Hypoxic reprogramming of vasculature relies on genetic, epigenetic, and metabolic circuitry, but the control points are unknown. In pulmonary arterial hypertension (PAH), a disease driven by hypoxia inducible factor (HIF)–dependent vascular dysfunction, HIF-2α promoted expression of neighboring genes, long noncoding RNA (lncRNA) histone lysine N -methyltransferase 2E-antisense 1 ( KMT2E-AS1 ) and histone lysine N-methyltransferase 2E ( KMT2E ). KMT2E-AS1 stabilized KMT2E protein to increase epigenetic histone 3 lysine 4 trimethylation (H3K4me3), driving HIF-2α–dependent metabolic and pathogenic endothelial activity. This lncRNA axis also increased HIF-2α expression across epigenetic, transcriptional, and posttranscriptional contexts, thus promoting a positive feedback loop to further augment HIF-2α activity. We identified a genetic association between rs73184087, a single-nucleotide variant (SNV) within a KMT2E intron, and disease risk in PAH discovery and replication patient cohorts and in a global meta-analysis. This SNV displayed allele (G)–specific association with HIF-2α, engaged in long-range chromatin interactions, and induced the lncRNA-KMT2E tandem in hypoxic (G/G) cells. In vivo, KMT2E-AS1 deficiency protected against PAH in mice, as did pharmacologic inhibition of histone methylation in rats. Conversely, forced lncRNA expression promoted more severe PH. Thus, the KMT2E-AS1 /KMT2E pair orchestrates across convergent multi-ome landscapes to mediate HIF-2α pathobiology and represents a key clinical target in pulmonary hypertension.

In Escherichia coli, the cytosolic chaperone SecB is responsible for the selective entry of a subset of precursor proteins into the Sec pathway. In vitro, SecB binds to a variety of unfolded substrates without apparent sequence specificity, but not native proteins. Selectivity has therefore been suggested to occur by kinetic partitioning of substrates between protein folding and SecB association. Evidence for kinetic partitioning is based on earlier observations that SecB blocks the refolding of the precursor form of maltose-binding protein (preMBP)5 and slow-folding maltose-binding protein (MBP) mutants, but not faster-folding mature wild-type MBP. In order to quantitatively validate the kinetic partitioning model, we have independently measured each of the rate constants involved in the interaction of SecB with refolding preMBP (a physiological substrate of SecB) and mature MBP. The measured rate constants correctly predict substrate folding kinetics over a wide range of SecB, MBP, and preMBP concentrations. Analysis of the data reveals that, for many substrates, kinetic partitioning is unlikely to be responsible for SecB-mediated protein export. Instead, the ability of SecB-bound substrates to continue folding while bound to SecB and their ability to interact with other components of the secretory machinery such as SecA may be key opposing determinants that inhibit and promote protein export, respectively.

Purpose Develop and test a flexible, scalable tool using interrupted time series (ITS) analysis to assess the impact of Food and Drug Administration (FDA) regulatory actions on drug use. Methods We applied the tool in the Sentinel Distributed Database to assess the impact of FDA's 2010 drug safety communications (DSC) concerning the safety of long-acting beta2-agonists (LABA) in adult asthma patients. We evaluated changes in LABA use by measuring the initiation of LABA alone and concomitant use of LABA and asthma controller medications (ACM) after the DSCs. The tool generated ITS graphs and used segmented regression to estimate baseline slope, level change, slope change, and absolute and relative changes at up to two user-specified time point (s) after the intervention. We tested the tool and compared our results against prior analyses that used similar measures. Results Initiation of LABA alone declined among asthma patients aged 18-45 years before FDA DSCs (-0.10% per quarter; 95%CI: -0.11% to -0.09%) and the downward trend continued after. Concomitant use of LABA and ACM was stable before FDA DSCs. After FDA DSCs, there was a small trend decrease of 0.006% per quarter (95% CI, -0.008% to -0.003%). We found similar results among those aged 46-64 years and patients with poorly-controlled asthma. Our results were consistent with previous studies, confirming the performance of the new tool. Conclusions We developed and tested a reusable ITS tool in real-world databases formatted to the Sentinel Common Data Model that can assess the impact of regulatory actions on drug use.

