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Am J Physiol Heart Circ Physiol 282: H1197-H1205, 2002. First published November 29, 2001; doi:10.1152/ajpheart.00483.2001
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Vol. 282, Issue 4, H1197-H1205, April 2002

Apoptosis in the left ventricle of chronic volume overload causes endocardial endothelial dysfunction in rats

Michael J. Cox1, Harpreet S. Sood1, Matthew J. Hunt1, Derrick Chandler1, Jeffrey R. Henegar2, Giorgio M. Aru3, and Suresh C. Tyagi1

1 Department of Physiology and Biophysics, 2 Department of Pathology, and 3 Department of Cardiothoracic Surgery, School of Medicine, University of Mississippi Medical Center, Jackson, Mississippi 39216


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The hypothesis is that chronic increases in left ventricular (LV) load induce oxidative stress and latent matrix metalloproteinase (MMP) is activated, allowing the heart to dilate in the absence of endothelial nitric oxide (NO) and thereby reduce filling pressure. To create volume overload, an arteriovenous (A-V) fistula was placed in male Sprague-Dawley rats. To decrease oxidative stress and apoptosis, 0.08 mg/ml nicotinamide (Nic) was administered in drinking water 2 days before surgery. The rats were divided into the following groups: 1) A-V fistula, 2) A-V fistula + Nic, 3) sham operated, 4) sham + Nic, and 5) control (unoperated); n = 6 rats/group. After 4 wk, hemodynamic parameters were measured in anesthetized rats. The heart was removed and weighed, and LV tissue homogeneates were prepared. A-V fistula caused an increase in heart weight, lung weight, and end-diastolic pressure compared with the sham group. The levels of malondialdehyde (MDA; a marker of oxidative stress) was 6.60 ± 0.23 ng/mg protein and NO was 6.87 ± 1.21 nmol/l in the LV of A-V fistula rats by spectrophometry. Nic treatment increased NO to 13.88 ± 2.5 nmol/l and decreased MDA to 3.54 ± 0.34 ng/mg protein (P = 0.005). Zymographic levels of MMP-2 were increased, as were protein levels of nitrotyrosine and collagen fragments by Western blot analysis. The inhibition of oxidative stress by Nic decreased nitrotyrosine content and MMP activity. The levels of tissue inhibitor of metalloproteinase-4 mRNA were decreased in A-V fistula rats and increased in A-V fistula rats treated with Nic by Northern blot analysis. TdT-mediated dUTP nick-end labeling-positive cells were increased in A-V fistula rats and decreased in fistula rats treated with Nic. Acetylcholine and nitroprusside responses in cardiac rings prepared from the above groups of rats suggest impaired endothelial-dependent cardiac relaxation. Treatment with Nic improves cardiac relaxation. The results suggest that an increase in the oxidative stress and generation of nitrotyrosine are, in part, responsible for the activation of metalloproteinase and decreased endocardial endothelial function in chronic LV volume overload.

nitric oxide; malondialdehyde; collagen degradation; tissue inhibitor of metalloproteinase; arteriovenous fistula; nicotinamide; NADH oxidase; nitrotyrosine; TUNEL; cardiac ring; acetylcholine; nitroprusside; stretch; contraction; relaxation; heart failure


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE EXTRACELLULAR MATRIX (ECM), particularly type I fibrillar collagen surrounding the cardiomyocytes, helps the cardiac muscle to synchronize contraction and relaxation during systole and diastole, respectively (22, 53, 62). To compensate for the increase in workload and to reduce the wall stress, the cardiac muscle undergoes hypertrophy. This leads to remodeling of the ECM (54). Remodeling implies synthesis and degradation of the ECM (52). The neutral matrix metalloproteinase (MMP) (a designer, architect, or tailor) remodels the ECM (33). In the normal myocardium, most of the MMP resides in latent form (59) and activated in chronic heart failure (55). The mechanism of MMP activation in chronic heart failure is not well understood. The MMP is regulated primarily at three stages: 1) at the transcription levels by multiple cytokines, growth factors, and neurohormones, including oxidative stress; 2) by their target tissue inhibitor of metalloproteinase (TIMP); and 3) by direct activation with oxidative stress and/or by proteolytic cleavage (33, 52). Alterations at any of these stages can lead to an imbalance in the composition and concentrations of MMP and TIMP and may lead to development of a disease state.

