Sulfide quinone oxidoreductase contributes to voltage sensing of the mitochondrial permeability transition pore.

Pathological opening of the mitochondrial permeability transition pore (mPTP) is implicated in the pathogenesis of many disease processes such as myocardial ischemia, traumatic brain injury, Alzheimer's disease, and diabetes. While we have gained insight into mPTP biology over the last several decades, the lack of translation of this knowledge into successful clinical therapies underscores the need for continued investigation and use of different approaches to identify novel regulators of the mPTP with the hope of elucidating new therapeutic targets. Although the mPTP is known to be a voltage-gated channel, the identity of its voltage sensor remains unknown. Here we found decreased gating potential of the mPTP and increased expression and activity of sulfide quinone oxidoreductase (SQOR) in newborn Fragile X syndrome (FXS) mouse heart mitochondria, a model system of coenzyme Q excess and relatively decreased mPTP open probability. We further found that pharmacological inhibition and genetic silencing of SQOR increased mPTP open probability in vitro in adult murine cardiac mitochondria and in the isolated-perfused heart, likely by interfering with voltage sensing. Thus, SQOR is proposed to contribute to voltage sensing by the mPTP and may be a component of the voltage sensing apparatus that modulates the gating potential of the mPTP.

Multicenter Population Pharmacokinetics of Fentanyl in Neonatal Surgical Patients Using Dried Blood Spot Specimen Collection Demonstrates Maturation of Elimination Clearance.

BACKGROUND: Fentanyl is widely used for analgesia and sedation in neonates, but pharmacokinetic (PK) analysis in this population has been limited by the relatively large sample volumes required for plasma-based assays. METHODS: In this multicenter observational study of fentanyl kinetics in neonates up to 42 weeks of postmenstrual age (PMA) who received fentanyl boluses and continuous infusions, dried blood spots were used for small-volume sampling. A population PK analysis was used to describe fentanyl disposition in term and preterm neonates. Covariates for the model parameters, including body weight, PMA, birth status (preterm or term), and presence of congenital cardiac disease, were assessed in a stepwise manner. RESULTS: Clearance was estimated to be greater than adult clearance of fentanyl and varied with weight. Covariate selection did not yield a significant relationship for age as a continuous or dichotomous variable (term or preterm, the latter defined as birth with PMA of <37 weeks) and clearance. CONCLUSIONS: A supra-allometric effect on clearance was determined during covariate analyses (exponential scaling factor for body weight >0.75), as has been described in population PK models that account for maturation of intrinsic clearance (here, predominantly hepatic microsomal activity) in addition to scaling for weight, both of which impact clearance in this age group.

Modeling propofol-induced cardiotoxicity in the isolated-perfused newborn mouse heart.

Infants and children are vulnerable to developing propofol infusion syndrome (PRIS) and young age is a risk factor. Cardiac involvement is often prominent and associated with death. However, the mechanisms of pediatric PRIS are poorly understood because of the paucity of investigation and lack of a gold standard animal model. Unfortunately, in vivo modeling of PRIS in a newborn mouse is not feasible and would be complicated by confounders. Thus, we focused on propofol-induced cardiotoxicity and aimed to develop an ex-vivo model in the isolated-perfused newborn mouse heart. We hypothesized that the model would recapitulate the key cardiac features of PRIS seen in infants and children and would corroborate prior in vitro observations. Isolated perfused newborn mouse hearts were exposed to a toxic dose of propofol or intralipid for 30-min. Surface electrocardiogram, ventricular contractile force, and oxygen extraction were measured over time. Real-time multiphoton laser imaging was utilized to quantify calcein and tetramethylrhodamine ethyl ester fluorescence. Propidium iodide uptake was assessed following drug exposure. A toxic dose of propofol rapidly induced dysrhythmias, depressed ventricular contractile function, impaired the mitochondrial membrane potential, and increased open probability of the permeability transition pore in propofol-exposed hearts without causing cell death. These features mimicked the hallmarks of pediatric PRIS and corroborated prior observations made in isolated newborn cardiomyocyte mitochondria. Thus, acute propofol-induced cardiotoxicity in the isolated-perfused developing mouse heart may serve as a relevant ex-vivo model for pediatric PRIS.

Assessment of Open Probability of the Mitochondrial Permeability Transition Pore in the Setting of Coenzyme Q Excess.

