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Angiotensin II Receptor Antagonist Blocks the Expression of Connexin43 Induced by Cyclical Mechanical Stretch in Cultured Neonatal Rat Cardiac Myocytes
J Mol Cell Cardiol 33, 691–698 (2001) doi:10.1006/jmcc.2000.1333, available online at http://www.idealibrary.com on
Angiotensin II Receptor Antagonist Blocks the Expression of Connexin43 Induced by Cyclical Mechanical Stretch in Cultured Neonatal Rat Cardiac Myocytes Kou-Gi Shyu, Chia-Chi Chen, Bao-Wei Wang and Peiliang Kuan The Division of Cardiology, Shin Kong Wu Ho-Su Memorial Hospital, 95 Wen-Chang Rd., Taipei 111, Taiwan (Received 17 October 2000, accepted in revised form 27 December 2000, published electronically 19 February 2001) K.-G. S, C.-C. C, B.-W. W P. K. Angiotensin II Receptor Antagonist Blocks the Expression of Connexin43 Induced by Cyclical Mechanical Stretch in Cultured Neonatal Rat Cardiac Myocytes. Journal of Molecular and Cellular Cardiology (2001) 33, 691–698. Mechanical forces have profound effects on cardiomyocytes. To test whether angiotensin II is a potential mediator of stretch-induced effects on gap junctions, we used the angiotensin II (AT1) receptor antagonist, losartan, to investigate the cyclical stretch-induced expression of connexin43 (Cx43), the major cardiac muscle gap junction channel protein. Cultured neonatal rat cardiomyocytes grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation, at 60 cycles/ min. The levels of Cx43 protein began to increase as early as 2 h after stretch was applied, reached a maximum of six-fold over the control by 24 h and remained at this level another 24 h (i.e. up to 48 h after stretch was applied). These increases of Cx43 protein at 24 h were largely (73%) and completely (100%) attenuated (P<0.001) by the addition (30 min before stretch) of 10 n and 100 n losartan, respectively. Similarly, the Cx43 mRNA levels in stretched cardiomyocytes rose 89% (P<0.01) above control (non-stretched cells) mRNA levels. This increase also was blocked by losartan. Cyclical stretch increased (and losartan decreased) the immunohistochemical labeling of Cx43 and significantly increased release of angiotensin II into the culture media from 7.5±0.6 ng/ ml to 23.8±1.0 ng/ml (P<0.01) after a 1 h stretch. These findings indicate that cyclical mechanical stretch augments angiotensin II production and Cx43 gene expression in cultured cardiomyocytes, partially through mediation of the AT1 receptors, and suggests interaction between the cardiomyocyte local rennin-angiotensin system and Cx43 in response to stretch. 2001 Academic Press K W: Stretch; Cardiomyocytes; Connexin43; Gap junctions; Angiotensin II; Antagonist.
Introduction Cardiac arrhythmias are common diseases and arrhythmias are often associated with significant morbidity and mortality. The molecular mechanisms of cardiac arrhythmia are barely known. Gap junctions play an important role in arrhythmogenesis.1,2 The cardiac myocytes express mul-
tiple gap junction proteins (connexins).3–5 Different connexin types have distinctive conductance, voltage dependence, and molecular permeability properties.6–8 Evaluation of the molecular controls of cardiac gap junction expression, assembly, and modification is crucial to understanding these processes. There is growing evidence to suggest that
Please address all correspondence to: Kou-Gi Shyu, Division of Cardiology, Department of Internal Medicine, Shin Kong Wu Ho-Su Memorial Hospital, 95 Wen-Chang Rd, Shih Lin, Taipei 111, Taiwan. Tel: 886-2-28332211; Fax: 886-2-28365775.
0022–2828/01/040691+08 $35.00/0
2001 Academic Press
692
K.-G. Shyu et al.
changes in pattern and velocity of conduction of myocardial electrical activity can affect cardiac rhythm and coordination of contraction.9 An abnormal coupling between cardiomyocytes through gap junctions is, therefore, increasingly considered an important factor in various pathophysiologic conditions including potentially life-threatening arrhythmias.10,11 Left ventricular end-diastolic pressure is elevated during acute myocardial infarction. The elevated end-diastolic pressure will stretch the myocardium, especially the infarct zone. Morphometric studies have demonstrated remodeling of gap junction in infarct border zones, which are critical sites for arrhythmogenesis.12 It has been shown that connexin43 is upregulated by cyclical stretch in the neonatal cardiac myocytes.13 However, how this upregulation occurs is not known. Mechanical stretch of cardiac myocytes causes direct release of angiotensin II14 and angiotensin II increases Cx43 expression in cultured cardiac myocytes.15 To date, there is no information in cardiac myocytes on the role of angiotensin II as a potential mediator of stretch-induced effects on gap junctions. The present study was designed to test whether a selective angiotensin II receptor antagonist could block cyclical stretch-induced connexin43 expression, and thereby imply a role for angiotensin this response.
anti-desmin antibody. The enriched myocytes were then subjected to cyclical stretch. Fibroblasts from the myocyte cultures were obtained by selective preplating. These fibroblasts were used at passages 1 to 3. For experiments, fibroblasts were plated into Flexcell I culture plates. Following overnight attachment, the fibroblast cultures were placed in serum-free medium (DMEM) and stretch experiments were performed 3 days later.
In vitro cyclical strain on cultured cardiac myocytes The Flexcell FX-2000 (Flexcell Co., McKeesport, PA, USA) strain unit consists of a vacuum unit linked to a valve controlled by a computer program. Cardiac myocytes and fibroblasts cultured on the flexible membrane base were subjected to cyclical stretch produced by this computer-controlled application of sinusoidal negative pressure. The flexible membranes supporting the cultured cells were deformed by a sinusoidal negative pressure with a peak level of ≅16 kPa at a frequency of 1 Hz (60 cycles per min) for various periods of time. After the stretch, the total RNA and protein from the stretched cells were collected for Northern ELISA and immunoprecipitation assay. In experiments involving inhibitors, the myocytes were pretreated with losartan, an angiotensin II (AT1) receptor antagonist for 30 min before cyclical stretch.
