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Keywords:

  • cardiac muscle;
  • connexin45;
  • embryonic stem cell;
  • gap junction

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

In early-stage heart, the cardiac impulse does not propagate through the specialized conduction system but spreads from myocyte to myocyte. We hypothesized that the gap junction protein connexin45 (Cx45) regulates early-stage contractions, because it is the only gap junction protein described in early hearts. Cx45-deficient (Cx45−/−) mice die of heart failure, concomitantly displaying other complex defects in the cardiovascular system. In order to determine the specific cardiac muscular function of Cx45, we created Cx45−/− embryonic stem (ES) cells to be differentiated into cardiac muscle in vitro. Unlike the coordinated contractions of wild-type cells, differentiated Cx45−/− cardiac myocytes showed high and irregular pulsation rates. Alterations of the electrophysiological properties of the Cx45−/− cardiac myocytes were indicated both by extracellular recording on planar multielectrode array probes and by intracellular Ca2+ recording of the fluorescent Ca2+ indicator fura-2. The in vitro system minimizes an influence of hemodynamic factors that complicate the phenotypes of Cx45−/− mice. Our results indicate that Cx45 is an essential connexin for coordinated conduction through early cardiac myocytes. © 2004 Wiley-Liss, Inc.

There are many forms of gap junction structures among vertebrate cardiac myocytes (Shibata and Yamamoto, 1979). Various configurations of the gap junction may be related to functional differences among species. Cx45 is one of the three gap junction proteins in mammalian cardiac myocytes (Lo, 2000). It has unique characteristics of low unitary conductance, strong voltage dependence, selective permeability, and pH sensitivity (Veenstra et al., 1994; Elfgang et al., 1995; Hermans et al., 1995; Moreno et al., 1995; Steiner and Ebihara, 1996; Barrio et al., 1997; Peracchia et al., 2003). It is expressed in almost all tissues during early embryogenesis, and then it undergoes consistent downregulation (Alcoléa et al., 1999; Kumai et al., 2000). Following this downregulation, Cx45 remains expressed at high levels in the cardiac conduction system (Coppen et al., 1999). In early cardiogenesis, Cx45 seems the only active connexin in the myocardium. Cx45−/− mice display conduction block in primitive cardiac myocytes (Kumai et al., 2000). However, other abnormalities related to endocardial cushion formation and vascular development have also been described (Krüger et al., 2000; Kumai et al., 2000). Impaired cardiac function should have a vicious effect on normal development of the vascular system, as the cardiovascular system constitutes a closed circuit. In addition, these structures are indirectly connected by a growth factor network (Nishii et al., 2001).

An experimental system that would enable analysis of the specific cardiac muscular function of Cx45 is therefore needed. As one approach, we have reported that mice lacking Cx45 conditionally in cardiac myocytes display a similar lethal phenotype to that of germline Cx45−/− mice (Nishii et al., 2003). This result indicates that one of the primary roles of Cx45 is undoubtedly to coordinate myocardial contractions in the embryo.

ES cells are able to differentiate into cardiac myocytes as a part of the amorphous embryoid body (Boheler et al., 2002). The differentiation course mimics normal development in vivo. Particularly, ES cell-derived cardiac myocytes show expression patterns of three cardiac gap junction proteins similar to embryonic ones in vivo (Oyamada et al., 1996). One of the probable advantages of this system is an ability to produce genetically modified cardiac myocytes. If disruption of a gene of interest causes early embryonic lethality, then this system provides a valuable opportunity for us to know the cardiac muscular function of the gene. In the case of Cx45, the in vitro differentiation system carries many advantages over analyzing the mutant embryos in vivo. Hydromechanical effect can be discounted and ES cells as proliferate, there is an abundance of cardiac myocytes available for experiments. Finally, this model is readily analyzed in culture dishes. We report here that Cx45−/− ES cell-derived cardiac myocytes display a conduction abnormality indicative of a requirement for Cx45 in early cardiac myocytes.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

In order to create an ES cell line lacking both of the Cx45 loci, we performed three rounds of selection in ES cells. The first step was a conventional gene targeting against the Cx45 locus of the wild-type CCE ES cell (Fig. 1B, lane 3). The second was neo-removal by transient expression of the Cre recombinase (lane 4) (Kumai et al., 2000). The third step was the retargeting of the line with the same targeting vector to replace the remaining wild-type allele (lane 1). This procedure minimized exposure to the selection drug G418.

