Replacement of Connexin40 by Connexin45 in the MouseImpact on Cardiac Electrical Conduction
From the Laboratoire de Génétique et Physiologie du Développement (UMR CNRS 6545) (S.A., T.J.-G., D.G.), Institut de Biologie du Développement de Marseille, Université de la Méditerranée, Marseille, France; Department of Medical Physiology (H.v.R., H.J.), University Medical Center, Utrecht, The Netherlands; Neurologia Experimental (D.G., L.B.), Hospital Ramón y Cajal, Madrid, Spain; Interuniversity Cardiology Institute (J.d.B.), Utrecht, The Netherlands; Department of Anatomy and Embryology (W.L.), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, and National Heart and Lung Institute (S.C.), Imperial College London, UK.0p, http://www.100md.com
Abstract0p, http://www.100md.com
Gap junction channels, required for the propagation of cardiac impulse, are intercellular structures composed of connexins (Cx). Cx43, Cx40, and Cx45 are synthesized in the cardiomyocytes, and each of them has a unique cardiac expression pattern. Cx40 knock-in Cx45 mice were generated to explore the ability of Cx45 to replace Cx40, and to assess the functional equivalence of these two Cxs that are both expressed in the conduction system. ECGs revealed that the consequences resulting from the biallelic replacement of Cx40 by Cx45 were an increased duration of the P wave, and a prolonged and fractionated QRS complex. Epicardial mapping indicated that the conduction velocities (CV) in the right atrium and the ventricular myocardium, as well as conduction through the AV node, were unaffected. The significant reduction of the CV in the left atrium would be the most likely cause of the P-wave lengthening. In the right ventricle, a changed and prolonged activation in sinus rhythm was found in homozygous mutant mice, which may explain the prolongation and splitting of the QRS complex. Electrical mapping of the His bundle branches revealed that this was due to slow conduction measured in the right branch. The CV in the left branch was unchanged. Therefore, in the absence of Cx40, the upregulation of Cx45 in the heart results in a normal impulse propagation in the right atrium, the AV node, and the left His bundle branch only.
Key Words: gap junction channels connexins cardiac conductionvaa, 百拇医药
Introductionvaa, 百拇医药
In the mammalian heart the electrical activity, spontaneously generated in the sinoatrial (SA) node, is propagated through the atria, then to the ventricular walls via specialized tissues that constitute the conduction system (CS). The CS includes the atrioventricular (AV) node, the His bundle, and the two bundle branches (BBs), which ramify into peripheral Purkinje fibers. Gap junction channels are responsible for the electrical coupling between the various types of myocytes, and they are required for the propagation of cardiac impulse.1vaa, 百拇医药
Gap junction channels are intercellular structures constituted by transmembrane proteins belonging to the connexin (Cx) family.2,3 Nineteen Cx genes have been identified in the mouse genome,4 but so far only four Cxs, Cx43, Cx40, Cx45, and Cx46, have been shown to be expressed in mammalian cardiomyocytes with each having a unique expression pattern in the adult heart.5 Hence, in the mouse heart, Cx43 is abundant in both the atrial and ventricular working myocytes, and the distal part of the CS; Cx40 is strongly expressed in the atrial working myocytes and the CS,6,7 whereas Cx45 is detected mainly in the SA node and the CS.8–10 Expression of Cx45 has been reported in the working ventricular myocardium11 but this finding has not been corroborated by other investigations.9,12 Lastly, Cx46 was detected in the rabbit SA node associated with Cx40 and Cx45.13,14
Analysis of Cx-deficient mice has provided evidence that Cx43, Cx40, and Cx45 are involved in both heart function and development. The Cx45 null mutation is lethal with the embryos dying in utero around embryonic day 10, 2 days after cardiac contractions are normally initiated.15,16 Several anomalies including defects in the endocardial cushions are thought to contribute to the lethal phenotype. Conditional knock-out (KO) mice in which Cx43 gene expression in the cardiomyocytes is reduced by about 90% have ventricular conduction velocity (CV) slowed by 50%. All these mice undergo sudden cardiac death from spontaneous ventricular arrhythmias by 2 months of age.17 Cx40KO mice are viable and fertile,18,19 and several studies have focused on impulse propagation in the hearts of these mice.20–25 Findings from these studies include impaired conduction at various levels of the CS, and increased incidence of inducible atrial tachyarrhythmias, indicating that Cx40 is an important determinant of impulse propagation in the atria and the AV conduction system. Recent investigations also suggest that Cx40 may play a role in cardiac morphogenesis.26
The analysis of KO mice has therefore provided important information on the in vivo functions of Cxs in heart. Another approach for analyzing the function of Cxs is to generate knock-in (KI) mice to assess the functional equivalence of two Cxs. Cx40 and Cx45 are both expressed in the mouse CS, albeit with very different levels of expression. Channels composed of Cx40 or Cx45 have quite different properties. Cx40 channels have a large unitary conductance (160 pS), in contrast with Cx45 channels, which have a small conductance ({approx} 30 pS).27,28 Macroscopic conductance of Cx40 channels is not very sensitive to the transjunctional voltage (Vj) as compared with that of Cx45 channels, which is very sensitive to Vj.29–32 Furthermore, both types of channels are differently regulated by phosphorylation.28,33 To assess the functional equivalence of Cx40 and Cx45, we have generated Cx40KICx45 mouse lines in which Cx45 is expressed in lieu and place of Cx40.
Materials and Methods81, 百拇医药
The strategy used to generate the transgenic mice and the techniques used for genotyping the ES cells and mice and for identifying expression of the transgene in the KI mice are described in detail in the expanded Materials and Methods in the online data supplement (available at http://www.circresaha.org). The expression of the transgene was assessed on three hearts from adult mice (8 to 12 weeks old), from three independent litters, for each type of experiment carried out (RT-PCR, Northern and Western blots, immunofluorescence), and for each genotype investigated. In addition, all experiments were performed with samples from both Sv129/CD1 and 129Sv/C57Bl/6 mice.81, 百拇医药
ECG recordings were performed on anesthetized mice as described previously.25,34 The hearts were dissected, connected to a Langendorff setup, and extracellular epicardial electrograms were recorded during sinus (SR) or paced rhythm.25,34Protocols for measuring epicardial activation and the conduction velocity (CV) in the various cardiac tissues including the BBs are described in the online data supplement. Multiple group comparisons were performed using ANOVA with LSD post hoc analysis using SPSS 10 for Macintosh. Values are mean±SEM. Values of P0.05 were considered statistically significant.
