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1.
Obes Surg ; 32(2): 463-471, 2022 02.
Article in English | MEDLINE | ID: mdl-34816355

ABSTRACT

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is common in patients with obesity. Liver biopsy (LB) can be routinely or selectively performed during bariatric surgery to identify patients with NASH. METHODS: Patients undergoing bariatric surgery between 2016 and 2020 at our institution were identified. Chart review identified patients undergoing concurrent LB. LB results were compared between patients undergoing routine LB and selective LB. Patient demographics and postoperative outcomes were compared between those who received LB and those who did not (non-LB). In the LB cohort, preoperative characteristics of patients with NASH were compared to those without NASH, and multivariable regression was used to identify predictors of NASH. RESULTS: Two thousand three hundred ninety-three patients were identified, of which 400 (16.7%) had liver biopsies (LB) and 1,993 (83.3%) did not (non-LB). Three hundred thirty LB were performed routinely, and 70 were selective. Compared to selective LB, routine LB identified significantly higher rates of steatosis (83.6% vs. 4.5%, p < 0.01), periportal inflammation (67.0% vs. 3.2%, p < 0.01), fibrosis (65.8% vs. 2.1%, p < 0.01), and NASH (10.9% vs. 1.5%, p < 0.01). There were no differences in postoperative complications, blood transfusions, readmissions, or reoperations between LB and non-LB. On multivariable regression, highest BMI > 40 (OR 2.85, 95% CI 1.43-5.67) and insulin-dependent diabetes (OR 4.83, 95% CI 1.70-13.69) were associated with a higher odds of NASH, while Black race was associated with lower odds (OR 0.25, 95% CI 0.09-0.65). CONCLUSIONS: Routine liver biopsies during bariatric surgery identify higher rates of advanced NAFLD compared to selective biopsies, and can be safely performed without an increased risk of postoperative complications.


Subject(s)
Bariatric Surgery , Non-alcoholic Fatty Liver Disease , Obesity, Morbid , Bariatric Surgery/methods , Biopsy , Humans , Liver/pathology , Non-alcoholic Fatty Liver Disease/complications , Obesity, Morbid/surgery , Postoperative Complications/epidemiology , Postoperative Complications/pathology
2.
Genome Instab Dis ; 2(4): 195-210, 2021.
Article in English | MEDLINE | ID: mdl-34723130

ABSTRACT

DNA-dependent protein kinase catalytic subunit DNA-PKcs/PRKDC is the largest serine/threonine protein kinase of the phosphatidyl inositol 3-kinase-like protein kinase (PIKK) family and is the most highly expressed PIKK in human cells. With its DNA-binding partner Ku70/80, DNA-PKcs is required for regulated and efficient repair of ionizing radiation-induced DNA double-strand breaks via the non-homologous end joining (NHEJ) pathway. Loss of DNA-PKcs or other NHEJ factors leads to radiation sensitivity and unrepaired DNA double-strand breaks (DSBs), as well as defects in V(D)J recombination and immune defects. In this review, we highlight the contributions of the late Dr. Carl W. Anderson to the discovery and early characterization of DNA-PK. We furthermore build upon his foundational work to provide recent insights into the structure of NHEJ synaptic complexes, an evolutionarily conserved and functionally important YRPD motif, and the role of DNA-PKcs and its phosphorylation in NHEJ. The combined results identify DNA-PKcs as a master regulator that is activated by its detection of two double-strand DNA ends for a cascade of phosphorylation events that provide specificity and efficiency in assembling the synaptic complex for NHEJ.

3.
J Gastrointest Oncol ; 12(4): 1308-1325, 2021 Aug.
Article in English | MEDLINE | ID: mdl-34532090

ABSTRACT

BACKGROUND: The burden of gastric cancer involving Hispanic patients in the United States is growing as both the population and the incidence of gastric cancer in this group increases. This burden is compounded by presentation with advanced disease and socioeconomic challenges shaping cancer care. We sought to describe the demographics, socioeconomic factors, treatment, and survival experience of Hispanic patients with gastric adenocarcinoma. METHODS: Patients with gastric adenocarcinoma diagnosed between 2004 and 2015 (n=90,737) in the National Cancer Database were retrospectively identified. Patients of Hispanic ethnicity were compared against non-Hispanic white patients. Surgical cohort was further analyzed, and 1:1 propensity score matching was used to balance covariates between Hispanic and non-Hispanic white surgical patients. Survival was compared using Kaplan-Meier method. Cox regression was used to determine prognostic factors for survival. RESULTS: Compared to non-Hispanic white patients, Hispanic patients are more likely to be younger, female, and healthier. They were more likely to be uninsured, reside in poorer neighborhoods and reside in areas with lower rates of education. Hispanic patients were more likely to live in a metropolitan area, travel shorter distances for healthcare, and receive treatment at an academic and high volume centers. Hispanic patients were more likely to have higher stage disease presentation, higher grade tumors, lymphovascular invasion, and poorly cohesive adenocarcinoma. Hispanic patients were more likely to receive surgery, but less likely to receive adjuvant therapy. In Cox regression of all patients, unmatched surgical patients, and matched surgical patients, Hispanic ethnicity was an independent prognostic factor of improved survival. CONCLUSIONS: Hispanic patients with gastric adenocarcinoma present with several unfavorable clinicopathologic and socioeconomic factors. Paradoxically, these patients demonstrate improved survival. Further study is warranted to characterize disease biology in this population.