Methods Western blot analysis Cardiac myocyte culture Hearts from 2–3-day-old neonatal Wistar rats were used for the study. The atrium and ventricle were divided and minced separately. The minced tissues were then subjected to trypsin (0.125%) digestions in a balanced salt solution. The disaggregated cells were collected by centrifugation at 300×g for 10 min. The cell pellet was resuspended in serumcontaining medium (80% F10 nutrient mixture, 20% fetal bovine serum and 1% penicillin-streptomycin) and plated into a Petri dish and kept for 2.5 h in a 5% CO2 atmosphere at 37°C to let the cells attach to the dish. The suspended non-attached myocytes in the medium were collected and plated at a density of 1.67×106 cells/well on to 6-well Flexcell I culture plates. After 2 days in culture, cells were transferred to serum-free medium (Ham’s F-12:DMEM;1:1) and maintained for another 2 days. Cultured myocytes thus obtained were >90% pure as revealed by observation of contractile characteristics with a light microscope and stained with
The myocytes were rinsed with PBS, scraped into PBS containing 10 g/100 ml aprotinin and then centrifuged (300×g) for 10 min at 4°C. The pellet was resuspended, homogenized in a Reporter Lysis Buffer (Promega Corp., Madison, WI, USA), and then centrifuged at 10 600×g for 20 min. Protein content of the supernatant was determined by the Bio-Rad Protein assay using bovine serum albumin as the standard. Equal amounts of protein extracts (15 g) were loaded into 12.5% SDS-polyacrylamide gels. Proteins were electroblotted onto nitrocellulose. The membranes were blocked overnight in 5% skim milk in PBS and incubated in a mouse monoclonal anti-Cx43 antibody (Transduction Laboratories, Inc., San Francisco, CA, USA) at a dilution of 1:250 in PBS containing 0.1% Tween 20 (TPBS). After five washes in TPBS, the blots were incubated with horseradish peroxidaseconjugated goat anti-mouse IgG which was diluted 1:3000 in TPBS. After being rinsed six times for 5 min each in TPBS, the blots were incubated for
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Figure 1 Representative Western blot for connexin43 (Cx43) in cardiac myocytes subjected to cyclical stretch by 20% in the absence (upper panel) or presence of 10 n (middle panel) and 100 n (lower panel) losartan and for various periods of time.
1 min in ECL solution and exposed to X-ray film. The membrane was detected with an enhanced chemiluminescence system (ECL, Amersham Corp., Buckinghamshire, England). Densitometric quantitation of signal intensity was then measured. Equal protein loading of the samples was further verified by staining with tubulin-specific monoclonal antibody.
Northern ELISA Total RNA was prepared by solubilizing cardiac myocytes in Ultraspec RNA kit (Biotecx Laboratory, Inc., Houston, TX, USA). Total RNA was collected and examined by mini-gel agarose electrophoresis. The poly A+ mRNA from total RNA was then isolated using the Oligotex mRNA SpinColumn according to manufacturer specification (QIAGEN Inc., Valencia, CA, USA). Biotin-labeling reagent was added to the aliquots of 1 g of mRNA. The biotin-labeled mRNA was precipitated by ethanol and then was quantified. The mRNA was added to the microtiter plate well after addition of hybridization buffer. The denatured digoxigenin(DIG)-labeled DNA probe, generated from rat Cx43 DNA or a 800-bp PstI fragment of the mouse -actin gene was added to the hybridization mix at 50°C, and spun at 43×g for 180 min. The hybridization mix was then transferred to the streptavidin-coated microtiter plate. Anti-DIG-peroxidase was added and shaken at room temperature for 30 min. The 3,3′,5,5′-tetramethylbenzidine substrate was added. The sample was then determined by absorbance at 450 nm with a reference wavelength at 690 nm (Boehringer Mannheim, Mannheim, Germany). The absorbance of Cx43 was normalized to that of -actin.
Figure 2 Quantitative analysis of Cx43 protein levels in cardiac myocytes as a result of cyclical stretch by 20% elongation. (A) Six-fold increases in Cx43 protein levels were found after cyclical stretch for various periods of time. The values from stretched cardiomyocytes have been normalized to values in control cells in each of four independent experiments. (B) Change of Cx43 protein expression in the presence of different concentrations of losartan after cyclical stretch for 24 h. The Cx43 protein levels by Western blot were adjusted to insure equal protein loading (equal loading of samples was verified by staining with tubulin-specific monoclonal antibody) and averaged for three to four independent experiments. ∗ P<0.005 v control (non-stretched cells). ∗∗ P<0.001 v control.
Immunohistochemical labeling of cultured myocytes After cyclical stretch, myocytes were rinsed with PBS, fixed for 20 min in 4% paraformaldehyde in PBS for 30 min at room temperature, rinsed with PBS, and then incubated with 0.3% H2O2 in methanol for 30 min at room temperature. The cells were rinsed with PBS and incubated in permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice. Cells were incubated in primary antibodies (anti-Cx43) at 1:100 dilution overnight
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K.-G. Shyu et al. Figure 3 (A) Representative Northern ELISA for connexin43 (Cx43) and -actin in cardiac myocytes subjected to cyclical stretch by 20% for various periods of time. (B) Fold increases in Cx43 mRNA as a result of cyclical stretch for various periods of time. The values from stretched cells have been normalized to matched actin measurements and then expressed as a ratio of normalized values to mRNA in unstretched cells (control). ∗ P<0.05 v control; ∗∗ P<0.01 v control.
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at 4°C, washed extensively, incubated with secondary antibodies (biotinylated anti-mouse IgG antibody) for 1 h, then incubated with avidin-peroxidase for an additional 1 h. The cells were finally examined with light microscope.
