bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dystrophin Insufficiency Causes Locomotor Dysfunction in a Spontaneously Occurring Pig Model
Authors: Spaulding Hannah , Kaiser Adrianne , Hess Andrew , Stalder Ken , Nonneman Dan , Ross Jason , Selsby Joshua 
Published: November 2023

Dystrophinopathies are caused by mutations in the dystrophin gene leading to a dystrophin deficiency (Duchenne muscular dystrophy) or insufficiency (Becker muscular dystrophy). These diseases are commonly modeled by the mdx mouse or other related mouse models as well as the Golden Retriever Muscular Dystrophy model. While these models have been useful, inherent limitations necessitate continued development of novel large animal models. The purpose of this investigation was to determine the extent to which a spontaneous dystrophin mutation would impair locomotion in a pig dystrophinopathy model. We performed a monthly gait analysis from 3–12 months of age in male pigs with a dystrophin mutation causing an insufficiency and healthy male littermates using a Zeno gait analysis system. This system consists of a 5 m pad with approximately 50,000 integrated force transducers, which allows sensitive detection of pressure during the gait cycle as the pig walks across the pad. Pigs were euthanized at 3 and 12 months of age and tissues collected for histological analyses. Histological quantifications revealed a 2‐fold increase (p<0.05) in centralized nuclei in affected pigs at 12 months of age and a 2‐fold increase (p<0.05) in centralized nuclei in affected pigs over time at 12 months compared to 3 months of age suggesting progressive disease‐related injury. By 9 months of age affected pigs developed a distinct walking gait. When considering movement in the front limbs, stride length was shortened by 6–10 cm (p<0.05) and by 12 months of age stride width was increased by 7 cm (p<0.05) in affected pigs compared to healthy pigs. In addition to a wider and shorter gait, the swing phase was decreased by 14% and the mean single support center of pressure distance was also decreased at 9 months of age by 36%. Similar results were discovered when measuring movement in the hind limbs and when comparing same side limbs and contralateral limbs. In total, these pigs appear to be moving with a shorter, wider gait with a slower march‐like movement pattern. This unique gait shares similarities with patients affected by dystrophinopathies who also have notable gait changes as the disease progresses. These data suggest that pigs affected with a spontaneously occurring dystrophin insufficiency develop a progressive muscle pathology that ultimately impacts locomotion, consistent with changes in dystrophinopathy patients. These data also contribute to a growing body of literature supporting the utility of this human‐sized model that accurately recapitulates the human disease.Support or Funding InformationThis work supported by NIH grant R21 NS079603.
Effect of Age on the Cerebrovascular Responses to the Initiation of Dynamic Exercise
Authors: Fisher James P , Ahmed Amar , Ainslie Phil N , Bain Anthony R , Wildfong Kevin W , Morris Laura , Flück Daniela 
Published: November 2023

The rapid hemodynamic changes induced by the initiation of dynamic exercise robustly challenge the regulatory processes which ensure that cerebral perfusion is appropriately maintained. We sought to determine whether age alters the cerebrovascular responses to the initiation of dynamic exercise.Ten young (mean±SD, 23±2 years, 2 women) and eight older (65±3 years, 1 women) individuals performed a 5‐minute bout of semi‐recumbent leg cycling exercise at 75W. Blood pressure, cardiac output, total peripheral resistance (Finometer) and middle cerebral artery blood flow velocity (MCAv: transcranial Doppler ultrasound) were monitored. Gosling pulsatility index (PI) was calculated as; (systolic MCAv–diastolic MCAv)/mean MCAv. Beat‐to‐beat data during exercise were linearly interpolated at 1 Hz.At baseline, MAP (mean±SEM, young, 87±3; older, 93±4 mmHg), MCAv (young, 57±2; older, 56±3 cm/s) and PI (young, 0.88±0.4; older, 0.92±0.4 cm/s) were not different in young and older groups (P>0.05), while TPR tended to be lower (young, 16±1; older, 21±2 mmHg/L·min−1, p=0.08) and CO tended to be higher (young, 5.6±0.4; older, 4.6±4 L·min−1; p=0.13) in the young. The initiation of exercise evoked a rapid and sustained increase in cardiac output and fall in total peripheral resistance, that was blunted in the older group. Blood pressure fell transiently at exercise onset, reaching a similar nadir (young, −9.9±2.8; older, −10.0±2.9 mmHg; p>0.05) after a similar duration (15±2; older, 13±2 s; p>0.05) in both groups. MCAv also fell transiently at the initiation of exercise (young, −5.3±1.5; older, −5.1±1.9 cm/s, p>0.05), but the nadir was reached later in the older group (young, 20±2; older, 29±2 s, p<0.05). PI increased rapidly at the onset of exercise in young individuals, but was elevated more sluggishly in older individuals.Collectively, these findings suggest that older individuals exhibit delayed cerebrovascular adjustments at the onset of exercise.Support or Funding InformationThis research was supported by a National Sciences and Engineering Research Council Discovery Grant and Canadian Research Chair in Cerebrovascular Physiology (PNA), the Swiss National Science Foundation (DF) and the British Heart Foundation (JPF).
The detection of the proteasomal proteolysis efficiency in C2C12 myoblast
Authors: Osana Shion , Suzuki Naoki , Aoki Masashi , Nagatomi Ryoichi 
Published: November 2023

