© 1996 American Heart Association, Inc.
Articles |
Functional and Structural Assessment of Intercellular Communication
Increased Conduction Velocity and Enhanced Connexin Expression in Dibutyryl cAMP–Treated Cultured Cardiac Myocytes
the Departments of Pathology, Medicine, and Pediatrics (B.J.D., E.C.B., J.E.S.), Washington University, St Louis, Mo, and the Department of Physiology (V.G.F., A.G.K.), University of Bern (Switzerland).
Correspondence to Jeffrey E. Saffitz, MD, PhD, Washington University Medical School, Department of Pathology, Box 8118, St Louis, MO 63110. E-mail saffitz@pathbox.wustl.edu.
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Abstract |
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Remodeling of conduction pathways in the hypertrophic response to myocardial injury is a potential mechanism leading to the development of anatomic substrates of lethal arrhythmias. To delineate the responsible mechanisms and to directly relate changes in intercellular coupling at gap junctions with electrophysiological alterations, we studied the effects of cAMP, a mediator of cardiac hypertrophy, on action potential conduction velocity and connexin expression in neonatal rat ventricular myocyte cultures. Conduction velocity was measured with an optical activation mapping technique in cells loaded with the voltage-sensitive dye RH-237. Action potentials were conducted 24% to 29% more rapidly (P<.005) after incubating cultures for 24 hours with the cAMP analogue dibutyryl cAMP (db-cAMP, 1 mmol/L). However, db-cAMP caused no change in the maximum rate of rise of the action potential upstroke, Vmax. Electron and immunofluorescence microscopy revealed a significant increase in the number and size of gap junctions in db-cAMP–treated cells. Immunoblotting showed that the total amounts of the ventricular gap junction proteins connexin43 and connexin45 (Cx43 and Cx45, respectively) increased 2- to 4-fold. Immunoprecipitation of metabolically labeled connexin proteins revealed a dose-dependent increase in the rate of Cx45 protein synthesis in myocytes exposed to db-cAMP (>2-fold after a 4-hour exposure) but no change in the Cx43 synthesis rate. Northern blot analysis demonstrated a time-dependent increase in the amount of Cx43 mRNA, with a maximum 3.3-fold increase after 4 hours of exposure to 1 mmol/L db-cAMP; cycloheximide did not block this effect. In contrast, Cx45 mRNA levels were not altered significantly after db-cAMP treatment. Thus, cAMP causes a significant increase in conduction velocity that appears to be attributable largely to enhanced expression of proteins responsible for intercellular communication. Cx43 and Cx45 levels appear to be upregulated by cAMP by disparate molecular mechanisms.
Key Words: gap junction • connexin • intercellular communication • ventricular myocyte • voltage-sensitive dye
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Introduction |
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Remodeling of intercellular connections at cardiac gap junctions is a potential mechanism leading to reentrant ventricular tachycardias and sudden death. Diverse myocardial disease states, including myocardial infarction, ventricular hypertrophy, and myocarditis, lead to structural alterations of myocardium and associated conduction abnormalities.1 2 3 4 5 The development of areas of discontinuous impulse propagation in structurally altered myocardium has been associated with both arrhythmogenesis1 3 6 7 and reduced expression of gap junction proteins.5 8 9 However, there has been no direct analysis of the effects of alterations of gap junctions on the conduction properties of myocardium.
Remodeling of gap junctions in cardiac tissue is undoubtedly a complex process involving perturbations of rates of connexin gene expression and connexin protein synthesis and degradation, as well as the rearrangements in spatial distribution of junctions. The specific mechanisms that initiate remodeling are incompletely understood but likely involve activation of signal transduction pathways triggered by mediators of hypertrophy. To further elucidate these mechanisms, we investigated the actions of one well-documented mediator, cAMP,10 11 in a widely used model system of the heart, neonatal rat ventricular myocyte cultures.12 13 The expression of the ventricular gap junction proteins Cx43 and Cx45 in this system has been previously described.14 The goal of the present study was to identify molecular mechanisms responsible for gap junction remodeling and to directly relate changes in connexin expression with alterations in conduction properties of cardiac myocytes.
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All reagents were obtained from Sigma Chemical Co unless otherwise indicated.
Isolation and Culture of Neonatal Rat Ventricular Myocytes
Primary cultures of 1- to 2-day-old neonatal rat ventricular myocytes were prepared as reported previously.12 14 15 Briefly, hearts were trimmed of atrial tissue and great vessels, minced on ice, and dissociated with collagenase. Cells were resuspended in medium supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 250 ng/mL amphotericin, and 0.1 mmol/L bromodeoxyuridine and seeded onto 60-mm polystyrene culture dishes without preplating. Culture medium was replenished daily; cultures were maintained for 72 hours. db-cAMP was dissolved in a DMSO vehicle (0.2% final DMSO concentration).
