Published online before print September 5, 2002, doi: 10.1161/01.RES.0000035854.11082.01
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© 2002 American Heart Association, Inc.
Integrative Physiology |
c-Jun N-Terminal Kinase Activation Mediates Downregulation of Connexin43 in Cardiomyocytes
From the Department of Cell Biology (X.G., X.W.), The Scripps Research Institute, La Jolla, Calif; the Departments of Pathology and Pediatrics (D.L.L., J.E.S.), Washington University, St. Louis, Mo; the Department of Physiology (B.G.P., Y.W.), University of Maryland School of Medicine, Baltimore; and the Department of Pharmacology (J.H.B.), University of California, San Diego.
Correspondence to Yibin Wang, PhD, Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201. E-mail ywang001{at}umaryland.edu
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Abstract |
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Loss of gap junctions and impaired intercellular communication are characteristic features of pathological remodeling in heart failure as a result of stress or injury, yet the underlying regulatory mechanism has not been identified. Here, we report that in cultured myocytes, rapid loss of the gap junction protein connexin43 (Cx43) occurs in conjunction with the activation of c-Jun N-terminal kinase (JNK), a stress-activated protein kinase, on stress stimulation. To investigate the specific role of JNK activation in the regulation of connexin in cardiomyocytes, an activated mutant of mitogen-activated protein kinase kinase 7 (mutant D), a JNK-specific upstream activator, was expressed in myocytes by adenovirus-mediated gene transfer. JNK activation in infected cardiomyocytes resulted in significant reduction of Cx43 expression at both mRNA and protein levels and impaired cell-cell communication. To evaluate the role of JNK in the regulation of Cx43 expression and gap junction structure in vivo, a Cre-LoxP–mediated gene-switch system was used to establish a transgenic animal model with targeted activation of JNK in ventricular myocardium. The transgenic hearts exhibited significant downregulation of Cx43 expression and loss of gap junctions in myocardium that may contribute to the cardiac dysfunction and premature death phenotype. Our report represents the first evidence, both in vitro and in vivo, implicating JNK as an important mediator of stress-induced Cx43 downregulation and impaired intercellular communication in the failing heart.
Key Words: gap junctions • connexin43 • c-Jun N-terminal kinase • stress response • heart failure
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Introduction |
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Pathological stress due to hypertension, ischemic injury, or myocarditis results in cardiac remodeling, which is initially compensatory but often progresses to heart failure. Intracellular signaling is thought to play critical roles in the remodeling process in response to various extracellular stimuli.1–5 Despite intense studies, it is still unclear whether and how specific signaling pathways mediate the development of distinct aspects of heart failure.
Efficient intercellular communication is essential for normal electromechanical coupling in the heart through cell-cell transmission of signaling molecules and organized propagation of the action potential. Alterations in the amount and distribution of the cardiac gap junction protein connexin43 (Cx43) can lead to altered conduction of current, predisposition to arrhythmias, uncoordinated contraction, and overall diminished myocardial function.6 Disrupted gap junction structure and decreased expression of Cx43 are common features of cardiac remodeling observed in a variety of animal heart failure models, including pressure overload in guinea pigs,7 ischemia in dogs,8 and pharmacologically induced pulmonary hypertension9 and lipopolysaccharide treatment10 in rats. More important, both hypertrophied and ischemic human left ventricles have decreased levels of Cx43.11 Reduced expression of cardiac Cx43 in heterozygous Cx43 knockout mice results in increased susceptibility to arrhythmias in response to ischemia.12 Recent studies using cardiac-restricted inactivation of the Cx43 gene have unequivocally demonstrated ventricular conduction slowing and arrhythmic sudden death caused by loss of myocardial Cx43 protein.13,14 Together, these studies suggest a critical role for gap junction defects and loss of Cx43 expression in the pathogenesis of sudden death. Although the Wnt-1/ß-catenin–mediated signaling pathway has been implicated in the activation of Cx43 transcription,15 the underlying regulatory mechanism involved in the loss of Cx43 expression in response to pathological stress is unknown.
