This Article Abstract Full Text (PDF) All Versions of this Article: 92/4/437    most recent 01.RES.0000059301.81035.06v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yao, J.-A. Articles by Wit, A. L. Search for Related Content PubMed PubMed Citation Articles by Yao, J.-A. Articles by Wit, A. L. Related Collections Electrophysiology Chronic ischemic heart disease Arrythmias-basic studies Ion channels/membrane transport

(Circulation Research. 2003;92:437.)
© 2003 American Heart Association, Inc.

Cellular Biology

Remodeling of Gap Junctional Channel Function in Epicardial Border Zone of Healing Canine Infarcts

Jian-An Yao, Wajid Hussain, Pravina Patel, Nicholas S. Peters, Penelope A. Boyden, Andrew L. Wit

From the Department of Pharmacology and Center for Molecular Therapeutics (J.-A.Y., P.A.B., A.L.W.), Columbia University, New York, NY; and Imperial College and St Mary’s Hospital (P.P., W.H., N.S.P.), London, UK.

Correspondence to Jianan Yao, MD, PhD, Dept of Pharmacology, Columbia University, 630 West 168th St, PH7W, New York, NY 10032. E-mail jy18{at}columbia.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The epicardial border zone (EBZ) of canine infarcts has increased anisotropy because of transverse conduction slowing. It remains unknown whether changes in gap junctional conductance (G_j) accompany the increased anisotropy. Ventricular cell pairs were isolated from EBZ and normal hearts (NZ). Dual patch clamp was used to quantify G_j. At a transjunctional voltage (V_j) of +10 mV, side-to-side G_j of EBZ pairs (9.2±3.4 nS, n=16) was reduced compared with NZ side-to-side G_j (109.4±23.6 nS, n=14, P<0.001). G_j of end-to-end coupled cells was not reduced in EBZ. Steady-state G_j of both NZ and EBZ showed voltage dependence, described by a two-way Boltzmann function. Half-maximal activation voltage in EBZ was shifted to higher V_j in positive and negative directions. Immunoconfocal planimetry and quantification showed no change in connexin43 per unit cell volume or surface area in EBZ. Decreased side-to-side coupling occurs in EBZ myocytes, independent of reduced connexin43 expression, and is hypothesized to contribute to increased anisotropy and reentrant arrhythmias.

Key Words: gap junction • myocardial infarction • arrhythmias

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
After coronary occlusion, a border zone of myocytes survives on the epicardial surface of healing canine infarcts, the epicardial border zone (EBZ).^1,2 The EBZ is characterized by reduced conduction velocity and increased anisotropy^1,2 associated with the occurrence of reentrant circuits and ventricular tachycardia.¹

Reduced sodium current^3,4 in EBZ myocytes may contribute to decreased conduction velocity. We studied gap junctional conductance in pairs of EBZ myocytes to determine if alterations occur and, therefore, might also contribute to changes in conduction and anisotropy. Connexin43 (Cx43) was quantified to determine if conductance changes were related to alterations in quantity of this gap junctional protein.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Preparation of Myocyte Pairs
Cell pairs were obtained from EBZ of infarcted canine left ventricle, 5 days after coronary occlusion. Surgical methods for occlusion^1,2 and enzymatic techniques for cell disaggregation⁴ have been described. EBZ tissue was removed from a region between the LAD and first circumflex branch that was visibly identified as infarct by its pale appearance (Figure 1), similar to the region sampled in previous studies of EBZ cells.^3,4 Because tachycardia was not induced nor reentry mapped, tissues did not come specifically from the central common pathway of reentrant circuits where we previously described redistribution of Cx43 gap junctions⁵ (Figure 1). For normal pairs (NZ), tissue from a similar region in noninfarcted hearts was used.

View larger version (39K): [in this window] [in a new window]   Figure 1. Diagrammatic representation of the epicardial surface of the anterior wall of the canine left ventricle (EBZ) that is infarcted by occlusion of the left anterior descending coronary artery (LAD). Margins of the infarct are labeled between LAD, base, and lateral left ventricle (LAT). Regions enclosed by solid, dashed, and dotted lines indicate the locations of the central common pathway of three reentrant circuits that were mapped in the experiments of Peters et al,5 where connexin43 was redistributed around the entire margins of the myocytes throughout the full thickness of the EBZ (50 to 200 cell layers). Shaded region is where tissue was obtained for this study. Scale bar=6 mm.

Studies were performed on both end-to-end– and side-to-side–coupled EBZ and NZ myocyte pairs, 2 to 8 hours after isolation. The morphological criterion for side-to-side coupling was greater than 50% contact of cell lengths. The criterion for end-to-end coupling was contact of more than 60% of the end-to-end surfaces between each of two paired cells and less than 10% contact of side-to-side cell surfaces.⁶ According to these criteria, about 60% of all cell pairs isolated were end-to-end coupled, and 40% were side-to-side coupled. End-to-end coupled pairs were easily identified under the optical microscope, because intercalated disks between the two cells could be seen clearly. It was sometimes difficult to identify a side-to-side coupled pair under the microscope because, on occasion, either a single large myocyte, or three myocytes coupled with each other without clear borders, resembled a cell pair. To verify that currents were recorded from a cell pair, we required that the macroscopic transjunctional conductance between paired cells (G_j) be blocked by halothane,⁷ particularly if there was a question concerning the presence of a cell pair. In 17 experiments, G_j was reversibly reduced by 91.7±3.1% at a transjunctional voltage (V_j) of +40 mV with 10 mmol/L halothane. Also, after experiments were completed, the supposed myocyte pairs were mechanically separated by moving the recording pipettes apart to confirm that there had been two coupled myocytes.

