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© 2001 American Heart Association, Inc.
Integrative Physiology |
Conduction Slowing and Sudden Arrhythmic Death in Mice With Cardiac-Restricted Inactivation of Connexin43
From the Section of Myocardial Biology, Cardiovascular Institute, Departments of Medicine (D.E.G., G.I.F.), Biochemistry and Molecular Biology (H.S., G.I.F.), and Physiology and Biophysics (G.I.F.), Mount Sinai School of Medicine, New York, NY; Department of Pharmacology (G.E.M., H.T., D.V.), SUNY Upstate Medical University, Syracuse, NY; Center for Cardiovascular Development (M.D.S.), Baylor College of Medicine, Houston, Tex; and UCSD-Salk NHLBI Program in Molecular Medicine (J.C., K.R.C.), Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, Calif. Present address for H.S. is Department of Vascular Biology, The Scripps Research Institute, La Jolla, Calif.
Correspondence to Dr Glenn I. Fishman, Mount Sinai School of Medicine, One Gustave L. Levy Pl, Box 1269, New York, NY 10029. E-mail fishmg01{at}doc.mssm.edu
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Abstract |
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Abstract—Cardiac arrhythmia is a common and often lethal manifestation of many forms of heart disease. Gap junction remodeling has been postulated to contribute to the increased propensity for arrhythmogenesis in diseased myocardium, although a causative role in vivo remains speculative. By generating mice with cardiac-restricted knockout of connexin43 (Cx43), we have circumvented the perinatal lethal developmental defect associated with germline inactivation of this gap junction channel gene and uncovered an essential role for Cx43 in the maintenance of electrical stability. Mice with cardiac-specific loss of Cx43 have normal heart structure and contractile function, and yet they uniformly (28 of 28 conditional Cx43 knockout mice observed) develop sudden cardiac death from spontaneous ventricular arrhythmias by 2 months of age. Optical mapping of the epicardial electrical activation pattern in Cx43 conditional knockout mice revealed that ventricular conduction velocity was significantly slowed by up to 55% in the transverse direction and 42% in the longitudinal direction, resulting in an increase in anisotropic ratio compared with control littermates (2.1±0.13 versus 1.66±0.06; P<0.01). This novel genetic murine model of primary sudden cardiac death defines gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate.
Key Words: gap junction • connexin43 • arrhythmia • conduction
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Introduction |
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Gap junction channels, composed of highly homologous proteins known as connexins in vertebrate species, have been implicated in diverse processes, including the electrotonic coupling of excitable tissues, such as cardiac muscle.1 2 Theoretical modeling3 4 5 and studies of connexin (Cx) expression in diseased human and experimental animal hearts suggest that altered gap junction expression or function may underlie the high incidence of arrhythmias associated with many forms of heart disease, including hypertrophic, ischemic, and dilated cardiomyopathies.6 7 8 9 10 11 12 13 14 Specifically, in diseased myocardium, Cx43, the major constituent of ventricular gap junctions, is often diminished in abundance and fails to preferentially localize to the intercalated disk. This pathologic process, referred to as gap junction remodeling, is hypothesized to be an essential component of the substrate promoting some atrial as well as lethal ventricular tachyarrhythmias.
Efforts to genetically define the role of gap junction channels in cardiac conduction and arrhythmogenesis in vivo have been limited by the perinatal lethal developmental phenotype observed in Cx43 knockout (KO) mice.15 Data on conduction properties in Cx43 heterozygous KO mice, which develop normally and have normal lifespans, have been contradictory, likely reflecting differing methodologies. Some investigators have reported conduction slowing,16 whereas others find no statistically significant electrophysiological differences compared with wild-type hearts.17 Regardless, hearts from Cx43 heterozygous mice seem to be abnormally susceptible to the development of ventricular tachycardia in response to acute ischemia, providing evidence, at least in isolated heart preparations, that altered gap junction expression may be proarrhythmic.11
To test whether Cx43 is essential for cardiac electrical stability and whether altered gap junction expression is arrhythmogenic in vivo, we conditionally inactivated the Cx43 gene exclusively in cardiomyocytes. Cardiac morphogenesis in conditional KO (CKO) mice is normal, demonstrating no intrinsic cardiomyocyte cell–autonomous requirement for Cx43 during heart development. Despite normal cardiac structure and contractile performance, Cx43 CKO mice show profound conduction defects and uniformly develop spontaneous ventricular arrhythmias and sudden cardiac death by 2 months of age. Thus, in contrast to all existing murine models of altered cardiac electrophysiology, primary derangements in Cx43 expression lead to formation of a highly arrhythmogenic substrate culminating in uniform sudden cardiac death.