Measurement of angiotenin II concentration Conditioned media from cardiac myocytes and fibrolasts subjected to cyclical stretch and those from control (unstretched) cells were collected for angiotensin II measurement. First, EDTA (1 mg/ml) and captopril (10 u) were added. After centrifugation at 1000 g at 4°C for 5 min, the supernatant was frozen at −20°C. The level of angiotensin II was measured by a quantitative sandwich enzyme immunoassay technique. The Angiotensin II immunoassay kit was purchased from Peninsula Laboratories (Peninsula Laboratories, Inc., Belmont, CA, USA). The lower limit of detection of angiotensin II was 0.05 ng/ml. The interassay variation was less than 5%.
Immunostaining of angiotensin II After cyclical stretch, myocytes and fibrolasts were rinsed with PBS, fixed for 20 min in 4% paraformaldehyde in PBS for 30 min at room temperature, rinsed with PBS, and then incubated with 0.3% H2O2 in methanol for 30 min at room temperature. The cells were rinsed with PBS and incubated in permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice. Cells were incubated in primary angiotensin II antibody (Santa Cruz Biotechnology, Inc., CA, USA) at 1:100 dilution for 45 min at room temperature, washed extensively, incubated with secondary antibodies (streptavidin peroxidase). After washing with PBS and blocking with H2O2, the cells were incubated with desmin monoclonal antibody (Biogenesis, Poole, England) at 1:100 dilution for 45 min at room temperature, washed extensively, incubated with secondary antibody (streptavidin alkaline
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phosphatase). The cells were finally examined with light microscope.
Statistical analysis The data were expressed as mean±... Statistical significance was evaluated using analysis of variance followed by Scheffe’s procedure. A value of P<0.05 was considered to denote statistical significance.
Results Effect of cyclical stretch on Cx43 protein expression To test the effect of cyclical mechanical stretch on Cx43 protein expression, cardiac myocytes were cyclically stretched for various periods of time and Cx43 levels subsequently measured. The mouse anti-Cx43 monoclonal antibody reacted well with rat protein extracts and recognized a single band. Cx43 protein expression was enhanced by 2 h after cyclical stretch, increased six-fold (to its maximum) by 24 h and stayed at this level until 48 h (Figs 1 and 2A).
Stretch-induced Cx43 in cardiac myocytes is angiotensin II receptor dependent To clarify the role of the endogenous renin– angiotensin system in stretch-induced Cx43 expression, the myocytes were stretched 20% for various periods in the presence or absence of losartan, an angiotensin II receptor antagonist. The losartan inhibited the stretch-induced Cx43 expression in a dose-dependent manner. As shown in Figures 1 and 2B, the stretch-induced increases of Cx43 protein at 24 h was attenuated by 73% (P<0.001) after addition of 10 n losartan. Pretreatment with 100 n losartan 30 min before stretch completely blocked the effect of cyclical stretch on Cx43 protein expression.
Figure 4 Representative microscopic images of the Cx43 immunoreactive signal in control cells (A) and cyclically stretched cardiac myocytes at 6 h (B) and 24 h (C) and after addition of losartan before cyclical stretch (D). The Cx43 signal appears as punctate spots (arrow) concentrated at sites of cellular apposition within the gap junction. Figure 5 Immunostaining of ventricular cardiac myocytes and non-myocytes (fibroblasts) with anti-angiotensin II and anti-desmin antibodies. (A) Staining by the anti-angiotensin II antibody shown as punctate spots (arrow) and antidesmin antibody shown as red color in the cardiac myocytes after cyclical stretch for 1 h. (B) Non-myocytes exhibited angiotensin II- and desmin-negative staining.
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Cyclical stretch enhances mRNA expression by cardiac myocytes The Northern ELISA showed that Cx43 message increased significantly after 4 h of stretch at 20% elongation (Fig. 3). The mRNA reached a maximal level after 6 h of stretch and then declined slightly at 24 h, but still was significantly higher than that of control without stretch. The -actin mRNA levels were relatively constant when cardiomyocytes were subjected to cyclical stretch. The stretch-induced increase in cardiac myocytes Cx43 mRNA was abolished by pretreatment with 100 n losartan. After addition of losartan at 100 n to the culture medium, the Cx43 mRNA level of myocytes stretched for 6 h decreased from 1.89±0.17-fold to 1.07±0.12-fold (normalized to control cells without stretch, n=4) (P<0.01).
Effects of cyclical stretch on Cx43 immunohistochemical labeling in cultured myocytes The anti-Cx43 antibody produces punctate labeling along the appositional membrane between cells. Cyclically stretched myocytes (when compared to unstretched myocytes) had increased Cx43 immunoreactive signal by 24 h (Fig. 4). This immunoreactive signal decreased after addition of losartan.
Cyclical stretch causes secretion of angiotensin II for cardiac myocytes We examined whether cyclical stretch causes release of angiotensin II into the culture medium. Aliquots of culture medium were collected from the dish after 1 h stretch and without stretch. After partially purifying the protein in the media, levels of angiotensin II were determined by ELISA using specific antibodies. The stretch significantly increased the angiotensin II in cardiac myocytes. The mean concentration of the angiotensin II rose from 7.5±0.6 to 23.8±1.0 ng/ml of medium (P<0.01) after a 1 h stretch. The level of angiotensin II in the conditioned medium of stretched and unstretched fibroblasts were not detectable.
Immunolocalization of angiotensin II The ventricular myocyte cultures used in these experiments contained 5% to 10% non-myocytes. To identify which cell type in the cardiac myocyte
culture contains angiotensin II, double immunostaining was performed using a polyclonal antibody against angiotensin II and a monoclonal antibody against desmin. As shown in Figure 5, the punctuated staining by the anti-angiotensin II antibody was located in the desmin-positive cells (which are cardiac myocytes). The fibroblasts exhibited negative staining for both desmin and angiotensin II antibodies.
