DOCSLIB.ORG

Mesoangioblasts from Ventricular Vessels Can Differentiate in Vitro Into Cardiac Myocytes with Sinoatrial-Like Properties

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mesoangioblasts from Ventricular Vessels Can Differentiate in Vitro Into Cardiac Myocytes with Sinoatrial-Like Properties Journal of Molecular and Cellular Cardiology 48 (2010) 415–423 Contents lists available at ScienceDirect Journal of Molecular and Cellular Cardiology journal homepage: www.elsevier.com/locate/yjmcc Original article Mesoangioblasts from ventricular vessels can differentiate in vitro into cardiac myocytes with sinoatrial-like properties Andrea Barbuti a,b,⁎, Beatriz G. Galvez c, Alessia Crespi a, Angela Scavone a, Mirko Baruscotti a,b, Chiara Brioschi a, Giulio Cossu c,d, Dario DiFrancesco a,b a Department of Biomolecular Sciences and Biotechnology, The PaceLab, University of Milano, via Celoria 26, 20133 Milan, Italy b Centro Interuniversitario di Medicina Molecolare e Biofisica Applicata (CIMMBA), University of Milan, Italy c Stem Cell Research Institute, San Raffaele Hospital, via Olgettina, 20132 Milan, Italy d University of Milano, Department of Biology, via Celoria 26, 20133 Milan, Italy article info abstract Article history: Cardiac mesoangioblasts (MABs) are a class of vessel-associated clonogenic, self-renewing progenitor cells, Received 17 June 2009 recently identified in the post-natal murine heart and committed to cardiac differentiation. Cardiomyocytes Received in revised form 7 September 2009 generated during cardiogenesis from progenitor cells acquire several distinct phenotypes, corresponding to Accepted 2 October 2009 different functional properties in diverse structures of the adult heart. Given the special functional relevance Available online 22 October 2009 to rhythm generation and rate control of sinoatrial cells, and in view of their prospective use in therapeutical applications, we sought to determine if, and to what extent, cardiac mesoangioblasts could also differentiate Keywords: Adult stem cells into myocytes with properties typical of mature pacemaker myocytes. We report here that a subpopulation Mesoangioblasts of cardiac mesoangioblasts, induced to differentiate in vitro into cardiomyocytes, do acquire a phenotype Pacemaker myocytes with specific mature pacemaker myocytes properties. These include expression of the HCN4 isoform of Funny current pacemaker (“funny”, f-) channels and connexin 45 (Cx45), as well as reduced expression of inwardly- HCN channels rectifying potassium channels. Furthermore, MAB-derived myocytes form agglomerates of pacing cells displaying stable rhythmic activity, and as in native cardiac pacemaker cells, f-channel modulation by autonomic transmitters contributes to control of spontaneous rate in differentiated mesoangioblasts. These data represent the first evidence for in vitro generation of pacemaker-like myocytes from proliferating non- embryonic stem/progenitor cells. © 2009 Elsevier Ltd. All rights reserved. 1. Introduction [9] and cardiac muscle [10], where they were shown to differentiate mainly to skeletal and cardiac myocytes, respectively. During cardiac embryogenesis, common early cardiac precursors In this latter study, we have shown that ventricle-derived and aorta- differentiate into diverse cardiovascular cell types according to derived MABs can differentiate spontaneously into contracting cardio- specificgeneprograms[1,2], and some of the transcriptional myocytes expressing cardiac-specific proteins and ion channels. During networks whose activation is linked to specification of different development cardiac precursors acquire different phenotypes, with cardiomyocytes have been elucidated [3,4]. specific functional features [11–13]. An important distinction exists Study of embryonic stem (ES) cell differentiation, which recapi- between cells of the cardiac pacemaker/conduction tissue, whose tulates early cardiac development, has shown generation of multiple contribution is mainly electrical (i.e. generation and propagation of the cardiac phenotypes from common progenitors [5,6]. Adult cardiac action potential) and