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Materials and Methods |
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Gene Targeting and Production of Cx43 CKO Mice
Embryonic stem (ES) cell manipulation and electroporation were performed according to established techniques.18 Blastocyst injection for the production of Cx43+/flox mice was performed in the Mouse Genetics Shared Resource Facility of the Mount Sinai School of Medicine.
Southern Blot Analysis and Polymerase Chain
Reaction
Southern blotting and polymerase chain reaction (PCR)
were performed according to established techniques. Southern blots were
probed with a 700-bp 3' flanking probe from a region outside of the
targeting vector.
Western Blot Analysis
Western blot analyses were performed with polyclonal
antibodies to Cx4319 and
Cx40 (Alpha Diagnostic). Blotting for Cx45 was performed with a
monoclonal antibody (directed against amino acids 354–367; Chemicon)
and embryonic mouse-heart lysate from embryonic day (E) 13.5 was used
as a positive control. For normalization of signals, blotting was also
performed with anti–ß-tubulin (Zymed Laboratories) or antisarcomeric
myosin heavy chain (MHC) (Developmental Studies Hybridoma Bank)
monoclonal antibodies, followed by blotting with horseradish
peroxidase–conjugated secondary antibody, chemiluminescent processing
(ECL, Amersham Pharmacia Biotech), and
autoradiography.
Histology, Immunofluorescence, and Confocal
Analysis
Hearts were removed from experimental mice at
predetermined times and flash-frozen in liquid nitrogen. Hearts were
later thawed and refrozen in O.C.T. freezing medium before sectioning.
Embryos at E12.5 were frozen directly in O.C.T. medium before
sectioning. Frozen sections were fixed in acetone followed by
immunostaining. Double staining of myocardial tissue with anti-Cx43
antisera and wheat germ agglutinin to visualize myocyte borders was
performed as previously
described.20 Cx43 was
detected using a polyclonal antibody (custom manufactured by Research
Genetics) directed against the same epitope used by Yamamoto et
al.19 Sections stained by
immunofluorescence were visualized by confocal microscopy. For the
evaluation of myocardial fibrosis, tissue was fixed in 4%
paraformaldehyde and embedded in paraffin, followed by staining with
modified Mason’s trichrome stain.
Telemetry Monitoring
For ambulatory electrocardiographic monitoring,
miniature telemetry transmitter devices (Data Sciences International)
were implanted as previously
described.21 The animals
were allowed 48 hours to recover from the surgery before telemetry
recordings were acquired. Recordings from CKO mice were continuous
beginning 48 hours after implantation until the time of
death.
Echocardiography
Echocardiography was performed under light anesthesia
with avertin using an Acuson Sequoia echocardiography machine and a
15-MHz linear probe. Measurements were performed online in a blinded
fashion. Fractional shortening (FS), left ventricular volumes, and
ejection fraction (EF) were calculated according to a standard formula,
as applied previously in
rodents.22 23
Optical Mapping of Ventricular
Activation
Mice were heparinized (heparin sodium 0.5 U/g
intraperitoneally) and killed by cervical dislocation, and hearts were
removed and perfused as previously
described.17 24
After 15 minutes of equilibration, the hearts were stained with a
voltage-sensitive dye (di-4-ANEPPS, Molecular Probes). Epicardial
pacing was performed with a unipolar electrode (stimuli at 1.5x
diastolic threshold) at a cycle length of 120 ms (control and MHC-CKO).
High-resolution optical mapping of voltage-dependent fluorescence was
performed on an upright Olympus microscope (BX50WI) with a reflected
light fluorescence attachment (BX-FLA). Excitation light from a
100-watt mercury arc lamp (Olympus) was filtered (480 to 550; dichroic
mirror 570 nm), and the emitted fluorescent light (>590 nm) was
projected onto a CCD camera (Dalsa). Images were acquired at 912
frames per second with 12-bit resolution from a 64x64-pixel array,
which provided a spatial resolution of 40 µm (4x objective, NA
0.28).
