bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative proteomics of collecting duct cells in bilateral ureteral obstruction‐induced nephrogenic diabetes insipidus (1137.4)
Authors: Khositseth Sookkasem , Somparn Poorichaya , Charngkaew Komgrid , Uawithya Panapat , Chen Shu‐hui , Pisitkun Trairak
Published: June 2021

The present study investigated the effects of early bilateral ureteral obstruction (BUO)‐induced nephrogenic diabetes insipidus (NDI) on rat inner medullary collecting duct (IMCD). Immunoblotting of IMCD isolated from rats after 4 hours of BUO showed a 42% decrease in total Aqp2 (p <0.05), 65.5% decrease in pS256‐AQP2 (p <0.001), 34.4 % decrease in pS261‐AQP2 (p <0.01), but no significant change in total UT‐A1 and pS486‐UT‐A1 abundances. Large‐scale identification and quantification of BUO‐affected proteins in IMCD was performedby label‐free LC‐MS/MS technique. Of 1172 proteins identified, 52 proteins were found to have significant changes in abundance in early BUO rats versus sham controls, with 40 proteins increased and 12 proteins decreased. Gene Ontology terms and pathway analysis revealed that BUO‐affected proteins were associated with focal adhesion (Flnc, Actn4, Lamb2, Lama5, Lamc1, Col4a2, Ctnnb1), cytoskeleton (Krt7, Actn4, Myh10, Tubb4a, Cryab, Ctnnb1), and generation of precursor metabolites and energy (Atp6v1e1, Atp5h, Atp5a1, Atp5b, Aco2, Mdh1, Uqcrc2). Immunohistochemistry studies demonstrated aggregation of filamentous actin in IMCD of BUO rats. These results provide potential clues for understanding the mechanisms responsible for BUO‐induced NDI.

Grant Funding Source: Supprted by Thailand Research Fund
Aquaporin‐2 interactome in rat inner medullary collecting duct (1137.5)
Authors: Chou Chung‐Lin , Hageman Daniel , Knepper Mark
Published: June 2021

Vasopressin regulates membrane trafficking of the water channel aquaporin‐2 (AQP2). We ran LC‐MS/MS analysis to identify AQP2 interacting proteins in native IMCD cells. Freshly isolated rat IMCD suspensions were treated with a vasopressin analog (dDAVP) or vehicle followed by in‐cell crosslinking using a homobifunctional crosslinker BSOCOES, detergent solubilization, AQP2 immunoprecipitation, SDS‐PAGE separation, and quantitative mass spectrometry of trypsin digests of gel pieces. The peptides passing stringent spectral quality thresholds were quantified (label‐free) to identify those with high signal:noise (AQP2 antibody/preimmune IgG > 4). The overall analysis identified a total of 89 replicable AQP2‐interacting proteins distributed throughout several cellular compartments (based on Gene Ontology analysis). 76 were found in dDAVP‐treated samples and 66 in vehicle‐treated samples (overlap 53). 16 have previously been found to undergo changes in phosphorylation in response to vasopressin including proteins involved in membrane trafficking (Lrba, Ndrg1, Sec61b), signaling (β‐catenin, inositol 3‐phosphate synthase, wolframin), cell‐cell adhesion (α‐catenin, β‐catenin, periplakin) and transport (Slc14a2, Atp1a1). Additionally, four distinct Rab proteins were identified (5C,7A,14,17). These findings provide extensive data for modeling vasopressin‐regulated AQP2 trafficking.

Grant Funding Source: Supported by NHLBI
Deciphering translational and transcriptional impairment in Roberts syndrome (1138.6)
Authors: Xu Baoshan , Bose Tania , Zueckert‐Gaudenz Karin , Gogol Madelaine , Gerton Jennifer
Published: June 2021

Roberts Syndrome (RBS) is an autosomal recessive disease characterized by prenatal growth retardation caused by mutations in the cohesin acetyltransferase ESCO2 gene. Previously we revealed that mutations in human ESCO2 impaired ribosomal RNA production and protein synthesis. We recently observed mTOR (mammalian target of rapamycin) signaling was strongly downregulated in RBS cells. Stimulation of the mTOR pathway with L‐leucine helped to partially rescue both protein synthesis and cell proliferation. To further address the transcriptional and translational deficits in RBS and their response to L‐leucine, we are performing ribosome profiling to monitor translation in vivo by deep sequencing of ribosome‐protected mRNA fragments. Western blot analysis shows that a number of 40S and 60S ribosome subunits are reduced in the RBS cells. Remarkably, global 5’TOP (5′terminal oligopyrimidine tract) mRNA translation, including 79 ribosome proteins and a panel of eukaryotic translation initiation factors, and most PRTE (Pyrimidine Rich Translational Element) genes show improved translation in RBS cells treated with the mTOR stimulant L‐leucine. The different expression of hundreds of genes is rescued with L‐leucine, suggesting the differential gene expression could be translation‐driven. In conclusion, our results uncover transcriptional and translational mechanisms that contribute to the etiology of RBS.

Grant Funding Source: Supported by Stowers Institute and Cornelia de Lange Syndrome Foundation Research Grants
Effects of di‐n‐butyl phthalate on neuron development (1139.1)
Authors: Feng Josh , Acey Roger
Published: June 2021

Plasticizers such as di‐n‐butyl phthalate (DBP) and bisphenol A (BPA) are well‐documented, ubiquitously distributed environmental contaminants. The effect of these compounds on human health has been controversial but research has strongly suggested they function as estrogen mimics or “endocrine disruptors”. We have developed a human umbilical cord mesenchymal stem cell (HUC‐SC) system as a model to predict relative toxicities of phthalate esters. The default pathway of these differentiating cells is the formation of neural tissue. Differentiating cells are exposed to the potential toxin for increasing periods of time, collected, and homogenized. Cell lysates are then analyzed by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) for changes in protein expression. Our first series of experiments looked at the effect of 10 nM DBP on undifferentiated HUC‐SCs. At this concentration of phthalate, there was no effect on cell proliferation, membrane integrity, or induction of apoptosis. Next, the HUC‐SCs were induced to differentiate in the presence or absence of 10 nM DBP. Cells were collected after 20, 24, 28, 32, and 48 hours of culture and analyzed by 2D‐PAGE. Comparison of control against phthalate‐treated cell extracts showed dramatic differences in protein expression. Preliminary analysis by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectroscopy (MALDI‐TOF MS) reveals two significant differentially‐expressed proteins: enolase, which suggests that DBP may be altering the neuron differentiation pathway, and heat shock protein β1, indicating that the cells are under stress. Future experiments will be to identify other differentially expressed proteins and determine their potential as biomarkers for evaluating the extent of DBP exposure.