the working myocardium, whose main function is stem cells, on the other hand, generate cardiomyocytes of the working to respond to electrical stimuli with a contraction. muscle [7], but their ability to give rise to multiple cardiac phenotypes An obvious question arising from the finding that cardiac MABs has not yet been demonstrated. express typical cardiac progenitor markers and have a high prolifera- MABs are vessel-associated, progenitor cells originally isolated in tive potency, is whether their differentiation gives rise to multiple the embryonic aorta as clonogenic, self-renewing and multipotent cardiac phenotypes. We specifically asked if, and to what extent, cells [8]. Recently, similar cells have been identified in adult skeletal MABs could differentiate into cardiomyocytes with a mature pacemaker/conduction tissue phenotype. Our investigation was prompted by the preliminary observation that spontaneous activity ⁎ Corresponding author. Department of Biomolecular Science and Biotechnology was often seen to arise in a fraction of differentiating MABs. University of Milano, Via Celoria 26, 20133 Milan, Italy. Tel.: +39 02 50314941; fax: +39 02 50314932. We found that in both ventricle-derived and aorta-derived MAB E-mail address: [email protected] (A. Barbuti). clones, expanded under suitable culture conditions and driven to 0022-2828/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2009.10.006 416 A. Barbuti et al. / Journal of Molecular and Cellular Cardiology 48 (2010) 415–423 differentiate in vitro into cardiomyocytes, a subpopulation of cells vector (Invitrogen). The constitutive promoter PCMV was then does possess features typical of mature pacemaker myocytes, such as removed (AseI/XhoI) to obtain the promoterless cGATA-6/GFP vector. spontaneous electrical activity, expression of HCN channels and Undifferentiated cardiac mesoangioblasts were transfected by connexin 45 (Cx45) and lack of the inward rectifying IK1 current. lipofectamine reagents (following manufacturer instructions) with the GATA6-GFP vector. Since GFP would be expressed only in cells that 2. Materials and methods actively transcribe the GATA-6 gene, cells were co-transfected with the pIREs2a-DsRed2 plasmid in order to evaluate transfection 2.1. Isolation and expansion of stem cells from murine ventricular vessels efficiency. The methods used for the identification, isolation, expansion of 2.5. Immunofluorescence and confocal analysis cardiac mesoangioblasts were as previously described [10]. Briefly, 2- to 4-week-old C57 mice were sacrificed and the hearts quickly removed. For immunofluorescence analysis, cells were grown onto 1% Ventricles were kept in DMEM without FCS and cut into small pieces gelatin-coated glass coverslips and fixed in formaldehyde (4%) for including large vessels. Pieces were placed in 1% gelatin B (Sigma)- 10 min on ice, and then rinsed for 20 min with PBS containing 0.1 mol/L coated 35 mm petri dishes and kept in DMEM solution supplemented glycine. The cells were permeabilized in PBS solution containing 1% with 20% FBS, 5 mmol/L L-glutamine and 100 U/mL penicillin, BSA and 0.3% Triton X-100 and incubated overnight at 4 °C with 0.07 mmol/L streptomycin at 37 °C (5% CO2). After 2–3weeks,ontop primary antibodies (anti-myosin slow 1:500, Sigma; anti-GATA-6 of a layer of fibroblast–like cells, small, round, phase-bright cells were 1:100, Abcam; anti-connexin 43 1:50, Chemicon; anti-connexin 45 identifiable. These cells were collected and progressively expanded as 1:50, Chemicon; anti-HCN1-4, 1:100, Alomone Labs, anti-β1 and anti- separate clones in 10% FBS-DMEM maintaining medium. β2 adrenergic receptors 1:200 and anti-muscarinic M2 receptors All experimental protocols conformed to guidelines for the care 1:200, Santa Cruz). Coverslips were then washed in PBS and incubated and use of laboratory animals as established by State (D.L. 116/1992) for 1 hour with fluorophore-conjugated secondary antibodies (anti and European directives (86/609/CEE). mouse-TRITC, anti rabbit-FITC, 1:1000, Molecular Probes; anti mouse- Cy5, 1:1000, Chemicon). When needed, rhodamin–phalloidin (1 U/ 2.2. Differentiation coverslip, Molecular Probes) was added and the cells were incubated for 30 min at room temperature. Primary and secondary antibodies Spontaneous differentiation was induced by lowering serum (FCS) were diluted in PBS. After a final washout with PBS, coverslips were concentration in the growth medium (DMEM) from 10% to 2%. After mounted with Vectashield mounting medium with DAPI (Vector). 