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Cardiovascular Research 2006 69(4):888-898; doi:10.1016/j.cardiores.2005.11.015
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Copyright © 2005, European Society of Cardiology

Opposing effect of p38 MAP kinase and JNK inhibitors on the development of heart failure in the cardiomyopathic hamster

Shiori Kyoi, Hajime Otani*, Seiji Matsuhisa, Yuzo Akita, Kimiko Tatsumi, Chiharu Enoki, Hiroyoshi Fujiwara, Hiroji Imamura, Hiroshi Kamihata and Toshiji Iwasaka

Cardiovascular Center, Kansai Medical University, Moriguchi City, Japan

* Corresponding author. Tel.: +81 6 6992 1001; fax: +81 6 6994 7022. Email address: otanih{at}takii.kmu.ac.jp

Received 28 April 2005; revised 18 October 2005; accepted 10 November 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: p38 MAP kinase (p38 MAPK) and c-Jun NH2-terminal kinase (JNK) have been implicated in the pathophysiology of heart failure. We investigated the effects of chronic treatment with p38 MAPK and JNK inhibitors on the development of heart failure in dilated cardiomyopathy (DCM) hamster heart.

Methods and results: BIO14.6 hamster hearts showed markedly increased p38 MAPK and JNK activities at 6 weeks of age when there was no significant increase in the area of fibrosis, heart weight/body weight, left ventricular (LV) chamber dilation and LV dysfunction. p38 MAPK and JNK activities were attenuated at 26 weeks of age and abolished at 40 weeks of age in BIO14.6 hamster hearts. BIO14.6 hamsters and the control BIOF1B hamsters were chronically treated (i.p.) with the p38 MAPK inhibitors, SB203580 (1 mg/kg/day) and FR167653 (3 mg/kg/day), or the JNK inhibitor, SP600125 (1 mg/kg/day) or vehicle for 20 weeks starting from 6 weeks of age. Treatment of BIO14.6 hamster hearts with SB203580 and FR167653 reduced the number of TUNEL-positive myocytes, the area of fibrosis and heart weight/body weight associated with a significant decrease of LV dimension and an increase in LV ejection fraction and LV contractility compared to the vehicle-treated counterpart. In contrast, treatment with SP600125 increased the number of TUNEL-positive myocytes and the area of interstitial fibrosis associated with aggravation of LV chamber dilation and LV dysfunction.

Conclusions: These results suggest that chronic treatment with p38 MAPK and JNK inhibitors produces opposing effects on the development of heart failure in the DCM hamster heart.

KEYWORDS p38 MAP kinase; JNK; Cardiomyopathy; Heart failure


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
A growing body of evidence suggests that p38 MAP kinase (p38 MAPK) and c-Jun NH2-terminal kinase (JNK) are the pivotal signal transducers involved in cardiac injury and protection [1]. The role of p38 MAPK activation in heart failure has been investigated in animal models targeted to p38 MAPK genes and by pharmacological tools that inhibit p38 MAPK. It has been demonstrated that transgenic overexpression of upstream kinases of p38 MAPK, MKK3bE and MKK6bE in ventricular myocytes produced a cardiomyopathic phenotype in mice [2]. In contrast, cardiac-specific p38{alpha} MAPK knockout mice developed cardiac dysfunction and heart dilation in response to pressure overload at an earlier stage than the wild-type mice [3]. On the other hand, chronic treatment with a p38{alpha} and p38β MAPK inhibitor, SB239063, prevented left ventricular (LV) hypertrophy and dysfunction in hypertensive rats [4]. Chronic treatment with another p38{alpha} and p38β MAPK inhibitor, RWJ-67657, improved cardiac function and attenuated LV remodeling in rats with myocardial infarction [5]. These previous studies have suggested that pharmacological approaches targeted to p38{alpha} and p38β MAPK isoforms may be beneficial in preventing heart failure in the animal model of hypertensive cardiomyopathy (CM) and myocardial infarction.

