bluuebunny/crossref_metadata_2025 huggingface dataset:
Correlation of ventricular arrhythmogenesis with neuronal remodeling in cardiac postganglionic parasympathetic neurons during the progression of heart failure after myocardial infarction
Authors: Zhang Dongze , Tu Huiyin , Wang Chaojun , Muelleman Robert L , Wadman Michael , Li Yu‐Long
Published: November 2023

Lethal ventricular arrhythmia has been a major cause of sudden cardiac death in patients with chronic heart failure (CHF). Autonomic dysfunction has been defined as a negative prognostic indicator for high mortality associated with lethal ventricular arrhythmia during the progression of CHF. Our previous study has demonstrated that expression and ion currents of N‐type Ca2+ channels in cardiac postganglionic parasympathetic (CPP) neurons located in intracardiac ganglia (ICG) are decreased in late stage of CHF rats. However, it is unclear whether electrical remodeling in CPP neurons contributes to ventricular arrhythmogenesis during the development of CHF. Here, we investigated relationship between ventricular arrhythmogenesis and neuronal remodeling in CPP neurons in coronary arterial ligation‐induced CHF rats. ECG data obtained from continuous telemetry recording in conscious rats showed that ventricular tachycardia/fibrillation (VT/VF) occurred in 1–3 and 12–14 weeks after coronary arterial ligation (60 % and 80%, respectively), whereas VT/VF did not occurred in 6–8 weeks after coronary arterial ligation. Additionally, as an index of ventricular vagal tone, the change of left ventricular pressure in response to vagal efferent nerve stimulation was blunted in 12 –14 weeks but not 1–3 weeks or 6–8 weeks after coronary arterial ligation. More importantly, the results from whole‐cell patch clamp recording demonstrated that N‐type Ca2+ currents in CPP neurons began to decrease in 6–8 weeks after coronary arterial ligation, and had a significant decrease in 12–14 weeks after coronary arterial ligation. Based on above results, we concluded that lethal ventricular arrhythmia mainly occurred in both early stage and late stage of CHF after myocardial infarction. Electrical remodeling in CPP neurons highly correlated with the occurrence of fatal ventricular arrhythmia in late stage of CHF. However, lethal ventricular arrhythmia in early stage of CHF was not attributed to N‐type Ca2+ currents in CPP neurons. These new findings suggest that improving ventricular vagal tone at late stage of CHF might be a potential therapeutic target for prevention of fatal ventricular arrhythmia and sudden cardiac death in patients with CHF.

Support or Funding Information

This study was supported by AHA Grant‐in‐Aid (15GRANT24970002)
Effect of Cortisol on Growth and Gene Expression of insulin‐like growth‐factor binding proteins (igfbps) in Atlantic Salmon Parr
Authors: Springer‐Miller Ryan , Chenoweth Damaris , Paskavitz Amanda , Regish Amy , McCormick Stephen , Breves Jason
Published: November 2023

Growth performance of teleost fishes, including salmonids, is principally controlled by the growth hormone (Gh)/insulin‐like growth‐factor (Igf) axis. Igfs interact with cognate binding proteins, termed Igf binding proteins (Igfbps), which modulate the biological activities of Igfs. While Atlantic salmon (Salmo salar) are known to express an expansive set of igfbp transcripts in several key metabolic tissues, the actual regulators of igfbp expression have not been identified. Given the well‐documented consequences of mounting a glucocorticoid stress response on growth, we hypothesized that cortisol inhibits anabolic processes by directing the dynamic expression of igfbps. We therefore examined changes in the activities of the Gh/Igf/Igfbp network of salmon parr implanted with cortisol (0, 10 and 40 μg/g body weight) and sampled after 3 or 14 days. We observed reductions in body mass and fork length following 3‐ and 14‐day treatments with cortisol at 40 μg/g. A dose dependent reduction in growth rate occurred by 14 days of treatment with cortisol, whereas hepatosomatic index was not affected. We will report igfbp transcript levels in liver, skeletal muscle and gill with an aim to resolve how activities within the Igfbp network are modulated by cortisol in juvenile Atlantic salmon.

Support or Funding Information

Supported by Start‐Up funds to J.P.B.
A Paradigm Shift: Benefits of Carbon Monoxide (CO) in Hypoxia‐Adapted Species
Authors: Tift Michael Scott , Jordan Peter , Lueker Timothy , Cabrales Pedro , Simonson Tatum , Heinrich Erica , Villafuerte Francisco , Meir Jessica , Crocker Daniel E , Moore Bradley , St. Leger Judy , Ponganis Paul J
Published: November 2023