Electrophysiological Recordings
An aliquot of resuspension solution containing myocytes was placed on a poly-L-lysine–coated coverslip at the bottom of a 1-mL superfusion chamber on the stage of a Nikon inverted microscope. A Nikon Coolpix 990 digital camera recorded morphology of studied cell pairs. Transjunctional currents were recorded with a double whole cell patch clamp method^8–10 using two independent patch clamp amplifiers (Axopatch 1D and 1C, Axon Instruments). Voltage-clamp protocol generation and data acquisition were controlled by computers equipped with A/D-D/A interfaces (Digidata 1320, Axon Instruments) and Pclamp8 software (Axon Instruments). Currents were low-pass filtered at 1 KHz with a Bessel filter (Frequency Devices) and recorded with a sampling interval of 2.5 ms.

To determine macroscopic transjunctional conductance (G_j), command voltages for cell 1 (or electrode 1), V₁, and cell 2 (or electrode 2), V₂, were held at 0 mV. Then V₁ was stepped to test voltages from -100 to +100 mV in 10 mV increments. Test pulse duration was 5 seconds with interpulse interval of 10 to 15 seconds. Current through electrode 1 (I₁) in response to V₁ steps divides into a component passing through the sarcolemmal membrane of cell 1 to ground and another component passing across the gap junctional membrane into cell 2.^8–10 Therefore, transjunctional current (I_j) can be recorded in cell 2. I_j can be expressed as I₂(1+r₂/R₂), where I₂ is the current through electrode 2, r₂ is the series resistance (R_s) of electrode 2, and R₂ is the nonjunctional membrane resistance. Because membrane potential of cell 2 remained unchanged, I_j can be approximated as I₂ when electrode 2 has a tight seal with the membrane of cell 2 (r₂/R₂ approaches 0).^8–10 G_j was calculated as I_j/V_j, where V_j is transjunctional voltage. V_j is influenced by the R_s of both electrodes (r₁ and r₂) and currents through them (I₁ and I₂).

After tight seals were formed, r₁ and r₂ were compensated electronically (60% to 80%). Uncompensated R_s was determined from the capacitance transient currents elicited by applying a -10-mV step from a holding voltage of 0 mV simultaneously to both cells, and calculated as R_s=/C_m, where is the time constant for the decaying phase of the capacitive transient, and C_m is cell membrane capacitance. V_j was thus corrected for the uncompensated R_s by the following equation: V_j=V₁-(r₁ · I₁+r₂ · I₂). In our experiments, r₁ and r₂ were 1.72±0.02 M (n=66) with a range of 0.8 to 3.1 M.

To minimize errors of conductance measurement,¹⁰ we used large-tipped (4 to 5 µm), low resistance (0.5 to 0.9 M) patch pipettes to reduce R_s and minimize the difference between the applied and the actual V_j. To reduce the effects of sarcolemmal membrane resistance on V_j, bath and pipette solutions were designed to minimize currents through nonjunctional ion channels (potassium, calcium, Na⁺-Ca²⁺ exchanger currents). Sodium current (I_Na) and residual L-type calcium current (I_CaL) were inactivated by holding membrane voltage at 0 mV. Bath solution was Ca²⁺- and K⁺-free and composed of (in mmol/L) NaCl 146, MgCl₂ 0.5, NiCl₂ 6, BaCl₂ 1, and CsCl 2, HEPES 5, dextrose 5.5, pH 7.3 adjusted with NaOH at 23±2°C. Pipette solution contained (in mmol/L) Cs-Aspartate 115, TEA-CL 20, EGTA 10, HEPES 10, ATP (Mg salt) 5, pH adjusted to 7.3 with CsOH.

For comparison between groups, G_j obtained at V_j +10 mV, at which G_j is close to its maximal level (see Results), was used so that voltage clamp errors caused by large currents through electrodes were minimized. Corrected V_j was 8.97±0.01 mV (n=66) at aV₁ of +10 mV.

To determine voltage dependence of G_j,¹¹ cell pairs with maximal G_j <50 nS and uncompensated R_s <1.6 M were selected. As a result, the difference between V₁ and the corrected V_j was less than 10% at V₁=100 mV. Steady-state conductance (G_j,ss) was measured at the end of each voltage step and normalized to the instantaneous conductance (G_j,in) measured at the beginning of the voltage step. The G_j,ss-V_j relationship was described using the two-way Boltzmann function: equation

where G_max is the maximum conductance, G_min is the sustained conductance at the end of voltage steps (also called voltage-insensitive residual conductance), V_o is the transjunctional voltage at the halfway between G_max and G_min (V_j at which G_j,ss=(G_j,max-G_j,min)/2), and A is a constant that defines the voltage sensitivity (see Table).

View this table: [in this window] [in a new window]   Table 1. Boltzmann Parameters for Gap Junctions in Normal and Infarct Hearts

Data sampling rate was reduced to 200 Hz by a digital filter in pClamp8 before analysis. Peakfit (SPSS Science) was used for fitting data with the Boltzmann function. Excel (Microsoft) and SigmaStat (SPSS Science) were used for mathematical and statistical analysis (unpaired t test, P<0.05 was considered significant). Summarized data are presented as mean±SE.

