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(Circulation. 1996;94:1513-1518.)
© 1996 American Heart Association, Inc.

Articles

Expression of Angiotensin-Converting Enzyme in Remaining Viable Myocytes of Human Ventricles After Myocardial Infarction

Seiji Hokimoto, MD; Hirofumi Yasue, MD; Kazuteru Fujimoto, MD; Hideyuki Yamamoto, MD; Koichi Nakao, MD; Koichi Kaikita, MD; Ryuzo Sakata, MD; Eishichi Miyamoto, MD

the Division of Cardiology (S.H., H.Yasue, K.F., K.N., K.K.) and the Department of Pharmacology (H.Yamamoto, E.M.), Kumamoto University School of Medicine, and the Division of Cardiovascular Surgery, Kumamoto Chuo Hospital (R.S.), Kumamoto, Japan.

Correspondence to Hirofumi Yasue, MD, Division of Cardiology, Kumamoto University School of Medicine, 1-1-1, Honjo, Kumamoto City, Kumamoto 860, Japan.

	Abstract

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Background Local ACE in the heart may be important in the pathophysiological state after myocardial infarction (MI). It is unknown, however, whether ACE is expressed in myocytes of the human heart.

Methods and Results Using a newly generated polyclonal antibody to a synthetic peptide corresponding to part of the human endothelial ACE sequence, we examined the localization of ACE in left ventricles of patients (n=10) with MI obtained at left ventricular aneurysmectomy or autopsy and in the hearts of control subjects at autopsy (n=10). The avidin–biotinylated peroxidase complex method was used for the immunohistochemical staining for ACE. In the left ventricles, positively stained myocytes for ACE were found in 8 of the 10 patients with MI. ACE immunoreactivity was seen in the remaining viable myocytes located near the infarct scar of the aneurysmal left ventricle and in nonmyocytes such as fibroblasts, macrophages, vascular smooth muscle cells, and endothelial cells within the scarred tissue. On the other hand, no immunoreactivity for ACE was detected in the ventricular myocytes of all control hearts obtained at autopsy.

Conclusions We observe immunohistochemical staining for ACE in the left ventricular myocytes of the region adjacent to the infarct scar and in nonmyocytes. These results indicate that ACE is markedly increased on the edge of the infarct scar and suggest that local ACE may be important in the ventricular remodeling after MI.

Key Words: angiotensin • myocardial infarction • aneurysm • immunohistochemistry

	Introduction

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The renin-angiotensin (RA) system has been shown to play an important role in the regulation of blood pressure and body fluid homeostasis.^{1 2} Besides this traditional concept that the RA system is a circulation-borne endocrine system, recent findings have demonstrated that the RA system is expressed locally in many tissues, including the heart.^{3 4 5} ACE inhibitors have been shown to prevent cardiac dilatation,⁶ to improve the ventricular function and the mortality rate of patients with low ejection fractions,^{7 8 9} and to reduce recurrence of myocardial infarction (MI) in patients with asymptomatic left ventricular dysfunction.¹⁰ These clinical data raise the possibility that the beneficial effects of ACE inhibitors in patients with heart failure or left ventricular hypertrophy may be due to interference with the activated local RA system and systemic endocrine and hemodynamic modulation. In this regard, recent studies have suggested that the local RA system plays an important role in the pathophysiological state of cardiovascular disorders such as vascular injury,^{11 12} hypertrophy,^{13 14} and heart failure after MI.^{15 16} It has been reported that the cardiac ACE mRNA level and activity are increased in left ventricular myocardium of patients with congestive heart failure.^{17 18} However, ACE mRNA level and activity were determined by use of tissue homogenate of the myocardium, and the cell types responsible for ACE production have not been identified in human heart after MI. In the present study, we detected ACE immunoreactivity in the remaining viable myocytes and in nonmyocytes of human heart after MI by immunohistochemical analysis.

	Methods

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Patients
Myocardial tissues from 20 patients with or without MI were investigated in this study. Ten autopsy cases (5 men and 5 women, 40 to 86 years of age [mean±SEM, 63.4±4.2 years]) were selected as control subjects on the basis of the following criteria: (1) death caused by noncardiac disease, (2) the absence of symptoms implying heart failure or rhythm disturbance during life, (3) the absence of hypertension during life, and (4) the absence of marked cardiomegaly (heart weight <350 g) or MI at autopsy. Specimens were obtained at autopsy within 2 hours after death at a room temperature of <20°C. The Table lists the characteristics of the control subjects.

