bluuebunny/crossref_metadata_2025 huggingface dataset:
Exercise training regulates autophagy in aorta
Authors: Okutsu Mitsuharu , Yamada Mami , Uto Taiki , Hokazono Chihiro
Published: November 2023

Objective

Atherosclerosis, an inflammatory vascular disease in large‐ and medium sized arteries, is characterized by the formation of atherosclerotic plaques. It has been demonstrated that exercise training decreases atherosclerotic plaque formation and reduces the levels of circulating pro‐inflammatory cytokines; but it is unknown how reduction of inflammatory cytokines by exercise contribute to the inhibition of atherosclerosis. Autophagy is a reparative and life‐sustaining process by which cytoplasmic components are sequestered in double‐membrane vesicles and degraded upon fusion with lysosomal compartments. Recent studies indicate that autophagy is stimulated in advanced atherosclerotic plaques by oxidized lipids, metabolic stress and inflammation. However, it is not clear whether exercise training modulates autophagy in arteries.

Methods

Here, we examined whether exercise training regulates autophagy in the aorta of male C57BL/6J and apolipoprotein E deficient mice. C57BL/6J were fed normal chow. Apolipoprotein E deficient mice were fed 45% high‐fat diet to induce atherosclerosis. Mice were randomly assigned into exercise training groups and sedentary group. Exercise training mice were individually housed in cages equipped with a running wheel for 16 weeks. Heart, aorta, blood and plantaris muscle were harvested at 24 hours after the last bout of exercise. To quantify atherosclerotic lesions, we stained aortic root sections with Oil Red O and hematoxylin‐eosin. Autophagy protein expression in aorta was analyzed by western blot. Serum inflammatory cytokines were measured by Proteome Profile Antibody Arrays.

Results

Voluntary running in C57BL/6J mice resulted in reduced autophagy flux (i.e., a combination of decreased LC3‐II/LC3‐I ratio, LC3‐II levels and tend to increase p62/sequestosome 1 protein content) in aorta. Exercise training significantly reduced atherosclerotic plaque area at the aortic root in apolipoprotein E deficient mice. Autophagy flux in the aorta was significantly lower in exercise trained apolipoprotein E deficient mice when compared to sedentary mice. Serum inflammatory cytokines in exercise trained apolipoprotein E deficient mice were significantly lower than sedentary mice.

Conclusions

These results suggest that the regular exercise‐mediated reduction of inflammatory cytokines is also associated with a potential decrease in autophagy levels in aorta, which may contribute to the attenuation of atherosclerosis.

Support or Funding Information

Grant‐in‐Aid for Challenging Exploratory Research (26560404) to Okutsu M.
Human umbilical cord mesenchymal stem cells attenuates heatstroke injury in rats
Authors: Lin Mao‐Tsun , Chang Ching‐Ping , Lin Cheng‐Hsien
Published: November 2023

Heatstroke reactions include hyperthermia, systemic inflammation, and multiple organ dysfunctions including brain ischemia and hypotension. We aimed to test whether human umbilical cord mesenchymal stem cells (hUC‐MSC) obtained from Meridigen Biotech company improves outcomes of heatstroke in rats. Adult male Sprague‐Dawley rats, under general anesthesia, were subjected to heat stress (42°C for 98 min) to induce heatstroke. Both the cerebral blood flow and the cellular levels of ischemia indicators in the hypothalamus were monitored during the entire experimentation. The serum levels of both lipid peroxidation and activated inflammation were determined by biochemical analyses. Heatstroke rats with vehicle treatment displayed (1) hyperthermia; (2) hypotension and bradycardia; (3) brain ischemia (e.g., decreased cerebral blood flow and increased hypothalamic levels of both glutamate and lactate/pyruvate); (4) systemic inflammation (e.g., increased serum levels of tumor necrosis factor‐alpha and interleukin‐6); and (5) lipid peroxidation (e.g., increased serum levels of malondialdehyde). Survival observations provide supportive evidence. hUC‐MSCs (1×106/ ml/ kg of body weight), but not vehicle (ml/ kg), when administered intravenously at 24 hours before heatstroke, significantly attenuated these heatstroke reactions. Mesenchymal stem cells derived from human umbilical cord promoted survival in rats after heatstroke, likely due to the ability of the cells to produce anti‐hyperthermia, anti‐hypotension, anti‐lipid peroxidation, and anti‐inflammation.
Effects of Dietary Creatine Monohydrate Supplementation and Resistance Training on Late Onset Doxorubicin‐Induced Skeletal Muscle Dysfunction and MyoD Expression
Authors: Kugler Benjamin A , Capps Michael J , Hydock David S
Published: November 2023