The role of JNK in myocyte injury and protection is controversial. Petrich et al. [6] demonstrated that targeted activation of JNK induces restrictive CM and conduction defects. In contrast, Sadoshima et al. [7] demonstrated that ablation of the MEKK1 gene abrogated JNK activation in pressure-overloaded mice heart and showed a severe CM phenotype and higher mortality than in the wild-type mice, suggesting that JNK may play a cardioprotective role under certain pathological conditions.

Despite the critical involvement of p38 MAPK and JNK in the pathogenesis of heart failure, little information is available about the effect of prophylactic use of p38 MAPK and JNK inhibitors on the animal model of dilated CM (DCM). Therefore, we investigated the effect of chronic treatment with p38 MAPK and JNK inhibitors on the development of heart failure in BIO14.6 hamsters when the drug administration was started before the emergence of heart failure. The results of the present study suggest that chronic treatment with p38 MAPK inhibitors prevents the development of heart failure, whereas a JNK inhibitor accelerates heart failure in the DCM hamster.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Animals
Male BIO14.6 hamsters that are devoid of {delta}-sarcoglycan gene [8] and the control BIOF1B hamsters at 5 weeks of age were obtained from BIO Breeders (Fitchburg, MA). All experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals (NIH publication no. 85-23, revised 1996) and approved by the institutional Committee of Animal Care and Use in Kansai Medical University (Moriguchi, Japan).

2.2 Materials
The p38{alpha} and p38β MAPK inhibitors, SB203580 ([4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole]) and FR167653 (1-[7-(4-fluorophenyl)-1,2,3,4-tetrahydro-8-(4-pyridyl)pyrazolo[5,1-c][1,2,4]triazin-2-yl]-2-phenylethanedione sulfate monohydrate), were obtained from Calbiochem (San Diego, CA), and was a gift from Fujisawa Pharmaceutical Co. (Osaka, Japan), respectively. The JNK inhibitor, SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one), was obtained from Calbiochem. These drugs were dissolved in dimethylsulfoxide (DMSO) and diluted with PBS to give a final concentration of 0.2 mg/ml for SB203580 and SP600125 and 0.6 mg/ml for FR167653 at 1% DMSO.

2.3 Chronic treatment with p38 MAPK and JNK inhibitors
To evaluate the effect of chronic treatment with p38 MAPK and JNK inhibitors on cardiac morphology and function, 20 BIOF1B and twenty BIO14.6 hamsters were randomly assigned to four treatment groups (n=5 in each group): vehicle, SB203580, FR167653 and SP600125. These drugs and the same amount of the vehicle were administered daily with an intraperitoneal injection for 20 weeks starting from 6 weeks of age. We used SB203580 at a dose of 1 mg/kg/day, because the same dose was used to inhibit p38 MAPK in the rat heart [9]. However, this dose could inhibit JNK activity [10]. Therefore, we employed another pyridinyl imidazole inhibitor, FR167653 (3 mg/kg/day), which has a slightly higher IC50 against p38{alpha} than SB203580 (0.71 µM vs. 0.56 µM) but has no other potential targets in a micromolar range [11]. IC50 of SP600125 against JNK1, 2 and 3 was <0.5 µM [12]. It has been demonstrated that SP600125 at a dose of 6 mg/kg abolished JNK activation in acute-overloaded rat heart [13]. However, we reduced its dose to 1 mg/kg/day, because higher concentrations of SP600125 could act as an inhibitor of MKK3 and MKK6, which are the upstream kinases of p38 MAPK [12].