The mitochondrial permeability transition pore (mPTP) is a voltage-gated, nonselective, inner mitochondrial membrane (IMM) mega-channel important in health and disease. The mPTP mediates leakage of protons across the IMM during low-conductance opening and is specifically inhibited by cyclosporine A (CsA). Coenzyme Q (CoQ) is a regulator of the mPTP, and tissue-specific differences have been found in CoQ content and open probability of the mPTP in forebrain and heart mitochondria in a newborn mouse model of fragile X syndrome (FXS, Fmr1 knockout). We developed a technique to determine the voltage threshold for mPTP opening in this mutant strain, exploiting the role of the mPTP as a proton leak channel. To do so, oxygen consumption and membrane potential (ΔΨ) were simultaneously measured in isolated mitochondria using polarography and a tetraphenylphosphonium (TPP+) ion-selective electrode during leak respiration. The threshold for mPTP opening was determined by the onset of CsA-mediated inhibition of proton leak at specific membrane potentials. Using this approach, differences in voltage gating of the mPTP were precisely defined in the context of CoQ excess. This novel technique will permit future investigation for enhancing the understanding of physiological and pathological regulation of low-conductance opening of the mPTP.

Altered brown adipose tissue mitochondrial function in newborn fragile X syndrome mice.

Brown adipose tissue (BAT) mitochondria generate heat via uncoupled respiration due to excessive proton leak through uncoupling proteins (UCPs). We previously found hyperthermia in a newborn mouse model of fragile X syndrome and excessive leak in Fmr1 KO forebrain mitochondria caused by CoQ deficiency. The inefficient thermogenic nature of Fmr1 mutant forebrain mitochondria was reminiscent of BAT metabolic features. Thus, we aimed to characterize BAT mitochondrial function in these hyperthermic mice using a top-down approach. Although there was no change in steady-state levels of UCP1 expression between strains, BAT weighed significantly less in Fmr1 mutants compared with controls. Fmr1 KO BAT mitochondria demonstrated impaired substrate oxidation, lower mitochondrial membrane potentials and rates of respiration, and CoQ deficiency. The CoQ analog decylubiquinone normalized CoQ-dependent electron flux and unmasked excessive proton leak. Unlike mutant forebrain, where such deficiency resulted in pathological proton leak, CoQ deficiency within BAT mitochondria resulted largely in abnormal substrate oxidation. This suggests that CoQ is important in BAT for uncoupled respiration to produce heat during development. Although our data provide further evidence of a link between fragile X mental retardation protein (FMRP) and CoQ biosynthesis, the results highlight the importance of CoQ in developing tissues and suggest tissue-specific differences from CoQ deficiency. Because BAT mitochondria are primarily responsible for regulating core body temperature, the defects we describe in Fmr1 KOs could manifest as an adaptive downregulated response to hyperthermia or could result from FMRP deficiency directly.

Modified Technique for the Use of Neonatal Murine Hearts in the Langendorff Preparation.

The use of the ex-vivo retrograde perfused heart has long been a cornerstone of ischemia-reperfusion investigation since its development by Oskar Langendorff over a century ago. Although this technique has been applied to mice over the last 25 years, its use in this species has been limited to adult animals. Development of a successful method to consistently cannulate the neonatal murine aorta would allow for the systematic study of the isolated retrograde perfused heart during a critical period of cardiac development in a genetically modifiable and low-cost species. Modification of the Langendorff preparation enables cannulation and establishment of reperfusion in the neonatal murine heart while minimizing ischemic time. Optimization requires a two-person technique to permit successful cannulation of the newborn mouse aorta using a dissecting microscope and a modified commercially available needle. The use of this approach will reliably establish retrograde perfusion within 3 min. Because the fragility of the neonatal mouse heart and ventricular cavity size prevents direct measurement of intraventricular pressure generated using a balloon, use of a force transducer connected by a suture to the apex of the left ventricle to quantify longitudinal contractile tension is necessary. This method allows investigators to successfully establish an isolated constant-flow retrograde-perfused newborn murine heart preparation, permitting the study of developmental cardiac biology in an ex-vivo manner. Importantly, this model will be a powerful tool to investigate the physiological and pharmacological responses to ischemia-reperfusion in the neonatal heart.

Mitochondrial ATP synthase c-subunit leak channel triggers cell death upon loss of its F1 subcomplex.

