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Cardiovascular Research 2005 65(1):230-238; doi:10.1016/j.cardiores.2004.08.013
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Copyright © 2004, European Society of Cardiology

Cardiac oxidative stress in acute and chronic isoproterenol-infused rats

Guo-Xing Zhanga, Shoji Kimuraa,*, Akira Nishiyamaa, Takatomi Shokojia, Matlubur Rahmana, Li Yaoa, Yukiko Nagaib, Yoshihide Fujisawab, Akira Miyatakeb and Youichi Abea

aDepartment of Pharmacology, Kagawa University Medical School, 1750-1 Ikenobe, Miki, Kagawa 761-0793, Japan
bResearch Equipment Center, Kagawa University Medical School, 1750-1 Ikenobe, Miki, Kagawa 761-0793, Japan

* Corresponding author. Tel.: +81 87 891 2125; fax: +81 87 891 2126. Email address: kimura{at}kms.ac.jp

Received 12 March 2004; revised 26 July 2004; accepted 24 August 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Sympathetic nervous system activity in the myocardium is increased in patients with heart failure. However, the in vivo mechanisms responsible for β-adrenoceptor-mediated cardiac hypertrophy or remodeling remain unclear. This study aimed to clarify the role of reactive oxygen species (ROS) in mitogen-activated protein (MAP) kinase activation and tissue remodeling of the heart of isoproterenol (ISO)-infused rats.

Methods and results: Different doses of ISO (up to1000 ng/kg/min) were given intravenously to conscious rats for 30 min. Phosphorylated MAP kinase levels (ERK1/2, JNK, p38) and lipid peroxidation were measured in the cardiac left ventricle, revealing the dose-dependent augmentation of MAP kinase phosphorylation and increased lipid peroxidation levels. Simultaneous treatment with 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (Tempol), a membrane-permeable radical scavenger, completely eliminated the increases of phosphorylated MAP kinases and their upstream elements (Raf-1, Rac-1, ASK-1) as well as the increases of cardiac lipid peroxidation induced by the highest dose of ISO infusion. In chronically ISO-infused rats (3 mg/kg/day, s.c. for 10 days), cardiac hypertrophy developed with accompanying increases of collagen content, whereas cardiac phosphorylated MAP kinases returned to normal. Tempol treatment prevented increases of collagen accumulation and type I collagen mRNA without any significant reduction of cardiac mass enlargement induced by chronic ISO infusion.

Conclusion: β-Adrenoceptor stimulation provokes cardiac oxidative stress. In the acute phase of ISO infusion, ROS are important activators of cardiac MAP kinase cascades; while, in the chronic phase, ROS may participate in cardiac remodeling, especially in respect to wall stiffness, based on fibrogenesis.

KEYWORDS Adrenergic agonists; MAP kinase; Oxygen radical


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Increased sympathetic nerve activity in the myocardium is a central feature of patients with heart failure [1–5], while cardiac hypertrophy is a major risk factor for the development of heart failure and sudden cardiac death. Therefore, to reduce mortality from cardiovascular diseases, elucidation of the mechanisms involved in cardiac hypertrophy is important. Cardiac remodeling is seen in isoproterenol (ISO) infused rats with severe myocardial hypertrophy accompanied by myocardial injury [6–9], and β-adrenoceptor-mediated apoptosis has also been demonstrated in cardiomyocytes [10,11]. Meanwhile, increased oxidative stress resulting from an increased cardiac generation of reactive oxygen species (ROS) is implicated in the progression of cardiac hypertrophy and heart failure [12–14]. However, the role of ROS in β-adrenoceptor stimulation-induced cardiac hypertrophy and remodeling is not clearly understood.

The mitogen-activated protein (MAP) kinase superfamily, which consists of extracellular signal-regulated kinases (ERK) 1/2 and three stress-responsive subfamilies, the c-Jun NH2-terminal kinases (JNKs), p38-kinases and ERK-5, is normally stimulated by growth factors and cellular stress or inflammatory cytokines [15,16]. There is increasing evidence that this pathway plays particular roles during the early development of cardiac hypertrophy [17–20]. In vitro studies have revealed that ISO stimulates ERKs in cultured cardiomyocytes of neonatal rats [21,22] and cardiac fibroblasts of adult rats [23]. Furthermore, there is evidence that isoprenaline stimulations increase protein synthesis and rapidly activate MAP kinases in cardiac myocytes [24]. An in vivo study also demonstrated that JNK and ERKs activities increase in cardiac tissues as a result of ISO stimulation [25]. Although both in vitro and in vivo evidence supports that β-adrenoceptor stimulation activates the MAP kinase superfamily, there is relatively little known about the relationship between ROS and MAP kinases in response to β-adrenoceptor stimulation.

