Cardiovascular Research

Cardiovascular Research 2002 55(4):757-767; doi:10.1016/S0008-6363(02)00457-1
© 2002 by European Society of Cardiology

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Copyright © 2002, European Society of Cardiology

Critical role of Rho-kinase pathway for cardiac performance and remodeling in failing rat hearts

Naohiko Kobayashi^*, Shigeo Horinaka, Shin-ichiro Mita, Shigefumi Nakano, Takeaki Honda, Kohtaro Yoshida, Tsutomu Kobayashi and Hiroaki Matsuoka

Department of Hypertension and Cardiorenal Medicine, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan

a-fukuda{at}dokkyomed.ac.jp

^* Corresponding author. Tel.: +81-282-87-2149; fax: +81-282-86-1596

Received 16 November 2001; accepted 19 April 2002

	Abstract

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

 
Objectives: Rho and Rho-kinase play a critical role in the regulation of cellular functions such as proliferation and migration. To elucidate the molecular mechanisms that regulate cardiac function and cardiovascular remodeling, we determined whether the signaling pathway through Rho is involved in Dahl salt-sensitive hypertensive rats with congestive heart failure (CHF) using a specific Rho-kinase inhibitor, Y-27632. Methods: Y-27632 was administered from the left ventricular hypertrophy stage (11 weeks) to the CHF stage (18 weeks) for 7 weeks. The left ventricular end-systolic pressure–volume relationship (contractility: E_es) was evaluated using a conductance catheter. Results: Downregulated E_es in the CHF stage was significantly ameliorated by Y-27632 treatment. Increased RhoA protein, Rho-kinase gene expression and myosin light chain phosphorylations in CHF rats were suppressed by Y-27632. Upregulated proto-oncogene c-fos gene expression in CHF rats was decreased by inhibiting Rho-kinase. In contrast, Y-27632 showed no effect on upregulated extracellular signal-regulated kinases (ERK) and p70S6 kinase phosphorylations, which were reported to be involved in protein synthesis. In the CHF stage, Y-27632 effectively inhibited vascular lesion formation such as medial thickness and perivascular fibrosis. Conclusions: These results suggest that differential activation of the Rho–Rho-kinase and the ERK–p70S6 kinase pathways may play a critical role in CHF, and the Rho–Rho-kinase pathway is involved in the pathogenesis of cardiac dysfunction and cardiovascular remodeling. Thus, inhibition of the Rho-kinase pathway may be at least a potential therapeutic strategy for CHF.

KEYWORDS Contractile function; G-proteins; Heart failure; Remodeling; Signal transduction

	1. Introduction

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

 
Congestive heart failure (CHF) begins with a cardiac insult such as long-standing hypertension, valvular disease, ischemic injury, or idiopathic myocardial dysfunction. The pathology of the end-stage failing human heart is characterized by myocyte loss, myocardial collagen accumulation and collagen fibril disruption, remodeling of the extracellular matrix, and disorganization of the cardiac myofibrils. Moreover, this maladaptive remodeling contributes to the diminished systolic performance as well as the decreased compliance of the failing human heart, and has been shown to be an important predictor of morbidity and mortality [1]. Therefore, identifying the pathway of molecular and cellular events that contribute to cardiovascular remodeling is likely to provide novel targets for preventing disease progression in heart failure. Extracellular signal-regulated kinases composed of p42ERK and p44ERK (ERK1/2), are one main subgroup of mitogen-activated protein kinases and are important mediators of the signal-transduction pathway responsible for cell differentiation and growth. Recent evidence suggests that ERK in cultured neonatal rat cardiac myocytes are rapidly activated by various extracellular stimuli, such as growth factors and other mitogens, and that they play a key role in cell growth and the regulation of various gene expressions [2]. On the other hand, p70S6 kinase, which leads to phosphorylation of the ribosomal S6 protein and increases the rate of translation of mRNAs containing a polypyrimidine tract, has been shown to activate in several cell types after either mitogenic stimulation, mechanical stretch, or integrin receptor engagement. Therefore, p70S6 kinase could have a key role on the load-induced hypertrophic growth process [3].