Discussion By using Western blot, Northern ELISA and immunohistochemical staining, we found that losartan, an angiotensin II (AT1) receptor antagonist blocks the expression of Cx43 induced by cyclical mechanical stretch in cultured cardiac myocytes. Myocardial cells respond to changes in the mechanical forces imposed on them with changes in myocardial tension in the short-term and with structural remodeling in the long-term. Tissue remodeling as a result of changes in mechanical load involves extensive cell-to-cell communication; therefore, mechanical forces may affect expression and formation of gap junctions.13,16 Changes in contractile activity are prerequisites of the structural remodeling of the myocardium that results from altered preload and afterload. Cyclical mechanical stretch directly causes elongation and alteration of orientation of cultured myocytes.17,18 The in vitro cyclical mechanical stretch model resembles hemodynamic overload in vivo.19 Therefore, we expected that cyclical mechanical stretch would alter the expression of gap junctions. The double immunostaining in the present study showed that angiotensin II was located in the myocytes, not in the fibroblasts. The angiotenisn II was detectable in the conditioned medium of cardiac myocytes, but not in the conditioned medium of fibroblasts. These results suggest that the cardiac myocytes are the major, if not the sole, source of stretch-induced release of angiotensin II. This phenomenon exhibited the autocrine or paracrine production in response to cyclical stretch. Recently, Zhuang et al. showed that a linear pulsatile stretch of 10% of resting length produced marked upregulation of Cx43 in cultured cardiac myocytes within 1 h.20 In this study, the upregulation of Cx43 protein determined by Western blotting was observed after 2 h of 20% cyclical stretch and maximum levels were reached at 24 h and maintained during the period between 24 to 48 h. Differences in culture method and stretch apparatus may explain the difference between these
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two studies. In the present study, 20% of maximum elongation was used to cyclically stretched the myocytes. This strain is within the range expected for physiological changes in blood pressure. This strain does not induce cell injury.19 It is not clear whether greater levels of stretch would cause an additional increase Cx43 content. However, if the strain is larger than 25% elongation, cyclical stretch of the silicone substrate can induce myocyte injury which may interfere the expression of Cx43. Mechanical load is an important factor in the process of cardiac hypertrophy21,22 and recent studies indicate that humoral factors such as angiotensin II23,24 also play an important role in hypertrophy and hyperplasia of the interstitial space in the heart. Static stretch induces hypertrophy of cardiomyocytes and release of angiotensin II in vitro.14 Dodge et al. demonstrated that angiotensin II upregulated Cx43 protein expression in cultured cardiomyocytes.15 In this study, we have shown that cyclical mechanical stretch released angiotensin II and enhanced Cx43 gene expression in cultured cardiomyocytes. The complete inhibition of stretchinduced Cx43 expression by losartan indicates a specific mechanical effect is occurring via the AT1 receptor. These results provide the first evidence for AT1 receptor mediated cyclical stretch-induced expression of Cx43 in cultured cardiomyocytes. Our result implied that angiotenin II is the mediator of stretch-induced Cx43 expression in cultured neonatal cardiomyocytes. These results also indicated that local renin–angiotensin system also might play an important role in the remodeling of myocardial gap junctions under pathophysiologic conditions. The experiment using cultured neonatal cardiac myocytes is an established model for cardiac growth. It is not clear whether the changes observed in the present study are part of a response associated with normal cardiac function or whether they are part of the pathophysiological insult of hypertension and cardiac hypertrophy and arrhythmogenesis. Given the very rapid increase in Cx43 expression at the gene and protein level,25 would it not be possible to achieve this type of experiment in freshly isolated cardiac myocytes or intact muscle preparations? In summary, this study revealed that cyclical mechanical stretch augments angiotensin II production and Cx43 expression from cultured cardiac myocytes, partially through the AT1 receptors, which suggests interaction of the local renin– angiotensin system and Cx43 in stretched cardiac myocytes. Signal transduction pathways activated by cyclical stretch under pathologic conditions may cause remodeling of conduction pathway and lead
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to the development of anatomic substrates of arrhythmias.
Acknowledgements This study was supported in part from grant of National Science Council, Taiwan, Republic of China (NSC 89-2320-B-341-002).
References 1. U PC, G PI, A A, F JJ, W AL. Structural and electrophysiological changes in the epicardial border zone of canine myocardial infarcts during infarct healing. Circ Res 1985; 56: 436–451. 2. S MS, D PC. Relating extracellular potentials and their derivatives to anisotropic propagation at a microscopic level in human cardiac muscle: evidence for electrical uncoupling of sideto-side fiber connections with increasing age. Circ Res 1986; 58: 356–371. 3. K HL, S JE, B EC. Cardiac myocytes express multiple gap junction proteins. Circ Res 1992; 70: 438–444. 4. K HL, L JG, B EC, G KG, S JE. Multiple connexins colocalize in canine ventricular myocyte gap junctions. Circ Res 1993; 73: 344–350. 5. D BJ, L JG, L PD, S JE, B EC. Expression of multiple connexins in cultured neonatal rat ventricular myocytes. Circ Res 1995; 76: 381–387. 6. V RD, W HZ, W EM, B EC. Multiple connexins confer distinct regulatory and conductance properties of gap junctions in developing heart. Circ Res 1992; 71: 1277–1283. 7. M AP, R MB, F GI, S DC. Gap junction channels: distinct voltage-sensitive and -insensitive conductance states. Biophs J 1994; 67: 113–119. 8. E C, E R, L-F H, B A, T O, K RA, H DF, W K. Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells. J Cell Biol 1995; 129: 805–817. 9. S MS, S CF. Altering the topology of gap junctions: A major therapeutic target for atrial fibrillation. Cardiovasc Res 1995; 30: 337–344. 10. J HJ, G D. The cardiac connection. News Physiol Sci 1991; 6: 34–40. 11. P NS, G CR, P-W PA, S NJ. Cardiac arrhythmogenesis and the gap junction. J Mol Cell Cardiol 1995; 27: 37–44. 12. L RA, S JE. Remodeling of ventricular conduction pathways in healed canine infarct border zones. J Clin Invest 1991; 87: 1594–1602. 13. W TL, T YZ, C H. Regulation of connexin43 gene expression by cyclical mechanical stretch in neonatal rat cardiomyocytes. Biochem Biophys Res Commun 2000; 267: 551–557.