BACKGROUND: The US Food and Drug Administration increasingly uses administrative databases to conduct surveillance of medications used during pregnancy. To assess adverse fetal effects, administrative records must be linked between the mother and infant. The Sentinel Initiative's Mother-Infant Linkage Table provides a large cohort of linked deliveries from national, regional, and public insurance claims in the United States for the US Food and Drug Administration to conduct surveillance. OBJECTIVE: This study aimed to describe baseline health conditions and prescription medication use during pregnancy in cohorts of women with a live-birth delivery linked and not linked to an infant. STUDY DESIGN: Live-birth deliveries in women aged 10 to 54 years with at least 391 days of medical and drug coverage before delivery were identified in the Sentinel Mother-Infant Linkage Table from 2000 to 2019. Two cohorts were created for analysis: deliveries linked to infant records (linked deliveries, n=2,320,805) and deliveries unable to be linked to an infant (not-linked deliveries, n=504,785). Baseline health conditions, pregnancy history, healthcare utilization, and pregnancy conditions were defined using International Classification of Diseases, Ninth Revision, and International Classification of Diseases, Tenth Revision, diagnosis and procedure codes. Medication exposure was identified in a 90-day prepregnancy period and in each trimester. RESULTS: Few notable differences were observed between the linked and not-linked deliveries except for maternal age and preterm birth; the not-linked cohort was 3.4 years younger on average and more likely to have a preterm delivery. At baseline among the linked deliveries, the most common conditions were depression and anxiety (5.2% each), acquired hypothyroidism (5.0%), pain conditions (3.9%), and asthma (2.8%). Among linked deliveries, 6.9% had evidence of gestational diabetes mellitus, 3.9% had gestational hypertension, and 4.5% had preeclampsia or eclampsia. The most commonly used medications during pregnancy in the linked deliveries were antibacterials (41.6%) and antiemetics (21.5%); similar medication use patterns were observed in the not-linked cohort. Age trends were observed for some medication groups; anti-infectives, pain medications, and antiemetics were more common in younger mothers, whereas endocrine medications were more common in older mothers. CONCLUSION: Similarities between the linked and not-linked cohorts suggested that the ability to link mother and infant records is not influenced by maternal health status. In the linked cohort, the prevalence of specific pregnancy complications and medication use were similar to previously reported national estimates. Some baseline comorbidities, such as obesity and smoking, may be underestimated in the Sentinel Mother-Infant Linkage.

Abstract Background In the United States, at least 50,000 emergency department visits for tick bite and an estimated 476,000 Lyme disease diagnoses occur annually, with incidence of both high among children. The majority of these healthcare visits occur in the northeastern and midwestern states having high Lyme disease incidence and during the summer and fall months, corresponding to peak opportunities for exposure to blacklegged ticks. Post-exposure prophylaxis (PEP) with a single dose of doxycycline can effectively prevent Lyme disease after a tick bite that is high risk for transmission of Lyme disease. We describe characteristics of patients with dispensings of single-dose doxycycline in a large US-based system that includes patients enrolled in private and public health insurance plans. Methods Single-dose doxycycline (≤200 mg) dispensings during January 2009 – February 2020 were identified for patients enrolled in seven Data Partners that contributed electronic healthcare data to the Food and Drug Administration Sentinel Distributed Database, including large national insurers, an integrated delivery care network, a state Medicaid, and the 100% Medicare fee-for-service plan. We examined patient and PEP dispensing characteristics by patient age, state of residence, and month of dispensing. Results We identified 408,897 patients with PEP (n=474,414 total dispensings) with a mean age of 60 years at first dispensing. Overall, there were 21 patients per 10,000 eligible members with PEP dispensings. Dispensings were less common in children (< 1 and 4 patients per 10,000 eligible members aged < 8 and 8-18 years, respectively). Most dispensings (72%) occurred in states with high incidence of Lyme disease. Seasonality of dispensings was bimodal, with most occurring during April – July and October – November (71 – 83%, by year). Conclusion Lyme disease PEP was relatively common and mirrored geographic and seasonal trends observed for ED visits for tick bites and Lyme disease diagnoses. However, we observed more PEP among older adults, and few dispensings among children. Despite healthcare visits for tick bites and Lyme disease occurring disproportionately among pediatric age groups, PEP appears to be underutilized in children. Disclosures All Authors: No reported disclosures

