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,1
* Department of Clinical Pharmacology, Niigata University of Pharmacy and Applied Life Sciences, Niigata City, 956-8603, Japan; Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305; Department of Homeostatic Regulation and Development, Niigata University School of Medicine, Niigata City, 951-8510, Japan; and First Department of Medicine, Niigata University School of Medicine, Niigata City, 951-8510, Japan
To whom requests for reprints should be addressed at 2 Department of Clinical Pharmacology, Niigata University of Pharmacy and Applied Life Sciences, Niigata City, 956-8603, Japan. E-mail: watanabe{at}nupals.ac.jp
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Abstract |
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Key Words: chymase inhibitor • cardiomyopathy • postmyocarditis rats • mast cells
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Introduction |
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Chymase mediates angiotensin (Ang) II generation primarily in hamsters, dogs, and humans (8). Ang II, in turn, stimulates hypertrophy of the myocardium and proliferation of interstitial fibroblasts and promotes cardiac remodeling in hamsters (9). Moreover, it has become clear that chymase possesses other functions such as induction of apoptosis in myocytes (10) and proliferation of noncardiomyocytes (i.e., fibrosis in neonatal rats’ cell culture [10]) and stimulation of the release of collagen III and transforming growth factor (TGF)-ß1 (7, 10). In an earlier study it was demonstrated that expression of the mast cell chymase gene is upregulated in the acute phase of viral myocarditis in mice, and it remains elevated in the late phase of heart failure; thus, chymase was implicated in the myocardial necrosis, inflammation, and remodeling process of myocarditis (11). Hence, it can be conceived that chymase might play an important role in the pathophysiology of postmyocarditis dilated cardiomyopathy (DCM) in rats.
Reports that have described chymase involvement in cardiac remodeling through pathways other than Ang II mediation prompted us to investigate the mechanism of chymase-mediated cardiac remodeling in a set-up that is independent of Ang II generation by chymase. Because rat chymase is unable to mediate Ang II generation (12–14), we decided to study the effect of TY-51469, a novel benzothiophenesulfonamide derivative with potent chymase inhibitory action, in rats with DCM after autoimmune myocarditis and its possible mechanism of action.
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Materials and Methods |
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TY-51469 was first dissolved in 100 mM NaOH, then diluted with 67 mM phosphate buffer for each dose, and used to fill an osmotic pump (Alza Corporation, Palo Alto, CA). An indwelling cannula (PE50: Becton Dickinson and Company, Parsippany, NJ) was implanted in the right jugular vein of each rat and was connected to an osmotic pump inserted within a subcutaneous pocket. The osmotic pump was maintained to release the drug solution continuously at a rate of 2.5 µl/hr for 28 days. Throughout the experiments, all the animals were treated in accordance with our institute’s guidelines for animal experimentation.
Hemodynamic and Echocardiographic Studies.
To obtain hemodynamic data, rats were anesthetized with 2% halothane in oxygen during the surgical procedures. A catheter-tip transducer (Miller SPR 249; Miller Instruments, Houston, TX) was introduced into the left ventricle through the right carotid artery for the determination of peak left ventricular pressure (LVP) and left ventricular end-diastolic pressure (LVEDP), and the rates of intraventricular pressure rise (max. dP/dt) and decline (min. dP/dt) were recorded as described previously (16). After instrumentation the concentration of halothane was reduced to 0.5% to minimize the anesthetic effect on hemodynamic parameters. Echocardiographic studies were carried out with a 7.5-MHz transducer (Aloka Inc., Tokyo, Japan). The left ventricular dimensions in diastole (LVDd) and systole (LVDs) and percent fractional shortening (FS) were estimated by using the M-mode measurements.
Histopathology.
The body weight (BW) of rats was noted just before anesthesia was induced. After the hemodynamic and echocardiographic analyses, the rats were sacrificed, and the myocardium was isolated and weighed to calculate the ratio of heart weight (HW) to BW. The excised myocardium was kept in formalin and the midventricle sections were then embedded with paraffin. The paraffinized midventricle sections stained with Azan-Mallory dye and a color image analyzer (CIA-102; Olympus, Tokyo, Japan) were used to quantify the area of myocardial fibrosis (blue areas were fibrotic, whereas red areas were myocardium). The results are presented as the ratio of the fibrotic area to the area of normal myocardium.
Measurement of Mean Cardiomyocyte Area.
