bluuebunny/crossref_metadata_2025 huggingface dataset:
Apoptosis Signal‐regulating Kinase 1 is associated with NADPH oxidase 1‐dependent signaling
Authors: Choi Hyehun , Lamb Fred S
Published: November 2023

Tumor necrosis factor‐α (TNFα) is pro‐inflammatory cytokine, linked to cardiovascular risk, including inflammatory vascular lesions. NADPH oxidase 1 (Nox1) is the primary source of superoxide activated by TNFα in vascular smooth muscle cells (VSMC). Apoptosis signal‐regulating kinase 1 (ASK1) is a MAPKKK that is activated by oxidation of associated thioredoxin. ASK1 in turn activtes JNK and p38 MAPKs and plays a role in vascular intimal hyperplasia. The relationship between ASK1 and Nox1‐mediated signaling in response to TNFα is poorly defined. We hypothesized that TNFα‐induced ASK1activation requires Nox1‐derived superoxide. We measured TNFα‐induced; endosomal superoxide (OxyBurst green), TNFα receptor (TNFR) endocytosis (TNFα‐biotin and FITC‐avidin), NF‐κB activation (luciferase reporter), and cell survival in VSMC. siRNA targeting ASK1 reduced TNFR endocytosis by 33%, TNFα‐mediated NF‐κB activation by 66 ± 0.7%, endosomal superoxide production by 80 ± 0.1%, and cell survival by 8.6 ± 2%. Immunoprecipitation‐western blot analysis was performed with ASK1 and the p22phox subunit of Nox1. ASK1 co‐immunoprecipitated with both Nox1 and its p22phox subunit. In Nox1 null cells, ASK1 protein expression was significantly higher than in wild type cells. Thus, ASK1 affects Nox1‐mediated inflammatory signaling in response to TNFα. These data provide a novel mechanism for Nox1‐dependent inflammatory effects in VSMC.

Support or Funding Information

American Heart Association (16SDG30610002)
The Effect of Hyperglycemia on the Expression of Stress Proteins of Endoplasmic Reticulum and Vascular Smooth Muscle Cell (VSMC) Migration from Normal Rats
Authors: Mejía‐Elizondo Rebeca , Hernández‐Méndez Aurelio , Chi‐Ahumada Erika , Espinosa‐Tanguma Ricardo
Published: November 2023

Diabetes mellitus (DM) is a chronic degenerative disease characterized by high glucose levels on blood, which is the leading cause of micro and macrovascular changes observed in this disease.

At cellular level DM promotes the misfolding of proteins in the endoplasmic reticulum (ER), a condition defined as ER stress. ER stress is characterized by an increase on the expression of some proteins, unfolded protein response (UPR), that promotes the decrease of the unfolded proteins translation and the increase in the expression of folding proteins or apoptosis when the damage is too great.

Since research efforts on macrovascular disease secondary to DM are focused on pathological changes on endothelial cells, we are interested in evaluating the changes that occur at the level of VSMC as it has been demonstrated that they play a fundamental role in the pathophysiological process.

On this research we obtained primary smooth muscle cell cultures from the thoracic aorta of normal rats by means of explant culture technique. The cells were characterized by immunofluorescence against muscle alpha‐actin and myosin and they were used on experiments were the cells were exposed to high glucose concentrations (15 mM) where we analyzed: proteins related to endoplasmic reticulum stress, expression of Ca+2 ATPase of the ER (SERCA2), cellular migration through the method of wound healing assay and the usage of the Boyden camera. Our results show that muscle cells exposed to 15 mM of glucose for 48 hours do not exhibit any changes on cell migration or in the expression of ATF‐6 and XBP‐1 proteins (analyzed by western blot), that we used as stress markers. However cells exposed for one week to 15 mM glucose showed an increase on cell migration as well as an increase on the expression of stress markers, which leads us to believe that these cells have a phenotype that is similar to those from diabetic rats. On the other hand the expression of SERCA2, analyzed by inmunoblot, seems to increase which suggests that the cells that are exposed to hyperglycemia over express SERCA2 to compensate for the damage on the ER being that the activity of this ATPase is fundamental to maintain a proper cell function. Overall these results show that the expression of smooth muscle cells to high glucose concentrations simulates the process that takes place in cells from diabetic animals, which allows us to have a study model of diabetes without inducing animals with drugs that produce severe side effects.

Support or Funding Information

Supported by Conacyt to RET: 223350
Age‐dependent Changes in Aortic Valve and Mitral Valve Function in Fibrillin‐mutant Mice
Authors: Olson Mariah F , Verzosa Grace C , Roos Carolyn M , Zhang Bin , Hagler Michael A , Thalji Nassir M , Miller Jordan D
Published: November 2023