2.4 Immunoblot analysis
BIOF1B and BIO14.6 hamsters at 6 weeks (n=5), 26 weeks (n=5) and 40 weeks (n=5) of age were treated with the vehicle for 5 days to determine the age-related change in p38 MAPK and JNK activities. To evaluate the effect of the p38 MAPK and JNK inhibitors on p38 MAPK and JNK activities, BIOF1B and BIO14.6 hamsters at 6 weeks of age were also treated with SB203580 (n=5), FR167653 (n=5) or SP600125 (n=5) for 5 days. Three hours after the last treatment with the vehicle or the drugs, the hamsters were anesthetized with sodium pentobarbital (100 mg/kg). The hearts were quickly excised, rinsed with ice-cold lactate Ringer solution and snap-frozen in liquid nitrogen. Frozen hearts were homogenized with a pre-chilled mortar and pestle in lysis buffer consisting (in mM) of 20 Tris (pH 7.5), 1 EDTA, 1 EGTA, 1 β-glycerolphosphate, 150 NaCl, 1 Na vanadate, 2.5 Na pyrophosphate, 4.5 MgCl2, 0.5 dithiothreitol (DTT), 1 phenylmethylsulfonyl fluoride (PMSF), 1% Triton X-100 and a protease inhibitor cocktail (Complete, Roche Diagnostic, Mannheim, Germany). The homogenate was centrifuged at 20,000 x g at 4 °C for 10 min and the supernatant was transferred into a new tube and stored at –80 °C until needed. Protein was determined by a BCA protein assay kit (Pierce Biotechnology Inc., Rockford, IL). In the animals not treated with p38 MAPK and JNK inhibitors, p38 MAPK and JNK activities were evaluated by measuring the phosphorylation level of p38 MAPK and JNK, respectively, while in those treated with p38 MAPK and JNK inhibitors, p38 MAPK and JNK activities were evaluated by phosphorylation level of MAPK-activated protein kinase 2 (MAPKAPK2) and c-Jun, the immediate downstream substrates for p38 MAPK and JNK, respectively. Immunoblotting for p38-MAPK, phospho-p38 MAPK, MAPKAPK2, phospho-MAPKAPK2, JNK, phospho-JNK, c-Jun and phospho-c-Jun was performed using mouse monoclonal antibodies (Cell Signaling, Beverly, MA), and the immunoreactive bands were quantified by densitometric analysis using the image analyzing software system Win Roof (Mitani Co., Fukui, Japan). The activity of p38 MAPK and JNK was expressed as the ratio of the phosphorylated kinases relative to total amount of the kinases. Inter-blot heterogeneity was normalized to the corresponding Coomassie blue stain signal as described previously [14].

2.5 Immunofluorescence microscopy
To identify the cell type responsible for the age-related change in p38 MAPK and JNK activities in the heart, frozen sections were obtained from the same hamster heart as used for immunoblot assay at 6 weeks, 26 weeks and 40 weeks of age and immunostained for phospho-p38 MAPK and phospho-JNK. These sections were cut at ~6 µm onto glass slides, incubated in acetone and hydrogen peroxide, rinsed with PBS, and blocked with 10% normal rabbit serum. The sections were incubated for 1 h at room temperature with mouse monoclonal anti-phospho-p38 MAPK antibodies or anti-phospho-JNK antibodies (Cell Signaling). They were then incubated for 2 h at room temperature with a fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin. The fluorescence staining was visualized using a confocal laser microscope (Fluoview, Olympus, Tokyo, Japan).

2.6 TUNEL assay
We employed in situ terminal transferase labeling (TUNEL) assay to evaluate the effect of the p38 MAPK and the JNK inhibitors on myocyte death. Although TUNEL assay is commonly used to identify apoptotic cells in situ, TUNEL assay detects double-and single-stranded DNA fragmentation that is also produced by necrotic DNA degradation [15]. Therefore, TUNEL assay was thought to be a measure of both apoptotic and necrotic myocyte death in our experimental model. BIOF1B and BIO14.6 hamsters at 6 weeks of age were treated with the vehicle, SB203580, FR16765 or SP600125 for 5 days, and the animal hearts were excised as described above. The frozen sections were double-stained for TUNEL (FITC fluorescence) using an assay kit (Intergen, Purchase, NY) and {alpha}-sarcomeric actin (tetrarhodamine isothiocyanate fluorescence) as described previously [16]. The section was visualized using a confocal laser microscope.

2.7 Echocardiography
To evaluate LV function in situ, the hamster was anesthetized with a mixture of ketamine, xylazine and acepromazine as described previously [7]. Echocardiography was performed using a SONOS-7500 (Philips Medical Systems, Andover, MA) equipped with a 6–15 MHz transducer (Model 21390A, Philips). M-mode measurements of LV internal diameter were made from more than three beats and averaged. Measurements of the LV end-diastolic diameter (LVEDD) were taken at the time of the apparent maximal LV diastolic dimension, while measurements of the LV end-systolic diameter (LVESD) were taken at the time of the most anterior systolic excursion of the posterior wall. LV ejection fraction (LVEF) was calculated according to the cubed method as described previously [7]. The echocardiographer was blinded to the groups.

