Cardiac Muscle Cell Hypertrophy and Apoptosis Induced by Distinct Members of the p38 Mitogen-activated Protein Kinase Family*

Yibin WangDagger , Shuang Huang§, Valerie P. Sah, John Ross Jr.Dagger , Joan Heller Brown, Jiahuai Han§, and Kenneth R. ChienDagger par **

From the Departments of Dagger  Medicine and  Pharmacology and the par  Center for Molecular Genetics, University of California at San Diego, La Jolla, California 92093 and the § Department of Immunology, The Scripps Research Institute, La Jolla, California 92121

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

p38 mitogen-activated protein (MAP) kinase activities were significantly increased in mouse hearts after chronic transverse aortic constriction, coincident with the onset of ventricular hypertrophy. Infection of cardiomyocytes with adenoviral vectors expressing upstream activators for the p38 kinases, activated mutants of MAP kinase kinase 3b(E) (MKK3bE) and MAP kinase kinase 6b(E) (MKK6bE), elicited characteristic hypertrophic responses, including an increase in cell size, enhanced sarcomeric organization, and elevated atrial natriuretic factor expression. Overexpression of the activated MKK3bE in cardiomyocytes also led to an increase in apoptosis. The hypertrophic response was enhanced by co-infection of an adenoviral vector expressing wild type p38beta , and was suppressed by the p38beta dominant negative mutant. In contrast, the MKK3bE-induced cell death was increased by co-infection of an adenovirus expressing wild type p38alpha , and was suppressed by the dominant negative p38alpha mutant. This provides the first evidence in any cell system for divergent physiological functions for different members of the p38 MAP kinase family. The direct involvement of p38 pathways in cardiac hypertrophy and apoptosis suggests a significant role for p38 signaling in the pathophysiology of heart failure.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

A variety of pathophysiological stimuli, such as myocardial infarction, hypertension, valvular diseases, viral myocarditis, and dilated cardiomyopathy can lead to an increase in cardiac workload and elevated mechanical stress on cardiomyocytes. In response to hemodynamic overload, an adaptive hypertrophic response is triggered, which is characterized by an increase in the mass and volume of individual myocytes, resulting in an increase of heart weight without an increase in the number of cardiomyocytes (reviewed in Refs. 1 and 2). During the hypertrophic response, cardiomyocytes activate a distinct pattern of gene expression that eventually results in qualitative and quantitative alterations in contractile protein content and the induction of an embryonic gene program (3, 4). As hemodynamic overload persists, the stressed heart enters a critical transition from compensatory hypertrophy to decompensated heart failure. Chamber dilatation, excitation-contraction uncoupling, abnormal interstitial morphology, sarcomeric disorganization, altered energy metabolism, and the loss of viable myocytes are common features found in end-stage failing hearts (5). Signaling molecules that transduce the signals from this extracellular stress to different cellular compartments play central roles in mediating the hypertrophic process and the transition to heart failure. Accordingly, the identification and characterization of these signaling molecules have been the focus of intense study in recent years (6).

One recently identified group of signaling molecules that mediates environmental stress responses in various cell types is the family of p38 mitogen-activated protein (MAP)1 kinases. The p38 MAP kinase activity is activated by dual phosphorylation on a Thr-Gly-Tyr motif in response to endotoxin, cytokines, physical stress (such as hyperosmolarity), and chemical stress (such as hydrogen peroxide) (7-12). In non-cardiac cells, p38 MAP kinases have been implicated in gene regulation, morphological alterations, and cell survival in response to various environmental stimuli (13-20). Recently, it has been reported that in ischemia/reperfusion-treated hearts, p38 MAP kinase activities are elevated in association with the onset of hypertrophy and programmed cell death (30, 31). In addition, p38 kinase activities are also significantly induced in transgenic mouse hearts expressing activated Ha-Ras(V12), correlating with the onset of cardiac hypertrophy.2 However, the specific function of p38 in the development of cardiac hypertrophy and cardiac cell apoptosis have not yet been directly demonstrated.

