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(Circulation. 2001;104:1194.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Heterogeneous Expression of Gap Junction Channels in the Heart Leads to Conduction Defects and Ventricular Dysfunction

David E. Gutstein, MD; Gregory E. Morley, PhD; Dhananjay Vaidya, PhD; Fangyu Liu, BS; Franklin L. Chen, BA; Heidi Stuhlmann, PhD; Glenn I. Fishman, MD

From the Section of Myocardial Biology and Departments of Medicine (D.E.G., G.E.M., F.L., F.L.C., G.I.F.), Physiology and Biophysics (G.I.F.), and Biochemistry and Molecular Biology (G.I.F.), Mount Sinai School of Medicine, New York, NY; the Department of Pharmacology, SUNY Upstate Medical University, Syracuse, NY (D.V.); and the Department of Vascular Biology, The Scripps Research Institute, La Jolla, Calif (H.S.).

Correspondence to Glenn I. Fishman, MD, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1269, New York, NY 10029. E-mail fishmg01{at}doc.mssm.edu

	Abstract

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Background—— Heterogeneous remodeling of gap junctions is observed in many forms of heart disease. The consequent loss of synchronous ventricular activation has been hypothesized to result in diminished cardiac performance. To directly test this hypothesis, we designed a murine model of heterogeneous gap junction channel expression.

Methods and Results—— We generated chimeric mice formed from connexin43 (Cx43)-deficient embryonic stem cells and wild-type or genetically marked ROSA26 recipient blastocysts. Chimeric mice developed normally, without histological evidence of myocardial fibrosis or hypertrophy. Heterogeneous Cx43 expression resulted in conduction defects, however, as well as markedly depressed contractile function. Optical mapping of chimeric hearts by use of voltage-sensitive dyes revealed highly irregular epicardial conduction patterns, quantified as significantly greater negative curvature of the activation wave front (-1.86±0.40 mm in chimeric mice versus -0.86±0.098 mm in controls; P<0.01; n=6 for each group). Echocardiographic studies demonstrated significantly reduced fractional shortening in chimeric mice (26.6±2.3% versus 36.5±1.6% in age-matched 129/SvxC57BL/6F1 wild-type controls; P<0.05).

Conclusions—— These data suggest that heterogeneous Cx43 expression, by perturbing the normal pattern of coordinated myocardial excitation, may directly depress cardiac performance.

Key Words: ion channels • contractility • conduction • genes • arrhythmia

	Introduction

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Gap junctions play a pivotal role in the coordinated excitation of the heart.¹ Formed from the assembly of a family of proteins known as connexins, gap junction channels in the myocardium are preferentially targeted to the intercalated disks between adjacent myocytes and account in part for anisotropic conduction in normal cardiac tissue.² In a wide spectrum of cardiac disease states, abnormalities in connexin subcellular localization is observed.^3–10 Increasing experimental evidence, including recent murine genetic studies, links this pathological remodeling of gap junctions to cardiac conduction abnormalities and rhythm disturbances.^8,11,12

Remodeling of gap junctions is often focal, a spatial pattern that might result in discrete areas of conduction defects within the myocardium.^7,13 Although experimental data are lacking, it has been hypothesized that remodeling could disrupt wave-front propagation, interfere with coordination of myocyte contraction, and diminish contractile performance.^7,14

Accordingly, in this study, we designed a model of heterogeneous gap junction expression in the heart to test the hypothesis that loss of synchronous ventricular activation diminishes cardiac contractile performance. We generated chimeric mice, formed from connexin 43 (Cx43)-deficient embryonic stem (ES) cells and wild-type or genetically marked ROSA26 recipient blastocysts, to model the heterogeneous pattern of gap junction expression typical of diseased myocardium. In contrast to the uniform conduction slowing in Cx43 conditional knockout mice,¹² optical maps of chimeric hearts with voltage-sensitive dye showed highly irregular epicardial activation profiles. Moreover, chimeric mice developed significant contractile defects, supporting the hypothesis that loss of synchronous electrical activation of the ventricular myocardium from gap junction remodeling leads to systolic dysfunction.

	Methods

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Gene Targeting and Production of Cx43^-/- Chimeric Mice
The generation of Cx43^+/flox ES cells (129/Sv) harboring a "floxed" Cx43 allele has been described previously.¹² To permanently inactivate this allele, we transiently expressed a Cre-GFP fusion protein¹⁵ in these Cx43^+/flox ES cells and excised the Cx43 open reading frame. Subsequently, a GFP-Neo fusion targeting construct was "knocked in" to the remaining wild-type Cx43 allele.

