Ion channel conductance can be influenced by electrostatic effects originating from fixed "surface" charges that are remote from the selectivity filter. To explore whether surface charges contribute to the conductance properties of Kir2.1 channels, unitary conductance was measured in cell-attached recordings of Chinese hamster ovary (CHO) cells transfected with Kir2.1 channels over a range of K + activities (4.6-293.5 mM) using single-channel measurements as well as nonstationary fluctuation analysis for low K + activities. K + ion concentrations were shown to equilibrate across the cell membrane in our studies using the voltage-sensitive dye DiBAC 4 (5). The dependence of γ on the K + activity (a K ) was fit well by a modified Langmuir binding isotherm, with a nonzero intercept as a K approaches 0 mM, suggesting electrostatic surface charge effects. Following the addition of 100 mM N-methyl-D-glucamine (NMG + ), a nonpermeant, nonblocking cation or following pretreatment with 50 mM trimethyloxonium (TMO), a carboxylic acid esterifying agent, the γ-a K relationship did not show nonzero intercepts, suggesting the presence of surface charges formed by glutamate or aspartate residues. Consistent with surface charges in Kir2.1 channels, the rates of current decay induced by Ba 2+ block were slowed with the addition of NMG or TMO. Using a molecular model of Kir2.1 channels, three candidate negatively charged residues were identified near the extracellular mouth of the pore and mutated to cysteine (E125C, D152C, and E153C). E153C channels, but not E125C or D152C channels, showed hyperbolic γ-a K relationships going through the origin. Moreover, the addition of MTSES to restore the negative charges in E53C channels reestablished wild-type conductance properties. Our results demonstrate that E153 contributes to the conductance properties of Kir2.1 channels by acting as a surface charge.
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Belinda K. Cornes;
Jennifer A. Brody;
Naghmeh Nikpoor;
Alanna C. Morrison;
Huan Chu;
Byung Soo Ahn;
Shuai Wang;
Marco Dauriz;
Joshua I. Barzilay;
Josee Dupuis;
Jose C. Florez;
Josef Coresh;
Richard A. Gibbs;
W.H. Linda Kao;
Ching-Ti Liu;
Barbara McKnight;
Donna Muzny;
James S. Pankow;
Jeffrey G. Reid;
Charles C. White;
Andrew D. Johnson;
Tien Y. Wong;
Bruce M. Psaty;
Eric Boerwinkle;
Jerome I. Rotter;
David S. Siscovick;
Robert Sladek;
James B. Meigs
Background-Common variation at the 11p11.2 locus, encompassing MADD, ACP2, NR1H3, MYBPC3, and SPI1, has been associated in genome-wide association studies with fasting glucose and insulin (FI). In the Cohorts for Heart and Aging Research in Genomic Epidemiology Targeted Sequencing Study, we sequenced 5 gene regions at 11p11.2 to identify rare, potentially functional variants influencing fasting glucose or FI levels. Methods and Results-Sequencing (mean depth, 38×) across 16.1 kb in 3566 individuals without diabetes mellitus identified 653 variants, 79.9% of which were rare (minor allele frequency <1%) and novel. We analyzed rare variants in 5 gene regions with FI or fasting glucose using the sequence kernel association test. At NR1H3, 53 rare variants were jointly associated with FI (P=2.73×10-3); of these, 7 were predicted to have regulatory function and showed association with FI (P=1.28×10-3). Conditioning on 2 previously associated variants at MADD (rs7944584, rs10838687) did not attenuate this association, suggesting that there are >2 independent signals at 11p11.2. One predicted regulatory variant, chr11:47227430 (hg18; minor allele frequency=0.00068), contributed 20.6% to the overall sequence kernel association test score at NR1H3, lies in intron 2 of NR1H3, and is a predicted binding site for forkhead box A1 (FOXA1), a transcription factor associated with insulin regulation. In human HepG2 hepatoma cells, the rare chr11:47227430 A allele disrupted FOXA1 binding and reduced FOXA1-dependent transcriptional activity. Conclusions-Sequencing at 11p11.2-NR1H3 identified rare variation associated with FI. One variant, chr11:47227430, seems to be functional, with the rare A allele reducing transcription factor FOXA1 binding and FOXA1- dependenttranscriptional activity.