Rho and Rho-kinase, a downstream target protein of small GTP-binding protein Rho, play crucial roles in various cellular functions, and mediating cellular events such as changes in cell morphology, cell motility, focal adhesions, and cytokinesis [4,5]. The possibility that Rho is involved in vascular proliferation and migration is suggested by the involvement of Rho in the growth of nonvascular cells in response to heterotrimeric G protein receptor stimulation and in the migration of endothelial cells in response to mechanical strain or tyrosine kinase growth factors [6]. Indeed, the Rho and Rho-kinase pathway is involved in DNA synthesis and migration in vascular smooth muscle cells (VSMCs) of rat aorta [7]. In addition, cardiomyocyte hypertrophy and myofibrillar assembly are blocked by inhibitory mutants of Rho-kinase, which suggests a role for this Rho effector in cellular growth responses [8]. A recent study demonstrated that Rho-kinase may play a role in angiotensin II (Ang II)-induced hypertrophic changes of VSMCs [9]. Furthermore, in vivo study, Rho-kinase was involved in hypertensive vascular diseases of spontaneously hypertensive rats (SHR) [10]. Therefore, inhibition of the Rho-kinase pathway may be useful in the treatment of arteriosclerotic cardiovascular diseases such as hypertensive heart failure. However, the molecular mechanism responsible for the Rho-kinase pathway in CHF remains to be determined. Recently, the agent Y-27632 was shown to specifically inhibit these Rho-dependent kinases [11]. To elucidate the potential cardioprotective effects of a specific Rho-kinase inhibitor, we evaluated the effects of Y-27632 on cardiac performance and cardiovascular remodeling, and also clarified the relation between the Rho–Rho-kinase and ERK–p70S6 kinase pathways in the failing heart of Dahl salt-sensitive (DS) hypertensive rats.

	2. Methods

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

 
2.1 Animal model and experimental designs
All procedures were performed in accordance with international standards on animal welfare. Male inbred DS and Dahl salt-resistant (DR) rats, which were originally obtained from Brookhaven National Laboratories (Upton, NY, USA) were bred and supplied by Eisai, After weaning, the rats were fed a diet containing 0.3% NaCl until the age of 6 weeks. Thereafter, they were fed a diet containing 8% NaCl (Oriental Bioservice Kanto). The rats were weighed, and their systolic blood pressure (SBP) was measured by the tail-cuff method (MK-1100, Muromachi Kikai) before feeding with 8% NaCl diet and at 1-week intervals thereafter. In DS rats fed an 8% NaCl diet after the age of 6 weeks, a stage of concentric left ventricular hypertrophy at 11 weeks was followed by a distinct stage of left ventricular failure with chamber dilatation at 18 weeks (DSCHF) [12,13]. Fourteen 11-week-old DS hypertensive rats were randomly divided into two groups (DSCHF-V: n = 6, and DSCHF-R: n = 8). An osmotic minipump (model 2ML4, Alzet) containing Y-27632 dissolved saline was implanted, and Y-27632 (DSCHF-R: 10 mg/kg per day, subdepressor dose; WelFide) or vehicle (DSCHF-V) were continuously infused from 11 to 18 weeks, and age-matched DR rats (DR-C, 18 weeks, n = 6) fed the same diet served as a control group (Fig. 1).

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Schematic representation of study groups. DR-C, Dahl salt-resistant rats (control group); DSCHF-V, failing heart of Dahl salt-sensitive hypertensive rats treated with vehicle; DSCHF-R, failing heart of Dahl salt-sensitive hypertensive rats treated with Y-27632.

 
2.2 Left ventricular pressure–volume relation
The chest was opened via a midline sternotomy, and the pericardium was dissected to expose the heart. The left ventricular end-systolic pressure–volume relationship (ESPVR) was modified for the conductance catheter technique as described previously [14]. Briefly, the conductance catheter was inserted into the LV through the apex and was pushed until the distal tip was placed into the ascending aorta along the longitudinal axis of the LV. A 3F catheter-tip micromanometer (SPR-524, Millar instruments) was also inserted into the LV from the apex. To change the preload, a snare was placed around the inferior vena cava. We recorded conductance volume and left ventricular pressure simultaneously during gradual inferior vena cava occlusion. Electrical signals were digitized through an analog-to-digital converter (AD12-8, Contec) at a sampling frequency of 1000 Hz with 12-bit resolution and stored on the personal computer (Dynabook SS 330, Toshiba). The points of the ESPVR were determined using an iterative technique reported previously [15]. In several consecutive pressure–volume loops, the points of each cardiac cycle with a maximum pressure-to-volume ratio were first determined. Linear regression of these points with expression.

yielded estimates for the slope, or end-systolic elastance (E_es), and volume–axis intercept (V₀), where P_es and V_es are end-systolic pressure and volume, respectively.

