- Email: [email protected]
Cardiac Fibrogenesis Following Infarction in Mice With Deletion of Inducible Nitric Oxide Synthase
Cardiac Fibrogenesis Following Infarction in Mice With Deletion of Inducible Nitric Oxide Synthase LI LU, MD; SUE SI CHEN, BA; AVIV HASSID, PHD; YAO SUN, MD, PHD
ABSTRACT: Background: Studies have shown that the absence of inducible nitric oxide synthase (iNOS) improves cardiac function and survival after myocardial infarction (MI). The responsible mechanisms, however, remain uncertain. Cardiac iNOS is significantly increased after MI, which is colocalized with fibrous tissue formation. Herein, we tested our hypothesis that iNOS is involved in the development of cardiac fibrosis. Methods: Wild-type and iNOS-knockout mice were subjected to MI by left coronary artery ligation. At week 1, 2, 3, and 4 post-MI, we addressed cardiac expression of profibrogenic mediator, growth of collagen-producing cells, collagen synthesis, and degradation. Results: In the infarcted myocardium of wild-type and iNOS-knockout mice, transforming growth factor (TGF)-1 expression was significantly increased, particularly in the early stage; myofibroblasts appeared and became abundant for over 4 weeks; matrix metalloproteinase-1 expression
was low, whereas tissue inhibitor of matrix metalloproteinase-1 was significantly elevated; type-I collagen mRNA was significantly increased and collagen was continuously accumulated. In the noninfarcted myocardium, TGF-1 and type-I collagen mRNA levels as well as collagen volume were also elevated, but less evident than infarcted myocardium. However, there was no significant difference in cardiac TGF-1 expression, myofibroblast population, collagen synthesis/degradation, and collagen volume between wild-type and iNOS-knockout mice with MI. Conclusion: The current study suggests that iNOS-induced nitric oxide production may not mediate cardiac fibrosis after MI. Thus, other mechanisms are involved in nitrosative stress-induced cardiac dysfunction after MI. KEY INDEXING TERMS: Myocardial infarction; Cardiac fibrosis; Nitric oxide; Mice. [Am J Med Sci 2008;335(6):431–438.]
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ture. Elucidating pathophysiologic characteristics and local regulating factors that account for cardiac fibrosis are of considerable importance. Nitric oxide is a molecule implicated in a variety of physiological and pathological processes. Its production depends on 3 isoforms of nitric oxide synthases, each having distinct tissue localization.4 – 6 After MI, inducible nitric oxide synthase (iNOS) activity is significantly increased in both infarcted and noninfarcted myocardium, which leads to enhanced nitric oxide production.7,8 Cells expressing iNOS are primarily macrophages in the infarcted myocardium and myocytes in the noninfarcted myocardium.7,8 The role of nitric oxide in the infarcted heart is not fully understood. Studies have shown that the absence of iNOS in gene knockout mice improves cardiac function and survival compared with wild-type animals after MI.9 –11 Studies have also shown that enhanced iNOS in the infarcted heart is colocalized with accumulated collagen. In the current study, we sought to determine whether iNOS-induced nitric oxide production is involved in the development of cardiac fibrosis that appeared in both infarcted and noninfarcted myocardium.
hronic heart failure has emerged as a major health problem in the past 2 decades. It appears most commonly in patients with myocardial infarction (MI). Ventricular remodeling is recognized to be a major determinant for the development of impaired ventricular function, leading to a poor prognosis. Structural remodeling after MI is characterized by scar formation in the infarcted myocardium as well as interstitial fibrosis and hypertrophy in noninfarcted sites.1–3 A particularly vexing aspect of such myocardial remodeling is its progressive na-
From the Division of Cardiovascular Diseases (LL, SSC), Department of Medicine; and Department of Physiology (AH), University of Tennessee, Health Science Center, Memphis, Tennessee. Submitted July 17, 2007; accepted in revised form August 1, 2007. Supported by the National Heart, Lung, and Blood Institute (RO1-HL077668 to YS). Correspondence: Yao Sun, MD, PhD, Division of Cardiovascular Diseases, Department of Medicine, University of Tennessee Health Science Center, 956 Court Ave. Rm B310, Memphis, TN 38163 (E-mail: [email protected]). THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
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The growth of extracellular matrix-producing cells, the balance between collagen synthesis and degradation, and the expression of profibrotic cytokines, particularly transforming growth factor (TGF)1, are the major determinants for cardiac fibrous tissue formation. Myofibroblasts are phenotypically transformed fibroblasts, which appear in the repairing sites of the infarcted heart and play a major role in cardiac collagen synthesis.12,13 Collagen degradation involves degradative enzymes, matrix metalloproteinase (MMPs), whereas their activity is controlled by tissue inhibitor of MMPs (TIMPs). By
using a MI model created by coronary artery ligation in wild-type and iNOS-knockout mice, we studied potential regulation of iNOS on the molecular and cellular events related to cardiac fibrosis in the infarcted heart, including the population of myofibroblasts, expression of TGF-1, type-I collagen, MMP-1, and TIMP-1 and collagen volume. Materials and Methods Animal Model Left ventricular anterior transmural MI was created in 8-weekold male wild-type C57BL/6J mice and iNOS-knockout mice (The
Figure 1. Cardiac immunohistochemical ␣-SMA labeling. Myofibroblasts are not present in the normal and noninfarcted myocardium. After MI, myofibroblasts (brown) become abundant at the infarcted myocardium in wild-type and iNOS-knockout mice at week 1 and remain evident at week 4 (magnification, 200⫻).