Sixteen percent of the myocardium is composed of capillary, including lumen and endothelium (18). The endothelium supplies oxygen and is also responsible for the generation of nitric oxide (NO) for relaxation of underlying cardiac muscle. The increase in pulse pressure during chronic volume overload may develop protracted cycles of ischemia-reperfusion (6, 37). The high oxygen concentration produces oxyradical: 2O2 + 2H2O = 2H2O2 + O<UP><SUB>2</SUB><SUP>−</SUP></UP> (toxic oxygen), dependent or independent of NADH/NAD oxidase (4), and masks the activity of superoxide dismutase and catalase (25, 28, 41). The production of oxyradical (O<UP><SUB>2</SUB><SUP>−</SUP></UP>·) is transient. However, superoxide, O<UP><SUB>2</SUB><SUP>−</SUP></UP>, and H2O2 (2OH-) are stable in the plasma as well as tissue. In volume overload, the capillary endothelial cell density is decreased (2, 29). The collagen content is decreased or near normal (29, 42). The mechanism of decreased endothelium and increased collagenolysis in chronic volume overload is unclear. Oxyradical species stimulate MMPs (39, 46, 58), and in vivo inhibition of NO production by N-nitro-L-arginine methyl ester increases MMP activity (38). In culture conditions, inhibition of cytokine-induced NO synthase reduced both expression and activity of MMPs (43). In contrast, cytokine-inducible MMPs in immortalized cells were not modified by NO synthase inhibition (19). The reasons for such diverse effects of NO on MMPs are not clear. However, a differential regulation of MMP release and activation in vivo versus in vitro may account for this discrepancy. The inhibition of NO production as well as the degradation of the ECM facilitate apoptosis (36, 48, 63). Oxidative stress instigates the decrease in endothelial NO availability (10, 65) as well as increases the levels of cytokines, growth factors, and neurohormones (11, 17, 26). This may further increase oxidative stress in which neurohormones such as angiotensin II (56) increase oxidative stress by decreasing the levels of bradykinin and prostaglandins; these molecules, otherwise, mediate antioxidation by increasing NO production (61). In parallel, angiotensin II also induces NADH/NAD oxidase (64). We hypothesized that the volume overload increases oxidative stress, leading to activate latent resident myocardial MMP. This causes collagenolysis, induces apoptosis, and impairs capillary endothelium and endocardium.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal protocol. Male Sprague-Dawley rats, 275-300 g, were obtained from Harlan Laboratories. Several models of volume overload, such as the conversion of pressure overload to volume overload by minoxidil and other drugs, have been used (51). Besides vasodilation, minoxidil also has a central effect (50). Also, creation of volume overload by mitral valve regurgitation secondary to chordal rupture (34) generates myocardial injury. On the other hand, an infrarenal arteriovenous (A-V) fistula creates an unambiguous model of chronic volume overload. In this model, arterial blood, below the kidneys, rapidly enters into the venous circulation and overloads the ventricle without contribution of the stimulation of circulating factors (16). To create an A-V fistula, rats were anesthetized with pentobarbital (50 mg/kg ip). A midline incision was performed to create the A-V fistula (16). The caudal vena cava ~1.5 cm below the renal arteries was identified. Blunt dissection was used to remove the overlying adventitia and expose the vessels, taking care not to disrupt the tissue connecting the vessels. Both vessels were then occluded proximal and distal to the intended puncture site, and an 18-gauge needle was inserted into the exposed abdominal aorta and advanced through the medial wall into the vena cava to create the fistula. The needle was withdrawn, and the ventral aortic puncture was sealed with cyanoacrylate. Creation of a successful A-V fistula was visually evident by the pulsatile flow of oxygenated blood into the vena cava. The abdominal musculature and skin incisions were closed in layers by standard techniques with absorbable suture and autoclips. The rats were kept warm and were monitored for any sign of morbidity for 2 h postsurgery before transport to the animal care unit. Sham rats were treated similarly except no punch was made. Rats were divided into the following experimental groups: 1) A-V fistula rats treated with nicotinamide (Nic; 0.67 mg/ml) in drinking water (A-V fistula + Nic), 2) sham-operated rats treated with Nic (sham + Nic), 3) A-V fistula rats, 4) sham operated (sham), and 5) control; n = 6 rats/group. To avoid early effect of oxidative stress, the Nic intervention, which began 2 days before A-V fistula or sham surgery, also continued for the entire duration of the experiment in these rats. This dose of Nic was based on the fact that the rat drink ~25 ml/day and ingests ~17 mg/day, and this concentration was enough to saturate all the binding sites on NADH/NAD oxidase (13) as well as poly(ADP-ribose) synthetase, an enzyme that can be activated by peroxynitrite and oxidants (7, 12), the two major pathways of oxyradical formation and apoptosis. All rats were exposed to a similar environment. Animal room temperature was maintained between 22 and 24°C. A 12:12-h light-dark cycle was maintained by artificial illumination. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center, Jackson, MI. All rats were given rat chow (deficient of niacin) and water ad libitum. Levels of water, food intake, and changes in body weight were measured every other day.

The rats were anesthetized with Inactin (100 mg/kg ip) at 4 wk. This anesthesia has minimal effect on cardiovascular function (9). To measure arterial pressure, a fluid-filled arterial catheter (polyethylene-50) was inserted into the left femoral artery. For the measurements of end-diastolic pressure (EDP), another catheter was advanced into the left ventricle (LV) through the right common carotid artery and aorta. The catheters were connected to pressure transducers (Micro-Med) positioned at the level of the heart. Ten minutes after the insertion of catheters, arterial pressure, heart rate, systolic and diastolic blood pressure (SBP and DBP, respectively), and EDP were recorded (32). The hearts were weighed. Because heart tissue was used for protein and mRNA isolation, the tissue was dissected under sterilized conditions. A portion of the LV was fixed in 10% zinc formalin for histology, immunostaining, and TdT-mediated dUTP nick-end labeling (TUNEL). For mRNA analysis, the tissue was processed within 10 min after death or frozen in liquid nitrogen before use. Total RNA was isolated using RNAZol solution. For the analysis of malondialdehyde (MDA) and NO, the tissue was homogenized in 10 mM Tris-Cl (pH 7.4); for nitrotyrosine, MMP, TIMP, and collagen degradation peptides, the LV tissue homogenate was prepared as previously described (55, 59). The total protein was measured by Bio-Rad dye-binding assay.

