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(Circulation Research. 2000;87:929.)
© 2000 American Heart Association, Inc.

Integrative Physiology

High-Resolution Optical Mapping of the Right Bundle Branch in Connexin40 Knockout Mice Reveals Slow Conduction in the Specialized Conduction System

Houman S. Tamaddon, Dhananjay Vaidya, Alexander M. Simon, David L. Paul, José Jalife, Gregory E. Morley

From the Department of Pharmacology (H.S.T., D.V., J.J., G.E.M.), SUNY Upstate Medical University, Syracuse, NY; the Department of Physiology (A.M.S.), University of Arizona, Tucson, Ariz; and the Department of Neurobiology (D.L.P.), Harvard Medical School, Boston, Mass.

Correspondence to Gregory E. Morley, PhD, Department of Pharmacology, SUNY Upstate Medical University, 766 Irving Ave, Syracuse, NY 13210. E-mail morleyg{at}mail.upstate.edu

	Abstract

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Abstract—Connexin40 (Cx40) is a major gap junction protein that is expressed in the His-Purkinje system and thought to be a critical determinant of cell-to-cell communication and conduction of electrical impulses. Video maps of the ventricular epicardium and the proximal segment of the right bundle branch (RBB) were obtained using a high-speed CCD camera while simultaneously recording volume-conducted ECGs. In Cx40^–/– mice, the PR interval was prolonged (47.4±1.4 in wild-type [WT] [n=6] and 57.5±2.8 in Cx40^–/– [n=6]; P<0.01). WT ventricular epicardial activation was characterized by focused breakthroughs that originated first on the right ventricle (RV) and then the left ventricle (LV). In Cx40^–/– hearts, the RV breakthrough occurred after the LV breakthrough. Additionally, Cx40^–/– mice showed RV breakthrough times that were significantly delayed with respect to QRS complex onset (3.7±0.7 ms in WT [n=6] and 6.5±0.7 ms in Cx40^–/– [n=6]; P<0.01), whereas LV breakthrough times did not change. Conduction velocity measurements from optical mapping of the RBB revealed slow conduction in Cx40^–/– mice (74.5±3 cm/s in WT [n=7] and 43.7±6 cm/s in Cx40^–/– [n=7]; P<0.01). In addition, simultaneous ECG records demonstrated significant delays in Cx40^–/– RBB activation time with respect to P time (P-RBB time; 41.6±1.9 ms in WT [n=7] and 55.1±1.3 ms in [n=7]; P<0.01). These data represent the first direct demonstration of conduction defects in the specialized conduction system of Cx40^–/– mice and provide new insight into the role of gap junctions in cardiac impulse propagation.

Key Words: optical mapping • specialized conduction system • knockout mice • connexin40

	Introduction

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Intercellular coupling via gap junction channels is an important determinant of impulse propagation in the heart.¹ These channels provide a low resistance pathway that is essential for the coordinated spread of electrical activation, which subsequently triggers the contraction of the heart. Recent studies have indicated that alterations in the expression pattern of cardiac connexin proteins may lead to abnormal electrical coupling and arrhythmias.^{2 3 4 5 6} Therefore, understanding the role of intercellular communication in impulse propagation is essential.

Three connexins, Cx40, Cx43, and Cx45, are thought to be involved in impulse propagation in the myocardium. Detailed immunolocalization studies have shown that each of these proteins has a unique pattern of expression in the adult heart;^{6 7 8} however, the functional role of connexin proteins in impulse propagation remains poorly understood. The presence and expression levels of these connexins vary considerably in cardiac tissues with different conduction properties.^{6 8 9} Cx45 is expressed in the atrioventricular node and proximal portion of the ventricular conduction system.⁷ Cx43 is expressed in both the atrial and ventricular myocardium.^{7 10 11} Immunohistochemistry studies in the mouse heart have indicated that Cx40 is expressed mainly in the atrial myocardium and His-Purkinje system.^{7 12 13} Previous studies have shown that deletion of Cx40 did not affect the expression of the other cardiac connexins or the gross structure of the heart.^{4 5} On the basis of these studies, it is expected that the targeted deletion of Cx40 will result in conduction deficits in both the atria and specialized conduction system.