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Jackson Laboratory Bar Harbor, Maine) by permanent ligation of the left coronary artery with silk ligature.14 Mice were anesthetized, intubated, and ventilated with a rodent respirator. After left thoracotomy, the heart was exposed and 7-0 silk suture placed around the left coronary artery. The vessel was ligated, the chest closed, and lungs reinflated using positive-end expiratory pressure. Animals were killed at week 1, 2, 3, and 4 (n ⫽ 10/time point/group). Sham-operated wild-type mice served as controls. Hearts were removed, rinsed in cold normal saline, frozen in isopentane with dry ice, and kept at ⫺80°C. Serial cryostat coronal sections were prepared for the following studies. This study was approved by the University of Tennessee Health Science Center Animal Care and Use Committee. In Situ Hybridization The localization and optical density of cardiac TGF-1, type-I collagen, MMP-1, and TIMP-1 mRNAs were detected by quantitative in situ hybridization. In brief, cardiac sections (16 m) were fixed in 4% formaldehyde for 10 minutes, washed with phosphate-buffered saline (PBS, pH 7.4), and incubated in 0.25% acetic anhydride in 0.1 M TE-HCl for 10 minutes. Sections were then hybridized overnight with (35S)-dATP-labeled DNA probes for TGF-1, TIMP-1, MMP-1, and type-I collagen at 450°C. The hybridized sections were then washed, dried, and subsequently exposed to Kodak Biomax x-ray film. After exposure, the film was developed. Quantitation of mRNA optical density (4 sections/ heart) was performed using a computer image analysis system (NIH Image, 1.60).15 Immunohistochemistry The population of myofibroblasts in the infarcted heart was detected by immunohistochemistry. Cardiac sections (6 m) were air dried, fixed in 10% buffered formalin for 5 minutes, and washed in PBS for 10 minutes. Sections were then incubated with the primary antibody against ␣-smooth muscle actin (SMA) (Sigma, St Louis, MO) for 1 hour at room temperature. Sections were then incubated with IgG-peroxidase conjugated secondary antibody (Sigma) for 1 hour at room temperature, washed in PBS for 10 minutes, and incubated with 0.5 mg/mL diaminobenzidine tetrahydrochloride 2-hydrate and 0.05% H2O2 for 5 minutes. Negative control sections were incubated with secondary antibody alone. All sections were counterstained with hematoxylin, dehydrated, mounted, and viewed by light microscopy.16 Interstitial myofibroblasts volume in the infarcted myocardium was determined by videodensitometry (NIH image 1.60) and was calculated as the sum of all interstitial ␣-SMA⫹ areas, divided by the sum of all areas of infarcted myocardium (4 sections/heart). Cardiac Morphology Cardiac sections (6 m) were prepared to determine the fibrillar collagen accumulation by collagen-specific picrosirius red staining and observed by light microscopy as previously reported.17 Collagen volume fraction of both the infarcted and noninfarcted myocardium was determined separately using a computer image analysis system (NIH image, 1.60) and was calculated as the sum of connective tissue areas, divided by the sum of connective tissue area and muscle area in all fields of infarcted or noninfarcted myocardium. Statistical Analysis Statistical analysis of in situ hybridization data, myofibroblasts, and collagen volume fraction was performed using analysis of variance. Values are expressed as mean ⫾ SEM with P ⬍ 0.05 considered significant. Multiple group comparisons among controls and each group were made by Scheffe F test.
Results Cardiac Myofibroblasts A hallmark of myofibroblasts is the expression of ␣-SMA. Figure 1 shows immunohistochemical ␣-SMA THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
Figure 2. Interstitial myofibroblast volume fraction in the infarcted myocardium of wild-type and iNOS-knockout mice at different stages post-MI.
labeling in control (sham-operated heart) and infarcted heart. Myofibroblasts were not observed in the control heart and noninfarcted myocardium (nonischemic area of an infarcted heart). After MI, myofibroblasts became evident in the infarcted myocardium at week 1 and remained abundant in the following weeks. The localization and population of myofibroblasts in the infarcted heart of iNOS-knockout mice were, however, similar to those found in wild-type mice. The quantitative data of myofibroblast volume fraction in the infarcted myocardium of wild-type and iNOS-knockout mice are shown in Figure 2. Cardiac TGF-1 Gene Expression As detected by quantitative in situ hybridization, low levels of cardiac TGF-1 mRNA were observed in sham-operated heart (Figure 3). After MI, TGF-1 mRNA was significantly increased at the site of MI in both wild-type and iNOS-knockout mice at week 1 and then gradually declined (Figure 3). TGF-1 mRNA was slightly increased at noninfarcted myocardium in wild-type and iNOS-knockout mice at week 1 and returned to normal levels in the following weeks. However, there was no significant difference in cardiac TGF-1 mRNA levels between wildtype and iNOS-knockout mice for the course of 4 weeks post-MI (Figure 4). Cardiac Type-I Collagen Gene Expression Normal myocardium contains low levels of type-I collagen mRNA (Figure 3). After MI, it was significantly elevated in the infarcted myocardium of both wild-type and iNOS-knockout mice at week 1 and gradually declined thereafter (Figure 3). In the noninfarcted myocardium, type-I collagen mRNA was slightly increased in wild-type and iNOS-knockout mice at week 1, but not in the following weeks as compared with controls. No significant difference in 433
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Figure 3. Cardiac TGF-1, type-I collagen, MMP-1, and TIMP-1 gene expression. Detected by in situ hybridization, low levels of TGF-1, type-I collagen, MMP-1, and TIMP-1 mRNAs are located in sham-operated heart. At week 1 post-MI, TGF-1, type-I collagen, and TIMP-I mRNA levels are largely increased in the infarcted myocardium of wild-type and iNOS-knockout mice, whereas MMP-1 mRNA levels remain low. TGF-1 and type-I collagen mRNA levels are also increased in noninfarcted myocardium at this stage.