The structural protein, fibrillin, is important for maintaining the strength and reliability of connective tissue found in blood vessels and heart valves. Mutations in fibrillin‐1 result in Marfan syndrome and leads to left‐sided heart valve regurgitation, aortic root dilatation and aortic aneurysm. While previous studies have focused predominantly on young fibrillin‐mutant mice, hemodynamically significant disease generally does not emerge in patients with fibrillin mutations until later in life. Thus, this study aims to evaluate molecular and functional changes in heart valves from aged fibrillin mutant mice. Wild‐type (+/+) or fibrillin mutant (+/C1037G) mice were fed normal chow diet for 3 months (young) or 14 months (old). Heart valve function, aortic dimensions, and cardiac function were evaluated using high‐resolution echocardiography, and gene expression was measured using qRT‐PCR. Consistent with previous reports, we found no evidence of mitral regurgitation in young mice of either genotype. In old +/C1037G mice, however, we did find evidence of mitral regurgitation (~5%). In contrast, aortic valve regurgitation was evident in a relatively large proportion of +/C1037G mice at both ages (~20% at both time points), and was not detected in any +/+ animals. Interestingly, left ventricular dimensions were not associated with the presence of mitral regurgitation, but aortic root dilatation was present in all mice with aortic valve regurgitation. To gain insight into the molecular underpinnings of these phenotypic changes, we measured expression of IL‐10 (an anti‐inflammatory gene) and Runx2 (a pro‐proliferative gene that drives matrix remodeling), both of which are genes previously implicated in the pathogenesis of mitral and aortic valve disease. In young animals, expression of IL‐10 in mitral valves tended to be increased in +/C1037G mice compared to +/+ littermates. With aging, IL‐10 increased substantially in mitral valves from in +/+ mice, and increased further in +/C1037G littermates. Similar trends were observed in expression of Runx2 in mitral valves from both genotypes of mice. While IL‐10 followed a similar pattern in aortic valve tissue from young and old mice of both genotypes, Runx2 was not significantly increased across any of the groups. Collectively, while our data suggest that there are substantial, potentially adaptive molecular changes that occur during development of regurgitant valvular diseases, aortic dilatation may be a key early factor driving development of aortic regurgitation. Future experimental work aimed at determining the relative mechanical and molecular contributions to development of heart valve regurgitation will be critical to development of novel therapeutic strategies in patients with syndromes secondary to fibrillin mutations.

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NHLBI (R01‐HL111121)
Integrity of intestinal mucosa and serum concentration of some metabolites in pigs exposed to acute and chronic heat stress
Authors: Morales Adriana , Vásquez Nydia , Gómez Tania , Ibarra Nely , Bernal Hugo , Rodríguez Luis Edgar , Cervantes Miguel
Published: November 2023

Heat stress (HS) is a condition that affects organisms when ambient temperature exceeds their thermoneutral (TN) zone. HS organisms try to increase their heat loss by reducing feed intake and changing blood irrigation to peripheral tissues instead of internal organs; other metabolic changes that can be analyzed by blood chemistry occur as well. A study was conducted during summer time in the Sonoran Desert, México, using the pig as a biological model to analyze the effect of acute and chronic HS on feed intake, blood chemistry, and intestinal epithelia integrity. Eighteen pigs (36 kg body weight) housed in individual cages inside a room with air conditioned equipment were used. Pigs were fed ad libitum a balanced diet and water was freely available all the time. Pigs were maintained under TN conditions (24 °C) during the first 10 d, at the end of which the air conditioned system was turned off, and pigs were exposed to natural diurnal fluctuations on ambient temperature, 28 °C (morning) and 38 °C (evening). Pigs were divided into three groups of six pigs each, one group was euthanized after 10 d under TN condition, the second group was euthanized on d2 of HS (acute HS, AHS), and the third group on d7 under HS (chronic, CHS). Immediately after euthanasia, the intestinal segments of duodenum, jejunum and ileum were collected. Blood samples were collected from each pig one hour before euthanasia; all pigs were fasted for 12 hours before sample collection. Feed intake in AHS and CHS pigs was reduced by about 38% (P<0.01) compared with TN. Acute HS pigs showed increased counts of lymphocytes, erythrocytes, and platelets (P<0.05). HS increased serum concentration of urea, BUN, uric acid, and triglycerides (P≤0.05); and reduced calcium concentration (P<0.05). Acute HS increased glucose, cholesterol, calcium, and phosphorous (P<0.05) compared to CHS. Histological analysis showed that duodenum villus height and crypt depth were reduced in CHS pigs (P<0.05) compared to TN conditions. The jejunal villus height and crypt depth were reduced (P<0.05) because of AHS and CHS, but the AHS damage in villus height was more severe. Also, ileal villus height and crypt depth were reduced (P<0.01) under CHS compared to TN. In general, the relation between villus height and crypt depth was lower in jejunum of AHS and higher in ileum of CHS pigs. In conclusion, these data show that HS significantly affects the intestinal epithelium and modifies the blood chemistry of pigs exposed to HS conditions.

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18 Convocatoria Interna, UABC.
G‐Hesperidin Supplementation Impairs the Beneficial Effects of Physical Exercise on the Body Composition, Biochemistry Profile and Oxidative Stress in Obese Rats
Authors: Priviero Fernanda B. M. , Gonçalves Tiago Tomazini , Lazaro Carolina Martins , De Mateo Fernanda Gonçalves , Campos Marcela Couto Brigagão , Claudino Mario Angelo , de Oliveira Carvalho Patrícia
Published: November 2023