Immunocytochemistry
Cx43 immunolabel was quantified in single myocytes from the same aliquot of cells from which cell pairs were obtained for electrophysiological studies. The immunolabeling protocol has been previously described.^5,12 It was adapted to give optimal labeling of cells to ensure maximal signal-to-noise ratio. Cells were fixed in 100% methanol and washed in phosphate-buffered saline before blocking in 1% bovine serum albumin (Sigma). The primary antibody was a mouse anti Cx43 IgG₁ raised against a synthetic peptide corresponding to positions 252 to 270 of rat Cx43 (Chemicon International). A secondary antibody tagged with CY3 fluorescent marker (Chemicon) was used. Cell pellets were resuspended in Citiflour Mounting Medium (Agar Scientific) and plated onto microscope slides. Immunolabeled myocytes were examined using a Leica TCS 4D two-channel laser scanning confocal microscope with an argon/krypton laser. The fluorescein isothiocyanate (FITC) filter detects auto fluorescence from whole myocytes, enabling cell volume measurement. Cx43 labeling was visualized using the tetramethyl rhodamine isothiocyanate (TRITC) filter. For each cell, serial highly confocal (1 µm depth) optical images at 1 µm intervals were recorded using the CY3 and FITC channels, giving a confluent series of 1 µm slices through the z-axis of the entire cell. This planimetry was performed for 20 randomly selected cells from each aliquot. Scion Image Software (Scion Corp) was used to measure immunolabeled Cx43 gap junctional area,¹² cell surface area, and cell volumes. Results are presented as mean±SE and were compared using unpaired t tests.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Gap Junctional Conductance in EBZ and NZ Myocyte Pairs
Microscopic images of typical side-to-side–coupled cells from NZ and EBZ are illustrated in Figures 2C and 2D, while those of end-to-end–coupled cells are shown in Figures 2E and 2F. NZ myocytes have smooth cell membranes, clear striations, and are rod-like in shape. As previously described, EBZ myocytes have rough "bumpy" membranes with blurred striations and small, dark, granules (lipid inclusions).^2,4

View larger version (100K): [in this window] [in a new window]   Figure 2. Representative current traces from side-to-side–coupled pairs from NZ (A) and EBZ (B). V1 of +10 and -10 mV (as marked) with duration of 5 seconds applied to cell 1, membrane voltage of cell 2 maintained at 0 mV. Positive (thin trace) and negative (thick trace) currents in cell 2 (Ij) correspond to negative (thin trace) and positives (thick trace) currents in cell 1, respectively. C and E, Images of side-to-side– and end-to-end–coupled pairs from NZ. D and F, Images of side-to-side– and end-to-end–coupled pairs from EBZ. Scale bars for images=20 µm.

Figure 2A shows representative I_j traces recorded from a side-to-side–coupled NZ cell pair (shown in Figure 2C). The I_j (recorded in cell 2) was generated by voltage steps of +10 mV (thick trace) and -10 mV (thin trace) applied to cell 1. In this example, I_j was 0.9 nA and G_j after correction of R_s was 101 nS. Figure 2B shows I_j recorded in a side-to-side–coupled EBZ cell pair (shown in Figure 2D) in response to the same voltage clamp protocol. I_j amplitude is decreased to 0.3 nA (with a G_j of 32 nS) at both positive and negative directions.

Figure 3 summarizes G_j obtained at +10 mV (near maximal conductance; see next section) for all groups. For NZ myocyte pairs, G_j was 109.4±23.6 nS (mean±SE, range of 24 to 338 nS) for side-to-side–coupled pairs, and 92.7±17.4 nS (16 to 297 nS) for end-to-end–coupled pairs (not significantly different from each other; P>0.05). In contrast, G_j of side-to-side–coupled EBZ myocytes (9.2±3.4 nS, range 0 to 63 nS) was significantly lower than G_j of end-to-end–coupled EBZ myocytes (82.6±20.5 nS, range 9 to 285 nS; P<0.01). G_j of side-to-side–coupled EBZ myocytes was also significantly lower than G_j of side to side NZ myocytes (P<0.001). G_j of end-to-end–coupled EBZ myocytes was not different from NZ end-to-end–coupled myocytes (P>0.05).

View larger version (16K): [in this window] [in a new window]   Figure 3. Steady-state transjunctional conductance (Gj) measured at a Vj of +10 mV in NZ and EBZ pairs. Open symbols, Individual measurements for side-to-side– and end-to-end–coupled cells. Filled symbols and vertical bars, Means and SE. Numbers of experiments in parentheses. *P<0.001 vs NZ.