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Figure 4. Temporal response of cardiac TGF-1, type-I collagen, MMP-1, and TIMP-1 gene expression in the infarcted myocardium of wild-type and iNOS-knockout mice. *P ⬍ 0.05 versus sham-operated controls.
cardiac type 1 collagen mRNA was observed between wild-type and iNOS-knockout mice at all time points (Figure 4). Cardiac MMP-1 And TIMP-1 Gene Expression The sham-operated heart contains low levels of MMP-1 mRNA (Figure 3). After MI, MMP-1 mRNA levels remained low in the infarcted and noninfarcted myocardium of both wild-type and iNOSknockout mice for the course of 4 weeks after MI (Figures 3 and 4). Compared with sham-operated heart, TIMP-1 mRNA was significantly elevated in the infarcted myocardium of wild-type and iNOS-knockout mice at week 1 and then gradually declined to normal levels (Figures 3 and 4). However, we did not observe significant difference in TIMP-1 mRNA levels in the infarcted heart between wild-type and iNOSknockout mice. Cardiac Collagen Volume By collagen-specific picrosirius red staining, we observed a small amount of collagen in the interstiTHE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
tial space of sham-operated heart. After MI, collagen started to accumulate in the infarcted myocardium at week 1 and continuously increased over the course of 4 weeks in both wild-type and iNOS-knockout mice (Figure 5). Increased collagen volume was also seen in the noninfarcted myocardium at week 4 (Figure 5), but not in earlier time points. Collagen volume in the infarcted heart of iNOS-knockout mice was, however, similar to those found in wildtype mice. The quantitative data of collagen volume fraction in the infarcted myocardium of wild-type and iNOS-knockout mice are shown in Figure 6. Discussion After MI, iNOS is significantly increased in the infarcted heart, leading to enhanced nitric oxide production in the early stage of MI.7–9 The increase in iNOS expression is temporally and spatially coincident with cardiac fibrogenetic response in the infarcted heart. The current study addressed the potential regulation of nitric oxide on molecular and 435
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Figure 5. Collagen in sham-operated and infarcted heart of wild-type mice. A small amount of collagen is present in the interstitial space of sham-operated heart. After MI, collagen starts to accumulate at the site of MI at week 1 and continues to build up at week 2, 3, and 4. Collagen volume is also slightly increased in noninfarcted myocardium at week 4 (magnification, 200⫻).
cellular events related to cardiac fibrogenesis that appears within the infarcted mouse heart. TGF-1 is a locally generated profibrogenic cytokine. It primarily suppresses collagen degradation and stimulates matrix producing cell proliferation and collagen synthesis in the repairing tissue, thereby leading to fibrous tissue formation. Excessive TGF-1 contributes to a pathologic excess of tissue fibrosis in various diseases. Studies from our and other laboratories have shown significantly increased TGF-1 synthesis within the infarcted and noninfarcted myocardium in rats.14,18,19 In the current study, we further observed significantly elevated TGF-1 gene expression particularly at the infarcted myocardium, indicating the involvement of TGF-1 in car436
diac fibrogenesis in mice. However, the depletion of iNOS does not affect cardiac TGF-1 expression. This finding suggests that nitric oxide may not play a role on TGF-1 synthesis in the infarcted mouse heart. Interstitial fibroblasts are responsible for collagen synthesis in the normal heart, and maintain cardiac collagen volume at 2% to 2.5% of total heart volume.16 However, studies have shown that myofibroblasts are the major cells responsible for fibrous tissue formation in the repairing tissue including the infarcted heart.13,20 Myofibroblasts possess the features of both fibroblasts and smooth muscle cells and actively produce collagen in the repairing tissue.12,13 These cells are primarily differentiated June 2008 Volume 335 Number 6
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Figure 6. Collagen volume fraction in the infarcted myocardium of both wild-type and iNOS-knockout mice.
from interstitial fibroblasts under the stimulation of TGF-1.21 In the current study, myofibroblasts appeared in the infarcted myocardium at week 1 and became abundant in the following weeks in wildtype and iNOS-knockout mice. These cells are colocalized with accumulated collagen, indicating that they are responsible for fibrous tissue formation in the infarcted mouse heart. The appearance and population of myofibroblasts in the infarcted myocardium, however, are similar in wild-type and iNOSknockout mice at all time points. This observation suggests that nitric oxide may not mediate the differentiation and proliferation of myofibroblasts in the infarcted heart. Myofibroblasts are, however, not seen in the noninfarcted myocardium in both wild-type and iNOS-knockout mice, suggesting that interstitial fibroblasts are contributory to the increased collagen volume in noninfarcted myocardium. Type-I collagen is the major extracellular component in the heart. The current study shows markedly increased type-I collagen gene expression in the infarcted myocardium of wild-type and iNOS-knockout mice, particularly in the early stage of MI. Enhanced cardiac collagen mRNA is anatomically coincident with myofibroblasts and accumulated collagen, indicating up-regulated collagen synthesis in the infarcted myocardium. Type-I collagen expression, however, is only increased in the noninfarcted myocardium at week 1, which leads to increased interstitial collagen volume in noninfarcted myocardium found in the later stage. Collagen synthesis and degradation coexist in the heart and their balance determines cardiac collagen volume. The current study shows low levels of MMP-1 mRNA in both control and infarcted mouse heart, suggesting that cardiac MMP synthesis remains low after MI. MMP activity is controlled by tissue MMP inhibitors, TIMPs.22 TIMPs function as an important regulatory brake on MMP activity by inhibition of the THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
