Departments of 1 Physiology and Biophysics, 2 Surgery, and 3 Medicine, University of Mississippi Medical Center, Jackson, Mississippi 39216; and 4 Case Western Reserve University and University Hospitals and Clinics, Cleveland, Ohio 44106
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ABSTRACT |
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Collagen degradation is required
for the creation of new integrin binding sites necessary for cell
survival. However, a complete separation between the matrix and the
cell leads to apoptosis, dilatation, and failure. Previous
studies have demonstrated increased metalloproteinase activity in the
failing myocardium. To test the hypothesis that disintegrin
metalloproteinase (DMP) is induced in human heart end-stage failure,
left ventricle tissue from ischemic cardiomyopathic (ICM,
n = 10) and dilated cardiomyopathic (DCM, n = 10) human hearts were obtained at the time of
orthotopic cardiac transplant. Normal (n = 5) tissue
specimens were obtained from unused hearts. The levels of reduced
oxygen species (ROS) were 12 ± 2, 25 ± 3, and 16 ± 2 nmol (means ± SE, P < 0.005) in normal, ICM, and
DCM, respectively, by spectrofluorometry. The percent levels of
endothelial cells were 100 ± 15, 35 ± 19, and 55 ± 11 in normal, ICM, and DCM, respectively, by CD31 labeling. The levels of
nitrotyrosine by Western analysis were significantly increased, and
endothelial nitric oxide (NO) by the Griess method was decreased in ICM
and DCM compared with normal tissue. The synthesis and degradation of
1-integrin and connexin 43 were significantly increased
in ICM and DCM compared with normal hearts by Western analysis. Levels
of DMP were increased, and levels of cardiac inhibitor of
metalloproteinase (CIMP) were decreased. Aggrecanase activity of DMP
was significantly increased in ICM and DCM hearts compared with normal.
These results suggest that the occurrence of cardiomyopathy is
significantly confounded by the increase in ROS, nitrotyrosine, and DMP
activity. This increase is associated with decreased NO, endothelial
cell density, and CIMP. In vitro, treatment of CIMP abrogated the DMP
activity. The treatment with CIMP may prevent degradation of integrin
and connexin and ameliorate heart failure.
nitric oxide; nitrotyrosine; capillary; endocardial; endothelial
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INTRODUCTION |
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CREATION OF INTEGRIN BINDING sites by collagen degradation in remodeling cardiac tissue is required for survival (16). However, complete loss of integrin signaling due to detachment from extracellular matrix (ECM) leads to myocyte apoptosis (12), cardiac dilatation, and the syndrome of heart failure. The mechanism of integrin-matrix disconnection in heart failure is unclear. We hypothesized that oxidative stress is a critical component of this process. Reduced oxygen species (ROS), both dependent and independent of inflammatory (and/or) mitochondrial NADH/NAD oxidase (2), mask the activity of superoxide dismutase and catalase (22). This instigates a decrease in endothelial nitric oxide (NO) availability and activates latent resident myocardial metalloproteinase (32) as well as increases the levels of cytokines, growth factors, and neurohormones (8). A vicious cycle of oxidative stress ensues in which neurohormones, such as angiotensin II (30), further increase oxidative stress by lowering the levels of bradykinin and prostaglandins. These latter two molecules mediate antioxidation by increasing NO production (33). In parallel, angiotensin II induces vascular NADH/NAD oxidase (35).
Sixteen percent of the myocardium is composed of capillaries, including the lumen and endothelium (11). The capillary cell density is negatively correlated with the muscle/fiber length in hypoxic (oxidative) hypertrophic myocardium (21). During protracted cycles of ischemia-reperfusion (17), volume overload states (19), and chronic heart failure, ROS are generated (5). Endothelial NO is the primary antioxidant to balance the ROS, and reduced endothelial cell density leads to a decrease in endothelial NO concentration (27). NO, in conjunction with thiols and oxygen radicals, generates nitrotyrosine and nitroarginine via the formation of peroxynitrite (26). By competing for the substrate of endothelial nitric oxide synthase (eNOS) in a feedback mechanism, nitroarginine blocks further NO generation. Therefore, the ability of oxidative stress to play a role in nitration of proteins and ligands is fundamentally important as a potential mechanism that modulates protein and ligand function.
The disintegrin metalloproteinase (DMP) is involved in cardiac development (14). It is unclear whether an increase in oxidative stress causes a decrease in endocardial endothelium and an increase in DMP. Therefore, the purpose of this study was to determine the role of oxidative stress, endothelial NO, and DMP in human heart failure.