Western Blot Analysis
Western blot analyses were performed with a rabbit polyclonal antibody to Cx43 and a mouse monoclonal antibody to tubulin (Zymed Laboratories, Inc) as previously described.^12,16

Histology
Hearts were fixed in 4% paraformaldehyde and embedded in paraffin. Sections were stained with modified Masson’s trichrome or hematoxylin and eosin.

Detection of ß-Galactosidase Expression by Whole-Organ X-Gal Staining
Cardiac chimerism with ROSA26 host embryos was evaluated by whole-organ X-gal staining as previously described.^17,18

Immunofluorescence and Confocal Analysis
Simultaneous immunodetection of Cx43 and visualization of myocyte borders within cardiac tissue with wheat-germ agglutinin was performed on frozen sections as previously described.^12,19

Optical Mapping of Ventricular Activation
High-resolution optical mapping of epicardial activation was performed with a voltage-sensitive dye (di-4-ANEPPS, Molecular Probes) in the absence of any motion reduction techniques as previously described.^12,20,21

Conduction Velocity and Wave-Front Curvature
Conduction velocities were calculated as previously described.²⁰ Wave-front curvature was calculated as follows. Unit vectors in the direction of local conduction are perpendicular to the local isochrones. According to the curvature theorem,²² the divergence of this unit vector field is equal to the local curvature of the wave front. Partial derivatives of this unit vector field were calculated by decomposing the unit vectors into their x and y components (U_x, U_y). The spatial derivatives of these components were taken by the methods described for calculating velocity vectors above. The curvature () is given by

High positive curvatures are expected in all hearts near the electrode during epicardial pacing. High negative curvatures, however, are expected only in areas in which the activation wave front negotiates an obstacle or where wave fronts collide. Hence, negative curvatures were quantified to reveal the presence of obstacles in the heart. Pixels with negative curvature were grouped in 0.1-mm bins of curvature, and a cumulative histogram of the negative curvatures was constructed. The value that accounted for 95% of the cumulative histogram was used as a measure of negative curvature.

Echocardiography
Echocardiography was performed as previously described at 3 months of age in wild-type 129/SvxC57BL/6 controls, Cx43^-/- chimeric mice with visible coat chimerism, and their littermates with no visible coat chimerism.¹² As an additional control, echocardiograms were also performed on 13-month-old chimeric mice formed from Cx43^+/flox (Cx43-expressing) 129/Sv ES cells injected into C57BL/6 host blastocysts. Measurements were performed online in a blinded fashion. Fractional shortening, left ventricular volumes, and ejection fraction were calculated as previously described.^23,24

Statistics
Data are expressed as mean±SEM. Conduction parameters were compared by a 2-tailed, unpaired Student’s t test (Microsoft Excel). Echocardiographic parameters were compared by ANOVA, followed by Fisher’s protected least significant difference test (StatView, SAS Institute). Because the intraventricular dimensions, volumes, fractional shortening, and ejection fraction are derived from only 2 independently measured parameters, we chose to test the intraventricular dimension in systole and fractional shortening for significance. Probability values of P<0.05 were considered statistically significant.

	Results

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Generation of Cx43^-/- Chimeric Mice
To generate Cx43-null ES cells, both alleles of the Cx43 gene were sequentially targeted by homologous recombination by use of a strategy diagrammed in Figure 1, A and B. The correct targeting events were confirmed by Southern blotting (Figure 1C), and the absence of Cx43 expression was demonstrated by Western analysis (Figure 1D). Several correctly targeted Cx43^-/- ES cell clones were identified, and 2 such clones were used in this study.