Grant Funding Source: Supported by the Doris A. Howell‐CSUPERB Research Scholars Award Program
Endemic and highly prevalent amyloid A amyloidosis in endangered island foxes (Urocyon littoralis) (1139.16)
Authors: Gaffney Patricia , Clifford Deana , Imai Denise , O'Brien Timothy , Nilsson Peter , Ghassemian Majid , Munson Linda , Sigurdson Christina
Published: June 2021

Critically endangered island foxes develop a systemic amyloidosis with an extraordinarily high prevalence of 32% (mean), and exceeding 40% in some subspecies. Amyloidosis has been recognized in foxes for 15 years. The organs most affected by amyloid are kidney, spleen, vessels, heart, adrenal gland, tongue, oropharynx, and skin. There was an increased risk of amyloidosis found in adults, the San Clemente island subspecies, captively housed foxes, and foxes with neoplasia. In amyloid‐rich tissue, the dominant insoluble protein was a 12‐kDa protein labeled by serum amyloid A (SAA) antibodies that were generated against canine SAA. Mass spectrometry on insoluble proteins from 9 amyloid‐laden samples revealed SAA as the dominant protein, which was not present in control samples. The SAA protein was composed of 111 amino acids and consisted of multiple isoforms, including isoforms unique amongst canids. SAA amyloidosis is typically associated with prolonged elevation of SAA in serum, and island foxes have a variety of chronic inflammatory diseases that may lead to elevated SAA levels. We found that conformationally sensitive amyloid binding probes known as luminescent conjugated polymers bind the island fox SAA amyloid and emit different spectra within different microanatomical locations in the kidney, suggesting there may be conformational or compositional differences in amyloid deposits.
Effect of Ang II on angiotensinogen expression levels in hypertensive rat astrocytes (1140.1)
Authors: Vudhya Gowrisankar Yugandhar , Clark Michelle
Published: June 2021

Objective: To investigate the effects of Angiotensin II (Ang II) on Angiotensinogen (AGT) mRNA and protein levels in spontaneous hypertensive rats (SHR) astrocytes. Background: AGT is the precursor molecule for the synthesis of Ang II produced by the renin angiotensin system (RAS). Astrocytes are the major source of AGT in the brain, and regulation of brain AGT levels alters blood pressure and electrolyte homeostasis. In this study, SHR were used as a genetic hypertension model and the results were compared to normotensive Wistar rats. Methods: Astrocytes isolated from neonatal rat’s cerebellum (CB) and brainstem (BS) regions were treated with 100nM Ang II, and the time‐dependent effect of Ang II on AGT protein and mRNA levels were measured using Western blotting and quantitative polymerase chain reaction techniques, respectively. Results: Ang II significantly increased AGT protein levels but, decreased AGT mRNA levels over most time points examined in BS and CB Wistar and SHR astrocytes. There was no significant difference between Ang II’s ability to affect AGT protein and mRNA levels in SHR as compared to Wistar astrocytes. Conclusions: These findings showed that Ang II regulates it’s synthesis by inducing basal AGT protein and mRNA levels. Further, the ability of astrocytes to produce AGT may not be contributing to the hypertensive state in the SHR model.

Grant Funding Source: Supported by Nova Southeastern University Chancellor's Faculty Research & Development Grant # 335872
The role of FXR in the regulation of phase‐I drug metabolizing gene expression during mouse liver maturation (1141.1)
Authors: Peng Lai , Cui Julia , Yoo Byunggil , Tien Yun‐chen , Lu Hong , Klaassen Curtis , Zhong Xiao‐bo
Published: June 2021

The functions of most Phase‐I drug metabolizing enzymes are not mature at birth, and their expression changes during liver maturation. Farnesoid X receptor (FXR) is critical for bile acid and lipid homeostasis in the liver. However, the role of FXR in regulating Phase‐I drug metabolizing gene expression during liver maturation is not clear. Hence, we applied RNA sequencing to provide a “true quantification” of mRNA expression of Phase‐I genes in both FXR‐null and its control (C57BL/6) mouse livers during development. Liver samples of male C57BL/6 and FXR‐null mice at 6 different ages from prenatal to adult were used to define the ontogenic profiles of 185 Phase‐I genes from 12 different families. Compared to control livers, the numbers of Phase‐I genes with significant changes in mRNA expressions in FXR‐null livers were 75, 82, 78, 89, 103, and 78 at the ages of day‐2, 1, 5, 20, 25 and day60, respectively. Differential expression during development occurred in genes from all 12 Phase‐I families, including hydrolysis: carboxylesterase (Ces), paraoxonase (Pon), epoxide hydrolase (Ephx); reduction: aldoketo reductase (Akr), quinone oxidoreductase (Nqo), dihydropyrimidine dehydrogenase (Dpyd); and oxidation: alcohol dehydrogenase (Adh), aldehyde dehydrogenase (Aldh), flavin monooxygenases (Fmo), molybdenum hydroxylase (Aox and Xdh), cytochrome P450 (P450), and cytochrome P450 oxidoreductase (Por). The expression levels of some genes (like Cyp7a1and genes from the Ces family) were affected at all ages, whereas others (like genes from Nqo and Fmo families) were altered only at specific ages during liver maturation. These results indicate an important role of FXR in developmental regulation of Phase‐I genes in liver.