Malondialdehyde. Biomarkers of oxidative stress such as isoprostane have been used in coronary artery disease (27), and MDA is used in chronic heart failure (14). Therefore, MDA and thiobarbituric acid-reactive species (TBARS) were measured by reacting the LV tissue homogenates prepared from control, sham, sham + Nic, A-V fistula, and A-V fistula + Nic rats. The fluorescence at 560 nm was measured when excited at 525 nm (24). 1,1,3,3-Tetraethoxypropane (Sigma) was used as a standard. The concentrations of TBARS in samples were measured by comparing with a series of standard solutions of tetraethoxypropane.

NADH oxidase. Superoxide released from the fresh LV tissue homogenate was measured using discontinuous assay (23). In brief, cytochrome c (100 µM; Sigma) in Krebs-Ringer buffer was prepared in two separate wells. In one well, 100 µg/ml superoxide dismutase (Sigma) were added. After 2 min at 37°C, 1-2 µg/ml phorbol 12-myristate 13-acetate (PMA) from a stock of 1 mg/ml in DMSO were added. After 1 h of incubation, tissue extract was removed by centrifugation at 400 g for 5 min at 4°C. The amount of cytochrome c reduced in the supernatant was measured at 550 nm. The superoxide dismutase-containing samples were used as reference. NADH oxidase was estimated in micromoles per gram of tissue using an extinction coefficient of 19 cm-1 · mM-1 at 550 nm (23).

Nitrate/nitrite. The concentrations of total NO in the LV tissue homogenate were estimated by measuring total nitrate/nitrite using a protocol kit (Cat. No. 22116) from Oxis Research (Portland, OR).

Nitrotyrosine. Nitrotyrosine in LV tissue homogenates was determined using mouse monoclonal anti-nitrotyrosine antibody (Upstate Biotechnology). Secondary anti-mouse alkaline phosphatase conjugate was used as a 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 it was loaded onto the gel and/or blot.

MMP-2. There are currently 18 known MMPs. Interstitial fibrillar collagen is the primary collagen in the heart, and MMP-2 and -9 degrade interstitial collagen as well as elastin (1, 44). To measure MMP-2 and -9 activity, zymography using 1% gelatin in SDS-PAGE as the impregnated substrate was performed on LV tissue homogenates prepared from the above groups of rats (55, 59).

Collagen and its degradation. The mRNA for collagen was measured by Northern blots using a collagen cDNA probe as previously described (55). The 18S RNA gene was used as a control. Because intrinsic activation of MMP cleaves fibrillar collagen to soluble 3/4 and 1/4 collagen fragments, therefore, the soluble collagen peptides were measured by immunoblot analysis using anti-collagen antibody in LV tissue homogenates. The bands below 100 kDa were identified as collagen cleavage products as previously described (55, 57).

TIMP-4. The levels of cardiospecific TIMP-4 mRNA were measured by Northern blot analysis using the cDNA probe isolated from RT-PCR. The primers for TIMP-4 that were used were as follows: sense, 5' -GTGACGAGAAGGAGGTGGATTCC-3'; and antisense, 5' -CTTGATGCAGGCAAAGAACTTGGC-3' (GenBank No. U76456). The isolated cDNA was radiolabeled by random primer labeling using [32P]ATP (57).

TUNEL and immunolabeling. To determine whether chronic volume overload causes capillary endothelial and endocardial injury, the serial tissue sections were labeled with TUNEL according to the instructions of the manufacturer (Oncogene Research Products, Fluorescein-FragELTM Cat No. QIA39) for identification of nicked DNA. Apoptosis was indexed by the counting TUNEL-positive cells per centimeter squared. Endothelial cells were characterized using FITC-labeled CD31 (PECAM-1) antibody (Sigma) and counted per centimeter squared in a fixed grid. Five randomly selected grids per tissue were analyzed. The numbers of cells were normalized with the cells in control tissue, and the percentage of endothelial cells were reported. The myocytes were identified by labeling with phosphatase-conjugated myosin antibody (Sigma).

Endocardial endothelial function. The hearts were arrested in diastole by injecting 0.2 ml/100 g body wt of 20% KCl solution (intravenously). The LV and right ventricle (RV) were separated. LV wall thickness and diameter (in mm) were measured by a digital micrometer. The "deli"-shaped LV rings were mounted in a tissue myobath (31, 60). One of the two mounted wires was connected to a force transducer. The ring was stretched and brought to resting tension, at which time 20 mM CaCl2 was added. At the maximum CaCl2 contraction, acetylcholine (endothelial dependent) or nitroprusside (endothelial independent) was added. The dose-response curves were generated. The percent relaxation was calculated based on 100% contraction to 20 mM CaCl2. The data were fitted to a nonlinear least-squares equation as follows: %relaxation = {A/[1 + expB(dose - C)]} + D, where A, B, C, and D are constants. The validity of measurements regarding endocardial endothelial function using deli-shaped LV rings has been established by measuring response to various cardiotonic agents (31, 60).

Statistical analysis. Values are given as means ± SE; n = 6 rats/group. Differences between groups were evaluated by using ANOVA followed by the Bonferroni post hoc test (47), focusing on the effects of volume overload (sham rats to A-V fistula rats) and treatment (A-V fistula + Nic rats compared with A-V fistula rats). P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hemodynamic parameters. The heart, LV, RV, and lung weights were increased in A-V fistula rats compared with sham controls. There was no significant change in the arterial pressure in the control and experimental groups. However, EDP was increased significantly in A-V fistula rats compared with sham. The levels of NADH oxidase activity were elevated in A-V fistula rats. Treatment with Nic regresses NADH oxidase and heart and lung weights and reduces LVEDP (Table 1).

