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

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.

Figure 1. Northern blot analysis of total RNA extracted from the HL-1 clones (indicated on top of the picture).

Membranes were hybridised with probes for NCX, L-type Ca²⁺ channel, T-type Ca²⁺ channel, RyR2 and SERCA2 as indicated on the left of the picture. Membranes were stained with ethidium bromide to visualize the ribosomal RNA bands 18S and 28S.

Figure 2. Northern and Western analysis of connexin expression in the HL-1 clones.

(A) Membranes were hybridised with probes for Cx40, Cx43 and Cx45 as indicated on the left of the picture. The three connexins were expressed at varying levels by all the HL-1 clones. Membranes were stained with ethidium bromide to visualize the ribosomal RNA bands 18S and 28S. (B) Western blot analysis of Cx40 and Cx43 (B). The Cx40 antibody detected a single band migrating at 40 kDa expressed at similar levels in all the HL-1 clones except clone 2. Cx43 analysis obtained a single band migrating at 43 kDa. As with Cx40, Cx43 was present at similar levels in all HL-1 clones with the exception of clone 2. The clone numbers for transcript analysis and protein expression are indicted at the top of the picture. (C) There is a positive correlation of connexin transcript to protein expression for Cx40 and Cx43.

Figure 3. Immunofluorescent labelling of α-actinin in the HL-1 clones.

Cells for clones 6 display the classic sarcomeric organisation in comparison to clone 2, where the α-actinin is associated with the cell membrane.

Figure 4. Co-localisation of Cx40, Cx43 and Cx45 in clone 6.

(A) Each connexin can be seen in the individual channels within the same area and the triple labelling image was generated by superimposing the image for each connexin subtype. (B) To demonstrate the varying degrees of co-localisation at each cell interface, sections 1 and 2 highlighted from triple labelling were separated into dual images of Cx40/Cx43, Cx40/Cx45, Cx43/Cx45 and triple image of Cx40/Cx43/Cx45 as indicated on the left panel of the image.

Figure 5. Action potential characteristics of HL-1 clones 3 and clone 6.

(A) Representative action potentials recorded in clone 3 and clone 6 under whole cell current clamp mode by successive increasing depolarising pulses. (B) Quantification of (AP) amplitude, duration at 90% repolarisation (APD90) and upstroke velocity dV/dt max in clones 3 and 6.

Figure 6. Current-voltage relationship of Na⁺ channels in HL-1 clones 2, 3 and 6.

(A) Representative sodium current recorded in whole cell voltage clamp mode. (B) Corresponding averaged current-voltage relationship for each clone (B).

Figure 7. Current-voltage relationship of Ca²⁺ channels in HL-1 clones 2, 3 and 6.

(A) Representative calcium current recorded in whole cell voltage clamp mode. (B) Corresponding averaged current-voltage relationship for each clone that show a specific peak current in each clone.

Figure 8. Ca²⁺ transient recordings from clones 2 and 6.

(A) Spontaneous rhythmic oscillations of [Ca²⁺]_i in clones 2 and 6 after staining with fluo-4. (B) Both clones had a comparable [Ca²⁺]_i release but there was a significant prolongation in the time to peak, time to 50% decay and time to 90% decay in clone 2 compared with clone 6.

Figure 9. Extracellular recordings of paced cellular preparations generated from MEAs.

(A) The MEA plate consists of 60 titanium nitride (TiN) electrodes. Each electrode has a diameter of 30 µM and the interelectrode distance from centre to centre is 200 µM. (B) Cells were seeded as confluent drop directly on the TiN electrodes. (C) Representative extracellular field potentials after line stimulation are shown in. (D) CV are calculated as a time delay between the artefact of stimulation and the resulting field potential against the respective distance of the electrodes from the origin of excitation in clones 2, 3 and 6 (E).

Figure 10. Effects of gap junctions uncoupling and the blocking of Na⁺ channels on CV and calcium transient parameters in clones 3 and 6.

(A) and (B) show a dose dependent decrease in CV upon the application of carbenoxolone and lidocaine respectively in both clones. (C) and (D) There was no significant difference in Ca²⁺ transient amplitude and time to peak in the presence of carbenoxolone and lidocaine in clone 6.