2.3 Reverse transcription-polymerase chain reaction (RT-PCR) analysis of Rho-kinase, c-fos and endothelial nitric oxide synthase (eNOS) mRNA
The RT-PCR was performed by the standard method with 1 µg of total RNA. First-strand cDNA was synthesized with random primers and Molony murine leukemia virus reverse transcriptase (Promega). PCR amplification was then performed with synthetic gene-specific primers for Rho-kinase (sense primer, 5'-GCA CAT GTA TGA AAA TGG ATG AAAC-3'; antisense primer, 5'-CAT AAT TTT GCT GTA GGT TCC TAC AAGT-3'), c-fos (sense primer, 5'-GGG ACA GCC TTT CCT ACT ACC ATT-3'; antisense primer, 5'-CGC AAA AGT CCT GTG TGT TGA-3'), and eNOS (sense primer, 5'-TCC AGT AAC ACA GAC AGT GCA-3'; antisense primer, 5'-CAG GAA GTA AGT GAG AGC-3') using a DNA PCR kit (Perkin Elmer) for 30 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and elongation at 72 °C for 1 min. GAPDH was used as the housekeeping gene. The reaction conditions were optimized to obtain reproducible and reliable amplification within the logarithmic phase of the reaction, as determined by preliminary experiments. The reaction was linear to 35 cycles with use of the ethidium bromide detection method. The intensity of each band was quantified using a densitometer [12,13,16,17].

2.4 Western blot analysis of RhoA and eNOS
LV was homogenized (25% w/v) in 10 mmol/l HEPES buffer, pH 7.4, containing 320 mmol/l sucrose, 1 mmol/l EDTA, 1 mmol/l DTT, 10 µg/ml leupeptin, and 2 µg/ml aprotinin at 0–4 °C with a polytron homogenizer. The homogenate was centrifuged at 3000 g for 5 min at 4 °C (eNOS), and then the supernatant was centrifuged at 100 000 g for 30 min to generate membrane and cytosolic fractions as described previously (RhoA) [18]. Protein concentrations were determined with bovine serum albumin as a standard protein. Equal amounts of protein from membrane and cytosolic fractions (RhoA) and the other supernatant samples (eNOS) were loaded in each lane of SDS–PAGE using 13 and 10% gels. The proteins in the gels were transferred electrophoretically to PVDF sheets for 1 h at 2 mA/cm² (40 V, 300 mA, overnight). The sheets were immunoblotted with an anti-RhoA and anti-eNOS antibody (Santa Cruz, Transduction Laboratories) in a buffer containing 10 mmol/l Tris–HCl, pH 7.5, 100 mmol/l NaCl, 0.1% Tween 20, and 5% skim milk followed by peroxidase-conjugated anti-mouse IgG (Amersham Life Science Inc). The RhoA and eNOS proteins transferred to the sheets were detected using the ECL immunoblotting detection system (Amersham Life Science) [12,13,19,20].

2.5 Western blot analysis of ERK1/2, p70S6 kinase and myosin light chain (MLC) phosphorylations
Left ventricular ERK1/2, p70S6 kinase and MLC phosphorylations were measured as described in detail previously [3,21]. Briefly, using rabbit polyclonal phospho-ERK1/2, phospho-p70S6 kinase and goat polyclonal phospho-MLC antibody (New England Biolabs, Santa Cruz, CA, USA) and anti-total ERK1/2, anti-total p70S6 kinase and anti-total MLC antibody (New England Biolabs) recognizing threonine-phosphorylated forms (active forms) of ERK1/2, p70S6 kinase and MLC, we measured left ventricular phosphorylated ERK1/2, p70S6 kinase and MLC proteins with Western blot analysis. Left ventricular protein extracts were boiled for 5 min in Laemmli sample buffer, then electrophoresed on an SDS–PAGE using 13% gels, and the separated proteins were electrophoretically transferred to Hybond–PVDF membranes. Complete protein transfer to the membrane was ensured by staining the gels with Coomassie blue. The membrane was incubated with phospho-specific ERK1/2, p70S6 kinase and MLC antibody for 1 h at room temperature, washed four times with TBS–T, and then incubated with horseradish peroxidase-conjugated donkey anti-rabbit and goat immunoglobulin (Amersham Life Sciences).

