bluuebunny/crossref_metadata_2025 huggingface dataset:
Strong fluid movement near mammalian skeletal muscle improves their stability for force and metabolic studies at 37°C
Authors: Renaud Jean‐Marc , Paquette Mathieu
Published: November 2023

Studying mammalian skeletal muscles at 37°C is challenging because of a high metabolic rate requiring rapid O2 diffusion and more importantly a high production rate of reactive oxygen species (ROS) requiring fast removal. Both problems can be circumvented when small muscles up to 10 mg wet weight are immersed in physiological solutions and superfused near the surface with rapid solution flow (10–15 ml/min). However, rapid superfusion is not feasible when using expensive drugs and radioactive markers representing conditions that require muscle incubation in small volume. The objective of this study was therefore to optimize the experimental conditions that will give rise to stable muscle preparations at 37°C when a rapid flow of physiological saline solution is not an option. We tested the hypothesis that in the absence of superfusion, one must create fluid movement in order to replace the convection effect of superfusion. Tetanic force can be maintained constant for hours when mouse FDB bundles (1–2 mg wet weight) were superfused whereas a continuous loss of force reaching 10% force by 25 min was observed when superfusion was interrupted. The decrease in tetanic force during fatigue, elicited with one contraction every sec for 3 min, was significantly faster and the final extent of the decrease greater in the absence than in the presence of superfusion. There was, on the other hand, no difference in the decrease in tetanic force between superfusion and gas bubbling both prior to fatigue and during fatigue. A lack of superfusion was also important for the glycogen content. Glycogen levels prior to fatigue were 119 μmoles glucose equivalent/g dry weight when superfusion was maintained while interrupting superfusion for 10 min reduced glycogen levels to 82 μmoles/g dry weight; representing a decrease of 37 μmoles/g dry weight. Following fatigue, glycogen levels were 77 μmoles/g dry muscle with superfusion and 43 μmoles/g dry weight in the absence of superfusion. Notably, the amount of glycogen depletion during fatigue was not different with and without superfusion, the values being 42 and 39 μmoles/g dry weight, respectively. The effect of interrupting superfusion on tetanic force was much greater following a first fatigue bout and 50 min recovery. That is, interrupting superfusion resulted in 35% decrease in tetanic force within 10 min, which was again prevented by gas bubbling or the addition of NAC or tiron, two known ROS scavengers. So, a lack of fluid movement near mammalian skeletal muscle muscles at 37°C results in large loss of tetanic force and glycogen. The loss of tetanic force can be prevented by applying gas bubbling near the muscle or by scavenging ROS.

Support or Funding Information

Study was supported by the National Science and Engineering Research Council of Canada (NRC)
Inhibition of Satellite Cells Proliferation with AraC Prolonged Survival of Adult Skeletal Muscle Fiber in Culture
Authors: Albadrani Hind Muteb , Renaud Jean Marc
Published: November 2023

The skeletal muscle is a dynamic tissue that has the ability to change and modify itself to fit the level of required activity; a phenomenon called muscle plasticity. Most studies of muscle plasticity are carried out in situ, a condition for which it is difficult to study and discern between the intrinsic properties of skeletal muscle, the myokines released by muscle fibers and the neurotrophic factors released by neurons innervating skeletal muscles that play various roles in the mechanisms of muscle plasticity. Another approach is to study the morphological and behavior of single adult muscle fibers under culture conditions for which one can fully control the level of activity and exogenous factors affecting muscle plasticity. However, single muscle fiber survival has been shown to be limited to a few days. The objective of this study was to optimize fiber survival in culture. Mouse FDB fibers were separated by trituration following a 3 hrs collagenase treatment at 37°C. When tested the day of the preparation, FDB fibers have normal morphology, normal resting membrane potential and calcium release during contraction. However, when maintained in culture at 37°C, they either degenerated or supercontracted and intact fibers are basically depleted within 5–7 days. Satellite cells are known to proliferate when fibers are maintained in culture and we tested whether an inhibition of their proliferation improves fiber survival. Exposing fibers under resting conditions to 50 μM AraC, a mitotic inhibitor, significantly improved fiber survival by significantly reducing the number of degenerating fibers. We also determined that i) MEM is a better medium than DMEM; ii) changing 50% of the culture medium every two days improved fiber survival compared to changing the medium every day; and iii) a collagen/laminin mixture as adhering substrate to improve fiber adhesion to glass coverslip gave rise to a better fiber survival than Matrigel. Together, the use of AraC, MEM, collagen/laminin and changing medium every two days resulted in having more than 60% of the fibers maintaining normal morphology up to 18 days. A large loss of fibers was then observed over the next 3 days primarily because of supercontraction. The results suggest i) that when satellite cells are allowed to proliferate they may contribute to the degeneration of fibers under resting conditions and ii) that the release of myokines by skeletal muscle fibers likely play a role in fiber survival (as removing 50% of medium every day lowered fiber survival).

Support or Funding Information

The study was supported by the National Science and Engineering Research Council of Canada (NSERC)
Perfluorooctanoic Acid Reduces Viability and Decreases Expression of Peroxisome Proliferator‐Activated Receptor Alpha (PPARα) and Estrogen Receptor Alpha (ERα) mRNA in MCF‐7 Human Breast Cancer Cells
Authors: Smith April N , Cannon Jennifer D
Published: November 2023