Counter to the typical idea that carbon monoxide (CO) is strictly toxic, low doses of CO 1) are naturally produced in the body through the regular turnover of heme in heme‐proteins (e.g. hemoglobin (Hb) and myoglobin), and 2) can elicit cytoprotective effects (e.g. anti‐inflammation, anti‐apoptosis). We hypothesized species with high Hb stores would have increased potential for elevated endogenous CO production. To address this, we measured levels of endogenous CO in the blood and end‐tidal breath samples of four species of marine mammals (beluga whales (n = 5), bottlenose dolphins (n = 7), elephant seals (n = 8) and killer whales (n = 9)) with a wide range of heme‐protein stores. In addition, we measured end‐tidal CO in non‐smokers from the Peruvian highlands who typically exhibit elevated Hb at high altitude (n = 48) as well as sea‐level residents (n = 18). Between the humans and marine mammals sampled in this study, there was a low Hb and low‐hypoxia group (i.e. low‐altitude humans, bottlenose dolphins and killer whales) and high‐Hb and chronic hypoxia group (i.e. high‐altitude humans, beluga whales and elephant seals). CO was measured in end‐tidal breath samples from all groups, and also in the blood from marine mammals to obtain carboxyhemoglobin (COHb) measurements. Here we show that Peruvian natives at high‐altitude exhibited significantly higher mean end‐tidal CO compared to their low‐altitude counterparts (4.8 ± 0.4 ppm vs 1.9 ± 0.7 ppm, respectively). The marine mammal species with high Hb concentrations (beluga whales and elephant seals) contained significantly higher CO in blood (1.8 ± 0.06 and 9.7 ± 1.9% COHb) and breath (7.4 ± 2.6 and 21.6 ± 2.6 ppm, respectively) than the other species which had almost undetectable levels. However, the elephant seals were unique in that they contained significantly higher mean blood and breath CO than all other groups (9.7% carboxyhemoglobin and 21.6ppm, respectively), which resembles values seen in chronic cigarette smokers (>6% carboxyhemoglobin, and >10 ppm, respectively). These results suggest elephant seals and native humans from high‐altitude represent excellent models for understanding the adaptive and potential cytoprotective benefits from moderate CO production and exposure.
Use of mTOR as a potential molecular marker to investigate the development of obesity in non‐model fish species
Authors: Spainhour Rebekah , Ball Jenna , Kobayashi Yass
Published: November 2023

Objectives of this study were to identify the genes encoding mechanistic target of rapamycin (mTOR) in channel catfish and examine their tissue distribution. mTOR plays a key role in the regulation of protein synthesis, cell growth, cell proliferation, and nutrient metabolism. Therefore, functions of mTOR have been investigated in association with obesity and diabetes development. In channel catfish, genetic selection toward growth causes the development of obese‐like phenotype, which suggests that channel catfish can serve as an alternative model to investigate obesity and diabetes development. However, little is known about the role of mTOR in channel catfish. Genes encoding mTOR were identified by screening the channel catfish genome database. The expression of various mTOR mRNA was examined in the cDNA of brain, spleen, trunk kidney, liver, Brockmann body and muscle tissues using RT‐PCR. With the exception of the brain, mTOR mRNA expression was detectable in all tissues examined at varied degree. The nucleotide sequence of amplicon corresponding mTOR (480 bp) was highly similar to those of other fish (>80%), as well as that of mice (>78%), and humans (>79%). Currently, we are investigating changes in expression of mTOR mRNA in relation to changes in food intake and genetic selection toward growth in channel catfish.

Support or Funding Information

Supported by the Kansas IDeA Network of Biomedical Research Excellence (P20GM103418).
Brief Exposure to Intermittent Hypoxia Prolongs QTc in Human Subjects, which is Abrogated with AT1a Receptor Blockade.
Authors: Jouett Noah , Bysani Kaethan , Cooley Daniel , Cutler Michael , Raven Peter , Smith Michael
Published: November 2023

Patients with Obstructive Sleep Apnea (OSA) frequently succumb to sudden cardiac death (SCD). Prolongation of the QTc, possibly via intermittent hypoxia (IH) mediated activation of angiotensin II type 1a receptors (ATR1a), predisposes towards SCD. Hence, the present study tested the hypothesis that (1) a brief exposure of IH lengthens QTc which (2) is abrogated with Losartan, an antagonist of ATR1a. Further, we evaluated how Losartan affects the relationship between direct measurements of muscle SNA (MSNA) and QTc via linear regression analyses during IH. Seven healthy human subjects were recruited with normal 12‐lead ECGs. Subjects ingested either placebo or 100 mg of Losartan 1 hour prior to experimentation, and then repeated the study on a separate day in a randomized, repeated measures design. 3‐lead ECG and MSNA (peroneal microneurography) were recorded throughout the study. Subjects were exposed to 20 minutes of IH, which was composed of 20 cycles of the following: 2–3 breaths of 95–100% N2, 20‐second end‐expiratory apnea, and 40‐second room‐air recovery. QTc (determined by Bazett's formula) averages were compared statistically using a repeated‐measures ANOVA. Losartan did not alter baseline QTc (P = 0.17). IH + Placebo significantly prolonged QTc (baseline: 359.8 ± 4.8 ms vs. IH: 368.3 ± 4.8 ms, P < 0.001). Losartan abrogated the IH‐mediated QTc prolongation (baseline: 364.5 ± 5.7 ms vs. IH: 367 ± 6.4 ms, P = 0.11). Furthermore, changes in MSNA explained only 6% of the variance in QTc during IH with Placebo, which increased to 22% with IH and Losartan (IH + Placebo: R2 = 0.06, IH + Losartan: R2: 0.22). In conclusion, IH prolongs QTc, which is prevented with Losartan . Furthermore, IH alters the relationship of MSNA and QTc, in part through activation of ATR1a. Future studies should evaluate the possible cardioprotective benefits of ATR1a antagonists in OSA patients.

Support or Funding Information

None
Renal denervation lowers mean arterial pressure in obese female Schlager mice but adversely affects glucose metabolism
Authors: Asirvatham‐Jeyaraj Ninitha , Banek Christopher T , Gauthier Madeline M , Han Ruijun , Razzoli Maria , Bartolomucci Alessandro , Panoskaltsis‐Mortari Angela , Osborn John W
Published: November 2023

