Skip Navigation

Cardiovascular Research 2001 50(1):34-45; doi:10.1016/S0008-6363(01)00203-6
© 2001 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Yoshida, H.
Right arrow Articles by Takeo, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yoshida, H.
Right arrow Articles by Takeo, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2001, European Society of Cardiology

Characterization of cardiac myocyte and tissue β-adrenergic signal transduction in rats with heart failure

Hiroyuki Yoshidaa, Kouichi Tanonakaa, Yuki Miyamotoa, Tsutomu Abea, Masaya Takahashia, Madhu B Anand-Srivastavab and Satoshi Takeoa,*

aDepartment of Pharmacology, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
bDepartment of Physiology, Faculty of Medicine, University of Montreal, Montreal, Canada

* Corresponding author. Tel.: +81-426-76-4583; fax: +81-426-76-5560 takeos{at}ps.toyaku.ac.jp

Received 4 October 2000; accepted 12 December 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The cellular basis of alterations in β-adrenergic signal transduction in rats with chronic heart failure (CHF) remains unclear. The aim of the present study was to examine this signal transduction system in isolated ventricular cardiomyocytes of rats with CHF. We focused on changes in the levels of stimulatory (Gs) and inhibitory G-proteins (Gi). Methods: CHF was induced in male Wistar rats by coronary artery ligation (CAL). Hemodynamic and biochemical parameters were measured 8 weeks after CAL. Alterations in contractile function and Ca2+ transients via β-adrenergic receptor signaling of cardiomyocytes isolated from rats with CHF were characterized by simultaneous measurements of cell shortening and fura-2 fluorescence intensity. Results: Coronary artery-ligated rats showed symptoms of CHF, such as decreased contractile function, increased left ventricular volume, decreased chamber stiffness, and about 40% infarct formation of the left ventricle, by 8 weeks after surgery. The contractile function and Ca2+ dynamics of cardiomyocytes from the rats with CHF remained normal under basal conditions. Only cardiac cell length was increased. The responses of peak shortening, fura-2 fluorescence ratio amplitude, and cAMP content to β-adrenoceptor stimulation were reduced in cardiomyocytes of the rats with CHF, whereas direct stimulation of adenylate cyclase did not affect the response of these variables. Cardiomyocyte Gs{alpha} protein was decreased, whereas no changes in Gi{alpha} proteins were seen in these cells. Increases in tissue Gs{alpha} and Gi{alpha} proteins in the scar zone were detected. The results on tissue levels of collagen and G-proteins in the viable left ventricle appeared to depend on the presence of nonmyocytes. Conclusions: The results suggest that impaired contractile function of cardiomyocytes is unlikely to account for global LV contractile dysfunction, and that down-regulation of β-adrenoceptors occurs in cardiomyocytes per se. The difference in changes of G-protein between the cardiomyocyte and myocardial tissue suggests an appreciable contribution of nonmyocytes to myocardial G-protein levels.

KEYWORDS Adrenergic (ant)agonists; Calcium (cellular); Contractile function; G-proteins; Heart failure; Myocytes; Signal transduction

This article is referred to in the Editorial by T.E. Hébert (pages 7–9) in this issue.


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The β-adrenergic signal transduction system is one of the major pathways mediating cardiac muscle contraction. This system includes mainly the β-adrenergic receptor, heterotrimeric G-proteins, adenylate cyclase, protein kinase A, and components of the contractile machinery. Several investigators have shown that chronic heart failure (CHF) induces a decrease in responsiveness to β-adrenergic receptor agonists as a result of an increased sympathetic activity [1,2]. This is considered to compensate for the development of contractile dysfunction including reduced cardiac output [3], down-regulation of β-adrenergic receptors [3], increases in levels of β-adrenoceptor kinase (β-ARK) and β-arrestin [4,5], and changes in levels of heterotrimeric G-proteins [6,7]. Several reports have shown alterations in cardiac membrane G-proteins in the development of heart failure. For example, increased expression of Gs{alpha} mRNA in failing hearts was found in some studies [8,9]; however, changes in Gs{alpha} levels and signal transduction via Gs{alpha} in failing hearts are controversial [6–9]. Furthermore, the Gi2{alpha} mRNA level was increased in human end-stage heart failure [7], and Gi2{alpha} protein was concomitantly increased in an experimental model of heart failure [10]. It remains, however, unclear whether these changes in G-proteins occur in cardiomyocytes per se or not. In addition, Peterson et al. showed that there was a marked increase in the expression and content of Gs{alpha} and Gi{alpha} in the myofibroblasts of the infarct scar and remnant cardiomyocytes bordering the scar in the rat heart 8 weeks after myocardial infarction [11]. This finding suggests that changes in myocardial G-proteins in CHF following acute myocardial infarction may be different between cardiomyocytes and the cardiac mass. To explore the cellular basis of the pathogenesis of the β-adrenergic signal transduction system in the failing heart, we examined contractile function and the signal transduction system of both whole heart and isolated cardiomyocytes of rats with chronic heart failure due to myocardial infarction.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Animals
Male Wistar rats (SLC, Hamamatsu, Japan), weighing 220–240 g, were used in the present study. The animals were maintained at 23±1°C, with a constant humidity of 55±5%, and a cycle of 12 h of light and 12 h of darkness, and had free access to food and tap water according to 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). The protocol of the study was approved by the Animal Care Committee of Tokyo University of Pharmacy and Life Science.

2.2 Induction of myocardial infarction
Myocardial infarction was produced in rats by occlusion of the left coronary artery at approximately 2 mm from its origin according to the method described previously [12]. Twenty-four hours after the operation, the rats were anesthetized with ether and their electrocardiograms (lead I) were then monitored. The rats that showed abnormal Q-wave (<1 mV) were considered to have developed acute myocardial infarction and were used for the following experiments. Sham-operated rats were treated similarly except that the coronary artery ligation was not performed.

Eight weeks after surgery, the rats underwent hemodynamic assessments. Some of these rats were used for cardiomyocyte studies on cell contraction, G-protein content, and cyclic nucleotides. Other rats were used to measure the pressure–volume relation and tissue G-protein content of the ventricles.