active species, thereby suppressing collagen degradation. We observed significantly elevated TIMP-1 expression at the site of infarction, suggesting that cardiac MMP activity is suppressed. These observations indicate that cardiac collagen synthesis is elevated, whereas collagen degradation is suppressed in the infarcted mouse heart. The imbalance of cardiac collagen turnover facilitates cardiac fibrous tissue accumulation, leading to cardiac fibrosis. However, our study has shown that in the infarcted heart, type-1 collagen, MMP-1 and TIMP-1 expression, and collagen accumulation in iNOS-knockout mice were similar to those found in wild-type mice. Thus, the effects of iNOS produced nitric oxide on collagen synthesis and degradation were not evident in the infarcted heart. In summary, the current study has examined the spatial and temporal fibrogenic responses in the infarcted mouse heart and potential regulation of nitric oxide in cardiac fibrogenesis. We observed increased TGF-1 expression, which is colocalized with appearance of myofibroblasts and activated collagen synthesis in the infarcted heart. Cardiac collagen degradation, however, is suppressed after MI. In iNOSknockout mice, however, myofibroblast population, profibrotic cytokine expression, collagen synthesis, and degradation as well as collagen volume in the infarcted heart were not significantly different from that in wild-type mice. Our findings suggest that iNOS-induced nitric oxide production may not mediate cardiac fibrosis after MI. It further suggests that other mechanisms are involved in nitrosative stress-induced cardiac dysfunction after MI. Acknowledgments The authors gratefully acknowledge the skillful technical assistance of Yuanjian Chen and Youde Jiang. References 1. Jugdutt BI. Ventricular remodeling after infarction and the extracellular collagen matrix: when is enough enough? Circulation 2003;08:1395– 403. 2. Ertl G, Frantz S. Healing after myocardial infarction. Cardiovasc Res 2005;66:22–32. 3. Anversa P, Li P, Zhang X. Ischemic myocardial injury and ventricular remodeling. Cardiovasc Res 1993;27:145–57. 4. Papapetropoulos A, Rudic RD, Sessa WC. Molecular control of nitric oxide synthases in the cardiovascular system. Cardiovasc Res 1999;43:509 –20. 5. Boucher JL, Moali C, Tenu JP. Nitric oxide biosynthesis, nitric oxide synthase inhibitors and arginase competition for L-arginine utilization. Cell Mol Life Sci 1999;55:1015–28. 6. Andrew PJ, Mayer B. Enzymatic function of nitric oxide synthases. Cardiovasc Res 1999;43:521–31. 7. Takimoto Y, Aoyama T, Keyamura R. Differential expression of three types of nitric oxide synthase in both infarcted and non-infarcted left ventricles after myocardial infarction in the rat. Int J Cardiol 2000;l76:135– 45. 8. Bing RJ, Suzuki H. Myocardial infarction and nitric oxide. Mol Cell Biochem 1996;160 –161:303– 6.
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9. Liu YH, Carretero OA, Cingolani OH. Role of inducible nitric oxide synthase in cardiac function and remodeling in mice with heart failure due to myocardial infarction. Am J Physiol Heart Circ Physiol 2005;289:H2616 –H2623. 10. Marfella R, Di Filippo C, Esposito K. Absence of inducible nitric oxide synthase reduces myocardial damage during ischemia reperfusion in streptozotocin-induced hyperglycemic mice. Diabetes 2004;53:454 – 62. 11. Feng Q, Lu X, Jones DL. Increased inducible nitric oxide synthase expression contributes to myocardial dysfunction and higher mortality after myocardial infarction in mice. Circulation 2001;104:700 – 4. 12. Desmouliere A, Gabbiani G. The role of the myofibroblast in wound healing and fibrocontractive diseases. In: Richard AF, editor. The molecular and cellular biology of wound repair. New York, NY: Plenum Press; 1996. p. 391– 423. 13. Sun Y, Weber KT. Angiotensin converting enzyme and myofibroblasts during tissue repair in the rat heart. J Mol Cell Cardiol 1996;28:851– 8. 14. Sun Y, Zhang JQ, Zhang J. Angiotensin II, transforming growth factor-beta1 and repair in the infarcted heart. J Mol Cell Cardiol 1998;30:1559 – 69. 15. Zhao W, Ahokas RA, Sun Y. ANG II-induced cardiac molecular and cellular events: role of aldosterone. Am J Physiol Heart Circ Physiol 2006;291:H336 –H343.
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16. Sun Y, Zhang JQ, Zhang J. Cardiac remodeling by fibrous tissue after infarction in rats. J Lab Clin Med 2000; 135:316 –23. 17. Sun Y, Ratjaska A, Weber KT. Inhibition of angiotensinconverting enzyme and attenuation of myocardial fibrosis by lisinopril in rats receiving angiotensin II. J Lab Clin Med 1995;126:95–101. 18. Youn TJ, Kim HS, Oh BH. Ventricular remodeling and transforming growth factor-beta 1 mRNA expression after nontransmural myocardial infarction in rats: effects of angiotensin converting enzyme inhibition and angiotensin II type 1 receptor blockade. Basic Res Cardiol 1999;94:246 –53. 19. Yue P, Massie BM, Simpson PC. Cytokine expression increases in nonmyocytes from rats with postinfarction heart failure. Am J Physiol 1998;275:H250 –H258. 20. Yano T, Miura T, Ikeda Y. Intracardiac fibroblasts, but not bone marrow derived cells, are the origin of myofibroblasts in myocardial infarct repair. Cardiovasc Pathol 2005;14:241– 6. 21. Ronty MJ, Leivonen SK, Hinz B. Isoform-specific regulation of the actin-organizing protein palladin during TGFbeta1-induced myofibroblast differentiation. J Invest Dermatol 2006;126:2387–96. 22. Kassiri Z, Khokha R. Myocardial extra-cellular matrix and its regulation by metalloproteinases and their inhibitors. Thromb Haemost 2005;93:212–9.