Voltage Dependence of Gap Junctions in EBZ and NZ
Voltage-dependent properties of gap junctions in cell pairs with G_j of <50 nS at +10 mV were determined. I_j was recorded from cell 2, whereas voltage steps from -100 to +100 mV were applied to cell 1 in 10-mV increments (Figure 4A). In both NZ (left) and EBZ (right), I_j generated by voltages >+40 mV or <-40 mV showed symmetrical decay phases, and current decay was faster as V_j became larger. These characteristics suggest that the gating processes of gap junction channels in canine ventricular myocytes are time- and voltage-dependent. The I_j-V_j relationships in side-to-side coupled myocytes are summarized in Figure 4B. In both NZ (left) and EBZ (right) pairs, instantaneous currents (I_j,in, current amplitude measured at the beginning of V_j steps; open circles) are linearly related to V_j over all applied voltages. Steady-state current (I_j,ss, current amplitude measured at the end of V_j steps; filled circles) also displays a linear relation to V_j but only between +30 and -30 mV. Current rectification is evident at V_j >±40 mV; I_j,ss starts to decline at V_j of ±70 mV. In EBZ pairs (right), reduction in amplitude of I_j,ss at V_j beyond ±70 mV was less than in NZ cells. For example, I_j,ss at V_j of +100 mV was reduced to 34.7±3.5% of I_j,in levels in NZ cells but was only reduced to 52.0±4.6% in EBZ cells (P<0.05 compared with NZ). Figure 4C shows I_j-V_j relationships of gap junctions in end-to-end–coupled myocytes. I_j,in were also linearly related to applied V_j in both NZ (left) and EBZ (right) myocytes (open circles). Similar to side-to-side–coupled cells, I_j,ss in end-to-end EBZ pairs (filled circles) was reduced less than that of NZ pairs, although the current amplitude in end-to-end–coupled EBZ cells was comparable to that in NZ cells. I_j,ss at V_j of +100 mV was reduced to 54.8±5.2% of I_j,in amplitude in EBZ versus a reduction to 36.8±3.7% in NZ (P<0.05). Thus, rectification of I_j at large V_j is weaker in EBZ myocytes.

View larger version (29K): [in this window] [in a new window]   Figure 4. Voltage dependence of Ij, NZ (left panels), and EBZ myocyte (right panels). A, Representative Ij traces corresponding to Vj of -100 to +100 mV (as marked) with steps of 10 mV. Data obtained from side-to-side–coupled pairs. B, Ij-Vj relationships for side-to-side–coupled NZ (n=5) and EBZ (n=6) pairs. Instantaneous currents (Ij,in, open circles) measured at beginning of voltage steps, and steady-state currents (Ij,ss, filled circles) measured at end. Symbols and vertical bars denote mean values and standard errors, respectively. C, Ij-Vj relationships for end-to-end–coupled NZ (n=8) and EBZ (n=6) cell pairs. *P<0.05, **P<0.01 vs Ij,in.

Because voltage-dependent properties of side-to-side and end-to-end gap junctions are similar in both EBZ and NZ pairs, data for the G_j,ss-V_j relationships were combined from both side-to-side– and end-to-end–coupled pairs within each group (Figure 5A). The G_j,ss-V_j relationships are bell-shaped for both NZ and EBZ pairs such that normalized G_j,ss reaches its maximum at V_j close to 0 mV (±10 mV, where G_j,ss=G_j,in), and then decreases symmetrically as V_j changes in either a positive or negative direction. Minimum conductance (maximum rectification) occurs at V_j near ±100 mV. The G_j,ss-V_j relationship could be described by a two-way Boltzmann function with the best-fit parameters (see Table). The half maximal activation voltage (V_o) in EBZ pairs was +79.2±2.1 mV as compared with +66.6±2.2 mV for the NZ group (P<0.001) in positive polarity, and -82.4±3.1 mV (EBZ) as compared with -68.7±2.2 (NZ) (P<0.001) in the negative polarity. A reduction in voltage sensitivity (A) in EBZ relative to NZ did not reach statistical significance (P>0.05). Voltage-insensitive residual conductance (G_min) in EBZ pairs was significantly increased compared with NZ (P<0.01).

View larger version (24K): [in this window] [in a new window]   Figure 5. A, Voltage dependence of Gj. Gj,ss was normalized to Gj,in and plotted against Vj. Data summarized from 12 NZ pairs (open circles, 5 side-to-side and 7 end-to-end) and 11 EBZ pairs (filled circles, 5 side-to-side and 6 end-to-end). Symbols and bars denote mean±SE. Smooth curves superimposed on data points are calculated based on a two-way Boltzmann function with best-fit results. See Table for a summary of parameters. *P<0.05, **P<0.01 vs NZ. B, Voltage dependence of time constants of current decay. Time courses of Ij decay fit with double exponential function (inset). Symbols stand for mean values of fast (f,) and slow (s) time constants for NZ and EBZ pairs, respectively. Vertical bars represent SE. Numbers of observations are in parentheses. *P<0.05 vs NZ.

In NZ cell pairs, the time course of current decay at V_j >±70 mV was best described by a double exponential function. The fast and slow time constants (_f and _s) became smaller as V_j increased (Figure 5B). For example, _f and _s at a V_j of -70 mV were 0.46±0.09 and 2.85±0.69 seconds (n=9), respectively. The _f and _s at a V_j of -100 mV were 0.15±0.04 (P<0.01 versus -70 mV) and 1.00±0.12 seconds (n=9, P<0.05 versus -70 mV), respectively. In EBZ pairs, the time course of current decay was prolonged although the voltage dependence of the time constants was similar to those in NZ cells. For example, _f and _s in EBZ pairs were 0.98±0.25 and 4.95±1.06 seconds (n=11) at -70 mV, and 0.32±0.18 (P<0.05 versus -70 mV) and 1.36±0.20 seconds (n=11, P<0.01 versus -70 mV) at -100 mV (P<0.05 for both _f and _s compared with NZ). These results indicate that the function of gap junctions in EBZ is modified in terms of voltage dependence as well as current inactivation kinetics.