2.3 Measurements of hemodynamic parameters and histological study
Eight weeks after the operation, the rats were anesthetized with nitrous oxide–oxygen (3:1) and 2.5% enflurane. Anesthesia was continued with a gas mixture of nitrous oxide and oxygen (3:1) containing 0.5% enflurane at the flow-rate of 1.2 l/min through a mask loosely placed on the nose. The pO2, pCO2, and pH of the arterial blood under these experimental conditions were found to be within physiological ranges; pO2, 90–108 mmHg, pCO2, 37–41 mmHg, and pH, 7.41–7.46. Hemodynamic parameters were measured as described previously [12].

After measurement of the hemodynamic parameters, the heart was isolated and sectioned into seven slices (1-mm thick) from the base to apex in a plane parallel to the artrioventricular groove. The slices were stained at 37°C for 5 min with 1% 2,3,5-triphenyltetrazolium chloride (TTC) in physiological saline, and the infarct areas were estimated according to the planimetric method [13].

2.4 Measurement of pressure–volume relation
To assess the passive pressure–volume characteristics of the left ventricle (LV), we arrested the hearts by injection of 1.0 ml/kg of 1 M KCl after measurement of hemodynamics, and quickly removed them. A double lumen catheter was inserted into the LV through the aorta. After removal of the right ventricle (RV), the atrioventricular groove was ligated tightly to prevent leakage of saline from the LV during infusion of saline. Saline was infused at a constant flow-rate of 0.48 ml/min after all residual fluid in the LV had been withdrawn by negative pressure. The LV pressure was continuously recorded until the pressure reached over 30 mmHg. Chamber stiffness constant was calculated from four segments: K1 (0–2.5 mmHg), K2 (2.5–10 mmHg), K3 (10–20 mmHg), and K4 (20–30 mmHg) as described previously [13]. K1 was determined from the slope of the linear portion of the pressure–volume curve. Other constants were calculated from the following equation

Formula
where K stands for K2, K3 and K4.

Left ventricular end-diastolic volume index (LVEDVI) was determined from the pressure–volume curve as the volume corresponding to the LVEDP measured in vivo.

2.5 Isolation of adult rat cardiomyocytes
Ventricular cardiomyocytes were isolated from Wistar rats (280–320 g) according to the modified method of Miyake et al. [14]. Briefly, after heparinization and anesthetization with 40 mg/kg sodium pentobarbital i.p., rat hearts were rapidly excised and perfused in the Langendorff manner with HEPES buffer contained 0.04% collagenase (Type II, Worthington, USA), 25 µM CaCl2, and 0.1% bovine serum albumin (BSA; Wako, Osaka, Japan). The HEPES buffer had the following composition (mM): NaCl, 130.0: KCl, 4.8: KH2PO4, 1.2: MgSO4, 1.2: CaCl2, 1.25: glucose, 11.0: sodium pyruvate, 5.0: and N-2-hydroxyethylpiperadine-N'-2-ethansulfonic acid (HEPES: Dojindo, Kumamoto, Japan), 10.0, pH 7.40. After perfusion, the ventricular tissue was chopped in a calcium-free HEPES buffer containing 1% BSA. Then calcium concentration in the suspension was increased gradually up to 1.25 mM. Approximately 1.5–2 million calcium-tolerant, rod-shaped cardiomyocytes were isolated from each heart. We term ventricular myocytes isolated from the coronary artery-ligated and sham-operated rats at the 8th week after the operation ‘CAL cells’ and ‘Sham cells’, respectively, in the following text.

2.6 Loading of cardiomyocytes with fluorescence probe
For assessment of Ca2+ dynamics parameters, isolated cardiomyocyte were loaded with 5 µM acetoxymethyl ester of fura-2 (fura-2/AM; Dojindo, Kumamoto, Japan), a membrane-permeable, calcium sensitive, fluorescent probe, by a 20-min exposure to the HEPES buffer described above at 25°C in darkness. To increase the solubility of the probe, we mixed 10 µl of 10% (w/v) Pluronic F-127 (Sigma, St. Louis, MO, USA), a detergent, with 25 µl of 1 mM fura-2/AM before incubation of the cells with the fluorescent probe. The final concentration of the detergent was 0.002% (w/v), which did not affect the contractile function of the cardiomyocytes.

2.7 Measurement of contraction and fluorescence
Measurements of contraction and calcium transient of the isolated cells were performed by the method described previously [14]. Cardiomyocytes loaded with the fluorescent probe were planted in the superfusion chamber (Warner Instrument, Hamden, CT, USA) on the stage of a phase-contrast microscope (Diaphot T-TMD; Nikon, Tokyo, Japan) connected to a detection system for fura-2 fluorescence (CAM-220; Jasco, Hachioji, Japan). First, the chamber was continuously superfused with the buffer in the absence of any agent at the flow-rate of 0.4 ml/min by means of an infusion pump (STC-523; Terumo, Tokyo, Japan). For simultaneous recording of fura-2 fluorescence and cell length, a single rod-shaped cardiomyocyte in the chamber was chosen in each experiment according to the following criteria [15]. The cardiomyocyte had clear striations and no blebs on its surface, and its spontaneous contraction was minimal (less than one contraction/min). The temperature of the perfusion buffer was maintained at 30±2°C throughout the experiments by means of a thermo-controller (TC344, Warner Instrument).

2.8 Intracellular cAMP
Measurement of intracellular cAMP content was performed according to a modified method of Ikenouchi et al. [16]. Cardiomyocytes were isolated as described above and suspended in 20 ml of HEPES buffer. The number of cardiomyocytes was adjusted to 3x104 in a volume of 0.5 ml buffer and placed in polypropylene tubes. Under microscopic observation, the ratio of rod-shaped cells in the cardiomyocyte suspension was 60–70%; the rest consisted of round-shaped cells. The cardiomyocytes in the tube were preincubated with 0.5 ml of 2 mM IBMX for 30 min at 32°C. Then 10 nM isoprenaline, 1 µM colforsin daropate or 0.7 mM ouabain was added, and the cells were incubated for 2, 5 or 5 min, respectively. The reaction mixture was centrifuged at 9000 g for 5 min at 4°C. The supernatant fluid was discarded, and 100 µl of 0.3 M perchloric acid (PCA)–0.1 mM EDTA Na2 was added under ice cooling. Then the cardiomyocytes were sonicated and centrifuged at 12 000 g for 20 min at 4°C. The supernatant fluid was neutralized by addition of 6 µl of 2.5 M K2CO3. Thereafter, the tube was centrifuged at 10 000 g for 5 min at 4°C. The supernatant fluid was stored at –20°C until assayed.