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ABSTRACT: Background: Studies have shown that the absence of inducible nitric oxide synthase (iNOS) improves cardiac function and survival after myocardial infarction (MI). The responsible mechanisms, however, remain uncertain. Cardiac iNOS is significantly increased after MI, which is colocalized with fibrous tissue formation. Herein, we tested our hypothesis that iNOS is involved in the development of cardiac fibrosis. Methods: Wild-type and iNOS-knockout mice were subjected to MI by left coronary artery ligation. At week 1, 2, 3, and 4 post-MI, we addressed cardiac expression of profibrogenic mediator, growth of collagen-producing cells, collagen synthesis, and degradation. Results: In the infarcted myocardium of wild-type and iNOS-knockout mice, transforming growth factor (TGF)-1 expression was significantly increased, particularly in the early stage; myofibroblasts appeared and became abundant for over 4 weeks; matrix metalloproteinase-1 expression
was low, whereas tissue inhibitor of matrix metalloproteinase-1 was significantly elevated; type-I collagen mRNA was significantly increased and collagen was continuously accumulated. In the noninfarcted myocardium, TGF-1 and type-I collagen mRNA levels as well as collagen volume were also elevated, but less evident than infarcted myocardium. However, there was no significant difference in cardiac TGF-1 expression, myofibroblast population, collagen synthesis/degradation, and collagen volume between wild-type and iNOS-knockout mice with MI. Conclusion: The current study suggests that iNOS-induced nitric oxide production may not mediate cardiac fibrosis after MI. Thus, other mechanisms are involved in nitrosative stress-induced cardiac dysfunction after MI. KEY INDEXING TERMS: Myocardial infarction; Cardiac fibrosis; Nitric oxide; Mice. [Am J Med Sci 2008;335(6):431–438.]
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ture. Elucidating pathophysiologic characteristics and local regulating factors that account for cardiac fibrosis are of considerable importance. Nitric oxide is a molecule implicated in a variety of physiological and pathological processes. Its production depends on 3 isoforms of nitric oxide synthases, each having distinct tissue localization.4 – 6 After MI, inducible nitric oxide synthase (iNOS) activity is significantly increased in both infarcted and noninfarcted myocardium, which leads to enhanced nitric oxide production.7,8 Cells expressing iNOS are primarily macrophages in the infarcted myocardium and myocytes in the noninfarcted myocardium.7,8 The role of nitric oxide in the infarcted heart is not fully understood. Studies have shown that the absence of iNOS in gene knockout mice improves cardiac function and survival compared with wild-type animals after MI.9 –11 Studies have also shown that enhanced iNOS in the infarcted heart is colocalized with accumulated collagen. In the current study, we sought to determine whether iNOS-induced nitric oxide production is involved in the development of cardiac fibrosis that appeared in both infarcted and noninfarcted myocardium.
hronic heart failure has emerged as a major health problem in the past 2 decades. It appears most commonly in patients with myocardial infarction (MI). Ventricular remodeling is recognized to be a major determinant for the development of impaired ventricular function, leading to a poor prognosis. Structural remodeling after MI is characterized by scar formation in the infarcted myocardium as well as interstitial fibrosis and hypertrophy in noninfarcted sites.1–3 A particularly vexing aspect of such myocardial remodeling is its progressive na-
From the Division of Cardiovascular Diseases (LL, SSC), Department of Medicine; and Department of Physiology (AH), University of Tennessee, Health Science Center, Memphis, Tennessee. Submitted July 17, 2007; accepted in revised form August 1, 2007. Supported by the National Heart, Lung, and Blood Institute (RO1-HL077668 to YS). Correspondence: Yao Sun, MD, PhD, Division of Cardiovascular Diseases, Department of Medicine, University of Tennessee Health Science Center, 956 Court Ave. Rm B310, Memphis, TN 38163 (E-mail: [email protected]). THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
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The growth of extracellular matrix-producing cells, the balance between collagen synthesis and degradation, and the expression of profibrotic cytokines, particularly transforming growth factor (TGF)1, are the major determinants for cardiac fibrous tissue formation. Myofibroblasts are phenotypically transformed fibroblasts, which appear in the repairing sites of the infarcted heart and play a major role in cardiac collagen synthesis.12,13 Collagen degradation involves degradative enzymes, matrix metalloproteinase (MMPs), whereas their activity is controlled by tissue inhibitor of MMPs (TIMPs). By
using a MI model created by coronary artery ligation in wild-type and iNOS-knockout mice, we studied potential regulation of iNOS on the molecular and cellular events related to cardiac fibrosis in the infarcted heart, including the population of myofibroblasts, expression of TGF-1, type-I collagen, MMP-1, and TIMP-1 and collagen volume. Materials and Methods Animal Model Left ventricular anterior transmural MI was created in 8-weekold male wild-type C57BL/6J mice and iNOS-knockout mice (The
Figure 1. Cardiac immunohistochemical ␣-SMA labeling. Myofibroblasts are not present in the normal and noninfarcted myocardium. After MI, myofibroblasts (brown) become abundant at the infarcted myocardium in wild-type and iNOS-knockout mice at week 1 and remain evident at week 4 (magnification, 200⫻).