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Yevgeniya E. Koshman;
Miensheng Chu;
Taehoon Kim;
Olivia Kalmanson;
Mariam Farjah;
Mohit Kumar;
William Lewis;
David L. Geenen;
Pieter de Tombe;
Paul H. Goldspink;
R. John Solaro;
Allen M. Samarel
Up-regulation and activation of PYK2, a member of the FAK family of protein tyrosine kinases, is involved in the pathogenesis of left ventricular (LV) remodeling and heart failure (HF). PYK2 activation can be prevented by CRNK, the C-terminal domain of PYK2. We previously demonstrated that adenoviral-mediated CRNK gene transfer improved survival and LV function, and slowed LV remodeling in a rat model of coronary artery ligation-induced HF. We now interrogate whether cardiomyocyte-specific, transgenic CRNK expression prevents LV remodeling and HF in a mouse model of dilated cardiomyopathy (DCM) caused by constitutively active Protein Kinase Cε (caPKCε). Transgenic (TG; FVB/N background) mice were engineered to express rat CRNK under control of the α-myosin heavy chain promoter, and crossed with FVB/N mice with cardiomyocyte-specific expression of caPKCε to create double TG mice. LV structure, function, and gene expression were evaluated in all 4 groups (nonTG FVB/N; caPKCε(+/-); CRNK(+/-); and caPKCε×CRNK (PXC) double TG mice) at 1, 3, 6, 9 and 12mo of age. CRNK expression followed a Mendelian distribution, and CRNK mice developed and survived normally through 12mo. Cardiac structure, function and selected gene expression of CRNK mice were similar to nonTG littermates. CRNK had no effect on caPKCε expression and vice versa. PYK2 was up-regulated ~6-fold in caPKCε mice, who developed a non-hypertrophic, progressive DCM with reduced systolic (Contractility Index=151±5 vs. 90±4s-1) and diastolic (Tau=7.5±0.5 vs. 14.7±1.3ms) function, and LV dilatation (LV Remodeling Index (LVRI)=4.2±0.1 vs. 6.0±0.3 for FVB/N vs. caPKCε mice, respectively; P<0.05 for each at 12mo). In double TG PXC mice, CRNK expression significantly prolonged survival, improved contractile function (Contractile Index=115±8s-1; Tau=9.5±1.0ms), and reduced LV remodeling (LVRI=4.9±0.1). Cardiomyocyte-specific expression of CRNK improves contractile function and slows LV remodeling in a mouse model of DCM.
Background: Neuro-immune interactions play a significant role in regulating the severity of inflammation. Our previous work demonstrated that neuropeptide Y (NPY) is upregulated in the enteric nervous system during murine colitis and that NPY knockout mice exhibit reduced inflammation. Here, we investigated if NPY expression during inflammation is induced by tumor necrosis factor (TNF), the main proinflammatory cytokine. Methods: Using primary enteric neurons and colon explant cultures from wild type and NPY knockout (NPY-/-) mice, we determined if NPY knockdown modulates TNF release and epithelial permeability. Further, we assessed if NPY expression is inducible by TNF in enteric neuronal cells and mouse model of experimental colitis, using the TNF inhibitors-etanercept (blocks transmembrane and soluble TNF) and XPro1595 (blocks soluble TNF only). Results: We found that enteric neurons express TNF receptors (TNFR1 and R2). Primary enteric neurons from NPY-/- mice produced less TNF compared with wild type. Further, TNF activated NPY promoter in enteric neurons through phospho-c-Jun. NPY-/- mice had decreased intestinal permeability. In vitro, NPY increased epithelial permeability through phosphatidyl inositol-3-kinase (PI3-K)-induced pore-forming claudin-2. TNF inhibitors attenuated NPY expression in vitro and in vivo. TNF inhibitor-treated colitic mice exhibited reduced NPY expression and inflammation, reduced oxidative stress, enhanced neuronal survival, and improved colonic motility. XPro1595 had more protective effects on neuronal survival and motility compared with etanercept. Conclusions: We demonstrate a novel TNF-NPY cross talk that modulates inflammation, barrier functions, and colonic motility during inflammation. It is also suggested that selective blocking of soluble TNF may be a better therapeutic option than using anti-TNF antibodies.