2.6 Histologic examination and evaluation of cardiovascular remodeling
Histological examination was studied as described in detail previously [12,13,16,17,19–21]. Briefly, excised hearts were perfused with physiological saline solution containing adenosine 10 µg/kg and nitroglycerin 10 µg/kg and then with 6% formaldehyde solution via retrograde infusion into the ascending aorta at a pressure of 90 mmHg. The LV was separated from the right ventricle, the atria, and the great vessels, and cut into five pieces perpendicular to the long axis. For light microscopy, 1.5-µm thick sections were cut using an ROM-380 microtome (Yamato Kohki, Saitama, Japan). Paraffin slices from each heart were mounted on glass slides and stained with hematoxylin–eosin and Masson's trichrome stains. All histopathological sections of each animal were examined using a 3CCD color video camera (DXC-930; Sony, Tokyo, Japan) mounted on a standard microscope (BHS-F; Olympus, Tokyo, Japan). Drawings of the limits of the vessels were made on the screen of a multiscan color computer display (model CPD-17SF7; Sony) and then digitized with a two-dimensional analysis system (Mac SCOPE; Mitani, Fukui, Japan) connected to a Macintosh computer system (Power Macintosh G3; Apple Computer, Cupertino, CA, USA). Histopathological findings of the myocardium and coronary arterioles were examined. We always measured the capillary density and cross-sectional surface area in the endocardium of the posterior portion of the left ventricular free wall. In this part of the heart, shrinkage was minimal and orientation of the myocardial fibers was similar from one heart to another. We analyzed five sites from each ventricle in all rats. To assess thickening of the coronary arterial wall and perivascular fibrosis, the transsectional images of the area of the total small arteriolar lumen 10⁴ µm² were studied. The inner border of the lumen and the outer border of the tunica media were traced in each arterial image with hematoxylin–eosin staining at x100 to x400 magnification, and the areas encircled by the tracings were calculated. In quantification, non-round vessels resulting from oblique transsection or branching were excluded, and only round vessels were studied. The wall-to-lumen ratio (the area of the vessel wall divided by the area of the total blood vessel lumen) was determined. The area of fibrosis immediately surrounding blood vessels was calculated, and perivascular fibrosis was determined as the ratio of the area of fibrosis surrounding the vessel wall to the total area of the vessel. To assess the area of myocardial fibrosis, the area of pathological collagen deposition was measured in the microscopic field of each Masson's trichrome-stained section. The ratio of the total area of fibrosis within the left ventricular myocardium to the total area of the left ventricular myocardium in each heart was calculated and used for analysis. The histopathological examination of the sections from each rat was carried out by an operator who was blinded to the treatment groups.

2.7 Statistical analysis
All results are expressed as the mean±S.E.M. The mean values were compared among the three groups using ANOVA followed by the Bonferroni test. Differences at P<0.05 were considered statistically significant. Calculations, including those of derived values, and statistical tests, were performed using the appropriate software (STATVIEW-J 4.5, Abacus Concepts) and a Power Macintosh computer system (G4, Apple Computer).

	3. Results

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

 
3.1 Systemic hemodynamics, body weight, and organ weight
Time-related changes in SBP among the three groups are shown in Fig. 2. Before feeding with 8% NaCl diet, SBP was 115±3 mmHg in DR-C, 118±4 mmHg in DSCHF-V and 116±4 mmHg in DSCHF-R. As shown in Table 1, SBP in DSCHF-V and DSCHF-R was similar and significantly higher than that in DR-C. Increased heart rate in DSCHF-V was significantly attenuated in DSCHF-R. Body weight in DSCHF-V was significantly decreased compared with DR-C, and significantly increased in DSCHF-R compared with DSCHF-V. The body-weight-corrected left ventricular mass of the DSCHF-V was significantly greater than that of the DR-C, and significantly attenuated in DSCHF-R compared with DSCHF-V after 7 weeks treatment with Y-27632. The body-weight-corrected lung and liver weight was significantly increased in DSCHF-V compared with the DR-C, and significantly decreased in DSCHF-R compared with the DSCHF-V after 7 weeks treatment with Y-27632.

