bluuebunny/crossref_metadata_2025 huggingface dataset:
Cough‐Induced Hyperventilation Does Not Elicit Prolonged Apnea During The Cough‐to‐Eupnea Transition Period
Authors: Musselwhite M , Pitts Teresa , Shen Tabitha , Rose Melanie , Poliacek Ivan , Morris Kendall , Davenport Paul , Bolser Donald
Published: June 2021

Repetitive cough induces profound hyperventilation with no loss of motor drive to the respiratory muscles. However, this hyperventilation routinely results in PC02 values of < 20 mmHg, and arterial pH of > 7.5. This cough‐induced derangement presents a challenge for the respiratory control system especially during the cough‐eupnea transition. We hypothesized that during this transition: a) there would be limited apnea, and b) the eupneic cycles would be significantly affected by the previous level of hyperventilation. Repetitive cough was elicited by mechanical stimulation of the intra‐thoracic trachea in anesthetized, spontaneously breathing cats (N = 6). Electromyograms were recorded from the diaphragm, parasternal, and internal oblique. During repetitive coughing, C02 levels dropped from a mean of 33.05 + 2.16 mmHg to 11.67 + 3.61 mmHg. The cough‐to‐eupnea transition was marked by hypopnea durations of 17.59 + 7.7 s. CO2 levels during cough trials had a small negative correlation with cough‐to‐eupnoea transition duration [r(88) = ‐.26, p < .05]. Furthermore, the eupneic CO2 levels had a moderate positive correlation with the lowest CO2 reached during the repetitive cough trials [r(88) = .41, p < .01]. These results support a significant reduction in apneic threshold during the cough‐to‐eupnea transition. This lowered apneic threshold likely functions to buffer cough induced hyperventilation; minimizing the probability of further oscillations in blood gasses and pH as the respiratory system returns to homeostasis. Supported by HL 103425 and HL 111215.
An In Vivo Anesthetic Model of Autoresuscitation Failure in Adult Rats
Authors: Krause Andrew , Nowak Zachary , Srbu Rebeka , Bell Harold
Published: June 2021

In studying the cardiorespiratory response to acute severe hypoxia, we observed that urethane anesthesia had deleterious effects on respiratory autoresuscitation. To investigate this effect further, we assessed the cardiorespiratory response to anoxia‐induced respiratory arrest (RA) in 3 groups of anesthetized, spontaneously breathing adult male rats. Three common injectables were used to achieve a surgical plane of anesthesia: ketamine‐xylazine (KET, n=10), pentobarbital (PEN, n=10), and urethane (URE, n=10). All KET and PEN animals successfully resuscitated from 4 consecutive RAs. In stark contrast, none of the URE animals were able to mount a successful autoresuscitation. Interestingly, only 4/10 URE animals were capable of expressing resuscitating breaths following the onset of RA. Arterial PO2 at RA was not different across groups (avg = 12.1 ± 2.6 mmHg). Duration of primary apnea was longest in the URE animals (141.90 ± 19.38) compared to either KET (54.30 ± 6.96) or PEN (68.35 ± 12.87) animals. The average and largest volumes of resuscitating breaths were smaller in the URE condition compared to either KET or PEN. These alterations are suggestive of a profound alteration of central control over switching between modes of respiratory rhythm generation. URE animals showed no clear aberrations in their cardiovascular responses to anoxia, with the exception of lower arterial pulse pressures compared to either KET or PEN animals at specific points following RA. Our results suggest that urethane anesthesia may provide a valuable model of catastrophic failure of the autoresuscitation process that will enable identification of physiological and neurobiological factors that predispose some individuals to sudden death during an acute bout of asphyxia.
Intraspinal Electrical Stimulation Reliably Triggers Respiratory Activity in the in situ Rodent Model
Authors: Samuel Kelara , Amirzadehasl Parisa , Schwanebeck Kelly , Denson Heather , Guarnieri Lauren , Molina Rene , Gonzalez‐Rothi Elisa , Fuller David , Baekey David
Published: June 2021

Cervical spinal cord injury impairs ventilation by preventing brainstem generated respiratory activity from reaching motor pools in the spinal cord which drive respiratory pump muscles. Epidural high frequency thoracic spinal cord stimulation is a method of producing respiratory efforts independent of centrally generated signals, which is effective at eliciting diaphragmatic EMG in anesthetized animal preparations. However, the exact spinal pathways and circuitry involved in producing these respiratory efforts are unknown. We hypothesize that targeted electrical stimulation within the spinal cord will identify pathways relaying the stimulus to the phrenic motoneurons. To investigate this, we monitored bilateral phrenic nerve activity (PNA) in the in situ rat preparation (n=6) and applied either bipolar epidural or intraspinal stimulation (ISS) (300ms, 100‐300Hz, 200μs pd) in the rostral thoracic spinal cord (T2‐6). Our initial findings demonstrated that epidural stimulation was only intermittently effective in eliciting PNA, while ISS reliably produced site and current dependent (10‐500μA) responses. We conclude that, in comparison to epidural stimulation, low current thoracic ISS more efficiently and reliably activates the phrenic motor circuit in the in situ model. Ongoing studies are focused on identifying the neural circuits activated by ISS. Funded by the UF College of Veterinary Medicine, APS UGSRF, and NIH 1R01NS080180‐01A1
Regional Genioglossus EMG Highlights Persistent Daytime Activation in OSA
Authors: Vranish Jennifer , Bailey E.Fiona
Published: June 2021