The gross structure of the adult mouse specialized conduction system has not been studied in any great detail.^{14 15} Some histological studies have demonstrated broad similarities between the structure of the specialized conduction system of the mouse and that of larger mammals.^{14 16} In the mouse, the atrioventricular (AV) bundle has been shown to give rise to a compact right bundle branch (RBB), whereas many left bundle branch (LBB) fibers originate progressively over a wide range. Studies of the canine intraventricular conduction system have shown that the fibers of the RBB and LBB insert into the interventricular septum and the ventricular free walls.^{16 17} Septal LBB fibers have previously been shown to initiate the first ventricular depolarization (Q wave) in humans and larger mammals, whereas septal RBB insertions contribute to later phases of ventricular activation.^{16 18 19}

The objective of this study was to characterize the electrophysiological consequences of the null mutation of Cx40 (Cx40^–/–) on patterns of ventricular activation as well as conduction in the RBB. To achieve these objectives, we have developed an imaging system that is capable of obtaining high-resolution optical maps of electrical excitation in the ventricles and from the proximal segment of the RBB. Our results indicate that deletion of Cx40 does not result in impaired conduction velocity (CV) within the ventricular myocardium. However, in all Cx40^–/– mice, epicardial breakthrough activation patterns during sinus rhythm and atrial pacing suggested slowed conduction in the RBB, which was confirmed by direct measurements of CV in the RBB. These data provide a detailed mechanism for the alterations recorded in surface ECGs from Cx40 deficient mice^{3 4 5 20} and provide important insight into basic mechanisms underlying impulse propagation in the heart.

	Materials and Methods

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All mapping studies were performed using Langendorff perfused mouse hearts in the absence of any motion reduction techniques on an upright microscope equipped with a cooled CCD camera (Dalsa Inc, CA-D1 128T). The right atrial appendage was paced to study activation patterns in intact left ventricle (LV) and right ventricle (RV) and on the exposed RBB. To confirm the location of the RBB, high-resolution images of acetylcholinesterase (AChE) staining were correlated with the optical mapping results. Ventricular conduction velocities (CV_max and CV_min) were studied by pacing the RV free wall. Ventricular conduction velocities were measured as described previously.^{21 22} CV in the RBB was determined by linear regression of activation time as a function of distance using the method of least squares.

Stimulus time (S) was defined as the beginning of the rectangular stimulus artifact obtained from volume-conducted ECGs, and ventricular activation (Q time) was defined as the first measurable deflection (above noise) of the QRS complex. LV and RV activation times were defined as the earliest optical breakthroughs identified on the LV and RV, respectively. RBB activation was defined as the time at which the first pixel on the RBB activated in the field of view (see Figure 5).

View larger version (45K): [in this window] [in a new window]   Figure 5. Figure 5. Acetylthiocholine staining in the RBB of WT mice correlates with optical signals, showing upstrokes that precede the QRS complex. A, Image of the right septal preparation in which the location of the RBB is demarcated by acetylthiocholine activity. B, All pixels that contained RBB activation are placed in register with the stained septal preparation. Clearly the 2 patterns correlate well. Upstrokes of pixels showing RBB activation (right inset) and ventricular activation (bottom inset) are shown. For display purposes, these signals were normalized to the ventricular upstroke, which causes the amplitude of the RBB upstroke to appear variable from pixel to pixel. Note that all RBB upstrokes preceded the ventricular activation and propagated from base to apex. C, Only the pixels used to measure conduction velocity, ie, RBB upstrokes with an SNR >4. The color bar beneath the panel and adjacent to the simultaneous ECG in panel D indicates the time of RBB activation. In this WT heart, the Q time is 3.3 ms after the activation time of the first pixel on the RBB. S indicates stimulus time; a, septal artery; PM, papillary muscle; ANT, anterior; POST, posterior; and CT, chordae tendineae.

An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.