To obtain a representative sequence of activation during epicardial pacing, 10 to 15 beats were averaged. No pharmacological or mechanical manipulations were used to limit motion. Because contraction begins during repolarization, it is possible to identify action potential upstrokes using dF/dtmax. Local fluorescence maxima and minima were determined within an 8-ms window centered on the dF/dtmax. A pixel was considered activated when its fluorescent signal exceeded 50% of this local fluorescence range.
Conduction Velocity
Conduction velocities were calculated as described
previously.17 Briefly, local
conduction velocities were calculated from the gradient of activation
times. Vectors near the stimulating electrode and at a distance from
the electrode were excluded to remove stimulus artifacts and the
effects of 3-dimensional propagation. In addition, vectors that
deviated >60 degrees from their neighboring sites were excluded from
analysis to remove the effect of wavefront collisions.
Statistics
Western blot densitometry, echocardiographic and
conduction velocity (CV) data are expressed as mean±SEM. Comparisons
between groups were performed with a 2-tailed
t test using Microsoft Excel
software. For analysis of CVmax,
CVmin, and anisotropic ratio, because only 2 of
these parameters can be considered independent, we chose to test
significance for CVmin and anisotropic ratio.
Kaplan-Meier survival curves for the CKO mice were constructed and
compared (with the logrank test) using StatView software.
P<0.05 was considered
statistically significant.
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Results |
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Conditional Inactivation of Cx43 in the Heart
In an attempt to circumvent the lethal developmental phenotype associated with germline deletion of Cx43 and explore the role of this gap junction channel protein in cardiac arrhythmogenesis in vivo, we used the Cre/loxP system to inactivate Cx43 expression exclusively in cardiomyocytes.25 We generated a targeting vector in which a floxed neomycin (Neo) resistance cassette was inserted into the single Cx43 intron and a third loxP site was inserted into exon 2 of the Cx43 gene, just downstream of the open reading frame (Figure 1A
). Genomic DNA from Neo-resistant ES cell
clones was first screened for homologous recombination by Southern
blotting
(Figure 1B) and secondarily screened by PCR for the presence
of the third loxP site
(Figure 1C). Of a total of 64 clones screened, 5 (7.8%) had
undergone homologous recombination and had the downstream loxP site.
The targeting event did not affect endogenous Cx43 gene
expression,26 because
wild-type and targeted ES cells expressed equivalent amounts of Cx43
protein
(Figure 1D). The floxed allele was successfully transmitted
through the germline and used to produce
Cx43flox/flox mice.
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50 bp larger than the corresponding wild-type sequence. D, Western blot depicting Cx43 protein expression in wild-type (lane 1) and targeted Cx43+/flox (lane 2) ES cell clones. Blotting for ß-tubulin was performed on the same membrane to indicate relative loading. Similar results were seen in 3 independently targeted ES cell lines.
Cx43 CKO Mice Develop Normally
Homozygous Cx43flox/flox
mice were crossed with strains of mice expressing Cre recombinase (Cre)
exclusively in cardiomyocytes. These included a transgenic strain in
which Cre was driven by regulatory elements from the 
-MHC
gene27 and a second strain
in which Cre has been knocked in to the myosin light chain 2v (MLC2v)
locus.28 Both the
-MHC–Cre:Cx43flox/flox CKO (MHC-CKO) and
the MLC2v-Cre:Cx43flox/flox CKO (MLC-CKO)
mice were born with the expected mendelian frequency and were grossly
indistinguishable from their non-KO littermates.
Efficient inactivation of Cx43 expression in the hearts of
CKO mice was examined by immunofluorescence. At both E12.5
(Figures 2A through 2C) and at 4 weeks postpartum
(Figures 2D through 2F), a marked reduction of Cx43 expression
was observed in CKO hearts, with extensive areas that were completely
devoid of Cx43 immunoreactivity.