The genioglossus (GG) is an extrinsic muscle of the human tongue that plays a critical role in preserving airway patency. Recent research has identified regional differences in muscle fiber type and myosin heavy chain expression giving rise to the possibility that the anterior and posterior GG may fulfill distinct functions. Here, we recorded GG EMG activity in anterior and posterior regions of the muscle, across upright and supine, and in involuntary (CPG‐driven) and volitionally modulated breathing tasks in healthy adults and adults with OSA. Using this step‐wise approach we tested the hypothesis that respiratory CPG‐driven inputs preferentially drive the posterior muscle region and that voluntary (i.e., motor cortex) inputs drive activation in the more anterior region of the muscle. Our results show clear differences in the magnitude of GG EMG (% of maximum) between anterior and posterior muscle regions (7.95±0.57 vs. 11.10±0.99, respectively; P < 0.001), between upright and supine (8.63±0.73 vs. 10.42±0.90, respectively; P = 0.008) and between volitionally modulated and CPG driven breathing or deep breathing (P < 0.001). Whereas an exclusively voluntary task results in greatest activation in the anterior tongue (P <0.001), volitionally modulated breathing tasks are associated with greatest activation in the posterior muscle (P <0.001). Preliminary findings indicate that whereas net drive to the GG is far greater in the OSA patient, the distribution of drive does not differ appreciably between the two groups. Our findings provide additional evidence that a nighttime breathing disorder impacts upon day‐time activation of a pharyngeal dilator muscle in a predictable fashion.
Bronchopulmonary Dysplasia‐associated Pulmonary Hypertension and Mutations in the DDAH1 Gene
Authors: Trittmann Jennifer , Gastier‐Foster Julie , Vieland Veronica , Klebanoff Mark , Chicoine Louis , Nelin Leif
Published: June 2021

Pulmonary hypertension (PH) is the most common comorbidity in bronchopulmonary dysplasia (BPD). Nitric oxide (NO) is produced by endothelial NO synthase (eNOS) in pulmonary endothelial cells and is a potent vasodilator. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of eNOS. Our group has recently published that ADMA levels are elevated in patients with BPD‐associated PH. ADMA is degraded by the enzyme Nᴳ,Nᴳ‐ dimethylarginine dimethylaminohydrolase (DDAH). We tested the hypothesis that there are single nucleotide polymorphisms (SNPs) in DDAH1 associated with PH in BPD. Neonates diagnosed with BPD were enrolled at Nationwide Children's Hospital. DNA was assayed for 34 SNPs in DDAH1. Calculated minor allele frequency (MAF) for cases and controls were compared using chi‐square. Assuming recessive inheritance, logistic regression was used to estimate the odds ratio. Among 157 patients with BPD, 53 (34%) also had echocardiographic evidence of PH. Gestational age, birth weight, gender, and ethnicity were not different between groups. DDAH1 rs11161637 SNP had a significantly (p=0.04) lower MAF in cases than in controls (0.25 vs. 0.34). DDAH1 rs11161637 was associated with a decreased risk (OR=0.27) of PH in this BPD cohort, although this did not reach statistical significance (p‐value=0.06). These results indicate that rs11161637 SNP of DDAH1 may be protective against the development of PH in patients with BPD. We speculate that the DDAH1 SNP may be a useful biomarker in developing predictive models for PH in patients with BPD.
GPR171, a Newly Deorphanized Hypothalamic G Protein‐Coupled Receptor is Involved in the Regulation of Reward‐Related Behaviors
Authors: Bobeck Erin , Gomes Ivone , Fujita Wakako , Devi Lakshmi
Published: June 2021

Recent neuropeptidomics studies have demonstrated that ProSAAS‐derived peptides, including BigLEN (b‐LEN) are abundant in the hypothalamus and enriched in the agouti related peptide (AgRP) neurons of the arcuate nucleus. Additional studies have implicated b‐LEN in acute feeding. Recently we deorphanized GPR171, as a G protein‐coupled receptor for b‐LEN. Overexpression of GPR171 leads to an increase and knockdown to a decrease in b‐LEN binding and signaling. In the hypothalamus GPR171 expression complements the expression of b‐LEN. Lentiviral shRNA‐mediated knockdown of GPR171 in the hypothalamus leads to alterations in food intake and metabolism. To further investigate the role of GPR171, we used a designer receptor exclusively activated by designer drugs (DREADD) approach to activate AgRP neurons leading to the release of b‐LEN (and other neuropeptides). As expected, activation of these neurons with clozapine‐N‐oxide leads to increases in food intake. Interestingly, this increase is attenuated by co‐administration of a GPR171‐selective small molecule antagonist. Given the known hedonic effects of activating these neuropeptide systems in the hypothalamus, we investigated additional reward‐related behaviors. We find that activation of AgRP neurons leads to a reduction in anxiety and depression. The specific role of b‐LEN/GPR171 in this phenomenon is currently being examined using GPR171 selective ligands. In addition, we find that stress alters GPR171 mRNA expresssion. In conclusion, GPR171 is the receptor for b‐LEN and plays a role in feeding and reward‐related behaviors.

This work was supported in part by NIH grants NS026880 and DA019521 to L.A.D and a NIDA postdoctoral training grant DA007135 to E.N.B.
Pregnenolone Sulfate as a Modulator of Synaptic Plasticity
Authors: Sugunan Kavitha , Singh Rumani , Badolato Ryan , Shetty Syona , Zambrano Mateo , Duval Kayla , Adams John , Luebke Jennifer , Kumaresan Vidhya , Farb David
Published: June 2021

Neurosteroids such as pregnenolone have been implicated in the pathophysiology of neuropsychiatric disorders such as schizophrenia and cannabinoid addiction (1, 2). Our laboratory discovered that the unique negatively charged steroid pregnenolone sulfate (PregS), the product of pregnenolone metabolism via a single sulfation step, modulates fast excitatory synaptic transmission by potentiating NMDA receptor activity in a fashion that is balanced by AMPA receptor inhibition at micromolar concentrations. We recently reported finding a novel high affinity response to PregS (EC50 = 2 pM) that increases intracellular [Ca2+] and CREB activation in cultured rat cortical neurons. Here, we report that PregS increases glutamate receptor and PSD95 colocalized puncta, presumably on dendritic spines of hippocampal neurons, suggesting that PregS increases surface‐postsynaptic AMPARs and NMDARs. Whole cell recordings of primary rat hippocampal neurons revealed that PregS increases the frequency of spontaneous excitatory postsynaptic currents, suggesting an increase in glutamate release from presynaptic terminals or an increase in the number of activatable synapses. The results indicate that PregS acts within the physiological range of endogenous concentrations (3) to recruit AMPA receptors to postsynaptic sites, thereby altering synaptic plasticity.