                              
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  		Table 1.   Hemodynamic parameters of the study groups

Effect of Nic on the oxidative stress in the LV of A-V fistula rats. The results suggest a robust increase in MDA in the LV of A-V fistula rats compared with sham controls. Treatment with Nic decreases the levels of MDA, suggesting a role of NADH oxidase generated oxidative stress in chronic volume overload (Fig. 1).


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  		Fig. 1.   The levels of malondialdehyde (MDA), a marker of oxidative stress, were determined by measuring changes in the fluorescence at 560 nm, when excited at 525 nm, due to the reaction between tissue homogenate and added thiobarbituric acid (TBA). In this reaction, TBA reacts with MDA in the homogenate and emits fluorescence at 560 nm. The typical kinetic plots of initial reaction and fluorescence generated at 560-nm emission when excited at 525-nm excitation between TBA and left ventricular (LV) tissue homogenates prepared from arteriovenous (A-V) fistula and sham + nicotinamide (Nic)-treated rats compared with buffer and reagents alone are shown in A. The same amount of total protein was added in each reaction mixture. B: 60 min after heating at 45°C, the fluorescence was recorded. On the basis of the standard curves generated for tetraethoxypropane (an acronym of MDA), the amounts of MDA were estimated. The amounts of MDA in tissue homogenates were normalized with total protein added into the reaction mixture and were measured as nanograms per milligram of protein. The following groups of rats are shown: sham operated (sham), sham + Nic, A-V fistula, and A-V fistula + Nic. *P = 0.05; **P = 0.005.

Effect of Nic on NO. The levels of NO were reduced in the LV of A-V fistula rats compared with sham controls. Nic treatment only partially restored the availability of NO in the LV of A-V fistula rats (Fig. 2).


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  		Fig. 2.   LV tissue homogenates from sham + Nic, A-V fistula, and A-V + Nic groups were analyzed for nitric oxide (NO) concentrations according to the Griess reaction, in which NO is converted to NO3. The color generated at 540 nm was measured. The NO3 was used as the standard. The concentration of NO was normalized with the total tissue used for the extraction. *P = 0.001; **P = 0.005.

Effect of Nic on nitrotyrosine levels in the LV of A-V fistula rats. The levels of nitrotyrosine were increased twofold in A-V fistula rats compared with controls. Treatment with Nic decreased the formation of nitrotyrosine in the LV of A-V fistula rats (Fig. 3).


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  		Fig. 3.   A: representative Western blot of nitrotyrosine in the LV tissue homogenates prepared from sham + Nic (lane 1), A-V fistula + Nic (lanes 2 and 3), and A-V fistula (lanes 4 and 5) groups. The specificity of anti-nitrotyrosine antibody was determined by immunoprecipitating the antigen in the LV tissue homogenates of A-V fistula rats (A-V-antbppt) before loading onto the gel (lane 6). B: bands labeled with nitrotyrosine were scanned and normalized with actin. *P = 0.001; **P = 0.03.

Effect of Nic on MMP activity in the LV of A-V fistula rats. The results suggested a twofold increase (P = 0.005) in 72-kDa MMP-2 and specific induction of 92-kDa MMP-9 in the LV of A-V fistula rats compared with controls. Treatment with Nic decreased MMP-2 activity to its basal level and inhibited completely the MMP-9 activity (Fig. 4). These results elicit specific regulation of MMP at 92 kDa by oxidative stress in chronic volume overload.


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  		Fig. 4.   A: levels of matrix metalloproteinase (MMP) activity in LV tissue homogenates prepared from sham + Nic (lane 1), A-V fistula +Nic (lanes 2 and 3), and A-V fistula (lanes 4 and 5) groups were measured by gelatin-zymography. B: lytic bands at 72 and 92 kDa were scanned and normalized with actin. *P < 0.005; **P < 0.05.

Collagen and TIMP-4. The levels of collagen transcripts at 4.6 and 5.7 kb increased compared with control. Nic treatment blocked the collagen induction to the basal level in A-V fistula rats (Fig. 5). The levels of TIMP-4 were significantly decreased in A-V fistula rats compared with sham controls. Treatment with Nic increased the level of TIMP-4 to that of the control (Fig. 5).


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  		Fig. 5.   A: LV collagen I and tissue inhibitor of metalloproteinase (TIMP)-4 mRNA analysis. Lanes 1 and 2, A-V + Nic; lanes 3 and 4, sham + Nic; lanes 5 and 6, A-V fistula. Top, 2 transcripts of collagen I at 5.7 and 4.6 kb. The same blots were stripped and reprobed with TIMP-4. Middle, levels of TIMP-4. Bottom, 18S RNA gene. B: scanned data for TIMP-4. *P = 0.001; **P = 0.002.

Endocardial endothelial cells. The endothelial cell labeling surrounding the cardiomyocytes was optimal in the hearts of sham control rats. However, the labeling was significantly (n = 6) decreased in the hearts of A-V fistula rats (Fig. 6). This suggests reduction in the endocardial endothelial cell density in A-V fistula hearts compared with sham controls.


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  		Fig. 6.   Representative endothelial antigen staining of the heart: labeling of deparaffinized fixed tissue sections of the heart from A-V fistula rat (A) compared with the heart from a sham rat (B); ×20 magnification. C: histographic presentation of the percentage of endothelial cells. *P = 0.001; **P = 0.001.