Resultsa$-^.[, http://www.100md.com
Generation of the KI Micea$-^.[, http://www.100md.com
KI mice were generated on two genetic backgrounds:129129/CD1 (50%/50%) and 129Sv/C57Bl/6 (50%/50%). No abnormal embryonic lethality was detected. The mice grew normally, were fertile, and had no obvious anatomical cardiac defects.a$-^.[, http://www.100md.com
Transcriptional and Translational Expressions of the Transgenea$-^.[, http://www.100md.com
The results revealed no significant difference dependent on the genetic background and were reproducible for each type of experiment performed.a$-^.[, http://www.100md.com
The expression of Cx40, Cx43, and transgenic Cx45 mRNAs in the atria of wild-type (wt, Cx40+/+), heterozygous (Cx40+/KICx45) and homozygous (Cx40KICx45/KICx45) mice was determined by semiquantitative RT-PCR and Northern blot experiments No endogenous Cx45 expression was detected (, but a gene-dependent gain of Cx45 mRNA, accompanied by a gene-dependent loss of Cx40 transcript was seen in Cx40+/KICx45 and Cx40KICx45/KICx45 mice. The expression level of Cx45 mRNA in Cx40KICx45/KICx45 mice was comparable to that of Cx40 mRNA in Cx40+/+ mice . The levels of expression of the transcript of the Cx43 gene in the three types of mice investigated were similar
fig.ommitted?4[w\, http://www.100md.com
Figure 1. Expression of the transgene. A, Diagram indicating the positions of the primers (arrows) used in RT-PCR experiments to amplify fragments from wild-type (WT) Cx40, transgenic Cx45, and endogenous WT Cx45 transcripts. Transcribed exons 1 and 2 (E1 and E2) of the Cx40 gene are represented as white boxes. Transcribed exons 1, 2, and 3 of the Cx45 gene are represented as dotted boxes but only E3 is indicated. The expected sizes of amplicons are indicated. B, Representative results of RT-PCR experiments performed with RNA extracted from the atria of Cx40+/+ (+/+), Cx40+/KICx45 (+/KI), and Cx40KICx45/KICx45 (KI/KI) 129Sv/CD1 mice. Similar results were obtained with 129Sv/C57Bl/6 mice. Amplicons of the expected sizes were detected in all samples investigated. The intensity of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signals indicated that equivalent amounts of synthesized cDNAs were analyzed. C, Representative results of Northern blot experiments performed with RNA extracted from atria of Cx40+/+ (+/+), Cx40+/KICx45 (+/KI), and Cx40KICx45/KICx45 (KI/KI) 129Sv/CD1 mice. Similar results were obtained with 129Sv/C57Bl/6 mice. Intensity of signals obtained with the hypoxanthine phosphoribosyl-transferase (HPRT) probe indicated that equal amounts of cDNA were analyzed. Exposure times: 3 days, 5 days, 1 day, and 12 hours after hybridization with Cx40, Cx45, Cx43, and HPRT probes, respectively. D, Representative results of Western blot experiments performed with samples of atrial tissue collected from Cx40+/+ (+/+), Cx40+/KICx45 (+/KI), and Cx40KICx45/KICx45 (KI/KI) 129Sv/CD1 mice. Similar results were obtained with 129Sv/C57Bl/6 mice. Intensity of the signals obtained after treatment with anti-desmin antibody (Des.) indicated that similar amounts of proteins were probed with each of the anti-Cx antibodies. Molecular mass of the detected proteins is indicated on the right.
Cx45 protein12 was correctly translated in the transgenic mice, as shown by Western blot experiments performed with atrial samples (D). The reduction of the level of expression of Cx40 protein in the atria of Cx40+/KICx45 mice (D) was associated with the detection of Cx45 protein in the same tissue. Cx40 protein was not detected in the atria samples of Cx40KICx45/KICx45 mice. In contrast, Cx45 protein was shown to be expressed in these same samples with a level of expression higher than that observed in the heterozygotes. The level of expression of Cx43 protein was unaffected by the expression of the transgene (D).\$fqqou, http://www.100md.com
The pattern of expression of Cx40, Cx45, and Cx43 was further investigated by immunofluorescence on sections of Cx40+/+ and Cx40KICx45/KICx45 mouse heart. No signal was detected in the atria of Cx40KICx45/KICx45 mice treated with anti-Cx40 antibodies (B’), or in the atria of Cx40+/+ mice treated with anti-Cx45 antibodies (C). In contrast, analysis of sections from atria of Cx40KICx45/KICx45 mice indicated that Cx45 was strongly expressed in this tissue with a pattern of expression similar to that of Cx40 in the samples of Cx40+/+ mice (compare D). The pattern of expression of Cx43, and the intensity of signals, were similar in the atria of both Cx40+/+ and Cx40KICx45/KICx45 mice (F). Cx40 and Cx45 proteins were detected in the cardiac CS of Cx40+/+ mice (F), as reported previously.5 The area of Cx45 labeling was larger than that of Cx40, corresponding to the so-called "extended conduction system,"9 and the intensity of signal was weaker than that detected for Cx40 protein (F). In the Cx40KICx45/KICx45 mice the pattern of expression of Cx45 delineated an extended CS similar to that seen in the Cx40+/+ mice (H). Cx40 was detected between the endothelial cells of the coronaries irrigating the cardiac walls of the Cx40+/+ mice (A). In the coronaries of Cx40KICx45/KICx45 mice, Cx45 had replaced Cx40, as expected (B).
fig.ommitted|2, http://www.100md.com
Figure 2. Expression pattern of Cxs associated with myocytes in the atria of WT and transgenic adult mice. Shown are micrographs of frozen sections from atria of Cx40+/+ (+/+) and Cx40KICx45/KICx45 129Sv/C57Bl/6 mice (KI/KI). Sections were treated with rabbit anti-Cx40 (A and B’), anti-Cx45 (C and D), or anti-Cx43 (E and F’) antibodies. A, B’, C, D, E, and F’, Immunofluorescence micrographs. B, C’, and F are Nomarski micrographs of sections shown in B’, C, and F’, respectively. Similar results were obtained with 129Sv/CD1 mutant mice. Bar=20 µm for A through F’.|2, http://www.100md.com
fig.ommitted|2, http://www.100md.com
Figure 3. Expression pattern of Cxs associated with myocytes in the ventricles of WT and transgenic adult mice. Shown are micrographs of frozen sections from the ventricles of Cx40+/+ (+/+) (A, D, E, and F) and Cx40KICx45/KICx45 129Sv/CD1 mice (KI/KI) (B, G, and H). Sections were treated with both rabbit anti-Cx40 antibodies (Cx40), and guinea pig anti-Cx45 antibodies (Cx45) (double-immunofluorescence technique). Superimposed Nomarski and immunofluorescence micrographs of sections of coronary vessels in the cardiac wall are presented in A and B. ec indicates endothelial cells; sm, smooth muscle cells; and m, myocytes. Cx40 detected between the endothelial cells of coronaries of Cx40+/+ mice (A, green signals) has been replaced by Cx45 in Cx40KICx45/KICx45 mice (B, red signals). D and G are Nomarski micrographs. en indicates endocardium; IVS, interventricular septum; and LVC, left ventricular chamber. E, F, and H are immunofluorescence micrographs. Position of the sections shown in these panels is indicated by a box in scheme C, which represents a mammalian heart. E and F, Subendocardial expression of Cx40 and Cx45, respectively, in a section (D) coming from the heart of a Cx40+/+ mouse. Expression of both Cxs overlaps in the His bundle branch, but weak Cx45 labeling is also observed in the vicinity of the bundle, in the IVS (arrows in F). In the Cx40KICx45/KICx45 mice (G and H), as in the Cx40+/+mice, Cx45 was detected in the His bundle branches (white arrow in H), and in the first layer cells of the septal working myocytes (black arrows in H). It was undetectable in the major portion of the septum (asterisks in H). Similar results were obtained with 129Sv/C57Bl/6 mutant mice. Bar in A=50 µm for A and B; bar in D=50 µm for D, E, and F; and bar in G=20 µm for G and H.
