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Cardiovascular Research 2004 62(2):397-406; doi:10.1016/j.cardiores.2004.01.015
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Copyright © 2004, European Society of Cardiology

Alterations of intercellular communication in neonatal cardiac myocytes from connexin43 null mice

Monique J Vinka, Sylvia O Suadicania, Delia M Vieirab, Marcia Urban-Maldonadoa, Yang Gaoa, Glenn I Fishmanb,c and David C Spray*,a,b

aDepartment of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
bDepartment of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA
cThe Leon H. Charney Division of Cardiology, New York University School of Medicine, New York, NY 10016, USA

* Corresponding author. Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. Tel.: +1-718-430-2537; fax: +1-718-430-8594. Email address: spray{at}aecom.yu.edu

Received 27 November 2003; revised 7 January 2004; accepted 9 January 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: To compare gap junction expression and intercellular coupling in wildtype neonatal cardiac myocytes to those from mice lacking the most abundant cardiac gap junction protein (connexin43, Cx43). Methods: Northern and Western blots compared connexin mRNA and protein levels, immunocytochemistry evaluated connexin distribution in neonatal Cx43 null(–/–), heterozygous(+/–) and wildtype(+/+) mouse hearts. Ca2+ imaging, dye coupling and electrophysiological methods evaluated intercellular communication. Results: Similar levels of Cx40 and Cx45 were detected in all genotypes, although in adult cardiac tissue from wildtype mice, Cx43 expression was higher than in heterozygotes. After culturing dissociated cells for 3–4 days, cardiocytes beat spontaneously; in Cx43(+/+) and (+/–) cultures, the beating was generally quite synchronous. In Cx43(–/–) mice, interbeat intervals were on average twice as long and more variable than in Cx43(+/+) or Cx43(+/–) cultures. Junctional conductance was lower by about 60% in Cx43(–/–) as compared to Cx43(+/–) and (+/+) littermates; Lucifer Yellow dye coupling was virtually absent in Cx43(–/–) cardiomyocytes but was comparably strong in wildtype and heterozygous siblings. Macroscopic junctional conductance measurements on Cx43(–/–) cardiocytes showed slightly stronger voltage sensitivity in these cells than in Cx43(+/+) cardiocytes. Unitary junctional conductance measurements revealed distinct populations of channels contributing to macroscopic conductance for Cx43(+/+) and Cx43(–/–) genotypes. Conclusions: In Cx43-deficient cardiac myocytes, the expression of other connexins only partially compensates for the functional loss, with dye coupling and spontaneous beating being strongly impaired.

KEYWORDS Gap junction; Nexus; Electrical coupling; Arrhythmia; Knockout mice; Cx43


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Cardiac tissue is composed of phenotypically distinct cell types, which are connected through gap junction channels into electrically syncytial communication compartments [1]. Although conduction of the cardiac action potential between individual cells is discontinuous and can be highly anisotropic due to geometric variables such as cell length and width, as well as the variable distribution and expression of gap junctions between heart cells [2], propagation of contraction in the working myocardium is normally spread through high conductance gap junctions connecting cells both in series and in parallel, so that conduction is macroscopically continuous, with high safety factor [3].

Connexin43 (Cx43) was the first gap junction protein found in the heart [4] and is the most abundant connexin in the working myocardium [1,5]. However, other connexins are also present; Cx40 is abundantly expressed in atrium and conduction system [6–10], Cx45 is expressed throughout the whole heart [7], preferentially in the outer zone of the His-Purkinje conduction system [11,12]. Cx37 is confined to endothelial cells [13,14], whereas Cx50 has only been reported in cardiac valves [15].

