Discontinuous Conduction in Mouse Bundle Branches Is Caused by Bundle-Branch Architecture
Background— Recordings of the electrical activity of mouse bundle branches (BBs) suggest reduced conduction velocity (CV) in the midseptal compared with the proximal part of the BB. The present study was performed to elucidate the mechanism responsible for this slowing of conduction.
Methods and Results— Hearts of 16 mice were isolated and Langendorff perfused. After the right and left ventricular free walls were removed, the extracellular activity of the BB was mapped with a 247-point electrode. Premature stimulation was used to estimate CV restitution in the BBs. Expression/distribution of connexin40 (Cx40), Cx43, and Cx45 was determined. Morphology of the conduction system was assessed by whole-mount acetylcholine esterase staining and in Cx40+/KI-GFP hearts. Effective CV in the midseptal part of the left and right BBs was reduced by 50% compared with the proximal BB. CV restitution in the proximal and midseptal parts of the BBs was similar. Myocytes labeled positive for Cx40 and Cx45 in the entire BB. Cx43 colocalized with Cx40 and Cx45 only in the very distal BB. Subcellular distribution of gap junctions differed between proximal and distal BBs. Geometry of the midseptal and distal BBs revealed on both sides a profuse network of interlacing fibers, whereas the proximal BB consisted of a single (right BB) or multiple (left BB) parallel fibers.
Conclusions— Comparison of connexin expression/distribution, geometry of the BBs, and CV characteristics suggests that increased path length for activation resulting from BB geometry is responsible for the apparently reduced CV in the midseptal BB of the mouse heart.
Received March 8, 2005; revision received July 12, 2005; accepted July 19, 2005.
In various mammalian hearts, gap junctions composed of connexin40 (Cx40), Cx43, and Cx45 have been identified in cardiomyocytes composing the ventricular conduction system.1–10
The structure of the specific conduction system in the right and left septal wall of the human11 and dog heart12 is similar to that of the mouse.13 The generation of connexin knockout mice has revealed the importance of the respective gap junction isoforms for propagation of the electrical impulse through the conduction system. Lack of Cx40 results in impaired conduction and conduction block in the bundle branch (BB).14,15 Depletion of Cx45, the other prominent isoform expressed in the conduction system, results in cardiac dilation with associated conduction failure and defective vascular development, which causes these mice to die in utero at 9.5 dpc.10,16
Although the significance of the different gap junction isoforms for cardiac impulse propagation has been shown, Clinical Perspective p 2244 data on the relation between conduction velocity (CV) in the conduction system, local expression and distribution of connexins, and geometry of the conductive tissue are lacking. CV within the different parts of the ventricular conduction system is not uniform. Data from a previous study in which we studied the effect of Cx40 deletion on conduction in the BBs suggested a reduced CV in the midseptal compared with the proximal part of the BB.15
The present study was performed to elucidate the mechanism that determines CV in the different parts of the conduction system.
Commercially available OLA-C57B/6 mice were studied. Sixteen mice between 4 and 6 months of age were used for the electrophysiological experiments; 8 of them were used for immunohistochemistry. To study the geometry of the conduction system, a transgenic mouse line was created in which eGFP was expressed under the control of the Cx40 promoter. Heterozygous Cx40+/KI-GFP mice were generated as described before by the Department of Genetics and Developmental Physiology, University de la Mediterranee, Marseille, France.13 The study conformed to the guiding principles of the American Physiological Society.
Preparation of the Hearts
Mice were anesthetized by an intraperitoneal injection of urethane (2.0 g/kg body weight). The heart was excised from the chest, prepared, and connected to a Langendorff perfusion setup as described before.15
Recording of Electrograms
Extracellular electrograms were recorded with a 247-point electrode (19×13 grid; interelectrode distance, 0.3 mm) mounted in a micromanipulator. Recordings were made in unipolar mode with regard to a reference electrode connected to the support of the heart. Electrograms were acquired with a custom-built 256-channel data acquisition system. Signals were band-pass filtered (low cutoff, 0.16 Hz [12 dB]; high cutoff, 1 kHz [6 dB]) and digitized with 16-bit resolution at a bit step of 2 μV and a sampling frequency of 2 or 4 kHz. The input noise of the system was 4 μV (peak-peak). For septal measurements, the right and left ventricular free walls were removed, and the electrode grid was positioned on the interventricular septum (IVS) just below the AV valves. Atrial pacing was performed at twice the stimulation threshold. Each stimulus train was composed of 16 basic stimuli (S1) of 150 ms, followed by 1 premature stimulus (S2) of 140 ms, which was decreased by 10-ms steps until the AV effective refractory period was reached.