Analysis of the Impulse Conduction in the Heartsj+, http://www.100md.com
Surface ECGssj+, http://www.100md.com
illustrates the ECG recordings of the 129Sv/CD1 mice. The values of the parameters of ECGs and their statistical analysis are summarized . No difference was found between the three genotypes investigated for the RR or the PQ intervals. However, the P wave duration was significantly increased from 9.1±0.3 ms in the Cx40+/+ mice to 10.7±0.5 ms in the Cx40KICx45/KICx45 mice, suggesting slower atrial activation or other mechanisms such as a delayed activation in the interatrial pathways or ectopic activation. The latter hypothesis is unlikely because the P-wave morphology was similar for all three genotypes. The QRS complexes were fractionated and their duration was increased from 10.8±0.4 ms in the Cx40+/+ mice to 14.0±0.6 ms in the Cx40KICx45/KICx45 mice, pointing to a delayed activation of the ventricles. The profiles of the ECGs of 129Sv/C57Bl/6 mice were similar to those of the 129Sv/CD1 mice (not shown). However the absolute values of the measured parameters were different from those of 129Sv/CD1 mice indicating a strain dependency, as expected.35 The P wave and the QRS complex durations were significantly increased in the Cx40KICx45/KICx45 129Sv/C57Bl/6 mice, as compared with the Cx40+/+ mice. Because the modifications identified were similar in the two genetic backgrounds, the experiments that followed were performed on one background only, 129Sv/CD1.
fig.ommittedhk*hm, 百拇医药
Figure 4. Representative ECGs recorded from Cx40+/+, Cx40+/KICx45, and Cx40KICx45/KICx45 mice. Genetic background, 129Sv/CD1. Prolongation and fractionation of the QRS complex was observed in the Cx40KICx45/KICx45 mice. Dotted lines represent isoelectric lines.hk*hm, 百拇医药
fig.ommittedhk*hm, 百拇医药
Table 1. Electrophysiological Parameters of Wild-Type and Mutant Micehk*hm, 百拇医药
Ventricular Activationhk*hm, 百拇医药
Epicardial mapping was performed in SR on Langendorff-perfused hearts to investigate the delayed activation of the ventricles in Cx40KICx45/KICx45 mice. shows representative activation maps of the right (A) and left ventricles (B) in SR. In the Cx40+/+ and Cx40+/KICx45 mice, the sites of first activation were found as breakthrough, apicolateral, or basolateral sites (see for the frequencies of events). In the Cx40KICx45/KICx45 mice no midventricular breakthrough activation was found, and the earliest activations were predominantly found at apical sites, indicating a different mode of ventricular activation in these animals.
fig.ommittedtf, 百拇医药
Figure 5. A and B, Representative examples of epicardial activation of the RV (A) and the LV (B) recorded in SR. Position of the electrode array on the epicardium is indicated in the pictograms. Activation times (ms) are indicated in A and B by color codes. In the maps shown in A, the sites of first activation were apicolateral, breakthrough, and basolateral sites for Cx40+/+, Cx40+/KICx45, and Cx40KICx45/KICx45 mice, respectively. Total epicardial activation times deduced from these maps were 5, 7, and 10 ms, respectively. In the maps shown in B the sites of first activation were basolateral and apicolateral sites for Cx40+/+, and Cx40+/KICx45 and Cx40KICx45/KICx45 mice, respectively. In these examples, the total epicardial activation times were 5, 6.5, and 6 ms, respectively. C, Representative curves of AV nodal conduction for the 3 groups of mice investigated. Relationship between the activation times of the RV is plotted as a function of the coupling interval of the premature stimulus. Black curves were drawn from experimental data; red curves are the best fits of the experimental data to online Equation 1 (available in the online data supplement).
The total activation time was calculated from all RV and LV activation maps recorded in SR by subtracting the latest activation times from the earliest ones. The activation time of the RV was significantly increased from 5.4±0.4 ms in Cx40+/+ mice to 9±0.7 ms (67%) in Cx40KICx45/KICx45 mice . The activation times of the LV of the mice of the three genotypes investigated were not significantly different.r5q9f(, http://www.100md.com
Atrioventricular Conduction.r5q9f(, http://www.100md.com
C shows examples of the AV delay as a function of the coupling interval of the premature stimulus (conduction curves). No significant differences were detected between the three groups of mice for neither the AV effective refractory period (ERP), the minimum AV delay at infinitely long coupling intervals (D), nor the time constant () ..r5q9f(, http://www.100md.com
Atrial Conduction.r5q9f(, http://www.100md.com
Activation maps of the paced right (RA) and left (LA) atria of the three groups of mice investigated are shown in B, respectively. In the RA, the CV was 30.8±1.8 cm/s for the Cx40+/+ mice and was not significantly changed in the Cx40+/KICx45 and Cx40KICx45/KICx45 mice (32.9±2.3 and 30.0±2.3 cm/s, respectively) . In the LA, there was a significant reduction of the CV, from 31.5±1.6 cm/s in Cx40+/+ mice to 24.3±2.2 cm/s in Cx40KICx45/KICx45 mice.
fig.ommitted^#4, 百拇医药
Figure 6. Representative paced activation maps of the RA and LA (A and B), the RV and LV (C and D), and the RBB and LBB (E and F) of Cx40+/+, Cx40+/KICx45, and Cx40KICx45/KICx45 mice. Position of the electrode array on the epicardium (A through D) or the septal myocardium (E and F) is indicated in the pictograms. Activation times are relative to the pacing stimulus in the maps of the RA, LA, RV, and LV and relative to the atrial activation in the maps of the RBB and LBB. CVs were calculated from these maps. Activation times (ms) are indicated by color codes. In the examples shown in A and B, the CVs in the RA and the LA were 36 and 29, 32 and 32, and 41 and 19 cm/s for Cx40+/+, Cx40+/KICx45, and Cx40KICx45/KICx45 mice, respectively. In the examples shown in C and D, the CVs in the RV and the LV were 40 and 38, 40 and 33, and 30 and 34 cm/s for Cx40+/+, Cx40+/KICx45, and Cx40KICx45/KICx45 mice, respectively. Note that the anisotropy of the conduction in the ventricles is independent of the genotype. CVs determined from the activation maps of the RBB and LBB illustrated in E (RS, right side of the septum) and F (LS, left side of the septum) were 42 and 47, 54 and 51, and 17 and 54 cm/s for Cx40+/+, Cx40+/KICx45, and Cx40KICx45/KICx45 mice, respectively. Note that the activation patterns in the right and left BB are asymmetrical. Electrograms recorded in 2 different sites of both bundle branches are shown for each genotype.