2.8 Isovolumic LV pressure measurements
In situ measurements of LV function may be modulated by a number of neurohumoral factors that are supposed to be increased in the CM hamsters associated with heart failure. Therefore, we measured LV function in the isolated heart. To this end, the hamster was anesthetized with pentobarbital sodium as described above. The heart was excised, mounted on a Langendorff perfusion apparatus and perfused at a constant mean pressure of 70–75 mm Hg using a Krebs–Henseleit bicarbonate buffer solution of the following composition (in mM): 118 NaCl, 4.7 KCl, 1.2 MgSO4, 25 NaHCO3, 1.2 KH2PO4, 1.8 CaCl2 and 11 glucose; pH 7.4 at 37 °C when equilibrated with a mixture of 95% O2–5% CO2 gas. Isovolumic LV function was monitored using a latex balloon-tipped catheter inserted into the LV through the left atrium and connected to a pressure transducer. Hemodynamic data were analyzed using a Biomedical Research System (LEG-1000, Nihon Kohden, Osaka, Japan). Coronary flow was measured by timed collection of the coronary effluent.

2.9 Histological analysis
To evaluate the morphological change and the degree of fibrosis of the heart, the isolated heart was perfusion-fixed with 10% formalin after measurements of isovolumic LV function, embedded in paraffin and sectioned at ~6 µm thickness. The section was stained with hematoxylin–eosin and gross morphology of the heart was viewed under a low power field (x 0.5). The area of fibrosis was identified by Masson trichrome staining and was quantified using Win Roof.

2.10 Statistical analysis
All numerical data are expressed as mean ± S.E. Statistical analysis was performed by Student's t-test to analyze the difference between two groups and one-way ANOVA followed by the Bonferroni post hoc test to compare the difference within the groups. The differences were considered significant at a p value of <0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Age-related change in p38 MAPK and JNK activities
It has been demonstrated that the earliest biological and morphological evidence of DCM in BIO14.6 hamsters develops at 5–6 weeks of age and more than 50% of the animals die before 40 weeks of age as a result of heart failure [17]. To delineate the relationship between p38 MAPK and JNK activities and the development of heart failure, we studied the age-related change in p38 MAPK and JNK activities in BIOF1B and BIO14.6 hamster hearts (Fig. 1A and B). p38 MAPK and JNK activities were markedly increased at 6 weeks of age in BIO14.6 hamster heart. These MAP kinase activities remained significantly activated in BIO14.6 hamster heart at 26 weeks of age. However, no significant increase in p38 MAPK and JNK activities was observed at 40 weeks of age in BIO14.6 hamster heart. There was no age-related change in p38 MAPK and JNK activities in BIOF1B hamster heart.


Figure 1
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  		Fig. 1 (A) Immunoblot analysis of total and phosphorylated form of p38 MAPK and JNK. Lower panel, representative immunoblot bands; upper panel, densitometric analysis. 6W, 26W and 40W represent 6 weeks, 26 weeks and 40 weeks of age, respectively. Each bar graph represents mean ± S.E. of five experiments. p-p38, phospho-p38 MAPK; p-JNK, phospho-JNK. *p<0.05 between BIOF1B (F1B) and BIO14.6 (14.6) hamster hearts. {dagger}p<0.05 compared to 6 weeks of age. (B) Representative immunofluorescence confocal images of phospho-p38 MAPK (a–d) and phospho-JNK (e–h). (a, e) BIOF1B hamster heart at 6 weeks of age; (b, f) BIO14.6 hamster heart at 6 weeks of age; (c, g) BIO14.6 hamster heart at 26 weeks of age; (d, h) BIO14.6 hamster heart at 40 weeks of age. Bars indicate 10 µm.

