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(Circulation Research. 2004;95:170.)
© 2004 American Heart Association, Inc.

Cellular Biology

Electrical Propagation in Synthetic Ventricular Myocyte Strands From Germline Connexin43 Knockout Mice

Philippe Beauchamp, Cécile Choby, Thomas Desplantez, Karin de Peyer, Karen Green, Kathryn A. Yamada, Robert Weingart, Jeffrey E. Saffitz, André G. Kléber

From the Department of Physiology (P.B., C.C., T.D, K.d.P., R.W., A.G.K.), University of Bern, Switzerland; and the Center for Cardiovascular Research and Departments of Pathology and Medicine (K.G., K.A.Y., J.E.S.), Washington University, St. Louis, Mo.

Correspondence to André G. Kléber, MD, Department of Physiology, University of Bern, Bühlplatz5, CH-3012 Bern, Switzerland. E-mail kleber{at}pyl.unibe.ch

	Abstract

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To characterize the role of connexin43 (Cx43) as a determinant of cardiac propagation, we synthesized strands and pairs of ventricular myocytes from germline Cx43^–/– mice. The amount of Cx43, Cx45, and Cx40 in gap junctions was analyzed by immunohistochemistry and confocal microscopy. Intercellular electrical conductance, g_j, was measured by the dual-voltage clamp technique (DVC), and electrical propagation was assessed by multisite optical mapping of transmembrane potential using a voltage-sensitive dye. Compared with wild-type (Cx43^+/+) strands, immunoreactive signal for Cx43 was reduced by 46% in Cx43^+/– strands and was absent in Cx43^–/– strands. Cx45 signal was reduced by 46% in Cx43^+/– strands and to the limit of detection in Cx43^–/– strands, but total Cx45 protein levels measured in immunoblots of whole cell homogenates were equivalent in all genotypes. Cx40 was detected in 2% of myocytes. Intercellular conductance, g_j, was reduced by 32% in Cx43^+/– cell pairs and by 96% in Cx43^–/– cell pairs. The symmetrical dependence of g_j on transjunctional voltage and properties of single-channel recordings indicated that Cx45 was the only remaining connexin in Cx43^–/– cells. Propagation in Cx43^–/– strands was very slow (2.1 cm/s versus 52 cm/s in Cx43^+/+) and highly discontinuous, with simultaneous excitation within and long conduction delays (2 to 3 ms) between individual cells. Propagation was abolished by 1 mmol/L heptanol, indicating residual junctional coupling. In summary, knockout of Cx43 in ventricular myocytes leads to very slow conduction dependent on the presence of Cx45. Electrical field effect transmission does not contribute to propagation in synthetic strands.

Key Words: Cx43 • Cx45 • very slow electrical propagation • discontinuous propagation • heptanol

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Connexin (Cx) proteins form intercellular channels enabling the intercellular exchange of ions and small molecules.¹ In the heart, they facilitate rapid, coordinated electrical excitation, a prerequisite for normal rhythmic contraction. Three different connexins, Cx43, Cx40, and Cx45, are expressed in heart.^2,3 Cx43, the most abundant connexin, is found in ventricular and atrial myocardium. Cx40 is expressed in atrial tissue and the cardiac conduction system. Although Cx45 expression in working ventricular myocardium is modest compared with Cx43, Cx45 is vital for early embryogenesis and cardiac development.^4,5 Cx45 is also expressed in the sinoatrial and atrioventricular nodes. It colocalizes with Cx40 in the conduction system,⁶ and with Cx43 in ventricular myocardium.⁷

The three cardiac connexins form channels with unique properties.⁸ Although multiple connexins are coexpressed in cardiac tissues, their interactions and contributions to electrical or metabolic function are not understood completely. Cx43 and Cx45 likely form heteromeric/heterotypic channels,^9–12 which may fulfill distinct functions. Cardiac diseases that lead to arrhythmias are associated with gap junction remodeling.^13–15 Therefore, knowledge about the specific roles of individual connexins in various cardiac regions is relevant for understanding arrhythmogenesis.