Ventricular Conduction(}iy]), 百拇医药
Examples of activation maps of the paced RV and LV are shown in D, respectively. RV and LV activation maps showed anisotropic conduction independent of the genotype. Both longitudinal and transversal CVs in the ventricles were similar in all three genotypes(}iy]), 百拇医药
Bundle Branch Conduction(}iy]), 百拇医药
Examples of RBB and LBB activation maps are shown in F, respectively. RBB activation was always recorded in a one-electrode wide strand, whereas LBB activation was electrically much wider and was always recorded in several electrode strands parallel to each other. In the Cx40+/+ mice, the CV in the RBB was 36.5±5.4 cm/s , and it was not significantly different in the Cx40+/KICx45 mice. Interestingly, in the Cx40KICx45/KICx45 mice, the CV in the RBB was significantly reduced by 46% to 19.2±2.9 ms. The CVs in the LBB were not significantly different between the three genotypes investigated .
Discussion:?v, 百拇医药
The targeted replacement of one Cx gene by another Cx gene has been performed for several Cxs.36–38 Analysis of the resulting transgenic mice has indicated that Cxs could have either unique or redundant functions, and has provided important clues regarding the role of Cxs in vivo. In this study, we have generated Cx40KICx45 mice, and assessed the involvement of Cx45 in the propagation of cardiac impulse in the absence of Cx40.:?v, 百拇医药
For both genetic backgrounds investigated, the major phenotypic effects of the biallelic replacement of Cx40 by Cx45 were an increased duration of the P wave, and a prolonged and fractionated QRS complex in the ECGs in vivo. Epicardial mapping indicated a changed and prolonged activation of the RV that could be attributed to slow conduction in the RBB. The CVs in ventricular working myocardium, and the conduction delay in the AV node, were unaffected. The duration of the P wave was increased by about 18% in the Cx40KICx45/KICx45 mice. No decrease of the CV in the RA was found in contrast to the LA in which a significant reduction of about 23% was observed. These data may explain the increase of the P-wave duration, although a delayed activation between the right and left atrium cannot be excluded, and indicate that Cx45 cannot fully replace the function of Cx40, at least in the LA. Studies on Cx40 KO mice have indicated that the increase in the duration of the P wave was higher, ranging from 30% to 56%,20,21 and was mainly due to a very significant reduction of the CV (30%) in the RA.21 These results, and the present data are suggestive of a chamber-specific role of Cx40. This hypothesis is strengthened by the fact that in the human heart the expression level of Cx40 is higher in the RA than in the LA.39 However, this differential expression remains to be demonstrated in the mouse heart.
No differences were found in the various parameters that characterize the AV conduction in the Cx40+/+, Cx40+/KICx45, and Cx40KICx45/KICx45 mice, indicating that the AV node functions normally when Cx40 is replaced by Cx45. Numerous studies have described an increase in the PR interval and the Wenckebach cycle length in the Cx40 KO mice,18–21 which indicated an impairment of the AV conduction. Our results indicate that the absence of Cx40 in the AV node can be corrected by the expression of Cx45 despite differences in their intrinsic properties.}y, 百拇医药
The delayed activation of the RV, but not that of the LV, during SR, which increases the activation time of the heart, accounts for the increase of the duration of the QRS complex. The CV in the ventricular working myocardium was unaffected by the replacement of Cx40 by Cx45, because Cx40 is not expressed in the ventricular working myocardium of the adult mouse heart. The slow CV measured in the RBB of Cx40KICx45/KICx45 mice, but not in the LBB, explains the abnormal activation of the RV, in these animals. In the absence of Cx40, Cx45 is the only known Cx which is expressed in the proximal parts of the bundle branches.5 Assuming that Cx45 was upregulated in a similar way in both bundle branches, its expression supports a normal conduction in the LBB, but fails to do so in the RBB, indicating that electrical and/or structural differences between LBB and RBB account for the observed effect. Analysis of mice in which one allele of the Cx40 gene was replaced by eGFP (Cx40+/KIeGFP mice) has shown that the LBB was in fact constituted with 20 or so parallel strands coming from the His bundle, and covering entirely the left side of the interventricular septum. In contrast, on the right flank the branch (RBB) emerging from the His bundle was constituted by only one thin fiber in its proximal part.40 Thus, the LBB is more extended and much more complex than the RBB, and this morphology is in agreement with the activation patterns which were recorded (F). Consequently, variations of the gap junctional coupling in the ventricular CS will affect RBB conduction more severely than LBB conduction. Furthermore, the relationship between the gap junctional coupling and the CV in a strand of model cells is not linear.41 At high levels of junctional conductance the CV saturates, and significant reductions of the conductance from very high values do not necessarily result in a significant reduction of the CV. In contrast, small reductions of the conductance from low values will lead to drastic reductions of the CV. If one assumes that the coupling is higher in the LBB than in the RBB, which is not unlikely because the CV is higher in the LBB (47.8 cm/s) than in the RBB (36.5 cm/s), a similar reduction of the intercellular coupling in both bundle branches would be more prominent in the RBB than in the LBB. The analysis of Cx40KO mice confirmed that the RBB is much more sensitive to changes in the Cx expression than the LBB. Indeed, the deletion of Cx40 without replacement resulted in conduction block in the RBB, but induced only a slowing down of the conduction in the LBB.25 The higher vulnerability of the RBB is also reflected in the clinical data that indicate that the frequency of conduction impairment in patients with cardiovascular diseases is much higher in the RBB than in the LBB (see for example Newby et al42).
In summary, the replacement of Cx40 by Cx45 resulted in the heart in a significant reduction of the CV in the LA and a normal CV in the RA. The conduction delay in the AV node was unaffected, whereas a partial loss of function became apparent in the RBB, but not in the LBB./gow6m, 百拇医药
Acknowledgments/gow6m, 百拇医药
This work was supported by the CNRS, the Université de la Méditerranée, the European Community (contract QLG1-1999-00516), The Netherlands Heart Foundation (grant 99-200), and the French Ministry of Education (ACI Biologie du Développement et Physiologie Intégrative). S.A. was supported (fellowships) by the Association Française contre les Myopathies, and the Groupe de Réflexion sur la Recherche Cardiovasculaire./gow6m, 百拇医药
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Coppen SR, Kodama I, Boyett MR, Dobrzynski H, Takagishi Y, Honjo H, Yeh HI, Severs NJ. Connexin45, a major connexin of the rabbit sinoatrial node, is co-expressed with connexin43 in a restricted zone at the nodal-crista terminalis border. J Histochem Cytochem. 1999; 47: 907–918.
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Gutstein DE, Morley GE, Tamaddon H, Vaidya D, Schneider MD, Chen J, Chien KR, Stuhlmann H, Fishman GI. Conduction slowing and sudden arrhythmic death in mice with cardiac-restricted inactivation of connexin43. Circ Res. 2001; 88: 333–339.x|ii/]e, 百拇医药
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Kirchhoff S, Nelles E, Hagendorff A, Krüger O, Traub O, Willecke K. Reduced cardiac conduction velocity and predisposition to arrhythmias in connexin40-deficient mice. Curr Biol. 1998; 8: 299–302.