Cells to be analyzed by optical recording of impulse propagation were seeded onto coverslips after preplating.15 Two types of coverslips and culture conditions were used: (1) Coverslips 22 mm in diameter were coated with type IV collagen to enhance cell adherence. These cells were cultured in M199 medium supplemented with 5% fetal calf serum. Impulse propagation by optical mapping has been extensively characterized in isotropic sheets of myocytes grown under these conditions.16 (2) A second set of cultures was maintained under serum-free conditions using PC-1 medium (Hycor Biochemical); this medium was also used for all subsequent experiments involving measurements of connexin expression. Because myocytes cultured under these conditions had a higher beating rate, optical mapping experiments were performed in the presence of cromakalim (10 µmol/L), an opener of the ATP-sensitive K+ channel,17 in order to reduce automaticity to a frequency that could be overdriven by stimulation (<2.5 Hz). Coverslips were coated with photoresist to cause cells to grow in patterned confluent strands 250 µm wide and 10 mm long, as previously described.15 In cultures grown under both conditions, we estimated that >95% of cultured cells were myocytes.
Optical Recording of Transmembrane Potential
Analysis of impulse propagation in myocyte cultures by multisite optical mapping has been previously described in detail.16 18 19 20 In brief, transmembrane action potentials were measured from the change in fluorescence of the voltage-sensitive dye RH-237 (Molecular Probes). The dye was prepared in a 2 mmol/L stock solution of DMSO and diluted in Tyrode's solution to yield a final concentration of 1.5 µmol/L. Before optical signals were recorded, cell cultures were superfused with the dye solution for 4 minutes. The recording system consisted of an inverted microscope (Axiovert 35M, Zeiss) equipped for epifluorescence with a mercury lamp light source. Excitation light was sent through a band-pass excitation filter (530 to 585 nm, Zeiss) and to the preparation via the microscope objective lens. Fluorescent emission light passed a dichroic mirror (600 nm) and a low pass–emitting filter (>615 nm). An electromechanical shutter and shutter driver (UniBlitz VS25 and UniBlitz T132, Vincent Associates) were used for timing the exposure.
The fluorescence emitted by the preparation was measured using 96 diodes from a 10x10 photodiode array (Centronic) located in the image plane of the microscope. The interdiode distance was 15 µm at x40 magnification. Photocurrents from the diodes were converted to voltage by custom-built current-to-voltage converters, amplified, multiplexed into 12 channels using a custom-made multiplexer, and digitized using three analog-to-digital converter cards (four channels, 1 MHz throughput, 12-bit resolution, NB-A2000, National Instruments) installed in a Quadra 840av computer (Apple Computer). The sampling rate was 25 kHz for each of the 96 channels. High-frequency noise was eliminated by digital filtration using a gaussian low-pass filter with a frequency cutoff of 1.5 kHz.20 Action potential upstrokes obtained simultaneously from the fluorescent changes at 96 diodes were differentiated, and local activation times were determined at 50% of the action potential amplitude using linear interpolation between the nearest sampling points.19 Conversion of dF/dtmax to Vmax was carried out by assuming an action potential amplitude of 100 mV. Activation maps were constructed using linear interpolation between the diodes.21 Propagation velocity for each recording was calculated using the average of the local activation times in the diode rows most proximal and distal to the stimulating electrode.
Immunofluorescent Labeling and Morphometric Analysis of Cultured Cells
After recording optical signals, myocytes on glass coverslips were rinsed with PBS, fixed for 20 minutes in 4% paraformaldehyde in PBS at room temperature, and permeabilized and blocked with 0.3% Triton X-100/3% normal goat serum/1% bovine serum albumin in PBS for 10 minutes at room temperature. The primary antibodies used in this study, a mouse monoclonal anti-Cx43 antibody (MAB3068, Chemicon International) and an affinity-purified polyclonal rabbit anti-Cx45 antiserum, have been extensively characterized in previous studies.14 22 23 Cells were incubated in diluted primary antisera overnight at 4°C, washed extensively, and then incubated with diluted secondary antibodies (Cy3-conjugated goat anti-mouse IgG and fluorescein-conjugated goat anti-rabbit IgG, Jackson ImmunoLabs) for 3 hours. The cells were examined with an inverted microscope (Axiovert 35M, Zeiss) equipped for epifluorescence with a mercury lamp light source. Quantification of immunofluorescent labeling was performed using a Molecular Dynamics Sarastro 2000 laser scanning confocal microscope. Random high-power fields of myocytes were selected under phase-contrast viewing conditions to eliminate selection bias. The number of pixels occupied by strong immunofluorescent signal in discrete regions of intercellular apposition was automatically counted for each field using the image acquisition software. The length-to-width ratios of cultured myocytes were determined from photographs of random cell culture fields by measuring the length of individual cells in both the axis parallel to action potential propagation and the axis perpendicular to action potential propagation.