(1) Marx et al. (2009) Neuropsychopharmacology 34:1885‐1903

(2) Vallée et al. (2014) Science 343(6166):94‐8

(3) Rustichelli et al. (2013) J Chromatogr B Analyt Technol Biomed Life Sci. 930:62‐9
Drug Discrimination in (+)‐Epibatidine‐Trained Monkeys: Agonists and Antagonists Effects of Nicotinic Drugs
Authors: Desai Rajeev , Doyle Michelle , Bergman Jack
Published: June 2021

The behavioral effects of nicotinic agonists and antagonists were studied in squirrel monkeys using a two‐lever drug discrimination procedure. Monkeys (n=4) were trained to discriminate i.m. injections of 0.001 mg/kg (+)‐epibatidine (EPI)–a α4β2 selective nicotinic agonist that is pharmacologically similar and structurally distinct from nicotine (NIC)–from saline on a 10‐response fixed‐ratio schedule of stimulus‐termination. Results show that high efficacy nicotinic agonists [(+)‐EPI, (‐)‐EPI, NIC] substituted fully for (+)‐EPI, whereas the highest doses of other nicotinic agonists produced intermediate levels of (+)‐EPI‐like discriminative‐stimulus (SD) effects [varenicline (VAR), cytisine (CYT)] or did not substitute for (+)‐EPI (lobeline). Minor tobacco alkaloids produced full (nornicotine, anabasine, myosmine, anatabine), intermediate (anabaseine) or no (cotinine) (+)‐EPI‐like effects. Pretreatment studies with nicotinic antagonists show that: a) mecamylamine (non‐selective) unsurmountably antagonized (+)‐EPI's effects; b) dihydro‐β‐erthroidine (α4β2‐selective) surmountably (>3‐fold rightward shift) blocked (+)‐EPI's effects; and c) methyllycaconitine (α7 selective) and hexamethonium (peripherally‐restrictive) failed to modify the SD effects of (+)‐EPI. In further studies, pretreatment with the partial nicotine agonists VAR and CYT did not block the SD effects of (+)‐EPI, and in fact, shifted the (+)‐EPI dose‐effect curve to the left (>3‐10‐fold). These results suggest that the SD effects of (+)‐EPI are mediated through a α4β2 nicotinic receptor subtype at which the partial agonists VAR and CYT do not exert partial antagonist actions (supported by NIH DA031231).
Effect of Dopamine D1 Receptor Activation on Extinction of Morphine CPP
Authors: Kobrin Kendra , Arena Danielle , Heinrichs Stephen , Kaplan Gary
Published: June 2021

The dopamine D1 receptor (D1R) mediates drug reward and morphology of accumbal neurons. We used a morphine conditioned place preference (CPP) mouse model to study the role of D1R in context‐reward associative learning and related dendritic morphology changes. To acquire CPP, a morphine group received saline or morphine 10 mg/kg s.c. on alternate days, while CPP controls received saline on all days, immediately before 8 daily place conditioning sessions. Post‐conditioning, morphine CPP mice expressed significantly higher place preference than saline mice. To induce extinction, mice were repeatedly exposed to the place conditioning environment in a drug‐free state. Morphine CPP mice received s.c. saline, 0.5 mg/kg SKF81297 (D1R agonist), or 0.8 mg/kg SKF81297 immediately after each extinction session, while controls received saline. After every two days of extinction training mice were tested for place preference. D1R agonist treatment inhibited extinction of place preference in a dose dependent manner. Mean place preference score of mice treated with the highest dose of agonist was double that of mice treated with saline. One day after the final place preference test brains were isolated and processed using Golgi‐Cox staining followed by digital tracing of accumbal neuron morphology. We previously showed that extinction of morphine CPP is associated with decreased dendritic complexity of neurons in the nucleus accumbens core. Since D1R agonist inhibits CPP extinction, we hypothesize that neurons from mice treated with 0.8 mg/kg SKF81297 will be prevented from undergoing extinction‐related changes, and will have higher dendritic complexity than controls.
Lipid‐derived aldehyde, acrolein, is a critical mediator of alcohol‐induced gut‐liver injury in alcoholic liver disease
Authors: Chen Wei‐Yang , Zhang Jingwen , Barve Shirish , McClain Craig , Joshi‐Barve Swati
Published: June 2021

Alcohol consumption can cause alcoholic liver disease (ALD). Chronic alcohol consumption causes a pro‐oxidant environment in the liver and increases hepatic lipid peroxidation. Acrolein (ACR) is the most reactive and toxic aldehyde generated through lipid peroxidation. ACR forms protein adducts and triggers endoplasmic reticulum (ER) stress and hepatocyte apoptosis, which are recognized etiologic factors in ALD. Moreover, recent evidence has established the critical role of the gut‐liver axis in ALD pathogenesis, wherein alcohol‐induced gut barrier dysfunction contributes to liver injury. This study investigates the pathogenic role of acrolein as a major mediator gut‐liver axis in ALD.