Several genetically engineered murine models of reduced expression of Cx43, Cx40, and Cx45 have been produced.^16–20 Mice with germline Cx43 knockout develop a cardiac malformation and die soon after birth,²¹ making analysis of conduction in intact hearts difficult. This had led to development of mice with conditional cardiac-specific Cx43 knockout (CKO).²² One CKO model produced using Cre-lox strategy exhibits a dramatic sudden death phenotype, apparently caused by ventricular tachycardia and fibrillation.²² However, 10% of ventricular myocytes in this CKO model expresses normal levels of Cx43,²² raising questions about the role of heterogeneous Cx43 expression in the pathogenesis of conduction abnormalities and arrhythmias. Chimeric mice with patchy Cx43 expression also exhibit spontaneous tachyarrhythmias (although less frequently than CKO mice) and areas of conduction abnormalities.²³

Elucidation of the precise relationship between Cx43 expression, intercellular coupling, and conduction delay is critical for understanding the role of altered connexin expression in arrhythmogenesis. Recently, we have created strands of murine cardiac myocytes in culture that exhibit action potentials and propagation properties similar to the intact animal.²⁴ This experimental tool has been used to analyze propagation of electrical impulses at high spatial resolution in strands of cells with heterozygous deletion of Cx43.²⁵ We observed that cardiac impulse propagation is relatively insensitive to a 50% reduction in Cx43 expression, in accordance with previous results from computer modeling.^26,27

In the present studies, we created strands of ventricular myocytes from germline Cx43^–/– mice. The goals were to assess conduction in ventricular tissue devoid of Cx43 and to characterize the nature and the role of other ventricular connexins.

	Materials and Methods

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Synthesis of Myocyte Strands of Specific Genotypes
The technique used to produce patterned growth of neonatal mouse ventricular myocytes has been described previously.²⁴ Hearts were obtained from mice maintained in an inbred colony (C57BL/6J; Jackson Laboratory; Bar Harbor, Me). Cx43^+/+, Cx43^+/–, and Cx43^–/– embryos were obtained at embryonic day 20 (E20). The genotype of each embryo was determined by polymerase chain reaction protocols modified from those of Reaume et al.²⁸ Ventricular tissue from individual hearts was minced and 1 cell suspension per heart was obtained by enzymatic digestion.²⁴ Cell suspensions (>10 000 cells/heart) were preplated to eliminate fibroblasts and seeded on coverslips to produce patterned growth.²⁹ Myocytes were grown in 5.5-mm-long channels of various widths (30 µm, 40 µm, and 60 µm). All experiments were performed on days 5 and 6 in culture. This study complied with the ethical principles of the Swiss Academy of Medical Science.

Immunohistochemistry, Confocal Microscopy, and Immunoblotting
Cultured cells were fixed in paraformaldehyde and immunostained with monospecific antibodies to quantify the amounts of Cx43, Cx45, and Cx40 at intercellular junctions as previously described.^24,30 Immunohistochemical analysis of Cx45 was performed using an anti-Cx45 antibody shown in previous studies to be monospecific.⁷ The amount of immunoreactive signal in discrete spots located at intercellular junctions was quantified using confocal microscopy and digital image processing algorithms validated in previous studies.³¹ When sufficient specific signal was present, it was measured and expressed as a percent of total tissue area. Cx45 protein expression was also measured by immunoblotting of whole cell homogenates prepared from Cx43^+/+, Cx43^+/–, and Cx43^–/– cultures as described previously.⁷

Dual-Voltage Clamp in Cell Pairs and Action Potential Recordings
Methods used to assess g_j in cell pairs have been described previously.³² In brief, freshly dispersed myocytes were plated onto glass coverslips coated with collagen. After 2 to 5 days in culture, spontaneously formed cell pairs were selected for measurement of transjunctional current, I_j, and junctional conductance, g_j=V_j/I_j, using dual-voltage clamp recordings at room temperature. Initially, the membrane potentials of both cells were clamped to the same voltage (V_j=V_2–V₁=0mV). Thereafter, voltage pulses of different amplitudes (up to 130 mV) and of either polarity were administered to cell 1 and I_j was measured in cell 2 (I_j=–I₂). Analyses that yielded maximal conductance, g_j,max, and the function of normalized conductance at steady state, g_j=f(V_j), were performed as previously described.³² Weakly coupled pairs and normally coupled pairs treated with heptanol (1 mmol/L) were used to determine single-channel events. Transmembrane action potentials were obtained in current clamp mode. Subthreshold voltage responses were used to calculate input resistance.³³

Optical Action Potential Measurement and Analysis of Propagation in Synthetic Strands
Staining of cell cultures with the voltage-sensitive dye RH237, multiple site optical recording of transmembrane potential with a light-sensitive diode array (10x10 diodes), and determination of conduction velocity, , have been described in detail.^24,34 Cell strands were stimulated at a site >1 mm from the recording site (cycle length 400 to 500 ms, rectangular pulse of 5 ms duration, 1.5-fold threshold strength). The spatial resolution of the system was 15 µm with a 40x objective, and 6 µm with a 100x objective. Time resolution between data points was 80 µs.