Activation maps were constructed from activation times determined with custom software based on Matlab (Mathworks Inc). The moment of maximal negative dV/dt in the unipolar electrograms was selected as the time of local activation. Activation times of at least 4 consecutive electrode terminals along lines perpendicular to intersecting isochronal lines were used to determine effective CV. The difference in CV restitution characteristics of the proximal and midseptal parts of the conduction system was derived from data obtained during premature stimulation. Statistical comparisons were performed with a paired Student t test using SPSS 11 for Macintosh. Values are given as mean±SEM. Values of P≤0.05 were considered statistically significant.
Immunohistochemistry and Histology
After the electrophysiological recordings, hearts were rapidly frozen in liquid nitrogen and stored at −80°C. Eight frozen hearts were serially sectioned in a frontal plane (to produce 4-chamber-view sections) of 10-μm thickness. Multiple sections taken from different levels (frontal to dorsal) of the IVS were incubated with primary antibodies directed against Cx40, Cx43, Cx45, α-actinin, β-catenin, desmin, and N-cadherin as reported previously.17 After immunolabeling, sections were mounted in Vectashield (Vector Laboratories) and examined with a light microscope equipped for epifluorescence (Nikon Optiphot-2) or by confocal laser scanning microscopy (Nikon RCM-8000 Real Time Laser Confocal Microscope). Staining for acetylcholine esterase (AchE) activity was performed on unfixed septa by incubation (37°C, dark) in 0.5 mmol/L 5-bromo-indoxyl-acetate, 5 mmol/L K3FeCN6, 5 mmol/L K4FeCN6, and 2 mmol/L MgCl2.
The following antibodies were used: rabbit polyclonal antibodies raised against Cx45 (kindly provided by Dr T.H. Steinberg, Washington University18), Cx40 (Chemicon), N-cadherin (Sigma), mouse monoclonal antibodies raised against Cx43 (Transduction Laboratories), desmin (Sanbio), α-actinin (Sigma), and β-catenin (Transduction Laboratories). Secondary antibodies (Texas Red and FITC-conjugated whole IgG) were purchased from Jackson Laboratories.
Delineation of the Ventricular Conduction System
Figure 1A schematizes the IVS with the different regions of the conduction system in the mouse heart. The length of the IVS measured from the valves until the very apical edge of the ventricular cavity ranges from 5 to 5.5 mm. We arbitrarily divided the BBs into 3 regions. P1 includes the area of IVS starting below the AV valves to 1.5 mm in the apical direction. In terms of conductive tissue, this area includes the common bundle (CB) and both BBs until just apical from the bifurcation. P2 is the midsection in between 1.5 mm below the valves to 4 mm below the valves; P3 includes the very distal (remaining) part of both BBs and the Purkinje fibers. For mapping of electrical activity, the electrode grid was positioned against the septum just below the AV valves (Figure 1B). In the mouse hearts, the grid completely covers the septum, and most basal electrodes record signals 0.5 mm below the valves (because a 0.5-mm rim, deprived of electrode terminals, surrounds the electrode).
A whole-mount AchE staining of the septum was performed to visualize the course of the BB. Figure 1C shows this course (in blue) of the left BB (LBB) running on the left septal surface. The LBB appeared as a network composed of multiple strands running rather parallel to the apex of the heart, thereby covering the whole width of left septal surface. Halfway along the septum, however, the fibers seem to branch and interconnect. Staining of the right septal surface did not reveal a clear signal of the right BB (RBB). Sections of stained septa, made perpendicular to the long axis, revealed that the blue staining of AchE activity was restricted to the endocardial and subendocardial cell layers (Figure 1D, P2 level). Non-specific border staining of damaged tissue was tested and excluded. Transverse cryosections taken at the level of the P1-P2 transition (just below the bifurcation) and immunolabeled for the presence of Cx40 confirmed the broad spreading of the LBB because Cx40 signals were found in subendocardial cells covering the entire width of the septum (Figure 1E). In a similar pattern, Cx40 signal was present in the Purkinje fibers at the opposite endocardial surface of the left ventricular free wall.