Electron Microscopy
Cultured myocytes were fixed and prepared for electron microscopy as previously described.2 Intercalated disk and gap junction profile lengths were measured in randomly photographed intercellular junctions to determine the relative number and size of gap junctions per unit intercalated disk length, as previously described.2
Immunoblotting of Connexin Proteins
Multiple myocyte cultures were treated with db-cAMP for selected intervals that were timed to allow simultaneous harvest of all treated and untreated culture dishes after a total of 
72 hours in culture. After exposure to db-cAMP, cells were rinsed with PBS, scraped into PBS containing 10 µg/100 mL aprotinin, and lysed by sonication on ice four times for 15 seconds each. Equal volumes of protein extracts from each dish were solubilized and electrophoresed on a 12.5% polyacrylamide/SDS gel. Proteins were transferred to Immobilon-P (Millipore) using a semidry transfer apparatus (BioRad) at 3 W for 1 hour. The membranes were blocked overnight in 2% gelatin/PBS and incubated in diluted primary antibody for 3 hours at room temperature. Anti-Cx43 was used at 1:1000 dilution, and anti-Cx45 was used at 1:200 dilution in T-PBS. After six 5-minute washes in T-PBS, the blots were incubated in secondary antibody (either goat anti-mouse IgG or goat anti-rabbit IgG conjugated to horseradish peroxidase, Boehringer Mannheim) diluted 1:2000 in T-PBS. After rinsing six times for 5 minutes each in T-PBS, the blots were incubated for 1 minute in ECL solution (Amersham) and exposed to x-ray film (Kodak). Densitometric quantification of signal intensity was performed as previously described14 ; background measurements of signal intensity were subtracted individually from each lane.
Immunoprecipitation and Determination of Rates of Protein Synthesis
Cultures were treated for selected intervals with db-cAMP, and during the final 2 hours, cells were incubated in methionine-depleted medium (1:1 DMEM/F-12) containing [35S]methionine (100 µCi/mL, Amersham). db-cAMP and labeling intervals were timed to permit simultaneous harvesting of db-cAMP–treated and control cells after a total culture interval of 72 hours. Protein extracts were prepared as previously described.14 23 Crude protein extracts were incubated with either 20 µL rPA-conjugated Sepharose beads (Pharmacia) and 5 µL anti-Cx45 antiserum or with 20 µL anti-mouse IgG–conjugated Sepharose beads (Pierce) and 3 µL anti-Cx43 antiserum for 2 hours at 4°C with agitation. The beads were collected by centrifugation, washed overnight, and then washed three times for 30 minutes in RIPA buffer (0.6% SDS, 1% Triton in PBS with aprotinin, Pefabloc [Boehringer Mannheim], sodium orthovanadate, and sodium fluoride, 10 µg/100 mL each) at 4°C, analyzed by SDS-PAGE on a 12.5% gel, and subjected to fluorography after treatment with ENH3ANCE (New England Nuclear).
RNA Isolation and Blotting
Total cellular RNA was prepared from cultured cells as described previously,24 separated on 1% agarose/formaldehyde gels, and transferred overnight to nylon membranes as previously described.25 Connexin transcripts were identified with specific probes generated from rat Cx43 and mouse Cx45 DNAs by random primer labeling (Boehringer Mannheim) with [
-32P]dCTP (Amersham) as previously described.26 27 The relative amount of sample loaded in each lane and the integrity of the RNA were monitored by ethidium bromide staining and by hybridization of blots with a probe for GAPDH.
Statistical Analysis
Control and test values were compared by unpaired t test using a statistical analysis software package (StatView, Abacus Concepts Inc). ANOVA was used to assess the significance of db-cAMP treatment from pooled data obtained from cells grown under different culture conditions. Results are reported as mean±SD. Differences between groups were considered significant at P<.05. The Bonferroni correction was used for multiple comparisons.
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db-cAMP Increases Conduction Velocity but Not Vmax
In the first series of experiments, conduction velocity was measured in cells cultured in M199 medium containing 5% fetal calf serum, conditions in which optical recording has been extensively characterized.19 20 21 The results of 34 optical measurements are presented in Fig 1
. Exposure to 1 mmol/L db-cAMP for 24 hours caused a 29% increase in mean propagation velocity, from 30.7±5.4 to 39.6±5.4 cm/s (P<.0001; Fig 1A, left). Vmax, calculated from the slope of the action potential upstroke, was unchanged (158.8±12.8 V/s, treated; 156.5±16.3 V/s, control; Fig 1B, left).