4-Hydroxy-2,2,6,6-tetramethyl piperidinoxyl (Tempol) is a membrane-permeable radical scavenger that exhibits potent antioxidant activity against superoxides as well as hydroxyl radicals [26,27]. The authors previously demonstrated that Tempol decreases vascular superoxide anion production in conscious chronic as well as acute Ang II-induced hypertensive rats [28–30]. This study investigates the relationship between the cardiac oxidative stress and MAP kinase cascade in acutely ISO-infused rats and determines the involvement of ROS in cardiac hypertrophy and collagen accumulation in chronically ISO-infused rats.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Animal preparation and treatment
Nine-week-old male Sprague–Dawley rats were used in this study. In the acute ISO infusion experiments, rats were intraperitoneally injected with pentobarbital (50 mg/kg), then polyethylene catheters were placed in the femoral artery for measurements of mean blood pressure (MBP) and heart rate (HR), and in femoral and carotid veins for drug administration. All catheters were tunneled through the subcutaneous tissue exteriorizing in the dorsal midcervical region of the neck, and then filled with heparin solution (1000 IU/ml). Animals were allowed to recover for 24 h before initiation of the experimental procedures. All experiments were performed on conscious rats. ISO at doses of 10, 100, and 1000 ng/kg/min was infused intravenously for 30 min. Propranolol (5 mg/kg i.v.), Tempol (30 mg/kg i.v. plus 0.5 mg/kg/min infusion) or 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (3-CP; a structurally related and inactive Tempol compound) was administrated 5 min before the start of ISO infusion. After 30 min ISO administration, the rats were sacrificed and their left ventricles were removed, quickly frozen in liquid nitrogen and stored at –80 °C.

In the chronic ISO infusion experiments, rats received ISO infusion at a rate of 3 mg/kg/day or vehicle (0.9% saline) by subcutaneously implanted osmotic minipumps (Alzet) for a period of 10 days. One ISO infused group received tap water containing Tempol at a concentration of 2.5 mmol/l. MBP and HR were measured serially using the tail-cuff method. On day 11, hearts were removed and weighed. A sample of each heart was then analyzed for superoxide anion production while the remainder was stored at –80 °C. Our investigations conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2. Measurement of superoxide anion production in cardiac tissue
After termination of the chronic ISO infusion experiment, superoxide anion production in the cardiac ventricular tissue was determined by the lucigenin chemiluminescence method as described previously [28]. Briefly, animals were sacrificed with an excess dose of sodium pentobarbital, after which ventricular tissue samples were quickly removed and placed in chilled bicarbonate buffer composed of (in mmol/l) 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25.0 NaHCO3, 5.5 glucose and 0.026 EDTA. The buffer was then bubbled continuously with 95% O2–5% CO2 to maintain a pH of 7.4, and allowed to equilibrate for 30 min at 37 °C. After equilibration, tissue samples were rinsed with pre-warmed (37 °C) modified Krebs–HEPES buffer composed of (in mmol/l) 119 NaCl, 20 HEPES, 4.6 KCl, 1.0 MgSO4, 0.15 Na2HPO4, 0.4 KH2PO4, 25 NaHCO3, 1.2 CaCl2 and 5.5 glucose (pH 7.4), then placed in 1 ml Krebs–HEPES buffer containing 20 µmol/l of lucigenin and equilibrated in the dark for 10 min at 37 °C. Chemiluminescence was recorded every 30 s for 15 min with a luminescence reader (BLR-301;Aloka) and expressed as counts per minute per milligram of dry tissue weight. After measurements, 50 mmol/l of Tiron was added to each sample, and the decreased value was evaluated as the net superoxide anion production.

2.3. Measurement of lipid peroxidation levels in the cardiac left ventricle
The details of the method used have been described previous [29]. Briefly, left ventricular tissue was homogenized in a buffer composed of 0.15 M KCl and 0.02 M Tris–HCl, with a pH of 7.4). The resultant homogenate was mixed with 15% trichloroacetic acid and 0.375% thiobarbituric acid then heated at 100 °C for 15 min. After cooling, the mixture was centrifuged at 12,000 x g for 10 min, after which the absorbance of the supernatant was measure at 535 nm. The amount of thiobarbituric acid reactive-substances (T-BARS) was determined by the malondialdehyde standard curve and expressed as nmol/g wet tissue.