Paraffin-embedded left ventricular tissue sections stained with hematoxylin-eosin were utilized to measure cardiomyocyte area. A high-power microscope aided by a camera and computer system (BH2-RFCA; Olympus) was used. We took the advantage of View Finder and Studio Lite software applications. View Finder helps in focusing, whereas Studio Lite is useful in the selection and capture of images at higher magnifications. The magnification fields were standardized with a micrometer. Short-axis and long-axis diameters of cardiomyocytes were measured in a blinded manner by using the standardized magnification (x400). At least 10 high-power fields were selected randomly from each slide. A minimum of 100 cardiomyocytes for each group was included to calculate the mean cardiomyocyte area. The unit of mean cardiomyocyte area was µm2.
Mast Cell Staining and Quantitation.
Histochemical staining with toluidine blue was performed to identify mast cells. Briefly, slides of paraffinized midventricle sections were dewaxed, rehydrated, incubated with 0.05% (w/v) toluidine blue for 30 min, and subsequently counter-stained with 0.01% (w/v) eosin for 1 min. Metachromatic staining of mast cell granules was useful in identifying these cells. Mast cell density (MCD) was quantified by counting the number of toluidine blue–positive mast cells per field (x100). At least 15 fields were included in each slide for counting.
Measurement of Histamine Concentration in Heart Tissue.
Histamine was extracted from heart tissue and estimated by using a fluorometer in accordance with the procedure described elsewhere (17). The extracted histamine from the cardiac homogenate formed a complex with orthophthaldialdehyde (OPT) (Sigma, Tokyo, Japan). The fluorescence of histamine-orthophthaldialdehyde fluorophor was maximized in the presence of citric acid and measured with a spectrofluorophotometer (Shimadzu, Kyoto, Japan) at 358 nm (activation wavelength) and 446 nm (fluorescence wavelength). The concentration of cardiac histamine was expressed as µg/g of wet heart tissue.
Quantitation of Chymase Activity in Myocardium.
Chymase activity was quantified by an enzymatic histochemical staining procedure adopted from a previous report (7). Formalin-fixed, paraffin-embedded midventricle sections were used for the detection of chymase activity with the help of a kit (Sigma). The slides were deparaffinized, rehydrated, and then incubated with naphthol AS-D chloroacetate in the presence of freshly formed diazonium salt and fast red violet as described in the procedure given by the manufacturer. Reddish pink coloration was regarded as a positive reaction for chymase activity. At least 100 fields were counted randomly in each slide, and the data were plotted as a graph.
Western Immunoblotting.
The myocardial tissue samples obtained from different groups were homogenized with lysis buffer. Protein concentrations in these homogenized samples were measured by the bicinchoninic acid method. For Western blot analysis, proteins were separated by SDS-PAGE and identified with the following antibodies to quantify the myocardial levels of various proteins: anti–rat aldosterone synthase (AS) (Chemicon International, Temecula, CA) and anti–cyclin D1 (Cell Signaling Technology, Beverly, MA) mouse monoclonal antibodies; anti–atrial natriuretic peptide (ANP) and anti–TGF-ß1 rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); and anti–collagen III, anti–stem cell factor (SCF), and anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) goat polyclonal antibodies (Santa Cruz Biotechnology). We used 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) used to separate the proteins to analyze AS, cyclin D1, SCF, and GAPDH. We used 15% SDS-PAGE for analysis of ANP and TGF-ß1, whereas 7.5% SDS-PAGE was used for analysis of collagen III. After incubation with primary antibody, the bound antibody was visualized with respective horseradish peroxidase (HRP)-coupled secondary antibody (Santa Cruz Biotechnology) and chemiluminescence-developing agents (ECL Plus, Amersham, Piscataway, NJ).
Immunohistochemistry.
Formalin-fixed, paraffin-embedded midventricle sections were used for immunohistochemical staining. After overnight incubation with anti–tumor necrosis factor (TNF)-
goat polyclonal antibody (Santa Cruz Biotechnology) at 4°C, the slides were washed in Tris-buffered saline, and then they were reacted with a respective HRP-conjugated secondary antibody (Santa Cruz Biotechnology) at room temperature for 45 minutes. The immunostaining was visualized with the use of diamino benzidine tetrahydrochloride, and the slides were counter-stained with hematoxylin. The slides were examined for brown coloration, as an index of immunopositivity, under a high-power light microscope. Quantitation of immunopositivity for TNF- was done by using higher magnification fields.
Estimation of the Inhibitory Potency of TY-51469 on Simian Chymase or Human Chymase.