The intracellular activation cascade for p38 MAP kinases under physiological conditions is still unclear, but several upstream MAP kinase kinases (MKKs) have been identified from in vitro analysis, including MKK3b and MKK6b (24-26). In the family of p38 MAP kinases, at least four isoforms have been identified thus far (8, 12, 27-29). Two well characterized isoforms, alpha  and beta , share extensive sequence similarity and a broad range of tissue distribution, including relatively high levels in the heart (8, 27). Although different isoforms of p38 have similar kinase activities in vitro on a given substrate, their specific functions under in vivo physiological conditions are largely unknown.

The present study was designed to critically assess the potential function of the p38 pathway in the onset of features that relate to cardiac muscle cell hypertrophy and failure. The p38 MAP kinase activities are activated during hypertrophy following in vivo pressure overload, suggesting their potential role in signal transduction of mechanical stimuli. To dissect specific functions of the p38 MAP kinases, we utilized recombinant adenoviruses to achieve efficient expression of the p38 signaling molecules in cultured cardiomyocytes, which allowed biochemical as well as morphological analysis on entire cell populations. Forced activation of p38beta activity results in characteristic features of hypertrophy, whereas the activation of p38alpha activity leads to the induction of myocyte apoptosis. The opposing effects of the p38 MAP kinase isoforms suggest that the activation of the p38 pathway may contribute to the development of hypertrophy and the transition to overt heart failure.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Transverse Aortic Constriction Surgery-- Transverse thoracic aortic constriction was performed as described previously (32, 33) on 8-week-old adult mice (C57/BL6XSJL, Jackson Laboratories). Briefly, in the anesthetized animals, a 7-0 nylon suture ligature was tied against a 27-gauge needle at the transverse aorta to produce a 65-70% constriction following the removal of the needle. At 4 h or 7 days after surgery, animals from the experimental and sham-operated groups were killed and the hearts removed. Ventricular chambers were weighed and quickly frozen in liquid nitrogen for protein extraction.

Recombinant Adenovirus Vectors-- Recombinant adenoviruses expressing activated MKK3bE, MKK6bE, wild type p38alpha , wild type p38beta and their corresponding dominant negative (TGY right-arrow AGF) mutants, p38alpha dn and p38beta dn, driven by a cytomegalovirus promoter were generated as described previously (22, 34). Similarly, recombinant adenoviruses expressing GFP and Ha-Ras-v were generated using cDNAs from pEGFP (CLONTECH) and mutant Ha-Ras(V12) (35). All recombinant adenoviruses were tested for transgene expression in cardiac myocytes by reverse transcriptase-polymerase chain reaction, Western blot, or kinase assays. The concentrated recombinant adenoviruses were prepared and titered as described (34).

Cardiomyocyte Culture and Adenoviral Infection-- Neonatal cardiomyocytes were prepared using a Percoll gradient method as described previously (36). Myocytes from 1-2-day-old Sprague-Dawley rats were plated in serum-containing medium (4:1 Dulbecco's modified Eagle's medium:medium 199, 10% horse serum, 5% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10 mM glutamine) overnight. Subsequently, the cells were changed into low serum medium containing 1% horse serum and 0.5% fetal bovine serum, and infected with adenoviruses at a multiplicity of infection of 50-100 particles/cell for 12 h. The cells were then cultured in serum-free medium for an additional 36-70 h before morphological or biochemical analysis.

MAP Kinase Assays-- Protein extracts from heart or myocytes were prepared and assayed for kinase activities, as described previously (37). Briefly, crushed frozen heart tissue or cells were harvested in lysis buffer (25 mM HEPES, pH 7.6, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 3 mM beta -glycerophosphate, 100 mM Na3VO4, 1% Nonidet P-40, 1 mM dithiothreitol, 10 µg/ml leupeptin, 10 µg/ml aprotinin and 1 mM phenylmethylsulfonyl fluoride. p38 kinases were immunoprecipitated using rabbit polyclonal anti-p38 antiserum (from J. Han (Scripps Research Institute, La Jolla, CA) and Santa Cruz Biotechnology, Inc.) conjugated to protein A-Sepharose. The kinase assays were then performed at 30 °C using [gamma -32P]ATP and myelin basic protein (Sigma) or glutathione S-transferase-ATF2 as a substrate. The phosphorylated substrate was separated by SDS-polyacrylamide gel electrophoresis, and visualized by autoradiography. The incorporated 32Pi in the substrate was quantified by radioanalytic scanning (AMBIS). A similar protocol was used to assay kinase activities of ERK1 and c-Jun N-terminal kinase (JNK) using myelin basic protein and c-Jun as substrates, as described previously (37).

