Author + information
- Received September 2, 1997
- Revision received May 4, 1998
- Accepted May 15, 1998
- Published online September 1, 1998.
- Stephen M. Dodge, MD∗,a,b,c,
- Michael A. Beardslee, MDa,b,c,
- Bruce J. Darrow, MD, PhD†,a,b,c,
- Karen G. Green, BAa,b,c,
- Eric C. Beyer, MD, PhD‡,a,b,c and
- Jeffrey E. Saffitz, MD, PhD, FACCa,b,c,* ()
- ↵*Address for correspondence: Dr. Jeffrey E. Saffitz, Department of Pathology, Box 8118, Washington University School of Medicine, 660 S. Euclid Avenue, Saint Louis, Missouri 63110
Objectives. To elucidate signal transduction pathways regulating expression of myocardial gap junction channel proteins (connexins) and to determine whether mediators of cardiac hypertrophy might promote remodeling of gap junctions, we characterized the effects of angiotensin II on expression of the major cardiac gap junction protein connexin43 (Cx43) in cultured neonatal rat ventricular myocytes.
Background. Remodeling of the distribution of myocardial gap junctions appears to be an important feature of anatomic substrates of ventricular arrhythmias in patients with heart disease. Remodeling of intercellular connections may be initiated by changes in connexin expression caused by chemical mediators of the hypertrophic response.
Methods. Cultures were exposed to 0.1 μmol/liter angiotensin II for 6 or 24 h, and Cx43 expression was characterized by immunoblotting, confocal microscopy and electron microscopy.
Results. Immunoblot analysis revealed a twofold increase in Cx43 content in cells treated for 24 h with angiotensin II (n = 4, p < 0.05). This response was inhibited by the presence of 1.0 μmol/liter losartan, an AT1-receptor blocker. Confocal and electron microscopy demonstrated enhanced Cx43 immunoreactivity and increases in the number and size of gap junction profiles in cells exposed to angiotensin II for 24 h. These effects were also blocked by losartan. Immunoprecipitation of Cx43 from cells metabolically labeled with [35S]methionine demonstrated 2.4- and 2.9-fold increases in Cx43 radioactivity after 6 and 24 h exposure to angiotensin II, respectively (p < 0.03 at each time point).
Conclusions. Angiotensin II up-regulates gap junctions in cultured neonatal rat ventricular myocytes by increasing Cx43 synthesis. Signal transduction pathways activated by angiotensin II under pathophysiologic conditions could initiate remodeling of conduction pathways, leading to the development of anatomic substrates of arrhythmias.
Reentrant ventricular tachycardias in patients with healed myocardial infarcts depend on regions of slow heterogeneous conduction and unidirectional conduction block that typically map to viable but structurally altered myocardium bordering the healed infarct scar (1–4). Although conduction through these regions is highly abnormal, intracellular action potential recordings in infarct border zone myocytes are typically normal or nearly normal (3,4). These observations indicate that derangements in conduction critical in reentrant arrhythmogenesis arise not primarily from abnormalities of active sarcolemmal ionic currents but rather from alterations in intercellular current transfer. This conclusion is supported by results of morphometric and immunofluorescence studies that have revealed extensive remodeling of the spatial distribution of gap junctions in myocardium bordering infarct scars (2,5–7). During the process of infarct healing, not only is necrotic muscle replaced by scar tissue, but viable myocardium at the edges of the infarct develops interstitial fibrosis characterized by accumulation of collagen bundles oriented parallel to the long axis of the myocytes (8,9). The normal network of extensive intercellular connections between neighboring myocytes in end-to-end and side-to-side apposition becomes remodeled such that there is a substantial loss of gap junctions between cells in side-to-side orientation (5,6). This rearrangement of connections impairs transverse propagation and creates regions of slow heterogeneous conduction and unidirectional conduction block (2,6).
Although peri-infarct myocytes eventually become interconnected by fewer and smaller gap junctions than normal cells once infarcts become fully healed, the initial events in this remodeling process probably involve dynamic changes in gap junction protein (connexin) expression and distribution. This suggestion is supported by the results of studies showing that during the active inflammatory phase of infarct healing, viable myocytes at the edge of the infarct lose the normal pattern of large gap junctions concentrated at ends of myocytes and exhibit a distinctly different pattern in which many small junctions become distributed uniformly over the cell surface (2,7). It is unknown whether connexin expression is increased in these myocytes during early infarct healing, but the marked rearrangement in gap junction distribution suggests that significant changes in connexin synthesis, assembly into channels and degradation may accompany the structural changes taking place in gap junctions. It seems likely that during this phase of infarct healing multiple signal transduction pathways are activated in both myocytes and nonmyocytic cells in the heart in response to cytokines and neurohumoral factors. These complex signaling events probably stimulate expression of multiple genes and alter protein synthesis and degradation dynamics, all of which eventually culminate in a chronic, anatomically stable state of tissue remodeling.