BackgroundThe loss of skeletal muscle mass in older adults, which is called sarcopenia, is a rapidly growing global health issue. The balance of muscle protein synthesis and degradation is the key point of the regulation of skeletal muscle mass. Previous reports showed that muscle‐specific deletion of a crucial proteasomal gene, Rpt3 (also known as Psmc4), resulted in sever skeletal muscle atrophy (Kitajima et al., 2014). Bortezomib and MG132, which are proteasome inhibitor induced a decrease in cellular amino acid levels (Suraweera et al., 2012, Zhang et al., 2014). We assume that proteasomal proteolysis play an important role in maintaining myocellular amino acid level. However, the effective detection of the proteasomal proteolysis method is not well established. One possibility of amino acids detection is click chemistry technology by using Azidohomoalanine (AHA). We therefore investigate whether click chemistry was able to detect the proteasomal proteolysis effectively. To examine this hypothesis, we used the AHA, which is methionine‐like chemical.MethodsMouse C2C12 myoblasts were cultured under proliferate conditions (37 °C under a humidified atmosphere containing 5% CO2) in high glucose DMEM supplemented with 10% fetal bovine serum and 100mg/ml penicillin streptomycin solution. C2C12 myoblasts stained using click chemistry technology. The medium was replaced with DMEM containing AHA without methionine for 3 hours. Next, We exposed proteasome inhibitor which is MG132 to C2C12 for 3h, 6h, 9h, 18h, 24h. Following fixation and permeabilization, cells were stained with green fluorescent Alexa Fluor 488 alkyne to detected the intracellular AHA intensity (arbitrary units).ResultsC2C12 myoblasts could be stained implied that click chemistry technology was available for C2C12 myoblasts. The intracellular AHA of normal C2C12 myoblasts decreased sequentially; on the other hand, the intracellular AHA level of proteasome inhibited C2C12 myoblasts did not change. Additionally the inhibition of proteasome induced cell death in C2C12 myoblasts. In summary, these data suggested that the AHA might be able to detect the proteasomal proteolysis effectively. This procedure may contribute to clarify more detail of the role of proteasomal proteolysis.ConclusionsWe concluded that the click chemistry technology is visualized sequential proteasomal proteolysis on C2C12 myoblasts.
MECHANISMS OF H2S ACTIVATION BY INTERMITTENT HYPOXIA
Authors: Yuan Guoxiang , Peng Ying‐Jie , Khan Shakil A. , Nanduri Jayasri , Kumar Ganesh K. , Prabhakar Nanduri R. 
Published: November 2023

H2S generated by the enzyme cystathionine‐ϒ‐lyase (CSE) is important for the carotid body activation by hypoxia. Chronic intermittent hypoxia (CIH) is a hallmark manifestation of sleep apnea. An enhanced carotid body chemoreflex has been implicated in causing sympathetic activation and hypertension in sleep apnea patients and in CIH treated rodents. Rodents exposed to CIH exhibit markedly increased carotid body response to hypoxia, which was attributed to increased generation of reactive oxygen species (ROS). But, how ROS mediates the carotid body activation by CIH is not known. We tested the hypothesis that CIH‐induced ROS activates CSE‐derived H2S production in the carotid body. Experiments were performed on adult rats, wild‐type and CSE null mice exposed to CIH for 10 days. H2S levels were markedly elevated in CBs of CIH treated rats and wild‐type mice. This effect was absent in rats treated with L‐propargyl glycine, an inhibitor of CSE and in mice deficient in CSE. These findings suggested that CIH increases H2S production by activating CSE. MnTMPyP, a ROS scavenger also blocked CIH‐induced H2S generation, suggesting that ROS activates CSE. However, studies on human embryonic kidney cells (HEK)‐293 cells heterologously expressing CSE showed that CSE is not a direct target of ROS. Instead, IH‐induced ROS inactivates heme oxygenase‐2, an enzyme that generates carbon monoxide (CO) by interfering with Cys265 (C265A) residue in the heme regulatory motif of the enzyme. As a consequence, maximal CO synthesis (Vmax) is reduced leading to decreased CO generation. The reduced CO generation in turn activates CSE by inhibiting the serine phosphorylation and thus increases H2S production. (Supported by NIH‐PO1‐HL90554).Support or Funding InformationNIH‐PO1‐HL90554
Bilateral Renal Cryodenervation Decreases Arterial Pressure and Plasma Renin Activity in Fructose‐Fed Sprague Dawley Rats
Authors: Soncrant Tyler , Beierwaltes William H. , Chen Haiping , Wu Min , Rossi Noreen F. 
Published: November 2023