View larger version (34K): [in this window] [in a new window]   Figure 1. Generation of Cx43-/- chimeric mice. A, Cre recombinase-mediated recombination in floxed Cx43 allele. NcoI polymorphisms on targeted, floxed allele (top construct) and floxed-out allele after Cre-mediated deletion of floxed region (bottom construct). Neo indicates neomycin; ORF, open reading frame. B, GFP-Neo knock-in targeting vector and strategy for generating homologous recombination. GFP-Neo fusion construct was used to replace native Cx43 sequence, eliminating Cx43 expression in this allele while conferring Neo resistance. IVS indicates intervening sequence; GFP, green fluorescent protein. C, Southern blotting of ES cell clones targeted with mutated Cx43 genomic constructs. Lanes contain NcoI-digested genomic DNA from wild-type (lane 1), Cx43flox/+ (lane 2), Cx43flox/- (lane 3), and 2 separate clones of Cx43-/-(GFPneo/FO) targeted ES cells (lanes 4 and 5). Wild-type (WT) allele produces a 6.5-kb band; floxed (Fl) allele a 5.4-kb band; floxed-out (FO) band is 4.3 kb; and GFP-Neo (GN) knock-in NcoI polymorphism is 3.4 kb. D, Western blot of Cx43 protein expression in wild-type, Cx43+/-, and Cx43-/- ES cell clones. Lane 1, protein from wild-type ES cells; lane 2, protein from Cx43+/-(+/GFPneo) ES cell clone; lanes 3 and 4, protein from Cx43-/-(GFPneo/FO) ES cell clones; 30 µg/lane of total protein was used. Blotting for ß-tubulin was performed on same membrane to indicate relative loading. E, Southern blotting of genomic tail DNA samples from Cx43-/-(GFPneo/FO) chimeric mice. In addition to wild-type NcoI polymorphisms in chimeric samples (lanes 1 and 3), bands from Cx43-null alleles are also seen (floxed-out and GFP-Neo bands). Only wild-type band is seen in sample from nonchimeric littermate (lane 2). F, Southern blotting of NcoI-digested genomic DNA from tail (T), heart (H), and liver (L) of a Cx43-/-(GFPneo/FO) chimeric mouse. Bands from wild-type and Cx43-null loci are seen in each lane.

Initial blastocyst injections were performed with C57BL/6 host embryos, allowing us to estimate the extent of chimerism by coat color. These injections resulted in 30 viable pups. Of these offspring, 7 had no visible coat chimerism, whereas 23 were visibly chimeric. The extent of chimerism estimated by coat color correlated well with results from Southern blotting of genomic DNA prepared from tail biopsies (Figure 1E). Within individual chimeric mice, the extent of chimerism was similar in tail, heart, and liver (Figure 1F).

To visualize the extent of cardiac chimerism, additional blastocyst injections were performed with ROSA-26 host embryos, which express ß-galactosidase in most cell types, allowing for whole-organ X-gal staining of the heart.²⁵ Compared with an adult nonchimeric ROSA-26 mouse heart, which appeared uniformly blue (Figure 2A), staining of a heart from a chimeric mouse derived from Cx43^-/- ES cells and ROSA-26 host embryos (Cx43^+/+) revealed a mosaic appearance, which was readily apparent on the epicardial surface of the heart (Figure 2B).

View larger version (63K): [in this window] [in a new window]   Figure 2. Visualization of cardiac chimerism. Whole-organ X-gal-stained ROSA-26 (A) and Cx43-/- chimeric hearts (B). Cx43-/- ES cells were injected into recipient blastocysts that carry ROSA-26 ß-galactosidase transgene. Blastocysts were then reimplanted into pseudopregnant females that carried chimeric embryos to term. Blue staining is evident in areas derived from ROSA-26 transgenic blastocyst. Areas without staining appear yellow and are derived from Cx43-/- ES cells.

To visualize chimerism at the cellular level, immunostaining was performed for Cx43, and the sarcolemma was demarcated with wheat germ agglutinin.¹⁹ Wild-type hearts showed the typical punctate pattern of Cx43 staining, localized at the intercalated disks (Figure 3, A and C). In chimeric hearts (Figure 3, B and D), patches of cardiac muscle with a wild-type staining pattern were adjacent to regions devoid of Cx43 staining, consistent with the heterogeneity visualized by X-gal staining.

View larger version (114K): [in this window] [in a new window]   Figure 3. Immunofluorescent staining for Cx43 in wild-type and Cx43-/- chimeric hearts. Hearts from wild-type (A, x10; C, x40) and chimeric (B, x10; D, x40) mice produced by injecting Cx43-/- ES cells into C57BL/6 recipient blastocysts were removed and frozen in OCT freezing medium. Sections were stained for Cx43 (green fluorescent stain), counterstained with wheat germ agglutinin (red fluorescent stain), and imaged with confocal microscopy. A wild-type staining pattern with abundant Cx43 signal mainly at intercalated disk is evident in A and C and throughout wild-type hearts. Chimeric hearts, conversely, have areas that are devoid of Cx43 staining adjacent to myocyte bundles, with a normal staining pattern (representative sections in B and D). Bar=200 µm for A and B, 50 µm for C and D.