2.4. Measurements of collagen content in the cardiac left ventricle
Hydroxyproline concentrations in the left ventricle were measured as an indicator of collagen accumulation. Each sample was dried and hydrolyzed by adding 6 mol/l HCl at 110 °C for 24 h. The hydrolysates were transferred to flasks and dried in a desiccator overnight to evaporate the hydrochloric acid. After dissolving the samples in 2 ml of 0.02 mol/l HCl, they were placed in 50 µl aliquots and applied to an amino acid analyzer (Amino Acid Analyzer Model 835; Hitachi) to determine the hydroxyproline concentrations. Because hydroxyproline is only incorporated into collagen and assuming that collagen contains 14% hydroxyproline, it is possible to calculate the total collagen content (nmol/mg dry weight).

2.5. Western blot for phosphorylated forms of MAP kinase cascade elements and CREB in the cardiac left ventricle
Frozen tissues were homogenized in cold lysis buffer consisting of 20 mM Tris–HCl, pH 7.4, 140 mM NaCl, 1% Triton-X, 10% Glycerol, 1 mM β-glycerophosphate, and 1 mM sodium orthovanadate. The homogenates were then subjected to sonication for 10 s on ice (repeated three times), followed by centrifugation for 30 min at 12,000 x g to remove cellular debris. The protein concentrations of the supernatants were measured with protein assay kits (Bio-Rad, CA, USA). Then equal amounts of protein (60 µg) from each sample were resolved on a 10% SDS-polyacrylamide gel by electrophoresis. Next, the proteins were transferred to nitrocellulose membranes. The membranes were blocked for 2 h at room temperature with 5% skimmed milk PBS and 0.1% Tween 20. Then the blots were incubated with primary antibodies (anti-phospho-ERK1/2, -p38, -JNK, Raf-1, Rac-1, ASK1 and CREB) overnight, followed by incubation for 1 h with a secondary antibody (HRP-conjugated anti-rabbit IgG, 1:2000). Immunoreactive bands were visualized using enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech) and quantified by densitometry in a linear range of film exposure using LAS-1000 plus (Fuji Film, Tokyo, Japan) and NIH image 1.61 software.

2.6. Real time PCR for RNA expression of type I collagen in the cardiac left ventricle
Total RNA was isolated from the left ventricle by guanidinium isothiocyanate-acid phenol extraction and quantified by measuring absorbance at 260 nm. One microgram of total RNA was used for reverse transcription, and specific messages of rat type I ({alpha}1) collagen were determined by real time PCR (prism7000; Applied Biosystems). The primer pairs for amplification of rat type I ({alpha}1) collagen cDNA, 5'-TCA CCT ACA GCA CGC TTG-3' for the forward, and 5'-GGT CTG TTT CCA GGG TTG-3' for the reverse were used. The primer pairs for rat GAPDH cDNA used were, 5'-TGA ACG GGA AGC TCA CTG G-3' for the forward, and 5'-TCC ACC ACC CTG TTG CTG TA-3' for the reverse.

2.7. Statistical analysis
All data are presented as the mean ± S.E.M. Statistical significance between more than two groups was tested using two-way ANOVA followed by the Newman–Keuls test or unpaired two-tailed Student's t-test as appropriate. P values of ≥ 0.05 were considered statistically significant.

2.8. Source of materials
ISO and propranolol were obtained from Wako (Osaka, Japan), and Tempol and 3-CP were purchased from Sigma (USA). Antibodies were purchased from Cell Signaling (Massachusetts, USA). All other chemicals were reagent grade and purchased from commercial sources and used without further purification.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Effects of acute ISO infusion on hemodynamics
Following the start of the ISO infusions, HR dose-dependently increased within 5 min, while MBP significantly decreased only with the highest ISO dose (Fig. 1). Propanolol alone significantly decreased HR, but did not affect MBP. Under propranolol treatment, the HR of the highest dose ISO-infused rats did not significantly differ from that of the saline-infused control rats (Table 1).


Figure 1
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  		Fig. 1 Dose-dependent hemodynamic changes during ISO infusion. Data are presented as the mean ± S.E.M. of four or more rats. *p<0.05 compared with saline-infused rats.