5 days of culture in differentiating medium most clones showed a high Fluorescence staining was analyzed by Video Confocal microscopy degree of cardiac differentiation based on myosin staining, and a few (ViCo Nikon). To test whether HCN2 and HCN1 antibodies were clones displayed several foci of spontaneously beating cells. Two functional, controls were performed in CHO cells transfected with the ventricle-derived clones (dubbed J2, J8) and an aorta-derived clone (I4) mouse HCN2 isoform and in murine ES cell-derived cardiomyocytes, were selected for further analysis due to their high rate of differentiation respectively, and results were positive; control experiments with into autorhythmic cells; all data presented here refer to these clones. secondary antibodies only were also carried out for all types of antibodies used, and resulted in no staining (data not shown). 2.3. RT-PCR analysis 2.6. Electrophysiology and data analysis RT-PCR analysis was carried out in both undifferentiated and differentiated MABs; in the latter case cells were kept in differenti- Action potentials were recorded from uniformly beating cell ation conditions for at least 5
Load more

Recommended publications
  • Transcriptome Pro Le of the Sinoatrial Ring Reveals Conserved and Novel Genetic Programs of the Zebra Sh Pacemaker
    Transcriptome Prole of the Sinoatrial Ring Reveals Conserved and Novel Genetic Programs of the Zebrash Pacemaker Rashid Minhas King's College London Faculty of Life Sciences and Medicine Henry Loeer-Wirth University of Leipzig - Interdisciplinary Centre for Bioinformatics Yusra Siddiqui International Institute of Molecular and Cell Biology in Warsaw: Miedzynarodowy Instytut Biologii Molekularnej i Komorkowej w Warszawie Tomasz Obrebski International Institute of Molecular and Cell Biology in Warsaw: Miedzynarodowy Instytut Biologii Molekularnej i Komorkowej w Warszawie Shikha Vhashist International Institute of Molecular and Cell Biology in Warsaw: Miedzynarodowy Instytut Biologii Molekularnej i Komorkowej w Warszawie Karim Abu Nahia International Institute of Molecular and Cell Biology in Warsaw: Miedzynarodowy Instytut Biologii Molekularnej i Komorkowej w Warszawie Aleksandra Paterek International Institute of Molecular and Cell Biology in Warsaw: Miedzynarodowy Instytut Biologii Molekularnej i Komorkowej w Warszawie Angelika Brzozowska International Institute of Molecular and Cell Biology in Warsaw: Miedzynarodowy Instytut Biologii Molekularnej i Komorkowej w Warszawie Lukasz Bugajski Nencki Institute of Experimental Biology: Instytut Biologii Doswiadczalnej im M Nenckiego Polskiej Akademii Nauk Katarzyna Piwocka Nencki Institute of Experimental Biology: Instytut Biologii Doswiadczalnej im M Nenckiego Polskiej Akademii Nauk Vladimir Korzh International Institute of Molecular and Cell Biology in Warsaw: Miedzynarodowy Instytut Biologii
    [Show full text]
  • Reciprocal Interaction Between IK1 and If in Biological Pacemakers: a Simulation Study
    bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.217232; this version posted July 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 2 3 4 5 Reciprocal interaction between IK1 and If in biological pacemakers: A simulation study 6 Running Title: Role of IK1 and If in bio-pacemaking 7 Yacong Li1,2, Kuanquan Wang1, Qince Li2,3, Jules C. Hancox4, Henggui Zhang2,3,5* 8 1 School of Computer Science and Technology, Harbin Institute of Technology, Harbin, Heilongjiang Province, China 9 2 Biological Physics Group, School of Physics and astronomy, The University of Manchester, Manchester, the Great Manchester, 10 UK 11 3 Peng Cheng Laboratory, Shenzhen, Guangdong Province, China 12 4 School of Physiology, Pharmacology and Neuroscience, Medical Sciences Building, University Walk, Bristol, UK 13 5 Key Laboratory of Medical Electrophysiology of Ministry of Education and Medical Electrophysiological Key Laboratory of 14 Sichuan Province, Institute of Cardiovascular Research, Southwest Medical University, Luzhou, Sichuan Province, China 15 16 * Correspondence: Henggui Zhang; [email protected] 17 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.217232; this version posted July 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