View larger version (25K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Time-related changes in systolic blood pressure in the DR-C, DSCHF-V, and DSCHF-R groups. DR-C, Dahl salt-resistant rats (control group); DSCHF-V, failing heart of Dahl salt-sensitive hypertensive rats treated with vehicle; DSCHF-R, failing heart of Dahl salt-sensitive hypertensive rats treated with Y-27632. Values are expressed as means±S.E.M. **, P<0.01 vs. DR-C.

 

View this table: [in this window] [in a new window]   Table 1 Effects of Y-27632 on systemic hemodynamics, body weight, and organ weight in Dahl rats

 
3.2 Left ventricular E_es, end-diastolic pressure, and end-systolic pressure in ESPVR
Left ventricular E_es in ESPVR was significantly lower in DSCHF-V than in DR-C (1178±106 vs. 2223±298 mmHg/ml, P<0.01), and significantly greater in DSCHF-R than in DSCHF-V (3745±276 mmHg/ml vs. DSCHF-V, P<0.01) (Figs. 3 and 4A). Left ventricular end-diastolic pressure in ESPVR was significantly increased in DSCHF-V compared with the DR-C (20.29±1.36 vs. 4.87±0.50 mmHg, P<0.01), and significantly decreased in DSCHF-R compared with the DSCHF-V (9.89±0.37 mmHg vs. DSCHF-V, P<0.01) (Fig. 4B). In ESPVR, left ventricular end-systolic pressure in DSCHF-V and DSCHF-R was similar and significantly higher than that in DR-C (DSCHF-V: 132.2±9.7, DSCHF-R: 137.0±9.3, vs. DR-C: 92.5±9.4 mmHg, P<0.05, respectively).

View larger version (31K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Effects of Y-27632 treatment on left ventricular Ees in ESPVR. Data are shown typical pressure–volume loop relationship in DR-C (A), DSCHF-V (B), and DSCHF-R (C). Left ventricular Ees in ESPVR was significantly lower in DSCHF-V than in DR-C, and significantly greater in DSCHF-R than in DSCHF-V. DR-C, Dahl salt-resistant rats (control group); DSCHF-V, failing heart of Dahl salt-sensitive hypertensive rats treated with vehicle; DSCHF-R, failing heart of Dahl salt-sensitive hypertensive rats treated with Y-27632; Ees, end-systolic elastance; ESPVR, end-systolic pressure–volume relationship.

 

View larger version (28K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Effects of Y-27632 treatment on left ventricular Ees (A) and end-diastolic pressure (B) in ESPVR. DR-C, Dahl salt-resistant rats (control group); DSCHF-V, failing heart of Dahl salt-sensitive hypertensive rats treated with vehicle; DSCHF-R, failing heart of Dahl salt-sensitive hypertensive rats treated with Y-27632; Ees, end-systolic elastance; ESPVR, end-systolic pressure–volume relationship. Values are expressed as means±S.E.M. **, P<0.01 vs. DR-C, , P<0.01 vs. DSCHF-V.

 
3.3 Involvement of RhoA and Rho-kinase expression and activity in the failing heart
The levels of RhoA protein in the LV were 3.8-fold (P<0.01) larger in DSCHF-V than in DR-C, and were 70% (P<0.01) lower in DSCHF-R than in DSCHF-V (Fig. 5A). Expressions of Rho-kinase mRNA were 4.3-fold (P<0.01) larger in DSCHF-V than in DR-C, and were 66% (P<0.01) lower in DSCHF-R than in DSCHF-V (Fig. 5B). In addition, to quantify the activity of Rho-kinase in failing heart, we performed Western blot analysis for phosphorylated MLC. The left ventricular phospho-MLC activity was 4.2-fold (P<0.01) larger in DSCHF-V than in DR-C, and 74% (P<0.01) lower in DSCHF-R than in DSCHF-V (Fig. 5C).

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Effects of Y-27632 treatment on RhoA protein (A), Rho-kinase mRNA (B), and phospho-MLC. Top panels are representative typical RT-PCR and Western blot bands. Bottom panels show percent of control of RhoA (A), phospho-MLC (C), and the mean densities of the Rho-kinase (B) bands in relation to GAPDH. Lanes: 1=DR-C, 2=DSCHF-V, and 3=DSCHF-R. Values are expressed as means±S.E.M. **, P<0.01 vs. DR-C; , P<0.01 vs. DSCHF-V.