Clinical studies have shown that, in addition to lowering arterial pressure (AP), renal denervation (RDNx) improves glucose metabolism in drug‐resistant hypertensive obese patients. One‐fourth of these patients were females. We have observed a similar arterial pressure response in preliminary studies in obese genetically hypertensive male Schlager mice (BPH/2J). Although sex differences may exist regarding RDNx therapy, the effect of RDNx on AP and glucose metabolism in females specifically has not previously been studied. We hypothesized that, similar to males, RDNx will decrease AP and improve glucose metabolism in high fat fed female Schlager mice. 8‐week old mice were fed either a low fat diet (LFD; 10 KCal% from fat) or high fat diet (HFD; 45 KCal% from fat) for 10 weeks. Fasting blood glucose were measured on week 11 of the protocol. Radiotelemeters were implanted in the femoral artery on week 12 of the protocol for measurement of ambulatory AP. Body weight, food intake, body‐composition and glucose metabolism were measured throughout the study. After measuring baseline AP, LFD and HFD groups were then divided into RDNx or Sham treatment and followed for 14 days. After 10 weeks of diet, HFD versus LFD fed mice had increased body weight (30±1 vs. 26±1, g) and fasting blood glucose (83±3 vs. 68±4, mg/dL). HFD increased the baseline AP (124±4 mmHg) compared to LFD mice (112±4 mmHg). RDNx normalized AP in HFD mice (107±3) but had no effect on AP in LFD mice (106±4). Compared to sham treated mice, RDNx had no effect on fasting blood glucose or the glucose tolerance in LFD mice. In contrast to our hypothesis, fasting blood glucose was higher in RDNx‐HFD (76±3) compared to Sham‐HFD (69±4) mice. Similarly, the area under the curve for the glucose tolerance test was higher in RDNx‐HFD (555 ± 51) compared to Sham‐HFD (426 ± 24) mice. We conclude that RDNx effectively lowers AP in obese genetically hypertensive female Schlager mice, but also results in adverse effects on glucose metabolism. The mechanisms mediating these responses are currently underway and identifying these mechanisms could help develop targeted therapies for obese hypertensive females by clearly avoiding the adverse effects.

Support or Funding Information

NHLBI R01 HL116476
Development of Tools for RNA interference (RNAi) of the engrailed Gene in Daphnia magna
Authors: Singer Davi , Beckman Matt
Published: November 2023

The legend of the cyclops has been a part of international folklore for centuries. Yet, we do not fully understand the developmental genetic basis for cyclopia. Daphnia magna, a water flea, is a naturally occurring cyclops which can serve as a model to study this phenomenon. In this study tools were created to examine the role of the engrailed gene in Daphnia eye development. In Drosophila melanogaster (fruit flies), silencing of the engrailed gene affects segmentation during early development. Additional studies suggest that engrailed is one candidate gene involved in eye field development. To understand the role of engrailed in Daphnia development, we sought to knockdown the gene's expression using RNA interference (RNAi). Using the online databases Uniprot and the wFleaBase we acquired the D. magna hedgehog mRNA sequence. Using this sequence we designed primers to amplify portions of the D. magna engrailed gene. Engrailed gene PCR products were then cloned into the pGC‐Blue vector. Work continues to clone the engrailed insert into the RNAi feeding vector L4440. The L4440 plasmid will be transformed into HT115 E.coli and fed to Daphnia. Future work will also include qPCR studies of the engrailed gene expression during key time‐points in Daphnia eye development and following RNAi knockdown. In summary, through this work tools are being developed to study the role of engrailed in cyclopean eye development. We believe that by leveraging in our studies, Daphnia magna, a true cyclopean organism that evolution built, we can gain a deeper understanding of the developmental genetic basis for midline disorders.

Support or Funding Information

We thank the Augsburg Biology Department, Augsburg McNair Program and the Augsburg LSAMP STEM program for supporting this work.
Letrozole and/or Fructose Induced‐Polycystic Ovarian Syndrome in Animal Model
Authors: Akintayo Christopher Oloruntoba , Anjola Johnson David
Published: November 2023

Polycystic ovarian syndrome (PCOS) is one of the most common causes of infertility among women, with about 10% of women affected in the general populace. The etiology of PCOS has not been fully elucidated; it is genetically based on ovarian disorder accompanied by excess production of androgen. The study was carried out to investigate in animal model Letrozole (L) and/or Fructose (F) induced polycystic ovarian syndrome using female Wistar rats. Ethical approval was obtained from National Institute for Pharmaceutical Research and Development (NIPRD) and the experiment conducted was in conformance with guidelines for experimental procedures as set forth in the Declaration of Helsinki and the APS Guiding Principals in the care and Use of Animals. Twenty virgin female rats were used and grouped into four groups of five rats each. Group served as the one control group, group two was treated with fructose (25% F mixed with 75% normal rodent diet), group three was treated with L (0.5mg/kg) and group four was treated with L and F. The experiment lasted for a period of 21 days. At the end of the experiment the animals were anesthetized with Thiopental (50mg/kg). Blood samples were obtained via cardiac puncture; sera obtained were analyzed for hormonal concentration of follicle stimulating hormone (FSH), luteinizing hormone (LH) and androgen (A) by ELISA technique. The ovaries, uteri and fallopian tubes were harvested and fixed in 10% formalin for histological evaluation. There was no significant increase in the % body weight in L and/or F treated rats compared with the control rats. Insignificant difference (p>0.05) in serum FSH was observed in groups treated with L (12.52±1.73 mIU/ml), F (11.22±1.76 mIU/ml) and L+F (11.72±1.83 mIU/ml) in comparison with their control counterpart (10.39±1.79 mIU/ml). However, insignificant differences were also observed in the serum levels of LH and A in L compared with the control group. The histological examination of the female reproductive organ (fallopian tube) of letrozole and/or fructose treated rats showed infiltration of inflammatory cells with severely congested epithelial cells and also hyperplasia of the tubular epithelial cells. Chronic intake of fructose and the use of letrozole in the treatment of cancer should be taken into consideration as both can cause imbalance in the level of androgen in females which was evident in the results obtained in our study.
Fetal brain sparing in a mouse model of chronic maternal hypoxia
Authors: Cahill Lindsay S , Zhou Yu‐Qing , Hoggarth Johnathan , Seed Mike , Macgowan Christopher K , Sled John G
Published: November 2023

Pregnancy complication may cause acute or chronic hypoxic stress to the fetus. The fetal response to acute hypoxia has been well characterized1 and is associated with brain sparing, a mechanism by which more oxygenated blood is directed to the brain at the expense of organs such as the liver and the lungs2–4. In contrast, the fetal response to chronic hypoxia is not well understood. In the present study, Doppler ultrasound was used to characterize the fetal hemodynamic responses to chronic maternal hypoxia during late gestation in pregnant mice.