Among the potential mediators implicated in ventricular remodeling after myocardial infarction is angiotensin II (10,11). Results of multiple clinical trials have shown that administration of angiotensin-converting enzyme inhibitors to patients after myocardial infarction improves cardiac function, prolongs survival and modulates ventricular remodeling (12–17). The specific mechanisms responsible may be multiple and probably involve both direct effects on the renin-angiotensin system and inhibition of kinin degradation. Angiotensin II has also been shown to elicit a hypertrophic response in cultured neonatal rat cardiac myocytes involving activation of many cardiac genes (18–23).
Work in our laboratory has focused on the hypothesis that during the development of anatomic substrates of arrhythmias, chemical mediators of the hypertrophic response activate synthesis of gap junction channel proteins, which we propose is an obligatory first step in the remodeling of sites of intercellular coupling. In previous studies (24), we showed that long-term exposure (up to 24 h) of cultured neonatal rat ventricular myocytes to the membrane-permeable cyclic adenosine 3′-5′ monophosphate (cAMP) analog dibutyryl cAMP increased expression of the ventricular gap junction channel protein connexin43 (Cx43) and also increased the speed of impulse propagation. To characterize further the potential role of mediators of hypertrophy in the development of anatomic substrates of arrhythmias, we performed the present studies to determine whether long-term exposure (24 h) of cardiac myocytes to angiotensin II affects expression of Cx43.
Materials and methods
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 (24,25). Briefly, hearts were trimmed of atrial tissue and great vessels, minced on ice and dissociated with collagenase. Cells were resuspended in PC-1 medium (Hycor Biochemical, Irvine, California) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 250 ng/ml amphotericin and 0.1 mmol/liter bromodeoxyuridine, and seeded onto 60-mm polystyrene culture dishes without preplating at a density of 1.5 × 105cells/cm2. Because this medium is serum-free, it contained no exogenous source of angiotensin, angiotensinogen or other growth factors. Culture medium was replenished daily. Cultures were maintained for 72 h before being treated for selected intervals with 0.1 μmol/liter angiotensin II and analyzed for the effects of connexin expression. In some experiments, the effects of 0.1 μmol/liter angiotensin II and 1.0 mmol/liter dibutyryl cAMP were compared by analyzing cultures exposed to either mediator.
Immunoblotting of Cx43
Multiple myocyte cultures were treated with angiotensin II or dibutyryl cAMP for 6 or 24 h. After exposure to mediators, cells were rinsed with phosphate-buffered saline (PBS), scraped into PBS containing 10 μg/ 100 ml aprotinin and lysed with sonication on ice with 4 bursts of 15 s each. Equal volumes of protein extracts from each dish were solubilized and electrophoresed on 12.5% polyacrylamide gels. Proteins were transferred to Immobilon-P (Millipore, Bedford, Massachusetts) using a semidry transfer apparatus (BioRad, Hercules, California) at 3 W for 1 h. The membranes were blocked overnight in 2% gelatin in PBS and incubated in a mouse monoclonal anti-Cx43 antibody (MAB 3068, Chemicon International, Temecula, California) at a dilution of 1:1,000 in phosphate-buffered saline containing 0.5% Triton X-100 (TPBS). After five washes in TPBS, the blots were incubated with horseradish peroxidase-conjugated goat antimouse immunoglobulin G (IgG) (Boehringer Mannheim, Indianapolis, Indiana) which was diluted 1:2,000 in TPBS. After being rinsed 6 times for 5 min each in TPBS, the blots were incubated for 1 min in ECL solution (Amersham, Arlington Heights, Illinois) and exposed to x-ray film (Kodak, New Haven, Connecticut). Densitometric quantification of signal intensity was performed as previously described (26). Background measurements of signal intensity were subtracted individually from each lane.