The intracellular cAMP content before and after an exposure to 10 nM isoprenaline, 1 µM colforsin daropate, or 0.7 mM ouabain was measured with the enzyme immunoassay kit (Biotrak, Amersham Life Science, Buckingham, UK).

2.9 Membrane preparations from whole heart
Myocardial membranes were prepared from the LV, septum, RV and infarct area according a modified method of McMahon [17]. The tissue was frozen in liquid nitrogen and then homogenized with a mortar and pestle. The homogenate was placed into five volumes of cold buffer (20 mM HEPES, 0.3 mM phenylmethylsulfonyl fluoride, 0.25 M sucrose, 1 mM DTT, 1 mM EGTA; pH 7.40), and then rehomogenized with a Potter homogenizer at a setting of 1000 rpm for 2 min (20 strokes). The homogenate was centrifuged at 1000 g at 4°C for 10 min. The supernatant fluid was recentrifuged at 100 000 g at 4°C for 20 min. The supernatant fluid after centrifugation was discarded, and the pellet was resuspended in a cold buffer to a protein concentration of 0.5 mg/ml. The protein concentration was determined by the method of Bradford [18] with bovine serum albumin used as the standard. The membrane preparations were stored at –80°C until assayed.

2.10 Membrane preparations from cardiomyocytes
Membranes were prepared from cardiomyocytes of the LV, septum, and RV of rats. The isolated cardiomyocytes were placed into five volumes of cold buffer (20 mM HEPES, 0.3 mM phenylmethylsulfonyl fluoride, 0.25 M sucrose, 1 mM DTT, 1 mM EGTA, pH 7.40), and then homogenized with a Potter homogenizer at the setting of 1000 rpm for 2 min (20 strokes). The procedure which followed was as described above.

2.11 Western blotting
Sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blotting were performed by the method previously described [19] with minor modifications. A 5-µg amount of membrane protein was electrophoretically separated using SDS–PAGE with 4 and 10% acrylamide in the stacking and separating gels, respectively. The separated proteins were then electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon, Millipore, Bedford, MA, USA) in a transfer apparatus (AE-6675, Atto, Japan) at 200 mA for 1 h. The membranes for analysis of Gs{alpha}, Gi1,2{alpha} and Gi3{alpha} were then incubated for 3 h with a 1:10 000 dilution of antibody RM/1, a 1:P10 000 dilution of antibody AS/7, and a 1:5000 dilution of antibody EC/2 (NENTM Life Science Products) [20] in phosphate-buffered saline (PBS) containing 10% Block Ace (Dainippon Pharm.) and 0.1% Tween 20, respectively. The membranes were subsequently washed in PBS containing 10% Block Ace and 0.1% Tween 20, and incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG1 (Sigma) at a dilution of 1:4000 in PBS containing 10% Block Ace and 0.1% Tween 20. Finally, the membranes were washed three times with PBS for 10 min and placed in the enhanced chemiluminescence (ECL) immunoblotting detection reagent (Amersham). The blots were finally exposed to X-ray film. Scanning of visualized immunoreactivity was performed on a Scan Touch (Nikon, Japan). Data were processed with a NIH IMAGE software.

2.12 Measurement of collagen content
Tissue collagen content was measured with a collagen staining kit (Cosmo Bio, Tokyo, Japan) according to a modified method of Lopez-De Leon and Rojkind [21]. The tissue was frozen in liquid nitrogen and then homogenized with a mortar and pestle. Ten milligrams of the homogenate was placed into 1 ml of 0.6 M PCA and then centrifuged at 10 000 g at room temperature for 3 min. The resultant pellet was washed and neutralized with 1 ml of PBS. The pellet was next incubated for 30 min at room temperature with a dye solution including 0.1% Sirius red F3BA and 0.1% Fast green FCF. Then the fluid was centrifuged at 10 000 g at room temperature for 3 min. The resultant pellet was washed with distilled water until the fluid was colorless. A 1-ml volume of 0.1 M NaOH in 50% methanol was then added and gently mixed until all the color was eluted from the tissue. The intensity of the eluted color was determined by a spectrophotometer at 540 (Sirius red F3BA) and 605 nm (Fast green FCF).

2.13 Agents
Colforsin daropate was kindly donated by Nihon Kayaku (Tokyo, Japan). Isoprenaline hydrochloride was purchased from Nacarai Tesque (Kyoto, Japan). These agents were dissolved in the perfusion buffer and diluted to desired concentrations with buffer. All solutions containing the agents were freshly prepared before the experiment.

2.14 Statistics
The results were expressed as means±S.E.M. The numbers of different preparations are indicated in the legends. Statistical significance of differences in myocyte study, Gs and Gi protein, cAMP, and collagen contents were estimated by using two-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison. Statistical significance of the pressure–volume relation was estimated by using two-way repeated-measures ANOVA. The results on hemodynamics, LVEDVI, and left ventricular chamber stiffness were statistically evaluated by the unpaired Student's t-test. Differences with a probability of 5% or less were considered to be statistically significant (P<0.05).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Hemodynamics and infarct size
Cardiac and hemodynamic parameters and infarct size of coronary artery-ligated and sham-operated rats at the 8th week after the operation are shown in Table 1. Significant decreases in body weight, LVSP, and ±dP/dt were observed 8 weeks after coronary artery ligation (CAL). The heart weight/body weight, lung weight/body weight and LVEDP were markedly increased after CAL. The infarct area of the coronary artery-ligated rat was approximately 42% of the left ventricular area 8 weeks after the operation. There was no infarct area in the LV of sham-operated rats.


View this table:
[in this window]
[in a new window]

  		Table 1 Cardiac and hemodynamic parameters in vivo and cardiomyocyte morphology of sham-operated rats (Sham) and rats with coronary artery ligation (CAL) at the 8th week after the operationa

 
3.2 Pressure–volume relations
Ventricular volumes measured at transmural pressures ranging from 2.5 to 30 mmHg were significantly increased in the potassium-arrested heart of the rats with CAL (Fig. 1). The substantial volume in the rats with CAL was increased at each pressure. The LV chamber stiffness constants, an index of ventricular stiffness, are summarized in Table 2. K0, K1, K2, K3 and K4 values were significantly decreased in the CAL group. LVEDVI, an index of LV chamber volume in the diastolic stage in vivo, was significantly increased with the rightward shift of pressure–volume curve (Fig. 1). LVEDVI values in sham and CAL groups were 0.370±0.063 ml/kg (n = 6) and 2.732±0.216 ml/kg (n = 6), respectively, and this difference was statistically significant (P<0.05).