The functional implications of redundant connexin expression in heart are incompletely resolved. However, expression of different connexins by different cell groups could provide strong coupling within each compartment, yet functional separation of the different cardiac compartments. For example, the initial reports that Cx43-expressing cells do not communicate with those expressing Cx40 suggested that such incompatibility could serve to insulate the conduction system from the working myocardium except at contact regions where compatible connexins are expressed [16–18]. Alternatively or in addition, differences in gating properties or selective permeability of gap junction channels formed of the various connexins could result in junctions that are differentially vulnerable to pathophysiological stimuli, electrically rectify to favor unidirectional transmission, or preferentially pass current-carrying ions, but not negatively charged metabolites [1]. For example, unitary conductance ({gamma}j) of Cx43 channels is increased under dephosphorylating conditions [19–21], whereas {gamma}j of other connexins is reduced or unchanged [20], the profoundly different voltage sensitivities of Cx43 and Cx45 result in strong rectification when cells expressing each connexin are heterotypically paired [22] and Cx43 channels are more permeable to anions than are channels formed of either Cx40 or Cx45 [23].

Transgenic mice have been generated in which Cx43 has been totally deleted [24], and in which deletion was targeted to cardiac tissue [25]. While both types of homozygous Cx43 null offspring survive embryogenesis, those with global Cx43 disruption exhibit right ventricular outflow tract obstruction, causing cyanosis and death at birth, precipitated by the closure of the shunts provided by the foramen ovale and ductus arteriosus [24]. By contrast, cardiac-specific disruption of Cx43 leads to ventricular arrhythmias and sudden cardiac death by 2 months of age [25]. The in utero survival of the total knockout, and the postnatal survival of the cardiac-targeted deletion, indicate that changes in cardiac conduction velocity and patterned contraction that presumably result from the absence of Cx43 are not immediately lethal, suggesting that other gap junction proteins contribute to cardiac conduction in these animals, although the lack of coupling through Cx43 channels in these mice apparently leads to a pronounced developmental defect. In the studies reported here, we have compared a variety of parameters measuring gap junction function and cardiac conduction in cultured cardiac myocytes from wildtype and Cx43 null(–/–) mice. These data show that there is minimal compensatory upregulation of other connexins in the Cx43(–/–) heart. However, whereas junctional conductance is reduced by only 60% in these animals, rhythmic beating and junctional permeability to Lucifer Yellow are greatly impaired. These results imply that the safety factor for cardiac conduction and the potential for normal tissue development and organization may be dependent on not only how many junctional channels are present between the cells, but also on the properties of the channels formed by the connexins that are expressed.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Mice
Mating pairs of Cx43 hemizygous [Cx43(+/–)] mice (C57Bl/GJ-Gja1tm1Kdr) were obtained from Jackson Laboratories (Bar Harbor, ME) and maintained in AAULC-accredited animal facilities. Genotypes of offspring were determined from polymerase chain reaction (PCR) on genomic tail DNA [26]. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2. Cell cultures
Within 15 min to 1 h after birth, hearts were removed from each littermate and either the whole heart or isolated atrial or ventricular region was individually dissociated and maintained in culture [27].

2.3. Indirect immunocytochemistry of cardiac myocytes in culture
Cardiac myocytes (generally at 80% confluence) were processed for immunocytochemistry using monoclonal anti-Cx40 (from D.L. Paul, Harvard) and rabbit polyclonal anti-Cx43 (from E.L. Hertzberg, Einstein; Ref. [28]) and anti-Cx45 (from T. Steinberg, Washington University) antibodies, as described previously in studies of astrocytes [26].

2.4. Dye coupling
Lucifer Yellow (5% wt/vol in 150 mM LiCl) was injected into cells through sharp microelectrodes and visualized as described previously [26].

2.5. Measurements of rates of spontaneous beating in culture
Intracellular Ca2+ levels were measured from cells and cell regions using the dual emission ratiometric Ca2+ indicator Indo-1 as described [27]. Data are presented here as ratios of intensities obtained at the two emission wavelengths (indicated as "Fluorescence ratio" on ordinates of graphs).