View this table: [in this window] [in a new window]   Table 1. Patient Profiles

Ten patients with MI (4 men and 6 women, 55 to 89 years of age [mean±SEM, 68.5±2.7 years]) were examined in this study. The diagnosis of MI was based on typical chest pain lasting >30 minutes, the appearance of Q waves, and elevation of serum cardiac enzymes. All patients had left ventricular aneurysms, and cardiac tissues were obtained at both left ventricular aneurysmectomy (n=7) and autopsy (n=3). The Table summarizes the clinical data for these 10 patients.

The study protocol was in agreement with the guidelines of the ethical committee of our institution, and written informed consent was obtained from each patient or the families of subjects.

Preparation of Antibody Against a Synthetic ACE Peptide
A peptide corresponding in sequence to part (residues 113 through 129, Thr-Asp-Pro-Gln-Leu-Arg-Arg-Ile-Ile-Gly-Ala-Val-Arg-Thr-Leu-Gly-Ser) of human endothelial ACE¹⁹ was synthesized by the solid-phase method and purified by high-performance liquid chromatography (Fujiya Bioscience Institute Inc). A polyclonal antibody to this ACE peptide was prepared by immunizing a rabbit with 1 mg peptide coupled to hemocyanin from keyhole limpet four times at 2-week intervals. The serum titer of the antibody was determined by ELISA with the use of 1 µg synthetic peptide as the antigen. After repeated immunizations, the antiserum was adjusted to 50% ammonium sulfate and stirred for 2 hours at 4°C. The precipitate obtained by centrifugation at 10 000g for 10 minutes was dissolved in PBS and dialyzed overnight at 4°C against the same buffer. The antibody was further purified from the dialyzed antiserum on an antigen-affinity column in which the synthetic peptide (4 mg) was coupled to 2 g epoxy-activated Sepharose 6B. After application of the dialyzed antiserum, the column was washed with PBS and PBS containing 2 mol/L NaCl. The specific antibody was eluted with 0.1 mol/L glycine buffer, pH 3.0, and collected in 1 mg/mL BSA solution and 0.1 mol/L Tris-HCl, pH 7.5. The purified antibody was suitable for immunohistochemistry and Western blotting.

Western Blot Analysis
As described previously,²⁰ membrane proteins from lung, kidney, and infarcted and control left ventricles were extracted by use of a modification of the method of Rubinstein et al.²¹ Membrane proteins of tissue homogenates (20 µg) were subjected to SDS-PAGE in 1.5-mm-thick 7% acrylamide slab gels.²² Proteins were then transferred to nitrocellulose membranes by semidry electroblotting. The membranes were incubated at room temperature for 1 hour with 4.5% skim milk in Tris-buffered saline (25 mmol/L Tris-HCl, pH 7.5, and 150 mmol/L NaCl) containing 0.2% Tween 20 to block nonspecific binding sites. Then, the membranes were incubated at room temperature for 4 hours with the rabbit polyclonal ACE antibody. After washing, the membranes were further incubated at room temperature for 2 hours with biotinylated goat anti-rabbit immunoglobulin. The membranes were washed and then incubated with avidin-biotin complex solution (Vectastain ABC kit, Vector Laboratories). Immunoreactive proteins were visualized with 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Co) as chromogen.

Immunohistochemical Analysis
Heart tissue specimens were fixed with Zamboni's fixative²³ for 6 hours and then washed sequentially with PBS containing 10% sucrose, PBS containing 15% sucrose, and finally PBS containing 20% sucrose for 8 hours each. After being rinsed in PBS containing 20% sucrose and 10% glycerol for 1 hour, specimens were embedded in OCT compound (Tissue-Tek, Miles Inc), quickly frozen in dry-ice acetone, and stored at -80°C until use. The avidin-biotinylated peroxidase complex method (Vectastain ABC kit, Vector Laboratories) was used for the immunohistochemical staining for ACE. Frozen tissue specimens were cut with a cryostat into 8-µm-thick sections, which were mounted on poly-L-lysine–coated slides. To block endogenous peroxidase activity, the sections were immersed in l5 mmol/L periodate solution for 10 minutes and then washed with PBS. The sections were incubated with the purified anti-ACE antiserum in a converted chamber at 4°C overnight and with biotinylated goat anti-rabbit IgG for 1 hour. The sections were extensively washed with PBS and then incubated with the avidin-biotin complex solution. In consecutive sections, myocardial cells were identified with a monoclonal antibody specific for both atrial and ventricular myosin light chain 1.^{24 25} Moreover, to identify the cell types responsible for ACE production within the infarct scar after MI, we used the specific monoclonal antibodies that recognize fibroblast, macrophage, and smooth muscle cells (5B5, KP-1, and HHF35, respectively; Dako A/S). Peroxidase activity was visualized by incubation with 3,3'-diaminobenzidine tetrahydrochloride and 0.03% H₂O₂ in 0.05 mol/L Tris-HCl, pH 7.6. Cell nuclei were counterstained with hematoxylin.