Accumulation of ACR adducts was seen in response to alcohol consumption in mouse livers and intestines. Acrolein adduct accumulation correlated with hepatic steatosis, JNK activation, ER stress, apoptosis and liver injury. We used cultured hepatic and intestinal cells to examine the direct in vitro effects of acrolein exposure in comparison to alcohol exposure. Alcohol‐induced in vivo intestinal effects were mimicked by ACR in vitro in intestinal Caco2 cells; specifically, ACR down‐regulated tight junction proteins, resulting in disruption of TEER (transepithelial electrical resistance), and increased FD‐4 permeability. In vitro acrolein exposure in hepatic cells triggered ER stress and induced apoptotic cell death, indicating that acrolein may mediate the effects of alcohol. Notably, these effects were attenuated by acrolein scavengers suggesting their therapeutic potential in ALD.
Toxicological study of β—HPC and innovative potential drug for metabolic syndrome
Authors: Mendoza‐Rivera Brissa , Reyes‐Esparza Jorge , De la Cruz‐Codero Ricardo , Rodriguez‐Fragoso Lourdes
Published: June 2021

β‐hydroxyphospho‐carnitine (β—HPC) has a pharmacological activity to decrease serum glucose, cholesterol and triglycerides, as well as, it is able to reduce body weight in obese rats. Then, these effects make to β—HPC a suitable drug for treating metabolic syndrome (MS). However, no data about its safety are available. Therefore, this study was focused to evaluate the toxicological effects of the β—HPC after a chronic administration. For the study we used male Wistar rats 120 gr. The experimental animals were divided into two groups of, a control group and other group receiving β—HPC (100 mg/kg) by oral gavage for 24 weeks. The results showed that there were no significant changes in body weight, bone marrow cell cycle, blood count parameters, blood chemistry, kidney and liver function in the control group and the group treated with the β—HPC. Furthermore, no morphological changes were observed in the structure of the different tissues analyzed. In conclusion, β‐HPC has not significant toxic effects. β—HPC could therefore be potentially used in the treatment of metabolic syndrome.
Involvement of Prostaglandins in the Pharmacological Actions of L‐Cysteine in Ocular Tissues
Authors: Nguyen Olivia , Robinson Jenaye , Mitchell Leah , Opere Catherine , Ohia Sunny , Njie‐Mbye Ya Fatou
Published: June 2021

Evidence from our laboratory demonstrates that hydrogen sulfide (H2S) can exert pharmacological actions in ocular tissues, an effect that can be blocked by inhibitors of cyclooxygenase (COX).

Purpose

To investigate whether H2S (using L‐cysteine as substrate) can alter prostaglandin E2 (PGE2) production in isolated bovine irides and retina.

Methods

Isolated bovine irides and retinae were incubated in oxygenated Krebs buffer for 30 mins, prior to treatment with different concentrations of L‐cysteine (1 nM ‐ 10 µM) and norepinephrine (NE, 1 µM). After incubation and termination of reaction, tissues were homogenized and both homogenates and incubation media were collected and prepared for PGE2 Enzyme‐immunoassay (EIA) using a well‐established methodology.

Results

In both bovine isolated irides and retinae, L‐cysteine elicited a concentration‐dependent (1 nM ‐ 10 µM) increase in PGE2 concentrations over basal levels. For instance, L‐cysteine (10nM; irides) and (10 µM; retina) produced an equi‐effective significant (p< 0.05) increase in PGE2 concentrations over basal levels. In bovine irides (but not retinae), L‐cysteine (10 µM) produced an increase in PGE2 levelsin the incubation media indicating a washout of prostanoids from tissues exposed to L‐cysteine. The positive control, NE (1µM) also caused an increase in PGE2 concentrations over basal levels in ocular tissues.

Conclusion

L‐cysteine can increase prostaglandin production in isolated bovine irides and retinae, affirming a role for these autacoids in the pharmacology of H2S in the eye.
Cardiac Myocyte‐Specific Overexpression of Caveolin‐3 Enhances Physiological Response to β‐Adrenergic Receptors
Authors: Busija Anna , Schilling Jan , Roth David , Insel Paul , Patel Hemal
Published: June 2021

Caveolins (Cavs) are scaffolding proteins essential to the formation of caveolae, membrane domains that compartmentalize numerous signaling molecules, including β‐adrenergic receptors (β‐ARs). Mice with cardiac myocyte (CM)‐specific overexpression of Cav‐3 (Cav‐3 OE) have more caveolae in CMs and are protected from ischemia‐reperfusion injury and other types of cardiac dysfunction. β1ARs are located throughout the plasma membrane but β2­ARs preferentially localize to caveolae, where they interact with downstream signaling effectors also located in caveolae; however the mechanism by which changes in Cav‐3 expression alter β‐AR response is unknown. Cav‐3 OE, by compartmentation of interdependent signaling components of the βAR response, may change the global and isoform‐specific physiological responses to agonism. Overall βAR and adenylyl cyclase V/VI expression in Cav‐3 OE myocytes is not altered, but the distribution of βAR signaling pathway proteins to caveolae is altered. Hearts treated with the β‐AR agonist isoproterenol (Iso) showed augmented inotropy (+dP/dt) and lusitropy (‐dP/dt) in Cav‐3 OE hearts relative to TGneg. CMs isolated from Cav‐3 OE mice have increased cAMP production in response to Iso. Moreover, agonist‐promoted responsivity of β1ARs is not changed in these hearts. Thus, increased cardiac expression of Cav‐3 enhances Iso‐mediated positive inotropy without altering­ β1AR selective response. We conclude that increased expression of Cav‐3 enhances cardiac function by enhancing responsivity of the heart in a manner independent of β1AR. Supported by NIH HL107200 and AHA 14PRE20460372.
Polyvinylpyrrolidone can be Used to Cost‐Effectively Increase the Viscosity of Culture Media
Authors: Denniss Steven , Rush James
Published: June 2021

The objective of this study was to examine of effect of polyvinylpyrrolidone(PVP) on the viscosity of Dulbecco's Modified Eagle's Medium (DMEM), a common media base used in in vitro cell culture and ex vivo organ culture experiments. The viscosity of solutions of DMEM with either 0, 2, 4, 6, 8, 9, or 10% PVP (40000 molecular weight) w/v prepared at 37 ͦ C were measured (n=4‐6 per solution) using a viscosimeter assembled using commercially‐available laboratory equipment (consisting of a syringe pump, pressure transducers, and rigid tubing). The viscosities (measured in centipoise) of the prepared medias were as follows: 1.141±0.012 (approximately that of water), 1.477±0.013, 2.010±0.005, 2.658±0.024, 3.501±0.016, 4.044±0.011 (approximately that of blood), and 4.453±0.022 for the 0, 2, 4, 6, 8, 9, and 10% PVP‐DMEM, respectively. There was a roughly linear increase in viscosity across this 0‐10% range of PVP concentrations. PVP did not alter the pH of the media at any concentration. The cost of increasing the viscosity of DMEM to that of blood (i.e. ~ 4.0 centipoise) would have been ~ 5x more expensive to accomplish if using dextran, a compound commonly used by experimenters to increase the viscosity of the media in their experiments. These data demonstrate that PVP may be a cost‐effective way to increase the viscosity of culture media.