Endocardial apoptosis. The endocardium of A-V fistula rats exhibited an increase in the number of TUNEL-positive cells compared with the sham controls. Treatment with Nic reduced the number of TUNEL-positive cells in A-V fistula rats (Fig. 7). The staining of serial tissue sections for endothelial cell antigen suggests ~70% apoptotic nuclei from endothelial and ~25% from nonendothelial.


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  		Fig. 7.   A: representative TdT-mediated dUTP nick-end labeling (TUNEL)-positive labeling in the LV of A-V fistula (a) and sham + Nic groups (b); ×20 magnification. B: relative counts of apoptotic nuclei. *P = 0.001; **P = 0.001.

Endocardial dysfunction. The magnitude of relaxation and acetylcholine response was significantly depressed in the endocardium of A-V fistula rats compared with the sham controls. Treatment with Nic partially improved the acetylcholine response in A-V fistula rats (Fig. 8B). The response to endothelial-independent nitroprusside was maintained in control and post-Nic treatment groups; however, not in A-V fistula rats (Fig. 8C). These data may suggest impairment of endothelial as well as nonendothelial cells post-A-V fistula.


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  		Fig. 8.   Endocardial endothelium response to acetylcholine. LV rings from sham + Nic, A-V fistula, and A-V + Nic groups of rats were precontracted with 20 mM CaCl2. Different doses of acetylcholine were added to the ring in a tissue myobath. The relaxation to acetylcholine was estimated as the percent remaining CaCl2 contraction. Data are an average of at least 6 animals. A: typical contractile response to CaCl2 and relaxation to 10-9, 10-8, and 10-7 M acetylcholine. B: acetylcholine dose-response curves representing the best fit of the data to a nonlinear least squares. C: nitroprusside dose-response curves representing the best fit of the data to a nonlinear least squares.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results suggest that oxidative stress generated by chronic volume overload causes a decrease in NO concentration, generates nitrotyrosine, activates latent resident myocardial MMPs, and instigates apoptosis and hypertrophy in the LV of A-V fistula rats. Treatment with Nic inhibits poly(ADP-ribose)synthetase, an enzyme induced by oxidants and peroxynitrite, and causes apoptosis (7, 12). Nic also inhibits NADH oxidase by competing for substrate binding (13). Nic decreases oxidative stress and improves LVEDP by ameliorating the oxidative stress-mediated remodeling.

The degree of severity of chronic heart failure is correlated with the oxidative stress as measured by an increase in the production of MDA (14). Our results suggest increased myocardial MDA in chronic volume overload (Fig. 1). MDA is a product of cell membrane lipid peroxidation and, therefore, a direct marker of oxidative cell toxicity (3, 15). The reduced endothelial cell density (Fig. 6) may lead to a decrease in endothelial NO concentration during chronic volume overload (49). Our results demonstrate decreased NO in the LV of A-V fistula rats (Fig. 2). One of the reasons of decreased levels of NO is that the NO in conjunction with thiols and oxygen radical generates nitrotyrosine (5, 20, 45). A study comparing the role of peroxynitrite in blood versus crystalloid cardioplegia demonstrated increase nitrotyrosine in the LV with crystalloid cardioplegia and associated decrease in diastolic function (40). Our results suggest increased nitrotyrosine contents (Fig. 3) in the LV with chronic volume overload.

It appears that the myocardium is well compensated at 4 wk of A-V fistula. However, it is known also that the myocardium continues to dilate and becomes thin. Brower et al. (8) demonstrated that pressure-volume curves continue to shift to the right even after 1 wk of A-V fistula. This can be explained by the following scenarios. The early MMP activity in the LV of volume overload by myocardial injury due to mitral valve regurgitation has been demonstrated (34). Janicki et al. (21) showed that the peak of MMP activity in the A-V fistula model occurring within the first week postoperatively is due, in part, to inflammatory infiltrate. The LV wall-to-LV diameter ratio was significantly lower in the A-V fistula group than in any other group (Table 1). This may support a role of increased MMP activity (Fig. 4) and decreased TIMP-4 (Fig. 5) in LV dilatation and wall thinning. Treatment with Nic tends to ameliorate the LV wall-to-LV diameter ratio, but insignificantly. The levels of collagen were increased in A-V fistula rats (Fig. 5). Similar results were also obtained by Namba et al. (35). Treatment with Nic reduces collagen expression to that of controls (Fig. 5). This may argue that the effects of Nic on LVEDP, LV dilatation, and LV hypertrophy might just as well represent some systemic effect(s) on fluid retention and/or venomotor tone, which in turn could markedly reduce ventricular preload. In other words, the effects of Nic as an antioxidant may rather be primarily due to ventricular unloading. However, our ex vivo culture, load-free experiments suggest that Nic decreases MMP activity and nitrotyrosine, in part, by decreasing oxidative stress (30). These results may suggest that decreasing oxidative stress may be an essential first step in amelioration of cardiac dysfunction in chronic volume overload.

The numbers of endothelial cells were decreased (Fig. 6) and the TUNEL-positive cells were increased in the endocardium of A-V fistula rats compared with the sham controls (Fig. 7). The treatment with Nic decreased the number of apoptotic cells (Fig. 7), although Nic inhibits oxidant-mediated myocyte apoptosis by inhibiting poly(ADP-ribose) synthase (7, 12). However, the decrease in the levels of MDA and nitrotyrosine contents by Nic does not necessarily suggest the improvement of NO-mediated cardiac relaxation; therefore, we measured acetylcholine and nitroprusside responses in cardiac rings (60) prepared from A-V fistula rats treated with and without Nic. Nitroprusside did not completely relax the heart from A-V fistula (Fig. 8C), suggesting apoptosis of nonendothelial cells as well.