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MATERIALS AND METHODS |
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Source of tissue. Human heart tissue was obtained at the time of orthotopic cardiac transplantation in the operating room at the University of Mississippi Medical Center. Because NO generation is greater in endocardium than in midmyocardium and the role of endocardial endothelium NO in the beating heart has been determined (18), endocardial tissue from failing hearts was examined in 10 patients with documented coronary artery disease and myocardial infarction (ischemic cardiomyopathy, ICM) and in 10 patients with idiopathic dilated cardiomyopathy (DCM). All patients were in either New York Heart Association functional class III or IV. Five normal control samples were obtained from unused hearts. The average age of the infarcts was 4 ± 1 yr. Tissue specimens from ICM, DCM, and normal hearts were collected and processed within 1-2 h (29). An Institutional Review Board waiver was obtained before collection of the tissue from explanted hearts.
In situ labeling. The standard deparaffinization protocol was used. Endothelial cells were labeled with mouse anti-CD31/platelet/endothelial cell adhesion molecule-1 FITC (1:200 dilution, Sigma Chemical) and identified by green fluorescence. To avoid tissue variability due to necrosis/apoptosis/fibrosis, the images were captured from each heart and stored in digital files. The 10-15 grids/heart were analyzed. The number of cells was referenced with normal tissue. Nitrotyrosine was labeled with monoclonal mouse anti-nitrotyrosine antibody (Upstate Biotechnology) and was detected with secondary antibody conjugated with FITC.
Levels of ROS and NO2/NO3.
Oxidative stress was assessed by measuring left ventricle (LV) tissue
ROS generated by incubating the tissue extract in 10 mM Tris-Cl (pH
7.4) with 2',7'-dichlorofluorescein (DCFH). DCFH acquires
fluorescence properties on reaction with ROS and induces oxidation of
DCFH yielding the fluorescent product dichlorofluorescein. Transient
oxyradical (O2 · ), stable ROS, superoxide,
O) are thereby produced. The product is
detected by an emission at 530 nm when excited at 485 nm as described
(36). The concentration of ROS was measured using oxidized
DCFH as standard. To determine the levels of NO, total
NO2/NO3 was measured by the Griess method, using a protocol kit (cat. no. #22116) from Oxis Research (Portland, OR). In this protocol, all available NO is converted to NO3
and measured by colorimetry; cadmium-based reduction of nitrate to nitrite is performed, and total nitrite is determined.
Preparation of cardiac tissue homogenate. The cardiac tissue extracts from respective hearts were prepared as described (32). The total protein in the samples was estimated using a Bio-Rad dye-binding assay.
Nitrotyrosine Western blot. Normally, NO functions as an antioxidant buffer against increased ROS. However, during oxidative stress, the availability of NO is decreased. This leads to a substantial increase in oxidative byproducts such as nitrotyrosine. Increased nitrotyrosine is associated with impaired cardiovascular function. To measure nitrotyrosine, Laemmli SDS-PAGE was prepared under reducing condition. After electrophoresis, gels were transferred onto nitrocellulose paper. The nonspecific sites were blocked by 5% fat-free milk. Nitrotyrosine was determined using anti-nitrotyrosine antibody (1:200 dilution; Upstate Biotechnology). The secondary alkaline phosphatase conjugate was used as the detection system. To establish the specificity of nitrotyrosine, anti-nitrotyrosine-agarose conjugate (Upstate Biotechnology) was used to immunoprecipitate the total nitrotyrosine content in the sample before loading onto gel/blots.
Cardiac inhibitor of metalloproteinase,
1-integrin, and connexin 43.
Because tissue inhibitor of metalloproteinase-4 (TIMP-4) is
primarily expressed in the heart (9), the levels of
cardiac inhibitor of metalloproteinase (CIMP) were measured using
anti-TIMP-4 antibody (1:200 dilution; Chemicon). For
1-intregrin, anti-
1-integrin (Chemicon) was used. For connexin 43, anti-connexin 43 antibody (Sigma) was used. All experiments were carried out by loading identical
amounts of total protein onto each lane. The bands in Western blots
were scanned by a Bio-Rad GS-700 densitometer. The relative band
intensity is reported.
Aggrecanase activity of DMP. Although specific DMP can be identified by immunoblotting, it does not demonstrate precisely whether the DMP is active or latent. Aggrecan is a substrate for disintegrin and metalloproteinase (28) and has been used to measure the activity of DMP. Ten micrograms of aggrecan was incubated with 10 µg of total protein from LV tissue extract in PBS containing 0.001% Triton X-100. After 2 h of incubation at 37°C, the incubates were separated on 10% SDS-PAGE. In controls, LV extract was loaded under identical conditions. Because other TIMPs can inhibit DMP (13) to determine whether CIMP can inhibit aggrecanase activity of cardiac DMP, extracts were preincubated with CIMP (10 µg, purified in our laboratory from human heart). The incubate was immunoprecipitated with anti-TIMP-4 (Chemicon) followed by conjugation with agarose beads before loading onto the gels. To specifically confirm DMP activity, extracts were incubated with anti-DMP (Chemicon) and secondary conjugated with agarose beads before loading onto the gels. To inhibit metalloproteinase activity of DMP, the extracts were preincubated with 1 mM EDTA before loading onto the gels. To quantitate the activity of DMP, the aggrecan degradation fragments on PAGE were scanned by densitometer, and means ± SE are reported.