The chemotherapy drug doxorubicin (DOX) has been shown to cause skeletal muscle dysfunction and reductions in MyoD gene expression. Creatine monohydrate (Cr) supplementation and resistance training (RT), however, have been shown to increase MyoD expression and improve skeletal muscle function, but their effects on DOX myotoxicity have yet to be explored. The aim of this study was to determine the effects of DOX treatment, RT, Cr, and combined Cr and RT on late‐onset DOX myotoxicity and MyoD expression. Male rats randomly assigned to control diet+sedentary+saline (SS), control diet+sedentary+DOX (SD), control diet+RT+DOX (RD), Cr+sedentary+DOX (CrSD), and Cr+RT+DOX (CrRD) were fed either standard rodent chow or a diet supplemented with 3% Cr for 40 days, and on day 14, animals received daily 1 mg/kg DOX injections for 12 consecutive days (12 mg/kg cumulative) or saline as a placebo. After injections, RD and CrRD were housed in cages where food and water were gradually raised to act as a RT model. Forelimb grip strength was assessed at baseline and throughout the 40‐day treatment period. Following drug treatments and dietary/exercise interventions, the right soleus (SOL) and extensor digitorum longus (EDL) were excised and analyzed for MyoD expression using Western blotting. From baseline to day 40, SS had an increase in grip strength (+20%, P<0.05), and this increase was not observed in SD, RD, or CrSD (+3%, +7%, and +2%, respectively, P>0.05); however, an increase in grip strength over the 40‐day period was observed in CrRD (+25%, P<0.05). In the SOL, no between group differences in MyoD expression were observed (P=0.093), and when compared to SS, MyoD expression in the SOL was 37% lower in SD. No trends in the nutrition and exercise interventions modulating MyoD expression were observed (−51%, −36%, and −64% vs. SS in RD, CrSD, CrRD, respectively). Similarly, no between group differences in EDL MyoD expression were observed (P=0.57) with SD, RD, CrSD, and CrRD expressing 46%, 42%, 21%, and 27% lower MyoD, respectively, than SS. Combined Cr and RT preserved grip strength following DOX treatment, but MyoD expression does not seem to be involved in this myoprotection. It is possible that myogenic regulatory factors other than MyoD may be involved in Cr and RT‐induced protection against DOX myotoxicity.
Recovery of Running Wheel Activity in Nursing Dams
Authors: Murphy Robert J , Welshhans Elizabeth A , O'Hagan Kathleen P
Published: November 2023

Rats provided with cage running wheels engage in daily spontaneous exercise (EX), and this model is used to study the impact of EX on maternal and fetal outcomes in normal and compromised pregnancies. Previously we reported that normal gestation in rats is marked by progressive decreases in exercise volume primarily through decreases in time rather than velocity. Here we report the recovery of EX over 5 weeks postgestation and address the hypothesis that the presence of nursing pups in the cage attenuates recovery of EX by dams. Female Sprague‐Dawley rats were housed individually with cage wheels and divided into Time Control (TC, N=9) and Pregnant groups (bred, N=17). The Pregnant group was subdivided into Pups‐Kept (N=9) and Pups‐Removed (N=8) groups at the time of parturition. Cage revolutions were detected by a bike computer and accumulated distance (m), time (min) and average velocity (m/min) were calculated for contiguous 24 hr periods. Daily data were averaged in bins of 7 days: Nonpregnant (1 bin), gestation (G; 3 bins), postgestation (PG, 3 bins) and postweaning (PW, 2 bins) for statistical analysis (ANOVA; Mean[SD]). Here we present the time data as the distance response was qualitatively similar. Average daily EX time was 164 (61) (n=26) in the nonpregnant state. EX time during 3rd week of gestation was reduced by 85% (10) in pregnancy (n=17) and 11% (19) in TC rats (P<0.001). Surprisingly, EX time (see table below) showed little increase moving from week 1 to week 2 (P=0.4) but was greater in the Pup‐Removed vs the Pup‐Kept groups as postgestation moved from week 2 to week 3 (Interaction contrast, P=0.04). Once the pups were weaned, the Pup‐Kept group increased EX time slightly more than the Pup‐Removed group (interaction contrast, P=0.049) and there was no further increase in EX time (P=0.9) during the 2nd week postweaning. EX time was not different in TC vs Preg groups at PW2 (P=0.6) although overall, EX time decreased 20% (41) over the ~60 day period of time (P=0.003). Surprisingly, the lack of pups had little effect on spontaneous EX in rat dams during the first 2 weeks postgestation, suggesting that metabolic energy conservation or time for nursing are probably not primary reasons for the slow recovery of exercise volume after parturition. Recovery of exercise volume accelerates during week 3 postgestation, and this recovery is accentuated in rats without nursing pups.

Support or Funding Information

Supported by Midwestern University

Daily wheel time in min averaged over 7 day periods

NP
PG1
PG2
PG3
W1
W2

TC
182 (57) N=9

125(64) N=6

P‐Kept
176 (70)
43(16)
44(18)
58(19)
114(48)
126(55)

P‐Remove
132(46)
55(30)
63(51)
102(54)
114(60)
101(31)

Mean(SD)

Interaction:
PG 2 vs 3, P=0.04
PG3 vs W1, P=0.05
All Rats;Vs NP, P<0.003
The effects of exercise on body composition and physical performance measures in college‐aged men and women with intellectual disabilities
Authors: Najm Nadia Marie Ghassan , Jesch Elliot D.
Published: November 2023

Prevention of chronic disease and improving the quality of life for adults with intellectual disabilities is an area of great concern among many professionals and families. Programs have been created to improve nutrition and physical fitness for individuals with an intellectual disability. However, adapting to the various disabilities and behaviors pose challenging circumstances that makes generating statistical norms about physiological adaptations within the population hard to obtain. The purpose of this study is to assess the physiological adaptations in response to a semester long group‐fitness program in college‐aged men and women with intellectual disabilities. Nine students (20.1± 1.67 y.o.) partook in three group fitness classes and two swim classes each week. Instructors of group fitness class were advised to conduct classes with minimal modifications from their regular classes for the general population. Additionally, students were encouraged to partake in regular physical activity of their choosing outside of the program.