Cx43^-/- Chimeric Mice Develop Normally
Of the 30 viable chimeric mice, all developed normally without signs of illness. On gross examination, visibly chimeric pups derived from C57BL/6 host embryos (n=23) differed from littermates with no visible chimerism (n=7) only in coat color and body weight, effects attributable to strain differences between the C57BL/6 hosts and the 129/Sv ES cells. Indeed, chimeric pups derived from ROSA-26 host embryos (129/Sv strain) appeared no different in coat color or body weight from their littermates with no detectable chimerism. On histological examination, there was no evidence of myocardial fibrosis or hypertrophy in chimeric mice (n=7) compared with age-matched controls (Figure 4). Of the cohort of 30 mice, 2 died unexpectedly, 1 with no visible chimerism at 5 months of age and a second with substantial chimerism at 2 months of age.

View larger version (87K): [in this window] [in a new window]   Figure 4. Histological evaluation of Cx43-/- chimeric hearts. Sections from 4% paraformaldehyde-fixed, paraffin-embedded wild-type (A) and Cx43-/- chimeric (B) hearts were stained with hematoxylin and eosin. Bar=50 µm.

Regional Conduction Deficits in Cx43^-/- Chimeric Mice
To determine the effects of patchy expression of Cx43 on cardiac impulse propagation, we performed optical mapping of epicardial activation patterns in visibly chimeric and control mouse hearts. In contrast to the smooth epicardial activation pattern typical of the normal murine heart (Figure 5B), 5 of 7 chimeric hearts showed discrete areas of conduction delay of varying magnitudes (Figure 5, C and E). To quantify the conduction abnormalities, we calculated negative curvature values as a measure of the extent of obstacles encountered during impulse propagation. Chimeric hearts showed significantly greater negative curvature than control hearts (-1.86±0.40 versus -0.86±0.098 mm, P<0.05; n=6 for each group). Interestingly, ventricular tachycardia occurred spontaneously in 2 of 7 isolated chimeric hearts (Figure 5F) but was not noted in any of the age-matched controls (n=6).

View larger version (71K): [in this window] [in a new window]   Figure 5. Arrhythmia and conduction studies in Cx43-/- chimeric mice. A, Anterior surface of heart indicating site of pacing (B and C) at right ventricular (RV) free wall and direction of activation wave front across anterior surface of heart toward left ventricular (LV) lateral wall. B, Epicardial pacing in control heart resulting in expected smooth epicardial activation pattern. C, Chimeric heart showing marked conduction defects during pacing at RV free wall (orientation as in A). D, Schematic illustrating orientation shown in E and F. As indicated, pacing site used in E is at LV lateral wall, and activation pattern is video-recorded as it traverses anterior wall toward septum. E, Chimeric heart showing modest conduction irregularities during pacing of LV lateral wall. F, Chimeric heart with spontaneous ventricular arrhythmia that occurred during pacing protocol. B, C, and E, Color scale bar=0 (red) to 17 (purple) ms; F, 0 to 24 ms. LA indicates left atrium; RA, right atrium; PA, pulmonary artery; and LAD, left anterior descending coronary artery.

Contractile Performance Is Diminished in Chimeric Hearts
We used echocardiography to investigate whether heterogeneous expression of Cx43 in the heart affected ventricular function (Figure 6). Fractional shortening was significantly decreased in visibly chimeric mice formed from Cx43-deficient 129/Sv ES cells and wild-type C57BL/6 blastocysts compared with both littermate mice with no visible chimerism and age-matched 129/SvxC57BL/6F1 controls (26.6±2.32%* versus 36.5±2.28% versus 36.5±1.59%, respectively; *P<0.05; Table). To control for the potential effect of strain chimerism itself on ventricular function (129/Sv ES cells in C57BL/6 hosts), we also performed echocardiograms on chimeric mice formed from Cx43^+/flox (Cx43-expressing) 129/Sv ES cells injected into C57BL/6 host blastocysts.¹² There was no significant decrease in fractional shortening in these chimeric mice (35.7±2.58%; n=5) compared with either control group.

View larger version (81K): [in this window] [in a new window]   Figure 6. Echocardiography of Cx43-/- chimeric mice. M-mode echocardiography in parasternal short-axis view was used to measure ventricular indices in wild-type (A) and Cx43-/- chimeric mice (B). Horizontal hash marks along top of each M-mode figure represent 0.2 second.