    [Show full text]
  • Electrophysiology and Electro-Physio-Pharmacology of Cardiac Cells.Docclass 2
    !1 ELECTRO-PHARMACO-PATHOPHYSIOLOGY OF CARDIAC ION CHANNELS AND IMPACT ON THE ELECTROCARDIOGRAM By ANDRÉS RICARDO PEREZ RIERA MD Key words: Ion channels – channelopathies – electrocardiogram RESTING POTENTIAL, ELECTROLYTIC CONCENTRATION, FAST AND SLOW FIBERS, ACTION POTENTIAL PHASES, SARCOLEMMAL AND INTRACELLULAR CHANNELS Introduction If we place both electrodes or wires (A and B) from a galvanometer (a device that records the difference in electric potential between two points) in the exterior (extracellular milieu) of a cardiac cell in rest or polarized, we would see that the needle of the device does not move (it indicates zero), because both electrodes are sensing the same milieu (extracellular). That is to say, there is no difference in potential between both ends of the galvanometer electrodes: Figure 1. !2 FIGURE 1 ILLUSTRATION OF TRANSMEMBRANE RESTING OR DIASTOLIC POTENTIAL IN A CARDIAC CELL AND MEASUREMENT WITH GALVANOMETER ! In rest, the extracellular milieu is predominantly positive in comparison to the intracellular one, as a consequence of positive charge (cations) predominance in the first in comparison to the intracellular one. What is the reason for the extracellular milieu to be predominantly positive in comparison to the intracellular? Reply: The reason lies in the greater concentration of proteins existing in the intracellular milieu in comparison to the extracellular one. Proteins have a double charge (positive or negative), and for this reason they are called amphoteric (amphoteric is any substance that can behave either as an acid or as a base, depending on the reactive agent present. If in the presence of an acid, it behaves as a base; if in the presence of a base, it behaves as an acid); therefore, in the intracellular pH negative charges are predominantly dissociated; i.e.
    [Show full text]
  • Cardiomyocyte Progenitor Cells As a Functional Gene Delivery Vehicle for Long-Term Biological Pacing
    Article Cardiomyocyte Progenitor Cells as a Functional Gene Delivery Vehicle for Long-Term Biological Pacing Anna M.D. Végh 1,2,†, A. Dénise den Haan 1,†, Lucía Cócera Ortega 1, Arie O. Verkerk 1, Joost P.G. Sluijter 3, Diane Bakker 1, Shirley van Amersfoorth 1,4, Toon A.B. van Veen 4, Mischa Klerk 1, Jurgen Seppen 5, Jacques M.T. de Bakker 1,4, Vincent M. Christoffels 6, Dirk Geerts 1,6, Marie José T.H. Goumans 2, Hanno L. Tan 1 and Gerard J.J. Boink 1,6,* 1 Heart Center, Clinical & Experimental Cardiology, Amsterdam University Medical Centers, Academic Medical Center; 1105 AZ Amsterdam, the Netherlands; [email protected] (A.M.D.V.); [email protected] (A.D.d.H.); [email protected] (L.C.O.); [email protected] (A.O.V.); [email protected] (D.B.); [email protected] (S.v.A.); [email protected] (M.K.); [email protected] (J.M.T.d.B.); [email protected] (D.G.); [email protected] (H.L.T.) 2 Cell and Chemical Biology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands; [email protected] 3 Department of Cardiology, Experimental Cardiology Laboratory, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands; [email protected] 4 Medical Physiology, University Medical Center Utrecht, 3584 CX Utrecht, the Netherlands; [email protected] 5 Tytgat Institute, Academic Medical Center; 1105 BK Amsterdam, the Netherlands; [email protected] 6 Department of Medical Biology, Amsterdam University Medical Centers, Academic Medical Center; 1105 AZ Amsterdam, the Netherlands; [email protected] * Correspondence: [email protected]; Tel.: +31-20-5669-111 † These authors contributed equally.