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P<.001, and
This experiment was then repeated with cells grown in a serum-free culture medium, PC-1, which was used in all subsequent experiments involving immunohistochemistry and measurements of connexin protein and mRNA expression. To reduce myocyte automaticity under serum-free conditions (which was too high endogenously for optical recording), cells were grown in 250-µm-wide myocyte strands, and the K+ channel opener cromakalim (10 µmol/L) was added to the superfusate during the optical recording experiments. The results of 46 such measurements are also shown in Fig 1
. The average conduction velocity was 37.7±10.7 cm/s in control cultures and 46.8±9.8 cm/s in db-cAMP–treated cultures, representing a 24% increase in conduction velocity (P<.005; Fig 1A, right). As with cells cultured in M199 medium, no difference in Vmax was observed between db-cAMP–treated and control cultures incubated in PC-1 medium (145.1±22.1 V/s, treated; 143.6±19.2 V/s, control; Fig 1B, right). There was a small but statistically significant decrease (8%, P<.003 by ANOVA) in the Vmax of recordings from cells (both treated and untreated) cultured in M199 compared with those cultured in PC-1.
Fig 2 shows representative optical recordings of electrical activity obtained from myocyte strands cultured in PC-1 medium. The isochrones, each encompassing an area of the culture that was activated during a 50-microsecond interval, are spaced farther apart in the map of the db-cAMP–treated culture. For the examples shown, the calculated conduction velocity was 37.9 cm/s in the control culture (Fig 2A) and 50.6 cm/s in the treated culture (Fig 2B).
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Treatment with db-cAMP did not cause any significant change in the length and width of cells. However, mean conduction velocities in both db-cAMP–treated and untreated cells were 18% and 23% higher, respectively, in myocyte strands cultured in PC-1 medium (P<.0001 by ANOVA). Because cellular anisotropy is known to affect conduction velocity,19 28 we determined the ratios of cell length to width in relation to the direction of activation in cells cultured under both conditions. Myocytes cultured in 250-µm-wide strands had a length-to-width ratio of 1.38 (n=235, P<.0001), indicating that these cells preferentially oriented along the long axis of the strand, parallel to the direction of electrical activation. In contrast, myocytes cultured in confluent monolayers had no significant axial preference (ratio, 1.05; n=242; P=.22). Thus, structural anisotropy of myocytes cultured in strands probably accounted for the faster conduction velocities observed in both control and db-cAMP–treated cultures grown in strands. However, regardless of the growth pattern, db-cAMP caused a significant increase in conduction velocity.
Expression of Gap Junction Proteins Is Increased in db-cAMP–Treated Cultures
The similarity of Vmax in both treated and untreated cultures suggests that the increased conduction velocity resulted not from increased active membrane properties, such as a greater Na+ channel density or activity, but rather from enhanced cell-to-cell electrical coupling. Therefore, we performed double-label immunohistochemical analysis of connexin expression in the identical culture regions shown in Fig 2, using antibodies specific for Cx43 (Fig 3A and 3C) and Cx45 (Fig 3B and 3D). Colocalization of Cx43 and Cx45 proteins was apparent, as has been previously reported.14 22 The amount of immunolabeled connexins was increased in cells treated with db-cAMP. Quantitative confocal analysis of 10 randomly selected fields each from db-cAMP–treated and control cultures revealed a 51% increase in the area of Cx43 staining per x40 field in the treated cultures, from 3.49±0.60x105 to 5.26±0.40x105 pixels (P<.0001). The amount of Cx43 and Cx45 immunolabeled also increased in db-cAMP–treated cultures grown in M199 medium (data not shown).
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We have previously demonstrated that Cx40, a gap junction protein expressed in atrial muscle and the cardiac conduction system,29 is not present in ventricular myocyte cultures.14 Using a previously characterized monospecific anti-Cx40 antibody,22 we confirmed that no Cx40 protein was detected by immunofluorescent labeling in control myocytes. Furthermore, we failed to detect Cx40 in db-cAMP–treated cells (1 mmol/L, 24 hours), indicating that db-cAMP did not induce the expression of Cx40 cells (data not shown).