In control sections, the purified anti-ACE antiserum was substituted with the corresponding antiserum preabsorbed with 10 µmol/L synthetic peptide or nonimmune rabbit serum.

Sensitivity and Evaluation of Immunostaining
To evaluate the sensitivity in immunohistochemical staining, we performed the immunostaining at various concentrations (an undiluted purified antibody and 1:100, 1:1000, and 1:5000 dilution) of the purified anti-ACE antiserum. To judge whether myocytes were stained, slides were evaluated independently by two different observers without knowledge of the clinical status, and the results were given as positive or negative.

	Results

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Specificity of the Polyclonal Antibody
Antiserum purified from an immunized rabbit showed a high titer (up to 1:1000) of anti-ACE antibody as determined by ELISA. Immunoblot analysis demonstrated that the polyclonal antibody to the peptide of human endothelial ACE recognized only a 170-kD protein in the homogenates of human kidney and lung (Fig 1B). These tissues are known to contain relatively abundant ACE with a molecular mass of 170 kD.²⁶ The 170-kD protein was observed in the homogenates of left ventricular tissues from patients with MI, whereas it was not detectable in those from control patients (Fig 1B). The immunoreactive band of 170 kD was not detected with the purified antiserum preabsorbed with 10 µmol/L synthetic peptide (Fig 1C) or with nonimmune rabbit serum (Fig 1D). These results indicate that the 170-kD protein in each tissue was ACE. A fine band with a molecular weight of 66 kD was sometimes observed not only with the immune serum but also with the preabsorbed or nonimmune serum. Thus, the 66-kD band was regarded as a nonspecific protein.

View larger version (71K): [in this window] [in a new window]   Figure 1. Western blot analysis (20 µg protein per lane) of the homogenates obtained from human kidney, lung, and the ventricles of control subjects and patients with myocardial infarction. A, Proteins stained with amido black; B, immunostaining with the anti-ACE antibody; C, immunostaining with the antibody preabsorbed with 1 mmol/L synthetic peptide; D, immunostaining with nonimmune rabbit serum. Lane 1, The homogenate of the control left ventricular tissue; lane 2, the homogenate of the aneurysmal left ventricular tissue; lanes 3 and 4, the homogenates of kidney and lung, respectively, obtained from a control subject at autopsy. A band of 170 kD disappeared with the preabsorbed antibody or nonimmune serum. A fine band was detected at a position of about 66 kD in each lane, and the band did not disappear with the preabsorbed antibody or nonimmune serum; therefore, it was regarded as a nonspecific product. The molecular mass standards are indicated in kilodaltons on the left.

Immunohistochemical Localization of Cardiac ACE
In all control hearts obtained at autopsy, no immunoreactivity for ACE was found in the myocytes of the left ventricles (Fig 2A), and positively stained myocytes for ACE were not recognized even with an undiluted purified antibody. On the other hand, immunostaining for ACE was strongly positive in the endothelial cells and in the endocardium of the left ventricles obtained from control patients (Fig 2B and 2C). No immunoreactivity for ACE was observed in consecutive sections incubated with the purified antiserum preabsorbed with 10 µmol/L synthetic peptide or with nonimmune rabbit serum (not shown).

View larger version (146K): [in this window] [in a new window]   Figure 2. Representative immunohistochemical stainings of left ventricular myocardium obtained at autopsy from a control subject without cardiac disorders and at a left ventricular aneurysmectomy after infarction. A, Myocytes were not immunostained with the anti-ACE antibody. B and C, ACE immunoreactivity was observed in endothelium (arrow in B) of the vessels and endocardium (arrow in C) of the left ventricle. D, The remaining viable myocytes located near the infarct scar were immunostained with anti-ACE antibody. E, In the serial section of D, myocytes were homogeneously immunostained with anti–myosin light chain 1 antibody. F, In the serial section of D and E, immunoreactivity was not detected with the purified antiserum preabsorbed with 10 µmol/L synthetic peptide. Photomicrographs of slides immunostained at a dilution of 1:100 of the anti-ACE antibody are presented. Magnification: A, x10; B and C, x80; and D through F, x13. Bars indicate 100 µm in A and D through F and 50 µm in B and C.