    [Show full text]
  • Mechanisms Underlying the Cardiac Pacemaker: the Role of SK4 Calcium-Activated Potassium Channels
    npg Acta Pharmacologica Sinica (2016) 37: 82–97 © 2016 CPS and SIMM All rights reserved 1671-4083/16 www.nature.com/aps Review Mechanisms underlying the cardiac pacemaker: the role of SK4 calcium-activated potassium channels David WEISBROD, Shiraz Haron KHUN, Hanna BUENO, Asher PERETZ, Bernard ATTALI* Department of Physiology & Pharmacology, Sackler Faculty of Medicine, Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 69978, Israel The proper expression and function of the cardiac pacemaker is a critical feature of heart physiology. The sinoatrial node (SAN) in human right atrium generates an electrical stimulation approximately 70 times per minute, which propagates from a conductive network to the myocardium leading to chamber contractions during the systoles. Although the SAN and other nodal conductive structures were identified more than a century ago, the mechanisms involved in the generation of cardiac automaticity remain highly debated. In this short review, we survey the current data related to the development of the human cardiac conduction system and the various mechanisms that have been proposed to underlie the pacemaker activity. We also present the human embryonic stem cell- derived cardiomyocyte system, which is used as a model for studying the pacemaker. Finally, we describe our latest characterization of the previously unrecognized role of the SK4 Ca2+-activated K+ channel conductance in pacemaker cells. By exquisitely balancing the inward currents during the diastolic depolarization, the SK4 channels appear to play a crucial role in human cardiac automaticity. Keywords: cardiac pacemaker; sinoatrial node; SK4 K+ channel; Ca2+ clock model; voltage clock model Acta Pharmacologica Sinica (2016) 37: 82−97; doi: 10.1038/aps.2015.135 Introduction studying the pacemaker, and the most recent characterization Normal cardiac function depends on the adequate timing of of the previously unrecognized role of the SK4 Ca2+-activated excitation and contraction in the various regions of the heart K+ channel conductance in pacemaker cells.