Hagendorff A, Schumacher B, Kirchhoff S, Luderitz B, Willecke K. Conduction disturbances and increased atrial vulnerability in connexin40-deficient mice analyzed by transesophageal stimulation. Circulation. 1999; 99: 1508–1515.dx3x-$, http://www.100md.com
Verheule S, van Batenburg CA, Coenjaerts FE, Kirchhoff S, Willecke K, Jongsma, HJ. Cardiac conduction abnormalities in mice lacking the gap junction protein connexin40. J Cardiovasc Electrophysiol. 1999; 10: 1380–1389.dx3x-$, http://www.100md.com
Tamaddon HS, Vaidya D, Simon AM, Paul DL, Jalife J, Morley GE. High-resolution optical mapping of the right bundle branch in connexin40 knockout mice reveals slow conduction in the specialized conduction system. Circ Res. 2000; 87: 929–936.dx3x-$, http://www.100md.com
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Bevilacqua LM, Simon AM, Maguire CT, Gehrmann J, Wakimoto H, Paul DL, Berul CI. A targeted disruption in connexin40 leads to distinct atrioventricular conduction defects. J Interv Card Electrophysiol. 2000; 4: 459–467.
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Bukauskas FF, Elfgang C, Willecke K, Weingart R. Biophysical properties of gap junction channels formed by mouse connexin40 in induced pairs of transfected human HeLa cells. Biophys J. 1995; 68: 2289–2298.|, http://www.100md.com
Van Veen TA, van Rijen HVM, Jongsma HJ. Electrical conductance of mouse connexin45 gap junction channels is modulated by phosphorylation. Cardiovasc Res. 2000; 46: 496–510.|, http://www.100md.com
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Abstract0p, http://www.100md.com
Gap junction channels, required for the propagation of cardiac impulse, are intercellular structures composed of connexins (Cx). Cx43, Cx40, and Cx45 are synthesized in the cardiomyocytes, and each of them has a unique cardiac expression pattern. Cx40 knock-in Cx45 mice were generated to explore the ability of Cx45 to replace Cx40, and to assess the functional equivalence of these two Cxs that are both expressed in the conduction system. ECGs revealed that the consequences resulting from the biallelic replacement of Cx40 by Cx45 were an increased duration of the P wave, and a prolonged and fractionated QRS complex. Epicardial mapping indicated that the conduction velocities (CV) in the right atrium and the ventricular myocardium, as well as conduction through the AV node, were unaffected. The significant reduction of the CV in the left atrium would be the most likely cause of the P-wave lengthening. In the right ventricle, a changed and prolonged activation in sinus rhythm was found in homozygous mutant mice, which may explain the prolongation and splitting of the QRS complex. Electrical mapping of the His bundle branches revealed that this was due to slow conduction measured in the right branch. The CV in the left branch was unchanged. Therefore, in the absence of Cx40, the upregulation of Cx45 in the heart results in a normal impulse propagation in the right atrium, the AV node, and the left His bundle branch only.
Key Words: gap junction channels connexins cardiac conductionvaa, 百拇医药
Introductionvaa, 百拇医药
In the mammalian heart the electrical activity, spontaneously generated in the sinoatrial (SA) node, is propagated through the atria, then to the ventricular walls via specialized tissues that constitute the conduction system (CS). The CS includes the atrioventricular (AV) node, the His bundle, and the two bundle branches (BBs), which ramify into peripheral Purkinje fibers. Gap junction channels are responsible for the electrical coupling between the various types of myocytes, and they are required for the propagation of cardiac impulse.1vaa, 百拇医药
Gap junction channels are intercellular structures constituted by transmembrane proteins belonging to the connexin (Cx) family.2,3 Nineteen Cx genes have been identified in the mouse genome,4 but so far only four Cxs, Cx43, Cx40, Cx45, and Cx46, have been shown to be expressed in mammalian cardiomyocytes with each having a unique expression pattern in the adult heart.5 Hence, in the mouse heart, Cx43 is abundant in both the atrial and ventricular working myocytes, and the distal part of the CS; Cx40 is strongly expressed in the atrial working myocytes and the CS,6,7 whereas Cx45 is detected mainly in the SA node and the CS.8–10 Expression of Cx45 has been reported in the working ventricular myocardium11 but this finding has not been corroborated by other investigations.9,12 Lastly, Cx46 was detected in the rabbit SA node associated with Cx40 and Cx45.13,14
Analysis of Cx-deficient mice has provided evidence that Cx43, Cx40, and Cx45 are involved in both heart function and development. The Cx45 null mutation is lethal with the embryos dying in utero around embryonic day 10, 2 days after cardiac contractions are normally initiated.15,16 Several anomalies including defects in the endocardial cushions are thought to contribute to the lethal phenotype. Conditional knock-out (KO) mice in which Cx43 gene expression in the cardiomyocytes is reduced by about 90% have ventricular conduction velocity (CV) slowed by 50%. All these mice undergo sudden cardiac death from spontaneous ventricular arrhythmias by 2 months of age.17 Cx40KO mice are viable and fertile,18,19 and several studies have focused on impulse propagation in the hearts of these mice.20–25 Findings from these studies include impaired conduction at various levels of the CS, and increased incidence of inducible atrial tachyarrhythmias, indicating that Cx40 is an important determinant of impulse propagation in the atria and the AV conduction system. Recent investigations also suggest that Cx40 may play a role in cardiac morphogenesis.26
The analysis of KO mice has therefore provided important information on the in vivo functions of Cxs in heart. Another approach for analyzing the function of Cxs is to generate knock-in (KI) mice to assess the functional equivalence of two Cxs. Cx40 and Cx45 are both expressed in the mouse CS, albeit with very different levels of expression. Channels composed of Cx40 or Cx45 have quite different properties. Cx40 channels have a large unitary conductance (160 pS), in contrast with Cx45 channels, which have a small conductance ({approx} 30 pS).27,28 Macroscopic conductance of Cx40 channels is not very sensitive to the transjunctional voltage (Vj) as compared with that of Cx45 channels, which is very sensitive to Vj.29–32 Furthermore, both types of channels are differently regulated by phosphorylation.28,33 To assess the functional equivalence of Cx40 and Cx45, we have generated Cx40KICx45 mouse lines in which Cx45 is expressed in lieu and place of Cx40.
Materials and Methods81, 百拇医药
The strategy used to generate the transgenic mice and the techniques used for genotyping the ES cells and mice and for identifying expression of the transgene in the KI mice are described in detail in the expanded Materials and Methods in the online data supplement (available at http://www.circresaha.org). The expression of the transgene was assessed on three hearts from adult mice (8 to 12 weeks old), from three independent litters, for each type of experiment carried out (RT-PCR, Northern and Western blots, immunofluorescence), and for each genotype investigated. In addition, all experiments were performed with samples from both Sv129/CD1 and 129Sv/C57Bl/6 mice.81, 百拇医药
ECG recordings were performed on anesthetized mice as described previously.25,34 The hearts were dissected, connected to a Langendorff setup, and extracellular epicardial electrograms were recorded during sinus (SR) or paced rhythm.25,34Protocols for measuring epicardial activation and the conduction velocity (CV) in the various cardiac tissues including the BBs are described in the online data supplement. Multiple group comparisons were performed using ANOVA with LSD post hoc analysis using SPSS 10 for Macintosh. Values are mean±SEM. Values of P0.05 were considered statistically significant.