 
3.2 Baseline morphological characteristics and hemodynamics
Gross morphology, heart weight/body weight and the area of fibrosis were not significantly different between BIOF1B and BIO14.6 hamster hearts at 6 weeks of age (Fig. 2A).


Figure 2
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  		Fig. 2 (A) Left panel, gross morphology of BIOF1B and BIO14.6 hamster hearts and heart weight/body weight. Right panel, Masson trichrome staining and the quantification of the percent area of fibrosis. Bars indicate 1 mm. Each bar graph represents mean ± S.E. of five samples. NS, not significant. (B) Echocardiography (upper panel) and isovolumic LV function (lower panel). LVEDD, LV end-diastolic dimension; LVESD, LV end-systolic dimension; LVEF, LV ejection fraction. Isovolumic LV function was measured in the isolated heart at LV end-diastolic pressure of 10 mm Hg. Filled bars, BIOF1B hamster; open bars, BIO14.6 hamster. Each bar graph represents mean ± S.E. of five hearts. NS, not significant.

 
Echocardiographic measurements showed no significant difference in LVEDD, LVESD and LVEF between BIOF1B and BIO14.6 hamster hearts (Fig. 2B). Heart rate (rpm) during echocardiography was 411 ± 10 in BIOF1B hamster and 407 ± 12 in BIO14.6 hamster. Isovolumic LV pressure measurements in the isolated heart also revealed no significant difference in heart rate, LVDP and LV dp/dt between these hamster hearts. Coronary flow (ml/min/g) in BIOF1B and BIO14.6 hamster hearts was not significantly different (17.3 ± 1.6 and 18.7 ± 1.5, respectively).

3.3 Effects of p38 MAPK and JNK inhibitors on p38 MAPK and JNK activities
SB203580, FR167653 and SP600125 were given for 5 days at 6 weeks of age when the activity of p38 MAPK and JNK was highest. The treatment with SB203580 and FR167653 but not SP600125 significantly inhibited p38 MAPK activity in BIO14.6 hamster heart (Fig. 3). On the other hand, JNK activity was significantly inhibited by SP600125 but not SB203580 and FR167653.


Figure 3
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  		Fig. 3 Immunoblot analysis of total and phosphorylated form of MAPKAPK2 and c-Jun. Lower panel, representative immunoblot bands; upper panel, densitometric analysis. BIOF1B (F1B) and BIO14.6 (14.6) hamsters at 6 weeks of age were treated with the vehicle (Veh), SB203580 (SB), FR16765 (FR) or SP600125 (SP) for 5 days. p-MK2, phospho-MAPKAPK2; p-c-Jun, phospho-c-Jun. Each bar graph represents mean ± S.E. of five hearts. *p<0.05 between BIOF1B and BIO14.6 hamster hearts; {dagger}p<0.05 compared to the vehicle treatment.

 
3.4 Effects of p38 MAPK and JNK inhibitors on myocyte death
TUNEL-positive myocytes were rarely found BIOF1B hamster heart at 6 weeks of age (Fig. 4A and C). The number of TUNEL-positive myocytes was significantly greater in BIO14.6 hamster heart (Fig. 4B and C). Treatment with SB203580 and FR167653 significantly reduced the number of TUNEL-positive myocytes, while treatment with SP600125 significantly increased the number of TUNEL-positive myocytes in BIO14.6 hamster heart.


Figure 4
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  		Fig. 4 TUNEL assay. Representative TUNEL (green) and {alpha}-sarcomeric actin (red) staining obtained from 6-week-old BIOF1B (A) and BIO14.6 hamster hearts (B). The arrow indicates TUEL-positive nucleus. Bar indicates 10 µm. (C) Quantitative analysis of TUNEL-positive myocytes. BIOF1B (F1B) and BIO14.6 (14.6) hamsters at 6 weeks of age were treated with the vehicle (Veh), SB203580 (SB), FR16765 (FR) or SP600125 (SP) for 5 days. Each bar graph represents mean ± S.E. of five hearts. *p<0.05 between BIOFIB and BIO14.6 hamster hearts; {dagger}p<0.05 compared to the vehicle treatment.