Statistical analysis. To minimize the variability of patient status within the category, the data are reported as means ± SE. To determine the significance of data, samples from ICM and DCM were compared with normal specimens using unpaired Student's t-test. P < 0.05 was considered significant.
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RESULTS |
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Clinical data.
The ejection fraction of DCM and ICM hearts was <35%. The LV volume
was twofold higher in DCM and ICM hearts compared with control hearts.
The results suggest there was LV volume overload in DCM and ICM hearts
(Table 1).
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Levels of ROS and NO2/NO3.
LV levels of ROS were significantly higher in both ICM and DCM compared
with controls, but the increase was greater in ICM compared with DCM
hearts. Similarly, the levels of NO were reduced in both ICM and DCM,
but the magnitude of change was smaller in DCM than ICM (Fig.
1).
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Endocardial endothelial cell density.
Positive labeling for CD31 was observed in endocardium of human heart,
and labeling was visualized in both vessels and capillaries. The number
of CD31-positive cells was significantly decreased in ICM and DCM
compared with normal hearts (Fig. 2),
suggesting that in ICM and DCM hearts, the capillary endothelial cell
density was decreased.
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Levels of nitrotyrosine.
The levels of nitrotyrosine were significantly increased in ICM and DCM
hearts. The in situ labeling of nitrotyrosine was associated with the
labeling of endothelial cells (Fig. 2 and Fig.
3, A and B). There
was a ~1.5-fold increase in the nitrotyrosine in ICM and DCM compared
with normal hearts (Fig. 3, C and D). These
results suggest an increase in protein nitration in failing human
heart.
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Levels of DMP and CIMP.
The representative analysis of serial Western blots of DMP and CIMP
(Fig. 4A) revealed a negative
correlation between the levels of DMP and CIMP. The ratio between DMP
and CIMP was robustly increased in ICM heart. The ratio was also
increased in DCM heart (Fig.
5B). The results
demonstrate an increase in the levels of DMP and a decrease in
the levels of CIMP in failing human myocardium.
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Levels of 1-integrin and connexin 43.
To determine whether integrin and cell-cell connections are disrupted
in failing human myocardium, the levels of
1-integrin and connexin 43 were measured by Western blot analysis. Although total
synthesis of integrin was increased in ICM and DCM hearts compared with
normal, the disruption of integrin was also increased, as suggested by
the appearance of fragments of
1 <100 kDa (Fig. 5). The
synthesis of connexin was also increased in ICM and DCM hearts compared
with normal tissue. The degradation of connexin was also increased, as
suggested by the appearance of fragments of connexin <45 kDa (Fig.
6). These results suggest induction and
degradation of integrin and connexin in human heart end-stage failure.
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Aggrecanase activity of DMP.
To determine whether DMP is activated, aggrecan degradation activity of
DMP was measured. The fragments of aggrecan were increased in LV
extracts incubated with aggrecan from DCM and ICM compared with
controls. Aggrecan migrates as a single band at 100 kDa. The incubation
with extract produces bands <60 kDa. These bands were not present in
the 5× concentrated LV homogenates (Fig.
7A). The incubation with CIMP,
anti-DMP antibody, and EDTA abolished the aggrecanase activity in the
extracts (Fig. 7B). These results suggest that DMP is
present in human heart and is activated in failing myocardium.
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DISCUSSION |
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The results suggest that end-stage heart failure is associated with a robust increase in ROS and activation of DMP. This phenotype is what would be expected if 1) collagen degradation normally supplies integrin ligands during cardiac muscle remodeling, and 2) complete collagen-integrin disconnect ensues and contributes to heart failure. The administration of CIMP may ameliorate ECM-cell detachment and heart failure.