 
3.4 Effect of a Rho-kinase inhibitor on c-fos mRNA
The levels of c-fos mRNA were 3.0-fold (P<0.01) larger in DSCHF-V than in DR-C, and were 59% (P<0.01) lower in DSCHF-R than in DSCHF-V (Fig. 6A).

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Effects of Y-27632 treatment on c-fos mRNA (A), ERK1/2 (B) and p70S6 kinase (C) phosphorylations. Top panels are representative typical RT-PCR and Western blot bands. Bottom panels show the mean densities of the c-fos mRNA (A) bands in relation to GAPDH, and percent of control of ERK1/2 (B) and p70S6 kinase (C) phosphorylations. Lanes: 1=DR-C, 2=DSCHF-V, and 3=DSCHF-R. Values are expressed as means±S.E.M. *, P<0.05; **, P<0.01 vs. DR-C; , P<0.01 vs. DSCHF-V.

 
3.5 Relation between Rho–Rho-kinase and ERK–p70S6 kinase pathway
To evaluate the relation between the Rho–Rho-kinase and ERK–p70S6 kinase pathways, we examined whether the Rho–Rho-kinase pathway was involved in ERK1/2 and p70S6 kinase activities in the failing heart. The left ventricular phospho-ERK1/2 activities were significantly higher in DSCHF-V than in DR-C, and the phosphorylation of ERK1/2 activation was not inhibited by Y-27632 (Fig. 6B). Moreover, similarly cardiac phospho-p70S6 kinase activity was significantly increased in DSCHF-V compared with DR-C, and the phospho-p70S6 kinase activation was not inhibited by Y-27632 (Fig. 6C).

3.6 Effect of a Rho-kinase inhibition on cardiovascular remodeling
The wall-to-lumen ratio was increased in DSCHF-V compared with DR-C but was significantly decreased by Y-27632 treatment (Fig. 7A,D–F). The degrees of perivascular fibrosis were significantly greater in DSCHF-V than in DR-C, and was also significantly decreased by Y-27632 treatment (Fig. 7B,D–F). Compared with DR-C, myocardial fibrosis was significantly greater in DSCHF-V, and it was significantly less in DSCHF-R than in DSCHF-V (Fig. 7C).

View larger version (105K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Effects of Y-27632 on cardiovascular remodeling in failing heart of Dahl salt-sensitive hypertensive rats. Wall-to-lumen ratio (A), perivascular fibrosis (B), and myocardial fibrosis (C) measured histopathologically. Values are expressed as means±S.E.M. **, P<0.01 vs. DR-C; , P<0.01 vs. DSCHF-V. Micrographs of small coronary arteries with Masson's trichrome stain for DR-C (D), DSCHF-V (E), and DSCHF-R (F). Bar, 100 µm.

 
3.7 Effect of Y-27632 on eNOS mRNA and protein levels
To evaluate the mechanisms of the beneficial effect of inhibiting the Rho-kinase pathway, expression of eNOS mRNA and protein was measured. Left ventricular eNOS mRNA levels were 57% (P<0.05) lower in DSCHF-V than in DR-C, and were 6.0-fold (P<0.01) larger in DSCHF-R than in DSCHF-V (Fig. 8A). The levels of eNOS protein in the LV were 54% (P<0.05) lower in DSCHF-V than in DR-C, and were 4.2-fold (P<0.01) larger in DSCHF-R than in DSCHF-V (Fig. 8B).

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 8 Effects of Y-27632 treatment on eNOS mRNA (A) and eNOS protein (B). Top panels are representative typical RT-PCR and Western blot bands. Bottom panels show the mean densities of the eNOS mRNA (A) bands in relation to GAPDH, and percent of control of eNOS protein (B). Lanes: 1=DR-C, 2=DSCHF-V, and 3=DSCHF-R. Values are expressed as means±S.E.M. *, P<0.05; **, P<0.01 vs. DR-C; , P<0.01 vs. DSCHF-V.

 

	4. Discussion

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

 
In the present study, we demonstrated that Rho and Rho-kinase pathway was involved in the pathogenesis of cardiac dysfunction and cardiovascular remodeling, and that inhibition of Rho-kinase plays a critical role in amelioration of the failing heart. The Rho–Rho-kinase and ERK–p70S6 kinase pathways may be independent of each other in the signaling of the failing heart. These results suggest that differential activation of the Rho–Rho-kinase and ERK–p70S6 kinase pathways may play a key role in the failing heart, and inhibiting the Rho-kinase pathway may be a useful therapeutic strategy for CHF (Fig. 9).

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 9 Schematic chart of signal transduction pathways in failing heart.

 
The Rho and Rho-kinase pathway plays an important role in regulation of VSMCs contraction and other cellular functions such as proliferation and migration. In vitro studies, Kuwahara et al. [22] evaluated the Rho–ROCK pathway in endothelin-1 (ET-1) induced hypertrophic signals in cardiac myocytes. They suggested that Y-27632 significantly suppressed ET-1-induced hypertrophic response: augmentation of natriuretic peptide gene expression, increase in protein synthesis and cell size, and myofibrillar reorganization. More recently, Sauzeau et al. [23] reported that human urotensin II-induced VSMC proliferation was inhibited by Y-27632 or TAT-C3, a RhoA inhibitor, indicating that RhoA and Rho-kinase mediate the stimulation of VSMC growth. Moreover, in vivo studies, Mukai et al. [10] examined the role of Rho-kinase in functional and structural alterations of hypertensive blood vessels in SHR. They concluded that upregulation of Rho-kinase plays a key role in the pathogenesis of hypertensive vascular disease. Furthermore, Hisaoka et al. [24] showed that a G-protein-coupled increase in myofilament Ca²⁺ sensitivity mediated through the Rho–Rho-kinase system is heavily involved in the mechanism underlying the enhanced vasoconstriction observed in heart failure produced by chronic rapid pacing. These results suggest that Rho and Rho-kinase may be involved in the pathogenesis of cardiac dysfunction and cardiovascular remodeling [10,24].

Activation of ERK1/2 and p70S6 kinase has been reported to be closely related to protein synthesis in VSMCs. We examined whether Rho-kinase might be involved in ERK–p70S6 kinase activation. Numaguchi et al. [25] reported that the Rho and Rho-kinase pathway regulated mechanical stretch-induced ERK1/2 activation in VSMCs. In contrast, in the present study, we observed that Y-27632 showed no effect on phosphorylation of ERK–p70S6 kinase activities. Takeda et al. [26] demonstrated that inhibition of Rho-kinase Y-27632 did not affect Ang II-induced ERK activation in VSMCs. In addition, Yamakawa et al. [9] reported that Y-27632 showed no effect on the ERK1/2 and p70S6 kinase phosphorylations induced by Ang II in VSMCs. These results suggested that the Rho–Rho-kinase and ERK–p70S6 kinase pathways may be independent of each other in the signaling of the failing heart. Thus, inhibiting the Rho-kinase pathway may play a key role in CHF.

In the present study, we showed that expression of the c-fos gene was upregulated in failing heart, and also that Y-27632 inhibited this gene expression. In cultured neonatal rat myocytes, passive stretch stimulates the immediate growth response of the induction of proto-oncogenes that is mediated by the autocrine release of Ang II [27]. In addition, Kent et al. [28] has reported that load-induced c-fos expression was Ang II-dependent in quiescent adult cardiocytes subjected to passive stretch. Recently, we have demonstrated that left ventricular angiotensin-converting enzyme (ACE) mRNA expression was increased in DSCHF-V [17]. Therefore, c-fos gene expression may increase in failing heart stage. Moreover, Yamakawa et al. [9] showed that Rho-kinase is partially involved in Ang II-induced c-fos gene expression in VSMCs. Furthermore, Ueyama et al. [29] reported that activated RhoA stimulated c-fos gene expression in myocardial cells. These results suggested that Ang-II-induced expression of the c-fos gene expression might be mediated through activation of Rho-kinase.

The agent Y-27632 has been shown to specifically inhibit Rho-dependent kinases (K_i=0.14 µM for p160ROCK: >100x selectivity vs. protein kinase C, cAMP-dependent protein kinase and MLC kinase) [11]. A new pyridine derivative, Y-27632, selectively inhibits smooth-muscle contraction by inhibiting the Ca²⁺-sensitization mechanism. This compound inhibited smooth muscle contraction both in vitro and in vivo, as well as the formation of stress fibres and focal adhesions induced by p160ROCK in cultured cells. Therefore, Y-27632 is a valuable tool for investigating the functions of p160ROCK in vivo and its pathophysiological implications. As well as modulating smooth-muscle contraction, the Rho-kinase pathway may help to regulate integrin-mediated cell adhesion and motility, which could be critical in processes such as tumor-cell metastasis and immunoactivation. Thus, Y-27632 should be useful for investigating the role of p160ROCK in these processes and may be clinically important [11].

The mechanisms of the cardioprotective effect of inhibiting Rho-kinase is unknown. In the present study, we showed that expression of eNOS mRNA and protein was upregulated by inhibiting Rho-kinase. Laufs et al. [30] showed that the upregulation of eNOS expression by hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor was mediated by inhibiting Rho GTPase. They suggested that Rho negatively regulated eNOS expression and that HMG-CoA reductase inhibitors upregulated eNOS expression by blocking Rho. These results suggest that the production of eNOS expression by inhibiting the Rho–Rho-kinase pathway may play a critical role in the cardioprotective effect of cardiac dysfunction and remodeling (Fig. 9).

Recent studies suggest that Rho-kinase is involved in the regulation of myofibrillar Ca²⁺ sensitivity in cardiac muscle. Sweeney et al. [31] showed that the regulatory light chain of myosin phosphorylation increases the Ca²⁺ sensitivity in skinned striated muscle preparations. In addition, Sanbe et al. [32] reported that using transgenic mice overexpressing nonphoshorylatable regulatory MLC, there was no shift of the myofibrillar Ca²⁺ sensitivity after treatment with MLC kinase, whereas the Ca²⁺ sensitivity increased depending on the treatment with MLC kinase in nontransgenic mice. These transgenic mice also showed enlargement of cardiac chambers. They concluded that the phosphorylation of regulatory MLC appeared to play an important role in maintaining normal cardiac function. Moreover, Suematsu et al. [33] reported that ₁-adrenoceptor-Gq signaling is upregulated in the failing myocardium to increase the myofibrillar Ca²⁺ sensitivity mainly through the RhoA–Rho kinase pathway rather than through the protein kinase C pathway. The increased Ca²⁺ sensitivity by the upregulated Gq signaling may be one of the abnormal regulatory mechanisms of contractility in the failing heart. These results suggest that MLC phosphorylation may play a key role in maintaining cardiac performance and remodeling.

The cardiac interstitium is composed of nonmyocyte cells and a structural protein network which plays a dominant role in governing the structure, architecture, and mechanical behavior of the myocardium. The heterogeneity in myocardial structure, created by the altered behavior of nonmyocyte cells, particularly cardiac fibroblasts which are responsible for myocardial collagen metabolism and fibrous tissue accumulation, may largely explain the appearance of diastolic and systolic myocardial failure. In addition, Ang II has been reported to be a hormonal stimulus of cardiac growth, a response that may involve myocyte hypertrophy as well as growth of nonmyocytes. Indeed, Ang II-induced cardiac hypertrophy is secondary to stimulated increases in nonmyocyte cellular growth [34]. Moreover, we have demonstrated that gene expression of type I collagen in the LV is upregulated in failing heart of DS rats [12,13]. These results suggest that possible confounding effects of nonmyocytes involved in cardiac function and cardiovascular structural changes.

In conclusion, we evaluated the cardioprotective effects of a specific Rho-kinase inhibitor, Y-27632, on cardiac performance and cardiovascular remodeling, and also clarified the relation between the Rho–Rho-kinase and ERK–p70S6 kinase pathways in the failing heart of Dahl salt-sensitive hypertensive rats. We demonstrated that the Rho and Rho-kinase pathway was involved in the pathogenesis of cardiac dysfunction and cardiovascular remodeling, and that inhibition of Rho-kinase plays a critical role in the failing heart [10,24]. The Rho–Rho-kinase and ERK–p70S6 kinase pathways may be independent of each other in the signaling of the failing heart. These results suggest that differential activation of the Rho–Rho-kinase and ERK–p70S6 kinase pathways may play a key role in the failing heart, and inhibiting the Rho-kinase pathway may be a useful therapeutic strategy for CHF.

Time for primary review 32 days.

	Acknowledgements

 
This study was supported in part by the Grant-in-Aid for Scientific Research (14570691) (C-2) from the Japan Society for the Promotion of Science (JSPS). We thank Kazumi Akimoto Ph.D. for technical assistance with RT-PCR, and Mrs. Noriko Suzuki for preparing and staining tissue sections for histological investigation, and Miss Yasuko Mamada for technical assistance.

	References

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

 

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