Resultsa$-^.[, http://www.100md.com
Generation of the KI Micea$-^.[, http://www.100md.com
KI mice were generated on two genetic backgrounds:129129/CD1 (50%/50%) and 129Sv/C57Bl/6 (50%/50%). No abnormal embryonic lethality was detected. The mice grew normally, were fertile, and had no obvious anatomical cardiac defects.a$-^.[, http://www.100md.com
Transcriptional and Translational Expressions of the Transgenea$-^.[, http://www.100md.com
The results revealed no significant difference dependent on the genetic background and were reproducible for each type of experiment performed.a$-^.[, http://www.100md.com
The expression of Cx40, Cx43, and transgenic Cx45 mRNAs in the atria of wild-type (wt, Cx40+/+), heterozygous (Cx40+/KICx45) and homozygous (Cx40KICx45/KICx45) mice was determined by semiquantitative RT-PCR and Northern blot experiments No endogenous Cx45 expression was detected (, but a gene-dependent gain of Cx45 mRNA, accompanied by a gene-dependent loss of Cx40 transcript was seen in Cx40+/KICx45 and Cx40KICx45/KICx45 mice. The expression level of Cx45 mRNA in Cx40KICx45/KICx45 mice was comparable to that of Cx40 mRNA in Cx40+/+ mice . The levels of expression of the transcript of the Cx43 gene in the three types of mice investigated were similar
fig.ommitted?4[w\, http://www.100md.com
Figure 1. Expression of the transgene. A, Diagram indicating the positions of the primers (arrows) used in RT-PCR experiments to amplify fragments from wild-type (WT) Cx40, transgenic Cx45, and endogenous WT Cx45 transcripts. Transcribed exons 1 and 2 (E1 and E2) of the Cx40 gene are represented as white boxes. Transcribed exons 1, 2, and 3 of the Cx45 gene are represented as dotted boxes but only E3 is indicated. The expected sizes of amplicons are indicated. B, Representative results of RT-PCR experiments performed with RNA extracted from the atria of Cx40+/+ (+/+), Cx40+/KICx45 (+/KI), and Cx40KICx45/KICx45 (KI/KI) 129Sv/CD1 mice. Similar results were obtained with 129Sv/C57Bl/6 mice. Amplicons of the expected sizes were detected in all samples investigated. The intensity of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signals indicated that equivalent amounts of synthesized cDNAs were analyzed. C, Representative results of Northern blot experiments performed with RNA extracted from atria of Cx40+/+ (+/+), Cx40+/KICx45 (+/KI), and Cx40KICx45/KICx45 (KI/KI) 129Sv/CD1 mice. Similar results were obtained with 129Sv/C57Bl/6 mice. Intensity of signals obtained with the hypoxanthine phosphoribosyl-transferase (HPRT) probe indicated that equal amounts of cDNA were analyzed. Exposure times: 3 days, 5 days, 1 day, and 12 hours after hybridization with Cx40, Cx45, Cx43, and HPRT probes, respectively. D, Representative results of Western blot experiments performed with samples of atrial tissue collected from Cx40+/+ (+/+), Cx40+/KICx45 (+/KI), and Cx40KICx45/KICx45 (KI/KI) 129Sv/CD1 mice. Similar results were obtained with 129Sv/C57Bl/6 mice. Intensity of the signals obtained after treatment with anti-desmin antibody (Des.) indicated that similar amounts of proteins were probed with each of the anti-Cx antibodies. Molecular mass of the detected proteins is indicated on the right.
Cx45 protein12 was correctly translated in the transgenic mice, as shown by Western blot experiments performed with atrial samples (D). The reduction of the level of expression of Cx40 protein in the atria of Cx40+/KICx45 mice (D) was associated with the detection of Cx45 protein in the same tissue. Cx40 protein was not detected in the atria samples of Cx40KICx45/KICx45 mice. In contrast, Cx45 protein was shown to be expressed in these same samples with a level of expression higher than that observed in the heterozygotes. The level of expression of Cx43 protein was unaffected by the expression of the transgene (D).\$fqqou, http://www.100md.com
The pattern of expression of Cx40, Cx45, and Cx43 was further investigated by immunofluorescence on sections of Cx40+/+ and Cx40KICx45/KICx45 mouse heart. No signal was detected in the atria of Cx40KICx45/KICx45 mice treated with anti-Cx40 antibodies (B’), or in the atria of Cx40+/+ mice treated with anti-Cx45 antibodies (C). In contrast, analysis of sections from atria of Cx40KICx45/KICx45 mice indicated that Cx45 was strongly expressed in this tissue with a pattern of expression similar to that of Cx40 in the samples of Cx40+/+ mice (compare D). The pattern of expression of Cx43, and the intensity of signals, were similar in the atria of both Cx40+/+ and Cx40KICx45/KICx45 mice (F). Cx40 and Cx45 proteins were detected in the cardiac CS of Cx40+/+ mice (F), as reported previously.5 The area of Cx45 labeling was larger than that of Cx40, corresponding to the so-called "extended conduction system,"9 and the intensity of signal was weaker than that detected for Cx40 protein (F). In the Cx40KICx45/KICx45 mice the pattern of expression of Cx45 delineated an extended CS similar to that seen in the Cx40+/+ mice (H). Cx40 was detected between the endothelial cells of the coronaries irrigating the cardiac walls of the Cx40+/+ mice (A). In the coronaries of Cx40KICx45/KICx45 mice, Cx45 had replaced Cx40, as expected (B).