	Results

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Conscious 6-Lead and Langendorff ECG
Figure 1 shows examples of 6-lead ECG recordings obtained from a conscious wild-type (WT) and a Cx40^–/– mouse. Although the heart rates of the 2 animals were similar (WT, 730 bpm; Cx40^–/–, 725 bpm), the PR interval was clearly prolonged in the Cx40^–/– mouse. In addition, as seen in this example, the QRS complexes recorded from Cx40^–/– mice were more notched than the WT mice, suggesting an altered ventricular activation sequence. The Table summarizes the ECG data obtained from 8 WT and 8 Cx40^–/– mice. On average, the PR interval was significantly prolonged from 35.1±1.5 ms in WT mice to 40.9±1.2 ms in Cx40^–/– mice (P<0.01). Significant differences were also found in the QRS duration (14.5±0.8 and 16.8±0.7 ms WT and Cx40^–/– mice, respectively; P<0.04). Moreover, sinus rhythm PR and RR intervals measured from Langendorff-perfused hearts were similar to the measurements in conscious mice (RR interval: 178.7±6.3 in WT [n=13] and 183±8.4 in Cx40^–/– [n=13], NS; PR interval: 34.7±0.9 in WT [n=13] and 44.5±1.4 in Cx40^–/– [n=13]; P<0.001). These data confirm the slower AV conduction that was reported from anesthetized Cx40^–/– animals.^{3 4 5}

View larger version (14K): [in this window] [in a new window]   Figure 1. Figure 1. ECGs of conscious WT and Cx40–/– mice. Examples of 6-lead ECGs obtained from a conscious control and a Cx40–/– mouse. Although the heart rates were similar in the 2 mice (730 and 725 bpm, WT and Cx40–/–, respectively), the PR interval was clearly prolonged, indicating atrioventricular defects.

View this table: [in this window] [in a new window]   Table 1. ECG Parameters in Conscious Cx40 Knockout Mice

Optical Mapping of Purkinje-Activated Ventricular Myocardium
To determine the mechanisms underlying the surface ECG alterations, ventricular epicardial activation patterns were recorded from the anterior surface of both WT and Cx40^–/– mice. Figure 2 shows color activation maps obtained during sinus rhythm and right atrial pacing at a cycle of 120 ms. In the left column, the WT activation pattern is characterized by 2 focused breakthroughs that originate on the free walls of the right and left ventricles. The breakthroughs form wavefronts that fully activate the field of view within 3 ms. In all WT mice during sinus rhythm, the RV breakthrough either preceded or occurred simultaneously with the LV breakthrough. These breakthrough sites represent 3D-wave propagation from Purkinje-muscle junctions (PMJ) originating from the RBB and LBB.²³ Similar activation patterns were observed in all WT hearts.

View larger version (70K): [in this window] [in a new window]   Figure 2. Figure 2. Epicardial activation patterns during sinus rhythm and atrial pacing. Iso- chrone maps during sinus rhythm and while pacing the atria at a BCL of 120 ms. Arrows (drawn by hand) pointing away from the breakthrough sites indicate the direction of wavefront propagation. WT animals showed 2 breakthrough sites on the anterior surface (near the apex of the RV and LV) that merge and activate the entire heart. The RV breakthrough preceded the LV breakthrough during sinus rhythm in all WT mice. Cx40–/– heart during sinus rhythm and atrial pacing at a BCL of 120 ms. During these rhythms, RV activation is grossly delayed and multiple breakthroughs are seen on the LV. Isochrone lines indicate that LV global activation propagates from the LV to the RV and eventually merges with the RV breakthrough. Note that the Cx40–/– mouse shows extensive RV breakthrough delay at a BCL of 120 ms.

In the right column, the activation patterns recorded from a Cx40^–/– mouse appear grossly different. Firstly, the focused LV breakthrough site seen in WT mice was replaced by a more diffuse and patchy activation pattern. In the example shown, the arrows indicate the direction of propagation from multiple breakthrough sites covering a large region of the LV. Secondly, the location of the first breakthrough site on both the LV and RV in Cx40^–/– mice was more variable than in WT mice. In this example, the first RV breakthrough in the Cx40^–/– heart occurred in the center of the RV free wall, whereas in other cases, the location of the first RV breakthrough site occurred elsewhere on the RV. Finally, in all Cx40^–/– mice the breakthrough activation sequence was reversed (ie, the RV breakthrough occurred after the first LV breakthrough). Note that in the example shown, the RV breakthrough during atrial pacing is delayed and is less apparent than during sinus rhythm.