Perfluorooctanoic acid (PFOA) is a synthetic fluoropolymer introduced into the environment post‐World War II as a component of a variety of industrial and consumer products. Human exposure occurs through these products, as well as through contaminated food, water, and air, or through occupational exposure. It is a very stable molecule with a half‐life of almost 4 years in humans. PFOA is a documented endocrine disruptor. It has been shown to decrease hormone production in some steroidogenic cells, however the effects of PFOA and its mechanism of action on hormone‐responsive cells remain unclear. The aims of this study were to determine the effects of PFOA on viability of MCF‐7 breast cancer cells as well as its effects on the expression of peroxisome proliferator‐activated receptors (PPARs) and estrogen receptors (ERs), both of which have defined roles in cell proliferation. Cells were treated with PFOA (100nm ‐ 100μM) for 24h to 48h before measuring cell viability using the CellTiter‐Blue Cell Viability Assay. The expression of PPARα, PPARβ, and PPARγ, as well as ERα and ERβ were determined by real‐time PCR. Cells treated with 100μM PFOA for 48h showed a significant, 25% decrease in cell viability (p<0.05) compared to untreated cells. This reduction in viability was preceded by a significant decline in both PPARα and ERα gene expression. This was observed in MCF‐7 cells treated with both 50μM and 100μM PFOA for 24h. There was no significant difference in the levels of PPARβ or PPARγ mRNA between PFOA treated and untreated cells, and the levels of ERβ mRNA were too low to be quantitated. These data suggest the PFOA‐induced decrease in cell viability in MCF‐7 cells is associated with a decrease in the expression of both PPARα and ERα mRNA.

Support or Funding Information

Augusta University's Center for Undergraduate Research and Department of Biological Sciences
Maternal Chronic Intermittent Hypoxia During Gestation Programs Hypercholesterolemia in the Offspring
Authors: Ciriello John , Iqbal Waseem , Hardy Daniel
Published: November 2023

Chronic intermittent hypoxia (CIH) is the underlying pathophysiological condition seen in individuals with obstructive sleep apnea. We have recently demonstrated that offspring exposed to maternal CIH during pregnancy exhibit reduced Liver X Receptor (LXR) protein leading to augmented expression of hepatic enzymes promoting gluconeogenesis. As LXR also plays an important role in cholesterol homeostasis, this study was done to determine the impact of diminished LXR expression on cholesterol handling by the liver in the offspring of CIH exposed pregnant mothers. Pregnant female Sprague‐Dawley rats were exposed daily to CIH (6.5% nadir; 18 cycles/h; 8h/day) from gestational day‐1 to day‐20. Control mothers were exposed to room air during pregnancy. Offspring were sacrificed at postnatal 6 and 12 weeks of age to assess early life and long‐term alterations in liver function. At both ages, male CIH offspring had significantly (p<0.05) higher total circulating cholesterol levels concomitant with significantly (p<0.05) greater body fat deposition compared to offspring exposed to gestational normoxia. Western blot analysis revealed that adult male CIH offspring at both ages had significantly (p<0.05) lower protein expression of the cholesterol catabolizing enzyme cholesterol 7α‐hydroxylase (CYP7A1), the cholesterol efflux transporter ATP‐binding cassette transporter G8 (ABCG8), and the low density lipoprotein receptor (LDLR) compared to the offspring exposed to gestational normoxia. Taken together, these data indicate that gestational CIH‐induced hypercholesterolemia in the offspring results from diminished expression of LXR target genes associated with cholesterol breakdown, excretion, and removal from the circulation.

Support or Funding Information

Supported in part by HSFO and CIHR
In the Short Term, Poor Nutrition Promotes Simple Steatosis without Inflammation in Adolescent Rats
Authors: Crawford Meli'sa , Liss Tyler , Calhoun Matthew , Sweazea Karen
Published: November 2023

The development of hepatic steatosis in adults has been linked to high caloric intake. However, less is known about the physiological consequences of this disease during adolescence. The present study examined the association of high caloric intake and hepatic steatosis in adolescent rats. Six‐week old male Sprague‐Dawley rats were fed one of the following diets: standard rodent chow (18.9% protein, 57.33% carbohydrates), high‐sucrose (HS: 20% protein, 70% carbohydrates (34.5% sucrose), 10% fat) or high‐fat (HF: 20% protein, 20% carbohydrates (6.8% sucrose), 60% fat) for 6 weeks. Following the diet, animals were euthanized (sodium pentobarbital; 200 mg/kg, i.p.) and livers extracted for analyses. An increase in Oil Red O stained areas of liver sections provided evidence for the development of steatosis in the HF fed animals that was confirmed by increased triglyceride concentrations in the liver as compared to animals fed the chow or HS diets (20.73 ± 2.09, 9.75 ± 0.52, and 10.76 ± 0.74 mg triglycerides/g tissue, respectively; p < 0.001). Despite the observed steatosis, western blot analyses showed no significant differences between groups in proteins associated with insulin signaling (p‐PI3K; p85, p=0.711; p55, p=0.740), gluconeogenesis (PEPCK‐C, p=0.333), immune response (XBP1; p=0.613) or inflammation (TLR‐4, p= 0.932; iNOS, p=0.675). The short feeding protocol used in the current study may explain the lack of measurable alterations in protein expression as others typically examine the effects of high caloric diets for 8–12 weeks before measuring changes. Together these findings suggest that short term high fat intake promotes simple steatosis in the absence of changes in insulin signaling or inflammation.

Support or Funding Information

This study was funded in part by new faculty start up funds, and Barrett, The Honors College, Arizona State University.
Delta‐Tocotrienol Regulates Lipid Metabolism in Livers of High Fat Fed Mice
Authors: Ramalingam Latha , Allen London , Scoggin Shane , Gurvinder Kaur , Tomison Michael D , Shen Chwan‐Li , Chung Eunhee , Dufour Jannette , Moustaid‐Moussa Naima
Published: November 2023

Non‐alcoholic fatty liver disease (NAFLD) affects more than 70% of patients with obesity. Several plant bioactive compounds with anti‐inflammatory properties are known to reduce steatohepatitis and associated metabolic complications. Hence we are investigating the regulation of hepatic metabolism by delta‐tocotrienol (δT3), member of the vitamin E family. We hypothesize that delta‐tocotrienol will reduce fatty liver and steatohepatitis through its anti‐inflammatory and lipid lowering properties. To test our hypothesis, C57BL/6J male mice were fed high fat (HF) diet with or without supplementation of increasing levels of δT3 (up to 1600mg/kg) for 14 weeks and compared to mice fed either low fat or metformin. Glucose tolerance tests revealed that mice supplemented with tocotrienol rapidly cleared glucose suggesting that liver plays a role in maintaining glucose homeostasis. By measuring expression of hepatic lipid metabolizing genes, we found that these changes were in part due to increased fatty acid oxidation and reduced fatty acid synthesis in the liver. Furthermore, hematoxylin and eosin staining of liver tissue sections demonstrated significant reductions in liver triglyceride content. Additional studies are ongoing to further understand mechanisms underlying regulation of lipid metabolism by delta‐tocotrienol. In conclusion, our results indicate that delta‐tocotrienol exerts beneficial effects in high fat diet‐induced steatohepatitis, which warrants further investigation.