Mitochondrial ATP synthase is vital not only for cellular energy production but also for energy dissipation and cell death. ATP synthase c-ring was suggested to house the leak channel of mitochondrial permeability transition (mPT), which activates during excitotoxic ischemic insult. In this present study, we purified human c-ring from both eukaryotic and prokaryotic hosts to biophysically characterize its channel activity. We show that purified c-ring forms a large multi-conductance, voltage-gated ion channel that is inhibited by the addition of ATP synthase F1 subcomplex. In contrast, dissociation of F1 from FO occurs during excitotoxic neuronal death suggesting that the F1 constitutes the gate of the channel. mPT is known to dissipate the osmotic gradient across the inner membrane during cell death. We show that ATP synthase c-subunit knock down (KD) prevents the osmotic change in response to high calcium and eliminates large conductance, Ca2+ and CsA sensitive channel activity of mPT. These findings elucidate the gating mechanism of the ATP synthase c-subunit leak channel (ACLC) and suggest how ACLC opening is regulated by cell stress in a CypD-dependent manner.

Propofol toxicity in the developing mouse heart mitochondria.

BACKGROUND: Propofol infusion syndrome (PRIS) is a potentially lethal consequence of long-term propofol administration. Children are vulnerable and cardiac involvement is often prominent and associated with mortality. We aimed to determine the mechanism of propofol toxicity in newborn mice, hypothesizing that propofol would induce discrete defects within immature cardiac mitochondria. METHODS: Newborn murine cardiac mitochondria were exposed to propofol or intralipid in vitro. Non-exposed mitochondria served as controls. Mitochondrial respiration and membrane potential (ΔΨ) were measured and respiratory chain complex kinetics were determined. RESULTS: Propofol and intralipid exerted biological activity in isolated mitochondria. Although intralipid effects were a potential confounder, we found that propofol induced a dose-dependent increase in proton leak and caused a defect in substrate oxidation at coenzyme Q (CoQ). These impairments prevented propofol-exposed cardiomyocyte mitochondria from generating an adequate ΔΨ. The addition of the quinone analog, CoQ0, blocked propofol-induced leak and increased Complex II+III activity. CONCLUSIONS: Propofol uncoupled immature cardiomyocyte mitochondria by inducing excessive CoQ-sensitive leak and interfered with electron transport at CoQ. The findings provide new insight into the mechanisms of propofol toxicity in the developing heart and may help explain why children are vulnerable to developing PRIS. IMPACT: Propofol uncouples immature cardiomyocyte mitochondria by inducing excessive coenzyme Q (CoQ)-sensitive proton leak. Propofol also interferes with electron transport at the level of CoQ. These defects provide new insight into propofol toxicity in the developing heart.

Prevalence of SARS-CoV-2 Positivity in Pediatric Surgical Patients Amid the First Wave of the COVID-19 Pandemic in New York City.

INTRODUCTION: New York State implemented an 11-week elective surgery ban in response to the coronavirus disease-2019 (COVID-19) pandemic, during which pediatric patients from the 10 New York Presbyterian network hospitals requiring urgent or emergent surgical procedures were cared for at Morgan Stanley Children's Hospital (MSCH). MATERIALS AND METHODS: Data was abstracted from the electronic medical record of all patients aged 0 to 20 years who had surgery at MSCH from March 23, 2020 to June 7, 2020. Comparative analysis of demographic and clinical data elements between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive and negative cohorts was conducted using the Fisher exact tests. RESULTS: A total of 505 surgical procedures were performed in 451 patients, with 32 procedures (6.3%) performed in 21 SARS-CoV-2-positive children. The prevalence of SARS-CoV-2 positivity in Medicaid beneficiaries was more than twice the prevalence in commercially insured (6.8% vs. 2.6%, P=0.04) children. SARS-CoV-2-positive patients were more likely to undergo multiple surgical procedures (23.8% vs. 7.2%, P=0.02), and to have higher American Society of Anesthesiologists (ASA) class designations (69.8% III to V vs. 47.4% I to II, P=0.03). There was no significant difference in the prevalence of SARS-CoV-2 positivity across sex, age, race, or ethnicity groups, or in emergent case status or surgical procedure type. Thirty-day mortality rate was <0.1% overall, with no deaths in the SARS-CoV-2-positive group. CONCLUSIONS: During the first wave of the COVID-19 pandemic in New York City, we found a higher prevalence of SARS-CoV-2 positivity in urgent/emergent pediatric surgical patients compared with other institutions in the United States. SARS-CoV-2-positive patients were more likely to be Medicaid beneficiaries, were clinically more complex, and had more surgical procedures.