Figure 1
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Left ventricular pressure–volume relationship in sham-operated rats ({circ}) and in rats with CAL (bullet). Eight weeks after surgery, the pressure–volume relationship had progressively shifted to the right.

 

View this table:
[in this window]
[in a new window]

  		Table 2 Left ventricular chamber stiffness of sham-operated rats (Sham) and rats with coronary artery ligation (CAL); values represents the mean±S.E.M. of six experiments

 
3.3 Morphology of isolated cardiomyocytes
Cardiomyocyte length of sham-operated rats and rats with CAL is summarized in Table 1. The left ventricular cardiomyocytes of rats with CAL were 15% longer than those of sham-operated rats, and the right ventricular cardiomyocytes of rats with CAL, 20% longer than those of sham-operated rats.

3.4 Simultaneous measurements of cell shortening and calcium transients
Simultaneous recordings of cell shortening and fura-2 fluorescence ratio are shown in Fig. 2. CAL cells showed a greater cell shortening and a slightly larger calcium transient than Sham cells. The peak calcium transient preceded the peak cell shortening.


Figure 2
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Typical simultaneous tracings of fura-2 fluorescence ratio signal (upper panel) and length after shortening (lower panel) of cardiomyocytes of Sham (A) and CAL cells (B). Solid lines show baseline values and dotted lines the responses to isoprenaline.

 
3.5 Contractile function of cardiomyocytes
To characterize the contractile function of isolated cells, we compared peak shortening, time to peak shortening, velocity of shortening, time to 50% relaxation, peak positive dL/dt, and peak negative dL/dt in CAL and Sham cells. The basal cardiomyocyte contraction is illustrated in Fig. 2. CAL cells exhibited a greater peak shortening and a slightly larger fura-2 fluorescence amplitude. However, when the peak shortening was expressed as a percent decrease to the baseline value, it did not differ between CAL and Sham cells (Table 3). There were no differences in any other contractile parameter between CAL and Sham cells. Furthermore, there were no differences in other contractile parameters between the left and right ventricular cells irrespective of the presence or absence of coronary artery ligation. However, CAL cells showed higher average values for most of the contractile parameters compared with the Sham cells.


View this table:
[in this window]
[in a new window]

  		Table 3 Contractile function of cardiomyocytes from left and right ventricles of rats with CAL and sham-operated rats at the 8th week after the operationa

 
3.6 Effects of inotropic agents on cell shortening
Fig. 3 shows the peak shortening in cardiomyocytes treated with 10 nM isoprenaline, 1 µM colforsin daropate, or 0.7 mM ouabain. There were no differences in the basal values for cell shortening between the left and right ventricular cells. In Sham cells, isoprenaline produced about 100% increase of the peak shortening (Fig. 3A). As shown in Fig. 2B, isoprenaline produced only a 50% increase in peak shortening in CAL cells. In contrast, colforsin daropate, which directly activates adenylate cyclase and thereby increases intracellular cyclic AMP concentrations, produced responses similar in degrees to those to isoprenaline in both CAL and Sham cells (Fig. 3B). Fig. 3C shows the effects of ouabain, a cardiotonic steroid that elicits a positive inotropic effect by a mechanism unrelated to the generation of cyclic AMP. Ouabain produced similar peak shortening in both CAL and Sham cells.


Figure 3
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Effects of 10 nM isoprenaline (A), 1 µM colforsin daropate (B), and 0.7 mM ouabain (C) on peak shortening of cardiomyocytes of the left and right ventricles of Sham ({square}) and CAL ({blacksquare}) cells. Each value represents the mean±S.E.M. of five to eight experiments. *, Significantly different from the sham-operated group (P<0.05).

 
3.7 Intracellular Ca2+ dynamics
To characterize the intracellular calcium transient of cardiomyocytes, we determined the fura-2 fluorescence ratio amplitude, time to peak fluorescence ratio, velocity of fluorescence ratio, time to 50% decrease, diastolic fluorescence ratio, and systolic fluorescence ratio in CAL and Sham cells (Table 4). The absolute amplitude of fura-2 fluorescence ratio of right ventricular myocytes was greater for CAL cells. However, there were no differences in any Ca2+ dynamics parameter between CAL and Sham cells. There were no differences in any Ca2+ dynamics parameter between the left and right ventricular cells irrespective of the presence or absence of coronary artery ligation.


View this table:
[in this window]
[in a new window]

  		Table 4 Calcium transients of cardiomyocytes isolated from left and right ventricles of rats with CAL and sham-operated rats at the 8th week after the operationa

 
3.8 Effects of inotropic agents on fura-2 fluorescence ratio amplitude
Fig. 4 shows the fura-2 fluorescence ratio amplitude in cardiomyocytes exposed to 10 nM isoprenaline, 1 µM colforsin daropate, or 0.7 mM ouabain. Treatment of Sham cells with isoprenaline, colforsin daropate, or ouabain resulted in an approximately 60% increase in the fura-2 fluorescence ratio amplitude. In contrast, treatment with isoprenaline increased the amplitude in CAL cells to a smaller degree than that for Sham cells (Fig. 2B and Fig. 4A). Colforsin daropate and ouabain increased the fura-2 fluorescence ratio amplitude similarly in CAL cells and Sham cells (Fig. 4B and C). Furthermore, there were no differences in fura-2 fluorescence ratio amplitude between the left and right ventricular cells irrespective of the presence or absence of coronary artery ligation.


Figure 4
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Effects of 10 nM isoprenaline (A), 1 µM colforsin daropate (B), and 0.7 mM ouabain (C) on fura-2 fluorescence ratio amplitude of cardiomyocytes of the left and right ventricles of Sham ({square}) and CAL ({blacksquare}) cells. Each value represents the mean±S.E.M. of five to eight experiments. *, Significantly different from the sham-operated group (P<0.05).