To delineate changes in the number and size of individual gap junctions, we examined cultured myocytes using the electron microscope. Representative micrographs are shown in Fig 4. Compared with myocytes cultured in PC-1 medium in the absence of db-cAMP (Fig 4, left), cells treated for 24 hours with 1 mmol/L db-cAMP showed a marked increase in the number of gap junctions within intercalated disks (Fig 4, right). The Table shows the number and lengths of gap junction profiles in the intercalated disks of control and treated cells. When comparable lengths of intercalated disks were examined, total gap junction length was 4-fold greater in db-cAMP–treated myocytes than in control myocytes. The increases in both the number and the total gap junction length per intercalated disk were highly significant (P<.0001 for both). Expressed as a percentage of total sarcolemma length, db-cAMP increased aggregate gap junction profile length 4.7-fold. The average length of each individual gap junction was 66% greater in the db-cAMP–treated myocytes (P<.01).
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The increase in amounts of Cx43 and Cx45 proteins in db-cAMP–treated myocytes was confirmed and quantified by immunoblot analysis. Parallel myocyte cultures were treated with 1 mmol/L db-cAMP for up to 24 hours. As shown in Fig 5A, Cx43 blotted as three bands migrating between 40 and 45 kD; a similar pattern has been previously reported.30 31 The intensity of labeling for Cx43 was increased 1.9- to 2-fold in extracts from myocytes treated with 1 mmol/L db-cAMP for each of the exposure times (see Fig 5C). Densitometric analysis of lighter exposures of the immunoblot shown in Fig 5A suggested that all three Cx43 bands were increased similarly as a result of the db-cAMP treatment. The blot shown in Fig 5A was stripped and reprobed with antibodies specific for Cx45 (Fig 5B). Cx45 protein migrated as two bands of 46 to 48 kD, as previously observed.14 23 32 33 The amount of Cx45 increased 2.6- to 3.8-fold in the treated samples, with the greatest increase observed after 4 hours (Fig 5C). Incubation for 24 hours with 1 mmol/L butyric acid, a by-product of db-cAMP hydrolysis, did not increase either Cx43 or Cx45 (data not shown).
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db-cAMP Causes a Transient Increase in Cx45 but Not Cx43 Synthesis Rate
To further elucidate the mechanisms by which connexin proteins accumulated, we measured the rates of Cx43 and Cx45 protein synthesis after timed exposure to 1 mmol/L db-cAMP. The rate of protein synthesis was determined by the amount of incorporation of [35S]methionine during a 2-hour interval immediately before cell lysis and immunoprecipitation of specific connexin proteins. We have previously shown that under identical culture conditions, the half-lives of metabolically labeled Cx43 and Cx45 are 1.9 and 2.9 hours, respectively.14 Thus, the amounts of radiolabeled connexin proteins isolated after a 2-hour labeling interval primarily reflect the rates of protein synthesis without any significant effect attributable to protein degradation. Fig 6A shows a representative fluorograph of immunoprecipitated Cx43. There was no significant difference in the amount of radiolabeled Cx43 at each time point. However, as shown in Fig 6B, Cx45 synthesis was noticeably increased during a 4-hour treatment with db-cAMP. A smaller effect was detected after an 8-hour exposure to db-cAMP, and the rate of synthesis returned to basal levels at the end of a 24-hour treatment. The results of four experiments were quantified using densitometry and are depicted graphically in Fig 6C. During the final 2 hours of a 4-hour treatment with db-cAMP, the rate of Cx45 synthesis in db-cAMP–treated myocytes was 2.1±0.56 times greater than that in the control myocytes (P<.0001).
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To exclude the possibility that butyric acid was responsible for the increase in Cx45 synthesis, we treated myocytes with 1 mmol/L butyric acid for 4 hours. As shown in Fig 7A, butyric acid did not mimic the action of db-cAMP. However, a second cAMP analogue, 8-bromo-cAMP, did cause a similar increase in Cx45 protein synthesis after a 4-hour treatment period (Fig 7B).
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Although most of the experiments in the present study were performed using relatively high concentrations of db-cAMP, we characterized the dose dependence of the cAMP-induced increase in Cx45 protein synthesis (Fig 8A). The amount of radiolabeled Cx45 increased as the concentration of db-cAMP was raised from 0 µmol/L (no treatment) to 1000 µmol/L. The results of three experiments were quantified using densitometry to generate the dose-response relation shown in Fig 8B. The apparent half-maximal effect occurred at a concentration of <10 µmol/L db-cAMP.