4.
Nat Commun ; 12(1): 1409, 2021 03 03.
Article in English | MEDLINE | ID: mdl-33658490

ABSTRACT

The lipid regulation of mammalian ion channel function has emerged as a fundamental mechanism in the control of electrical signalling and transport specificity in various cell types. In this work, we combine molecular dynamics simulations, mutagenesis, and electrophysiology to provide mechanistic insights into how lipophilic molecules (ceramide-sphingolipid probe) alter gating kinetics and K+ currents of hERG1. We show that the sphingolipid probe induced a significant left shift of activation voltage, faster deactivation rates, and current blockade comparable to traditional hERG1 blockers. Microseconds-long MD simulations followed by experimental mutagenesis elucidated ceramide specific binding locations at the interface between the pore and voltage sensing domains. This region constitutes a unique crevice present in mammalian channels with a non-swapped topology. The combined experimental and simulation data provide evidence for ceramide-induced allosteric modulation of the channel by a conformational selection mechanism.


Subject(s)
Ceramides/metabolism , Ether-A-Go-Go Potassium Channels/chemistry , Ether-A-Go-Go Potassium Channels/metabolism , Ceramides/chemistry , Ceramides/pharmacology , Electrophysiology/methods , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Ether-A-Go-Go Potassium Channels/genetics , HEK293 Cells , Humans , Molecular Dynamics Simulation , Mutagenesis, Site-Directed
5.
Prog Biophys Mol Biol ; 163: 87-108, 2021 08.
Article in English | MEDLINE | ID: mdl-33035590

ABSTRACT

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a key member of the phosphatidylinositol-3 kinase-like (PIKK) family of protein kinases with critical roles in DNA-double strand break repair, transcription, metastasis, mitosis, RNA processing, and innate and adaptive immunity. The absence of DNA-PKcs from many model organisms has led to the assumption that DNA-PKcs is a vertebrate-specific PIKK. Here, we find that DNA-PKcs is widely distributed in invertebrates, fungi, plants, and protists, and that threonines 2609, 2638, and 2647 of the ABCDE cluster of phosphorylation sites are highly conserved amongst most Eukaryotes. Furthermore, we identify highly conserved amino acid sequence motifs and domains that are characteristic of DNA-PKcs relative to other PIKKs. These include residues in the Forehead domain and a novel motif we have termed YRPD, located in an α helix C-terminal to the ABCDE phosphorylation site loop. Combining sequence with biochemistry plus structural data on human DNA-PKcs unveils conserved sequence and conformational features with functional insights and implications. The defined generally progressive DNA-PKcs sequence diversification uncovers conserved functionality supported by Evolutionary Trace analysis, suggesting that for many organisms both functional sites and evolutionary pressures remain identical due to fundamental cell biology. The mining of cancer genomic data and germline mutations causing human inherited disease reveal that robust DNA-PKcs activity in tumors is detrimental to patient survival, whereas germline mutations compromising function are linked to severe immunodeficiency and neuronal degeneration. We anticipate that these collective results will enable ongoing DNA-PKcs functional analyses with biological and medical implications.


Subject(s)
DNA-Activated Protein Kinase , DNA-Binding Proteins , DNA/metabolism , DNA-Activated Protein Kinase/genetics , DNA-Activated Protein Kinase/metabolism , DNA-Binding Proteins/metabolism , Humans , Nuclear Proteins/metabolism , Phosphorylation , Phylogeny
7.
J Mol Cell Cardiol ; 85: 71-8, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25986146

ABSTRACT

In Europe, ivabradine has recently been approved to treat patients with angina who have intolerance to beta blockers and/or heart failure. Ivabradine is considered to act specifically on the sinoatrial node by inhibiting the If current (the funny current) to slow automaticity. However, in vitro studies show that ivabradine prolongs phase 3 repolarization in ventricular tissue. No episodes of Torsades de Pointes have been reported in randomized clinical studies. The objective of this study is to assess whether ivabradine blocked the hERG1 current. In the present study we discovered that ivabradine prolongs action potential and blocks the hERG current over a range of concentrations overlapping with those required to block HCN4. Ivabradine produced tonic, rather than use-dependent block. The mutation Y652A significantly suppressed pharmacologic block of hERG by ivabradine. Disruption of C-type inactivation also suppressed block of hERG1 by ivabradine. Molecular docking and molecular dynamics simulations indicate that ivabradine may access the inner cavity of the hERG1 via a lipophilic route and has a well-defined binding site in the closed state of the channel. Structural organization of the binding pockets for ivabradine is discussed. Ivabradine blocks hERG and prolongs action potential duration. Our study is potentially important because it indicates the need for active post marketing surveillance of ivabradine. Importantly, proarrhythmia of a number of other drugs has only been discovered during post marketing surveillance.


