Original Article
Atorvastatin ameliorates cardiac fibrosis and improves left ventricular diastolic function in hypertensive diastolic heart failure model rats Hirokuni Akahori a, Takeshi Tsujino b, Yoshiro Naito a, Mika Matsumoto a, Naoko Sasaki b, Toshihiro Iwasaku a, Akiyo Eguchi a, Hisashi Sawada a, Shinichi Hirotani a, and Tohru Masuyama a
Objective: Clinical studies have suggested the beneficial effects of statin therapy on diastolic heart failure. However, the mechanism of the beneficial effects of statin on diastolic heart failure remains unknown. We examined the effect of atorvastatin on the cardiac function of Dahl salt-sensitive rat, a model of hypertensive diastolic heart failure. Methods: Dahl salt-sensitive rats were divided into three groups: the low-salt group (given standard diet), the highsalt group (given 8% NaCl diet from 7 weeks of age), and the high-salt R atorvastatin (HS R Ato) group (given 8% NaCl diet from 7 weeks of age and atorvastatin from 17 weeks of age). We evaluated left ventricular hypertrophy (LVH), fibrosis, and function by using echocardiography and histology. We also examined the expression of molecules related to fibrosis in the hearts of Dahl salt-sensitive rats and cultured rat cardiac fibroblasts. Results: Left ventricular hypertrophy, diastolic dysfunction, and cardiac fibrosis were observed in the high-salt group. Atorvastatin ameliorated cardiac fibrosis and normalized left ventricular diastolic function without altering blood pressure. Atorvastatin also decreased the expression of heat shock protein 47 (HSP47), an essential chaperone for type 1 collagen processing, without changing in expression of transforming growth factor beta. In rat cardiac fibroblast cells, atorvastatin also reduced HSP47 level induced by transforming growth factor beta. The effect of atorvastatin was reversed by mevalonate and geranylgeranyl-pyrophosphate and mimicked by Rho kinase inhibitor. Conclusion: Atorvastatin administration ameliorates cardiac fibrosis and improves left ventricular diastolic function in Dahl salt-sensitive rats. Lowering HSP47 by atorvastatin via inhibition of Rho–Rho kinase pathway is suggested as a mechanism. Keywords: cardiac fiblosis, diastolic heart failure, heat shock protein 47, statin Abbreviations: Ato, atorvastatin; Fpp, farnesylpyrophosphate; GGpp, geranylgeranylpyrophosphate; HF, heart failure; HR, heart rate; HSP, heat shock protein; LVH, left ventricular hypertrophy; MMP,
matrix metalloproteinase; TGF, transforming growth factor; TIMP, tissue inhibitor of metalloproteinase
INTRODUCTION
H
eart failure (HF) is one of the most common causes of cardiovascular morbidity and mortality [1–4]. A substantial proportion of patients with HF have diastolic heart failure [5]. There is a growing recognition that congestive heart failure caused by diastolic dysfunction is common and causes significant morbidity and mortality. The 3-hydroxy-3-methylgutaryl-CoA reductase inhibitors, commonly referred to as statins, are potent inhibitors of cholesterol biosynthesis. Recently, evidence of statin is steadily increasing regarding the beneficial, lipid-independent ‘pleiotropic’ effects on cardiovascular diseases [6–8]. Stains have indeed been suggested to affect alternative target, preventing the synthesis of important isoprenoid intermediates such as farnesylpyrophosphate (Fpp) and geranylgeranyl-pyrophosphate (GGpp) [9]. Statins would inhibit posttranslational lipid modification of a variety of intracellular signaling proteins including Ras and Rho [10]. Recent clinical and animal studies have suggested the beneficial effects of statin therapy on diastolic heart failure [11–13]. However, the mechanism of the beneficial effects of statin on diastolic heart failure remains unknown. Heat shock protein 47 (HSP47) is a basic glycoprotein that binds collagen, and it has a pivotal role as a collagenspecific molecular chaperon [14]. Recent studies have shown that inhibition of HSP47 is useful as a treatment of fibrotic disease such as pulmonary fibrosis, peritoneal fibrosis, and liver fibrosis [15–17]. Moreover, HSP47 is Journal of Hypertension 2014, 32:1534–1541 a Cardiovascular Division, Department of Internal Medicine, Hyogo College of Medicine, Nishinomiya and bDepartment of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, Kobe, Japan
Correspondence to Takeshi Tsujino, MD, PhD, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, Kobe, Japan, 1-3-6 Minatojima, Chuo-ku, Kobe, 650-8530, Japan. Tel: +81 78 304 3182; fax: +81 78 304 2812; e-mail: [email protected] Received 1 June 2012 Revised 8 December 2013 Accepted 19 February 2014 J Hypertens 32:1534–1541 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. DOI:10.1097/HJH.0000000000000184
1534
www.jhypertension.com
Volume 32 Number 7 July 2014
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Use of atorvastatin in hypertensive diastolic heart failure
associated with fibrosis following myocardial infarction and has been localized in atherosclerotic arteries [18]. However, a role of HSP47 in diastolic dysfunction has not been elucidated. Dahl salt-sensitive rat model is a well established model of hypertensive diastolic heart failure [19,20]. We performed animal studies using Dahl salt-sensitive rats to evaluate the effect of atorvastatin on diastolic heart failure in hypertensive hearts with established left ventricular hypertrophy (LVH). We also investigated the effects of atorvastatin on the expression fibrosis-related genes including HSP47.