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db-cAMP Increases the Amount of Cx43 mRNA but Not Cx45 mRNA
Total cellular RNA was harvested from cultures treated for up to 24 hours with 1 mmol/L db-cAMP and analyzed by Northern blotting. Fig 9A shows a representative blot hybridized simultaneously with radiolabeled probes specific for Cx43, Cx45, and GAPDH, a constitutively expressed transcript. These three transcripts can be distinguished according to size; in separate experiments, each probe hybridized with a single band (data not shown). Cx43 mRNA was noticeably increased in amount in the treated samples, whereas Cx45 mRNA showed only a moderate transient increase when normalized to the amount of the control GAPDH transcript. The results of three experiments were quantified using densitometry and are shown in Fig 9B. The maximum increase in Cx43 mRNA occurred after 4 hours of exposure to 1 mmol/L db-cAMP, at which point the amount of Cx43 transcript was 3.3±1.1 times greater than in the control condition (P=.0005).
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To ensure that the effect of db-cAMP on Cx43 mRNA was not caused by butyric acid, RNA was harvested from cells incubated with DMSO, 1 mmol/L db-cAMP, or 1 mmol/L butyric acid for 4 hours. As shown in Fig 10A, butyric acid did not induce an increase in Cx43 mRNA.
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To determine whether the increase in Cx43 mRNA caused by db-cAMP required synthesis of new protein factors, we performed Northern blotting on RNA samples from cultures incubated with 10 µmol/L cycloheximide. As demonstrated in Fig 10B, Cx43 mRNA was upregulated after 4 hours of treatment with 1 mmol/L db-cAMP, and cycloheximide did not block this effect. In fact, the increase in Cx43 mRNA after 4-hour db-cAMP treatment was 40% greater in the presence of cycloheximide (as quantified by densitometry). Thus, new protein synthesis was not required for db-cAMP to induce increased levels of Cx43 mRNA.
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Discussion |
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The results of the present study indicate that db-cAMP causes a marked increase in expression of the ventricular gap junction channel proteins Cx43 and Cx45 in cultured neonatal rat ventricular myocytes. The upregulation of connexins and enhanced accumulation of these proteins within gap junctions were associated with a 24% to 29% increase in the conduction velocity in myocyte cultures without any change in Vmax. Because conduction velocity is determined by several independent factors, including both active and passive membrane properties as well as cell size and shape, we will consider the potential impact of changes in each of these parameters individually.
Determinants of Conduction Velocity
Number of Gap Junction Channels in Myocyte Membranes
Our results strongly suggest that increased gap junction protein expression is largely responsible for the increase we have observed in action potential conduction velocity. Computer models predict that halving intercellular resistance could increase conduction velocity by up to 41%.34 Taken together, the results of immunoblotting (total cellular connexin protein), confocal microscopy (amount of connexins in gap junctions), and electron microscopy (amount of gap junctions per unit intercalated disk length) all indicate a significant increase in gap junctional area, which could account for the observed increase in conduction velocity.
Increased gap junctional area may also occur independent of enhanced connexin synthesis. For example, intercellular communication in a mouse mammary tumor cell line increased fourfold after a 24-hour 8-bromo-cAMP treatment, with increased immunofluorescent labeling of Cx43 at cell surfaces but no corresponding change in the amount of total Cx43 protein.35 This finding suggests that cAMP may mobilize intracellular connexin to be assembled into cell surface channels at gap junctions.
Biophysical Properties of Individual Gap Junction Channels
There is strong evidence that acute exposure to cAMP analogues causes changes in both the single-channel conductance of gap junction channels and the total junctional conductance between communicating cell pairs, resulting in increased cell-to-cell communication.36 37 38 39 However, these effects are temporary, and junctional conductance reverts to control levels within minutes after the removal of cAMP from the cell medium. In the present study, all conduction velocity measurements were obtained in the absence of cAMP; cultures were perfused with Tyrode's solution for at least 15 minutes before the earliest optical recordings. Therefore, it is unlikely that acute cAMP-induced changes in the biophysical characteristics of gap junction channels contributed to the increase in conduction velocity observed in our experiments.
Morphology
Because conduction velocity is inversely related to the square root of the cell surface-to-volume ratio, morphological change could contribute to the increased conduction velocity in the treated cells. However, treatment with db-cAMP before optical recording did not significantly change the average length or width of cultured myocytes grown in confluent sheets or strands. The apparent absence of morphological changes suggests that this mechanism did not play a significant role.
Active Membrane Properties
In our measurements of conduction velocity under two different culture conditions, we found no change in Vmax in myocyte cultures treated for 24 hours with db-cAMP. In a continuous or only moderately discontinuous conducting medium, Vmax is a qualitative index for the inward Na+ current.40 Because Vmax did not increase in db-cAMP–treated myocytes, it is unlikely that alterations in Na+ channel conductance played a significant role in increasing the conduction velocity in these cells. Although little is known about changes in the properties of Na+ channels in myocytes exposed to cAMP analogues for as long as 24 hours, previous reports have demonstrated a decrease in the Na+ current of myocytes and cardiac muscle fibers treated with cAMP for brief periods.41 42 43 Thus, it seems unlikely that changes in active membrane properties contributed importantly to the changes in conduction velocity observed in our experiments.
