Disparate Effects of Deficient Expression of Connexin43 on Atrial and Ventricular Conduction
Evidence for Chamber-Specific Molecular Determinants of Conduction
Background—Myocardial conduction depends on intercellular transfer of currrent at gap junctions. Atrial myocytes express three different gap junction channel proteins—connexin43 (Cx43), connexin45 (Cx45), and connexin40 (Cx40)—whereas ventricular myocytes express only Cx43 and Cx45. However, the physiological roles of individual connexins are unknown. We have previously shown that mice heterozygous for a null mutation in the gene encoding Cx43 (Cx43+/− mice) express 50% of the normal amount of Cx43 in ventricular myocardium and exhibit marked slowing of ventricular conduction.
Methods and Results—To determine whether atrial conduction is affected in Cx43+/− mice, we measured atrial conduction velocity in isolated hearts, performed detailed ECG and electrophysiological studies in intact animals, and determined the amount of cardiac connexins in atrial and ventricular tissue. Ventricular conduction velocity was reduced by 38% in Cx43+/− mice compared with wild-types, but atrial conduction velocity in the same hearts was normal. QRS duration was significantly greater in Cx43+/− mice than in wild-types, but P-wave duration and amplitude did not differ. Atrial expression of Cx43 was reduced by 50%.
Conclusions—These results indicate that Cx43 is a principal conductor of intercellular current in the ventricle because ventricular conduction is significantly slowed when Cx43 content is reduced by only 50%. In contrast, a similar reduction in Cx43 content in atrial muscle has no effect on atrial conduction, suggesting that Cx40 (which is expressed in atrial but not ventricular myocytes) is a major electrical coupling protein in atrial muscle. Thus, Cx43 and Cx40 may be chamber-specific determinants of myocardial conduction.
Reentry is a principal mechanism of both atrial and ventricular tachyarrhythmias.1 2 In many of these arrhythmias, slow conduction and unidirectional conduction block appear to be related to derangements in intercellular electrical coupling at gap junctions, which determine how current spreads from one myocyte to another.3 4 5 6 Like other differentiated cells, cardiac myocytes express multiple connexins, proteins that form gap junction channels, but different cardiac tissues express different connexin phenotypes.7 8 9 Ventricular myocytes express connexin (Cx)43 and Cx45. Atrial myocytes express both Cx43 and Cx45 but also express another protein, Cx40. Each of these proteins forms intercellular channels with unique biophysical properties when expressed in “communication-deficient” cell lines.10 However, the specific roles of these connexins as determinants of the conduction properties of functionally distinct cardiac tissues are unknown.
We recently analyzed the effects of deficient expression of Cx43 on ventricular conduction in mice with targeted deletion of Cx43 produced by Reaume et al.11 Mice homozygous for the Cx43 null mutation die soon after being born, apparently due to a malformation of the right ventricular outflow tract that obstructs blood flow to the lungs.11 We discovered, however, that heterozygotes, which survive and breed without apparent abnormalities, exhibit slow ventricular conduction not related to any differences in action potential parameters of ventricular myocytes, gross or microscopic changes in the structure of the ventricular wall, or alterations in expression of other cardiac connexins.12 We now report that atrial conduction is unaffected in mice heterozygous for the Cx43 null mutation (Cx43+/− mice) even though both atrial and ventricular muscle express Cx43 abundantly, its level in both tissues is diminished by ≈50% in heterozygotes, and ventricular conduction velocity is reduced by ≈40%. These findings suggest that Cx43 is the principal conductor of intercellular current in the ventricle, but Cx40 appears to be a major conductor of intercellular current in atrial muscle. These proteins could, therefore, be chamber-specific targets of drugs designed to selectively modulate conduction in patients with serious atrial or ventricular arrhythmias.
Cx43 Mutant Mice
Studies were performed on animals produced in our mouse colony using breeders originally purchased from the Jackson Laboratories (Bar Harbor, ME). Mice were housed in barrier facilities under standard conditions. All mice were maintained in an inbred background (C57BL/6). The genotypes of all mice were determined by polymerase chain reaction using primer sequences and protocols identical to those of Reaume et al.11
Homogenates of atrial and ventricular myocardium were prepared from 6 to 8 individual adult Cx43+/+ and +/− hearts. Samples containing 30 μg of total protein from ventricular homogenates and 15 μg of total protein from atrial homogenates were resolved by SDS-polyacrylamide gel electrophoresis, transferred to Immobilon-P membranes (Millipore Corp), and incubated with polyclonal rabbit antibodies shown previously to be monospecific for Cx43, Cx45, or Cx40.3 Immunoreactivity was detected by chemiluminescence (ECL; Amersham) as previously described,9 12 and signals were quantified by densitometry.12 Cx43, Cx45, and Cx40 signal intensities in Cx43+/− atrial samples were compared with the use of ANOVA with corresponding signals in Cx43+/+ atrial samples, which were normalized to a value of 1.0.