Obesity is a worldwide public health problem and its treatment includes limited dietary intake of saturated fats and physical activity. Obesity may be accompanied by metabolic changes and may cause several organic dysfunctions such as vascular dysfunctions due to reduced bioavailability of vasodilatory substances such as nitric oxide (NO). There are evidences that the reduced vasodilatory substances are related to increased reactive oxygen species (ROS), which leads to an oxidative stress. Additionally, the intake of antioxidant flavonoids has been largely proposed as a therapy for metabolic and cardiovascular disorders. Therefore, the aim of this work was to evaluate the effects of G‐ hesperidin supplementation (an antioxidant flavonoid) associated (or not) with physical exercise, on the body composition, lipidic metabolism, oxidative status and vascular function of obese rats. Obesity was induced by a hyperlipidic diet plus fructose given to 4 weeks‐old male Wistar rats, lasting 12 weeks. Physical training and/or antioxidant treatment started along with the 8th week of life and lasted 8 weeks further. It was evaluated the body compositon, lipid profile, oxidative status and mesenteric artery was removed for functional evaluation. After 12 weeks of hyperlipidic diet, rats presented higher body weight gain and body fat accumulation when compared to rats fed with standard chow. Treatment with G‐hesperidin did not change body weight gain and fat depot. Only physical training was able to reduce the body weight gain and fat accumulation. Furthermore, physical training modulated the oxidative status by increasing SOD activity, total antioxidant capacity and reducing lipid peroxidation. Treatment with G‐hesperidin did not affect oxidative status and when associated to physical training, decreased changes elicited by physical training. Hyperlipidic diet caused endothelial dysfunction, since the potency of acetylcholine was reduced in all groups fed with hyperlipidic diet. Treatment with G‐hesperidin and physical exercise did not alter the potency of acetylcholine. Potency of sodium nitroprusside was not changed by hyperlipidic diet whereas it was potentiated by physical training. Our results show that, in experimental model of obesity, physical exercise is able to modify body composition and biochemical parameters and improve general oxidative status of rats fed with hyperlipidic diet. Treatment with antioxidant flavonoid G‐hesperidin inhibited the benefit promoted by physical exercise.

Support or Funding Information

FAPESP ‐ Fundação de Amparo à Pesquisa do Estado de São Paulo.
Regulation of microtubule dynamics by suppression of microtubule assembly kinetics
Authors: Gardner Melissa K.
Published: November 2023

TPX2 is a widely conserved microtubule‐associated protein that is required for mitotic spindle formation and function. In this study, we explored a potential role of TPX2 in regulating microtubule assembly kinetics, which could lead to downstream consequences for its effects on the parameters of microtubule dynamic instability. We found that TPX2 acts to suppress tubulin subunit off‐rates during microtubule assembly and disassembly. This allowed for the support of unprecedentedly slow rates of plus‐end microtubule growth, and to a dramatically reduced microtubule shortening rate. These changes in microtubule dynamics could be explained in computational simulations by a moderate increase in tubulin‐tubulin bond strength upon TPX2 association with the microtubule lattice, which in turn acts to reduce the departure rate of tubulin subunits from the microtubule ends. Further, our simulations naturally reproduced the suppression of catastrophe events in TPX2 as a direct result of the suppressed tubulin subunit off‐rates, likely due to increased retention of GTP‐tubulin subunits at the microtubule tip, which would lead to a larger GTP‐cap size. Thus, the direct suppression of tubulin subunit off‐rates by TPX2 during microtubule growth and shortening could provide a molecular mechanism to explain downstream changes in the parameters of microtubule dynamic instability in the presence of TPX2.

Support or Funding Information

NIGMS, National Institutes of Health
Mechanics of the Actin Cytoskeleton
Authors: Gardel Margaret
Published: November 2023

Networks of the semi‐flexible biopolymer actin are essential determinants of the mechanical behaviors of eukaryotic cells. Actin networks have revealed novel mechanical behaviors not observed in physical biopolymers, arising both from its semi‐flexibility and mechanochemical activity of constituents. I will discuss current understanding of how the structure and mechanics of materials constructed from F‐actin is controlled. I will also describe a new liquid‐like phase of cross‐linked actin. In this liquid phase, the surface tension of the actin droplets can be controlled by cross‐link concentration and this surface tension can be used to drive contractility in the absence of enzymatic activity. Our results provide a strategy for designing a novel class of materials and demonstrate a new putative mechanism of mechanical sensing and shape change in eukaryotic cells.
Moderate Exerciwse Buffers Against Endocrine and Behavioral Consequences of Social and Environmental Stress
Authors: Watanasriyakul Withayapon , Cox Miranda , Woodbury Matthew , Colburn William , Wardwell Joshua , McNeal Neal , Grippo Angela J.
Published: November 2023