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14. S JI, X Y, S HS, I S. Autocrine release of angiotenin II mediates stretch-induced hypertrophy of cardiac yachts in vitro. Cell 1993; 75: 977–984. 15. D SM, B MA, D BJ, G KG, B EC, S JE. Effects of angiotensin II on expression of the gap junction channel protein connexin43 in neonatal rat ventricular myocytes. J Am Coll Cardiol 1998; 32: 800–807. 16. K RR, G M, D E, S MN, R SM, U R, P DJ, F K, P J, P-W PA, S NJ. Downregulation of immunodetectable connexin43 and decreased gap junction size in the pathogenesis of chronic hibernation in the human left ventricle. Circulation 1998; 97: 651–660. 17. T L, M B, B TK. Effects of cyclic mechanical stimulation of the cellular components of the heart: in vitro. In Vitro Cell Dev Biol 1988; 24: 53–58. 18. S JL, V HH. Mechanically induced orientation of adult rat cardiac myocytes in vitro. In Vitro Cell Dev Biol 1990; 26: 905–914. 19. S KG, C JJ, S NL, W DL, C H, L WP, L CC. Regulation of human cardiac myosin heavy chain genes by cyclical mechanical stretch in cultured cardiocytes. Biochem Biophys Res Commun 1995; 210: 567–573.
20. Z J, Y KA, S JE, K AG. Pulsatile stretch remodels cell-to-cell communication in cultured myocytes. Circ Res 2000; 87: 316–322. 21. K I, K Y, K T, S Y, K M, H E, T F, Y Y. Mechanical loading stimulates cell hypertrophy and specific gene expression in cultured rat cardiac myocytes: possible role of protein kinase C activation. J Biol Chem 1991; 266: 1265–1268. 22. S J, J L, T T, K TJ, I S. Molecular characterization of the stretch-induced adaptation of cultured cardiac cells: an in vitro model of load-induced cardiac hypertrophy. J Biol Chem 1992; 267: 10551–10560. 23. S J, I S. Molecular characterization of angiotensin II-induced hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts: critical role of AT1 receptor subtype. Circ Res 1993; 73: 413–423. 24. S WW, B GW, D DE, C KM, C KC, B KM. Angiotensin II is mitogenic in neonatal rat cardiac fibroblasts. Circ Res 1993; 72: 1245–1254. 25. B MA, L JG, B EC, S JE. Rapid turnover of connexin43 in the adult rat heart. Circ Res 1998; 83: 629–635.
Angiotensin II Receptor Antagonist Blocks the Expression of Connexin43 Induced by Cyclical Mechanical Stretch in Cultured Neonatal Rat Cardiac Myocytes Kou-Gi Shyu, Chia-Chi Chen, Bao-Wei Wang and Peiliang Kuan The Division of Cardiology, Shin Kong Wu Ho-Su Memorial Hospital, 95 Wen-Chang Rd., Taipei 111, Taiwan (Received 17 October 2000, accepted in revised form 27 December 2000, published electronically 19 February 2001) K.-G. S, C.-C. C, B.-W. W P. K. Angiotensin II Receptor Antagonist Blocks the Expression of Connexin43 Induced by Cyclical Mechanical Stretch in Cultured Neonatal Rat Cardiac Myocytes. Journal of Molecular and Cellular Cardiology (2001) 33, 691–698. Mechanical forces have profound effects on cardiomyocytes. To test whether angiotensin II is a potential mediator of stretch-induced effects on gap junctions, we used the angiotensin II (AT1) receptor antagonist, losartan, to investigate the cyclical stretch-induced expression of connexin43 (Cx43), the major cardiac muscle gap junction channel protein. Cultured neonatal rat cardiomyocytes grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation, at 60 cycles/ min. The levels of Cx43 protein began to increase as early as 2 h after stretch was applied, reached a maximum of six-fold over the control by 24 h and remained at this level another 24 h (i.e. up to 48 h after stretch was applied). These increases of Cx43 protein at 24 h were largely (73%) and completely (100%) attenuated (P<0.001) by the addition (30 min before stretch) of 10 n and 100 n losartan, respectively. Similarly, the Cx43 mRNA levels in stretched cardiomyocytes rose 89% (P<0.01) above control (non-stretched cells) mRNA levels. This increase also was blocked by losartan. Cyclical stretch increased (and losartan decreased) the immunohistochemical labeling of Cx43 and significantly increased release of angiotensin II into the culture media from 7.5±0.6 ng/ ml to 23.8±1.0 ng/ml (P<0.01) after a 1 h stretch. These findings indicate that cyclical mechanical stretch augments angiotensin II production and Cx43 gene expression in cultured cardiomyocytes, partially through mediation of the AT1 receptors, and suggests interaction between the cardiomyocyte local rennin-angiotensin system and Cx43 in response to stretch. 2001 Academic Press K W: Stretch; Cardiomyocytes; Connexin43; Gap junctions; Angiotensin II; Antagonist.
Introduction Cardiac arrhythmias are common diseases and arrhythmias are often associated with significant morbidity and mortality. The molecular mechanisms of cardiac arrhythmia are barely known. Gap junctions play an important role in arrhythmogenesis.1,2 The cardiac myocytes express mul-
tiple gap junction proteins (connexins).3–5 Different connexin types have distinctive conductance, voltage dependence, and molecular permeability properties.6–8 Evaluation of the molecular controls of cardiac gap junction expression, assembly, and modification is crucial to understanding these processes. There is growing evidence to suggest that
Please address all correspondence to: Kou-Gi Shyu, Division of Cardiology, Department of Internal Medicine, Shin Kong Wu Ho-Su Memorial Hospital, 95 Wen-Chang Rd, Shih Lin, Taipei 111, Taiwan. Tel: 886-2-28332211; Fax: 886-2-28365775.