Conduction velocity was calculated along a strand segment of 105 µm. In Cx43^–/– strands in which conduction delays were confined to cell borders, an additional estimate of mean conduction velocity was obtained by dividing the average cell length (44 µm)²⁴ by the average conduction delay.

Statistical Analysis
Student t test was used for pairwise comparisons. Analysis of variance was used for multiple comparisons between genotypes. Variables are expressed as means±SD.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Immunohistochemistry of Cx Distribution in Synthetic Strands of Cx43^–/– Myocytes
Immunohistochemical analysis of Cx43 expression was performed in 3 Cx43^+/+ cultures, 3 Cx43^+/– cultures, and 6 Cx43^–/– cultures. All strands of each genotype showed a normal appearance and regular contractions when viewed by light microscopy. Immunohistochemical staining showed a reduction in the area occupied by Cx43 immunoreactive signal of 46% in Cx43^+/– preparations, caused mainly by a reduction in number of gap junctions (Table 1) as previously reported.²⁵ Cx43 immunoreactive signal was undetectable in all Cx43^–/– preparations, consistent with the complete absence of Cx43 in these cells (Figure 1A through 1D).

View this table: [in this window] [in a new window]   Table 1. Quantitative Immunofluorescence of WT Cx43+/+, HZ Cx43+/–, and Knock Out Cx43–/– Mice Cultures

View larger version (115K): [in this window] [in a new window]   Figure 1. A through D, Immunohistochemical analysis of Cx43 and Cx45 in Cx43+/+ and Cx43–/– myocyte strands. A, Confocal section of a synthetic Cx43+/+ strand stained with anti-Cx43 antibody. Note distribution of gap junctions at regular intervals around the cell perimeters. B, Immunostaining of a synthetic Cx43–/– strand with anti-Cx43 antibody revealed absence of Cx43 immunofluorescent signal. C, Confocal section of a Cx43+/+ strand stained with anti-Cx45 antibody. D, Bright-field picture of a Cx43–/– strand stained with anti-Cx45 antibody. Bars=10 µm. E, Immunoblot of total cell homogenate from neonatal Cx43+/+ (WT), Cx43+/–, (HT) and Cx43–/– (KO) ventricular myocyte cultures probed with anti-Cx45 antibody. Immunoblot of the same membrane probed with anti-GAPDH antibody is shown on the bottom trace.

Cx45 expression has been demonstrated previously in Cx43^+/+, Cx43^+/–, and Cx43^–/– neonatal mouse ventricular myocyte cultures.^7,35 To confirm these observations, we performed Cx45 immunohistochemical staining in 2 Cx43^+/+, 4 Cx43^+/–, and 6 Cx43^–/– cultures. Compared with Cx43^+/+ strands, the area occupied by Cx45 signal was decreased by 49% in Cx43^+/– strands (Table 1). This was caused by a decrease in the number of individual gap junctions containing Cx45. Cx45 signal at gap junctions was apparent in all Cx43^–/– cultures as tiny regular spots around the cell perimeter in amounts too low to reliably quantify (Figure 1D). Despite the marked differences in Cx45 signal in gap junctions, total Cx45 protein levels, determined by immunoblotting whole cell homogenates, were equivalent in Cx43^+/+, Cx43^+/–, and Cx43^–/– cultures (Figure 1E and Table 1). This observation confirmed results obtained in neonatal Cx43^–/– hearts.⁷

Although working ventricular myocytes do not express Cx40, immunostaining with specific anti-Cx40 antibodies was performed in 4 Cx43^–/– and 2 Cx43^+/+ cultures to determine whether cultures from minced ventricles included conduction system cells, which are known to be coupled by Cx40. In 26 microscopic fields from 4 Cx43^–/– cultures, 2.4±4.0% of all cells expressed Cx40. These occurred as individual cells interspersed among myocytes showing no Cx40 signal. In Cx43^+/+ cultures, 3.1±4.4% of all cells were Cx40-positive. The presence of this small number of Cx40-positive cells was considered to have no significant effect on impulse propagation.