 

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  		Table 1 Mean blood pressure (upper; mm Hg) and heart rate (lower; bpm) during isoproterenol infusion

 
3.2. Effects of acute ISO infusion on cardiac lipid peroxidation
The effect of acute ISO administrations on oxidative stress in the heart was examined by measurements of cardiac T-BARS content. Thirty minutes of intravenous ISO infusion resulted in dose-dependent increases in cardiac lipid peroxidation levels (Fig. 2). Propranolol alone had no effect on basal lipid peroxidation in the heart, but completely prevented the increasing levels induced by the highest dose of ISO infusion (Fig. 2).


Figure 2
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  		Fig. 2 Dose-dependent increases in lipid peroxidation in the heart as a result of acute ISO treatment. Lipid peroxidation levels in the heart were measured 30 min after treatment. Data are presented as the mean ± S.E.M. of four or more rats. *p<0.05 compared with saline-infused rats. {dagger}p<0.05 compared with ISO (1000)-infused rats.

 
3.3. Effects of acute ISO infusion on cardiac MAP kinase activation
Intravenous ISO infusions for 30 min increased the phosphorylation of all the MAP kinases examined (ERK, p38 and JNK) in a dose-dependent manner (Fig. 3). The highest dose of ISO infusion increased the phosphorylation of cardiac ERK1/2, JNK, and p38 MAP kinases by 6.7-, 3.4-, and 8.2-fold, respectively, compared to controls. Propranolol alone had no effect on basal MAP kinase phosphorylation in the heart, but eliminated the augmentation of all MAP kinase phosphorylation induced by the highest dose of ISO (Fig. 3).


Figure 3
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  		Fig. 3 Dose-dependent increases in phosphorylated MAP kinases in the heart as a result of acute ISO treatment. The phosphorylated MAP kinases were measured 30 min after treatment. Upper: representative blots are shown. Lower: densitometric analysis of the phosphorylated MAP kinases. The mean value of each MAP kinase from the saline-infused rats is represented as 1. Data are presented as the mean ± S.E.M. of four or more rats. *p<0.05 compared with saline-infused rats. {dagger}p<0.05 compared with ISO (1000)-infused rats.

 
3.4. Effects of Tempol and 3-CP on the cardiac MAP kinase cascades and lipid peroxidation levels
To assess whether the increased ROS generation in heart tissue as a result of acute ISO infusion augments cardiac MAP kinase activity, the effects of Tempol and its inactive compound, 3-CP, on the ISO-induced phosphorylation of cardiac MAP kinase cascade elements were examined. Tempol and 3-CP had no significant effect on the hemodynamic changes elicited by ISO infusion (Table 1). Pre- and simultaneous treatment with Tempol completely suppressed increased cardiac lipid peroxidation (Fig. 4), as well as the augmented phosphorylation of all cardiac MAP kinases and their upstream elements, Rac-1, Raf-1 and ASK-1 (Fig. 5) induced by the highest dose of ISO infusion, however, 3-CP did not. Phosphorylated CREB, which increased with ISO, was not affected by Tempol or 3-CP treatment (Fig. 5).


Figure 4
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  		Fig. 4 Effects of Tempol and 3-CP on ISO-induced lipid peroxidation in the heart. Lipid peroxidation levels in the heart were measured 30 min after treatment. ISO infusion dose: 1000 ng/kg/min. Data are presented as the mean ± S.E.M. of four or more rats. *p<0.05 compared with saline-infused rats. {dagger}p<0.05 compared with ISO (1000)-infused rats.

 

Figure 5
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  		Fig. 5 Effects of Tempol and 3-CP on the ISO-induced phosphorylation of MAP kinase cascade elements in the heart. ISO infusion dose: 1000 ng/kg/min. The phosphorylated forms of the MAP kinase cascade elements were measured 30 min after treatment. Upper: representative blots are shown. Lower: densitometric analysis of the phosphorylated forms. The mean value of each element from the saline-infused rats is represented as 1. Data are presented as the mean ± S.E.M. of four or more rats. *p<0.05 compared with saline-infused rats. {dagger}p<0.05 compared with ISO-infused rats.

 
3.5. Effects of Tempol on oxidative stress and hypertrophy in the hearts of chronically ISO infused rats
With chronic ISO infusions, HR was maintained at high levels throughout the experimental period, while MBP increased significantly during ISO infusion compared with the saline-infused rats (Fig. 6A). Tempol treatment had no effects on ISO induced hemodynamic changes. Cardiac hypertrophy increased by 25% (heart-to-body weight ratio) at day 10 compared to saline-infused rats, while Tempol did not significantly affect on the development of ISO induced cardiac hypertrophy (Fig. 6B). Superoxide generation of cardiac tissue estimated by lucigenin chemiluminescence and cardiac lipid peroxidation in chronic ISO-infused rats increased by 3- and 2-fold, respectively, as compared with those levels in sham rats, which were significantly suppressed by the treatment of Tempol (Fig. 6C). Cardiac collagen content as well as type I collagen mRNA expression in chronic ISO infused rats increased, and was reversed by treatment with Tempol (Fig. 6D).


