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. 2009 Mar;13(3):562-73.
doi: 10.1111/j.1582-4934.2008.00361.x.

Impulse conduction and gap junctional remodelling by endothelin-1 in cultured neonatal rat ventricular myocytes

Y Reisner  1 , G Meiry, N Zeevi-Levin, D Y Barac, I Reiter, Z Abassi, N Ziv, S Kostin, J Schaper, M R Rosen, O Binah
Affiliations

Impulse conduction and gap junctional remodelling by endothelin-1 in cultured neonatal rat ventricular myocytes

Y Reisner et al. J Cell Mol Med. 2009 Mar.

Abstract

Endothelin-1 (ET-1) is an important contributor to ventricular hypertrophy and failure, which are associated with arrhythmogenesis and sudden death. To elucidate the mechanism(s) underlying the arrhythmogenic effects of ET-1 we tested the hypothesis that long-term (24 hrs) exposure to ET-1 impairs impulse conduction in cultures of neonatal rat ventricular myocytes (NRVM). NRVM were seeded on micro-electrode-arrays (MEAs, Multi Channel Systems, Reutlingen, Germany) and exposed to 50 nM ET-1 for 24 hrs. Hypertrophy was assessed by morphological and molecular methods. Consecutive recordings of paced activation times from the same cultures were conducted at baseline and after 3, 6 and 24 hrs, and activation maps for each time period constructed. Gap junctional Cx43 expression was assessed using Western blot and confocal microscopy of immunofluorescence staining using anti-Cx43 antibodies. ET-1 caused hypertrophy as indicated by a 70% increase in mRNA for atrial natriuretic peptide (P < 0.05), and increased cell areas (P < 0.05) compared to control. ET-1 also caused a time-dependent decrease in conduction velocity that was evident after 3 hrs of exposure to ET-1, and was augmented at 24 hrs, compared to controls (P < 0.01). ET-1 increased total Cx43 protein by approximately 40% (P < 0.05) without affecting non- phosphorylated Cx43 (NP-Cx43) protein expression. Quantitative confocal microscopy showed a approximately 30% decrease in the Cx43 immunofluorescence per field in the ET-1 group (P < 0.05) and a reduced field stain intensity (P < 0.05), compared to controls. ET-1-induced hypertrophy was accompanied by reduction in conduction velocity and gap junctional remodelling. The reduction in conduction velocity may play a role in ET-1 induced susceptibility to arrhythmogenesis.