Pregnant CD‐1 mice were randomly assigned to either a chronic hypoxia (n=13) (11% O2 from gestational day E14.5 to E17.5) or control (n=13) group. Doppler ultrasound measurements were acquired at E17.5 (full term for CD‐1 dams is E18.5) of the middle cerebral artery, ductus venosus, main pulmonary artery, ductus arteriosus, and the umbilical artery. An acute hypoxic condition (8% O2 for 3 minutes) was used to challenge the fetus and assess its capacity to compensate for additional hypoxia stress. Fetal organ growth was assessed using ex vivo magnetic resonance imaging (MRI).

The fetuses exposed to chronic maternal hypoxia weighed 15% less than controls, indicating that the hypoxia‐exposed fetuses failed to reach their growth potential. Fetal cerebral blood flow was elevated by 31%, ductus venosus blood flow was 69% higher and pulmonary blood flow was decreased by 74% in the fetuses exposed to chronic hypoxia compared to controls. This demonstrates that the brain sparing response persists during chronic fetal hypoxia and is mediated by “streaming”, where highly oxygenated blood preferentially flows through the ductus venosus towards the cerebral circulation, bypassing the liver and the lungs. Consistent with these changes in blood flow, the brain volume is preserved while the lung and liver volumes decreased by 27 and 22% respectively in fetuses exposed to chronic hypoxia compared to controls.

With an acute hypoxic challenge, the cerebral and ductus venosus blood flow increased in control fetuses. The elevated flow was achieved by an increased fetal heart rate and vasodilation. In contrast, the blood flow in the fetuses exposed to maternal hypoxia decreased when challenged. The decrease was accompanied by a drop in fetal heart rate, suggesting the fetus can no longer compensate for the decreased oxygen transport across the placenta. This highlights that fetuses exposed to chronic maternal hypoxia are at increased risk of fetal demise caused by an acute episode of hypoxia; for example, those that commonly occur during labour and delivery.

Support or Funding Information

Funded by Canadian Institutes of Health Research Grant MOP130403.
Prenatal food restriction causes adaptive changes in the metabolic regulation of reproductive physiology
Authors: Rhinehart Erin M
Published: November 2023

Reproduction is the most energetically expensive undertaking that a female can perform, making energy the most important factor regulating reproduction. Reproductive physiology is sensitive to fluctuations in energy availability, and entrainment of female reproductive efforts with energy availability is especially critical because of the large amounts of energy required for gestation and lactation. To optimize reproductive fitness in an environment with reduced food availability, physiological mechanisms are needed to connect oxidizable fuel availability with reproductive physiology. When prenatal food availability is restricted, indicators of reduced maternal energy availability reach the fetus. These signals purportedly prepare the offspring for a food insecure postnatal environment. According to this idea, the energy availability in the intrauterine environment organizes the developmentally plastic mechanisms responsible for energy homeostasis, which would necessarily impact the regulation of reproduction. Therefore, we hypothesized that gestationally nutrient restricted, low birth weight (LBW) offspring would exhibit changes in energy balance and reproductive physiology that would improve survival and reproductive success in a food insecure postnatal environment. To test this hypothesis, we used a well‐characterized model of gestational nutrient restriction where pregnant rats were fed ad libitum or 50% of their normal caloric intake from embryonic day 10 to 21 and all offspring were cross‐fostered to non‐restricted dams for lactation. Consistent with previous studies, restricted female offspring were born LBW (5.6 ± 0.08 vs. 6.5 ± 0.06, p<0.01). LBW offspring also had decreased hypothalamic gonadotropin releasing hormone (GnRH) protein expression at postnatal day (PND) 1 and exhibited rapid catch‐up growth during lactation. LBW female offspring had earlier vaginal opening (33.5 ± 0.2 vs. 38.2 ± 0.2 PND, p<0.01), demonstrating early pubertal onset. AT PND 60–90, LBW female offspring had elongated estrous cycles (6.1 ± 0.4 vs. 4.7 ± 0.3, p<0.05) and significant alterations in ovarian histology. For example, ovaries from young adult LBW females were larger (3.6 ± 0.7 vs. 6.5 ± 0.6 mm2, p<0.05) due to large antral, cyst‐like follicles. By 8 months of age, 80% of LBW offspring were in persistent estrous and had stopped cycling, compared to 0% of control offspring. These data indicate that prenatal energy availability has a significant impact on the regulation of reproductive physiology. Early pubertal onset, in particular, may be an adaptive mechanism to ensure reproductive success in a food‐insecure postnatal environment, but early puberty has significant health ramifications. Therefore, a more thorough understanding of the mechanisms responsible for gestational programming of the metabolic regulation of reproduction should have a significant impact on treatment of female reproductive problems such as polycystic ovary syndrome.