In vivo inhibition of NO production by
NG-nitro-L-arginine methyl
ester increases matrix metalloproteinase (MMP) activity
(20) and instigates left ventricle hypertrophy
(15). Inactivation of MMP by NO may result from
1) inhibition of redox-sensitive MMP gene activation
(31), 2) direct induction of CIMP
(31), or 3) blockade of the metal ion active
site in the latent form of MMP. The neutralization of NO by oxyradical
may activate MMP (32) and generate nitrotyrosine,
suggesting a rate of MMP activation similar to the rate of NO oxidation
(Fig. 8). Despite the induction of eNOS
and inducible nitric oxide synthase (iNOS) in the failing myocardium
(10), the bioavailability of NO is reduced (Fig. 1),
nitrotyrosine is generated (Fig. 3), and MMPs are activated (29). In vitro studies have demonstrated that cardiac
myocytes also synthesize iNOS (3, 25). Therefore, it may
be difficult to differentiate in vivo NO from endothelium and/or
myocyte. However, studies have demonstrated the role of impaired
endothelium in the reduction of cardiac relaxation (7) as
well as suggested that the endothelial NO attenuates the myocyte
contractile function (4). Therefore, it is most likely
that in vivo endothelial NO modulates myocyte contractile function.
Moreover, unlike large epicardial arteries, the capillary endothelium
is without a smooth muscle layer, and, therefore, endothelial NO may
diffuse faster across the interstitium to interact with ECM components
than the NO from large vessels. Also, it is likely that the flow (i.e., volume)-mediated oxidative stress may impair endothelial NO before myocyte NO.
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There is an inverse relationship between muscle/fiber length and
capillary cell density (21). Our results suggest a
decrease in capillary endocardial endothelial cell density (Fig. 2)
associated with an increase in ROS (Fig. 1). The generation of
nitrotyrosine (Fig. 3) exceeds the bioavailability of NO (Fig. 1).
Previous studies from our laboratory have demonstrated increased MMP
activity in ICM and DCM hearts (29). The increase in ROS
and decrease in NO are associated with activation of DMP (Figs. 4 and
7). The synthesis and degradation of 1-integrin are
increased (Fig. 5). ECM is composed of integrin and cell-cell junction
proteins. Others have suggested that during hypertrophy, more cell
junctions are formed (34). This leads to increased
communications as compensatory response of cells in pressure and volume
overload hypertrophy. We demonstrate that both synthesis and
degradation of connexin 43 are induced in heart failure (Fig. 6),
suggesting upregulation of gap junction remodeling.
Perivascular, microvascular interstitial endocardial fibrosis, and dilatation are primary entities manifested in the chronic failing myocardium. It is a paradox that fibrosis and dilatation track together. This can be explained by the fact that during increases in preload or afterload and especially in the absence of NO, the latent resident myocardial metalloproteinases are activated (Fig. 8) in an attempt to dilate the heart and to reduce the wall stress. Because the metalloproteinase degrades ultrastructural collagen and elastin (1, 24) more rapidly than oxidized collagen, and because the turnover of ultrastructural collagen and elastin is remarkably lower than oxidized stiffer collagen (23), the ultrastructural collagen and elastin are replaced by oxidatively modified stiffer collagen. Consequently, the cardiac wall stress is increased. In addition, the activation of DMP leads to complete ECM-cell separation and may instigate apoptosis. It is known that the isolation of adult cardiomyocytes by disruption of surrounding ECM by collagenase instigates cell swelling (6). Added, these myocytes do not survive after 2-3 h. It is a paradox that in many remodeling tissues, such as heart, collagen degradation, to provide new integrin-binding sites, is required for survival (16). However, complete disconnection from ECM leads to apoptosis (12). This study contributes to our understanding of the importance of oxidative stress in cardiac remodeling, structure, and function, by defining their link to NO metabolism.
Limitation. The hearts from DCM were homogeneous. However, the ICM hearts were heterogeneous. To be consistent, a part of endomyocardium (i.e., inner wall) was dissected from DCM hearts. The infarcted, thinned, and dilated regions of endomyocardium were taken from ICM hearts. Although antibodies are made for whole molecules, they also cross-react with degraded fragments. This may suggest that the degradation does not alter the antigen epitope. Moreover, the specificity of anti-nitrotyrosine, integrin, and connexin 43 was established by immunoprecipitating the antigen before loading onto the gel. This eliminated the specific bands related to the respective antigens.
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ACKNOWLEDGEMENTS |
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The authors greatly appreciate the generous help of Dr. Indria Rao and Yolanda Smith, Department of Pathology, University of Mississippi Medical Center, for in situ labeling and histological staining.
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FOOTNOTES |
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This work was supported in part by National Institutes of Health Grants GM-48595 and HL-51971, American Heart Association-Mississippi Affiliate, and Kidney Care Foundation of Mississippi.
Address for reprint requests and other correspondence: S. C. Tyagi, Dept. of Physiology and Biophysics, Univ. of Mississippi Medical Center, 2500 N. State St., Jackson, MS 39216-4505 (E-mail: styagi{at}physiology.umsmed.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajplung.00001.2002
Received 4 January 2002; accepted in final form 26 March 2002.
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