Connexin43 in EBZ and NZ Myocytes
Images of typical Cx43 immunolabeled myocytes from EBZ and NZ are shown in Figure 6. Planimetry of 180 cells from 9 EBZ preparations and 200 cells from 10 NZ preparations showed that cell volume of EBZ cells (36887±2504 µm³) was not significantly different from NZ cells (39570±2296 µm³; P<0.44). EBZ cells displayed a reduction in Cx43 immunolabel per cell (2454±382 µm² versus 3811±436 µm²; P<0.04). When corrected for cell volume, Cx43 immunolabel was not significantly reduced (NZ 0.104±0.01 µm²/µm³ versus EBZ 0.07±0.01 µm²/µm³; P<0.07). There was also no significant difference in cell surface area (NZ 5549±288 µm² versus EBZ 5142±271 µm²; P<0.34) or in Cx43 area per unit cell surface area (NZ 0.75±0.09 µm²/µm² versus EBZ 0.48±0.08 µm²/µm²; P<0.06).

View larger version (20K): [in this window] [in a new window]   Figure 6. Cx43 staining (reddish fluorescence) of an NZ (A) and EBZ (B) myocyte, respectively. C and D, 2nd slice and 8th slice of the EBZ myocyte shown in B. Scale bar=25 µm.

The ratio of Cx43 immunolabel located at the ends and sides of myocytes (longitudinal/transverse Cx43 label distribution) was also not significantly different in EBZ cells compared with NZ cells (0.45±0.058 versus 0.47±0.055; P=0.86). However, despite the lack of quantitative change, the Cx43 along the sides of 18% of EBZ cells was redistributed from normal small transversely oriented clusters (Figure 6A), to longitudinally arrayed streaks of Cx43 labeling (Figure 6B), as previously shown in cells immediately abutting the necrotic infarct and constituting the central common pathway.⁵

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Pathological Alterations in Gap Junctions
Information on pathological alterations of gap junctional electrophysiology as a cause of conduction abnormalities and arrhythmias is limited. Increased longitudinal resistance related to slowing of conduction, partly a consequence of decreased cell coupling, has been shown in papillary muscle during acute ischemia.¹³ Also, decreased conduction in multicellular preparations from hypertrophied hearts results from increased junctional resistance.¹⁴ Methods used in those studies did not permit determination of G_j nor resolution of increased R_j to side-to-side or end-to-end connections. G_j decreased in cell pairs from hearts after 30 minutes of coronary occlusion and was related to increased intracellular Ca²⁺ and decreased intracellular pH.¹⁵ Alterations in G_j associated with other pathologies have not been described.

Electrical Properties of Canine NZ and EBZ Gap Junctions
To determine if G_j was altered in EBZ, we studied adult canine ventricular myocyte pairs, a preparation that had not previously been investigated. To minimize errors of conductance measurement inherent in investigations of adult myocyte pairs, we used large-tipped patch pipettes to reduce R_s. However, errors in G_j measurements, particularly of high conductance junctions, cannot be totally eliminated due to current-passing limits of the recording pipettes.¹⁰

Macroscopic conductance of canine NZ gap junctions was 24 to 338 nS for side-to-side–coupled cells and 16 to 297 nS for end-to-end–coupled cells at a V_j of 10 mV (maximal values). G_j reported for mammalian adult ventricular myocytes has varied from 26 to 3073 nS, depending on species and recording methodology.^9,15,16–18 Our data are similar to rabbit ventricular myocyte pairs, studied using similar methods and experimental conditions.¹⁷

We demonstrated that adult canine myocyte gap junctions from normal hearts have voltage-dependent properties, not described in earlier studies on adult myocyte pairs.^9,16,18–20 G_j had the characteristic bell-shaped voltage dependence, similar to that of expressed connexin43 channels.^21,22 Failure to show voltage dependence in previous studies may have been secondary to inaccurate control of V_j because of uncompensated R_s.

In EBZ, there was a significant decrease in coupling conductance in the side-to-side direction compared with NZ. No difference in coupling conductance of end-to-end connections was found between EBZ and NZ pairs. However, because end-to-end G_j was relatively high in both groups, differences between the groups may have been obscured by errors in the methodology described above.¹⁰ G_j in EBZ also had bell-shaped voltage dependence. However, EBZ showed weaker responses to changes in voltage.

Relationship of Changes in G_j to Changes in Cx43
Redistribution of Cx43 around the cell perimeter occurs in myocytes abutting necrotic infarct and constituting the reentrant circuit central common pathway in EBZ.⁵ We did not induce reentrant tachycardia to locate the central common pathway in this study and did not, therefore, subselect a myocyte population for Cx43 redistribution. A majority (82%) of myocytes that we studied did not have redistribution of Cx43 characteristic of the central common pathway because they likely came from outside of this region (see Figure 1), nor did they show a reduction in amount of Cx43 as previously described for more prolonged ischemia.¹²

Although there was no quantitative alteration in end-to-side ratio of Cx43 distribution, indicating that reduction of transverse G_j occurred independently of a decrease in Cx43 immunolabel, distribution of Cx43 label along transverse membranes was altered in 18% of myocytes, changing from normal small transversely oriented clusters to longitudinally arrayed streaks. Any relationship between this redistribution and altered conductance is unknown, but a relationship between the pattern of this redistribution across EBZ and reentry has been shown.⁵ It is also possible that some gap junctions on the lateral sides of EBZ myocytes are not functional.²³

Changes in other connexins may also be related to altered EBZ conductance. Connexin45 has been detected in ventricular myocardium.²⁴ Immunolabeling for Cx45 (Cx45 antibody, Chemicon International) in frozen tissue sections of NZ and EBZ have shown indistinguishably scant or absent labeling (unpublished data, 2002), suggesting that significant upregulation of this connexin does not occur in EBZ.