METHODS Animals Male Dahl salt-sensitive rats (SLC Japan, Shizuoka, Japan) were fed a standard diet (0.3% NaCl) from weaning until 7 weeks. Afterward, rats were randomized to normal lowsalt diet (0.3% NaCl) group [(low-salt group) n ¼ 10], highsalt diet (8% NaCl) group [(high-salt group) n ¼ 10], or highsalt diet (8% NaCl) with administration of atorvastatin from 17 weeks of age [high-salt þ atorvastatin group (HS þ Ato group) n ¼ 10]. Atorvastatin (20 mg/kg per day) or vehicle was administrated orally by gavage from 17 weeks of age. The statin dose was based on a previous report showing a beneficial pleiotropic vascular effect in a hypertensive model [21]. Rats were maintained on a 12-h light/dark cycle and had free access to food and water. All of our experimental procedures were approved by the Animal Research Committee of Hyogo College of Medicine.
Assessments of blood pressure monitoring and urinary date Three rats for each group are chronically instrumented with telemetry probes (TA11-PA40; Data Sciences International, Minnesota, USA) for continuous monitoring of mean arterial pressure and heart rate (HR) without physical restriction. Twenty-four-hour urine samples were collected in metabolic cages for measuring urinary volume, protein, and creatinine levels.
Echocardiography Rats were anesthetized with ketamine HCl (50 mg/kg) and xylazine HCl (10 mg/kg), and evaluated by transthoracic echocardiographic studies. We measured left ventricular cavity size and wall thickness, and calculated left ventricular ejection fraction (LVEF) with a 12-MHz phased-array transducer (ACUSON; Mochida Siemens Medical Systems, Tokyo, Japan). We also recorded pulsed Doppler mitral flow velocity pattern, and measured peak early diastolic filling velocity (E), peak filling velocity at atrial contraction (A), their ratio (E/A), and deceleration time as previously described [22]. Moreover, we used velocity vector imaging to evaluate diastolic function. Velocity vector imaging represents the heart motion as the vector by tracking the myocardial motion with the speckle tracking technology and has been developed to evaluate diastolic function [23].
Cell cultures and treatments Primary cultures of neonatal rat cardiac fibroblasts were prepared. The culture medium was Dulbecco’s Modified Journal of Hypertension
Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) supplemented with 5% calf serum. The cells were seeded at 378C in an atmosphere of 5% CO2. Atorvastatin (10 mmol/l) was added to the medium of confluent cardiac fibroblasts in the absence of calf serum before stimulation with transforming growth factor beta (TGF-b) (10 ng/ml). To address the statin site of action along the mevalonate pathway, Fpp (5 mmol/l), GGpp (5 mmol/l), and mevalonate (200 mmol/l) were added to the medium containing atorvastatin. In additional experiments, fasudil hydrochloride (10 mg/ml), Rho kinase inhibitor, was added to the medium instead of atorvastatin to confirm the involvement of specific isoprenoids in the mevalonate pathway.