Subject(s)
Benzazepines/pharmacology , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/antagonists & inhibitors , Myocytes, Cardiac/physiology , Potassium Channel Blockers/pharmacology , Action Potentials , Animals , Benzazepines/chemistry , Binding Sites , Dose-Response Relationship, Drug , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels/chemistry , Humans , Inhibitory Concentration 50 , Ivabradine , Lipid Bilayers/chemistry , Mice , Molecular Docking Simulation , Myocytes, Cardiac/drug effects , Potassium Channel Blockers/chemistry , Protein Structure, Secondary , Protein Structure, Tertiary
8.
Biophys J ; 108(6): 1400-1413, 2015 Mar 24.
Article in English | MEDLINE | ID: mdl-25809253

ABSTRACT

Activators of hERG1 such as NS1643 are being developed for congenital/acquired long QT syndrome. Previous studies identify the neighborhood of L529 around the voltage-sensor as a putative interacting site for NS1643. With NS1643, the V1/2 of activation of L529I (-34 ± 4 mV) is similar to wild-type (WT) (-37 ± 3 mV; P > 0.05). WT and L529I showed no difference in the slope factor in the absence of NS1643 (8 ± 0 vs. 9 ± 0) but showed a difference in the presence of NS1643 (9 ± 0.3 vs. 22 ± 1; P < 0.01). Voltage-clamp-fluorimetry studies also indicated that in L529I, NS1643 reduces the voltage-sensitivity of S4 movement. To further assess mechanism of NS1643 action, mutations were made in this neighborhood. NS1643 shifts the V1/2 of activation of both K525C and K525C/L529I to hyperpolarized potentials (-131 ± 4 mV for K525C and -120 ± 21 mV for K525C/L529I). Both K525C and K525C/K529I had similar slope factors in the absence of NS1643 (18 ± 2 vs. 34 ± 5, respectively) but with NS1643, the slope factor of K525C/L529I increased from 34 ± 5 to 71 ± 10 (P < 0.01) whereas for K525C the slope factor did not change (18 ± 2 at baseline and 16 ± 2 for NS1643). At baseline, K525R had a slope factor similar to WT (9 vs. 8) but in the presence of NS1643, the slope factor of K525R was increased to 24 ± 4 vs. 9 ± 0 mV for WT (P < 0.01). Molecular modeling indicates that L529I induces a kink in the S4 voltage-sensor helix, altering a salt-bridge involving K525. Moreover, docking studies indicate that NS1643 binds to the kinked structure induced by the mutation with a higher affinity. Combining biophysical, computational, and electrophysiological evidence, a mechanistic principle governing the action of some activators of hERG1 channels is proposed.


Subject(s)
Cresols/metabolism , Cresols/pharmacology , Ether-A-Go-Go Potassium Channels/metabolism , Phenylurea Compounds/metabolism , Phenylurea Compounds/pharmacology , Animals , Binding Sites , Cell Line , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels/genetics , Fluorometry , Humans , Membrane Potentials/physiology , Molecular Docking Simulation , Molecular Dynamics Simulation , Mutation , Oocytes , Patch-Clamp Techniques , Protein Structure, Secondary , Transfection , Xenopus laevis
9.
Circ Arrhythm Electrophysiol ; 8(2): 420-8, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25648353

ABSTRACT

BACKGROUND: N629D KCNH2 is a human missense long-QT2 mutation. Previously, we reported that the N629D/N629D mutation embryos disrupted cardiac looping, right ventricle development, and ablated IKr activity at E9.5. The present study evaluates the role of KCNH2 in vasculogenesis. METHODS AND RESULTS: N629D/N629D yolk sac vessels and aorta consist of sinusoids without normal arborization. Isolated E9.5 +/+ first branchial arches showed normal outgrowth of mouse ERG-positive/α-smooth muscle actin coimmunolocalized cells; however, outgrowth was grossly reduced in N629D/N629D. N629D/N629D aortas showed fewer α-smooth muscle actin positive cells that were not coimmunolocalized with mouse ERG cells. Transforming growth factor-ß treatment of isolated N629D/N629D embryoid bodies partially rescued this phenotype. Cultured N629D/N629D embryos recapitulate the same cardiovascular phenotypes as seen in vivo. Transforming growth factor-ß treatment significantly rescued these embryonic phenotypes. Both in vivo and in vitro, dofetilide treatment, over a narrow window of time, entirely recapitulated the N629D/N629D fetal phenotypes. Exogenous transforming growth factor-ß treatment also rescued the dofetilide-induced phenotype toward normal. CONCLUSIONS: Loss of function of KCNH2 mutations results in defects in cardiogenesis and vasculogenesis. Because many medications inadvertently block the KCNH2 potassium current, these novel findings seem to have clinical relevance.