Determination of g_j in Cell Pairs
Electrical properties of gap junctions in spontaneously formed pairs of Cx43^+/+, Cx43^+/–, and Cx43^–/– cells were determined by dual-voltage clamp recordings. Voltage pulses of ±10mV amplitude were administered to cell 1 to produce transjunctional gradients, V_j, and associated noninactivating gap junction currents, I_j, to determine the maximal gap junction conductance, g_j,max. As shown in Figure 2 and Table 2, g_j in Cx43^+/– and Cx43^–/– cell pairs was reduced to 68% and 4% of the Cx43^+/+ level, respectively.

View larger version (43K): [in this window] [in a new window]   Figure 2. Determination of gap junction conductance in pairs of Cx43+/+, Cx43+/–, and Cx43–/– cells using the dual-voltage clamp technique. Heterozygote deletion of Cx43 produced a 32.6% decrease in gj. Cx43-null cell pairs exhibited a 96.2% decrease in gj. Inset, A representative spontaneously formed cell pair together with the tips of the patch pipettes. Note the irregular contours of the cells and the relatively small areas of cell-to-cell contact.

View this table: [in this window] [in a new window]   Table 2. Electrophysiological Parameters of Wild-Type Cx43+/+, Heterozygous Cx43+/–, and Knock Out Cx43–/– Mice Cultures

Application of V_j pulses of increasing amplitude leads to progressive inactivation of g_j. Currents at the beginning and end of each V_j pulse, I_j,inst, and I_j,ss (instantaneous and steady-state currents, respectively), are useful to characterize the properties and connexin composition of gap junction channels. We determined the amplitudes of I_j,inst and I_j,ss for each I_j signal. Figure 3A and Table 2 summarize normalized g_j values plotted versus V_j in Cx43^–/– and Cx43^+/+ cell pairs. The fit of the data to the Boltzmann equation³⁶ at negative and positive V_j revealed that V_j,0, corresponding to V_j at half maximal inactivation, was smaller in Cx43^–/– than in Cx43^+/+ cell pairs (43.6mV versus 64.7mV).

View larger version (31K): [in this window] [in a new window]   Figure 3. A, Relationships between normalized gap junction conductance at steady state, gj,ss, and the transjunctional voltage, Vj, determined in Cx43+/+ () and Cx43–/– ()cell pairs. B, Single gap junction channel currents, Ij, recorded from Cx43+/+ and Cx43–/– cell pairs during 2 pulses of alternating Vj applied across intercellular junctions. The short dashes denote the levels of the main conductance states, and the long dashes indicate the levels of subconductance states. Left panel, Ij from a channel in a Cx43+/+ pair at Vj=±70 mV (upper trace) and ±130 mV (bottom trace). Right panel, Ij recorded from 2 channels in a Cx43–/– cell pair (upper and lower traces) at Vj=±90 mV. The consistent unitary conductance (gj value) of 30 pS is characteristic of channels formed entirely by Cx45. Note the asymmetrical behavior of Ij on reversal of Vj polarities in channels recorded in Cx43+/+ cell pairs.

Single-channel recordings from Cx43^+/+ and Cx43^–/– cell pairs revealed marked differences between genotypes (Figure 3B). Two distinct conductance levels were observed in Cx43^–/– cell pairs, suggesting the presence of a single gap junction channel type exhibiting a main open state at 33±5 pS (n=66) and a residual state at 13±5 pS (n=47). In contrast, a wide variety of conductances was observed in Cx43^+/+ cell pairs (range, 7 to 66 pS; n=141), rendering it difficult to attribute the values to 2 conductance states only. Moreover, changing the polarity of V_j pulses produced a change in single-channel conductance, which is characteristic of heterotypic channels (see Discussion).