Under pathological stress, a number of intracellular signaling pathways are activated in ventricular myocytes, including c-Jun N-terminal kinase (JNK), a stress-activated protein kinase.16,17 JNKs, along with p38 and extracellular signal–regulated kinases, constitute the highly conserved mitogen-activated protein kinase family of signaling molecules. Each of these distinct but interconnected pathways are activated on dual phosphorylation of both threonine and tyrosine residues by upstream mitogen-activated protein kinase kinases (MKKs).18 The parallel signaling cascades are distinguished in part by their respective phosphorylation motifs, their downstream targets, and the stimuli to which they are primarily responsive.19 Earlier studies have demonstrated robust activation of JNK in cardiomyocytes in response to a variety of cellular stresses20 and in intact hearts in response to ischemia/reperfusion21 and chronic hemodynamic overload (Y. Wang, J.H. Brown, unpublished data, 1998). Furthermore, earlier studies have also identified a critical role for JNK in the process of cardiac hypertrophy both in vitro22,23 and in vivo, 24 suggesting that the JNK-mediated signaling pathway contributes to pathological remodeling in stressed hearts. However, it is still unclear whether and how JNK activation leads to specific aspects of cardiac remodeling.
In the present study, we demonstrate that stress stimulation in cardiac myocytes causes rapid downregulation of the gap junction protein Cx43 expression correlated with JNK activation. Specific activation of JNK activities in cardiomyocytes was sufficient to induce significant loss of Cx43 expression and impairment of cell-cell communication. To investigate the role of JNK pathway in pathological remodeling in vivo, we generated transgenic animals with targeted JNK activation in ventricular myocardium with the use of a Cre-LoxP–mediated gene-switch system. JNK activation in transgenic myocardium also led to significant loss of Cx43 expression and decreased the numbers of gap junctions, which likely contributed to the development of heart failure and premature death in the transgenic animals. In summary, our data provide the first in vitro and in vivo evidence identifying JNK as an important signaling pathway mediating Cx43 downregulation and impairment of intercellular communication in cardiac myocytes under pathological stresses.
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Materials and Methods |
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Cardiomyocyte Culture and Adenoviral Infection
Neonatal ventricular cardiomyocytes were dissociated from 1- to 2-day-old Sprague-Dawley rats, as described previously.25 Myocytes were plated overnight in media containing DMEM/medium 199 (4:1) supplemented with 10% horse serum, 5% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10 mmol/L glutamine. Subsequently, the cells were irradiated and incubated with serum-free media and infected with adenoviruses at a multiplicity of infection of 50 to 100 particles per cell. The cells were then incubated in serum-free media for an additional 2 days before harvesting and analysis. Experiments with the JNK inhibitor SP600125 (SP, Calbiochem) were carried out in serum-free media supplemented with 1 mg/mL BSA and 0.5% dimethyl sulfoxide to improve solubility of the compound.26 SP was added to culture media 30 minutes before anisomycin treatment and 12 hours after adenovirus infection.
Dye Transfer Assay
Gap junctional communication in primary myocyte cultures was assayed by microinjection of 5% Lucifer yellow CH dye in 0.1 mol/L LiCl solution. Cells were visualized by using an inverted phase-contrast/epifluorescence microscope (Carl Zeiss, Inc). Five minutes after dye injection, the number of dye-labeled cells directly adjacent to the microinjected dye-loaded cells was determined to represent cell-cell coupling properties. For each treatment condition, 10 cells were microinjected in each of 3 dishes.
Generation of Transgenic Animals
The cloning of an 
-myosin heavy chain–flox–MKK7D construct (Figure 4A) followed steps similar to those previously described,27 except that the cDNA fragment expressing human MKK7 (mutant D) was inserted into the pflox vector.22 Transgenic animals harboring the floxed-MKK7D transgene were identified by Southern blots and polymerase chain reaction, as described,27 and colonies were established in a Black Swiss background. The floxed-MKK7D mice were then bred with mice possessing the gene coding for Cre recombinase under the regulation of myosin light chain-2v (MLC-2v/Cre), a kind gift from Ju Chen, University of California, San Diego.28 The GFP-expressing floxed-MKK7D and the Cre-expressing MLC-2v/Cre mice have no abnormal cardiac phenotype according to functional and molecular analysis29 (B. Petrich, unpublished data, 2002). The double-transgenic offspring (MKK7D) of the floxed-MKK7D mice and MLC-2v/Cre mice were identified by polymerase chain reaction from tail DNA as described earlier27 and were analyzed between 6 to 8 weeks after birth by using age/sex-matched littermates with genotypes of floxed-MKK7D, MLC-2v/Cre, and wild type as controls.