The following are the mean differences in measurements of: height (0.078 ± 0.175 inches), weight (4.033 ± 4.656 pounds), BMI (0.367 ± 0.879), percent fat mass (0.28± 0.81%), and the Sit and Reach flexibility test (−0.41 ± 3.006 inches). All measurements were found to be non‐significant, suggesting that a non‐modified fitness program is ineffective at improving physiological adaptions.
Effects of environmental sodium ion concentration on the activity of Na/K‐ATPase. H‐ATPase and Glutamate Dehydrogenase from the gills of fathead minnows (Pimephales promelas)
Authors: Packer Randall K. , Monroe Ian , Wentworth Simon A. , Garvin Jeffrey L.
Published: November 2023

Na+/K+ ATPase on the basolateral membrane of gill epithelial cells actively transports Na+ from the cell interior to the blood, creating the electrochemical gradient that may help drive secondary active transporters in the apical membrane essential for Na+ influx. H+‐ATPase on apical membranes has been hypothesized to facilitate Na+ uptake. In the renal proximal tubules of mammals glutamate dehydrogenase, GDH, along with phosphate‐dependent glutaminase, deamminates glutamine. Ammonium ions generated exit the apical cell membrane in exchange for Na+ entering the cell via NEH3. We hypothesized that Na/K ATPase, H‐ATPase and GDH activity would be higher in fish acclimated to 1.2 mEq/l [Na+] versus 124 mEq/l environmental [Na+]. Fathead minnows were acclimated to 1.2, 3.2 or 124mE/l Na+ for a minimum of 28 days. Na/K ATPase, H‐ATPase and GDH activities were measured in homogenates of gill tissue. Na/K‐ATPase activity from fish acclimated to 1.4 mE/l (1.99± 0.19 μEq/g protein·hr−1) was significantly (P<0.05) higher than in fish acclimatized to 124 mEq/l (1.41±1.34 μEq/g protein·hr−1). Similarly, GDH activity increased significantly, from 12.32±1.59 to 24.81±2.54 nanoEq/g protein·hr−1g in fish acclimated to 1.2 mEq/l compared to those acclimated to 124 mQ/l Na+. In contrast, H+‐ATPase activity was essentially zero (0.1 ± 0.06 μmoles/mg protein/hr) in fish acclimated to 1.2 mEq/l [Na+] and increased to 0.58±0.10 μmoles/mg protein/hr in fish acclimated to 124 mEq/l [Na+]. These data do not support a role for H+ ATPase in gill Na+ uptake at low environmental [Na+] but do suggest that at low environmental [Na+] ammonium ions generated from deamination of glutamine might exchange at the apical gill membrane for sodium ions entering the cell via NEH3.

Support or Funding Information

Supported by the Columbian College of Arts and Sciences, GWU
Historical Perspective of Therapeutic Electricity
Authors: Dias Daniel Penteado Martins , Fazan Rubens , Salgado Helio Cesar
Published: November 2023

The development and employment of effective methods to treat clinical conditions has affected life quality and expectancy worldwide. There are records showing that the Egyptian civilization, 4,000 years ago, used electricity from electric fish for treating pathologies. Later, Luigi Galvani proposed the concept of bioelectricity in 1780, when he observed that if two metallic instruments touched the sciatic nerve of a toad, there was contraction of the musculature of the hind limbs. The electrical stimulation of tissues has developed noticeably over the past decades. Shelden (1966) has employed electrical stimulation of a branch of the trigeminal nerve for treating an acute pain. It is believed, for instance, that electrical stimulation of neural pathways promotes pain relief by blocking the transmission of physiological electrical signals. The development of new forms of treatment, based on electrical stimulation, depends on three steps: 1) identification of the target tissue; 2) determination of the stimulation pattern; 3) development of appropriate device for the application. Among the clinical conditions currently treated with electrical stimulation we could highlight: depression, epilepsy, hypertension, chronic lower back pain, obesity, and obstructive sleep apnea. Although it seems a challenging technique, there are recent reports (2012) showing that more than 100,000 patients have already been implanted with electrical stimulators in the vagus nerve to treat a variety of diseases. It is clear that the technique is extremely promising but requires joint work by clinicians, neuroscientists and engineers to develop effective protocols for chronic electrical stimulation of tissues.

Support or Funding Information

FAPESP (Processes n. 2013/20549‐7 and 2015/20463‐0).
Purification and Subsequent Binding Assay of B‐Cell Activating Factor's Receptor (BAFF‐R) to an Oligonucleotide Aptamer (BAFF‐R1)
Authors: Inman Christopher Fredric
Published: November 2023

In Non‐Hodgkin's Lymphoma (NHL), it has been shown that the proteins B‐cell activating factor (BAFF), and its receptor (BAFF‐R) are overexpressed by cancerous cells. Upon BAFF trimerization and binding to BAFF‐R on the cellular membrane of malignant B‐cells, the complex endocytoses and starts a signaling cascade that leads to the constitutive expression of oncogenes including STAT3, a transcription factor that causes increased rates of survival and proliferation. This mechanism of action potentially accounts for one possible reason for why these cells resist apoptotic signals. It has also been shown that an RNA aptamer (BAFF‐R1) binds to BAFF‐R with high specificity and high affinity. BAFF‐R1 may be used to transport small interfering RNAs (siRNAs) that can knockout the overexpressed oncogenes through the RNA silencing complex (RISC). The knockout of oncogene mRNA leads to decreased rates of malignant cell survival, and therefore the mechanism of how BAFF‐R1 binds to BAFF‐R for transport into the cell is an interesting area of research and could result in a potential targeted therapy for NHL.

To conduct in vitro research involving the interactions between the RNA aptamer and BAFF‐R, it was necessary to synthesize a truncated, extracellular loop of the BAFF‐R protein because integral membrane proteins are difficult to work with. BAFF‐R expression was induced in BL21 E. coli through the pGEX‐KG vector and purified by affinity column chromatography. BAFF‐R was cleaved off the immobilized GST fusion protein with thrombin.