Subject(s)
Abnormalities, Drug-Induced/prevention & control , Embryonic Stem Cells/drug effects , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Ether-A-Go-Go Potassium Channels/genetics , Fetal Death , Heart Defects, Congenital/prevention & control , Mutation, Missense , Neovascularization, Physiologic/drug effects , Phenethylamines/toxicity , Potassium Channel Blockers/toxicity , Sulfonamides/toxicity , Transforming Growth Factor beta/pharmacology , Vascular Malformations/prevention & control , Abnormalities, Drug-Induced/embryology , Abnormalities, Drug-Induced/genetics , Abnormalities, Drug-Induced/metabolism , Animals , Cells, Cultured , ERG1 Potassium Channel , Embryo Culture Techniques , Embryonic Stem Cells/metabolism , Ether-A-Go-Go Potassium Channels/metabolism , Gene Expression Regulation, Developmental , Genotype , Heart Defects, Congenital/chemically induced , Heart Defects, Congenital/embryology , Heart Defects, Congenital/genetics , Heart Defects, Congenital/metabolism , Humans , Mice, 129 Strain , Mice, Transgenic , Morphogenesis/drug effects , Phenotype , Signal Transduction , Vascular Malformations/chemically induced , Vascular Malformations/embryology , Vascular Malformations/genetics , Vascular Malformations/metabolism
10.
Pediatr Dev Pathol ; 18(1): 80-3, 2015.
Article in English | MEDLINE | ID: mdl-25437309

ABSTRACT

We present the case of a 14-year-old female who experienced several episodes of reversible altered mental status triggered by hypoglycemia. Following endocrine investigation, she was diagnosed with insulinoma. Insulinoma, a rare, differentiated, and functioning neuroendocrine tumor of the pancreas overproduces insulin, thus leading to hypoglycemic episodes. Conventional imaging failed to detect the lesion; therefore, arterial calcium stimulation with venous sampling (ASVS) was used for preoperative localization. The patient recovered without complications after surgical enucleation of the tumor. The ASVS is a useful method for localizing insulinomas when conventional imaging techniques fail, and can help reduce morbidities associated with surgical excision.


Subject(s)
Arteries/metabolism , Calcium/blood , Calcium/metabolism , Hypoglycemia/diagnosis , Hypoglycemia/metabolism , Insulinoma/diagnosis , Veins/metabolism , Adolescent , Blood Glucose/analysis , Cell Proliferation , Female , Humans , Hypoglycemia/blood , Hypoglycemia/therapy , Insulinoma/blood , Insulinoma/therapy , Neuroendocrine Tumors/diagnosis
11.
Exp Physiol ; 98(2): 462-72, 2013 Feb.
Article in English | MEDLINE | ID: mdl-22848083

ABSTRACT

Heart failure is associated with a low-grade and chronic cardiac inflammation that impairs function; however, the mechanisms by which this sterile inflammation occurs in structural heart disease remain poorly defined. Cardiac-specific heterozygous overexpression of the calcineurin transgene (CNTg) in mice results in cardiac hypertrophy, inflammation, apoptosis and ventricular dilatation. We hypothesized that activation of the Nlrp3 inflammasome, an intracellular danger-sensing pathway required for processing the pro-inflammatory cytokine interleukin-1ß (IL-1ß), may contribute to myocardial dysfunction and disease progression. Here we report that Nlrp3 mRNA was increased in CNTg mice compared with wild-type. Consistent with inflammasome activation, CNTg animals had increased conversion of pro-caspase-1 to cleaved and activated forms, as well as markedly increased serum IL-1ß. Blockade of IL-1ß signalling via chronic IL-1 receptor antagonist therapy reduced cardiac inflammation and myocyte pathology in CNTg mice, resulting in improved systolic performance. Furthermore, genetic ablation of Nlrp3 in CNTg mice reduced pro-inflammatory cytokine maturation and cardiac inflammation, as well as improving systolic performance. These findings indicate that activation of the Nlrp3 inflammasome in CNTg mice promotes myocardial inflammation and systolic dysfunction through the production of pro-inflammatory IL-1ß. Blockade of IL-1ß signalling with the IL-1 receptor antagonist reverses these phenotypes and offers a possible therapeutic approach in the management of heart failure.


Subject(s)
Cardiomyopathies/immunology , Carrier Proteins/metabolism , Heart Failure/immunology , Inflammasomes/metabolism , Inflammation Mediators/metabolism , Interleukin-1beta/metabolism , Myocarditis/immunology , Myocardium/immunology , Animals , Calcineurin/genetics , Calcineurin/metabolism , Cardiomyopathies/drug therapy , Cardiomyopathies/genetics , Cardiomyopathies/pathology , Cardiomyopathies/physiopathology , Carrier Proteins/genetics , Caspase 1/metabolism , Disease Models, Animal , Enzyme Activation , Enzyme Precursors , Heart Failure/genetics , Heart Failure/pathology , Heart Failure/physiopathology , Heart Failure/prevention & control , Inflammasomes/deficiency , Inflammasomes/genetics , Inflammation Mediators/blood , Interleukin 1 Receptor Antagonist Protein/pharmacology , Interleukin-1beta/blood , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Myocarditis/genetics , Myocarditis/pathology , Myocarditis/physiopathology , Myocarditis/prevention & control , Myocardium/pathology , NIH 3T3 Cells , NLR Family, Pyrin Domain-Containing 3 Protein , Receptors, Interleukin-1/antagonists & inhibitors , Receptors, Interleukin-1/metabolism , Recovery of Function , Signal Transduction , Systole , Time Factors , Ventricular Function, Left
12.
J Pharmacol Exp Ther ; 342(2): 441-52, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22573844