Support or Funding Information

NSF CAREER IOS‐1350448
Firing properties of pre‐inspiratory neurons are altered in female rats exposed to chronic intermittent hypoxia
Authors: Souza George Miguel P R , Amorim Mateus R , Moraes Davi JA , Machado Benedito H
Published: November 2023

Female rats submitted to chronic intermittent hypoxia (CIH) presented a reduced time of inspiration, sympathetic overactivity and hypertension. The increase in sympathetic nerve discharge in CIH‐female rats is associated to an increase in the inspiratory modulation of sympathetic activity. In this context, we hypothesized that inspiratory neurons at the brainstem have change their activity in CIH‐female rats, which in turn, modulates the presympathetic neurons producing an increase in sympathetic activity during inspiration. To test this hypothesis, juvenile female Wistar rats (P20–21) underwent intermittent hypoxia for 10 days. One day after CIH, we performed the extracellular single unit recordings of respiratory neurons at the ventral surface of the medulla using the ventral approach of the working heart‐brainstem preparation. All the experimental protocols were approved by the institutional ethical committee (#091/2013). Pre‐inspiratory neurons (Pre‐I/I) of CIH‐female rats (n=16) have reduced their firing frequency (58.3 ± 9 vs 107.6 ± 12Hz, p=0.0027) compared to Pre‐I/I neurons of control (n=15). Synaptic blockade demonstrated that Pre‐I/I neurons (n=3) have auto‐depolarization property. Ramp‐I, Early‐I and Late‐I neurons presented no changes in their firing properties after CIH. Regarding the expiratory neurons, post‐inspiratory neurons (Post‐I) have increased the firing time relative to duration of expiration (92.2 ± 2 vs 81.3 ± 3 % of total duration of expiration, p=0.0127) in CIH‐female rats (n=15) compared to control (n=17). We conclude that CIH produces a reduction in the activity of pacemaker Pre‐I/I neurons that could explain the reduced time of inspiration and the increase in inspiratory modulation of sympathetic activity in CIH‐female rats. These findings contribute to a better understanding of the respiratory network activity associated with sympathetic overactivity and hypertension in female rats exposed to CIH.

Support or Funding Information

Fapesp, CAPES and CNPq
Ventral Medullary Inspiratory Neurons Recordings in Rats Submitted to Sino‐aortic Denervation
Authors: Amorim Mateus R. , Souza George Miguel P. R. , Bonagamba Leni G. H. , Machado Benedito H.
Published: November 2023

There is experimental evidence showing that pre‐sympathetic neurons in the ventrolateral medulla have their firing frequency modulated by the respiratory network. After baroreceptors afferents removal [sino‐aortic denervation (SAD)], awake rats present normal levels of sympathetic activity and mean arterial pressure. However, the mechanisms involved in the respiratory modulation of the sympathetic activity after SAD remain unclear. In this study we hypothesized that rats present changes in the firing frequency of the inspiratory neurons after SAD. To test this hypothesis, male juvenile Wistar rats underwent to SAD or Sham surgery and on the 3rd day after the surgery they were surgically prepared for the in situ preparation. We recorded the activities of phrenic and thoracic sympathetic nerves as well as the single unit extracellular activities from the inspiratory neurons in the ventral medulla (CEUA #093/2013). The duration of inspiration significantly increased in SAD rats (P > 0.05). During inspiration, the firing frequency of Ramp‐I [Sham (n = 11) and SAD (n = 7, 52 ± 11 vs 56 ± 14 Hz)], Pre‐I / I [Sham (n = 13) and SAD (n = 11, 82 ± 15 vs 99 ± 20 Hz] and Late‐I neurons [Sham (n = 8) and SAD (n = 6, 8 ± 3 vs 9 ± 2 Hz] were not different between groups (P > 0.05). The data showed that the increase in time of inspiration observed in SAD rats was not associated with changes in the baseline firing frequency of the inspiratory neurons. However, these findings do not rule out the possibility that post‐inspiratory neurons are driving the activity of the pre‐sympathetic neurons after SAD. Further experiments are required to characterize the electrophysiological firing properties of the post‐inspiratory neurons in SAD rats.

Support or Funding Information

FAPESP, CAPES and CNPq
Effects of lipopolysaccharide injection on in vivo breathing pattern in developing rats
Authors: Johnson Rhaya L. , Murray Samuel , Wilson Christopher G.
Published: November 2023

Breathing pattern irregularities, which are common problems in pre‐term infants, are often exacerbated by infection. These irregularities can manifest as apneas, tachypnea, and periodic breathing. We have previously shown that lipopolysaccharide (LPS) injected into the airway of neonatal rats (postnatal days 10 to 12) causes up‐regulation of early pro‐inflammatory cytokines and blunts the response to an acute hypoxia challenge. Furthermore, we are using vagal nerve stimulation (VNS), an FDA approved treatment for epilepsy and depression, in an effort to reduce neuroinflammation in the brainstem regions for respiratory control. We have assessed cytokine expression in response to LPS injection using immunohistochemistry and mRNA quantification in nTS, XII, PBC.

Our preliminary data suggests that LPS injection causes a blunted hypoxic ventilatory response to 10% oxygen (10 min, balance N2). Sham animals had a 46% increase in breath rate from normoxia to hypoxia, whereas LPS animals only had a 26% increase in breath rate from normoxia to hypoxia (n = 3 for each). LPS injection also significantly increases the expression of IL‐6 (p < 0.001) and is hypothesized to increase TNFa and IL‐1b in the respiratory control regions of the brainstem. VNS slows the baseline breath rate (13% lower than sham) over the two hour recovery period. The hypoxic ventilatory response begins to recover (31% change from normoxia to hypoxia) though the increase in breath rate with hypoxia is not as large as with sham animals. VNS does reduce cytokines IL‐6 and TNFa (p<0.5) in the nTS and XII.
EVIDENCE FOR ACE‐2 UBIQUITINATION AND PROTEASOMAL PROCESSING IN LUNG EPITHELIUM
Authors: Abdul‐Hafez Amal , Markey Jonathan , Uhal Bruce
Published: November 2023

Background and Aim

The angiotensin converting enzyme 2 (ACE‐2) was shown to play a protective role in the fibrogenesis and inflammation of many organs including liver and lung. Previous studies from our lab showed that ACE‐2 is downregulated in fibrotic conditions of the adult and neonatal human lung. The exact mechanisms of ACE‐2 downregulation and protein degradation in lung cells still remain unknown. This study aims to investigate whether ACE‐2 is regulated by ubiquitination/proteasome degradation pathways in the lung.