Figure 6
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  		Fig. 6 Effects of cardiac hypertrophy and oxidative stress in chronically ISO-infused rats. (A) Mean blood pressure and heart rate of rats during chronic ISO infusion using an osmotic mini-pump at a dose of 3 mg/kg/day. Open square, open circle and closed circle mean sham control, ISO infused and Tempol treated ISO infused rats, respectively. (B) Ratio of heart and left ventricle to body weight in chronically ISO-infused rats. (C) Superoxide generation (left) and lipid peroxidation levels (right) in the heart of chronically ISO-infused rats. (D) Cardiac collagen content (left; expressed as hydroxyproline) and type I collagen mRNA expression (right) of chronically ISO-infused rats. All data represent mean ± S.E.M. of six or more rats. *p<0.05 compared with saline-infused sham rats. {dagger}p<0.05 compared with ISO-infused rats.

 
3.6. Cardiac MAPK in the hearts of chronically ISO infused rats
Cardiac MAP kinase phosphorylation in chronically ISO-infused rats was also estimated to reveal no significant changes in the MAP kinase family members examined (ERK1/2, JNK and p38) at day 11 among the three groups–saline-infused sham, ISO-infused, and Tempol-treated ISO-infused rats (Fig. 7).


Figure 7
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  		Fig. 7 Phosphorylated MAP kinases in the heart of chronically ISO infused rats. Analyses were performed on tissue obtained from rats that received ISO infusion for 10 days with or without Tempol treatment. Upper: representative blots are shown. Lower: densitometric analysis of phosphorylated MAP kinases. The mean value of each MAP kinase from the saline-infused rats is represented as 1. Data are presented as the mean ± S.E.M. of four rats.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
This study demonstrates that acute administrations of ISO to conscious rats induced dose-dependent increases in cardiac lipid peroxidation and ERK1/2, p38 and JNK MAP kinase phosphorylation via β-adrenoceptor. These changes were blocked by pretreatments of Tempol, a radical scavenger. However, in chronically ISO-infused rats, phosphorylated MAK kinase levels in the heart did not change compared to the sham rats despite increased cardiac ROS generation.

Recently, the role of oxidative stress in cardiovascular disease has been characterized. Many of the deleterious cellular phenotypes presented in hypertrophied and failing myocardium might be attributed to ROS and oxidative stress [31]. Experimental studies with antioxidants support the hypothesis that oxidative stress is pathogenic in myocardial remodeling and failure. Dhalla et al. [32] found that the transition from compensated hypertrophy to failure in guinea pigs with pressure-overload due to aortic constriction was prevented by vitamin E administrations. Similarly, dimethylthiourea, a hydroxy radical scavenger, prevented chamber dilation and pump dysfunction in infarct mouse hearts [33]. ROS induce serious oxidative damage on cells and tissues through the Fenton or Haber–Weiss reactions. It is also well established that ROS act as important mediators for intracellular signalings in a variety of cells leading to changes in gene expression; for example, through MAP kinase activation. In neonatal rat ventricular myocytes, it has been shown that ERK1/2, p38, and JNK MAP kinases are activated by H2O2 [34]. In addition, Sano et al. demonstrated that exogenous H2O2 activates these three MAP kinases in cardiac fibroblasts [35]. However, relatively little is known about oxidative stress in response to β-adrenoceptor stimulation in in vivo conditions.

The p38 and JNK are recognized as stress inducible MAP kinases. Furthermore, Rac/cdc42, members of the Rho family, and ASK-1 are thought to be responsible for the intracellular signaling required to activate these MAP kinases. Clerk et al. [18] showed that phenylephrine and endotheline induced the rapid activation of Rac-1 leading to the augmentation of p38 MAP kinase phosphorylation. The present study showed that the ASK-1 and Rac/cdc42 pathways are both activated in the hearts of conscious acutely ISO-infused rats. Furthermore, Tempol but not 3-CP abolished the ISO-induced phosphorylation of JNK and p38 MAP kinases as well as their upstream elements, ASK-1 and Rac-1/cdc42, indicating that β-adrenoceptor activation rapidly provoked oxidative stress in the heart (estimated by cardiac lipid peroxidation) and induced stress inducible MAP kinase activation. Importantly, this oxidative stress inducible pathway leading to JNK activation has been suggested to participate in apoptosis signaling through β-adrenoceptor in adult rat ventricular myocytes [36].