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Jackson Laboratory Bar Harbor, Maine) by permanent ligation of the left coronary artery with silk ligature.14 Mice were anesthetized, intubated, and ventilated with a rodent respirator. After left thoracotomy, the heart was exposed and 7-0 silk suture placed around the left coronary artery. The vessel was ligated, the chest closed, and lungs reinflated using positive-end expiratory pressure. Animals were killed at week 1, 2, 3, and 4 (n ⫽ 10/time point/group). Sham-operated wild-type mice served as controls. Hearts were removed, rinsed in cold normal saline, frozen in isopentane with dry ice, and kept at ⫺80°C. Serial cryostat coronal sections were prepared for the following studies. This study was approved by the University of Tennessee Health Science Center Animal Care and Use Committee. In Situ Hybridization The localization and optical density of cardiac TGF-1, type-I collagen, MMP-1, and TIMP-1 mRNAs were detected by quantitative in situ hybridization. In brief, cardiac sections (16 m) were fixed in 4% formaldehyde for 10 minutes, washed with phosphate-buffered saline (PBS, pH 7.4), and incubated in 0.25% acetic anhydride in 0.1 M TE-HCl for 10 minutes. Sections were then hybridized overnight with (35S)-dATP-labeled DNA probes for TGF-1, TIMP-1, MMP-1, and type-I collagen at 450°C. The hybridized sections were then washed, dried, and subsequently exposed to Kodak Biomax x-ray film. After exposure, the film was developed. Quantitation of mRNA optical density (4 sections/ heart) was performed using a computer image analysis system (NIH Image, 1.60).15 Immunohistochemistry The population of myofibroblasts in the infarcted heart was detected by immunohistochemistry. Cardiac sections (6 m) were air dried, fixed in 10% buffered formalin for 5 minutes, and washed in PBS for 10 minutes. Sections were then incubated with the primary antibody against ␣-smooth muscle actin (SMA) (Sigma, St Louis, MO) for 1 hour at room temperature. Sections were then incubated with IgG-peroxidase conjugated secondary antibody (Sigma) for 1 hour at room temperature, washed in PBS for 10 minutes, and incubated with 0.5 mg/mL diaminobenzidine tetrahydrochloride 2-hydrate and 0.05% H2O2 for 5 minutes. Negative control sections were incubated with secondary antibody alone. All sections were counterstained with hematoxylin, dehydrated, mounted, and viewed by light microscopy.16 Interstitial myofibroblasts volume in the infarcted myocardium was determined by videodensitometry (NIH image 1.60) and was calculated as the sum of all interstitial ␣-SMA⫹ areas, divided by the sum of all areas of infarcted myocardium (4 sections/heart). Cardiac Morphology Cardiac sections (6 m) were prepared to determine the fibrillar collagen accumulation by collagen-specific picrosirius red staining and observed by light microscopy as previously reported.17 Collagen volume fraction of both the infarcted and noninfarcted myocardium was determined separately using a computer image analysis system (NIH image, 1.60) and was calculated as the sum of connective tissue areas, divided by the sum of connective tissue area and muscle area in all fields of infarcted or noninfarcted myocardium. Statistical Analysis Statistical analysis of in situ hybridization data, myofibroblasts, and collagen volume fraction was performed using analysis of variance. Values are expressed as mean ⫾ SEM with P ⬍ 0.05 considered significant. Multiple group comparisons among controls and each group were made by Scheffe F test.
Results Cardiac Myofibroblasts A hallmark of myofibroblasts is the expression of ␣-SMA. Figure 1 shows immunohistochemical ␣-SMA THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
Figure 2. Interstitial myofibroblast volume fraction in the infarcted myocardium of wild-type and iNOS-knockout mice at different stages post-MI.
labeling in control (sham-operated heart) and infarcted heart. Myofibroblasts were not observed in the control heart and noninfarcted myocardium (nonischemic area of an infarcted heart). After MI, myofibroblasts became evident in the infarcted myocardium at week 1 and remained abundant in the following weeks. The localization and population of myofibroblasts in the infarcted heart of iNOS-knockout mice were, however, similar to those found in wild-type mice. The quantitative data of myofibroblast volume fraction in the infarcted myocardium of wild-type and iNOS-knockout mice are shown in Figure 2. Cardiac TGF-1 Gene Expression As detected by quantitative in situ hybridization, low levels of cardiac TGF-1 mRNA were observed in sham-operated heart (Figure 3). After MI, TGF-1 mRNA was significantly increased at the site of MI in both wild-type and iNOS-knockout mice at week 1 and then gradually declined (Figure 3). TGF-1 mRNA was slightly increased at noninfarcted myocardium in wild-type and iNOS-knockout mice at week 1 and returned to normal levels in the following weeks. However, there was no significant difference in cardiac TGF-1 mRNA levels between wildtype and iNOS-knockout mice for the course of 4 weeks post-MI (Figure 4). Cardiac Type-I Collagen Gene Expression Normal myocardium contains low levels of type-I collagen mRNA (Figure 3). After MI, it was significantly elevated in the infarcted myocardium of both wild-type and iNOS-knockout mice at week 1 and gradually declined thereafter (Figure 3). In the noninfarcted myocardium, type-I collagen mRNA was slightly increased in wild-type and iNOS-knockout mice at week 1, but not in the following weeks as compared with controls. No significant difference in 433
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Figure 3. Cardiac TGF-1, type-I collagen, MMP-1, and TIMP-1 gene expression. Detected by in situ hybridization, low levels of TGF-1, type-I collagen, MMP-1, and TIMP-1 mRNAs are located in sham-operated heart. At week 1 post-MI, TGF-1, type-I collagen, and TIMP-I mRNA levels are largely increased in the infarcted myocardium of wild-type and iNOS-knockout mice, whereas MMP-1 mRNA levels remain low. TGF-1 and type-I collagen mRNA levels are also increased in noninfarcted myocardium at this stage.