ACE immunoreactivity was observed in the left ventricular myocardium from patients with MI (Fig 2D). Myocardial cells were identified with antibody raised against myosin light chain 1 (Fig 2E). The remaining viable myocytes within the infarct scar showed positive staining for ACE in 5 of the 7 ventricles obtained at aneurysmectomy. ACE was detected in myocytes located in the proximity of the infarcted tissue of the three hearts with MI obtained at autopsy, and an extensive search in the left ventricular tissue remote from the infarction failed to reveal positively stained myocytes. In addition, the immunostaining at various concentrations of the purified anti-ACE antiserum showed that ACE immunoreactivity was observed in myocytes on the edge of the infarct scar from patients with MI at a dilution of 1:1000 but not at a dilution of 1:5000. No immunoreactivity for ACE was observed in consecutive sections immunostained with the purified antiserum preabsorbed with 10 µmol/L synthetic peptide (Fig 2F) or with nonimmune serum (not shown).

To identify more precisely nonmyocytes that contain ACE within the scar tissue after MI, we performed the immunostainings of scar tissue in consecutive sections using the specific monoclonal antibodies that recognize fibroblasts (Fig 3C), macrophages (Fig 3F), and smooth muscle cells (Fig 3I). In the infarct scar of the left ventricles, nonmyocytes positively stained for ACE were found for all patients with MI, including both aneurysmectomy and autopsy cases. As shown in Fig 3, immunopositive cells for ACE were observed in the left ventricular infarct scar, and positive cells in each infarct region were identified as fibroblasts (Fig 3A and 3B), macrophages (Fig 3D and 3E), and smooth muscle cells and vascular endothelial cells (Fig 3G and 3H, respectively). Smooth muscle cells of newly generated vessels and fibroblasts positively stained for ACE were observed in the scar tissue of all patients with MI and in macrophages in 7 of the 10 patients with infarction (except for patients 11, 16, and 17 in the Table). Immunostaining over a range of antibody concentrations showed that ACE immunoreactivity in nonmyocytes was detected at a dilution of 1:1000 but not at a dilution of 1:5000.

View larger version (155K): [in this window] [in a new window]   Figure 3. Representative immunohistochemical stainings of serial sections of the left ventricular scar tissue obtained from a patient with myocardial infarction. A, D, and G, Immunoreactivity was strongly detected in the infarct scar with the anti-ACE antibody. B, E, and H, Higher-magnification view of A, D, and G, respectively. C, F, and I, Fibroblasts, macrophages, and smooth muscle cells were immunostained with the specific monoclonal antibody in the serial section of A, D, and G, respectively. In H, an arrowhead and arrow represent ACE-positive smooth muscle cells and endothelial cells, respectively. Photomicrographs of slides immunostained at a dilution of 1:100 of the anti-ACE antibody are presented. Magnification: A, C, D, F, G, and I, x16; B, E, and H, x80. Bars indicate 100 µm in A, C, D, F, G, and I and 50 µm in B, E, and H.

	Discussion

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There are two isozymes of ACE, one produced by endothelium and several other somatic tissues (somatic ACE) and a smaller protein produced only by developing spermatozoa (testis ACE).^{26 27} Several studies demonstrated that the molecular weight of ACE extracted from human kidney or lung was 140²⁸ or 170 kD.²⁶ In this study, the immunogen that we used to make polyclonal antibody was a synthetic peptide corresponding to part of the human endothelial ACE,¹⁹ which was composed of 17 amino acids. We examined the specificity of the polyclonal antibody by Western blotting performed on membrane proteins extracted from the human lung and kidney, which were supposed to contain high levels of ACE,²⁹ and detected a 170-kD band. Thus, the antibody developed in this study recognizes the human ACE protein, and our results were obtained with the highly specific anti-ACE antibody.