Mild chronic environmental stress has been associated with emotional disturbances and negative physiological changes, such as hypothalamic‐pituitary‐adrenal (HPA) dysfunction. This dysfunction may lead to cardiovascular diseases as well as psychological disorders such as depression. Further, social and environmental stressors may have synergistic damaging effects. Socially isolated individuals who also experience chronic stress may be more susceptible to depression and HPA axis dysregulation. However, physical activity has been shown to combat the detrimental effects of stress; as elevated mood, decreased plasma glucose, and increased metabolism are beneficial outcomes of exercise. The current study examined the potential stress buffering effects of exercise against social isolation and chronic mild stress (CMS) using a prairie vole model. Prairie voles are a socially monogamous rodent species with a similar social structure to humans, including forming social bonds and living in family groups. Adult female prairie voles were separated from a same‐sex sibling and housed individually for 4 weeks. During the final 2 weeks of the isolation period, all animals underwent a CMS paradigm involving mild unpredictable stressors. Furthermore, during this 2‐week period, half of the voles were allowed access to a running wheel (vs. sedentary control). At the end of the isolation period, all animals underwent a forced swim test (FST) and an elevated plus maze (EPM), to assess depressive‐ and anxiety‐behaviors, respectively. In addition, plasma was collected for corticosterone (CORT) assay. A high variability of activity was noted in the Exercise group; therefore, animals were split into 3 subgroups: low (< 0.01 km/day), moderate (0.01 – 3.5 km/day), and high (> 3.5 km/day). Voles in the Exercise group displayed significantly lower levels of immobility during the FST, and spent more time in the open arms of the EPM compared to animals in the Sedentary group, indicative of reduced depressive‐ and anxiety‐behaviors, respectively. Voles in the Exercise group had significantly lower plasma CORT levels compared to voles in the Sedentary group. Interestingly, animals that moderately exercised displayed less immobility during the FST and had lower CORT levels compared to animals in the low and high exercise subgroups. The current study provides evidence that physical activity can have protective effects against social isolation as well as CMS. Moreover, targeting the optimum level of activity may have additional benefits, as animals with moderate exercise levels exhibited fewer behavioral and endocrine signs of depression and anxiety. Thus, moderate exercise may lead to superior behavioral and physiological outcomes as a buffer against social and environmental stressors in humans.

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HL112350
Effects of Repeated Whole Body Heating on Sleep Architecture and Chronic Lower Back Pain
Authors: Smith Caroline J. , Collier Scott R. , Clarke Megan M. , Bouldin Erin L. , Uher David T.
Published: November 2023

Chronic lower back pain (CLBP) is recognized as a critical public health problem, yet understanding and treating chronic pain conditions remains challenging. Poor sleep quality, including frequent sleep disturbances and prolonged sleep onset latency, are some of the most commonly reported complaints in individuals suffering chronic pain conditions. Our aim was to investigate the effects of repeated systematic, whole body heating (WBH) on sleep architecture and pain symptoms in individuals experiencing CLBP. Eight CLBP sufferers (49±7 yrs; 4 male, 4 female) with no known sleep disturbances completed baseline measures (BL) of sleep architecture utilizing an ambulatory sleep monitor (Sleep profiler™) and McGill Pain Questionnaires. Seven consecutive evenings of whole body heating (WBH) to a 0.8°C core temperature (Tcore) rise were conducted using a water perfused suit (48°C water). Two subjects completed a control protocol utilizing 34°C water, in randomized order and following a 3 month washout period. McGill pain questionnaires and ambulatory sleep measures were recorded each evening throughout the protocol, and 48 hours and 2 weeks following. Sleep architecture was assessed using stages N1, N2, N3 and REM. Pain scores were significantly reduced from 22 at BL to 14 after 5 day of WBH (p<0.05), and remained significantly lower for the remainder of the heating protocol and at the 48 hour follow‐up (p<0.05). Whilst pain was reduced following heating on all days, the initial 4 days and the 2‐week follow‐up were not significantly different from baseline. Sleep architecture showed a significant increase in REM from BL to day 1 of heating (1.20±0.3 Vs. 1.77±0.97 hrs; P=0.044), with all other days approaching significance. N3 approached significance following all heating days and at the 48h follow‐up (p>0.05). Stage N1 decreased from baseline to heating day 7 (0.40±0.23 Vs. 0.30 0.25 hrs, p=0.06), which was close to significance. Taken together these preliminary data suggest the potential for WBH as a non‐pharmacological method to improve sleep architecture and reduce pain symptoms in CLBP patients.

Support or Funding Information

None.
Muscle‐kidney crosstalk through microRNA‐29 in chronic kidney disease mouse
Authors: Wang Xiaonan H , Escobar Elizabeth M , Zhang Aiqing , Klein Janet D.
Published: November 2023

Kidney fibrosis is a progressive process that ultimately leads to end‐stage renal failure. Muscle wasting is a serious complication of chronic kidney disease (CKD) and contributes substantially to the morbidity and mortality of patients. Exosomes, natural carriers of many signal molecules including microRNA (miR), mediate organ to organ communication. MiR‐29 has been suggested a therapeutic target for muscle wasting by our studies (Wang JANS 2011), and also has anti‐fibrosis character. We hypothesized that miR‐29 has benefic for both unilateral ureteral obstruction (UUO)‐induced muscle wasting and kidney fibrosis through exosomes‐medicated muscle‐kidney crosstalk.

UUO was induced by left ureteral ligation. NanoSight instrument was used to quantity exosomes. The miR deep sequences assay and qPCR were used to identify microRNA. Western blot was used for protein quantification. Immunohistochemistry was used to identify renal fibrosis.

Using Nanosight measurement we found that serum‐derived exosomes size is larger in UUO mice than controls. The miRNA deep sequencing showed that that miR‐29a‐5p was significantly increased in CKD mouse. However, the miR‐29a expression decreased in both skeletal and kidney of UUO vs controls. Uremic serum enhanced exosome secretion miR‐29a in cultured C2C12 skeletal muscle cells. We using adeno‐associated virus (AAV) to overexpress miR‐29a through tibialis anterior muscle injection. Exogenous miR‐29a increased muscle cross‐section area and decreased UUO‐induced upregulation of atrogin‐1 and MuRF1 in muscle. Interestingly, kidney fibrosis also partial depressed in miR‐29a overexpressing UUO mice using Masson staining. We further confirmed that alpha smooth muscle actin, vimentin and fibronectin and collagen 1α were decreased by exogenous miR‐29a in the kidney of UUO mice. In a cell culture model, we identify that exosome containing miR‐29a from skeletal muscle cells, can transfer miR‐29 to human kidney cells and attenuated uremic serum‐induced upregulation of alpha smooth muscle actin. This give us an indirectly evidence of muscle‐kidney crosstalk.