Simian chymase used was obtained from the heart of rhesus monkey (Charles River Japan Inc.) through purification in accordance with a human heart chymase purification method (18), and human chymase used was obtained from silkworms infected with baculovirus in which a gene that encoded human chymase had been integrated (19). Chymase activity was measured by reacting free His-Leu together with Ang II and OPT to prepare a fluorescent derivative and then by determining the amount quantitatively by means of a fluorophotometer.
First, 3.6 µmol of TY-51469 was weighed in a test tube and was dissolved in 3 ml of dimethyl sulfoxide (DMSO). The DMSO solution was diluted 1000-fold with 20 mM Tris-hydrochloric acid buffer solution (pH 8.0) that contained 0.01% Triton X-100 and 0.5 M potassium chloride to prepare a 1.2 x 10–6 M solution. It was successively diluted with the buffer solution to prepare TY-51469 solutions whose concentrations ranged from 1.2 x 10–6 M to 1.2 x 10–9 M. To 500 µl of TY-51469 solution of each concentration or buffer solution we added 50 µl of an enzyme solution, and this underwent 10 minutes of preincubation at 37°C. Then, 50 µl of 0.1 mM Ang I solution was added to initiate a reaction. Human angiotensin I (Sigma) was employed as Ang I. The enzyme solution to be used for the reaction was adjusted to hydrolyze about 60% of the substrate under the experimental conditions, and the reaction wherein a buffer solution contained no enzyme was carried out as a blind test. After 120 minutes of incubation at 37°C, the reaction was terminated by adding 900 µl of 15% trichloroacetic acid. Thereafter, the reaction mixture was centrifuged at 3,000 rpm for 10 minutes at 4°C, and 2 ml of 2 M sodium hydroxide and 1 ml of methanol were added to 1 ml of the resulting supernatant. Then 100 µl of the methanol solution that contained 1.2 mg of N-acetyl-L-cysteine and 1 mg/ml OPT was added; thus, a derivatization reaction was initiated. After the reaction mixture was left standing for exactly 1 hour, the fluorescence intensity at the fluorescence wavelength of 502 nm under the excitation wavelength of 304 nm was measured. The measurement was repeated twice for each sample and blind test. The fluorescence intensity obtained by subtracting the average value at the blind test from the average value thereof was determined as chymase activity. An enzymatic reaction in which a buffer solution instead of a TY-51469 solution was used served as a control, and the inhibitory ratio of chymase activity was determined as a percentage by using the chymase activity of the control to divide the difference between the activity at the addition of TY-51469 and the chymase activity of the control. On the basis of each inhibitory ratio, we calculated the IC50 value (i.e., the concentration at which 50% of the activity was inhibited).
Stability Study of TY-51469 in Rat Plasma.
Seven-week-old male Sprague-Dawley rats were anesthetized with ether in overnight fasting conditions. By abdominal incision, blood was collected from the abdominal aorta by using a heparinized disposable plastic syringe. The blood was centrifuged under cooling to collect supernatant plasma. The collected plasma was frozen and stored at –30°C and was melted before use. After TY-51469 was dissolved in DMSO, the solution was added to 200 µl of plasma to achieve a concentration of 10 µg/ml and then incubated at 37°C. After 60 minutes, the mixture was acidified by adding 200 µl of 0.1 M hydrochloric acid and then extracted twice with 2 ml of ethyl acetate. The resulting organic layer was evaporated to dryness under a nitrogen gas flow, and the residue was dissolved in 200 µl of acetonitrile to prepare a sample solution. In contrast, the test compound was dissolved in a 1% acetonitrile solution of DMSO to achieve a concentration of 10 µg/ml and to serve as a control solution. The sample solution and control solution were investigated by high-performance liquid chromatography. The residual ratio (%) was determined by dividing the peak area of the sample solution with the peak area of the control solution
Toxicity Study of TY-51469 in Rats.
The 2-week intravenous repeated-dose toxicity study of TY-51469 was conducted in male Sprague-Dawley rats at daily doses of 0 (control), 20, and 60 mg/kg.
Statistical Analysis.
Data are presented as the mean ± the standard error of the mean (SEM). Statistical assessment of the groups was performed by one-way analysis of variance, which was followed by the Tukey or Bonferroni methods of posthoc analysis. The Kruskal-Wallis test was used to analyze the survival times (in the present study data for multiple groups were available). Differences were considered significant at P < 0.05.
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Stability of TY-51469 in Rat Plasma.
The compound TY-51469 showed 100% stability in rat plasma at 40°C for as long as 1 hour.
Results of Toxicity Studies.