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To quantify the reduction in Cx43 expression, Western blot
analysis was performed. At 4 weeks of age, compared with control
littermates, Cx43 expression was reduced by 95% in the MHC-CKO mice
(P=0.027; n=3 MHC-CKO mice and
4 controls) and by 86% in MLC-CKO mice
(P<0.01; n=4 MLC-CKO and 4
controls), consistent with the immunofluorescence data
(Figure 3A). There was no apparent compensatory increase in
the expression of other connexins known to be expressed in myocytes. By
Western blot analysis, Cx40 levels in both the MHC-CKO and MLC-CKO
strains were unchanged compared with littermate controls
(Figure 3B). Cx45 expression was below the limits of
detection in both CKO and control hearts, in agreement with previous
studies of normal murine
heart.29 In addition, no
expression of Cx40 or Cx45 was detected by immunofluorescence in
working ventricular myocardium (not shown).
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Histological examination of CKO hearts at several time points revealed no evidence of the right ventricular outflow tract (RVOT) obstruction phenotype associated with germline ablation of Cx43. Both at 1 week after birth (n=2 CKO and 3 Cre- littermate hearts) and in older mice at 8 weeks of age (n=3 CKO and 3 Cre- littermate hearts), the ventricular muscle appeared entirely normal, without fibrosis, hypertrophy, or myofibrillar disarray (not shown).
Contractile performance, left ventricular chamber sizes, and
wall thicknesses, as assessed by echocardiography in 1-month-old
MHC-CKO mice, were no different
than in non-KO littermates
(Table).
Thus, Cre recombinase, driven by regulatory elements from either the
-MHC or MLC2v genes, efficiently and specifically inactivated Cx43
expression in the myocyte compartment of the heart, with no discernible
compensatory effect on the abundance of other cardiac connexins.
Moreover, there appeared to be no intrinsic cardiomyocyte
cell–autonomous requirement for Cx43 during heart development. This
conclusion is consistent with evidence suggesting a primary neural
crest origin of the Cx43 KO
phenotype.30
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Sudden Cardiac Death in Cx43 CKO Mice
Although the Cx43 CKO mice appeared morphologically and
functionally normal, they began to die suddenly at 2 to 3 weeks of age
(Figure 4). Within the first 2 months of life, 13 of 13
MLC-CKO mice and 15 of 15 MHC-CKO mice died suddenly, all without
previous signs of illness. In contrast, there was no mortality among
the
Cre-:Cx43flox/flox
littermates (n=25) during the first 6 months of life.
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We implanted miniaturized telemetry transmitter devices in
four 5-week-old MHC-CKO mice to record the cardiac rhythm during
episodes of sudden death.21
During the continuous recording period, all 4 MHC-CKO mice were in
normal sinus rhythm, with no evidence of ventricular ectopy. In 3 of
the mice, we successfully captured the abrupt onset of spontaneous
ventricular tachyarrhythmias, confirming that the deaths were
arrhythmic in nature
(Figure 5). In the fourth mouse, the recording device was
inadvertently inactive at the time of death.
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Abnormal Conduction Properties and Arrhythmias
in MHC-CKO Hearts
To directly examine the consequences of loss of Cx43 on
ventricular conduction, we optically mapped electrical activity in the
hearts of MHC-CKO mice and littermate controls using a
voltage-sensitive dye. In hearts from 5- to 8-week-old control mice
(n=6;
Figure 6B), the normal anisotropic pattern of electrical
activation was
observed,17 21
without evidence of conduction abnormalities or spontaneous
arrhythmias. In contrast, hearts from 5- to 8-week-old MHC-CKO mice
demonstrated markedly abnormal conduction parameters
(Figure 6C). Six of the 10 MHC-CKO mouse hearts tested
developed spontaneous polymorphic ventricular tachyarrhythmias that
could not be terminated by high-frequency pacing. The remaining MHC-CKO
hearts initially were in normal sinus rhythm and could be paced.
However, conduction parameters in these hearts were markedly abnormal.