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Protein Preparation and Immunoblotting
Whole hearts were excised from mice and snap-frozen in liquid nitrogen. To obtain protein extracts, left ventricles were pulverized on dry ice and homogenized in cell lysis buffer containing 20 mmol/L Tris (pH 7.4), 150 µmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L ß-glycerophosphate, 1 mmol/L Na3VO4, 1 mg/mL leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride. Protein from cultured cardiomyocytes was prepared in the same lysis buffer after brief sonication and centrifugation. Western blots were performed by loading equal amount of proteins, as indicated in figure legends, with the use of polyclonal antibodies to JNK, phospho-JNK, phospho-c-Jun (Ser63, Cell Signaling, Inc), MKK7 (Santa Cruz Biotechnology), Cx43 (Zymed), and connexin45 (Cx45, gift from Dr T. Steinberg, Washington University, St Louis, Mo).30 Protein signals were detected by use of the Pico West System (Pierce, Inc) and exposure to autoradiographic film and were quantified with the use of Scion NIH Image software.
RNA Preparation and Northern Blot Analysis
Total RNA was obtained from left ventricular tissue and from cultured cardiomyocytes with the use of Trizol reagent (Life Technologies) according to the manufacturer’s protocol. Northern blot analysis was performed with the use of 32P-labeled cDNA probes,31 and hybridization signals were visualized and quantified by using a PhosphoImager system and ImageQuant software (Molecular Dynamics).
Immunohistochemistry for Connexins
Myocytes on chamber slides were fixed with 4% paraformaldehyde in PBS for 15 minutes, followed by 3 washes with PBS. Immunostaining procedures were then followed as previously described.32 Immunofluorescent labeling of Cx43 and Cx45 in intact hearts was performed as described previously.33 Briefly, whole hearts were perfusion-fixed with 10% formalin, embedded in paraffin, and sectioned at a thickness of 6 µm. Immunoreactive signal was detected with polyclonal Cx43 (Zymed) or Cx45 (gift from Dr T. Steinberg30) antibodies and a CY3-conjugated secondary antibody. Confocal immunofluorescence microscopy and quantitative analysis of immunoreactive signal was performed by using a Sarastro model 2000 confocal laser scanning microscope (Molecular Dynamics). Cx43 signal was measured as the percentage of cell area as described.34
Electron Microscopy
Hearts were rapidly excised, and the major left ventricular papillary muscles of the mitral valve and a portion of epicardium from the lateral left ventricular wall were removed. The tissue was fixed and embedded as described.34 Ultrathin (50- to 100-nm) sections were cut in a plane parallel to the long axis of the myocytes and analyzed according to methods used in previous studies.34 Ten regions from control papillary muscle and epicardium were analyzed, and 20 regions from transgenic hearts were analyzed because of the marked reduction in gap junctions in transgenic hearts. The total number of gap junction profiles was counted in each region and expressed as a function of total tissue area.
Statistical Analysis
Results are expressed as mean±SD. Statistical significance was assessed by the Student paired 2-tailed t test, with a value of P<0.05 considered to be statistically significant.
