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Detection and Measurement of Cardiac Ion Channels

  • Gwilym M. Morris
  • Mark R. Boyett
  • Joseph Yanni
  • Rudolf Billeter
  • Halina Dobrzynski
Chapter

Abstract

Since the 1950s, technological advances have forged molecular biology into one of the most powerful fields of science. The primary molecular specializations of the cardiac conduction system are a lower expression of the fast Na+ channel (Nav1.5), the background K+ channel (Kir2.1), and the high conductance connexin (Cx43), but with a higher expression of the pacemaker channel (HCN4) and the alternative L-type Ca2+ channel (Cav1.3). Therefore, it is possible to investigate gene transcription and protein expression for these channels. This chapter describes the use of in situ hybridization, qPCR, and immunohistochemistry to study cardiac ion channel expression.

Keywords

Acetic Anhydride Laser Capture Microdissection Target cDNA Cardiac Conduction System Primer Dime Formation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

BCIP

5-bromo-4-chloro-3-indolyl phosphate

BSA

bovine serum albumin

Cav1.3

L-type Ca2+ channel in the heart, mainly found in the cardiac conduction system

cDNA

complementary DNA

CT

critical threshold

Cx43

main gap junction channel in the heart

DAPI

4′,6-diamidino-2-phenylindole

DNA

deoxyribonucleic acid

DNAse

deoxyribonuclease

EDTA

ethylenediaminetetraacetic acid

FITC

fluorescein isothiocyanate

HCN4

main ion channel responsible for the pacemaker current in the heart, I f

IgG

immunoglobulin G

IgM

immunoglobulin M

Kir2.1

inward rectifier K+ channel, responsible for the resting potential in the heart

mRNA

messenger RNA

Nav1.5

cardiac Na+ channel

N-BT

nitro blue tetrazolium

OCT

optimal cutting temperature compound

PBS

phosphate-buffered saline

PFA

paraformaldehyde

PVA

polyvinylalcohol

qPCR

quantitative polymerase chain reaction

RNA

ribonucleic acid

RNAse

ribonuclease

rRNA

ribosomal RNA

RyR2

sarcoplasmic reticulum Ca2+ release channel in heart

SSC

sodium chloride and sodium citrate solution

Tm

melting temperature

UTP

uridine-5′-triphosphate

References

  1. 1.
    Astbury WT. Molecular biology or ultrastructural biology? Nature 1961; 190:1124.PubMedCrossRefGoogle Scholar
  2. 2.
    Tellez JO, Dobrzynski H, Greener ID, et al. Differential expression of ion channel transcripts in atrial muscle and sinoatrial node in rabbit. Circ Res 2006; 319:105–114.Google Scholar
  3. 3.
    Dobrzynski H, Boyett MR, Anderson RH. New insights into pacemaker activity. Promoting understanding of sick sinus syndrome. Circulation 2007; 115:1921–1932.PubMedCrossRefGoogle Scholar
  4. 4.
    Boyett MR, Tellez JO, Dobrzynski H. The sinoatrial node: its complex structure and unique ion channel gene program. Chapter 12: Cardiac Electrophysiology: From cell to bedside. 5th edition. Zipes DP, Jalife J, editors. Philadelphia: Saunders, Elsevier Science 2009; 127–138.Google Scholar
  5. 5.
    Chandler NJ, Greener ID, Tellez JO, et al. Molecular architecture of the human sinus node: insights into the function of the cardiac pacemaker. Circulation 2009; 119:1562–1575.PubMedCrossRefGoogle Scholar
  6. 6.
    Greener ID, Tellez J, Dobrzynski H, et al. Ion channel transcript expression at the rabbit atrioventricular conduction axis. Circulation (Arrhythmia and Electrophysiology) 2009; 2:305–315.Google Scholar
  7. 7.
    Yanni J, Boyett MR, Anderson RH, et al. The extent of the specialized atrioventricular ring tissues. Heart Rhythm 2009; 6:672–680.PubMedCrossRefGoogle Scholar
  8. 8.
    Mahmood R, Mason I. In-situ hybridization of radioactive riboprobes to RNA in tissue sections. Methods Mol Biol 2008; 461:675–686.PubMedCrossRefGoogle Scholar
  9. 9.
    Küpper H, Seib LO, Sivaguru M, et al. A method for cellular localization of gene expression via quantitative in situ hybridization in plants. Plant J 2007; 50:159–175.PubMedCrossRefGoogle Scholar
  10. 10.
    Braissant O, Wahli W. A simplified in situ hybridization protocol using non radioactively labelled probes to detect abundant and rare mRNAs on tissue sections. Biochemica 1998; 1:10–16.Google Scholar
  11. 11.
    De Block M, Debrouwer D. RNA-RNA in situ hybridization using digoxigenin-labelled probes: the use of high molecular weight polyvinyl alcohol in the alkaline phosphatise indoxyl-nitroblue tetrazolium reaction. Anal Biochem 1993; 215:86–89.PubMedCrossRefGoogle Scholar
  12. 12.
    Pallansch L, Beswick H, Talian J, et al. Use of an RNA folding algorithm to choose regions for amplification by the polymerase chain reaction. Anal Biochem 1990; 185:57–62.PubMedCrossRefGoogle Scholar
  13. 13.
    Brattelid T, Tvwit K, Birkeland JAK, et al. Expression of mRNA encoding G protein-coupled receptors involved in congestive heart failure. A quantitative RT-PCR study and the question of normalisation. Basic Res Cardiol 2007; 102:198–208.PubMedCrossRefGoogle Scholar
  14. 14.
    Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002; 3:1–12.CrossRefGoogle Scholar
  15. 15.
    Beasley EM, Myers RM, Cox DR, et al. Statistical refinement of primer design parameters. In: Innis MA, Gelfand DH, Sninssky JJ, editors. PCR applications: protocols for functional genomics. London: Academic Press, 1999; 55–71.Google Scholar
  16. 16.
    Smith A, Burton JA Colour atlas of histological staining techniques. London: Wolfe Medical Publications Ltd., Sackville Press Billericay Ltd., 1977.Google Scholar
  17. 17.
    Kuhn DE, Roy S, Radtke J, et al. Laser microdissection and capture of pure cardiomyocytes and fibroblasts from infarcted heart regions: perceived hyperoxia induces p21 in peri-infarct myocytes. Am J Physiol 2007; 292:H1245–1253.Google Scholar
  18. 18.
    Murray J, Oquendo CE, Willis JH, et al. Monitoring oxidative and nitrative modification of cellular proteins; a paradigm for identifying key disease related markers of oxidative stress. Adv Drug Deliv Rev 2008; 60:1497–1503.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Gwilym M. Morris
  • Mark R. Boyett
  • Joseph Yanni
  • Rudolf Billeter
  • Halina Dobrzynski
    • 1
  1. 1.Cardiovascular Medicine, School of MedicineUniversity of ManchesterManchesterUK

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