An electrophoretic mobility shift assay was performed to generate a binding or saturation curve from which the dissociation constant may be determined. Samples of constant amounts of radiolabeled BAFF‐R1 aptamer and varying amounts of BAFF‐R protein were prepared, and as the protein concentration increased, the base RNA band decreased in intensity and a shifted band representing the aptamer bound to BAFF‐R increased in intensity. By comparing these relative intensities, the saturation curve may be prepared, and the dissociation constant of aptamer binding may be determined.

In the future, point mutations will be performed on BAFF‐R and the mutant proteins will be subjected to further electrophoretic mobility shift assays to determine the critical amino acid residues responsible for aptamer binding.

Support or Funding Information

Dr. Monica Lares provided the support for this project as an opportunity for undergraduate research.
Doxycycline Blocks Matrixmetalloproteinase‐9 and Attenuates Blood‐Brain Barrier dysfunction and Hyperpermeability after Traumatic Brain Injury
Authors: Robinson Bobby Darnell , Lomas Angela , Shaji Chinchusha Anasooya , Isbell Claire Larson , Tharakan Binu
Published: November 2023

Traumatic brain injury (TBI) is a global issue affecting approximately 10 million people annually. Brain edema that occurs after TBI leads to challenging clinical scenarios, including elevated intracranial pressure (ICP), decreased cerebral blood flow, poor tissue oxygenation, and brain herniation. Currently, treatment modalities exist to somewhat address symptoms of cerebral edema, but there are no effective medical therapies able to modulate the underlying pathophysiology responsible for TBI‐induced microvascular “leak.” The purpose of this study is to demonstrate that doxycycline inhibits matrixmetalloproteinase‐9 (MMP‐9) and reduces microvascular hyperpermeability in TBI in vivo. Molecular docking simulation was performed with AutoDock 4.2.6 using Lamarckian genetic algorithm. Each docking experiment was a composite of 100 independent executions. MMP‐9 and doxycycline structures were obtained from Protein Databank and DrugBank respectively. Molecular modeling showed doxycycline spontaneous binding to the MMP‐9 active site. For in vivo studies, C57BL/6 mice were injected via tail vein with 0.1 mL of 50 mg/mL FITC‐dextran prior to injury as an intravascular tracer. A mild‐moderate TBI (velocity of 0.50 m/s, contact time of 0.1 milliseconds, and depth of 2 millimeters) was performed with the Leica controlled cortical impactor after craniotomy. Sham mice had only craniotomy performed. The doxycycline group was given 20 mg/kg of doxycycline intravenously 10 minutes after injury. Intravital microscopy of brain pial microvasculature was performed 10 minutes after injury up to 70 minutes after injury and the data was analyzed. The difference in fluorescence intensity between the intravascular space and the interstitial space was calculated as a measure of BBB permeability. There was no significant difference in permeability among the groups at the initial time point of 10 mins after injury. There was a significant increase in TBI vs sham group and a decrease in hyperpermeability when compared between the TBI and TBI plus doxycycline groups (p<0.05; ANOVA followed by Bonferroni's multiple comparisons test). There was no significant difference between the Sham group and TBI plus doxycycline group. The brain tissue was harvested from each mouse and ELISA of brain tissue extract demonstrated increase in interleukin‐1β (IL‐1β) between sham and TBI (p<0.05) but no difference in the TBI and TBI + Doxycycline groups, suggesting that doxycycline has no effects on tissue IL‐1β levels upstream. Doxycycline spontaneously binds to MMP‐9 and its active site evidenced by the molecular docking studies. Mice treated with doxycycline following TBI had enhanced BBB integrity/decreased microvascular hyperpermeability. Doxycycline may be a viable treatment in TBI to protect the BBB and decrease brain edema when established in patients.
Androgens alter mitophagy in the refed metabolic state
Authors: Steiner Jennifer L , Rossetti Michael L , Gordon Bradley Scott
Published: November 2023

We previously showed that depletion of androgens via castration increased markers of autophagy activation (i.e. increased LC3 II/I ratio and reduced p62 protein content) in the tibialis anterior (TA) following nutrient consumption. Importantly, this androgen‐mediated effect occurred despite no differences in measures of known putative autophagy regulatory factors. Because of this, it is likely that the sustained elevation in autophagy‐activation was specific to a cellular organelle(s) rather than global autophagy. As others have shown that castration reduced skeletal muscle oxidative capacity, we sought to determine whether autophagic clearance of mitochondria, termed mitophagy, could be contributing to the sustained increase in autophagic markers. To answer this, mice were randomized to sham or castration surgery. After 8 weeks of recovery, all mice were fasted overnight. The next morning, mice remained fasted or were refed for 30 min, and sacrificed 4.5 hr following completion of the refeeding. In the TA muscle, castration reduced mitochondrial protein content as the mitochondria specific proteins cytochrome c oxidase subunit IV (COX IV) and voltage‐dependent anion channel (VDAC) were decreased. An inverse relationship was observed between the protein content of either COX IV or VDAC with p62, suggesting enhanced mitophagy. Castration also reduced the protein expression of the mitophagy regulatory protein, BCL2/adenovirus E1B 19 kDa protein‐interacting protein 3 (BNIP3), despite no change in BNIP3 mRNA suggesting enhanced turnover of the protein during mitophagy. Indeed, direct relationships were observed between the protein content of BNIP3 and either p62, COX IV, or VDAC supporting a role of BNIP3 in castration‐induced mitophagy. Additional analysis also supports a role of Pink1 and Parkin as castration enhanced the mRNA levels of each without a corresponding change in the protein expression, consistent with enhanced turnover of these proteins during mitophagy. Lastly, fasting conditions that enhance global autophagy prevented the castration‐induced reduction in mitochondrial proteins and enhancement of mitophagy markers. In all, these data support a model in which androgens prevent BNIP3/Pink1/Parkin‐mediated mitophagy in skeletal muscle during the refed, but not the fasted, metabolic state.