Results of toxicity studies showed a good profile, even when doses of 20 or 60 mg/kg were administered intravenously. There were no undesired effects on survival rate, clinical signs, BW, and food and water consumption. Ophthalmologic, hematologic, and gross pathological examination showed no abnormalities. Decreases in serum potassium and increases in urine N-acetyl-ß-D-glucosaminidase excretion were noted in the 60 mg/kg/day group. Increases in absolute submandibular gland weight were recorded for the 20 and 60 mg/kg/day groups without any apparent morphologic changes in the submandibular glands. Histopathological examination revealed the regeneration of renal tubules characterized by cytoplasmic basophilia, increased nucleus-to-cytoplasm ratio, and lymphocytic infiltration in kidneys in the 60 mg/kg/day group. An increase in infiltration of eosinophils in the stomach was observed in one of four rats in the 60 mg/kg/day group. Although these changes may be related to TY-51469 treatment, the degree of these changes was minimal or mild.
Survival Rates.
Six (40%) of 15 rats in group V, 4 (27%) of 15 rats in group CYI-0.1, and 3 (20%) of 15 rats in group CYI-1 died between days 30 and 56 of immunization. The survival rate of group CYI-1 was significantly higher than that of group V (P < 0.05). The survival rate of group CYI-0.1 was also improved in comparison with that of group V; however, this difference did not attain statistical significance. The normal rats were healthy and alive throughout the protocol.
Myocardial Fibrosis.
Fibrosis in the DCM hearts was clearly visible as blue rather than red (normal) regions of the heart (Fig. 1B
). Group V showed a significant increase in the percent area of myocardial fibrosis in relation to that of group N (Fig. 1A, B, and Fig. 2A). The percent area of myocardial fibrosis was lower in group CYI-1 than in group CYI-0.1 (Fig. 1C, D, and Fig. 2A). Of the treatment groups, group CYI-1 showed a statistically significant reduction in the percent area of fibrosis when compared with that in group V; however, group CYI-0.1 also showed a trend of decline (Fig. 2A).
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). In contrast, the mean cardiomyocyte area was significantly decreased in a dose-dependent manner with chymase inhibitor treatment (Fig. 2C).
Cardiac MCD.
Cardiac MCD was significantly greater in the group V samples than in the group N samples (Fig. 1E, F, and Fig. 2D). Treatment with the chymase inhibitor decreased the cardiac MCD in comparison to that in vehicle-treated rats. The reduction in cardiac MCD was higher in the myocardial sections of group CYI-1 than in those of group CYI-0.1 (Fig. 1G, H, and Fig. 2D).
Histamine Concentration in Heart Tissue.
The concentration of histamine in wet heart tissue has been designated as an index of cardiac MCD. The concentration of histamine in wet heart tissue of group V was significantly higher than that of group N (Fig. 2E). In the groups CYI-0.1 and CYI-1, the histamine concentration was less than that in group V (Fig. 2E). In these two treatment groups, the group CYI-1 attained a statistically significant difference in comparison with group V (Fig. 2E).
Chymase Activity in Myocardium.
Midventricle sections from group V showed greater chymase activity than did those of group N (Fig. 1I, J, and Fig. 2F), whereas chymase inhibitor treatment attenuated it dose-dependently (CYI-0.1 [Fig. 1K, Fig. 2F]; CYI-1 [Fig. 1L, Fig. 2F]).
TNF- Levels in the Myocardium.
Immunostaining showed greater myocardial levels of TNF- in the midventricle sections of group V than in those of group N (Fig. 1M, N, and Fig. 2G). A dose-dependent reduction was observed in the treatment groups (CYI-0.1 and CYI-1). However, group CYI-1, but not group CYI-0.1, attained a statistically significant difference (Fig. 1O, P, and Fig. 2G).
Myocardial Functional Parameters.
Hemodynamic and echocardiographic analyses revealed the systolic and diastolic function of the myocardium (Fig. 3). The systolic functional parameters (LVP, max. dP/dt, and percent FS) were significantly decreased. The LVDs was significantly greater in group V than in group N, whereas a dose-dependent improvement was observed in the treatment groups (Fig. 3A, C, F, and G). The diastolic parameters LVEDP and LVDd were increased in group V in relation to those in group N, whereas treatment with the chymase inhibitor decreased them dose-dependently (Fig. 3B, E). LVEDP values showed statistical significance between groups, but LVDd readings did not attain statistical significance as shown in Figure 3B and 3E. At the same time, min. dP/dt, another diastolic parameter, was significantly improved in a dose-dependent manner with chymase inhibitor treatment, and was decreased in rats with DCM in comparison with their normal counterparts (Fig. 3D).