    [Show full text]
  • Current Aspects of the Basic Concepts of the Electrophysiology of the Sinoatrial Node
    Journal of Electrocardiology 57 (2019) 112–118 Contents lists available at ScienceDirect Journal of Electrocardiology journal homepage: www.jecgonline.com Review Current aspects of the basic concepts of the electrophysiology of the sinoatrial node Andrés Ricardo Pérez-Riera, MD PhD a,⁎, Raimundo Barbosa-Barros, MD b, Rodrigo Daminello-Raimundo, PhD a, Luiz Carlos de Abreu, PhD a,d, Kjell Nikus, MD PhD c a Laboratório de Metodologia de Pesquisa e Escrita Científica, Faculdade de Medicina do ABC, Santo André, São Paulo, Brazil b Coronary Center of the Hospital de Messejana Dr. Carlos Alberto Studart Gomes, Fortaleza, Ceará, Brazil c Heart Center, Tampere University Hospital and Faculty of Medicine and Health Technology, Tampere University, Finland d Graduate Entry Medical School, University of Limerick, Limerick, Ireland article info abstract Keywords: Cardiac pacemaker cells, also named P-cells (pale cytoplasm, pacemaker, phylogenetically primitive), including Ion channels cells of the sinoatrial node, are heterogeneous in size, morphology, and electrophysiological characteristics. Sinus node The exact extent to which these cells differ electrophysiologically in the human heart is unclear, yet it is critical Calcium clock for the understanding of normal cellular function. In this review, we describe major ionic currents and Ca2+ Sarcoplasmic reticulum clocks acting on Ca2+ release in the sarcoplasmic reticulum. We also explain the external regulation of the heart rate controlled by the two branches of the autonomic (involuntary) nervous system: the sympathetic and the parasympathetic nervous system. Vagal stimulus causes bradycardia, rapid and short-duration modula- tion, and controls rapid responses, and increases heart rate variability. A typical example is constituted by phasic or respiratory sinus arrhythmia, characterized by pronounced vagal activity, more frequent in children and young individuals.
    [Show full text]
  • Regulation of Cardiac Voltage Gated Potassium Currents in Health and Disease
    REGULATION OF CARDIAC VOLTAGE GATED POTASSIUM CURRENTS IN HEALTH AND DISEASE DISSERTATION Presented in Partial Fulfillment of the Requirements for The Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Arun Sridhar, M.S. ****** The Ohio State University 2007 Dissertation Committee: Dr. Cynthia A. Carnes, Pharm.D, PhD Approved By: Dr. Robert L. Hamlin, DVM, PhD ______________________ Dr. Sandor Gyorke, PhD Advisor Dr. Mark T. Ziolo, PhD Graduate Program in Biophysics ABSTRACT Cardiovascular disease (CVD) is a major cause of mortality and morbidity worldwide. CVD accounts for more deaths than all forms of cancer in the United States. Hypertension, Heart Failure and Atrial Fibrillation are the most common diagnosis, hospitalization cause and the sustained cardiac arrhythmia respectively in the US. Sudden cardiac death is the one of the most common causes of cardiovascular mortality after myocardial infarction, and a common cause of death in heart failure patients. This has been attributed to the development of ventricular tachyarrhythmias. In addition, most forms of acquired CVD have been shown to produce electrophysiological changes due to very close interactions between structure, signaling pathways and ion channels. Due to the increased public heath burden caused by CVD, a high impetus has been placed on identifying novel therapeutic targets via translational research. Identification of novel therapeutic targets to treat heart failure and sudden death is underway and is still in a very nascent stage. In addition, ion channel blockers, more specifically “atrial-specific” ion channel blockers have proposed to be a major therapeutic target to treat atrial fibrillation without the risk of ventricular pro- arrhythmia.
    [Show full text]
  • Electric Pulse Current Stimulation Increases Electrophysiological
    EXPERIMENTAL AND THERAPEUTIC MEDICINE 11: 1323-1329, 2016 Electric pulse current stimulation increases electrophysiological properties of If current reconstructed in mHCN4-transfected canine mesenchymal stem cells YUANYUAN FENG1,2, SHOUMING LUO1, PAN YANG1 and ZHIYUAN SONG1 1Department of Cardiology, Southwest Hospital, Third Military Medical University, Chongqing 400038; 2Department of Cardiology, The 401 Hospital of PLA, Qingdao, Shandong 266071, P.R. China Received December 7, 2014; Accepted January 28, 2016 DOI: 10.3892/etm.2016.3072 Abstract. The 'funny' current, also known as the If current, transcription-quantitative polymerase chain reaction and play a crucial role in the spontaneous diastolic depolarization western blot analyses. Transmission electron micrographs also of sinoatrial node cells. The If current is primarily induced by confirmed the increased gap junctions in group B. Collectively, the protein encoded by the hyperpolarization-activated cyclic these results indicated that reconstructed If channels may have nucleotide-gated channel 4 (HCN4) gene. The functional If a positive regulatory role in EPCS induction. The cMSCs channel can be reconstructed in canine mesenchymal stem cells transfected with mHCN4 induced by EPCS may be a source (cMSCs) transfected with mouse HCN4 (mHCN4). Biomimetic of effective biological pacemaker cells. studies have shown that electric pulse current stimulation (EPCS) can promote cardiogenesis in cMSCs. However, Introduction whether EPCS is able to influence the properties of the If current reconstructed in mHCN4-transfected cMSCs remains The sinoatrial node (SAN) is located in the right dorsal wall unclear. The present study aimed to investigate the effects of of the right atrium, and serves as the primary pacemaker for EPCS on the If current reconstructed in mHCN4-transfected initiating the heartbeat and controlling the rate and rhythm of cMSCs.