Grant Funding Source: Supported by the NIEHS [Grant R01ES‐019487] and NIGMS [Grant R01GM‐087376]
Silencing of ClC‐3 protects against preadipocyte apoptosis in type 2 diabetes (1142.6)
Authors: Wang Guan‐Lei , Huang Yun‐Ying , Zhang Feng , Zhu Xiao‐Nan , Guan Yong‐Yuan
Published: June 2021

OBJECTIVE: ClC‐3 chloride channel, plays an important role in the regulation of cell volume,proliferation and apoptosis. Preadipocytes of type 2 diabetes show a tendency to enhanced apoptosis. We hypothesized that there is a relation between ClC‐3 and insulin resistance, and one underlying mechanism may be associated with the modulation of free fatty acid (FFA) ‐induced preadipocyte apoptosis by ClC‐3.

METHODS: Type 2 diabetes mellitus (T2DM) models in ClC‐3 KO mice and rats were induced by fed with high‐sucrose‐high‐fat diet combined with a low dose of streptozocin. The cellular apoptosis was detected by Annexin V‐PI staining.

RESULTS: Compared with the wild‐type T2DM models, ClC‐3 knockout T2DM mice displayed an improvement in insulin resistance, a decrease in serum plasma free fatty acid (FFA) levels and preadipocyte apoptosis in epididymal fat pad, as evidenced by significant decrease in homeostatic model assessment of insulin resistance (HOMA‐IR) and flow cytometry. In T2DM rat models, we further found that ClC‐3 protein in adipose tissue is increased, and was positively correlated to HOMA‐IR, but was not the case in liver and skeletal muscle. We further found that palmitate‐induced preadipocyte apoptosis in vitro was parallel to a significant increase in the endogenous expression of ClC‐3 protein. Transfection with ClC‐3 siRNA decreased apoptosis and activation of caspase‐3 and 9, whereas increased ratio of Bcl‐2/Bax in palmitate‐treated preadipocytes. ClC‐3 affected apoptosis mainly through regulating ER stress signaling proteins such as Grp78, CHOP and ATF4.

CONCLUSION: We concluded that chloride channel ClC‐3 modulates palmitate‐induced apoptosis in preadipocytes via the ER stress pathway. ClC‐3 in adipocyte tissue may be involved in the development of type 2 diabetes.

Grant Funding Source: Supported by National Natural Science Foundation of China (No. 81370897,No. 813111115 and No. 812300
Manganese accumulates in leaves of radishes grown in manganese supplemented soils (1143.10)
Authors: Clarke‐Lambert Shellyann , Stapleton Melissa , Ruddock Karl , Skeete Dereck
Published: June 2021

Manganese (Mn) is a naturally occurring element, essential in trace amounts for living organisms, but is potentially toxic in high concentrations. Certain occupations including mining, welding and steel manufacturing can expose workers to chronically high levels of airborne Mn, leading to a clinical condition known as Manganism, which has Parkinson like symptoms. Recent studies report excess dietary Mn can impair immune and reproductive functions in birds. Previously we showed Mn is present in some commercially available fertilizers. We hypothesize plants grown in soils high in Mn or supplemented with fertilizers containing Mn will accumulate Mn in leaves and fruits. To test this we grew radishes in soils supplemented with fertilizers containing Mn, as well as in soil without added Mn. Samples (0.5 g) of each of the fertilizers as well as radish leaves were digested with HNO3 in a CEM Discovery Microwave Digester. Digested samples were analyzed for Mn levels using electrothermal vaporization with deuterium lamp background correction in a Perkin Elmer AA800 Atomic Absorption spectrophotometer with a THGA graphite furnace. We found that leaves from plants grown in soils supplemented with Mn accumulated Mn up to about 104 µg/gm tissue. The control leaves contained 25.28 µg/gm of Mn, which was significantly less than all the experimental groups. On the other hand, the soil that contained fertilizer had a significantly higher concentration of Mne than the plant tissue. The highest concentration of Mn recorded in the soil with Mn supplement fertilizer was 1709 µg/gm, while the lowest concentration was 1280 µg/gm. The study shows that plants will accumulate Mn from the soil and that use of fertilizers with high concentrations of Mn will increase the accumulations, possible creating a situation where animals and people ingesting the fruits and vegetables might be subjected to elevated Mn levels.

Grant Funding Source: Supported by 0516041071 of NYSDOE and 0622197 of the DUE Program of NSF
Alleviation of neuronal oxidative stress by thioredoxin through PDE2 inhibition (1143.13)
Authors: Klabnik Jonathan , O'Donnell James
Published: June 2021

Purpose Reactive oxygen species (ROS) are a normal byproduct of metabolism within the cell. Normally the cell is able to sequester these species by the various redox agents but in some cases, the stress can be too great for the system as ROS is prone to act in positive feedback manner. This redox imbalance then places the cell into oxidative stress where severe consequences arise, such as protein damage and even apoptosis. Recent studies suggest this can have global effects on CNS function and overall health. The working hypothesis of this study is that the use of PDE2 inhibitors can be used to upregulate redox agents and return ROS to homeostatic levels. The rationale behind this is that is has been shown thioredoxin upregulation is downstream of the mediator Protein Kinase G (PKG) leading to reduced ROS levels. One way to increase PKG levels is to promote cGMP, which can be done through inhibition of phosphodiesterases (PDE), which catalyze the degradation of cyclic nucleotides. The idea of the use of PDE2 versus other inhibitors is that reported expression levels found relative to available PKG expression compared to other cGMP‐metabolizing PDEs. Previous work done by our lab has shown that PDE2 inhibitors were able to decrease ROS activity as well as total antioxidant capacity. Presently, however, it is not known the exact mechanism of those results and this research hopes to illustrate a possible mechanism.

Results Currently, the results indicate that, indeed, PDE2 inhibition by the use of Bay 60‐7550 is involved in increasing thioredoxin response in a dose‐dependent manner. This upregulation also correlates with a decrease in overall ROS and peroxynitrite levels.