Compared with control hearts, CV in these 4 MHC-CKO hearts was
substantially reduced in all directions, as visualized by optical
mapping. CV was reduced most drastically in the transverse
(CVmin) direction (control
CVmin=0.38±0.02 m/s; MHC-CKO
CVmin=0.17±0.02 m/s;
P<0.001;
Figure 6D), presumably reflecting the lessening of
rotational anisotropy attributable to extreme uncoupling between
epicardial and deeper cell
layers.31 32
CVmax decreased from 0.62±0.02 m/s in controls
to 0.36±0.05 m/s in MHC-CKO mice (significance was not tested). As a
result, the anisotropic ratio
(CVmax/CVmin) was
significantly increased in the CKO hearts (1.66±0.06 in control hearts
versus 2.1±0.13 in MHC-CKO hearts;
P<0.01;
Figure 6D).
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The MHC-CKO hearts were highly susceptible to spontaneous
ventricular arrhythmias, which occurred before or during the standard
pacing protocol
(Figure 6E
). Interestingly, within the group of MHC-CKO
hearts, spontaneous arrhythmias at explantation that prevented pacing
were more common in mice older than 7 weeks of age (3 of 3 MHC-CKO
hearts) than in those younger than 7 weeks (3 of 7 MHC-CKO hearts).
There were no age-dependent differences in conduction parameters in the
control group. In total, 8 of 10 MHC-CKO mice studied had spontaneous
ventricular arrhythmias, compared with 0 of 6
controls.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the past decade, several animal and human studies have described alterations in the abundance or subcellular localization of connexin proteins in arrhythmogenic cardiac conditions, a process referred to as gap junction remodeling.7 8 9 10 12 However, determining the unique contribution of gap junctional remodeling to the substrate for cardiac arrhythmias has been difficult, inasmuch as the diseased heart invariably shows a multitude of structural and functional perturbations. In the present study, by specifically ablating the expression of Cx43 in the myocardium, we sought to circumvent the lethal developmental defect caused by germline deletion of Cx43 and address this question directly.
The first novel finding in this study is the demonstration
that there is no intrinsic cardiomyocyte cell–autonomous requirement
for Cx43 during heart development. A similar conclusion was made for
the RXR gene when inactivated by crossing with the same MLC2v-Cre mouse
used in this study.28
Whether recombination was catalyzed in the Cx43 floxed mice with Cre
recombinase expressed under the transcriptional control of the MLC2v or
MHC regulatory elements, where Cx43 expression is inactivated no
later than E12.5, heart development proceeded normally, without the
RVOT phenotype observed with germline knockout of
Cx43.15 Thus, the lethal
developmental defect resulting from global inactivation of Cx43 seems
to result from a nonmyocyte lineage, consistent with previous
experimental evidence suggesting a primary neural-crest origin of this
phenotype.30 33
Lineage-restricted inactivation of Cx43 using strains of mice
expressing Cre recombinase preferentially in the cardiac neural crest,
such as Wnt-134 or
Pax3,35 may unequivocally
answer this question. In addition, targeted ablation of Cx43 in
additional lineages will likely provide additional insight into the
tissue-specific functions of this widely expressed gap junction channel
protein.
By circumventing the lethal RVOT phenotype caused by germline deletion of Cx43 with the conditional strategy, we were able to examine the consequences of loss of Cx43 exclusively in the myocardium. Despite normal heart structure and contractile performance, Cx43 CKO mice uniformly developed sudden cardiac death, apparently from spontaneous ventricular tachycardia. These data support the critical role of the gap junction channel in maintaining cardiac electrical stability. Indeed, it seems that loss of Cx43 function, even in the absence of other identifiable structural cardiac abnormalities, can lead to a substrate in which spontaneous, lethal cardiac arrhythmias are inevitable.
Although CV is markedly slowed in the Cx43 CKO mice, successful impulse propagation is maintained, at least until the abrupt onset of lethal ventricular tachyarrhythmias. This observation is consistent with modeling suggesting that the safety factor for conduction is paradoxically increased with reduced gap junctional coupling.5 Because only scattered myocytes express Cx43 in the CKO mice, our data suggest that the myocardium must be coupled by low-level expression of gap junction channels formed from other connexin isoforms.