During increased oxidative tension in the LV, a number of events take place that lead to increased LVEDP. Our results suggest that the decreased NO concentration and increased MMP activity instigate apoptosis. This leads to endocardial endothelial dysfunction, hypertrophy, and an increase in LVEDP. Although collagen synthesis was increased in volume overload, the degradation of newly synthesized ultrastructural collagen was increased. The MMP-9 degrades collagen and elastin and leads to decreased collagen III-to-I and elastin-to-collagen ratios. This impairs synchronization of the myocytes and leads to increase end-diastolic volume and development of pressure. Treatment with Nic ameliorates the oxidative stress-mediated increase in LVEDP during chronic volume overload.


    ACKNOWLEDGEMENTS

This work was supported in part by National Institutes of Health grants GM-48595 and HL-51971, by the American Heart Association, Mississippi Affiliate, and by the Kidney Care Foundation of Mississippi.


    FOOTNOTES

First published November 29, 2001;10.1152/ajpheart.00483.2001

Address for reprint requests and other correspondence: S. C. Tyagi, Univ. of Mississippi Medical Center, Dept. of Physiology and Biophysics, 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.

Received 2 June 2001; accepted in final form 26 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aimes, RT, and Quigley JP. MMP-2 is an interstitial collagenase. J Biol Chem 270: 5872-5876, 1995.

2.   Amann, K, Breitbach M, Ritz E, and Mall G. Myocyte/capillary mismatch in the heart of uremic patients. J Am Soc Nephrol 9: 1018-1022, 1998.

3.   Ambrosio, G, and Flaherty JJ. Membrane lipid peroxidation induced by oxygen radical generation at reflow in reperfused heart. J Clin Invest 87: 2056-2066, 1991.

4.   Babior, BM. NADPH oxidase: an update. Blood 93: 1464-1476, 1999.

5.   Beckman, JS, Beckman TW, Chen J, Marshall PA, and Freeman BA. Apparent hydroxy radical production by peroxinitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA 87: 1620-1624, 1990.

6.   Belch, JJF, Chopra M, Hutchinson S, Lorimer R, Sturrock RD, Forbes CD, and Smith WE. Free radical pathology in chronic arterial disease. Free Radc Biol Med 6: 375-378, 1989.

7.   Brower, GL, Henegar JR, and Janicki JS. Temporal evaluation of LV remodeling and function in rats with chronic volume overload. Am J Physiol Heart Circ Physiol 271: H2071-H2078, 1996.

8.   Bowes, J, McDonald MC, Piper J, and Thiemermann C. Inhibitors of poly (ADP-ribose)synthetase protect rats cardiomyocytes against oxidant stress. Cardiovasc Res 41: 126-134, 1999.

9.   Buelke, SJ, Holson JF, Bazare JJ, and Young JF. Comparative stability of physiological parameters during sustained anesthesia in rats. Lb Animal Sci 28: 157-162, 1978.

10.   Carr, A, and Frei B. The role of natural antioxidants in preserving the biological activity of endothelial-derived nitric oxide. Free Radic Biol Med 28: 1806-1814, 2000.

11.   Chen, CY, Huang YL, and Lin TH. Association between oxidative stress and cytokine production in nickel-treated rats. Arch Biochem Biophys 356: 127-132, 1998.

12.   Cuzzocrea, S, Zingarell B, and Cuputi AP. Role of peroxynitrite and poly(ADP-ribose)synthetase activation in cardiovascular de-arrangement induced by zymosan in the rats. Life Sci 63: 923-933, 1998.

13.   Dang, PM, Johnson JL, and Babior BM. Binding of nicotinamide adenine dinucleotide phosphate to the tetratricopeptide repeat domains at the N-terminals of p67PHOX, a subunit of the leukocyte nicotinamide adenine dinucleotide phosphate oxidase. Biochemistry 39: 3069-3075, 2000.

14.   Diaz-Velez, CR, Garcia-Castineiras S, Mendoza-Ramos E, and Hernandez-Lopez E. Increased malondialdehyde in peripheral blood of patients with congestive heart failure. Am Heart J 131: 146-152, 1996.

15.   Draper, HH, Dhanakoti SN, Hadley M, and Piche PA. Malondialdehyde in biological systems. In: Cellular Antioxidant Defense Mechanisms, edited by Chow CK.. Boca Raton, FL: CRC, 1988, vol. 2, p. 97-109.

16.   Garcia, R, and Diebold S. Simple, rapid, and effective method of producing aortocaval shunts in the rats. Cardiovasc Res 24: 430-432, 1990.

17.   Givertz, MM, and Colucci WS. New targets for heart-failure therapy: endothelin, inflammatory cytokines, a oxidative stress. Lancet 352: SI34-SI38, 1998.

18.   Hoppeler, H, and Kayar SR. Capillary and oxidative capacity of muscles. News Physiol Sci 3: 113-116, 1998.

19.   Horton, JR, Udo WE, Precht P, Balakir R, and Hasty K. Cytokine inducible MMP expression in immortalized rat chondrocytes is independent of NO stimulation. In Vitro Cell Devel Biol Animal 34: 378-384, 1998.