Conclusion The implications of this research can shed light on a novel way to treat patients suffering from diseases that are largely associated with ROS activity, such as Parkinson’s disease or autoimmune disorders.
Ginsenoside Rg1 protects hydrogen peroxide‐induced PC12 cell death: involvement of NMMHC IIA‐NF‐κB/p65 pathway (1143.15)
Authors: Kou Junping , Liu Qian , Guan Teng , Wang Yan , Lv Yanni , Huang Yalin , Cao Zhengyu , Yu Boyang
Published: June 2021

Neuronal oxidative stress is a primary mechanism of neuron loss and neurological dysfunction in several CNS diseases. Our previous study demonstrated that ginsenoside Rg1 (Rg1) afforded neuroprotection against oxidative injury by attenuating the over‐activation of NF‐kappaB/p65, ERK1/2 and Akt signalling pathways. To further investigate how Rg1 regulates the NF‐kappaB/p65 activation, we explored the molecular targets for Rg1 through which Rg1 exerts neuroprotective activity. Using the serial affinity chromatography, we identified the non‐muscle myosin heavy chain IIA (NMMHC IIA) as one of the Rg1 specific binding proteins in PC12 cells. To understand the Rg1‐NMMHC IIA binding complex structure, we employed computer‐aided homology modelling to illustrate how the small molecule interacts with the protein based on the structure of the NMMHC IIA modelled with 1BR2 and 1YV3 (PDB coded). The pharmacological inhibitor of NMMHC IIA, blebbistatin, and NMMHC IIA ShRNA both blocked Rg1’s neuroptection on oxidative stress‐induced NF‐kappaB/p65 activation and cell death suggesting NMMHC IIA was a potential target for Rg1 neuroprotection against neuronal oxidative stress. In addition, Rg1 normalized the oxidative stress‐induced NMMCH IIA over‐expression in PC12 cells. Considered these data together suggested that NMMHC IIA‐NF‐kappaB/p65 pathway involved in oxidative stress‐induced cell death. This novel identified pathway may potentially explain the neuroprotective mechanism of Rg1 against oxidative damage in several CNS diseases. Our results therefore may help to understand the pathophysiology of oxidative stress and the molecular mechanisms of Rg1 neuroprotection.
Discovery and characterization of GPR6 inverse agonists (1144.11)
Authors: Wood Michael , Tanaka Shigeru , Ryberg Erik , Norris Tyrrell , Hastings Richard , Bostwick James , Greasley Peter , Raddatz Rita , Sakai Norio , Cross Alan
Published: June 2021

GPR6 is a Class A G protein‐coupled receptor and a member of a highly related subset that includes GPR3 and GPR12. Although GPR6 is a constitutively‐active orphan GPCR, it has been reported that some signaling activity may be enhanced by lipids. Following transient transfection of the rat GPR6 ortholog into HEK293s cells, surface expression of GPR6 was confirmed using Fluorescence Activated Cell Sorting. A corresponding increase in intracellular cAMP was also detected, consistent with constitutive activity and Gα,s coupling. A high‐throughput, random screen of approximately 130,000 compounds was then performed. Compounds from two chemical series (benzimidamides and 2‐naphthimidamides) were identified as putative inverse agonists of GPR6. Representative compounds demonstrated concentration‐dependent inhibition of cAMP production. The inverse agonism of cellular cAMP production correlated with inhibition of constitutive GTPγS activity in membranes derived from cells expressing GPR6. Anadamide‐stimulation of rGPR6 elevated cellular cAMP levels and was also inhibited by the inverse agonists. Several exemplar compounds were further characterized and found to be selective against other members of the subclass (i.e., GPR3 and GPR12) and other fatty acid GPCRs (i.e., CB1, CB2, and GPR55). Three compounds from the larger chemical class (2‐naphthimidamides) were evaluated for the ability to reverse neurite outgrowth in a native preparation and found to block GPR6‐induced sprouting. The identification and characterization of inverse agonists of GPR6 should enable further investigation into the role of this brain specific GPCR as a potential drug target for psychiatric or neurologic conditions.
Antidepressant potential of organic cation transporter blockade: the role of norepinephrine (1144.5)
Authors: Mitchell Nathan , Horton Rebecca , Vitela Melissa , Gould Georgianna , Koek Wounter , Daws Lynette
Published: June 2021

Depression is a major health problem for which most patients are not effectively treated. This underscores a need to identify new targets for the development of antidepressants with improved efficacy. Our previous studies have shown that blockade of organic cation transporters (OCTs) and the plasma membrane monoamine transporter (PMAT) with decynium‐22 (D‐22) can produce antidepressant‐like effects, which are correlated with the ability of D‐22 to inhibit serotonin (5‐HT) clearance in brain, when the 5‐HT transporter (SERT) is pharmacologically or genetically compromised. In vitro studies show that OCT3 takes up norepinephrine (NE) more efficiently than 5‐HT, which raises the possibility that D‐22 might enhance the antidepressant‐like actions of drugs that block NE uptake via the NE transporter (NET). Using in vivo chronoamperometry, an electrochemical technique which allows second by second recording of extracellular biogenic amine concentrations, we show D‐22 increases time to clear NE from the extracellular space, and show D‐22 potentiates the effects of NET blockers venlafaxine (VEN) and desipramine (DMI) to increase clearance time of NE in the dentate gyrus and CA3 of the hippocampus. We next investigated if D‐22 can enhance the antidepressant‐like effects of VEN/DMI. Both VEN and DMI reduced immobility on the tail suspension test (TST), a test commonly used to assay antidepressant‐like activity of drugs in mice. Unlike the enhancing effect of D‐22 on increasing clearance time of NE, D‐22 did not enhance the antidepressant‐like effect of VEN/DMI in wild‐type mice. Ongoing studies are investigating the antidepressant‐like effect of D22 in SERT KO and heterozygous mice.