Methods

Human lung epithelial cell line (A549) and mouse lung epithelial cell line (MLE12) were cultured and cell lysates were used for immunoprecipitation using ACE‐2 antibodies. The immunoprecipitate was used to detect ubiquitination. Several proteasome inhibitors (Lactacystin, clasto‐Lactacystin β‐lactone, MG132, HMB‐Val‐Ser‐Leu‐VE, and Epoxomicin) were used on MLE12 cell culture to detect the effects on ACE‐2 protein bands by western blotting and to immunoprecipitate ACE‐2 and detect the effect on the ubiquitination of ACE‐2. Several dosing of the proteasome inhibitor MG132 on MLE12 cells and of clasto‐Lactacystin β‐lactone on A549 cells were used to detect changes in ACE‐2 bands by western blotting.

Results

Immunoprecipitation of ACE‐2 revealed ubiquitination of 3 major bands (150, 45, 40 kDa) detected by anti‐ubiquitin antibodies in MLE12 cells, while ubiquitination of ACE2 was markedly noted in 50 kDa band of A549 cells. On MLE12 cells, all proteasome inhibitors resulted in various degrees of increased total ubiquitinated protein levels. Lactacystin and clasto‐Lactacystin β‐lactone showed increased ACE‐2 isoform band at 70 kDa and were associated with the most increase in total ubiquitinated protein levels, more noted in clasto‐Lactacystin β‐lactone treatment than Lactacystin. Additionally, increasing doses of MG132 treatment on MLE12 cells resulted in a dose dependent increase of the 70 kDa ACE‐2 band with a corresponding decrease in 40 kDa band. Similarly, treatment of A549 cells with increasing doses of clasto‐Lactacystin β‐lactone resulted in a dose dependent decrease in the 50 kDa band.

Conclusion

These data present the first evidence suggesting ubiquitination and proteasome dependent degradation of ACE‐2 in lung cells in a species‐dependent pattern. Future work will be directed to detect the roles of this ubiquitination/proteasome degradation in fibrotic lung diseases.

Support or Funding Information

PHS HL‐45136; Department of Pediatrics, Michigan State University
Novel inflammatory mechanisms in the regulation of airway hyperresponsiveness: regulation of airway smooth muscle Kv7 channel expression by REST.
Authors: Brueggemann Lyubov I. , Zhang Chonyu , Kakad Priyanka P. , Cribbs Leanne L. , Byron Kenneth L.
Published: November 2023

Inflammation contributes to airway hyperresponsiveness (AHR) in asthma, but the cellular mechanisms by which this occurs are not well understood. In previous research, inflammatory mediators were found to upregulate repressor element 1‐silencing transcription factor (REST) in neurons, and consequently, REST mediated transcriptional suppression of neuronal KCNQ genes, which was associated with decreased M‐current density and increased neuronal excitability. Functional binding sites for REST have been identified in both KCNQ4 and KCNQ5 genes which encode Kv7.4 and Kv7.5 potassium channels expressed in airway smooth muscle cells (ASMCs). We found that Kv7 currents and KCNQ gene expression were both decreased in ovalbumin‐(OA‐) sensitized guinea pigs (a model for allergic asthma) relative to saline‐treated control guinea pigs. Although REST has not been reported to be expressed in ASMCs, we found that it is expressed and that REST protein levels are significantly higher in the nuclei of ASMCs from sensitized guinea pigs relative to saline control guinea pigs. Furthermore, serum from the OA‐sensitized animals, but not from control animals, increased nuclear REST immunostaining in ASMCs of control animals. In previous research, serum from sensitized guinea pigs or from asthmatic humans was shown to induce features of AHR in ASMCs, and features of AHR could also be induced by treating human ASMCs with purified inflammatory mediators, including IL‐4 and IL‐13. We exposed strips of human trachealis tissue to IL‐13 overnight and observed a significant increase in nuclear REST expression in ASMCs, supporting a specific role for IL‐13 and providing evidence that this pathway is active in human ASMCs. These findings provide new evidence for an inflammatory mechanism that might account for airway smooth muscle hyper‐excitability in asthma.

Support or Funding Information

The project described was supported by Award Number I01BX007080 from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development.
Resveratrol Protects Against Hyperoxia‐Induced Airway Hyperreactivity in Neonatal Rats
Authors: SOPI RAMADAN B , Reçica Shkëlzen , Lajçi Azem , Kryeziu Islam , Thaçi Qëndrim , Dreshaj Ismail A , Basholli Mimoza
Published: November 2023

Neonatal hyperoxia is associated with airway hyperreactivity and lung injury. We tested the hypothesis that supplementation of resveratrol will overcome the increased contractile responses of tracheal smooth muscle exposed to hyperoxia. Tracheal cylinders were obtained from Wistar rat pups (P12) exposed to hyperoxia (≥ 95% O2) or room air for seven days. These cylinders were used to study contractile responses of airway smooth muscle (ASM) evoked by methacholine (MCh, 10‐8 – 10‐4 M) in absence or presence of resveratrol (100 μM). MCh induced dose‐dependent contractile responses of ASM. The contractile responses towards MCh were significantly higher in preparations obtained from animals exposed to hyperoxia as compared to those obtained from control animals exposed to room air. The enhanced contractile responses in hyperoxic tissues were significantly attenuated in presence of resveratrol. The results of this study revealed that resveratrol diminishes the airway hyperreactivity induced by hyperoxia and we speculate that the use of resveratrol might be an effective therapeutic approach to prevent the adverse effects of hyperoxia.