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Figure 4. Temporal response of cardiac TGF-1, type-I collagen, MMP-1, and TIMP-1 gene expression in the infarcted myocardium of wild-type and iNOS-knockout mice. *P ⬍ 0.05 versus sham-operated controls.
cardiac type 1 collagen mRNA was observed between wild-type and iNOS-knockout mice at all time points (Figure 4). Cardiac MMP-1 And TIMP-1 Gene Expression The sham-operated heart contains low levels of MMP-1 mRNA (Figure 3). After MI, MMP-1 mRNA levels remained low in the infarcted and noninfarcted myocardium of both wild-type and iNOSknockout mice for the course of 4 weeks after MI (Figures 3 and 4). Compared with sham-operated heart, TIMP-1 mRNA was significantly elevated in the infarcted myocardium of wild-type and iNOS-knockout mice at week 1 and then gradually declined to normal levels (Figures 3 and 4). However, we did not observe significant difference in TIMP-1 mRNA levels in the infarcted heart between wild-type and iNOSknockout mice. Cardiac Collagen Volume By collagen-specific picrosirius red staining, we observed a small amount of collagen in the interstiTHE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
tial space of sham-operated heart. After MI, collagen started to accumulate in the infarcted myocardium at week 1 and continuously increased over the course of 4 weeks in both wild-type and iNOS-knockout mice (Figure 5). Increased collagen volume was also seen in the noninfarcted myocardium at week 4 (Figure 5), but not in earlier time points. Collagen volume in the infarcted heart of iNOS-knockout mice was, however, similar to those found in wildtype mice. The quantitative data of collagen volume fraction in the infarcted myocardium of wild-type and iNOS-knockout mice are shown in Figure 6. Discussion After MI, iNOS is significantly increased in the infarcted heart, leading to enhanced nitric oxide production in the early stage of MI.7–9 The increase in iNOS expression is temporally and spatially coincident with cardiac fibrogenetic response in the infarcted heart. The current study addressed the potential regulation of nitric oxide on molecular and 435
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Figure 5. Collagen in sham-operated and infarcted heart of wild-type mice. A small amount of collagen is present in the interstitial space of sham-operated heart. After MI, collagen starts to accumulate at the site of MI at week 1 and continues to build up at week 2, 3, and 4. Collagen volume is also slightly increased in noninfarcted myocardium at week 4 (magnification, 200⫻).
cellular events related to cardiac fibrogenesis that appears within the infarcted mouse heart. TGF-1 is a locally generated profibrogenic cytokine. It primarily suppresses collagen degradation and stimulates matrix producing cell proliferation and collagen synthesis in the repairing tissue, thereby leading to fibrous tissue formation. Excessive TGF-1 contributes to a pathologic excess of tissue fibrosis in various diseases. Studies from our and other laboratories have shown significantly increased TGF-1 synthesis within the infarcted and noninfarcted myocardium in rats.14,18,19 In the current study, we further observed significantly elevated TGF-1 gene expression particularly at the infarcted myocardium, indicating the involvement of TGF-1 in car436
diac fibrogenesis in mice. However, the depletion of iNOS does not affect cardiac TGF-1 expression. This finding suggests that nitric oxide may not play a role on TGF-1 synthesis in the infarcted mouse heart. Interstitial fibroblasts are responsible for collagen synthesis in the normal heart, and maintain cardiac collagen volume at 2% to 2.5% of total heart volume.16 However, studies have shown that myofibroblasts are the major cells responsible for fibrous tissue formation in the repairing tissue including the infarcted heart.13,20 Myofibroblasts possess the features of both fibroblasts and smooth muscle cells and actively produce collagen in the repairing tissue.12,13 These cells are primarily differentiated June 2008 Volume 335 Number 6
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Figure 6. Collagen volume fraction in the infarcted myocardium of both wild-type and iNOS-knockout mice.
from interstitial fibroblasts under the stimulation of TGF-1.21 In the current study, myofibroblasts appeared in the infarcted myocardium at week 1 and became abundant in the following weeks in wildtype and iNOS-knockout mice. These cells are colocalized with accumulated collagen, indicating that they are responsible for fibrous tissue formation in the infarcted mouse heart. The appearance and population of myofibroblasts in the infarcted myocardium, however, are similar in wild-type and iNOSknockout mice at all time points. This observation suggests that nitric oxide may not mediate the differentiation and proliferation of myofibroblasts in the infarcted heart. Myofibroblasts are, however, not seen in the noninfarcted myocardium in both wild-type and iNOS-knockout mice, suggesting that interstitial fibroblasts are contributory to the increased collagen volume in noninfarcted myocardium. Type-I collagen is the major extracellular component in the heart. The current study shows markedly increased type-I collagen gene expression in the infarcted myocardium of wild-type and iNOS-knockout mice, particularly in the early stage of MI. Enhanced cardiac collagen mRNA is anatomically coincident with myofibroblasts and accumulated collagen, indicating up-regulated collagen synthesis in the infarcted myocardium. Type-I collagen expression, however, is only increased in the noninfarcted myocardium at week 1, which leads to increased interstitial collagen volume in noninfarcted myocardium found in the later stage. Collagen synthesis and degradation coexist in the heart and their balance determines cardiac collagen volume. The current study shows low levels of MMP-1 mRNA in both control and infarcted mouse heart, suggesting that cardiac MMP synthesis remains low after MI. MMP activity is controlled by tissue MMP inhibitors, TIMPs.22 TIMPs function as an important regulatory brake on MMP activity by inhibition of the THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