 
3.5 Effects of chronic treatment with p38 MAPK and JNK inhibitors on myocardial fibrosis
The area of fibrosis in the LV myocardium was significantly greater in BIO14.6 hamster heart than BIOF1B hamster heart at 26 weeks of age (Fig. 5). Chronic treatment with SB203580 and FR167653 significantly decreased the area of fibrosis in the LV myocardium, while chronic treatment with SP600125 significantly increased the area of fibrosis in BIO14.6 hamster heart. The area of fibrosis was not affected by chronic treatment with these drugs in BIOF1B hamster heart.


Figure 5
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  		Fig. 5 Area of fibrosis at 26 weeks of age. BIOF1B (F1B) and BIO14.6 (14.6) hamsters at 6 weeks of age were treated with the vehicle (Veh), SB203580 (SB), FR16765 (FR) or SP600125 (SP) for 20 weeks. Bar indicates 1 mm. Each bar graph represents mean ± S.E. of five hearts. *p<0.05 between BIOFIB and BIO14.6 hamster hearts; {dagger}p<0.05 compared to the vehicle treatment.

 
3.6 Effects of chronic treatment with p38 MAPK and JNK inhibitors on gross morphology of the heart and heart weight/body weight
BIO14.6 hamster heart at 26 weeks of age showed dilation of the LV chamber and thinning of the LV wall (Fig. 6). Nevertheless, heart weight/body weight was significantly increased in BIO14.6 hamster heart compared with BIOF1B hamster heart at the same age. Chronic treatment with SB203580 and FR167653 ameliorated dilation of the LV chamber and thinning of the LV wall and inhibited the increase in heart weight/body weight in BIO14.6 hamster heart. Chronic treatment with SP600125 aggravated dilation of the LV chamber and thinning of the LV wall without further increasing heart weight/body weight in BIO14.6 hamster heart. Gross morphology of the heart and heart weight/body weight was not affected by chronic treatment with these drugs in BIOF1B hamster.


Figure 6
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  		Fig. 6 Gross morphology and heart weight/body weight at 26 weeks of age. BIOF1B (F1B) and BIO14.6 (14.6) hamsters at 6 weeks of age were treated with the vehicle (Veh), SB203580 (SB), FR16765 (FR) or SP600125 (SP) for 20 weeks. Bar indicates 1 mm. Each bar graph represents mean ± S.E. of five hearts. *p<0.05 between BIOF1B and BIO14.6 hamster hearts.

 
3.7 Effects of chronic treatment with p38 MAPK and JNK inhibitors on LV function
LVEDD and LVESD were significantly increased in BIO14.6 hamster heart at 26 weeks of age compared to BIOF1B hamster heart (Fig. 7A and B). This was associated with a significant decrease in LVEF in BIO14.6 hamster heart (Fig. 7C). Chronic treatment with SB203580 and FR167653 mitigated the change in LVEDD and LVESD and improved LVEF in BIO14.6 hamster heart. On the contrary, chronic treatment with SP600125 further increased LVEDD and LVESD and decreased LVEF in BIO14.6 hamster heart. Heart rate (rpm) during echocardiography was not significantly different between the groups (BIOF1B-Vehicle, 395 ± 10; BIOF1B-SB203580, 390 ± 9; BIOF1B-FR167653, 398 ± 12; BIOF1B-SP600125, 396 ± 7; BIO14.6-Vehicle, 379 ± 10; BIO14.6-SB203580, 389 ± 9; BIO14.6-FR167653, 391 ± 10; BIO14.6-SP600125, 375 ± 8).


Figure 7
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  		Fig. 7 LV function at 26 weeks of age. BIOF1B (F1B) and BIO14.6 (14.6) hamsters at 6 weeks of age were treated with the vehicle (Veh), SB203580 (SB), FR16765 (FR) or SP600125 (SP) for 20 weeks. (A) Echocardiographic measurements of LV end-diastolic dimension (LVEDD); (B) LV end-systolic dimension (LVESD); (C) LV ejection fraction (LVEF). (D) Isovolumic measurements of heart rate; (E) LV developed pressure (LVDP); (F) LV dp/dt. Isovolumic LV function was measured in isolated and perfused heart at LV end-diastolic pressure of 10 mm Hg. Each bar graph represents mean ± S.E. of five experiments. *p<0.05 between BIOF1B and BIO14.6 hamster hearts; {dagger}p<0.05 compared to the vehicle treatment.