We identified a small but significant decrease in Vmax and an increase in conduction velocity when we compared optical recordings from cultures grown in PC-1 medium with those from cells grown in M199. Although it is possible that this result reflects differences in the expression or function of ion channel proteins under the two conditions, the use of cromakalim during the optical recording from cultured strands grown in PC-1 medium could be responsible for the observed effect. Thus, differences in Vmax could theoretically be explained by a hyperpolarization of the resting membrane potential in the presence of cromakalim. These hyperpolarized cells could produce action potentials with greater total amplitude than cells not treated with cromakalim. By normalizing optical recordings of action potentials obtained both in the absence and presence of cromakalim to a single fixed scale, we may have introduced a scaling error that could account for the observed difference in Vmax. It should be noted, however, that conduction velocity calculations were independent of normalization because the time course of each measured action potential was unchanged.
It is unclear whether cromakalim contributed to the observed increase in conduction velocity in myocytes cultured in PC-1 medium. As discussed above, an increase in action potential amplitude secondary to hyperpolarization would be expected to increase conduction velocity. Conversely, an increase in membrane conductance and hyperpolarization due to cromakalim would be expected to increase the amount of excitatory current necessary to reach threshold and, therefore, to decrease the conduction velocity.44 The same arguments apply to any potential changes in resting membrane conductance brought about by cAMP.
Regulation of Connexin Expression by db-cAMP
The amount of both Cx43 and Cx45 proteins expressed in myocytes treated with db-cAMP doubled after 24 hours. However, it is apparent that different intracellular mechanisms are responsible for these parallel effects.
Expression of Connexin mRNA
After treatment with db-cAMP, the amount of Cx43 mRNA in cultured myocytes increased in a time-dependent manner. The greatest effect was observed after 4 hours, at which time Cx43 transcript was present at more than three times the normal concentration. These results are consistent with previous findings in several (but not all) types of cultured cells.35 45 It is unclear whether Cx43 mRNA is increased because of enhanced transcription or stabilization of the transcript; both mechanisms have been implicated in cAMP-dependent regulation of connexin mRNAs.45 46 The selective upregulation of Cx43 mRNA (compared with Cx45, which was only modestly increased) raises the possibility that specific promoter or enhancer sequences near the Cx43 gene mediate this effect. Interestingly, the addition of cycloheximide, an inhibitor of protein synthesis, magnified the cAMP-induced upregulation of Cx43 transcript, consistent with previous findings in hepatoma cell cultures.45 This indirectly suggests the modification of existing proteins, perhaps through phosphorylation/dephosphorylation events, as part of an intracellular signaling mechanism beginning with the production of cAMP.
Expression of Connexin Protein
The amounts of both Cx43 and Cx45 proteins increased in myocytes treated with db-cAMP. By measuring the rate of methionine incorporation into newly synthesized connexin protein, we were able to estimate the rate of connexin protein synthesis at selected times after the onset of db-cAMP treatment. Accumulation of Cx45 protein was associated with a dose-dependent increase in the rate of Cx45 protein synthesis. That the increase in Cx45 synthesis was transient may have accounted for the observation that the amount of total cellular Cx45 (but not Cx43) protein peaked after 4 hours of treatment with db-cAMP. The observed increase in Cx45 protein synthesis, without a corresponding increase in Cx45 mRNA level, suggests the possibility that Cx45 may be upregulated posttranscriptionally. Posttranscriptional regulation has been implicated previously in the expression of gap junction proteins in both regenerating rat liver hepatocytes (Cx32 and Cx26) and cultured rat liver epithelial cells (Cx43).47 48 Further studies will be required to describe the regulation of gap junction protein and RNA expression more fully.
Conduction velocity measurements were obtained after 24 hours of treatment with db-cAMP, at which time both immunofluorescent and electron microscopic analysis demonstrated a significant increase in the number and size of gap junctions. At this time point, levels of Cx43, the major cardiac connexin, were maximal. It is true, however, that maximal expression of Cx45 occurred earlier. Further experiments will be required to relate in detail the relative contributions of changes in individual connexins to propagation.
Cx43 and Cx45 are the only known gap junction proteins expressed in ventricular myocytes; Cx40, expressed in atrial muscle and the cardiac conduction system, is absent from the working ventricle of mammalian myocytes.29 In the present study, treatment with db-cAMP did not induce the expression of Cx40.