Immunohistochemistry and confocal microscopy
Myocytes on glass coverslips were rinsed with PBS, fixed for 20 min 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 min at room temperature. Cells were incubated with mouse monoclonal anti-Cx43 antibody (MAB 3068, Chemicon International) overnight at 4°C, washed extensively and then incubated with CY3-conjugated goat antimouse IgG (Jackson Immunolabs, West Grove, Pennsylvania) for 3 h. Quantification of immunofluorescent labeling was performed using a Molecular Dynamics Sarastro 2000 laser scanning confocal microscope (Sunnyvale, California). Random high-power fields of myocytes were selected for image analysis under phase-contrast viewing conditions. The number of pixels occupied by the high intensity immunofluorescent signal in discrete regions of intercellular apposition was automatically counted in each field using the image analysis software. The high intensity of this signal, which appeared to localize exclusively to gap junctions, was likely due to the extremely dense packing of connexin molecules in gap junction channel arrays. The image analysis protocol eliminated lower intensity immunofluorescent signal within cells that probably included both specific Cx43 signals in intracellular sites and nonspecific signals. Confocal data were expressed as the number of pixels that exhibited strong immunofluorescent signals divided by the total number of pixels occupied by cells.
Angiotensin II-treated and control cultures were fixed and prepared for electron microscopy as previously described (5). Intercalated disk and gap junction profile lengths and total sarcolemma lengths and myocyte areas were measured in randomly photographed groups of interconnected myocytes to determine the relative number and size of gap junctions per unit intercalated disk length, unit sarcolemma length and unit cell area, as previously described (5).
Metabolic labeling and immunoprecipitation assays
Cultures were treated for 6 or 24 h with angiotensin II or dibutyryl cAMP and, during the final 2 h of the treatment interval, were also incubated in methionine-depleted medium containing [35S]methionine (100 μCi/ml, Amersham, Arlington Heights, Illinois), as previously described (24,25). At the completion of the treatment interval, cells were washed to remove unincorporated radioactivity. Protein extracts were prepared as previously described and incubated with 20 μl antimouse IgG-conjugated Sepharose D beads (Pierce, Rockford, Illinois) and 3 μl anti-Cx43 mouse monoclonal antibody for 2 h at 4°C with agitation. The beads were collected by centrifugation, washed overnight and then washed three times for 30 min in radioimmunoprecipitation assay buffer (0.6% sodium dodecyl sulfate [SDS], 1% Triton X-100 in PBS with aprotinin, Pefabloc [Boehringer Mannheim, Indianapolis, Indiana], sodium orthovanadate and sodium fluoride, 10 μg/100 ml each) at 4°C. Radiolabeled proteins were separated by sodium dodecyl phosphate polyacrylamide gel electrophoresis (SDS–PAGE) on 12.5% gels and analyzed by fluorography after treatment with ENH3ANCE (New England Nuclear).
Results are reported as mean ± SD. Differences between groups were considered significant at p < 0.05. Control and test values in cultures treated with angiotensin II for selected intervals were compared by one way analysis of variance with Fisher’s protected least significant difference post hoc testing using a statistical analysis software package (Statview, Abbacus Concepts, Inc, Berkeley, California). Electron microscopic data were compared with unpaired ttests.
Effects of angiotensin II on Cx43 content in cultured myocytes
To determine whether the total amount of Cx43 protein changed in cultures exposed to angiotensin II, total protein from control and treated cultures was separated by SDS–PAGE and analyzed by immunoblotting. Figure 1shows a representative immunoblot and quantitative results from four separate experiments in which Cx43 band intensities were analyzed densitometrically and normalized to the value measured in untreated, control cultures. Cx43 migrated as two major bands at approximately 46 and 43 kDa. These bands correspond to the positions of phosphorylated isoforms of Cx43 (27), which comprises the majority of Cx43 in cardiac myocytes (28). Exposure of myocytes to 0.1 μmol/liter angiotensin for 6 h resulted in a modest (<1.5-fold) increase in Cx43 content that did not achieve statistical significance. However, exposure of myocytes to angiotensin for 24 h resulted in a twofold increase in Cx43 content (p < 0.05). The increase in Cx43 content after 24 h of angiotensin exposure was blocked by losartan.
Effects of angiotensin II on gap junctions in cultured myocytes
To determine whether the increased content of Cx43 in cells treated with angiotensin II was associated with changes in gap junctions, we performed immunofluorescence studies. Control cultures and cultures treated with 0.1 μmol/liter angiotensin II for 24 h were incubated with monoclonal antibodies against Cx43 and analyzed by quantitative laser scanning confocal microscopy. Figure 2shows representative confocal images and Figure 3shows the results of quantitative digital image analysis of immunostained control and angiotensin II-treated myocytes in four separate experiments. Treatment of myocytes with angiotensin II for 24 h caused an increase in Cx43 immunoreactive signal compared with untreated control myocytes as judged by inspection of confocal images (Fig. 2). Quantitative analysis revealed a twofold increase in the area occupied by Cx43 immunoreactive signals (Fig. 3), consistent with the results of immunoblotting shown in Figure 1. The increase in the immunoreactive signal area was blocked completely when cultures were incubated with angiotensin in the presence of losartan.