2.6. Electrophysiological studies
The dual whole cell voltage clamp technique with patch pipettes was used to measure properties of junctional channels between pairs of cardiac myocytes obtained from wildtype, heterozygous, and Cx43(–/–) animals, as recently described [29].

For single channel recordings, cell pairs with low gj were chosen or gj was reversibly reduced to low levels by applying 3 mM halothane to the bathing medium (in mM NaCl:140, CsCl:4, CaCl2:2, MgCl2:1, HEPES:5, KCl:4, dextrose:5, pyruvate:2, BaCl2:1, pH 7.4); halothane reduces gap junction channel open probability but does not affect unitary conductance [30]. Histograms of unitary conductances ({gamma}j=ij/Vj) were obtained from 30 to 244 unambiguously discrete events in each cell pair; normalized frequencies of {gamma}j values in different cell pairs were obtained as events of each size and are displayed as the means±standard errors of normalized {gamma}j values for all pairs of Cx43(+/+) and Cx43(–/–) cardiac myocytes. Histogram values were fitted with Gaussian distributions using Peakfit software (Jandel). In some experiments, data were acquired and analyzed using Pclamp6 programs (Axon Instruments, Sunnyvale, CA) [29].

2.7. Northern blot analysis
Total RNA was isolated from individual whole hearts as described [26]. Probes used were full length (1.3 kb) coding region of rat Cx43 (from E.C. Beyer, Washington University), rat Cx40 (from D.L. Paul, Harvard) and Cx45 (from K. Willecke, Bonn, Germany). 18S and 28S ribosomal RNA probes were from Ambion (Austin, TX); β-actin was generated by RT-PCR from mouse brain as described [26].

2.8. Western blot analyses
Hearts collected on ice in PBS from Cx43(+/+) and Cx43(+/–) adult mice were homogenized and sonicated and Western blots performed as described [26], using polyclonal Cx43 antibody from E.L. Hertzberg.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Northern blot analyses of connexin expression in Cx43(–/–), Cx43(+/–) and Cx43(+/+) littermates
To determine whether the absence of Cx43 led to compensatory expression of other cardiac connexins, Northern blots on hearts from each genotype were performed using connexin-specific probes (Fig. 1A). Densitometric scans for Cx40 and Cx43 levels normalized to β-actin in three litters are shown in Fig. 1B,C, and average values for Cx45 in two litters in Fig. 1D. Cx43 mRNA was absent in neonatal Cx43(–/–) heart and reduced by about 23% in Cx43(+/–); other connexin mRNA levels were similar among genotypes.


Figure 1
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  		Fig. 1 Northern blot analyses of connexin expression in neonatal Cx43(–/–), Cx43(+/–) and Cx43(+/+) WT mice. (A) Hearts from one litter were combined according to genotype and hybridized for Cx43, Cx40, Cx45 and β-actin. (B–D) Histograms of densitometric tracings of Northern blots from cardiac tissues in three litters for Cx43 and Cx40 and two litters for Cx45 after normalization to β-actin. Low but non-zero densitometric value for Cx43 in KO represents background.

 
3.2. Northern and Western blot analyses of Cx43 expression in Cx43(+/–) and Cx43(+/+) adults
Previous studies reported approximately 50% reduction in Cx43 mRNA levels in adult Cx43(+/–) hearts [31] and brains [26]. To examine both Cx43 mRNA and protein levels, hearts of mice of each genotype were bisected. Northern blot analyses on four samples are illustrated in Fig. 2A and normalized densitometric values are provided in Fig. 2B; mean normalized densitometric value was 2.1-fold higher in Cx43(+/+) than in Cx43(+/–) (p=0.026). Representative Western blots for four wildtype and three Cx(+/–) hearts are illustrated in Fig. 2C and densitometric values shown in Fig. 2D. Mean normalized densitometric value for Cx43 was 1.4-fold higher in eight Cx43(+/+) hearts than in seven heterozygous hearts (p=0.023). Thus, both Cx43 mRNA and protein levels are reduced in the hearts of Cx43(+/–) mice.