Support or Funding Information

Supported by a research grant from American River Nutrition
Hypoglycaemia Modulates The Patterns Of Ventilatory Responses Evoked By Chemoreceptor Stimulation
Authors: Coney Andrew Mark , Mobley Alastair , Ray Clare
Published: November 2023

Hypoglycaemia (HG) evokes a counter‐regulatory response to restore arterial blood glucose levels. Additionally, HG has also been shown to evoke an increase in ventilation whilst maintaining normocapnia suggesting the ventilation remained matched to metabolism. Whilst it has been shown that HG induces an increase in CO2 sensitivity, there is a lack of data on how HG may modulate the pattern of breathing in normoxia as well as during chemoreceptor‐mediated responses. Whole body plethysmography (WBP) allows the measurement of respiratory variables in conscious unanesthetised animals under baseline conditions as well as during hypercpanic or hypoxic challenges.

WBP was used to measure the ventilatory responses to air breathing (21% O2), acute hypoxia (10% O2) and hypercapnia (8% CO2) in male Wistar rats that had free access to food, were fasted overnight or were made hypoglycaemic by insulin injection. Blood glucose levels tended to be lower after fasting but not significantly (8±0.8mM vs 6.8±0.5mM), however, insulin injection did induce a significant HG (3.8±0.5mM). HG induced an increase in response to hypercapnia primarily by increasing tidal volume changes. Further, HG induced an increase in expiratory time (Te) that was abolished during hypercapnia but remained present during hypoxia with no changes in inspiratory time (TI). The TI:TE ratio increased in both hypercapnia and hypoxia, however, the magnitude of increase was only dependent on glycaemic state with hypercapnia.

HG induces changes in baseline ventilation but also interacts with the response to a hypercapnic stimulus but not a hypoxic stimulus. The dependency on blood glucose levels of the TI:TE increase during hypercapnia may reflect the coordinated neuroendocrine responses seen during the counter‐regulatory response to HG and matching ventilation to an increased metabolic rate.

Support or Funding Information

A. Mobley carried out this work in support of his MRes degree
Sensitizing Effects of Chronic Pretreatment with Tumor Necrosis Factor Alpha on Vagal Pulmonary Afferent Sensitivity in Mice
Authors: Lin Ruei‐Lung , Lee Lu‐Yuan
Published: November 2023

Tumor necrosis factor alpha (TNFα), a pro‐inflammatory cytokine, is known to play an important role in the pathogenesis of allergic asthma. Inhalation of TNFα can also induce airway hyperresponsiveness in healthy human subjects, but underlying mechanisms are not fully understood. A recent study in our lab has demonstrated that TNFα pretreatment induced airway inflammation and a sustained elevation of pulmonary chemoreflex sensitivity (JAP 114: 1536–43, 2013), suggesting a possible involvement of pulmonary C‐fibers, but the direct evidence is lacking. The present study investigated the effects of chronic pretreatment with TNFα on the sensitivity of vagal pulmonary afferents in anesthetized open‐chest mice, using the single‐fiber recording technique. TNFα (10 μg/kg) and vehicle were administered by intra‐tracheal instillation in treated (TNF) and control (Veh) groups, respectively. Our results showed: 1) The peak activity of pulmonary C‐fibers to a bolus right‐atrial injection of capsaicin (Cap; 0.5 μg/kg) was significantly greater in TNF group (6.52 ± 1.34 impulses/s, n=10) than Veh group (2.10 ± 0.56 impulses/s, n=12; p<0.05). 2) The same dose of Cap evoked a burst of discharge (2.44 ± 0.71 impulses/s, n=8) in 75% (6/8) of rapidly adapting receptors (RARs) studied in TNF group, but did not stimulate any of the RARs (0/9) in Veh group. 3) This sensitizing effect of TNFα was further demonstrated in the enhanced response of Ca2+ influx to Cap in isolated vagal pulmonary sensory neurons using the Ca2+ imaging technique, and this effect was completely absent in the mice lacking both TNF receptors (p55/p75‐null mice). In conclusion, chronic pretreatment with TNFα induced a sensitizing effect on the Cap sensitivity in both pulmonary C‐fibers and RARs, and the action was mediated through TNF receptors. These sensitizing effects of TNFα may contribute, at least in part, to the pathogenesis of airway hyperresponsiveness induced by this cytokine.