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Results |
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Cellular Stress Decreases Cx43 Expression in Cardiomyocytes
Because Cx43 is reported to be markedly downregulated in diseased hearts, we first investigated whether Cx43 expression is also affected under stress conditions in cultured cardiomyocytes. Neonatal rat cardiomyocytes were treated with 50 ng/mL anisomycin (a protein synthesis inhibitor) to simulate stress conditions in cultured myocytes as reported.20 The treated myocytes were analyzed at various time points for JNK activity and Cx43 expression. In agreement with previous findings,20 anisomycin treatment induced a robust but transient activation of JNK activity (Figures 1A and 1B). After JNK activation by anisomycin treatment, Cx43 expression was significantly downregulated to 56% of untreated controls within 60 minutes and further downregulated to <41% at 90 minutes after stress stimulation. Similar to an earlier report,20 JNK activity was also rapidly induced by treatment with 0.5 mol/L sorbitol (an osmotic stressor), and the induction was sustained for >90 minutes. Again, a significant reduction of Cx43 protein was observed in sorbitol-treated cells within a time frame similar to that for anisomycin treatment (Figures 1C and 1D). These results suggest that cellular stress induced by different mechanisms can lead to rapid loss of Cx43 protein in correlation with JNK activation.
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Cx43 Downregulation and Impaired Cell-Cell Coupling Occur in Response to JNK Activation in Cultured Myocytes
To determine whether JNK activation plays a role in stress-induced Cx43 downregulation, an adenovirus vector expressing activated mutant MKK7D was used to infect the cardiomyocytes in culture. In a previous report, we demonstrated that expression of the MKK7D mutant in neonatal rat cardiomyocytes resulted in specific induction of JNK activity without affecting extracellular signal–regulated kinase and p38 pathways (Wang et al22 and Figure 2A). Expression of MKK7D and specific activation of JNK activities in cultured myocytes led to >90% reduction in Cx43 protein content compared with that in untreated control cells (Figure 2B). As expected, the loss of Cx43 protein content in JNK-activated cardiomyocytes was also reflected in a significant decrease in Cx43 immunoreactive signal at cell-cell junctions (Figure 2C). In addition, a near 60% reduction in Cx43 mRNA (Figure 2D) was observed in MKK7D-expressing cells, demonstrating that activation of JNK is sufficient to reduce Cx43 expression at both transcriptional and posttranscriptional levels in cardiomyocytes. To determine the functional effect of JNK-mediated Cx43 downregulation on intercellular communication, a dye transfer assay was used to measure the level of coupling between adjacent myocytes in culture. In MKK7D-expressing myocytes, minimal dye transfer to neighboring cells was observed. In contrast, dye transfer was detected in an average of 9 neighboring cells in untreated or LacZ-expressing controls (Figure 2E). Therefore, JNK activation is sufficient to mediate Cx43 downregulation at both mRNA and protein levels and leads to impaired intercellular coupling in cultured myocytes.
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Attenuation of JNK-Induced Cx43 Downregulation With the JNK-Specific Inhibitor SP
To confirm our observation and further demonstrate the specificity of JNK-induced Cx43 downregulation, we treated cultured myocytes with SP, a compound previously reported to be a highly specific inhibitor of JNK.26 Treatment of cultured myocytes expressing MKK7D with SP decreased the levels of JNK activation, as shown by Western blot analysis using a phosphospecific c-Jun antibody (Figure 3A). Western blot analysis of Cx43 shows the inhibition of JNK activity with 50 µmol/L SP–attenuated Cx43 downregulation in MKK7D-expressing cells (Figure 3B). We tested the dependence of stress-induced Cx43 downregulation on JNK signaling by pretreating cells with SP for 30 minutes before 90 minutes of incubation with anisomycin (as in Figure 1). Pretreatment with the JNK inhibitor substantially blocked the anisomycin-induced Cx43 decrease (Figure 3C). Although SP was unable to completely block JNK activation, perhaps because of its low solubility at higher concentrations, the levels of JNK inhibition achieved were closely correlated to the degree of attenuation of Cx43 downregulation (Figure 3). Together, these data demonstrate the specificity in JNK-induced Cx43 downregulation and support a critical role for JNK signaling in the regulation of Cx43 expression in response to cellular stresses.