Isoflurane and low-level carbon monoxide exposures increase expression of pro-survival miRNA in neonatal mouse heart.

Anesthetics such as isoflurane are known to cause apoptosis in the developing mammalian brain. However, isoflurane may have protective effects on the heart via relieving ischemia and downregulating genes related to apoptosis. Ischemic preconditioning, e.g. through the use of low levels of carbon monoxide (CO), has promise in preventing ischemia-reperfusion injury and cell death. However, it is still unclear how it either triggers the stress response in neonatal hearts. For this reason, thirty-three microRNAs (miRNAs) known to be differentially expressed following anesthesia and/or ischemic or hypoxic heart damage were investigated in the hearts from neonatal mice exposed to isoflurane or low level of CO, using an air-exposed control group. Only miR-93-5p increased with isoflurane exposure, which may be associated with the suppression of cell death, autophagy, and inflammation. By contrast, twelve miRNAs were differentially expressed in the heart following CO treatment. Many miRNAs previously shown to be responsible for suppressing cell death, autophagy, and myocardial hypertrophy were upregulated (e.g., 125b-3p, 19-3p, and 21a-5p). Finally, some miRNAs (miR-103-3p, miR-1a-3p, miR-199a-1-5p) which have been implicated in regulating energy balance and cardiac contraction were also differentially expressed. Overall, this study demonstrated that CO-mediated miRNA regulation may promote ischemic preconditioning and cardioprotection based on the putative protective roles of the differentially expressed miRNAs explored herein and the consistency of these results with those that have shown positive effects of CO on heart viability following anesthesia and ischemia-reperfusion stress.

The newborn Fmr1 knockout mouse: a novel model of excess ubiquinone and closed mitochondrial permeability transition pore in the developing heart.

BACKGROUND: Mitochondrial permeability transition pore (mPTP) closure triggers cardiomyocyte differentiation during development while pathological opening causes cell death during myocardial ischemia-reperfusion and heart failure. Ubiquinone modulates the mPTP; however, little is known about its mechanistic role in health and disease. We previously found excessive proton leak in newborn Fmr1 KO mouse forebrain caused by ubiquinone deficiency and increased open mPTP probability. Because of the physiological differences between the heart and brain during maturation, we hypothesized that developing Fmr1 KO cardiomyocyte mitochondria would demonstrate dissimilar features. METHODS: Newborn male Fmr1 KO mice and controls were assessed. Respiratory chain enzyme activity, ubiquinone content, proton leak, and oxygen consumption were measured in cardiomyocyte mitochondria. Cardiac function was evaluated via echocardiography. RESULTS: In contrast to controls, Fmr1 KO cardiomyocyte mitochondria demonstrated increased ubiquinone content and decreased proton leak. Leak was cyclosporine (CsA)-sensitive in controls and CsA-insensitive in Fmr1 KOs. There was no difference in absolute mitochondrial respiration or cardiac function between strains. CONCLUSION: These findings establish the newborn Fmr1 KO mouse as a novel model of excess ubiquinone and closed mPTP in the developing heart. Such a model may help provide insight into the biology of cardiac development and pathophysiology of neonatal heart failure. IMPACT: Ubiquinone is in excess and the mPTP is closed in the developing FXS heart. Strengthens evidence of open mPTP probability in the normally developing postnatal murine heart and provides new evidence for premature closure of the mPTP in Fmr1 mutants. Establishes a novel model of excess CoQ and a closed pore in the developing heart. Such a model will be a valuable tool used to better understand the role of ubiquinone and the mPTP in the neonatal heart in health and disease.

An isolated retrograde-perfused newborn mouse heart preparation.

The Langendorff-perfused model is a powerful tool to study biological responses in the isolated heart in the absence of confounders. The model has been adapted recently to enable study of the isolated mouse heart and the effects of genetic manipulation. Unfortunately, the small size and fragility of the mouse heart pose significant challenges, limiting application of the Langendorff model to the study of adult mice. Cardiac development is a complex and dynamic process that is incompletely understood. Thus, establishing an isolated-perfused heart model in the newborn mouse would be an important and necessary advance. Here we present a method to successfully cannulate and perfuse the isolated newborn murine heart. We describe the basic and fundamental physiological characteristics of the ex-vivo retrograde-perfused beating neonatal heart in wild-type C57Bl/6 male mice. Our approach will enable future study of the physiological and pharmacological responses of the isolated immature murine heart to enhance knowledge of how developmental cardiac biology impacts health and disease.•The Langendorff model is a powerful tool to study the heart without confounders.•An isolated-perfused newborn murine heart model has yet to be established.•We demonstrate the first successful isolated neonatal murine heart preparation.