fig.ommitted|2, http://www.100md.com
Figure 2. Expression pattern of Cxs associated with myocytes in the atria of WT and transgenic adult mice. Shown are micrographs of frozen sections from atria of Cx40+/+ (+/+) and Cx40KICx45/KICx45 129Sv/C57Bl/6 mice (KI/KI). Sections were treated with rabbit anti-Cx40 (A and B’), anti-Cx45 (C and D), or anti-Cx43 (E and F’) antibodies. A, B’, C, D, E, and F’, Immunofluorescence micrographs. B, C’, and F are Nomarski micrographs of sections shown in B’, C, and F’, respectively. Similar results were obtained with 129Sv/CD1 mutant mice. Bar=20 µm for A through F’.|2, http://www.100md.com
fig.ommitted|2, http://www.100md.com
Figure 3. Expression pattern of Cxs associated with myocytes in the ventricles of WT and transgenic adult mice. Shown are micrographs of frozen sections from the ventricles of Cx40+/+ (+/+) (A, D, E, and F) and Cx40KICx45/KICx45 129Sv/CD1 mice (KI/KI) (B, G, and H). Sections were treated with both rabbit anti-Cx40 antibodies (Cx40), and guinea pig anti-Cx45 antibodies (Cx45) (double-immunofluorescence technique). Superimposed Nomarski and immunofluorescence micrographs of sections of coronary vessels in the cardiac wall are presented in A and B. ec indicates endothelial cells; sm, smooth muscle cells; and m, myocytes. Cx40 detected between the endothelial cells of coronaries of Cx40+/+ mice (A, green signals) has been replaced by Cx45 in Cx40KICx45/KICx45 mice (B, red signals). D and G are Nomarski micrographs. en indicates endocardium; IVS, interventricular septum; and LVC, left ventricular chamber. E, F, and H are immunofluorescence micrographs. Position of the sections shown in these panels is indicated by a box in scheme C, which represents a mammalian heart. E and F, Subendocardial expression of Cx40 and Cx45, respectively, in a section (D) coming from the heart of a Cx40+/+ mouse. Expression of both Cxs overlaps in the His bundle branch, but weak Cx45 labeling is also observed in the vicinity of the bundle, in the IVS (arrows in F). In the Cx40KICx45/KICx45 mice (G and H), as in the Cx40+/+mice, Cx45 was detected in the His bundle branches (white arrow in H), and in the first layer cells of the septal working myocytes (black arrows in H). It was undetectable in the major portion of the septum (asterisks in H). Similar results were obtained with 129Sv/C57Bl/6 mutant mice. Bar in A=50 µm for A and B; bar in D=50 µm for D, E, and F; and bar in G=20 µm for G and H.
Analysis of the Impulse Conduction in the Heartsj+, http://www.100md.com
Surface ECGssj+, http://www.100md.com
illustrates the ECG recordings of the 129Sv/CD1 mice. The values of the parameters of ECGs and their statistical analysis are summarized . No difference was found between the three genotypes investigated for the RR or the PQ intervals. However, the P wave duration was significantly increased from 9.1±0.3 ms in the Cx40+/+ mice to 10.7±0.5 ms in the Cx40KICx45/KICx45 mice, suggesting slower atrial activation or other mechanisms such as a delayed activation in the interatrial pathways or ectopic activation. The latter hypothesis is unlikely because the P-wave morphology was similar for all three genotypes. The QRS complexes were fractionated and their duration was increased from 10.8±0.4 ms in the Cx40+/+ mice to 14.0±0.6 ms in the Cx40KICx45/KICx45 mice, pointing to a delayed activation of the ventricles. The profiles of the ECGs of 129Sv/C57Bl/6 mice were similar to those of the 129Sv/CD1 mice (not shown). However the absolute values of the measured parameters were different from those of 129Sv/CD1 mice indicating a strain dependency, as expected.35 The P wave and the QRS complex durations were significantly increased in the Cx40KICx45/KICx45 129Sv/C57Bl/6 mice, as compared with the Cx40+/+ mice. Because the modifications identified were similar in the two genetic backgrounds, the experiments that followed were performed on one background only, 129Sv/CD1.
fig.ommittedhk*hm, 百拇医药
Figure 4. Representative ECGs recorded from Cx40+/+, Cx40+/KICx45, and Cx40KICx45/KICx45 mice. Genetic background, 129Sv/CD1. Prolongation and fractionation of the QRS complex was observed in the Cx40KICx45/KICx45 mice. Dotted lines represent isoelectric lines.hk*hm, 百拇医药
fig.ommittedhk*hm, 百拇医药
Table 1. Electrophysiological Parameters of Wild-Type and Mutant Micehk*hm, 百拇医药
Ventricular Activationhk*hm, 百拇医药
Epicardial mapping was performed in SR on Langendorff-perfused hearts to investigate the delayed activation of the ventricles in Cx40KICx45/KICx45 mice. shows representative activation maps of the right (A) and left ventricles (B) in SR. In the Cx40+/+ and Cx40+/KICx45 mice, the sites of first activation were found as breakthrough, apicolateral, or basolateral sites (see for the frequencies of events). In the Cx40KICx45/KICx45 mice no midventricular breakthrough activation was found, and the earliest activations were predominantly found at apical sites, indicating a different mode of ventricular activation in these animals.
fig.ommittedtf, 百拇医药
Figure 5. A and B, Representative examples of epicardial activation of the RV (A) and the LV (B) recorded in SR. Position of the electrode array on the epicardium is indicated in the pictograms. Activation times (ms) are indicated in A and B by color codes. In the maps shown in A, the sites of first activation were apicolateral, breakthrough, and basolateral sites for Cx40+/+, Cx40+/KICx45, and Cx40KICx45/KICx45 mice, respectively. Total epicardial activation times deduced from these maps were 5, 7, and 10 ms, respectively. In the maps shown in B the sites of first activation were basolateral and apicolateral sites for Cx40+/+, and Cx40+/KICx45 and Cx40KICx45/KICx45 mice, respectively. In these examples, the total epicardial activation times were 5, 6.5, and 6 ms, respectively. C, Representative curves of AV nodal conduction for the 3 groups of mice investigated. Relationship between the activation times of the RV is plotted as a function of the coupling interval of the premature stimulus. Black curves were drawn from experimental data; red curves are the best fits of the experimental data to online Equation 1 (available in the online data supplement).
The total activation time was calculated from all RV and LV activation maps recorded in SR by subtracting the latest activation times from the earliest ones. The activation time of the RV was significantly increased from 5.4±0.4 ms in Cx40+/+ mice to 9±0.7 ms (67%) in Cx40KICx45/KICx45 mice . The activation times of the LV of the mice of the three genotypes investigated were not significantly different.r5q9f(, http://www.100md.com
Atrioventricular Conduction.r5q9f(, http://www.100md.com
C shows examples of the AV delay as a function of the coupling interval of the premature stimulus (conduction curves). No significant differences were detected between the three groups of mice for neither the AV effective refractory period (ERP), the minimum AV delay at infinitely long coupling intervals (D), nor the time constant () ..r5q9f(, http://www.100md.com
Atrial Conduction.r5q9f(, http://www.100md.com
Activation maps of the paced right (RA) and left (LA) atria of the three groups of mice investigated are shown in B, respectively. In the RA, the CV was 30.8±1.8 cm/s for the Cx40+/+ mice and was not significantly changed in the Cx40+/KICx45 and Cx40KICx45/KICx45 mice (32.9±2.3 and 30.0±2.3 cm/s, respectively) . In the LA, there was a significant reduction of the CV, from 31.5±1.6 cm/s in Cx40+/+ mice to 24.3±2.2 cm/s in Cx40KICx45/KICx45 mice.
fig.ommitted^#4, 百拇医药
Figure 6. Representative paced activation maps of the RA and LA (A and B), the RV and LV (C and D), and the RBB and LBB (E and F) of Cx40+/+, Cx40+/KICx45, and Cx40KICx45/KICx45 mice. Position of the electrode array on the epicardium (A through D) or the septal myocardium (E and F) is indicated in the pictograms. Activation times are relative to the pacing stimulus in the maps of the RA, LA, RV, and LV and relative to the atrial activation in the maps of the RBB and LBB. CVs were calculated from these maps. Activation times (ms) are indicated by color codes. In the examples shown in A and B, the CVs in the RA and the LA were 36 and 29, 32 and 32, and 41 and 19 cm/s for Cx40+/+, Cx40+/KICx45, and Cx40KICx45/KICx45 mice, respectively. In the examples shown in C and D, the CVs in the RV and the LV were 40 and 38, 40 and 33, and 30 and 34 cm/s for Cx40+/+, Cx40+/KICx45, and Cx40KICx45/KICx45 mice, respectively. Note that the anisotropy of the conduction in the ventricles is independent of the genotype. CVs determined from the activation maps of the RBB and LBB illustrated in E (RS, right side of the septum) and F (LS, left side of the septum) were 42 and 47, 54 and 51, and 17 and 54 cm/s for Cx40+/+, Cx40+/KICx45, and Cx40KICx45/KICx45 mice, respectively. Note that the activation patterns in the right and left BB are asymmetrical. Electrograms recorded in 2 different sites of both bundle branches are shown for each genotype.