ABSTRACT

Loss-of -function mutations in human ether-a-go-go-related gene 1 (hERG1) is associated with life-threatening arrhythmias. hERG1 activators are being developed as treatments for acquired or genetic forms of long QT syndrome. The locations of the putative binding pockets for activators are still being elucidated. In silico docking of the activator 1,3-bis-(2-hydroxy-5-trifluoromethylphenyl)-urea (NS1643) to an S1-S6 transmembrane homology model of hERG1 predicted putative binding sites. The predictions of the in silico docking guided subsequent in vitro mutagenesis and electrophysiological measurements. The novel interacting site for NS1643 is predicted around Asn629 at the outer mouth of the channel. The applied N629H mutation is the sole amino acid replacement in the literature that abrogates the NS1643-induced left shift of the V(1/2) of activation. In contrast, both N629T and N629D showed pharmacologic responses similar to wild type. Another important interacting pocket is predicted at the intracellular surface in the S4-S5 linker. Mutagenesis of the residues critical to interactions in this pocket had major effects on the pharmacologic response to NS1643. The inward conductance elicited by hyperpolarization of D540K hERG1 was abrogated by NS1643 treatment, suggesting that it alters the inward movement of the S4 segment. The neighboring E544L mutation markedly exaggerated tail-current responses to NS1643. However, an L564A substitution inhibited drug response. Structure-guided mutagenesis identified widespread clusters of amino acids modulating drug-induced shifts in inactivation; such modulation may reflect allosteric changes in tertiary structure. Model-guided mutagenesis led to the discovery of a range of novel interacting residues that modify NS1643-induced pharmacologic responses.


Subject(s)
Cresols/chemistry , Cresols/pharmacology , Ether-A-Go-Go Potassium Channels/genetics , Ether-A-Go-Go Potassium Channels/metabolism , Phenylurea Compounds/chemistry , Phenylurea Compounds/pharmacology , Binding Sites , Dose-Response Relationship, Drug , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels/chemistry , HEK293 Cells , Humans , Models, Molecular , Mutagenesis, Site-Directed , Mutation , Protein Structure, Tertiary
13.
Biophys J ; 96(9): 3600-10, 2009 May 06.
Article in English | MEDLINE | ID: mdl-19413965

ABSTRACT

hERG1 is a member of the cyclic nucleotide binding domain family of K(+) channels. Alignment of cyclic nucleotide binding domain channels revealed an evolutionary conserved sequence HwX(A/G)C in the S5 domain. We reasoned that histidine 562 in hERG1 could play an important structure-function role. To explore this role, we created in silica models of the hERG1 pore domain based on the KvAP crystal structure with Rosetta-membrane modeling and molecular-dynamics simulations. Simulations indicate that the H562 residue in the S5 helix spans the gap between the S5 helix and the pore helix, stabilizing the pore domain, and that mutation at the H562 residue leads to a disruption of the hydrogen bonding to T618 and S621, resulting in distortion of the selectivity filter. Analysis of the simulated point mutations at positions 562/618/621 showed that the reciprocal double mutations H562W/T618I would partially restore the orientation of the 562 residue. Matching hydrophobic interactions between mutated W562 residue and I618 partially compensate for the disrupted hydrogen bonding. Complementary in vitro electrophysiological studies confirmed the results of the molecular-dynamics simulations on single mutations at positions 562, 618, and 621. Experimentally, mutations of the H562 to tryptophan produced a functional channel, but with slowed deactivation and shifted V(1/2) of activation. Furthermore, the double mutation T618I/H562W rescued the defects seen in activation, deactivation, and potassium selectivity seen with the H562W mutation. In conclusion, interactions between H562 in the S5 helix and amino acids in the pore helix are important determinants of hERG1 potassium channel function, as confirmed by theory and experiment.


Subject(s)
Ether-A-Go-Go Potassium Channels/chemistry , Ether-A-Go-Go Potassium Channels/metabolism , Protein Conformation , Amino Acid Sequence , Cell Line , Computer Simulation , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels/genetics , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Membrane Potentials/physiology , Models, Molecular , Molecular Sequence Data , Mutagenesis , Patch-Clamp Techniques , Potassium/metabolism , Protein Structure, Secondary , Sequence Alignment , Sequence Homology, Amino Acid , Structure-Activity Relationship
14.
Circ Res ; 103(12): 1483-91, 2008 Dec 05.
Article in English | MEDLINE | ID: mdl-18948620