View this table: [in this window] [in a new window]   Table 1. Echocardiographic Measurements in Cx43-/- Chimeric Mice

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Gap junction remodeling has been observed in a variety of cardiomyopathic conditions, including hibernating myocardium, infarction, and dilated cardiomyopathy.^3–10 These observations have led to the hypothesis that gap junction remodeling may contribute not only to conduction defects but also to wall motion abnormalities.¹⁴ Indeed, human myocardial biopsy specimens have demonstrated focally reduced gap junction immunoreactivity in ischemic or hibernating regions, in which ventricular wall motion is often locally decreased.⁷ Mechanistically, regional dysregulation of Cx43 in acquired cardiomyopathies is theorized to cause a loss of coordinated contraction and ultimately ventricular dysfunction. Furthermore, delayed activation in regions with a relative paucity of Cx43 could lead to increased systolic wall stress in those regions and progressive ventricular dilatation, in a manner akin to that described in infarct expansion.²⁶

To date, the evidence linking gap junction remodeling with ventricular dysfunction has been correlative. Accordingly, the primary goal of this study was to test whether heterogeneous expression of Cx43 in the heart influenced cardiac contractile performance. We therefore generated chimeric mice composed of variable admixtures of Cx43-null and wild-type cells throughout all tissues of the body. Immunohistochemical analyses demonstrated patches of cells of each genetic background throughout the myocardium of chimeric mice, without morphological abnormalities, myocardial fibrosis, or hypertrophy.

Reflecting the heterogeneous expression of Cx43, chimeric mice showed markedly abnormal epicardial activation profiles, which we quantified by calculating negative curvature. The major finding in this study is that the chimeric mice developed significant systolic dysfunction, whereas control mice with normal Cx43 expression had preserved ventricular function. Because contractile performance is normal when Cx43 expression is uniformly inactivated in the myocardium,¹² our findings suggest that it is the heterogeneity of electrical activation that accounts for the systolic dysfunction.

Conceivably, chimeric mice formed from ES cells and blastocysts of different strains might lead to hearts in which individual myocytes have intrinsic differences in contractility, resulting in a myopathic syndrome.²⁷ Therefore, we also performed echocardiography on chimeras formed from Cx43-expressing (floxed) 129/Sv ES cells injected into C57BL/6 embryos. On the basis of these studies, the heterogeneous loss of Cx43 in the myocardium, rather than strain chimerism, appears to account for the contractile dysfunction. Nonetheless, additional studies of contractility in individual myocytes from chimeric hearts will be of interest to confirm the absence of intrinsic alterations in performance at the cellular level.

There are several limitations of the chimeric mice as a surrogate for gap junction remodeling in human cardiomyopathies. The extent of uncoupling between genetically modified myocytes in the chimeras and the anatomic distribution of these cells within the chimeric hearts are both likely to differ substantially from the remodeling process associated with acquired forms of heart disease. In addition, although a relationship between the degree of chimerism and the extent of conduction abnormalities and systolic dysfunction may well exist, we did not generate sufficient numbers of chimeric animals in this study to reach this conclusion. Furthermore, Cx43 chimerism may also affect diastolic function, although the techniques we used do not allow for such an investigation.

In summary, the results of the present study provide the first evidence directly linking heterogeneous expression of gap junction channels in the heart to both conduction defects and systolic dysfunction. Because gap junction abnormalities have been observed in many forms of cardiac disease, restoration of normal intercellular coupling may well serve as a novel approach to diminish lethal arrhythmias and improve cardiac function. Strategies to improve intercellular coupling in the diseased heart could include methods designed to upregulate Cx43 or other connexins normally expressed at lower levels in the heart.

	Acknowledgments

 
This study was supported in part by grants HL-04222 (Dr Gutstein), HL-39707 (Dr Morley), and HL-30557 and HL-64757 (Dr Fishman) from the NIH and an AHA Scientist Development Grant (Dr Morley). Confocal laser scanning microscopy (CLSM) was performed at the Mount Sinai School of Medicine (MSSM)-CLSM core facility, supported with funding from an NIH Shared Instrumentation grant (1-SS10-RR-09145-01) and NSF Major Research Instrumentation grant (DBI-9724504). Blastocyst injection for the production of Cx43^-/- mice was performed in the Mouse Genetics Shared Resource Facility of the MSSM. The authors thank Dr John T. Fallon, Dr Kevin Kelley, Dr Scott Henderson, Stacey Rentschler, Philip J. Mulieri, Veronica Gulle, and Samantha Buckley. The authors are grateful to Dr Andrew L. Wit for his thoughtful review of the manuscript.

Received February 6, 2001; revision received May 9, 2001; accepted May 9, 2001.

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