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Figure 1. Characterization of Cx45−/− ES cells. A: Cx45-targeting strategy. Detailed descriptions on the targeting construct have been described in Kumai et al. (2000). The targeting vector was introduced into the neominus variant of the Cx45+/− ES cells. Resultant recombinant clones were selected by G418. Sc, SacI. B: Southern blot analysis of SacI-digested genomic DNAs isolated from ES cells with a 3′ flanking probe. Cx45−/− (lane 1), wild-type (lane 2), neo-plus Cx45+/− (lane 3), and neo-minus Cx45+/− (lane 4). C: Immunofluorescence representing the absence of Cx45 in Cx45−/− ES cells. The ES cells expressed another major connexin, Cx43. Composite images are presented in the third column. The signals surrounding the ES cell colonies are the autofluorescence from feeder cells. D: Blastocysts injected with the Cx45−/− ES cells (E3.5) were cultured in microdrops, photographed daily, then fixed and stained with X-gal to reveal the injected cells' fate. Bars = 50 μm.

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The ES cell differentiation procedure followed the reported protocol with minor modifications (Oyamada et al., 1996; Boheler et al., 2002). In brief, ES cells were cultivated in hanging drops in an ES cell culture medium without leukemia-inhibitory factor. The day when the hanging drop culture was started was counted as day 0. On day 3, the embryoid bodies were transferred onto cover glasses or planar multielectrode array probes (Panasonic, Tokyo, Japan) in culture plates. The differentiated cardiac myocytes started beating from day 12 onward (Fig. 2A). Days 12–14 were chosen for experiments in this study because the differentiated cardiac myocytes seemed to represent the ones roughly at the lethal stage of the Cx45−/− mice (Krüger et al., 2000; Kumai et al., 2000; Boheler et al., 2002).

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Figure 2. Differentiation of ES cells into cardiac myocytes. A: Numbers of embryoid bodies with contracting cardiac myocytes increased consistently from day 12 onward in our experimental system. In particular, they increased greatly between days 14 and 15. The percentages are presented as line graphs. Numbers indicate days after the differentiation procedure took place. B: RT-PCR analysis of whole embryoid bodies. CCE, wild-type undifferentiated ES cells. Numbers indicate differentiation days as in A. Faint Cx45 signals in the days 6, 8, and 10 Cx45−/− embryoid bodies are likely due to contaminated feeder cells. C: Cx43 and cardiac troponin T double immunostaining at day 13. Cx43 was detected diffusely in the embryoid bodies, while cardiac troponin T was confined to the sarcomeres. Bar = 50 μm. D: Electron microscopy at day 13 showing the early-stage immature cardiac myocytes where bundles of myofibrils (arrows) were present in both genotypes. Bar = 0.2 μm.

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Southern blotting, immunofluorescence, electron microscopy, X-gal staining, and RT-PCR were performed as previously reported (Kumai et al., 2000). Primer sequences for α- and β-MHC have been described (Robbins et al., 1990; Oyamada et al., 1996). Here, 100 ng of total RNA was used for 30 cycles of amplification for each reaction. Blastocyst culture was done as described previously (Nishii et al., 1999), with the ES cell culture medium lacking leukemia inhibitory factor to promote differentiation.

For measurements of [Ca2+]i, cells grown onto coverslips were loaded for 20–240 min at 37°C with fura-2 AM (0.5 μmol/L) in the culture medium (Goto et al., 1998). The coverslips were mounted on a thermostated chamber at 37°C located on the stage of an inverted microscope and continuously perfused with Krebs solution oxygenated with 95% O2/5% CO2. Contracting areas were magnified, and fura-2 fluorescence of the field (i.e., dozens of cells were included in the field) was detected. [Ca2+]i was monitored as the ratios of fluorescence emitted at 510 nm with alternating excitation at 340 and 380 nm using a fluorescence and contractility recording system (MyoCam and Photo-Multiplier system with Galvo-Driven HyperSwitch Dual Excitation Light Source; IonOptix, Milton, MA).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