Support or Funding Information

Supported by MEST
Effects of perinatal nicotine or inflammation exposure on ventilation in the developing rat model
Authors: Williams Paul Allen , Wilson Christopher
Published: November 2023

The regulation of ventilation is critical in maintaining homeostasis and ensuring survival in the face of environmental and developmental changes. Previous studies have shown that regulatory control of breathing changes during early life with a sensitive developmental window between 10 and 13 days post birth (P10 to P13) [Li et al., J Physiol, 577:957–770, 2006]. Other investigators have shown that perinatal nicotine exposure can alter ventilation, long‐term facilitation, neurotransmission, and neuronal excitability in neonatal rats. Recent studies have also suggested that perinatal inflammation can alter respiratory rhythm and auto‐resuscitation in neonatal rats. However, very little is known about the effects of perinatal nicotine or inflammation exposure before, during, and after this P10 to P13 sensitive period. We hypothesized that perinatal exposure to nicotine or inflammation would alter the maturation of breathing pattern in postnatal rat pups with a maximum effect circa the P10 to P13 sensitive window. To test this hypothesis, we exposed pregnant dams to either nicotine (6mg/kg/day) or lipopolysaccharide (LPS, 0.1 mg/kg) and then used whole‐body plethysmography to study the breathing pattern of the rat pups. Sets of three pregnant adult Sprague‐Dawley dams were obtained at E3, at E5 all three were implanted with osmotic pumps (Alzet) with two animals receiving saline and the third nicotine. Later at E15 i.p. injections were administered to the nicotine‐exposed dam and another dam receiving 0.5ml saline with the third dam receiving 0.1mg/kg of LPS. At postnatal ages P1, P3, P5, P7, P10, P12, P13, P17, & P21, we performed whole‐body plethysmography exposing the rat pup to 10 minutes each of room air (baseline), 10% oxygen, 50% oxygen, and room air to assess recovery of breathing pattern. Respiratory rate, expiratory time, inspiratory time, sample and Shannon entropy were quantified using custom software (BASS, https://github.com/drcgw/BASS). Preliminary data showed that the nicotine and inflammation groups have a 50% higher respiratory rate compared to control at P1. Consistent with previous studies, the respiratory rate was similar for the three groups (< 5% difference across groups) at P3 to P12. At P12 and P13 the control group had a 35% higher respiratory rate than either nicotine or inflammation, then at P17 and P21 the control group had approximately a 20% higher respiratory rate than the nicotine or inflammation groups. Our results suggest that there are persistent age‐dependent changes in ventilation due to perinatal exposure to nicotine or inflammation.
Hypercapnic and hypoxic ventilatory response in streptozotocin‐model of Alzheimer's disease during wakefulness
Authors: Vicente Mariane C , Bícego Kênia C , Carrettiero Daniel C , Gargaglioni Luciane H
Published: November 2023

Neurodegenerative diseases such as Alzheimer's may impair breathing function; however, how these breathing deficits occur is not completely understood. Therefore, it is important to evaluate how Alzheimer's disease (AD) can alter breathing activity. In light of this, we used an animal model for sporadic AD induced by intracerebroventricular (icv) injection of streptozotocin (STZ, 4 mg/kg) in adult male Wistar rats. After 30 days of STZ treatment, memory impairment (using Y‐maze and Barnes maze tests) was analyzed to validate STZ model. Then, ventilation (VE) was measured by whole‐body plethysmograph (barometric technique) during wakefulness and sleep under normocapnia, hypercapnia and hypoxia. STZ treatment caused learning and memory impairment and increased hypercapnia ventilatory response during wakefulness (VE STZ= 2460.9 ± 83.7 vs VE Vehicle= 1758.5 ± 234.3 mL.Kg−1.min−1) due to an effect on both tidal volume and breathing frequency. No difference was observed in the respiratory variables between STZ and vehicle groups during room air conditions (VE STZ = 775.5 ± 56.1 vs VE Vehicle= 651.3 ± 56.1 mL.Kg−1.min−1) and hypoxia (VE STZ = 1073,2 ± 53,7 vs VE Vehicle =1085.9 ± 127.2 mL.Kg−1.min−1) in wakefulness. Our data suggest that sporadic AD animals present an increased CO2 but no O2 chemosensitivity.

Support or Funding Information

FAPESP and CNPq
Optogenetic Activation of Leptin‐Receptor Expressing Neurons in the Region of the Dorsomedial Hypothalamus Increases Respiratory Motor Output in Mice
Authors: Chang Zheng , Kowal Anthony S. , Sekerkova Gabriella , McKenna Kevin E. , Martina Marco , McCrimmon Donald R
Published: November 2023