Support or Funding Information

NIH Grant F32 AA‐023422 to JLS
Discovery of a Cell‐penetrating Peptide via Heparin‐binding from Pereskia bleo
Authors: Loo Shining , Kam Antony , Tam James P
Published: November 2023

Pereskia bleo or rose cactus belongs to the Cactaceae family and is commonly found in South‐east Asia. Traditionally, it is used for cancer treatment, inflammatory diseases and wound healing1. Wound healing involves the extracellular matrix and a complex network of protein‐protein interactions2. Protein‐protein interactions is a vast underexplored space for drug targeting. Disruption of intracellular protein‐protein interactions is largely limited in the ability of molecules capable of specific entry via receptor‐mediated mechanisms and non‐specific entry by membrane penetrations. Extracellular matrices, particularly glycosaminoglycans have been exploited to increase the efficiency of intracellular uptake through non‐receptor mediated entry mechanisms3. Heparin/heparain sulfate, commonly found in the extracellular matrix, can be involved in the cellular uptake of cell‐penetrating peptides3. Here we report the discovery from Pereskia bleo, bleotide pB1, a novel heparin‐binding peptide with cell‐penetrating capabilities. Bleotide pB1 was isolated and purified by a series of liquid chromatography. Transcriptomic and proteomic analyses showed that bleotide pB1 is a 36‐residue peptide with a cystine‐knot arrangement. Similar to known cystine‐knot peptides, bleotide pB1 has high metabolic stability4. Bleotide pB1 is not cytotoxic and hemolytic at concentrations up to 100 μM. Using heparin affinity chromatography and in silico modeling; our results demonstrated that bleotide pB1 is a heparin‐binding peptide. To examine the cell‐penetrating properties of bleotide pB1, N‐terminal TAMRA‐conjugated bleotide pB1 was synthesized by solid‐phase peptide synthesis and oxidative‐folded to the native state. Using flow cytometry and confocal microscopy, we show that TAMRA‐pB1 can be internalized into living cells. CHO‐K1 (wild type) and pgsA‐745 (glycosaminoglycan‐deficient) cells were used to show that the cellular uptake of TAMRA‐pB1 is dependent on glycosaminoglycan expressions. Taken together, this study reports the discovery of a novel heparin‐binding hyper‐stable peptide with cell penetrating properties which could be useful for drug development.

Support or Funding Information

This project was supported in part by the National Research Foundation (NRF‐CRP8‐2011‐05) of the Prime Minister's Office of Singapore.
CFTR‐Associated ATP Release in Neonatal and Adult Skeletal Myoblasts
Authors: Wu Tsz Hin Elton , Ballard Heather Janet
Published: November 2023

ATP release from contracting skeletal muscle cells stimulates extracellular adenosine formation, which contributes to vasodilation. We previously showed that 1) lactic acid (which is formed by contracting muscle) lowers intracellular pH and stimulates ATP release, and 2) cystic fibrosis transmembrane conductance regulator (CFTR) is activated by low pH and it is involved in acidosis‐induced ATP release.

Patients with Cystic Fibrosis commonly have impaired muscle function and reduced exercise tolerance. ATP is a large diameter molecule, and CFTR chloride channel is too narrow for passage of ATP: we investigated whether connexins and/or pannexins were involved in CFTR‐associated ATP release from rat L6 skeletal myoblast cell line (a model for neonatal muscle) and human skeletal muscle myoblasts (HSMM; a model for adult muscle).

We measured protein expression with western blot, ATP release by the luciferase assay and large diameter channel opening by Lucifer yellow (LY) uptake.

L6 myoblasts expressed Connexin 43 strongly: LY uptake was increased by Ca2+‐free medium; niflumic acid (NA) or lanthanum (Lan3+) inhibited LY uptake in Ca2+‐free medium, suggesting that connexins function as membrane channels in L6 myoblasts. HSMM expressed Pannexin 1 strongly: high K+ stimulated LY uptake, which was inhibited by probenecid (PB) or 10mM carbenoxolone (CBX), indicating that pannexins functioned as membrane channels in adult myoblasts.

Lactic acid (LA) increased LY uptake and ATP release in both cell types, confirming that both neonatal and adult muscle cells are capable of acidosis‐induced ATP release. In L6 cells, LA‐induced ATP release was inhibited by NA or Lan3+, but not by PB or 10mM CBX, indicating that it was mediated by connexins but not pannexins. In HSMMs, either Lan3+ or Fast Green‐FCF (FG) could inhibit LA‐induced ATP release, suggesting that either connexins or pannexins could mediate ATP efflux.

Forskolin (forsk), alone or with either IBMX or AMP‐PNP, an ATP analog that locks CFTR in its open state, strongly stimulated ATP release from HSMMs and L6 cells, confirming that CFTR activation could stimulate ATP release from neonatal or adult myoblasts. In HSMMs, forsk/AMP‐PNP‐induced ATP release was inhibited by FG but not Lan3+, suggesting that CFTR activation stimulates ATP release through pannexins but not connexins. In L6 cells, forsk/AMP‐PNP‐stimulated ATP release was about 75% inhibited by Lan3+, suggesting that it is largely mediated by connexins.

