Standards for preclinical research and publications in developmental anaesthetic neurotoxicity: expert opinion statement from the SmartTots preclinical working group.

In March 2019, SmartTots, a public-private partnership between the US Food and Drug Administration and the International Anesthesia Research Society, hosted a meeting attended by research experts, anaesthesia journal editors, and government agency representatives to discuss the continued need for rigorous preclinical research and the importance of establishing reporting standards for the field of anaesthetic perinatal neurotoxicity. This group affirmed the importance of preclinical research in the field, and welcomed novel and mechanistic approaches to answer some of the field's largest questions. The attendees concluded that summarising the benefits and disadvantages of specific model systems, and providing guidance for reporting results, would be helpful for designing new experiments and interpreting results across laboratories. This expert opinion report is a summary of these discussions, and includes a focused review of current animal models and reporting standards for the field of perinatal anaesthetic neurotoxicity. This will serve as a practical guide and road map for novel and rigorous experimental work.

Physical and functional interaction sites in cytoplasmic domains of KCNQ1 and KCNE1 channel subunits.

The cardiac potassium IKs current is carried by a channel complex formed from α-subunits encoded by KCNQ1 and ß-subunits encoded by KCNE1. Deleterious mutations in either gene are associated with hereditary long QT syndrome. Interactions between the transmembrane domains of the α- and ß-subunits determine the activation kinetics of IKs. A physical and functional interaction between COOH termini of the proteins has also been identified that impacts deactivation rate and voltage dependence of activation. We sought to explore the specific physical interactions between the COOH termini of the subunits that confer such control. Hydrogen/deuterium exchange coupled to mass spectrometry narrowed down the region of interaction to KCNQ1 residues 352-374 and KCNE1 residues 70-81, and provided evidence of secondary structure within these segments. Key mutations of residues in these regions tended to shift voltage dependence of activation toward more depolarizing voltages. Double-mutant cycle analysis then revealed energetic coupling between KCNQ1-I368 and KCNE1-D76 during channel activation. Our results suggest that the proximal COOH-terminal regions of KCNQ1 and KCNE1 participate in a physical and functional interaction during channel opening that is sensitive to perturbation and may explain the clustering of long QT mutations in the region.NEW & NOTEWORTHY Interacting ion channel subunits KCNQ1 and KCNE1 have received intense investigation due to their critical importance to human cardiovascular health. This work uses physical (hydrogen/deuterium exchange with mass spectrometry) and functional (double-mutant cycle analyses) studies to elucidate precise and important areas of interaction between the two proteins in an area that has eluded structural definition of the complex. It highlights the importance of pathogenic mutations in these regions.

Comparative effectiveness of opioid replacement agents for neonatal opioid withdrawal syndrome: a systematic review and meta-analysis.

OBJECTIVE(S): To compare short-term treatment outcomes of opioid pharmacotherapy for neonatal opioid withdrawal syndrome (NOWS). STUDY DESIGN: PubMed/MEDLINE, Embase, PsycINFO, and The Cochrane Library were searched from inception through September 30, 2018. Primary outcome was treatment duration (LOT). Secondary outcomes included hospitalization duration (LOS) and rate of adjunct drug needed (RAD). RESULTS: Of 753 publications, 11 studies met inclusion criteria. There was no difference in LOT (WMD -1.39 [-5.79 to -3.01] days, I2 82%) or LOS (WMD -1.48 [-5.75 to -2.79] days, I2 92%) between morphine and methadone. RAD with morphine was higher (RR 1.51 [1.35-1.69], I2 0%). Buprenorphine was associated with shorter LOT (WMD 7.70 [0.88-14.53] days, I2 76%) and LOS (WMD 5.61 [-0.01 to -11.24] days, I2 60%) compared with morphine, in addition to methadone according to two cohort studies. CONCLUSIONS: Methadone had superior primary treatment success compared with morphine. Buprenorphine was associated with the shortest overall durations of treatment and hospitalization.

Using Neuroimaging to Study the Effects of Pain, Analgesia, and Anesthesia on Brain Development.

Neuroimaging has been increasingly used as a modality to study the impact of pain, analgesia, and anesthetics on pediatric neurodevelopment. The sixth biennial Pediatric Anesthesia Neurodevelopmental Assessment (PANDA) Symposium addressed the 2016 US Food and Drug Administration drug safety warning regarding the potential neurotoxic effects of commonly used anesthetic and sedative medications in children, and included a session discussing the use of various neuroimaging techniques, to detect structural, metabolic, and functional brain changes that can occur with exposure to pain and to anesthetic medications. The presenters concluded that advanced multimodal magnetic resonance imaging techniques are useful in detecting the aforementioned changes, which were found to be pain-specific and anesthetic agent-specific.

Engaging Stakeholders to Promote Safe Anesthesia and Sedation Care in Young Children.