 
3.9 cAMP content of cardiomyocytes and effects of inotropic agents
The basal cAMP content of CAL cells was about 50% lower than that of Sham cells. When the effects of 10 nM isoprenaline, 1 µM colforsin daropate, and 0.7 mM ouabain on cAMP production in Sham and CAL cells were examined (Fig. 5), it was found that treatment with ouabain did not increase the cAMP levels in either cells. Treatment with isoprenaline increased the cAMP levels in Sham cells, and it also increased the cAMP levels of CAL cells to a smaller, but statistically significant, degree. Treatment with colforsin daropate increased the cAMP levels in both cells to a similar extent.


Figure 5
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Effects of 10 nM isoprenaline, 1 µM colforsin daropate, and 0.7 mM ouabain on cAMP content of cardiomyocytes of the LVs of sham-operated rats ({square}) and rats with CAL ({blacksquare}). Each value represents the mean±S.E.M. of five to six experiments. *, Significantly different from the corresponding sham-operated group (P<0.05).

 
3.10 Changes in Gs{alpha} protein of the ventricles and cardiomyocytes of animals with CAL and sham-operated animals
The amounts of Gs{alpha} depicted in Fig. 6B and D are the sum of the 45- and 52-kDa peptides (Fig. 6A and C). Fig. 6B and D shows tissue and cardiomyocyte Gs{alpha} protein content in the LV, septum, and RV in animals with CAL and in sham-operated animals. There was no difference in tissue Gs{alpha} protein content between the viable LV, septum and RV of the sham-operated rats. In the coronary artery-ligated rats, Gs{alpha} protein in the scar tissue increased by approximately 1.4-fold as compared with that in the LV of the same animal. There was no change in tissue Gs{alpha} protein in the viable LV of the rats with coronary artery ligation, but tissue Gs{alpha} protein in the septum and RV was decreased.


Figure 6
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Immunoblot analysis of Gs{alpha} in the membrane fraction of the ventricle and cardiomyocyte. Representative Western blots in the upper panels indicate specific 52- and 45-kDa bands for Gs{alpha} in the membrane fraction of the ventricle (A) and cardiomyocytes (C). Quantified data for Gs{alpha} protein content of the scar, LV (viable left), septum, and RV (B) and of cardiomyocytes isolated from the left (viable left), septum, and RV (D) are shown in the lower panels. Each value represents the mean±S.E.M. of six experiments. *, Significantly different from the corresponding sham-operated group (P<0.05).

 
Gs{alpha} protein content of cardiomyocytes from the LV, septum and RV of the sham-operated rats and rats with CAL were measured; no difference in cellular Gs{alpha} protein content was found in the sham-operated rats. The levels of Gs{alpha} protein of the viable LV, septum and RV decreased in CAL cells (P<0.0001, two-way ANOVA).

3.11 Changes in Gi1,2{alpha} proteins of the ventricles and cardiomyocytes of animals with CAL and sham-operated animals
A band migrating at 40 kDa, identified as Gi1,2{alpha}, was detected by using the Gi1,2{alpha} antibody as shown in Fig. 7A and C. Tissue Gi1,2{alpha} protein of the viable LV, septum and RV was not altered in the sham-operated animal (Fig. 7B). There was a significant difference in cardiac Gi1,2{alpha} content between sham-operated and coronary artery-ligated animals (P = 0.0024, two-way ANOVA). Tissue Gi1,2{alpha} increased by approximately 7.9-, 3.4-, 1.7- and 1.5-fold in the respective scar, viable LV, septum and RV as compared with the corresponding tissues of sham-operated rats.


Figure 7
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Immunoblot analysis of Gi1,2{alpha} in the membrane fraction of the ventricle and cardiomyocytes. Representative Western blots in the upper panels indicate specific 40 kDa band for Gi1,2{alpha} in the membrane fraction of the ventricle (A) and cardiomyocytes (C). Quantified data for Gi1,2{alpha} protein content of the scar, LV (viable left), septum, and RV (B) and of cardiomyocytes isolated from the LV (viable left), septum, and RV (D) are shown in the lower panels. Each value represents the mean±S.E.M. of six experiments. *, Significantly different from the corresponding sham-operated group (P<0.05).

 
Fig. 7D shows Gi1,2{alpha} protein content of Sham and CAL cells isolated from the viable LV, septum and RV. There were no significant differences in the cardiomyocyte Gi1,2{alpha} content among the viable LV, septum, and RV or between the sham-operated and coronary artery-ligated animals.

3.12 Changes in Gi3{alpha} proteins of the ventricles and cardiomyocytes of animals with CAL and sham-operated animals
A band migrating at 41 kDa, identified as Gi3{alpha}, was detected with Gi3{alpha} antibody, as shown in Fig. 8A and C. Tissue Gi3{alpha} protein of the viable LV, septum and RV was not altered in the sham-operated animal (Fig. 8B). There was a significant difference in cardiac Gi3{alpha} content between sham-operated and coronary artery-ligated animals (P = 0.0001, two-way ANOVA). Tissue Gi3{alpha} increased by approximately 9.9-, 4.4-, 1.7- and 1.4-fold in the scar, viable LV, septum and RV, respectively, as compared with the corresponding tissues of sham-operated rats.


Figure 8
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8 Immunoblot analysis of Gi3{alpha} in the membrane fraction of the ventricle and cardiomyocytes. Representative Western blots in the upper panels indicate specific 41 kDa band for Gi3{alpha} in the membrane fraction of the ventricle (A) and cardiomyocytes (C). Quantified data for Gi3{alpha} protein content of the scar, LV (viable left), septum, and RV (B) and of cardiomyocytes isolated from the LV (viable left), septum, and RV (D) are shown in the lower panels. Each value represents the mean±S.E.M. of six experiments. *, Significantly different from the corresponding sham-operated group (P<0.05).

 
As in the case of Gi1,2{alpha}, Gi3{alpha} content of cardiomyocytes isolated from the viable LV, septum and RV of sham-operated animals or animals with CAL showed no significant differences among these locations or between the sham-operated and coronary artery-ligated animals.

3.13 Changes in cardiac collagen content in cardiac tissue of animals with CAL and sham-operated animals
The cardiac collagen content is shown in Fig. 9. In the sham-operated rats, cardiac collagen content did not differ among the LV, septum, and RV. A marked increase in the collagen content in the scar tissue was seen, and a significant increase in the collagen content was observed in the viable LV of the rat with CAL. However, the collagen content of the septum and RV between the sham-operated rats and the rats with CAL was the same.