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). However, the statistical significance in the treatment groups differed with different proteins as indicated in Figure 4.
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Discussion |
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Chymase inhibition prevents myocardial fibrosis and improves cardiac function in experimental animals (7, 20). In the chronic phase of autoimmune myocarditis (from day 28 of myosin immunization), the occurrence of fibrosis and hypertrophy is an important cause of myocardial dysfunction. To define the role of chymase in the remodeling process after autoimmune myocarditis, we examined fibrosis and hypertrophy. We also measured levels of cyclin D1, because it has been designated an index of fibrogenesis in heart failure (21). Treatment with the chymase inhibitor decreased the percent area of fibrosis in the myocardial sections that underwent Azan-Mallory staining, the myocardial levels of TGF-ß1 and collagen III (the molecular markers of fibrosis), and fibrogenesis as evident from reduced cyclin D1 levels. The ratio of HW to BW, the mean cardiomyocyte area, and ANP, a molecular marker of hypertrophy, were decreased with chymase inhibitor treatment; these decreases indicated a reduction in hypertrophy. Chymase inhibition suppressed myocardial remodeling and ultimately led to improvement in myocardial function as shown by our results. Because rat chymase lacks the ability to generate Ang II (12–14), we found no significant difference in myocardial Ang II levels among the treated and untreated groups (data not shown). Documented evidence suggests that increased expression of AS in the myocardium of rats after myocardial infarction (MI) contributes to post-MI ventricular remodeling (22). It has been suggested that an increase in myocardial AS and aldosterone is involved in myocardial remodeling (23). Hence, finding the involvement of myocardial AS in the present scenario might suggest a new explanation for the mechanism of the chymase-mediated remodeling. Experimentally, increased aldosterone induces pericoronary inflammation and cardiac fibrosis (23). When we analyzed mast cells in myocardial sections, usually we found them adjacent to coronary blood vessels (Fig. 2E–H). Degranulation of mast cells is an integral part of the pathogenesis in this animal model in which chymase is also released. The contribution of chymase in stimulating AS expression and thereby increasing the aldosterone levels in the myocardium to cause the deleterious effect in the postmyocarditis hearts cannot be ruled out. Moreover, our data showing the dose-dependent reduction in the myocardial AS levels with chymase inhibitor treatment also suggest this possibility. However, these notions should be cautiously interpreted for further steps because the discrepancy about the myocardial AS (24) and elevation in myocardial AS levels may not necessarily lead to the increase in the production of myocardial aldosterone.
Treatment with the chymase inhibitor decreased the degranulating cardiac mast cells, MCD, and histamine concentration. Infiltration, proliferation, maturation (i.e., increased in size and filled with complete granules), and degranulation of mast cells are associated with the progression of myocarditis to heart failure in our model (17, 25). We saw colocalized mast cells in the inflammatory infiltration in postmyocarditis hearts, which suggests that smoldering inflammation exists in postmyocarditis hearts and that the inflammation is an integral part of disease progression (25). Consequently, there is a possibility that the release of inflammatory cytokines from mast cells was suppressed by the chymase inhibitor in the early phase of treatment and decreased the recruitment of inflammatory cells, including mast cells. Our results and hypothesis emulate those of the report of He et al., which declared that chymase inhibitors are potent mast cell stabilizers (26). On the basis of our results, we consider that the effects of the chymase inhibitor in the prevention of heart failure were achieved partly through the suppression of mast cell activity.
Cardiac-specific overexpression of TNF- in mice caused left ventricular hypertrophy and dilation with a concomitant increase in matrix metalloproteinase activity (27). SCF has been suggested to play an important role in the fibrotic cascade in cardiomyopathy (5). Thus, our results showed that the reduction in myocardial levels of TNF- and SCF with chymase inhibition may have importance in suppressing the molecular signaling pathways of remodeling events.
In summary, the inhibition of the chymase pathophysiological pathway, independent of Ang II involvement, contributes to the suppression of myocardial fibrosis, hypertrophy, and residual inflammation, thereby leading to the improvement of myocardial function and prevention of heart failure in rats with DCM after autoimmune myocarditis. Finally, it can be concluded that inhibition of chymase reduces the pathogenesis of DCM in rats.
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Footnotes |
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1 These authors contributed equally to this work. 
Received for publication March 19, 2007. Accepted for publication May 29, 2007.
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