Figure 2
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  		Fig. 2 Comparison of Northern (A, B) and Western blots (C, D) of hearts from adult Cx43(+/–) and Cx43(+/+) mice. For the experiments illustrated, hearts of four age matched female mice of each genotype were divided for subsequent Northern and Western blot analyses (one heterozygote protein sample was lost). Although Northern blot analyses revealed variability, mean normalized densitometric values for these and three additional analyses were 0.47±0.12 for Cx43(+/–) and 1.0±0.1 for Cx43(+/+) mice (p= 0.026). For Western blots, Cx43 was more abundant in all Cx43(+/+) samples than in any of the Cx43(+/–) samples; mean normalized densitometric values for these and additional samples revealed that intensity was 0.73±0.03 for seven Cx43(+/–) samples (p=0.023).

 
3.3. Connexin expression patterns in cultured wildtype and Cx43-null cardiac myocytes
Immunostaining of cultured cardiac myocytes from wildtype mice with Cx43 antibodies revealed abundant linear and punctate staining as well as diffuse cytoplasmic immunoreactivity (Fig. 3A,B). Cx40 antibodies also recognized appositional membranes between wildtype myocytes (Fig. 3C,D), as did Cx45 immunostaining (Fig. 3E,F). Immunostaining of cultured cardiac myocytes from Cx43(–/–) mice did not detect Cx43 in these cells (Fig. 3G,H), whereas staining of appositional regions of the cells in cultures was apparent using both Cx40 (Fig. 3I,J) and Cx45 (Fig. 3K,L) antibodies. We conclude from these experiments that both Cx40 and Cx45 are components of gap junctions in neonatal cardiac myocytes from wildtype and Cx43-null mice.


Figure 3
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  		Fig. 3 Immunostaining for Cx43 (A, B, G, H), Cx40 (C, D, I, J) and Cx45 (E, F, K, L) in cardiac myocytes prepared from wildtype (A–F) and Cx43(–/–) mice (G–L). Cx43 was prominent at interfaces between wildtype myocytes (arrows, A) and also intracellularly (A, B), Cx43 was absent in Cx43(–/–) cells (G, H). Cx40 (C, D, I, J) and Cx45 (E, F, K, L) were detectable in cells cultured from both genotypes as linear appositional distributions (arrows, C, I, E, K).

 
3.4. Beat rate and rhythmicity in cultured cardiac myocytes from wildtype and Cx43(–/–) mice
Phase contrast observations of cultured cardiac myocytes from Cx43(+/+), Cx43(+/–) and Cx43(–/–) littermates revealed that cardiocytes began to beat spontaneously within 24 h after plating, with high synchrony in WT cultures at 48–72 h. In order to quantitatively compare synchrony and rate of spontaneous beating in cultures from littermates with different Cx43 genotypes, intracellular Ca2+ levels were measured using the ratiometric Ca2+ indicator Indo-1. Spontaneous changes in intracellular Ca2+ resolved relatively stable systolic and diastolic Ca2+ levels (Fig. 4) and permitted analysis of the mean frequency of beating and the average interbeat intervals. Such measurements obtained simultaneously from numerous cardiac myocytes within the same field are illustrated in Fig. 4A for Cx43(+/+) myocytes and in Fig. 4B for Cx43(–/–) littermates. These recordings show prominent differences. Calculations of the interbeat intervals (IBI) of 38 beating cell clusters from two litters of wildtype mice showed a mean IBI of 0.85±0.08 s, corresponding to a beat rate of about 71/min. Cx43(–/–) cardiocytes, however, beat more slowly, with an average IBI of 1.51±0.99 s (n=42), corresponding to a beat rate of about 40/min. Even more striking than this 1.75-fold difference in beat rate was the variability in IBI. Cell clusters and even adjacent cells from Cx43(–/–) hearts showed beating that was only poorly synchronized, in contrast to the virtual simultaneous beating throughout wildtype cultures. This variance or dispersion in IBI was 12 times higher in Cx43(–/–) than in Cx43(+/+) cardiocytes.