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Sarah T. Plummer;
Christoph P. Hornik;
Hamilton Baker;
Gregory A. Fleming;
Susan Foerster;
Matthew Ferguson;
Andrew C. Glatz;
Russel Hirsch;
Jeffrey P. Jacobs;
Kyong-Jin Lee;
Alan B. Lewis;
Jennifer S. Li;
Mary Martin;
Diego Porras;
Wolfgang A. K. Radtke;
John F. Rhodes;
Julie A. Vincent;
Jeffrey D. Zampi;
Kevin D. Hill
Objectives: Aortic arch reconstruction in children with single ventricle lesions may predispose to circulatory inefficiency and maladaptive physiology leading to increased myocardial workload. We sought to describe neoaortic anatomy and physiology, risk factors for abnormalities, and impact on right ventricular function in patients with single right ventricle lesions after arch reconstruction. Methods: Prestage II aortic angiograms from the Pediatric Heart Network Single Ventricle Reconstruction trial were analyzed to define arch geometry (Romanesque [normal], crenel [elongated] , or gothic [angular]), indexed neoaortic dimensions, and distensibility. Comparisons were made with 50 single-ventricle controls without prior arch reconstruction. Factors associated with ascending neoaortic dilation, reduced distensibility, and decreased ventricular function on the 14-month echocardiogram were evaluated using univariate and multivariable logistic regression. Results: Interpretable angiograms were available for 326 of 389 subjects (84%). Compared with controls, study subjects more often demonstrated abnormal arch geometry (67% vs 22%, P < .01) and had increased ascending neoaortic dilation (Z score 3.8 ± 2.2 vs 2.6 ± 2.0, P < .01) and reduced distensibility index (2.2 ± 1.9 vs 8.0 ± 3.8, P < .01). Adjusted odds of neoaortic dilation were increased in subjects with gothic arch geometry (odds ratio [OR], 3.2 vs crenel geometry, P < .01) and a right ventricle-pulmonary artery shunt (OR, 3.4 vs Blalock–Taussig shunt, P < .01) but were decreased in subjects with aortic atresia (OR, 0.7 vs stenosis, P < .01) and those with recoarctation (OR, 0.3 vs no recoarctation, P = .04). No demographic, anatomic, or surgical factors predicted reduced distensibility. Neither dilation nor distensibility predicted reduced right ventricular function. Conclusions: After Norwood surgery, the reconstructed neoaorta demonstrates abnormal anatomy and physiology. Further study is needed to evaluate the longer-term impact of these features.