Exogenous exosome/miR29a through tibialis anterior muscle injection not only attenuated skeletal muscle atrophy but also ameliorate UUO‐induced renal fibrosis through exosome‐mediated muscle kidney cross talk.

Support or Funding Information

This work was supported by NIAMS R01 AR060268 (30971471) to X.W; This research project was supported in part (production of AAV virus) by the Viral Vector Core of the Emory Neuroscience NINDS Core Facilities grant, P30NS055077. This study was also supported in part (AAV virus and luciferase constructs) by the Emory Integrated Genomics Core (EIGC), which is subsidized by the Emory University School of Medicine and is one of the Emory Integrated Core Facilities under NIH Award Number UL1TR000454.
Characterization of Zinc Finger Protein 593 (Zfp593) in Skeletal Muscle
Authors: McLeod Marc , Waddell David
Published: November 2023

Skeletal muscle atrophy is a debilitating medical condition which can arise due to aging, cancer, corticosteroid use, and denervation. In order to better characterize the molecular genetic events of neurogenic atrophy, the gastrocnemius muscle was isolated from mice following 3 days and 14 days of sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue in order to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that zinc finger protein 593 (Zfp593) is expressed in skeletal muscle and is significantly induced in response to denervation. Furthermore, Western blot analysis confirmed that Zfp593 is expressed at the protein level in C2C12 mouse muscle cells. In order to characterize the transcriptional regulation of Zfp593, fragments of the proximal promoter located immediately upstream from the start of transcription were cloned and fused to a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells alone or in combination with MyoD1 or myogenin expression plasmids, resulting in a differential effect on reporter activity. Furthermore, analysis of the promoter region of Zfp593 has revealed four conserved E‐box elements, which are known myogenic regulatory factor (MRF) binding sites. Zfp593 is predicted to possess a zinc finger domain and is believed to function as a transcription factor. Furthermore, two predicted putative nuclear localization signal sequences have been identified in the Zfp593 protein sequence, further suggesting that this protein is localized to the nucleus. Therefore, in order to determine the sub‐cellular localization of Zfp593 in muscle cells, the Zfp593 cDNA was isolated from C2C12 cells, cloned into a green fluorescent protein (GFP) expression plasmid and transfected into proliferating C2C12 cells. Transfected cells were visualized by confocal microscopy revealing that Zfp593 is localized to the nucleus of proliferating myoblasts. The discovery that Zfp593 is expressed in skeletal muscle combined with the observation that this gene, a potential modulator of gene expression itself, is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of skeletal muscle wasting.

Support or Funding Information

The work was supported by University of North Florida Transformational Learning grants to D.W.
Unconventional SNARE in the ER 1 (Use1) is Expressed in Skeletal Muscle and is Upregulated in Response to Skeletal Muscle Atrophy
Authors: Tran Kien , Waddell David
Published: November 2023

Muscle atrophy, or a loss in muscle size and strength, results from various physiological conditions and occurs when protein degradation outpaces protein synthesis. Unconventional SNARE in the ER 1 (Use1) protein, in which SNARE stands for soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor, was recently found to be highly upregulated in skeletal muscle cells undergoing atrophy. However, the mechanism of how Use1 is transcriptionally regulated and its potential role in skeletal muscle atrophy is not clear. Interestingly, we have cloned four distinct protein encoding splice‐variants in skeletal muscle, but the significance and functions of these isoforms remains to be determined. It is known that Use1 functions in the endomembrane system (EMS) and that it could play a role in ER stress and the unfolded protein response (UPS) as a consequence of skeletal muscle atrophy. Therefore, in order to simulate atrophy‐induced ER stress in cell culture, we treated cells with Tucamycin (TN) or Thapsigargin (TG) to determine if Use1 expression is impacted in response to UPS activation. If TN or TG induced ER stress results in an increase in Use1 protein, or an isoform switch, it may suggest that Use1 is an important marker of atrophy‐induced ER stress and that it could play an important role in the progression of skeletal muscle wasting. Since muscle atrophy may contribute to increased morbidity and mortality in response to chronic disease states, it is important to gain a better understanding of the factors that participate in the molecular cascade of muscle atrophy, which could lead to the identification of new therapeutic targets for the treatment and prevention of muscle wasting.