20.   Huie, RE, and Padjama S. The reaction of NO with superoxide. Free Radic Res Commun 18: 195-199, 1993.

21.   Janicki, JS, Brower GL, Henegar JR, and Wang L. Ventricular remodeling in heart failure: the role of myocardial collagen. Adv Exp Med Biol 382: 239-245, 1995.

22.   Janicki, JS, Tyagi SC, Matsubara BB, and Campbell SE. Structural and functional consequences of myocardial collagen remodeling. In: Cardiac Adaptation and Failure, edited by Hori M, Maruyama Y, and Reneman RS.. Tokyo: Springer-Verlag, 1994, p. 279-289.

23.   Jones, TG, and Hancock JT. Assay of plasma membrane NADPH oxidase. Methods Enzymol 233: 222-229, 1994.

24.   Knight, JA, Pieper RK, and McClellan L. Specificity of the thiobarbituric acid reaction: its use in studies of lipid peroxidation. Clin Chem 34: 2433-1438, 1988.

25.   Lawrence, RA, and Burk RF. Species, tissue and subcellular distribution of selenium dependent, glutathione peroxidase activity. J Nutr 108: 211-215, 1978.

26.   Laycock, SK, McMurray J, Kane KA, and Parratt JR. Effects of chronic norepinephrine administration on cardiac function in rats. J Cardiovasc Pharmacol 26: 584-589, 1995.

27.   Mehrabi, MR, Ekmekcioglu C, Tatzber F, Oguogho A, Ullrich R, Morgan A, Tamaddon F, Grimm M, Glogar HD, and Simzinger H. The isoprostane, 8-epi-PGF2 alpha is accumulated in coronary arteries isolated from patients with coronary heart disease. Cardiovasc Res 43: 492-499, 1999.

28.   McCord, JM, and Fridovich I. Superoxide dismutase:an enzymatic function for erythrocuprein (hemocuprein). J Biol Chem 244: 6049-6055, 1969.

29.   Michel, J-B, Salzmann J-L, Nlom MO, Bruneval P, Barres D, and Camilleri JP. Morphometric analysis of collagen network and plasma perfused capillary bed in the myocardium of rats during evolution of cardiac hypertrophy. Basic Res Cardiol 81: 142-154, 1986.

30.   Mujumdar, VS, Aru GM, and Tyagi SC. Induction of oxidative stress by homocyst(e)ine impairs endothelial function. J Cell Biochem 82: 491-500, 2001.

31.   Mujumdar, VS, Smiley LM, and Tyagi SC. Activation of matrix metalloproteinase dilates and decreases cardiac tensile strength. Int J Cardiol 79: 277-286, 2001.

32.   Mujumdar, VS, and Tyagi SC. Temporal regulation of ECM components in transition from compensatory hypertrophy to decompensatory heart failure. J Hypertens 17: 261-270, 1999.

33.   Nagase, H, and Woessner JF. Matrix metalloproteinase. J Biol Chem 274: 21491-21494, 1999.

34.   Nagatomo, Y, Carabello BA, Coker ML, McDermott PJ, Nemoto S, Hamawaki M, and Spinale FG. Differential effects of pressure or volume overload on myocardial MMP levels and inhibitory control. Am J Physiol Heart Circ Physiol 278: H151-H161, 2000.

35.   Namba, T, Tsutsui H, Tagawa H, Takahashi M, Saito K, Kozai T, Usui M, Imanaka-Yoshida K, Imaizumi T, and Takeshita A. Regulation of fibrillar collagen gene expression and protein accumulation in volume-overloaded cardiac hypertrophy. Circulation 95: 2448-2454, 1997.

36.   Ono, Y, Ono H, Matsuoka H, Fujimori T, and Frohlich ED. Apotosis, coronary arterial remodeling, and myocardial infarction after nitric oxide inhibition. Hypertension 34: 609-616, 1999.

37.   Prasad, K, Gupta JB, Kalra J, Lee P, Mantha SV, and Bharadwaj B. Oxidative stress as a mechanism of cardiac failure in chronic volume overload in canine model. J Mol Cell Cardiol 28: 375-385, 1996.

38.   Radomski, A, Sawicki G, Olson DM, and Radomski MW. The role of nitric oxide and metalloproteinases in the pathogenesis of hyperoxia-induced lung injury in newborn rats. Br J Pharmacol 125: 1455-1462, 1998.

39.   Rajagopalan, S, Meng XP, Ramasamy S, Harrison DG, and Galis ZS. Reactive oxygen species produced by macrophage-derived foam cells regulate the activity of vascular MMP in vitro. J Clin Invest 98: 2572-2579, 1996.

40.   Ronson, RS, Thourani VH, Ma XL, Katzmark SL, Han D, Zhao ZQ, Nakamura M, Guyton RA, and Venten-Johansen J. Peroxynitrite, the breakdown product of nitric oxide, is beneficial in blood cardioplegia but injurious in crystalloid cardioplegia. Circulation 100: II384-II391, 1999.

41.   Roos, D, Wening RS, and Wyss SR. Protection of human neutrophils by endogenous catalase:studies with cells from catalase-deficient individuals. J Clin Invest 65: 1515-1522, 1980.

42.   Ruzicka, M, Keeley FW, and Leenen FHH The renin-angiotensin system and volume overload-induced changes in cardiac collagen and elastin. Circulation 90: 1989-1996, 1994.

43.   Sasaki, K, Hattori T, Fujisawa T, Takahashi K, Inoue H, and Takigawa M. Nitric oxide mediates IL-1 induced gene expression of MMPs and bFGF in cultured rabbit articular chondrocytes. J Biochem 123: 431-439, 1998.