These data suggest that CFTR‐associated ATP release from muscle cells can be mediated by connexins or pannexins. Further investigation is required to determine the mechanism by which CFTR regulates the opening of connexin and pannexin channel.
The role of hydrogen peroxide production in skeletal muscle dysfunction following nerve crush.
Authors: Staunton Caroline Amy , Pollock Natalie , Vasilaki Aphrodite , Barrett‐Jolley Richard , McArdle Anne , Jackson Malcolm J
Published: November 2023

Age‐related loss of muscle mass and function greatly influences the quality of life of older people. Denervation, mitochondrial hydrogen peroxide (H202) production and elevated pro‐inflammatory cytokines levels are thought to play a substantial role in this muscle dysfunction and to be key contributors to frailty in the elderly.

A substantial body of evidence links reactive oxygen species (ROS), and in particular mitochondrial H202 production, with skeletal muscle dysfunction. This research area has been limited by the availability of accurate and specific methods for H202 detection. We have therefore examined the use of HyPer2 as a specific fluorescent probe for H202 in muscle in vivo following transfection using rAAV6.

The aim of this study was (1) to use intra‐vital imaging to examine morphological changes in the structure of peripheral axons and neuromuscular junctions following peroneal nerve crush in Thy1‐CFP mice, and (2) to monitor in parallel the levels of H202 in the cytosol of denervated muscle fibres in muscles transfected with HyPer2‐Cyto in a rAAV6 vector at 3, 7 and 21 days post‐nerve injury using intra‐vital imaging. Peroneal nerve crush was used as a model that potentially reflects the ageing process due to the reversible nature of the denervation. To complement the rAAV6‐HyPer2‐Cyto in vivo study, mitochondrial H202 generation was also assessed using the Amplex red assay ex vivo in small bundles of permeabilised muscle fibres from the anterior tibilas (AT) muscles from Thy1‐CFP mice which underwent the same nerve crush procedure.

Following the peroneal nerve crush an increased H202 content was seen in fibres of the anterior tibialis muscle of adult mice in vivo compared with sham control mice using the rAAV6‐Hyper2‐Cyto probe. The temporal profile of changes in H202 content was obtained over a 21 day period post nerve crush by repeated examination of the same mice.

This work expands on previous studies using a peroneal axotomy model where adult mice were shown to have an increased mitochondrial peroxide production ex vivo2 and data from our laboratory that have further demonstrated that this peroxide generation by muscle remained significantly elevated after full denervation. Further data also suggest that increased mitochondrial peroxide generation may mediate cytokine production by muscle cells1 and our group has shown that following denervation, mRNA from AT muscles show IL‐6, MCP1 and RANTES are significantly increased from control mice.

These data provide additional insight into the generation of H2O2 in muscle following nerve damage and together with other data will allow further exploration of how changes in neuromuscular junction integrity prevent neuromuscular transmission and the relationship of this to muscle H202 levels.

Support or Funding Information

This work was supported by the UK Medical Research Council.
Cigarette smoke exposure induces diaphragm atrophy that partially regresses following cessation
Authors: Foster Andrew , Stampfli Martin R. , Simpson Jeremy A.
Published: November 2023

Atrophy is well known to occur in the diaphragm and limb muscles of patients with chronic obstructive pulmonary disease (COPD), a disease predominantly caused by cigarette smoke exposure. Interestingly, non‐COPD smokers also exhibit limb muscle atrophy; a finding that implicates cigarette smoke as a cause of atrophy. This has been substantiated in animal models demonstrating that limb muscle atrophy occurs early following cigarette smoke exposure and prior to overt lung dysfunction. These studies also indicate that pro‐atrophy pathways are partially reversed upon the cessation of cigarette smoke exposure suggesting possible regression of atrophy. However, the effect of cigarette smoke exposure and cessation, on diaphragm muscle atrophy, has not been investigated. Thus, the overall objective of this project is to investigate the effect of cigarette smoke exposure on the development of diaphragm atrophy in mice. We hypothesize that cigarette smoke exposure in mice will induce diaphragm atrophy; further, will be reversed following cessation.

Methods

BALB/c mice were repeatedly exposed to room air or cigarette smoke twice a day, 5 days/week. To assess the pathological development of diaphragm atrophy, mice were exposed to either 8 or 24 weeks of cigarette smoke. Following initial exposure, a subset of mice were switched to room air for an additional 8 weeks to evaluate the effect of cigarette smoke cessation. All groups were compared to non‐smoking controls. At the time of sacrifice, mice were formalin fixed and diaphragm tissue was removed for histological evaluation of diaphragm myocyte cross sectional area (CSA).

Results

In comparison to non‐smoking controls, diaphragm CSA was reduced by 40% following 8 weeks cigarette smoke exposure and did not worsen by 24 weeks. Median diaphragm CSA in 8 and 24 week exposure groups were 220um2 and 215um2 respectively, which was significantly reduced from 370um2 in non‐smoking controls (p<0.05). In mice switched to room air, diaphragm CSA was reduced by 20% as compared to non‐smoking controls; 298um2 vs. 370um2 respectively.

Conclusions

Here we demonstrate that diaphragm atrophy is induced early following cigarette smoke exposure and does not worsen with prolonged experience. Further, our data indicate that the cessation of cigarette smoke exposure partially regresses diaphragm atrophy. In future work we aim to identify the intracellular mechanisms through which diaphragm atrophy develops in response to cigarette smoke exposure.