0022–2828/01/040691+08 $35.00/0
2001 Academic Press
692
K.-G. Shyu et al.
changes in pattern and velocity of conduction of myocardial electrical activity can affect cardiac rhythm and coordination of contraction.9 An abnormal coupling between cardiomyocytes through gap junctions is, therefore, increasingly considered an important factor in various pathophysiologic conditions including potentially life-threatening arrhythmias.10,11 Left ventricular end-diastolic pressure is elevated during acute myocardial infarction. The elevated end-diastolic pressure will stretch the myocardium, especially the infarct zone. Morphometric studies have demonstrated remodeling of gap junction in infarct border zones, which are critical sites for arrhythmogenesis.12 It has been shown that connexin43 is upregulated by cyclical stretch in the neonatal cardiac myocytes.13 However, how this upregulation occurs is not known. Mechanical stretch of cardiac myocytes causes direct release of angiotensin II14 and angiotensin II increases Cx43 expression in cultured cardiac myocytes.15 To date, there is no information in cardiac myocytes on the role of angiotensin II as a potential mediator of stretch-induced effects on gap junctions. The present study was designed to test whether a selective angiotensin II receptor antagonist could block cyclical stretch-induced connexin43 expression, and thereby imply a role for angiotensin this response.
anti-desmin antibody. The enriched myocytes were then subjected to cyclical stretch. Fibroblasts from the myocyte cultures were obtained by selective preplating. These fibroblasts were used at passages 1 to 3. For experiments, fibroblasts were plated into Flexcell I culture plates. Following overnight attachment, the fibroblast cultures were placed in serum-free medium (DMEM) and stretch experiments were performed 3 days later.
In vitro cyclical strain on cultured cardiac myocytes The Flexcell FX-2000 (Flexcell Co., McKeesport, PA, USA) strain unit consists of a vacuum unit linked to a valve controlled by a computer program. Cardiac myocytes and fibroblasts cultured on the flexible membrane base were subjected to cyclical stretch produced by this computer-controlled application of sinusoidal negative pressure. The flexible membranes supporting the cultured cells were deformed by a sinusoidal negative pressure with a peak level of ≅16 kPa at a frequency of 1 Hz (60 cycles per min) for various periods of time. After the stretch, the total RNA and protein from the stretched cells were collected for Northern ELISA and immunoprecipitation assay. In experiments involving inhibitors, the myocytes were pretreated with losartan, an angiotensin II (AT1) receptor antagonist for 30 min before cyclical stretch.
Methods Western blot analysis Cardiac myocyte culture Hearts from 2–3-day-old neonatal Wistar rats were used for the study. The atrium and ventricle were divided and minced separately. The minced tissues were then subjected to trypsin (0.125%) digestions in a balanced salt solution. The disaggregated cells were collected by centrifugation at 300×g for 10 min. The cell pellet was resuspended in serumcontaining medium (80% F10 nutrient mixture, 20% fetal bovine serum and 1% penicillin-streptomycin) and plated into a Petri dish and kept for 2.5 h in a 5% CO2 atmosphere at 37°C to let the cells attach to the dish. The suspended non-attached myocytes in the medium were collected and plated at a density of 1.67×106 cells/well on to 6-well Flexcell I culture plates. After 2 days in culture, cells were transferred to serum-free medium (Ham’s F-12:DMEM;1:1) and maintained for another 2 days. Cultured myocytes thus obtained were >90% pure as revealed by observation of contractile characteristics with a light microscope and stained with
The myocytes were rinsed with PBS, scraped into PBS containing 10 g/100 ml aprotinin and then centrifuged (300×g) for 10 min at 4°C. The pellet was resuspended, homogenized in a Reporter Lysis Buffer (Promega Corp., Madison, WI, USA), and then centrifuged at 10 600×g for 20 min. Protein content of the supernatant was determined by the Bio-Rad Protein assay using bovine serum albumin as the standard. Equal amounts of protein extracts (15 g) were loaded into 12.5% SDS-polyacrylamide gels. Proteins were electroblotted onto nitrocellulose. The membranes were blocked overnight in 5% skim milk in PBS and incubated in a mouse monoclonal anti-Cx43 antibody (Transduction Laboratories, Inc., San Francisco, CA, USA) at a dilution of 1:250 in PBS containing 0.1% Tween 20 (TPBS). After five washes in TPBS, the blots were incubated with horseradish peroxidaseconjugated goat anti-mouse IgG which was diluted 1:3000 in TPBS. After being rinsed six times for 5 min each in TPBS, the blots were incubated for
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Figure 1 Representative Western blot for connexin43 (Cx43) in cardiac myocytes subjected to cyclical stretch by 20% in the absence (upper panel) or presence of 10 n (middle panel) and 100 n (lower panel) losartan and for various periods of time.
1 min in ECL solution and exposed to X-ray film. The membrane was detected with an enhanced chemiluminescence system (ECL, Amersham Corp., Buckinghamshire, England). Densitometric quantitation of signal intensity was then measured. Equal protein loading of the samples was further verified by staining with tubulin-specific monoclonal antibody.
Northern ELISA Total RNA was prepared by solubilizing cardiac myocytes in Ultraspec RNA kit (Biotecx Laboratory, Inc., Houston, TX, USA). Total RNA was collected and examined by mini-gel agarose electrophoresis. The poly A+ mRNA from total RNA was then isolated using the Oligotex mRNA SpinColumn according to manufacturer specification (QIAGEN Inc., Valencia, CA, USA). Biotin-labeling reagent was added to the aliquots of 1 g of mRNA. The biotin-labeled mRNA was precipitated by ethanol and then was quantified. The mRNA was added to the microtiter plate well after addition of hybridization buffer. The denatured digoxigenin(DIG)-labeled DNA probe, generated from rat Cx43 DNA or a 800-bp PstI fragment of the mouse -actin gene was added to the hybridization mix at 50°C, and spun at 43×g for 180 min. The hybridization mix was then transferred to the streptavidin-coated microtiter plate. Anti-DIG-peroxidase was added and shaken at room temperature for 30 min. The 3,3′,5,5′-tetramethylbenzidine substrate was added. The sample was then determined by absorbance at 450 nm with a reference wavelength at 690 nm (Boehringer Mannheim, Mannheim, Germany). The absorbance of Cx43 was normalized to that of -actin.