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Establishing Transgenic Mice With Targeted Activation of JNK in Heart via Cre-LoxP–Mediated Gene Switch
To investigate whether JNK activation is also sufficient to downregulate Cx43 in myocardium in vivo, we generated transgenic mice expressing MKK7D under the regulation of the murine cardiac-specific -myosin heavy chain promoter. Because of an early lethal phenotype among transgenic founder animals (Y. Wang, unpublished observations), we first established a floxed-MKK7D transgenic line that expressed only green fluorescent protein (GFP). This transgenic line showed strong and uniform GFP expression in the myocardium but had neither detectable expression of the MKK7D transgene nor JNK activation (data not shown). No abnormal phenotype was detected in cardiac morphology, function, or gene expression (B.G. Petrich, unpublished data, 2002). The floxed-MKK7D mice were bred with previously established MLC-2v/Cre mice.28 In double-transgenic offspring, a significant level of MKK7D expression was detected (Figure 4B), whereas GFP expression was diminished as a result of Cre-LoxP–mediated DNA recombination (B.G. Petrich, unpublished data, 2002). The expression of MKK7D in these double-transgenic animals (MKK7D) led to marked induction of JNK activities in the ventricular myocardium, as determined by anti–phospho-JNK antibodies (Figure 4C).
Activation of JNK In Vivo Downregulates Cx43 Expression in Myocardium
To determine the effects of JNK activation on Cx43 expression in the transgenic hearts, Cx43 mRNA and protein in the transgenic ventricular myocardium were measured by Northern blots and Western blots. As shown in Figure 5A, Cx43 mRNA was significantly reduced in MKK7D transgenic hearts relative to control hearts (28.6±12.5 from MKK7D hearts versus 100±14.0 from control hearts, P<0.05), a level comparable to that observed in cultured myocytes with the use of Adv-MKK7D (Figure 2D). Cx43 protein was reduced to 
13% in MKK7D-expressing transgenic hearts relative to control hearts (Figure 5B), also comparable to the near 10-fold reduction seen in cultured myocytes (Figure 2A). By quantitative immunohistochemistry, a 94% reduction of Cx43 staining in the transgenic hearts was observed uniformly across the entire region of the ventricle (Figure 5D; percent tissue area occupied by Cx43 staining in MKK7D versus control hearts, 0.08±0.03% versus 1.44±0.39%, respectively [P<0.0005]). In contrast, Cx45 expression was not upregulated in transgenic hearts (Figure 6) but showed a modest trend of reduction, particularly at the cell-cell junction of intercalated disks, as determined by immunostaining (Figure 6C). No significant change in Cx40 expression was observed (data not shown). These results suggest that specific JNK activation in vivo led to marked downregulation of Cx43 in intact hearts and that the loss of Cx43 was not compensated by upregulation of other connexins.
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Activation of JNK In Vivo Leads to Loss of Gap Junctions in Myocardium and Causes Premature Death
The reduction of Cx43 expression was associated with a near 5-fold reduction in the number of gap junction profiles observed by electron microscopy in left ventricular epicardium and an almost 10-fold reduction in papillary muscle preparations (Figure 7A). Other than containing a paucity of gap junctions, adhesive junctions at intercalated disks appeared intact in transgenic myocardium, further suggesting the specificity of the gap junction defects caused by JNK activation. No ventricular hypertrophy, chamber dilation, or interstitial collagen induction was observed in the transgenic ventricle (Figure 7B; B.G. Petrich, Y. Wang, unpublished data, 2002). However, the transgenic animals died prematurely between 6 to 8 weeks of age (Figure 7C), suggesting that loss of Cx43 and gap junctions in the heart may contribute to the development of heart failure as a result of JNK activation.
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Discussion |
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In the present study, we have provided several lines of evidence implicating the stress-activated protein kinase JNK as an important intracellular signaling mediator in the loss of Cx43 expression in the heart under stress conditions. First, the activation of JNK activity in cultured myocytes in response to acute stress stimulation is correlated with the downregulation of Cx43. Second, specific activation of JNK in myocytes leads to significant reduction of Cx43 expression at both mRNA and protein levels and diminishes cell-cell coupling. Third, partial inhibition of JNK activities in response to the expression of upstream activators and to cellular stress attenuates Cx43 downregulation. Last, targeted activation of JNK activities in transgenic ventricles results in a marked decrease in Cx43 expression at both mRNA and protein levels, in a loss of Cx43 immunoreactive signal at cell-cell junctions, and in a reduction in ultrastructurally identified gap junctions at intercalated disks. The transgenic animals died prematurely as a result of JNK activation in the heart. Thus, these findings provide the first in vitro and in vivo evidence implicating the JNK-mediated pathway as an important signaling mechanism for the loss of gap junction protein and function in response to stresses.