Background-Tumor necrosis factor (TNF) levels are associated with risk for heart failure (HF). The soluble TNF type 1 (sTNF-R1) and type 2 (sTNF-R2) receptors are elevated in patients with manifest HF, but whether they are associated with risk for incident HF is unclear. Methods and Results-Using Cox proportional hazard models, we examined the association between baseline levels of sTNF-R1 and sTNF-R2 with incident HF risk among 1285 participants of the Health, Aging, and Body Composition Study (age, 74.0±2.9 years; 51.4% women; 41.1% black). At baseline, median (interquartile range) of TNF, sTNF-R1, and sTNF-R2 levels was 3.14 (2.42-4.06), 1.46 (1.25-1.76), and 3.43 (2.95-4.02) ng/mL, respectively. During a median follow-up of 11.4 (6.9-11.7) years, 233 (18.1%) participants developed HF. In models controlling for other HF risk factors, TNF (hazard ratio [HR], 1.28; 95% confidence interval [CI], 1.02-1.61 per log2 increase) and sTNF-R1 (HR, 1.68; 95% CI, 1.15-2.46 per log2 increase), but not sTNF-R2 (HR, 1.15; 95% CI, 0.80-1.63 per log2 increase), were associated with a higher risk for HF. These associations were consistent across whites and blacks (TNF, sTNF-R1, sTNF-R2; interaction P=0.531, 0.091, and 0.795, respectively) and in both sexes (TNF, sTNF-R1, sTNF-R2; interaction P=0.491, 0.672, and 0.999, respectively). TNF-R1 was associated with a higher risk for HF with preserved versus reduced ejection fraction (HR, 1.81; 95% CI, 1.03-3.18; P=0.038 for preserved versus HR, 0.90; 95% CI, 0.56-1.44; P=0.667 for reduced ejection fraction; interaction P=0.05). Conclusions-In older adults, elevated levels of sTNF-R1 are associated with increased risk for incident HF. However, addition of TNF-R1 to the previously validated Health ABC HF risk model did not demonstrate material improvement in net discrimination or reclassification.
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Corinne E. Camalier;
Ming Yi;
Li-Rong Yu;
Brian L. Hood;
Kelly A. Conrads;
Young Jae Lee;
Yiming Lin;
Laura M Garneys;
Gary Francis Bouloux;
Matthew R. Young;
Timothy D. Veenstra;
Robert M. Stephens;
Nancy H. Colburn;
Thomas P. Conrads;
George R Beck Jr
Recent studies have suggested that changes in serum phosphate levels influence pathological states associated with aging such as cancer, bone metabolism, and cardiovascular function, even in individuals with normal renal function. The causes are only beginning to be elucidated but are likely a combination of endocrine, paracrine, autocrine, and cell autonomous effects. We have used an integrated quantitative biology approach, combining transcriptomics and proteomics to define a multi-phase, extracellular phosphate-induced, signaling network in pre-osteoblasts as well as primary human and mouse mesenchymal stromal cells. We identified a rapid mitogenic response stimulated by elevated phosphate that results in the induction of immediate early genes including c-fos. The mechanism of activation requires FGF receptor signaling followed by stimulation of N-Ras and activation of AP-1 and serum response elements. A distinct long-term response also requires FGF receptor signaling and results in N-Ras activation and expression of genes and secretion of proteins involved in matrix regulation, calcification, and angiogenesis. The late response is synergistically enhanced by addition of FGF23 peptide. The intermediate phase results in increased oxidative phosphorylation and ATP production and is necessary for the late response providing a functional link between the phases. Collectively, the results define elevated phosphate, as a mitogen and define specific mechanisms by which phosphate stimulates proliferation and matrix regulation. Our approach provides a comprehensive understanding of the cellular response to elevated extracellular phosphate, functionally connecting temporally coordinated signaling, transcriptional, and metabolic events with changes in long-term cell behavior.
Objective: Locomotor adaptation enables safe, efficient navigation among changing environments. We investigated how healthy young (HYA) and older (HOA) adults and persons with Parkinson's disease (PD) adapt to walking on a split-belt treadmill, retain adapted gait parameters during re-adaptation, and store aftereffects to conventional treadmill walking.
Methods: Thirteen PD, fifteen HYA, and fifteen HOA walked on a split-belt treadmill for ten minutes with one leg twice as fast as the other. Participants later re-adapted to the same conditions to assess retention of the split-belt gait pattern. After re-adaptation, we assessed aftereffects of this pattern during conventional treadmill walking.