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Figures

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Treatment of NRVM with ET-1 for 24 hrs causes hypertrophy. (A) Representative myocytes photographed at baseline (left) and at 24 hrs (right), from control (top panel) and ET-1 treated (bottom panel) cultures. To illustrate the changes in myocyte size, the perimeters of the cells were traced manually. (B) Morphometric analysis of the same myocytes followed throughout a 24 hrs period, using light microscopy. The figure shows a summary of the changes in myocytes size. The results are expressed as percent change from control. Control, n= 16 cell groups; ET-1, n= 24 cell groups, *P < 0.05 versus ET-1 cultures.
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Treatment of NRVM with ET-1 for 24 hrs causes hypertrophy: increase in cellular area and ANP mRNA levels. (A) Immunofluorescence staining for α-actinin from control (left panel) and ET-1 (right panel) cultures. (B) Summary of field α-actinin staining intensity. Control, n= 9 fields; ET-1, n= 8 fields, P < 0.05 versus Control. (C) mRNA levels of ANP. The upper panel depicts representative gels of ANP mRNA (top) and actin (bottom). The lower panel shows quantitative densitometric analysis of control and ET-1 cultures (24 hrs). Each value was divided by its corresponding actin value, and normalized to control. Control, n= 3 cultures; ET-1, n= 3 cultures. *P < 0.05.
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Percent capture of control and ET-1 treated cultures at baseline, 3, 6 and 24 hrs. The histograms depict the percent of cultures that were captured by pacing at both time-points. Control, n= 12 cultures; ET-1, n= 18 cultures.
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Effects of ET-1 on activation and conduction velocity in paced NRVM cultures, determined by the micro-electrode-array (MEA) data acquisition system. (A) Representative activation maps serving as a visual representation of the activation sequence, recorded at baseline, 6 and 24 hrs in paced Control and ET-1-treated cultures. The intersections of the straight lines mark the location of the recording electrodes. The map activation time (the time duration between first and last activations) is represented by the lower scale at the bottom of the map. The colour strip below the map represents the colour spectrum and its scaling according to time. Colour-coding: red – early; blue -late. Isochronal lines are overlaid on the maps and are spaced 1 ms apart. Notice the changes in the inter-isochronal spacing as an indication of conduction velocity changes. The calculated conduction velocities are shown below the activation maps. (B) The effect of ET-1 on conduction velocity. Mean conduction velocities in Control and in ET-1 exposed cultures paced at a BCL of 500 ms. The values shown are normalized to baseline conduction velocity which was set as 1. Control, n= 15 cultures; ET-1, n= 17 cultures. P= 0.004 for the trend differences between Control and ET-1. *P < 0.05 compared to baseline in the Control group. #P < 0.05 compared to baseline in the ET-1 group.
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Effects of ET-1 on activation and conduction velocity in NRVM cultures that could not be paced. Representative activation maps serving as a visual representation of the activation sequence, recorded at baseline, 6 and 24 hrs in spontaneously beating Control (A) and ET-1 treated (B) cultures. The intersections of the straight lines mark the location of the recording electrodes. The map activation time (the time duration between first and last activations) is represented by the lower scale at the bottom of the map. The colour strip below the map represents the colour spectrum and its scaling according to time. Colour coding: red – early; blue – late. Isochronal lines are overlaid on the maps and are spaced 1 ms apart. Notice the changes in the inter-isochronal spacing as an indication of conduction velocity changes. The calculated conduction velocities are shown below the activation maps.
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6
The effects of pacing on conduction velocity in Control, and ET-1-treated cultures. (A) Activation maps from a culture stimulated at BCLs of 250, 400 and 1000 ms. Each activation map represents propagation of one action potential. The map activation time (the time duration between first and last activations) is represented by the lower scale at the bottom of the map. The colour strip below the map represents the colour spectrum and its scaling according to time. Colour coding: red – early; blue – late. (B) Three spikes recorded from electrode #12 (denoted by the red circles in A) at BCL = 1000 ms (blue trace), 400 ms (red trace) and 250 ms (black trace). The spikes are overlaid and synchronized relative to the stimulus artefact occurrence. (C) The relationships between conduction velocity and BCL. The conduction velocity values were normalized to the values at BCL = 300 ms. n= 23 cultures, P < 0.001, one-way ANOVA analysis. (D) Conduction velocity values at baseline and at 24 hrs, from Control (n= 5), ET-1 treated cultures that captured pacing (n= 4) and ET-1 cultures unresponsive to pacing (n= 6). *P < 0.005 for the interaction between treatment versus time. In the non-capturing cultures beating spontaneously, the conduction velocity values were rate-corrected according to the rate correction equation discussed in the text.
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The effect of ET-1 on Cx43 protein expression. (A) Representative Western blots from Control and ET-1 cultures. Culture lysates were probed for total-Cx43 (upper panel) and NP-Cx43 (middle panel). Upper and lower arrows indicate the positions of the 44–46 and 41-kD bands, respectively. (B) Quantitative densitometric analysis of total-Cx43 in Control (n= 16 cultures) and ET-1 (n= 5 cultures). *P < 0.05 versus Control. (C) Quantitative densitometric analysis of NP-Cx43 in control (n= 16 cultures) and ET-1 (n= 5 samples). Equivalency of loading was verified with an antibody against actin (lower panel). Each value was normalized to its corresponding actin value (A, lower panel). Each sample is a pooling of two to three cultures.
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The effect of ET-1 on the morphological arrangement of Cx43 in NRVM cultures: quantitative confocal microscopy analysis of immunostained preparations. (A) Represen tative confocal images of a control culture and ET-1 treated culture. (B) The number of Cx43 gap junctions (GJ) per microscopic field. (C) Mean fluorescence intensity (FI) of Cx43 (expressed as percent change from control) per microscopic field. In (B) and (C): Control, n= 8 fields; ET-1, n= 7 fields, *P < 0.05 compared to control cultures.
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