Cx43 is the major component of gap junctions in the ventricle and plays an essential role in electrical and metabolic coupling between adjacent cardiomyocytes. In myocardium, conductance through gap junctions is one of the major determinants of cardiac conduction velocity. Under physiological conditions, effective coupling ensures uniform propagation of the action potential (wave front) in the myocardium and coordinated contraction during each cardiac cycle. Diminished expression of Cx43 increases gap junctional resistance, decreases conduction velocity, and enhances dispersion of action potential duration, all of which can contribute to arrhythmogenesis caused by both focal and reentrant mechanisms.6 Indeed, in chronically diseased human hearts, reduced expression of Cx43 has been observed as a common feature that is associated with increased arrhythmias.11,35,36 Genetically engineered mice possessing 50% Cx43 protein levels compared with wild-type mice exhibit increased propensity toward arrhythmic activity in response to acute ischemia.12 Similarly, targeted inactivation of Cx43 in myocardium leads to increased arrhythmia activity and sudden death.13,14 On the other hand, uncoupling from injured myocytes may provide a protective benefit to neighboring cells, as in the case of myocardial infarction. In fact, loss of Cx43 is one of the earliest changes to occur around the infarct zone after myocardial infarction and may help to reduce infarct size and insulate the injured area from neighboring cells.35,37 Cx43 has a relatively short half-life (1.3 hours) in the heart, further suggesting that tight temporal regulation of its expression levels may be important.38 Therefore, linking a major stress-induced signaling pathway, JNK, with Cx43 downregulation provides a plausible mechanism for regulating the dynamic changes in Cx43 expression and gap junction structure and function in stressed hearts. The transgenic animals established in the present study should provide a valuable model system to further investigate the pathophysiological role of JNK-induced loss of Cx43 in cardiac dysfunction and arrhythmogenesis.
In JNK-activated myocytes, a significant reduction in Cx43 mRNA expression (40% of normal) was observed by Northern blot analysis. Dupont et al36 have recently reported a similar level of cardiac Cx43 mRNA reduction in end-stage human heart failure. Interestingly, activation of activator protein-1, a well characterized downstream target of JNK, has been shown to be necessary for maximal Cx43 promoter activity in human myometrial smooth muscle.39 In addition, aortic smooth muscle cells have been shown to significantly increase steady-state levels of Cx43 mRNA in response to hypertension,40 a known activator of JNK.41 Together, these experiments suggest that the Cx43 promoter is subject to JNK-mediated regulation in a cell-type–specific manner. Further studies are needed to identify the necessary cis-regulatory elements and trans-acting protein factors that mediate the JNK-induced suppression of Cx43 transcription in cardiac muscle cells.
Immunocytochemistry of Cx43 in cultured myocytes showed a significant loss of Cx43 staining at the cell-cell junction (Figure 2A), although the magnitude of the reduction did not appear to be as much as that observed by immunoblot analysis (Figure 2C), perhaps because of background staining in cultured myocytes. All other evidence from both in vivo and in vitro protein analysis suggested a near 90% reduction in Cx43 protein contents on JNK activation. Therefore, JNK is most likely involved in Cx43 regulation at the posttranscriptional level. Although the regulation of both synthesis and degradation probably plays a role in Cx43 homeostasis,42 further studies will be needed to establish the specific mechanisms of JNK-mediated Cx43 downregulation, including changes in Cx43 protein synthesis, degradation, and membrane assembly.
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Acknowledgments |
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This study was sponsored in part by grants from the NIH (Drs Y. Wang, Saffitz, and Brown), an Intramural Research Award from University of Maryland School of Medicine (Dr Y. Wang), and an NIH Predoctoral Fellowship from the Interdisciplinary Training Program in Muscle Biology (Dr Petrich). The authors wish to thank Haiying Pu and William Kraft for their excellent technical assistance.
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Footnotes |
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*Both authors contributed equally to this study.
Received December 26, 2001; revision received August 2, 2002; accepted August 21, 2002.
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