Electrical Propagation in Synthetic Strands of Cx43^+/+, Cx43^+/–, and Cx43^–/– Ventricular Myocytes
Multisite optical mapping of transmembrane potential was performed in 10 different cell cultures from Cx43^–/– mice, and in 3 Cx43^+/+ and 8 Cx43^+/– cultures (Table 2). Propagation in Cx43^+/+ and Cx43^+/– strands was fast and continuous. As observed previously,^24,25 there was a slight difference in propagation velocity between Cx43^+/+ and Cx43^+/– strands (52±1 cm/s in Cx43^+/+ and 48±9 cm/s in Cx43^+/– strands), but this was not statistically significant (Table 2). A small difference was also seen in maximal upstroke velocity of the transmembrane potential in Cx43^+/+ and Cx43^+/– strands, confirming previous measurements.²⁵

Propagation in Cx43^–/– strands was dramatically different than in Cx43^+/+ and Cx43^+/– strands, as illustrated in Figure 4. Figure 4A depicts upstrokes of transmembrane action potentials measured during propagation from left to right along a strand distance of 105 µm with a resolution of 15 µm per measuring diode. The upstrokes are clustered in 4 groups with prolonged conduction delays (1587 µs to 2489 µs) between groups. This type of highly discontinuous conduction is typical of cardiac tissue with a very low degree of intercellular coupling, in which excitation within individual cells is virtually instantaneous and delays are localized to cell borders.^27,37 At a resolution of 15 µm (Figure 4A), the light-sensing diodes overlap cell borders leading to upstrokes with multiple components. To assess the topology of the conduction delay more accurately, experiments were performed in 4 cultures at the maximal magnification of 100x (resolution of 6 µm). Figure 4C and 4D show the progress of propagation from one single cell to the next. Action potentials occurred virtually simultaneously at multiple sites within a single cell, whereas a very large conduction delay occurred at the cell border. A histogram of intercellular delays is shown in Figure 4B. A major population of delays occurred with a peak value of 2250 µs. A second smaller population had a peak delay of 5250 µs, and a few very large delays were observed as well. Full conduction block, confined to local cell-to-cell contacts that presumably contained no gap junctions, is the most likely explanation for the presence of the second smaller peak. In this case, the delay between adjacent cells was determined by the sum of the cell-to-cell delays along the detour path of the excitation wave.

View larger version (52K): [in this window] [in a new window]   Figure 4. Propagation of electrical excitation in a Cx43–/– strand. A, Micrograph (top) of a synthetic strand with overlaid grid showing the positions of measuring diodes. Colors of circles correspond to clustered action potential upstrokes (bottom). Note very large conduction delays between action potential clusters. B, Histogram of conduction delays at cell borders. C, Synthetic Cx43–/– strand imaged at high resolution (6 µm). Cell borders are outlined for clarity. D, Propagation of action potential upstrokes at high resolution. The colors correspond to the measuring sites in C. Note clustering and simultaneous excitation of action potentials within individual cells and a conduction delay of >1 ms at the cell border.

Conduction velocity, , calculated from the conduction time along a distance of 105 µm was 2.1 cm/s in Cx43^–/– cultures. This was more than an order of magnitude slower than in Cx43^+/– and Cx43^+/+ cultures (Table 2). A second, independent estimate of conduction velocity in Cx43^–/– cultures, the ratio of the mean cell length and the average conduction delay (2460 µs, first population in Figure 4B), yielded a velocity of 1.8 cm/s.

In a recent theoretical study, it was shown that slow electrical propagation could occur under highly specialized conditions in the absence of gap junctional coupling (see Discussion). To determine whether this occurred in Cx43^–/– strands, we performed 3 experiments in which propagation was studied in the presence and absence of the uncoupling agent heptanol (1 mmol/L).³⁸ As shown in Figure 5, application of 1 mmol/L heptanol abolished propagation within 30 seconds. Washout of heptanol restored electrical propagation within a few seconds, indicating that very slow propagation in Cx43^–/– strands required the presence of functional gap junction channels. Higher concentrations of heptanol (3 mmol/L) have been reported to decrease I_Ca,, I_K1,³⁹ and I_Na.⁴⁰ We therefore tested the effect of 1 mmol/L heptanol on action potentials in single Cx43^+/+, Cx43^+/–, and Cx43^–/-– cells. Exposure to heptanol for 60 seconds in 6 experiments (2 Cx43^+/+, 2 Cx43^+/–, 2 Cx43^–/–) had no significant effect on action potential amplitude (126±4 mV versus 128±8 mV control) or input resistance (2.6±0.7 G, versus 3.1±0.9 G control), whereas action potential duration at 50% repolarization was significantly decreased (66±76 ms versus 37±26 ms control). These observations ruled out the possibility that depression of action potential upstroke or decreased membrane resistance contributed to the propagation block observed in Cx43^–/– strands 30 seconds after application of heptanol.