Synchronized Ventricular ECG Recordings
To gain insight into the differences between RV and LV breakthrough activation patterns, RV and LV activation times were correlated with the Q time obtained from simultaneous volume-conducted ECG records. Figure 3 shows the relation between the Q time and the optically recorded RV and LV times while pacing the right atria at a basic cycle length (BCL) of 120 ms. Figures 3A and 3B show examples of these times and their relationship for a WT and Cx40^–/– mouse, respectively. Clearly, in the Cx40^–/– mouse, the RV activation time is delayed much more than the LV activation time relative to onset of the QRS complex. Figure 3C summarizes all right atrial stimulus (S) to Q time measurements obtained at the fastest BCL resulting in 1:1 AV capture (54.6±1.2 in WT [n=6] and 61.5±3.2 in Cx40^–/– [n=6]) and at BCL=120 ms (47.4±1.4 in WT [n=6] and 57.5±2.8 in Cx40^–/– [n=6]; P<0.01 between genotypes; P<0.02 between cycle lengths). The increase in the S-Q interval is consistent with the ECG data obtained in conscious mice (see Table). Figure 3D shows a bar graph summarizing our measurements of Q to LV time (4.6±0.4 in WT [n=6] and 5.2±0.5 in Cx40^–/– [n=6]; NS) and Q to RV time (BCL=120: 3.7±0.7 in WT [n=5] and 6.5±0.7 in Cx40^–/– [n=6]; P<0.01) at a constant BCL of 120 ms. These data indicate that the RV but not LV time is significantly delayed with respect to the Q time in the Cx40^–/– mice. Figure 3E illustrates the interval changes between the 2 genotypes. Clearly, in Cx40^–/– mice, only the delayed LV breakthrough is fully accounted for by the late arrival of the Q wave.

View larger version (22K): [in this window] [in a new window]   Figure 3. Figure 3. Ventricular synchronized ECG recordings from WT and Cx40–/– mice. A and B, Volume-conducted ECGs recorded from the same WT and Cx40–/– hearts as shown in Figure 2 during atrial pacing at 120 ms. Note that the RV and LV sequence is reversed in panel B (Cx40–/–), compared with panel A. The LV breakthrough time is not significantly different with respect to the Q time. C, S-Q (PR) intervals are significantly different with respect to genotype and pacing frequency. D, Only Q-RV interval is significantly different between WT and Cx40–/–. No cycle-dependent differences were found in the Q-LV and Q-RV intervals between the fastest 1:1 AV capture and 120 ms. E, Schematic of AV conduction marking the stimulus time (), RV activation (), LV activation (•), and Q (*) times for both WT (top) and Cx40–/– (bottom). Whereas the S-Q interval increase accounts for the delayed arrival of the LV breakthrough (c=a), it does not fully account for the later arrival of the RV breakthrough (b>a). P<0.02, P<0.01.

Ventricular Conduction Measurements
The delayed arrival of the RV breakthrough is likely attributable to slowed conduction in the RBB; however, to exclude the possible contribution of conduction defects within the ventricular myocardium, epicardial conduction patterns and velocities were determined. Figure 4 shows activation maps obtained from the RV of a WT (panel A) and a Cx40^–/– (panel B) mouse while pacing the ventricles directly at a BCL of 120 ms. Pixels were systematically excluded near the pacing electrode (<1 mm) to remove any stimulus artifacts and at a distance (>3 mm) to exclude potential wavefront collisions and 3D-wave propagation.²² Clearly, the anisotropic conduction pattern in the Cx40^–/– was similar to that of WT. Figure 4C shows mean CV_max and CV_min for all WT and Cx40^–/– mice tested. No statistical differences were found between the 2 genotypes. Similar results were obtained at a BCL of 90 ms (data not shown). These data indicate that Cx40 does not significantly contribute to impulse propagation within the ventricular myocardium, which is in agreement with multielectrode²⁰ and immunolocalization studies for Cx40.⁷

View larger version (35K): [in this window] [in a new window]   Figure 4. Figure 4. Epicardial ventricular conduction velocities. A and B, Ventricular activation isochrone maps of the anterior aspect of a WT and Cx40–/– heart. Note that the anisotropic propagation pattern in the ventricle allows for the identification of CVmax and CVmin. C, Scatter plot of these conduction velocity measurements. No statistically significant changes were found.