 
Isovolumic measurements of LV function showed significantly lower heart rate, LVDP, and LV dp/dt in BIO14.6 hamster heart compared to BIOF1B hamster heart at 26 weeks of age (Fig. 7D–F). There was a rightward shift in the volume–pressure curve in BIO14.6 hamster heart, but peak systolic pressure and LV dp/dt were always lower in BIO14.6 hamster heart at any given LVEDP (data not shown). Chronic treatment with SB203580 and FR167653 significantly improved heart rate, LVDP and LV dp/dt in BIO14.6 hamster heart. In contrast, chronic treatment with SP600125 further decreased heart rate, LVDP and LV dp/dt in BIO14.6 hamster heart. Coronary flow (ml/min/g) was comparable between BIOF1B and BIO14.6 hamster hearts (17.4 ± 1.4 and 17.2 ± 1.3, respectively). Chronic treatment with these drugs did not affect any parameters of LV function in BIOF1B hamsters.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
4.1 Activation of p38 MAPK and JNK precedes development of heart failure
We showed that p38 MAPK and JNK had already been highly activated in BIO14.6 hamster heart at 6 weeks of age when there was no significant difference in heart weight/body weight, the area of fibrosis, LV dimension and LV function compare to BIOF1B hamster heart at the same age. Activation of these MAP kinases was attenuated during the progression of heart failure and abolished at the end-stage heart failure. These observations suggest that the activation of p38 MAPK and JNK may be causally involved in the development of heart failure in BIO14.6 hamster.

4.2 Chronic treatment with p38 MAPK and JNK inhibitors produces opposing effects on myocyte death
We have investigated the effect of the p38 MAPK and JNK inhibitors on myocyte death in BIO14.6 hamster heart, because increased myocyte death has gained a mechanistic implication in the development of heart failure [18]. Inhibition of p38 MAPK by treatment with SB203580 or FR167653 decreased TUNEL-positive myocytes, while inhibition of JNK produced an opposing effect. The role of p38 MAPK and JNK in myocyte life and death is controversial. A growing body of evidence obtained from in vitro studies suggests that activation of p38 MAPK promotes mitochondria-mediated cell death pathways [19]. In contrast, cardiac-specific p38{alpha} MAPK knockout mice developed cardiac dysfunction and heart dilation in response to pressure overload at an earlier stage than the wild-type mice [3]. However, the same p38{alpha} MAPK knockout mice were found to be resistant to ischemia/reperfusion injury [20]. It has also been reported that neonatal rat cardiomyocytes expressing a dominant negative p38{alpha} are resistant to lethal simulated ischemia [21]. Thus, p38{alpha} MAPK appears to act as a cardioprotective and detrimental kinase depending on the type of cardiac stress. Since BIO14.6 hamster heart was not subjected to pressure-overload stress but was supposed to undergo recurrent ischemia [22], the pathophysiology of heart failure in BIO14.6 hamster heart is more akin to that of ischemia/reperfusion injury. In contrast to well-known pro-apoptotic role of p38{alpha} MAPK, p38β MAPK has been implicated in cardiomyocyte survival [23]. Although no information is available regarding the relative abundance of p38{alpha} and p38β MAPK in BIO14.6 hamster heart, the favorable effect of SB203580 and FR167653 on myocyte survival in BIO14.6 hamster heart tends to suggest that p38{alpha} MAPK is a predominant isoform activated in this hamster heart. The observation that SP600125 increased myocyte death is odd in light of most of acute in vitro studies, which argue in favor of a pro-apoptotic role of JNK in myocytes [19]. However, the fact that JNK activation mediates cardioprotection by brief heat stress and ischemic preconditioning [9,24,25] suggests that JNK activation is necessary to trigger cardioprotective signal transduction and protects myocytes from apoptotic and necrotic death in the DCM hamster heart.