Funded by NSERC.
Tensile Loading Induces Translational Signaling in Peripheral Nerve
Authors: Shah Sameer , Love James , Lovering Richard
Published: June 2021

Tension is a key determinant of neuronal growth and function. Though nerve stretch (strain) is often associated with damage, moderate strain at a low strain rate encourages axonal outgrowth. To understand the interplay between nerve tension and biological signaling, and as an early step to assess the potential of mechanical loading for peripheral nerve regeneration, we examined the activation of signaling pathways associated with local synthesis of new proteins in stretched nerves. Sciatic nerves of anesthetized rats were exposed bilaterally and strained 0% or 20% for 6 hrs. EMG was used to assess neuromuscular conduction latency, and quantitative immunoblotting was used to assess activation of mTOR/S6 signaling pathways (ratio of phospho to total mTOR/S6), the expression of beta‐actin, neurofilaments (NF), tubulin, and myelin basic protein, and NF phosphorylation (ratio of phospho to total NF). Repeated‐measures ANOVA/Sidak's post‐hoc was used to test the effect of strain on EMG parameters and protein expression. Stretch for 6 hours did not alter compound muscle action potential latency (p>0.9). mTOR and S6 activation were significantly increased in stretched nerves by 40% each (p<0.02), indicating a strong local translational response. Significant increases in expression of beta‐actin (125%; p<0.01) and myelin basic protein (60%; p<0.01) and in NF phosphorylation (55%; p<0.03) with stretch suggest structural remodeling in neuronal and myelinating cells to accommodate the demand for increased volume and structural integrity in lengthened nerves. Nerve stretch did not impair function and may provide a novel approach to peripheral nerve repair.
CD38‐Dependent Ca2+ Signaling Mechanisms in Pulmonary Arterial Smooth Muscle is Altered in Hypoxic Pulmonary Hypertension
Authors: Lee Suengwon , Paudel Omkar , Sham James
Published: June 2021

Prolonged exposure to hypoxia causes chronic hypoxic pulmonary hypertension (CHPH), but the underlying mechanism is not fully understood. Accumulating evidence suggests aberrant Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). This study sought to characterize the effect of CH on the expression and functions of CD38, a multi‐functional enzyme that produces the Ca2+ mobilizing messengers cyclic ADP‐ribose and nicotinic acid adenine dinucleotide phosphate, in PASMCs. CD38 protein and mRNA were upregulated in pulmonary arteries (PAs) of CH rats. It was accompanied by significant increase in NADase activity, cADPR and NAADP levels. Angiotensin II‐induced Ca2+ release (AICR), which is mediated in part by CD38, was significantly increased in PASMCs of CH rats, and the increase was completely abolished by the CD38 inhibitor nicotinamide. Endothelium‐denuded PA of 1 week CH rat exhibited spontaneous rhythmic contraction, which was not observed in PA of normoxic rat. The spontaneous contractions were blocked by inhibitors of the CD38‐dependent pathways. CH‐induced CD38 upregulation was due to direct effect of hypoxia on PASMCs. Exposure of cultured PASMCs to hypoxia for 3 days caused significant increase in CD38 expression and enhancement in AICR, which was also suppressed by nicotinamide. CD38 upregulation in hypoxia exposed PASMCs was inhibited by the specific NFAT‐inhibitor VIVIT and cyclosporine A, indicating that it is mediated by the calcineurin/NFAT‐pathway. Our results indicate that CH has a profound effect on CD38‐dependent Ca2+ signaling in PASMCs, and these changes may play an important role in the development of CHPH. (supported by HL075134 and AHA GIA)
Sex‐specific consequences of neonatal stress on laryngeal chemoreflex stimulation in rat pups: Contribution of excitatory currents onto key brainstem regions
Authors: Baldy Cécile , Chamberland Simon , Kinkead Richard
Published: June 2021

In preterm infants and rat pups, the presence of liquids or solids in the airways stimulates the laryngeal chemoreflex (LCR) which results in prolonged apneas, O2 desaturations, and bradycardias with life threatening consequences. We previously showed that neonatal stress interferes with respiratory control development and aggravates the bradycardias evoked by LCR stimulation. Here, we addressed underlying mechanisms by testing the hypothesis that neonatal stress augments synaptic inputs converging onto the vagal motor nucleus (NX), a key brainstem region regulating heart rate. Experiments were performed on 14‐15 days old pups that were either undisturbed (controls) or subjected to neonatal maternal separation (NMS; 3h/day from P3 to P12). Because the impact of NMS is greater in males, experiments were performed on pups from both sexes. Brainstem slices from the obex area were obtained and whole‐cell voltage clamp recordings were performed on single NX neurons. Miniature EPSCs were revealed by TTX application. The frequency and amplitude of spontaneous and miniature EPSCs were measured. NMS augments the frequency but not the amplitude of spontaneous EPSCs. TTX application reduced the frequency and amplitude of currents. These effects were greater in NMS rats but did not differ between sexes. This suggests that NMS increases the activity level of incoming excitatory inputs to NX neurons. We conclude that in NMS pups, the increase in excitatory inputs converging onto the vagal motor nucleus contribute to the augmented bradycardia following LCR stimulation; however, the lack of sexual dimorphism in these effects suggest that other mechanisms and/or structures are responsible for sex‐specific effects of NMS on the LCR.
Neurons in the Pontine Medial Parabrachial (PB) Region Play a Key Role In the Control of Breathing Frequency
Authors: Zuperku Edward , Prkic Ivana , Stucke Astrid , Miller Justin , Hopp Francis , Stuth Eckehard
Published: June 2021