Immunohistochemical Assay-- Cells were fixed in 3.7% formaldehyde and permeabilized in 0.3% Triton X-100. The atrial natriuretic factor (ANF) protein was detected using rabbit anti-rat alpha -ANF polyclonal antibody (Peninsula Laboratories) and fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody (Amersham Life Science). The F-actin was detected using rhodamine-conjugated phalloidin (Sigma). The tropomyosin was detected using a monoclonal antibody CH1 (a kind gift from Dr. Jim Lin, University of Iowa) and a tetramethylrhodamine isothiocyanate-conjugated goat anti-mouse secondary antibody (Amersham Life Science).

Ribonuclease Protection Assays-- The ANF and EF-1alpha mRNA were detected using a Direct-Protect kit according to manufacturer's recommendations (AMBION). Briefly, the rat ANF cDNA (pGEM-ANF) (54) or EF-1alpha cDNA (45) was linearized with XhoI or DdeI and used as templates for generation of radiolabeled riboprobes using polymerase T7 and SP 6, respectively. The protected fragments were separated on a 7% denaturing polyacrylamide gel, visualized by autoradiography, and quantitated by radioactive scanning (AMBIS).

Cell Survival Assay and Apoptosis Analysis-- Cell survival was analyzed using the 3-(4,5-dimethylthiaziazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) method (38) as reported previously. Briefly, myocytes were cultured in triplicate in 24-well tissue culture plates and infected with adenoviruses as described above. The cells were then stained with 500 µg/ml MTT (Sigma) for 4 h, and the positively stained cells were counted as living cells. For analysis of apoptotic cells, a DNA fragmentation assay was performed as described previously using a DNA isolation kit (Purogene) and standard agarose gel electrophoresis (49). Fragmented and condensed nuclei in apoptotic cells were also identified by staining cultured cells with Hoescht 33258 dye as described (49).

Statistical Analysis-- Analyses between two groups were performed using unpaired two-tailed t tests, with p values less than 0.001 as being significantly different.

    RESULTS
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Abstract
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Procedures
Results
Discussion
References

In Vivo Activation of the p38 MAP Kinase Activities during Pressure Overload-induced Hypertrophy-- To establish the functional role of the p38 pathway in the development of cardiac hypertrophy, we measured the p38 MAP kinase activity in mouse hearts that had been exposed to pressure overload following transverse aortic constriction (TAC). Previous studies have established that chronic TAC in mice can induce several conserved phenotypic features of ventricular hypertrophy with concentric enlargement of the ventricular chamber, an increase in heart weight/body weight ratio, and a concomitant activation of immediate early genes and embryonic marker genes (such as ANF) expression (32, 33). In hearts, isolated 4 h after TAC, the p38 MAP kinase activities were not significantly different from the basal level activity (Fig. 1). Seven days after surgery, however, a marked elevation of p38 activity was observed in TAC animal hearts as compared with those from the sham-operated group. Activation of the p38 MAP kinase activities during the development of hypertrophy suggested a potential role for this pathway in mediating defined features of the hypertrophic response.


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Fig. 1.   p38 MAP kinase activities in the pressure overloaded murine heart after chronic transverse aortic constriction. The left ventricles were dissected from TAC and sham-operated mice and then frozen, and 100-µg protein samples were assayed for p38 kinase activity using myelin basic protein as a substrate as described under "Experimental Procedures." The data represent the fold of induction versus sham-operated groups ± standard error from 6-12 animals as indicated (p < 0.0001).

Expression of p38 Signaling Molecules in Cultured Cardiomyocytes by Adenoviral Vector-mediated Gene Transfer-- To study the function of the p38 pathway in neonatal cardiac myocytes, we utilized recombinant adenoviruses as an efficient gene delivery vector to express various p38 signaling molecules (22, 34, 45). As demonstrated using a recombinant adenovirus expressing the green fluorescent protein (GFP) as a reporter, greater than 90% of the myocytes expressed the transgene, a much higher level of expression as compared with conventional calcium phosphate methods (Fig. 2A). The constitutively activated mutants of two upstream activators for the p38 kinases, MKK3bE and MKK6bE, as well as the wild type and dominant negative mutants of the p38 MAP kinase alpha  and beta  isoforms, were constructed in recombinant adenoviruses (22), and their expression in infected cardiac myocytes was detected at comparable levels by Western blot analysis (Fig. 2B). When cardiomyocytes were infected with vectors expressing MKK3bE and MKK6bE, the endogenous p38 MAP kinase activities were induced 12.2-fold and 3.0-fold, respectively (Fig. 2C). In contrast, the endogenous JNK activity and ERK activity were not activated by either virus (data not shown). This result was consistent with previous studies that have established MKK3b and MKK6b as specific upstream activators of the p38 MAP kinases (26, 27).


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Fig. 2.   Overexpression of p38 MAP kinase signaling molecules in cardiomyocytes via recombinant adenoviral gene transfer. A, cardiomyocytes were infected with a replication-defective adenovirus expressing GFP at a multiplicity of infection of 50 viral particles/cell (a and b) or transfected with 2 µg of pCMV/GFP plasmid by a CaPO4 method. Twenty-four hours later, cells were photographed under phase contrast microscopy in a light field (a and c) or under fluorescein isothiocyanate fluorescence (b and d). B, protein extracts from 100,000 cardiomyocytes were analyzed by Western blot to measure the relative expression of the p38 signaling molecules. The hemagglutinin-tagged MKK3bE and MKK6bE were detected by a monoclonal anti-hemagglutinin antibody (Boehringer Mannheim) and the FLAG-tagged wild type and mutant p38 kinases (arrowheads) were detected by a monoclonal anti-FLAG antibody (Eastman Kodak Co.) using standard procedures recommended by the suppliers. C, a p38 kinase assay was utilized with glutathione S-transferase-ATF2 as a substrate in cells treated with a GFP vector (Control), MKK3bE, or MKK6bE vectors, as indicated. The fold induction in kinase activity was measured from radioactive signals of the phosphorylated substrate.

Activation of the p38 Pathway in Cardiomyocytes Induces Several Independent Features of the Hypertrophic Response and Cell Death-- To assess the effects of the activated p38 pathway on cardiomyocytes, a number of independent effects on cellular morphology were assayed including surface area, F-actin organization, and sarcomere organization. The expression of a marker of the hypertrophic response, ANF, was also monitored by immunohistochemistry and RNase protection assays. In comparison to uninfected cells, MKK3bE- and MKK6bE-infected cells displayed an increase in cell surface area, enhanced organization of sarcomeric units with increased nonstriated myofibrils, and induction of ANF expression (Table I and Fig. 3). Levels of ANF mRNA, as quantified by RNase protection, were elevated approximately 2.9-fold by MKK3bE and 4.6-fold by MKK6bE (Fig. 4). These are all well characterized features of myocardial cell hypertrophy in this in vitro assay system induced by other bona fide hypertrophic stimuli (3, 4). To determine whether the effects of MKK3bE and MKK6bE were indeed mediated by the p38 MAP kinases, cardiomyocytes were treated with SB202190, a pyridinyl imidazole compound that specifically inhibits p38 kinase activity (12, 22). As shown in Figs. 3 and 4, addition of the p38 inhibitor suppressed both morphological changes and ANF expression in either MKK3bE- or MKK6bE-infected myocytes. Based on morphological and biochemical criteria, these results suggested that activation of the p38 MAP kinase pathway was sufficient to induce hypertrophy in cardiomyocytes.

                              
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Table I
Morphological analysis of cardiac myocytes infected with adv vectors expressing signaling molecules in the p38 MAP kinase pathway
Cell surface area represents the mean value of 40 cells in each group. S.E., standard error. *, significant difference in comparison with MKK6bE alone, p < 0.001. Sarcomere organization was measured by staining for actin filament and tropomyosin. ANF expression was measured by immunohistochemical staining of ANF protein, counting 10%-50% positive as +/-, >50% positive as +, and >80% positive as ++.


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Fig. 3.   Actin filament organization and ANF expression in cultured cardiac muscle cells. Neonatal cardiac myocytes were cultured and treated with adenoviral constructs, as described under "Experimental Procedures." Fifty hours after infection, cells were stained for F-actin by phalloidin (a, c, e, g, i, and k) or ANF protein (b, d, f, h, j, and l) by immunocytochemistry. Top panel, cardiac myocytes were infected with no virus and with a control vector which directed the expression of lacZ. Center panel, myocytes were infected with MKK3bE virus alone or in the presence of 20 mM SB202190 (Calbiochem, San Diego, CA), as indicated. Bottom panel, myocytes were infected with MKK6bE alone or in the presence of the same p38 inhibitor. All pictures were photographed at the same magnification. Positive staining for ANF protein was identified as a "ringlike" green fluorescent signal at the perinuclear regions of the myocytes.


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Fig. 4.   RNase protection assay for the expression of ANF mRNA. A, total RNA was isolated from approximately 500,000 myocytes treated in the absence of viral vector (C, lane 1) or a vector that directs the expression of the activated mutant Ha-Ras (H-Ras, lane 2). They were used as negative and positive controls, respectively. RNA samples from myocytes treated with MKK3bE (lanes 3-8) and MKK6bE (lanes 9-14) vectors, in combination with other p38 viruses or the p38 inhibitor were used as indicated. The lower levels of the EF-1a mRNA signal in lanes 3 and 7 resulted from the induction of cell death by MKK3bE expression. The ANF mRNA induction was determined by comparison with the control samples following normalization to the EF-1alpha mRNA level. These data are representative of at least three independent experiments.

Further observations at a later time point indicated that infection of MKK3bE also led to a significant increase in cell death (Fig. 5A). Using the MTT staining method as a quantitative cell survival assay, we estimated that the number of viable myocytes dropped to approximately 50% 72 h after infection as compared with control samples (Fig. 5C). In contrast, MKK6bE did not appear to induce myocyte death (Fig. 5B) even though its expression level was comparable with that of the MKK3bE in myocytes (Fig. 2B).


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Fig. 5.   Viability of cardiac muscle cells infected with adenoviruses expressing p38 signaling molecules. A and B, morphology of cultured cardiac muscle cells treated with different viral vectors as indicated was examined 72 h after infection following phase contrast microscopy. C, the viability of virus treated myocytes, as shown in A, was quantitated by a MTT method. The striped bars represent the percentage of viable cells, referring to the untreated samples as 100%. The error bars represent the standard error from three different experiments.

Differential Effects of p38alpha and p38beta on Cardiac Muscle Cells-- Although it has been suggested from previous in vitro studies that p38alpha and p38beta isoforms may have preferential upstream activators and different downstream target substrates (27, 28, 29), their specific functions under physiological conditions has not yet been demonstrated. To dissect the specific roles of these two isoforms in mediating cardiac muscle cell hypertrophy and cell death, adenoviruses expressing the wild type and dominant mutants of p38alpha or p38beta were co-infected along with recombinant adenoviruses that direct the expression of MKK3bE or MKK6bE.

Overexpression of the wild type p38alpha or p38beta isoform alone in myocytes had little effect, with the exception of a modest increase in cell surface area that was occasionally associated with adenoviral infection (Table I).3 Overexpression of wild type p38alpha in MKK3bE-infected cells, however, disrupted the hypertrophic morphology (Fig. 5A), partially inhibited ANF induction from 2.9-fold to 1.8-fold (Fig. 4), reduced the cell surface area to basal levels (Table I), and led to a marked induction of cell death (Fig. 5C). Although p38alpha did not appear to significantly affect the size and survival of the MKK6bE-infected cardiomyocytes (Fig. 5B, Table I), the ANF mRNA induction was significantly reduced from 4.6-fold to 2.8-fold (Fig. 4). In contrast, overexpression of wild type p38beta augmented the MKK3bE-induced hypertrophy (Fig. 5A), and further induced the ANF mRNA expression from 2.9-fold to 3.9-fold (Fig. 4). Forced activation of p38beta also promoted cell survival in the MKK3bE-infected myocytes, as indicated by the MTT assay (Fig. 5C). In MKK6bE-infected cells, overexpression of wild type p38beta also had a positive, albeit marginal, effect on the already highly elevated ANF mRNA levels, increasing its expression from 4.6- to 5.4-fold (Fig. 4). The surface area was also further increased from 2.67-fold over basal levels to 4.42-fold (Table I). From these results, we postulated that activation of p38beta was able to induce several features of the hypertrophic response and also promoted survival of cardiac myocytes, whereas activation of p38alpha was able to antagonize such an effect and resulted in cell death.

To further support the distinctive roles of p38 isoforms in cardiac myocytes, dominant negative mutants (TGY right-arrow AGF) of p38alpha (p38alpha dn) and p38beta (p38beta dn) were also co-expressed along with MKK3bE and MKK6bE (27). Overexpression of the p38alpha dominant negative mutant in MKK3bE-infected cardiomyocytes did not suppress the hypertrophic response, as indicated by cell surface area (in Table I), cellular morphology (Fig. 5A), and ANF expression (Fig. 4, lane 6). Suppressing p38alpha activity, however, increased the survival of MKK3bE-infected myocytes (Fig. 5C), therefore confirming the role of p38alpha in apoptotic induction. In contrast, overexpression of the p38beta dominant negative mutant in MKK3bE-infected cells abolished the characteristic features of the hypertrophic phenotype, including a decrease in cell surface area (in Table I), disruption of cellular morphology (Fig. 5B), and a reduction in ANF expression (Fig. 4, lane 7). Suppression of p38beta activity also led to a significant increase in cell death (Fig. 5C), suggesting its role in promoting survival of myocytes. Similar effects were also observed in MKK6bE-infected cells on ANF expression (Fig. 4, lanes 12 and 13), but to a much lesser extent on cellular morphology and cell survival (see Table I and Fig. 5B). These data further support the notion that activation of p38alpha was able to induce cell death and suppress hypertrophy, whereas activation of p38beta was able to induce hypertrophy and promote cell survival. The opposing effects of the two p38 isoforms were best demonstrated in the MKK3bE-infected cells, whereas the MKK6bE-infected cells were not affected by the different p38 isoforms to a similar extent.

p38alpha -mediated Cardiac Muscle Cell Death Involves Apoptotic Pathways-- To determine whether the observed cell death in myocytes involved apoptosis, a programmed genetic process, we performed DNA fragmentation assays to detect the presence of internucleosomal laddering in the genomic DNA, which is the hallmark of apoptosis (see Fig. 6). DNA fragmentation was observed in samples from myocytes that were infected with the MKK3bE vector, and the DNA laddering was significantly induced in samples from myocytes co-infected with wild type p38alpha or the dominant negative mutant of p38beta vectors. In comparison, DNA laddering was not detected from control myocytes or myocytes infected with MKK6bE. We also analyzed the integrity of myocyte nuclei by Hoescht dye staining in myocytes. Chromosomal condensation and fragmentation of nuclei, another characteristic feature of apoptotic cells, was also observed in a high percentage of tropomyosin-positive cardiac muscle cells co-infected with MKK3bE and wild type p38alpha or the dominant negative mutant p38beta (shown by arrows in Fig. 7). Taken together, these data suggested that cell death induced by the activation of the p38 MAP kinase pathway was an apoptotic process.


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Fig. 6.   DNA fragmentation assays on viral treated myocytes. Genomic DNA samples were prepared from untreated myocytes (Control) or virus treated cells, as indicated, at different time points and analyzed for the presence of internucleosomal cleavage by standard electrophoresis on a 2% agarose gel. MW, 1-kb molecular weight marker.


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Fig. 7.   Differential effects of p38alpha and p38beta isoforms on the apoptosis of MKK3bE-infected myocytes. Myocytes were treated in the absence of viruses (a and b), or viral vectors directing the expression of GFP (c and d), MKK3bE alone (e and f), or MKK3bE in combination with various p38 vectors as indicated (g-n), or MKK3bE in the presence of the p38 inhibitor SB202190 (o and p). The myocytes were identified using a monoclonal antibody CH-1 against tropomyosin. The myocyte nuclei were stained with Hoescht 33258 dye. Arrows indicate chromatin condensation and nuclear fragmentation in apoptotic cells. Photographs for Hoescht- and CH-1-stained cells were produced from the same field.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Cardiac hypertrophy is an adaptive process to cellular stress that involves changes in both gene expression and sarcomeric organization (1, 2). It is believed to be mediated by signaling molecules that transduce the stress signals from the environment into different cellular compartments (for a review, see Ref. 39). In this report, we document that the p38 MAP kinase activities are induced during the onset of in vivo hypertrophy in an experimental pressure overload model. In cultured cardiomyocytes, activation of the p38 pathway induces several independent characteristic features of myocyte hypertrophy, including an increase in cell surface area, enhanced sarcomeric organization, and expression of an embryonic marker gene, ANF. In the case of MKK3bE-infected myocytes, activation of the p38 pathway is also able to induce cell death. In addition, we have been able to dissect the specific roles of the p38 family members and demonstrate that the p38beta isoform mediates the hypertrophic response, whereas the p38alpha isoform is involved in an apoptotic process.

The distinct phenotypes of hypertrophy and cell death in MKK3bE-infected cardiac muscle cells appears to be dictated by the balance of the relative activity between two different p38 isoforms. This observation could be the result of a quantitative difference between the ability of the two isoforms to activate a common signaling pathway or a qualitative difference in the activation of divergent bona fide pathways for apoptosis and hypertrophy. In other cell types, the p38alpha kinase has been implicated as part of the Fas-induced apoptotic pathway involving ICE/Ced-3 proteases, suggesting a direct role for the p38 kinase in apoptotic responses (21, 22). Our data with the dominant negative mutants of p38alpha and p38beta also supports a qualitative difference between the p38alpha - and p38beta -mediated responses in apoptosis and hypertrophy, respectively. The final outcome of p38 activation in myocytes, either programmed cell death or hypertrophy, may be determined by the competing downstream pathways as suggested by previous studies of other MAP kinase pathways (48). Interestingly, MKK3b- and MKK6b-induced responses are differentially affected by the overexpression of p38 molecules. Both MKK3b and MKK6b are able to phosphorylate and activate different p38 kinases in vitro, although p38beta has been suggested as a preferred substrate for MKK6b rather than MKK3b (25, 26). Therefore, the difference between the effects of MKK3bE and MKK6bE in cardiomyocytes may result from their different specificities to various members of the p38 MAP kinase family. This conclusion remains to be tested when isoform-specific monoclonal antibodies for p38 kinases become available.

Activation of gene expression and the change of cellular morphology in p38 activated cardiac myocytes could be mediated by several distinct down-stream target molecules that have been identified in other cell types. A number of transcription factors, including ATF-2, ELK-1, CREB (13, 14), and MEF-2C, have been shown to be activated upon phosphorylation by p38 (16). Although the involvement of these transcriptional factors in hypertrophy is unclear, several of these factors, particularly MEF-2C, are known to play important roles in regulating cardiac gene expression and development (15). p38 can also activate some members of the small heat-shock proteins, including hsp25 and hsp27, through phosphorylation of MAPKAP kinase 2/3 (11, 18, 19, 20). Interestingly, it has been shown in non-cardiac cells that p38-mediated activation of hsp27 can induce F-actin reorganization and vinculin recruitment to the focal adhesion complex (46). Additional studies are needed to identify the specific activators as well as downstream targeting molecules of different p38 isoforms and to dissect out the relationship among the effectors of this signaling pathway in vivo.