RNA preparation and quantitative real-time reverse-transcription PCR Total RNA was extracted from the tissue using TRIzol reagent (Invitrogen, Carlsbad, California, USA). DNase-treated RNA was reverse-transcribed into cDNA using random primers (Applied Biosystems, Foster City, California, USA). Quantitative RT-PCR was performed with an Applied Biosystems 7900 real-time PCR System with TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assays (Applied Biosystems) as previously described [24]. TaqMan Gene Expression Assays were used as primers and probes for each gene were as follows: TGF-b (assay ID Rn99999016_m1), collagen type 1 (assay ID Rn00801649_m1), HSP47 (assay ID Rn00567777_m1), and GAPDH (assay ID Rn99999916_s1). glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.
Western blot analysis The total protein homogenate (50 mg) from the heart was separated by SDS-PAGE and transferred onto polyvinylidene fluoride membrane. The membrane was incubated with antibodies against rabbit anti-collagen type 1 (Rockland, Gilbertsville, Pennsylvania, USA; dilution 1 : 1000), rabbit anti-HSP47 (Assay Designs, Ann Arbor, Michigan, USA; dilution 1 : 1000), and mouse anti-b-actin (Abcam, Cambridge, UK; dilution 1 : 1000). The expression levels of proteins were detected by an enhanced chemilumiescence kit (Thermo Scientific, Waltham, Massachusetts, USA). Quantification of the intensity of the bands was normalized with that for b-actin.
Zymography of matrix metalloproteinase activity and ELISA for tissue inhibitor of metalloproteinase Left ventricular myocardial samples were homogenized in 2 ml of an ice-cold extraction buffer containing cacodylic acid (10 mmol/l), NaCl (0.15 mol/l), ZnCl (20 mmol/l), NaN3 (1.5 mmol/l), and 0.01% Triton X-100 (pH 5.0). The myocardial extracts were loaded onto SDS-PAGE gel containing 1 mg/ml gelatin. The gelatinolytic activity was tested as specific matrix metalloproteinase (MMP) activity by adding EDTA and phenylmethylsulfonyl fluoride. The MMP subtypes were distinguished according to each molecular weight. Tissue inhibitor of metalloproteinase (TIMP)-1 (TIMP1 Rat ELISA Kit; Abcam Japan, Tokyo, Japan) and TIMP-2 (Enzyme-linked Immunosorbent Assay Kit For www.jhypertension.com
1535
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Akahori et al.
Tissue Inhibitors of Metalloprpteinase2 (TIMP2); Uscn Life Science Inc, Wuhan, China) were measured by ELISA kit.
RESULTS
Histological analysis
Mean blood pressure (MBP) was significantly higher in Dahl salt-sensitive rats fed a high-salt diet than in those fed a normal diet. There was no difference in MBP between the high-salt group and the HS þ Ato group (Fig. 1). HR did not differ among three groups. Urine protein excretion increased in Dahl salt-sensitive rats fed a high-salt diet, and there was no difference in urine protein excretion between the highsalt group and the HS þ Ato group (Table 1).
Physiological parameters and urinary protein excretion
The organs were harvested, dissected, and weighed after the rats were euthanized. The weight of the total heart was normalized to the tibial length. The left ventricle was fixed with buffered 4% paraformaldehyde, embedded in paraffin, and cut into 4-mm-thick sections. Sections were stained with Masson trichrome for the evaluation of fibrosis. Quantitative measurement of the area of fibrosis was calculated using computer-aided planimetry and expressed as a percentage of the total surface area of the tissue section. The area of interstitial fibrosis was identified after excluding the vessel area from the region of interest, as the ratio of interstitial fibrosis to the total tissue area.
Organ weight Body weight was not different at 23 weeks between the three groups, but left ventricular weight-to-tibial length ratio was significantly higher in the high-salt and the HS þ Ato groups than in the low-salt group. The difference in the weight between wet and dry lungs was significantly larger in the high-salt group than in the low-salt group and the HS þ Ato group (Table 1).
Statistical analysis All statistical analyses were performed using commercially available statistical software (Dr SPSS II for Windows; SPSS Japan Inc, Tokyo, Japan). Values are reported as the mean standard error of the mean. Statistical analysis was performed using one-way ANOVA or Student’s t test. Differences among three groups were assessed by posthoc Bonferroni tests. Differences were considered significant when the probability value was
Atorvastatin ameliorates cardiac fibrosis and improves left ventricular diastolic function in hypertensive diastolic heart failure model rats Hirokuni Akahori a, Takeshi Tsujino b, Yoshiro Naito a, Mika Matsumoto a, Naoko Sasaki b, Toshihiro Iwasaku a, Akiyo Eguchi a, Hisashi Sawada a, Shinichi Hirotani a, and Tohru Masuyama a
Objective: Clinical studies have suggested the beneficial effects of statin therapy on diastolic heart failure. However, the mechanism of the beneficial effects of statin on diastolic heart failure remains unknown. We examined the effect of atorvastatin on the cardiac function of Dahl salt-sensitive rat, a model of hypertensive diastolic heart failure. Methods: Dahl salt-sensitive rats were divided into three groups: the low-salt group (given standard diet), the highsalt group (given 8% NaCl diet from 7 weeks of age), and the high-salt R atorvastatin (HS R Ato) group (given 8% NaCl diet from 7 weeks of age and atorvastatin from 17 weeks of age). We evaluated left ventricular hypertrophy (LVH), fibrosis, and function by using echocardiography and histology. We also examined the expression of molecules related to fibrosis in the hearts of Dahl salt-sensitive rats and cultured rat cardiac fibroblasts. Results: Left ventricular hypertrophy, diastolic dysfunction, and cardiac fibrosis were observed in the high-salt group. Atorvastatin ameliorated cardiac fibrosis and normalized left ventricular diastolic function without altering blood pressure. Atorvastatin also decreased the expression of heat shock protein 47 (HSP47), an essential chaperone for type 1 collagen processing, without changing in expression of transforming growth factor beta. In rat cardiac fibroblast cells, atorvastatin also reduced HSP47 level induced by transforming growth factor beta. The effect of atorvastatin was reversed by mevalonate and geranylgeranyl-pyrophosphate and mimicked by Rho kinase inhibitor. Conclusion: Atorvastatin administration ameliorates cardiac fibrosis and improves left ventricular diastolic function in Dahl salt-sensitive rats. Lowering HSP47 by atorvastatin via inhibition of Rho–Rho kinase pathway is suggested as a mechanism. Keywords: cardiac fiblosis, diastolic heart failure, heat shock protein 47, statin Abbreviations: Ato, atorvastatin; Fpp, farnesylpyrophosphate; GGpp, geranylgeranylpyrophosphate; HF, heart failure; HR, heart rate; HSP, heat shock protein; LVH, left ventricular hypertrophy; MMP,
matrix metalloproteinase; TGF, transforming growth factor; TIMP, tissue inhibitor of metalloproteinase
INTRODUCTION
H
eart failure (HF) is one of the most common causes of cardiovascular morbidity and mortality [1–4]. A substantial proportion of patients with HF have diastolic heart failure [5]. There is a growing recognition that congestive heart failure caused by diastolic dysfunction is common and causes significant morbidity and mortality. The 3-hydroxy-3-methylgutaryl-CoA reductase inhibitors, commonly referred to as statins, are potent inhibitors of cholesterol biosynthesis. Recently, evidence of statin is steadily increasing regarding the beneficial, lipid-independent ‘pleiotropic’ effects on cardiovascular diseases [6–8]. Stains have indeed been suggested to affect alternative target, preventing the synthesis of important isoprenoid intermediates such as farnesylpyrophosphate (Fpp) and geranylgeranyl-pyrophosphate (GGpp) [9]. Statins would inhibit posttranslational lipid modification of a variety of intracellular signaling proteins including Ras and Rho [10]. Recent clinical and animal studies have suggested the beneficial effects of statin therapy on diastolic heart failure [11–13]. However, the mechanism of the beneficial effects of statin on diastolic heart failure remains unknown. Heat shock protein 47 (HSP47) is a basic glycoprotein that binds collagen, and it has a pivotal role as a collagenspecific molecular chaperon [14]. Recent studies have shown that inhibition of HSP47 is useful as a treatment of fibrotic disease such as pulmonary fibrosis, peritoneal fibrosis, and liver fibrosis [15–17]. Moreover, HSP47 is Journal of Hypertension 2014, 32:1534–1541 a Cardiovascular Division, Department of Internal Medicine, Hyogo College of Medicine, Nishinomiya and bDepartment of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, Kobe, Japan
Correspondence to Takeshi Tsujino, MD, PhD, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, Kobe, Japan, 1-3-6 Minatojima, Chuo-ku, Kobe, 650-8530, Japan. Tel: +81 78 304 3182; fax: +81 78 304 2812; e-mail: [email protected] Received 1 June 2012 Revised 8 December 2013 Accepted 19 February 2014 J Hypertens 32:1534–1541 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. DOI:10.1097/HJH.0000000000000184
1534
www.jhypertension.com
Volume 32 Number 7 July 2014
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Use of atorvastatin in hypertensive diastolic heart failure
associated with fibrosis following myocardial infarction and has been localized in atherosclerotic arteries [18]. However, a role of HSP47 in diastolic dysfunction has not been elucidated. Dahl salt-sensitive rat model is a well established model of hypertensive diastolic heart failure [19,20]. We performed animal studies using Dahl salt-sensitive rats to evaluate the effect of atorvastatin on diastolic heart failure in hypertensive hearts with established left ventricular hypertrophy (LVH). We also investigated the effects of atorvastatin on the expression fibrosis-related genes including HSP47.
METHODS Animals Male Dahl salt-sensitive rats (SLC Japan, Shizuoka, Japan) were fed a standard diet (0.3% NaCl) from weaning until 7 weeks. Afterward, rats were randomized to normal lowsalt diet (0.3% NaCl) group [(low-salt group) n ¼ 10], highsalt diet (8% NaCl) group [(high-salt group) n ¼ 10], or highsalt diet (8% NaCl) with administration of atorvastatin from 17 weeks of age [high-salt þ atorvastatin group (HS þ Ato group) n ¼ 10]. Atorvastatin (20 mg/kg per day) or vehicle was administrated orally by gavage from 17 weeks of age. The statin dose was based on a previous report showing a beneficial pleiotropic vascular effect in a hypertensive model [21]. Rats were maintained on a 12-h light/dark cycle and had free access to food and water. All of our experimental procedures were approved by the Animal Research Committee of Hyogo College of Medicine.
Assessments of blood pressure monitoring and urinary date Three rats for each group are chronically instrumented with telemetry probes (TA11-PA40; Data Sciences International, Minnesota, USA) for continuous monitoring of mean arterial pressure and heart rate (HR) without physical restriction. Twenty-four-hour urine samples were collected in metabolic cages for measuring urinary volume, protein, and creatinine levels.
Echocardiography Rats were anesthetized with ketamine HCl (50 mg/kg) and xylazine HCl (10 mg/kg), and evaluated by transthoracic echocardiographic studies. We measured left ventricular cavity size and wall thickness, and calculated left ventricular ejection fraction (LVEF) with a 12-MHz phased-array transducer (ACUSON; Mochida Siemens Medical Systems, Tokyo, Japan). We also recorded pulsed Doppler mitral flow velocity pattern, and measured peak early diastolic filling velocity (E), peak filling velocity at atrial contraction (A), their ratio (E/A), and deceleration time as previously described [22]. Moreover, we used velocity vector imaging to evaluate diastolic function. Velocity vector imaging represents the heart motion as the vector by tracking the myocardial motion with the speckle tracking technology and has been developed to evaluate diastolic function [23].
Cell cultures and treatments Primary cultures of neonatal rat cardiac fibroblasts were prepared. The culture medium was Dulbecco’s Modified Journal of Hypertension
Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) supplemented with 5% calf serum. The cells were seeded at 378C in an atmosphere of 5% CO2. Atorvastatin (10 mmol/l) was added to the medium of confluent cardiac fibroblasts in the absence of calf serum before stimulation with transforming growth factor beta (TGF-b) (10 ng/ml). To address the statin site of action along the mevalonate pathway, Fpp (5 mmol/l), GGpp (5 mmol/l), and mevalonate (200 mmol/l) were added to the medium containing atorvastatin. In additional experiments, fasudil hydrochloride (10 mg/ml), Rho kinase inhibitor, was added to the medium instead of atorvastatin to confirm the involvement of specific isoprenoids in the mevalonate pathway.
RNA preparation and quantitative real-time reverse-transcription PCR Total RNA was extracted from the tissue using TRIzol reagent (Invitrogen, Carlsbad, California, USA). DNase-treated RNA was reverse-transcribed into cDNA using random primers (Applied Biosystems, Foster City, California, USA). Quantitative RT-PCR was performed with an Applied Biosystems 7900 real-time PCR System with TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assays (Applied Biosystems) as previously described [24]. TaqMan Gene Expression Assays were used as primers and probes for each gene were as follows: TGF-b (assay ID Rn99999016_m1), collagen type 1 (assay ID Rn00801649_m1), HSP47 (assay ID Rn00567777_m1), and GAPDH (assay ID Rn99999916_s1). glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.
Western blot analysis The total protein homogenate (50 mg) from the heart was separated by SDS-PAGE and transferred onto polyvinylidene fluoride membrane. The membrane was incubated with antibodies against rabbit anti-collagen type 1 (Rockland, Gilbertsville, Pennsylvania, USA; dilution 1 : 1000), rabbit anti-HSP47 (Assay Designs, Ann Arbor, Michigan, USA; dilution 1 : 1000), and mouse anti-b-actin (Abcam, Cambridge, UK; dilution 1 : 1000). The expression levels of proteins were detected by an enhanced chemilumiescence kit (Thermo Scientific, Waltham, Massachusetts, USA). Quantification of the intensity of the bands was normalized with that for b-actin.
Zymography of matrix metalloproteinase activity and ELISA for tissue inhibitor of metalloproteinase Left ventricular myocardial samples were homogenized in 2 ml of an ice-cold extraction buffer containing cacodylic acid (10 mmol/l), NaCl (0.15 mol/l), ZnCl (20 mmol/l), NaN3 (1.5 mmol/l), and 0.01% Triton X-100 (pH 5.0). The myocardial extracts were loaded onto SDS-PAGE gel containing 1 mg/ml gelatin. The gelatinolytic activity was tested as specific matrix metalloproteinase (MMP) activity by adding EDTA and phenylmethylsulfonyl fluoride. The MMP subtypes were distinguished according to each molecular weight. Tissue inhibitor of metalloproteinase (TIMP)-1 (TIMP1 Rat ELISA Kit; Abcam Japan, Tokyo, Japan) and TIMP-2 (Enzyme-linked Immunosorbent Assay Kit For www.jhypertension.com
1535
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Akahori et al.
Tissue Inhibitors of Metalloprpteinase2 (TIMP2); Uscn Life Science Inc, Wuhan, China) were measured by ELISA kit.
RESULTS
Histological analysis
Mean blood pressure (MBP) was significantly higher in Dahl salt-sensitive rats fed a high-salt diet than in those fed a normal diet. There was no difference in MBP between the high-salt group and the HS þ Ato group (Fig. 1). HR did not differ among three groups. Urine protein excretion increased in Dahl salt-sensitive rats fed a high-salt diet, and there was no difference in urine protein excretion between the highsalt group and the HS þ Ato group (Table 1).
Physiological parameters and urinary protein excretion
The organs were harvested, dissected, and weighed after the rats were euthanized. The weight of the total heart was normalized to the tibial length. The left ventricle was fixed with buffered 4% paraformaldehyde, embedded in paraffin, and cut into 4-mm-thick sections. Sections were stained with Masson trichrome for the evaluation of fibrosis. Quantitative measurement of the area of fibrosis was calculated using computer-aided planimetry and expressed as a percentage of the total surface area of the tissue section. The area of interstitial fibrosis was identified after excluding the vessel area from the region of interest, as the ratio of interstitial fibrosis to the total tissue area.
Organ weight Body weight was not different at 23 weeks between the three groups, but left ventricular weight-to-tibial length ratio was significantly higher in the high-salt and the HS þ Ato groups than in the low-salt group. The difference in the weight between wet and dry lungs was significantly larger in the high-salt group than in the low-salt group and the HS þ Ato group (Table 1).
Statistical analysis All statistical analyses were performed using commercially available statistical software (Dr SPSS II for Windows; SPSS Japan Inc, Tokyo, Japan). Values are reported as the mean standard error of the mean. Statistical analysis was performed using one-way ANOVA or Student’s t test. Differences among three groups were assessed by posthoc Bonferroni tests. Differences were considered significant when the probability value was