active species, thereby suppressing collagen degradation. We observed significantly elevated TIMP-1 expression at the site of infarction, suggesting that cardiac MMP activity is suppressed. These observations indicate that cardiac collagen synthesis is elevated, whereas collagen degradation is suppressed in the infarcted mouse heart. The imbalance of cardiac collagen turnover facilitates cardiac fibrous tissue accumulation, leading to cardiac fibrosis. However, our study has shown that in the infarcted heart, type-1 collagen, MMP-1 and TIMP-1 expression, and collagen accumulation in iNOS-knockout mice were similar to those found in wild-type mice. Thus, the effects of iNOS produced nitric oxide on collagen synthesis and degradation were not evident in the infarcted heart. In summary, the current study has examined the spatial and temporal fibrogenic responses in the infarcted mouse heart and potential regulation of nitric oxide in cardiac fibrogenesis. We observed increased TGF-1 expression, which is colocalized with appearance of myofibroblasts and activated collagen synthesis in the infarcted heart. Cardiac collagen degradation, however, is suppressed after MI. In iNOSknockout mice, however, myofibroblast population, profibrotic cytokine expression, collagen synthesis, and degradation as well as collagen volume in the infarcted heart were not significantly different from that in wild-type mice. Our findings suggest that iNOS-induced nitric oxide production may not mediate cardiac fibrosis after MI. It further suggests that other mechanisms are involved in nitrosative stress-induced cardiac dysfunction after MI. Acknowledgments The authors gratefully acknowledge the skillful technical assistance of Yuanjian Chen and Youde Jiang. References 1. Jugdutt BI. Ventricular remodeling after infarction and the extracellular collagen matrix: when is enough enough? Circulation 2003;08:1395– 403. 2. Ertl G, Frantz S. Healing after myocardial infarction. Cardiovasc Res 2005;66:22–32. 3. Anversa P, Li P, Zhang X. Ischemic myocardial injury and ventricular remodeling. Cardiovasc Res 1993;27:145–57. 4. Papapetropoulos A, Rudic RD, Sessa WC. Molecular control of nitric oxide synthases in the cardiovascular system. Cardiovasc Res 1999;43:509 –20. 5. Boucher JL, Moali C, Tenu JP. Nitric oxide biosynthesis, nitric oxide synthase inhibitors and arginase competition for L-arginine utilization. Cell Mol Life Sci 1999;55:1015–28. 6. Andrew PJ, Mayer B. Enzymatic function of nitric oxide synthases. Cardiovasc Res 1999;43:521–31. 7. Takimoto Y, Aoyama T, Keyamura R. Differential expression of three types of nitric oxide synthase in both infarcted and non-infarcted left ventricles after myocardial infarction in the rat. Int J Cardiol 2000;l76:135– 45. 8. Bing RJ, Suzuki H. Myocardial infarction and nitric oxide. Mol Cell Biochem 1996;160 –161:303– 6.
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9. Liu YH, Carretero OA, Cingolani OH. Role of inducible nitric oxide synthase in cardiac function and remodeling in mice with heart failure due to myocardial infarction. Am J Physiol Heart Circ Physiol 2005;289:H2616 –H2623. 10. Marfella R, Di Filippo C, Esposito K. Absence of inducible nitric oxide synthase reduces myocardial damage during ischemia reperfusion in streptozotocin-induced hyperglycemic mice. Diabetes 2004;53:454 – 62. 11. Feng Q, Lu X, Jones DL. Increased inducible nitric oxide synthase expression contributes to myocardial dysfunction and higher mortality after myocardial infarction in mice. Circulation 2001;104:700 – 4. 12. Desmouliere A, Gabbiani G. The role of the myofibroblast in wound healing and fibrocontractive diseases. In: Richard AF, editor. The molecular and cellular biology of wound repair. New York, NY: Plenum Press; 1996. p. 391– 423. 13. Sun Y, Weber KT. Angiotensin converting enzyme and myofibroblasts during tissue repair in the rat heart. J Mol Cell Cardiol 1996;28:851– 8. 14. Sun Y, Zhang JQ, Zhang J. Angiotensin II, transforming growth factor-beta1 and repair in the infarcted heart. J Mol Cell Cardiol 1998;30:1559 – 69. 15. Zhao W, Ahokas RA, Sun Y. ANG II-induced cardiac molecular and cellular events: role of aldosterone. Am J Physiol Heart Circ Physiol 2006;291:H336 –H343.
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16. Sun Y, Zhang JQ, Zhang J. Cardiac remodeling by fibrous tissue after infarction in rats. J Lab Clin Med 2000; 135:316 –23. 17. Sun Y, Ratjaska A, Weber KT. Inhibition of angiotensinconverting enzyme and attenuation of myocardial fibrosis by lisinopril in rats receiving angiotensin II. J Lab Clin Med 1995;126:95–101. 18. Youn TJ, Kim HS, Oh BH. Ventricular remodeling and transforming growth factor-beta 1 mRNA expression after nontransmural myocardial infarction in rats: effects of angiotensin converting enzyme inhibition and angiotensin II type 1 receptor blockade. Basic Res Cardiol 1999;94:246 –53. 19. Yue P, Massie BM, Simpson PC. Cytokine expression increases in nonmyocytes from rats with postinfarction heart failure. Am J Physiol 1998;275:H250 –H258. 20. Yano T, Miura T, Ikeda Y. Intracardiac fibroblasts, but not bone marrow derived cells, are the origin of myofibroblasts in myocardial infarct repair. Cardiovasc Pathol 2005;14:241– 6. 21. Ronty MJ, Leivonen SK, Hinz B. Isoform-specific regulation of the actin-organizing protein palladin during TGFbeta1-induced myofibroblast differentiation. J Invest Dermatol 2006;126:2387–96. 22. Kassiri Z, Khokha R. Myocardial extra-cellular matrix and its regulation by metalloproteinases and their inhibitors. Thromb Haemost 2005;93:212–9.
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