ABSTRACT

Loss-of-function mutations in the human ERG1 potassium channel (hERG1) frequently underlie the long QT2 (LQT2) syndrome. The role of the ERG potassium channel in cardiac development was elaborated in an in vivo model of a homozygous, loss-of-function LQT2 syndrome mutation. The hERG N629D mutation was introduced into the orthologous mouse gene, mERG, by homologous recombination in mouse embryonic stem cells. Intact homozygous embryos showed abrupt cessation of the heart beat. N629D/N629D embryos die in utero by embryonic day 11.5. Their developmental defects include altered looping architecture, poorly developed bulbus cordis, and distorted aortic sac and branchial arches. N629D/N629D myocytes from embryonic day 9.5 embryos manifested complete loss of I(Kr) function, depolarized resting potential, prolonged action potential duration (LQT), failure to repolarize, and propensity to oscillatory arrhythmias. N629D/N629D myocytes manifest calcium oscillations and increased sarcoplasmic reticulum Ca(+2) content. Although the N629D/N629D protein is synthesized, it is mainly located intracellularly, whereas +/+ mERG protein is mainly in plasmalemma. N629D/N629D embryos show robust apoptosis in craniofacial regions, particularly in the first branchial arch and, to a lesser extent, in the cardiac outflow tract. Because deletion of Hand2 produces apoptosis, in similar regions and with a similar final developmental phenotype, Hand2 expression was evaluated. Robust decrease in Hand2 expression was observed in the secondary heart field in N629D/N629D embryos. In conclusion, loss of I(Kr) function in N629D/N629D cardiovascular system leads to defects in cardiac ontogeny in the first branchial arch, outflow tract, and the right ventricle.


Subject(s)
Ether-A-Go-Go Potassium Channels/genetics , Gene Expression Regulation, Developmental/genetics , Homozygote , Mutation, Missense/genetics , Ventricular Dysfunction, Right/embryology , Ventricular Dysfunction, Right/genetics , Amino Acid Substitution/genetics , Animals , Asparagine/genetics , Aspartic Acid/genetics , Cardiac Output/genetics , ERG1 Potassium Channel , Female , Heart Ventricles/embryology , Heart Ventricles/metabolism , Long QT Syndrome/embryology , Long QT Syndrome/genetics , Long QT Syndrome/mortality , Mice , Mice, Mutant Strains , Pregnancy , Ventricular Dysfunction, Right/mortality
15.
Am J Physiol Heart Circ Physiol ; 295(3): H1122-H1131, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18621856

ABSTRACT

Transgenic overexpression of calcineurin (CN/Tg) in mouse cardiac myocytes results in hypertrophy followed by dilation, dysfunction, and sudden death. Nitric oxide (NO) produced via inducible NO synthase (iNOS) has been implicated in cardiac injury. Since calcineurin regulates iNOS expression, and since phenotypes of mice overexpressing iNOS are similar to CN/Tg, we hypothesized that iNOS is pathogenically involved in cardiac phenotypes of CN/Tg mice. CN/Tg mice had increased serum and cardiac iNOS levels. When CN/Tg-iNOS(-/-) and CN/Tg mice were compared, some phenotypes were similar: extent of hypertrophy and fibrosis. However, CN/Tg-iNOS(-/-) mice had improved systolic performance (P < 0.001) and less heart block (P < 0.0001); larger sodium current density and lower serum TNF-alpha levels (P < 0.03); and less apoptosis (P < 0.01) resulting in improved survival (P < 0.0003). To define tissue origins of iNOS production, chimeric lines were generated. Bone marrow (BM) from wild-type or iNOS(-/-) mice was transplanted into CN/Tg mice. iNOS deficiency restricted to BM-derived cells was not protective. Calcineurin activates the local production of NO by iNOS in cardiac myocytes, which significantly contributes to sudden death, heart block, left ventricular dilation, and impaired systolic performance in this murine model of cardiac hypertrophy induced by the overexpression of calcineurin.


Subject(s)
Calcineurin/genetics , Calcineurin/physiology , Cardiomegaly/genetics , Cardiomegaly/pathology , Cell Death/physiology , Myocytes, Cardiac/enzymology , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/physiology , Animals , Apoptosis/genetics , Apoptosis/physiology , Arrhythmias, Cardiac/genetics , Arrhythmias, Cardiac/physiopathology , Cardiomyopathy, Dilated/genetics , Cardiomyopathy, Dilated/pathology , Death, Sudden , Echocardiography , Enzyme-Linked Immunosorbent Assay , Heart Block/genetics , Heart Block/physiopathology , Immunohistochemistry , In Situ Nick-End Labeling , Mice , Mice, Knockout , Mice, Transgenic , Myocytes, Cardiac/physiology , Nitric Oxide/metabolism , Sodium Channels/physiology , Survival Analysis , Tumor Necrosis Factor-alpha/metabolism
16.
Rep Prog Phys ; 70(5)2007 May 01.
Article in English | MEDLINE | ID: mdl-34996300