    [Show full text]
  • Pacemaker Current If in Adult Canine Cardiac Ventricular Myocytes
    2910 Journal of Physiology (1995), 485.2, pp. 469-483 469 Pacemaker current if in adult canine cardiac ventricular myocytes Hangang Yu, Fang Chang and Ira S. Cohen * Department of Physiology and Biophysics, Health Sciences Center, State University of New York, Stony Brook, NY 11794-8661, USA 1. Single cells enzymatically isolated from canine ventricle and canine Purkinje fibres were studied with the whole-cell patch clamp technique, and the properties of the pacemaker current, if, compared. 2. Steady-state if activation occurred in canine ventricular myocytes at more negative potentials (-120 to -170 mV) than in canine Purkinje cells (-80 to -130 mV). 3. Reversal potentials were obtained in various extracellular Nae (140, 79 or 37 mM) and K+ concentrations (25, 9 or 5-4 mM) to determine the ionic selectivity of 4i in the ventricle. The results suggest that this current was carried by both sodium and potassium ions. 4. The plots of the time constants of if activation against voltage were 'bell shaped' in both canine ventricular and Purkinje myocytes. The curve for the ventricular myocytes was shifted about 30 mV in the negative direction. In both ventricular and Purkinje myocytes, the fully activated I-Vrelationship exhibited outward rectification in 5A4 mm extracellular K+. 5. Calyculin A (0 5 /M) increased 4 by shifting its activation to more positive potentials in ventricular myocytes. Protein kinase inhibition by H-7 (200 /LM) or H-8 (100 uM) reversed the positive voltage shift of if activation. This effect of calyculin A also occurred when the permeabilized patch was used for whole-cell recording.
    [Show full text]
  • Pacemaker Channel Dysfunction in a Patient with Sinus Node Disease
    Pacemaker channel dysfunction in a patient with sinus node disease Eric Schulze-Bahr, … , Olaf Pongs, Dirk Isbrandt J Clin Invest. 2003;111(10):1537-1545. https://doi.org/10.1172/JCI16387. Article Cardiology The cardiac pacemaker current If is a major determinant of diastolic depolarization in sinus nodal cells and has a key role in heartbeat generation. Therefore, we hypothesized that some forms of “idiopathic” sinus node dysfunction (SND) are related to inherited dysfunctions of cardiac pacemaker ion channels. In a candidate gene approach, a heterozygous 1-bp deletion (1631delC) in exon 5 of the human HCN4 gene was detected in a patient with idiopathic SND. The mutant HCN4 protein (HCN4-573X) had a truncated C-terminus and lacked the cyclic nucleotide–binding domain. COS-7 cells transiently transfected with HCN4-573X cDNA indicated normal intracellular trafficking and membrane integration of HCN4-573X subunits. Patch-clamp experiments showed that HCN4-573X channels mediated If-like currents that were insensitive to increased cellular cAMP levels. Coexpression experiments showed a dominant-negative effect of HCN4- 573X subunits on wild-type subunits. These data indicate that the cardiac If channels are functionally expressed but with altered biophysical properties. Taken together, the clinical, genetic, and in vitro data provide a likely explanation for the patient’s sinus bradycardia and the chronotropic incompetence. Find the latest version: https://jci.me/16387/pdf Pacemaker channel dysfunction in a patient with sinus node disease
    [Show full text]
  • Expression of Connexin 43, Ion Channels and Ca2+-Handling Proteins in Rat Pulmonary Vein Cardiomyocytes