The electrophysiological mechanisms leading to the lethal
ventricular arrhythmias and the factors accounting for the timing of
sudden cardiac death in the CKO mice are uncertain. Cellular uncoupling
resulting from loss of Cx43 gap junction channels might unmask ectopic
foci or trigger arrhythmias by enhancing the generation of early
afterdepolarizations
(EADs).36 Computer modeling
studies indicate that moderate decreases in junctional coupling
predispose to the generation of EADs, whereas higher levels of
junctional resistance limit their
propagation.37 Thus, the
residual level of junctional coupling in the CKOs may allow for the
generation and propagation of EADs. Indeed, a pause-dependent EAD may
account for the initiation of the arrhythmia shown in
Figure 5, top.
By enhancing dispersion of action potential duration (APD) and repolarization, reduced cell coupling also predisposes to the formation of unidirectional block and reentry.36 38 Modeling of a multicellular theoretical fiber suggests that decreased gap junction coupling, although causing a paradoxical increase in the safety factor as CV decreases, also may facilitate microreentry.5 Furthermore, on the basis of in vitro studies of dissociated myocytes,39 it has been theorized that extreme conduction slowing in combination with geometrical factors may permit reentrant excitation to occur in extremely small areas of cardiac tissue. These factors in combination with the progressive growth of individual myocytes after birth, which is predicted to increase the discontinuous nature of propagation, may also play a role in the time course of the arrhythmic phenotype.3
In recent years, increasing attention has focused on the complex interactions between passive and active membrane properties. The Cx43 CKO mice provide a novel experimental system to examine these relationships in a multicellular preparation, particularly with respect to formation of a substrate that clearly enhances the propensity for spontaneous arrhythmias. For example, theoretical simulations by Rudy and colleagues5 36 38 predict that reduced cellular coupling markedly increases APD dispersion and also renders action potential propagation increasingly dependent on the L-type calcium current. Moreover, Laurita et al40 have presented evidence that APD restitution is influenced by cell-to-cell coupling. While recognizing the significant differences in the shape and ionic components of the action potential in various species, additional analyses of impulse propagation in intact hearts or multicellular fibers from wild-type and homozygous Cx43 CKO mice may yield experimental data that can be directly compared with predictions based on theoretical modeling. In addition, with the availability of the Cx43 CKO mice, one can envision increasingly complex genetic strategies in which both the extent of cellular coupling and the magnitude of specific sarcolemmal currents are simultaneously modified, thereby modeling the complex electrophysiological derangements typically observed in diseased myocardium.
In summary, the results of the present study provide strong evidence that loss of Cx43 expression may serve as a critical event in the formation of the arrhythmogenic substrate. Indeed, altered gap junction channel expression in the heart seems sufficient to induce spontaneous ventricular tachycardia with complete penetrance. Moreover, in contrast to other murine models associated with premature cardiovascular mortality, Cx43 CKO mice develop uniform sudden cardiac death in the absence of cardiac dysfunction and morphological abnormalities. Because gap junction remodeling has been described in many forms of human cardiac disease, restoration of normal intercellular coupling in the myopathic heart may well serve as a novel target in the treatment of patients at risk for lethal ventricular arrhythmias.
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Acknowledgments |
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This study was supported in part by grants HL04222 (to D.E.G.), HL39707 (to G.E.M.), and HL38449, HL30557, and HL64757 (to G.I.F.) from the National Institutes of Health and an Established Investigator Award from the American Heart Association (to G.I.F.). Confocal laser scanning microscopy was performed at the Mount Sinai School of Medicine–Confocal Laser Scanning Microscopy core facility, supported with funding from National Institutes of Health shared instrumentation grant (1 SS10 RR0 9145-01) and National Science Foundation Major Research Instrumentation grant (DBI-9724504). The authors acknowledge the kind gift of mouse Cx43 genomic DNA from Dr Janet Rossant (Samuel Lunenfeld Research Institute, Toronto, Ontario) and anti-Cx43 antisera from Dr Elliot L. Hertzberg (Albert Einstein College of Medicine, Bronx, NY). We would like to thank Dr John T. Fallon, Dr Kevin Kelley (Mount Sinai Mouse Genetics Shared Resource Facility), Dr Scott Henderson (the Mount Sinai School of Medicine–Confocal Laser Scanning Microscopy core facility), Fang-Yu Liu, Jie Zhang, Veronica Gulle, Samantha Buckley, and Maya Srinivas for their help on this project.
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Footnotes |
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Original received August 31, 2000; revision received December 13, 2000; accepted December 13, 2000.
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