Additional information about the course of the BB was obtained from Cx40+/KI-GFP mice. In those mice, in which 1 allele encoding Cx40 was replaced by eGFP, 3D visualization of Cx40-positive tissue using fluorescent illumination was performed as described previously.13 Figure 2 shows the course of the RBB and LBB. Because of the high resolution of this technique, in addition to the LBB, we were able to identify the RBB. The most proximal part of the RBB (solid arrowhead 1 in Figure 2) is identified at the top of the IVS just below the valve (solid circle). In the apical direction, the bundle continues as a single compact bunch of fibers. At the midseptal area, the bundle fans out into a subendocardial network of interlacing fibers (arrowhead 2). Several asterisks indicate the septal artery and its ramifications which are GFP positive because of Cx40 expression by vascular endothelial cells.1 The architecture of the LBB is more complex because the proximal part consists of multiple fibers running parallel from base to apex. In the midseptal area, however, these fibers ramify in a profuse network of interlacing fibers similar to that found in the RBB. The course of the LBB closely resembles that obtained with whole-mount AchE staining (compare with Figure 1C).
Connexin Expression in the Ventricular Conduction System
Sections of the IVS were labeled with antibodies raised against Cx40, Cx43, and Cx45. As described previously, myocytes in the septal working myocardium (SWM) show abundant Cx43 labeling but are negative for Cx40. In the CB, labeling of Cx40 and Cx45 can be detected, whereas Cx43 is absent. Cx43 colocalizes with Cx45 at the interface between the CB and the SWM where a thin sheet of connective tissue exists that is discontinuous and penetrated by myocytes (data shown elsewhere15).
No colocalization of Cx40 and Cx43 (Figure 3A and 3C) or Cx45 and Cx43 (Figure 3B) was observed in cells forming the border zone of the proximal BB (level P1) and the SWM. In this area, cells were positive for either Cx40 and Cx45 (BB) or Cx43 (SWM). Although a small amount of collagen was present between the 2 cell types, it is unclear whether this completely separates the bundle from the septum. Further apically, the expression pattern remained similar as shown by Cx43 and Cx40 double labeling (Figure 3C, level P2). In the very distal parts of both BBs, colocalization of Cx40 and Cx43 (Figure 3D, level P3) and of Cx45 and Cx43 was present. Here, cells positive for Cx40 or Cx45 were occasionally neighbored by cells positive for Cx40/Cx45 and Cx43. No qualitative differences between the RBB and LBB were observed with regard to the expression of the connexin isoforms.
Connexin Distribution in the Ventricular Conduction System
In ventricular SWM, most gap junctions are located in the intercalated discs (IDs) forming the longitudinal cell borders. In contrast, in P1 and P2 of the BBs, labeling with Cx40 (Figure 3A and 3C) and Cx45 (Figure 3B) revealed a pattern of small steps (like a staircase) covering the cells. Double labeling of Cx40 with desmin (Figure 3E) or of Cx40 and α-actinin (Figure 3F) showed that Cx40 not only colocalized at the longitudinal IDs where desmin staining showed the highest intensity (Figure 3E, arrowheads) but also was found between these longitudinal IDs. This suggests that ID-like structures in the ventricular conduction system are not located exclusively at the longitudinal cell borders. Labeling of other ID-associated proteins like β-catenin (anchors the cytoskeleton to the adherence junctions; Figure 4A) and N-cadherin (component of the adherence junctions; Figure 4B) revealed a similar pattern in the proximal bundle.