Additional angiotensin-treated and control cultures were examined by electron microscopy. Figure 4shows representative electron microscopic images of intercalated disk regions connecting control myocytes and myocytes treated for 24 h with angiotensin II, and Table 1shows morphometric data from two sets of treated and control cultures. A total of 80 myocytes in 13 fields from angiotensin-treated cultures and 66 myocytes in 8 fields from control cultures were analyzed. Both the number and size of individual gap junction profiles were increased in cells treated with angiotensin II for 24 h. There was no apparent increase in the total length of intercalated disk profiles per unit myocyte area. These data imply that angiotensin II had a relatively selective effect on gap junctions without affecting other junctional components. However, we did not perform morphometric analyses of other organelles such as sarcomeres, mitochondria or components of the sarcoplasmic reticulum. The changes in gap junctions may be only part of a more generalized hypertrophic growth response induced by angiotensin II.
Effects of angiotensin II on Cx43 synthesis
To identify potential mechanisms responsible for the increase in Cx43 content caused by angiotensin II, we performed metabolic labeling studies to compare the rates of Cx43 synthesis in control cells and treated cells. Cells were exposed to angiotensin II or to dibutyryl cAMP for 6 or 24 h. During the final 2 h of each exposure interval, the cells were incubated with [35S]methionine. The cells were lysed and the amount of radioactivity incorporated into Cx43 was measured with quantitative immunoprecipitation assays. Because the half-life of Cx43 in cultured neonatal rat ventricular myocytes is approximately 3 h (25,29), the amount of radioactivity incorporated into Cx43 during a 2-h labeling interval was assumed to be determined mainly by the rate of Cx43 synthesis.
Figure 5shows a representative immunoprecipitation assay and quantitative results from four separate metabolic labeling and immunoprecipitation experiments. The amount of radiolabeled Cx43 increased by 2.4- and 2.9-fold in cells treated with angiotensin II for 6 or 24 h, respectively (p < 0.03 for each compared to controls). This effect was blocked by losartan. These results indicate that although the total content of Cx43 had not increased by a significant amount after a 6-h exposure to angiotensin II, the rate of incorporation of [35S]methionine into Cx43 had increased significantly during the final 2 h of a 6-h exposure to angiotensin. The increased rate of incorporation persisted such that it was nearly threefold greater than control values during the final 2 h of a 24-h exposure to angiotensin II, by which time the total content of Cx43 had doubled. In contrast, and as previously observed (24), there was no increase in incorporation of radioactivity into Cx43 during a 2-h metabolic labeling interval in cells treated with dibutyryl cAMP for 6 or 24 h. Thus, angiotensin II but not cAMP increases the rate of Cx43 protein synthesis in cultured neonatal rat ventricular myocytes.
Regulation of connexin expression by mediators of the cardiac hypertrophic response
The results of this study indicate that exposure of neonatal ventricular myocytes to angiotensin II for 24 h enhances expression of the principal ventricular gap junction protein, Cx43, and leads to an increase in the number and size of gap junctions. Because angiotensin has been implicated as an important mediator of ventricular remodeling after myocardial infarction, our results suggest that one potential feature of the remodeling process involves dynamic changes in gap junction protein expression and distribution, giving rise to anatomic substrates of ventricular arrhythmias. In support of this concept are results of recent clinical trials showing a significant reduction in the incidence of sudden death or fatal myocardial infarcts in postinfarct patients treated with angiotensin-converting enzyme inhibitors (15,30,31).
In previous studies (24), we showed that exposure of cultured neonatal myocytes to cAMP increases Cx43 content and the number and size of gap junctions. The results of the present study, combined with these previous observations (24), suggest that activation of at least two major signal transduction pathways, those involving increased intracellular cAMP levels and those stimulated by angiotensin II, enhance expression of Cx43 and increase the number and size of gap junctions. The dynamic nature of these changes suggests that remodeling of conduction pathways during early phases of infarct healing is an active process involving enhanced connexin expression and rearrangements of gap junction distributions rather than being a purely passive process in which some junctions become disrupted by the accumulation of interstitial fibrosis and others are preserved.