Epicardial Conduction Velocity Measurements
Conduction velocity was measured on the atrial and ventricular epicardial surfaces in superfused and perfused adult hearts. Hearts of adult mice were rapidly excised and placed in oxygenated cardioplegic solution (Plegisol; Abbott Labs) at 4°C. In initial studies, the conduction velocity of paced beats was measured in atria isolated from adult Cx43+/− and +/+ animals. The atria were separated from the ventricles by cutting the ventricles below the atrioventricular (AV) groove. The isolated atria were placed in a 7-mL tissue bath with continuous superfusion of oxygenated Krebs-Henseleit buffer at 31°C at a flow rate of 12 mL/min. A temperature of 31°C was chosen to slow the spontaneous heart rate and thereby facilitate pacing. At this temperature, conduction velocity may also be slowed, but because all mice were studied under identical conditions, relative conduction velocities in Cx43+/− and +/+ preparations could be determined. The velocity of atrial conduction was measured on the surface of the right atrial appendage, the largest atrial structure, by placing an extracellular electrode array consisting of 16 bipolar pairs on the appendage parallel to its long axis. The distance between bipolar pairs and the distance between electrodes within a pair were both 200 μm. The right atrial appendage had a maximal length of 3 to 4 mm from its tip to the origin of the inferior vena cava. The size and shape of the atrial structures were the same in Cx43+/− and +/+ mice. A pacing electrode produced from wire (75 μm in diameter) tapered to a fine tip was placed at the distal tip of the appendage.
In subsequent experiments, the conduction velocities of paced beats in the atria and ventricles of the same hearts were measured. Hearts isolated from adult Cx43+/− and +/+ animals were perfused with Krebs-Henseleit buffer at 31°C via an aortic cannula at a flow rate of 1.0 to 1.2 mL/min while simultaneously being superfused with the same buffer at a flow rate of 12 mL/min as previously described.12 The linear electrode array was placed on the anterior surface of each adult heart along the maximum apical-basal dimension. Care was taken to place the electrode array at the same location in each heart in an orientation roughly parallel to the left anterior descending coronary artery. We have shown previously that in this orientation, the electrode array is approximately parallel to the longitudenal fiber axis and that ventricular epicardial fiber orientation and curvature do not differ in Cx43+/− and +/+ mice.12 The pacing electrode was located at the ventricular apex. After completion of electrophysiological measurements of ventricular activity, atrial conduction velocity was measured on the right atrial appendage as described above. Electrograms were recorded on a multichannel computerized data acqusition system at a sampling rate of 3000 Hz. At this sampling rate, temporal resolution was <1.0 ms, which is sufficient for measuring rapid conduction velocities over short distances. Activation times were defined by determining the maximum absolute amplitude of each electrogram (peak criterion) as previously described,12 and the average conduction velocity was calculated by linear regression relating interelectrode distance to activation times. The slope of the regression line was the average conduction velocity.
ECG and Electrophysiological Studies
These studies were performed in anesthetized adult mice according to the methods of Berul et al.13 Mice were anesthetized by intraperitoneal administration of pentobarbital and ketamine (0.033 mg/g each). The surface six-lead ECG was recorded from subcutaneous 27-gauge needles in each limb. Pacing electrode catheters (CIBer mouse EP catheter; NuMed Inc) were placed transvenously into the right atrium and right ventricular apex for intracardiac electrogram recording, pacing, and programmed electrical stimulation. ECG channels were amplified (0.1 mV/cm) and filtered between 10 and 100 Hz. ECG parameters were calculated according to standard criteria. High-fidelity electrogram signals were acquired at a sampling rate of 400/s, which is 10 times the frequency of a standard ECG recorder. Sinus node function was evaluated by indirectly measuring sinus node recovery time by pacing for 30 seconds at cycle lengths of 200, 150, and 100 ms and measuring the duration of the return cycle. AV-His-Purkinje conduction times (defined as the total conduction time between the right atrial and right ventricular electrodes) were assessed during rapid atrial pacing at rates up to 1200 bpm. Because the majority of this conduction time involved AV nodal conduction, potential small differences in electrode position had a negligible impact on this parameter. The minimum cycle length required to maintain 1:1 AV conduction, the Wenckebach paced cycle length, and the maximum paced cycle length causing 2:1 AV block were also determined. Programmed right atrial stimulation was performed at two paced drive rates to determine effective refractory periods. Single and double extrastimulation techniques down to a minimum coupling interval of 40 ms were performed in an attempt to induce atrial arrhythmias. Ventricular burst pacing was performed at rates of 250 to 1200 bpm to assess retrograde ventriculoatrial (VA) conduction, including measurements of VA Wenckebach block rates and ventricular pacing exit block (2:1 capture block). Ventricular effective refractory periods were determined using programmed stimulation at two paced drive rates using single extrastimuli. Double and triple extrastimuli were delivered in an attempt to induce ventricular arrhythmias.