RBB Optical Mapping
To determine the contribution of Cx40 in impulse propagation in the specialized conduction system, high-resolution optical maps of the proximal segment of the RBB were obtained. Figure 5A is an image of the right septal wall where the RBB has been stained with acetylthiocholine iodide, which precipitates in the presence of AChE activity. Figure 5B shows a color activation map of the RBB superimposed on the stained septal preparation. Optical signals recorded from the right septum are shown in the 2 insets. Voltage-dependent fluorescence recorded from pixels on the RBB is expected to originate from both the specialized conduction system and the underlying ventricular myocardium, giving rise to 2 temporally distinct action potential upstrokes. Therefore, during antegrade propagation, the first upstroke should result from a wave of activation through the RBB, whereas the second should result from the subsequent activation of the ventricular myocardium. Pixels not on the RBB are expected to show a single upstroke representing endocardial activation. The time sequence plots shown in the insets to the right and below panel B were obtained from pixels falling on the RBB and away from the RBB, respectively. Clearly, 2 action potential upstrokes are seen in all of the traces in the right inset, whereas only 1 action potential upstroke is seen in the bottom inset. In addition, these pixels showed activation that propagated from base to apex, consistent with antegrade propagation through the RBB. Figure 5C shows the pixels that were identified using a signal-to-noise ratio (SNR) criterion (>4) and the activation times from these pixels were used to measure RBB conduction velocity. Gaps in the activation sequence were attributable to uneven staining of the endocardium. The trace in Figure 5D shows a volume-conducted ECG obtained during the optical recording. The color bar above the trace demarcates the beginning and end of RBB activation. Thus, the RBB optical upstrokes preceded the QRS complex by 3 ms and propagated distally toward the apex, which is again characteristic of RBB activation. These data represent the first optical recordings obtained from the specialized conduction system in the right ventricle.

Figure 6 shows a comparison between the activation patterns recorded from the RBB of a WT (panel A) and a Cx40^–/– (panel B) mouse. These maps depict the similarities and differences of the RBB activation in the 2 mice. In both maps, activation begins near the RV base, distal to the septal leaflet of the tricuspid valve, crosses the septum along the main septal artery, and extends across the base of the anterior papillary muscle. In the Cx40^–/– example shown, a smaller segment of the RBB was analyzed because of the early arrival of the underlying septal activation. Fusion of the RBB and ventricular action potential upstrokes occurred at distal sites on the bundle, obscuring RBB upstrokes. This merging of the upstrokes may be attributable to either delayed RBB activation, resulting from slow conduction in the RBB, or earlier septal activation. These possibilities are studied below. Figure 6C displays the RBB conduction velocity in these 2 hearts. A >50% slowing of CV is apparent in the Cx40^–/– heart. Summary of the RBB conduction velocity measurements is shown in Figure 6D. Clearly, the plots show a significant slowing of CV in the RBB of Cx40^–/– mice. No cycle length dependence could be demonstrated at the cycle lengths tested. These data indicate that the slower CV contributed to the merging of RBB and ventricular action potentials upstrokes.

View larger version (59K): [in this window] [in a new window]   Figure 6. Figure 6. Conduction velocity of the Cx40–/– and WT RBB. A, WT mouse. B, Cx40–/– mouse. C, Linear regression comparison from the WT in panel A (CV=0.72 mm/ms, R2=0.87) and the Cx40–/– in panel B (CV=0.36 mm/ms, R2=0.91). Linear regression calculations were performed with time as the dependent variable. a indicates septal artery; PM, papillary muscle; ANT, anterior; and POST, posterior. Overall Cx40–/– (n=7) conduction velocity was 50% of WT (n=7) mice (P<0.001) at both cycle lengths tested (120 and 160 ms), as seen in panel D.

RBB-Synchronized ECG Recordings
To quantify the extent of RBB delay in relation to ventricular activation, RBB activation times were compared with Q time. Figures 7A and 7B illustrate synchronized volume-conducted ECG recordings from WT and Cx40^–/– mice. Variable QRS morphologies were seen in the dissected RV preparations from both WT and Cx40^–/–. Figure 7C summarizes S to RBB time and indicates a prolongation in Cx40^–/– mice compared with WT mice (BCL=120 ms: 41.6±1.9 ms in WT [n=7] and 55.1±1.3 ms in Cx40^–/– [n=7]; BCL=160 ms: 38.7±1.5 ms in WT [n=7] and 50.6±1.5 ms in Cx40^–/– [n=7]; P<0.001 between genotypes; P<0.03 between cycle lengths). RBB to Q time measured in WT (BCL=120 ms: 4.30±0.8 ms; [n=7]) and Cx40^–/– (BCL=120 ms: 1.2±0.5 ms; [n =7]) mice is shown in Figures 7D. Cx40^–/– mice have a significantly shorter RBB to Q time (P<0.001). It is important to note that although the mean RBB to Q time was positive for Cx40^–/– mice, 1 mouse showed RBB activation that occurred after the onset of the QRS complex. Figure 7E summarizes the interval changes seen between genotypes. Clearly, the delayed RBB activation that occurs in Cx40^–/– mice is not fully accounted for by the late arrival of the Q wave (ie, the S-RBB increase is greater than the RBB-Q increase). Thus, the additional delay accounting for this difference must exist proximal to the optically mapped region of the RBB.

