyr1]apelin-13(1-12) was detected by immunostaining in human heart and lung localized to the endothelium. Expression was undetectable in lung from patients with pulmonary arterial hypertension. In human heart [Pyr1]apelin-13(1-12) (pKi = 8.04 ± 0.06) and apelin-13(F13A) (pKi = 8.07 ± 0.24) competed with [125I]apelin-13 binding with nanomolar affinity, 4-fold lower than for [Pyr1]apelin-13 (pKi = 8.83 ± 0.06) whereas apelin-17 exhibited highest affinity (pKi = 9.63 ± 0.17). The rank order of potency of peptides to inhibit forskolin-stimulated cAMP was apelin-17 (pD2 = 10.31 ± 0.28) > [Pyr1]apelin-13 (pD2 = 9.67 ± 0.04) ≥ apelin-13(F13A) (pD2 = 9.54 ± 0.05) > [Pyr1]apelin-13(1-12) (pD2 = 9.30 ± 0.06). The truncated peptide apelin-13(R10M) retained nanomolar potency (pD2 = 8.70 ± 0.04) but shorter fragments exhibited low micromolar potency. In a ß-arrestin recruitment assay the rank order of potency was apelin-17 (pD2 = 10.26 ± 0.09) >> [Pyr1]apelin-13 (pD2 = 8.43 ± 0.08) > apelin-13(R10M) (pD2 = 8.26 ± 0.17) > apelin-13(F13A) (pD2 = 7.98 ± 0.04) ≥ [Pyr1]apelin-13(1-12) (pD2 = 7.84 ± 0.06) >> shorter fragments (pD2 < 6). [Pyr1]apelin-13(1-12) and apelin-13(F13A) contracted human saphenous vein with similar sub-nanomolar potencies and [Pyr1]apelin-13(1-12) was a potent inotrope in paced mouse right ventricle and human atria. [Pyr1]apelin-13(1-12) elicited a dose-dependent decrease in blood pressure in anesthetized rat and dose-dependent increase in forearm blood flow in human volunteers. Conclusions: We provide evidence that ACE2 cleaves [Pyr1]apelin-13 to [Pyr1]apelin-13(1-12) and this cleavage product is expressed in human cardiovascular tissues. We have demonstrated biological activity of [Pyr1]apelin-13(1-12) at the human and rodent apelin receptor in vitro and in vivo. Our data show that reported enhanced ACE2 activity in cardiovascular disease should not significantly compromise the beneficial effects of apelin based therapies for example in PAH.

Exploring alternative routes for oxygen administration.

BACKGROUND: Hypoxemia may compromise cell metabolism and organ function. Supplemental oxygen (O2) at high concentrations may prove ineffective, and issues relating to hyperoxia, barotrauma, mechanical ventilation, and extracorporeal oxygenation are well documented. Old reports suggest the potential safety and efficacy of alternative routes for O2 administration, such as intravenous or intestinal. We re-explored these routes in rat models of hypoxemia. METHODS: Hypoxemia was induced in spontaneously breathing, anesthetized rats by breathing a hypoxic gas mix (FiO2 0.1). Pilot studies infusing pure O2 gas caused early death, likely due to pulmonary embolism. Instead, rats (n = 6/group) were given intravenous O2 via a continuous infusion of pre-oxygenated Hartmann's solution (10 ml/kg/h) for 3 h with normal Ringer's lactate used in control animals. In separate experiments (n = 8/group), bowel intraluminal oxygenation was assessed with pure O2 administered through a cannula placed into the jejunal lumen at a dose of a 15 ml/kg bolus followed by a continuous infusion of 50 ml/kg/h; no treatment was given to controls. Echocardiography, arterial blood gas analysis, mean arterial pressure, muscle and liver tPO2, muscle microvascular perfused vessel density, and urine output were measured. RESULTS: Administration of oxygenated Hartmann's solution (PO2 of solution at end-experiment = 87.5 ± 1.7 kPa) was safe but did not increase either systemic or tissue oxygenation. Similarly, the administration of bowel O2 was safe but did not improve neither systemic nor liver oxygenation. CONCLUSIONS: In this rat model of hypoxemia, the intravenous infusion of gaseous O2 was unfeasible as it induced early mortality. Although safe, both intravenous infusion of oxygenated Hartmann's solution and bowel O2 administration were unable to improve arterial or tissue oxygenation.