Confocal analysis (stacks of 0.2-μm optical sections) of the organization of IDs in the proximal BB confirmed that the IDs exist as multiple small stairs covering the sarcolemma (Figure 4C). Contrarily, following the signal in the subsequent optical sections revealed that labeling profiles in SWM cells were much larger and remain on the longitudinal cell borders. More distally (P3), the step-like morphology of the IDs was nearly absent, and labeling of all 3 connexins covered the cells in small profiles (as shown in Figure 3D for Cx40 and Cx43).
Figure 5A and 5B summarizes the expression and subcellular distribution of gap junction proteins in the different regions of the mouse ventricular conduction system.
Conduction of the Electrical Impulse
To determine the CV of the electric impulse in the BB, we recorded electric activity of the septum with the multielectrode positioned right below the AV valves during sinus rhythm. Figure 6 shows typical examples of such recordings. Figure 6A and 6C shows individual electrograms recorded from the right ventricular septum (the selected electrodes are marked A and C in the activation map in Figure 6E). The onset of remote atrial activation was defined as t=0 ms (open arrow). Solid arrows indicate BB electrograms that are followed by a large deflection of septal activation. Activation times of the BB signals in electrograms were used to construct an activation map for the RBB (Figure 6E). Typically, the RBB is found as a 1- to 2-electrode-wide strand (0.3 to 0.6 mm) of which signals are recorded until approximately halfway in the P2 region. Similarly, electrograms of the left ventricular septum are shown (Figure 6B and 6D). The LBB activation map (Figure 6F) shows a much wider activation pattern (5 to 8 electrodes, 1.5 to 2.4 mm). The electrical asymmetry of the RBB and LBB closely correlates with the anatomic asymmetry shown in Figures 1C and 2⇑. Furthermore, as shown in Figure 2, the RBB is a single strand in the upper septal region, whereas the LBB is composed of multiple parallel strands in this area. The electric signals also reflected this anatomic difference. A single BB deflection was found in electrodes on the RBB, whereas LBB signals often exhibit multiple or fractionated deflections (multiple arrowheads in Figure 6D), indicating asynchronous parallel conduction in the multiple strands of the LBB.
In both the RBB and LBB, proximal CV (P1) was significantly faster compared with more distal parts (P2). CV in the RBB was 39.6±2.4 cm/s in P1 versus 22.4±1.0 cm/s in P2 (n=8; P<0.001). In the LBB, CV was 67.2±3.5 cm/s in P1 versus 33.4±2.8 cm/s in P2 (n=9; P<0.001).
Conduction delay in the proximal and midseptal part of the BBs during premature stimulation is illustrated in Figure 7. The top panels show typical examples of RBB (Figure 7A) and LBB (Figure 7B) activation patterns at S1-S1 pacing. Activation patterns of BB activation remained virtually the same after premature stimulation. However, sporadically, especially in maps of the LBB, we observed regions outside the main path where activation delay locally increased. Figure 7C and 7D shows that activation delay increases exponentially with decreasing S1-S2 interval. Figure 7E and 7F shows, however, that the difference in activation time between P1 and P2 did not change with decreasing S1-S2 interval (on average, 0.06±0.04 ms/10 ms; P=0.24; n=5 for RBB; and 0.03±0.04 ms/10 ms; P=0.48; n=5 for LBB), indicating similar CV restitution for both parts of the BBs.
Septal and Ventricular Activation
Figure 8 shows 2 examples of BB, septal, and epicardial activation patterns during sinus rhythm. Earliest activation of the right septum in Figure 8A arises at a basal site, suggesting that the septum is activated directly via the interconnection of the CB and the septum.15 The activation pattern of the left septum reveals earliest activation in the mid septum (at t=72 ms) near a site where Purkinje activity was recorded 7 ms earlier. From this site, septal activation spreads more or less centrifugally, suggesting that the septum is activated via the LBB. The Table shows that septal activation was initiated by the LBB or RBB in only 47% and 14% of the cases, respectively. The right ventricular septum revealed earliest activation near the apex in only 1 heart. In Figure 8B, activation of both the right and left septa seems to be initiated by the CB.