Figure 9
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 9 Myocardial collagen content of rats with coronary artery ligation and sham-operated rats. The collagen content was significantly increased in the viable LV of the rat with CAL. Each value represents the mean±S.E.M. of four experiments. *, Significantly different from the corresponding sham-operated group (P<0.05).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In the present study, we showed that the left coronary artery ligation consistently produced approximately 42% infarction of the LV 8 weeks after the operation. The left coronary artery ligation resulted in a reduction in the left ventricular systolic pressure and its positive and negative dP/dt, and a rise in the left ventricular end-diastolic pressure 8 weeks after the operation. The pressure–volume curve of the isolated heart was shifted to the right in rats with CAL. This model also showed decreases in cardiac output and stroke volume indices and an increase in heart and lung weight 8 weeks after the operation [12]. Thus, the data suggest that CHF with low cardiac output and diastolic contractile dysfunction had developed in the coronary artery-ligated rats by this period.

An increase in the length of the left and right ventricular cardiomyocytes was observed in rats with CAL, indicating hypertrophy of cardiac cells per se. The difference in the extent of hypertrophy between the two ventricles may be attributable to difference in loading conditions of the ventricles: the LV was predominantly volume-overloaded whereas the RV was predominantly pressure-overloaded [22].

Baseline values for contractile and Ca2+ dynamics parameters did not differ between CAL and Sham cells. Previous studies have shown conflicting results regarding the contractile function of cardiomyocytes in rats with myocardial infarction. Capasso and Anversa [23] showed the lower percent cell shortening and slower velocity of cell shortening of the left ventricular myocytes isolated from the rats 1 week after myocardial infarction. Li et al. [24] reported the reduced kinetics of cell shortening and the decreased systolic calcium levels at different time periods ranging from 6 h to 1 month after myocardial infarction. In contrast, Anand et al. [25] showed no difference in the contractile function and intracellular Ca2+ movement in cardiomyocytes isolated from an area remote from the infarct zone at different time periods ranging from 1 to 6 weeks after myocardial infarction. A similar trend in the contractile function was observed in the cardiomyocytes isolated from the noninfarcted region at 1 week after acute myocardial infarction [26]. In this sense the observation of Melillo et al. [27] should be noted, i.e. that the left ventricular myocytes isolated from the segment adjacent to the infarct area showed contractile dysfunction, whereas those from the segment remote from the infarct area did not show it. Since we used cardiomyocytes isolated from the segment remote from the infarct area in the present study, our findings of the normal cellular contractile function in the CAL cells are consistent with those of the above investigators. This observation suggests that abnormalities of myofilament Ca2+ sensitivity are unlikely present and that cellular contractile function cannot account for contractile dysfunction observed in the cardiac mass.

One possible explanation for the cardiac dysfunction is a myocardial infarction-induced increase in the interstitial collagen in the extracellular matrix of the viable myocardium. Such an increase is considered to restrain the shortening of cardiac cells or cause reductions in the force transmission and cell-to-cell mechanical coupling [28]. In heart failure, changes in the extracellular matrix proteins have also been reported [29,30]. Such changes in non-myocyte morphology may eventually lead to the development of heart failure. We also observed an appreciable increase in the collagen content of the viable LV of rats with CHF in the present study.

To explore the pathogenesis of signal transduction of the β-adrenoceptor/adenylate cyclase/cAMP system in cardiac cells of the failing heart, we examined the effects of several pharmacological agents on cardiac cell contraction. CAL cells exhibited a reduced positive inotropic effect of isoprenaline, which acts via stimulation of membrane β-adrenergic receptors, as compared with Sham cells (Fig. 3A). In contrast, colforsin daropate, a direct stimulant of adenylate cyclase, increased the peak shortening, fura-2 fluorescence ratio amplitude, and cAMP content in CAL cells to an extent similar to that in Sham cells (Figs. 3B and 4BGo). Similarly, the direct stimulation of adenylate cyclase [31] could produce a positive inotropic effect on CAL cells even under conditions where the cardiac responsiveness to β-adrenoceptor agonist was diminished. These results imply that the ability of adenylate cyclase to produce cAMP was not impaired in this failing heart. Sanbe and Takeo [32] have reported that a direct adenylate cyclase stimulant, colforsin daropate, but not a β1-stimulant, denopamine, elicited an increase in cardiac output of the failing heart to an extent similar to that of the sham-operated rat. This observation suggests that the primary defect is attributable either to signal transduction from β-adrenergic receptor to adenylate cyclase, or to down-regulation of adrenergic β-receptor in cardiac cells. Sanbe and Takeo [33] also reported the reduction in the number of β1-adrenergic receptors in homogenates of the viable myocardium of this experimental model. In addition, we observed an appreciable decrease in the Gs{alpha} protein levels in CAL cells of the viable LV, septum, and RV. Thus, it is likely that the reduction in β-adrenergic responsiveness is due to the desensitization of the remaining viable myocardium including a shortage of Gs proteins in the signal transduction system and that down-regulation of β-adrenoceptors may occur in cardiomyocytes per se because nonmyocyte contributes to cardiac contractility to a minor degree.

Our results showed that the response of CAL cells to ouabain, which exerts inotropic effects by the mechanism independent of cyclic AMP, i.e. an increase in [Ca2+]i, was the same as that of Sham cells. This finding indicates that, under appropriate conditions, the viable working cells of the failing rat heart can generate normal contractile function (Figs. 3C and 4CGo). These results suggest that the response of myofilament to intracellular Ca2+ concentration may be normal in this experimental model. The difference in cardiac cell function between rats with CAL and sham-operated rats may be attributable only to their cell lengths. Thus, we conclude that longer cardiac cells in CAL animals can produce larger cell shortening.