Grant Funding Source: Supported by MH093320 (LD & WK), MH064489 (LD), NARSAD (LD) and UL1RR025767 (R25 START UP).
Functional characterization of human α7 nicotinic acetylcholine receptor genetic variants (1147.4)
Authors: Baez‐Pagan Carlos , Holder‐Viera Mileyshmi , Santana‐Rodriguez Zuleirys , Aponte‐Santiago Nicole , Aviles‐Pagan Emir , Lasalde‐Dominicci Jose
Published: June 2021

The α7 nicotinic acetylcholine receptor (nAChR) is a ligand‐gated ion channel that is abundantly expressed throughout the central nervous system (CNS) and has been linked to Alzheimer’s disease, Parkinson’s, and Schizophrenia. In addition, the α7 nAChR has been identified in non‐neuronal cells such as macrophages, where it plays an essential role in the control of inflammation through the cholinergic anti‐inflammatory pathway; and cancer cells, where its activation promotes tumor growth, angiogenesis, metastasis, and cancer stem cell proliferation. Consistent with its protagonistic role in various diseases, the α7 nAChR is extensively studied by the pharmaceutical industry. The CHRFAM7A gene, a partial duplication of the gene encoding the α7 nAChR (CHRNA7), encodes dupα7, which is a protein that was recently demonstrated to act as negative regulator of the α7 functionality. The objective of the research presented herein was to examine whether genetic variants of the CHRNA7 gene could result in α7 receptors with significant changes in functionality, expression, and proneness to dupα7 regulation. To achieve this objective, two‐electrode voltage clamp, cell‐attached patch clamp and confocal microscopy experiments were performed using Xenopus laevis oocytes as expression system. We found that genetic variants of the CHRNA7 gene can result in significant changes in functionality and expression of the α7 nAChR in addition to significantly affecting the capacity of dupα7 to negatively regulate α7. In conclusion, our results demonstrate that genetic variants of the CHRNA7 gene can modify the functionality, expression, and regulation of the α7 nAChR, and should thus be considered when designing drugs that target this important receptor.

Grant Funding Source: Supported by NIMH grant # P30MH075673‐07 and NIMHD grant # 8U54MD007587‐03.
Matrix metalloproteinase system is involved in cardiac extracellular matrix remodeling during pregnancy and postpartum (1152.1)
Authors: Sonanez‐Organis Jose , Limon‐Miranda Sarai , Salazar‐Enriquez Diana , Muniz‐Murguia Jesus , Sanchez‐Pastor Enrique , Ramirez‐Archila Mario , Castro‐Rodriguez Elena , Moran‐Palacio Edgar , Virgen‐Ortiz Adolfo
Published: June 2021

Matrix metalloproteinases (MMPs) and their natural inhibitors, the tissue inhibitors metalloproteinases (TIMPs) are the principle enzymes that regulate collagen remodeling, and play an important role in the determination of the collagen architecture of the tissue. Collagens (Col) are the most abundant proteins of the extracellular matrix (ECM) and the major structural component of connective tissues. The contribution of MMPs to heart remodeling during pregnancy and postpartum is not well defined. In the present study, we investigated changes in fibrosis, MMPs (MMP ‐1, ‐2 and ‐9) and TIMP‐1, along with Col (Col ‐I and III) expression during late pregnancy (21 days) and postpartum (7 days) in rat heart. Col ‐I and ‐III mRNA and protein expression increased during pregnancy and decreased to basal levels after 7 days of postpartum in left and right ventricle, respectively. Moreover, TIMP‐1 protein expression also increased during pregnancy and decreased in postpartum, while MMPs expression decreased in pregnancy. Results suggest that MMP system contribute to remodeling of cardiac extracellular matrix during pregnancy and postpartum in rats.
Scleraxis over‐expression promotes the cardiac myofibroblast phenotype (1152.13)
Authors: Roche Patricia , Bagchi Rushita , Aroutiounova Nina , Czubryt Michael
Published: June 2021

Myofibroblasts are the primary mediators of cardiac remodeling, and their persistence in the myocardium is implicated in the development of fibrosis. Thus, inhibiting myofibroblast differentiation represents a novel approach for treating cardiac fibrosis. Scleraxis is a transcription factor required for development of collagen rich tissues. Interestingly, the same stimuli that induce differentiation also cause increased scleraxis levels in vitro and in vivo. Thus, we hypothesized that scleraxis promotes the myofibroblast phenotype. To test this, we over‐expressed scleraxis via adenovirus in primary cardiac fibroblasts (CFs) and measured changes in key myofibroblast markers. We observed that scleraxis over‐expression in CFs caused increased mRNA and protein levels of these markers. Since myofibroblasts are also distinguished from fibroblasts by their contractility, we used a contraction assay to determine the functional implications of increased marker levels. We found that over‐expressing scleraxis in CFs caused a significant increase in contraction. However, over‐expression of a dominant negative DNA binding‐deficient scleraxis mutant did not induce contraction, indicating a requirement for intact scleraxis. These results demonstrate that scleraxis promotes the cardiac myofibroblast phenotype, implicating scleraxis as a potential target for the intervention of cardiac fibrosis.

Grant Funding Source: Supported by Canadian Institute of Healthy Research, St. Boniface Hospital Foundation
Hyperglycemia suppresses cardiomyocyte bioenergetic reserve independent of O‐GlcNAcylation (1155.5)
Authors: Dassanayaka Sujith , Readnower Ryan , Long Bethany , Aird Allison , Salabei Joshua , Hill Bradford , Jones Steven
Published: June 2021

Hyperglycemia, a hallmark of diabetes, can augment flux through accessory glucose metabolism pathways, such as the hexosamine biosynthetic pathway, which produces the sugar donor for the β‐O‐linked‐N‐acetylglucosamine (O‐GlcNAc) post‐translational modification of proteins. We examined whether hyperglycemia‐induced elevations in O‐GlcNAcylation of cultured neonatal rat cardiomyocytes (NRCMs) affected mitochondrial function using extracellular flux analysis. Hyperglycemia (33 mmol/L) augmented O‐GlcNAcylation and suppressed mitochondrial reserve capacity (p<0.05); however, an osmotic control recapitulated the effect of hyperglycemia‐suppressed reserve capacity (p<0.05) without increasing O‐GlcNAcylation. Furthermore, neither adenoviral overexpression nor pharmacologic inhibition of either OGA or OGT altered mitochondrial reserve capacity. Specific interrogation of individual electron transport Complex II function showed that hyperglycemia reduced Complex II mediated oxygen consumption rate (OCR), which, again was recapitulated by an osmotic control. Interestingly, pharmacologic augmentation of O‐GlcNAcylation increased Complex II OCR (p<0.05). Thus, hyperglycemia may perturb mitochondrial function independent of O‐GlcNAcylation, thereby questioning the purported causal relationship between O‐GlcNAcylation and diabetic cardiomyocyte dysfunction.