Carbon monoxide (CO) remains the most common cause of human poisoning. The consequences of CO poisoning include cardiac dysfunction, brain injury, and death. CO causes toxicity by binding to hemoglobin and by inhibiting mitochondrial cytochrome c oxidase (CcO), thereby decreasing oxygen delivery and inhibiting oxidative phosphorylation. We have recently developed a CO antidote based on human neuroglobin (Ngb-H64Q-CCC). This molecule enhances clearance of CO from red blood cells in vitro and in vivo. Herein, we tested whether Ngb-H64Q-CCC can also scavenge CO from CcO and attenuate CO-induced inhibition of mitochondrial respiration. Heart tissue from mice exposed to 3% CO exhibited a 42 ? 19% reduction in tissue respiration rate and a 33 ? 38% reduction in CcO activity compared with unexposed mice. Intravenous infusion of Ngb-H64Q-CCC restored respiration rates to that of control mice correlating with higher electron transport chain CcO activity in Ngb-H64Q-CCC?treated compared with PBS-treated, CO-poisoned mice. Further, using a Clark-type oxygen electrode, we measured isolated rat liver mitochondrial respiration in the presence and absence of saturating solutions of CO (160 ?m) and nitric oxide (100 ?m). Both CO and NO inhibited respiration, and treatment with Ngb-H64Q-CCC (100 and 50 ?m, respectively) significantly reversed this inhibition. These results suggest that Ngb-H64Q-CCC mitigates CO toxicity by scavenging CO from carboxyhemoglobin, improving systemic oxygen delivery and reversing the inhibitory effects of CO on mitochondria. We conclude that Ngb-H64Q-CCC or other CO scavengers demonstrate potential as antidotes that reverse the clinical and molecular effects of CO poisoning.

BACKGROUND:Physical function decreases with age, and though bioenergetic alterations contribute to this decline, the mechanisms by which mitochondrial function changes with age remains unclear. This is partially because human mitochondrial studies require highly invasive procedures, such as muscle biopsies, to obtain live tissue with functional mitochondria. However, recent studies demonstrate that circulating blood cells are potentially informative in identifying systemic bioenergetic changes. Here, we hypothesize that human platelet bioenergetics reflect bioenergetics measured in muscle biopsies.METHODS & RESULTS:We demonstrate that maximal and ATP-linked respiratory rate measured in isolated platelets from older adults (86-93 years) correlates significantly with maximal respiration (r = 0.595; P = 0.003) measured by muscle biopsy respirometry and maximal ATP production (r = 0.643; P = 0.004) measured by 31P-MRS respectively, in the same individuals. Comparison of platelet bioenergetics in this aged cohort to platelets from younger adults (18-35 years) shows aged adults demonstrate lower basal and ATP-linked respiration. Platelets from older adults also show enhanced proton leak, which is likely due to increased protein levels of uncoupling protein 2, and correlates with increased gate speed in this cohort (r = 0.58; P = 0.0019). While no significant difference in glycolysis was observed in older adults compared to younger adults, platelet glycolytic rate correlated with fatigability (r = 0.44; P = 0.016).CONCLUSIONS:These data advance the mechanistic understanding of age-related changes in mitochondrial function. Further, they suggest that measuring platelet bioenergetics provides a potential supplement or surrogate for muscle biopsy measurement and may be a valuable tool to study mitochondrial involvement in age-related decline of physical function.

GCN5L1 regulates protein acetylation and mitochondrial energy metabolism in diverse cell types. In the heart, loss of GCN5L1 sensitizes the myocardium to injury from exposure to nutritional excess and ischemia/reperfusion injury. This phenotype is associated with the reduced acetylation of metabolic enzymes and elevated mitochondrial reactive oxygen species (ROS) generation, although the direct molecular targets of GCN5L1 remain largely unknown. In this study, we sought to determine the mechanism by which GCN5L1 impacts energy substrate utilization and mitochondrial health. We find that hypoxia and reoxygenation (H/R) leads to a reduction in cell viability and Akt phosphorylation in GCN5L1 knockdown AC16 cardiomyocytes, in parallel with elevated glucose utilization and impaired fatty acid use. We demonstrate that glycolysis is uncoupled from glucose oxidation under normoxic conditions in GCN5L1-depleted cells. We show that GCN5L1 directly binds to the Akt-activating mTORC2 component Rictor, and that loss of Rictor acetylation is evident in GCN5L1 knockdown cells. Finally, we show that restoring Rictor acetylation in GCN5L1-depleted cells reduces mitochondrial ROS generation and increases cell survival in response to H/R. These studies suggest that GCN5L1 may play a central role in energy substrate metabolism and cell survival via the regulation of Akt/mTORC2 signaling.