Relationship to Increased Anisotropy and Reentry in the EBZ
In EBZ of healing canine infarcts, lines of functional block that bound the central common pathway of reentrant circuits causing tachycardia develop in regions of high anisotropy caused by slow transverse propagation at the interface between myocytes with structurally remodeled gap junctions (see previous section) and normally distributed gap junctions.^1,5 Slowing of transverse impulse propagation in regions of reentrant circuits outside the central common pathway (outer pathways), attributed to increased anisotropy, also facilitates the occurrence of reentry.¹ The decrease in transverse G_j that we have shown provides a possible electrophysiological mechanism for increased anisotropy and slowing of transverse conduction. However, we did not directly assess the influence of reduced gap junction conductance on conduction slowing or enhanced anisotropy. A more direct demonstration of such a role for decreased transverse G_j might be obtained from measuring cell-to-cell coupling in the in situ heart.²⁵

Other important factors are also likely to change anisotropy, such as arrangement of myocardial fiber bundles and properties of the extracellular environment.²⁶ An increased extracellular resistance accompanies conduction slowing during acute ischemia.¹³ It is uncertain how changes in extracellular space of EBZ at 5 days affect extracellular resistance and anisotropy. Although new collagen deposition is not yet significant,⁵ swelling and edema occur.² Extracellular resistance and conduction velocity are sensitive to changes in volume of the interstitial space.²⁷ Cell size also influences anisotropic properties,²⁸ but myocytes of the EBZ have similar cell size as normal.⁴

The pathophysiological role of altered EBZ gap junction voltage dependence is uncertain. When myocytes are depolarized in pathological conditions, voltage sensitivity may play a protective role by closing junctional channels, isolating the damaged region electrically from healthy tissue.²⁹ Because gap junctions in EBZ were less sensitive to transjunctional voltage, such isolation would be less severe and permit continuation of cell communication.

	Acknowledgments

 
This work was supported by grants HL-30557 and HL-66140; Heart Lung and Blood Institute, National Institutes of Health, and by the British Heart Foundation grant RG/2000003.