Support or Funding Information

Canadian Thoracic Society/ Canadian Lung Association
Muscle ULK1 and ULK2 modulate different aspects of autophagy and are required for atrophy during fasting
Authors: Mere Caleb , Fuqua Jordan , Blomme Jay , de Melo Vitor , Kronemberger Ana , Lira Vitor A
Published: November 2023

(Macro)autophagy, hereafter referred to as autophagy, is an evolutionarily conserved catabolic process involving in the degradation of cellular proteins and structures sequestered into a vesicle known as an autophagosome. Impairments of autophagy have been implicated in contractile and/or metabolic deficiencies in muscle diseases, obesity, diabetes, and aging; however, regulation of skeletal muscle autophagy is poorly understood at the molecular level. Here, we examined the role of the two partially homologous unc‐51 like autophagy activating kinases 1 and 2 (ULK1 and ULK2) in modulating autophagy and myofiber atrophy during fasting via a miRNA‐specific knockdown of these proteins in mouse skeletal muscle. Suppression of ULK1 caused LC3‐I (the non‐autophagosomal‐interacting form of LC3) to accumulate in fasted muscle without changes in LC3 mRNA, indicating an impairment in the step of LC3‐I conversion into LC3‐II (the autophagosomal‐interacting form of LC3). Similar trends were observed with non‐autophagosomal and autophagosomal forms of other LC3‐like proteins (GABARAP, GABARAPL1, GABARAPL2) suggesting a specific role for ULK1 in autophagy activation. Knockdown of ULK2 did not affect the different forms of LC3 and LC3‐like proteins suggesting that ULK2 does not regulate autophagy activation. However, it led to accumulation of ubiquitinated proteins, and the autophagy receptors p62 and NBR‐1, irrespective of their mRNA, under both basal and fasting conditions. Since autophagy receptors bind to and are degraded together with ubiquitinated proteins, these findings are consistent with impaired involvement of receptors and consequent deficient cargo recognition by autophagy. Of note, reduced expression of both ULK1 and ULK2 was required to diminish myofiber atrophy during fasting, indicating their synergistic role in mediating fiber atrophy in this model. Altogether, these results uncover fundamental divergent roles for ULK1 and ULK2 in modulating autophagy and its target recognition/selectivity in muscle. Future studies expanding the molecular signature of autophagy activation and selectivity will be instrumental to identify novel targets for therapy in conditions associated with autophagy deficiency in muscle.

Support or Funding Information

American Heart Association Grant 16SDG30360001 (V.L.)
ICAM‐1 Augments Myoblast Adhesion and Fusion through Homophilic Trans‐Interactions and GTPase Rac Activity
Authors: Martin Ryan , Springer Evan , Leffler Maxwell , Woelmer Bryce , Leaman Douglas , Pizza Frank
Published: November 2023

The purpose of the study was to identify mechanisms through which intercellular adhesion molecule‐1 (ICAM‐1) augments the adhesive and fusogenic properties of myoblasts. Immuno‐precipitation and myoblast adhesion assays using recombinant ICAM‐1, as well as cell mixing experiments using ICAM‐1+ and control myoblasts, demonstrated that homophilic trans‐interactions for ICAM‐1 serve as a mechanism through which ICAM‐1 augments myoblast‐myoblast adhesion. Homophilic trans‐interactions for ICAM‐1 were found to trigger myoblast fusion through the signaling function of the cytoplasmic domain of ICAM‐1, GTPase Rac activity, and actin‐based lamellipodia formation. In conclusion, our findings demonstrate that ICAM‐1 mediated myoblast adhesion to myoblasts and myotubes occurs through homophilic trans‐interactions of ICAM‐1, which in turn facilitates myoblast fusion through the activation of GTPase Rac.
Autonomic cardiovascular response during post‐occlusive reactive hyperaemia test comparing healthy and sickle cell anemic patients
Authors: Carrazana‐Escalona Ramón , Sanchez‐Hechavarria Miguel Enrique , Planas‐Rodríguez Maylet , García Adolfo Fernández , Cutiño‐Clavel Ileana , Pascau‐Simon Alexander , Martín‐González Juan Manuel , Connes Philippe
Published: November 2023

Introduction

Although the sickle cell disease (sicklemia) is characterized by significant m icrocirculatory dysfunction, in is not clear whether regulatory physiological phenomena associated with hemodynamic response during reactive hyperaemia post ‐occlusive test are affected.

Objective

We sought to determine autonomic cardiovascular responses during reactive hyperaemia post‐occlusive test in healthy and sicklemic patients.

Methods

Using a quasi‐experimental design with 25 healthy subjects and 18 sicklemic patients, we studied the effects of reactive hyperaemia on vascular and cardiac variables. Following 10 min for acclimation, we evaluated 5 min before (basal), 10 min during post‐occlusive reactive hyperaemia test on the lower right limb, while performing Pulse Rate Variability (PRV), obtained by the Pulse Peak In tervals of the photoplethysmographic signal (AngiodinÂ® PD 3000 device) on the first finger of the lower right limb. Pulse Peak Intervals were analyzed using frequency (Welch) and time‐frequency (Wavelet) methods of the bands of high frequency (HF: 0.15–0.4) and low frequency (LF: 0.04–0.15), as well as temporal and nonlinear (Sample Entropy) analysis.

Results

We observe an increase (p<0,05) in the basal state of the suggestive indicators of sympathetic (LF, LF/HF) activity in post‐occlusive reactive hyperaemia test during the 5 first minutes, which tend to be lower in the last 5 minutes of recuperation besides a significant increase in all indicators of the PRV existed in the basal state in healthy regarding sicklemic patients. With time‐frequency analysis, we found that the LF/HF peak of response to the post‐occlusive reactive hyperaemia test occurs more rapidly in healthy compared to sicklemic patients.