Grant Funding Source: Supported by NIH and AHA
Limb specific comparison of flow‐mediated dilation in overweight, pre‐menopausal women (1158.1)
Authors: Barlow Matthew , Restaino Robert
Published: June 2021

The dilatory responses of the brachial artery as compared to the popliteal artery have been found to be equal in healthy young women and reduced in normal aging in healthy, post‐menopausal women (Parker et al. 2006). However, limb‐specific differences have not been investigated among young overweight, premenopausal women who are at a greater propensity for advanced cardiovascular aging. The hypothesis is that overweight women will exhibit limb specific differences in artery dilation with reduced dilation in the popliteal artery as compared to the brachial artery. Artery dilatory responses were assessed using flow‐mediated dilation (FMD) in 18 women (9 controls, 9 overweight) aged 24.7+1.29, non‐smokers, and sedentary. Significant difference were found between groups including % body fat, BMI, weight, waist to hip ratio, fasting blood glucose and oxLDL. A between group comparison of percent dilation revealed that the popliteal artery in overweight participants were significantly lower than control (p<0.05). We conclude that young, metabolically compromised women exhibit a limb specific difference in dilation of the upper and lower extremities as compared to healthy controls, showing possible complications of oxidative stress within the vasculature.

Grant Funding Source: HSI STEM – Engaging Students in STEM Careers Award Number: P031C110114
Peroxisomal biogenesis occurs in response to obesity and to a high lipid environment in human skeletal muscle (1159.5)
Authors: Huang Tai‐Yu , Zheng Donghai , Muller‐Borer Barbara , Collins Maria , Noland Robert , Funai Katsuhiko , Hickner Robert , Cortright Ronald
Published: June 2021

Obesity is associated with elevated levels of lipids and reductions in mitochondrial fatty acid oxidation in skeletal muscle, both of which are associated with insulin resistance. Known almost exclusively for their actions in liver, peroxisomes are critical subcellular compartments essential for chain‐shortening very long‐ and long‐chain fatty acids to acyl‐carnitines which are hypothesized to permit CPT‐1 independent entry into the mitochondria for subsequent oxidation. We hypothesize that peroxisomes proliferate in human skeletal muscle as an adaptive response to a high lipid environment to facilitate mitochondrial fatty acid oxidation. Assays for peroxisomal membrane protein 70 (PMP70) and peroxisomal biogenesis factor PEX 19 protein content, and peroxisomal‐mitochondrial co‐localization were assessed in vastus lateralis from obese and lean humans and after lipid incubation in human skeletal muscle primary myotubes. Results: 1) A peroxisomal‐mitochondrial co‐localization was identified in human skeletal muscle 2) Peroxisomal abundance (PEX 19) was elevated (P<0.05) with obesity and 3) lipid oversupply leads to induction of the peroxisomal marker PMP70 (P<0.05) in human primary myotubes. Conclusion: Peroxisomes co‐localize with mitochondria and compared to leans, their abundance in skeletal muscle from obese individuals is significantly elevated and is responsive to lipid oversupply.
The impact of aging on mitochondrial proton leaks in human skeletal muscle (1159.9)
Authors: Porter Craig , Hurren Nicholas , Sidossis Labros , Børsheim Elisabet
Published: June 2021

Proton leaks are thought to account for 20‐25% of mitochondrial oxygen consumption. It has been postulated that mitochondrial proton cycling in senescent tissue may reduce free radical production. Here, we determined mitochondrial respiratory control in skeletal muscle biopsies obtained from young and older individuals. 6 young (31±3 yrs) and 10 older (58±7 yrs) subjects participated in this study. Mitochondrial respiration was determined in permeabilized myofibers after the addition of substrates, ADP and oligomycin. Thereafter, coupling control ratios were calculated. Coupled mitochondrial respiration tended to be higher in muscle from young vs. older subjects (38.0±3.1 vs. 31.7±2.8 pmol/mg/sec, P=0.16). ADP sensitivity was similar between groups, whereas oligomycin sensitivity was significantly lower in the older vs. young group (P<0.05). Mitochondrial ADP sensitivity is preserved in skeletal muscle of older individuals. However, mitochondria of older individuals are less sensitive to the ATP synthase inhibitor oligomycin. These data suggest that inner membrane proton conductance is elevated in skeletal muscle mitochondria with aging.

Grant Funding Source: Supported by NIH (1R01 AG033761, P30 AG024832, 1 U54RR026141) and NIDRR (H133P110012) grants.
GQ‐16, a novel partial PPARγ ligand, decreases visceral adiposity and induces the expression of thermogenesis‐related genes in mice with obesity and insulin resistance induced by diet (1160.8)
Authors: Coelho Michella , Lima Caroline , Royer Carine , Pitta Ivan , Amato Angélica , Neves Francisco
Published: June 2021

Thiazolidinediones are used for the treatment of type 2 diabetes (T2DM) and their therapeutic actions are mediated via activation of PPARγ. Despite their efficacy, their use is limited by side effects such as weight gain. We have previously identified a novel PPARγ ligand with similar anti‐diabetic efficacy as rosiglitazone (RSG), but without weight gain.