Figure 2 Quantitative analysis of Cx43 protein levels in cardiac myocytes as a result of cyclical stretch by 20% elongation. (A) Six-fold increases in Cx43 protein levels were found after cyclical stretch for various periods of time. The values from stretched cardiomyocytes have been normalized to values in control cells in each of four independent experiments. (B) Change of Cx43 protein expression in the presence of different concentrations of losartan after cyclical stretch for 24 h. The Cx43 protein levels by Western blot were adjusted to insure equal protein loading (equal loading of samples was verified by staining with tubulin-specific monoclonal antibody) and averaged for three to four independent experiments. ∗ P<0.005 v control (non-stretched cells). ∗∗ P<0.001 v control.
Immunohistochemical labeling of cultured myocytes After cyclical stretch, myocytes were rinsed with PBS, fixed for 20 min in 4% paraformaldehyde in PBS for 30 min at room temperature, rinsed with PBS, and then incubated with 0.3% H2O2 in methanol for 30 min at room temperature. The cells were rinsed with PBS and incubated in permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice. Cells were incubated in primary antibodies (anti-Cx43) at 1:100 dilution overnight
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K.-G. Shyu et al. Figure 3 (A) Representative Northern ELISA for connexin43 (Cx43) and -actin in cardiac myocytes subjected to cyclical stretch by 20% for various periods of time. (B) Fold increases in Cx43 mRNA as a result of cyclical stretch for various periods of time. The values from stretched cells have been normalized to matched actin measurements and then expressed as a ratio of normalized values to mRNA in unstretched cells (control). ∗ P<0.05 v control; ∗∗ P<0.01 v control.
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at 4°C, washed extensively, incubated with secondary antibodies (biotinylated anti-mouse IgG antibody) for 1 h, then incubated with avidin-peroxidase for an additional 1 h. The cells were finally examined with light microscope.
Measurement of angiotenin II concentration Conditioned media from cardiac myocytes and fibrolasts subjected to cyclical stretch and those from control (unstretched) cells were collected for angiotensin II measurement. First, EDTA (1 mg/ml) and captopril (10 u) were added. After centrifugation at 1000 g at 4°C for 5 min, the supernatant was frozen at −20°C. The level of angiotensin II was measured by a quantitative sandwich enzyme immunoassay technique. The Angiotensin II immunoassay kit was purchased from Peninsula Laboratories (Peninsula Laboratories, Inc., Belmont, CA, USA). The lower limit of detection of angiotensin II was 0.05 ng/ml. The interassay variation was less than 5%.
Immunostaining of angiotensin II After cyclical stretch, myocytes and fibrolasts were rinsed with PBS, fixed for 20 min in 4% paraformaldehyde in PBS for 30 min at room temperature, rinsed with PBS, and then incubated with 0.3% H2O2 in methanol for 30 min at room temperature. The cells were rinsed with PBS and incubated in permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice. Cells were incubated in primary angiotensin II antibody (Santa Cruz Biotechnology, Inc., CA, USA) at 1:100 dilution for 45 min at room temperature, washed extensively, incubated with secondary antibodies (streptavidin peroxidase). After washing with PBS and blocking with H2O2, the cells were incubated with desmin monoclonal antibody (Biogenesis, Poole, England) at 1:100 dilution for 45 min at room temperature, washed extensively, incubated with secondary antibody (streptavidin alkaline
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phosphatase). The cells were finally examined with light microscope.
Statistical analysis The data were expressed as mean±... Statistical significance was evaluated using analysis of variance followed by Scheffe’s procedure. A value of P<0.05 was considered to denote statistical significance.
Results Effect of cyclical stretch on Cx43 protein expression To test the effect of cyclical mechanical stretch on Cx43 protein expression, cardiac myocytes were cyclically stretched for various periods of time and Cx43 levels subsequently measured. The mouse anti-Cx43 monoclonal antibody reacted well with rat protein extracts and recognized a single band. Cx43 protein expression was enhanced by 2 h after cyclical stretch, increased six-fold (to its maximum) by 24 h and stayed at this level until 48 h (Figs 1 and 2A).
Stretch-induced Cx43 in cardiac myocytes is angiotensin II receptor dependent To clarify the role of the endogenous renin– angiotensin system in stretch-induced Cx43 expression, the myocytes were stretched 20% for various periods in the presence or absence of losartan, an angiotensin II receptor antagonist. The losartan inhibited the stretch-induced Cx43 expression in a dose-dependent manner. As shown in Figures 1 and 2B, the stretch-induced increases of Cx43 protein at 24 h was attenuated by 73% (P<0.001) after addition of 10 n losartan. Pretreatment with 100 n losartan 30 min before stretch completely blocked the effect of cyclical stretch on Cx43 protein expression.
Figure 4 Representative microscopic images of the Cx43 immunoreactive signal in control cells (A) and cyclically stretched cardiac myocytes at 6 h (B) and 24 h (C) and after addition of losartan before cyclical stretch (D). The Cx43 signal appears as punctate spots (arrow) concentrated at sites of cellular apposition within the gap junction. Figure 5 Immunostaining of ventricular cardiac myocytes and non-myocytes (fibroblasts) with anti-angiotensin II and anti-desmin antibodies. (A) Staining by the anti-angiotensin II antibody shown as punctate spots (arrow) and antidesmin antibody shown as red color in the cardiac myocytes after cyclical stretch for 1 h. (B) Non-myocytes exhibited angiotensin II- and desmin-negative staining.
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Cyclical stretch enhances mRNA expression by cardiac myocytes The Northern ELISA showed that Cx43 message increased significantly after 4 h of stretch at 20% elongation (Fig. 3). The mRNA reached a maximal level after 6 h of stretch and then declined slightly at 24 h, but still was significantly higher than that of control without stretch. The -actin mRNA levels were relatively constant when cardiomyocytes were subjected to cyclical stretch. The stretch-induced increase in cardiac myocytes Cx43 mRNA was abolished by pretreatment with 100 n losartan. After addition of losartan at 100 n to the culture medium, the Cx43 mRNA level of myocytes stretched for 6 h decreased from 1.89±0.17-fold to 1.07±0.12-fold (normalized to control cells without stretch, n=4) (P<0.01).