ABSTRACT

A review of the experimental and theoretical determinations of the anomalous magnetic moment of the muon is given. The anomaly is defined bya= (g- 2)/2, where the Landég-factor is the proportionality constant that relates the spin to the magnetic moment. For the muon, as well as for the electron and tauon, the anomalyadiffers slightly from zero (of the order 10-3) because of radiative corrections. In the Standard Model, contributions to the anomaly come from virtual 'loops' containing photons and the known massive particles. The relative contribution from heavy particles scales as the square of the lepton mass over the heavy mass, leading to small differences in the anomaly fore, µ and τ. If there are heavy new particles outside the Standard Model which couple to photons and/or leptons, the relative effect on the muon anomaly will be ∼ (mµ/me)2≈ 43 × 103larger compared with the electron anomaly. Because both the theoretical and experimental values of the muon anomaly are determined to high precision, it is an excellent place to search for the effects of new physics or to constrain speculative extensions to the Standard Model. Details of the current theoretical evaluation and of the series of experiments that culminates with E821 at the Brookhaven National Laboratory, are given. At present the theoretical and the experimental values are known with a similar relative precision of 0.5 ppm. There is, however, a 3.4 standard-deviation difference between the two, strongly suggesting the need for continued experimental and theoretical study.

17.
Heart Rhythm ; 2(8): 860-6, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16051125

ABSTRACT

BACKGROUND: Environmental and genetic factors interact to define susceptibility to drug-induced long QT syndrome. Although erythromycin induces long QT syndrome, substantial variability exists with regard to its incidence. OBJECTIVES: Because fever frequently results in empiric antibiotic usage, we assessed whether temperatures over the range from 36 degrees to 42 degrees C determined responsiveness to erythromycin (100 micromol/L). METHODS: I(hERG) was recorded in mammalian cells, and action potentials were recorded in neonatal ventricular mouse myocytes. RESULTS: Erythromycin (100 micromol/L) produced no block of I(hERG) at 22 degrees C but produced significant block at 37 degrees C. Extent of block of I(hERG) increased linearly (r = 0.46, P < .01) as temperature increased between 36 degrees C and 42 degrees C. To assess physiologic relevance, action potential duration (APD) was recorded at temperatures between 36 degrees C and 42 degrees C in neonatal ventricular myocytes. Significantly greater prolongation of APD by erythromycin was observed at 42 degrees C compared with 37 degrees C. To assess whether transmembrane diffusion of erythromycin was the rate-limiting step for block of I(hERG) at 22 degrees C, erythromycin was applied within the patch pipette. Under these conditions, erythromycin rapidly blocked I(hERG) even at 22 degrees C. The F656C mutation in the distal S6 of KCNH2 completely abrogated block of I(hERG) measured at 37 degrees C. CONCLUSION: Progressively greater block of hERG and prolongation of APD by erythromycin was observed at temperatures between 36 and 42 degrees C. Temperature-dependent block of I(hERG) is explained by temperature-dependent access of erythromycin to the intracellular binding site at F656.


Subject(s)
Action Potentials/drug effects , Body Temperature , Erythromycin/pharmacology , Ether-A-Go-Go Potassium Channels/drug effects , Myocytes, Cardiac/drug effects , Animals , Cation Transport Proteins/drug effects , Disease Susceptibility , ERG1 Potassium Channel , Electrophysiology , Gene Expression Regulation, Neoplastic/drug effects , Long QT Syndrome/chemically induced , Mice , Time Factors
18.
Cardiovasc Res ; 61(2): 268-77, 2004 Feb 01.
Article in English | MEDLINE | ID: mdl-14736543

ABSTRACT

OBJECTIVE: The long QT syndrome, N629D HERG mutation, alters the pore selectivity signature sequence, GFGN to GFGD. Heterologous co-expression of N629D and the wildtype HERG resulted in a relative loss of the selectivity of K+ over Na+, but its physiologic relevance has not been assessed in cardiac myocytes. METHODS AND RESULTS: Accordingly, N629D was overexpressed, via adenoviral gene transfer, in cardiomyocytes derived from mouse stem cells. Three IKr phenotypes were observed: (1) the wildtype-like IKr showed inward rectification and a positive tail current; (2) the N629D-like IKr showed outward rectification and an inward tail current; and (3) intermediate IKr showed a small outward tail current. Action potentials (AP) were paired with the IKr measurements in each cell. Resting membrane potential (RMP) was critically dependent on the IKr phenotype. The resting membrane potential of the cells was -61 +/- 5 mV (n=40) in wildtype, -63 +/- 3 mV (n=18) in wildtype-like IKr phenotype, -30 +/- 2 mV (n=12) in N629D-like and -47 +/- 2 mV (n=24) in intermediate phenotype (p<0.00001). Triggered action potential durations (APD) were: 62 +/- 12 ms (n=6) in wildtype, 65 +/- 11 ms (n=6) in wildtype-like IKr phenotypes and 106 +/- 10 ms (n=6) (p<0.01) in intermediate IKr phenotypes. Lowering [K+]o hyperpolarized wildtype cells and cells with a wildtype-like IKr phenotype, but depolarized those with intermediate phenotype (from -45 +/- 1 to -35 +/- 0.5 mV (n=12), p<0.01). In 6 of 12 cells, with intermediate phenotype, the hypokalemia-induced depolarization resulted in triggered activity. TTX suppressed this triggered activity. CONCLUSION: Overexpression of N629D in cardiomyocytes derived from stem cells results in phenotypic variability in IKr, which was the critical determinant of the resting membrane potential, action potential duration and arrhythmogenic response to low [K+]o.