Support or Funding Information

Supported by NIH grants HL‐96914 and UL1TR0000117
The Src Family Tyrosine Kinase fyn Protects Mice from Inflammation‐induced Lung Injury
Authors: Jin Yi D , Liu Yusen , Trittmann Jennifer K , Chicoine Louis G , Chen Bernadette , Nelin Leif D
Published: November 2023

Endothelial cell apoptosis is an early event in the development of inflammatory lung diseases such as acute respiratory distress syndrome (ARDS) and bronchopulmonary dysplasia (BPD). In studies utilizing cultured human pulmonary endothelial cells we have shown that the Src family tyrosine kinase (STK) yes activated caspase‐3, while the STK fyn inhibited caspase‐3 activation. Therefore, in this study we tested the hypothesis that mice deficient in fyn will have more severe lipopolysaccharide (LPS)‐induced pulmonary apoptosis and lung injury than will similarly treated wild‐type mice. Wild‐type and fyn−/− mice (all on the same 129S1/SvImJ background) were given LPS 10 mg/kg intraperitoneally or saline for 24 hours and lungs were harvested for determination of proteins related to apoptosis. LPS treatment resulted in greater levels of cleaved caspase‐8, cleaved caspase‐9, cleaved caspase‐3, and p21 in the lungs from fyn−/− mice than in the lungs from wild‐type mice. In a second set of studies, lung sections from wild‐type and fyn−/− mice were stained for platelet endothelial cell adhesion molecule‐1 (PECAM‐1), we found that the fyn−/− mice had less PECAM‐1 expression, thicker alveolar septa, and more leukocyte infiltration than did wild‐type mice. In another set of studies wild‐type and fyn−/− mice were given LPS 2 mg/kg intratracheally or an equal volume of saline, and 24 hours later lung mechanics were measured using a Flexivent system (SCIREQ, Montreal, Canada). The fyn−/− mice had significantly (p<0.01) lower lung compliance (0.030 ± 0.003 ml/cmH2O) than did wild‐type mice (0.040 ± 0.001 ml/cmH2O). The fyn−/− mice also had significantly (p<0.05) higher tissue elastance (32.6 ± 3.1 cmH2O/mL) than did wild‐type mice (23.8 ± 0.8 cmH2O/mL). The fyn−/− mice had greater (p = 0.046) lung resistance (0.67±0.06 cmH2O/ml/s) than did wild‐type mice (0.55±0.02 cmH2O/ml/s). These results demonstrate that mice lacking fyn develop more apoptosis and more severe lung injury following LPS exposure than do wild‐type mice. These findings demonstrate that fyn is an important negative regulator of LPS‐induced apoptosis and the resultant lung injury in a mouse model of inflammatory lung diseases such as ARDS.
Inhibition of Equilibrative Nucleoside Transporters Attenuated Pseudomonas Aeruginosa‐Induced Acute Lung Injury
Authors: Chambers Eboni , Borgas Diana , Blackburn Michael , Rounds Sharon , Lu Qing
Published: November 2023

RATIONALE

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), causes acute respiratory failure with 40% mortality. We and others have previously demonstrated that adenosine is increased during tissue injury and protects against ALI through activation of adenosine receptors. Pseudomonas aeruginosa infections possess high mortality due to antibiotic resistance and there is compounded risk for ALI in patients with compromised innate immunity. However, it is unknown whether altering adenosine signaling can prevent or treat P. aeruginosa‐induced ALI. Extracellular and intracellular adenosine concentrations are regulated by equilibrative nucleoside transporters (ENTs). We hypothesized that increased extracellular adenosine by inhibition of ENT1 attenuates P. aeruginosa‐induced ALI.

METHODS

To determine the effects of inhibition of ENT1 on P. aeruginosa‐induced ALI, both ENT1 inhibitors and null mice were used. P. aeruginosa was given to mice via intratracheal instillation. To elucidate the mechanism that underlies the effects of ENT1 inhibition on P. aeruginosa‐induced ALI, both agonists and antagonists directed against adenosine receptors A2AR and A2BR were used.

RESULTS

P. aeruginosa caused ALI in C57BL/6 mice in a dose dependent manner. Pharmacological inhibition of ENT1 by NBTI significantly attenuated P. aeruginosa‐induced ALI, as assessed by wet‐to‐dry lung weight, bronchoalveolar lavage (BAL) protein levels, BAL inflammatory cells and cytokine expression. This protective effect of NBTI was abolished when mice received an antagonist against either A2AR or A2BR. Additionally, agonists against A2AR or A2BR mimics NBTI blunting P. aeruginosa‐induced ALI. These results suggest that NBTI protects against P. aeruginosa‐induced ALI via adenosine‐mediated activation of both A2AR and A2BR. Interestingly, ENT1 null mice did not protect against P. aeruginosa‐induced ALI, suggesting an involvement of a compensatory mechanism.

CONCLUSION

Inhibition of nucleoside transporters may be a novel approach to prevent and treat P. aeruginosa‐ induced ALI.

Support or Funding Information

This work was in part supported by NIGMS P20GM103652 to S. Rounds and Q. Lu, VA Merit Review to S. Rounds, ATS/PHA research grant to Q. Lu, and NIEHS T32 ES007272 to E. Chambers.
Toll‐like Receptor 7 on Airway Nerves Causes Bronchodilation
Authors: Drake Matthew Georgenson , Proskocil Becky J , Lebold Katherine , Blum Emily , Fryer Allison , Jacoby David
Published: November 2023

BACKGROUND

Toll‐like receptor 7 (TLR7) detects viral single‐stranded RNA and triggers an innate immune response. We found that TLR7 agonists also induce a rapid and potent bronchodilation in the lung.

OBJECTIVE

To evaluate TLR7 expression and function in the lung.

METHODS

TLR7 expression was evaluated in lungs of Hartley guinea pigs and human trachea and primary bronchi (from deceased donors) using immunohistochemistry and RT‐PCR. TLR7's effect on bronchoconstriction was assessed using an organ bath system. Airways were contracted with methacholine (3 μM) and treated with TLR7 agonist R837 (10–300 μM). Some airways were pre‐treated with nitric oxide synthase inhibitor (L‐NMMA) or TLR7 antagonist IRS661. To differentiate the role of specific TLR7‐expressing airway nerve subtypes, some airways were cultured with or without attached sensory nerve ganglia for 48 hours prior to physiology experiments.