Aims: Asthma, characterized by airway obstruction and hyper-responsiveness, is more severe and less responsive to treatment in obese subjects. While alterations in mitochondrial function and redox signaling have been implicated in asthma pathogenesis, it is unclear whether these mechanisms differ in lean versus obese asthmatics. In addition, we previously demonstrated that circulating platelets from asthmatic individuals have altered bioenergetics; however, it is unknown whether platelet mitochondrial changes reflect those observed in airway epithelial cells. Herein we hypothesized that lean and obese asthmatics show differential bioenergetics and redox signaling in airway cells and that these alterations could be measured in platelets from the same individual. Results: Using freshly isolated bronchial airway epithelial cells and platelets from lean and obese asthmatics and healthy individuals, we show that both cell types from obese asthmatics have significantly increased glycolysis, basal and maximal respiration, and oxidative stress compared with lean asthmatics and healthy controls. This increased respiration was associated with enhanced arginine metabolism by arginase, which has previously been shown to drive respiration. Inducible nitric oxide synthase (iNOS) was also upregulated in cells from all asthmatics. However, due to nitric oxide synthase uncoupling in obese asthmatics, overall nitric oxide (NO) bioavailability was decreased, preventing NO-dependent inhibition in obese asthmatic cells that was observed in lean asthmatics. Innovation and Conclusion: These data demonstrate bioenergetic differences between lean and obese asthmatics that are, in part, due to differences in NO signaling. They also suggest that the platelet may serve as a useful surrogate to understand redox, oxidative stress and bioenergetic changes in the asthmatic airway.

GCN5L1 regulates mitochondrial protein acetylation, cellular bioenergetics, reactive oxygen species (ROS) generation, and organelle positioning in a number of diverse cell types. However, the functional role of GCN5L1 in the heart is currently unknown. As many of the factors regulated by GCN5L1 play a major role in ischemia-reperfusion (I/R) injury, we sought to determine if GCN5L1 is an important nexus in the response to cardiac ischemic stress. Deletion of GCN5L1 in cardiomyocytes resulted in impaired myocardial post-ischemic function and increased infarct development in isolated work-performing hearts. GCN5L1 knockout hearts displayed hallmarks of ROS damage, and scavenging of ROS restored cardiac function and reduced infarct volume in vivo. GCN5L1 knockdown in cardiac-derived AC16 cells was associated with reduced activation of the pro-survival MAP kinase ERK1/2, which was also reversed by ROS scavenging, leading to restored cell viability. We therefore conclude that GCN5L1 activity provides an important protection against I/R induced, ROS-mediated damage in the ischemic heart.

Background: Pulmonary hypertension (PH) is a deadly vascular disease with poorly defined molecular origins. While evidence is emerging in regard to dysregulation of long non-coding RNAs (lncRNAs) i...

Although acute exposure to hypoxia can disrupt metabolism, longer-term exposure may normalize glucose homeostasis or even improve glucose disposal in the presence of obesity. We examined the effects of two-week exposure to room air (Air), continuous 10% oxygen (C10%), and 12 hr nocturnal periods of 10% oxygen (N10%) on glucose disposal, insulin responsiveness, and mitochondrial function in lean and obese C57BL/6J mice. Both C10% and N10% improved glucose disposal relative to Air in lean and obese mice without evidence of an increase in insulin responsiveness; however, only the metabolic improvements with N10% exposure occurred in the absence of confounding effects of weight loss. In lean mice, N10% exposure caused a decreased respiratory control ratio (RCR) and increased reactive oxygen species (ROS) production in the mitochondria of the muscle and liver compared to Air-exposed mice. In the absence of hypoxia, obese mice exhibited a decreased RCR in the muscle and increased ROS production in the liver compared to lean mice; however, any additional effects of hypoxia in the presence of obesity were minimal. Our data suggest that the development of mitochondrial inefficiency may contribute to metabolic adaptions to hypoxia, independent of weight, and metabolic adaptations to adiposity, independent of hypoxia.

Supplementary figures. (ZIP 623 kb)