High consumption of fructose containing foods is increasingly prevalent. Fructose intake is associated with metabolic syndrome including hypertension. Fructose upregulates renal sodium and chloride reabsorption. When combined with high salt diet, fructose‐fed rats display increased arterial pressure and renal sympathetic nerve activity (RSNA). We tested the hypothesis that bilateral renal denervation using cryo‐techniques will decrease arterial pressure and plasma renin activity (PRA) in fructose‐fed rats on high salt diet. Male Sprague Dawley rats were instrumented with radiotransmitters for heart rate and arterial pressure. They were placed on 20% fructose (FF) in their drinking water and 0.4% NaCl diet. After 1 week they were maintained on 0.4 % (LS) or switched to 4% NaCl (HS) diet. One week later, rats were subjected either to bilateral denervation by freeze (−150°C)‐thaw × 3 (cryoDNX) or sham denervation (shamDNX). After 7–10 days, aortic blood was taken for PRA and renal cortex tested for norepinephrine content to verify denervation. Mean arterial pressure (MAP) increased by 9.6 ± 1.0 Hg in FF‐HS vs 0.8 ± 0.7 in FF‐LS rats. MAP decreased from 114.8 ± 2.9 to 108.5 ± 2.0 mmHg in FF‐HS‐cryo but did not change in FF‐HS‐sham rats, 115.6 ± 2.6 mmHg. PRA was higher in FF‐LS‐shamDNX 35.1 ± 13.2 vs FF‐HS‐shamDNX 10.9 ± 5.5 ng AngI/hr/ml and decreased significantly after cryodenervation in both groups. These findings support the concept that high fructose associated with high salt diet leads to renin dependent hypertension which is mediated, at least in part, by increased renal sympathetic nerve activity.Support or Funding InformationVA Merit Award to NFR
The Effect Of Dietary Amino Acids On Chronic Kidney Disease Progression in Rats
Authors: Pillai Samyuktha Muralidharan , Verrey François 
Published: November 2023

Chronic kidney disease (CKD) affects over 10% of the population (Troidle, 2014) and is on the rise. An increase in the protein intake, one of the characteristics of a western‐type diet, is thought to have a detrimental effect by increasing the Renal Plasma Flow (RPF), inducing hyperfiltration and increasing acid load. In addition to these, the source of the protein i.e. animal or vegetal (Goraya and Wesson, 2016) and consequently its amino acid content, might also play a role in the progression of the disease. The actual mechanisms by which these proteins / amino acids lead to increased Glomerular Filtration Rate (GFR) and to kidney function deterioration is not well understood.In our experiments, we address the question of which amino acids, or groups of amino acids, might affect the progression of renal disease.To address this question, we utilized the well‐established 5/6th Nephrectomy (5/6th Nx) model. 5/6th Nx Wistar Han rats were randomly divided into groups, receiving either the control diet (18% protein), or one of different diets, in each case containing 8% protein supplemented with 10% amino acid (aa) mix (Essential aas, Non‐essential aas, Branched chain aas or all aas in the same proportion as in the protein mix). Both GFR and RPF were measured in free moving animals, GFR transcutaneously using FITC‐sinistrin, and RPF by using radiolabelled para‐aminohippurate (PAH). None of the modified diets caused any decrease in body weight, food or water consumption in the different groups. However, the pace of RPF and GFR alteration was diet‐dependent, with the slowest change in animals receiving the EAA diet, and the most dramatic one in the animals on BCAAs. The kidney of these BCAA receiving rats also showed the strongest increase in smooth muscle actin and collagen mRNA expression in their kidney, as expected for a higher level of inflammation and fibrosis. The SMA levels were increased 2‐fold and the collagen levels were increased more than 3‐fold compared to animals on the control diet. Taken together, these results suggest that high levels of BCAAs contained in the diet have a deleterious effect on the progression of CKD.Support or Funding InformationThis research/project was supported by the NCCR Kidney. CH funded by the Swiss National Science Foundation
Novel CRISPR/Cas9 mouse knock‐in model containing an alpha‐actinin 4 mutation associated with focal segmental glomerulosclerosis recapitulated human kidney injury and provides insight into how aberrations in cytoskeleton lead to podocyte dysfunction
Authors: Feng Di , Krishnan Ramaswamy , Benjamin Ava , Pollak Martin R. 
Published: November 2023