RESULTS

Airway parasympathetic nerves, and sensory nerves that originate in in the nodose, jugular, dorsal root and esophageal ganglia all express TLR7. TLR7 agonist rapidly dilated pre‐contracted human and guinea pig airways. TLR7‐mediated bronchodilation was blocked by TLR7 antagonist and by inhibition of nitric oxide production. Bronchodilation was unchanged in airways whose sensory nerves had undergone degeneration, suggesting parasympathetic nerves alone are sufficient for TLR7‐induced bronchodilation.

CONCLUSIONS

TLR7 is expressed by airway parasympathetic and sensory nerves. TLR7 mediates a rapid and potent bronchodilation. These data suggest that TLR7 is a potential bronchodilatory therapeutic target in humans.

Support or Funding Information

Funding Source: This project was supported by NIH grants HL121254, HL124165, HL113023, AR061567.
Cyclic AMP pathways leading to immediate‐early gene activation in neuroendocrine cells: relevance to neurotransmitter signaling through the D1 dopamine and PAC1 neuropeptide receptors
Authors: Eiden Lee E , Elkahloun Abdel , Samal Babru B , Eiden Maribeth V , Emery Andrew C
Published: November 2023

Gs‐coupled GPCR signaling in the NS‐1 pheochromocytoma cell line leads to the activation of three distinct cyclic AMP sensors, Epac, PKA and NCS/Rapgef2 mediate distinct cellular outputs: p38‐dependent growth arrest; CREB‐dependent cell survival and neuron‐specific gene expression; and ERK‐dependent neuritogenesis, respectively (Emery et al., Sci. Sig. 6: ra51, 2013; Emery et al., JBC 289: 10126, 2014). Expression of D1 dopamine receptors from a stably‐integrated, CMV promoter‐driven expression cassette in NS‐1 cells causes cyclic AMP elevation and activation of all three of these pathways, leading to D1 receptor‐dependent activation of all three downstream pathways, as previously reported for activation of the endogenous PAC1hop receptor by the neuropeptide PACAP. To investigate which of the three pathways might mediate cAMP‐dependent immediate‐early gene (IEG) transcription relevant to neurotransmitter action in vivo, we examined genome‐wide transcriptional regulation by cyclic AMP in NS‐1 cells, using microarray analysis. Treatment of NS‐1 cells for one hour with the cell‐permeant cyclic AMP analog 8‐CPT‐cAMP (100 micromolar), an equipotent activator of Epac2, PKA and NCS/Rapgef2 (Emery et al., JBC 289: 10126, 2014) resulted in significant up‐regulation of 174 mRNAs. Of these, ten encoded transcription factors, and six of these encoded IEGs: Ier‐3/Egr3, Zif268/Egr1, Nr4a1, Nr4a3, c‐Fos, and JunB. Cyclic AMP‐dependent up‐regulation of the Ier‐3/Egr2 and Zig268/Egr1 genes, but not the Nr1a1, Nr4a3, or JunB genes, was ERK‐dependent (blocked by the MEK inhibitor U0126, at 10 micromolar), while up‐regulation of the c‐Fos gene appeared to be partially ERK‐dependent. These results suggest that regulation of IEG expression by D1 and PAC1 receptor stimulation may involve two cAMP‐dependent pathways, one mediated by NCS/Rapgef2, and the other by either Epac or PKA. These novel signaling pathways may be relevant to IEG regulation by dopamine in the striatum in vivo (Gerfen et al., J. Neurosci. 15: 8167, 1995) and by the stress‐regulatory neuropeptide PACAP in hypothalamus and extended amygdala (Stroth and Eiden, Neuroscience 165: 1025, 2010; Emery and Eiden, FASEB J. 26: 3199, 2012).

Support or Funding Information

Supported by NIMH‐IRP project ZO1‐MH002386
Rutin attenuates high cholesterol diet‐induced hepatotoxicity in rat model via regulation of tumor suppressor genes; p53 and caspase‐3 and P21
Authors: Alhoshani Ali , Hafez Mohammed M
Published: November 2023

Hypercholesterolemia‐induced oxidative stress has been implicated in the pathogenesis of various disease including cardiovascular disorders and non‐alcoholic fatty liver disease (NAFLD). Tumor suppressor genes such as p53 play a central role in NAFLD. p53 activation facilitates apoptosis, oxidative stress, steatosis and injury. Levels of p21 often determine the cellular response to different stress induced by drug and others. Caspase‐3‐mediated cleavage of p21. This study was designed to investigate the potential protective effects of dietary antioxidant rutin (RT) combination with high cholesterol diet (HCD) induced hepatotoxicity in male Wistar albino rats. Twenty four male Wistar rats were used in the present study. Animals were divided into four groups; (1) control, (2) RT, (3) HCD and (4) HCD+RT. Animals received freshly prepared experimental diets for 6 consecutive weeks. In plasma, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were estimated. In hepatic tissue, gene expressions of p53 and caspase‐3 and P21 were measured using real time PCR. Rats fed with HCD for 6 weeks had significant increase in plasma ALT and AST levels compared to control and HCD+RT groups. In the current study, HCD increase p53 and caspase‐3 mRNA expression and reduced the mRNA expression P21. Rutin in combination with HCD induced a significant protective effect against the alteration in p53 and caspase‐3 and P21 genes expression. In conclusion, our findings demonstrated that rutin could attenuate the hepatotoxicity induced by HCD in rat model via regulation of tumor suppressor genes.
Role of neuronal signaling effector hydrogen sulfide (H2S) and sulfhydration in Huntington's disease
Authors: Paul Bindu Diana , Snyder Solomon H.
Published: November 2023