This study shows that (1) CV in the midseptal parts of both BBs is substantially reduced compared with the CV measured in the proximal BB; (2) CV restitution is similar in the proximal and midseptal parts of the BBs; (3) expression of Cx40 and Cx45 is found throughout the ventricular conduction system, whereas Cx43 is expressed only in the very distal BB and SWM; and (4) connexin isoform distribution in the proximal BB differs from that found in the distal BB and the SWM.
Impulse Propagation in BB of the Mouse Heart
CV in the proximal BBs compares well with previously reported velocities.13–15,19 The reduction in CV by half for both the left and right BBs, however, is an intriguing finding. Main parameters for conduction in the specific conduction system are electric coupling, cell shape and size, excitability,20–23 and tissue architecture.
Comparing the expression pattern of the 3 connexin isoforms reveals Cx40 and Cx45 in the proximal BB and Cx40, Cx43, and Cx45 in the very distal BB. The expression of Cx40 and Cx45 throughout the complete ventricular conduction system has been reported by others.8,9 However, expression of Cx43 does not appear until the level of P3, the very distal parts of the BB and the Purkinje fibers. This expression pattern of Cx43 differs from the pattern previously described for larger mammals like humans and cows in which Cx43 expression was already detected in P1, just below the bifurcation.24 The reduction in CV in the midseptal part of the BB cannot be attributed to the additional expression of Cx43 because this arises only at the very distal end where we were unable to record electrical activity of the BB, nor can the reduction be explained by differences in the expression of Cx40 and Cx45, which was similar in both regions.
In adult working myocardial cells, Cx43 (atria and ventricles) and Cx40 (atria) are expressed predominantly in large IDs found at the longitudinal cell borders where they colocalize with N-cadherin and desmoplakin.25,26 This polarized distribution favors longitudinal over transverse conduction and thus gives rise to anisotropic conduction.27 Myocytes composing the conduction system differ in several aspects from working myocardial cells. First, myocytes of the BBs and SWM express different connexins. Second, labeling profiles of connexins in myocytes of the BB seem smaller than neighboring cardiomyocytes in the SWM. Third, the subcellular distribution of Cx40 and Cx45 in myocytes forming the proximal and midseptal parts (P1 and P2) and of Cx40, Cx43, and Cx45 in the distal part (P3) of both the LBB and RBB differs substantially from the distribution of Cx43 gap junctions in myocytes of the SWM.
In P1 and P2 of the conduction system, expression of Cx40 and Cx45 seems to extend like multiple small step-like regions over the entire sarcolemma. This labeling pattern was also observed for other ID-related proteins such as N-cadherin and β-catenin. Desmin staining, however, revealed the characteristic pattern of cross-striations with highest the labeling intensity found only at the longitudinal IDs. We propose that the described step-like pattern found with the various ID-associated proteins could represent multiple small processes at sites where a BB myocyte interacts with adjacent BB myocytes. This might indicate that BB myocytes are intensively connected electrically to many adjacent myocytes in a pattern different from that found in the working myocardium.
On the contrary, more apical in the BB (P3), the remarkable ID structures are not as pronounced as those found in P1 and P2. At this site, labeling is diffuse and apparently not clustered in IDs at the longitudinal cell border; rather, it covers the entire cell surface in small dots. This labeling pattern in P3 of the mouse BB conforms to the distribution of gap junctions in the complete BB of larger mammals like humans and cows.24 In addition, in rat BB, labeling for both connexins and other ID-associated proteins revealed this rather diffuse labeling over the entire sarcolemma, although the highest intensity was found at the longitudinal cell borders (data not shown).
Because the distribution of Cx40 and Cx45 is similar in P1 and P2, it cannot explain the difference in CV.
Differences in cell size and shape between myocytes in the BB and those in the SWM have been reported in studies using rabbits and pigs.28 However, in mice, we and others could not detect such differences (this study and another29). Staining with the nuclear dye Hoechst 33258 of sections serial to those used for immunolabeling did not reveal differences between the proximal and midseptal BB or between the BB and SWM with respect to the number of stained nuclei/distance, which suggests comparable cell dimensions. Thus, cell size is unlikely to account for the differences in CV in the proximal and midseptal parts of the BB.