Nonmyocytes including fibroblasts, smooth muscle cells, and endothelial cells constitute two-thirds of the myocardial cell population in terms of number [34,35]. Unlike cardiomyocytes, nonmyocytes can proliferate even in adult hearts. Upon exposure to various stresses, such as systemic overload and myocardial infarction, cardiac myocytes increase in size, whereas cardiac fibroblasts increase in number and produce extracellular matrix proteins, such as collagen and fibronectin [36,37]. Apparently, the formation of scar in the LV represents an increase in the production of nonmyocytes, as predicted by a large increase in tissue collagen content in the LV of rats with CHF. The viable LV, an area adjacent to the scar tissue, may also contain a considerable number of nonmyocytes. In contrast, there was no increase in collagen content of the septum and RV, indicating a minor production of nonmyocytes in these regions. We observed decreases in cardiomyocyte Gs{alpha} content in the viable LV, septum and RV, and in tissue Gs{alpha} content in the septum, and RV. Since the tissue Gs{alpha} content in the scar zone, where a great number of nonmyocytes had proliferated after myocardial infarction, was markedly increased, the reduction in Gs{alpha} protein of the septum and RV of CAL rats may represent a substantial decrease in this protein in these regions. With respect to alterations in Gi{alpha} protein in the failing heart, there were no alterations in this protein in CAL cells of the LV, septum, and RV, whereas an appreciable increase in Gi{alpha} protein of the scar and viable LV was seen without a significant increase in tissue levels of this protein in the septum and RV. These observations show a significant contribution of nonmyocytes to the alterations in the tissue Gs{alpha} and Gi{alpha} protein contents in the failing heart.

It is generally considered that an excessive accumulation of extracellular matrix proteins is one of the signs of the left ventricular remodeling, eventually leading to cardiac dysfunction [38]. Assessment of the molecular mechanism for proliferation of cardiac fibroblasts is thus meaningful. Several investigators reported that nonmyocytes regulate the development of cardiomyocyte hypertrophy at least partially via endothelin-1 and cardiotrophin-1 secretion [39,40]. Our findings of the increase in Gi{alpha} protein levels in tissues without changes in the protein levels in cardiomyocytes and the increase in tissue collagen content may predict a contribution of nonmyocytes to induction and/or development of cardiac hypertrophy.

There are a number of limitations to the experimental design of current study that should be taken into account when considering the results. The population of cardiomyocytes examined may not be representative of cells in CAL hearts. Undamaged cardiomyocytes may have better tolerated the cardiomyocyte isolation process and may, therefore, constitute a higher proportion of the total number of cells than in the whole heart. Cardiomyocytes were selected on their ability to contract, which may also bias the selection in favour of undamaged cardiomyocytes. It should also be remembered that isolated cardiomyocytes are unloaded and therefore differ significantly from the in vivo state.

In summary, our study showed that global LV systolic and diastolic dysfunction of the rats with CHF was not attributed to reduced contractile function of the cardiomyocytes. The response to β-adrenoceptor stimulation was reduced in cardiac cells isolated from animals with CHF, whereas the direct response of adenylate cyclase was preserved. There was a decrease in the level of Gs{alpha} protein in cardiomyocytes of the failing heart. These findings suggest that severe defects in the signal transduction from β-adrenoceptor to adenylate cyclase such as β-adrenoceptor down-regulation and shortage of Gs{alpha} protein may exist in the failing cardiomyocytes.

Time for primary review 21 days.