Molecular Mechanisms Linking Autonomic Dysfunction and Impaired Cardiac Contractility in Critical Illness.

OBJECTIVES: Molecular mechanisms linking autonomic dysfunction with poorer clinical outcomes in critical illness remain unclear. We hypothesized that baroreflex dysfunction alone is sufficient to cause cardiac impairment through neurohormonal activation of (nicotinamide adenine dinucleotide phosphate oxidase dependent) oxidative stress resulting in increased expression of G-protein-coupled receptor kinase 2, a key negative regulator of cardiac function. DESIGN: Laboratory/clinical investigations. SETTING: University laboratory/medical centers. SUBJECTS: Adult rats; wild-type/nicotinamide adenine dinucleotide phosphate oxidase subunit-2-deficient mice; elective surgical patients. INTERVENTIONS: Cardiac performance was assessed by transthoracic echocardiography following experimental baroreflex dysfunction (sino-aortic denervation) in rats and mice. Immunoblots assessed G-protein-coupled receptor recycling proteins expression in rodent cardiomyocytes and patient mononuclear leukocytes. In surgical patients, heart rate recovery after cardiopulmonary exercise testing, time/frequency measures of parasympathetic variables were related to the presence/absence of baroreflex dysfunction (defined by spontaneous baroreflex sensitivity of <6 ms mm Hg). The associations of baroreflex dysfunction with intraoperative cardiac function and outcomes were assessed. MEASUREMENTS AND MAIN RESULTS: Experimental baroreflex dysfunction in rats and mice resulted in impaired cardiac contractility and upregulation of G-protein-coupled receptor kinase 2 expression. In mice, genetic deficiency of gp91 nicotinamide adenine dinucleotide phosphate oxidase subunit-2 prevented upregulation of G-protein-coupled receptor kinase 2 expression in conditions of baroreflex dysfunction and preserved cardiac function. Baroreflex dysfunction was present in 81 of 249 patients (32.5%) and was characterized by lower parasympathetic tone and increased G-protein-coupled receptor kinase 2 expression in mononuclear leukocytes. Baroreflex dysfunction in patients was also associated with impaired intraoperative cardiac contractility. Critical illness and mortality were more frequent in surgical patients with baroreflex dysfunction (relative risk, 1.66 [95% CI, 1.16-2.39]; p = 0.006). CONCLUSIONS: Reduced baroreflex sensitivity is associated with nicotinamide adenine dinucleotide phosphate oxidase subunit-2-mediated upregulation of G-protein-coupled receptor kinase 2 expression in cardiomyocytes and impaired cardiac contractility. Autonomic dysfunction predisposes patients to the development of critical illness and increases mortality.

Hypothermia protects brain mitochondrial function from hypoxemia in a murine model of sepsis.

Sepsis is commonly associated with brain dysfunction, but the underlying mechanisms remain unclear, although mitochondrial dysfunction and microvascular abnormalities have been implicated. We therefore assessed whether cerebral mitochondrial dysfunction during systemic endotoxemia in mice increased mitochondrial sensitivity to a further bioenergetic insult (hyoxemia), and whether hypothermia could improve outcome. Mice (C57bl/6) were injected intraperitoneally with lipopolysaccharide (LPS) (5 mg/kg; n = 85) or saline (0.01 ml/g; n = 47). Six, 24 and 48 h later, we used confocal imaging in vivo to assess cerebral mitochondrial redox potential and cortical oxygenation in response to changes in inspired oxygen. The fraction of inspired oxygen (FiO2) at which the cortical redox potential changed was compared between groups. In a subset of animals, spontaneous hypothermia was maintained or controlled hypothermia induced during imaging. Decreasing FiO2 resulted in a more reduced cerebral redox state around veins, but preserved oxidation around arteries. This pattern appeared at a higher FiO2 in LPS-injected animals, suggesting an increased sensitivity of cortical mitochondria to hypoxemia. This increased sensitivity was accompanied by a decrease in cortical oxygenation, but was attenuated by hypothermia. These results suggest that systemic endotoxemia influences cortical oxygenation and mitochondrial function, and that therapeutic hypothermia can be protective.