Administration of the anorexigenic hormone leptin to rodents increases energy expenditure with a parallel increase in respiratory motor output in order to maintain arterial blood gas and acid‐base balance. This occurs at least in part through activation of CNS neurons that express the long form of the leptin receptor (LepRb). LepRb expressing neurons are concentrated in several clusters, most notably in the hypothalamus and medulla. Previous studies have linked respiratory motor stimulation to activation of LepRb neurons in the medulla. In this study we sought to determine whether hypothalamic neurons also contribute to the respiratory stimulation. Specifically, LepRb neurons are concentrated in the dorsomedial nucleus of the hypothalamus (DMH) and activation of DMH neurons is known to stimulate breathing. However, whether LepRb expressing neurons contribute to the stimulation is not known. We have initiated studies to determine whether DMH LepRb neurons contribute to the respiratory stimulation using mice that express channelrhodopsin‐2 (ChR2) in LepRb neurons. Mice were anesthetized and an optical fiber (200 um diameter) conducting 473 nm wavelength light was used to activate the ChR2‐LepRb neurons. Light intensity was 4 mW, below the threshold for nonspecific activation or damage to tissue in the vicinity of the fiber. For stimulation, 3 min (or shorter) trains of light pulses at 5, 10, 20 and 50 Hz (≤10 ms pulse width) were used. In parallel control experiments, areas dorsal or lateral to the DMH were stimulated. Activating the DMH elicited a progressive increase in respiratory motor output, consisting primarily of an increase in the peak amplitude of integrated phrenic nerve activity with little change in the number of phrenic bursts per minute. The phrenic amplitude continued to increase after the end of the stimulation, averaging 189 +/− 23% of control (n = 3) at 30 min and 212 +/−17% (n = 2) at 60 min after the end of stimulation. Our preliminary conclusion is that the DMH contributes significantly to the leptin‐mediated stimulation of breathing.

Support or Funding Information

We are grateful for the support from NIH grant HL122921.
Basal Ventilatory Activity in Conscious Freely‐moving Spontaneously Breathing 6‐OHDA SN‐lesioned Parkinson's Disease Rat Model
Authors: Wadolowski Robert M , Wadolowski Steven B , Collins William F , Solomon Irene C
Published: November 2023

Respiratory dysfunction in Parkinson's Disease (PD) patients manifests as a variety of altered breathing patterns that are suggested to result from impairment of central respiratory control. Ongoing work in our laboratory has recently identified time‐dependent alterations in basal inspiratory motor output and chemical control of breathing in the 6‐hydroxydopamine (6‐OHDA) neurotoxin‐induced unilateral substantia nigra (SN) lesion rat model. Our studies, however, have been limited to examination of these ventilatory behaviors at specified time points in acute terminal experiments; thus, the time course underlying the progression of the changes observed is not known. Here, we begin to address this issue by examining the basal respiratory phenotype using whole body plethysmography before and weekly for 4‐weeks after administration of 6‐OHDA into the SN of adult female Sprague‐Dawley rats; vehicle injected rats served as a control. Our preliminary data from 6‐OHDA SN‐lesioned rats (compared to vehicle control rats) suggest that there is a progressive reduction in tidal volume (TV) with a slight decrease in peak expiratory flow as indicated by a reduced EF50 and relaxation time albeit these changes are fairly modest. In addition, 6‐OHDA SN‐lesioned rats appear to exhibit a progressive reduction in variability in most of the time series respiratory parameters examined (e.g., TV, expiratory volume, EF, PIF, PEF, MV). These observations suggest that in conscious freely‐moving 6‐OHDA SN‐lesioned rats, ventilatory behaviors undergo a progressive alteration; however, the 4‐week time course studied here was only able to identify limited changes. We suggest that additional experiments examine a longer post‐lesion time period in order to more fully characterize the time course underlying the progression of ventilatory impairment in this PD rat model.

Support or Funding Information

Thomas Hartman Center for Parkinson's Disease Research at Stony Brook University
Gasotransmitters in Sleep Apnea
Authors: Zhang Xiuli , Peng Yingjie , Irina Chupikova , Gridina Anna , Nanduri Jayasri , Kumar Ganesh , Snyder Solomon H. , Nanduri Prabhakar
Published: November 2023

Sleep apnea is a highly prevalent respiratory disease, affecting an estimated 10% of adult human population. Sleep apneas are characterized by periodic cessations of breathing during sleep and are associated with several sequelae including hypertension and stroke. Clinical studies suggest that abnormal carotid body (CB) activity is one of the major drivers of sleep apnea. Because gaseous molecules, carbon monoxide (CO) and hydrogen sulfide (H2S) are important determinants of CB activity we hypothesized that aberrations in their signaling could lead to sleep apnea. We tested this possibility in mice deficient in heme oxygenase‐2 (HO‐2), which catalyzes the endogenous production of CO. Basal breathing was monitored in unsedated 6‐to‐9 months old wild‐type and HO‐2−/− mice by plethysmography. A majority of the HO‐2−/− mice (40/70 mice, 57%) had 20 or more apnea events per hour. In contrast, only 2/40 wild‐type mice (5%) exhibited such frequent apneas. The apnea index was higher in 6–9 month old mice compared to 6–9 weeks old HO‐2−/− mice. Fifty‐six percent of the HO‐2−/− mice (39/70) had a hypopnea index of > 80 (breathing events with ≥ 30% reduction in tidal volume per hour), whereas only 2.5% of the wild‐type mice (1/40 mice) displayed a similar hypopnea index. Apneas and hyponeas were remarkably higher in non‐rapid eye movement (NREM) and REM sleep. Further characterization of apneas revelead that HO‐2−/− mice experience both obstructive and central apneas, and the incidence of former was greater than the latter. Hyperoxia, which inhibits the CB activity reduced, whereas hypoxia, which stimulates the CB activity increased the incidence of apneas. We identified hydrogen sulfide (H2S), as a major molecule driving apneas. Genetically ablating the H2S‐synthesizing enzyme cystathionine‐g‐lyase (CSE) or pharmacologic blockade of CSE normalized breathing in HO‐2−/− mice. These observations demonstrate that dysregulated CO and H2S signaling in the CB leads to apneas.

Support or Funding Information

Supported by NIH‐UH3‐HL90554 and PO1‐HL90554