We performed immunohistochemical staining for ACE in the left ventricles of patients with MI obtained by left ventricular aneurysmectomy or at autopsy and in the hearts of control subjects at autopsy. To distinguish between myocytes and nonmyocytes, immunostaining was carried out in serial sections with the monoclonal antibody that is specific for myosin light chain 1,^{24 25} and confirmed the localization of ACE in myocytes. In previous studies, it has been reported that both ACE mRNA level and activity are increased in left ventricular myocardium of patients with heart failure, including MI, and they have been determined by use of tissue homogenates of the myocardium.^{17 18} Thus, the participation of cardiomyocytes in ACE induction has been unknown in the failing human heart after MI. In the in vitro study, ACE protein was detected in the cultured neonatal and adult rat cardiac myocytes and fibroblasts,^{30 31} and through immunohistochemical study, the expression of ACE was observed in fibroblasts and macrophages of the rat heart after infarction^{32 33} and in endothelial cells and neointimal smooth muscle cells after vascular injury.¹² In the present study, we detected ACE immunoreactivity in the myocytes of human heart after MI. These results are consistent with our report²⁰ that ACE activity is increased in left ventricular aneurysm of patients after MI and suggest the contribution of the remaining viable myocytes to the increased ACE activity in aneurysmal tissue. Moreover, ACE immunoreactivity was observed in the remaining viable myocytes of the region adjacent to the infarct scar but not in the myocytes remote from the infarction of the hearts with MI obtained at autopsy, and by immunohistochemistry with various concentrations of the anti-ACE antibody, myocytes and nonmyocytes in the infarct scar were immunostained at a dilution of 1:1000 but not at a dilution of 1:5000. These results indicate that the distribution of ACE is confined to the remaining viable myocytes on the edge of the infarct scar and suggest the possibility that there may be no apparent difference in response of ACE expression among the aneurysmal tissue samples of MI hearts examined in this study, although quantification by immunohistochemistry is not accurate. We previously reported that the expression of atrial or fetal-type myosin light chain 1 in human ventricles with MI was strong in the surviving myocytes of the ventricular aneurysm compared with those in the noninfarcted ventricles and that its reexpression occurred as one of the regional responses to increased load.²⁴ Likewise, the regional ACE expression in postinfarct remodeling may be regulated by local wall stress as a possible stimulus because the aneurysmal left ventricular wall near the scarred tissue is very thin and wall tension is considered to be high. It is demonstrated that the factor released by mechanical stretch of cardiac ventricular myocytes in vitro is angiotensin II and that the autocrine secretion of angiotensin II plays a critical role in the stretch-induced hypertrophic response in the absence of neuronal or hormonal factors.³⁴ This report may support, at least partially, our hypothesis that regional ventricular loading may activate the RA system in cardiac myocytes after MI.

In scar tissue after infarction, ACE-positive fibroblasts, macrophages, and newly generated vascular smooth muscle cells were observed. Vascular endothelial cells were always positive for ACE staining in both control and infarcted hearts. The appearance of ACE-positive cells in infarct scar is consistent with the findings that ACE activity was increased in scar tissue of rat heart after MI.³⁵

Locally synthesized cardiac angiotensin II through the increased ACE activity in left ventricles may indirectly augment cardiac systolic function through facilitation of norepinephrine release from sympathetic nerve terminals³⁶ because angiotensin II may not have positive inotropic effects in ventricular human myocardium.³⁷ Angiotensin II may also induce myocardial hypertrophy¹⁴ and collagen production,^{38 39} and these effects may cause deterioration of cardiac function in patients after MI over long periods. Recent studies also demonstrate that ACE inhibitor treatment in stroke-prone spontaneously hypertensive rats improved cardiac function through the inhibition of bradykinin degradation,⁴⁰ and it is possible that the beneficial effects of ACE inhibitors in heart failure may be partially due to the inhibition of bradykinin degradation. ACE inhibition in the heart tissue may thus lead to improvement of the ventricular function and mortality rate in patients with heart failure.

A recent study demonstrated that the ACE mRNA level is higher in myocytes isolated from rats with ischemic cardiomyopathy than in normal myocytes.⁴¹ These results support our finding that the protein levels of ACE detected with the specific antibody are increased in the remaining viable myocytes on the edge of the infarct scar compared with the normal myocytes. In that report,⁴¹ however, ACE mRNA was detected in myocytes of normal rat heart, even though it was very low, and these results differ from our finding that ACE immunoreactivity was not detected in normal myocytes. To evaluate the sensitivity in immunohistochemical staining, we performed the immunostaining at various concentrations of the anti-ACE antiserum, and no immunoreactivity was detected even with an undiluted anti-ACE antibody in the left ventricular myocytes of all control hearts. However, we cannot exclude the possibility that the lack of ACE immunoreactivity in normal myocytes in the present study may be due to the low sensitivity of the anti-ACE antibody.

In conclusion, we have demonstrated that ACE immunoreactivity was seen in the left ventricular myocytes of the region adjacent to the infarct scar and in nonmyocytes such as fibroblasts, macrophages, vascular smooth muscle cells, and endothelial cells. These results indicate that ACE is markedly increased on the edge of the infarct scar and suggest that local ACE may be important in the ventricular remodeling after MI.

	Acknowledgments

 
This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science and Culture, and for biomedical research from the Smoking Research Foundation, Japan. We wish to acknowledge Dr Y. Miyauchi, First Department of Surgery, Kumamoto University School of Medicine, for providing tissue samples.

Received January 31, 1996; revision received April 3, 1996; accepted April 15, 1996.

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