Figure 4
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  		Fig. 4 Synchronous contractions in wildtype (A) and Cx43 null (B) mouse heart cell cultures, revealed by fluctuations in Ca2+concentrations during beating, which were captured at 30-ms intervals using the ratiometric indicator Indo-1. Different symbols represent individual myocyte Ca2+ levels.

 
3.5. Lucifer Yellow permeability of gap junctions in wildtype and Cx43(–/–) cardiac myocytes
Lucifer Yellow dye transfer was used to compare gap junction permeance to small molecules in cardiac myocytes from Cx43(–/–), Cx43(+/–) and Cx43(+/+) littermates. Representative photomicrographs from one experiment are illustrated in Fig. 5A–D and a histogram displaying the number of cells to which dye spread in each cardiocyte genotype is presented in the panel to the right (Fig. 5E). Mean number of cells to which dye spread in 15 injections each in Cx43(–/–), Cx43(+/–) and Cx43(+/+) cultures was 0.6±0.2, 2.5±0.3 and 2.5±0.4, respectively; similar difference in coupling of Cx43(–/–) and Cx43(+/+) cultures was obtained in another experiment (2.1±0.4, n=11 vs. 0.3±0.2, n=30). The difference in strength of dye coupling between Cx43 null and both heterozygous and wildtype cardiocytes was statistically significant (p<0.01), whereas dye coupling strength in Cx43(+/–) and Cx43(+/+) myocytes was not. We conclude from these studies that dye coupling between Cx43 null myocytes is strongly impaired, whereas it is similarly strong in heterozygous and wildtype myocytes.


Figure 5
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  		Fig. 5 Coupling strength in WT and Cx43(–/–) myocytes. (A–E) Lucifer Yellow dye coupling in cultures of wildtype (A, C) and Cx43 knockout (B, D) heart cells obtained from sibling mice. As shown in the graph (E), dye coupling in knockout mouse heart cells was significantly less extensive than in wildtypes (p<0.05). Black arrowheads in C, D indicate the injected cell; white arrows in C indicate coupled neighbors. (F, G) Macroscopic junctional conductances measured in WT and Cx43(–/–) cardiocytes. High conductance values were more often measured in wildtype myocytes, whereas many Cx43(–/–) cardiac myocytes were coupled weakly or not at all, with junctional conductances of 0–2 nS. The average conductance of Cx43 (–/–) cell pairs was 4.2±0.6 nS (n=62; see histogram G), less than half the value of the wildtypes (12.5±3 nS, n=22) or heterozygotes (11.1±1.6 nS, n=20, not illustrated).

 
3.6. Macroscopic junctional conductance and its voltage sensitivity in pairs of cardiac myocytes from wildtype and Cx43(–/–) mice
Electrophysiological measurements were performed to investigate the functional aspects of junctional conductance between wildtype and between Cx43 null cardiac myocytes (Fig. 5F,G; mean values given in legend). As for dye coupling measurements, junctional conductances (gj) were significantly lower for Cx43(–/–) pairs than for either Cx43(+/–) or wildtypes, whereas heterozygotes did not differ significantly from wildtypes.