The pontine control of the eupneic breathing pattern remains enigmatic. The pons facilitates I‐phase termination but its role in controlling the E‐phase is not well understood. We provide evidence that neurons in a small PB region (PBR) control breathing frequency (fB) via changes in TE. Multichannel neuronal recording with highly localized microinjections into the PBR were performed in decerebrate, vagotomized dogs during isocapnic hyperoxia. The phrenic neurogram was used to assess fB responses. Tachypneic responses (via decreases in TE) induced by microinjections of AMPA located the fB‐sensitive region in the medial PB complex (4.5‐5.5 mm lateral to midline; 3.0‐4.3 mm ventral to dorsal surface; 1‐2 mm caudal to the inferior colliculus; 10th‐90th PCTL; n=30). Microinjections of neurotransmitter /modulator agonists and antagonists showed that excitation of PBR neurons increase fB, while decreases in neuronal activity decrease fB, even to the point of apnea. Plots of fB vs. neuronal discharge frequency (Fn) before and during induced fB responses are linear with high correlation values (r > 0.75). Also associated with the AMPA‐induced tachypneic response is an increase in pre‐I activity of preBötzinger I‐neurons, suggesting that PBR neuronal excitation is relayed to the rhythm‐generating network. Thus, neurons in the medial PB region appear to play a primary role in fB control, and drugs that affect neuronal activity in this region have a major impact on breathing. Supt. by VA grant I01BX000721.
Orexinergic Modulation of Hypercapnic and Hypoxic Ventilatory Response in Toads
Authors: Fonseca Elisa , Dias Mirela , Bicego Kenia , Gargaglioni Luciane
Published: June 2021

Orexins (or hypocretins) play an important role in the modulation of respiratory control in mammals, but there are no data describing the role of the orexinergic system in the peripheral and central chemoreception of non‐mammalian vertebrates. Thus, the aim of this study was to examine the localization of orexin‐immunoreactive neurons in the brain of toads (Rhinella schneideri) and to investigate the contribution of orexin receptor‐1 (OX1R) to the hypoxic (5%O2) and hypercarbic (5%CO2) ventilatory responses during light and dark phases by intracerebroventricular (i.c.v.) injections of SB‐334867 (OX1R selective antagonist; dose: 5 and 10mM). Our results demonstrated that the SB‐334867 (10mM) attenuated the ventilatory response to hypercarbia during the dark phase in 54.8% by acting on tidal volume (vehicle: 172.9±5.3% of baseline; SB‐334867: 114.2±9.8% of baseline) and breathing frequency (vehicle: 218.3±24.1% of baseline; SB‐334867: 134.8±7.8% of baseline), while during the light phase, attenuated the ventilatory response to hypoxia in 51.6% by acting on tidal volume only (vehicle: 230.9±33.4% of baseline; SB‐334857: 127.5±4.9% of baseline). We conclude that inR. schneideri,orexinergic neurons are located in the suprachiasmatic nucleus and OX1R contributes to hypercarbic and hypoxic chemoreflex. Financial support: FAPESP, INCT‐FisComp and CNPq.
Novel Insights into Anatomical and Functional Roles of Nucleus Retroambiguus in Respiratory Function
Authors: Jones Sarah , Stanić Davor , Dutschmann Mathias
Published: June 2021

Purpose

The study aims to elucidate the role and contribution of nucleus retroambiguus (NRA) in generating or modulating the respiratory central pattern generator (rCPG). Previously we identified a putative rhythmogenic area 1.5 mm caudal to obex in the rat in‐situ perfused brainstem‐spinal cord preparation. Histological analysis indicated the putative rhythmogenic cell population resides at the level of the pyramidal decussation, therefore likely to overlap with the NRA. We investigated possible involvement of NRA in rhythmogenesis anatomically and functionally.

Methods

Anatomical tracing: Anterograde neuronal tracer, biotinylated dextran amine, was injected into the NRA. Respiratory areas which contained labelled terminal fields received the retrograde tracer Fast Blue in subsequent experiments. Functional testing: Glutamate microinjections were made in a grid formation across the NRA region in the rat in‐situ perfused brainstem spinal cord preparation; with whole nerve recordings taken from phrenic and vagus nerves.

Results

Anterograde and retrograde tracing revealed anatomical connectivity between the NRA and respiratory nuclei, including the pre‐Bötzinger complex, parafacial respiratory group and Kölliker Fuse nucleus. Glutamate mapping in‐situ revealed two distinct responses to pharmacological stimulation. A tachypneic response was seen predominantly overlapping the NRA whilst a breath hold response was seen more frequently in the periphery, dorsal and lateral to the NRA.

Conclusion

Anatomical evidence shows that NRA can drive respiratory modulation via ascending projections to the rCPG. Therefore the NRA is not just a bulbospinal relay, but rather an integral part of the rCPG.