   Acknowledgements
 
The authors wish to thank Dr. L.D. Frye, Ph.D. for editing the text.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Hasking G.J, Esler M.D, Jennings G.L, et al. Norepinephrine spillover to plasma in patients with congestive heart failure: evidence of increased overall and cardiorenal sympathetic nervous activity. Circulation (1986) 73:615–621.[Abstract/Free Full Text]
  2. Swedberg K, Viquerat C, Rouleau J.L, et al. Comparison of myocardial catecholamine balance in congestive heart failure and in angina pectoris without heart failure. Am. J. Cardiol. (1984) 54:783–786.[CrossRef][Web of Science][Medline]
  3. Brodde O.-E. Pathophysiology of the β-adrenoceptor system in chronic heart failure: consequences for treatment with agonists, partial agonist or antagonists? Eur. Heart J. (1991) 12(Suppl. F):54–62.[Abstract/Free Full Text]
  4. Ungerer M, Bohm M, Elce J.S, et al. Altered expression of β-adrenergic receptor kinase and β1-adrenergic receptors in the failing human heart. Circulation (1993) 87:454–463.[Abstract/Free Full Text]
  5. Ungerer M, Parruti G, Bohm M, et al. Expression of β-arrestins and β-adrenergic receptor kinases in the failing human heart. Circ. Res. (1994) 74:206–213.[Abstract/Free Full Text]
  6. Brodde O.-E, Michel M.C, Zerkowski H.-R. Signal transduction mechanisms controlling cardiac contractility and their alterations in chronic heart failure. Cardiovasc. Res. (1995) 30:570–584.[Free Full Text]
  7. Eschenhagen T, Mende U, Nose M, et al. Increased messenger RNA level of the inhibitory G protein {alpha} subunit Gi{alpha}-2 in human end-stage heart failure. Circ. Res. (1992) 70:688–696.[Abstract/Free Full Text]
  8. Feldman A.M, Cates A.E, Bristow M.R, et al. Altered expression of {alpha}-subunits of G proteins in failing human hearts. J. Mol. Cell Cardiol. (1989) 21:359–365.[CrossRef][Web of Science][Medline]
  9. Sethi R, Elimban V, Chapman D, et al. Differential alterations in left and right ventricular G-proteins in congestive heart failure due to myocardial infarction. J. Mol. Cell Cardiol. (1998) 30:2153–2163.[CrossRef][Web of Science][Medline]
  10. Shi B, Heavner J.E, McMahon K.K, et al. Dynamic changes in G{alpha}i-2 levels in rat hearts associated with impaired heart function after myocardial infarction. Am. J. Physiol. (1995) 269:H1073–H1079.[Web of Science][Medline]
  11. Peterson D.J, Ju H, Hao J, et al. Expression of Gi-2{alpha} and Gs{alpha} in myofibroblasts localized to the infarct scar in heart failure due to myocardial infarction. Cardiovasc. Res. (1999) 41:575–585.[Abstract/Free Full Text]
  12. Sanbe A, Tanonaka K, Hanaoka Y, et al. Regional energy metabolism of failing hearts following myocardial infarction. J. Mol. Cell Cardiol. (1993) 25:995–1013.[CrossRef][Web of Science][Medline]
  13. Fletcher P.J, Pfeffer J.M, Pfeffer M.A, et al. Left ventricular diastolic pressure–volume relations in rats with healed myocardial infarction: effects on systolic function. Circ. Res. (1981) 49:618–626.[Abstract/Free Full Text]
  14. Miyake R, Yoshida H, Tanonaka K, et al. Characterization of positive inotropic effect of colforsin daropate hydrochloride, a water soluble forskolin derivative, in isolated adult rat cardiomyocytes. Can. J. Physiol. Pharmacol. (1999) 77:225–234.[CrossRef][Web of Science][Medline]
  15. Spurgeon H.A, Stern M.D, Baartz G, et al. Simultaneous measurement of Ca2+, contraction, and potential in cardiac myocytes. Am. J. Physiol. (1990) 258:H574–H586.[Web of Science][Medline]
  16. Ikenouchi H, Kangawa K, Matsuo H, et al. Negative inotropic effect of adrenomedullin in isolated adult rabbit cardiac ventricular myocytes. Circulation (1997) 95:2318–2324.[Abstract/Free Full Text]
  17. McMahon K.K. Developmental changes of the G proteins-muscarinic cholinergic receptor interactions in rat heart. J. Parmacol. Exp. Ther. (1989) 251:372–377.[Abstract/Free Full Text]
  18. Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. (1976) 72:248–254.[CrossRef][Web of Science][Medline]
  19. Hayashi H, Sato K, Kuruhara Y, et al. Microsphere embolism-induced changes in noradrenaline release in the cerebral cortex in rats. Brain Res. (1998) 783:241–248.[CrossRef][Web of Science][Medline]
  20. Anand-Srivastava M.B. Enhanced expression of inhibitory guanine nucleotide regulatory protein in spontaneously hypertensive rats. Biochem. J. (1992) 288:79–85.[Web of Science][Medline]
  21. Lopez-De Leon A, Rojkind M. A simple micromethod for collagen and total protein determination in formalin-fixed paraffin-embedded sections. J. Histochem. Cytochem. (1985) 33:737–743.[Abstract]
  22. Zimmer H.-G, Gerdes A.M, Lortet S, et al. Changes in heart function and cardiac cell size in rats with chronic myocardial infarction. J. Mol. Cell Cardiol. (1990) 22:1231–1243.[CrossRef][Web of Science][Medline]
  23. Capasso J.M, Anversa P. Mechanical performance of spared myocytes after myocardial infarction in rats: effects of captopril treatment. Am. J. Physiol. (1992) 263:H841–H849.[Web of Science][Medline]
  24. Li P, Park C, Micheletti R, et al. Myocyte performance during evolution of myocardial infarction in rats: effects of propionyl-L-carnitine. Am. J. Physiol. (1995) 268:H1702–H1713.[Web of Science][Medline]
  25. Anand I.S, Liu D, Chugh S.S, et al. Isolated myocyte contractile function is normal in postinfarct remodeled rat heart with systolic dysfunction. Circulation (1997) 96:3974–3984.[Abstract/Free Full Text]
  26. Lefroy D.C, Crake T, Monte F.D, et al. Angiotensin II and contraction of isolated myocytes from human, guinea pig, and infarcted rat hearts. Am. J. Physiol. (1996) 270:H2060–H2069.[Medline]
  27. Melillo G, Lima J.A.C, Judd R.M, et al. Intrinsic myocyte dysfunction and tyrosine kinase pathway activation underlie the impaired wall thickening of adjacent regions during postinfarct left ventricular remodeling. Circulation (1996) 93:1447–1458.[Abstract/Free Full Text]
  28. Weber K.T, Pick R, Janicki J.S, et al. Inadequate collagen tethers in dilated cardiopathy. Am. Heart J. (1988) 116:1641–1646.[CrossRef][Web of Science][Medline]
  29. Hein S, Kostin S, Heling A, et al. The role of the cytoskeleton in heart failure. Cardiovasc. Res. (2000) 45:273–278.[Abstract/Free Full Text]
  30. Tyagi S.C. Dynamic role of extracellular matrix metalloproteinases in heart failure. Cardiovasc. Pathol. (1998) 7:153–159.[CrossRef][Web of Science]
  31. Fujita A, Takahira T, Hosono M, et al. Improvement of drug-induced cardiac failure by NKH477, a novel forskolin derivative, in the dog heart–lung preparation. Jpn J. Pharmacol. (1992) 58:375–381.[Medline]
  32. Sanbe A, Takeo S. Effects of NKH477, a water-soluble forskolin derivative, on cardiac function in rats with chronic heart failure after myocardial infarction. J. Pharmacol. Exp. Ther. (1995) 274:120–126.[Abstract/Free Full Text]
  33. Sanbe A, Takeo S. Long-term treatment with angiotensin I-converting enzyme inhibitors attenuates the loss of cardiac β-adrenoceptor responses in rats with chronic heart failure. Circulation (1995) 92:2666–2675.[Abstract/Free Full Text]
  34. Brilla C.G, Maisch B, Weber K.T. Myocardial collagen matrix remodeling in arterial hypertension. Eur. Heart J. (1992) 13(suppl_D):24–32.
  35. Eghbali M. Cardiac fibroblasts: function of gene expression, and phenotypic modulation. Basic Res. Cardiol. (1992) 87(suppl 2):183–189.[Web of Science][Medline]
  36. Nawata J, Ohno I, Isoyama S, et al. Differential expression of {alpha}1,{alpha}3 and {alpha}5 integrin subunits in acute and chronic stages of myocardial infarction in rats. Cardiovasc. Res. (1999) 43:371–381.[Abstract/Free Full Text]
  37. Heling A, Zimmermann R, Kostin S, et al. Increased expression of cytoskeletal, linkage, and extracellular proteins in failing human myocardium. Circ. Res. (2000) 86:846–853.[Abstract/Free Full Text]
  38. Weber K.T. Extracellular matrix remodeling in heart failure: A role of de novo Angiotensin II generation. Circulation (1997) 96:4065–4082.[Free Full Text]
  39. Harada M, Itoh H, Nakagawa O, et al. Significance of ventricular myocytes and nonmyocytes interaction during cardiocyte hypertrophy: evidence for endothelin-1 as a paracrine hypertrophic factor from cardiac nonmyocytes. Circulation (1997) 96:3737–3744.[Abstract/Free Full Text]
  40. Kuwahara K, Saito Y, Harada M, et al. Involvement of cardiotrophin-1 in cardiac myocyte–nonmyocyte interactions during hypertrophy of rat cardiac myocytes in vitro. Circulation (1999) 100:1116–1124.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Yoshida, H.
Right arrow Articles by Takeo, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yoshida, H.
Right arrow Articles by Takeo, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?