Homogenates of atrial and ventricular myocardium were analyzed by immunoblotting and quantitative densitometry to measure the relative amounts of cardiac connexins in adult Cx43+/− and wild-type (Cx43+/+) hearts. Levels of Cx43 were reduced by approximately one half in the atria of Cx43+/− animals compared with wild-types (Fig 1⇓). However, the atrial content of Cx40 and Cx45 was similar in Cx43+/− and +/+ animals. We also confirmed that ventricular Cx43 expression was reduced by ≈50% without a change in the expression of Cx45, the other ventricular connexin (Fig 1⇓).
The conduction velocity of paced beats was first measured in atria isolated from adult Cx43+/− and +/+ animals. The atria were separated from the ventricles by cutting the ventricles below the AV groove. Because the atria remained attached to the AV valve rings, they retained their shape and could be maintained in viable condition for several hours in a superfused preparation. No difference was observed in the velocity of conduction of paced beats in the right atrial appendage of Cx43+/− and +/+ animals (Fig 2A⇓). We next performed experiments to compare directly the conduction velocities of paced beats in the atria and ventricles of the same hearts. There was no difference in atrial conduction velocity (Fig 2B⇓ and 2C⇓) in perfused, intact Cx43+/− and +/+ hearts. However, in the same hearts, the conduction velocity of paced ventricular beats was 38% slower in Cx43+/− compared with Cx43+/+ hearts (Fig 2B⇓ and 2C⇓).
To further characterize ECG and electrophysiological features in Cx43+/− and +/+ mice, we performed detailed studies in situ using techniques developed by Berul et al13 to characterize mouse cardiac electrophysiology. Selected measurements are shown in the Table⇓, and representative ECG recordings are shown in Fig 3⇓. The only ECG difference observed was significant prolongation of the QRS interval in Cx43+/− compared with Cx43+/+ animals. This difference was predicted by the slow ventricular conduction demonstrated with epicardial extracellular recordings. Electrophysiological studies included measurements of the minimum cycle length required to maintain 1:1 AV conduction (the Wenckebach paced cycle length) and the maximum paced cycle length causing 2:1 AV block. When these parameters were measured in the anterograde (AV) direction by pacing at the right atrium and recording at the right ventricular apex, no differences between Cx43+/− and +/+ groups were observed. However, when studies were repeated in the retrograde (VA) direction, conduction block occurred at a greater cycle length in Cx43+/− animals despite the fact that the same electrode sites were used. This difference can be explained by the slow ventricular conduction in Cx43+/− hearts, which becomes more apparent when the ventricles are activated at an ectopic site (ie, the right ventricular apex) during studies of VA conduction than when the ventricles are activated via the His-Purkinje system during AV conduction studies. This conclusion is supported by the significantly wider QRS complexes observed during VA pacing in Cx43+/− compared with Cx43+/+ hearts (paced QRS duration in the Table⇓).