Funding: Melbourne University
Coupled Oscillators Underlying Inspiration and Post‐Inspiration
Authors: Anderson Tatiana , Garcia Alfredo , Ramirez Jan
Published: June 2021

At rest, mammalians generate two phases of breathing: inspiration (I) and post‐inspiration (PI). These phases are thought to be generated by interacting inhibitory neurons in the Bӧtzinger complex (BӧtC) and preBӧtzinger complex (preBӧtC). Indeed transverse slices containing only the preBӧtC fail to generate PI and generate only I based on rhythmogenic neurons that express the transcription factor Dbx1. We employed a horizontal slice (HS) that keeps BӧtC and preBӧtC regions intact bilaterally, and two transverse slices at the levels of the BӧtC and preBӧtC, respectively. In the HS, the level of the BӧtC spontaneously generates PI population activity and is stimulated by norepinephrine. We show that the Dbx1 expression pattern extends throughout the preBӧtC and BӧtC areas forming a contiguous excitatory rhythmogenic column. Isolating the two areas retains respiratory rhythmic activity in both transverse slices. Photo‐stimulation of channelrhodopsin expressed in Dbx1 positive cells evokes network bursts in both preBӧtC and BӧtC transverse slices. Our data indicate that the two rhythms can function as independent oscillators and are coupled to generate I and PI in the HS. We conclude that the preBӧtC is not the only excitatory kernel, but instead forms an excitatory column containing the preBӧtC and BӧtC regions. Similar rhythm generating mechanisms determine rhythm generation along this column and phase relationships are established by GABAergic inhibition.
Transcriptional Control of Oscillatory Homocysteine Homeostasis by Nuclear Receptor SHP
Authors: yang zhihong , Lee Sangmin , Wang Li
Published: June 2021

[Purpose]Hyperhomocysteinemia (HHcy) is often associated with metabolic diseases. This study aims at identifying transcriptional control of oscillatory homocysteine homeostasis.

[Methods]Wild‐type (WT), Shp‐/‐, Bhmt‐/‐, and Bhmt‐/‐Shp‐/‐mice were used. HHcy was induced by alcohol‐ binge or chow diet supplied with drinking water containing 0.18% of DL‐Hcy. Serum and livers were collected over a 24h light/dark cycle. Assays: RNA‐seq, metabolomics (LC/MS, GC/MS), qPCR, adenovirus transduction, transfection, luciferase reporter assay, ChIP assay, Co‐IP, Western blots, and enzymatic assays.

[Results]Loss of small heterodimer partner (SHP) induced a robust expression of betaine‐homocysteine S‐methyltransferase (Bhmt) and cystathionine γ‐lyase (Cth), and caused rhythmic alterations of other genes in the Hcy metabolic pathway. The induction of Bhmt and Cthwas blocked by Shp reexpression in Shp‐/‐liver. Consequently, the oscillatory productions of betaine, choline, cystathionine, cysteine, SAH and SAM, GSSG and GSH, were modulated by Shp‐deficiency or Shp‐reexpression. Forkhead box A1 (FoxA1) activated Bhmt and Cth promoter activities and expression, which was inhibited by Shp. Ethanol‐binge or homocysteine induced HHcy, glucose intolerance and ER stress response in WT mice, which was diminished in Shp‐/‐mice. However, ethanol‐binge triggered macrovesicular fatty changes in Bhmt‐/‐ and Bhmt‐/‐/Shp‐/‐ mice, but not in Shp‐/‐ mice.

Conclusions

The study sheds light on a new regulatory module to control one‐carbon metabolism in the liver, which may have significant clinical implications.

[Grant support]NIH DK080440, AHA 13GRNT14700043, VA Merit Award 1I01BX002634.
Cardiac Peroxisome Proliferator‐activated Receptor‐β Activity is Up‐regulated in the Absence of Muscle RING Finger‐3, and Exaggerates High‐fat Diet‐induced Cardiomyopathy
Authors: Quintana Megan , He Jun , Sullivan Jenyth , Han Yipin , Stansfield William , Willis Monte
Published: June 2021

Diabetic cardiomyopathy is characterized by structural and functional alterations leading to cardiac hypertrophy and heart failure. These changes are regulated in part by PPAR signaling and subsequent changes in fatty acid metabolism. We recently identified that mice lacking ubiquitin ligase Muscle RING Finger‐3 (MuRF3‐/‐) have increased cardiac PPAR‐β activity in vivo. We hypothesized that challenging MuRF3‐/‐ mice with a high‐fat diet, where lipids act as PPAR ligands, would enhance the severity of diabetic cardiomyopathy. MuRF3‐/‐ mice and strain‐matched controls were fed a high‐fat diet. Serum insulin and glucose were measured every two weeks. Total cholesterol and serum triglycerides (TG) were measured at baseline and 26 weeks. Echocardiography was performed every three weeks. MuRF3‐/‐ mice have less blood glucose after 26 weeks on high fat diet (HFD) as compared to strain‐matched controls (168.8±6.2 vs. 240.2±15.5 mg/dL, P<0.05) and increased fasting insulin levels relative to controls (4.7±0.3 vs. 3.4±0.2 ng/ml, P<0.05). Knockout mice do not show differences in fasting total cholesterol or serum TG compared to wild‐type mice at any timepoint. By echocardiography, MuRF3‐/‐ mice have depressed fractional shortening at 6 weeks HFD compared to wild‐type mice (43.6±6.1% vs. 49.2±3.5%, P<0.05). Mice lacking MuRF3 demonstrate exaggerated cardiomyopathy in the presence of high‐fat diet. Increased PPAR‐β activity and fatty acid metabolism are known contributors to diabetic cardiomyopathy. Taken together, these findings corroborate a novel role for MuRF3 in protecting against the development of diabetic cardiomyopathy.
Identification and Characterization of Disease Mechanisms in MEGF10 Myopathy
Authors: Saha Madhurima , Mitsuhashi Satomi , Kang Peter
Published: June 2021