Effects of cyclical stretch on Cx43 immunohistochemical labeling in cultured myocytes The anti-Cx43 antibody produces punctate labeling along the appositional membrane between cells. Cyclically stretched myocytes (when compared to unstretched myocytes) had increased Cx43 immunoreactive signal by 24 h (Fig. 4). This immunoreactive signal decreased after addition of losartan.
Cyclical stretch causes secretion of angiotensin II for cardiac myocytes We examined whether cyclical stretch causes release of angiotensin II into the culture medium. Aliquots of culture medium were collected from the dish after 1 h stretch and without stretch. After partially purifying the protein in the media, levels of angiotensin II were determined by ELISA using specific antibodies. The stretch significantly increased the angiotensin II in cardiac myocytes. The mean concentration of the angiotensin II rose from 7.5±0.6 to 23.8±1.0 ng/ml of medium (P<0.01) after a 1 h stretch. The level of angiotensin II in the conditioned medium of stretched and unstretched fibroblasts were not detectable.
Immunolocalization of angiotensin II The ventricular myocyte cultures used in these experiments contained 5% to 10% non-myocytes. To identify which cell type in the cardiac myocyte
culture contains angiotensin II, double immunostaining was performed using a polyclonal antibody against angiotensin II and a monoclonal antibody against desmin. As shown in Figure 5, the punctuated staining by the anti-angiotensin II antibody was located in the desmin-positive cells (which are cardiac myocytes). The fibroblasts exhibited negative staining for both desmin and angiotensin II antibodies.
Discussion By using Western blot, Northern ELISA and immunohistochemical staining, we found that losartan, an angiotensin II (AT1) receptor antagonist blocks the expression of Cx43 induced by cyclical mechanical stretch in cultured cardiac myocytes. Myocardial cells respond to changes in the mechanical forces imposed on them with changes in myocardial tension in the short-term and with structural remodeling in the long-term. Tissue remodeling as a result of changes in mechanical load involves extensive cell-to-cell communication; therefore, mechanical forces may affect expression and formation of gap junctions.13,16 Changes in contractile activity are prerequisites of the structural remodeling of the myocardium that results from altered preload and afterload. Cyclical mechanical stretch directly causes elongation and alteration of orientation of cultured myocytes.17,18 The in vitro cyclical mechanical stretch model resembles hemodynamic overload in vivo.19 Therefore, we expected that cyclical mechanical stretch would alter the expression of gap junctions. The double immunostaining in the present study showed that angiotensin II was located in the myocytes, not in the fibroblasts. The angiotenisn II was detectable in the conditioned medium of cardiac myocytes, but not in the conditioned medium of fibroblasts. These results suggest that the cardiac myocytes are the major, if not the sole, source of stretch-induced release of angiotensin II. This phenomenon exhibited the autocrine or paracrine production in response to cyclical stretch. Recently, Zhuang et al. showed that a linear pulsatile stretch of 10% of resting length produced marked upregulation of Cx43 in cultured cardiac myocytes within 1 h.20 In this study, the upregulation of Cx43 protein determined by Western blotting was observed after 2 h of 20% cyclical stretch and maximum levels were reached at 24 h and maintained during the period between 24 to 48 h. Differences in culture method and stretch apparatus may explain the difference between these
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two studies. In the present study, 20% of maximum elongation was used to cyclically stretched the myocytes. This strain is within the range expected for physiological changes in blood pressure. This strain does not induce cell injury.19 It is not clear whether greater levels of stretch would cause an additional increase Cx43 content. However, if the strain is larger than 25% elongation, cyclical stretch of the silicone substrate can induce myocyte injury which may interfere the expression of Cx43. Mechanical load is an important factor in the process of cardiac hypertrophy21,22 and recent studies indicate that humoral factors such as angiotensin II23,24 also play an important role in hypertrophy and hyperplasia of the interstitial space in the heart. Static stretch induces hypertrophy of cardiomyocytes and release of angiotensin II in vitro.14 Dodge et al. demonstrated that angiotensin II upregulated Cx43 protein expression in cultured cardiomyocytes.15 In this study, we have shown that cyclical mechanical stretch released angiotensin II and enhanced Cx43 gene expression in cultured cardiomyocytes. The complete inhibition of stretchinduced Cx43 expression by losartan indicates a specific mechanical effect is occurring via the AT1 receptor. These results provide the first evidence for AT1 receptor mediated cyclical stretch-induced expression of Cx43 in cultured cardiomyocytes. Our result implied that angiotenin II is the mediator of stretch-induced Cx43 expression in cultured neonatal cardiomyocytes. These results also indicated that local renin–angiotensin system also might play an important role in the remodeling of myocardial gap junctions under pathophysiologic conditions. The experiment using cultured neonatal cardiac myocytes is an established model for cardiac growth. It is not clear whether the changes observed in the present study are part of a response associated with normal cardiac function or whether they are part of the pathophysiological insult of hypertension and cardiac hypertrophy and arrhythmogenesis. Given the very rapid increase in Cx43 expression at the gene and protein level,25 would it not be possible to achieve this type of experiment in freshly isolated cardiac myocytes or intact muscle preparations? In summary, this study revealed that cyclical mechanical stretch augments angiotensin II production and Cx43 expression from cultured cardiac myocytes, partially through the AT1 receptors, which suggests interaction of the local renin– angiotensin system and Cx43 in stretched cardiac myocytes. Signal transduction pathways activated by cyclical stretch under pathologic conditions may cause remodeling of conduction pathway and lead
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to the development of anatomic substrates of arrhythmias.
Acknowledgements This study was supported in part from grant of National Science Council, Taiwan, Republic of China (NSC 89-2320-B-341-002).
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