Alpha‐actinin 4 (ACTN4) is a cytoskeleton protein that crosslinks actin filaments, helps anchor the actin cytoskeleton to focal adhesions, and provides structural support for cells. Mutations in ACTN4 cause a highly penetrant form of focal segmental glomerulosclerosis (FSGS) in humans, a type of kidney injury characterized by proteinuria and podocyte dysfunction. We previously found that disease‐causing mutant K255E ACTN4 increases podocyte contractility in response to injurious circulating stimuli and increased extracellular matrix stiffness. We hypothesize that this increased contractility is pathologic, rendering mutant podocytes unable to adapt to the dynamic mechanical stresses that they experience in the glomerulus while maintaining the filtration barrier. Here we test this hypothesis by subjecting primary podocytes derived from a novel mutant mouse model to repetitive stretch and measuring their response. Using CRISPR/Cas9 technology, we first developed a new mouse model Actn4 K256E (a mutation analogous to the FSGS‐causing K255E mutation in humans) in a pure FVB background. Compared with WT littermates, homozygous mutant mice developed significant proteinuria consistent with the human phenotype. Traction force microscopy was then used to study the contractile forces exerted by mutant and WT podocytes on their underlying substrates in response to stretch. Each podocyte was subjected to five intermittent 4s stretches, with a 3 min recovery period between stretches during which contraction force was continuously measured. Whereas WT podocytes were able to consistently recover their initial contraction forces after each stretch, mutant podocytes on average demonstrated higher initial contraction forces that drastically deteriorated without recovery over time. Additionally, mutant podocytes developed obvious cracks in their cytoskeletons as visualized by immunofluorescence staining – a finding largely absent in WT podocytes. These results represent the net effect of aberrant cytoskeleton remodeling contributed by mutant ACTN4, leading to abnormalities of podocyte structure and function that may underlie the development of FSGS.Support or Funding InformationR37DK059588 and T32DK007199
Chronic alterations in blood pH affect fasting‐state amino acid oxidation and myofibrillar and albumin protein synthesis in healthy young men
Authors: Moore Daniel R. , Lysecki Paul , Breen Leigh , Burd Nicholas A. , Smith Kenneth , Atherton Philip J. , Phillips Stuart M. 
Published: November 2023

Blood pH is typically held within a narrow range in healthy individuals. However, pathological conditions (e.g. chronic kidney disease) or net acid loads associated with Western diets and/or aging can induce shifts in acid‐base homeostasis that may alter whole body and muscle protein kinetics that, over time, could lead to changes in lean body mass. Therefore, we aimed to determine the effect of chronic alterations in blood pH over physiological and pathophysiological ranges on muscle and whole body protein metabolism in vivo in humans. After 7‐d supplementation in a randomized, crossover fashion with 0.15g/kg/d of NH4Cl (acid, A), KHCO3 (base, B), or KCl (control, C), 8 healthy males (22±1y; BMI: 27.8±1.9) received a primed constant [13C]leucine infusion to measure whole body leucine metabolism and myofibrillar (MYO) and albumin (ALB) protein synthesis in the fasted state. Supplementation predictably altered venous blood pH (A<C<B; P ≤ 0.051; range = 7.26–7.41) but had no effect (P>0.05) on whole body leucine turnover, leucine rate of appearance, or non‐oxidative leucine disposal (estimate of protein synthesis). Although there were no differences (P>0.05) between conditions, leucine oxidation was negatively (r = −0.57; P<0.01) whereas leucine net balance was positively (r = 0.54; P<0.01) correlated with blood pH. There was a trend (P=0.07) towards differences in MYO between conditions. MYO was positively correlated with blood pH (r = 0.47; P<0.05). There was no effect of blood pH on mTORSer2448, 4E‐BP1Thr37/46, or eEF2Thr52 phosphorylation (P>0.05). S6K1Thr389 phosporylation was lower in A compared to B and C and its phosphorylated‐state was correlated with blood pH (r = 0.52; P=0.02) and tended to relate to MYO (r = 0.44; P=0.07). Given this, metabolic acidosis may suppress MYO in part via an impairment in mRNA translation initiation. There was no difference (P>0.05) between conditions in candidate markers of muscle protein breakdown (i.e. total ubiquitin‐conjugated proteins and 55‐kDa actin fragment). ALB was lower in A compared to B (P<0.05) and, similar to MYO, was correlated with blood pH (r = 0.51; P=0.01). Our data demonstrate that blood pH has a modifiable effect on whole body, muscle, and albumin protein kinetics. It is possible that normalization of acid‐base balance towards an alkolytic state will help mitigate the characteristic loss of lean body mass in populations at risk of clinical and subclinical metabolic acidosis through the attenuation of oxidative amino acid losses and an enhancement of myofibrillar protein synthetic rates.Support or Funding InformationSupported by the Natural Sciences and Engineering Research Council of Canada
CENTRAL TRPV4 AND WARMTH‐DEFENCE RESPONSE IN WISTAR RATS
Authors: Bícego Kenia Cardoso , Scarpellini Carolina Silveira , Almeida Maria Camila , Gargaglioni Luciane Helena 
Published: November 2023