There was no difference between Cx43+/− and +/+ animals in P-wave duration or amplitude (Table⇑). These data provide independent confirmation of results of studies in vitro indicating that atrial conduction in Cx43+/− mice is normal. No differences were observed between Cx43+/− and +/+ animals in atrial effective refractory periods measured at different paced drive rates, nor was a significant difference seen in ventricular effective refractory period. Heart rate, sinus node recovery time, PR interval, and QT interval were similar in Cx43+/− and +/+ mice, suggesting that diminished expression of Cx43 in heterozygotes does not significantly affect sinus node or AV node function, or repolarization during normal sinus rhythm. No arrhythmias were induced in Cx43+/− or +/+ mice when either the atria or ventricles were subjected to aggressive extrastimulation protocols.13
These results indicate that although both atrial and ventricular muscle express abundant amounts of the predominant cardiac connexin, Cx43, only ventricular muscle exhibits slowing of conduction when the expression of Cx43 in both tissues is diminished by ≈50% because of the presence of a single null allele in the Cx43 gene. The marked slowing of ventricular conduction in mice in which Cx43 expression is diminished by only 50% suggests that Cx43 plays a major role as a conductor of intercellular current in ventricular muscle. Atrial and ventricular muscle express other connexins. Cx45 is present in both tissues in roughly equal amounts based on previous immunoblotting studies.7 8 9 Its level of expression is not altered in either atrial or ventricular muscle in Cx43+/− animals. Thus, expression of a normal amount of Cx45 does not prevent ventricular conduction slowing when 50% of ventricular Cx43 is deleted. In contrast to the ventricle, atrial muscle expresses another connexin, Cx40, in addition to Cx43 and Cx45. In expression systems, Cx40 forms channels characterized by greater unitary conductance than Cx43 or Cx45 channels.10 Cx40 expression in the heart appears to be limited to atrial muscle and components of the specialized cardiac conduction system.8 9 14 The identical atrial conduction velocities observed in Cx43+/− and +/+ mice suggest that the presence of Cx40 can prevent development of a conduction phenotype in atrial myocardium when Cx43 expression is reduced by half. Thus, Cx40 appears to be a major conductor of intercellular current in atrial muscle. These results suggest that there are chamber-specific molecular determinants of intercellular coupling in atrial and ventricular muscle and provide the first evidence of which we are aware that different connexin phenotypes confer functional specificity rather than mere biological redundancy.
Histological examination and preliminary ultrastructural analysis of atrial and ventricular tissues in Cx43+/− and +/+ mice have revealed no obvious differences in tissue structure, nor has interstitial fibrosis been identified. However, high-resolution quantitative studies have not been performed. Furthermore, the continuous sheet of epicardial muscle of the murine right atrial appendage is very thin and in many regions is composed of only a few myocyte layers. For this reason, it has been technically difficult to define the orientation of atrial epicardial fibers located under the recording electrode array. Because both atrial and ventricular conduction velocities were measured on the epicardial surface after initiation of paced beats on the surface, conduction pathways were undefined. It will be necessary in future studies to precisely characterize tissue structure to determine whether deletion of a single Cx43 allele affects structural determinants of conduction, including myocyte size and shape, distribution of gap junctions, volume and configuration of the extracellular space, and orientation and curvature of muscle bundles.
The two most important types of arrhythmias affecting patients with heart disease are ventricular tachycardia, often associated with sudden cardiac death, and atrial fibrillation, which occurs in up to 10% of elderly subjects and has been associated with 65% of the strokes in the elderly population.1 2 15 16 In both of these arrhythmias, reentrant mechanisms dependent on the development of slow, discontinuous conduction and unidirectional conduction block appear to be of critical importance.1 2 3 4 5 6 In many patients with ventricular tachycardia, zones of abnormal conduction have been localized by mapping procedures to areas of fibrotic myocardium in which intercellular electrical coupling of ventricular myocytes is altered because of redistribution of gap junctions.5 17 18 Similar changes in gap junction distribution have been described in the atria in association with aging.19 Pharmacological therapy of atrial fibrillation and reentrant ventricular tachycardia is not always effective. Because of the potential role of changes in electrical coupling in the pathogenesis of conduction abnormalities critical to the initiation and maintenance of reentrant arrhythmias, targeting of antiarrhythmic drugs to gap junction channels to modulate electrical coupling could be effective in preventing these arrhythmias. The expression of chamber-specific molecular determinants of coupling in atrial and ventricular myocardium therefore provides an opportunity to target drugs selectively to modulate either atrial or ventricular conduction without affecting conduction in the other chamber. Although there are no currently available compounds that specifically modulate gap junctional conductance and this approach is speculative, it would appear to be an attractive strategy for the development of a new class of antiarrhythmic drugs to treat patients with atrial or ventricular arrhythmias.
This work was supported by National Institutes of Health grants HL-50598, HL-45466, and HL-03607; a Grant-in-Aid from the American Heart Association; and the Council on Clinical Cardiology. We thank Susan Johnson for secretarial assistance.
- Received June 6, 1997.
- Revision received September 4, 1997.
- Accepted October 6, 1997.
- Copyright © 1998 by American Heart Association
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