Methodologic considerations and limitations
Our studies were performed in cultures of neonatal rat ventricular myocytes using angiotensin concentrations similar to those used in previous studies by others (18,20,21,23). It is possible that the effects of angiotensin II could be different in neonatal cells that are actively synthesizing muscle-specific genes than in fully developed adult myocytes. However, there is evidence to suggest that angiotensin II induces a hypertrophic response in adult myocardium. For example, chronic infusion of subpressor amounts of angiotensin II causes ventricular hypertrophy in rats without changing blood pressure (32), and cardiac hypertrophy can be prevented by angiotensin-converting enzyme inhibitors in rats with abdominal aortic constriction (33). These observations, combined with clinical results of converting enzyme inhibitor therapy in patients with heart disease, suggest that angiotensin II acts as an endogenous growth factor in heart muscle and that it elicits a hypertrophic response, albeit by one or more direct and/or indirect mechanisms.
Angiotensin II has been shown to stimulate a hyperplastic response in cardiac fibroblasts that may also express Cx43 (20,34). It is possible, therefore, that some of the effects observed in cultures exposed to angiotensin II could have been due to changes in Cx43 expression in fibroblasts. However, contamination by fibroblasts in our cultures was modest (<10%) and fibroblast growth was inhibited by including 0.1 mmol/liter bromodeoxyuridine in the culture medium. Furthermore, the results of confocal immunofluorescence microscopy and electron microscopic morphometry demonstrated directly that cardiac myocyte gap junctions increased in size and number in response to angiotensin II. Although a change in fibroblast Cx43 content probably did not contribute significantly to the increase in total Cx43 signal measured in immunoblots, recent evidence has suggested that the hypertrophic growth response induced by angiotensin in cardiac myocytes may be mediated, at least in part, by a paracrine mechanism involving release of endothelin-1 by cardiac fibroblasts (35). We are not aware of reports on the effects of endothelin-1 on connexin expression by cardiac myocytes. Future studies will be required to sort out the complex interactions between cardiac myocytes and fibroblasts in the development of anatomic substrates for arrhythmias in heart disease.
Signal transduction pathways regulating connexin expression
The specific signal transduction pathways responsible for the effects of angiotensin II on Cx43 expression are not known. Angiotensin has been shown to activate both protein kinase C and mitogen-activated protein kinase pathways in neonatal cardiac myocytes (23), either of which could have played a role in increasing Cx43 expression. It is likely, however, that the responsible pathways activated by angiotensin II are distinct from those activated by cAMP, suggesting that multiple pathways may affect connexin expression. Further evidence that angiotensin and cAMP cause accumulation of Cx43 by disparate mechanisms comes from the results of metabolic labeling studies. We observed a marked effect of angiotensin II on the rate of Cx43 protein synthesis as demonstrated by a nearly threefold increase in the amount of [35S]methionine incorporated into Cx43 during a 2-h pulse interval that coincided with the final 2 h of a 24-h exposure of myocytes to angiotensin II. Interestingly, although the increase in total Cx43 content (approximately twofold) was equivalent in myocytes exposed to either angiotensin II or dibutyryl cAMP (24)for 24 h, no increase in Cx43 synthesis rate was observed in cells treated with cAMP. These observations suggest that accumulation of Cx43 in response to prolonged cAMP exposure is related to diminished Cx43 proteolysis. This potential mechanism is consistent with results of earlier studies demonstrating that cAMP delays the disappearance of gap junctions in freshly isolated pairs of rat hepatocytes (36), in which loss of intercellular coupling typically occurs with a time course similar to the measured half-life of the major hepatocyte gap junction protein, Cx32 (37).
The results of this study suggest that connexin expression is activated during a generalized hypertrophic response and may be regulated by multiple signal transduction pathways and involve different molecular mechanisms. A more detailed understanding of these pathways and mechanisms could provide specific targets for new therapies designed to limit structural alterations that create anatomic substrates of lethal ventricular arrhythmias in patients with heart disease.
We thank Susan Johnson for secretarial assistance.
↵∗ Present address: Department of Internal Medicine, University of Minnesota School of Medicine, Minneapolis, Minnesota 55410.
↵† Present address: Department of Internal Medicine, Columbia-Presbyterian Medical Center, New York, New York 10032.
↵‡ Present address: Section of Pediatric Hematology/Oncology, University of Chicago Children’s Hospital, Chicago, Illinois 60637.
☆ This study was supported by National Institutes of Health Grants HL50598 and HL45466.
- cyclic adenosine 3′-5′ monophosphate
- immunoglobulin G
- phosphate-buffered saline
- sodium dodecyl phosphate polyacrylamide gel electrophoresis
- phosphate-buffered saline containing 0.5% Triton X-100
- Received September 2, 1997.
- Revision received May 4, 1998.
- Accepted May 15, 1998.
- American College of Cardiology
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