Peripheral TRPV4 is activated and triggers warmth‐defense responses at ambient temperatures (Tas) of 26–30°C in non‐genetically modified rats. TRPV4 also can be found in medial preoptic area (MPA), region known by being involved with activation of heat loss mechanisms. Then, the aim of this study was to verify if central TRPV4 in the MPA plays a role in the control of body temperature (Tb) affecting autonomic and behavioral thermoeffectors. To this end, under two different Tas (21 e 28°C), the body temperature (Tb) of rats was measured after TRPV4 in MPA were antagonized by HC‐067047. Metabolic rate, heat loss index (HLI) and preferred temperature were also recorded after central blockage of TRPV4. The antagonism of central TRPV4 caused a further increase in the Tb of rats (p< 0.001) exposed to 28°C, but not to those ones exposed to 21°C, compared to vehicle‐treated animals. The rise on Tb under 28°C was accompanied by an augment in oxygen consumption (p= 0.007) and by reduction of HLI (p=0.041). Lastly, the cold seeking behaviour observed in control animals after exposure to warm Tas for 2 hours (28–30°C) was inhibited by the microinjection of the antagonist of TRPV4 into MPA (p= 0.01). Thus, our results indicate that central TRPV4 plays a role in thermoregulation in warm condition, affecting both autonomic and behavioral heat loss responses.Support or Funding InformationFAPESP (15/04849‐6)
Medullary reticular GABAergic neurons that mediate hunger responses induced by hypothalamic neuropeptide Y
Authors: Nakamura Yoshiko , Yanagawa Yuchio , Morrison Shaun F , Nakamura Kazuhiro 
Published: November 2023

Hunger stimulates release of neuropeptide Y (NPY) in the paraventricular hypothalamus (PVH) from nerves derived from the arcuate nucleus. The NPY action in the PVH reduces energy expenditure through inhibition of brown adipose tissue (BAT) thermogenesis and stimulates food intake—hunger responses to survive starvation. However, the central circuit mechanism by which the NPY‐mediated hunger signaling from the hypothalamus coordinates the autonomic and somatic motor systems to reduce energy expenditure and to drive the consummatory responses is unknown. In this study, using rats and mice, we found that hypothalamic action of NPY activates GABAegic neurons in the intermediate and parvicellular reticular nuclei (IRt/PCRt) of the medulla oblongata that project to the rostral medullary raphe, which contains sympathetic premotor neurons controlling BAT thermogenesis. Selective stimulation of GABAergic neurons in the IRt/PCRt with a chemogenetic technique inhibited cooling‐induced BAT thermogenesis. Inactivation of IRt/PCRt neurons eliminated the inhibitory effect of NPY action in the PVH on cooling‐induced BAT thermogenesis. Intriguingly, the GABAergic IRt/PCRt neurons innervating the BAT sympathetic premotor region also innervated the masticatory motor region, and stimulation of the IRt/PCRt elicited mastication and increased feeding as well as inhibition of BAT thermogenesis. These results demonstrate a hunger response central circuit in which NPY‐triggered signaling from the hypothalamus activates GABAergic IRt/PCRt neurons, which then inhibit the thermogenic sympathetic premotor drive to BAT to reduce energy expenditure and stimulate a masticatory motor drive for feeding.Support or Funding InformationGrants‐in‐Aid for Scientific Research from MEXT, Japan (16H05128, 15H05932 & 15K21744 to KN, 26290002 & 15H01415 to YY, 26860159 to YN); NIH grant (R01NS40987 to SFM); Takeda Science Foundation (KN & YY); Uehara Memorial Foundation (KN).