Regulation of cAMP and Connexins During Cardiac Hypertrophy
It is well established that cAMP, or diverse factors that increase the intracellular concentration of cAMP, produce a hypertrophic phenotype in cardiac cells.49 50 However, the regulation of cAMP levels during different stages of the hypertrophic process is complex. The concentration of cAMP in cardiac myocytes appears to be increased during the early stages of hypertrophy10 11 but becomes decreased in chronically hypertrophied heart muscle and failing myocardium.51 52 Expression of Cx43 protein appears to follow a similar pattern. For example, Cx43 protein was found to increase in amount by 59% (as determined by immunofluorescent labeling) in guinea pig ventricular myocytes 3 weeks after renal artery clipping,53 whereas in chronic myocardial disease states, including chronic hypertrophy9 54 and healed myocardial infarction,8 9 myocardial Cx43 protein content is reduced by 40% to 70%. This reduction in gap junction channel protein coincides with decreased conduction velocity in ischemic and chronically hypertrophied myocardium.55 56 57 Little is presently known about the expression of Cx45 protein under these conditions.
Whereas cardiac hypertrophy is certainly a multifactorial disease process, results of the present study and others suggest that connexin proteins and cAMP are both increased as part of an adaptive response initiating the hypertrophic process. As the heart decompensates in response to severe and/or prolonged hemodynamic or metabolic stress, both cAMP and Cx43 become downregulated, resulting in decreased conduction velocity and increased arrhythmogenicity. Genetic or pharmacological strategies designed to increase connexin protein expression or function could therefore be effective in improving cardiac conduction and reducing morbidity and mortality in patients with chronic heart disease.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by National Institutes of Health grants HL-50598 and HL-45466 and grants from the Swiss National Science Foundation and the Swiss Heart Foundation. Dr Beyer is an Established Investigator of the American Heart Association. We thank Regula Fluckiger-Labrada and Karen Green for invaluable technical assistance. We also thank Dr Stephan Rohr for expert assistance with the technique of patterned cell culture.
Received January 16, 1996; accepted April 5, 1996.
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J. E. Saffitz, R. B. Schuessler, and K. A. Yamada Mechanisms of remodeling of gap junction distributions and the development of anatomic substrates of arrhythmias Cardiovasc Res, May 1, 1999; 42(2): 309 - 317. [Full Text] [PDF]
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M. A. Beardslee, J. G. Laing, E. C. Beyer, and J. E. Saffitz Rapid Turnover of Connexin43 in the Adult Rat Heart Circ. Res., September 21, 1998; 83(6): 629 - 635. [Abstract] [Full Text] [PDF]
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S. M. Dodge, M. A. Beardslee, B. J. Darrow, K. G. Green, E. C. Beyer, and J. E. Saffitz Effects of angiotensin II on expression of the gap junction channel protein connexin43 in neonatal rat ventricular myocytes J. Am. Coll. Cardiol., September 1, 1998; 32(3): 800 - 807. [Abstract] [Full Text] [PDF]
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S. T Rapundalo Cardiac protein phosphorylation: functional and pathophysiological correlates Cardiovasc Res, June 1, 1998; 38(3): 559 - 588. [Abstract] [Full Text] [PDF]
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J. G Laing, P. N Tadros, K. Green, J. E Saffitz, and E. C Beyer Proteolysis of connexin43-containing gap junctions in normal and heat-stressed cardiac myocytes Cardiovasc Res, June 1, 1998; 38(3): 711 - 718. [Abstract] [Full Text] [PDF]
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R. R. Kaprielian, M. Gunning, E. Dupont, M. N. Sheppard, S. M. Rothery, R. Underwood, D. J. Pennell, K. Fox, J. Pepper, P. A. Poole-Wilson, et al. Downregulation of Immunodetectable Connexin43 and Decreased Gap Junction Size in the Pathogenesis of Chronic Hibernation in the Human Left Ventricle Circulation, February 24, 1998; 97(7): 651 - 660. [Abstract] [Full Text] [PDF]
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S. R. Coppen, E. Dupont, S. Rothery, and N. J. Severs Connexin45 Expression Is Preferentially Associated With the Ventricular Conduction System in Mouse and Rat Heart Circ. Res., February 9, 1998; 82(2): 232 - 243. [Abstract] [Full Text] [PDF]
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R. C. Pimentel, K. A. Yamada, A. G. Kleber, and J. E. Saffitz Autocrine Regulation of Myocyte Cx43 Expression by VEGF Circ. Res., April 5, 2002; 90(6): 671 - 677. [Abstract] [Full Text] [PDF]
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