44.   Senior, RM, Griffin GL, Eliszar CJ, Shapiro SD, Goldberg GI, and Welgus HG. Human 92- and 72-kilodalton type IV collagenases are elastases. J Biol Chem 266: 7870-7875, 1991.

45.   Simon, DI, Mullins ME, Jia L, Gaston B, Singel DL, and Stamler JS. Polynitrosylated proteins: characterization, bioactivity and functional consequences. Proc Natl Acad Sci USA 93: 4736-4741, 1996.

46.   Siwik, DA, Pagano PJ, and Colucci WS. Oxidative stress regulates collagen synthesis and MMP activity in cardiac fibroblasts. Am J Physiol Cell Physiol 280: C53-C60, 2001.

47.   Tarone, RE. A modified Bonferroni method for discrete data (Abstract). Biometrics 46: 515, 1990.

48.   Thomas, SE, Andoh TF, Pichler RH, Shankland SJ, Couser WG, Bennett WM, and Johnson RJ. Accelerated apotosis characterizes cyclosporine-associated interstitial fibrosis. Kidney Int 53: 897-908, 1998.

49.   Treasure, CB, Klein JL, Vita JA, Manoukian SV, Renwick GH, Selwyn AP, Ganz P, and Alexander RW. Hypertension and LVH are associated with impaired endothelium-mediated relaxation in human coronary resistance vessels. Circulation 87: 86-93, 1993.

50.   Tsoporis, J, and Leenen FHH Effects of arterial vasodilators on cardiac hypertrophy and sympathetic activity in rats. Hypertension 11: 376-386, 1988.

51.   Turcani, M, and Jacob R. Minoxidil accelerates heart failure development in rats with ascending aortic constriction. Can J Physiol Pharmacol 76: 613-620, 1998.

52.   Tyagi, SC. Proteinases and myocardial extracellular matrix turnover. Mol Cell Biochem 168: 1-12, 1997.

53.   Tyagi, SC. Physiology of extracellular matrix: cardiovascular adaptation and remodeling. Pathophysiology 7: 177-182, 2000.

54.   Tyagi, SC, Bheemanathini VS, Mandi S, Reddy HK, and Voelker DJ. Role of extracellular matrix metalloproteinases in cardiac remodeling. Heart Failure Rev 1: 73-80, 1996.

55.   Tyagi, SC, Haas SJ, Kumar SG, Reddy HK, Voelker DJ, Hayden MR, Demmy TL, Schmaltz RA, and Curtis JJ. Post-trancriptional regulation of extracellular matrix metalloproteinase in human heart end-stage failure secondary to ischemic cardiomyopathy. J Mol Cell Cardiol 28: 1415-1428, 1996.

56.  Tyagi SC, Hayden MR, and Hall JE. Role of angiotensin in angiogenesis and cardiac fibrosis in heart failure. In: Angiotensin II Receptor Blockade: Physiological and Clinical Implications, edited by Dhalla NS, Zahradka P, Dixon IMC, and Beamish RE. Boston, MA: Kluwer Academic, Progress in Experiential Cardiology, 1998, vol. 2, p. 537-549.

57.   Tyagi, SC, Kumar SG, Voelker DJ, Reddy HK, Janicki JS, and Curtis JJ. Differential gene expression of extracellular matrix components in dilated cardiomyopathy. J Cell Biochem 63: 185-198, 1996.

58.   Tyagi, SC, Kumar SG, and Borders S. Reduction-oxidation (redox) state regulation of extracellular matrix metalloproteinases and tissue inhibitors in cardiac normal and transformed fibroblast cells. J Cell Biochem 61: 139-151, 1996.

59.   Tyagi, SC, Ratajska A, and Weber KT. Myocardial matrix metalloproteinases: localization and activation. Mol Cell Biochem 126: 49-59, 1993.

60.   Tyagi, SC, Smiley LM, and Mujumdar VS. Homocysteine impairs endocardial endothelial function. Can J Physiol Pharmacol 77: 95-957, 1999.

61.   Varin, R, Mulder P, Tamion F, Richard V, Henry JP, Lallemand F, Lerebours G, and Thuillez C. Improvement of endothelial function by chronic ACEI in heart failure: role of NO, prostanoids, oxidant stress and bradykinin. Circulation 102: 351-356, 2000.

62.   Weber, KT, Sun Y, Tyagi SC, and Cleutjens J. Collagen network of the myocardium: function, structural remodeling and regulatory mechanisms. J Mol Cell Cardiol 26: 279-292, 1994.

63.   Werb, Z, Askenas J, MacAuley A, and Wiesen JF. ECM remodeling as a regulator of stromal-epithelial interactions during mamary gland develpment, involution and carcinogenesis. Braz J Med Biol Res 29: 1087-1097, 1996.

64.   Zhang, H, Schmeisser A, Garlichs CD, Plotze K, Damme V, Muge A, and Daniel WG. Angiotensin II-induced superoxide anion generation in human vascular endothelial cells:role of membrane-bound NADH/NADPH-oxidases. Cardiovasc Res 44: 215-222, 1999.

65.   Zhou, XJ, Laszik Z, Wang XQ, Silva FG, and Vaziri ND. Association of renal injury with increased oxygen free radical activity and altered NO metabolism in chronic experimental hemosiderosis. Lab Invest 80: 1905-1914, 2000.

Am J Physiol Heart Circ Physiol 282(4):H1197-H1205
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