Previously, we established a germline heterozygous Cx45-deficient (Cx45+/−) ES cell clone (Kumai et al., 2000). In this study, the clone was retargeted with the same targeting vector (Fig. 1A and B). The resultant Cx45−/− ES cell clone lacked Cx45 immunoreactivity, but expressed Cx43 (Fig. 1C). The cells were indistinguishable from the wild-type ES cells in electron microscopy, had the normal chromosome number of 40, and became embryoid bodies consisting of all three germ layers (not shown). Making chimeric mice was our first aim, because conduction block was expected at the borders of the mosaic in the heart. However, neither live mice nor postimplantation embryos were found with any ES cell contribution through extensive blastocyst injections. In order to examine the fate of injected cells, a blastocyst culture system was carried out (Fig. 1D). We found that Cx45−/− ES cells never got mixed with recipient inner cell masses (n = 36). Cx45+/− ES cells also displayed a similar incompatibility, though 3 out of 17 injected cases successfully yielded mixed inner cell masses (not shown). The difference was significant (P = 0.005, χ2-test).

The established Cx45−/− ES cells were subjected to in vitro differentiation into cardiac myocytes (Fig. 2). More than 500 Cx45−/− embryoid bodies, as well as wild-type Cx45+/+ embryoid bodies, were prepared for experiments. Spontaneously beating cardiac myocytes were found at equivalent efficiency in both genotypes, and the timing of initiation of contraction was not different (Fig. 2A). In RT-PCR, they displayed almost the same expression patterns of cardiac markers and connexins, except for Cx45 and its reporter nls-lacZ (Fig. 2B). The cells equally expressed Cx43 and cardiac troponin T (Fig. 2C) and had similar ultrastructure (Fig. 2D), gene expression patterns, and morphology.

Contractions of the Cx45−/− differentiated cardiac myocytes, however, appeared to be peculiar (see supplemental video clips available at: http://www.interscience.wiley.com/jpages/0003-276X/suppmat). In wild-type cardiac myocytes, contractions were coordinated within beating cardiac cell masses. Though we found multiple beating cell masses in 5–10% of the embryoid bodies, they were separated for more than one cell diameter from one another, and the contractions were coordinated within each cell mass in the wild-type cardiac myocytes. In contrast, when the beating Cx45−/− cardiac myocytes were magnified, we could always find cells contracting irrespective of abutting cells, even if the contractions of the beating cell mass looked coordinated as a whole. Additionally, rates of contraction of Cx45−/− cardiac myocytes were high and irregular, indicating a change of electrophysiological property, which we further pursued.

We performed extracellular field potential recording on planar multielectrode array probes (Igelmund et al., 1999; Oka et al., 1999). Three independent traces on each genotype showed similar results and representative traces are presented in Figure 3. Irregular and disorganized electrical activities of Cx45−/− cardiac myocytes were noted (Fig. 3B). In the wild-type cardiac myocytes, depolarization of cells on electrodes 1 and 2 related with simultaneous reverse potentials on other electrodes (Fig. 3C). This may indicate that cells on those electrodes are electrically coupled (Mitzdorf, 1985). On the other hand, in the Cx45−/− cardiac myocytes, depolarization on electrodes 6 and 7 had no effect on other electrodes. This may indicate electrical uncoupling. [Ca2+]i levels of the beating cardiac myocytes also indicated difference in their ionic environment (Fig. 4). The pulse rate was high in Cx45−/− cardiac myocytes. The wave forms of the Cx45−/− traces were broader, which was revealed by comparing T1/2 value of each genotype.

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Figure 3. Surface field potential data recorded on planar multielectrode array probes. A: Representative photograph of a plated embryoid body on a planar multielectrode array probe. Contracting cardiac myocytes appear preferentially around the core of the embryoid bodies (dark shadow in this photograph). Each electrode is laid 300 μm apart. B: A part of recordings of each electrode is presented. Locations where each trace is presented correspond to the place where each electrode was set. Note that the wild-type field potentials were coordinated among the electrodes, while the Cx45−/− potentials were not. At least two distinct contracting groups of cells are discerned in the Cx45−/− recording. C: Magnified views of graphs 1–10 marked in B. Field potentials triggered by depolarization on electrodes 1 and 2 (arrow in Wild) corresponded to simultaneous reverse potentials on electrodes 3–5 in the wild-type recording. In contrast, field potentials triggered by depolarization on electrodes 6 and 7 (arrow in Cx45−/−) did not provoke any change in electrodes 8–10 in the Cx45−/− recording. When cells on electrodes 4, 5, or 9 depolarized (arrowheads), similar differences were noted.