View larger version (21K): [in this window] [in a new window]   Figure 7. Figure 7. RBB synchronized ECG recordings from WT and Cx40–/– mice. A and B, Volume-conducted ECGs where the Q (*) and RBB () times are shown. Note that the RBB activation time is delayed relative to Q time. C, Significant S-RBB interval increase in Cx40–/– compared with WT while pacing at BCL of 120 ms. D, RBB-Q interval is significantly less than in the Cx40–/– mice (n=7) compared with WT mice (n=7). No cycle-dependent differences were detected in RBB-Q intervals between BCLs of 120 and 160 ms (data not shown). E, Schematic of AV conduction marking the stimulus (), RBB (), and Q (*) times for both WT (top) and Cx40–/– (bottom) mice. The RBB-Q interval increase does not fully account for the S-RBB increase measured in Cx40–/– mice (b>a). indicates RBB time; *Q time. P<0.03, P<0.001.

His-Bundle Branching and LBB Conduction Time
To better understand the relation of the LBB conduction time and the Q time, calculations were made using RBB activation maps to estimate the activation time where branching of the common His bundle occurs (B time; see online Figure 2; available in an online data supplement at http://www.circresaha.org). Assuming that the branch point is 1.5 mm proximal to the optically mapped region of the RBB (see Lev et al¹⁴ for histological description of branch point), the B time can be extrapolated from the RBB conduction velocity measurements and the B to Q time can be determined. Thus, the B to Q time delay provides an estimate of conduction time in the LBB fibers that give rise to the start of the QRS complex. From these estimates, B to Q time was not significantly different between the Cx40^–/– and WT mice (BCL=120 ms: 6.4±0.8 in WT [n=7] and 5±0.5 in Cx40^–/– [n=7]; BCL=160 ms: 6.9±0.7 in WT [n=7] and 5.5±0.7 in Cx40^–/– [n=7]; NS). Therefore, no detectable conduction defect was found in the LBB using this model. This finding is in agreement with LV free-wall activation times, which also indicate nondetectable LBB conduction delays in Cx40^–/– mice.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Impulse propagation in cardiac tissue is the result of many parameters functioning in concert, such as cell excitability,²⁴ intercellular coupling,^{25 26 27 28} and tissue geometry.^{29 30} Perturbation of any of these parameters during cardiac disease is associated with slowing of conduction and increased risk of cardiac arrhythmias. Previous reports characterizing the role of intercellular coupling have relied on pharmacological interventions to modulate gap junctional conductance^{25 26 31} ; hence, those studies have the limitation of nonspecific pharmacological effects. On the other hand, the development of genetic engineering technology has provided a new approach to investigate the role of specific proteins in cardiac disease in the absence of pharmacological interventions.^{32 33 34} In particular, targeted deletion of connexin proteins offers the potential to investigate the specific roles of these proteins in impulse propagation in the mammalian heart.^{4 5 35} Because the deletion of Cx40 is not associated with gross cardiac malformations within the myocardium or the His-bundle branches,⁵ the Cx40^–/– mouse provides an elegant model to study the effects of cellular uncoupling on impulse propagation.

ECG Intervals
The ECG data obtained from conscious Cx40^–/– mice confirm the PR and QRS prolongation that has been previously reported in anesthetized mice.^{4 5} In this study, we focused on the role of Cx40 in the specialized conduction system and ventricular myocardium. However, Cx40 is also expressed in the atria. Other studies have reported in some Cx40^–/– mice slower atrial conduction velocities, prolonged P-wave durations, longer sinus node recovery times, and atrial arrhythmias.^{3 4} Together, these studies provide supporting evidence that Cx40 is an important determinant of impulse propagation in the atria as well as the specialized conduction system.