Conclusions

The sympathetic cardiovascular response during post‐occlusive reactive hyperaemia test in sicklemic patients is smaller and slower than in healthy subjects, which suggest that in this disease there are physiopathologic phenomena that affect regional vascular responsiveness to ischemia.

Support or Funding Information

None
Association Between Telomere Length and Autonomic Functions: Applying Psychological and Physiological Measures
Authors: Kim Nahyun , Sung Jin Young , Kim Dae‐Kwang
Published: November 2023

Telomeres are protective chromosomal structures that are important in maintaining genomic stability. Telomere shortening results in genomic instability and contributes to aging and carcinogenesis. Despite the health risks posed by this condition, few studies have examined the relationship of telomere length (TL) to autonomic functions. In this study, we examined the association between TL and autonomic functions in Korean adolescents. A cross‐sectional study was performed with high school adolescents in a Korean city. Self‐report questionnaires were used to assess psychological measures (stress and anxiety levels), and blood samples were analyzed for physiological parameters (plasma catecholamine levels) of the participants. TL measurements were made using a qPCR‐based technique, and relative TL was calculated as the telomere/single copy (T/S) ratio. Data were analyzed using descriptive analysis and Pearson's correlation coefficient with SPSS/WIN 22.0. We found that T/S ratio was positively correlated with plasma epinephrine and norepinephrine levels (r=.225, p=.001 and r=.187, p=.005). However, psychological stress and anxiety levels were not significantly associated with adolescents' T/S ratio. Our physiological results indicate that alteration of autonomic functions may induce TL shortening in adolescents. We believe that our findings increase the understanding of the health effects of autonomic functions. Moreover, they highlight the need for intervention strategies to prevent TL shortening due to autonomic imbalance.

Support or Funding Information

This research was supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2016R1D1A3B03934143).
Role of GABA Receptors in the Postganglionic Regulation of Salivation
Authors: Lee Joseph , Giovannucci David
Published: November 2023

The salivary gland secretes saliva components critical for oral health. Autonomic neural activity controls the secretory output of acini, the basic units of saliva production. This process can be disrupted in patients by benzodiazepine treament, leading to the severe oral condition of xerostomia, or dry mouth. Neural inputs to the acini are primarily cholinergic and noradrenergic, but the discovery of gamma‐aminobutyric acid (GABA) and its receptors in the salivary glands hints at an unexplored mechanism for benzodiazepine‐induced xerostomia. In the current study, biochemical, live‐cell imaging, immunofluorescence and electrophysiological methods were used to elucidate the GABAergic system in the mouse parotid gland and define its effect on acinar regulation. RT‐PCR identified mRNA transcripts for multiple GABA receptor subunits in enzymatically dispersed acinar cells and immunofluorescence microscopy indicated the expression of GABA receptors in mouse parotid tissue. However, both live cell calcium imaging and whole‐cell recordings of acinar cells revealed no acute impact of GABA or benzodiazepine treatment on cellular physiology. In contrast, immunohistochemical analysis of the autonomic autonomic ganglia and postganglionic innervations to the parotid gland aligned with the identification and characterization of GABA‐evoked chloride currents in a subset of neurons recorded from primary ganglionic cultures. Our data suggest GABA has no direct effect on the parotid acini but may downregulate acinar function by inhibiting neurotransmitter release from autonomic neural inputs.

Support or Funding Information

This work was funded by National Institutes of Health National Institute of Dental and Craniofacial Research Grant DE‐023418 and bridge funds from the University of Toledo College of Medicine.
Rats deficient in tryptophan hydroxylase 2 (SSTph2−/− rats) show altered pulmonary neuroendocrine cells (PNECs), reduced resting ventilation and altered hypoxic and hypercapnic chemoreflexes
Authors: Mouradian Gary C , Dillard Matthew , Kaplan Kara , Thuku Gabriel , Uche Nnamdi , Hodges Matthew R
Published: November 2023

Sudden Infant Death Syndrome (SIDS) is a leading cause of infant mortality from 1–12 months of age, and is thought to occur due to cardiorespiratory failure during sleep. The leading hypothesis as to the causes of SIDS is that abnormalities in the central serotonergic (5HT) system are underlying biological factors contributing to SIDS, but other studies report hypertrophy and hyperplasia of serotonergic lung airway chemosensors called neuroepithelial bodies (NEB) in SIDS. Herein, we tested the hypothesis that rats deficient in tryptophan hydroxylase 2 (TPH2; SSTph2−/− rats), which fail to synthesize brain 5HT, would display NEB hypertrophy and hyperplasia during post‐natal development. We further tested the hypothesis that SSTph2−/− rats would show altered ventilatory chemoreflexes during development, potentially due to altered airway chemoreceptor activity. Cryosections (20μm thick) of inflation fixed lungs were co‐stained for CGRP and Synaptophysin, standard markers of NEBs, and DAPI. Blinded imaging and subsequent analyses indicate a greater number of NEB cells in Tph2−/− rats but a decrease in overall NEB cell size compared to WT rats in 1–2, 12–13, and 18–22 day old groups. These changes correlated with significantly lower weight‐normalized minute ventilation in Tph2−/− rats during room air, hypercapnic, and hypoxic challenges at P12–13, 14–16, and 18–20 day old groups. In conclusion, loss of central serotonin appears to directly or indirectly alter the morphology of peripheral airway 5HT chemosensors.

Support or Funding Information

NIH HL R01 122358