Characterisation of connexin expression and electrophysiological properties in stable clones of the HL-1 myocyte cell line

PLoS One. 2014 Feb 28;9(2):e90266. doi: 10.1371/journal.pone.0090266. eCollection 2014.

Abstract

The HL-1 atrial line contains cells blocked at various developmental stages. To obtain homogeneous sub-clones and correlate changes in gene expression with functional alterations, individual clones were obtained and characterised for parameters involved in conduction and excitation-contraction coupling. Northern blots for mRNAs coding for connexins 40, 43 and 45 and calcium handling proteins (sodium/calcium exchanger, L- and T-type calcium channels, ryanodine receptor 2 and sarco-endoplasmic reticulum calcium ATPase 2) were performed. Connexin expression was further characterised by western blots and immunofluorescence. Inward currents were characterised by voltage clamp and conduction velocities measured using microelectrode arrays. The HL-1 clones had similar sodium and calcium inward currents with the exception of clone 2 which had a significantly smaller calcium current density. All the clones displayed homogenous propagation of electrical activity across the monolayer correlating with the levels of connexin expression. Conduction velocities were also more sensitive to inhibition of junctional coupling by carbenoxolone (∼ 80%) compared to inhibition of the sodium current by lidocaine (∼ 20%). Electrical coupling by gap junctions was the major determinant of conduction velocities in HL-1 cell lines. In summary we have isolated homogenous and stable HL-1 clones that display characteristics distinct from the heterogeneous properties of the original cell line.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Action Potentials / physiology*
  • Animals
  • Calcium Channels, L-Type / genetics
  • Calcium Channels, L-Type / metabolism
  • Calcium Channels, T-Type / genetics
  • Calcium Channels, T-Type / metabolism
  • Cell Line
  • Clone Cells
  • Connexin 43 / genetics
  • Connexin 43 / metabolism*
  • Connexins / genetics
  • Connexins / metabolism*
  • Excitation Contraction Coupling / physiology
  • Gap Junctions / physiology*
  • Gene Expression
  • Heart Atria / cytology
  • Heart Atria / metabolism
  • Heart Conduction System / physiology
  • Humans
  • Mice
  • Myocytes, Cardiac / cytology
  • Myocytes, Cardiac / metabolism*
  • Ryanodine Receptor Calcium Release Channel / genetics
  • Ryanodine Receptor Calcium Release Channel / metabolism
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases / genetics
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases / metabolism
  • Sodium-Calcium Exchanger / genetics
  • Sodium-Calcium Exchanger / metabolism

Substances

  • Calcium Channels, L-Type
  • Calcium Channels, T-Type
  • Connexin 43
  • Connexins
  • GJA1 protein, mouse
  • Ryanodine Receptor Calcium Release Channel
  • Sodium-Calcium Exchanger
  • connexin 40
  • connexin 45
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases