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1Institut für Pathophysiologie, Zentrum für Innere Medizin, and 2Institut für Physiologische Chemie, Universitätsklinikum Essen, 45122 Essen, Germany
Submitted 2 December 2002 ; accepted in final form 31 May 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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and -
were increased in failing compared with normal hearts (149,441 ± 38,381 and 170,430 ± 32,952 vs. 68,815 ± 28,984 and 81,788 ± 22,774 AU, respectively, both P < 0.05). In failing compared with normal hearts, total and phosphorylated JNK46 and JNK54 MAPK were increased, whereas total and phosphorylated ERK MAPK remained unchanged. In pacing-induced HF, p38 and JNK MAPK phosphorylation as well as p38 MAPK activity was increased. Further studies will have to define whether or not chronic specific blockade of MAPK activity can interfere with apoptosis/fibrosis and thereby attenuate the progression of HF.
fibrosis; apoptosis; hypertrophy
In the heart, MAPKs such as ERK, JNK, and p38 are expressed (7, 8, 13). The - (8), 
- (28), and 
-isoforms (16) of p38 MAPK have been identified, whereas the existence of the p38 MAPK -isoform has been suggested (20) but not yet convincingly proven.
The consequences of activation of myocardial MAPKs are still incompletely understood. In isolated cardiomyocytes, activation of ERK MAPK appears to be involved in hypertrophic growth (4, 35) and protection against apoptosis induced by either osmotic load (11) or -adrenergic stimulation (29). Cardiomyocyte transfection with a constitutively active mitogen kinase kinase (MKK) specifically activating JNK MAPK (MKK-7) induces hypertrophy (36). Also, a potential role for JNK MAPK activation in apoptosis has been demonstrated in cardiomyocytes (22). In contrast, in mice with aortic banding, activation of JNK MAPK prevents apoptosis and inflammation (26) while having no effect (26) or even suppressing cardiomyocyte growth (21). Overexpression of p38 MAPK- in murine cardiomyocytes increases apoptosis, and overexpression of p38 MAPK- induces hypertrophy (36). Similarly, in mice in vivo that overexpress MKK-3b and -6b (18) or TAK 1 (40), which subsequently leads to activation of p38 MAPK, the extent of myocardial fibrosis is increased; the remaining cardiomyocytes display signs of hypertrophy, and, over time, the left ventricular (LV) ejection fraction is decreased. In rats in vivo, hypertension-induced myocardial hypertrophy is associated with increased p38 MAPK phosphorylation and activity (33, 38), although with progression toward severe heart failure, p38 MAPK phosphorylation and subsequently activity return toward baseline values (2).
In biopsies from explanted human hearts, ERK, JNK, and p38 MAPK activities were unaltered in hearts with signs of hypertrophy only (12) but increased in failing hearts secondary to dilated cardiomyopathy or ischemic heart disease (10, 12). However, these findings are still controversial, because in other studies ERK MAPK activity remained unaltered (6, 7) and p38 MAPK activity was decreased in explanted failing hearts (6, 16, 32). The latter three studies analyzed p38 MAPK activity only in heart failure secondary to dilated cardiomyopathy (6, 32) or only one specific p38 MAPK isoform (p38 MAPK-) activity (16). Thus part of the controversy regarding p38 MAPK might relate to the underlying cause of heart failure or the analyzed isoform.
We therefore studied MAPK phosphorylation (ERK, JNK, and p38) and/or activity (p38 MAPK- and -) in pacing-induced heart failure.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Male Chinchilla bastard rabbits weighing 3.1 ± 0.2 kg were anesthetized, initially with ketamine (50 mg/kg)-xylazine (3 mg/kg) and then followed by propofol (12–25 ml/h)-fentanyl (0.003 mg/kg), intubated, and ventilated using a Dräger UV2 ventilator (Lübeck, Germany) with 70% room air and 30% oxygen. After a left thoracotomy, a pacing lead was sutured onto the apical region of the left ventricle. The pacing lead was connected to a pacemaker (Medtronic; Düsseldorf, Germany), which was implanted subcutaneously. The chest was closed in layers and evacuated with a Bülau drainage. The tracheal tube was removed after spontaneous breathing was assured. The rabbits were placed on an antibiotic regimen (12 mg/kg enrofloxacin) for 3 days, and postoperative analgesia was performed with buprenorphin (0.03 mg/kg). After instrumentation, the rabbits were allowed to recover for 7–10 days. Thereafter, heart failure was induced in eight rabbits by rapid LV pacing at a rate of 400 beats/min for 3 wk (5). Heart failure was evident from clinical signs, such as ascites and cachexia, and echocardiographic parameters, such as an increase in LV end-diastolic diameter and a reduction of LV systolic shortening fraction. Echocardiography was performed using a 7-MHz sector phase array transducer with two-dimensional real-time and M-mode acquisition (Supervision 7000, Toshiba; Neuss, Germany). LV function was measured in the short-axis view at baseline and after 3 wk of pacing, with the rabbits in the conscious state and the pacer turned off for at least 60 min. At the end of the study, the rabbits were euthanized, and two to three tissue samples from the LV free wall were rapidly stored in liquid nitrogen for later analysis. Other samples were fixed in formalin and embedded in paraffin. Eight rabbits underwent surgery without pacing and served as shams.
Histology. Myocardial fibrosis was determined using Masson-Goldner trichrome staining, and the extent of fibrosis was expressed as a percentage of the field of view (3 fields of 0.075 mm2 each). Myocyte cross-sectional area was measured in hematoxylin and eosin-stained tissue sections (3 fields of 0.075 mm2 each). Apoptosis was assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique (In Situ Cell Death Detection Kit, La Roche Diagnostics; Mannheim, Germany) and counterstaining with bisbenzimide (HOE-33342, Sigma; Taufkirchen, Germany) and phalloidin (Sigma) (9). TUNEL-positive cardiomyocyte nuclei were counted using fluorescence microscopy (Leica; Bensheim, Germany) and expressed as TUNEL-positive cardiomyocyte nuclei per millimeter squared (Fig. 1).
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Caspase-3 activity. To confirm the findings on apoptosis by a second method, the activity of caspase-3 was determined with the use of a fluorescent substrate. In brief, the frozen tissues were homogenized with 25 mM HEPES (pH 7.25) containing 0.1% CHAPS, 5 mM MgCl2, 10 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, and 0.5% Triton X-100. The homogenate was then centrifuged at 10,000 g for 10 min. The supernatant (200 µl) was incubated with 10 µM of the caspase-3 substrate Ac-DEVD-AMC (Alexis) for 90 min at 20°C in buffer containing 25 mM HEPES (pH 7.25), 0.1% CHAPS, 10 mM dithiothreitol, 1 mM EDTA, 1 mM PMSF, 10% saccharose, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 µg/ml pepstatin A. At the end of the incubation, substrate cleavage was monitored fluorometrically using a spectrophotometer (Fluoroscan II, Labsystems; Helsinki, Finland) with an excitation wavelength of 355 nm and an emission wavelength of 460 nm. Data are expressed as arbitrary units per microgram of protein. The total protein content was determined by the BCA Protein Assay Kit (Pierce; Rockford, IL).
MAPK phosphorylation. The tissue samples were weighed, diluted with sample buffer (1:60; containing 2% sodium dodecyl sulfate, 50 mM dithiothreitol, 10% glycerol, 0.1% bromphenol blue, and 62.5 mM Tris; pH 6.8 at 25°C), and homogenized. The homogenates were boiled for 5 min, cooled on ice, and centrifuged at 14,000 g for 5 min at 4°C. Aliquots (20 µl) of the supernatants from each experiment were loaded in parallel on two 10% PAGE-SDS gels. The proteins were separated by electrophoresis (25 µA for 2 h at 4°C), and the separated proteins were transferred to nitrocellulose membranes by electroblotting (40 V overnight at 4°C). The membranes were blocked with Tris-buffered saline (TBS; containing 20 mM Tris and 120 mM NaCl at 25°C) and 5% nonfat dry milk for 90 min and washed four times with TBS containing 0.05% Tween 20 (TTBS) for 10 min. The resulting blots were incubated overnight with antiserum recognizing either ERK MAPK, JNK MAPK, or p38 MAPK (Table 1). The blots were then washed four times with 80 ml of washing buffer (150 mM NaCl, 0.1% Tween 20, and 50 mM Tris; pH 7.4 at 25°C) and incubated for 1 h with a secondary antibody. After the blots were washed four more times with buffer, detection was performed by enhanced chemiluminescence. The resulting autoradiographs were analyzed by quantitative two-dimensional densitometry using commercially available software (Herolab; Wiesloch, Germany).
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p38 MAPK activity. The tissue samples were weighed, diluted with cell lysis buffer (1:10, with 1 mM PMSF added; Cell Signaling Technology; Beverly, MA), sonicated four times for 5 s on ice, and centrifuged at 14,000 g for 10 min at 4°C. A monoclonal mouse anti-p38 MAPK- or anti-p38 MAPK- antibody (Table 1) was then used to selectively immunoprecipitate the respective isoform of p38 MAPK from each supernatant. The resulting immunoprecipitates were incubated with ATF-2 fusion protein in the presence of ATP and kinase buffer for 30 min at 30°C to phosphorylate ATF-2. Phosphorylation of ATF-2 at Thr71 was measured by Western blotting using a phosphorylated ATF-2 (Thr71) antibody (Table 1).
To prove that ATF-2 phosphorylation was related to p38 MAPK, in a subset of samples (n = 8), immunoprecipitates were incubated with SB-203580 (10 or 20 µM) before ATF-2 fusion protein and ATP were added.
Statistical analysis. Values are expressed as means ± SE. Statistical comparison of all data before and after 3 wk of pacing between the two groups was performed by two-way ANOVA. When a significant overall effect was detected, individual mean values were compared using Bonferroni's method. All other parameters obtained at 3 wk in sham and heart failure rabbits were compared by unpaired t-test. Statistical significance was accepted for a P value < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Histology. In failing hearts, the number of TUNEL-positive cardiomyocyte nuclei (Fig. 1), caspase-3 activity, and extent of fibrosis were increased compared with normal hearts (Table 3). The cross-sectional area of the remaining viable myocytes in failing hearts was increased compared with normal hearts (Table 3).
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MAPK. Total p38 MAPK did not differ between failing and normal hearts. However, p38 MAPK phosphorylation was significantly increased (Table 4), and p38 MAPK- and p38 MAPK- activities were increased in failing compared with normal hearts (Fig. 2 and Table 4). The activities of p38 MAPK- (by 91.5 ± 1.0%) and p38 MAPK- (by 99.7 ± 0.2%) were almost completely inhibited in the presence of 20 µM SB-203580.
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The increase in LV end-diastolic diameter from baseline to 3 wk correlated with the extent of p38MAPK phosphorylation (Fig. 3), and, in turn, systolic shortening fraction inversely correlated with the extent of p38 MAPK phosphorylation (Table 5).
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The number of TUNEL-positive cardiomyocyte nuclei (Fig. 4), extent of fibrosis, and myocyte cross-sectional area correlated with the extent of p38 MAPK phosphorylation (Table 5).
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Both total and phosphorylated JNK46 and JNK54 MAPK were increased in failing compared with normal hearts (Table 4). The number of TUNEL-positive cardiomyocyte nuclei, extent of fibrosis, and myocyte cross-sectional area correlated with the absolute amount of phosphorylated JNK46 MAPK and JNK54 MAPK (Table 5).
Total and phosphorylated ERK42 MAPK remained unchanged (Table 4).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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and - activities. In contrast, ERK MAPK phosphorylation remains unaltered. Methodological considerations. In the present study, LV end-diastolic diameter, which probably reflects increased LV stretch, was increased from 15.6 ± 0.7 to 18.8 ± 0.7 mm after 3 wk of rapid LV pacing (Table 2), as were p38 MAPK phosphorylation and activity (Table 4 and Fig. 2). In isolated rat cardiomyocytes (17) and rat hearts (33, 38), increased stretch induced an increase in p38 MAPK phosphorylation and activity, and in the present study in vivo, the increase in LV end-diastolic diameter correlated with the extent of p38 MAPK phosphorylation (Fig. 3). However, the scatter of the data also supports the notion that apart from increased LV stretch, other factors contributed to the increased p38 MAPK phosphorylation, for example, an increase in cardiac norepinephrine (14, 19) or endothelin (30) concentrations, which have been demonstrated to occur in this heart failure model.
Part of the MAPK activation could have been secondary to acutely increased LV stretch (17, 33, 38) by turning off the pacer before tissue collection. The reduction of heart rate from 400 beats/min to the resting heart rate of 270 beats/min indeed increased LV enddiastolic diameter in failing hearts from 17.2 ± 0.7 to 18.1 ± 0.9 mm (5.5 ± 0.7%) within 30 min, as measured in three additional heart failure rabbits. Whereas acute LV stretch activated p38 MAPK within minutes in normal hearts, stretch had, however, no impact on the already activated p38 MAPK in failing hearts (33), arguing against the idea of acute changes in loading conditions being responsible for p38 MAPK activation in the present study.
MAPK. In hypertensive rats, ERK MAPK activity was increased (15, 33), whereas JNK MAPK activity remained unaltered (33). The latter finding was, however, contrasted by results obtained in mice with aortic banding, in which JNK MAPK activity was increased for a time period of up to 2 wk (26). p38 MAPK phosphorylation and subsequently p38 MAPK activity were increased in hypertensive rats but once again decreased during the progression of heart failure from a compensated to a decompensated state (2). In the present study, both JNK and p38 MAPK phosphorylation were increased, whereas ERK MAPK phosphorylation remained unchanged, in failing hearts. Because MAPK phosphorylation and p38 MAPK activity were analyzed only at a single time point, i.e., after 3 wk of LV pacing, when all rabbits had clinical signs of severe heart failure and a severely impaired LV shortening fraction, we cannot rule out that at a different stage of heart failure JNK and p38 MAPK phosphorylation and activity would have been differentially activated. However, more recently, increased p38 MAPK and JNK phosphorylation were measured in a rabbit model of pacing-induced heart failure with only moderate LV dysfunction (25), suggesting also that at earlier stages of heart failure development MAPK is activated.
In ischemia-reperfusion, a differential activation of p38 MAPK- and - has been demonstrated, with increased p38 MAPK- activity and decreased p38 MAPK- activity (27). Also, in explanted hearts from patients with terminal heart failure, only p38 MAPK- activity was detectable and decreased compared with normal hearts. In the present study, however, both p38 MAPK- and - activities were detected in normal hearts and increased during heart failure. Because p38 MAPK- is thought to mediate myocardial hypertrophy (18, 36), its activity might therefore be increased only during a state of compensated heart failure but is possibly decreased once more when heart failure is decompensated.
Apoptosis. One explanation for the progression of LV dysfunction in heart failure relates to an increased level of apoptosis (1, 23), and MAPKs are involved in the regulation of apoptotic processes. A potential role of JNK MAPK activation in apoptosis induction is supported by findings that a dominant negative mutant of JNK MAPK can prevent DNA fragmentation and caspase activation in cardiac myocytes (34) and that Rac-1-induced JNK MAPK activation causes DNA fragmentation (22). Also, overexpression of p38 MAPK- in mouse cardiomyocytes increased apoptosis (36). In contrast, activation of ERK MAPK protected against apoptosis induced by either osmotic load (11) or -adrenergic stimulation (29). Thus, in the present study, activation of JNK and p38 MAPK in the presence of unaltered ERK MAPK activity would favor apoptotic cell death, and indeed the number of apoptotic cardiomyocyte nuclei was increased in failing hearts.
Hypertrophy. In isolated cardiomyocytes, activation of either ERK MAPK (4, 35), JNK MAPK (36), or p38 MAPK- (36) induced hypertrophy. In contrast, simultaneous activation of JNK and p38 MAPK inhibited cardiomyocyte growth (36). In the present study, JNK and p38 MAPK were activated in failing hearts, and surviving cardiomyocytes displayed an increase in cross-sectional area. This finding appears to somewhat contradict the results obtained in isolated cardiomyocytes (36); however, the use of myocardial biopsies for MAPK analysis, as done in the present study, does not allow us to differentiate whether activation of certain MAPKs occurs homogeneously within all cell types (cardiomyocytes, fibroblasts, endothelial cells) or whether a differential activation of MAPK occurs among different cell types (cardiomyocytes vs. fibroblasts) or even between adjacent cardiomyocytes.
Future directions. The present model of pacing-induced heart failure is characterized not only by atrophic (increase in the number of TUNEL-positive cardiomyocyte nuclei and extent of fibrosis) but also hypertrophic (increase in cross-sectional myocyte area) processes. The simultaneous increase of both JNK and p38 MAPK phosphorylation makes it impossible to analyze a causal involvement of one or other MAPKs in the process of apoptosis or hypertrophy. Such a causal involvement of different MAPKs or their isoforms can only be established in future studies using specific MAPK inhibitors. So far, use of a nonspecific p38 MAPK- and - inhibitor (SB-239063) revealed a significant reduction in LV hypertrophy and dysfunction and an increased survival rate in spontaneously hypertensive stroke-prone rats, outlining the importance of p38 MAPK in the development of heart failure (2).
In conclusion, in pacing-induced heart failure, JNK and p38 MAPK are activated, the number of apoptotic cardiomyocytes is increased, and the remaining cardiomyocytes are hypertrophied. Future studies will have to prove whether selective blockade or activation of certain MAPKs can delay the onset or attenuate the progression of heart failure and thereby improve its prognosis.
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FOOTNOTES |
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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.
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REFERENCES |
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