Differences in CV might also result from reduced upstroke velocities of the action potential in midseptal regions of the BB compared with the proximal region. However, this is unlikely in view of the data from Anumonwo et al,30 which indicate that upstroke velocity is actually higher in distal parts of the BB. Furthermore, simulation studies have shown that reducing excitability reduces the steepness of the CV restitution curve.31 As shown in Figure 7, CV restitution was almost identical in the proximal and midseptal regions of the LBB and RBB. That we sporadically observed regions outside the main path where activation delay increased locally suggests an effect of load mismatch in the network at those sites. Depending on the architecture and fiber diameter of the interlacing bundles, conduction delay can be expected after premature stimulation in such a network.
Geometry of the Ventricular Conduction System
In the mouse, the proximal LBB (P1) consists of multiple parallel-orientated fibers, whereas the RBB is tiny and consists of only 1 or occasionally 2 fibers (this study and that by Miquerol13). The observed fractionation in the LBB but not RBB electrograms fits well with the observed differences in geometry and can be explained by asynchronous conduction in the parallel fibers of the LBB.
In the midseptal (P2) region, on both the left and the right sides, intense branching of the proximal BB is observed, which correlates with the site of BB conduction slowing. The change in BB architecture might explain the reduction in CV in 2 ways: Activation delay caused by load mismatch at bundle discontinuities resulting from branching, or increased path length resulting from the profuse network of interlacing fibers, leading to an underestimation of CV. The fact that premature stimulation does not affect BB activation delay more in P2 compared with P1 (cf Figure 7) strongly favors the hypothesis of increased path length. In case of generalized load mismatch, delay in P2 would increase progressively with decreasing S1-S2.32 However, premature stimulation suggests a role of load mismatch at some local sites outside the main path.
In summary, the data indicate that the reduction in CV in the midseptal part of the BB is most likely due to the profuse network of fibers in this part of the specific conduction system.
BBs play a major role in BB reentry arrhythmias. Conduction abnormalities in the BB and the Purkinje network, together with or even without left ventricular dysfunction, seem to be the most critical requirement for the development of these arrhythmias.33–35 Given the similarity of BB architecture between mice and humans,11,13 the activation delay that occurs in the network of interlacing fibers in the midseptal portion of the BB in mice might also be present in the human heart, thus acting as an additional factor that favors the initiation of reentry in patients. Besides, additional conduction delay resulting from load mismatch in the profuse network may arise under pathological conditions.
Further support that our observations are not a special feature of the mouse heart but are more general can be distilled from the work by Lazarra et al.36 In that study, recordings were made from the septum with bipolar electrodes of adjacent fine Teflon-coated stainless steel wires. From recorded deflections caused by activation in the bundle/Purkinje network, activation maps were constructed. These maps showed that CV in the proximal part is faster than in the more distal area where the network becomes more outspoken. To the best of our knowledge, no such data on human hearts are available.
This study was supported by the Netherlands Organization for Scientific Research (grant 916.36.012).
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Rapid propagation of cardiac excitation through the Purkinje system is critical to normal cardiac activation. The Purkinje system is also involved in the genesis of uncommon forms of idiopathic left ventricular tachycardia, ectopy that can initiate ventricular fibrillation in susceptible patients and when damaged can give rise to a macroreentrant type of ventricular tachycardia known as BB reentry. This study of the structure and conduction of the mouse Purkinje system shows the delicate branching structure. Surprisingly, conduction velocity in the midseptal part of both right and left BBs is substantially slower than in the more proximal portion of the Purkinje system. Evidence is provided that conduction slowing in the midseptal part of the bundle branches is due to the intense sprouting of the bundles in this area, resulting in increased path length for activation resulting from the profuse network of interlacing fibers. This increased path length may facilitate reentry, suggesting a potential mechanism for arrhythmias that may emerge when the Purkinje system is injured. Furthermore, disease processes that reduce coupling between cells may produce mismatches in electrical loading (impedance mismatch) that might slow conduction, promote conduction block, or facilitate automaticity in branching networks. Given the similarity of the BB architecture between mice and humans, the activation delay that occurs in the network of interlacing fibers in the midseptal portion of the BBs might also be present in the human heart. If so, it may be a factor in the genesis of arrhythmias.