Two other groups of MAP kinases mediate signal transduction in parallel with the p38 pathway, including ERK and JNK pathways (25-27, 40-44). Previous studies have documented that ERK activation is not sufficient to initiate a hypertrophic response in vitro (44), whereas in vivo its activation is not associated with the hypertrophic phenotype in the Ras transgenic mice (37). On the other hand, the JNK pathway is also activated in the Ras transgenic animals and its activation is essential for a hypertrophic response in vitro (37). It is highly likely that p38 and JNK are both required to generate a hypertrophic or an apoptotic response in overloaded hearts. Therefore, the potential interactions between the p38 pathway and the JNK or other signaling pathways in cardiac muscle cells is worthy of further investigation.

The finding that a stress-activated signaling pathway may play direct roles in inducing apoptosis has significant implications. There is an increasing body of evidence which suggests that apoptotic cells are a clear feature of heart failure in various animal models, in ischemia/reperfusion-treated hearts, as well as in human end-stage failing hearts (recently reviewed in Ref. 47). Programmed cell death may therefore serve as one of the underlined mechanisms for the transition from hypertrophy to decompensated heart failure. The implication of p38alpha in apoptosis of cardiomyocytes thereby provides a potential signaling pathway for such an apoptotic response. It will become of interest to determine if p38 pathways play any role in the cell survival effects mediated by cardiotrophin-1 (49) via GP130-dependent pathways (50). It will also be of interest to determine if manipulation of the p38 MAP kinase pathway and their downstream target molecules in vivo would have an effect in animal models of heart failure that are associated with apoptosis.

In conclusion, activation of p38 MAP kinase activities during hypertrophy, and the opposing effects of hypertrophy and cell death mediated by the two members of p38 MAP kinase family suggest a potential role of the p38 pathway in the onset of hypertrophy and heart failure. As presented in Fig. 8, a working model can now be constructed whereby hemodynamic stress, as a result of mechanical overload or chronic ischemia, can activate the p38 MAP kinase activities, which subsequently contribute to the hypertrophic response in the initial compensatory phase. As the stress stimulus persists, the balance between hypertrophic and apoptotic signaling is disrupted and, as a consequence, cardiomyocytes lose cellular viability and structural integrity and enter the cell death pathway. The loss of contractile function and viable cells eventually places more stress on the surviving myocytes and initiates the irreversible deterioration of cardiac function, resulting in overt heart failure. The recent development of miniaturized physiological technology (51), strategies for conditional transgenesis and ventricular chamber-restricted gene targeting in the murine heart (54), and genetically based mouse models of distinct forms of concentric and asymmetric hypertrophy (52) and failure (53) should allow a rigorous assessment of the in vivo validity of this model.


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Fig. 8.   The proposed role of p38 in cardiac hypertrophy and heart failure in response to hemodynamic stress. The p38alpha and p38beta MAP kinases are placed as components of the molecular response to hemodynamic stress in the pathological signaling pathway for cardiac hypertrophy and heart failure.

    ACKNOWLEDGEMENTS

We thank Dr. Lan Mao for surgical assistance and Mahmoud Itani for technical help. We are in debt to members of the Chien laboratory for their input and critical review of this manuscript.

    FOOTNOTES

* This work was supported by grants from the American Heart Association (AHA) and National Institutes of Health (NIH) (to K. R. C.), by AHA Grant-in-aid 95007690 and NIH Grants GM51417 and AI 41637 (to J. H.), and by NIH Grants HL28143 and HL46345 (to J. H. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: Dept. of Medicine and Center for Molecular Genetics, Mail Box 0613-C, 9500 Gilman Dr., University of California at San Diego, La Jolla, CA 92093. Tel.: 619-534-4801; Fax: 619-534-8081; E-mail: kchien{at}ucsd.edu.

1 The abbreviations used are: MAP, mitogen-activated protein; TAC, transverse aortic constriction; MKK, MAP kinase kinase; ANF, atrial natriuretic factor; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; GFP, green fluorescent protein; MTT, 3-(4,5-dimethylthiaziazol-2-yl)2,5-diphenyl tetrazolium bromide.

2 J. J. Hunter, M. Shimizu, J. Brown, V. P. Sah, K. Gottshall, C. Milano, R. Lefkowitz, J. H. Brown, and K. R. Chien, submitted for publication.

3 Y. Wang and K. R. Chien, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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