4.3 Chronic treatment with p38 MAPK and JNK inhibitors produces opposing effects on LV remodeling and LV dysfunction
The increase in myocyte death was associated with an increase in the area of fibrosis and dilation of the LV chamber in BIO14.6 hamster heart at 26 weeks of age. LV function as evaluated by echocardiography was significantly decreased in BIO14.6 hamster heart compared to the control hamster heart. In particular, ex vivo measurements of LV function showed that heart rate and isovolumic LV contractility were markedly lower in BIO14.6 hamster heart. These ex vivo measurements of LV function preclude the influence of positive inotropic and chronotropic effects mediated by neurohumoral factors. Thus, these results suggest that inherent LV contractile function was significantly preserved by treatment with p38 MAPK inhibitors and markedly deteriorated by treatment with the JNK inhibitor in the DCM hamster heart.

Chronic treatment with SB203580 and FR167653 ameliorated myocardial fibrosis and LV chamber dilation and reduced heart weight/body weight, while SP600125 aggravated myocardial fibrosis and LV chamber dilation and increased heart weight/body weight in BIO14.6 hamster heart. The role of p38 MAP kinase and JNK in myocyte hypertrophy has been a considerable matter of debate. It has been demonstrated that p38 MAPK activation increases cell size of myocytes through sarcomeric reorganization and induction of muscle-specific genes [26]. In addition, the mice with a dominant negative form of p38{alpha} or p38β MAPK developed cardiac hypertrophy in response to pressure overload [27], suggesting that either p38{alpha} or p38β MAPK alone is sufficient to induce cardiac hypertrophy. Our study using p38{alpha} and p38β MAPK inhibitors demonstrating reduced heart weight/body weight does not address which isoform of p38 MAPK or whether both of them are necessary for myocyte hypertrophy in BIO14.6 hamster heart. On the other hand, our results showing chronic inhibition of JNK increased heart weight/body weight seem to corroborate the notion provided by Sadoshima et al. [7] that JNK does not play a major role in cardiac hypertrophy.

4.4 Study limitations and clinical implications
The present study employed BIO14.6 hamster lacking the {delta}-sarcoglycan gene [8]. The loss of {delta}-sarcoglycan causes instability of the sarcolemma and disruption of the membrane by mechanical stress, leading to myocyte death. Consequent loss of functional myocardium and hypertrophy of the remaining myocytes is a pathogenic feature of the early phase of DCM in BIO14.6 hamster. It might be questioned whether similar pathophysiology is involved in DCM in human subjects. It has become apparent that human DCM is a disease of the cytoskeleton and sarcolemma. Many but not all patients with hereditary forms of DCM possess mutation of genes encoding cytoskeletal and membrane skeletal proteins such as dystrophin, {delta}-sarcoglycan and desmin, which participate in maintaining the integrity of the sarcolemma and cytoskeleton [28]. Moreover, viral myocarditis is an important etiology of human DCM and this acquired form of DCM is caused by cleavage of dystrophin [29]. Therefore, common mechanisms may be involved in the development of heart failure in DCM of BIO14.6 hamster and human.

Currently, administration of angiotensin converting enzyme inhibitors, angiotensin II type-1 receptor antagonists and β-blockers is a standard therapy for heart failure. However, blockade of all the neurohumoral system is a formidable task. In addition, blockade of neurohumoral receptors inhibits all the downstream signal transduction pathways that are involved in both cardioprotection and injury. Thus, selective blockade of injurious pathways may be a preferable choice of treatment for heart failure. In this context, p38 MAPK inhibitors may represent a promising tool in preventing the development of heart failure in patients with DCM. Furthermore, our study demonstrating that p38 MAPK activity was highly increased at the compensated stage of heart failure but was attenuated along with the progression of heart failure suggests the importance of prophylactic use of p38 MAPK inhibitors in these patients. Future clinical trials using p38 MAPK inhibitors would disclose the efficacy of this novel class of drug in the treatment of DCM.


   Acknowledgements
 
This work was supported in part by The Promotion and Mutual Aid Corporation for Private Schools of Japan and Research Grant from the Ministry of Education, Science and Culture of Japan.


   Notes
 
Time for primary review 18 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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