Hydrogen sulfide (H2S) has joined the ranks of the gasotransmitters, nitric oxide and carbon monoxide as a signaling molecule that modulates a wide spectrum of physiological processes. H2S signals via sulfhydration, wherein it mediates the conversion of −SH groups of reactive cysteine residues on target proteins to −SSH or persulfide groups. H2S is synthesized by cystathionine gamma lyase (CSE), cystathionine beta synthase (CBS) and 3‐mercaptopyruvate sulfurtransferase of the reverse transsulfuration pathway. Aberrant H2S metabolism is involved in the progressive neurodegeneration seen in Huntington's disease (HD). HD is a devastating condition and is currently considered to be an incurable disease of the nervous system. New treatment options are highly sought after. We asked how normal healthy neural development is influenced by the differential interplay of huntingtin versus mutant huntingtin (mHtt) function vis‐à‐vis CSE and CBS action in the nervous system. Specifically, we sought to understand how the modalities of mutant transformation of huntingtin at the genetic and epigenetic levels affect mHtt effects on H2S action. We chose cell culture and mouse models of HD to study their manifestation on the progressive neurodegeneration observed alongside the cognitive and motor deficits that are characteristic of human HD patients. We have shown that loss of CSE mediates neurodegeneration and disease progression in HD. It appears that the neuronal redox effector functions of H2S are differentially modulated by the action of mHtt on CSE and CBS thereby affecting disease progression in HD by variable regulatory mechanisms.

Representative Recognition/Honor

ASBMB/jbc Herbert Tabor Young Investigator Award (May 2015) at the third European Conference on the Biology of Hydrogen Sulfide at Athens, Greece.

Support or Funding Information

United States Public Health Service Grant MH18501 to S.H.S, CHDI Foundation
Cardiomyocyte Ogt limits ventricular dysfunction in mice following pressure overload without affecting hypertrophy
Authors: Dassanayaka Sujith , Brainard Robert E , Watson Lewis J , Long Bethany W , Brittian Kenneth R , DeMartino Angelica M , Aird Allison L , Kilfoil Peter J , Muthusamy Senthilkumar , Hamid Tariq , Prabhu Sumanth D , Jones Steven P
Published: November 2023

The myocardial response to pressure overload involves coordination of multiple transcriptional, posttranscriptional, and metabolic cues. Previous studies show that one such metabolic cue, O‐linked beta‐N‐acetylglucosamine (O‐GlcNAc), is elevated in the pressure‐overloaded heart, and the increase in O‐GlcNAcylation is required for cardiomyocyte hypertrophy in vitro. Yet, it is not clear whether and how O‐GlcNAcylation participates in the hypertrophic response in vivo. Here, we addressed this question using patient samples and a preclinical model of heart failure. Protein O‐GlcNAcylation levels were increased in myocardial tissue from heart failure patients compared with normal patients. To test the role of O‐GlcNAc transferase (OGT) in the heart, we subjected cardiomyocyte‐specific, inducibly deficient Ogt (i‐cmOgt−/−) mice and Ogt competent littermate wild type (WT) mice to transverse aortic constriction (TAC). Deletion of cardiomyocyte Ogt significantly decreased O‐GlcNAcylation and exacerbated ventricular dysfunction, without producing widespread changes in metabolic transcripts. Although some changes in hypertrophic (elevated Nppa, reduction in GATA4) and fibrotic signaling (elevated Tgfb2, reduction in Fgf2) were noted, there were no histological differences in hypertrophy or fibrosis. We next determined whether significant differences were present in i‐cmOgt−/− cardiomyocytes from surgically‐naïve mice. Interestingly, markers of cardiomyocyte dedifferentiation (Acta2, Nkx 2–5) were elevated in Ogt deficient cardiomyocytes. Although no significant differences in cardiac dysfunction were apparent after recombination, it is possible that such changes in dedifferentiation markers could reflect a larger phenotypic shift within the Ogt deficient cardiomyocytes. We conclude that cardiomyocyte Ogt is not required for cardiomyocyte hypertrophy in vivo; however, loss of Ogt may exert subtle phenotypic differences in cardiomyocytes that sensitize the heart to pressure overload‐induced ventricular dysfunction. Because diabetes is often associated with altered O‐GlcNAcylation, our findings could have implications for the diabetic heart.

Support or Funding Information

Dr. Jones has been supported by grants from the NIH (R01 HL083320, R01 HL094419, P20 RR103492, and P01 HL078825). Mr. Dassanayaka holds an American Heart Association Predoctoral Fellowship – Great Rivers Affiliate (14PRE19710015).
Cardiomyocyte Glucose Handling is Influenced by β‐adrenergic and AMPK Regulation of Glycogen and Autophagy in Metabolic Stress Settings
Authors: Mellor Kimberley M , Varma Upasna , Stevens Ellie , Delbridge Lea MD
Published: November 2023

Autophagy disturbance and glycogen mishandling in the heart play a role in metabolic stress settings such as diabetes. We have recently demonstrated that an autophagy process specific for glycogen (‘glycophagy’), which is distinct from macro‐autophagy, is modulated by metabolic stress and is an important regulator of glycogen content in the heart. Intracellular glucose flux through these pathways has an important role in glycosylation‐related post‐translational modifications (e.g. O‐GlcNAcylation and advanced glycation end products). The aim of this study was to elucidate β‐adrenergic and AMPK signaling regulation of glycogen handling and autophagy in the heart.

Excised hearts (male Sprague Dawley 8 weeks) were perfused with 10−6M isoproterenol (β agonist) or control buffer for 5 or 60 minutes, snap frozen, homogenized and protein extracted. Neonatal rat ventricular myocytes (NRVMs) cultured in 5mM or 30mM glucose were exposed to AICAR (AMPK agonist) for 30 minutes, lysed and frozen. Protein expression was assessed by western blot and glycogen content was assessed by amyloglucosidase assay.