Support or Funding Information

The work was support by University of North Florida Transformational Learning Opportunity grants to D.W.
Skeletal muscle amino acid pool following the administration of Doxorubicin chemotherapy
Authors: Fabris Sergio , MacLean David
Published: November 2023

The purpose of the current study was to determine the effect of Doxorubicin (DOX) administration on the skeletal muscle amino acids. Male Sprague Dawley rats received either a 1.5 or 4.5 mg/kg dose of DOX and grouped into endpoints every 24 hrs up to 192 hrs. Total amino acids (TAA), essential amino acids (EAA) and branched‐chain amino acids (BCAA) were quantified in the plantaris (PLANT), white gastrocnemius (WG) and soleus (SOL) muscle groups. Following the administration of either 1.5 or 4.5 mg/kg dose, TAA concentrations in each muscle group were significantly increased throughout the experiment when compared to control. However, there were no consistent differences in TAA concentrations when compared between doses. These results were also observed in the EAA concentrations. Subsequent to the administration of the 1.5 mg/kg dose, BCAA were significantly increased in the PLANT, WG and SOL after 48 hrs, compared to control. Similarly, the 4.5 mg/kg dose increased BCAA after 48 hrs compared to control but returned to baseline after 120, 72 and 168 hrs in the PLANT, WG and SOL, respectively. To our knowledge, these data represent for the first time the effect of DOX administration on amino acid availability in skeletal muscle. As such, this study provides important insight in the role of DOX therapy on amino acid metabolism.

Support or Funding Information

Supported by NSERC.
Myostatin Inhibition as a Therapeutic Strategy for Skeletal Muscle Myopathies–Effects on Nuclear Organisation
Authors: Ross Jacob Alexander , Levy Yotam , Lawlor Michael W , Ochala Julien
Published: November 2023

Skeletal myopathies are a broad spectrum of genetic disorders that affect muscle, and are caused by mutations in a variety of genes required for muscle function and cellular homeostasis. Actin is an essential component of the contraction machinery. Mutations in actin‐associated proteins cause nemaline myopathy, which is characterized by the presence of protein aggregates, as well as muscle fiber atrophy, contractile dysfunction and weakness. At present, there are no effective therapies for nemaline myopathy.

Myostatin is a secreted factor responsible for negatively regulating muscle growth. Inhibition of myostatin (MI) results in muscle fiber hypertrophy, and has received much interest as a possible therapeutic strategy for a variety of muscle diseases. Previous work has been carried out in two mouse models of nemaline myopathy with mutations in ACTA1 (skeletal muscle actin): an H40Y mutant, in which MI confers only modest therapeutic benefits; and a D286G mutant, in which MI is able to induce marked hypertrophy and functional improvements. We aim to investigate the reasons for this differing response.

Muscle fibers are giant cells which require hundreds of internal nuclei to generate sufficient gene products. It has been hypothesized that insufficient myonuclear density is a pathological factor in a variety of muscle diseases, leading to reduced expression of proteins required for e.g. contraction and energy metabolism. We hypothesized that nuclear density might also be a pathological factor in ACTA1 mutants, and that differences in this parameter might explain the variable responses to MI. Thus, using confocal microscopy and single muscle fibers, we assessed nuclear number and organization in 3 dimensions, in mutants with and without MI treatment.

We find that nuclei are positioned irregularly in the severe H40Y mutant. MI treatment is able to rescue this defect, and cause insertion of extra nuclei; yet these effects are not accompanied by significant hypertrophy. No underlying nuclear defects were observed in the milder D286G mutant; MI treatment did not alter nuclear arrangement but caused marked hypertrophy. Nuclear organisation defects will also be discussed in light of our findings in patients with ACTA1 mutations. These findings suggest that aberrant nuclear arrangement may be a pathological factor in diseases of muscle contraction. MI treatment may not be an effective treatment when force production and/or pathology is too severe. Unravelling the mechanisms of myostatin action at the subcellular level is of importance for the design, modulation and improvement of therapies.

Support or Funding Information

Medical Research Council, UK
Carotid Body Chemoreflex–Dependent Activation Of Epigenetic Mechanisms
Authors: Nanduri Jayasri , Peng Ying‐Jie , Wang Ning , Khan Shakil A , Semenza Gregg L , Prabhakar Nanduri R
Published: November 2023

Epigenetic regulation by DNA methylation leads to a long‐lasting suppression of gene expression. We recently reported that long‐term (30 days) exposure of rats to intermittent hypoxia (LT‐IH) results in DNA methylation‐dependent suppression of genes encoding anti‐oxidant enzymes (AOEs) in the adrenal medulla, leading to persistent hypertension that does not resolve after the cessation of LT‐IH (Nanduri J. et al. J. Physiol. 2016). LT‐IH–induced DNA methylation was due to increased activity of DNA methyltransferases (Dnmts) resulting from increased Dnmt1 and Dnmt3b protein levels, with no changes in mRNA levels. In the present study, we assessed the mechanisms underlying the induction of increased Dnmt protein levels by LT‐IH. We found that LT‐IH increases the activity of AKT kinases. Treatment of LT‐IH rats with a pan‐AKT inhibitor (GSK690693) prevented the induction of AKT activity and Dnmt protein expression, abolished the suppression of AOE gene expression. LT‐IH‐induced AKT activation required the generation of reactive oxygen species in the adrenal medulla. LT‐IH is sensed by the carotid body, which activates the sympathetic nervous system to stimulate the adrenal medulla. Selective ablation of carotid body inhibited LT‐IH induced oxidative stress, Dnmt activity and DNA hypermethylation of AOE in AM. Overall, our data indicate that LT‐IH induces carotid body chemoreflex‐dependent ROS‐AKT signaling in the adrenal medulla, which triggers epigenetic regulation of AOE genes that alters redox homeostasis leading to persistent elevations of plasma catecholamines and blood pressure.