View larger version (30K): [in this window] [in a new window]   Figure 5. Effect of the uncoupling agent heptanol (1 mmol/L) on propagated excitation in a Cx43–/– strand. Top, Micrograph (top) of a synthetic strand with color-coded measuring diodes corresponding to locations of colored action potential upstrokes on the bottom. Bottom, Discontinuous propagation between 2 cells in the absence of heptanol with a delay of 1524 µs (left traces). Propagated excitation is blocked after 30 seconds of heptanol application (middle traces) and fully restored 30 seconds after heptanol washout.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The purpose of this study was to assess the effect of deletion of Cx43 in ventricular tissue on electrical propagation, to identify other connexins in impulse propagation, and to evaluate the possibility of field effect in cell-to-cell transmission of electrical impulses. Because germline Cx43^–/– mice die soon after birth, ventricular strands were synthesized from hearts excised 1 day before birth.

Immunohistochemical analysis showed complete absence of Cx43 signal in Cx43^–/– cells and confirmed 50% reduction of Cx43 signal in gap junctions in Cx43^+/– cells.^7,25 This is different than Cx43 conditional knockouts in which deletion of Cx43 by the Cre/lox system occurs in 90% of myocytes.^22,41 As observed previously in neonatal and adult mouse hearts,⁷ the area occupied by Cx45 immunofluorescent signal varied in cells with different Cx43 expression levels. Cx45 signal was reduced by 49% in Cx43^+/– strands and apparent only as small fluorescent spots in Cx43^–/– strands. These changes probably reflect a decrease in the size of gap junctional plaques caused by deficiency of Cx43 (which is, by far, the predominant connexin expressed in both adult and neonatal ventricular myocytes), rather than a real decrease in Cx45 content in gap junctions.

Dual-voltage clamp experiments were performed to quantify the decrease of g_j in Cx43^–/– cells and characterize properties of remaining connexins. We found that g_j in Cx43^–/– cell pairs was reduced by 96% compared with Cx43^+/+ cells. This is similar to results in pairs of adult myocytes from conditional Cx43 knockouts,²² but larger than the decrease reported for Cx43^–/– perinatal myocytes.⁴² In our study, the absolute value of g_j was significantly smaller than in myocytes of adult conditional knockout animals. A likely explanation is that neonatal cells form cell–cell contacts via small cytoplasmic processes (see Figure 2), whereas adult myocytes are coupled via larger junctions.

Both the dependence of g_j on transjunctional voltage V_j and recordings of single-channel events strongly suggested that Cx45 was the sole connexin responsible for cell-to-cell communication in Cx43^–/– strands. Boltzmann parameters V_j,0 and g_j,min in Cx43^–/– cell pairs were comparable to those of perinatal Cx43^–/– myocytes⁴² and closely resembled those of mouse Cx45 channels expressed in HeLa cells.¹¹ However, they were somewhat larger than those of murine Cx45 expressed in N2A cells.¹² The larger values of V_j,0 and g_j,min could be caused by background currents in cardiac cells that are absent in transfected cells. Single-channel recordings in Cx43^–/– cell pairs showed a consistent, small main state conductance of 33 pS, which is highly characteristic of Cx45.¹² In contrast, single-channel recordings in Cx43^+/+ preparations showed a wide range of conductances that were dependent on the V_j polarity. This suggests the presence of different conductance states (main state, residual state, substates) and channel types (homotypic Cx43, heteromeric/heterotypic Cx43/Cx45) and confirms that gap junction channels in Cx43^+/+ ventricular myocytes can be heterotypic and heteromeric.¹⁰