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Figure 4. [Ca2+]i recording by fura-2. Top: Representative traces of 340/380 ratios of fura-2 fluorescence. Absolute values of the signals were similar in both genotypes but aligned for simplicity. Bottom: Scatter diagram of pulse rate versus T1/2 value of recording on each embryoid body. T1/2 represents a duration in which [Ca2+]i exceeds its half-maximum. Each value showed significant difference (pulse: P = 0.003; T1/2: P = 0.00002; two-tailed Student's t-test).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

In this study, Cx45−/− ES cells were generated and differentiated into cardiac myocytes. Contractions of differentiated Cx45−/− cardiac myocytes were irregular, and their beating rates were higher than control myocytes. These parameters were examined by extracellular field potential and intracellular Ca2+ recordings.

The Cx45−/− ES cells were indistinguishable from the wild-type counterparts in many aspects. They had the normal chromosome number and could be differentiated into all three germ layers. Nevertheless, these cells could not be integrated into chimeras (Fig. 1D). We have reported that only one out of eight Cx45+/− ES cell clones went into germline (Kumai et al., 2000). The chimerism was not high (not shown). Though it is speculative at present, there may be a protein whose expression is controlled by Cx45 in a gene dosage manner and whose function is important in cell adhesion.

Embryonic tissues strongly express Cx43 as well as Cx45, and most of the function of Cx45 may be compensated by Cx43. We found the differentiated cardiac myocytes mostly expressed Cx43 (Fig. 2C). Therefore, one possibility is that the conduction abnormality of Cx45−/− cardiac myocytes may indicate a unique regulatory function of Cx45, or Cx43-Cx45 heterotypic/heteromeric channels (Elenes et al., 2001; Martinez et al., 2002). Alternatively, lack of Cx45 may lead to decrease of total amount of functional gap junctions, causing conduction block. We found many patches of Cx43-absent areas (Fig. 2C). The abnormal conduction of Cx45−/− cardiac myocytes may preferentially occur in such areas.

Detecting difference of the wave forms in field potential recording may be useful for coarse assessment of electrical coupling because the technique is easily carried out. Moreover, long-term recording is possible because it does not injure the cells. Further, the difference in [Ca2+]i levels reported here may indicate a change in electrical coupling. The broader wave form of Cx45−/− traces suggests at least a change of ionic environment, which can affect contraction rates (Fig. 4). Further characterization of these electrophysiological properties is beyond the scope of this study.

The broadness of the [Ca2+]i levels of Cx45−/− cardiac myocytes seems more evident in cells with lower beating rates (Fig. 4, bottom). Slow beating is a character of primitive cardiac myocytes (Boheler et al., 2002). Therefore, the data may indicate a requirement of Cx45 in cardiac myocytes at early embryonic stages.

Spontaneous activity of early-stage cardiac myocytes is not generated by transmembrane ion currents, but by intracellular Ca2+ oscillations (Viatchenko-Karpinski et al., 1999). Here, the importance of gap junctional communication in early cardiac myocytes is clear. Together with our recent report on conditional Cx45−/− mice in cardiac myocytes (Nishii et al., 2003), the importance of Cx45 was confirmed both in vivo and in vitro. The Cx45−/− ES cell differentiation system is especially suited for revealing the precise mechanism of arrhythmogenesis in vitro.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

The authors thank Dr. Ken Shimono for his constructive comments.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

The Supplementary Material referred to in this article can be found at the Anatomical Record website ( http://www.interscience.wiley.com/jpages/0003-276X/suppmat ).

FilenameFormatSizeDescription
Cx45-KO.mov13341KSupporting Information file Cx45-KO.mov
wild.mov13342KSupporting Information file wild.mov

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