Results: Persons with PD exhibited step length asymmetry throughout many adaptation and adaptive learning conditions. Early adaptation was similar across groups, though HYA and HOA continued to adapt into late adaptation while PD did not. Despite pervasive step length asymmetry among conditions which were symmetric in HYA and HOA, persons with PD demonstrated significant step length aftereffects during conventional treadmill walking after split-belt walking.
Conclusions: Though they may exhibit a default asymmetry under various walking conditions, persons with PD can adapt and store new walking patterns. Significance: Locomotor adaptation therapy may be effective in ameliorating asymmetric gait deficits in persons with PD.
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Lihe Chen;
Jae Wook Lee;
Chung-Lin Chou;
Anil V. Nair;
Maria A. Battistone;
Teodor G. Paunescu;
Maria Merkulova;
Sylvie Breton;
Jill W. Verlander;
Susan M Wall;
Dennis Brown;
Maurice B. Burg;
Mark A. Knepper
Prior RNA sequencing (RNA-seq) studies have identified complete transcriptomes for most renal epithelial cell types. The exceptions are the cell types that make up the renal collecting duct, namely intercalated cells (ICs) and principal cells (PCs), which account for only a small fraction of the kidney mass, but play critical physiological roles in the regulation of blood pressure, extracellular fluid volume, and extracellular fluid composition. To enrich these cell types, we used FACS that employed well-established lectin cell surface markers for PCs and type B ICs, as well as a newly identified cell surface marker for type A ICs, c-Kit. Single-cell RNA-seq using the IC- and PC-enriched populations as input enabled identification of complete transcriptomes of A-ICs, B-ICs, and PCs. The data were used to create a freely accessible online gene-expression database for collecting duct cells. This database allowed identification of genes that are selectively expressed in each cell type, including cell-surface receptors, transcription factors, transporters, and secreted proteins. The analysis also identified a small fraction of hybrid cells expressing aquaporin-2 and anion exchanger 1 or pendrin transcripts. In many cases, mRNAs for receptors and their ligands were identified in different cells (e.g., Notch2 chiefly in PCs vs. Jag1 chiefly in ICs), suggesting signaling cross-talk among the three cell types. The identified patterns of gene expression among the three types of collecting duct cells provide a foundation for understanding physiological regulation and pathophysiology in the renal collecting duct.
High salt, Ang II (angiotensin II), and reactive oxygen species enhance progression of chronic kidney disease. We tested the hypothesis that a high salt intake generates specific reactive oxygen species to enhance Ang II contractions of afferent arterioles from mice with reduced renal mass (RRM). C57BL/6 mice were subjected to surgical RRM or sham operations and received 6% or 0.4% NaCl salt diet for 3 months. Ang II contractions were measured in perfused afferent arterioles and superoxide (O2-) and hydrogen peroxide (H2O2) by fluorescence microscopy. RRM enhanced the afferent arteriolar gene expression for p47phox and neutrophil oxidase (NOX) 2 and high salt intake in RRM mice enhanced gene expression for angiotensin type 1 receptors, POLDIP2 and NOX4 and reduced catalase. High salt in mice with RRM enhanced arteriolar O2- and H2O2 generation and maximal contractions to Ang II (10-6 mol/L) that were dependent on O2- because they were prevented by gene deletion of p47phox and on H2O2 because they were prevented by transgenic smooth muscle cell expression of catalase (tgCAT-SMC) and POLDIP2 gene deletion. Three months of tempol normalized arteriolar reactive oxygen species and Ang II contractions. However, arteriolar contractions to lower concentrations of Ang II (10-8 to 10-11 mol/L) were paradoxically inhibited by H2O2 and POLDIP2. In conclusion, both O2- from p47phox/NOX2 and H2O2 from NOX4/POLDIP2 enhance maximal arteriolar Ang II contractions from RRM mice during high salt, but H2O2 and NOX4/POLDIP2 reduce the sensitivity to lower concentrations of Ang II by >100-fold. Tempol prevents all of these changes in function.