The dependence of the macroscopic junctional conductance (gj) on transjunctional voltage (Vj) was determined in Cx43(–/–) myocytes by applying >5-s pulses of increasing voltages to one cell of a pair and plotting gj (normalized to that obtained at small voltages; Gj) as a function of Vj. At low voltages, junctional current was stable during the pulses, whereas at higher voltages, junctional currents declined to reach steady state levels over a time course of several seconds or less (Fig. 6A). Even at the highest Vj values, however, a voltage insensitive component remained (gmin, 34). Mean Gj values obtained from five pairs of Cx43(–/–) cardiac myocytes are plotted as a function of Vj in Fig. 6B. These data points were compared to the Boltzmann equations obtained previously for gap junctions formed by cardiac connexins (Cx43: 19, Cx40: 32, and Cx45: 22). The differences in V0, A and gmin/gmax suggest that junctional conductance is slightly more sensitive to Vj in Cx43(–/–) than in wildtype cardiac myocytes. Presumably, this difference reflects a different complement of connexins forming the gap junctions between cardiac myocytes of the two genotypes.


Figure 6
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  		Fig. 6 The dependence of macroscopic junctional conductance (Gj) on transjunctional voltage (Vj) in Cx43 null cardiac myocytes. Top: Junctional currents in response to transjunctional voltage steps ±100 mV in 20-mV increments in a Cx43 null cell pair. Bottom: Steady state conductances (mean±S.E.M.) normalized to initial conductances (Gj) from five pairs of Cx43(–/–) myocytes are plotted as a function of transjunctional voltage. Lines represent Boltzmann curves reported previously for the three cardiac connexins expressed in mammalian cells [21,24,44].

 
3.7. Unitary conductances of gap junction channels in wildtype and Cx43(–/–) cardiac myocytes
Generally, gj was too high to resolve single channel currents and in order to do so, cells were exposed to 3 mM halothane, which reversibly reduced gj to reveal single channels. Examples of such single channel recordings are shown in Fig. 7A,B, with a driving force of 40 mV. Amplitudes of each channel opening or closing in six pairs of WT myocytes (two litters) and 17 pairs of Cx43(–/–) myocytes (four litters) were digitized and these values plotted in histograms shown in Fig. 7C,D. The histogram obtained from wildtype cell cultures (1872 events), when fitted to a Gaussian distribution, revealed peaks at 54.0±0.4, 96.3±1.5, 137.3±5.4 and 167.9±5.5 pS (mean±S.E.), with areas under the curves of respectively 40%, 43%, 11% and 6% (Fig. 7C). In Cx43(–/–) cultures, fitting of the amplitude histograms (2461 events) revealed two peaks, corresponding to 55.6±1.1 and 156.9±1.6 pS with areas of 35% and 65% (Fig. 7D); although not statistically separable, this larger peak appeared to have components centered at about 140 and 170–180 pS, similar to the amplitudes of the two larger peaks seen in wildtypes (compare Fig. 7C,D). When the histograms obtained from wildtype and Cx43 null myocytes are compared, the prominent difference is the absence in Cx43 null myocytes of events comprising the 96-pS population, representing 43% of the total events in wildtype cardiocytes.


Figure 7
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  		Fig. 7 Unitary conductances ({gamma}j) between pairs of WT and Cx43(–/–) cardiac myocytes. (A, B) Examples of unitary conductance measurements from Cx43 null cardiac myocytes, obtained under conditions when junctional conductance was reduced with halothane application. Note that both very large (A) and rather small (B) {gamma}j values were obtained. Transjunctional voltage in both recordings was 40 mV. (C, D) Histograms showing the relative frequency of events of unitary conductances at 40 mV transjunctional voltages recorded between wildtype (C) and Cx43 KO (D) cardiomyocyte pairs, where curves represent Gaussian best fits.