    EXPERIMENTAL AND THERAPEUTIC MEDICINE 12: 3233-3241, 2016 Expression of connexin 43, ion channels and Ca2+-handling proteins in rat pulmonary vein cardiomyocytes YAQIONG XIAO1, XUE CAI2, ANDREW ATKINSON2, SUNIL JIT LOGANTHA2, MARK BOYETT2 and HALINA DOBRZYNSKI2 1Department of Critical Care Medicine, Peking University International Hospital, Beijing 102206, P.R. China; 2Institute of Cardiovascular Sciences, Faculty of Medical and Human Sciences, University of Manchester, Manchester M13 9NT, UK Received March 25, 2016; Accepted July 1, 2016 DOI: 10.3892/etm.2016.3766 Abstract. Atrial fibrillation (AF) is the most common cardiac of AF, with a 5‑fold increased risk of stroke (3). In addition, AF arrhythmia. AF is thought to be triggered by ectopic beats, carries a 3‑fold increased risk of heart failure (4,5), and a 2‑fold originating primarily in the myocardial sleeves surrounding increased risk of both dementia (6) and mortality (3,7). the pulmonary veins (PVs). The mechanisms underlying Clinical AF can be categorized as paroxysmal, persistent these cardiac arrhythmias remain unclear. To investigate or permanent (7). Paroxysmal AF, caused by focal drivers, this, frozen sections of heart and lung tissue from adult rats particularly in the ectopic sites in myocardial sleeves around without arrhythmia were obtained in different planes, stained the pulmonary veins (PVs), can be eliminated by catheter with Masson's trichrome, and immunolabeled for connexin 43 ablation (8,9). The proximal tunica media of the PVs, typically (Cx43), caveolin‑3 (Cav3), hyperpolarization‑activated cyclic referred to as the myocardial sleeve, is the primary source of nucleotide‑gated channel 4 (HCN4), Nav1.5, Kir2.1, and the supraventricular ectopic activity.
    [Show full text]
  • Ion Channels Product Listing | Edition 1
    Ion Channels Product Listing | Edition 1 Western Honey Bee Apis mellifera A source of Apamin Products by Gating Mechanism: • Ligand-gated Ion Channels • Voltage-gated Ion Channels • Other Ion Channels Tocris Product Listing Series Introduction Ion channels are pore-forming proteins present in the plasma membrane of most cells, as well as the intracellular membranes surrounding many organelles, allowing the passage of ions across the membrane. They are essential for maintaining resting membrane potential, generating action potentials and other electrical signaling, and regulating cell volume. Ion channels have a fundamental role in physiological processes such as T cell activation, muscle contraction, insulin release from pancreatic β cells and transport of nutrients and ions across epithelial cells. A huge variety of ion channels exist, exhibiting selective permeability to particular ions and having different gating mechanisms. Ion channels may be classified according to their ion selectivity or gating mechanism, i.e. how the flow of ions is controlled. We have followed the IUPHAR classification of ion channels for the purposes of this listing and grouped the products into three main sections according to gating mechanism: Ligand-gated Ion Channels, Voltage- gated Ion Channels, and Other Channels. Contents Ligand-gated Ion Channels 3 Potassium Channels 17 Ca2+-Activated Potassium Channels 17 5-HT3 4 Inward Rectifier Potassium (K ) Channels 18 Acetylcholine Nicotinic Receptors 4 ir Two-P Potassium Channels 19 Nicotinic (α7) Receptors 4
    [Show full text]