Ventricular Activation in the Absence of Cx40
The null mutation of Cx40 resulted in obvious differences in both RV and LV activation patterns and times. Unlike WT mice, the LV of Cx40^–/– mice showed multiple breakthrough sites on the anterior surface. In addition, the first breakthrough on the RV varied more in location than that of the LV and showed significant delays with respect to the first LV breakthrough. Because of the broader distribution of left compared with RBB fibers in mice,¹⁴ conduction failures resulting from cellular uncoupling in the absence of Cx40 is expected to manifest as islands of epicardial activations surrounded by areas of quiescence. This patchy failure of conduction may occur at branching Purkinje fiber sites or at PMJs, where source-sink mismatches exist because of a change in the geometry in the propagating pathway.²⁹

Effects on the safety factor for propagation attributable to a reduction in intercellular coupling and abrupt changes in tissue geometry have been studied in patterned cell cultures.^{27 29 36} In the whole heart, the best-known example of current-to-load mismatch is that of the PMJ.^{37 38} At PMJs, depolarizing current (source) originating from a small Purkinje fiber has to provide enough excitatory current for a large ventricular mass of tissue (sink). Studies conducted on this subject have shown that successful propagation is indeed challenged by this transition.^{36 38}

Indeed, in larger mammals, which show a larger spatial distribution of LBB fiber insertions, a patchy and diffuse epicardial activation pattern is seen in the LV but not the RV.^{17 39} In our experiments, large delays in the RV breakthrough times resulted in significant changes in the sequence of RV epicardial activation attributable to a sweeping wave of activation from the LV. Hence, we cannot exclude the possibility that patchy patterns of conduction block also exist in the RV of the mouse heart. On the other hand, from our data, these isolated, local propagation failures do not provide a satisfactory mechanism for the overall delayed activation of the RV in Cx40^–/– mice.

Conduction Slowing in the RBB of Cx40^–/– Mice
Using newly developed high-resolution imaging techniques, RBB activation was quantitatively measured to determine the source of RV activation delays. Qualitatively, the activation pattern of the RBB in the Cx40^–/– mice was similar to that seen in the WT. In both cases, activation began near the septal leaflet of the tricuspid valve and continued distally to the base of the anterior papillary muscle. However, quantitative measurements of RBB conduction velocities showed a significant slowing in Cx40^–/– mice. Therefore, the lack of Cx40 in the RBB resulted in significant conduction slowing, which additionally resulted in delayed RV free-wall activation.

During optical mapping, it was noticed that RV septal activation occurred before the complete activation of the RBB in the Cx40^–/– mice. Moreover, simultaneous ECGs indicated that the RBB in the Cx40^–/– mice activated later with respect to the start of the QRS complex. Because the LBB is presumed to activate the septum and cause the first deflection of the QRS complex, both the Q-time and RV septal activation represent surrogate markers of LBB activation.^{16 18} Thus, both the optical and ECG measurements suggest that the RBB is more affected than the LBB via these surrogate markers. With improved technology and strides in optical mapping techniques, future left septal mapping studies in Cx40^–/– mice may directly demonstrate the role of Cx40 in the LBB.

Role of Connexin45
The presence of Cx45 in the His-Purkinje system cannot be forgotten.⁷ In Cx40^–/– mice, the existence of successful conduction through the RBB and LBB provides reason to implicate Cx45 as the remaining intercellular coupler in these tissues. Moreover, the lack of a detectable delay in the LV conduction system in Cx40^–/– mice leads to speculation that Cx45 is maintaining near normal conduction through this safer pathway. It is also important to note that because the RBB is anatomically thinner and action potential durations are longer than those in the LBB,^{16 40 41} it is expected that a lower safety factor for antegrade conduction exists through the right pathway.^{36 42}

	Acknowledgments

 

This work was supported by Program Project Grant PO1 HL 39707 from the National Heart, Lung and Blood Institute. We would like to thank Dr Mario Delmar for his contribution during the initial phase of this study. We would also like to thank Matthew Brunson and Robert Morton for providing excellent technical support.

Received August 29, 2000; revision received September 19, 2000; accepted September 19, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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