Mutations in MEGF10 have recently been found to cause a rare congenital recessive myopathy, MEGF10 myopathy / early‐onset myopathy with areflexia, respiratory distress, and dysphagia (EMARDD). MEGF10 is an orphan receptor with 17 EGF‐like domains in the extracellular region. MEGF10 has multiple tyrosine residues in its cytoplasmic region that are potential sites of tyrosine phosphorylation. We previously showed that the p.C774R mutation is more severe than the p.C326R mutation due to differential impairment of tyrosine phosphorylation primarily of Y1030. We hypothesize that MEGF10 is a positive regulator of myoblast proliferation and migration in skeletal muscles in part due to involvement in the Wnt signaling pathway, and that this mechanism is impaired in MEGF10 myopathy. siRNA knockdown of endogenous MEGF10 in C2C12 cells caused significantly reduced proliferation compared to untreated C2C12 cells. Overexpression of wild type and mutant MEGF10 in C2C12 mouse myoblasts led to increased proliferation with wild type overexpression, whereas overexpressed p.C326R MEGF10 showed normal levels of proliferation, and overexpressed p.C774R MEGF10 showed reduced proliferation. Cell migration patterns were impaired in C2C12 cells that were subjected to shRNA knockdown of MEGF10 compared to wild type C2C12 cells. Preliminary proteomics studies suggest that MEGF10 interacts with components of the Wnt signaling pathway. These data indicate that MEGF10 plays an important role in myoblast proliferation and migration, and that impairment of these mechanisms lead to human muscle disease. Further studies will explore crucial details of MEGF10 signaling in these contexts.
Discovery of Distinct Molecular Pathways Affected in Mild and Severe Troponin T‐Related Cardiomyopathies
Authors: Gilda Jennifer , Lai Xianyin , Witzmann Frank , Gomes Aldrin
Published: June 2021

Familial hypertrophic cardiomyopathy (FHC) is a genetic heart disease associated with arrhythmias, heart failure and sudden cardiac death. It is known that FHC is primarily caused by sarcomeric protein mutations; however the molecular mechanisms that lead to impaired cardiac function are not well understood. Troponin T (TnT) mutations are the third most common cause of FHC and cause the greatest portion of FHC deaths. To gain insight into the pathways affected in FHC, hearts from two TnT FHC models (I79N and R278C) were investigated by proteomic and metabolomic analysis. The I79N mutation, like most FHC mutations, increases myofilament calcium (Ca) sensitivity, while R278C does not affect Ca sensitivity and has milder cardiac effects. Of approximately 1200 proteins identified, expression of 53 and 76 proteins was changed in I79N and R278C hearts relative to wild‐type hearts respectively. Three major systems affected were the proteasome, antioxidant systems and metabolic pathways. Further investigation by Western blotting and biological assays revealed impaired proteasome function in I79N hearts, as well as increased oxidative stress, antioxidant expression and antioxidant capacity, suggesting higher stress levels in I79N hearts. The results also suggest that glycolysis and galactose metabolism are accelerated in I79N hearts, while fatty acid metabolism appears to be upregulated in R278C hearts, suggesting different energy substrate utilization in mild and severe TnT‐related FHC. Our data suggest that altered metabolism and proteasome dysfunction are important mechanisms in severe TnT‐related FHC.
Thyroid hormone‐induced cardioprotection is lost in AT2R null mice
Authors: da Silva Ivson , Lima Vanessa , Ferreira Julio Cesar , Santos Robson Augusto , Barreto‐Chaves Maria Luiza
Published: June 2021

Thyroid hormones (TH) positively affect the cardiovascular system. We have recently reported that high TH levels activate cardiac renin angiotensin system (RAS), suggesting a possible crosstalk between them. Here we hypothesize that angiotensin 2 receptor (AT2R) mediates cardioprotection‐induced TH during ex vivo ischemia/reperfusion (I/R). Mice were randomly assigned into four different groups: Wild‐type (WT) and AT2R knockout (KO) daily treated (over 14 days) with either saline or TH (T3, 0.7 µg/Kg, i.p.). Hearts were retrograde perfused using a Langendorff apparatus. The lack of AT2R abolished the positive effects of TH on LVDP (100 ± 2.58% vs. 75.28 ± 4.85%), +dP/dt (100 ± 4.01% vs. 85.61 ± 5.03%) and ‐dP/dt (100 ± 3.68% vs. 82.27 ± 5.2%) seen in WT animals upon I/R. AT2RKO mice also presented increased LDH release compared to TH‐treated WT group (253.5±19.82 vs. 121.32±19.02). Finally, the lost in cardioprotection seen in AT2RKO mice was paralleled by increased mitochondrial H2O2 release compared to WT animals, suggesting a possible role of cardiac mitochondria on AT2R‐mediated cardioprotection. In conclusion, our preliminary results suggest a crucial role of AT2R during the TH‐induced cardioprotection. We are currently trying to understand the molecular mechanisms evolved in this phenomenon. Financial Support: CNPq/FAPESP (2013/04703‐6).
Substrate Stiffness Regulates Cardiac Fibroblast Phenotype in Volume Overload Induced Heart Failure
Authors: Childers Rachel , Gooch Keith , Lucchesi Pamela
Published: June 2021

Objective

Determine whether substrate stiffness contributes to the hypo‐fibrotic phenotype of cardiac fibroblasts (CFs) isolated from a volume overload heart failure model.

Methods

Primary (P0) CFs were isolated from left ventricle (LV) tissue of sham operated (control) rats and rats subjected to 4 weeks of volume overload surgically induced by Aortocaval Fistula (ACF). CFs were plated on polyacrylamide gels at a range of stiffness (0.2 kPa to 50 kPa) similar to LV stiffness in vivo. The alignment of f‐actin and αSMA was assessed by immunostaining and used as an indicator of the pro‐fibrotic myofibroblast phenotype. ImageJ was used to quantify fluorescent intensity and morphology. Changes in gene expression were assessed by qRT‐PCR.

Results

Substrate stiffness differentially affected CF morphology and αSMA expression. CFs on soft gels had a smaller spread area; CFs on stiff gels displayed stellate morphologies and αSMA aligned stress fibers (Fig A). Compared to sham, there was ~50% reduction in ACF CFs differentiation towards a myofibroblast phenotype (Fig B) on stiff substrates. Substrate stiffness elicited significantly different patterns of αSMA (~5‐fold), BNP (~3‐fold) and PPAR‐γ (~12‐fold for sham; ~6‐fold for ACF) mRNA expression between sham and ACF (FigC‐E).

Conclusions

This study provides evidence that substrate stiffness plays an important role in phenotype regulation by affecting cytoskeletal organization and mRNA expression. The changes in αSMA, BNP, and PPAR‐γ could provide some explanation for the distinct phenotype in ACF.

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