 
Because Cx43 is believed to contribute more substantially to junctional communication in ventricles than atria, we hypothesized that recordings from myocytes from these regions in Cx43 null mice might reveal functional expression of the other connexins. Cardiac myocytes were separately cultured from atrium and ventricle in Cx43(–/–) mice from three litters. Macroscopic voltage sensitivity was generally stronger and currents recorded at higher transjunctional voltages declined with slightly more rapid kinetics in ventricular than atrial Cx43(–/–) myocytes, as illustrated in Fig. 8A,B. Whereas unitary conductances recorded from ventricular cell pairs were generally small (40–55 pS illustrated in Fig. 8C), channels recorded in atrial cell pairs generally exhibited currents corresponding to unitary conductance of >150 pS (slope conductance illustrated in Fig. 8D is 170 pS). Nevertheless, these features were not absolute; {gamma}j values about 120 pS were occasionally seen in Cx43(–/–) ventricular myocytes, and 40–55 pS channels were observed in many of the atrial myocyte pairs. As was the case for recordings obtained from whole heart cultures, a channel population corresponding to 95 pS was totally lacking from cell pairs obtained from both cardiac regions from Cx43 null mice.


Figure 8
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  		Fig. 8 Properties of gap junctions between ventricular (A, C) and atrial (B, D) myocytes from Cx43(–/–) mice. (A) Representative recording from Cx43(–/–) ventricular myocyte pair in response to 15-mV incremental voltage steps from 0 to ±90 mV; note rapid current relaxations in response to 45<Vj<–45 mV. (B) Macroscopic junctional currents in a pair of atrial myocytes in response to 20-mV steps from 0 to ±100 mV exhibit slower relaxations, beginning at 60<Vj<–60 mV. (C) Single channel currents recorded in a Cx43(–/–) ventricular myocyte pair in response to Vj=100 mV; note that events are generally ~50–55 pS in amplitude. (D) Recording from Cx43(–/–) atrial myocyte pair in response to ±100 mV Vj ramp, showing the large amplitude unitary current fluctuations characteristic of these cells; slope conductance indicated by line is 170 pS.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
Mice in which Cx43 has been genetically ablated through homologous recombination [25] offer the opportunity to evaluate the extent to which Cx43 contributes to normal propagation as well as to evaluate the functional properties of the other connexins expressed in the heart. Cx43-deficient mice die at birth, due to a developmental defect leading to hypertrophy and hyperplasia of the right ventricular outflow tract. Despite this abnormality, however, the hearts of these animals still beat rather rhythmically, though not synchronously throughout the tissue, suggesting that gap junction proteins in addition to Cx43 may partially compensate for the loss of intercellular connections.

Our Northern blot results indicate that neonatal myocytes from each genotype expressed Cx40 and Cx45 mRNA, with no major changes in compensation for the lack of Cx43 detected in the Cx43 null mice. Quantitative studies on adult hearts showed that level of Cx43 mRNA in the heterozygotes was 47% that expressed in wildtypes, agreeing with reports of an approximately two-fold difference in Cx43 mRNA levels in wildtype and heterozygous hearts [31] and in adult brain [26], as is expected based on gene dosage. The Western blot analyses on adult hearts confirmed the approximately two-fold differences in levels of Cx43 protein between heterozygous and wildtype mouse hearts.

The functional impact of the 50% reduction in Cx43 on impulse propagation in heterozygotes has become controversial. Although slowing of ventricular conduction was reported using electrical measurements [31], optical mapping has not confirmed this [32]. Moreover, the high safety factor for conduction in the normal heart [33] would not be expected to be compromised substantially by a 50% reduction in gj (for further discussion, see Refs. [34,35]). Cx43(–/–)l cardiocytes were found to beat more irregularly and on average more slowly than wildtypes. Treatment with agents that upregulate Cx43 has been reported to accelerate beating in wildtype myocytes [36], and both the slowing and asynchrony in Cx43(–/–) cultures might be due to less efficient entrainment of beating as a consequence of reduced coupling to endogenous pacemaking cells in the cultures.

Lucifer Yellow dye transfer, which is normally strong between wildtype myocytes, was virtually absent from Cx43(–/–) mice. Although the loss of dye coupling is attributable in part to the reduction in junctional conductance (see below), this deficiency may be exaggerated by the properties of the remaining gap junction channels, because Cx40 and Cx45 channels are less permeant to anions than is Cx43 [23]. The implications of this charge selectivity for the function of the Cx43(–/–) heart may be to restrict diffusion of negatively charged second messenger molecules (see below).