An important aspect of any research endeavor is engaging various stakeholders to work toward the common goal of pushing knowledge forward about the question at hand. Research into pediatric anesthetic neurotoxicity could benefit greatly from interventions designed to improve the efforts and dedication of government agencies, pharmaceutical companies, research communities, and most importantly, patients. The Pediatric Anesthesia Neurodevelopment Assessment (PANDA) symposium is a biennial meeting where updates in research in the field are presented, and issues relevant to the community are discussed in round table discussions. Here, we summarize a discussion that took place at the 2018 meeting regarding new methods of engaging various stakeholders, as well as perspectives from other stakeholders. Topics discussed included an online portal to better reach patients, experiences with a public-private partnership, steps by the National Institutes of Health to improve engagement with research and improve the dissemination of results, and the experiences of the United States Food and Drug Administration attempting to improve stakeholder engagement following the passage of a new law to promote drug development. The round table discussion provided interesting insights into a critical research topic, and shared first-hand experience of attempts to improve engagement with a variety of stakeholders.

Anesthesia Exposure in Children: Practitioners Respond to the 2016 FDA Drug Safety Communication.

In December 2016, the US Food and Drug Administration (FDA) issued a drug safety warning stating that 11 commonly used anesthetic and sedative medications had potential neurotoxic effects when used in children under the age of 3 years and in pregnant women during the third trimester. A panel presentation at the sixth biennial Pediatric Anesthesia Neurodevelopmental Assessment (PANDA) symposium addressed the FDA announcement in a session entitled "Anesthesia Exposure in Children During Surgical and Non-Surgical Procedures: How Do We Respond to the 2016 FDA Drug Safety Communication?" Panelists included representatives from pediatric anesthesiology, obstetrics, pediatric surgery, and several pediatric surgical subspecialties. Each panelist was asked to address the following questions: How has the FDA labelling change affected your clinical practice including patient discussions, timing, and frequency of procedures? Has your professional society provided any guidelines for this discussion? Has there been any discussion of this topic at your national meetings? The panelists provided important perspectives specific to each specialty, which generated a lively discussion and a detailed response from the Deputy Director of the Division of Anesthesia and Addiction of the FDA describing the FDA procedures that led to this drug safety warning.

Binding interface of cardiac potassium channel proteins identified by hydrogen deuterium exchange of synthetic peptides.

Three synthetic peptides, derived from the human potassium channel proteins Ether-a-go-go-related gene (HERG), KCNQ1, and KCNE1, were investigated by hydrogen deuterium exchange coupled with electron-transfer dissociation mass spectrometry at single residue resolution. Each amino acid residue in the first half of the HERG peptide incorporated deuterons with a higher rate than those in the second half of the peptide, consistent with the nuclear magnetic resonance structure of this peptide, with amino acids 1-10 being a flexible coil, whereas amino acids 11-24 are a stable amphipathic helix. The binding interface of KCNQ1 and KCNE1 was determined by comparing the difference of sequential fragment ions before and after binding. The residues determined to be involved in binding were consistent with a cysteine cross-linking study and confirmed by double mutant cycle analysis.

A dual mechanism for I(Ks) current reduction by the pathogenic mutation KCNQ1-S277L.

BACKGROUND: The hereditary long QT syndrome is characterized by prolonged ventricular repolarization that can be caused by mutations to the KCNQ1 gene, which encodes the α subunits of the cardiac potassium channel complex that carries the I(Ks) current (the ß subunits are encoded by KCNE1). In this study, we characterized a deleterious variant, KCNQ1-S277L, found in a patient who presented with sudden cardiac death in the presence of cocaine use. METHODS: The KCNQ1-S277L mutation was analyzed via whole-cell patch clamp, confocal imaging, surface biotinylation assays, and computer modeling. RESULTS: Homomeric mutant KCNQ1-S277L channels were unable to carry current, either alone or with KCNE1. When co-expressed in a 50/50 ratio with WT KCNQ1, current density was reduced in a dominant-negative manner, with the residual current predominantly wild type. There was no change in the activation rate and minimal changes to voltage-dependent activation for both KCNQ1 current and I(Ks) current. Immunofluorescence confocal imaging revealed reduced surface expression of mutant KCNQ1-S277L, which was biochemically confirmed by surface biotinylation showing a 44% decrease in mutant surface expression. Expression of KCNQ1-S277L with human ether-a-go-go-related gene (HERG) did not significantly affect HERG protein or current density compared to KCNQ1-WT co-expression. CONCLUSION: The KCNQ1-S277L mutation causes biophysical defects that result in dominant-negative reduction in KCNQ1 and I(Ks) current density, and a trafficking defect that results in reduced surface expression, both without affecting HERG/I(Kr) . KCNQ1-S277L mutation in the proband resulted in defective channels that compromised repolarization reserve, thereby enhancing the arrhythmic susceptibility to pharmacological blockage of I(Kr) current.

Analysis of the interactions between the C-terminal cytoplasmic domains of KCNQ1 and KCNE1 channel subunits.