ATP Synthase c-Subunit Leak Causes Aberrant Cellular Metabolism in Fragile X Syndrome.

Loss of the gene (Fmr1) encoding Fragile X mental retardation protein (FMRP) causes increased mRNA translation and aberrant synaptic development. We find neurons of the Fmr1-/y mouse have a mitochondrial inner membrane leak contributing to a "leak metabolism." In human Fragile X syndrome (FXS) fibroblasts and in Fmr1-/y mouse neurons, closure of the ATP synthase leak channel by mild depletion of its c-subunit or pharmacological inhibition normalizes stimulus-induced and constitutive mRNA translation rate, decreases lactate and key glycolytic and tricarboxylic acid (TCA) cycle enzyme levels, and triggers synapse maturation. FMRP regulates leak closure in wild-type (WT), but not FX synapses, by stimulus-dependent ATP synthase ß subunit translation; this increases the ratio of ATP synthase enzyme to its c-subunit, enhancing ATP production efficiency and synaptic growth. In contrast, in FXS, inability to close developmental c-subunit leak prevents stimulus-dependent synaptic maturation. Therefore, ATP synthase c-subunit leak closure encourages development and attenuates autistic behaviors.

Inefficient thermogenic mitochondrial respiration due to futile proton leak in a mouse model of fragile X syndrome.

Fragile X syndrome (FXS) is the leading known inherited intellectual disability and the most common genetic cause of autism. The full mutation results in transcriptional silencing of the Fmr1 gene and loss of fragile X mental retardation protein (FMRP) expression. Defects in neuroenergetic capacity are known to cause a variety of neurodevelopmental disorders. Thus, we explored the integrity of forebrain mitochondria in Fmr1 knockout mice during the peak of synaptogenesis. We found inefficient thermogenic respiration due to futile proton leak in Fmr1 KO mitochondria caused by coenzyme Q (CoQ) deficiency and an open cyclosporine-sensitive channel. Repletion of mitochondrial CoQ within the Fmr1 KO forebrain closed the channel, blocked the pathological proton leak, restored rates of protein synthesis during synaptogenesis, and normalized the key phenotypic features later in life. The findings demonstrate that FMRP deficiency results in inefficient oxidative phosphorylation during the neurodevelopment and suggest that dysfunctional mitochondria may contribute to the FXS phenotype.

Report on the Sixth Pediatric Anesthesia Neurodevelopmental Assessment (PANDA) Symposium, "Anesthesia and Neurodevelopment in Children".

On April 14 and 15, 2018, the Sixth Biennial Pediatric Anesthesia Neurodevelopmental Assessment (PANDA) Symposium convened at Columbia University Medical Center and New York Presbyterian/Morgan Stanley Children's Hospital of New York. Since its inception over 10 years ago, the PANDA Symposium has served as a key forum for clinicians, researchers, and other major stakeholders to gather and review the current state of preclinical and clinical research related to anesthetic neurotoxicity in the developing brain. It has also served as an important venue for participants to gain insight and leverage support from various public and private regulatory bodies. Goals of this year's meeting included assessments of how current knowledge has evolved, endeavors to develop common outcome measures, and formulations of future directions for research and policy. The Symposium program highlighted a diverse body of cutting-edge work, from results of preclinical and clinical studies to updates in clinical practice and policymaking.

Data on the effect of sex on the size, cellular content, and neuronal density of the developing brain in mice exposed to isoflurane and carbon monoxide.

The data presented here detail the changes in size, cellular content, and neuronal density of the developing brain over time with respect to sex in C57Bl/6 mice following neonatal exposure to isoflurane, carbon monoxide, or their combination. Specifically, brain weight- and brain volume-to-body weight ratios are presented, representative immunoblots of whole brain cell-specific protein content are depicted, and quantification of the number of neurons in the primary somatosensory cortex and CA3 region of the hippocampus are shown. Three discrete postnatal time points are represented: P7 (prior to exposure), P14 (one-week post exposure), and P42-56 (5-7 weeks post exposure). Major findings from the data presented here are reported in the manuscript "Carbon Monoxide Incompletely Prevents Isoflurane-induced Defects in Murine Neurodevelopment" (Wang et al., in press) [1].