Received August 13, 2002; revision received January 22, 2003; accepted January 22, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Dillon SM, Allessie MA, Ursell PC, Wit AL. Influences of anisotropic tissue structure on reentrant circuits in the epicardial border zone of subacute canine infarcts. Circ Res. 1988; 63: 182–206.[Abstract/Free Full Text]
2. Ursell PC, Gardner PI, Albala A, Fenoglio JJ Jr, Wit AL. Structural and electrophysiological changes in the epicardial border zone of myocardial infarcts during infarct healing. Circ Res. 1985; 56: 436–451.[Abstract/Free Full Text]
3. Pu J, Balser JR, Boyden PA. Lidocaine action on Na⁺ currents in ventricular myocytes from the epicardial border zone of the infarcted heart. Circ Res. 1998; 83: 431–440.[Abstract/Free Full Text]
4. Lue WM, Boyden PA. Abnormal electrical properties of myocytes from chronically infarcted canine heart: alterations in V_max and the transient outward current. Circulation. 1992; 85: 1175–1188.[Abstract/Free Full Text]
5. Peters NS, Coromilas J, Severs NJ, Wit AL. Disturbed connexin43 gap junctional distribution correlates with the location of reentrant circuits in the epicardial border zone of healing canine infarcts that cause ventricular tachycardia. Circulation. 1997; 95: 988–996.[Abstract/Free Full Text]
6. Saffitz JE, Davis LM, Darrow BJ, Kanter HL, Laing JG, Beyer EC. The molecular basis of anisotropy: role of gap junctions. J Cardiovasc Electrophysiol. 1995; 6: 498–510.[Medline] [Order article via Infotrieve]
7. Burt JM, Spray DC. Volatile anesthetics block intercellular communication between neonatal rat myocardial cells. Circ Res. 1989; 65: 829–837.[Abstract/Free Full Text]
8. Neyton J, Trautmann A. Single-channel currents of an intercellular junction. Nature. 1985; 317: 331–335.[CrossRef][Medline] [Order article via Infotrieve]
9. Weingart R. Electrical properties of the nexal membrane studied in rat ventricular cell pairs. J Physiol. 1986; 370: 267–284.[Abstract/Free Full Text]
10. Wilders R, Jongsma HJ. Limitations of the dual voltage clamp method in assaying conductance and kinetics of gap junction channels. Biophys J. 1992; 63: 942–953.[Abstract/Free Full Text]
11. Wang HZ, Li J, Lemanski LF, Veenstra RD. Gating of mammalian cardiac gap junction channels by transjunctional voltage. Biophys J. 1992; 63: 139–151.[Abstract/Free Full Text]
12. Peters NS, Green CR, Poole-Wilson PA, Severs NJ. Reduced content of connexin43 gap junctions in ventricular myocardium from hypertrophied and ischemic human hearts. Circulation. 1993; 88: 864–875.[Abstract/Free Full Text]
13. Kleber AG, Riegger CB, Janse MJ. Electrical uncoupling and increase of extracellular resistance after induction of ischemia in isolated, arterially perfused rabbit papillary muscle. Circ Res. 1987; 61: 271–279.[Abstract/Free Full Text]
14. Cooklin M, Wallis WRJ, Sheridan DJ, Fry CH. Changes in cell-to-cell electrical coupling associated with left ventricular hypertrophy. Circ Res. 1997; 80: 765–771.[Abstract/Free Full Text]
15. Kieval RS, Spear JF, Moore EN. Gap junctional conductance in ventricular myocyte pairs isolated from postischemic rabbit myocardium. Circ Res. 1992; 71: 127–136.[Abstract/Free Full Text]
16. White RL, Spray DC, Campos de Carvalho AC, Wittenberg BA, Bennett MVL. Some electrical and pharmacological properties of gap junctions between adult ventricular myocytes. Am J Physiol. 1985; 249: C447–C455.[Medline] [Order article via Infotrieve]
17. Verheule S, van Kempen MJA, teWelscher PHJA, Kwak BR, Jongsma HJ. Characterization of gap junction channels in adult rabbit atrial and ventricular myocardium. Circ Res. 1997; 80: 673–681.[Abstract/Free Full Text]
18. Kameyama M. Electrical coupling between ventricular paired cells isolated from guinea-pig heart. J Physiol. 1983; 336: 345–357.[Abstract/Free Full Text]
19. Noma A, Tsuboi N. Dependence of junctional conductance on proton, calcium, and magnesium ions in cardiac paired cells of guinea-pig. J Physiol. 1987; 382: 193–211.[Abstract/Free Full Text]
20. Rüdisüli A, Weingart R. Electrical properties of gap junction channels in guinea-pig ventricular cell pars revealed by exposure to heptanol. Pflügers Arch. 1989; 415: 95–98.[CrossRef][Medline] [Order article via Infotrieve]
21. Spray DC, Moreno AP, Campos de Carvalho AC. Biophysical properties of the human cardiac gap junction channel. Brazil J Med Biol Res. 1993; 26: 541–552.[Medline] [Order article via Infotrieve]
22. Moreno AP, Rook MB, Fishman GI, Spray DC. Gap junction channels: distinct voltage-sensitive and insensitive conductance states. Biophys J. 1994; 67: 113–119.[Abstract/Free Full Text]
23. Matsushita T, Oyamada M, Fujimoto K, Yasuda Y, Masuda S, Wada Y, Oka T, Takamatsu T. Remodeling of cell-cell and cell-extracellular matrix interactions at the border zone of rat myocardial infarcts. Circ Res. 1999; 85: 1046–1055.[Abstract/Free Full Text]
24. Johnson CM, Kanter EM, Green KG, Laing JG, Betsuyaku T, Beyer EC, Steinberg TH, Saffitz JE, Yamada KA. Redistribution of connexin45 in gap junctions of connexin43-deficient hearts. Cardiovasc Res. 2002; 53: 921–935.[Abstract/Free Full Text]
25. Akar FG, Roth BJ, Rosenbaum DS. Optical measurement of cell-to-cell coupling in intact heart using subthreshold electrical stimulation. Am J Physiol. 2001; H533–H542.
26. Kleber AG, Janse MJ, Fast VG. Normal and abnormal conduction in the heart. In: Page E, Fozzard HA, Solaro RJ, Solaro J, eds. Handbook of Physiology, Section 2: The Cardiovascular System, Vol 1: The Heart. New York, NY: Oxford Press; 2001: 455–530.
27. Fleischhauer J, Lehmann L, Kleber AG. Electrical resistances of interstitial and microvascular space as determinants of the extracellular field and velocity of propagation in ventricular myocardium. Circulation. 1995; 92: 587–594.[Abstract/Free Full Text]
28. Spach MS, Heidlage JF, Dolber PC, Barr RC. Electrophysiological effects of remodeling cardiac gap junctions and cell size: experimental and model studies of normal cardiac growth. Circ Res. 2000; 86: 302–311.[Abstract/Free Full Text]
29. Jongsma HJ, Rook MB. Morphology and electrophysiology of cardiac gap junction channels. In: Zipes DP, Jalife J, eds. Cardiac Electrophysiology: From Cell to Bedside. Philadelphia, Pa: Saunders; 1995: 115–126.