Ventricular Conduction(}iy]), 百拇医药
Examples of activation maps of the paced RV and LV are shown in D, respectively. RV and LV activation maps showed anisotropic conduction independent of the genotype. Both longitudinal and transversal CVs in the ventricles were similar in all three genotypes(}iy]), 百拇医药
Bundle Branch Conduction(}iy]), 百拇医药
Examples of RBB and LBB activation maps are shown in F, respectively. RBB activation was always recorded in a one-electrode wide strand, whereas LBB activation was electrically much wider and was always recorded in several electrode strands parallel to each other. In the Cx40+/+ mice, the CV in the RBB was 36.5±5.4 cm/s , and it was not significantly different in the Cx40+/KICx45 mice. Interestingly, in the Cx40KICx45/KICx45 mice, the CV in the RBB was significantly reduced by 46% to 19.2±2.9 ms. The CVs in the LBB were not significantly different between the three genotypes investigated .
Discussion:?v, 百拇医药
The targeted replacement of one Cx gene by another Cx gene has been performed for several Cxs.36–38 Analysis of the resulting transgenic mice has indicated that Cxs could have either unique or redundant functions, and has provided important clues regarding the role of Cxs in vivo. In this study, we have generated Cx40KICx45 mice, and assessed the involvement of Cx45 in the propagation of cardiac impulse in the absence of Cx40.:?v, 百拇医药
For both genetic backgrounds investigated, the major phenotypic effects of the biallelic replacement of Cx40 by Cx45 were an increased duration of the P wave, and a prolonged and fractionated QRS complex in the ECGs in vivo. Epicardial mapping indicated a changed and prolonged activation of the RV that could be attributed to slow conduction in the RBB. The CVs in ventricular working myocardium, and the conduction delay in the AV node, were unaffected. The duration of the P wave was increased by about 18% in the Cx40KICx45/KICx45 mice. No decrease of the CV in the RA was found in contrast to the LA in which a significant reduction of about 23% was observed. These data may explain the increase of the P-wave duration, although a delayed activation between the right and left atrium cannot be excluded, and indicate that Cx45 cannot fully replace the function of Cx40, at least in the LA. Studies on Cx40 KO mice have indicated that the increase in the duration of the P wave was higher, ranging from 30% to 56%,20,21 and was mainly due to a very significant reduction of the CV (30%) in the RA.21 These results, and the present data are suggestive of a chamber-specific role of Cx40. This hypothesis is strengthened by the fact that in the human heart the expression level of Cx40 is higher in the RA than in the LA.39 However, this differential expression remains to be demonstrated in the mouse heart.
No differences were found in the various parameters that characterize the AV conduction in the Cx40+/+, Cx40+/KICx45, and Cx40KICx45/KICx45 mice, indicating that the AV node functions normally when Cx40 is replaced by Cx45. Numerous studies have described an increase in the PR interval and the Wenckebach cycle length in the Cx40 KO mice,18–21 which indicated an impairment of the AV conduction. Our results indicate that the absence of Cx40 in the AV node can be corrected by the expression of Cx45 despite differences in their intrinsic properties.}y, 百拇医药
The delayed activation of the RV, but not that of the LV, during SR, which increases the activation time of the heart, accounts for the increase of the duration of the QRS complex. The CV in the ventricular working myocardium was unaffected by the replacement of Cx40 by Cx45, because Cx40 is not expressed in the ventricular working myocardium of the adult mouse heart. The slow CV measured in the RBB of Cx40KICx45/KICx45 mice, but not in the LBB, explains the abnormal activation of the RV, in these animals. In the absence of Cx40, Cx45 is the only known Cx which is expressed in the proximal parts of the bundle branches.5 Assuming that Cx45 was upregulated in a similar way in both bundle branches, its expression supports a normal conduction in the LBB, but fails to do so in the RBB, indicating that electrical and/or structural differences between LBB and RBB account for the observed effect. Analysis of mice in which one allele of the Cx40 gene was replaced by eGFP (Cx40+/KIeGFP mice) has shown that the LBB was in fact constituted with 20 or so parallel strands coming from the His bundle, and covering entirely the left side of the interventricular septum. In contrast, on the right flank the branch (RBB) emerging from the His bundle was constituted by only one thin fiber in its proximal part.40 Thus, the LBB is more extended and much more complex than the RBB, and this morphology is in agreement with the activation patterns which were recorded (F). Consequently, variations of the gap junctional coupling in the ventricular CS will affect RBB conduction more severely than LBB conduction. Furthermore, the relationship between the gap junctional coupling and the CV in a strand of model cells is not linear.41 At high levels of junctional conductance the CV saturates, and significant reductions of the conductance from very high values do not necessarily result in a significant reduction of the CV. In contrast, small reductions of the conductance from low values will lead to drastic reductions of the CV. If one assumes that the coupling is higher in the LBB than in the RBB, which is not unlikely because the CV is higher in the LBB (47.8 cm/s) than in the RBB (36.5 cm/s), a similar reduction of the intercellular coupling in both bundle branches would be more prominent in the RBB than in the LBB. The analysis of Cx40KO mice confirmed that the RBB is much more sensitive to changes in the Cx expression than the LBB. Indeed, the deletion of Cx40 without replacement resulted in conduction block in the RBB, but induced only a slowing down of the conduction in the LBB.25 The higher vulnerability of the RBB is also reflected in the clinical data that indicate that the frequency of conduction impairment in patients with cardiovascular diseases is much higher in the RBB than in the LBB (see for example Newby et al42).
In summary, the replacement of Cx40 by Cx45 resulted in the heart in a significant reduction of the CV in the LA and a normal CV in the RA. The conduction delay in the AV node was unaffected, whereas a partial loss of function became apparent in the RBB, but not in the LBB./gow6m, 百拇医药
Acknowledgments/gow6m, 百拇医药
This work was supported by the CNRS, the Université de la Méditerranée, the European Community (contract QLG1-1999-00516), The Netherlands Heart Foundation (grant 99-200), and the French Ministry of Education (ACI Biologie du Développement et Physiologie Intégrative). S.A. was supported (fellowships) by the Association Française contre les Myopathies, and the Groupe de Réflexion sur la Recherche Cardiovasculaire./gow6m, 百拇医药
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