We measured propagation in strands of Cx43^–/– ventricular myocytes for the first time. Propagation in Cx43^–/– ventricular strands differed significantly from propagation in Cx43^+/+ and Cx43^+/– strands in 2 respects. First, conduction velocity was reduced by 25-fold to 2 cm/s, a value described previously as "very slow conduction."⁴³ In the heart in vivo, such slow velocity is observed only in the AV node.⁴³ Second, propagation was remarkably discontinuous, with virtually simultaneous excitation within individual cells and a mean conduction delay of >2 ms between cells. In synthetic strands of neonatal rat ventricular myocytes with normal Cx43 expression, delays across gap junctions during continuous steady-state propagation are typically 80 µs. Thus, a >20-fold increase in conduction time across gap junctions occurs in the Cx43^–/– phenotype.⁴⁴ This discontinuous type of propagation occurs at a higher margin of safety than normal propagation,²⁷ caused by recurrent alternations of low and high electrical resistances^27,45 formed by the low cytoplasmic resistance in series with the high resistance of Cx45 gap junctions. The same type of conduction, leading to a comparable degree of conduction slowing, has been produced experimentally in neonatal rat myocyte strands by the presence of structural discontinuities and/or by partial uncoupling with drugs.^37,46 However, one unavoidable limitation of chemical uncoupling agents is that development of uncoupling leading to very slow conduction is transient and unstable.³⁷ In the present studies, a steady-state level of uncoupling, sufficient to cause very slow but stable conduction, was produced experimentally for the first time, allowing comparison with g_j measurements in corresponding cell pairs and with theoretical simulations of low coupling states. Our results agree with theoretical computations^26,27 and prove that stable impulse propagation can be maintained, albeit slowly, despite a >95% decrease in g_j.

Although our measurements of g_j and propagation velocity agree closely, propagation velocity measured in intact ventricles in Cx43^–/– conditional knockouts was decreased to only 50% of normal.⁴¹ The reason for the marked discrepancy in conduction velocity in CKO hearts versus germline Cx43-null neonatal myocyte strands is not clear, and any explanations remain speculative. One important reason may be that gene deletion is incomplete in conditional knockout strategies using Cre-lox, and10% of ventricular myocytes express normal levels of Cx43.²² The consequences of this unavoidable heterogeneity in Cx43 expression in conditional Cx43 knockout hearts must be considered in terms of normal cellular connections in ventricular myocardium. For example, an individual left ventricular myocyte in adult canine myocardium is connected to an average of 11.3 neighboring myocytes.⁴⁷ A comparable degree of intercellular connectivity has been found in murine myocardium. In synthetic strands identical to those used in the present study, an individual neonatal mouse myocyte is connected 6.5 neighbors, which extrapolates to 16.6 neighbors in 3-dimensional tissue.²⁴ Thus, an average myocyte in a conditional Cx43 knockout heart may be connected to at least 1 neighbor that expresses wild-type levels of Cx43. This extent of low resistance connections within a poorly coupled syncytium should be considered as a potential explanation of the relatively high velocity values observed in Cx43 conditional knockout hearts.

As recently shown in computer simulations, field effect transmission can theoretically occur in the absence of cell-to-cell coupling by gap junction channels. However, successful field effect transmission required localization of >95% of Na⁺ channels at cell poles and the presence of very high resistance intercellular clefts. It is difficult to test this mechanism in an experimental setting that faithfully reproduces these conditions. Moreover, the presence of Na⁺ channels associated with T-tubules⁴⁸ speaks against localization of the totality of Na⁺ channels at intercalated disks. Results of the present experiments clearly ruled out field-effect transmission as a mechanism contributing to slow propagation in strands of neonatal murine myocytes.

The specific biological roles of each of the cardiac connexins are not completely understood. In the normal heart, Cx45 may contribute to slow propagation in the AV node and protect the sinus node from the large impedance load exerted by atrial tissue.⁴³ Although Cx45 is the dominant connexin in early heart development,⁴⁹ its role in the neonatal and adult ventricle remains unclear. Our results indicate clearly that Cx43 is the dominant ventricular coupling protein. Cx45 alone cannot maintain ventricular propagation at a velocity required for rapid, physiological depolarization and synchronized contraction. Cx45 differs substantially from other connexins in amino acid composition and molecular functions of the C-terminus.^1,50 Further experiments will be needed to clarify the specific role of Cx45 in ventricular myocardium.

	Acknowledgments

 
Supported by the Swiss National Science Foundation (A.G.K. and R.W.), the Swiss Heart Foundation (A.G.K.), the Swiss University Conference, and NIH grants HL58507 (J.E.S.) and HL66350 (K.A.Y.)

	Footnotes

 
Original received November 25, 2003; resubmission received April 20, 2004; revised resubmission received May 26, 2004; accepted May 27, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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