This article has been cited by other articles:

				T. Sato, T. Ohkusa, H. Honjo, S. Suzuki, M.-a. Yoshida, Y. S. Ishiguro, H. Nakagawa, M. Yamazaki, M. Yano, I. Kodama, et al. Altered expression of connexin43 contributes to the arrhythmogenic substrate during the development of heart failure in cardiomyopathic hamster Am J Physiol Heart Circ Physiol, March 1, 2008; 294(3): H1164 - H1173. [Abstract] [Full Text] [PDF]

				M. Ruiz-Meana, A. Rodriguez-Sinovas, A. Cabestrero, K. Boengler, G. Heusch, and D. Garcia-Dorado Mitochondrial connexin43 as a new player in the pathophysiology of myocardial ischaemia-reperfusion injury Cardiovasc Res, January 15, 2008; 77(2): 325 - 333. [Abstract] [Full Text] [PDF]

				A. L. Wit and H. S. Duffy Drug development for treatment of cardiac arrhythmias: targeting the gap junctions Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H16 - H18. [Full Text] [PDF]

				F. G. Akar, R. D. Nass, S. Hahn, E. Cingolani, M. Shah, G. G. Hesketh, D. DiSilvestre, R. S. Tunin, D. A. Kass, and G. F. Tomaselli Dynamic changes in conduction velocity and gap junction properties during development of pacing-induced heart failure Am J Physiol Heart Circ Physiol, August 1, 2007; 293(2): H1223 - H1230. [Abstract] [Full Text] [PDF]

				S. Nattel, A. Maguy, S. Le Bouter, and Y.-H. Yeh Arrhythmogenic Ion-Channel Remodeling in the Heart: Heart Failure, Myocardial Infarction, and Atrial Fibrillation Physiol Rev, April 1, 2007; 87(2): 425 - 456. [Abstract] [Full Text] [PDF]

				C. Cabo and P. A. Boyden Heterogeneous gap junction remodeling stabilizes reentrant circuits in the epicardial border zone of the healing canine infarct: a computational study Am J Physiol Heart Circ Physiol, December 1, 2006; 291(6): H2606 - H2616. [Abstract] [Full Text] [PDF]

				R. D. Veenstra Gap junction heterogeneity in reentrant ventricular tachycardia Cardiovasc Res, November 1, 2006; 72(2): 196 - 197. [Full Text] [PDF]

				C. Cabo, J. Yao, P. A. Boyden, S. Chen, W. Hussain, H. S. Duffy, E. J. Ciaccio, N. S. Peters, and A. L. Wit Heterogeneous gap junction remodeling in reentrant circuits in the epicardial border zone of the healing canine infarct Cardiovasc Res, November 1, 2006; 72(2): 241 - 249. [Abstract] [Full Text] [PDF]

				A. T. Yan, A. J. Shayne, K. A. Brown, S. N. Gupta, C. W. Chan, T. M. Luu, M. F. Di Carli, H. G. Reynolds, W. G. Stevenson, and R. Y. Kwong Characterization of the Peri-Infarct Zone by Contrast-Enhanced Cardiac Magnetic Resonance Imaging Is a Powerful Predictor of Post-Myocardial Infarction Mortality Circulation, July 4, 2006; 114(1): 32 - 39. [Abstract] [Full Text] [PDF]

				A. E. Pollard and R. C. Barr Cardiac microimpedance measurement in two-dimensional models using multisite interstitial stimulation Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H1976 - H1987. [Abstract] [Full Text] [PDF]

				W. R. Mills, N. Mal, F. Forudi, Z. B. Popovic, M. S. Penn, and K. R. Laurita Optical mapping of late myocardial infarction in rats Am J Physiol Heart Circ Physiol, March 1, 2006; 290(3): H1298 - H1306. [Abstract] [Full Text] [PDF]

				M. Shah, F. G. Akar, and G. F. Tomaselli Molecular Basis of Arrhythmias Circulation, October 18, 2005; 112(16): 2517 - 2529. [Abstract] [Full Text] [PDF]

				S. Baba, W. Dun, C. Cabo, and P. A. Boyden Remodeling in Cells From Different Regions of the Reentrant Circuit During Ventricular Tachycardia Circulation, October 18, 2005; 112(16): 2386 - 2396. [Abstract] [Full Text] [PDF]

				L. Li, V. Nikolski, D. W. Wallick, I. R. Efimov, and Y. Cheng Mechanisms of enhanced shock-induced arrhythmogenesis in the rabbit heart with healed myocardial infarction Am J Physiol Heart Circ Physiol, September 1, 2005; 289(3): H1054 - H1068. [Abstract] [Full Text] [PDF]

				B. D. Stuyvers, W. Dun, S. Matkovich, V. Sorrentino, P. A. Boyden, and H. E.D.J. ter Keurs Ca2+ Sparks and Waves in Canine Purkinje Cells: A Triple Layered System of Ca2+ Activation Circ. Res., July 8, 2005; 97(1): 35 - 43. [Abstract] [Full Text] [PDF]

				V. Sharma, R. C. Susil, and L. Tung Paradoxical Loss of Excitation with High Intensity Pulses during Electric Field Stimulation of Single Cardiac Cells Biophys. J., April 1, 2005; 88(4): 3038 - 3049. [Abstract] [Full Text] [PDF]

				L. Formigli, F. Francini, A. Tani, R. Squecco, D. Nosi, L. Polidori, S. Nistri, L. Chiappini, V. Cesati, A. Pacini, et al. Morphofunctional integration between skeletal myoblasts and adult cardiomyocytes in coculture is favored by direct cell-cell contacts and relaxin treatment Am J Physiol Cell Physiol, April 1, 2005; 288(4): C795 - C804. [Abstract] [Full Text] [PDF]

				X. Lin, J. Gemel, E. C. Beyer, and R. D. Veenstra Dynamic model for ventricular junctional conductance during the cardiac action potential Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1113 - H1123. [Abstract] [Full Text] [PDF]

				R. J. Filion and A. S. Popel Intracoronary administration of FGF-2: a computational model of myocardial deposition and retention Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H263 - H279. [Abstract] [Full Text] [PDF]