Support or Funding Information

(Supported by NIH‐PO1‐HL90554).
T‐type Calcium Channels Mediate the Augmented Calcium Influx in Carotid Body Glomus Cells by Chronic Intermittent Hypoxia
Authors: Chupikova Irina , Makarenko Vladislav , Peng Ying‐Jie , Ahmmed Gias , Khan Shakil , Nanduri Jayasri , Kumar Ganesh , Fox Aaron , Prabhakar Nanduri
Published: November 2023

Chronic intermittent hypoxia (CIH) is a hallmark manifestation of sleep apnea. In the present study, we examined the effects of CIH on intracellular calcium responses of carotid body glomus cells to acute hypoxia and assessed the underlying mechanisms. Glomus cells were harvested from adult rats, wild‐type and Cav 3.2 null mice treated with 10 days of room air (control) or CIH. Intracellular calcium levels were monitored by single cell imaging with calcium sensitive dye Fura‐2. Hypoxia (pO2 ~40 mmHg) increased intracellular calcium in glomus cells from control room air treated rats and wild‐type mice, and this response was markedly augmented in CIH treated glomus cells. The augmented intracellular calcium response to CIH was prevented 25μM of TTA‐A2 a specific inhibitor of T‐type calcium channels, in rat glomus cells. Previous studies showed that CaV3.2 is the major T‐type calcium channel in the carotid body glomus cells. Cav 3–2 null glomus cells displayed remarkable absence of augmented calcium response to CIH. Analysis of the underlying mechanisms revealed that CIH had no effect on CaV3.2 mRNA levels in the carotid body. Studies on human embryonic kidney‐293 cells stably expressing CaV3.2 showed that CIH increases CaV3.2 calcium currents and this effect was associated with increased CaV3.2 protein expression in the plasma membrane but not in the cytosol. Either a ROS scavenger or brefeldin‐A, an inhibitor of protein trafficking, prevented the effects of CIH on CaV3.2 currents and the increased protein expression of CaV 3.2 in the plasma membrane. These findings suggest that CIH leads to an augmented calcium influx via ROS‐dependent facilitation of CaV3.2 protein trafficking to the plasma membrane.

Support or Funding Information

Supported by NIH‐PO1‐HL90554
Chronic inhibition of melanocortin 4 receptor (MC4R) attenuates hypertension‐induced by intermittent hypoxia
Authors: do Carmo Jussara M. , da Silva Alexandre A. , Moak Sydney P. , Wang Zhen , Hall John E.
Published: November 2023

Chronic intermittent hypoxia (CIH) is associated with increased sympathetic nervous system activity and blood pressure (BP). We have previously demonstrated that the central nervous system (CNS) melanocortin 4 receptor (MC4R) plays a key role in regulating sympathetic nervous system (SNS) activity and BP. However, the importance of MC4R in mediating the chronic pressor effect of CIH is still unknown. In this study we examined if activation of CNS MC4R contributes to CIH‐induced hypertension. Sprague‐Dawley rats were instrumented with intracerebroventricular (ICV) cannula in the lateral ventricle for continuous infusion of MC4R antagonist (SHU‐9119, 1 nmol/h, 0.5 ml/h) and telemetry probe for measurement of mean arterial pressure (MAP) and heart rate (HR) 24‐hrs/day. Untreated (n=6) and SHU‐9119 treated rats were exposed to CIH (7% O2, 8 hrs/day from 8:00–16:00 hours for 5 consecutive days). CIH reduced food intake (16±1 to 14±1 g) leading to a net cumulative decrease in food intake of −18 g, whereas SHU‐9119 increased food intake (15±1 to 46±3 g) and prevented CIH‐induced reduction in food intake. No changes were observed in plasma glucose levels. CIH induced hypertension was also attenuated by MC4R antagonism (average MAP increase of 10±1 vs. 6±1 mmHg for untreated and treated rats, respectively). In addition, CIH abolished the bradycardia effects of chronic MC4R infusion (−24±5 – SHU‐9119 alone and −2±4 bpm combined SHU‐9119 plus CIH). These results show that the activation of CNS melanocortin system contributes to elevated BP during CIH.

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(NHLBI‐PO1HL51971, NIGMS‐ P20GM104357 and U54GM115428)
Sympathetic Nerve Activity during Development of Deoxycorticosterone Acetate‐Salt Hypertension in Rat
Authors: Yoshimoto Misa , Shiwa Yuki , Setoguchi Shiori , Kawamura Shiho , Miki Kenju
Published: November 2023

Excess salt intake is associated with hypertension. There is increasing evidence that many forms of hypertension are initiated and maintained by elevated sympathetic nerve activity (SNA). Thus, SNA may play a critical role in development of hypertension induced by a high salt diet. However, the changes in SNA during development of hypertension induced by high salt intake remain unclear. The aim of this study was to examine a role for SNA in causing hypertension in a rat model of deoxycorticosterone acetate (DOCA)‐salt hypertension. Male Wistar rats were chronically instrumented with bipolar electrodes for measurement of renal SNA (RSNA) and lumbar SNA (LSNA), and a telemeter was used for measurement of arterial pressure (AP). The time course of changes in AP, heart rate, RSNA, and LSNA were measured continuously and simultaneously for 4 days prior and 17 days after induction of DOCA (100 mg/rat) with a high sodium diet (4% NaCl) in intact and uni‐nephrectomized (uni‐NX) rats. The onset of DOCA‐salt loading caused a progressive increase in AP over 17 days in both intact and uni‐NX rats; this effect was greatest in uni‐NX rats. MAP increased progressively due to DOCA‐salt loading in both intact and uni‐NX rats, while RSNA decreased slightly and LSNA remained unchanged. These data indicate that RSNA and LSNA are not critical for initiation and development of hypertension in DOCA‐salt rats.
Lipid Raft Residency is Required for Renal Dopamine D1 Receptor Activity
Authors: Tiu Andrew Chua , Asico Laureano D , Zheng Xiaoxu , Jose Pedro A , Villar Van Anthony M
Published: November 2023