CREB is activated through phosphorylation at Ser133 by various cellular signals, including ERK1/2 via p90RSK, p38 via MSK-1, calmodullin kinase II, Akt and also protein kinase A (PKA). In this study, Tempol had no influence on the ISO-induced hemodynamic changes or phosphorylation of CREB. Although the involvement of PKA in p38 MAP kinase activation via β2 adrenoceptor has been shown in adult cardiomyocytes by Zhang et al. [37], it is possible that PKA does not contribute to the ROS sensitive activation of p38 MAP kinases through β adrenoceptor stimulation, at least in an in vivo conscious condition.

The ERK1/2 kinases have been extensively investigated and are thought to play an important roles during hypertrophic responses of cardiomyocytes both in vitro [21–24] and in vivo [25,38,39], although the activation of ERKs does not always lead to cardiomyocyte hypertrophy [40]. Beside transactivation of the tyrosine kinase receptor, β-adrenoceptor coupled with G(s)-mediated ERK1/2 MAP kinase activation is attributed to the Rap/B-Raf pathway via PKA in neural cells, whereas involvement of the Ras/Raf1pathway has been also shown via β2-adrenoceptor coupled with G(i) in some circumstances [41]. Yanazaki et al. [22] showed that in neonatal cardiomyocytes, ISO activated ERK1/2 through the Ras/Raf1pathway, which was enhanced synergistically by the addition of a {alpha}-adrenoceptor agonist. We followed this using conscious rats and reveal that phosphorylated ERK1/2 MAP kinases in the heart increase with ISO infusion; furthermore that the activation of these ERKs is also ROS sensitive. It is noteworthy that under basal condition β-adrenoreceptors do not seem to contribute to cardiac MAP kinase activation, since the blockade of β-adrenoreceptors had no significant effect on the basal phosphorylation of cardiac MAP kinases.

The therapeutic applications of ROS scavengers to experimental cardiac hypertrophy developed by pressure-overloading [32] or infarction [33] have shown reductions of cardiac mass and the preservation of cardiac function; indicating a significant role of ROS in cardiac structural remodeling under these pathophysiological status. We showed increases of oxidative stress during the development of cardiac hypertrophy accompanied with enhanced collagen accumulation in chronic ISO infusion. Tempol treatment normalized the levels of lipid peroxidation in and superoxide generation from the cardiac tissue of ISO infused rats without any significant effect on hemodynamics. Although the hypertrophic response of the heart induced by chronic ISO infusion was not affected, cardiac collagen accumulation and RNA expression of type I collagen were significantly suppressed by Tempol treatment. Siwik et al. [42] showed suppression of collagen biosynthesis by ROS in cultured cardiac fibroblasts. Nevertheless, from the results of this in vivo study, it may be possible that ROS play important roles in fibrosclerotic structural alterations but not in compensatory hypertropic adaptations of the heart as seen in chronic ISO infused rats.

In contrast to the acute effects, chronic ISO infusion did not result in any changes in phosphorylated levels of the cardiac MAP kinase family despite the enhancement of oxidative stress in the heart. The results indicate that the redox-sensitive activation of cardiac MAP kinase cascades by the β-adrenoceptor agonist is not persistent; these results are concordant with a recent investigation in which MAP kinase activation was observed only in acute, not chronic, Angiotensin II-infused hypertensive rats [29,30,38]. An equal number of reports have described the importance of MAP kinases on hypertrophy and extracellular-matrix protein production [43]. However, this study did not ascertain whether Tempol treatment completely suppressed redox-sensitive cardiac MAP kinase activation during the early phase of ISO infusion. Further studies will be necessary to evaluate the relation between MAP kinases and cardiac remodeling from the viewpoint of oxidative stress.

In conclusion, the current study demonstrates that cardiac oxidative stress increases in response to β-adrenoceptor stimulation, and that cardiac MAP kinase activation triggered by the β-adrenoceptor agonist is mediated through ROS generation. In the chronic phase of ISO infusion, increased ROS play important roles in extracellular matrix biosynthesis, which may lead to the alteration of wall stiffness and affect cardiac function.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan.


   Notes
 
Time for primary review 26 days


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

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