Objective: To evaluate the effects of different doses of GQ‐16 on adiposity and gene expression in mice with insulin resistance and obesity induced by diet.

Methods: Mice were fed normal‐fat (NFD) or high‐fat (HFD) diet since weaning. At the age of 16 wk, they received GQ‐16 (5, 10 or 20mg/kg/d), RSG (4mg/kg/d) or vehicle by gavage. White adipose tissue (WAT) pads were excised for determination of adiposity and gene expression by qPCR.

Results (P<0.05, 4 mice/group): In HFD‐treated mice, weight gain was reduced by treatment with 5, 10 and 20 mg/kg/d of GQ‐16, but increased by RSG. GQ‐16 (10 and 20 mg/kg/d) decreased blood glucose similarly to RSG. In addition, GQ‐16 treatment (20 mg/kg/d) reduced WAT pads, whereas RSG treatment increased them. Brown fat‐selective genes were upregulated in WAT in response to GQ‐16.

Conclusions: Lower doses of GQ‐16 decreased blood glucose and visceral adiposity. This latter finding might possibly reflect increased thermogenesis, since GQ‐16 induced the expression of thermogenesis‐related genes, and reinforces the potential of GQ‐16 as a new strategy to treat obese patients with T2DM.

Grant Funding Source: CNPq
Regulation of leucyl‐tRNA synthetase and RagB expression in human skeletal muscle by essential amino acids (1161.4)
Authors: Carlin Matthew , Tanner Ruth , Agergaard Jakob , Jalili Thunder , Askew E , McClain Donald , Supiano Mark , Marcus Robin , LaStayo Paul , Drummond Micah
Published: June 2021

Essential amino acids (EAA) are a potent stimulator of mammalian target of rapamycin (mTORC1) signaling and protein synthesis. However, the molecular regulators upstream of mTORC1 signaling that are stimulated by EAA are not well described especially in human skeletal muscle. Leucyl tRNA Synthetase (LRS) and RagB have recently been shown to be important in mTORC1 activation. The purpose of this study was to determine if the LRS‐ and RagB‐mTORC1 complexes and LRS and RagB gene and protein expression are altered following acute EAA ingestion in healthy human skeletal muscle. Muscle biopsies (vastus lateralis) were obtained from 8 young adults (4M, 4F) in the fasted state (basal) and 1 and 3h after EAA (13g; 2.4g Leu) ingestion. Co‐immunoprecipates were used to determine LRS‐ and RagB‐mTORC1 protein complexes and whole muscle homogenates were analyzed for LRS and RagB gene and protein expression and mTOR phosphorylation. We found that EAA increased RagB protein abundance and mTOR phosphorylation 1h following EAA ingestion (P<0.05). However EAA ingestion did not alter LRS‐ or RagB‐mTORC1 complexes, LRS protein expression or change LRS and RagB gene expression 1 or 3h‐post EAA. We conclude that LRS‐ and RagB‐mTORC1 complexes are not altered 1 and 3h‐post EAA. However, RagB protein increased after EAA ingestion in healthy human skeletal muscle, which may be a mechanism to promote protein anabolism.

Grant Funding Source: Supported by K01 AG038556 and UL1 TR000105
PPARβ/δ is not required by PGC‐1α to enhance skeletal muscle oxidative metabolism (1164.3)
Authors: Pérez‐Schindler Joaquín , Vargas‐Fernández Elyzabeth , Santos Gesa , Svensson Kristoffer , Wahli Walter , Handschin Christoph
Published: June 2021

Impaired skeletal muscle metabolism is a hallmark of numerous, chronic metabolic diseases. Exercise training efficiently improves skeletal muscle metabolic fitness, increases mitochondrial biogenesis and exerts beneficial therapeutic effects. Aberrant expression of metabolic‐related genes in skeletal muscle is thought to contribute to the development and progression of such pathologies. The peroxisome proliferator‐activated receptor γ (PPARγ) coactivator‐1α (PGC‐1α) and the nuclear receptor PPARβ/δ have both been independently shown to increase skeletal muscle oxidative capacity and exercise performance. Therefore, current understanding is that the PGC‐1α‐PPARβ/δ axis plays a pivotal role in the regulation of skeletal muscle metabolism. However, the interplay and hierarchical regulation of these two proteins remains unclear, with limited research conducted in vivo. In the present study, we demonstrate that the disruption of the PGC‐1α‐PPARβ/δ axis in mouse skeletal muscle does not affect body composition, whole body metabolism and glucose homeostasis under basal conditions. Importantly, PPARβ/δ seems dispensable for most of the effects of PGC‐1α on VO2 and lactate metabolism during exercise as well as the enhancement of skeletal muscle oxidative metabolism. Therefore, these results indicate that PPARβ/δ is not an essential partner of PGC‐1α in the control of skeletal muscle remodeling.

Grant Funding Source: Supported by the Swiss National Science Foundation
Aging alters muscle reflex control of autonomic cardiovascular responses to rhythmic contractions in humans (1165.8)
Authors: Sidhu Simranjit , Weavil Joshua , Venturelli Massimo , Rossman Matthew , Gmelch Benjamin , Bledsoe Amber , Jessop Jacob , Richardson Russell , Amann Markus
Published: June 2021

We investigated the influence of aging on the role of group III/IV muscle afferents in determining circulatory responses to physical activity. Six old (OLD; 70 ± 2 years) and 8 young (YNG; 24 ± 2 years) males performed single‐leg knee extensor exercise (14 W / 30 W / 80% max) under control conditions and with lumbar intrathecal fentanyl impairing feedback from µ‐opioid receptor‐sensitive lower limb muscle afferents. Cardiac output (Q), leg perfusion pressure (PP), leg vascular conductance (LVC), and femoral blood flow (QL) were collected continuously. There were no hemodynamic differences between conditions at rest. Fentanyl blockade during exercise did not affect Q in OLD (P=0.2); however, secondary to significant decreases in heart rate and stroke volume, Q was attenuated by ~20% in YNG (P<0.05). Furthermore, in the face of a similar 5‐7% reduction in PP (P<0.05) during exercise with fentanyl blockade in both groups, LVC increased ~13% in OLD, but decreased ~10% in YNG (both P<0.05). The net effect of these blockade‐induced changes was an unaltered QL response to exercise in OLD, contrasting the significant 15‐17% decrease in YNG. In conclusion, healthy aging alters muscle reflex control of autonomic circulatory responses to exercise. While muscle afferent feedback remains critical in regulating PP, their contribution to central (Q) and peripheral (QL) hemodynamic responses might diminish with age.