Ion channel subunits encoded by KCNQ1 and KCNE1 produce the slowly activating K+ current (IKs) that plays a central role in myocardial repolarization. The KCNQ1 alpha-subunit and the KCNE1 beta-subunit assemble with their membrane-spanning segments interacting, resulting in transformation of channel activation kinetics. We recently reported a functional interaction involving C-terminal portions of the two subunits with ensuing regulation of channel deactivation. In the present study, we provide evidence characterizing a physical interaction between the KCNQ1-CT (KCNE1 C-terminus) and the KCNE1-CT (KCNE1 C-terminus). When expressed in cultured cells, the KCNE1-CT co-localized with KCNQ1, co-immunoprecipitated with KCNQ1 and perturbed deactivation kinetics of the KCNQ1 currents. Purified KCNQ1-CT and KCNE1-CT physically interacted in pull-down experiments, indicating a direct association. Deletion analysis of KCNQ1-CT indicated that the KCNE1-CT binds to a KCNQ1 region just after the last transmembrane segment, but N-terminal to the tetramerization domain. SPR (surface plasmon resonance) corroborated the pull-down results, showing that the most proximal region (KCNQ1 amino acids 349-438) contributed most to the bimolecular interaction with a dissociation constant of approximately 4 microM. LQT (long QT) mutants of the KCNE1-CT, D76N and W87F, retained binding to the KCNQ1-CT with comparable affinity, indicating that these disease-causing mutations do not alter channel behaviour by disruption of the association. Several LQT mutations involving the KCNQ1-CT, however, showed various effects on KCNQ1/KCNE1 association. Our results indicate that the KCNQ1-CT and the KCNE1-CT comprise an independent interaction domain that may play a role in IKs channel regulation that is potentially affected in some LQTS (LQT syndrome) mutations.

Functional interactions between KCNE1 C-terminus and the KCNQ1 channel.

The KCNE1 gene product (minK protein) associates with the cardiac KvLQT1 potassium channel (encoded by KCNQ1) to create the cardiac slowly activating delayed rectifier, I(Ks). Mutations throughout both genes are linked to the hereditary cardiac arrhythmias in the Long QT Syndrome (LQTS). KCNE1 exerts its specific regulation of KCNQ1 activation via interactions between membrane-spanning segments of the two proteins. Less detailed attention has been focused on the role of the KCNE1 C-terminus in regulating channel behavior. We analyzed the effects of an LQT5 point mutation (D76N) and the truncation of the entire C-terminus (Delta70) on channel regulation, assembly and interaction. Both mutations significantly shifted voltage dependence of activation in the depolarizing direction and decreased I(Ks) current density. They also accelerated rates of channel deactivation but notably, did not affect activation kinetics. Truncation of the C-terminus reduced the apparent affinity of KCNE1 for KCNQ1, resulting in impaired channel formation and presentation of KCNQ1/KCNE1 complexes to the surface. Complete saturation of KCNQ1 channels with KCNE1-Delta70 could be achieved by relative over-expression of the KCNE subunit. Rate-dependent facilitation of K(+) conductance, a key property of I(Ks) that enables action potential shortening at higher heart rates, was defective for both KCNE1 C-terminal mutations, and may contribute to the clinical phenotype of arrhythmias triggered by heart rate elevations during exercise in LQTS mutations. These results support several roles for KCNE1 C-terminus interaction with KCNQ1: regulation of channel assembly, open-state destabilization, and kinetics of channel deactivation.

Cardioprotective effects of K ATP channel activation during hypoxia in goldfish Carassius auratus.

The activation of ATP-sensitive potassium (K ATP) ion channels in the heart is thought to exert a cardioprotective effect under low oxygen conditions, possibly enhancing tolerance of environmental hypoxia in aquatic vertebrates. The purpose of this study was to examine the possibility that hypoxia-induced activation of cardiac K ATP channels, whether in the sarcolemma (sarcK ATP) or mitochondria (mitoK ATP), enhances viability in cardiac muscle cells from a species highly tolerant of low oxygen environments, the goldfish Carassius auratus. During moderate hypoxia (6-7 kPa), the activation of sarcK ATP channels was indicated by a reduction in transmembrane action potential duration (APD). This response to hypoxia was mimicked by the NO-donor SNAP (100 micromol l(-1)) and the stable cGMP analog 8-Br-cGMP, but abolished by glibenclamide or l-NAME, an inhibitor of NO synthesis. The mitoK ATP channel opener diazoxide did not affect APD. Isolated ventricular muscle cells were then incubated under normoxic and hypoxic conditions. Cell viability was decreased in hypoxia; however, the negative effects of low oxygen were reduced during simultaneous exposure to SNAP, 8-Br-cGMP, and diazoxide. The cardioprotective effect of diazoxide, but not 8-Br-cGMP, was reduced by the mitoK ATP channel blocker 5-HD. These data suggest that hypoxia-induced activation of sarcK ATP or mitoK ATP channels could enhance tolerance of low-oxygen environments in this species, and that sarcK ATP activity is increased through a NO and cGMP-dependent pathway.