Lipid rafts (LR) are plasma membrane microdomains enriched in cholesterol and sphingolipids that serve as a platform for an effective G protein‐coupled receptor (GPCR) signaling. The renal dopamine D1 receptor (D1R) partitions to the LR in human renal proximal tubule cells and interacts with the LR protein marker caveolin‐1. We tested the hypothesis that the LR is crucial for the organization and signal transduction of the renal D1R. Palmitoylation, a process that involves the formation of thioesters between palmitate and cysteine residues which then serve as membrane anchors, promotes LR partitioning of a few GPCRs. The human D1R has palmitoylation sites at two cysteine residues at positions 347 & 351. Thus, we mutated and converted the cysteine residues to alanine (347C>A & 351C>A) and evaluated their distribution and activity in human renal proximal tubule cells. Through biotinylation and sucrose gradient ultracentrifugation experiments, we found that the mutant D1R 347A and 351A still targeted to the plasma membrane but failed to localize in the LR, unlike the endogenous wild‐type D1R which partitioned to lipid and non‐lipid raft microdomains, its normal plasma membrane distribution. A di‐leucine mutant (344L>G & 345L>A), used as a control, was observed to mis‐ target the plasma membrane. The plasma membrane distribution of the mutant and endogenous wild‐type D1R was confirmed via confocal microscopy. The cAMP response to the D1R/D5R agonist fenoldopam was blunted in D1R 347A and 351A mutants, reflecting the requirement for LR residency for normal renal D1R function. We next evaluated the importance of LR in the kidneys of C57Bl/6J mice by infusing methyl‐β‐cyclodextrin (β‐MCD; a LR disruptor by depletion of LR cholesterol) subcapsularly for 7 days into the kidneys of C57Bl/6J mice; methyl‐ α‐cyclodextrin (α‐MCD) and vehicle were used as negative controls. We found that chronic β‐MCD treatment increased the systolic blood pressure (118±12 mm Hg, measured under anesthesia), similar to that observed in Drd1 knockout mice. Treatment with α‐MCD and vehicle did not change the blood pressure (98±1 mm Hg and 98±1 mm Hg, respectively) of C57Bl/6J mice. Confocal microscopic examination of the chronically treated kidneys revealed the disappearance of the LR without affecting the brush border architecture, and the redistribution of the D1R and its regulatory proteins (e.g., GRK4 and SNX19) from the brush border to the cytoplasm, indicating the importance of the LR in the proper deployment and activity of renal D1R. Our data underline the crucial role of D1R residency in LR membrane microdomains for its full activity.

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The work was funded by grants from the US National Institutes of Health, P01HL074940, P01HL068686, R01HL092196, R37HL023081, R01DK039308, and DK090918.
High salt intake modulates adiposity and insulin sensitivity via endothelin‐1
Authors: Speed Joshua S
Published: November 2023

Factors associated with sodium homeostasis, such as the renin angiotensin system and sympathetic nerve activity, contribute to lipid metabolism and overall metabolic balance. Endothelin‐1 (ET‐1), a peptide released in response to high sodium intake can promote lipolysis in cultured adipocytes via the ET‐1 type A (ETA) receptor, while activation of the ET‐1 type B (ETB) receptor has been shown to inhibit the actions of insulin on adipocytes. The goal of the current study was to determine if high salt in a take affects metabolic parameters, including body weight (BW), adiposity, plasma insulin, triglycerides, and glucose, and to determine if loss of ETB receptor function improves these markers in a rat model lacking ETB receptors. Age matched control and ETB deficient rats were fed a normal (NS) or high (HS) salt diet for 2 weeks, and euthanized every 4 hours to observe circadian patterns of plasma parameters. After two weeks of HS intake, BW was significantly lower in both genotypes compared to NS fed rats. In control rats, epididymal fat pad mass was not significantly different between NS and HS groups (1.37±0.06 vs. 1.46±0.05 g respectively; n=6); however, ETB deficient rats had significantly less epididymal fat compared to NS fed ETB deficient rats (1.56±0.06 vs. 1.18±0.06 g, NS vs. HS; n=6). No significant differences were observed in plasma glucose, triglycerides, or free fatty acids between genotype and/or diet at any time point over the 24 hour period. Interestingly, circadian variation in plasma insulin was abolished in HS fed animals of both genotypes, and was significantly lower in both NS and HS fed ETB deficient rats compared to controls. These data support the hypothesis that activation of the ET‐1 system during HS intake modulates adipose deposition and insulin sensitivity, via the ETB receptor.

Support or Funding Information

This work is funded by NHLBI grant # K99HL127178.