Grant Funding Source: HL_103786; HL‐116579
The C‐terminal tail of aquaporin 1 modulates β‐catenin expression in pulmonary arterial smooth muscle cells (1175.2)
Authors: Yun Xin , Jiang Haiyang , Lai Ning , Shimoda Larissa
Published: June 2021

PASMC proliferation and migration are important contributors to the vascular remodeling that occurs during hypoxic pulmonary hypertension. Hypoxia increases the expression of AQP1, a member of the water channel family of proteins, in rat PASMCs and we previously showed that AQP1 controls proliferation and migration in PASMCs via a mechanism that includes the C‐terminal tail but is independent of water transport. β‐catenin, part of the Wnt signaling pathway, activates the transcription of crucial target genes controlling proliferation. In melanoma and endothelial cells, Lin‐7, a cell junction protein, interacts with AQP1 to regulate β‐catenin levels. Thus, we explored whether β‐catenin, Lin‐7 and AQP1 interact in PASMCs and, if so, whether the C‐terminal tail of AQP1 was required. Rat PASMCs were harvested, plated onto culture dishes and infected with adenoviral constructs expressing green fluorescent protein (AdGFP), the control for infection, wild‐type AQP1 (AdAQP1) or AQP1 lacking the cytoplasmic C‐terminal tail (AdAQP1CT). Compared to AdGFP cells, infection with AdAQP1, but not AdAQP1CT, increased PASMC migration and proliferation and β‐catenin protein expression. Interestingly, immunoblot analysis revealed that Lin‐7 was not expressed in rat PASMCs. Our results suggest that increased AQP1 levels upregulate β‐catenin protein levels via a mechanism requiring the AQP1 C‐terminal tail but independent of Lin‐7.

Grant Funding Source: AHA0755479
Mitochondrial bioenergetics and permeability response in rat pulmonary microvascular endothelial cells in culture (1176.1)
Authors: Merker Marilyn , Townsley Mary , Bongard Robert
Published: June 2021

Hyperoxia causes lung endothelial injury and depresses mitochondrial complex I (CI) activity. In isolated rat lung perfused with a physiological saline solution (5.5 mM glucose), CI inhibitors decrease lung tissue ATP and increase endothelial permeability (measured as Kf), effects that are overcome using a quinone (CoQ1) (Free Radic Biol Med. doi: 10.1016/j.freeradbiomed.2013.07.040). To unmask the mitochondrial contribution to pulmonary endothelial cell culture permeability responses, PMVEC were treated with the CI inhibitor rotenone (0.5 µM) in 0.2 mM glucose without or with CoQ1 (10 µM). In PMVEC monolayers, rotenone decreased ATP from 18.4 ± 1.6 (mean ± SE) to 4.6 ± 0.8 nmol/mg protein, decreased O2 consumption from 2.6 ± 0.1 to 0.3 ± 0.1 nmol/min/mg protein and increased permeability to FITC‐dextran from 8.3 ± 0.7 to 14.3 ± 0.4 nl/min/cm2. When CoQ1 was present with rotenone, ATP (17.4 ± 1.4 nmol/mg protein), O2 consumption (1.6 ± 0.2 nmol/min/mg protein) and permeability (10.1 ± 1.2 nl /min/cm2) were maintained. CoQ1 also prevented rotenone‐induced mitochondrial depolarization. Protective effects of CoQ1 on ATP, O2 consumption and mitochondrial membrane potential were blocked by inhibitors of the quinone mediated cytosol‐mitochondria electron shuttle. We conclude that, as in the lung, mitochondrial bioenergetics can be a determinant of the PMVEC permeability response.

Grant Funding Source: Supported by Dept of Veterans Affairs
Whole body plethysmography measurement of respiratory function of mice in vivo (1178.9)
Authors: Quindry John , Ballmann Christopher , Selsby Joshua
Published: June 2021

Applied physiology research commonly incorporates mouse models, but in vivo physiologic measures in conscious rodents can be difficult to collect. Since recommendations for standardized whole body plethysmography (WBP) use do not currently exist, the current study employed WBP to quantify pulmonary function in conscious mice. Two month old Male C57BL6 and MDX mice (n=8/group) were housed on a reverse light cycle and examined on consecutive days using a calibrated WBP (Fine Pointe unrestrained WBP, Buxco Electronics). Multiple measurements such as time of day (8 am, 12 pm, 3 pm, 6:30 pm), environment lighting influence, and day‐to‐day reproducibility as well as chamber habituation time required for stable data collection plateau were recorded. Fine Pointe software was used to determine respiratory rate, tidal volume, minute ventilation, peak inspiratory flow, peak expiratory flow, inspiratory time, expiratory time, and relaxation time between breaths. Statistical observance of respiratory chamber habituation occurred between 5‐10 mins from the start of collection for all variables measured. Reverse light cycle housed mice exhibited significant time of day pulmonary function differences, performing more forceful and less frequent breaths during the middle of the day (12 pm & 3 pm). Significant differences were observed for WBP performed in lighted vs. darkened conditions, with the latter eliciting more forceful breathing. Day‐to‐day reproducibility was significant (p<0.05) with a range of from r2=0.58 to r2=0.62 for measures collected. Findings indicate good reproducibility of WBP is possible provided investigators collect data after at least a 10 minute habituation period and standardize time of day and room lighting used for data collection. This work supported by the Duchenne Alliance and its member foundations.

Grant Funding Source: Duchenne Alliance and member foundations