Macroscopic conductance between Cx43(–/–) cardiocytes was found to be less than half that in wildtype cardiocytes, supporting the Northern blot results showing lack of compensatory expression of other cardiac connexins. Recordings of single channel openings and closings revealed that both wildtype and Cx43(–/–) myocytes exhibited a wide range of unitary conductance values. However, Gaussian fitting of the amplitude histograms of the channel sizes revealed discrete populations of channels in both genotypes. For wildtype myocytes, 43% of the events were in the 96-pS conductance category. Studies on gap junction channels expressed exogenously indicate that Cx43 channels display mainstate conductances of about 90 and 110 pS and substrate conductance of about 30 pS [37]. Because our experiments were performed using low driving forces, at which substrate currents are rarely detected, we interpret the 96-pS events in wildtype myocytes as most likely representing mainstate conductances of homomeric Cx43 channels, which are absent in the Cx43 null mice. Expression of Cx40 in mammalian cells leads to channels with mainstate conductances in the 135–180-pS range and substrate conductances about 40–60 pS [38–40]. Such events predominate in the Cx43 null myocytes, and it is possible that they account for most of the non-Cx43 activity in wildtypes.

There are several other possible explanations for the channel sizes recorded in these studies, involving the potential formation of heterotypic or heteromeric hemichannels. For example, Cx43 readily forms heterotypic channels with homomeric Cx45 hemichannels, with perhaps an even higher affinity for each other than for themselves; the unitary conductance of such hybrids is about 50 pS [22]. Moreover, Cx43 and Cx40, which were initially reported to be incompatible for heterotypic pairing [16,17,41] have more recently been shown to form such channels, with unitary conductances of about 60 and 100 pS, when the Cx43 or Cx40 side was stepped positively [18]. Whether Cx45/Cx40 hybrids form has not yet been demonstrated; however, the predicted conductance of such a pairing would be about 50–60 pS, and channels of this size recorded in cultured cardiac myocytes have been interpreted as possibly representing such heterotypic channels [42]. It is interesting that the unitary conductances of such heterotypic combinations are in the range of the smallest unitary conductances observed in these studies on wildtype and Cx43(–/–) myocytes. An additional possibility is that connexins may mix within a hemichannel to form heteromeric channels, with a wide variety of predicted unitary conductances [43–45].

The deficiencies in intercellular coupling that we have quantified in cardiac myocytes from Cx43(–/–) mice indicate that dye coupling is more profoundly disturbed than is junctional conductance. These mice are characterized by a profound hypertrophy of the right ventricular outflow tract, leading to pulmonary stenosis and perinatal lethality [24]. While it is conceivable that abnormal impulse propagation in the developing ventricles might contribute to abnormal alignment of the ventricular fibres, the severity of this abnormality implies the disruption of an early event in ontogeny. Using mouse mutants in which nonfunctional Cx43 was targeted to ventricular precursor cells, Cecilia Lo's group has suggested that the causative defect is retarded migration of neural crest derivatives [46,47]. Thus, the missing function in Cx43(–/–) cells that would have been provided by Cx43 gap junctions may be the signal that coordinates migration. Although the identity of such a signal is unknown, the profoundly reduced Lucifer Yellow permeability that we have found in Cx43(–/–) cardiocytes suggests that intercellular diffusion of anionic morphogens might be impaired. Conceivably, the loss of such signaling could have catastrophic consequences, leading to the retarded migration of requisite components of the cardiovascular system to their appropriate sites.


   Acknowledgements
 
Supported in part by NIH grants HL38449, HL73732, and NS41282 and Beatrice A. Parvin Grant-in-Aid and Participating Laboratory Awards from the New York Chapter of the American Heart Association to DCS and MJV.


   Notes
 
Time for primary review 31 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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