Subject(s)
Cation Transport Proteins/genetics , Long QT Syndrome/metabolism , Myocytes, Cardiac/metabolism , Point Mutation , Potassium Channels, Voltage-Gated , Potassium Channels/genetics , Action Potentials , Adenoviridae/genetics , Animals , Cell Differentiation , Cells, Cultured , Ether-A-Go-Go Potassium Channels , Genetic Vectors/pharmacology , Green Fluorescent Proteins , Luminescent Proteins/genetics , Membrane Potentials , Mice , Patch-Clamp Techniques , Potassium Channels/metabolism , Stem Cells/cytology , Transduction, Genetic/methods
19.
Cardiovasc Res ; 57(3): 642-50, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12618226

ABSTRACT

OBJECTIVES: We hypothesized that exposure of N629D/wildtype channels to transient increases in [K(+)](o) could alter the conformation of the outer vestibule and thus reverse the disease phenotype. N629D is a recently described mutation of the HERG1 gene that causes familial long QT syndrome. This mutation alters the pore signature sequence resulting in loss of K(+) selectivity. Previous studies have reported that enforced occupancy of [K(+)](o) at sites near the selectivity filter alters the conformation/folding of the outer vestibule of the Kv2.1 channel. METHODS: Since the long QT syndrome is manifest in individuals who are heterozygous for this HERG trait, we co-expressed N629D and the wildtype at equimolar concentrations. RESULTS: Co-expression of N629D/wildtype in Xenopus oocytes and mammalian cells resulted in a channel with a positive shift in reversal potential and a loss in the outward tail current, relative to the wildtype. Exposure of the N629D/wildtype to transient increases in [K(+)](o) from 5 to 40 mM/l changed the tail current from inward to outward during repolarization and restored the reversal potential to values similar to the wildtype. These findings in Xenopus oocytes were also seen when N620D/wildtype channels were expressed in mammalian cells. These [K(+)](o)-dependent changes persisted for hours after the [K(+)](o) was returned to 2.5 mM. This potential therapeutic effect began with increases in [K(+)](o) from 2.5 to 5 mM. CONCLUSIONS: This study reports a novel therapeutic strategy and mechanism to partially restore physiologic function in this HERG LQTS mutation.


Subject(s)
Cation Transport Proteins , Long QT Syndrome/genetics , Mutation , Potassium Channels, Voltage-Gated , Potassium Channels/genetics , Potassium/physiology , Animals , Electrophysiology , Ether-A-Go-Go Potassium Channels , Female , Long QT Syndrome/physiopathology , Oocytes/metabolism , Patch-Clamp Techniques , Phenotype , Potassium/pharmacology , Potassium Channels/drug effects , Potassium Channels/physiology , Xenopus
20.
Mol Cell Biol ; 23(6): 1856-62, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12612061

ABSTRACT

The ERG1 gene encodes a family of potassium channels. Mutations in human ERG1 lead to defects in cardiac repolarization, referred to as the long QT syndrome. Through homologous recombination in mouse embryonic stem cells the ERG1 B potassium channel transcript was eliminated while the ERG1 A transcript was maintained. Heterologous expression of ERG1 isoforms had previously indicated that the deactivation time course of ERG1 B is 10-fold more rapid than that of ERG1 A. In day-18 fetal +/+ myocytes, I(Kr) exhibited two time constants of deactivation (3,933 +/- 404 and 350 +/- 19 ms at -50 mV), whereas in age-matched ERG1 B(-/-) mice the rapid component was absent. Biexponential deactivation rates (2,039 +/- 268 and 163 +/- 43 ms at -50 mV) were also observed in adult +/+ myocytes. In adult ERG1 B(-/-) myocytes no I(Kr) was detected. Electrocardiogram intervals were similar in +/+ and -/- mice. However, adult -/- mice manifested abrupt spontaneous episodes of sinus bradycardia (>100 ms of slowing) in 6 out of 21 mice. This phenomenon was never observed in +/+ mice (0 out of 16). We conclude that ERG1 B is necessary for I(Kr) expression in the surface membrane of adult myocytes. Knockout of ERG1 B predisposes mice to episodic sinus bradycardia.


Subject(s)
Cation Transport Proteins , DNA-Binding Proteins , Ion Transport/genetics , Myocardium/metabolism , Potassium Channels, Voltage-Gated , Potassium Channels/genetics , Potassium/metabolism , Trans-Activators , Age Factors , Animals , Arrhythmia, Sinus , Bradycardia , Disease Models, Animal , ERG1 Potassium Channel , Electrocardiography , Ether-A-Go-Go Potassium Channels , Gene Targeting , Heart Ventricles , Ion Transport/drug effects , Long QT Syndrome , Mice , Mice, Knockout , Patch-Clamp Techniques , Phenethylamines/pharmacology , Potassium Channel Blockers/pharmacology , Potassium Channels/deficiency , Potassium Channels/physiology , Protein Isoforms/deficiency , Protein Isoforms/genetics , Protein Isoforms/physiology , RNA, Messenger/biosynthesis , Sulfonamides/pharmacology
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