bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationThis 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 InformationThe 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 InformationThe 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 InformationSupported 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 InformationMedical 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 InformationSupported 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.Support or Funding Information(NHLBI‐PO1HL51971, NIGMS‐ P20GM104357 and U54GM115428)
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.Support or Funding InformationThe 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 InformationThis work is funded by NHLBI grant # K99HL127178.