Isoproterenol‐induced β‐adrenergic activation was evidenced by an increased phospho(Ser14)‐ to total glycogen phosphorylase ratio after 5min of isoproterenol perfusion (62% increase p<0.05). Glycophagy markers GABARAPL1 and acid α glucosidase were markedly increased by β‐adrenergic signaling activation (3‐fold and 1.5‐fold increase respectively, p<0.05) consistent with depletion of glycogen content (71% lower after 5min and fully depleted after 60min Isoproterenol perfusion, p<0.05). Macro‐autophagy markers, LC3BII:I ratio and p62 protein expression were not changed in the isoproterenol perfusion groups. Inactivation of glycogen synthase (phosphorylation‐Ser641) was only evident after 60min isoproterenol perfusion. AICAR‐induced activation of AMPK attenuated high glucose‐induced glycogen accumulation in NRVMs with no change in glycogen phosphorylase activation, suggesting an independent and parallel role for glycophagy in glycogen management.

This is the first study to show that β‐adrenergic signaling positively regulates glycophagy. These findings suggest that β‐adrenergic regulation of glycophagy is distinct from macro‐autophagy, demonstrating that autophagy sub‐types are influenced differentially by signaling modalities. Involvement of AMPK signaling in glycophagy induction is evident and further investigation of glycophagy in cardiac metabolic stress settings is now warranted. These studies seek to understand the relationship between glycophagic throughput and intracellular glucose exposure to functionally detrimental post‐translational modifications.

Support or Funding Information

Marsden Fund Project Grant, FRDF University of Auckland Project Grant
Mechanism of Atrial Fibrillation is Related to Changes of Type IV Collagen in Rheumatic Heart Valvular Disease
Authors: Jiang Yi‐Yao , Yang Qin , Liu Xiao‐Cheng , He Guo‐Wei
Published: November 2023

Purpose

Atrial fibrillation (AF) is a common complication in heart valvular disease and is associated with morphology and cell ultrastructure abnormality. We aimed to study the distribution of collagen types and to investigate the relationship between type IV collagen changes in the tissue of the right atrium and the development of permeant AF in patients with mitral valvular disease.

Methods

The atrial appendage obtained from patients with mitral valvular disease associated with either sinus rhythm (SR, n=26, right atrial tissue) or permanent AF (AF ≥6 months, n=36, right and left atrial tissue). One part of the sample was used for histological analysis, including iTRAQ proteomics, immunohistochemistry, immunofluorescence and transmission electron microscope; the other part was analyzed by qRT‐PCR and western blotting to observe the molecular level of type IV collagen.

Results

iTRAQ proteomic study showed that type IV collagen was up‐regulated in right and left atrial tissue. Type IV collagen was located around cardiomyocytes, especially on vascular basement membrane and myocardial cell membrane. The fluorescence intensity of type IV collagen increased in AF patients. Electron microscope revealed that there was a large number of immature fibrocytes among myocytes, likely immature type collagen IV. qRT‐PCR and western blots showed that the mRNA and protein of type IV collagen were significantly elevated in AF patients, especially in the left atrial tissue.

Conclusions

Our study identified that the change of type IV collagen in the atrial appendage is related to the development of AF in the mitral valvular disease. The present study suggests that abnormality of the type IV collagen may play an important role in the structure and electrical signal in the heart valvular disease associated with AF.

Support or Funding Information

Supported by National Natural Science Foundation of China (No.81170148), Zhejiang Provincial Natural Science Foundation (LY15H020008), Hangzhou Science and Technology Projects (20140633B12), National Science and Technology Major Project (2013ZX09303004‐005), & Tianjin Binhai Key Platform for Creative Research Program (2012‐BH110004), Binhai New Area Health Bureau (2012BWKZ008,2014BWKY002, 2014BWKY010), Tianjin Health Bureau (2013KZ009,2014KZ005).
Cav1.2 channel oligomerization amplifies Ca2+ influx during β‐adrenergic stimulation
Authors: Dixon Rose Ellen , Santana Luis Fernando
Published: November 2023

L‐type Cav1.2 channels are ubiquitously expressed in the surface membranes of excitable cells where they regulate myriad processes including muscle contraction, neuronal excitability, hormone secretion and gene expression. Recently, we reported that Cav1.2 channels form clusters that undergo dynamic, reciprocal, allosteric interactions. This ‘functional coupling’ facilitates Ca2+ influx by increasing activity (NPo) of adjoined channels and occurs through C‐terminal‐to‐C‐terminal interactions. Channel‐to‐channel interactions are initiated by binding of incoming Ca2+ to CaM and proceed through Ca2+/CaM binding to the Cav1.2 pre‐IQ domain. FRET experiments revealed that coupling fades as [Ca2+]i decreases, but persists longer than the current that evoked it, providing evidence for ‘molecular memory’. This creates a ‘primed state’ which may have a role during repetitive stimulations, such as those that occur during periods of increased heart rate – stimulated for instance by exercise or the fight‐or‐flight response. This ‘memory’ could be beneficial in generating a positive inotropic effect. In the present study we used FRET, Bimolecular Fluorescence Complementation (BiFC), electrophysiology and Ca2+ imaging to investigate the effects of β‐adrenergic signaling on Cav1.2 interactions. Optical and electrophysiological examination of single channel activity in mouse ventricular myocytes or transiently transfected tsA‐201 cells revealed increased coupling of Cav1.2 channels after application of the β‐agonist isoproterenol (iso; 100 nM) or the activator of adenylyl cyclase, forskolin (10 μM). BiFC experiments in tsA‐201 cells co‐transfected with Cav1.2 channels tagged with the N‐ or C‐terminal half of the split Venus fluorescent protein (Cav1.2‐VN and Cav1.2 ‐VC) revealed increased channel‐channel interactions stimulated by iso or forskolin and inhibited by the PKA inhibitor (PKI). These data suggest that β‐adrenergic stimulation amplifies Cav1.2 channel activity by increasing/prolonging Cav1.2 channel interactions.

Support or Funding Information

This work was supported by grants from the AHA (15SDG25560035) and NIH (HL085870 and HL085686).
TRPV1‐Caveolin 1 direct association and its regulation of NO production: implications for vascular regulation
Authors: DelloStritto Daniel , Fahmy Joseph , Connell Patrick , Geldenhyus Werner , Bratz Ian
Published: November 2023

Caveolin‐1 (Cav‐1), a scaffolding protein responsible for the formation of caveolae, is ubiquitously expressed, but predominately in endothelial cells. Moreover, Cav‐1 is known to associate with several ion channels including various Kv and TRP subtypes. It is well established when Cav‐1 and endothelial nitric oxide synthase (eNOS) are bound; Cav‐1 exerts an inhibitory effect on eNOS activity and NO production until a Ca2+ dependent signal untethers the proteins. Our lab and others have shown that TRPV1 regulates eNOS activity and NO production; specifically, via an AKT‐CaMKII‐AMPK signaling cascade. Therefore, we hypothesize that TRPV1, Cav‐1, and eNOS form a protein complex in endothelial cells responsible for the regulation of NO production. Examination of the TRPV1 amino acid sequence revealed 4 potential Cav‐1 binding motifs (where Ø represents any aromatic amino acid and X represents any amino acid): ØXXØXXXXØØ, ØXXØXXXØ, ØXXXØXXØ, and ØXXXØXØ. Using computational modeling we confirmed potential interactions between TRPV1 and Cav‐1. Staining bovine aortic endothelial cells (BAEC) demonstrate TRPV1 and Cav‐1 co‐localization. Further verification of a direct interaction between Cav‐1 and TRPV1 was established using co‐immunoprecipitation. Treatment of BAECs with capsaicin decreased the association between Cav‐1 and eNOS resulting in increased NO production. Interestingly, this treatment also results in the increased complex formation of Cav‐1 with TRPV1. In conclusion, this is the first evidence of a direct interaction between TRPV1 and Cav‐1 and its implications in the regulation of eNOS and vascular function.
Acid Sensing Ion Channel 1 Contributes to Endothelium‐Dependent Vasodilation in Mesenteric Arteries
Authors: Garcia Selina , Herbert Lindsay , Resta Thomas C , Jernigan Nikki L
Published: November 2023

Our laboratory has previously shown that acid sensing ion channel 1 (ASIC1) is expressed in pulmonary arterial smooth muscle and contributes to agonist‐induced Ca2+ influx and vasoconstriction. However, the contribution of ASIC1 to vasoreactivity in the systemic circulation is unknown. To address this question, we simultaneously measured changes in arterial wall Ca2+ and inner diameter in response to increasing concentrations of phenylephrine (PE; 10−8‐10−5 M) or endothelin‐1 (ET‐1; 10−11 – 10−8 M) in isolated, pressurized mesenteric resistance arteries. To examine the role of ASIC1 in agonist‐induced vasoconstriction, these experiments were completed in the presence or absence of the selective ASIC1 antagonist, psalmotoxin 1 (PcTX1; 20 nM), applied either abluminally or to both luminal and abluminal surfaces of the artery. Inhibition of ASIC1 did not alter PE‐induced vasoconstriction, but augmented vasoconstriction in response to lower concentrations of ET‐1 when PcTX1 was present in the lumen. These data suggest that, in contrast to vasoconstrictor actions of ASIC1 in the pulmonary circulation, ASIC1 limits agonist‐induced vasoconstriction in the systemic circulation. Therefore, we hypothesized that endothelial expression of ASIC1 contributes to mesenteric arterial dilation. To test this hypothesis, mesenteric arteries were pre‐constricted to ~50% of baseline inner diameter with the thromboxane analog, U‐46619 (10−7 M), and vasodilatory responses to increasing concentrations of acetylcholine Ach (ACh; 10−9 ‐ 10−5 M) were assessed in the presence or absence of PcTX1 in the lumen. ACh‐induced vasodilation was significantly blunted in the presence of PcTX1. We conclude that ASIC1 contributes to endothelium‐dependent vasodilation in mesenteric arteries. These data demonstrate differing roles of ASIC1 in regulation of pulmonary and systemic vasoreactivity.

Support or Funding Information

NIH NHLBI ROI HL111084 (N.L. Jernigan)

NIH NHLBI T32 HL007736 (B.R. Walker)
Aerobic exercise training increases testosterone production in the testis in OLETF rats
Authors: Kumagai Hiroshi , Zempo‐Miyaki Asako , Myoenzono Kanae , Maeda Seiji
Published: November 2023

Testosterone is a male sex hormone and low circulating testosterone levels are associated with cardiovascular diseases, type 2 diabetes mellitus. Obesity results in reduced circulating testosterone levels. We have demonstrated that lifestyle modifications (increased physical activity and calorie restriction) can increase circulating testosterone levels in obese men and increased physical activity is important for lifestyle modification‐induced increase in circulating testosterone levels. However, it is unclear the mechanism underlying regular exercise‐induced increase in circulating testosterone levels.

The aim of this study was to investigate the effect of aerobic exercise training on testosterone production in the testis in male Otsuka Long Evans Tokushima Fatty (OLETF) rat.

OLETF rats (12 weeks of age) were assigned randomly into exercise group (n = 8) and control group (n = 8). In the exercise group, rats ran on the treadmill for 8 weeks (for 60 min at 25 m/min, 5 days/week). After the 8‐week aerobic exercise training, we analyzed circulating and testis testosterone levels by EIA, and the protein expression of 17β‐HSD, enzyme regulating testosterone, in the testis by western blotting.

After the 8‐week aerobic exercise training, body mass was significantly lower in the exercise group than control group. Circulating and testis testosterone levels were significantly higher in the exercise group than control group. Also, the protein expression of 17β‐HSD in the testis was higher in the exercise group than control group.

In the present study, circulating and testis testosterone levels increased after the 8‐week aerobic exercise training in OLETF rat. Furthermore, the protein expression of 17β‐HSD in the testis in OLETF rat increased after aerobic exercise training. Therefore, the 8‐week aerobic exercise training increases testosterone and 17β‐HSD productions in the testis in OLETF rat, thereby contributing to the increased circulating testosterone level after exercise training.